Compositions targeting epidermal growth factor receptor and methods for making and using the same

The disclosure describes antibody binding domains for cluster of differentiation 3 T cell receptor (CD3), antibody binding domains for epidermal growth factor receptor (EGFR), cleavable linker sequences, and protease-activatable bispecific fusion proteins such as protease-activatable T cell engagers, as well as uses and methods of treatment.

1. 11. A polypeptide comprising the amino acid sequence of SEQ ID NO:1000.
2. 22. The polypeptide of claim 1, which consists of the amino acid sequence of SEQ ID NO: 1000.
3. 33. A pharmaceutical composition, comprising the polypeptide of claim 1 and one or more pharmaceutically suitable excipients.
4. 44. A method of treating a cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 3, wherein the cancer is characterized with expression of epidermal growth factor receptor (EGFR).
5. 55. The method of claim 4, wherein the cancer is selected from the group consisting of lung cancer, colorectal cancer, head and neck cancer, breast cancer, pancreatic cancer, brain cancer, liver cancer, kidney cancer, ovarian cancer, prostate cancer, esophageal cancer, cervical cancer, and bladder cancer.
6. 66. A polynucleotide encoding the polypeptide of claim 1.
7. 77. A vector, comprising the polynucleotide of claim 6 and a recombinant regulatory sequence operably linked to the polynucleotide.
8. 88. A host cell, comprising the vector of claim 7.

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 18/636,855, filed Apr. 16, 2024, which claims priority to U.S. Provisional Patent Application Ser. No. 63/459,828, filed Apr. 17, 2023; and 63/463,273, filed May 1, 2023; the contents of which are hereby incorporated by reference in their entireties.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on Feb. 25, 2025, is named 385484.xml and is 2,959,226 bytes in size.
BACKGROUND
The epidermal growth factor receptor (EGFR), also known as also known as ErbB1 and HER1, is a receptor tyrosine kinase that is involved in cell proliferation. The overexpression or aberrant activity of EGFR is associated with numerous cancers and is therefore an attractive target for therapeutic intervention. While approved therapies exist, their utility can be hampered by toxicity and/or low stability.
There is a long-felt and yet unmet need for therapeutic intervention of tumors that express EGFR, including stable antibody-based therapeutics that have an improved therapeutic index.
BRIEF DESCRIPTION
The present disclosure provides, among other things, antigen-binding molecules with binding specificity to EGFR, antigen-binding molecules with binding specificity to CD3, as well as bispecific antigen-binding molecules that bind both EGFR and CD3 for use in therapeutic settings in which specific targeting and T cell-mediated killing of EGFR-expressing cells is desired. Aspects disclosed herein address a long-felt unmet need for EGFR-targeting cancer therapeutics, including T cell engagers (TCEs) that have an increased therapeutic index. Aspects of the present disclosure also address the long-felt and yet unmet need for the therapeutic intervention of immunologically cold tumors, e.g., solid tumors, that express EGFR.
In one aspect, the disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds epidermal growth factor receptor (EGFR) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3), wherein the first antigen binding domain comprises: a VH domain comprising a CDR1 amino acid sequence of GGSVSSGDYYWT (SEQ ID NO: 562), a CDR2 amino acid sequence of HIYYSGNTNYNPSLKS (SEQ ID NO: 563), and a CDR3 amino acid sequence of DRVTGAFDI (SEQ ID NO: 564); and at least one of: a proline (P) residue at position 40 in FR2 (alternately referred to as amino acid residue 42 relative to SEQ ID NO: 450), a valine (V) residue at position in position 67 in FR3 (alternately referred to as amino acid residue 69 relative to SEQ ID NO: 450), a valine (V) residue at position 71 in FR3 (alternately referred to as amino acid residue 73 relative to SEQ ID NO: 450), an asparagine (N) residue at position 76 in FR3 (alternately referred to as amino acid residue 78 relative to SEQ ID NO: 450), a valine (V) residue at position 89 in FR3 (alternately referred to as amino acid residue 94 relative to SEQ ID NO: 450), an alanine (A) residue at position 93 in FR3 (alternately referred to as amino acid residue 98 relative to SEQ ID NO: 450), and/or a leucine (L) residue at position 108 in FR4 (alternately referred to as amino acid residue 114 relative to SEQ ID NO: 450), wherein the FR numbering is according to Kabat; and a VL domain comprising a CDR1 amino acid sequence of QASQDISNYLN (SEQ ID NO: 565), a CDR2 amino acid sequence of DASNLET (SEQ ID NO: 566), a CDR3 amino acid sequence of QHFDHLPLA (SEQ ID NO: 567); and wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or EGFR, and wherein the protease-cleavable release segment is cleavable by at least one protease that is present in a tumor.
In some embodiments, the VH domain comprises an asparagine (N) residue at position 76 in FR3. In some embodiments, the VH domain comprises alanine (A) residue at position 93 in FR3. In some embodiments, the VH domain comprises a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, and an alanine (A) residue at position 93 in FR3. In some embodiments, the VH domain comprises a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and a leucine (L) residue at position 108 in FR4.
In some embodiments, the VL domain comprises at least one of: a tyrosine (Y) residue at position 87 in FR3 (alternately referred to as amino acid residue 87 relative to SEQ ID NO: 451) and/or a glutamine (Q) residue at position 100 in FR4 (alternately referred to as amino acid residue 100 relative to SEQ ID NO: 451), wherein the FR numbering is according to Kabat. In some embodiments, the VL domain comprises a tyrosine (Y) residue at position 87 in FR3 and a glutamine (Q) residue at position 100 in FR4.
In some embodiments, the VH domain comprises an amino acid sequence of QVQLQX1X2GX3GLX4KPSETLSLTCX5VX6GGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS, wherein X1 corresponds to E or Q; X2 corresponds to S or W; X3 corresponds to P or A; X4 corresponds to V or L; X5 corresponds to T or A; and X6 corresponds to S or Y (SEQ ID NO: 576); and the VL domain comprises an amino acid sequence of X1X2X3TQSPX4X5LSX6SX7GX8RX9TX10X11CQASQDISNYLNWYQQKPGX12APX13LLIYDASNLET GX14PX15RFSGSGSGTDFTX16TISX17LX18PEDX19AX20YYCQHFDHLPLAFGQGTKVEIK, wherein X1 corresponds to D or E; X2 corresponds to Q or V; X3 corresponds to M or L; X4 corresponds to S. G, or A; X5 corresponds to S or T; X6 corresponds to L or A; X7 corresponds to P or V; X8 corresponds to D or E; X9 corresponds to V or A; X10 corresponds to I or L; X11 corresponds to T or S; X12 corresponds to K or Q; X13 corresponds to K or R; X14 corresponds to V or I; X15 corresponds to S. D. or A; X16 corresponds to F or L; X17 corresponds to S or R; X18 corresponds to Q or E; X19 corresponds to I or F; and X20 corresponds to T or V (SEQ ID NO: 577).
In one aspect, the disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3), wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or EGFR, wherein the protease-cleavable release segment is not capable of being cleaved by legumain in human plasma, or wherein legumain cleaves the protease-cleavable release segment in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In one aspect, the disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds epidermal growth factor receptor (EGFR) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3), wherein the chimeric polypeptide has a melting temperature (Tm) of greater than 62° C. and/or a thermostability ratio of greater than 0.5 at 62° C.; wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or EGFR, and wherein the protease-cleavable release segment is cleavable by at least one protease that is present in a tumor.
In some embodiments, the Tm is determined by differential scanning fluorimetry (DSF).
In some embodiments, the thermostability ratio is determined by: i) incubating an input amount of a chimeric polypeptide at 62° C. for 30 minutes thereby denaturing a fraction of the input amount of chimeric polypeptide; ii) measuring an amount of monomeric chimeric polypeptide remaining following step i); and iii) dividing the amount of monomeric chimeric polypeptide by the input amount of the chimeric polypeptide to generate the thermostability ratio.
In some embodiments, the amount of monomeric chimeric polypeptide is measured by mass spectrometry.
In one aspect, the disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds a cancer cell antigen and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3), wherein the second antigen binding domain comprises: a VH domain comprising a CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), a CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and a CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and a VL domain comprising a CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), a CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and a CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6), wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or the cancer cell antigen, and wherein the protease-cleavable release segment is cleavable by at least one protease that is present in a tumor.
In some embodiments, the second antigen binding domain comprises: (i) the VL domain comprising the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF SGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127); and (ii) the VH domain comprising the amino acid sequence of









(SEQ ID NO: 126)



EVOLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGK



 



GLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMN



 



SLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS.






In some embodiments, the cancer cell antigen is human alpha 4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, EGFR, HER2, HER3, HER4, PD-L1, prostate-specific membrane antigen (PSMA), CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16 BhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3; 9—O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, GD2, carbonicanhydrase IX, CD44v6, Sonic Hedgehog (Shh), Wue-1, plasma cell antigen 1, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, 6-transmembrane epithelial antigen of prostate (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6, desmoglein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA-125), Müellerian inhibitory substance receptor type II (MISIIR), sialylated Tn antigen (sTN), fibroblast activation antigen (FAP), endosialin (CD248), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMPs, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, or EphA2.
In some embodiments, the cancer cell antigen is EGFR.
In some embodiments, chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (first antigen binding domain)-(second antigen binding domain)-(linker)-(mask polypeptide), (second antigen binding domain)-(first antigen binding domain)-(linker)-(mask polypeptide), (mask polypeptide)-(linker)-(first antigen binding domain)-(second antigen binding domain), or (mask polypeptide)-(linker)-(second antigen binding domain)-(first antigen binding domain), wherein each—is a covalent connection or a polypeptide linker.
In some embodiments, the mask polypeptide is an extended length non-natural polypeptide (ELNN).
In some embodiments, the linker further comprises a spacer.
In some embodiments, the protease-cleavable release segment is fused to the bispecific antibody domain via the spacer.
In some embodiments, the spacer is characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
In some embodiments, the spacer is from 9 to 14 amino acids in length.
In some embodiments, the spacer comprises at least 4 types of amino acids selected from the group consisting of G. A. S. T, E, and P.
In some embodiments, the amino acids of the spacer consists of A, E, G, S. P. and/or T.
In some embodiments, the spacer is cleavable by a non-mammalian protease.
In some embodiments, the non-mammalian protease is Glu-C.
In some embodiments, the spacer comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.
In some embodiments, the spacer comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES (SEQ ID NO: 96) or GTATPESGPG (SEQ ID NO:97).
In some embodiments, the protease-cleavable release segment comprises an amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N. In some embodiments, X is S.
In some embodiments, chimeric polypeptide comprises a first mask polypeptide joined to the first antigen binding domain via a first linker wherein the first linker comprises a first protease cleavable release segment (RS1) cleavable by at least one protease present in a tumor; and a second mask polypeptide joined to the second antigen binding domain via a second linker wherein the second linker comprises a second protease cleavable release segment (RS2) cleavable by at least one protease present in a tumor.
In some embodiments, chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (Mask1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(Mask2), (Mask1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(Mask2), (Mask2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(Mask1), or (Mask2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(Mask1), wherein each—is, individually, a covalent bond or a polypeptide linker.
In some embodiments, the first mask polypeptide is a first ELNN (ELNN1) and the second mask polypeptide is a second ELNN (ELNN2).
In some embodiments, chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(ELNN2), (ELNN1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(ELNN2), (ELNN2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(ELNN1), or (ELNN2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(ELNN1), wherein each—is, individually, a covalent bond or a polypeptide linker.
In some embodiments, Linker1 further comprises a first spacer (Spacer1).
In some embodiments, Linker2 further comprises a second spacer (Spacer2).
In some embodiments, RS1 is fused to the bispecific antibody domain via Spacer1 and/or RS2 is fused to the bispecific antibody domain via Spacer2.
In some embodiments, chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(RS1)-(Spacer1)-(first antigen binding domain)-(second antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(Spacer1)-(second antigen binding domain)-(first antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN2)-(RS2)-(Spacer2)-(first antigen binding domain)-(second antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), or (ELNN2)-(RS2)-(Spacer2)-(second antigen binding domain)-(first antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), wherein each—is a, individually, covalent bond or a polypeptide linker.
In some embodiments, Spacer 1 and/or the Spacer2 is characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
In some embodiments, Spacer 1 and/or the Spacer2 is from 9 to 14 amino acids in length.
In some embodiments, Spacer 1 and/or the Spacer2 comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
In some embodiments, the amino acids of Spacer1 and/or the Spacer2 consists of A, E, G, S, P, and/or T.
In some embodiments, Spacer 1 and/or the Spacer2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.
In some embodiments, Spacer 1 and/or the Spacer2 comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES (SEQ ID NO:96) or GTATPESGPG (SEQ ID NO:97).
In some embodiments, the amino acid sequence of the first ELNN is between 250 amino acids and 350 amino acids in length, and wherein the amino acid sequence of the second ELNN is between 500 amino acids and 600 amino acids in length.
In some embodiments, the amino acid sequence of the first ELNN is 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is 582 amino acids in length.
In some embodiments, RS1 and/or RS2 comprises an amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N. In some embodiments, X is S.
In one aspect, the disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that has binding specificity to a cancer cell antigen, and a second antigen binding domain that has binding specificity to an effector cell antigen expressed on an effector cell, wherein the chimeric polypeptide further comprises a first ELNN joined to the first antigen binding domain via a first linker comprising a first protease-cleavable release segment (RS1) positioned between the first ELNN and the first antigen binding domain such that the first ELNN is capable of reducing the binding of the first antigen binding domain to the cancer cell antigen, wherein the RS1 is cleavable by at least one protease that is present in a tumor, wherein the chimeric polypeptide further comprises a second ELNN joined to the second antigen binding domain via a second linker comprising second protease-cleavable release segment (RS2) positioned between the second ELNN and the second antigen binding domain such that the second ELNN is capable of reducing the binding of the first antigen binding domain to the effector cell antigen, wherein the RS2 is cleavable by at least one protease that is present in a tumor, wherein the first ELNN has a shorter amino acid sequence than the second ELNN, and wherein the cancer cell antigen is EGFR.
In some embodiments, chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(ELNN2), (ELNN1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(ELNN2), (ELNN2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(ELNN1), or (ELNN2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(ELNN1), wherein each—is, individually, a covalent bond or a polypeptide linker.
In some embodiments, Linker1 further comprises a first spacer (Spacer 1).
In some embodiments, Linker2 further comprises a second spacer (Spacer2).
In some embodiments, RS1 is fused to the bispecific antibody domain via Spacer1 and/or RS2 is fused to the bispecific antibody domain via Spacer2.
In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(RS1)-(Spacer1)-(first antigen binding domain)-(second antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(Spacer1)-(second antigen binding domain)-(first antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN2)-(RS2)-(Spacer2)-(first antigen binding domain)-(second antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), or (ELNN2)-(RS 2)-(Spacer2)-(second antigen binding domain)-(first antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), wherein each—is a, individually, covalent bond or a polypeptide linker.
In one aspect, the disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, comprising the formulas that comprises from the N-terminal side to the C-terminal side: Formula 1: (Mask1)-(RS1)-(Spacer1)-(first antigen binding domain)-[antibody domain linker]-(second antigen binding domain); Formula 2: (first antigen binding domain)-[antibody domain linker]-(second antigen binding domain)-(Spacer2)-(RS2)-(Mask2); or Formula 3: (Mask1)-(RS1)-(Spacer1)-(first antigen binding domain)-[antibody domain linker]-(second antigen binding domain)-(Spacer2)-(RS2)-(Mask2), wherein, the first antigen binding domain has binding specificity to a cancer cell antigen; the second antigen binding domain has binding specificity to an effector cell antigen expressed on an effector cell; each—comprises, individually, a covalent connection or a polypeptide linker; the Mask1 is a polypeptide that is capable of reducing binding of the first antigen binding domain to its target; the Mask2 is a polypeptide that is capable of reducing binding of the second antigen binding domain to its target; if the chimeric polypeptide comprises Formula I then the Spacer1 consists of A, E, G, S. P, and/or T residues, if the chimeric polypeptide comprises Formula 2 then the Spacer2 consists of A, E, G, S. P. and/or T residues, and if the chimeric polypeptide comprises Formula 3 then the Spacer1 and/or the Spacer2 consists of A, E, G. S. P. and/or T residues; and wherein the cancer cell antigen is EGFR.
In some embodiments, each—is, individually, a covalent connection. In some embodiments, each—is, individually, a covalent bond. In some embodiments, each—is a peptide bond. In some embodiments, each—is, individually, a polypeptide linker of no more than 5 amino acids.
In some embodiments, the second antigen binding domain has binding specificity to human CD3 and cynomolgus monkey CD3. In some embodiments, the second antigen binding domain has binding specificity to human CD3. In some embodiments, the effector cell antigen is cluster of differentiation 3 T cell receptor (CD3). In some embodiments, the CD3 is CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta. In some embodiments, the CD3 is CD3 epsilon.
In some embodiments, the Mask1 is a first ELNN and the Mask2 is a second ELNN.
In some embodiments, the Spacer 1 and/or the Spacer2 is characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S. T, E, and P.
In some embodiments, the Spacer 1 and/or the Spacer2 is from 9 to 14 amino acids in length.
In some embodiments, the Spacer 1 and/or the Spacer2 comprises at least 4 types of amino acids selected from the group consisting of G. A. S. T. E, and P.
In some embodiments, the amino acids of the Spacer1 and/or the Spacer2 consists of A, E, G, S, P, and/or T.
In some embodiments, the Spacer1 and/or the Spacer2 is cleavable by a non-mammalian protease.
In some embodiments, the non-mammalian protease is Glu-C.
In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.
In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES (SEQ ID NO:96) or GTATPESGPG (SEQ ID NO:97).
In some embodiments, the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN.
In some embodiments, the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN.
In some embodiments, the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN.
In some embodiments, the amino acid sequence of the first ELNN is between 250 amino acids and 350 amino acids in length, and wherein the amino acid sequence of the second ELNN is between 500 amino acids and 600 amino acids in length.
In some embodiments, the amino acid sequence of the first ELNN is 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is 582 amino acids in length.
In some embodiments, the first antigen binding domain comprises a first antibody or an antigen-binding fragment thereof, and wherein the second antigen binding domain comprises a second antibody or an antigen-binding fragment thereof.
In some embodiments, the first antigen binding domain is a Fab, an scFv, or an ISVD.
In some embodiments, the second antigen binding domain is a Fab, an scFV, or an ISVD.
In some embodiments, the ISVD is a VHH domain.
In some embodiments, the first antigen binding domain is an scFV.
In some embodiments, the second antigen binding domain is an scFV.
In some embodiments, there is an antibody domain linker between the first antigen binding domain and the second antigen binding domain.
In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table A or B.
In some embodiments, the antibody domain linker consists of G and S amino residues.
In some embodiments, the antibody domain linker is 6-12 residues in length.
In some embodiments, the antibody domain linker comprises the amino acid sequence









(SEQ ID NO: 87)



GGGGS



or



 



(SEQ ID NO: 125)



GGGGSGGGS.






In some embodiments, the first antigen binding domain and/or the second antigen binding domain comprise an scFv comprising a VL domain, a VH domain, and a linker between the VL domain and the VH domain, wherein the linker consists of A, E, G, S, P, and/or T residues.
In some embodiments, the linker is characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
In some embodiments, the linker between the VL domain and the VH domain is from 25 to 35 amino acids in length.
In some embodiments, the linker between the VL domain and the VH domain comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
In some embodiments, the amino acids of the linker between the VL domain and the VH domain consists of A, E, G, S, P, and/or T.
In some embodiments, the linker between the VL domain and the VH domain is cleavable by a non-mammalian protease.
In some embodiments, the non-mammalian protease is Glu-C.
In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).
In some embodiments, the second antigen binding domain comprises the following CDRs: a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSX1GAVTX2SNYAN (SEQ ID NO:8023), wherein X1 corresponds to T or N, and X2 corresponds to T or S; a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP (SEQ ID NO:4); a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX4NLWV (SEQ ID NO:8024), wherein X4 corresponds to S or P; a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX8TYAMN (SEQ ID NO:8025), wherein X8 corresponds to S or N; a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRX10KX11NX12YATYYADSVKX13 (SEQ ID NO: 8026), wherein X10 corresponds to T or S. X11 corresponds to R or Y, X12 corresponds to D or N, and X13 corresponds to G or D; a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HX14NFGNSYVSWFAX15 (SEQ ID NO:8027), wherein X14 corresponds to E or G, and X15 corresponds to H or Y.
In some embodiments, the second antigen binding domain comprises: a VH domain comprising a CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), a CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and a CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and a VL domain comprising a CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), a CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and a CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6).
In some embodiments, the second antigen binding domain comprises: a VH domain comprising an amino acid sequence of EVOLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYA DSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126); and a VL domain comprising an amino acid sequence of









(SEQ ID NO: 127)



ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ



 



APRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVY



 



YCALWYPNLWVFGGGTKLTVL






In some embodiments, the first antigen binding domain comprises the following CDRs: a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QASQDISNYLN (SEQ ID NO:565); a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to DASNLET (SEQ ID NO:566); a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QHFDHLPLA (SEQ ID NO:567); a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GGSVSSGDYYWT (SEQ ID NO:562); a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HIYYSGNTNYNPSLKS (SEQ ID NO:563); and a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to DRVTGAFDI (SEQ ID NO:564).
In some embodiments, the VH domain comprises at least one of: a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and/or a leucine (L) residue at position 108 in FR4, wherein the FR numbering is according to Kabat. In some embodiments, the VH domain comprises an asparagine (N) residue at position 76 in FR3. In some embodiments, the VH domain comprises alanine (A) residue at position 93 in FR3. In some embodiments, the VH domain comprises a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, and an alanine (A) residue at position 93 in FR3. In some embodiments, the VH domain comprises a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and a leucine (L) residue at position 108 in FR4.
In some embodiments, the VL domain comprises at least one of: a tyrosine (Y) residue at position 87 in FR3 and/or a glutamine (Q) residue at position 100 in FR4, wherein the FR numbering is according to Kabat. In some embodiments, the VL domain comprises a tyrosine (Y) residue at position 87 in FR3 and a glutamine (Q) residue at position 100 in FR4.
In some embodiments, the first antigen binding domain comprises a VH domain comprising an amino acid sequence of SEQ ID NO: 576 and a VL domain comprising an amino acid sequence of SEQ ID NO: 577.
In some embodiments, the first antigen binding domain comprises: i) a VH domain comprising an amino acid sequence of SEQ ID NO: 468 and a VL domain comprising an amino acid sequence of SEQ ID NO: 469; ii) a VH domain comprising an amino acid sequence of SEQ ID NO: 466 and a VL domain comprising an amino acid sequence of SEQ ID NO: 467; iii) a VH domain comprising an amino acid sequence of SEQ ID NO: 490 and a VL domain comprising an amino acid sequence of SEQ ID NO: 491; iv) a VH domain comprising an amino acid sequence of SEQ ID NO: 492 and a VL domain comprising an amino acid sequence of SEQ ID NO: 493; v) a VH domain comprising an amino acid sequence of SEQ ID NO: 514 and a VL domain comprising an amino acid sequence of SEQ ID NO: 515; vi) a VH domain comprising an amino acid sequence of SEQ ID NO: 516 and a VL domain comprising an amino acid sequence of SEQ ID NO: 517; vii) a VH domain comprising an amino acid sequence of SEQ ID NO: 538 and a VL domain comprising an amino acid sequence of SEQ ID NO: 539; or viii) a VH domain comprising an amino acid sequence of SEQ ID NO: 540 and a VL domain comprising an amino acid sequence of SEQ ID NO: 541.
In some embodiments, the VL domain is N-terminal to the VH domain. In some embodiments, the VL domain is C-terminal to the VH domain.
In some embodiments, the second antigen binding domain comprises a scFV comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:







(SEQ ID NO: 128)


ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL


 


IGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLW


 


VFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQLVESG


 


GGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRND


 


YATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGN


 


SYVSWFAHWGQGTLVTVSS.






In some embodiments, the first antigen binding domain comprises a scFV comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:







(SEQ ID NO: 449)


DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY


 


DASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAF


 


GQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPG


 


LVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNT


 


NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDI


 


WGQGTLVTVSS.






In some embodiments, the RS comprises a protease cleavage site is cleavable by at least one protease listed in Table 6.
In some embodiments, the RS comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 7a.
In some embodiments, the RS is cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.
In some embodiments, the RS is not cleavable by legumain.
In some embodiments, the RS is not cleavable by legumain in human blood, plasma, or serum.
In some embodiments, the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours.
In some embodiments, the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.
In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, the RS1 and/or RS2 comprises protease cleavage is cleavable by at least one protease listed in Table 6.
In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 7a.
In some embodiments, the RS1 and/or RS2 is cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.
In some embodiments, the RS1 and/or RS2 is not cleavable by legumain.
In some embodiments, the RS1 and/or RS2 is not cleavable by legumain in human blood, plasma, or serum.
In some embodiments, the RS1 and/or RS2 is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours.
In some embodiments, the RS1 and/or RS2 is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.
In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
In some embodiments, the RS1 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
In some embodiments, the RS2 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
In some embodiments, RS1 and/or RS2 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSASHTPAGLTGP (SEQ ID NO: 7628).
In some embodiments, the RS1 and the RS2 are the same.
In some embodiments, the RS1 and the RS2 are different.
In some embodiments, the first ELNN and the second ELNN are each individually characterized in that: (i) at least 90% of each of the first ELNN's and the second ELNN's amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and (ii) each comprises at least 3 types of amino acids selected from the group consisting of G, A. S. T. E, and P.
In some embodiments, the first ELNN and the second ELNN are each individually further characterized in that: (i) each comprises at least 100 amino acid residues; (ii) each comprises a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acids in length, wherein the plurality of non-overlapping sequence motifs comprise a set of non-overlapping sequence motives, wherein each non-overlapping sequence motive of the set of non-overlapping sequence motifs is repeated at least two times in the ELNN.
In some embodiments, the plurality of non-overlapping sequence motifs comprises at least one non-overlapping sequence motif that occurs only once within the ELNN.
In some embodiments, the non-overlapping sequence motifs comprise one of or any combination of the sequence motifs listed in Table 1.
In some embodiments, the non-overlapping sequence motifs comprise at least 2, 3, or 4 of the sequence motifs listed in Table 1.
In some embodiments, the non-overlapping sequence motifs comprise any one of or any combination of GTSTEPSEGSAP (SEQ ID NO:189), GTSESATPESGP (SEQ ID NO:188), GSGPGTSESATP (SEQ ID NO:8028), GSEPATSGSETP (SEQ ID NO:187), GSPAGSPTSTEE (SEQ ID NO: 186), and GTSPSATPESGP (SEQ ID NO:8029).
In some embodiments, each of the first ELNN and the second ELNN comprises at least 4 types of amino acids selected from the group consisting of G. A. S. T. E, and P.
In some embodiments, the amino acids of each of the first ELNN and the second ELNN consists of A, E, G, S, P, and/or T.
In some embodiments, the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is between 250 amino acids and 350 amino acids in length, and wherein the amino acid sequence of the second ELNN is between 500 amino acids and 600 amino acids in length. In some embodiments, the amino acid sequence of the first ELNN is 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is 582 amino acids in length.
In some embodiments, the first ELNN and/or the second ELNN comprises an amino acid sequence that is at least 85% identical to an amino acid sequence listed in Table 3a or 3b.
In some embodiments, the first ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:







(SEQ ID NO: 8021)


ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTS


 


ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPA


 


GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG


 


SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA


 


TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP


 


TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT


 


P.






In some embodiments, the second ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:







(SEQ ID NO: 8022)


ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT


 


SGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS


 


EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT


 


STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE


 


SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES


 


GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG


 


PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP


 


GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG


 


TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS


 


PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS


 


PSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST


 


EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA.






In some embodiments, the chimeric polypeptide comprises one or more barcode fragments.
In some embodiments, the chimeric polypeptide comprises two or more barcode fragments.
In some embodiments, each barcode fragment is different from every other barcode fragment.
In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that are releasable from the chimeric polypeptide upon complete digestion the chimeric polypeptide by a non-mammalian protease.
In some embodiments, the non-mammalian protease is Glu-C.
In some embodiments, the chimeric polypeptide comprises a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG (SEQ ID NO:8030). SGSETPGT (SEQ ID NO:8031), and GTSESATP (SEQ ID NO:8032).
In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences: SGPE.SGPGXnSGPE.SGPG (SEQ ID NO:8033). SGPE.SGPGXnATPE.SGPG (SEQ ID NO: 8034). SGPE.SGPGXnGTSE.SATP (SEQ ID NO:8036). SGPE.SGPGXnTTPE.SGPG (SEQ ID NO: 8037). SGPE.SGPGXnSTPE.SGPG (SEQ ID NO:8038). SGPE.SGPGXnGTPE.SGPG (SEQ ID NO: 8039). SGPE.SGPGXnGTPE.TPGS (SEQ ID NO:8040). SGPE.SGPGXnGTPE.TPGS (SEQ ID NO: 8040). SGPE.SGPGXnSGSE.TGTP (SEQ ID NO:8041), SGPE.SGPGXnGTPE.GSAP (SEQ ID NO: 8042). SGPE.SGPGXnEPSE.SATP (SEQ ID NO:8043). ATPE.SGPGXnSGPE.SGPG (SEQ ID NO: 8044). ATPE.SGPGXnATPE.SGPG (SEQ ID NO:8045), ATPE.SGPGXnGTSE.SATP (SEQ ID NO: 8047). ATPE.SGPGXnTTPE.SGPG (SEQ ID NO:8049). ATPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8051). ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:8053). ATPE.SGPGXnGTPE.TPGS (SEQ ID NO: 8055). ATPE.SGPGXnSGSE.TGTP (SEQ ID NO:8056), ATPE.SGPGXnGTPE.GSAP (SEQ ID NO: 8057). ATPE.SGPGXnEPSE.SATP (SEQ ID NO:8058), GTSE.SATPXnSGPE.SGPG (SEQ ID NO: 8059). GTSE.SATPXnATPE.SGPG (SEQ ID NO:8060), GTSE.SATPXnGTSE.SATP (SEQ ID NO: 8061). GTSE.SATPXnTTPE.SGPG (SEQ ID NO:8062). GTSE.SATPXnSTPE.SGPG (SEQ ID NO: 8063). GTSE.SATPXnGTPE.SGPG (SEQ ID NO:8064), GTSE.SATPXnGTPE.TPGS (SEQ ID NO: 8065). GTSE.SATPXnSGSE.TGTP (SEQ ID NO:8066). GTSE.SATPXnGTPE.GSAP (SEQ ID NO: 8067). GTSE.SATPXnEPSE.SATP (SEQ ID NO:8068). TTPE.SGPGXnSGPE.SGPG (SEQ ID NO: 8069). TTPE.SGPGXnATPE.SGPG (SEQ ID NO:8070). TTPE.SGPGXnGTSE.SATP (SEQ ID NO: 8071). TTPE.SGPGXnTTPE.SGPG (SEQ ID NO:8072). TTPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8074). TTPE.SGPGXnGTPE.SGPG (SEQ ID NO:8075). TTPE.SGPGXnGTPE.TPGS (SEQ ID NO: 8076). TTPE.SGPGXnSGSE.TGTP (SEQ ID NO:8077). TTPE.SGPGXnGTPE.GSAP (SEQ ID NO: 8078). TTPE.SGPGXnEPSE.SATP (SEQ ID NO:8079), STPE.SGPGXnSGPE.SGPG (SEQ ID NO: 8080). STPE.SGPGXnATPE.SGPG (SEQ ID NO:8081), STPE.SGPGXnGTSE.SATP (SEQ ID NO: 8082). STPE.SGPGXnTTPE.SGPG (SEQ ID NO:8083). STPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8084). STPE.SGPGXnGTPE.SGPG (SEQ ID NO:8086). STPE.SGPGXnGTPE.TPGS (SEQ ID NO: 8087). STPE.SGPGXnSGSE.TGTP (SEQ ID NO:8088). STPE.SGPGXnGTPE.GSAP (SEQ ID NO: 8089). STPE.SGPGXnEPSE.SATP (SEQ ID NO:8090). GTPE.SGPGXnSGPE.SGPG (SEQ ID NO: 8091). GTPE.SGPGXnATPE.SGPG (SEQ ID NO:8092), GTPE.SGPGXnGTSE.SATP (SEQ ID NO: 8093). GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:8094). GTPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8096). GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:8098). GTPE.SGPGXnGTPE.TPGS (SEQ ID NO: 8100). GTPE.SGPGXnSGSE.TGTP (SEQ ID NO:8101). GTPE.SGPGXnGTPE.GSAP (SEQ ID NO: 8102). GTPE.SGPGXnEPSE.SATP (SEQ ID NO:8103), GTPE.TPGSXnSGPE.SGPG (SEQ ID NO: 8104). GTPE.TPGSXnATPE.SGPG (SEQ ID NO:8105), GTPE.TPGSXnGTSE.SATP (SEQ ID NO: 8106). GTPE.TPGSXnTTPE.SGPG (SEQ ID NO:8107), GTPE.TPGSXnSTPE.SGPG (SEQ ID NO: 8108). GTPE.TPGSXnGTPE.SGPG (SEQ ID NO:8109). GTPE.TPGSXnGTPE.TPGS (SEQ ID NO: 8110). GTPE.TPGSXnSGSE.TGTP (SEQ ID NO:8111), GTPE.TPGSXnGTPE.GSAP (SEQ ID NO: 8113), GTPE.TPGSXnEPSE.SATP (SEQ ID NO:8114), SGSE.TGTPXnSGPE.SGPG (SEQ ID NO: 8115). SGSE.TGTPXnATPE.SGPG (SEQ ID NO:8116). SGSE.TGTPXnGTSE.SATP (SEQ ID NO: 8117). SGSE.TGTPXnTTPE.SGPG (SEQ ID NO:8118). SGSE.TGTPXnSTPE.SGPG (SEQ ID NO: 8119). SGSE.TGTPXnGTPE.SGPG (SEQ ID NO:8120). SGSE.TGTPXnGTPE.TPGS (SEQ ID NO: 8121). SGSE.TGTPXnSGSE.TGTP (SEQ ID NO:8122). SGSE.TGTPXnGTPE.GSAP (SEQ ID NO: 8123). SGSE.TGTPXnEPSE.SATP (SEQ ID NO:8124). GTPE.GSAPXnSGPE.SGPG (SEQ ID NO: 8125), GTPE.GSAPXnATPE.SGPG (SEQ ID NO:8126). GTPE.GSAPXnGTSE.SATP (SEQ ID NO: 8127). GTPE.GSAPXnTTPE.SGPG (SEQ ID NO:8128), GTPE.GSAPXnSTPE.SGPG (SEQ ID NO: 8129). GTPE.GSAPXnGTPE.SGPG (SEQ ID NO:8130), GTPE.GSAPXnGTPE.TPGS (SEQ ID NO: 8131). GTPE.GSAPXnSGSE.TGTP (SEQ ID NO:8132), GTPE.GSAPXnGTPE.GSAP (SEQ ID NO: 8133). GTPE.GSAPXnEPSE.SATP (SEQ ID NO:8134). EPSE.SATPXnSGPE.SGPG (SEQ ID NO: 8136). EPSE.SATPXnATPE.SGPG (SEQ ID NO:8137). EPSE.SATPXnGTSE.SATP (SEQ ID NO: 8138). EPSE.SATPXnTTPE.SGPG (SEQ ID NO:8139), EPSE.SATPXnSTPE.SGPG (SEQ ID NO: 8140). EPSE.SATPXnGTPE.SGPG (SEQ ID NO:8141). EPSE.SATPXnGTPE.TPGS (SEQ ID NO: 8142). EPSE.SATPXnSGSE.TGTP (SEQ ID NO:8143). EPSE.SATPXnGTPE.GSAP (SEQ ID NO: 8144), or EPSE.SATPXnEPSE.SATP (SEQ ID NO:8145), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50.
In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences: SGPE.SGPGXnATPE.SGPG (SEQ ID NO:8035). ATPE.SGPGXnGTSE.SATP (SEQ ID NO: 8048). ATPE.SGPGXnTTPE.SGPG (SEQ ID NO:8050), ATPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8052). ATPE.SGPGXnATPE.SGPG (SEQ ID NO:8046), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO: 8054). ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:8054). ATPE.SGPGXnATPE.SGPG (SEQ ID NO: 8046). GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:8099), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8097). GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:8095), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8097). GTPE.TPGSXnSGSE.TGTP (SEQ ID NO:8112), GTPE.GSAPXnEPSE.SATP (SEQ ID NO: 8135), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:8054), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO: 8054), ATPE.SGPGXnATPE.SGPG (SEQ ID NO:8046), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO: 8054), TTPE.SGPGXnTTPE.SGPG (SEQ ID NO:8073), or STPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8085), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 30.
In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 3 to 7. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is 4.
In some embodiments, Xn is PGTGTSAT (SEQ ID NO:8146), PGSGPGT (SEQ ID NO:8147), PGTTPGTT (SEQ ID NO:8148), PGTPPTST (SEQ ID NO:8149), PGTSPSAT (SEQ ID NO:8150), PGTGSAGT (SEQ ID NO:8151), PGTGGAGT (SEQ ID NO:8152), PGTSPGAT (SEQ ID NO:8153), PGTSGSGT (SEQ ID NO:8154), PGTSSAST (SEQ ID NO:8155), PGTGAGTT (SEQ ID NO:8156), PGTGSTST (SEQ ID NO:8157), GSEPATSG (SEQ ID NO:8158), APGTSTEP (SEQ ID NO:8159), PGTAGSGT (SEQ ID NO:8160), PGTSSGGT (SEQ ID NO:8161), PGTAGPAT (SEQ ID NO:8162), PGTPGTGT (SEQ ID NO:8163), PGTGGPTT (SEQ ID NO:8164), or PGTGSGST (SEQ ID NO:8165).
In some embodiments, X11 is TGTS (SEQ ID NO:8166), SGP. TTPG (SEQ ID NO:8167), TPPT (SEQ ID NO:8168), TSPS (SEQ ID NO:8169), TGSA (SEQ ID NO:8170), TGGA (SEQ ID NO: 8171), TSPG (SEQ ID NO:8172), TSGS (SEQ ID NO:8173), TSSA (SEQ ID NO:8174), TGAG (SEQ ID NO: 8175), TGST (SEQ ID NO:8176), EPAT (SEQ ID NO:8177), GTST (SEQ ID NO:8178), TAGS (SEQ ID NO:8179), TSSG (SEQ ID NO:8180), TAGP (SEQ ID NO:8181), TPGT (SEQ ID NO: 8182), TGGP (SEQ ID NO:8183), or TGSG (SEQ ID NO:8184).
In some embodiments, neither the N-terminal amino acid nor the C-terminal amino acid of the chimeric polypeptide is included in a barcode fragment.
In some embodiments, the chimeric polypeptide comprises an ELNN with a non-overlapping sequence motif that occurs only once within the ELNN, wherein the ELNN further comprises a barcode fragment that includes at least part of the non-overlapping sequence motif that occurs only once within the ELNN.
In some embodiments, the chimeric polypeptide comprises a first ELNN with a first barcode fragment and a second ELNN with a second barcode fragment, wherein neither the first barcode fragment nor the second barcode fragment includes a glutamate that is immediately adjacent to another glutamate, if present, in the ELNN that contains the barcode fragment.
In some embodiments, at least one of the barcode fragments comprises a glutamate at the C-terminus thereof.
In some embodiments, at least one of the barcode fragments has an N-terminal amino acid that is immediately preceded by a glutamate in the chimeric polypeptide.
In some embodiments, the glutamate that precedes the N-terminal amino acid of the barcode fragment is not immediately adjacent to another glutamate.
In some embodiments, at least one of the barcode fragments does not include a second glutamate at a position other than the C-terminus of the barcode fragment unless the second glutamate is immediately followed by a proline.
In some embodiments, the chimeric polypeptide comprises a single polypeptide chain, wherein the chimeric polypeptide comprises a barcode fragment that is at a position within the polypeptide chain that is from 10 to 200 amino acids or from 10 to 125 amino acids from the N-terminus or the C-terminus of the chimeric polypeptide.
In some embodiments, the first ELNN is at the N-terminal side of the bispecific antibody domain, and wherein the first barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the N-terminus of the chimeric polypeptide.
In some embodiments, the second ELNN is at the C-terminal side of the bispecific antibody domain, and wherein the second barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.
In some embodiments, at least one of the barcode fragments is at least 4 amino acids in length. In some embodiments, at least one of the barcode fragments is from 4 to 20, from 5 to 15, from 6 to 12, or from 7 to 10 amino acids in length.
In some embodiments, each mask polypeptide comprises one barcode fragment that is listed in Table 2 or disclosed in Table 3a.
In some embodiments, the chimeric polypeptide comprises a barcode fragment comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SGPGSGPGTSE (SEQ ID NO:78) or SGPGTSPSATPE (SEQ ID NO:79).
In some embodiments, the chimeric polypeptide comprises one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGSGPGTSE (SEQ ID NO:78) and one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGTSPSATPE (SEQ ID NO: 79).
In some embodiments, the barcode fragment consists of A, E, G, S, P, and/or T residues.
In some embodiments, the barcode fragment is part of a mask peptide.
In some embodiments, the mask peptide is the first ELNN or the second ELNN.
In one aspect, the disclosure provides a chimeric polypeptide, comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:







(SEQ ID NO: 1000)


ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSE


 


SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS


 


PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT


 


STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES


 


GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE


 


GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA


 


SHTPAGLTGPGTSESATPESDIQMTQSPSSLSASVGDRVTITCQASQDIS


 


NYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP


 


EDIATYYCQHFDHLPLAFGQGTKVEIKSESATPESGPGTSPGATPESGPG


 


TSESATPQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPP


 


GKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA


 


VYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQEPSLTVSPGGTVT


 


LTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL


 


EGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGP


 


GTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFS


 


TYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNT


 


LYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATP


 


ESGPGEAGRSASHTPAGLTGPATPESGPGTSESATPESGPGSPAGSPTST


 


EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


 


GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT


 


STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP


 


ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP


 


SEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT


 


STEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


 


TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


 


GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS


 


PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE


 


SATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS


 


PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGE


 


PEA.






In some embodiments, the chimeric polypeptide comprises the following amino acid sequence:







(SEQ ID NO: 1000)


ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSE


 


SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS


 


PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT


 


STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES


 


GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE


 


GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA


 


SHTPAGLTGPGTSESATPESDIQMTQSPSSLSASVGDRVTITCQASQDIS


 


NYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP


 


EDIATYYCQHFDHLPLAFGQGTKVEIKSESATPESGPGTSPGATPESGPG


 


TSESATPQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPP


 


GKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA


 


VYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQEPSLTVSPGGTVT


 


LTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL


 


EGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGP


 


GTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFS


 


TYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNT


 


LYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATP


 


ESGPGEAGRSASHTPAGLTGPATPESGPGTSESATPESGPGSPAGSPTST


 


EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


 


GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT


 


STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP


 


ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP


 


SEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT


 


STEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


 


TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


 


GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS


 


PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE


 


SATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS


 


PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGE


 


PEA.






In one aspect, the disclosure provides a pharmaceutical composition comprising the chimeric polypeptide described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
In one aspect, the disclosure provides an injection device comprising the pharmaceutical composition described herein. In some embodiments, injection device comprises a syringe.
In one aspect, the disclosure provides a polynucleotide sequence encoding the chimeric polypeptide described herein.
In one aspect, the disclosure provides an expression vector comprising the polynucleotide sequence encoding the chimeric polypeptide described herein.
In one aspect, the disclosure provides a host cell comprising the expression vector comprising the polynucleotide sequence encoding the chimeric polypeptide described herein.
In one aspect, the disclosure provides a method of producing the chimeric polypeptide described herein. In some embodiments, the method further comprises isolating the chimeric polypeptide from a host cell.
In one aspect, the disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of the chimeric polypeptide described herein to the subject.
In some embodiments, the cancer comprises a solid tumor.
In some embodiments, the cancer is a carcinoma, a sarcoma, or a melanoma.
In some embodiments, the cancer expresses EGFR.
In some embodiments, the cancer overexpresses EGFR.
In some embodiments, the cancer comprises cells that express, on average, at least 3,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; 100,000; or 200,000EGFR proteins per cell.
In some embodiments, the cancer comprises cells having one or more oncogenic mutations in an EGFR gene.
In some embodiments, the cancer comprises cells having an EGFR gene amplification.
In some embodiments, the cells comprise a 2 to 5-fold, 2 to 10-fold, 2 to 15-fold, 2 to 30-fold, 2 to 50-fold, 3 to 5-fold, 3 to 10-fold, 3 to 15-fold, 3 to 30-fold, 3 to 50-fold, 5 to 10-fold, 5 to 15-fold, 5 to 30-fold, or 5 to 50-fold increase in EGFR gene copy number as compared to a non-cancerous cell of the same tissue type.
In some embodiments, the cancer is lung cancer, colorectal cancer, head and neck cancer, breast cancer, pancreatic cancer, brain cancer, liver cancer, kidney cancer, ovarian cancer, prostate cancer, esophageal cancer, cervical cancer, or bladder cancer.
In some embodiments, the cancer is lung cancer.
In some embodiments, the lung cancer is non-small cell lung cancer.
In some embodiments, the cancer is colorectal cancer.
In some embodiments, the cancer is head and neck squamous cell carcinoma.
In some embodiments, the cancer is breast cancer.
In some embodiments, the cancer is triple-negative breast cancer.
In some embodiments, the cancer is brain cancer.
In some embodiments, the brain cancer is glioblastoma.
In some embodiments, the method further comprises administering a checkpoint inhibitor to the subject.
In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
In some embodiments, the checkpoint inhibitor is pembrolizumab or cemiplimab.
In one aspect, the disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds EGFR, comprising: a VH domain comprising a CDR1 amino acid sequence of GGSVSSGDYYWT (SEQ ID NO: 562), a CDR2 amino acid sequence of HIYYSGNTNYNPSLKS (SEQ ID NO: 563), and a CDR3 amino acid sequence of DRVTGAFDI (SEQ ID NO: 564); and at least one of: a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and/or a leucine (L) residue at position 108 in FR4, wherein the FR numbering is according to Kabat; and a VL domain comprising a CDR1 amino acid sequence of QASQDISNYLN (SEQ ID NO: 565), a CDR2 amino acid sequence of DASNLET (SEQ ID NO: 566), a CDR3 amino acid sequence of QHFDHLPLA (SEQ ID NO: 567).
In some embodiments, the VH domain comprises an asparagine (N) residue at position 76 in FR3. In some embodiments, the VH domain comprises alanine (A) residue at position 93 in FR3. In some embodiments, the VH domain comprises a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, and an alanine (A) residue at position 93 in FR3. In some embodiments, the VH domain comprises a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and a leucine (L) residue at position 108 in FR4.
In some embodiments, the VL domain comprises at least one of: a tyrosine (Y) residue at position 87 in FR3 and/or a glutamine (Q) residue at position 100 in FR4, wherein the FR numbering is according to Kabat. In some embodiments, the VL domain comprises a tyrosine (Y) residue at position 87 in FR3 and a glutamine (Q) residue at position 100 in FR4.
In some embodiments, antibody or an antigen-binding fragment comprises a VH domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 576; and a VL domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 577.
In some embodiments, the antibody comprises: i) a VH domain comprising an amino acid sequence of SEQ ID NO: 468 and a VL domain comprising an amino acid sequence of SEQ ID NO: 469; ii) a VH domain comprising an amino acid sequence of SEQ ID NO: 466 and a VL domain comprising an amino acid sequence of SEQ ID NO: 467; iii) a VH domain comprising an amino acid sequence of SEQ ID NO: 490 and a VL domain comprising an amino acid sequence of SEQ ID NO: 491; iv) a VH domain comprising an amino acid sequence of SEQ ID NO: 492 and a VL domain comprising an amino acid sequence of SEQ ID NO: 493; v) a VH domain comprising an amino acid sequence of SEQ ID NO: 514 and a VL domain comprising an amino acid sequence of SEQ ID NO: 515; vi) a VH domain comprising an amino acid sequence of SEQ ID NO: 516 and a VL domain comprising an amino acid sequence of SEQ ID NO: 517; vii) a VH domain comprising an amino acid sequence of SEQ ID NO: 538 and a VL domain comprising an amino acid sequence of SEQ ID NO: 539; or viii) a VH domain comprising an amino acid sequence of SEQ ID NO: 540 and a VL domain comprising an amino acid sequence of SEQ ID NO: 541.
In one aspect, the disclosure provides an anti-CD3 antibody or an antigen-binding fragment thereof, comprising the following CDRs: a VH domain comprising a CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), a CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and a CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and a VL domain comprising a CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), a CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and a CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6).
In some embodiments, the VL domain comprises the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF SGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127); and the VH domain comprises the amino acid sequence of







(SEQ ID NO: 126)


EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR


 


IRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR


 


HENFGNSYVSWFAHWGQGTLVTVSS.






Various features of this disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a schematic representation of an exemplary EGFR-targeted paTCE.
FIG. 2 depicts a schematic representation of a proposed mechanism of action of the exemplary paTCEs of the disclosure.
FIG. 3 depicts a schematic representation of fully unmasked paTCE (a uTCE) and singly masked metabolites paTCE (1x-N) and paTCE (1x-C) from an exemplary paTCE as shown in FIG. 1.
FIG. 4 depicts a schematic representation of an antibody framework screen. To identify anti-EGFR antigen-binding fragments with improved properties, the CDRs of a donor anti-EGFR antibody, panitumumab, were grafted in a combinatorial manner into the framework regions from approved monoclonal antibody therapies. A paTCE library having the anti-EGFR antigen-binding fragments was screened for stability, expression, and binding.
FIG. 5A-FIG. 5D depicts the results from a screen of EGFR-targeted paTCEs for thermal stability (FIG. 5A), binding affinity (FIG. 5B), and a thermostability ratio representing the amount of thermostable monomer remaining at 62° C. as compared to the amount of protein input (FIG. 5C). FIG. 5D depicts a simulated ribbon structure of an anti-EGFR antibody fragment associated with EGFR.
FIG. 6 depicts PTE score evaluations using internal PTE algorithm v22 for anti-CD3 antibody fragments.
FIG. 7A depicts an alignment of the RSR-2295 and RSR-3213 amino acid sequences and proteases capable of cleaving them. FIG. 7B depicts in vitro protease digestion of paTCEs employing RSR-2295 or RSR-3213. The RSR-3213 sequence is modified to substantially reduce cleavage by legumain.
FIG. 8A and FIG. 8B depict relative plasma stability of paTCEs employing RSR-2295 or RSR-3213, measured at Day 0 and Day 7. In FIG. 8A. RSR-2295 employed the SCy5.5 fluorophore and RSR-3213 employed the Scy7.5 fluorophore. In FIG. 8B, the RSR-2295 employed the Scy7.5 fluorophore and RSR-3213 employed the Scy5.5 fluorophore. FIG. 8C depicts the observed cleavability in vivo from tumor homogenates from 3 different mouse tumor models. For each set of bar graphs (i.e., % 1×-C, % 1×-N, % uTCE), each bar from left to right represents B1, B2, B3, B4, A1, A2, A3, A4, 43-1, 43-2, 43-3, and 43-4. B1-B4 represent 4 different mice from a first tumor model (NCI-N87). A1-A4 represent 4 different mice from a second tumor model (HT-29). 43-1-43-4 represent 4 different mice from a third tumor model (HT-55). FIG. 8D depicts the % of total for the 3 metabolites plus the paTCE (paTCE, 1×-N, 1×-C, and uTCE) when employing RSR-2295 or RSR-3213.
FIG. 9 depicts relative tumor uptake of paTCEs employing RSR-2295 or RSR-3213. The plasma: tumor ratio was calculated in 3 different mouse tumor models (4 mice per tumor model). There is a “Mouse 1” for each of the 3 different tumor models, a “Mouse 2” for each of the 3 different tumor models, a “Mouse 3” for each of the different tumor models, and a “Mouse 4” for each of the 3 different tumor models.
FIG. 10A-FIG. 10C depicts cytotoxicity curves for exemplary donor cells HT-29 (FIG. 10A), MDA-MB-231 (FIG. 10B), and A-431 (FIG. 10C)
FIG. 11A-FIG. 11D depicts in vitro cytokine induction assays from a representative HT-29 donor. The induction of IFNγ (FIG. 11A), TNFα (FIG. 11B), IL-6 (FIG. 11C), and IL-10 (FIG. 11D) are shown.
FIG. 12 depicts expression of CD69, CD25, and PD-1 expression in CD4+ and CD8+ T cells.
FIG. 13 depicts in vitro plasma stability of AMX-525 from samples in healthy human donors, human cancer donors (8 pancreatic, 2 head and neck, 4 ovarian), healthy cynomolgus monkeys, healthy mice, and tumor-bearing mice (HT-29-implanted CDX).
FIG. 14 depicts tumor growth curves in mice bearing HT-29 tumors.
FIG. 15 depicts tumor growth curves in mice bearing LoVo tumors.
FIG. 16 depicts immunohistochemistry (IHC) images and corresponding quantification of CD8+ T cells in tumor tissue from a LoVo xenograft mouse model.
FIG. 17 depicts IHC images and corresponding quantification of CD4+ T cells in tumor tissue from a LoVo xenograft mouse model.
FIG. 18 depicts IHC images and corresponding quantification of PD-L1 expression in tumor tissue from a LoVo xenograft mouse model.
FIG. 19 depicts tumor growth curves in mice bearing MDA-MB-231 tumors.
FIG. 20 depicts the efficacy of AMX-525 as indicated by tumor growth curves in mice bearing SK—OV-3 ovarian tumors. NSG-MHC I/II DKO mice were inoculated subcutaneously with SK—OV-3 tumor cells (Day 0), engrafted with PBMCs (Day 18), and treated with the indicated test articles on days denoted by the arrows.
FIG. 21 depicts the efficacy of the combination of AMX-525 and an anti-PD-1 antibody,



Pembrolizumab, as indicated by tumor growth curves in mice bearing SK—OV-3 ovarian tumors. NSG-MHC I/II DKO mice were inoculated subcutaneously with SK—OV-3 tumor cells (Day 0), engrafted with PBMCs (Day 18), and treated with the indicated test articles on days denoted by the arrows.
DETAILED DESCRIPTION
There is a significant unmet need in cancer therapeutics for an EGFR-targeted bispecific treatment modality that is efficacious against solid tumors, particularly solid tumors that are present in an immunologically cold microenvironment. While TCEs have been shown to be effective in inducing remission in certain cancers, they have not led to the development of widespread therapeutics due to their extreme potency and on target, off tumor toxicities in healthy tissues.
Without being bound by any scientific theory. TCEs form a bridge between T cells and tumor cells and activate T cell-mediated killing of the tumor cells and further initiating a cytokine amplification cascade. The cytokine amplification cascade can promote further killing of tumor cells and potentially provide long term immunity. T cells activated by TCEs release cytolytic perforin/granzymes in a manner that is independent of antigen-MHC recognition. This creates a two-fold response: direct tumor cell death and amplification of tumor killing through initiation of a powerful cytokine response from the tumor cells. The direct tumor cell death results in release of tumor antigens. The cytokine response may include, among others, increased interferon-y which stimulates CD8 T cell activity and stimulates antigen presentation by APCs; increased IL2 which causes increased proliferation of activated T-cells, and increased CXCL9 and 10 response which increases T cell recruitment. Together the release of tumor antigens and the initiation of the cytokine response results in activation of the endogenous T cell response which potentially causes epitope spreading to induce long term immunity.
One toxicity challenge with TCEs arises out the fact that many tumor targets are, to some extent, also expressed in healthy tissue, and normal cells also can produce the cytokines response resulting in cytokine release syndrome (CRS). These two powerful responses of health tissue to T cell activation by TCEs often results in an overall lack of acceptable therapeutic index for these agents.
The present disclosure provides protease-activatable TCEs (paTCEs) that address an unmet need and are superior in one or more aspects including enhanced terminal half-life, improved stability, targeted delivery, and/or improved therapeutic ratio with reduced toxicity to healthy tissues compared to conventional antibody therapeutics or bispecific antibody therapeutics that are active upon injection.
Included herein are compounds, compositions and methods that overcome the drawbacks in the existing TCEs by providing paTCEs that target EGFR (referred to herein as EGFR-paTCEs and exemplified as AMX-525).
AMX-525 comprises the amino acid sequence set forth as SEQ ID NO: 1000. Without being bound by any scientific theory, the paTCEs described herein are understood to exploit the dysregulated protease activity present in tumors vs. healthy tissues, enabling expansion of the therapeutic index. The paTCE core comprises antigen binding domains; one targets CD3 and the other targets EGFR. The two antigen-binding domains may, in exemplary embodiments, be in two different antibody formats (such as, e.g., a single chain antibody fragment (scFv) and a VHH), or the same antibody format (such as, e.g., scFvs). Many different antibody fragments or formats may be used.
In some embodiments, an EGFR-targeting paTCE comprises a first portion that is an scFv that binds to EGFR and a second portion that is an scFv that binds to CD3. One or more (e.g., two) unstructured polypeptide masks are attached to the core. In some embodiments, these unstructured polypeptide masks sterically reduce target engagement of either the tumor target and/or CD3, and also extend protein half-life. In some embodiments, the unstructured polypeptide masks are extended length non-natural polypeptides (ELNNs).
In some embodiments, the properties of ELNNs also minimize the potential for immunogenicity, as their lack of stable tertiary structures disfavors antibody binding, and the absence of hydrophobic, aromatic, and positively charged residues that serve as anchor residues for peptide MHC II binding reduces the potential for T cell epitopes.
In some embodiments, protease cleavage sites at the base of the ELNN or ELNNs enable proteolytic activation of paTCEs in the tumor microenvironment, unleashing a smaller, highly potent TCEs that are capable redirecting cytotoxic T cells to kill target-expressing tumor cells. In some embodiments, in healthy tissues, where protease activity is tightly regulated, paTCEs remain predominantly inactive, thus expanding the therapeutic index compared to unmasked TCEs.
In some embodiments, in addition to localized activation, the short half-life of the unmasked TCE form further widens the therapeutic index while providing the potency of T-cell immunity to improve the eradication of solid tumors. In some embodiments, the release sites used in the paTCEs can be cleaved across a broad array of tumors by proteases that are collectively involved in every cancer hallmark (growth; survival and death; angiogenesis; invasion and metastasis; inflammation; and immune evasion). Thus, TCE activity of the paTCEs is localized to tumors by exploiting the enhanced protease activity that is upregulated in all stages of cancer and tumor development but is tightly regulated in healthy tissues.
Terminology
As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof, unless the context clearly dictates otherwise.
Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B.” “A or B.” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and C”; “A and B”; “B and C”; “A” (alone); “B” (alone); and “C” (alone).
It is understood that wherever aspects are described herein with the language “comprising.” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.
Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, the term indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±10%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.01%.
With respect to naturally occurring compounds, the term “isolated” refers to a compound (i.e., a polypeptide or polynucleotide) that is not in its native state (e.g., free to varying degrees from components that naturally accompany the compound in nature). No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. “Isolate” and “isolated” may also denote a degree of separation from an original source or surrounding, depending on context.
The term “polypeptide” refers to any polymer of two or more amino acids. Thus, the terms peptide, dipeptide, tripeptide, oligopeptide, protein, amino acid chain, or any other term used to refer to a chain of two or more amino acids, is included within the definition of “polypeptide.” The term “polypeptide” also encompasses an amino acid polymer that has been modified (e.g., by post-translational modification), for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Depending on context, the term “polypeptide” may also be used to refer to a protein comprising two or more polymers of two or more amino acids.
A “host cell” includes an individual cell (e.g., in culture) which that comprises an exogenous polynucleotide. Host cells may include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to naturally occurring or genetically engineered variation.
A “fusion” or “chimeric” polypeptide or protein comprises a first polypeptide portion linked to a second polypeptide portion with which it is not naturally linked in nature. In some embodiments, the portions may normally exist in separate proteins and are brought together in the fusion polypeptide; they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide; or the portions may be brought together from different sources. In some embodiments, a fusion or chimeric protein comprises two or more moieties that do not occur in nature (e.g., are created, designed, or otherwise generated by humans, such as binding domains, masks, linkers, barcodes, and other polypeptides provided herein). A chimeric protein may be created, for example, by chemical synthesis, or by recombinant expression (e.g., comprising creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship).
“Conjugated”, “linked.” “fused.” and “fusion” may be used interchangeably herein, depending on context. These terms may refer to the covalent joining together of two more chemical (e.g., polypeptide) elements or components, by whatever means including chemical conjugation or recombinant means.
As known in the art, “sequence identity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. Similarly, “sequence identity” between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide to the sequence of a second polynucleotide. The terms “% identical”, “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids (as applicable) which are identical in an optimal alignment between the sequences to be compared. Said percentage may be purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. For example, the optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using the algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website. In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, −2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the parameters are set, of course, such that the percentage of identity is calculated over the full-length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
As used herein, the terms “mask polypeptide”, “mask”, and “masking moiety” refer to a polypeptide that is capable of reducing the binding of an antigen binding domain (e.g., an antibody) to the target antigen in the context of a fusion protein (such as a chimeric polypeptide) provided herein. Exemplary mask polypeptides include, but are not limited to, the ELNN polypeptides described herein. Additional mask polypeptides include albumin, polypeptides consisting of proline, serine and alanine, coiled-coil domains, albumin binding domains, Fc domains, and binding domains with specificity to conserved regions of an antibody variable domain. Mask polypeptides are described in further detail in Lucchi et al. (ACS Cent Sci. 2021 May 26; 7 (5): 724-738).
As used herein, the terms “ELNN polypeptides” and “ELNNs” are synonymous and refer to extended length polypeptides comprising non-naturally occurring, substantially non-repetitive sequences (e.g., polypeptide motifs) that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. ELNN polypeptides include unstructured hydrophilic polypeptides comprising repeating motifs of 6 natural amino acids (G, A. P. E. S, and/or T). In some embodiments, an ELNN polypeptide comprises multiple motifs of 6 natural amino acids (G, A. P. E. S. T), wherein the motifs are the same or comprise a combination of different motifs. In some embodiments, ELNN polypeptides can confer certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties when linked to proteins, including T-cell engagers as disclosed herein. Such desirable properties may include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics, as well as improved therapeutic index. ELNN polypeptides are known in the art, and non-limiting descriptions relating to and examples of ELNN polypeptides known as XTEN® polypeptides are available in Schellenberger et al., (2009) Nat Biotechnol 27 (12): 1186-90; Brandl et al., (2020) Journal of Controlled Release 327:186-197; and Radon et al., (2021) Advanced Functional Materials 31, 2101633 (pages 1-33), the entire contents of each of which are incorporated herein by reference.
In some embodiments, the repetitiveness of an ELNN sequence refers to the 3-mer repetitiveness and can be measured by computer programs or algorithms or by other means known in the art. In some embodiments, the 3-mer repetitiveness of an ELNN may be assessed by determining the number of occurrences of the overlapping 3-mer sequences within the polypeptide. For example, a polypeptide of 200 amino acid residues has 198 overlapping 3-amino acid sequences (3-mers), but the number of unique 3-mer sequences will depend on the amount of repetitiveness within the sequence. In some embodiments, the score can be generated (hereinafter “subsequence score”) that is reflective of the degree of repetitiveness of the 3-mers in the overall polypeptide sequence. In this context, “subsequence score” means the sum of occurrences of each unique 3-mer frame across a 200 consecutive amino acid sequence of the polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence. Examples of such subsequence scores derived from the first 200 amino acids of repetitive and non-repetitive polypeptides are presented in Example 73 of International Patent Application Publication No. WO 2010/091122 A1, which is incorporated by reference in its entirety.
In some embodiments, and in the context of ELNNs, a “substantially non-repetitive sequence,” refers to an ELNN sequence, wherein (1) there are few or no instances of four identical amino acids in a row in the ELNN sequence and wherein (2) the ELNN has a subsequence score (defined in the preceding paragraph herein) of 12, or 10 or less or that there is not a pattern in the order, from N- to C-terminus, of the sequence motifs that constitute the polypeptide sequence.
The term “single chain variable fragment” (scFV) corresponds to an antigen binding domain composed of at least one heavy chain variable domain (VH) linked to at least one light chain variable domain (VL). The VH and VL may be linked with any art recognized linker, including, but not limited to, SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO:81). In some embodiments, the scFV comprises, from N-terminus to C-terminus, a VH domain and a VL domain. In other embodiments, the scFv comprises, from N-terminus to C-terminus, a VL domain and a VH domain. Tandem scFvs, such as divalent scFvs (di-scFvs), are scFvs including multiple scFvs linked in tandem. Di-scFvs include two VH and two VL domains, each scFvs having either the same or differing (e.g., bispecific) target specificity. In some embodiments, an scFv described herein is a monovalent scFv or a divalent scFv.
The term “immunoglobulin single variable domain” (ISVD), defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins (e.g. monoclonal antibodies) or their fragments (such as Fab, Fab′, F(ab′)2, scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation, whereas in an ISVD only 3 CDRs from a single domain are contributing to the antigen binding site formation.
In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab′)2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.
In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH, a single VHH or single VL domain.
As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).
An immunoglobulin single variable domain (ISVD) can for example be a heavy chain ISVD, such as a VH, VHH, including a camelized VH or humanized VHH. In some embodiments, it is a VHH, including a camelized VH or humanized VHH. Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.
For example, the immunoglobulin single variable domain may be a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a “dAb” or dAb (or an amino acid sequence that is suitable for use as a dAb); other single variable domains, or any suitable fragment of any one thereof.
In some embodiments, the immunoglobulin single variable domain may be a NANOBODY® molecule or a suitable antigen-binding fragment thereof. NANOBODY® is a registered trademark of Ablynx N.V.
“VHH domains”, also known as VHHs, VHH regions, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin variable domain of “heavy chain antibodies” (i.e., of “antibodies devoid of light chains”; Hamers-Casterman et al. Nature 363:446-448, 1993). The term “VHH domain” has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH domains,” “VH regions”, and “VHs”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains”, “VL regions”, and “VLs”). For a further description of VHHs, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).
A “vector” is a nucleic acid molecule that transfers an inserted nucleic acid molecule into and/or between host cells. In some embodiments, a vector self-replicates in an appropriate host. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be used for the transcription of mRNA that is translated into a polypeptide(s). In some embodiments, an “expression system” is a suitable host cell comprising an expression vector that can function to yield a desired expression product.
The terms “treatment” or “treating.” and “ameliorating” may be used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit. By “therapeutic benefit” is meant eradication or amelioration of the underlying disorder being treated. In some embodiments, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease condition such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. In some embodiments, a therapeutic benefit comprises slowing or halting the growth of one or more tumors. In some embodiments, a therapeutic benefit comprises reducing the size of one or more tumors. In some embodiments, a therapeutic benefit comprises eradicating one or more tumors from a subject. In some embodiments, a therapeutic benefit comprises effecting the death of cancer cells.
As used herein, the term “therapeutically effective amount” refers to an amount of a biologically active agent (such as a fusion protein provided herein, e.g., as part of a pharmaceutical composition), that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. The disease condition can refer to a disorder or a disease, e.g., cancer or a symptom of cancer.
Antigen Binding Domains, Cleavage Sequences, Barcode Fragments, and Fusion Polypeptides
The present disclosure provides, inter alia, new and useful anti-EGFR antibodies, new and useful anti-CD3 antibodies, cleavage sequences, barcode fragments, and fusion proteins comprising the same. Included herein are fusion polypeptides comprising (i) one or more mask polypeptides (such as ELNNs), (ii) a bispecific antibody (BsAb, e.g., a TCE) linked to the mask polypeptide(s), and (iii) one or more protease-cleavable release segments (RS), wherein an RS is positioned between the mask polypeptide(s) and the BsAb.
In some embodiments, anti-EGFR antibodies provided herein include a VH domain comprising the sequence: QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSL KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS (SEQ ID NO: 468), and a VL domain comprising the sequence:







(SEQ ID NO: 469)


DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD


 


ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQ


 


GTKVEIK.






In some embodiments, anti-CD3 antibodies provided herein comprise a VH domain comprising the CDRs of a VH domain comprising the sequence:







(SEQ ID NO: 126)


EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR


 


IRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR


 


HENFGNSYVSWFAHWGQGTLVTVSS






and/or a VL domain comprising the CDRs of a VL domain comprising the sequence:








(SEQ ID NO: 127)


ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLI


 


GGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVF


 


GGGTKLTVL.






Also provided are BsAbs comprising, e.g., anti-EGFR antibodies and/or anti-CD3 antibodies disclosed herein. In some embodiments, the bispecific antibodies comprise the VH and VL regions of an anti-EGFR antibody region disclosed herein. In some embodiments, the BsAbs comprise the VH and VL regions of an anti-CD3 antibody disclosed herein. In some embodiments, the BsAbs comprise an anti-EGFR scFv region comprising a VH and VL pair disclosed herein and an anti-CD3 scFV comprising a VH and VL pair disclosed herein. In some embodiments, the BsAbs are TCEs.
In some embodiments, the fusion polypeptide comprises a first ELNN (such as an ELNN described herein). In some embodiments, the polypeptide further comprises a second ELNN (such as an ELNN described herein). In some embodiments, the polypeptide comprises an ELNN at or near its N-terminus (an “N-terminal ELNN”). In some embodiments, the polypeptide comprises an ELNN at or near its C-terminus (a “C-terminal ELNN”). In some embodiments, the polypeptide comprises both an N-terminal ELNN and a C-terminal ELNN.
In some embodiments, a fusion polypeptide comprises a BsAb and a first ELNN is attached to the N-terminus of the BsAb by a first RS and a second ELNN is attached to the C-terminus of the BsAb by a second RS. In some embodiments, each RS is cleavable by a protease mentioned herein. In some embodiments, each RS comprises an RS sequence disclosed herein. In some embodiments, the fusion polypeptide is a paTCE.
Included herein are polypeptide sequences that may be used, e.g., to link one polypeptide moiety to another within a fusion protein. For example, useful linkers are provided that are cleaved by multiple proteases but not legumain. In some embodiments, such linkers may be used outside the context of antibodies such as those described herein.
In some embodiments, a fusion polypeptide (e.g., one or more ELNNs of a paTCE and/or another portion of a fusion polypeptide such as a linker or spacer sequence) can comprise one or more barcode fragments (e.g., as described herein) releasable (e.g., configured to be released) the fusion polypeptide upon cleavage or digestion of the fusion polypeptide (e.g., a paTCE) by a protease. In some embodiments, the protease is a non-mammalian protease. In some embodiments, each barcode fragment differs in sequence and molecular weight from all other peptide fragments (including all other barcode fragments if present) that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease, thereby making it unique and making its presence detectable through techniques such as mass spectrometry.
Extended Recombinant Polypeptides (ELNNs)
Chain Length and Amino Acid Composition
In some embodiments, an ELNN comprises at least 100, or at least 150 amino acids. In some embodiments, an ELNN is from 100 to 3,000, or from 150 to 3,000 amino acids in length. In some embodiments, an ELNN is from 100 to 1,000, or from 150 to 1,000 amino acids in length. In some embodiments, an ELNN is at least (about) 100, at least (about) 150, at least (about) 200, at least (about) 250, at least (about) 300, at least (about) 350, at least (about) 400, at least (about) 450, at least (about) 500, at least (about) 550, at least (about) 600, at least (about) 650, at least (about) 700, at least (about) 750, at least (about) 800, at least (about) 850, at least (about) 900, at least (about) 950, at least (about) 1,000, at least (about) 1,100, at least (about) 1,200, at least (about) 1,300, at least (about) 1,400, at least (about) 1,500, at least (about) 1,600, at least (about) 1,700, at least (about) 1,800, at least (about) 1,900, or at least (about) 2,000 amino acids in length. In some embodiments, an ELNN is at most (about) 100, at most (about) 150, at most (about) 200, at most (about) 250, at most (about) 300, at most (about) 350, at most (about) 400, at most (about) 450, at most (about) 500, at most (about) 550, at most (about) 600, at most (about) 650, at most (about) 700, at most (about) 750, at most (about) 800, at most (about) 850, at most (about) 900, at most (about) 950, at most (about) 1,000, at most (about) 1,100, at most (about) 1,200, at most (about) 1.300, at most (about) 1,400, at most (about) 1,500, at most (about) 1,600, at most (about) 1,700, at most (about) 1,800, at most (about) 1,900, or at most (about) 2,000 amino acids in length. In some embodiments, an ELNN has (about) 100, (about) 150, (about) 200, (about) 250, (about) 300, (about) 350, (about) 400, (about) 450, (about) 500, (about) 550, (about) 600, (about) 650, (about) 700, (about) 750, (about) 800, (about) 850, (about) 900, (about) 950, (about) 1,000, (about) 1,100, (about) 1.200, (about) 1,300, (about) 1,400, (about) 1,500, (about) 1,600, (about) 1,700, (about) 1,800, (about) 1,900, or (about) 2.000 amino acids in length, or of a range between any two of the foregoing. In some embodiments, at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P). In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P). In some embodiments, an ELNN comprises at least 3 different types of amino acids selected from the group consisting of G. A. S. T. E, and P. In some embodiments, an ELNN comprises at least 4 different types of amino acids selected from the group consisting of G, A. S. T. E, and P. In some embodiments, an ELNN comprises at least 5 different types of amino acids selected from the group consisting of G, A. S. T. E, and P. In some embodiments, an ELNN consists of amino acids selected from the group consisting of G, A. S. T. E, and P. In some embodiments, an ELNN comprises G. A. S. T. E, or P amino acids. In some embodiments, an ELNN (e.g., ELNN1, ELNN2, etc.) is characterized in that: (i) it comprises at least 100, or at least 150 amino acids; (ii) at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P); and (iii) it comprises at least 4 different types of the amino acids from G, A, S. T. E, or P. As used herein, the term “glutamate” is a synonym for “glutamic acid,” and refers to the glutamic acid residue whether or not the side-chain carboxyl is deprotonated. In some embodiments, the ELNN-containing fusion polypeptide comprises a first ELNN and a second ELNN. In some embodiments, the sum of the total number of amino acids in the first ELNN and the total number of amino acids in the second ELNN is at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids.
Non-Overlapping Sequence Motif
In some embodiments, the ELNN comprises, or is formed from, a plurality of non-overlapping sequence motifs. In some embodiments, at least one of the non-overlapping sequence motifs is recurring (or repeated at least two times in the ELNN). In some embodiments, the ELNN comprises at least one other non-overlapping sequence motif that is non-recurring (or found only once within the ELNN). In some embodiments, the plurality of non-overlapping sequence motifs comprises (a) a set of (recurring) non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least two times in the ELNN; and (b) a non-overlapping (non-recurring) sequence motif that occurs (or is found) only once within the ELNN. In some embodiments, each non-overlapping sequence motif is from 9 to 14 (or 10 to 14, or 11 to 13) amino acids in length. In some embodiments, each non-overlapping sequence motif is 12 amino acids in length. In some embodiments, the plurality of non-overlapping sequence motifs comprises a set of non-overlapping (recurring) sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is (1) repeated at least two times in the ELNN; and (2) is between 9 and 14 amino acids in length. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprise 12-mer sequence motifs identified herein by SEQ ID NOs: 179-200 and 1715-1722 in Table 1. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprise 12-mer sequence motifs identified herein by SEQ ID NOs: 186-189 in Table 1. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprise at least two, at least three, or all four of 12-mer sequence motifs of SEQ ID NOs: 186-189 in Table 1. In some embodiments, an ELNN further comprises a sequence other than a 12-mer sequence motif shown in Table 1. In some embodiments, an ELNN comprises a sequence that is not in Table 1 such as ASSATPESGP (SEQ ID NO:8185), GSGPGTSESATP (SEQ ID NO:8028), or GTSESATP (SEQ ID NO:8032). In some embodiments, an ELNN comprises a sequence that is not in Table 1 such as ATPESGP (SEQ ID NO:8186), GTSPSATPESGP (SEQ ID NO:8029), or GTSESAGEPEA (SEQ ID NO:8187). In some embodiments, an ELNN comprises a barcode sequence.







TABLE 1







Exemplary 12-Mer Sequence Motifs for


Construction of ELNNs











Motif Family*
Amino Acid Sequence
SEQ ID NO.







AD
GESPGGSSGSES
 182



 



AD
GSEGSSGPGESS
 183



 



AD
GSSESGSSEGGP
 184



 



AD
GSGGEPSESGSS
 185



 



AE, AM
GSPAGSPTSTEE
 186



 



AE, AM, AQ
GSEPATSGSETP
 187



 



AE, AM, AQ
GTSESATPESGP
 188



 



AE, AM, AQ
GTSTEPSEGSAP
 189



 



AF, AM
GSTSESPSGTAP
 190



 



AF, AM
GTSTPESGSASP
 191



 



AF, AM
GTSPSGESSTAP
 192



 



AF, AM
GSTSSTAESPGP
 193



 



AG, AM
GTPGSGTASSSP
 194



 



AG, AM
GSSTPSGATGSP
 195



 



AG, AM
GSSPSASTGTGP
 196



 



AG, AM
GASPGTSSTGSP
 197



 



AQ
GEPAGSPTSTSE
 198



 



AQ
GTGEPSSTPASE
 199



 



AQ
GSGPSTESAPTE
 200



 



AQ
GSETPSGPSETA
 179



 



AQ
GPSETSTSEPGA
 180



 



AQ
GSPSEPTEGTSA
 181



 



BC
GSGASEPTSTEP
1715



 



BC
GSEPATSGTEPS
1716



 



BC
GTSEPSTSEPGA
1717



 



BC
GTSTEPSEPGSA
1718



 



BD
GSTAGSETSTEA
1719



 



BD
GSETATSGSETA
1720



 



BD
GTSESATSESGA
1721



 



BD
GTSTEASEGSAS
1722







*Denotes individual motif sequences that, when used together in various permutations, results in a “family sequence”






Unstructured Polypeptide Confirmation

In various embodiments, an ELNN component (or the ELNN components) of a fusion protein has an unstructured conformation under physiological conditions, regardless of the length (e.g., extended length) of the polymer. For example, the ELNN is characterized by a large conformational freedom of the peptide backbone. In some embodiments, the ELNN is characterized by a lack of long-range interactions as determined by NMR. In some embodiments, the present disclosure provides ELNNs that, under physiologic conditions, resemble the structure of denatured sequences largely devoid in secondary structure. In some embodiments, the ELNNs can be substantially devoid of secondary structure under physiologic conditions. “Largely devoid,” as used in this context, means that less than 50% of the ELNN amino acid residues of the ELNN contribute to secondary structure as measured or determined by the means described herein. “Substantially devoid,” as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the ELNN amino acid residues of the ELNN sequence do not contribute to secondary structure, as measured or determined by the means described herein.
A variety of methods have been established in the art to discern the presence or absence of secondary and tertiary structures in a given polypeptide. In some embodiments, ELNN secondary structure can be measured spectrophotometrically, e.g., by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm). Secondary structure elements, such as alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra. Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13:222-45) and the Garnier-Osguthorpe-Robson (“GOR”) algorithm (Garnier J, Gibrat J F, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553), as described in US Patent Application Publication No. 20030228309A1 (the entire contents of which are incorporated herein by reference). For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as the total and/or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation (which lacks secondary structure).
In some embodiments, the ELNNs used in a fusion protein composition can have an alpha-helix percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. In some embodiments, the ELNNs of the fusion protein compositions can have a beta-sheet percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. In some embodiments, the ELNNs of the fusion protein compositions can have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. In some embodiments, the ELNNs of the fusion protein compositions will have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2%. In some embodiments, the ELNNs of the fusion protein compositions can have a high degree of random coil percentage, as determined by a GOR algorithm. In some embodiments, an ELNN can have at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% random coil, as determined by a GOR algorithm.
Net Charge
In some embodiments, the ELNN polypeptides can have an unstructured characteristic imparted by incorporation of amino acid residues with a net charge and/or reducing the proportion of hydrophobic amino acids in the ELNN sequence. The overall net charge and net charge density may be controlled, e.g., by modifying the content of charged amino acids in the ELNNs. In some embodiments, the net charge density of the ELNN of the compositions may be above +0.1 or below-0.1 charges/residue. In some embodiments, the net charge of a ELNN can be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more.
Since most tissues and surfaces in a human or animal have a net negative charge, the ELNNs can optionally be designed to have a net negative charge to minimize non-specific interactions between the ELNN containing compositions and various surfaces such as blood vessels, healthy tissues, or various receptors. Not to be bound by a particular theory, an ELNN may adopt open conformations due to electrostatic repulsion between individual amino acids of the ELNN polypeptide that individually carry a high net negative charge and that are distributed across the sequence of the ELNN polypeptide. Such a distribution of net negative charge in the extended sequence lengths of ELNN can lead to an unstructured conformation that, in turn, can result in an effective increase in hydrodynamic radius. Accordingly, in some embodiments the ELNNs contain glutamic acid such that the glutamic acid is at about 8, 10, 15, 20, 25, or even about 30% of the amino acids in the sequences. The ELNN of the compositions of the present disclosure generally have no or a low content of positively charged amino acids. In some embodiments the ELNN may have less than about 10% amino acid residues with a positive charge, or less than about 7%, or less than about 5%, or less than about 2% amino acid residues with a positive charge. However, the present disclosure contemplates polypeptides where a limited number of amino acids with a positive charge, such as lysine, may be incorporated into an ELNN, e.g., to permit conjugation between the epsilon amine of the lysine and a reactive group on a peptide, a linker bridge, or a reactive group on a drug or small molecule to be conjugated to the ELNN backbone.
In some embodiments, an ELNN may comprise charged residues separated by other residues such as serine or glycine, which may lead to better expression or purification behavior. Based on the net charge, ELNNs of the subject compositions may have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In some embodiments, the ELNN will have an isoelectric point between 1.5 and 4.5. In some embodiments, an ELNN incorporated into an paTCE fusion protein carries a net negative charge under physiologic conditions contributes to the unstructured conformation and reduced binding of the ELNN component to mammalian proteins and tissues.
As hydrophobic amino acids can impart structure to a polypeptide, in some embodiments the content of hydrophobic amino acids in the ELNN is less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. In some embodiments, an ELNN has no hydrophobic amino acids. In some embodiments, the amino acid content of methionine and tryptophan in the ELNN component of a paTCE fusion protein is less than 5%, or less than 2%, and most preferably less than 1%. In some embodiments, the ELNN has a sequence that has less than 10% amino acid residues with a positive charge, or less than about 7%, or less that about 5%, or less than about 2% amino acid residues with a positive charge, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 10% of the total ELNN sequence. In some embodiments, the ELNN has no methionine or tryptophan residues.
Increased Hydrodynamic Radius
In some embodiments, the ELNN can have a high hydrodynamic radius, conferring a corresponding increased Apparent Molecular Weight to the paTCE fusion protein which incorporates the ELNN. The linking of ELNNs to BsAb (e.g., TCE) sequences can result in paTCE compositions that can have increased hydrodynamic radii, increased Apparent Molecular Weight, and increased Apparent Molecular Weight Factor compared to BsAbs (e.g., TCEs) not linked to an ELNN. For example, in some therapeutic applications in which prolonged half-life is desired, one or more ELNNs with a high hydrodynamic radius are incorporated into a fusion protein comprising a BsAb (e.g., a TCE) to effectively enlarge the hydrodynamic radius of the fusion protein beyond the glomerular pore size of approximately 3-5 nm (corresponding to an apparent molecular weight of about 70 kDa) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv. Drug Deliv. Rev. 55:1261-1277), resulting in reduced renal clearance of circulating proteins. In some embodiments, the hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape and compactness. Not to be bound by a particular theory, the ELNN may adopt open conformations due to electrostatic repulsion between individual charges of the peptide or the inherent flexibility imparted by the particular amino acids in the sequence that lack potential to confer secondary structure. In some embodiments, the open, extended and unstructured conformation of the ELNN polypeptide has a greater proportional hydrodynamic radius compared to polypeptides of a comparable sequence length and/or molecular weight that have secondary and/or tertiary structure, such as typical globular proteins. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. In some embodiments, the addition of increasing lengths of ELNN results in proportional increases in the parameters of hydrodynamic radius, Apparent Molecular Weight, and Apparent Molecular Weight Factor, permitting the tailoring of paTCE to desired characteristic cut-off Apparent Molecular Weights or hydrodynamic radii. Accordingly, in some embodiments, the paTCE fusion protein can be configured with an ELNN such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm. In some embodiments, the large hydrodynamic radius conferred by the ELNN in an paTCE fusion protein can lead to reduced renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal half-life, an increase in mean residence time, and/or a decrease in renal clearance rate.
In some embodiments, an ELNN (or multiple ELNNs, such as two ELNNs) of a chosen length and sequence can be selectively incorporated into a paTCE to create a fusion protein that will have, under physiologic conditions, an Apparent Molecular Weight of at least about 150 kDa, or at least about 300 kDa, or at least about 400 kDa, or at least about 500 kDa, or at least about 600 kDa, or at least about 700 kDa, or at least about 800 kDa, or at least about 900 kDa, or at least about 1000 kDa, or at least about 1200 kDa, or at least about 1500 kDa, or at least about 1800 kDa, or at least about 2000 kDa, or at least about 2300 kDa or more. In some embodiments, an ELNN (or multiple ELNNs, such as two ELNNs) of a chosen length and sequence can be selectively linked to a BsAb (e.g., a TCE) to result in a paTCE fusion protein that has, under physiologic conditions, an Apparent Molecular Weight Factor of at least 3, alternatively of at least 4, alternatively of at least 5, alternatively of at least 6, alternatively of at least 7, alternatively of at least 8, alternatively of at least 9, alternatively of at least 10, alternatively of at least 15, or an Apparent Molecular Weight Factor of at least 20 or greater. In some embodiments, the paTCE fusion protein has, under physiologic conditions, an Apparent Molecular Weight Factor that is about 4 to about 20, or is about 6 to about 15, or is about 8 to about 12, or is about 9 to about 10 relative to the actual molecular weight of the fusion protein. In some embodiments, the fusion polypeptide exhibits an apparent molecular weight factor under physiological conditions that is greater than about 6.
Increased Terminal Half-Life
In some embodiments, a fusion polypeptide comprising an ELNN (such as a paTCE) has a terminal half-life that is at least two-fold longer, or at least three-fold longer, or at least four-fold longer, or at least five-fold longer, compared to a corresponding biologically active polypeptide that is not linked to the ELNN. In some embodiments, the (fusion) polypeptide has a terminal half-life that is at least two-fold longer compared to the biologically active polypeptide not linked to the ELNN.
In some embodiments, administration of a therapeutically effective amount of a paTCE fusion protein to a subject in need thereof results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold or more spent within a therapeutic window for the fusion protein compared to the corresponding BsAb (e.g., TCE) not linked to the ELNN(s) when administered at a comparable dose to a subject.
In some embodiments, a TCE released from a paTCE upon protease cleavage comprises one or more short polypeptides (e.g., about 30, 25, 20, 15, 14, 13, 12, 11, 10, or less amino acids in length) that has no amino acids other than G. A. P. E. S. and/or T. For example, a short polypeptide that has no amino acids other than G, A. P. E. S. and/or T might be incorporated into one or more spacer or linker sequences of the TCE, and/or a portion of one or more spacers or linkers that remain part of the TCE after cleavage. In some embodiments, a TCE that is released from a paTCE comprises a GTSESATPES (SEQ ID NO:96) on the N-terminal side (e.g., the closest amino acid of the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid positions of the N-terminal amino acid or the sequence includes the N-terminus) of the TCE. In some embodiments, a TCE that is released from a paTCE comprises a GTATPESGPG (SEQ ID NO: 97) on the C-terminal side (e.g., the closest amino acid of the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid positions of the N-terminal amino acid or the sequence includes the N-terminus) of the TCE. In some embodiments, a TCE comprises an internal linker (e.g., between a VL region and a VH region of a scFV) that comprises a polypeptide sequence with no amino acids other than G, A, P. E. S, and/or T, such as SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).
Low Immunogenicity
In some embodiments, the present disclosure provides compositions in which the ELNNs have a low degree of immunogenicity or are substantially non-immunogenic. Several factors can contribute to the low immunogenicity of an ELNN, e.g., the substantially non-repetitive sequence, the unstructured conformation, the high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, and the low degree or lack of epitopes in the ELNN.
One of ordinary skill in the art will understand that, in general, polypeptides having highly repetitive short amino acid sequences (e.g., wherein a 200 amino acid-long sequence contain on average 20 repeats or more of a limited set of 3- or 4-mers) and/or having contiguous repetitive amino acid residues (e.g., wherein 5- or 6-mer sequences have identical amino acid residues) have a tendency to aggregate or form higher order structures or form contacts resulting in crystalline or pseudo-crystalline structures.
In some embodiments, a ELNN sequence is substantially non-repetitive, wherein (1) the ELNN sequence has no three contiguous amino acids that are identical amino acid types, unless the amino acid is serine, in which case no more than three contiguous amino acids can be serine residues; and wherein (2) the ELNN contains no 3-amino acid sequences (3-mers) that occur more than 16, more than 14, more than 12, or more than 10 times within an at least 200 amino acid-long sequence of the ELNN (e.g., the entire span of an ELNN that is at least amino acids long). Without being bound by any scientific theory, such substantially non-repetitive sequences have less tendency to aggregate and, thus, enable the design of long-sequence ELNNs with a relatively low frequency of charged amino acids that would be likely to aggregate if the sequences or amino acid residues were otherwise more repetitive.
Conformational epitopes can be formed by regions of protein surfaces that are composed of multiple discontinuous amino acid sequences of a protein antigen. Without being bound by any scientific theory, the precise folding of the protein may bring these sequences into well-defined, stable spatial configurations or epitopes that can be recognized as “foreign” by the host humoral immune system, resulting in the production of antibodies to the protein and/or triggering a cell-mediated immune response. In the latter case, the immune response to a protein in an individual is heavily influenced by T-cell epitope recognition that is a function of the peptide binding specificity of that individual's HLA-DR allotype. Engagement of an MHC Class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation may lead to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
Without being bound by any scientific theory, the ability of a peptide to bind a given MHC Class II molecule for presentation on the surface of an APC (antigen presenting cell) may depend on a number of factors; most notably its primary sequence. In some embodiments, a lower degree of immunogenicity may be achieved by designing ELNNs that resist antigen processing in antigen presenting cells, and/or choosing sequences that do not bind MHC receptors well. In some embodiments, ELNN-containing fusion proteins have substantially non-repetitive ELNN polypeptides designed to reduce binding with MHC II receptors, as well as to avoid formation of epitopes for T-cell receptor or antibody binding, resulting in a low degree of immunogenicity. Without being bound by any scientific theory, avoidance of immunogenicity is, in part, a direct result of the conformational flexibility of ELNNs; i.e., the lack of secondary structure due to the selection and order of amino acid residues. For example, of particular interest are sequences having a low tendency to adapt compactly folded conformations in aqueous solution or under physiologic conditions that could result in conformational epitopes. The administration of fusion proteins comprising ELNNs, using conventional therapeutic practices and dosing, would generally not result in the formation of neutralizing antibodies to the ELNNs, and may also reduce the immunogenicity of BsAb (e.g., TCE) fusion partners in paTCE compositions.
In some embodiments, the ELNNs utilized in the subject fusion proteins can be substantially free of epitopes recognized by human T cells. The elimination of such epitopes for the purpose of generating less immunogenic proteins has been disclosed previously; see for example WO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated by reference herein. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281:95-108). Of particular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences. This can be achieved by testing direct repeats of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to 15-mer and, in particular, 9-mer sequences that are not human, and then altering the design of the ELNN sequence to eliminate or disrupt the epitope sequence. In some embodiments, the ELNNs are substantially non-immunogenic by the restriction of the numbers of epitopes of the ELNN predicted to bind MHC receptors. With a reduction in the numbers of epitopes capable of binding to MHC receptors, there is a concomitant reduction in the potential for T cell activation as well as T cell helper function, reduced B cell activation or upregulation and reduced antibody production. The low degree of predicted T-cell epitopes can be determined by epitope prediction algorithms such as, e.g., TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17:555-61), as shown in Example 74 of International Patent Application Publication No. WO 2010/144502 A2, which is incorporated by reference in its entirety. Aspects of the TEPITOPE score of a given peptide frame within a protein are disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555). The score ranges over at least 20 logs, from about 10 to about −10 (corresponding to binding constraints of 10e10 kD to 10e−10 kD), and can be reduced by avoiding hydrophobic amino acids that can serve as anchor residues during peptide display on MHC, such as M, I, L, V, or F. In some embodiments, an ELNN component incorporated into a paTCE does not have a predicted T-cell epitope at a TEPITOPE score of about −5 or greater, or −6 or greater, or −7 or greater, or −8 or greater, or at a TEPITOPE score of −9 or greater. As used herein, a score of “−9 or greater” would encompass TEPITOPE scores of 10 to −9, inclusive, but would not encompass a score of −10, as −10 is less than −9.
In some embodiments, the ELNNs, including those incorporated into the subject paTCE fusion proteins, can be rendered substantially non-immunogenic by the restriction of known proteolytic sites from the sequence of the ELNN, reducing the processing of ELNN into small peptides that can bind to MHC II receptors. In some embodiments, the ELNN sequence can be rendered substantially non-immunogenic by the use a sequence that is substantially devoid of secondary structure, conferring resistance to many proteases due to the high entropy of the structure. Accordingly, the reduced TEPITOPE score and elimination of known proteolytic sites from the ELNN may render the ELNN compositions, including the ELNN of the paTCE fusion protein compositions, substantially unable to be bound by mammalian receptors, including those of the immune system. In some embodiments, an ELNN of a paTCE fusion protein can have >100 nM kD binding to a mammalian receptor, or greater than 500 nM kD, or greater than 1 μM kD towards a mammalian cell surface or circulating polypeptide receptor.
Additionally, the substantially non-repetitive sequence and corresponding lack of epitopes of such embodiments of ELNNs can limit the ability of B cells to bind to or be activated by the ELNNs. In some embodiments, while an ELNN can make contacts with many different B cells over its extended sequence, each individual B cell may only make one or a small number of contacts with an individual ELNN. As a result, ELNNs typically may have a much lower tendency to stimulate proliferation of B cells and thus an immune response. In some embodiments, the paTCE may have reduced immunogenicity as compared to the corresponding BsAb (e.g., TCE) that is not fused to a mask polypeptide such as an ELNN. In some embodiments, the administration of up to three parenteral doses of a paTCE to a mammal may result in detectable anti-paTCE IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In some embodiments, the administration of up to three parenteral doses of an paTCE to a mammal may result in detectable anti-BsAb (e.g., TCE) IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In some embodiments, the administration of up to three parenteral doses of an paTCE to a mammal may result in detectable anti-ELNN IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In some embodiments, the mammal can be, e.g., a mouse, a rat, a rabbit, cynomolgus monkey, or human. In some embodiments, the mammal is a human.
An additional feature of certain ELNNs with substantially non-repetitive sequences relative to those less non-repetitive sequences (such as one having three contiguous amino acids that are identical) can be that non-repetitive ELNNs form weaker contacts with antibodies (e.g., monovalent interactions), thereby resulting in less likelihood of immune clearance such that the paTCE compositions can remain in circulation for an increased period of time.
In some embodiments, a biologically active polypeptide (such as a BsAb, e.g., a TCE) comprising an ELNN is less immunogenic compared to the fusion polypeptide not linked to any ELNN, wherein immunogenicity is ascertained by measuring production of IgG antibodies that selectively bind to the biologically active polypeptide after administration of comparable doses to a subject.
Barcode Fragment
In some embodiments, a polypeptide (e.g., a fusion polypeptide or a portion thereof such as an ELNN) comprises one or more barcode fragments (e.g., a first, second, or third barcode fragment) releasable from the polypeptide upon digestion by a protease. In some embodiments, the protease is a non-mammalian protease. In some embodiments, the protease is a prokaryotic protease. As used herein, the term “barcode fragment” (or “barcode.” or “barcode sequence”) can refer to either the portion of the polypeptide cleavably fused within the polypeptide, or the resulting peptide fragment released from the polypeptide.
In some embodiments, a barcode fragment (1) is a portion of an ELNN that includes at least part of the (non-recurring, non-overlapping) sequence motif that occurs (or is found) only once within the ELNN; and (2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon cleavage or complete digestion of the polypeptide by the protease.
In some embodiments, a barcode fragment does not include the N-terminal amino acid or the C-terminal amino acid of the fusion polypeptide. As described herein, in some embodiments, a barcode fragment is releasable (e.g., configured to be released) upon Glu-C digestion of the fusion polypeptide. In some embodiments, a barcode fragment is in an ELNN and does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in the ELNN. In some embodiments, a barcode fragment has a glutamic acid at its C-terminus. One of ordinary skill in the art will understand that the C-terminus of a barcode fragment can refer to the “last” (or the most C-terminal) amino acid residue within the barcode fragment, when cleavably fused within a polypeptide (such as an ELNN), even if other non-barcode amino acid residues are positioned C-terminal to the barcode fragment within the polypeptide (e.g., ELNN). In some embodiments, a barcode fragment has an N-terminal amino acid that is immediately preceded by a glutamic acid residue. In some embodiments, the glutamic acid residue that precedes the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, a barcode fragment does not include a (second) glutamic acid residue at a position other than the C-terminus of the barcode fragment unless the glutamic acid is immediately followed by a proline. In some embodiments, a barcode fragment is positioned a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is from 10 to 150, or 10 to 125 amino acids. In some embodiments, a barcode fragment is positioned within, or at a location of, 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids from the N-terminus of the polypeptide, or at a location in a range between any of the foregoing. In some embodiments, a barcode fragment is positioned within 200, within 150, within 100, or within 50 amino acids of the N-terminus of the polypeptide. In some embodiments, a barcode fragment is positioned at a location that is between 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from the N-terminus of the polypeptide. In some embodiments, a barcode fragment is positioned within, or at a location of, 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids from the C-terminus of the polypeptide, or at a location in a range between any of the foregoing. In some embodiments, a barcode fragment is positioned within 200, within 150, within 100, or within 50 amino acids of the C-terminus of the polypeptide. In some embodiments, a barcode fragment is positioned at a location that is between 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from the C-terminus of the polypeptide. In some embodiments, a barcode fragment (BAR) is characterized in that: (i) it does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in the ELNN; (ii) it has a glutamic acid at its C-terminus; (iii) it has an N-terminal amino acid that is immediately preceded by a glutamic acid residue; and (iv) it is positioned a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is from 10 to 150 amino acids, or from 10 to 125 amino acids in length. In some embodiments, a barcode fragment is in an ELNN and (i) does not include the N-terminal amino acid or the C-terminal amino acid of the polypeptide; (ii) does not include a glutamic acid that is immediately adjacent to another glutamic acid in the ELNN; (iii) has a glutamic acid at its C-terminus; (iv) has an N-terminal amino acid that is immediately preceded by a glutamic acid residue; and (v) is positioned a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is from 10 to 150, or 10 to 125 amino acids in length. In some embodiments, the glutamic acid residue that precedes the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, a barcode fragment does not include a glutamic acid residue at a position other than the C-terminus of the barcode fragment unless the glutamic acid is immediately followed by a proline. Depending on context herein and when referring to placement within a polypeptide sequence, the term “distance” can refer to the number of amino acid residues from the N-terminus of the polypeptide to the most N-terminal amino acid residue of the barcode fragment, or from the C-terminus of the polypeptide to the most C-terminal amino acid residue of the barcode fragment. In some embodiments, for a barcoded ELNN fused to a biologically active polypeptide, at least one barcode fragment (or at least two barcode fragments, or three barcode fragments) contained in the barcoded ELNN is positioned at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300 amino acids from the biologically active polypeptide. In some embodiments, a barcode fragment is at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids in length. In some embodiments, a barcode fragment is at least 4 amino acids in length. In some embodiments, a barcode fragment is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids in length, or in a range between any of the foregoing values. In some embodiments, a barcode fragment is between 4 and 20, between 5 and 15, between 6 and 12, or between 7 and 10 amino acids in length. In some embodiments, a barcode fragment comprises an amino acid sequence identified herein by SEQ ID NOs: 68-79 and SEQ ID NOs: 1010−1027 in Table 2.







TABLE 2







Exemplary Barcode Fragments Releasable Upon


Glu-C Digest








Amino Acid Sequence
SEQ ID NO:





SPATSGSTPE
  68


 


GSAPATSE
  69


 


GSAPGTATE
  70


 


GSAPGTE
  71


 


PATSGPTE
  72


 


SASPE
  73


 


PATSGSTE
  74


 


GSAPGTSAE
  75


 


SATSGSE
  76


 


SGPGSTPAE
  77


 


SGPGSGPGTSE
  78


 


SGPGTSPSATPE
  79


 


SGPGTGTSATPE
1010


 


SGPGTTPGTTPE
1011


 


SGPGTPPTSTPE
1012


 


SGPGTGSAGTPE
1013


 


SGPGTGGAGTPE
1014


 


SGPGTSPGATPE
1015


 


SGPGTSGSGTPE
1016


 


SGPGTSSASTPE
1017


 


SGPGTGAGTTPE
1018


 


SGPGTGSTSTPE
1019


 


TPGSEPATSGSE
1020


 


GSAPGTSTEPSE
1021


 


SGPGTAGSGTPE
1022


 


SGPGTSSGGTPE
1023


 


SGPGTAGPATPE
1024


 


SGPGTPGTGTPE
1025


 


SGPGTGGPTTPE
1026


 


SGPGTGSGSTPE
1027









In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that are releasable from the chimeric polypeptides described herein upon complete digestion the chimeric polypeptide by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.
In some embodiments, the chimeric polypeptides disclosed herein comprises a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG (SEQ ID NO:8030), SGSETPGT (SEQ ID NO:8031), and GTSESATP (SEQ ID NO:8032).
In some embodiments, the chimeric polypeptides disclosed herein comprises at least one of the following amino acid sequences: PE.GSXnPE.SG (SEQ ID NO:8188), PE.GSXnSE.GG (SEQ ID NO: 8189), PE.GSXnSE.TG (SEQ ID NO:8191), PE.GSXnSE.SA (SEQ ID NO:8192), PE.SGXnPE.SG (SEQ ID NO:8193), PE.SGXnSE.GG (SEQ ID NO:8195), PE.SGXnSE.TG (SEQ ID NO: 8196), PE.SGXnSE.SA (SEQ ID NO:8197), and PE.TPXnPE.SG (SEQ ID NO:8199), PE.TPXnSE.GG (SEQ ID NO:8200), PE.TPXnSE.TG (SEQ ID NO:8201), PE.TPXnSE.SA (SEQ ID NO: 8203), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptides disclosed herein comprises at least one of the following amino acid sequences: PE.SGXnPE.SG (SEQ ID NO:8194), PE.GSXnSE.GG (SEQ ID NO:8190), PE.TPXnSE.TG (SEQ ID NO:8202), PE.SGXnSE.SA (SEQ ID NO:8198). In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is any integer from 5 to 15. In some embodiments, X11 is SGPGTGTSATPE (SEQ ID NO:1010), SGPGSGPGTSE (SEQ ID NO:78), SGPGTTPGTTPE (SEQ ID NO:1011), SGPGTPPTSTPE (SEQ ID NO:1012), SGPGTSPSATPE (SEQ ID NO: 79), SGPGTGSAGTPE (SEQ ID NO:1013), SGPGTGGAGTPE (SEQ ID NO:1014), SGPGTSPGATPE (SEQ ID NO:1015), SGPGTSGSGTPE (SEQ ID NO:1016), SGPGTSSASTPE (SEQ ID NO: 1017), SGPGTGAGTTPE (SEQ ID NO:1018), SGPGTGSTSTPE (SEQ ID NO:1019), TPGSEPATSGSE (SEQ ID NO:1020), GSAPGTSTEPSE (SEQ ID NO:1021), SGPGTAGSGTPE (SEQ ID NO: 1022), SGPGTSSGGTPE (SEQ ID NO:1023), SGPGTAGPATPE (SEQ ID NO:1024), SGPGTPGTGTPE (SEQ ID NO:1025), SGPGTGGPTTPE (SEQ ID NO:1026), or SGPGTGSGSTPE (SEQ ID NO:1027).
In some embodiments, a chimeric polypeptide comprises at least one of the following amino acid sequences: SGPE.SGPGXnSGPE.SGPG (SEQ ID NO:8033), SGPE.SGPGXnATPE.SGPG (SEQ ID NO:8034), SGPE.SGPGXnGTSE.SATP (SEQ ID NO:8036), SGPE.SGPGXnTTPE.SGPG (SEQ ID NO:8037), SGPE.SGPGXnSTPE.SGPG (SEQ ID NO:8038), SGPE.SGPGXnGTPE.SGPG (SEQ ID NO:8039), SGPE.SGPGX1GTPE.TPGS (SEQ ID NO:8040), SGPE.SGPGXnGTPE.TPGS (SEQ ID NO:8040), SGPE.SGPGXnSGSE.TGTP (SEQ ID NO:8041), SGPE.SGPGXnGTPE.GSAP (SEQ ID NO:8042), SGPE.SGPGXnEPSE.SATP (SEQ ID NO:8043), ATPE.SGPGXnSGPE.SGPG (SEQ ID NO:8044), ATPE.SGPGXnATPE.SGPG (SEQ ID NO:8045), ATPE.SGPGXnGTSE.SATP (SEQ ID NO:8047), ATPE.SGPGXnTTPE.SGPG (SEQ ID NO:8049), ATPE.SGPGXnSTPE.SGPG (SEQ ID NO:8051), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:8053), ATPE.SGPGXnGTPE.TPGS (SEQ ID NO:8055), ATPE.SGPGXnSGSE.TGTP (SEQ ID NO:8056), ATPE.SGPGXnGTPE.GSAP (SEQ ID NO:8057), ATPE.SGPGXnEPSE.SATP (SEQ ID NO:8058), GTSE.SATPXnSGPE.SGPG (SEQ ID NO:8059), GTSE.SATPXnATPE.SGPG (SEQ ID NO:8060), GTSE.SATPXnGTSE.SATP (SEQ ID NO:8061), GTSE.SATPXnTTPE.SGPG (SEQ ID NO:8062), GTSE.SATPXnSTPE.SGPG (SEQ ID NO:8063), GTSE.SATPXnGTPE.SGPG (SEQ ID NO:8064). GTSE.SATPXnGTPE.TPGS (SEQ ID NO:8065). GTSE.SATPXnSGSE.TGTP (SEQ ID NO:8066). GTSE.SATPXnGTPE.GSAP (SEQ ID NO:8067). GTSE.SATPXnEPSE.SATP (SEQ ID NO:8068). TTPE.SGPGXnSGPE.SGPG (SEQ ID NO:8069). TTPE.SGPGXnATPE.SGPG (SEQ ID NO:8070). TTPE.SGPGXnGTSE.SATP (SEQ ID NO:8071). TTPE.SGPGXnTTPE.SGPG (SEQ ID NO:8072). TTPE.SGPGXnSTPE.SGPG (SEQ ID NO:8074). TTPE.SGPGXnGTPE.SGPG (SEQ ID NO:8075). TTPE.SGPGXnGTPE.TPGS (SEQ ID NO:8076). TTPE.SGPGXnSGSE.TGTP (SEQ ID NO:8077). TTPE.SGPGXnGTPE.GSAP (SEQ ID NO:8078), TTPE.SGPGXnEPSE.SATP (SEQ ID NO:8079). STPE.SGPGXnSGPE.SGPG (SEQ ID NO:8080). STPE.SGPGXnATPE.SGPG (SEQ ID NO:8081). STPE.SGPGXnGTSE.SATP (SEQ ID NO:8082). STPE.SGPGXnTTPE.SGPG (SEQ ID NO:8083), STPE.SGPGXnSTPE.SGPG (SEQ ID NO:8084). STPE.SGPGXnGTPE.SGPG (SEQ ID NO:8086). STPE.SGPGXnGTPE.TPGS (SEQ ID NO:8087). STPE.SGPGXnSGSE.TGTP (SEQ ID NO:8088), STPE.SGPGXnGTPE.GSAP (SEQ ID NO:8089), STPE.SGPGXnEPSE.SATP (SEQ ID NO:8090). GTPE.SGPGXnSGPE.SGPG (SEQ ID NO:8091). GTPE.SGPGXnATPE.SGPG (SEQ ID NO:8092), GTPE.SGPGXnGTSE.SATP (SEQ ID NO:8093). GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:8094). GTPE.SGPGXnSTPE.SGPG (SEQ ID NO:8096). GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:8098). GTPE.SGPGXnGTPE.TPGS (SEQ ID NO:8100). GTPE.SGPGXnSGSE.TGTP (SEQ ID NO:8101). GTPE.SGPGXnGTPE.GSAP (SEQ ID NO:8102), GTPE.SGPGXnEPSE.SATP (SEQ ID NO:8103). GTPE.TPGSXnSGPE.SGPG (SEQ ID NO:8104), GTPE.TPGSXnATPE.SGPG (SEQ ID NO:8105), GTPE.TPGSXnGTSE.SATP (SEQ ID NO:8106). GTPE.TPGSXnTTPE.SGPG (SEQ ID NO:8107). GTPE.TPGSXnSTPE.SGPG (SEQ ID NO:8108), GTPE.TPGSXnGTPE.SGPG (SEQ ID NO:8109), GTPE.TPGSXnGTPE.TPGS (SEQ ID NO:8110). GTPE.TPGSXnSGSE.TGTP (SEQ ID NO:8111), GTPE.TPGSXnGTPE.GSAP (SEQ ID NO:8113). GTPE.TPGSXnEPSE.SATP (SEQ ID NO:8114). SGSE.TGTPXnSGPE.SGPG (SEQ ID NO:8115), SGSE.TGTPXnATPE.SGPG (SEQ ID NO:8116). SGSE.TGTPXnGTSE.SATP (SEQ ID NO:8117), SGSE.TGTPXnTTPE.SGPG (SEQ ID NO:8118). SGSE.TGTPXnSTPE.SGPG (SEQ ID NO:8119), SGSE.TGTPXnGTPE.SGPG (SEQ ID NO:8120), SGSE.TGTPXnGTPE.TPGS (SEQ ID NO:8121). SGSE.TGTPXnSGSE.TGTP (SEQ ID NO:8122), SGSE.TGTPXnGTPE.GSAP (SEQ ID NO:8123). SGSE.TGTPXnEPSE.SATP (SEQ ID NO:8124), GTPE.GSAPXnSGPE.SGPG (SEQ ID NO:8125), GTPE.GSAPXnATPE.SGPG (SEQ ID NO:8126), GTPE.GSAPXnGTSE.SATP (SEQ ID NO:8127). GTPE.GSAPXnTTPE.SGPG (SEQ ID NO:8128), GTPE.GSAPXnSTPE.SGPG (SEQ ID NO:8129). GTPE.GSAPXnGTPE.SGPG (SEQ ID NO:8130), GTPE.GSAPXnGTPE.TPGS (SEQ ID NO:8131). GTPE.GSAPXnSGSE.TGTP (SEQ ID NO:8132), GTPE.GSAPXnGTPE.GSAP (SEQ ID NO:8133). GTPE.GSAPXnEPSE.SATP (SEQ ID NO:8134). EPSE.SATPXnSGPE.SGPG (SEQ ID NO:8136). EPSE.SATPXnATPE.SGPG (SEQ ID NO:8137). EPSE.SATPXnGTSE.SATP (SEQ ID NO:8138). EPSE.SATPXnTTPE.SGPG (SEQ ID NO:8139), EPSE.SATPXnSTPE.SGPG (SEQ ID NO:8140), EPSE.SATPXnGTPE.SGPG (SEQ ID NO:8141), EPSE.SATPXnGTPE.TPGS (SEQ ID NO:8142), EPSE.SATPXnSGSE.TGTP (SEQ ID NO:8143), EPSE.SATPXnGTPE.GSAP (SEQ ID NO:8144), or EPSE.SATPXnEPSE.SATP (SEQ ID NO:8145), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences:









(SEQ ID NO: 8035)



SGPE.SGPGXnATPE.SGPG,



 



(SEQ ID NO: 8048)



ATPE.SGPGXnGTSE.SATP,



 



(SEQ ID NO: 8050)



ATPE.SGPGXnTTPE.SGPG,



 



(SEQ ID NO: 8052)



ATPE.SGPGXnSTPE.SGPG,



 



(SEQ ID NO: 8046)



ATPE.SGPGXnATPE.SGPG,



 



(SEQ ID NO: 8054)



ATPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8054)



ATPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8046)



ATPE.SGPGXnATPE.SGPG,



 



(SEQ ID NO: 8099)



GTPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8097)



GTPE.SGPGXnSTPE.SGPG,



 



(SEQ ID NO: 8095)



GTPE.SGPGXnTTPE.SGPG,



 



(SEQ ID NO: 8097)



GTPE.SGPGXnSTPE.SGPG,



 



(SEQ ID NO: 8112)



GTPE.TPGSXnSGSE.TGTP,



 



(SEQ ID NO: 8135)



GTPE.GSAPXnEPSE.SATP,



 



(SEQ ID NO: 8054)



ATPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8054)



ATPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8046)



ATPE.SGPGXnATPE.SGPG,



 



(SEQ ID NO: 8054)



ATPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8073)



TTPE.SGPGXnTTPE.SGPG,



or



 



(SEQ ID NO: 8085)



STPE.SGPGXnSTPE.SGPG,






wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 30. In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 3 to 7. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is 4. In some embodiments, n is any integer from 5 to 15. In some embodiments, wherein X11 is PGTGTSAT (SEQ ID NO:8146), PGSGPGT (SEQ ID NO:8147), PGTTPGTT (SEQ ID NO:8148), PGTPPTST (SEQ ID NO:8149), PGTSPSAT (SEQ ID NO:8150), PGTGSAGT (SEQ ID NO:8151), PGTGGAGT (SEQ ID NO:8152), PGTSPGAT (SEQ ID NO:8153), PGTSGSGT (SEQ ID NO:8154), PGTSSAST (SEQ ID NO:8155), PGTGAGTT (SEQ ID NO:8156), PGTGSTST (SEQ ID NO:8157), GSEPATSG (SEQ ID NO:8158), APGTSTEP (SEQ ID NO:8159), PGTAGSGT (SEQ ID NO:8160), PGTSSGGT (SEQ ID NO:8161), PGTAGPAT (SEQ ID NO:8162), PGTPGTGT (SEQ ID NO:8163), PGTGGPTT (SEQ ID NO:8164), or PGTGSGST (SEQ ID NO:8165). In some embodiments, Xn is TGTS (SEQ ID NO:8166), SGP, TTPG (SEQ ID NO:8167), TPPT (SEQ ID NO: 8168), TSPS (SEQ ID NO:8169), TGSA (SEQ ID NO:8170), TGGA (SEQ ID NO:8171), TSPG (SEQ ID NO: 8172), TSGS (SEQ ID NO:8173), TSSA (SEQ ID NO:8174), TGAG (SEQ ID NO:8175), TGST (SEQ ID NO:8176), EPAT (SEQ ID NO:8177), GTST (SEQ ID NO:8178), TAGS (SEQ ID NO: 8179), TSSG (SEQ ID NO:8180), TAGP (SEQ ID NO:8181), TPGT (SEQ ID NO:8182), TGGP (SEQ ID NO: 8183), or TGSG (SEQ ID NO:8184).

In some embodiments, barcodes are designed to have improved analytical properties. In some embodiments, such barcodes can be released with relatively modest concentrations of a non-mammalian protease such as Glu-C. This facilitates better detection, e.g., through LC/MS, and also allows measurement of peptides that are generated from the cleavable linker thereby allowing a measurement of cleavage products using, e.g., LC/MS.
In some embodiments of fusion proteins comprising an ELNN, the fusion protein has a single polypeptide chain, and the polypeptide chain comprises a barcode fragment that is at a position within the polypeptide chain that is from 10 to 200 amino acids or from 10 to 125 amino acids from the N-terminus or the C-terminus of the polypeptide chain. In some embodiments, a fusion protein (such as a paTCE) comprises a first ELNN and a second ELNN, the first ELNN is at the N-terminal side of the bispecific antibody domain, and the first barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the N-terminus of the fusion protein. In some embodiments, the second ELNN is at the C-terminal side of the bispecific antibody domain, and the second barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.
In some embodiments, an ELNN further comprises one or more additional barcode fragments, wherein the one or more additional barcode fragments each differs in sequence and molecular weight from all other peptides fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease. In some embodiments, a barcoded ELNN comprises only one barcode fragment. In some embodiments, a barcoded ELNN comprises a set of barcode fragments, comprising a first barcode fragment, such as those described herein. In some embodiments, the set of barcode fragments comprises a second barcode fragment (or a further barcode fragment), such as those described herein. In some embodiments, the set of barcode fragments comprises a third barcode fragment, such as those described herein.
A set of barcode fragments fused within an N-terminal ELNN can be referred to as an N-terminal set of barcodes (an “N-terminal set”). A set of barcode fragments fused within a C-terminal ELNN can be referred to as a C-terminal set of barcodes (a “C-terminal set”). In some embodiments, the N-terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the N-terminal set further comprises a third barcode fragment. In some embodiments, the C-terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the C-terminal set further comprises a third barcode fragment. In some embodiments, the polypeptide comprises a set of barcode fragments that includes a first barcode fragment, a further (second) barcode fragment, and at least one additional barcode fragment, wherein each barcode fragment of the set of barcode fragments (1) is a portion of the second ELNN and (2) differs in sequence and molecular weight from all other peptides fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease.
Included herein is a mixture comprising a plurality of polypeptides of varying length; the mixture comprising a first set of polypeptides and a second set of polypeptides. In some embodiments, each polypeptide of the first set of polypeptides comprises a barcode fragment that (a) is releasable from the polypeptide by digestion with a protease and (b) has a sequence and molecular weight that differs from the sequence and molecular weight of all other fragments that are releasable from the first set of polypeptides. In some embodiments, the second set of polypeptides lack the barcode fragment of the first set of polypeptides (e.g., due to truncation). In some embodiments, both the first set of polypeptides and the second set of polypeptides each comprise a reference fragment that (a) is common to the first set of polypeptides and the second set of polypeptides and (b) releasable by digestion with the protease. In some embodiments, the ratio of the first set of polypeptides to polypeptides comprising the reference fragment is greater than 0.70. In some embodiments, the ratio of the first set of polypeptides to polypeptides comprising the reference fragment is greater than 0.80, 0.90, 0.95, or 0.98. In some embodiments, the reference fragment occurs no more than once in each polypeptide of the first set of polypeptides and the second set of polypeptides. In some embodiments, the protease is a protease that cleaves on the C-terminal side of glutamic acid residues. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the polypeptides of varying lengths comprise polypeptides comprising at least one ELNN, such as any described herein. In some embodiments, the first set of polypeptides comprises a full-length polypeptide, wherein the barcode fragment is a portion of the full-length polypeptide. In some embodiments, the full-length polypeptide is a (fusion) polypeptide, such as any described hereinabove or described anywhere else herein. In some embodiments, the polypeptides of varying lengths in a mixture differ from one another due to N-terminal truncation, C-terminal truncation, or both N- and C-terminal truncation of a full-length polypeptide. In some embodiments, the first set of polypeptides and the second set of polypeptides may differ in one or more pharmacological properties.
The present disclosure also provides methods for assessing, in a mixture comprising polypeptides of varying length, a relative amount of a first set of polypeptides in the mixture to a second set of polypeptides in the mixture, wherein (1) each polypeptide of the first set of polypeptides shares a barcode fragment that occurs once and only once in the polypeptide and (2) each polypeptide of the second set of polypeptides lacks the barcode fragment that is shared by polypeptides of the first set, wherein individual polypeptides of both the first of polypeptides and the second set of polypeptides each comprises a reference fragment. In some embodiments, the methods comprise contacting the mixture with a protease to produce a plurality of proteolytic fragments that result from cleavage of the first set of polypeptides and the second set of polypeptides, wherein the plurality of proteolytic fragments comprise a plurality of reference fragments, and a plurality of barcode fragments. In some embodiments, the methods can further comprise determining a ratio of the amount of barcode fragments to the amount of reference fragments, thereby assessing the relative amounts of the first set of polypeptides to the second set of polypeptides. In some embodiments, the barcode fragment occurs no more than once in each polypeptide of the first set of polypeptides. In some embodiments, the reference fragment occurs no more than once in each polypeptide of the first set of polypeptides and the second set of polypeptides. In some embodiments, the plurality of proteolytic fragments comprises a plurality of reference fragments, and a plurality of barcode fragments. In some embodiments, the protease cleaves the first and second sets of polypeptides (or the polypeptides of varying length) on the C-terminal side of glutamic acid residues that are not followed by a proline residue. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the step of determining a ratio of the amount of barcode fragments to the amount of reference fragments comprises identifying barcode fragments and reference fragments from the mixture after it has been contacted with the protease. In some embodiments, the barcode fragments and the reference fragments are identified based on their respective masses. In some embodiments, the barcode fragments and the reference fragments are identified via mass spectrometry.
In some embodiments, the barcode fragments and reference fragments are identified via liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the step of determining a ratio of the barcode fragments to the reference fragments comprises isobaric labeling. In some embodiments, the step of determining a ratio of the barcode fragments to the reference fragments comprises spiking the mixture with one or both of an isotope-labeled reference fragment and an isotope labeled barcode fragment. In some embodiments, the polypeptides of varying lengths comprise polypeptides that comprise at least one ELNN, as described hereinabove or described anywhere else herein. In some embodiments, the ELNN is characterized in that (i) it comprises at least 100, or at least 150 amino acids; (ii) at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P); and (iii) it comprises at least 4 different types of amino acids that are G, A. S. T. E, or P. In some embodiments, the barcode fragment, when present, is a portion of the ELNN. In some embodiments, the mixture of polypeptides of varying lengths comprises a polypeptide as any described hereinabove or described anywhere else herein. In some embodiments, the polypeptides of varying length comprise a full-length polypeptide and truncated fragments thereof. In some embodiments, the polypeptides of varying length consist essentially of the full-length polypeptide and truncated fragments thereof. In some embodiments, the polypeptides of varying lengths in a mixture differ from one another due to N-terminal truncation, C-terminal truncation, or both N- and C-terminal truncation of a full-length polypeptide. In some embodiments, the full-length polypeptide is a polypeptide as described hereinabove or described anywhere else herein. In some embodiments, the ratio of the amount of barcode fragments to reference fragments is greater than 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, or 0.99.
Isobaric Labeling-Based Quantification of Peptides
In some embodiments, isobaric labeling can be used for determining a ratio of the barcode fragments to the reference fragments. Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics, wherein peptides or proteins (or portions thereof) are labeled with various chemical groups that are isobaric (identical in mass) but vary in terms of distribution of heavy isotopes around their structure. In some embodiments, these tags, commonly referred to as tandem mass tags, are designed so that the mass tag is cleaved at a specific linker region upon high-energy collision-induced dissociation (CID) during tandem mass spectrometry, thereby yielding reporter ions of different masses. Some of the most common isobaric tags are amine-reactive tags.
Exemplary Barcoded ELNN Polypeptides
Included herein are ELNNs comprising barcode fragments that are portions of the ELNNs.
Amino acid sequences of exemplary barcoded ELNNs, containing one barcode (e.g., SEQ ID NOs: 8002-8003, 8005-8009, and 8013-8022), or two barcodes (e.g., SEQ ID NOS: 8001, 8004, and 8012), or three barcodes (e.g., SEQ ID NO: 8011), are illustrated in Table 3a. In some embodiments, among these exemplary barcoded ELNNs, 12 (SEQ ID NOs: 8001-8003, 8008-8009, 8011, 8015-8019, and 8022) are to be fused to a biologically-active protein (such as a TCE) at the C-terminal of the biologically-active protein, and 10 (SEQ ID NOS: 8004-8007, 8010, 8012-8014, 8020, and 8021) are to be fused at the N-terminal of the biologically-active protein. In some embodiments, the ELNN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 8001-8022 in Table 3a.







TABLE 3a







Exemplary Barcoded ELNNs











SEQ ID
ELNN
# of

Total #


NO.
Type
Barcode(s)
Amino Acid Sequence
of AAs





8001
C-terminal
2
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS
 864



ELNN

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG






SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP






ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS






TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS






APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTE






PSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE






GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS






EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT






STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG






SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP






ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG






PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA






TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG






TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE






GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS






ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS






APGTSESATPESGPGTSESATPESGPGftabTSESATPESGPG






SEPATSGPTESGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE






GSAPGTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTS






TEPSEGSAPGEPEA



 


8002
C-terminal
1
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS
 864



ELNN

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG






SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP






ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS






TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS






APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTE






PSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE






GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS






EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT






STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG






SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP






ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG






PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA






TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG






TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE






GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS






ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS






APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA






TSGPTESGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP






GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS






EGSAPGEPEA



 


8003
C-terminal
1
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS
 864



ELNN

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG






SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP






ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS






TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS






APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTE






PSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE






GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS






EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT






STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG






SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP






ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG






PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA






TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG






TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE






GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS






ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS






APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA






TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP






GTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS






EGSAPGEPEA



 


8004
N-terminal
2
ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESA
 288



ELNN

TPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETP






GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT






PESGPGESPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGS






PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE






SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA






GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP



 


8005
N-terminal
1
ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESA
 288



ELNN

TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG






TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP






ESGPGESPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSP






AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES






GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG






SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP



 


8006
N-terminal
1
ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESA
 288



ELNN

TPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETP






GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT






PESGPGEEPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGS






PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE






SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA






GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP



 


8007
N-terminal
1
ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESA
 288



ELNN

TPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETP






GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT






PESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS






PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE






SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA






GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP



 


8008
C-terminal
1
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS
 864



ELNN

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG






SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP






ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS






TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGS






APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTE






PSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE






GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS






EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT






STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG






SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP






ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG






PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA






TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG






TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE






GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS






ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS






APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA






TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP






GTESTPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS






EGSAPG



 


8009
C-terminal
1
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
 576



ELNN

TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG






TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT






STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS






ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS






APGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG






SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP






GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT






PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS






EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS






TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE






SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE






EGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSES






ATPESGPGTSTEPSEGSAPG



 


8010
N-terminal
2
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS
1152



ELNN

PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE






SGPGSTPAESGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE






ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST






EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTE






PSEGSAPGTSESATPESGPGSEPATSGSTETPGTSTEPSEGSA






PGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGS






PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG






TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE






GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS






TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES






GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES






ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE






GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS






GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT






STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS






ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST






EPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESG






PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEP






SEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPG






TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE






GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS






ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST






EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG






SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP






GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS






EGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS



 


8011
C-terminal
3
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS
1152



ELNN

PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE






SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE






SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE






EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEP






SEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG






TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPT






STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS






TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS






APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE






PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP






GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESAT






PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGT






SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS






ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST






EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET






PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP






SEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPG






SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE






GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTS






TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS






APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES






ATPESGPGSEPATSGSETPGSEPATSGSTETPGSPAGSPTSTE






EGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS






PTSTEEGTSTEPSEGSAPGTATESPEGSAPGTSESATPESGP






GTSTEPSEGSAPGTSAESATPESGPGSEPATSGSETPGTSTE






PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS



 


8012
N-terminal
2
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
 864



ELNN

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPATSESATPESGPGS






EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESASPE






SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST






EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA






PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP






SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEG






TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE






GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS






TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS






APGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA






TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP






GSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESAT






PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT






SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG






SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE






SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA






PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT






SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG






TSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE






GSAP



 


8013
N-terminal
1
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
 864



ELNN

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGS






ESATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPE






SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST






EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA






PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP






SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEG






TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE






GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS






TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS






APGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA






TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP






GSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESAT






PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT






SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG






SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE






SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA






PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT






SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG






TSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE






GSAP



 


8014
N-terminal
1
SPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATP
 292



ELNN

ESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPGT






SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE






SGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA






GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG






PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS






PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP



 


8015
C-terminal
1
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA
 582



ELNN

TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG






TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT






STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS






ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS






APGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG






SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP






GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT






PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS






EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS






TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE






SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE






EGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETPGTSES






ATPESGPGTSTEPSEGSAPGEPEA



 


8016
C-terminal
1
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG
 576



ELNN

SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSE






GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS






TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES






GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES






ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE






GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS






EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS






EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS






TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA






GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG






PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSESAT






SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG






SEPATSGSETPGTSESA



 


8017
C-terminal
1
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS
 576



ELNN

GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT






STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG






SAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP






ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG






PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT






SGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG






TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE






GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE






PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES






GPGTSTEPSEGSAPGTSESASPESGPGSPAGSPTSTEEGSPAG






SPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP






GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS






GSETPGTSESATPESGP



 


8018
C-terminal
1
GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT
 576



ELNN

STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG






SAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP






ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG






PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT






SGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG






TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE






GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE






PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES






GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAG






SPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP






GTSESATPESGPGSEPATSGSTETGTSESATPESGPGSEPAT






SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG






TSESATPESGPGSEPATS



 


8019
C-terminal
1
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT
 576



ELNN

SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG






SAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSE






SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA






PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP






SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG






TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG






SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP






AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES






GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES






ATPESGPGSEPATSGSETPGTSESASPESGPGTSTEPSEGSAP






GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT






PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT






SESATPESGPGTSESAT



 


8020
N-terminal
1
ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESA
 294



ELNN

TPESGPGSEPATSGSETPGTSESATPESGPGSTPAESGSETPG






TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP






ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSP






AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES






GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG






SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP



 


8021
N-terminal
1
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSE
 294



ELNN

SATPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS






TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE






TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTE






PSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP






GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS






EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP



 


8022
C-terminal
1
ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGP
 582



ELNN

GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPS






EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT






STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE






SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE






SATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE






EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP






SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG






SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT






STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP






AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES






GPGTSESATPESGPGTSPSATPESGPGSEPATSGSETPGSEP






ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA






PGSEPATSGSETPGTSESAGEPEA









In some embodiments, a barcoded ELNN can be obtained by making one or more mutations to existing ELNN, such as any listed in Table 3b, according to one or more of the following criteria: to minimize the sequence change in the ELNN, to minimize the amino acid composition change in the ELNN, to substantially maintain the net charge of the ELNN, to substantially maintain (or improve) low immunogenicity of the ELNN, and to substantially maintain (or improve) the pharmacokinetic properties of the ELNN. In some embodiments, the ELNN sequence has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 601-659 listed in Table 3b. In some embodiments, the ELNN sequence, having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) but less than 100% sequence identity to any of SEQ ID NOs: 601-659 listed in Table 3b, is obtained by one or more mutations (e.g., less than 10, less than 8, less than 6, less than 5, less than 4, less than 3, less than 2 mutations) of the corresponding sequence from Table 3b. In some embodiments, the one or more mutations comprise deletion of a glutamic acid residue, insertion of a glutamic acid residue, substitution of a glutamic acid residue, or substitution for a glutamic acid residue, or any combination thereof. In some embodiments, where the ELNN sequence differs from, but has at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity to, any one of SEQ ID NOs: 601-659 listed in Table 3b, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the ELNN sequence and the corresponding sequence of Table 3b involve deletion of a glutamic acid residue, insertion of a glutamic acid residue, substitution of a glutamic acid residue, or substitution for a glutamic acid residue, or any combination thereof. In some such embodiments, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the ELNN sequence and the corresponding sequence of Table 3b involve a substitution of a glutamic acid residue, or a substitution for a glutamic acid residue, or both.
The “a substitution of a first amino acid,” as used herein, refers to replacement of the first amino acid residue with a second amino acid residue, resulting in the second amino acid residue taking its place at the substitution position in the obtained sequence. For example, “a substitution of glutamic acid” refers to replacement of the glutamic acid (E) residue for a non-glutamic acid residue (e.g., serine(S)).







TABLE 3b







Exemplary Existing ELNNs for Engineering into Barcoded ELNN(s)









ELNN

SEQ ID


Name
Amino Acid Sequence
NO





AE144
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
601



APGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATP




ESGPGSEPATSGSETPGTSTEPSEGSAP



 


AE144_1A
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP
602



GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST




EEGTSESATPESGPGTSTEPSEGSAPG



 


AE144_2A
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
603



GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS




APGTSESATPESGPGTSESATPESGPG



 


AE144_2B
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP
604



GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS




APGTSESATPESGPGTSESATPESGPG



 


AE144_3A
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
605



GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS




APGSPAGSPTSTEEGTSTEPSEGSAPG



 


AE144_3B
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
606



GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS




APGSPAGSPTSTEEGTSTEPSEGSAPG



 


AE144_4A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
607



GTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST




EEGTSESATPESGPGTSTEPSEGSAPG



 


AE144_4B
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
608



GTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST




EEGTSESATPESGPGTSTEPSEGSAPG



 


AE144_5A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP
609



GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSPAGSPTSTEEGSPAGSPTSTEEG



 


AE144_6B
TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETP
610



GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSE




TPGTSESATPESGPGTSTEPSEGSAPG



 


AE288_1
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
611



GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP




ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA




TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST




EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



 


AE288_2
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS
612



APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE




GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA




GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP



 


AE576
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
613



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP




AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG




TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP



 


AE624
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTS
614



TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE




GSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESA




TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST




EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT




STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE




GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS




APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT




STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT




SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA




GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP



 


AE864
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
615



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP




AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG




TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST




EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS




PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP




GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



 


AE865
GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG
616



SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP




TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP




SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP




ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS




PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP




GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS




APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP




ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS




PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES




ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS




TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG




SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP




GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



 


AE866
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
617



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP




AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG




TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST




EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS




PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP




GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG



 


AE1152
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
618



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP




AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG




TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST




EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS




PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP




GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP




ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS




EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG




SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE




SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT




STEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



 


AE144A
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
619



GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES




GPGSPAGSPTSTEEGSPAGSPTSTEEGS



 


AE144B
SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
620



GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST




EEGSPAGSPTSTEEGTSTEPSEGSAPG



 


AE180A
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG
621



SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP




ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS




EPATS



 


AE216A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES
622



ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS




ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG




TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT



 


AE252A
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA
623



TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST




EPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS




EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP




GTSESATPESGPGTSTEPSE



 


AE288A
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP
624



ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS




EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP




GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE




TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA



 


AE324A
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP
625



SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE




SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT




SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP




GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST




EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS



 


AE360A
PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG
626



SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE




SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT




SESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE




GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE




TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG




SETPGTSESAT



 


AE396A
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAG
627



SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSE




SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT




STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP




GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES




GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE




GSAPGTSTEPS



 


AE432A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES
628



ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP




AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG




TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP




GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP




ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGSEPATS



 


AE468A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES
629



ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS




TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG




TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP




GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE




GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT




SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES




AT



 


AE504A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG
630



SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP




ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS




PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP




GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS




APGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESA




TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST




EPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS



 


AE540A
TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST
631



EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT




STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP




GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES




GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE




GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA




TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES




ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS




ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG




TSTEPSEGSAPGTSTEP



 


AE576A
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES
632



ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS




TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG




SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP




GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES




GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG




SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS




GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES




ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP




AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA



 


AE612A
GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAG
633



SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST




EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGS




PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP




GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST




EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATP




ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS




EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG




SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE




SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT




STEPSEGSAPGSEPATSGSETPGTSESAT



 


AE648A
PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP
634



SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP




ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT




STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP




GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES




ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS




TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPG




SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP




GTSESAT



 


AE684A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTE
635



PSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTS




ESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG




TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE




GSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA




TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP




ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS




EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP




GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE




TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS



 


AE720A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS
636



TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG




TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP




GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE




TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE




GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST




EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGT




SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP




GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE




GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT




SGSETPGSPAGSPTSTEEGTSTE



 


AE756A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS
637



TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG




TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP




GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE




TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE




GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST




EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGT




SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP




GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE




GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT




SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES



 


AE792A
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES
638



ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS




TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG




TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE




GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS




APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT




STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT




SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA




GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS




EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP




GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST




EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP




ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS




EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS



 


AE828A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES
639



ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTS




TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG




TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP




GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE




GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP




SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE




SATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS




PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP




GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST




EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT




STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT




SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP




ATSGSETPGTSESAT



 


AE869
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE
640



GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS




PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE




PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE




PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG




SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP




GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS




APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP




ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS




PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES




ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS




TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG




SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP




GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGR



 


AE144_R1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
641



PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP




AGSPTSTEEGTSESATPESGPGTESASR



 


AE288_R1
SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
642



STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP




GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES




GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE




GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSASR



 


AE432_R1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
643



PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP




AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG




TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP




GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES




GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE




GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP




SEGSAPGSPAGSPTSTEEGTESASR



 


AE576_R1
SAGSPTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT
644



STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST




EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS




PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP




GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSASR



 


AE864_R1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
645



PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP




AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG




TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP




GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES




GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE




GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP




SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE




SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS




PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP




GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP




ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA




TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST




EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTESASR



 


AE712
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
646



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP




AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG




TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST




EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS




PAGSPTSTEAHHH



 


AE864_R2
GSPGAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE
647



PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP




AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG




TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP




GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES




GPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE




GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP




SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE




SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS




PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP




GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP




ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA




TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST




EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTESASR



 


AE288_3
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
648



GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST




EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT




STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT




SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG



 


AE284
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
649



GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP




ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA




TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST




EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE



 


AE292
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
650



GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST




EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT




STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT




SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA




P



 


AE864_2
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG
651



TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE




GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSG




SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGS




PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTE




PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP




AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG




SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE




GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE




GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS




PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES




ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS




TEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAAEPEA



 


AE867
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
652



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP




AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG




TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST




EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS




PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP




GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAAEPEA



 


AE867_2
SPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG
653



SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP




TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP




SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP




ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS




PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP




GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS




APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP




ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS




PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES




ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS




TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG




SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP




GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG



 


AE868
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
654



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP




AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG




TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA




TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST




EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS




PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP




GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS




APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAAEPEA



 


AE144_7A
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
655



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAP



 


AE292
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP
656



GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST




EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT




STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT




SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA




P



 


AE293
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
657



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPEGAAEPE




A



 


AE300
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
658



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP




AGAAEPEA



 


AE584
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
659



APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT




STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS




EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA




TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP




AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG




TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP




GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES




GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPT




STEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGAAEPE




A









In some embodiments, for constructing the sequence of a barcoded ELNN, amino-acid mutations are performed on ELNN of intermediate lengths to those of Table 3b, as well as ELNN of longer lengths than those of Table 3b, such as those in which one or more 12-mer motifs of Table 1 are added to the N- or C-terminus of a general-purpose ELNN of Table 3b.
Additional examples of existing ELNNs that can be used according to the present disclosure are disclosed in U.S. Patent Publication Nos. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1, 2011/0077199 A1, or 2011/0172146 A1, or International Patent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1. WO 2011028344 A2, WO 2014/011819 A2, or WO 2015/023891.
In some embodiments, a barcoded ELNN fused within a polypeptide chain adjacent to the N-terminus of the polypeptide chain (“N-terminal ELNN”) can be attached to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus to facilitate the purification of the fusion polypeptide. In some embodiments, a barcoded ELNN fused within a polypeptide chain at the C-terminus of the polypeptide chain (“C-terminal ELNN”) can be comprise or be attached to the sequence EPEA at the C-terminus to facilitate the purification of the fusion polypeptide. In some embodiments, the fusion polypeptide comprises both an N-terminal barcoded ELNN and a C-terminal barcoded ELNN, wherein the N-terminal barcoded ELNN is attached to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus; and wherein the C-terminal barcoded ELNN is attached to the sequence EPEA at the C-terminus, thereby facilitating purification of the fusion polypeptide, for example, to at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% purity by chromatography methods known in the art, including but not limited to IMAC chromatography, C-tagXL affinity matrix, and other such methods.
A barcode fragment, as described herein, can be cleavably fused within the ELNN and releasable (i.e., configured to be released) from the ELNN upon digestion of the polypeptide by a protease. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves on the C-terminal side of glutamic acid residues that are not followed by proline. In some embodiments, a barcoded ELNN (an ELNN that contains barcode fragment(s) therewithin) is designed to achieve high efficiency, precision and accuracy of the protease digestion. For example, in some embodiments, adjacent Glu-Glu (EE) residues in an ELNN sequence can result in varying cleavage patterns upon Glu-C digestion. Accordingly, when Glu-C protease is used for barcode release, the barcoded ELNN or the barcode fragment(s) may not contain any Glu-Glu (EE) sequence. Additionally, a di-peptide Glu-Pro (EP) sequence, if present in the fusion polypeptide, may not be cleaved by Glu-C protease during the barcode release process.
Structural Configuration of Activatable Tces
In some embodiments, a fusion protein comprises a single BsAb in the form of a TCE and a single ELNN. In some embodiments, such a fusion protein can have at least the following permutations of configurations, each listed in an N- to C-terminus orientation: (TCE)-(ELNN); (ELNN)-(TCE); (TCE)-(Linker)-(ELNN); and (ELNN)-(Linker)-(TCE).
In some embodiments, the fusion protein comprises a C-terminal ELNN and, optionally, a linker (such as one described herein, e.g., in Table C) between the ELNN and the TCE. In some embodiments, such a fusion protein can be represented by Formula I (depicted N- to C-terminus):

(TCE)-(Linker)-(ELNN)  (I),

wherein the TCE is as described herein; Linker is a linker sequence (such as one described herein, e.g., in Table C) comprising between 1 to about 50 amino acid residues that can optionally include a TCE release segment (e.g., as described herein); and the ELNN can be any ELNN described herein.

In some embodiments, the fusion protein comprises an N-terminal ELNN and, optionally, a linker (such as one described herein, e.g., in Table C) between the ELNN and the TCE. In some embodiments, such a fusion protein can be represented by Formula II (depicted N- to C-terminus):

(ELNN)-(Linker)-(TCE)  (II),

wherein TCE is as described herein; Linker is a linker sequence (such as one described herein, e.g., in Table C) comprising between 1 to about 50 amino acid residues that can optionally include a TCE release segment (e.g., as described herein); and ELNN can be any ELNN described herein.

In some embodiments, the fusion protein comprises both an N-terminal ELNN and a C-terminal ELNN. In some embodiments, such a fusion protein can be represented by Formula III:

(ELNN)-(Linker)-(TCE)-(Linker)-(ELNN)  (III)

wherein TCE is as described herein; each Linker is, individually, a linker sequence (such as one described herein, e.g., in Table C) having between 1 to about 50 amino acid residues that can optionally include a TCE release segment (e.g., as described herein); and each ELNN can be, individually, any ELNN described herein.

The present disclosure provides BsAbs (e.g., TCEs) comprise one or more sequences disclosed herein in any one of Tables 5a-5f.
Of particular interest are BsAbs (e.g., TCEs) for which an increase in a pharmacokinetic parameter, increased solubility, increased stability, masking of activity, or some other enhanced pharmaceutical property is sought, or those BsAbs (e.g., TCEs) for which increasing the terminal half-life would improve efficacy, and/or safety. Thus, the paTCE fusion protein compositions are prepared with various objectives in mind, including improving the therapeutic efficacy of the TCE by, for example, increasing the in vivo exposure or the length that the TCE remains within the therapeutic window when administered to a subject, compared to a TCE not linked to any ELNNs.
It will be appreciated that various amino acid substitutions (especially conservative amino acid substitutions) can be made in a bispecific sequence to create variants without departing from the spirit of the present disclosure with respect to the biological activity or pharmacologic properties of, e.g., a TCE. Examples of conservative substitutions for amino acids in polypeptide sequences are shown in Table 4. In addition, variants can also include, for instance, polypeptides wherein one or more amino acid residues are added or deleted at the N- or C-terminus of the full-length native amino acid sequence of a TCE that retains at least a portion of the biological activity of the native peptide.
In some embodiments, sequences that retain at least about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more of the activity compared to the corresponding original TCE sequence would be considered suitable for inclusion in the subject paTCE. In some embodiments, a TCE found to retain a suitable level of activity can be linked to one or more ELNN polypeptides, having at least about 80% sequence identity (e.g., at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity) to a sequence from Tables 3a-3b.







TABLE 4







Exemplary conservative amino acid substitutions










Original Residue
Exemplary Substitutions






Ala (A)
val; leu; ile



Arg (R)
lys; gin; asn



Asn (N)
gin; his; lys; arg



Asp (D)
Glu



Cys (C)
Ser



Gln (Q)
Asn



Glu (E)
Asp



Gly (G)
Pro



His (H)
asn: gin: lys: arg



Ile (I)
leu; val; met; ala; phe: norleucine



Leu (L)
norleucine: ile: val; met; ala: phe



Lys (K)
arg: gin: asn



Met (M)
leu; phe; ile



Phe (F)
leu: val: ile; ala



Pro (P)
gly



Ser (S)
thr



Thr (T)
ser



Trp (W)
tyr



Tyr(Y)
trp: phe: thr: ser



Val (V)
ile; leu; met; phe; ala; norleucine









The present disclosure provides ELNNylated TCEs (such as paTCEs) that target EGFR, wherein TCE is a bispecific antibody (e.g., a bispecific TCE) that specifically binds to EGFR with one portion of the bispecific TCE and CD3 with the other portion of the bispecific TCE.
In some embodiments, the ELNNylated TCE comprises (1) a first portion comprising a first binding domain and a second binding domain, and (2) a second portion comprising a release segment, and (3) a third portion comprising an unstructured polypeptide mask (also sometimes referred to herein as a masking moiety).
In some embodiments, the ELNNylated TCE comprises the configuration of Formula Ia (depicted N-terminus to C-terminus):

(first portion)-(second portion)-(third portion)  (Ia)

    
    
        wherein first portion is a bispecific antibody domain comprising two antigen binding domains as noted above wherein the first binding domain has specific binding affinity to EGFR (e.g., as expressed on a cancer cell) and the second binding domain has specific binding affinity to a CD3 (e.g., as expressed on an effector cell); the second portion comprises a release segment (RS) capable of being cleaved by a mammalian protease; and the third portion is a masking moiety that serves to mask the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that is cleavable by a protease that is present in a tumor microenvironment.
    
    


In some embodiments in which the first portion comprises two binding domains that each comprise a VL and VH, the first portion binding domains can be in the order (VL-VH) 1-(VL-VH)2, wherein “1” and “2” represent the first and second binding domains, respectively, or (VL-VH) 1-(VH-VL)2, or (VH-VL) 1-(VL-VH)2, or (VH-VL) 1-(VH-VL)2, wherein the paired binding domains are linked by a polypeptide linker (e.g., as described herein).
In some embodiments, the domain that binds EGFR is an scFv comprising a VH and a VL.
In some embodiments, the first portion binding domains comprise sequences provided in Tables 5a-5f, wherein Tables 5a-5e show sequences that bind CD3 and Table 5f show sequences that bind to EGFR; the RS sequence comprises a sequence provided in Tables 7a-7b (e.g., as described herein); and the masking moiety is an ELNN. In some embodiments, the masking moiety is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence comprising the group of sequences set forth in Tables 3a-3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, the portions are linked by chemical conjugation.
In some embodiments, the fusion protein comprises the configuration of Formula IIa (depicted N-terminus to C-terminus):

(third portion)-(second portion)-(first portion)  (IIa)

    
    
        wherein first portion is a bispecific comprising two antigen binding domains wherein the first binding domain has specific binding affinity to a EGFR (e.g., as expressed on a cancer cell) and the second binding domain has specific binding affinity to CD3 (e.g., as expressed on an effector cell); the second portion comprises a release segment (RS) capable of being cleaved by a mammalian protease; and the third portion is a masking moiety that serves to mask the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that is universally cleavable in a tumor microenvironment.
    
    


In some embodiments in which the first portion comprises two binding domains that each comprise a VL and VH, the first portion binding domains can be in the order (VL-VH) 1-(VL-VH)2, wherein “1” and “2” represent the first and second binding domains, respectively, or (VL-VH) 1-(VH-VL)2, or (VH-VL) 1-(VL-VH)2, or (VH-VL) 1-(VH-VL)2, wherein the paired binding domains are linked by a polypeptide linker (e.g., as described herein).
In some embodiments, the domain that binds EGFR is an scFv comprising a VH and a VL.
In some embodiments, the first portion binding domains comprise sequences provided in Tables 5a-6f, wherein Tables 5a-e show sequences that bind CD3 and Table 5f shows sequences that bind to EGFR; the RS sequence comprises a sequence provided in Tables 7a-7b (e.g., as described herein); and the masking moiety is an ELNN. In some embodiments, the masking moiety is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence comprising the group of sequences set forth in Tables 3a-3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, the portions are linked by chemical conjugation.
In some embodiments, a paTCE composition comprises the configuration of Formula IIIa (depicted N-terminus to C-terminus):

(fifth portion)-(fourth portion)-(first portion)-(second portion)-(third portion)  (IIIa)

    
    
        wherein first portion is a bispecific comprising two antigen binding domains wherein the first binding domain has specific binding affinity to a EGFR (e.g., as expressed on a cancer cell) and the second binding domain has specific binding affinity to CD3 (e.g., as expressed on an effector cell); the second portion comprises a release segment (RS) capable of being cleaved by a mammalian protease; and the third portion is a masking moiety that serves to mask the biological properties of the bispecific antibody domain; the fourth portion comprises a release segment (RS) capable of being cleaved by a mammalian protease which may be identical or different from the second portion; and the fifth portion is a masking moiety that may be identical or may be different from the third portion.
    
    


In some embodiments in which the first portion comprises two binding domains that each comprise a VL and VH, the first portion binding domains can be in the order (VL-VH) 1-(VL-VH)2, wherein “1” and “2” represent the first and second binding domains, respectively, or (VL-VH) 1-(VH-VL)2, or (VH-VL) 1-(VL-VH)2, or (VH-VL) 1-(VH-VL)2, wherein the paired binding domains are linked by a polypeptide linker (e.g., as described herein).
In some embodiments, the domain that binds EGFR is an scFv comprising a VH and a VL.
In some embodiments, the first portion binding domains comprise sequences provided in Tables 5a-5f, wherein Tables 5a-5e show sequences that bind CD3 and Table 5f shows sequences that bind to EGFR; each RS sequence comprises, individually, a sequence provided in Tables 7a-7b (e.g., as described herein); and each masking moiety is, individually, an ELNN. In some embodiments, each masking moiety is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence comprising the group of sequences set forth in Tables 3a-3b. In some embodiments, the paTCE is a recombinant fusion protein. In some embodiments, one or more portions of the paTCE are linked by chemical conjugation.
Provided herein are compositions that advantageously provide EGFR-targeted bispecific therapeutics that have more selectivity, greater half-life, and result in less toxicity and fewer side effects once they are cleaved by proteases found in the target tissues or tissues rendered unhealthy by a disease, such that the subject compositions have improved therapeutic index compared to bispecific antibody compositions known in the art. Such compositions are useful in the treatment of cancer. In some embodiments, when a paTCE is in proximity to a target tissue or cell bearing or secreting a protease capable of cleaving the RS, the bispecific binding domains are liberated from the ELNN(s) by the action of protease(s), removing a steric hindrance barrier, and rendering the TCE freer to exert its pharmacologic effect. This property is particularly advantageous in treating immunologically cold tumors that express EGFR. In some embodiments, a paTCE provided herein is activated at in a target tissue, wherein the target tissue is a solid tumor of an organ or system.
Binding Domains
In some embodiments, a binding domain provided herein comprises one or more full-length antibodies or one or more antigen-binding fragments thereof. Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides comprising a portion or portions of an antibody that specifically bind to an antigen. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. The terms binding domain and antibody domain are used interchangeably herein.
In some embodiments, single chain binding domains are used, such as but not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2, linear antibodies, single domain antibodies, VHHs, single-chain antibody molecules (scFv), and diabodies capable of binding ligands or receptors associated with effector cells and antigens of diseased tissues or cells that are cancers, tumors, or other malignant tissues.
In some embodiments, the binding domain is a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds to a first target and a second antigen binding domain that specifically binds to a second target. In some embodiments, the first antigen binding domain is a first antigen binding fragment (e.g., an scFv or an ISVD, such as a VHH) and the second antigen binding domain is a second antigen binding fragment (e.g., an scFv or an ISVD, such as a VHH).
In some embodiments, an antigen binding fragment (AF) (e.g., a first antigen binding fragment (AF1), and/or a second antigen binding fragment (AF2)) can (each independently) be a chimeric, a humanized, or a human antigen-binding fragment. The antigen binding fragment (AF) (e.g., a first antigen binding fragment (AF1), and/or a second antigen binding fragment (AF2)) can (each independently) be an Fv, Fab, Fab′, Fab′-SH, linear antibody, VHH, or scFv.
In some embodiments, one or both antigen binding fragments (e.g., the first and/or second antigen binding fragments) can be configured as an (Fab′)2 or a single chain diabody. In some embodiments, the bispecific antibody comprises a first binding domain with binding specificity to a cancer cell marker and a second binding domain with binding specificity to an effector cell antigen. In some embodiments, the binding domain for the tumor cell target is a variable domain of a T cell receptor that has been engineered to bind MHC that is loaded with a peptide fragment of a protein that is overexpressed by tumor cells.
In some embodiments, a paTCE is designed with consideration of the location of the target tissue protease as well as the presence of the same protease in healthy tissues not intended to be targeted, as well as the presence of the target ligand in healthy tissue but a greater presence of the ligand in unhealthy target tissue, in order to provide a wide therapeutic window. A “therapeutic window” refers to the difference between the minimal effective dose and the maximal tolerated dose for a given therapeutic composition. In some embodiments, to help achieve a wide therapeutic window for a TCE, the binding domains of the TCE are shielded by the proximity of a masking (e.g., ELNN) moiety or moieties such that the binding affinity of the intact composition for one, or both, of the ligands is reduced compared to the composition that has been cleaved by a mammalian protease, thereby releasing the first portion from the shielding effects of the masking moiety.
In some embodiments, a complete antigen recognition and binding site comprises a dimer of one heavy chain variable domain (VH) and one light chain variable domain (VL). Within each VH and VL chain are three complementarity determining regions (CDRs) that interact to define an antigen binding site on the surface of the VH-VL dimer; the six CDRs of a binding domain confer antigen binding specificity to the antibody or single chain binding domain. Framework sequences flanking the CDRs have a tertiary structure that is essentially conserved in native immunoglobulins across species, and the framework residues (FR) serve to hold the CDRs in their appropriate orientation. In some embodiments, a constant domain is not required for binding function but may aid in stabilizing VH-VL interaction. In some embodiments, a binding site can be a pair of VH-VL, VH—VH or VL-VL domains either of the same or of different immunoglobulins, however it is generally preferred to make single chain binding domains using the respective VH and VL chains from the parental antibody. In some embodiments, the order of VH and VL domains within the polypeptide chain is not limiting, provided the VH and VL domains are arranged so that the antigen binding site can properly fold. Thus, in some embodiments, a single chain binding domains comprising a VH and a VL (e.g., in an scFv) can have the VH and VL arranged as VL-VH or VL-VH.
In some embodiments, the arrangement of the V chains may be VH (cancer cell surface antigen)-VL(cancer cell surface antigen)-VL(effector cell antigen)-VH (effector cell antigen), VH (cancer cell surface antigen)-VL(cancer cell surface antigen)-VH (effector cell antigen)-VL(effector cell antigen), VL(cancer cell surface antigen)-VH (cancer cell surface antigen)-VL(effector cell antigen)-VH (effector cell antigen), VL(cancer cell surface antigen)-VH (cancer cell surface antigen)-VH (effector cell antigen)-VL(effector cell antigen), VHH (cancer cell surface antigen)-VH (effector cell antigen)-VL(effector cell antigen), VHH (cancer cell surface antigen)-VL(effector cell antigen)-VH (effector cell antigen), VL(cancer cell surface antigen)-VH (cancer cell surface antigen)-VHH (effector cell antigen), or VH (cancer cell surface antigen)-VL(cancer cell surface antigen)-VHH (effector cell antigen).
In some embodiments, the following orders are possible: VH (effector cell antigen)-VL(effector cell antigen)-VL(cancer cell surface antigen)-VH (cancer cell surface antigen), VH (effector cell antigen)-VL(effector cell antigen)-VH (cancer cell surface antigen)-VL(cancer cell surface antigen), VL(effector cell antigen)-VH (effector cell antigen)-VL(cancer cell surface antigen)-VH (cancer cell surface antigen), VL(effector cell antigen)-VH (effector cell antigen)-VH (cancer cell surface antigen)-VL(cancer cell surface antigen), VHH (effector cell antigen)-VH (cancer cell surface antigen)-VL(cancer cell surface antigen), VHH (effector cell antigen)-VL(cancer cell surface antigen)-VH (cancer cell surface antigen), VL(effector cell antigen)-VH (effector cell antigen)-VHH (cancer cell surface antigen), or VH (effector cell antigen)-VL(effector cell antigen)-VHH (cancer cell surface antigen).
As used herein. “N-terminally to” or “C-terminally to” and grammatical variants thereof denote relative location within the primary amino acid sequence rather than placement at the absolute N- or C-terminus of the bispecific single chain antibody. Hence, as a non-limiting example, a first binding domain which is “located C-terminally to” a second binding domain denotes that the first binding is located on the carboxyl side of the second binding domain within a bispecific single chain antibody, and does not exclude the possibility that an additional sequence, for example a linker and/or an ELNN, a His-tag, or another compound such as a radioisotope, is located at the C-terminus of the bispecific single chain antibody.
In some embodiments, a paTCE comprises a first portion comprising a first binding domain and a second binding domain wherein each of the binding domains is an scFv and wherein each scFv comprises one VL and one VH. In some embodiments, the first binding domain is an scFv that binds CD3 and the second binding domain is an scFv that binds EGFR. In some embodiments, the paTCE compositions comprise a first portion comprising a first binding domain and a second binding domain wherein one of the binding domains is an scFV and the other binding domain is a VHH. In some embodiments, a paTCE comprises a first portion comprising a first binding domain and a second binding domain wherein the binding domains are in a diabody configuration and wherein one domain comprises one VL region and one VH region and the other domain comprises one VL region and one VH region. Exemplary VH and VL of CD3-binding domains are shown in Tables 5a-5e. Exemplary VH and VL of EGFR-binding domains are shown in Table 5f.
In non-limiting examples, a TCE can comprise a sequence that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an antibody sequence identified herein. In some embodiments, a TCE comprises a bispecific sequence (e.g., a BsAb) comprising a first binding domain and a second binding domain, wherein the first binding domain has specific binding affinity to a tumor-specific marker or a cancer cell antigen, and exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to paired VL and VH sequences of an anti-EGFR antibody disclosed herein in Table 5f; and wherein the second binding domain has specific binding affinity to an effector cell, and exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to paired VL and VH sequences of an anti-CD3 antibody disclosed herein in any of Tables 5a-5e.
In some embodiments, a TCE can comprise a binding domain (e.g., a VH and/or VL amino acid sequence) of or derived from an anti-CD3 antibody. Non-limiting examples of anti-CD3 antibodies include OKT3 (also called muromonab) and humanized anti-CD3 monoclonal antibody (hOKT31 (Ala-Ala)) (KC Herold et al., New England Journal of Medicine 346:1692-1698. 2002), as well as fragments and derivatives thereof that selectively bind to CD3. Additional examples are described in U.S. Pat. Nos. 5,885,573; 6,491,916; and US Patent Application Publication No. 2021/0054077-A1, the entire contents of each of which are incorporated herein by reference. Additional non-limiting examples of anti-CD3 antibody sequences include those of pasotuxizumab (also known as AMG-212) and acapatamab (also known as AMG-160).
In some embodiments, a TCE can comprise a binding domain (e.g., a VH and/or VL amino acid sequence) of or derived from an anti-EGFR antibody. Non-limiting examples of anti-EGFR antibody sequences include those of panitumumab and cetuximab.
The present disclosure provides antigen binding domains that bind EGFR. The present disclosure provides scFvs that bind EGFR (e.g., an scFv having a paired VH and VL of Table 5f). The present disclosure further provides nucleic acids encoding the antigen binding domains (e.g., scFvs) or polypeptides as well as vectors, hosts and methods to produce these antigen binding domains or polypeptides. Also provided are multispecific polypeptides comprising an antigen binding domain that binds EGFR according to the present disclosure and at least one CD3 binding domain, including paTCEs. Included are methods for treatment making use of the antigen binding domains or polypeptides according to the present disclosure.
Also provided is a nucleic acid molecule encoding the antigen binding domains (e.g., an scFv) or polypeptide of the present disclosure or a vector comprising the nucleic acid.
The present disclosure also relates to a non-human host or host cell transformed or transfected with the nucleic acid or vector that encodes an antigen binding domains (e.g., an scFv) or polypeptide disclosed herein.
The present disclosure furthermore relates to compositions comprising an antigen binding domains (e.g., an scFv) or polypeptide disclosed herein, such as a pharmaceutical composition.
Included herein is a method for producing an antigen binding domains (e.g., an scFv) or polypeptide as disclosed herein, the method comprising the steps of:

    
    
        a. expressing, in a host cell or host organism or in another expression system, a nucleic acid sequence encoding the antigen binding domains (e.g., an scFv) or polypeptide; optionally followed by:
        b. isolating and/or purifying the antigen binding domains (e.g., an scFv) or polypeptide.
    
    


Provided herein are compositions and polypeptides comprising an antigen binding domains (e.g., an scFv) for use as a medicament. In some embodiments, the polypeptide or composition is for use in the treatment of a proliferative disease. In some embodiments, the proliferative disease is cancer.
The present disclosure also provides a method of treatment comprising the step of administering a composition or polypeptide comprising an antigen binding domains (e.g., an scFv) to a subject in need thereof. In some embodiments, the method of treatment is for treating a proliferative disease. In some embodiments, the proliferative disease is cancer.
Included herein are composition and polypeptides comprising an antigen binding domains (e.g., an scFv) for use in the preparation of a medicament. In some embodiments, the medicament is used in the treatment of a proliferative disease. In some embodiments, the proliferative disease is cancer.
In some embodiments, the structure of each of the VH or VL of an antigen binding domain (e.g., scFv) sequence can be considered to be comprised of four framework regions (“FRs”), which are referred to in the art and herein as “Framework region 1” (“FR1”); as “Framework region 2” (“FR2”); as “Framework region 3” (“FR3”); and as “Framework region 4” (“FR4”), respectively; which framework regions are interrupted by three complementary determining regions (“CDRs”), which are referred to in the art and herein as “Complementarity Determining Region 1” (“CDR1”); as “Complementarity Determining Region 2” (“CDR2”); and as “Complementarity Determining Region 3” (“CDR3”), respectively.
In some embodiments, technology provided herein uses antigen binding domains (e.g., scFvs) that can bind to EGFR. In the context of the present technology, “binding to” a certain target molecule has the usual meaning in the art as understood in the context of antibodies and their respective antigens.
As will be clear from the further description above and herein, the antigen binding domain (e.g., scFv) of the present technology can be used as “building blocks” to form polypeptides of the present technology, e.g., by suitably combining them with other groups, residues, moieties or binding units, in order to form compounds or fusion proteins as described herein (such as, without limitations, the bi-/tri-/tetra-/multivalent and bi-/tri-/tetra-/multispecific polypeptides of the present technology described herein), which combine within one molecule one or more desired properties or biological functions.
The terms “specificity”, “binding specifically” or “specific binding” refer to the number of different target molecules, such as antigens, from the same organism to which a particular binding unit, such as an antigen binding domain (e.g., scFv), can bind with sufficiently high affinity (see below). “Specificity”, “binding specifically” or “specific binding” are used interchangeably herein with “selectivity”, “binding selectively” or “selective binding”. Binding units, such as scFvs, preferably specifically bind to their designated targets.
The specificity/selectivity of a binding unit can be determined based on affinity. The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as by the KD which is expressed in units of mol/liter (or M).
The affinity is a measure for the binding strength between a moiety and a binding site on the target molecule: the lower the value of the kD, the stronger the binding strength between a target molecule and a targeting moiety.
Typically, binding units used in the present technology (such as scFvs) will bind to their targets with a kD of 10−5 to 10−12 moles/liter or less, and preferably 10−7 to 10−12 moles/liter or less and more preferably 10−8 to 10−12 moles/liter.
In some embodiments, a kD value greater than 10−4 mol/liter is considered nonspecific. In some embodiments, a kD value less than 10−4 mol/liter is considered specific.
The kD for biological interactions, such as the binding of antibody sequences to an antigen, which are considered specific are typically in the range of 10000 nM or 10 μM to 0.001 nM or 1 μM or less.
Accordingly, specific/selective binding may mean that-using the same measurement method, e.g., SPR-a binding unit (or polypeptide comprising the same) binds to EGFR with a kD value of 10−5 to 10−12 moles/liter or less and binds to different targets with a kD value greater than 10−4 moles/liter.
Specific binding to a certain target from a certain species does not exclude that the binding unit can also specifically bind to the analogous target from a different species. For example, specific binding to human EGFR does not exclude that the binding unit (or a polypeptide comprising the same) can also specifically bind to EGFR from cynomolgus monkeys.
Specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
The dissociation constant may be, e.g., the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned below.
The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al. 2001, Intern. Immunology 13:1551-1559). The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding kon, koff measurements and hence kD values. This can for example be performed using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991 Biotechniques 11:620-627), Johnsson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnnson et al. (1991, Anal. Biochem. 198:268-277).
Another well-known biosensor technique to determine affinities of biomolecular interactions is bio-layer interferometry (BLI) (see for example Abdiche et al. 2008, Anal. Biochem. 377:209-217). The term “bio-layer Interferometry” or “BLI”, as used herein, refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and affinities can be determined. BLI can for example be performed using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).
Alternatively, affinities can be measured in Kinetic Exclusion Assay (KinExA) (see for example Drake et al. 2004, Anal. Biochem., 328:35-43), using the KinExAR platform (Sapidyne Instruments Inc, Boise, USA). The term “KinExA”, as used herein, refers to a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules. Equilibrated solutions of an antibody/antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).
The GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5:1765-74).
In some embodiments, a paTCE comprises a first binding domain that is an scFv and a second binding domain that is an scFv. In some embodiments, the first scFv comprises VL and VH domains and specificity binds to an effector cell antigen (such as CD3), and the second scFv specifically binds a cancer cell antigen (such as EGFR). In some embodiments, the scFv comprises six CDRs. In some embodiments, the scFv that comprises VH and VL regions comprising amino acid sequences that are at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to, or are identical to, paired VL and VH sequences of an anti-CD3 antibody identified in Table 5a. In some embodiments, the scFv comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region of paired VL and VH sequences of an anti-CD3 antibody identified in Table 5a. In some embodiments, the scFv is derived from an anti-EGFR antibody identified as the antibodies set forth in Table 5f. In some embodiments, the scFv comprises VH and VL regions comprising amino acid sequences that are at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to, or is identical to, a VH and VL sequence disclosed in Table 5f. In some embodiments, the VH and VL comprise a CDR-1 region, a CDR-2 region, and a CDR-3 region of a VH and VL sequence in Table 5f.
In some embodiments, a paTCE comprises a first binding domain that is an scFv and a second binding domain that is also an scFv. In some embodiments, the scFvs comprise VL and VH domains that are derived from monoclonal antibodies with binding specificity to the tumor-specific marker or an antigen of a cancer cell and effector cell antigen, respectively. In some embodiments, the first and second binding domains each comprise six CDRs derived from monoclonal antibodies with binding specificity to a cancer cell marker, such as a tumor-specific marker and effector cell antigens, respectively. In some embodiments, the first and second binding domains of the first portion of the subject compositions can have 3, 4, 5, or 6 CDRs within each binding domain. In some embodiments, a paTCE comprises a first binding domain and a second binding domain wherein each comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, wherein each of the regions is derived from a monoclonal antibody capable of binding a tumor-specific marker or an antigen of a cancer cell, and an effector cell antigen, respectively.
In some embodiments, the second binding domain comprises VH and VL regions derived from a monoclonal antibody capable of binding human CD3. In some embodiments, the second binding domain comprises a scFv that comprises VH and VL regions wherein each VH and VL regions exhibit at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or is identical to paired VL and VH sequences of an anti-CD3 antibody identified in Table 5a. In some embodiments, the second domain comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, wherein each of the regions is derived from a monoclonal antibody identified herein as the antibodies set forth in Table 5a. In some embodiments, the VH and/or VL domains can be configured as scFvs or diabodies.
In some embodiments, a paTCE comprises a first binding domain that is a diabody and a second binding domain that is also a diabody. In some embodiments, the diabodies comprise VL and VH domains that are derived from monoclonal antibodies with binding specificity to the tumor-specific marker or an antigen of a cancer cell and the effector cell antigen, respectively.
In some embodiments, the present disclosure provides a paTCE composition, wherein the diabody second binding domain comprises VH and VL regions wherein each of the VH and VL regions exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or is identical to the VL and a VH sequence of the huUCHT1 antibody of Table 5a. In some embodiments, the diabody second domain of the composition is derived from an anti-CD3 antibody described herein. In some embodiments, the anti-CD3 diabody is linked to an anti-EGFR-binding scFv sequence disclosed herein.
Methods to measure binding affinity and/or other biologic activity of an antigen binding domain can be those disclosed herein or methods generally known in the art. For example, the binding affinity of a binding pair (e.g., antibody and antigen), denoted as kD, can be determined using various suitable assays including, but not limited to, radioactive binding assays, non-radioactive binding assays such as fluorescence resonance energy transfer and surface plasmon resonance (SPR, Biacore), and enzyme-linked immunosorbent assays (ELISA), kinetic exclusion assay (KinExA®) or as described in the Examples. An increase or decrease in binding affinity, for example the increased binding affinity of a TCE that has been cleaved to remove a masking moiety compared to the paTCE with the masking moiety attached, can be determined by measuring the binding affinity of the TCE to its target binding partner with and without the masking moiety.
Measurement of half-life of a subject chimeric assembly can be performed by various suitable methods. For example, the half-life of a substance can be determined by administering the substance to a subject and periodically sampling a biological sample (e.g., biological fluid such as blood or plasma or ascites) to determine the concentration and/or amount of that substance in the sample over time. The concentration of a substance in a biological sample can be determined using various suitable methods, including enzyme-linked immunosorbent assays (ELISA), immunoblots, and chromatography techniques including high-pressure liquid chromatography and fast protein liquid chromatography. In some cases, the substance may be labeled with a detectable tag, such as a radioactive tag or a fluorescence tag, which can be used to determine the concentration of the substance in the sample (e.g., a blood sample or a plasma sample. The various pharmacokinetic parameters are then determined from the results, which can be done using software packages such as SoftMax Pro software, or by manual calculations known in the art.
In addition, the physicochemical properties of the paTCE compositions may be measured to ascertain the degree of solubility, structure, and retention of stability. Assays of the subject compositions are conducted that allow determination of binding characteristics of the binding domains towards a ligand, including affinity and binding constants (kD, kon and koff), the half-life of dissociation of the ligand-receptor complex, as well as the activity of the binding domain to inhibit the biologic activity of the sequestered ligand compared to free ligand (IC50 values). The term “EC50” refers to the concentration needed to achieve half of the maximum biological response of the active substance, and is generally determined by ELISA or cell-based assays, including the methods of the Examples described herein.
Anti-CD3 Binding Domains
Also provided are anti-CD3 antibodies, fragments thereof, and fusion proteins comprising such antibodies and/or fragments.
In some embodiments, the present disclosure provides paTCE compositions comprising a binding domain of a first portion with binding affinity to T cells. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody that binds CD3. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody to CD3 epsilon and/or CD3 delta. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody to CD3 epsilon. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody to CD3 delta. Exemplary, non-limiting examples of VL and VH sequences of monoclonal antibodies to CD3 are presented in Table 5a. In some embodiments, the present disclosure provides a paTCE comprising a binding domain with binding affinity to CD3 comprising anti-CD3 VL and VH sequences set forth in Table 5a. In some embodiments, the present disclosure provides a paTCE comprising a binding domain of the first portion with binding affinity to CD3epsilon comprising anti-CD3epsilon VL and VH sequences set forth in Table 5a. In some embodiments, the present disclosure provides a paTCE composition, wherein a binding domain of the first portion comprises an scFv that comprises VH and VL regions wherein each VH and VL regions exhibit at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or is identical to paired VL and VH sequences of the huUCHT1 anti-CD3 antibody of Table 5a. In some embodiments, the present disclosure provides a paTCE composition comprising a binding domain with binding affinity to CD3 comprising the CDR-L1 region, the CDR-L2 region, the CDR-L3 region, the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region, wherein each is derived from the respective anti-CD3 VL and VH sequences set forth in Table 5a. In some embodiments, the present disclosure provides a paTCE composition comprising a binding domain with binding affinity to CD3 comprising an CDR-L1 region of RSSNGAVTSSNYAN (SEQ ID NO: 1), an CDR-L2 region of GTNKRAP (SEQ ID NO: 4), an CDR-L3 region of ALWYPNLWV (SEQ ID NO: 6), an CDR-H1 region of GFTFSTYAMN (SEQ ID NO: 12), an CDR-H2 region of RIRTKRNNYATYYADSVKG (SEQ ID NO: 13), and an CDR-H3 region of HENFGNSYVSWFAH (SEQ ID NO: 10). In some embodiments, the present disclosure provides a paTCE composition comprising a binding domain with binding affinity to CD3 comprising an CDR-L1 region of RSSNGAVTSSNYAN (SEQ ID NO: 1), an CDR-L2 region of GTNKRAP (SEQ ID NO: 4), an CDR-L3 region of ALWYPNLWV (SEQ ID NO: 6), an CDR-H1 region of GFTFSTYAMN (SEQ ID NO: 12), an CDR-H2 region of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and an CDR-H3 region of HENFGNSYVSWFAH (SEQ ID NO: 10).
The CD3 complex is a group of cell surface molecules that associates with the T-cell antigen receptor (TCR) and functions in the cell surface expression of TCR and in the signaling transduction cascade that originates when a peptide: MHC ligand binds to the TCR. Without being bound by any scientific theory, typically, when an antigen binds to the T-cell receptor, the CD3 sends signals through the cell membrane to the cytoplasm inside the T cell. This causes activation of the T cell that rapidly divide to produce new T cells sensitized to attack the particular antigen to which the TCR was exposed. The CD3 complex is comprised of the CD3epsilon molecule, along with four other membrane-bound polypeptides (CD3-gamma, -delta, and/or -zeta). In humans, CD3-epsilon is encoded by the CD3E gene on Chromosome 11. The intracellular domains of each of the CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) that serve as the nucleating point for the intracellular signal transduction machinery upon T cell receptor engagement.
A number of therapeutic strategies modulate T cell immunity by targeting TCR signaling, particularly the anti-human CD3 monoclonal antibodies (mAbs) that are widely used clinically in immunosuppressive regimes. The CD3-specific mouse mAb OKT3 was the first mAb licensed for use in humans (Sgro, C. Side-effects of a monoclonal antibody, muromonab CD3/orthoclone OKT3: bibliographic review. Toxicology 105:23-29. 1995) and is widely used clinically as an immunosuppressive agent in transplantation (Chatenoud, Clin. Transplant 7:422-430, (1993); Chatenoud, Nat. Rev. Immunol. 3:123-132 (2003); Kumar, Transplant. Proc. 30:1351-1352 (1998)), type 1 diabetes, and psoriasis. Importantly, anti-CD3 mAbs can induce partial T cell signaling and clonal anergy (Smith, JA, Nonmitogenic Anti-CD3 Monoclonal Antibodies Deliver a Partial T Cell Receptor Signal and Induce Clonal Anergy J. Exp. Med. 185:1413-1422 (1997)). OKT3 has been described in the literature as a T cell mitogen as well as a potent T cell killer (Wong, JT. The mechanism of anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-T cell bridging. Transplantation 50:683-689 (1990)). In particular, the studies of Wong demonstrated that by bridging CD3 T cells and target cells, one could achieve killing of the target and that neither FcR-mediated ADCC nor complement fixation was necessary for bivalent anti-CD3 MAB to lyse the target cells.
OKT3 exhibits both a mitogenic and T-cell killing activity in a time-dependent fashion; following early activation of T cells leading to cytokine release, upon further administration OKT3 later blocks all known T-cell functions. It is due to this later blocking of T cell function that OKT3 has found such wide application as an immunosuppressant in therapy regimens for reduction or even abolition of allograft tissue rejection. Other antibodies specific for the CD3 molecule are disclosed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50, WO2005/118635 and WO2007/033230 describe anti-human monoclonal CD3 epsilon antibodies, U.S. Pat. No. 5,821,337 describes the VL and VH sequences of murine anti-CD3 monoclonal Ab UCHT1 (muxCD3, Shalaby et al., J. Exp. Med. 175, 217-225 (1992) and a humanized variant of this antibody (hu UCHT1), and U.S. patent application No. 20120034228 discloses binding domains capable of binding to an epitope of human and non-chimpanzee primate CD3 epsilon chain.
In some embodiments, an anti-CD3 antibody domain comprises a VH region comprising the sequence EVOLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYA DSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311), or the CDRs thereof, and a VL region comprising the sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF SGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), or the CDRs thereof.
In some embodiments, an anti-CD3 antibody domain comprises a VH region comprising the sequence EVOLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYA DSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126), or the CDRs thereof, and a VL region comprising the sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF SGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127), or the CDRs thereof.







TABLE 5a







Anti-CD3 Monoclonal Antibodies and Sequences













Clone
Antibody


SEQ ID

SEQ ID


Name
Name
Target
VH Sequence
NO.
VL Sequence
NO.





huOKT3

CD3
QVQLVQSGGGVVQP
301
DIQMTQSPSSLSASV
351





GRSLRLSCKASGYT

GDRVTITCSASSSVS






FTRYTMHWVRQAP

YMNWYQQTPGKAP






GKGLEWIGYINPSR

KRWIYDTSKLASGV






GYTNYNQKVKDRF

PSRFSGSGSGTDYTF






TISRDNSKNTAFLQ

TISSLQPEDIATYYC






MDSLRPEDTGVYFC

QQWSSNPFTFGQGT






ARYYDDHYCLDYW

KLQITR






GQGTPVTVSS





 


huUCHT1

CD3
EVQLVESGGGLVQP
302
DIQMTQSPSSLSASV
352





GGSLRLSCAASGYS

GDRVTITCRASQDIR






FTGYTMNWVRQAP

NYLNWYQQKPGKA






GKGLEWVALINPYK

PKLLIYYTSRLESGV






GVSTYNQKFKDRFT

PSRFSGSGSGTDYTL






ISVDKSKNTAYLQM

TISSLQPEDFATYYC






NSLRAEDTAVYYCA

QQGNTLPWTFGQG






RSGYYGDSDWYFD

TKVEIK






VWGQGTLVTVSS





 


hu12F6

CD3
QVQLVQSGGGVVQP
303
DIQMTQSPSSLSASV
353





GRSLRLSCKASGYT

GDRVTMTCRASSSV






FTSYTMHWVRQAP

SYMHWYQQTPGKA






GKGLEWIGYINPSS

PKPWIYATSNLASG






GYTKYNQKFKDRF

VPSRFSGSGSGTDYT






TISADKSKSTAFLQM

LTISSLQPEDIATYYC






DSLRPEDTGVYFCA

QQWSSNPPTFGQGT






RWQDYDVYFDYW

KLQITR






GQGTPVTVSS





 


mOKT3

CD3
QVQLQQSGAELARP
304
QIVLTQSPAIMSASP
354





GASVKMSCKASGY

GEKVTMTCSASSSV






TFTRYTMHWVKQR

SYMNWYQQKSGTS






PGQGLEWIGYINPSR

PKRWIYDTSKLASG






GYTNYNQKFKDKA

VPAHFRGSGSGTSYS






TLTTDKSSSTAYMQ

LTISGMEAEDAATY






LSSLTSEDSAVYYCA

YCQQWSSNPFTFGS






RYYDDHYCLDYWG

GTKLEINR






QGTTLTVSS





 


MT103
blinatumomab
CD3
DIKLQQSGAELARP
305
DIQLTQSPAIMSASP
355





GASVKMSCKTSGYT

GEKVTMTCRASSSV






FTRYTMHWVKQRP

SYMNWYQQKSGTS






GQGLEWIGYINPSR

PKRWIYDTSKVASG






GYTNYNQKFKDKA

VPYRFSGSGSGTSYS






TLTTDKSSSTAYMQ

LTISSMEAEDAATY






LSSLTSEDSAVYYCA

YCQQWSSNPLTFG






RYYDDHYCLDYWG

AGTKLELK






QGTTLTVSS





 


MT110
solitomab
CD3
DVQLVQSGAEVKKP
306
DIVLTQSPATLSLSP
356





GASVKVSCKASGYT

GERATLSCRASQSV






FTRYTMHWVRQAP

SYMNWYQQKPGKA






GQGLEWIGYINPSR

PKRWIYDTSKVASG






GYTNYADSVKGRF

VPARFSGSGSGTDYS






TITTDKSTSTAYMEL

LTINSLEAEDAATYY






SSLRSEDTATYYCA

CQQWSSNPLTFGG






RYYDDHYCLDYWG

GTKVEIK






QGTTVTVSS





 


CD3.7

CD3
EVQLVESGGGLVQP
307
QTVVTQEPSLTVSPG
357





GGSLKLSCAASGFT

GTVTLTCGSSTGAV






FNKYAMNWVRQAP

TSGYYPNWVQQKP






GKGLEWVARIRSKY

GQAPRGLIGGTKFL






NNYATYYADSVKD

APGTPARFSGSLLGG






RFTISRDDSKNTAYL

KAALTLSGVQPEDE






QMNNLKTEDTAVY

AEYYCALWYSNRW






YCVRHGNFGNSYIS

VFGGGTKLTVL






YWAYWGQGTLVTV








SS





 


CD3.8

CD3
EVQLVESGGGLVQP
308
QAVVTQEPSLTVSP
358





GGSLRLSCAASGFT

GGTVTLTCGSSTGA






FNTYAMNWVRQAP

VTTSNYANWVQQK






GKGLEWVGRIRSKY

PGQAPRGLIGGTNK






NNYATYYADSVKG

RAPGVPARFSGSLL






RFTISRDDSKNTLYL

GGKAALTLSGAQPE






QMNSLRAEDTAVY

DEAEYYCALWYSN






YCVRHGNFGNSYV

LWVFGGGTKLTVL






SWFAYWGQGTLVT








VSS





 


CD3.9

CD3
EVQLLESGGGLVQP
309
ELVVTQEPSLTVSPG
359





GGSLKLSCAASGFT

GTVTLTCRSSTGAV






FNTYAMNWVRQAP

TTSNYANWVQQKP






GKGLEWVARIRSKY

GQAPRGLIGGTNKR






NNYATYYADSVKD

APGTPARFSGSLLGG






RFTISRDDSKNTAYL

KAALTLSGVQPEDE






QMNNLKTEDTAVY

AEYYCALWYSNLW






YCVRHGNFGNSYV

VFGGGTKLTVL






SWFAYWGQGTLVT








VSS





 


CD3.10

CD3
EVKLLESGGGLVQP
310
QAVVTQESALTTSP
360





KGSLKLSCAASGFT

GETVTLTCRSSTGA






FNTYAMNWVRQAP

VTTSNYANWVQEK






GKGLEWVARIRSKY

PDHLFTGLIGGTNK






NNYATYYADSVKD

RAPGVPARFSGSLIG






RFTISRDDSQSILYLQ

DKAALTITGAQTED






MNNLKTEDTAMYY

EAIYFCALWYSNLW






CVRHGNFGNSYVS

VFGGGTKLTVL






WFAYWGQGTLVTV








SS





 


CD3.228

CD3
EVQLVESGGGIVQP
311
ELVVTQEPSLTVSPG
361





GGSLRLSCAASGFT

GTVTLTCRSSNGAV






FSTYAMNWVRQAP

TSSNYANWVQQKP






GKGLEWVGRIRTK

GQAPRGLIGGTNKR






RNNYATYYADSVK

APGTPARFSGSLLGG






GRFTISRDDSKNTVY

KAALTLSGVQPEDE






LQMNSLKTEDTAVY

AVYYCALWYPNLW






YCVRHENFGNSYVS

VFGGGTKLTVL






WFAHWGQGTLVTV








SS





 


CD3.23

CD3
EVQLLESGGGIVQPG
102
ELVVTQEPSLTVSPG
101





GSLKLSCAASGFTF

GTVTLTCRSSNGAV






NTYAMNWVRQAPG

TSSNYANWVQQKP






KGLEWVARIRSKYN

GQAPRGLIGGTNKR






NYATYYADSVKDR

APGTPARFSGSLLGG






FTISRDDSKNTVYLQ

KAALTLSGVQPEDE






MNNLKTEDTAVYY

AVYYCALWYPNLW






CVRHENFGNSYVS

VFGGGTKLTVL






WFAHWGQGTLVTV








SS





 


CD3.24

CD3
EVQLLESGGGIVQPG
102
ELVVTQEPSLTVSPG
103





GSLKLSCAASGFTF

GTVTLTCRSSNGEV






NTYAMNWVRQAPG

TTSNYANWVQQKP






KGLEWVARIRSKYN

GQAPRGLIGGTIKR






NYATYYADSVKDR

APGTPARFSGSLLGG






FTISRDDSKNTVYLQ

KAALTLSGVQPEDE






MNNLKTEDTAVYY

AVYYCALWYPNLW






CVRHENFGNSYVS

VFGGGTKLTVL






WFAHWGQGTLVTV








SS





 


CD3.30

CD3
EVQLQESGGGIVQP
105
ELVVTQEPSLTVSPG
104





GGSLKLSCAASGFT

GTVTLTCRSSNGAV






FNTYAMNWVRQAP

TSSNYANWVQQKP






GKGLEWVARIRSKY

GQAPRGLIGGTNKR






NNYATYYADSVKD

APGTPARFSGSSLGG






RFTISRDDSKNTVYL

KAALTLSGVQPEDE






QMNNLKTEDTAVY

AVYYCALWYPNLW






YCVRHENFGNSYVS

VFGGGTKLTVL






WFAHWGQGTLVTV








SS





 


CD3.31

CD3
EVQLQESGGGIVQP
105
ELVVTQEPSLTVSPG
106





GGSLKLSCAASGFT

GTVTLTCRSSNGAV






FNTYAMNWVRQAP

TSSNYANWVQQKP






GKGLEWVARIRSKY

GQAPRGLIGGTNKR






NNYATYYADSVKD

APGTPARFSGSLLGG






RFTISRDDSKNTVYL

SAALTLSGVQPEDE






QMNNLKTEDTAVY

AVYYCALWYPNLW






YCVRHENFGNSYVS

VFGGGTKLTVL






WFAHWGQGTLVTV








SS





 


CD3.32

CD3
EVQLQESGGGIVQP
105
ELVVTQEPSLTVSPG
107





GGSLKLSCAASGFT

GTVTLTCRSSNGAV






FNTYAMNWVRQAP

TSSNYANWVQQKP






GKGLEWVARIRSKY

GQAPRGLIGGTNKR






NNYATYYADSVKD

APGTPARFSGSSLGG






RFTISRDDSKNTVYL

SAALTLSGVQPEDE






QMNNLKTEDTAVY

AVYYCALWYPNLW






YCVRHENFGNSYVS

VFGGGTKLTVL






WFAHWGQGTLVTV








SS





 


CD3.33

CD3
EVQLQESGGGLVQP
111
ELVVTQEPSLTVSPG
110





GGSLKLSCAASGFT

GTVTLTCRSSTGAV






FNTYAMNWVRQAP

TTSNYANWVQQKP






GKGLEWVARIRSKY

GQAPRGLIGGTNKR






NNYATYYADSVKD

APGTPARFSGSSLGG






RFTISRDDSKNTAYL

SAALTLSGVQPEDE






QMNNLKTEDTAVY

AEYYCALWYSNLW






YCVRHGNFGNSYV

VFGGGTKLTVL






SWFAYWGQGTLVT








VSS





 


CD3.318

CD3
EVQLVESGGGIVQP
126
ELVVTQEPSLTVSPG
127





GGSLRLSCAASGFT

GTVTLTCRSSNGAV






FSTYAMNWVRQAP

TSSNYANWVQQKP






GKGLEWVGRIRTK

GQAPRGLIGGTNKR






RNDYATYYADSVK

APGTPARFSGSLLEG






GRFTISRDDSKNTLY

KAALTLSGVQPEDE






LQMNSLKTEDTAVY

AVYYCALWYPNLW






YCVRHENFGNSYVS

VFGGGTKLTVL






WFAHWGQGTLVTV








SS





*underlined sequences, if present, are CDRs within the VL and VH






In some embodiments, the disclosure relates to antigen binding fragments (AF) having specific binding affinity for an effector cell antigen.
Various AF that bind effector cell antigens, particularly CD3 on T cells, have particular utility for pairing with an antigen binding fragment with binding affinity to EGFR antigens associated with a diseased cell or tissue in composition formats in order to recruit and effect effector cell-mediated cell killing of the diseased cell or tissue.
Binding specificity to the antigen of interest can be determined by complementarity determining regions, or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity is determined by light chain CDRs and heavy chain CDRs. A given combination of heavy chain CDRs and light chain CDRs provides a given binding pocket that confers greater affinity and/or specificity towards an effector cell antigen as compared to other reference antigens. The resulting bispecific compositions which on the one hand bind to an effector cell antigen and on the other hand bind to an antigen on the diseased cell or tissue, having a first antigen binding fragment to EGFR linked by a short, flexible peptide linker to a second antigen binding fragment with binding specificity to an effector cell antigen are bispecific, with each antigen binding fragment having specific binding affinity to their respective ligands.
It will be understood that in such compositions, an AF directed against EGFR of a disease tissue is used in combination with an AF directed towards an effector cell marker in order to bring an effector cell in close proximity to the cell of a disease tissue in order to effect the cytolysis of the cell of the diseased tissue. Further, the first antigen fragment (AF1) and the second antigen fragment (AF2) are incorporated into the specifically designed polypeptides comprising cleavable release segments and ELNN segments in order to confer inactive characteristics on the compositions that becomes activated by release of the fused AF1 and AF2 upon the cleavage of the release segments when in proximity to the disease tissue having proteases capable of cleaving the release segments in one or more locations in the release segment sequence.
In some embodiments, the AF2 of the subject compositions has binding affinity for an effector cell antigen expressed on the surface of a T cell. In some embodiments, the AF2 of the subject compositions has binding affinity for CD3. In some embodiments, the AF2 of the subject compositions has binding affinity for a member of the CD3 complex, which includes in individual form or independently combined form all known CD3 subunits of the CD3 complex; for example, CD3 epsilon. CD3 delta, CD3 gamma, and CD3 zeta. In some embodiments, the AF2 has binding affinity for CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.
In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to cluster of differentiation 3 T cell receptor (CD3), comprising the following CDRs: a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSXnGAVTX2SNYAN (SEQ ID NO:8023), wherein X1 corresponds to T or N, and X2 corresponds to T or S; a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP (SEQ ID NO:4); a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX4NLWV (SEQ ID NO: 8024), wherein X4 corresponds to S or P; a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX8TYAMN (SEQ ID NO:8025), wherein X8 corresponds to S or N; a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRX10KX1NX12YATYYADSVKX13 (SEQ ID NO:8026), wherein X10 corresponds to T or S, X11 corresponds to R or Y, X12 corresponds to D or N, and X13 corresponds to Gor D; a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HX14NFGNSYVSWFAX15 (SEQ ID NO:8027), wherein X14 corresponds to E or G, and X15 corresponds to H or Y.
In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to cluster of differentiation 3 T cell receptor (CD3), comprising the following CDRs: a VL region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN (SEQ ID NO:1); a VL region CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP (SEQ ID NO:4); a VL region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV (SEQ ID NO: 6); a VH region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN (SEQ ID NO: 12); a VH region CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNDYATYYADSVKG (SEQ ID NO:14); and a VH region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HENFGNSYVSWFAH (SEQ ID NO:10).
In some embodiments, the antigen binding domain comprises the following FRs: a VL region FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTC (SEQ ID NO:51); a VL region FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVQQKPGQAPRGLIG (SEQ ID NO:52); a VL region FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTPARFSGSLLEGKAALTLSGVQPEDEAVYYC (SEQ ID NO: 403); a VL region FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL (SEQ ID NO:59); a VH region FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVOLVESGGGIVQPGGSLRLSCAAS (SEQ ID NO: 400); a VH region FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG (SEQ ID NO: 401); a VH region FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR (SEQ ID NO:404); and a VH region FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS (SEQ ID NO:67).
In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to CD3, comprising: a VL region comprising three VL CDRs, wherein the three VL CDRs comprise the CDR1, CDR2, and CDR3 of a VL region comprising the following amino acid sequence: ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF SGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127); and a VH region comprising three VH CDRs, wherein the three VH CDRs comprise the CDR1, CDR2, and CDR3 of a VH region comprising the following amino acid sequence:







(SEQ ID NO: 126)


EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG


 


RIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC


 


VRHENFGNSYVSWFAHWGQGTLVTVSS.






In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to cluster of differentiation 3 T cell receptor (CD3), comprising the following CDRs: a VL region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN (SEQ ID NO:1); a VL region CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP (SEQ ID NO:4); a VL region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV (SEQ ID NO: 6); a VH region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN (SEQ ID NO:12); a VH region CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG (SEQ ID NO:13); and a VH region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HENFGNSYVSWFAH (SEQ ID NO: 10).
In some embodiments, the antigen binding domain comprises the following FRs: a VL region FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTC (SEQ ID NO:51); a VL region FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVQQKPGQAPRGLIG (SEQ ID NO:52); a VL region FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC (SEQ ID NO: 53); a VL region FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL (SEQ ID NO:59); a VH region FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVOLVESGGGIVQPGGSLRLSCAAS (SEQ ID NO: 400); a VH region FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG (SEQ ID NO: 401); a VH region FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR (SEQ ID NO:402); and a VH region FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS (SEQ ID NO:67).
In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to CD3, comprising: a VL region comprising three the VL CDRs, wherein the three VL CDRs comprise the CDR1, CDR2, and CDR3 of a VL region comprising the following amino acid sequence:







(SEQ ID NO: 8204)


ELVVTQEPSLTVSPGGTVTLTCRSSX1GAVTX2SNYANWVQQKPGQAPRG


 


LIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX3YYCALWYX4N


 


LWVFGGGTKLTVL,






wherein X1 corresponds to T or N, X2 corresponds to T or S, X3 corresponds to E or V, and X4 corresponds to S or P; and a VH region comprising three VH CDRs, wherein the three VH CDRs comprise the CDR1, CDR2, and CDR3 of a VH region comprising the following amino acid sequence: EVQLXnESGGGX6VQPGGSLX-LSCAASGFTFX8TYAMNWVRQAPGKGLEWVX,RIRXJOKX1NNY ATYYADSVKX12RFTISRDDSKNTX13YLQMNX14LKTEDTAVYYCVRHX1SNFGNSYVSWFAX16W GQGTLVTVSS (SEQ ID NO:8205), wherein X5 corresponds to V or L, X6 corresponds to I or L, X7 corresponds to R or K, X8 corresponds to S or N, X9 corresponds to Gor A, X10 corresponds to T or S, X11 corresponds to R or Y, X12 corresponds to G or D, X13 corresponds to V or A, X14 corresponds to S or N, X15 corresponds to E or G, and X16 corresponds to H or Y.

In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to CD3, comprising: a VL region comprising three VL CDRs, wherein the three VL CDRs comprise the CDR1, CDR2, and CDR3 of a VL region comprising the following amino acid sequence: ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF SGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361); and a VH region comprising three VH CDRs, wherein the three VH CDRs comprise the CDR1, CDR2, and CDR3 of a VH region comprising the following amino acid sequence:







(SEQ ID NO: 311)


EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG


 


RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC


 


VRHENFGNSYVSWFAHWGQGTLVTVSS.






In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to CD3, comprising a VL region amino acid sequence SEQ ID NO/VH region amino acid sequence SEQ ID NO pair selected from the group consisting of: 896/897; 902/903; 700/701; 702/703; 716/717; 718/719; 728/729; 736/737; 738/739; 740/741; 742/743; 744/745; 746/747; 748/749; 750/751; 752/753; 754/755; 756/757; 758/759; 760/761; 762/763; 764/765; 766/767; 774/775; 776/777; 790/791; 792/793; 798/799; 800/801; 806/807; 808/809; 814/815; 816/817; 822/823; 824/825; or 826/867.
In some embodiments, the present disclosure provides an antigen binding fragment (e.g., AF1 or AF2) that binds to the CD3 protein complex that has enhanced stability compared to CD3 binding antibodies or antigen binding fragments known in the art. In some embodiments, a CD3 antigen binding fragment of the disclosure is designed to confer a higher degree of stability on the chimeric bispecific antigen binding fragment compositions into which they are integrated, leading to improved expression and recovery of the fusion protein, increased shelf-life and enhanced stability when administered to a subject. In some embodiments, an anti-CD3 AF of the present disclosure has a higher degree of thermal stability compared to certain CD3-binding antibodies and antigen binding fragments known in the art. In some embodiments, an anti-CD3 AF of the present disclosure has a higher degree of thermal stability compared to SP34 or an antigen binding fragment thereof. In some embodiments, an anti-CD3 AF of the present disclosure has a higher degree of thermal stability compared to CD3.9 and/or CD3.23 as disclosed in PCT International Patent Application Publication No. WO2021263058, the entire content of which is hereby incorporated herein by reference. In some embodiments, the anti-CD3 AF of the present disclosure is less immunogenic in a human compared to certain CD3-binding antibodies and antigen binding fragments known in the art. In some embodiments, an anti-CD3 AF of the present disclosure is less immunogenic than SP34 or an antigen binding fragment thereof. In some embodiments, an anti-CD3 AF of the present disclosure is less immunogenic than CD3.9 and/or CD3.23 as disclosed in PCT International Patent Application Publication No. WO2021263058, the entire content of which is hereby incorporated herein by reference. In some embodiments, the degree to which an AF is immunogenic is determined by an immunogenicity prediction method such as TEPITOPEpan (described in Zhang et al. PLoS One. 2012; 7 (2): 30483. doi: 10.1371/journal.ponc.0030483, PMID: 22383964, the entire content of which is incorporated herein by reference) or NetMHCpan-4.1 and NetMHCIIpan-4.0 (each described in Reynisson et al., Nucleic Acids Res 2020; 48 (W1): W449-W454. doi: 10.1093/nar/gkaa379., PMID: 32406916, the entire content of which is hereby incorporated herein by reference). In some embodiments, the anti-CD3 AF utilized as components of the chimeric bispecific antigen binding fragment compositions into which they are integrated exhibit favorable pharmaceutical properties, including high thermostability and low aggregation propensity, resulting in improved expression and recovery during manufacturing and storage, as well promoting long scrum half-life. Biophysical properties such as thermostability are often limited by the antibody variable domains, which differ greatly in their intrinsic properties. High thermal stability is often associated with high expression levels and other desired properties, including being less susceptible to aggregation (Buchanan A, et al. Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression. MAbs 2013; 5:255). In some embodiments, thermal stability is determined by measuring the “melting temperature” (Tm), which is defined as the temperature at which half of the molecules are denatured. The melting temperature of each heterodimer is indicative of its thermal stability. In vitro assays to determine Tm are known in the art, including methods described in the Examples, below. The melting point of the heterodimer may be measured using techniques such as differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of the heterodimer may be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9), or as described in the Examples, below.
In some embodiments of the polypeptides of this disclosure, the antigen binding fragment (e.g., AF1 or AF2) can exhibit a higher thermal stability than an anti-CD3 binding fragment consisting of a sequence of SEQ ID NO: 206 (see Table 5c), as evidenced in an in vitro assay by a higher melting temperature (Tm) of the first antigen binding fragment relative to that of the anti-CD3 binding fragment; or upon incorporating the first antigen binding fragment into a test bispecific antigen binding domain, a higher Tm of the test bispecific antigen binding domain relative to that of a control bispecific antigen binding domain, wherein the test bispecific antigen binding domain comprises the first antigen binding fragment and a reference antigen binding fragment that binds to an antigen other than CD3; and wherein the control bispecific antigen binding domain consists of the anti-CD3 binding fragment consisting of the sequence of SEQ ID NO:206 (see Table 5e) and the reference antigen binding fragment. In some embodiments, the melting temperature (Tm) of the first antigen binding fragment can be at least 2° C. greater, or at least 3° C. greater, or at least 4° C. greater, or at least 5° C. greater than the Tm of the anti-CD3 binding fragment consisting of the sequence of SEQ ID NO: 206 (see Table 5c).
In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an antigen binding fragment (AF) that specifically bind human CD3. The antigen binding fragment (AF) can specifically bind human CD3. In some embodiments, the antigen binding fragment (AF) can bind a CD3 complex subunit identified herein as CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta unit of CD3. The antigen binding fragment (AF) can bind a CD3 epsilon fragment of CD3. In some embodiments, the antigen binding fragment (AF) can specifically bind human CD3 with a binding affinity (kD)) constant between about 10 nM and about 400 nM, or between about 50 nM and about 350 nM, or between about 100 nM and 300 nM, as determined in an in vitro antigen-binding assay comprising a human CD3 antigen. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an antigen binding fragment (AF) that specifically binds human CD3 with a binding affinity (KD) weaker than about 10 nM, or about 50 nM, or about 100 nM, or about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 350 nM, or weaker than about 400 nM as determined in an in vitro antigen-binding assay. For clarity, an antigen binding fragment (AF) with a kD of 400 binds its ligand more weakly than one with a kD of 10 nM. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an antigen binding fragment (AF) that specifically binds human CD3 with at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker binding affinity than an antigen binding fragment consisting of an amino acid sequence of Table 5a-e, as determined by the respective binding affinities (kD) in an in vitro antigen-binding assay.
In some embodiments, the present disclosure provides bispecific polypeptides comprising an antigen binding fragment (AF) that exhibits a binding affinity to CD3 (anti-CD3 AF) that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold at weaker relative to that of an anti-EGFR AF embodiments described herein that are incorporated into the subject polypeptides, as determined by the respective binding affinities (kD) in an in vitro antigen-binding assay.
The binding affinity of the subject compositions for the target ligands can be assayed, e.g., using binding or competitive binding assays, such as Biacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in U.S. Pat. No. 5,534,617, assays described in the Examples herein, radio-receptor assays, or other assays known in the art. The binding affinity constant can then be determined using standard methods, such as Scatchard analysis, as described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19) 12): 487, or other methods known in the art.
In some embodiments, the present disclosure provides an antigen binding fragment (AF) that binds to CD3 (anti-CD3 AF) and is incorporated into a chimeric, bispecific polypeptide composition that is designed to have an isoelectric point (pI) that confers enhanced stability on the composition compared to corresponding compositions comprising CD3 binding antibodies or antigen binding fragments known in the art. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise AF that bind to CD3 (anti-CD3 AF) wherein the anti-CD3 AF exhibits a pI that is between 6.0 and 6.6, inclusive. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise AF that bind to CD3 (anti-CD3 AF) wherein the anti-CD3 AF exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the pI of a reference antigen binding fragment (e.g., consisting of a sequence shown in SEQ ID NO: 206 (see Table 5e)). In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an AF that binds to CD3 (anti-CD3 AF) fused to another AF that binds to a EGFR antigen (anti-EGFR AF) wherein the anti-CD3 AF exhibits a pI that is within at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pI of the AF that binds EGFR antigen or an epitope thereof. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an AF that binds to CD3 (anti-CD3 AF) fused to an AF that binds to a EGFR antigen (anti-EGFR AF) wherein the AF exhibits a pI that is within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pI of the anti-CD3 AF. It is specifically intended that by such design wherein the pI of the two antigen binding fragments are within such ranges, the resulting fused antigen binding fragments will confer a higher degree of stability on the chimeric bispecific antigen binding fragment compositions into which they are integrated, leading to improved expression and enhanced recovery of the fusion protein in soluble, non-aggregated form, increased shelf-life of the formulated chimeric bispecific polypeptide compositions, and enhanced stability when the composition is administered to a subject. In some embodiments, having the two AFs (the anti-CD3 AF and the anti-EGFR AF) within a relatively narrow pI range of may allow for the selection of a buffer or other solution in which both the AFs (anti-CD3 AF and anti-EGFR AF) are stable, thereby promoting overall stability of the composition. In some embodiments, the antigen binding fragment (AF) can exhibit an isoelectric point (pI) that is less than or equal to 6.6. In some embodiments, the antigen binding fragment (AF) can exhibit an isoelectric point (pI) that is between 6.0 and 6.6, inclusive. In some embodiments, the antigen binding fragment (AF) can exhibit an isoelectric point (pI) that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pI of a reference antigen binding fragment consisting of a sequence shown in SEQ ID NO: 206 (see Table 5c). In some embodiments, the antigen binding fragment (AF) can specifically bind human CD3 with a binding affinity (kD) constant between about between about 10 nM and about 400 nM (such as determined in an in vitro antigen-binding assay comprising a human CD3 antigen). In some embodiments, the antigen binding fragment (AF) can specifically bind human CD3 with a binding affinity (kD)) of less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM, or less than about 350 nM, or less than about 400 nM (such as determined in an in vitro antigen-binding assay). In some embodiments, the antigen binding fragment (AF) can exhibit a binding affinity to CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker relative to that of an antigen binding fragment consisting of an amino acid sequence of SEQ ID NO: 206 (see Table 5c) (such as determined by the respective binding affinities (KD) in an in vitro antigen-binding assay).
In some embodiments, the VL and VH of the antigen binding fragments are fused by relatively long linkers, consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that, when joined together, have a flexible characteristic. In some embodiments, the VL and VH of any of the scFv embodiments described herein are linked by a relatively long linker having the sequence SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the VL and VH of any of the scFv embodiments described herein are linked by relatively long linkers of hydrophilic amino acids having the sequences GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 82), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 83), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 84), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 85). In some embodiments, the AF1 and AF2 are linked together by a short linker of hydrophilic amino acids having 3, 4, 5, 6, or 7 amino acids. In some embodiments, the short linker sequences are identified herein as the sequences SGGGGS (SEQ ID NO: 86), GGGGS (SEQ ID NO: 87), GGSGGS (SEQ ID NO: 88), GGS, or GSP. In some embodiments, the disclosure provides compositions comprising a single chain diabody in which after folding, the first domain (VL or VH) is paired with the last domain (VH or VL) to form one scFv and the two domains in the middle are paired to form the other scFv in which the first and second domains, as well as the third and last domains, are fused together by one of the foregoing short linkers and the second and the third variable domains are fused by one of the foregoing relatively long linkers. In some embodiments, the selection of the short linker and relatively long linker is to prevent the incorrect pairing of adjacent variable domains, thereby facilitating the formation of a single chain configuration comprising the VL and VH of the first antigen binding fragment and the second antigen binding fragment.







TABLE 5b







Exemplary CD3 CDR Sequences











CDR

SEQ


Antibody Domain
REGION
Amino Acid Sequence
ID NO:





3.23, 3.30, 3.31, 3.32, 3.228, 3.318
CDR-L1
RSSNGAVTSSNYAN
 1


 


3.24
CDR-L1
RSSNGEVTTSNYAN
 2


 


3.33, 3.9
CDR-L1
RSSTGAVTTSNYAN
 3


 


3.23, 3.30, 3.31, 3.32, 3.9, 3.33,
CDR-L2
GTNKRAP
 4


3.228, 3.318





 


3.24
CDR-L2
GTIKRAP
 5


 


3.23, 3.24, 3.30, 3.31, 3.32, 3.228,
CDR-L3
ALWYPNLWV
 6


3.318





 


3.33, 3.9
CDR-L3
ALWYSNLWV
 7


 


3.23, 3.24, 3.30, 3.31, 3.32, 3.9,
CDR-H1
GFTFNTYAMN
 8


3.33





 


3.228, 3.318
CDR-H1
GFTFSTYAMN
12


 


3.23, 3.24, 3.30, 3.31, 3.32, 3.9,
CDR-H2
RIRSKYNNYATYYADSVKD
 9


3.33





 


3.228
CDR-H2
RIRTKRNNYATYYADSVKG
13


 


3.318
CDR-H2
RIRTKRNDYATYYADSVKG
14


 


3.23, 3.24, 3.30, 3.31, 3.32, 3.228,
CDR-H3
HENFGNSYVSWFAH
10


3.318





 


3.9, 3.33
CDR-H3
HGNFGNSYVSWFAY
11
















TABLE 5c







Exemplary CD3 FR Sequences











FR

SEQ ID


Antibody Domain
REGION
Amino Acid Sequence
NO:





3.23, 3.24, 3.30, 3.31,
FR-L1
ELVVTQEPSLTVSPGGTVTLTC
 51


3.32, 3.9, 3.33, 3.228,





3.318





 


3.23, 3.24, 3.30, 3.31,
FR-L2
WVQQKPGQAPRGLIG
 52


3.32, 3.9, 3.33, 3.228,





3.318





 


3.23, 3.24, 3.228
FR-L3
GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC
 53


 


3.30
FR-L3
GTPARFSGSSLGGKAALTLSGVQPEDEAVYYC
 54


 


3.31
FR-L3
GTPARFSGSLLGGSAALTLSGVQPEDEAVYYC
 55


 


3.32
FR-L3
GTPARFSGSSLGGSAALTLSGVQPEDEAVYYC
 56


 


3.9
FR-L3
GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC
 57


 


3.33
FR-L3
GTPARFSGSSLGGSAALTLSGVQPEDEAEYYC
 58


 


3.318
FR-L3
GTPARFSGSLLEGKAALTLSGVQPEDEAVYYC
403


 


3.23, 3.24, 3.30, 3.31,
FR-L4
FGGGTKLTVL
 59


3.32, 3.9, 3.33, 3.228,





3.318





 


3.228, 3.318
FR-H1
EVQLVESGGGIVQPGGSLRLSCAAS
400


 


3.23, 3.24
FR-H1
EVQLLESGGGIVQPGGSLKLSCAAS
 60


 


3.30, 3.31, 3.32
FR-H1
EVQLQESGGGIVQPGGSLKLSCAAS
 61


 


3.33
FR-H1
EVQLQESGGGLVQPGGSLKLSCAAS
 62


 


3.9
FR-H1
EVQLLESGGGLVQPGGSLKLSCAAS
 63


 


3.23, 3.24, 3.30, 3.31,
FR-H2
WVRQAPGKGLEWVA
 64


3.32, 3.9, 3.33





 


3.228, 3.318
FR-H2
WVRQAPGKGLEWVG
401


 


3.23, 3.24, 3.30, 3.31,
FR-H3
RFTISRDDSKNTVYLQMNNLKTEDTAVYYCVR
 65


3.32





 


3.9, 3.33
FR-H3
RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR
 66


 


3.228
FR-H3
RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR
402


 


3.318
FR-H3
RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR
404


 


3.23, 3.24, 3.30, 3.31,
FR-H4
WGQGTLVTVSS
 67


3.32, 3.9, 3.33, 3.228,





3.318
















TABLE 5d







Exemplary CD3 VL & VH Sequences










Antibody


SEQ


Domain
REGION
Amino Acid Sequence
ID NO:





3.23
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
101




APRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVY





YCALWYPNLWVFGGGTKLTVL



 


3.23, 3.24
VH
EVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKG
102




LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNN





LKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS



 


3.24
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYANWVQQKPGQ
103




APRGLIGGTIKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYY





CALWYPNLWVFGGGTKLTVL



 


3.30
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
104




APRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYY





CALWYPNLWVFGGGTKLTVL



 


3.30, 3.31,
VH
EVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGK
105


3.32

GLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMN





NLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS



 


3.31
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
106




APRGLIGGTNKRAPGTPARFSGSLLGGSAALTLSGVQPEDEAVYY





CALWYPNLWVFGGGTKLTVL



 


3.32
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
107




APRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAVYY





CALWYPNLWVFGGGTKLTVL



 


3.9
VL
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQ
108




APRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYY





CALWYSNLWVFGGGTKLTVL



 


3.9
VH
EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGK
109




GLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMN





NLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS



 


3.33
VL
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQ
110




APRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYY





CALWYSNLWVFGGGTKLTVL



 


3.33
VH
EVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGK
111




GLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMN





NLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS



 


3.228
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
361




APRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVY





YCALWYPNLWVFGGGTKLTVL



 


3.228
VH
EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG
311




LEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSL





KTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS



 


3.318
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
127




APRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYY





CALWYPNLWVFGGGTKLTVL



 


3.318
VH
EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG
126




LEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSL





KTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS
















TABLE 5e







Exemplary CD3 scFv Sequences









Antibody

SEQ ID


Domain
Amino Acid Sequence
NO:





3.23
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK
201



RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL




GATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGF




TFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTV




YLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS



 


3.24
ELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYANWVQQKPGQAPRGLIGGTIKR
202



APGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLG




ATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFT




FNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTV




YLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS



 


3.30
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK
203



RAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL




GATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGF




TFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTV




YLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS



 


3.31
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK
204



RAPGTPARFSGSLLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL




GATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGF




TFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTV




YLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS



 


3.32
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK
205



RAPGTPARFSGSSLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL




GATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGF




TFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTV




YLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS



 


3.9
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNK
206



RAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL




GATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGF




TFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTA




YLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS



 


3.33
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNK
207



RAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLG




ATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFT




FNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTA




YLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS



 


4.11
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPS
208



GVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGLWVFGGGTKLTVLG




ATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGF




TFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQ




MNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS



 


4.12
QAGLTQPPSASGTPGQRVTLSCSGSYSNIGTYYVYWYQQLPGTAPKLLIYSNDQRL
209



SGVPDRFSGSKSGTSASLAISGLQSEDEAAYYCAAWDDSLNGWAFGGGTKLTVLG




ATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGF




TFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQ




MNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS



 


4.13
QPGLTQPPSASGTPGQRVTLSCSGRSSNIGSYYVYWYQHLPGMAPKLLIYRNSRRP
210



SGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDDSLKSWVFGGGTKLTVLG




ATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGF




TFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQ




MNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS



 


4.14
QSVLTQPPSASGTPGQRVTISCSGSSSNIGTNYVYWYQQFPGTAPKLLIYSNNQRPS
211



GVPDRFSGSKSGTSGSLAISGLQSEDEADYSCAAWDDSLNGWVFGGGTKLTVLGA




TPPETGAETESPGETTGGSAESEPPGEGQVQLVQWGGGLVKPGGSLRLSCAASGFT




FSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQ




MNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS



 


4.15
QPGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPS
212



GVPDRLSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGA




TPPETGAETESPGETTGGSAESEPPGEGQVQLVQWGGGLVKPGGSLRLSCAASGFT




FSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQ




MNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS



 


4.16
QAVLTQPPSASGTPGQRVTISCSGSSSNIGSYYVYWYQQVPGAAPKLLMRLNNQR
213



PSGVPDRFSGAKSGTSASLVISGLRSEDEADYYCAAWDDSLSGQWVFGGGTKLTV




LGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAAS




GFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLY




LQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS



 


4.17
QAGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPS
214



GVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDASLSGWVFGGGTKLTVLGA




TPPETGAETESPGETTGGSAESEPPGEGEVQLVQWGGGLVKPGGSLRLSCAASGFT




FSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQ




MNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS



 


3.228
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK
215



RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL




SESATPESGPGTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFT




FSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVY




LQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS



 


3.318
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK
128



RAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLS




ESATPESGPGTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTF




STYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYL




QMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS









Anti-EGFR Binding Domains

Also provided are anti-EGFR antibodies, fragments thereof, and fusion proteins comprising such antibodies and/or fragments.
In some embodiments, the present disclosure provides paTCE compositions comprising a first portion binding domain with binding affinity to the tumor-specific marker EGFR and a second binding domain that binds to an effector cell antigen, such as CD3 antigen.
In some embodiments, the first portion binding domain is an scFv domain, comprising a VH domain and a VL domain. Non-limiting examples of VH and VL domain sequences are provided in Table 5f. In some embodiments, the binding domain with binding affinity for the tumor-specific marker EGFR is an scFv domain comprising a VH and VL domain, listed in Table 5f. In some embodiments, the binding domain with binding affinity for EGFR is a scFv domain comprising three CDRs from a VH domain listed in Table 5f and three CDRs from a VL listed in Table 5f.
In some embodiments, the present disclosure provides a paTCE composition comprising a first portion binding domain with binding affinity to the tumor-specific marker EGFR comprising anti-EGFR VH and VL sequences set forth in Table 5f. In some embodiments, the binding has a kD value of about 10−10 to 10−7 M, as determined in an in vitro binding assay. In some embodiments, the binding has a KD value of about 1-10 nM, as determined in an in vitro binding assay. In some embodiments, the binding has a KD value of about 2 nM, as determined in an in vitro binding assay. It is specifically contemplated that the paTCE composition can comprise any one of the binding domains disclosed herein or sequence variants thereof so long as the variants exhibit binding specificity for the described antigen.







TABLE 5f







Anti-EGFR VH and VL Sequences












Antibody
AC

SEQ ID

SEQ ID


Name
Number
VH Sequence
NO:
VL Sequence
NO:





EGFR.2
AC2876
QVQLQESGPGLVKPS
450
DIQMTQSPSSLSA
451


Donor

ETLSLTCTVSGGSVSS

SVGDRVTITCQAS



antibody

GDYYWTWIRQSPGK

QDISNYLNWYQQ





GLEWIGHIYYSGNTN

KPGKAPKLLIYD





YNPSLKSRLTISIDTS

ASNLETGVPSRFS





KTQFSLKLSSVTAAD

GSGSGTDFTFTISS





TAIYYCVRDRVTGAF

LQPEDIATYFCQH





DIWGQGTMVTVSS

FDHLPLAFGGGT







KVEIK



 


EGFR.29
AC2877
QVQLVQSGAEVKKP
452
DIQMTQSPSSLSA
453




GASVKVSCKASGGS

SVGDRVTITCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGKAPKLLIYD





GNTNYNPSLKSRVTS

ASNLETGVPSRFS





TRDTSISTAYMELSRL

GSGSGTDFTFTISS





RSDDTVVYYCARDR

LQPEDIATYYCQ





VTGAFDIWGQGTLVT

HFDHLPLAFGQG





VSS

TKVEIK



 


EGFR.30
AC2878
QVQLVQSGAEVKKP
454
DIQMTQSPSSLSA
455




GASVKVSCKASGGS

SVGDRVTITCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGKAPKLLIYD





GNTNYNPSLKSRVT

ASNLETGVPSRFS





MTRDTSTSTVYMELS

GSGSGTDFTFTISS





SLRSEDTAVYYCARD

LQPEDIATYYCQ





RVTGAFDIWGQGTL

HFDHLPLAFGQG





VTVSS

TKVEIK



 


EGFR.31
AC2879
QVQLVQSGAEVKKP
456
DIQMTQSPSSLSA
457




GSSVKVSCKASGGSV

SVGDRVTITCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GQGLEWMGHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSRVTIT

ASNLETGVPSRFS





ADESTSTAYMELSSL

GSGSGTDFTFTISS





RSEDTAVYYCARDR

LQPEDIATYYCQ





VTGAFDIWGQGTLVT

HFDHLPLAFGQG





VSS

TKVEIK



 


EGFR.32
AC2880
EVQLLESGGGLVQPG
458
DIQMTQSPSSLSA
459




GSLRLSCAASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVSHIYYSGNT

KPGKAPKLLIYD





NYNPSLKSRFTISRDN

ASNLETGVPSRFS





SKNTLYLQMNSLRAE

GSGSGTDFTFTISS





DTAVYYCAKDRVTG

LQPEDIATYYCQ





AFDIWGQGTLVTVSS

HFDHLPLAFGQG







TKVEIK



 


EGFR.33
AC2881
QVQLVESGGGVVQP
460
DIQMTQSPSSLSA
461




GRSLRLSCAASGGSV

SVGDRVTITCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GKGLEWVAHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSRFTISR

ASNLETGVPSRFS





DNSKNTLYLQMNSL

GSGSGTDFTFTISS





RAEDTAVYYCARDR

LQPEDIATYYCQ





VTGAFDIWGQGTLVT

HFDHLPLAFGQG





VSS

TKVEIK



 


EGFR.34
AC2882
EVQLVESGGGLVQPG
462
DIQMTQSPSSLSA
463




GSLRLSCAASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVAHIYYSGN

KPGKAPKLLIYD





TNYNPSLKSRFTISRD

ASNLETGVPSRFS





NAKNSLYLQMNSLR

GSGSGTDFTFTISS





AEDTAVYYCARDRV

LQPEDIATYYCQ





TGAFDIWGQGTLVTV

HFDHLPLAFGQG





SS

TKVEIK



 


EGFR.35
AC2883
EVQLVESGGGLVQPG
464
DIQMTQSPSSLSA
465




GSLRLSCAASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVSHIYYSGNT

KPGKAPKLLIYD





NYNPSLKSRFTISRDN

ASNLETGVPSRFS





SKNTLYLQMNSLRAE

GSGSGTDFTFTISS





DTAVYYCARDRVTG

LQPEDIATYYCQ





AFDIWGQGTLVTVSS

HFDHLPLAFGQG







TKVEIK



 


EGFR.36
AC2884
QVQLQQWGAGLLKP
466
DIQMTQSPSSLSA
467




SETLSLTCAVYGGSV

SVGDRVTITCQAS





SSGDYYWTWIRQPPG

QDISNYLNWYQQ





KGLEWIGHIYYSGNT

KPGKAPKLLIYD





NYNPSLKSRVTISVD

ASNLETGVPSRFS





TSKNQFSLKLSSVTA

GSGSGTDFTFTISS





ADTAVYYCARDRVT

LQPEDIATYYCQ





GAFDIWGQGTLVTVS

HFDHLPLAFGQG





S

TKVEIK



 


EGFR.37
AC2885
QVQLQESGPGLVKPS
468
DIQMTQSPSSLSA
469




ETLSLTCTVSGGSVSS

SVGDRVTITCQAS





GDYYWTWIRQPPGK

QDISNYLNWYQQ





GLEWIGHIYYSGNTN

KPGKAPKLLIYD





YNPSLKSRVTISVDTS

ASNLETGVPSRFS





KNQFSLKLSSVTAAD

GSGSGTDFTFTISS





TAVYYCARDRVTGA

LQPEDIATYYCQ





FDIWGQGTLVTVSS

HFDHLPLAFGQG







TKVEIK



 


EGFR.38
AC2886
EVQLVQSGAEVKKP
470
DIQMTQSPSSLSA
471




GESLKISCKGSGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQMP

QDISNYLNWYQQ





GKGLEWMGHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSQVTIS

ASNLETGVPSRFS





ADKSISTAYLQWSSL

GSGSGTDFTFTISS





KASDTAMYYCARDR

LQPEDIATYYCQ





VTGAFDIWGQGTLVT

HFDHLPLAFGQG





VSS

TKVEIK



 


EGFR.39
AC2887
QVQLVQSGSELKKPG
472
DIQMTQSPSSLSA
473




ASVKVSCKASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





QGLEWMGHIYYSGN

KPGKAPKLLIYD





TNYNPSLKSRFVFSL

ASNLETGVPSRFS





DTSVSTAYLQICSLK

GSGSGTDFTFTISS





AEDTAVYYCARDRV

LQPEDIATYYCQ





TGAFDIWGQGTLVTV

HFDHLPLAFGQG





SS

TKVEIK



 


EGFR.40
AC2888
QVQLVQSGVEVKKP
474
DIQMTQSPSSLSA
475




GASVKVSCKASGGS

SVGDRVTITCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGKAPKLLIYD





GNTNYNPSLKSRVTL

ASNLETGVPSRFS





TTDSSTTTAYMELKS

GSGSGTDFTFTISS





LQFDDTAVYYCARD

LQPEDIATYYCQ





RVTGAFDIWGQGTL

HFDHLPLAFGQG





VTVSS

TKVEIK



 


EGFR.41
AC2889
QVQLVQSGAEVKKP
476
DIQMTQSPSSLSA
477




GASVKVSCKASGGS

SVGDRVTITCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGKAPKLLIYD





GNTNYNPSLKSRVTS

ASNLETGVPSRFS





TRDTSISTAYMELSRL

GSGSGTDFTLTIS





RSDDTVVYYCARDR

SLQPEDFATYYC





VTGAFDIWGQGTLVT

QHFDHLPLAFGQ





VSS

GTKVEIK



 


EGFR.42
AC2890
QVQLVQSGAEVKKP
478
DIQMTQSPSSLSA
479




GASVKVSCKASGGS

SVGDRVTITCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGKAPKLLIYD





GNTNYNPSLKSRVT

ASNLETGVPSRFS





MTRDTSTSTVYMELS

GSGSGTDFTLTIS





SLRSEDTAVYYCARD

SLQPEDFATYYC





RVTGAFDIWGQGTL

QHFDHLPLAFGQ





VTVSS

GTKVEIK



 


EGFR.43
AC2891
QVQLVQSGAEVKKP
480
DIQMTQSPSSLSA
481




GSSVKVSCKASGGSV

SVGDRVTITCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GQGLEWMGHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSRVTIT

ASNLETGVPSRFS





ADESTSTAYMELSSL

GSGSGTDFTLTIS





RSEDTAVYYCARDR

SLQPEDFATYYC





VTGAFDIWGQGTLVT

QHFDHLPLAFGQ





VSS

GTKVEIK



 


EGFR.44
AC2892
EVQLLESGGGLVQPG
482
DIQMTQSPSSLSA
483




GSLRLSCAASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVSHIYYSGNT

KPGKAPKLLIYD





NYNPSLKSRFTISRDN

ASNLETGVPSRFS





SKNTLYLQMNSLRAE

GSGSGTDFTLTIS





DTAVYYCAKDRVTG

SLQPEDFATYYC





AFDIWGQGTLVTVSS

QHFDHLPLAFGQ







GTKVEIK



 


EGFR.45
AC2893
QVQLVESGGGVVQP
484
DIQMTQSPSSLSA
485




GRSLRLSCAASGGSV

SVGDRVTITCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GKGLEWVAHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSRFTISR

ASNLETGVPSRFS





DNSKNTLYLQMNSL

GSGSGTDFTLTIS





RAEDTAVYYCARDR

SLQPEDFATYYC





VTGAFDIWGQGTLVT

QHFDHLPLAFGQ





VSS

GTKVEIK



 


EGFR.46
AC2894
EVQLVESGGGLVQPG
486
DIQMTQSPSSLSA
487




GSLRLSCAASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVAHIYYSGN

KPGKAPKLLIYD





TNYNPSLKSRFTISRD

ASNLETGVPSRFS





NAKNSLYLQMNSLR

GSGSGTDFTLTIS





AEDTAVYYCARDRV

SLQPEDFATYYC





TGAFDIWGQGTLVTV

QHFDHLPLAFGQ





SS

GTKVEIK



 


EGFR.47
AC2895
EVQLVESGGGLVQPG
488
DIQMTQSPSSLSA
489




GSLRLSCAASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVSHIYYSGNT

KPGKAPKLLIYD





NYNPSLKSRFTISRDN

ASNLETGVPSRFS





SKNTLYLQMNSLRAE

GSGSGTDFTLTIS





DTAVYYCARDRVTG

SLQPEDFATYYC





AFDIWGQGTLVTVSS

QHFDHLPLAFGQ







GTKVEIK



 


EGFR.48
AC2896
QVQLQQWGAGLLKP
490
DIQMTQSPSSLSA
491




SETLSLTCAVYGGSV

SVGDRVTITCQAS





SSGDYYWTWIRQPPG

QDISNYLNWYQQ





KGLEWIGHIYYSGNT

KPGKAPKLLIYD





NYNPSLKSRVTISVD

ASNLETGVPSRFS





TSKNQFSLKLSSVTA

GSGSGTDFTLTIS





ADTAVYYCARDRVT

SLQPEDFATYYC





GAFDIWGQGTLVTVS

QHFDHLPLAFGQ





S

GTKVEIK



 


EGFR.49
AC2897
QVQLQESGPGLVKPS
492
DIQMTQSPSSLSA
493




ETLSLTCTVSGGSVSS

SVGDRVTITCQAS





GDYYWTWIRQPPGK

QDISNYLNWYQQ





GLEWIGHIYYSGNTN

KPGKAPKLLIYD





YNPSLKSRVTISVDTS

ASNLETGVPSRFS





KNQFSLKLSSVTAAD

GSGSGTDFTLTIS





TAVYYCARDRVTGA

SLQPEDFATYYC





FDIWGQGTLVTVSS

QHFDHLPLAFGQ







GTKVEIK



 


EGFR.50
AC2898
EVQLVQSGAEVKKP
494
DIQMTQSPSSLSA
495




GESLKISCKGSGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQMP

QDISNYLNWYQQ





GKGLEWMGHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSQVTIS

ASNLETGVPSRFS





ADKSISTAYLQWSSL

GSGSGTDFTLTIS





KASDTAMYYCARDR

SLQPEDFATYYC





VTGAFDIWGQGTLVT

QHFDHLPLAFGQ





VSS

GTKVEIK



 


EGFR.51
AC2899
QVQLVQSGSELKKPG
496
DIQMTQSPSSLSA
497




ASVKVSCKASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





QGLEWMGHIYYSGN

KPGKAPKLLIYD





TNYNPSLKSRFVFSL

ASNLETGVPSRFS





DTSVSTAYLQICSLK

GSGSGTDFTLTIS





AEDTAVYYCARDRV

SLQPEDFATYYC





TGAFDIWGQGTLVTV

QHFDHLPLAFGQ





SS

GTKVEIK



 


EGFR.52
AC2900
QVQLVQSGVEVKKP
498
DIQMTQSPSSLSA
499




GASVKVSCKASGGS

SVGDRVTITCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGKAPKLLIYD





GNTNYNPSLKSRVTL

ASNLETGVPSRFS





TTDSSTTTAYMELKS

GSGSGTDFTLTIS





LQFDDTAVYYCARD

SLQPEDFATYYC





RVTGAFDIWGQGTL

QHFDHLPLAFGQ





VTVSS

GTKVEIK



 


EGFR.53
AC2901
QVQLVQSGAEVKKP
500
EIVLTQSPGTLSLS
501




GASVKVSCKASGGS

PGERATLSCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGQAPRLLIYDA





GNTNYNPSLKSRVTS

SNLETGIPDRFSG





TRDTSISTAYMELSRL

SGSGTDFTLTISR





RSDDTVVYYCARDR

LEPEDFAVYYCQ





VTGAFDIWGQGTLVT

HFDHLPLAFGQG





VSS

TKVEIK



 


EGFR.54
AC2902
QVQLVQSGAEVKKP
502
EIVLTQSPGTLSLS
503




GASVKVSCKASGGS

PGERATLSCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGQAPRLLIYDA





GNTNYNPSLKSRVT

SNLETGIPDRFSG





MTRDTSTSTVYMELS

SGSGTDFTLTISR





SLRSEDTAVYYCARD

LEPEDFAVYYCQ





RVTGAFDIWGQGTL

HFDHLPLAFGQG





VTVSS

TKVEIK



 


EGFR.55
AC2903
QVQLVQSGAEVKKP
504
EIVLTQSPGTLSLS
505




GSSVKVSCKASGGSV

PGERATLSCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GQGLEWMGHIYYSG

KPGQAPRLLIYDA





NTNYNPSLKSRVTIT

SNLETGIPDRFSG





ADESTSTAYMELSSL

SGSGTDFTLTISR





RSEDTAVYYCARDR

LEPEDFAVYYCQ





VTGAFDIWGQGTLVT

HFDHLPLAFGQG





VSS

TKVEIK



 


EGFR.56
AC2904
EVQLLESGGGLVQPG
506
EIVLTQSPGTLSLS
507




GSLRLSCAASGGSVS

PGERATLSCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVSHIYYSGNT

KPGQAPRLLIYDA





NYNPSLKSRFTISRDN

SNLETGIPDRFSG





SKNTLYLQMNSLRAE

SGSGTDFTLTISR





DTAVYYCAKDRVTG

LEPEDFAVYYCQ





AFDIWGQGTLVTVSS

HFDHLPLAFGQG







TKVEIK



 


EGFR.57
AC2905
QVQLVESGGGVVQP
508
EIVLTQSPGTLSLS
509




GRSLRLSCAASGGSV

PGERATLSCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GKGLEWVAHIYYSG

KPGQAPRLLIYDA





NTNYNPSLKSRFTISR

SNLETGIPDRFSG





DNSKNTLYLQMNSL

SGSGTDFTLTISR





RAEDTAVYYCARDR

LEPEDFAVYYCQ





VTGAFDIWGQGTLVT

HFDHLPLAFGQG





VSS

TKVEIK



 


EGFR.58
AC2906
EVQLVESGGGLVQPG
510
EIVLTQSPGTLSLS
511




GSLRLSCAASGGSVS

PGERATLSCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVAHIYYSGN

KPGQAPRLLIYDA





TNYNPSLKSRFTISRD

SNLETGIPDRFSG





NAKNSLYLQMNSLR

SGSGTDFTLTISR





AEDTAVYYCARDRV

LEPEDFAVYYCQ





TGAFDIWGQGTLVTV

HFDHLPLAFGQG





SS

TKVEIK



 


EGFR.59
AC2907
EVQLVESGGGLVQPG
512
EIVLTQSPGTLSLS
513




GSLRLSCAASGGSVS

PGERATLSCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVSHIYYSGNT

KPGQAPRLLIYDA





NYNPSLKSRFTISRDN

SNLETGIPDRFSG





SKNTLYLQMNSLRAE

SGSGTDFTLTISR





DTAVYYCARDRVTG

LEPEDFAVYYCQ





AFDIWGQGTLVTVSS

HFDHLPLAFGQG







TKVEIK



 


EGFR.60
AC2908
QVQLQQWGAGLLKP
514
EIVLTQSPGTLSLS
515




SETLSLTCAVYGGSV

PGERATLSCQAS





SSGDYYWTWIRQPPG

QDISNYLNWYQQ





KGLEWIGHIYYSGNT

KPGQAPRLLIYDA





NYNPSLKSRVTISVD

SNLETGIPDRFSG





TSKNQFSLKLSSVTA

SGSGTDFTLTISR





ADTAVYYCARDRVT

LEPEDFAVYYCQ





GAFDIWGQGTLVTVS

HFDHLPLAFGQG





S

TKVEIK



 


EGFR.61
AC2909
QVQLQESGPGLVKPS
516
EIVLTQSPGTLSLS
517




ETLSLTCTVSGGSVSS

PGERATLSCQAS





GDYYWTWIRQPPGK

QDISNYLNWYQQ





GLEWIGHIYYSGNTN

KPGQAPRLLIYDA





YNPSLKSRVTISVDTS

SNLETGIPDRFSG





KNQFSLKLSSVTAAD

SGSGTDFTLTISR





TAVYYCARDRVTGA

LEPEDFAVYYCQ





FDIWGQGTLVTVSS

HFDHLPLAFGQG







TKVEIK



 


EGFR.62
AC2910
EVQLVQSGAEVKKP
518
EIVLTQSPGTLSLS
519




GESLKISCKGSGGSVS

PGERATLSCQAS





SGDYYWTWVRQMP

QDISNYLNWYQQ





GKGLEWMGHIYYSG

KPGQAPRLLIYDA





NTNYNPSLKSQVTIS

SNLETGIPDRFSG





ADKSISTAYLQWSSL

SGSGTDFTLTISR





KASDTAMYYCARDR

LEPEDFAVYYCQ





VTGAFDIWGQGTLVT

HFDHLPLAFGQG





VSS

TKVEIK



 


EGFR.63
AC2911
QVQLVQSGSELKKPG
520
EIVLTQSPGTLSLS
521




ASVKVSCKASGGSVS

PGERATLSCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





QGLEWMGHIYYSGN

KPGQAPRLLIYDA





TNYNPSLKSRFVFSL

SNLETGIPDRFSG





DTSVSTAYLQICSLK

SGSGTDFTLTISR





AEDTAVYYCARDRV

LEPEDFAVYYCQ





TGAFDIWGQGTLVTV

HFDHLPLAFGQG





SS

TKVEIK



 


EGFR.64
AC2912
QVQLVQSGVEVKKP
522
EIVLTQSPGTLSLS
523




GASVKVSCKASGGS

PGERATLSCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGQAPRLLIYDA





GNTNYNPSLKSRVTL

SNLETGIPDRFSG





TTDSSTTTAYMELKS

SGSGTDFTLTISR





LQFDDTAVYYCARD

LEPEDFAVYYCQ





RVTGAFDIWGQGTL

HFDHLPLAFGQG





VTVSS

TKVEIK



 


EGFR.65
AC2913
QVQLVQSGAEVKKP
524
EIVLTQSPATLSLS
525




GASVKVSCKASGGS

PGERATLSCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGQAPRLLIYDA





GNTNYNPSLKSRVTS

SNLETGIPARFSG





TRDTSISTAYMELSRL

SGSGTDFTLTISSL





RSDDTVVYYCARDR

EPEDFAVYYCQH





VTGAFDIWGQGTLVT

FDHLPLAFGQGT





VSS

KVEIK



 


EGFR.66
AC2914
QVQLVQSGAEVKKP
526
EIVLTQSPATLSLS
527




GASVKVSCKASGGS

PGERATLSCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGQAPRLLIYDA





GNTNYNPSLKSRVT

SNLETGIPARFSG





MTRDTSTSTVYMELS

SGSGTDFTLTISSL





SLRSEDTAVYYCARD

EPEDFAVYYCQH





RVTGAFDIWGQGTL

FDHLPLAFGQGT





VTVSS

KVEIK



 


EGFR.67
AC2915
QVQLVQSGAEVKKP
528
EIVLTQSPATLSLS
529




GSSVKVSCKASGGSV

PGERATLSCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GQGLEWMGHIYYSG

KPGQAPRLLIYDA





NTNYNPSLKSRVTIT

SNLETGIPARFSG





ADESTSTAYMELSSL

SGSGTDFTLTISSL





RSEDTAVYYCARDR

EPEDFAVYYCQH





VTGAFDIWGQGTLVT

FDHLPLAFGQGT





VSS

KVEIK



 


EGFR.68
AC2916
EVQLLESGGGLVQPG
530
EIVLTQSPATLSLS
531




GSLRLSCAASGGSVS

PGERATLSCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVSHIYYSGNT

KPGQAPRLLIYDA





NYNPSLKSRFTISRDN

SNLETGIPARFSG





SKNTLYLQMNSLRAE

SGSGTDFTLTISSL





DTAVYYCAKDRVTG

EPEDFAVYYCQH





AFDIWGQGTLVTVSS

FDHLPLAFGQGT







KVEIK



 


EGFR.69
AC2917
QVQLVESGGGVVQP
532
EIVLTQSPATLSLS
533




GRSLRLSCAASGGSV

PGERATLSCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GKGLEWVAHIYYSG

KPGQAPRLLIYDA





NTNYNPSLKSRFTISR

SNLETGIPARFSG





DNSKNTLYLQMNSL

SGSGTDFTLTISSL





RAEDTAVYYCARDR

EPEDFAVYYCQH





VTGAFDIWGQGTLVT

FDHLPLAFGQGT





VSS

KVEIK



 


EGFR.70
AC2918
EVQLVESGGGLVQPG
534
EIVLTQSPATLSLS
535




GSLRLSCAASGGSVS

PGERATLSCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVAHIYYSGN

KPGQAPRLLIYDA





TNYNPSLKSRFTISRD

SNLETGIPARFSG





NAKNSLYLQMNSLR

SGSGTDFTLTISSL





AEDTAVYYCARDRV

EPEDFAVYYCQH





TGAFDIWGQGTLVTV

FDHLPLAFGQGT





SS

KVEIK



 


EGFR.71
AC2919
EVQLVESGGGLVQPG
536
EIVLTQSPATLSLS
537




GSLRLSCAASGGSVS

PGERATLSCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVSHIYYSGNT

KPGQAPRLLIYDA





NYNPSLKSRFTISRDN

SNLETGIPARFSG





SKNTLYLQMNSLRAE

SGSGTDFTLTISSL





DTAVYYCARDRVTG

EPEDFAVYYCQH





AFDIWGQGTLVTVSS

FDHLPLAFGQGT







KVEIK



 


EGFR.72
AC2920
QVQLQQWGAGLLKP
538
EIVLTQSPATLSLS
539




SETLSLTCAVYGGSV

PGERATLSCQAS





SSGDYYWTWIRQPPG

QDISNYLNWYQQ





KGLEWIGHIYYSGNT

KPGQAPRLLIYDA





NYNPSLKSRVTISVD

SNLETGIPARFSG





TSKNQFSLKLSSVTA

SGSGTDFTLTISSL





ADTAVYYCARDRVT

EPEDFAVYYCQH





GAFDIWGQGTLVTVS

FDHLPLAFGQGT





S

KVEIK



 


EGFR.73
AC2921
QVQLQESGPGLVKPS
540
EIVLTQSPATLSLS
541




ETLSLTCTVSGGSVSS

PGERATLSCQAS





GDYYWTWIRQPPGK

QDISNYLNWYQQ





GLEWIGHIYYSGNTN

KPGQAPRLLIYDA





YNPSLKSRVTISVDTS

SNLETGIPARFSG





KNQFSLKLSSVTAAD

SGSGTDFTLTISSL





TAVYYCARDRVTGA

EPEDFAVYYCQH





FDIWGQGTLVTVSS

FDHLPLAFGQGT







KVEIK



 


EGFR.74
AC2922
EVQLVQSGAEVKKP
542
EIVLTQSPATLSLS
543




GESLKISCKGSGGSVS

PGERATLSCQAS





SGDYYWTWVRQMP

QDISNYLNWYQQ





GKGLEWMGHIYYSG

KPGQAPRLLIYDA





NTNYNPSLKSQVTIS

SNLETGIPARFSG





ADKSISTAYLQWSSL

SGSGTDFTLTISSL





KASDTAMYYCARDR

EPEDFAVYYCQH





VTGAFDIWGQGTLVT

FDHLPLAFGQGT





VSS

KVEIK



 


EGFR.75
AC2923
QVQLVQSGSELKKPG
544
EIVLTQSPATLSLS
545




ASVKVSCKASGGSVS

PGERATLSCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





QGLEWMGHIYYSGN

KPGQAPRLLIYDA





TNYNPSLKSRFVFSL

SNLETGIPARFSG





DTSVSTAYLQICSLK

SGSGTDFTLTISSL





AEDTAVYYCARDRV

EPEDFAVYYCQH





TGAFDIWGQGTLVTV

FDHLPLAFGQGT





SS

KVEIK



 


EGFR.76
AC2924
QVQLVQSGVEVKKP
546
EIVLTQSPATLSLS
547




GASVKVSCKASGGS

PGERATLSCQAS





VSSGDYYWTWVRQA

QDISNYLNWYQQ





PGQGLEWMGHIYYS

KPGQAPRLLIYDA





GNTNYNPSLKSRVTL

SNLETGIPARFSG





TTDSSTTTAYMELKS

SGSGTDFTLTISSL





LQFDDTAVYYCARD

EPEDFAVYYCQH





RVTGAFDIWGQGTL

FDHLPLAFGQGT





VTVSS

KVEIK



 


EGFR.81
AC2925
QVQLVESGGGVVQP
548
DIQMTQSPSSLSA
549




GRSLRLSCAASGGSV

SVGDRVTITCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GKGLEWVAHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSRLTISR

ASNLETGVPSRFS





DNSKNTLYLQMNSL

GSGSGTDFTLTIS





RAEDTAVYYCVRDR

SLQPEDFATYFCQ





VTGAFDIWGQGTLVT

HFDHLPLAFGQG





VSS

TKVEIK



 


EGFR.82
AC2926
EVQLVESGGGLVQPG
550
DIQMTQSPSSLSA
551




GSLRLSCAASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVAHIYYSGN

KPGKAPKLLIYD





TNYNPSLKSRLTISRD

ASNLETGVPSRFS





NAKNSLYLQMNSLR

GSGSGTDFTLTIS





AEDTAVYYCVRDRV

SLQPEDFATYFCQ





TGAFDIWGQGTLVTV

HFDHLPLAFGQG





SS

TKVEIK



 


EGFR.83
AC2927
QVQLVQSGAEVKKP
552
DIQMTQSPSSLSA
553




GSSVKVSCKASGGSV

SVGDRVTITCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GQGLEWMGHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSRLTITA

ASNLETGVPSRFS





DESTSTAYMELSSLR

GSGSGTDFTLTIS





SEDTAVYYCVRDRV

SLQPEDFATYFCQ





TGAFDIWGQGTLVTV

HFDHLPLAFGQG





SS

TKVEIK



 


EGFR.84
AC2928
QVQLVESGGGVVQP
554
DIQMTQSPSSLSA
555




GRSLRLSCAASGGSV

SVGDRVTITCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GKGLEWVAHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSRLTISR

ASNLETGVPSRFS





DNSKNTLYLQMNSL

GSGSGTDFTFTISS





RAEDTAVYYCVRDR

LQPEDIATYFCQH





VTGAFDIWGQGTLVT

FDHLPLAFGQGT





VSS

KVEIK



 


EGFR.85
AC2929
EVQLVESGGGLVQPG
556
DIQMTQSPSSLSA
557




GSLRLSCAASGGSVS

SVGDRVTITCQAS





SGDYYWTWVRQAPG

QDISNYLNWYQQ





KGLEWVAHIYYSGN

KPGKAPKLLIYD





TNYNPSLKSRLTISRD

ASNLETGVPSRFS





NAKNSLYLQMNSLR

GSGSGTDFTFTISS





AEDTAVYYCVRDRV

LQPEDIATYFCQH





TGAFDIWGQGTLVTV

FDHLPLAFGQGT





SS

KVEIK



 


EGFR.86
AC2930
QVQLVQSGAEVKKP
558
DIQMTQSPSSLSA
559




GSSVKVSCKASGGSV

SVGDRVTITCQAS





SSGDYYWTWVRQAP

QDISNYLNWYQQ





GQGLEWMGHIYYSG

KPGKAPKLLIYD





NTNYNPSLKSRLTITA

ASNLETGVPSRFS





DESTSTAYMELSSLR

GSGSGTDFTFTISS





SEDTAVYYCVRDRV

LQPEDIATYFCQH





TGAFDIWGQGTLVTV

FDHLPLAFGQGT





SS

KVEIK



 


EGFR.87
AC2931
QVQLQESGPGLVKPS
560
DIQMTQSPSSLSA
561




ETLSLTCTVSGGSVSS

SVGDRVTITCQAS





GDYYWTWIRQSPGK

QDISNYLNWYQQ





GLEWIGHIYYSGNTN

KPGKAPKLLIYD





YNPSLKSRLTISIDTS

ASNLETGVPSRFS





KTQFSLKLSSVTAAD

GSGSGTDFTFTISS





TAIYYCVRDRVTGAF

LQPEDIATYFCQH





DIWGQGTLVTVSS

FDHLPLAFGQGT







KVEIK









In certain embodiments, an anti-EGFR VH domain comprises an amino acid sequence of QVQLQX1X2GX3GLX4KPSETLSLTCXsVX6GGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS, wherein X1 corresponds to E or Q; X2 corresponds to S or W; X3 corresponds to P or A; X4 corresponds to V or L; X5 corresponds to T or A; and X6 corresponds to S or Y (SEQ ID NO: 576); and an anti-EGFR a VL domain comprises an amino acid sequence of X1IX2X3TQSPX4XSLSX6SX-GX8RXnTX10X1CQASQDISNYLNWYQQKPGX12APX13LLIYDASNLET GX14PX15RFSGSGSGTDFTX16TISX17LX18PEDX19AX20YYCQHFDHLPLAFGQGTKVEIK, wherein X1 corresponds to D or E; X2 corresponds to Q or V; X3 corresponds to M or L; X4 corresponds to S, G, or A; X5 corresponds to S or T; X6 corresponds to L or A; X7 corresponds to P or V; X8 corresponds to D or E; X9 corresponds to V or A; X10 corresponds to I or L; X11 corresponds to T or S; X12 corresponds to K or Q; X13 corresponds to K or R; X14 corresponds to V or I; X15 corresponds to S, D, or A; X16 corresponds to F or L; X17 corresponds to S or R; X18 corresponds to Q or E; X19 corresponds to I or F; and X20 corresponds to T or V (SEQ ID NO: 577);
Each EGFR antibody recited in Table 5f contains the following CDR sequences:









HCDR1



(SEQ ID NO: 562)



GGSVSSGDYYWT


 



HCDR2



(SEQ ID NO: 563)



HIYYSGNTNYNPSLKS


 



HCDR3



(SEQ ID NO: 564)



DRVTGAFDI


 



LCDR1



(SEQ ID NO: 565)



QASQDISNYLN


 



LCDR2



(SEQ ID NO: 566)



DASNLET


 



LCDR3



(SEQ ID NO: 567)



QHFDHLPLA






In some embodiments, the disclosure provides an anti-EGFR antibody VH region comprising the following CDRs: a VH region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GGSVSSGDYYWT (SEQ ID NO: 562); a VH region CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HIYYSGNTNYNPSLKS (SEQ ID NO: 563); and a VH region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to DRVTGAFDI (SEQ ID NO:564).
In some embodiments, the disclosure provides an anti-EGFR antibody VL region comprising the following CDRs: a VL region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QASQDISNYLN (SEQ ID NO: 565); a VL region CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to DASNLET (SEQ ID NO:566); and a VL region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QHFDHLPLA (SEQ ID NO:567).
In some embodiments, the anti-EGFR antibody VH region comprises the following framework regions (FRs): a VH region FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO:8206); a VH region FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WIRQPPGKGLEWIG (SEQ ID NO: 8207); a VH region FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO:8208); and a VH region FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS (SEQ ID NO:67).
In some embodiments, the anti-EGFR antibody VL region comprises the following framework regions (FRs): a VL region FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:8209); a VL region FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WYQQKPGKAPKLLIY (SEQ ID NO:8210); a VL region FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO:8211); and a VL region FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGQGTKVEIK (SEQ ID NO:8212).
In some embodiments, the disclosure provides an anti-EGFR antibody VH region comprising the sequence QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNT NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS (SEQ ID NO: 468), or the CDRs thereof; and an anti-EGFR antibody VL region comprising the sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGS GSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIK (SEQ ID NO: 469), or the CDRs thereof.
In some embodiments, the disclosure provides an anti-EGFR binding domain (e.g., scFv) comprising a sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to







(SEQ ID NO: 449)


DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD


 


ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQ


 


GTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVK


 


PSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNP


 


SLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGT


 


LVTVSS.






In some embodiments, the VL and VH of the antigen binding fragments (e.g., of Table 5f) are fused by relatively long linkers, consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that, when joined together, have a flexible characteristic. In some embodiments, the VL and VH of any of the scFv embodiments described herein (e.g., of Table 5f) are linked by a relatively long linker having the sequence SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the VL and VH of any of the scFv embodiments described herein are linked by relatively long linkers of hydrophilic amino acids having the sequences GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 82), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 83), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 84), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 85). In some embodiments, the AF1 and AF2 are linked together by a short linker of hydrophilic amino acids having 3, 4, 5, 6, or 7 amino acids. In some embodiments, the short linker sequences are identified herein as the sequences SGGGGS (SEQ ID NO: 86), GGGGS (SEQ ID NO: 87), GGSGGS (SEQ ID NO: 88), GGS, or GSP. In some embodiments, the disclosure provides compositions comprising a single chain diabody in which after folding, the first domain (VL or VH) is paired with the last domain (VH or VL) to form one scFv and the two domains in the middle are paired to form the other scFv in which the first and second domains, as well as the third and last domains, are fused together by one of the foregoing short linkers and the second and the third variable domains are fused by one of the foregoing relatively long linkers. In some embodiments, the selection of the short linker and relatively long linker is to prevent the incorrect pairing of adjacent variable domains, thereby facilitating the formation of a single chain configuration comprising the VL and VH of the first antigen binding fragment and the second antigen binding fragment.
In some embodiments, the present disclosure provides an antigen binding fragment (e.g., AF1 or AF2) that binds to EGFR that has enhanced stability compared to EGFR binding antibodies or antigen binding fragments known in the art. In some embodiments, an EGFR antigen binding fragment of the disclosure is designed to confer a higher degree of stability on the chimeric bispecific antigen binding fragment compositions into which they are integrated, leading to improved expression and recovery of the fusion protein, increased shelf-life and enhanced stability when administered to a subject. In some embodiments, an anti-EGFR AF of the present disclosure has a higher degree of thermal stability compared to certain EGFR-binding antibodies and antigen binding fragments known in the art. In some embodiments, an anti-EGFR AF of the present disclosure has a higher degree of thermal stability compared to an antigen binding fragment comprising the VH and VL of panitumumab. In some embodiments, an anti-EGFR AF of the present disclosure has a higher degree of thermal stability compared to EGFR.2 as disclosed in PCT International Patent Application Publication No. WO/2020/264208. In some embodiments, the anti-EGFR AF of the present disclosure is less immunogenic in a human compared to certain EGFR-binding antibodies and antigen binding fragments known in the art. In some embodiments, an anti-EGFR AF of the present disclosure is less immunogenic than antigen binding fragment comprising the VH and VL of panitumumab. In some embodiments, an anti-EGFR AF of the present disclosure is less immunogenic than EGFR.2 as disclosed in PCT International Patent Application Publication No. WO/2020/264208. In some embodiments, the degree to which an AF is immunogenic is determined by an immunogenicity prediction method such as TEPITOPEpan (described in Zhang et al. PLoS One. 2012; 7 (2): c30483. doi: 10.1371/journal.pone.0030483, PMID: 22383964, the entire content of which is incorporated herein by reference) or NetMHCpan-4.1 and NetMHCIIpan-4.0 (each described in Reynisson et al., Nucleic Acids Res 2020; 48 (W1): W449-W454. doi: 10.1093/nar/gkaa379., PMID: 32406916, the entire content of which is hereby incorporated herein by reference). In some embodiments, the anti-EGFR AF utilized as components of the chimeric bispecific antigen binding fragment compositions into which they are integrated exhibit favorable pharmaceutical properties, including high thermostability and low aggregation propensity, resulting in improved expression and recovery during manufacturing and storage, as well promoting long serum half-life. Biophysical properties such as thermostability are often limited by the antibody variable domains, which differ greatly in their intrinsic properties. High thermal stability is often associated with high expression levels and other desired properties, including being less susceptible to aggregation (Buchanan A, et al. Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression. MAbs 2013; 5:255). In some embodiments, thermal stability is determined by measuring the “melting temperature” (Tm), which is defined as the temperature at which half of the molecules are denatured. The melting temperature of each heterodimer is indicative of its thermal stability. In vitro assays to determine Tm are known in the art, including methods described in the Examples, below. The melting point of the heterodimer may be measured using techniques such as differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of the heterodimer may be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9), or as described in the Examples, below.
In some embodiments of the polypeptides of this disclosure, the antigen binding fragment (e.g., AF1 or AF2) can exhibit a higher thermal stability than an anti-EGFR binding fragment comprising a VH of SEQ ID NO: 450 and a VL of SEQ ID NO: 451 (see Table 5f), as evidenced in an in vitro assay by a higher melting temperature (Tm) of the first antigen binding fragment relative to that of the anti-EGFR binding fragment; or upon incorporating the first antigen binding fragment into a test bispecific antigen binding domain, a higher Tm of the test bispecific antigen binding domain relative to that of a control bispecific antigen binding domain, wherein the test bispecific antigen binding domain comprises the first antigen binding fragment and a reference antigen binding fragment that binds to an antigen other than EGFR; and wherein the control bispecific antigen binding domain consists of the anti-EGFR binding fragment comprising a VH of SEQ ID NO: 450 and a VL of SEQ ID NO: 451 (see Table 5f) and the reference antigen binding fragment. In some embodiments, the melting temperature (Tm) of the first antigen binding fragment can be at least 2° C. greater, or at least 3° C. greater, or at least 4° C. greater, or at least 5° C. greater than the Tm of the anti-EGFR binding fragment comprising a VH of SEQ ID NO: 450 and a VL of SEQ ID NO: 451 (see Table 5f). In some embodiments, the melting temperature (Tm) of the first antigen binding fragment can be 2° C. to 15° C. greater, or 3° C. to 15° C. greater, or 4° C. to 15° C. greater, or 5° C. to 15° C. greater than the Tm of the anti-EGFR binding fragment comprising a VH of SEQ ID NO: 450 and a VL of SEQ ID NO: 451 (see Table 5f).
In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an antigen binding fragment (AF) that specifically bind human EGFR. The antigen binding fragment (AF) can specifically bind human EGFR. In some embodiments, the antigen binding fragment (AF) can specifically bind human EGFR with a binding affinity (KD) constant between about 10 nM and about 400 nM, or between about 50 nM and about 350 nM, or between about 100 nM and 300 nM, as determined in an in vitro antigen-binding assay comprising a human EGFR antigen. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an antigen binding fragment (AF) that specifically binds human EGFR with a binding affinity (kD)) weaker than about 10 nM, or about 50 nM, or about 100 nM, or about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 350 nM, or weaker than about 400 nM as determined in an in vitro antigen-binding assay. For clarity, an antigen binding fragment (AF) with a kD of 400 binds its ligand more weakly than one with a kD of 10 nM. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an antigen binding fragment (AF) that specifically binds human EGFR with at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker binding affinity than an antigen binding fragment consisting of an amino acid sequence of Table 5f, as determined by the respective binding affinities (KD) in an in vitro antigen-binding assay.
In some embodiments, the present disclosure provides bispecific polypeptides comprising an antigen binding fragment (AF) that exhibits a binding affinity to EGFR (anti-EGFR AF) that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold at weaker relative to that of an anti-EGFR AF embodiments described herein that are incorporated into the subject polypeptides, as determined by the respective binding affinities (kD) in an in vitro antigen-binding assay.
The binding affinity of the subject compositions for the target ligands can be assayed, e.g., using binding or competitive binding assays, such as Biacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in U.S. Pat. No. 5,534,617, assays described in the Examples herein, radio-receptor assays, or other assays known in the art. The binding affinity constant can then be determined using standard methods, such as Scatchard analysis, as described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19) 12): 487, or other methods known in the art.
In some embodiments, the present disclosure provides an antigen binding fragment (AF) that binds to EGFR (anti-EGFR AF) and is incorporated into a chimeric, bispecific polypeptide composition that is designed to have an isoelectric point (pI) that confers enhanced stability on the composition compared to corresponding compositions comprising EGFR binding antibodies or antigen binding fragments known in the art. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise AF that bind to EGFR (anti-EGFR AF) wherein the anti-EGFR AF exhibits a pI that is between 6.0 and 6.6, inclusive. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise AF that bind to EGFR (anti-EGFR AF) wherein the anti-EGFR AF exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the pI of a reference antigen binding fragment. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an AF that binds to EGFR (anti-EGFR AF) fused to another AF that binds to a CD3 antigen (anti-CD3 AF) wherein the anti-EGFR AF exhibits a pI that is within at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pI of the AF that binds CD3 antigen or an epitope thereof. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an AF that binds to EGFR (anti-EGFR AF) fused to an AF that binds to a CD3 antigen (anti-CD3 AF) wherein the AF exhibits a pI that is within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pI of the anti-EGFR AF. It is specifically intended that by such design wherein the pI of the two antigen binding fragments are within such ranges, the resulting fused antigen binding fragments will confer a higher degree of stability on the chimeric bispecific antigen binding fragment compositions into which they are integrated, leading to improved expression and enhanced recovery of the fusion protein in soluble, non-aggregated form, increased shelf-life of the formulated chimeric bispecific polypeptide compositions, and enhanced stability when the composition is administered to a subject. In some embodiments, having the two AFs (the anti-EGFR AF and the anti-CD3 AF) within a relatively narrow pI range of may allow for the selection of a buffer or other solution in which both the AFs (anti-EGFR AF and anti-CD3 AF) are stable, thereby promoting overall stability of the composition. In some embodiments, the antigen binding fragment (AF) can exhibit an isoelectric point (pI) that is less than or equal to 6.6. In some embodiments, the antigen binding fragment (AF) can exhibit an isoelectric point (pI) that is between 6.0 and 6.6, inclusive.
Unless otherwise specified, numbering of amino acid residues in the variable domain of antibody domain, antigen binding domain, or fragment thereof described herein is according to the Kabat numbering scheme. The Kabat numbering for EGFR.2 VH (SEQ ID NO: 450) and VL (SEQ ID NO: 451) is provided below.







TABLE 5g







Kabat numbering of EGFR.2 VH (SEQ


ID NO: 450) and VL (SEQ ID NO: 451)













VH
VH

VL
VL



Position
Position

Position
Position



relative
according

relative
according


VH
to SEQ ID
to Kabat
VL
to SEQ ID
to Kabat


residue
NO: 450
numbering
residue
NO: 451
numbering















Q
1
 1
D
1
1


V
2
 2
I
2
2


Q
3
 3
Q
3
3


L
4
 4
M
4
4


Q
5
 5
T
5
5


E
6
 6
Q
6
6


S
7
 7
S
7
7


G
8
 8
P
8
8


P
9
 9
S
9
9


G
10
10
S
10
10


L
11
11
L
11
11


V
12
12
S
12
12


K
13
13
A
13
13


P
14
14
S
14
14


S
15
15
V
15
15


E
16
16
G
16
16


T
17
17
D
17
17


L
18
18
R
18
18


S
19
19
V
19
19


L
20
20
T
20
20


T
21
21
I
21
21


C
22
22
T
22
22


T
23
23
C
23
23


V
24
24
Q
24
24


S
25
25
A
25
25


G
26
26
S
26
26


G
27
27
Q
27
27


S
28
28
D
28
28


V
29
29
I
29
29


S
30
30
S
30
30


S
31
31
N
31
31


G
32
32
Y
32
32


D
33
33
L
33
33


Y
34
34
N
34
34


Y
35
35
W
35
35


W
36
  35A
Y
36
36


T
37
  35B
Q
37
37


W
38
36
Q
38
38


I
39
37
K
39
39


R
40
38
P
40
40


Q
41
39
G
41
41


S
42
40
K
42
42


P
43
41
A
43
43


G
44
42
P
44
44


K
45
43
K
45
45


G
46
44
L
46
46


L
47
45
L
47
47


E
48
46
I
48
48


W
49
47
Y
49
49


I
50
48
D
50
50


G
51
49
A
51
51


H
52
50
S
52
52


I
53
51
N
53
53


Y
54
52
L
54
54


Y
55
53
E
55
55


S
56
54
T
56
56


G
57
55
G
57
57


N
58
56
V
58
58


T
59
57
P
59
59


N
60
58
S
60
60


Y
61
59
R
61
61


N
62
60
F
62
62


P
63
61
S
63
63


S
64
62
G
64
64


L
65
63
S
65
65


K
66
64
G
66
66


S
67
65
S
67
67


R
68
66
G
68
68


L
69
67
T
69
69


T
70
68
D
70
70


I
71
69
F
71
71


S
72
70
T
72
72


I
73
71
F
73
73


D
74
72
T
74
74


T
75
73
I
75
75


S
76
74
S
76
76


K
77
75
S
77
77


T
78
76
L
78
78


Q
79
77
Q
79
79


F
80
78
P
80
80


S
81
79
E
81
81


L
82
80
D
82
82


K
83
81
I
83
83


L
84
82
A
84
84


S
85
  82A
T
85
85


S
86
  82B
Y
86
86


V
87
 82C
F
87
87


T
88
83
C
88
88


A
89
84
Q
89
89


A
90
85
H
90
90


D
91
86
F
91
91


T
92
87
D
92
92


A
93
88
H
93
93


I
94
89
L
94
94


Y
95
90
P
95
95


Y
96
91
L
96
96


C
97
92
A
97
97


V
98
93
F
98
98


R
99
94
G
99
99


D
100
95
G
100
100


R
101
96
G
101
101


V
102
97
T
102
102


T
103
98
K
103
103


G
104
99
V
104
104


A
105
100 
E
105
105


F
106
 100A
I
106
106


D
107
101 
K
107
107


I
108
102 





W
109
103 





G
110
104 





Q
111
105 





G
112
106 





T
113
107 





M
114
108 





V
115
109 





T
116
110 





V
117
111 





S
118
112 





S
119
113 









Linkers and Spacers Between Antibody Regions in Bispecific Antibodies

In some embodiments of the polypeptides of this disclosure, a pair of the light chain variable region (VL) and the heavy chain variable region (VH) of an antigen binding fragment can be linked by a linker, or a long linker (e.g., of hydrophilic amino acids). In some embodiments, a first antigen binding fragment (AF1) (e.g., an scFv domain, such as an anti-EGFR scFv domain) and a second antigen binding fragment (AF2) (e.g., an scFv, such as an anti-CD3 scFv) are linked by linker, or a long linker (e.g., of hydrophilic amino acids). In some embodiments, a linker linking the light chain variable region (VL) and the heavy chain variable region (VH) of an antigen binding fragment (e.g., a first antigen binding fragment (AF1) and/or a second antigen binding fragment (AF2)), can (each independently) comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in Table A. In some embodiments, a linker linking the light chain variable region (VL) and the heavy chain variable region (VH) of an antigen binding fragment (e.g., a first antigen binding fragment (AF1) and/or a second antigen binding fragment (AF2)), can (each independently) comprise an amino acid sequence identical to a sequence set forth in Table A. In some embodiments of the polypeptides of this disclosure, two antigen binding fragments (e.g., a first and a second antigen binding fragments) can be fused together by a peptide linker, or a short linker. In some embodiments, the peptide linker linking two antigen binding fragments (e.g., a first and a second antigen binding fragments), can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in Table B. In some embodiments, the peptide linker linking two antigen binding fragments (e.g., a first and a second antigen binding fragments), can comprise an amino acid sequence identical to a sequence set forth in Table B. In some cases, the first antigen binding fragment is a single-chain variable fragment (scFv). In some cases, the second antigen binding fragment is a single-chain variable fragment (scFv). The two single-chain variable fragments of the first and second antigen binding fragments can be linked together by the peptide linker. In some embodiments of the polypeptides of this disclosure, the linker used to link the scFv of the first antigen binding fragment (e.g., an anti-EGFR scFv) and the linker used to link the VL and VH of the second antigen binding fragment (e.g., an anti-CD3 scFv) can be GGGGSGGGS (SEQ ID NO: 125) of Table A. In other embodiments, the linker used to link the VL and VH of an antigen binding fragment (e.g., an anti-CD3 scFv) can be SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the disclosure provides polypeptides comprising a single chain diabody in which after folding, the first domain (VL or VH) is paired with the last domain (VH or VL) to form one scFv and the two domains in the middle are paired to form the other scFv in which the first and second domains, as well as the third and last domains, are fused together by a short linker of hydrophilic amino acids identified herein by the sequences set forth in Table B and the second and the third variable domains are fused by a long linker identified in Table A. In some embodiments, the selection of the short linker and long linker is to prevent the incorrect pairing of adjacent variable domains, thereby facilitating the formation of the single chain configuration comprising the VL and VH of the first binding moiety and the second binding moiety.







TABLE A







Intramolecular Long Linkers










Linker 

SEQ



#
Name
ID
Amino Acid Sequence





L1
(G4S)3
112
GGGGSGGGGSGGGGS


 


L2
MT110_
113
GEGTSTGSGGSGGSGGAD



18




 


L3
MT103_
114
VEGGSGGSGGSGGSGGVD



18




 


L4
UCHT1_
115
RTSGPGDGGKGGPGKGPG



29

GEGTKGTGPGG


 


L5
Y30
116
GSGEGSEGEGGGEGSEGE





GSGEGGEGEGSG


 


L6
Y32
117
TGSGEGSEGEGGGEGSEG





EGSGEGGEGEGSGT


 


L7
G1_30_
118
GATPPETGAETESPGETT



3

GGSAESEPPGEG


 


L8
G9_30_
119
GSAAPTAGTTPSASPAPP



1

TGGSSAAGSPST


 


L9
Y30_
120
GEGGESGGSEGEGSGEGE



modi-

GGSGGEGESEGG



fied




 


L10
G1_30_
121
STETSPSTPTESPEAGSG



1

SGSPESPSGTEA


 


L11
G1_30_
122
PTGTTGEPSGEGSEPEGS



2

APTSSTSEATPS


 


L12
G1_30_
123
SESESEGEAPTGPGASTT



4

PEPSESPTPETS


 


L13
UCHT1_
124
PEGGESGEGTGPGTGGEP



modi-

EGEGGPGGEGGT



fied
















TABLE B







Intermolecular Short Linkers










Name
Amino Acid Sequence






S-1
GGGGSGGGS (SEQ ID NO: 125)


 



S-2
SGGGGS (SEQ ID NO: 86)


 



S-3
GGGGS (SEQ ID NO: 87)


 



S-4
GGS


 



S-5
GSP









Spacers & TCE Release Segments
Included herein are fusion proteins comprising TCE components that either becomes biologically active or have an increase in biological activity upon release from an ELNN by cleavage of an optional cleavage sequence incorporated within optional spacer sequences into the fusion protein, e.g., as described herein.
In some embodiments, the spacer may be provided to enhance expression of the fusion protein from a host cell and/or to decrease steric hindrance such that the TCE component may assume its desired tertiary structure and/or interact appropriately with its target molecule. For spacers and methods of identifying desirable spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879, specifically incorporated by reference herein. In some embodiments, the spacer comprises one or more peptide sequences that are between 1 to 50 amino acid residues in length, or about 1 to 25 residues, or about 1 to 10 residues in length. Spacer sequences, exclusive of cleavage sites, can comprise any of the 20 natural L amino acids, and will preferably comprise hydrophilic amino acids that are sterically unhindered that can include, but not be limited to, glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P). In some embodiments, the spacer can be a polyglycine or polyalanine, or predominately a mixture of combinations of glycine and alanine residues. In some embodiments, the spacer polypeptide exclusive of a cleavage sequence is substantially devoid of secondary structure. In some embodiments, one or both spacer sequences in a paTCE fusion protein composition may each further contain a cleavage sequence, which may be identical or may be different, wherein the cleavage sequence may be acted on by a protease to release the TCE from the fusion protein.







TABLE C







Exemplary Spacers between a Release 


Segment and a Bispecific Antibody 


Domain










Amino Acid Sequence
SEQ ID NO:






STEPS
89


 



SATPESGPGT
90


 



ATSGSETPGT
91


 



GTAEAASASG
92


 



STEPSEGSAPGTS
93


 



SGPGTS
94


 



GTSTEPS
95


 



GTSESATPES
96


 



GTATPESGPG
97









In some embodiments of the polypeptides of this disclosure, a release segment (RS) (e.g., a first release segment (RS1), a second release segment (RS2), etc.) can be fused to a bispecific antibody domain (BsAb) by a spacer. In some embodiments, a spacer can (each independently) comprise at least 4 types of amino acids that are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P). In some embodiments, the peptides of this disclosure can comprise a first release segment fused to the bispecific antibody domain via a first spacer and a second release segment fused to the bispecific antibody domain via a second spacer. In some embodiments, a spacer (e.g., a first spacer, a second spacer, etc.) can (each independently) comprise an amino acid sequence having at least (about) 80%, at least (about) 90%, or 100% sequence identity to a sequence set forth in Table C. In some embodiments, the spacer (e.g., the first spacer, the second spacer, etc.) can (each independently) comprise an amino acid sequence identical to a sequence set forth in Table C.
In some embodiments, the incorporation of the cleavage sequence into a fusion protein is designed to permit release of a TCE that becomes active or more active upon its release from one or more ELNNs. In some embodiments, the cleavage sequences are located sufficiently close to the TCE sequences, generally within 18, or within 12, or within 6, or within 2 amino acids of the TCE sequence terminus, such that any remaining residues attached to the TCE after cleavage do not appreciably interfere with the activity (e.g., such as binding to a receptor) of the TCE yet provide sufficient access to the protease to be able to effect cleavage of the cleavage sequence. In some embodiments, the cleavage site is a sequence that can be cleaved by a protease endogenous to the mammalian subject such that a paTCE can be cleaved after administration to a subject. In such cases, the paTCE can serve as a circulating depot for the TCE. Examples of cleavage sites contemplated herein include, but are not limited to, a polypeptide sequence cleavable by a mammalian endogenous protease listed in Table 6.
In some embodiments, a paTCE fusion protein comprises spacer sequences that comprise one or more cleavage sequences configured to release the TCE from the fusion protein when acted on by a protease. In some embodiments, a spacer sequence does not comprise a cleavage sequence. In some embodiments, the one or more cleavage sequences can be a sequence having at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%) sequence identify to a sequence from Table 7a or 7b.
In some embodiments, the disclosure provides TCE release segment polypeptides (or release segments (RSs)) that are substrates for one or more mammalian proteases associated with or produced by disease tissues or cells found in proximity to disease tissues. Such proteases can include, but not be limited to the classes of proteases such as metalloproteinases, cysteine proteases, aspartate proteases, and serine proteases, including, but not limited to, the proteases of Table 6. The RSs are useful for, amongst other things, incorporation into the subject recombinant polypeptides, conferring an inactive format that can be activated by the cleavage of the RSs by mammalian proteases. As described herein, the RSs are incorporated into the subject recombinant polypeptide compositions, linking the incorporated binding moieties to the ELNN (exemplary configurations of which are described herein) such that upon cleavage of the RSs by action of the one or more proteases for which the RSs are substrates, the binding moieties and ELNN are released from the composition and the binding moieties, no longer shielded by the ELNN, regain their full potential to bind their ligands.







TABLE 6







Proteases of Target Tissues








Class of Proteases
Protease





Metalloproteinases
Meprin



Neprilysin (CD10)



PSMA



BMP-1



A disintegrin and metalloproteinases (ADAMs)



ADAM8



ADAM9



ADAM10



ADAM12



ADAM15



ADAM17 (TACE)



ADAM19



ADAM28 (MDC-L)



ADAM with thrombospondin motifs (ADAMTS)



ADAMTS1



ADAMTS4



ADAMTS5



Matrix Metalloproteinases (MMPs)



MMP-1 (Collagenase 1)



MMP-2 (Gelatinase A)



MMP-3 (m1)



MMP-7 (Matrilysin 1)



MMP-8 (Collagenase 2)



MMP-9 (Gelatinase B)



MMP-10 (Stromelysin 2)



MMP-11(Stromelysin 3)



MMP-12 (Macrophage elastase)



MMP-13 (Collagenase 3)



MMP-14 (MT1-MMP)



MMP-15 (MT2-MMP)



MMP-19



MMP-23 (CA-MMP)



MMP-24 (MT5-MMP)



MMP-26 (Matrilysin 2)



MMP-27 (CMMP)


Cysteine Proteases
Legumain



Cysteine cathepsins



Cathepsin B



Cathepsin C



Cathepsin K



Cathepsin L



Cathepsin S



Cathepsin X


Aspartate Proteases
Cathepsin D



Cathepsin E



Secretase


Serine Proteases
Urokinase (uPA)



Tissue-type plasminogen activator (tPA)



Plasmin



Thrombin



Prostate-specific antigen (PSA, KLK3)



Human neutrophil elastase (HNE)



Elastase



Tryptase



Type II transmembrane serine proteases (TTSPs)



DESC1



Hepsin (HPN)



Matriptase



Matriptase-2



TMPRSS2



TMPRSS3



TMPRSS4 (CAP2)



Fibroblast Activation Protein (FAP)



kallikrein-related peptidase (KLK family)



KLK4



KLK5



KLK6



KLK7



KLK8



KLK10



KLK11



KLK13



KLK14









In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a first release segment (RS1) sequence having at least 88%, or at least 94%, or 100% sequence identity, when optimally aligned, to a sequence identified in Table 7a, wherein the RS1 is a substrate for one or more mammalian proteases. In some embodiments, the RS is further engineered to remove a legumain cleavage site. In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a RS1 and a second release segment (RS2) sequence, each having at least 88%, or at least 94%, or 100% sequence identity, when optimally aligned, to a sequence identified herein by the sequences set forth in Table 7a, wherein the RS1 and the RS2 each are a substrate for one or more mammalian proteases. In some embodiments, the RS1 and RS2 each do not serve as substrates for legumain.
In some embodiments, disclosure provides activatable recombinant polypeptides comprising a first RS (RS1) sequence having at least 90%, at least 93%, at least 97%, or 100% identity, when optimally aligned, to a sequence identified in Table 7b, wherein the RS1 is a substrate for one or more mammalian proteases. In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a RS1 and a second release segment (RS2) sequence, each having at least 88%, or at least 94%, or 100% sequence identity, when optimally aligned, to a sequence identified herein by the sequences set forth in Table 7b, wherein the RS1 and the RS2 are each a substrate for one or more mammalian proteases (e.g., at one, two, or three cleavage sites within each release segment sequence). In some embodiments of activatable recombinant polypeptides comprising RS1 and RS2, the two release segments can be identical. In some embodiments of activatable recombinant polypeptides comprising RS1 and RS2, the two release segments can be different.
The present disclosure contemplates release segments that are substrates for one, two or three different classes of proteases that are metalloproteinases, cysteine proteases, aspartate proteases, or serine proteases, including the proteases of Table 6. In some embodiments, a paTCE comprises RSs (e.g., RS1 and RS2) that serve as substrates for one or more proteases found in close association with or are co-localized with tumors or cancer cells, and upon cleavage of the RSs, the binding moieties that are otherwise shielded by ELNNs of the paTCE (and thus have a lower binding affinity for their respective ligands) are released from the ELNNs and regain their full potential to bind target and effector cell ligands. In some embodiments, a paTCE comprises RSs (e.g., RS1 and RS2), that each comprise an amino acid sequence that is a substrate for one or more cellular proteases located within a targeted cell, including but not limited to a protease of Table 6. In some embodiments, RSs are substrates for two or three classes of proteases that cleave different portions of each RS. In some embodiments, each RS that is a substrate for two, three, or more classes of proteases has two, three, or more distinct cleavage sites, but cleavage by a single protease nevertheless results in the release of the binding moieties from an ELNN.
In some embodiments, an RS of the disclosure for incorporation into a fusion protein (such as a paTCE) is a substrate for one or more proteases including but not limited to meprin, neprilysin (CD10), PSMA. BMP-1, A disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, ADAM12. ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with thrombospondin motifs (ADAMTS), ADAMTS1. ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (MMP-3, stromelysin 1), matrix metalloproteinase-7 (MMP-7, Matrilysin 1), matrix metalloproteinase-8 (MMP-8, collagenase 2), matrix metalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase-10 (MMP-10, stromelysin 2), matrix metalloproteinase-11 (MMP-11, stromelysin 3), matrix metalloproteinase-12 (MMP-12, macrophage clastase), matrix metalloproteinase-13 (MMP-13, collagenase 3), matrix metalloproteinase-14 (MMP-14, MT1-MMP), matrix metalloproteinase-15 (MMP-15, MT2-MMP), matrix metalloproteinase-19 (MMP-19), matrix metalloproteinase-23 (MMP-23, CA-MMP), matrix metalloproteinasc-24 (MMP-24, MT5-MMP), matrix metalloproteinasc-26 (MMP-26, matrilysin 2), matrix metalloproteinase-27 (MMP-27, CMMP), legumain, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X, cathepsin D, cathepsin E, secretase, urokinase (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, prostate-specific antigen (PSA, KLK3), human neutrophil elastase (HNE), elastase, tryptase, Type II transmembrane serine proteases (TTSPs), DESC1, hepsin (HPN), matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activation protein (FAP), kallikrein-related peptidase (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14. In some embodiments, the RS is a substrate for ADAM17. In some embodiments, the RS is a substrate for BMP-1. In some embodiments, the RS is a substrate for cathepsin. In some embodiments, the RS is a substrate for HtrA1. In some embodiments, the RS is a substrate for legumain. In some embodiments, the RS is a substrate for MMP-1. In some embodiments, the RS is a substrate for MMP-2. In some embodiments, the RS is a substrate for MMP-7. In some embodiments, the RS is a substrate for MMP-9. In some embodiments, the RS is a substrate for MMP-11. In some embodiments, the RS is a substrate for MMP-14. In some embodiments, the RS is a substrate for uPA. In some embodiments, the RS is a substrate for matriptase. In some embodiments, the RS is a substrate for MT-SP1. In some embodiments, the RS is a substrate for neutrophil elastase. In some embodiments, the RS is a substrate for thrombin. In some embodiments RS is a substrate for TMPRSS3. In some embodiments, the RS is a substrate for TMPRSS4. In some embodiments, the RS of the subject recombinant polypeptide compositions is a substrate for at least two proteases including but not limited to legumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In some embodiments, the RS of the subject recombinant polypeptide compositions is a substrate for legumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In specific embodiments, the RS of the subject recombinant polypeptide compositions is not a substrate for legumain. In some embodiments, the RS of the subject recombinant polypeptide compositions is a substrate for uPA, matriptase (also known as MT-SP1 and ST14), MMP2, MMP7, MMP9, and MMP14. In some embodiments, the RS of the subject recombinant polypeptide compositions is substrate for uPA, matriptase, MMP2, MMP7, MMP9, and MMP14 but not legumain.







TABLE 7a







TCE Release Segment Sequences.









Name
Amino Acid Sequence
SEQ ID NO





RSR-1517
EAGRSANHEPLGLVAT
7001


 


BSRS-A1-1
ASGRSTNAGPSGLAGP
7002


 


BSRS-A2-1
ASGRSTNAGPQGLAGQ
7003


 


BSRS-A3-1
ASGRSTNAGPPGLTGP
7004


 


VP-1
ASSRGTNAGPAGLTGP
7005


 


RSR-1752
ASSRTTNTGPSTLTGP
7006


 


RSR-1512
AAGRSDNGTPLELVAP
7007


 


RSR-1517
EAGRSANHEPLGLVAT
7008


 


VP-2
ASGRGTNAGPAGLTGP
7009


 


RSR-1018
LFGRNDNHEPLELGGG
7010


 


RSR-1053
TAGRSDNLEPLGLVFG
7011


 


RSR-1059
LDGRSDNFHPPELVAG
7012


 


RSR-1065
LEGRSDNEEPENLVAG
7013


 


RSR-1167
LKGRSDNNAPLALVAG
7014


 


RSR-1201
VYSRGTNAGPHGLTGR
7015


 


RSR-1218
ANSRGTNKGFAGLIGP
7016


 


RSR-1226
ASSRLTNEAPAGLTIP
7017


 


RSR-1254
DQSRGTNAGPEGLTDP
7018


 


RSR-1256
ESSRGTNIGQGGLTGP
7019


 


RSR-1261
SSSRGTNQDPAGLTIP
7020


 


RSR-1293
ASSRGQNHSPMGLTGP
7021


 


RSR-1309
AYSRGPNAGPAGLEGR
7022


 


RSR-1326
ASERGNNAGPANLTGF
7023


 


RSR-1345
ASHRGTNPKPAILTGP
7024


 


RSR-1354
MSSRRTNANPAQLTGP
7025


 


RSR-1426
GAGRTDNHEPLELGAA
7026


 


RSR-1478
LAGRSENTAPLELTAG
7027


 


RSR-1479
LEGRPDNHEPLALVAS
7028


 


RSR-1496
LSGRSDNEEPLALPAG
7029


 


RSR-1508
EAGRTDNHEPLELSAP
7030


 


RSR-1513
EGGRSDNHGPLELVSG
7031


 


RSR-1516
LSGRSDNEAPLELEAG
7032


 


RSR-1524
LGGRADNHEPPELGAG
7033


 


RSR-1622
PPSRGTNAEPAGLTGE
7034


 


RSR-1629
ASTRGENAGPAGLEAP
7035


 


RSR-1664
ESSRGTNGAPEGLTGP
7036


 


RSR-1667
ASSRATNESPAGLTGE
7037


 


RSR-1709
ASSRGENPPPGGLTGP
7038


 


RSR-1712
AASRGTNTGPAELTGS
7039


 


RSR-1727
AGSRTTNAGPGGLEGP
7040


 


RSR-1754
APSRGENAGPATLTGA
7041


 


RSR-1819
ESGRAANTGPPTLTAP
7042


 


RSR-1832
NPGRAANEGPPGLPGS
7043


 


RSR-1855
ESSRAANLTPPELTGP
7044


 


RSR-1911
ASGRAANETPPGLTGA
7045


 


RSR-1929
NSGRGENLGAPGLTGT
7046


 


RSR-1951
TTGRAANLTPAGLTGP
7047


 


RSR-2295
EAGRSANHTPAGLTGP
7048


 


RSR-2298
ESGRAANTTPAGLTGP
7049


 


RSR-2038
TTGRATEAANLTPAGLTGP
7050


 


RSR-2072
TTGRAEEAANLTPAGLTGP
7051


 


RSR-2089
TTGRAGEAANLTPAGLTGP
7052


 


RSR-2302
TTGRATEAANATPAGLTGP
7053


 


RSR-3047
TTGRAGEAEGATSAGATGP
7054


 


RSR-3052
TTGEAGEAANATSAGATGP
7055


 


RSR-3043
TTGEAGEAAGLTPAGLTGP
7056


 


RSR-3041
TTGAAGEAANATPAGLTGP
7057


 


RSR-3044
TTGRAGEAAGLTPAGLTGP
7058


 


RSR-3057
TTGRAGEAANATSAGATGP
7059


 


RSR-3058
TTGEAGEAAGATSAGATGP
7060


 


RSR-2485
ESGRAANTEPPELGAG
7061


 


RSR-2486
ESGRAANTAPEGLTGP
7062


 


RSR-2488
EPGRAANHEPSGLTEG
7063


 


RSR-2599
ESGRAANHTGAPPGGLTGP
7064


 


RSR-2706
TTGRTGEGANATPGGLTGP
7065


 


RSR-2707
RTGRSGEAANETPEGLEGP
7066


 


RSR-2708
RTGRTGESANETPAGLGGP
7067


 


RSR-2709
STGRTGEPANETPAGLSGP
7068


 


RSR-2710
TTGRAGEPANATPTGLSGP
7069


 


RSR-2711
RTGRPGEGANATPTGLPGP
7070


 


RSR-2712
RTGRGGEAANATPSGLGGP
7071


 


RSR-2713
STGRSGESANATPGGLGGP
7072


 


RSR-2714
RTGRTGEEANATPAGLPGP
7073


 


RSR-2715
ATGRPGEPANTTPEGLEGP
7074


 


RSR-2716
STGRSGEPANATPGGLTGP
7075


 


RSR-2717
PTGRGGEGANTTPTGLPGP
7076


 


RSR-2718
PTGRSGEGANATPSGLTGP
7077


 


RSR-2719
TTGRASEGANSTPAPLTEP
7078


 


RSR-2720
TYGRAAEAANTTPAGLTAP
7079


 


RSR-2721
TTGRATEGANATPAELTEP
7080


 


RSR-2722
TVGRASEEANTTPASLTGP
7081


 


RSR-2723
TTGRAPEAANATPAPLTGP
7082


 


RSR-2724
TWGRATEPANATPAPLTSP
7083


 


RSR-2725
TVGRASESANATPAELTSP
7084


 


RSR-2726
TVGRAPEGANSTPAGLTGP
7085


 


RSR-2727
TWGRATEAPNLEPATLTTP
7086


 


RSR-2728
TTGRATEAPNLTPAPLTEP
7087


 


RSR-2729
TQGRATEAPNLSPAALTSP
7088


 


RSR-2730
TQGRAAEAPNLTPATLTAP
7089


 


RSR-2731
TSGRAPEATNLAPAPLTGP
7090


 


RSR-2732
TQGRAAEAANLTPAGLTEP
7091


 


RSR-2733
TTGRAGSAPNLPPTGLTTP
7092


 


RSR-2734
TTGRAGGAENLPPEGLTAP
7093


 


RSR-2735
TTSRAGTATNLTPEGLTAP
7094


 


RSR-2736
TTGRAGTATNLPPSGLTTP
7095


 


RSR-2737
TTARAGEAENLSPSGLTAP
7096


 


RSR-2738
TTGRAGGAGNLAPGGLTEP
7097


 


RSR-2739
TTGRAGTATNLPPEGLTGP
7098


 


RSR-2740
TTGRAGGAANLAPTGLTEP
7099


 


RSR-2741
TTGRAGTAENLAPSGLTTP
7100


 


RSR-2742
TTGRAGSATNLGPGGLTGP
7101


 


RSR-2743
TTARAGGAENLTPAGLTEP
7102


 


RSR-2744
TTARAGSAENLSPSGLTGP
7103


 


RSR-2745
TTARAGGAGNLAPEGLTTP
7104


 


RSR-2746
TTSRAGAAENLTPTGLTGP
7105


 


RSR-2747
TYGRTTTPGNEPPASLEAE
7106


 


RSR-2748
TYSRGESGPNEPPPGLTGP
7107


 


RSR-2749
AWGRTGASENETPAPLGGE
7108


 


RSR-2750
RWGRAETTPNTPPEGLETE
7109


 


RSR-2751
ESGRAANHTGAEPPELGAG
7110


 


RSR-2754
TTGRAGEAANLTPAGLTES
7111


 


RSR-2755
TTGRAGEAANLTPAALTES
7112


 


RSR-2756
TTGRAGEAANLTPAPLTES
7113


 


RSR-2757
TTGRAGEAANLTPEPLTES
7114


 


RSR-2758
TTGRAGEAANLTPAGLTGA
7115


 


RSR-2759
TTGRAGEAANLTPEGLTGA
7116


 


RSR-2760
TTGRAGEAANLTPEPLTGA
7117


 


RSR-2761
TTGRAGEAANLTPAGLTEA
7118


 


RSR-2762
TTGRAGEAANLTPEGLTEA
7119


 


RSR-2763
TTGRAGEAANLTPAPLTEA
7120


 


RSR-2764
TTGRAGEAANLTPEPLTEA
7121


 


RSR-2765
TTGRAGEAANLTPEPLTGP
7122


 


RSR-2766
TTGRAGEAANLTPAGLTGG
7123


 


RSR-2767
TTGRAGEAANLTPEGLTGG
7124


 


RSR-2768
TTGRAGEAANLTPEALTGG
7125


 


RSR-2769
TTGRAGEAANLTPEPLTGG
7126


 


RSR-2770
TTGRAGEAANLTPAGLTEG
7127


 


RSR-2771
TTGRAGEAANLTPEGLTEG
7128


 


RSR-2772
TTGRAGEAANLTPAPLTEG
7129


 


RSR-2773
TTGRAGEAANLTPEPLTEG
7130


 


RSR-3213
EAGRSASHTPAGLTGP
7628
















TABLE 7b







Release Segment Sequences













SEQ





ID



Name
Amino Acid Sequence
NO:






RSN-0001
GSAPGSAGGYAELRMG
7131




GAIATSGSETPGT



 



RSN-0002
GSAPGTGGGYAPLRMG
7132




GGAATSGSETPGT



 



RSN-0003
GSAPGAEGGYAALRMG
7133




GEIATSGSETPGT



 



RSN-0004
GSAPGGPGGYALLRMG
7134




GPAATSGSETPGT



 



RSN-0005
GSAPGEAGGYAFLRMG
7135




GSIATSGSETPGT



 



RSN-0006
GSAPGPGGGYASLRMG
7136




GTAATSGSETPGT



 



RSN-0007
GSAPGSEGGYATLRMG
7137




GAIATSGSETPGT



 



RSN-0008
GSAPGTPGGYANLRMG
7138




GGAATSGSETPGT



 



RSN-0009
GSAPGASGGYAHLRMG
7139




GEIATSGSETPGT



 



RSN-0010
GSAPGGTGGYGELRMG
7140




GPAATSGSETPGT



 



RSN-0011
GSAPGEAGGYPELRMG
7141




GSIATSGSETPGT



 



RSN-0012
GSAPGPGGGYVELRMG
7142




GTAATSGSETPGT



 



RSN-0013
GSAPGSEGGYLELRMG
7143




GAIATSGSETPGT



 



RSN-0014
GSAPGTPGGYSELRMG
7144




GGAATSGSETPGT



 



RSN-0015
GSAPGASGGYTELRMG
7145




GEIATSGSETPGT



 



RSN-0016
GSAPGGTGGYQELRMG
7146




GPAATSGSETPGT



 



RSN-0017
GSAPGEAGGYEELRMG
7147




GSIATSGSETPGT



 



RSN-0018
GSAPGPGIGPAELRMGG
7148




TAATSGSETPGT



 



RSN-0019
GSAPGSEIGAAELRMG
7149




GAIATSGSETPGT



 



RSN-0020
GSAPGTPIGSAELRMGG
7150




GAATSGSETPGT



 



RSN-0021
GSAPGASIGTAELRMG
7151




GEIATSGSETPGT



 



RSN-0022
GSAPGGTIGNAELRMG
7152




GPAATSGSETPGT



 



RSN-0023
GSAPGEAIGQAELRMG
7153




GSIATSGSETPGT



 



RSN-0024
GSAPGPGGPYAELRMG
7154




GTAATSGSETPGT



 



RSN-0025
GSAPGSEGAYAELRMG
7155




GAIATSGSETPGT



 



RSN-0026
GSAPGTPGVYAELRMG
7156




GGAATSGSETPGT



 



RSN-0027
GSAPGASGLYAELRMG
7157




GEIATSGSETPGT



 



RSN-0028
GSAPGGTGIYAELRMG
7158




GPAATSGSETPGT



 



RSN-0029
GSAPGEAGFYAELRMG
7159




GSIATSGSETPGT



 



RSN-0030
GSAPGPGGYYAELRMG
7160




GTAATSGSETPGT



 



RSN-0031
GSAPGSEGSYAELRMG
7161




GAIATSGSETPGT



 



RSN-0032
GSAPGTPGNYAELRMG
7162




GGAATSGSETPGT



 



RSN-0033
GSAPGASGEYAELRMG
7163




GEIATSGSETPGT



 



RSN-0034
GSAPGGTGHYAELRMG
7164




GPAATSGSETPGT



 



RSN-0035
GSAPGEAGGYAEARMG
7165




GSIATSGSETPGT



 



RSN-0036
GSAPGPGGGYAEVRMG
7166




GTAATSGSETPGT



 



RSN-0037
GSAPGSEGGYAEIRMG
7167




GAIATSGSETPGT



 



RSN-0038
GSAPGTPGGYAEFRMG
7168




GGAATSGSETPGT



 



RSN-0039
GSAPGASGGYAEYRMG
7169




GEIATSGSETPGT



 



RSN-0040
GSAPGGTGGYAESRMG
7170




GPAATSGSETPGT



 



RSN-0041
GSAPGEAGGYAETRMG
7171




GSIATSGSETPGT



 



RSN-0042
GSAPGPGGGYAELAMG
7172




GTRATSGSETPGT



 



RSN-0043
GSAPGSEGGYAELVMG
7173




GARATSGSETPGT



 



RSN-0044
GSAPGTPGGYAELLMG
7174




GGRATSGSETPGT



 



RSN-0045
GSAPGASGGYAELIMG
7175




GERATSGSETPGT



 



RSN-0046
GSAPGGTGGYAELWM
7176




GGPRATSGSETPGT



 



RSN-0047
GSAPGEAGGYAELSMG
7177




GSRATSGSETPGT



 



RSN-0048
GSAPGPGGGYAELTMG
7178




GTRATSGSETPGT



 



RSN-0049
GSAPGSEGGYAELQMG
7179




GARATSGSETPGT



 



RSN-0050
GSAPGTPGGYAELNMG
7180




GGRATSGSETPGT



 



RSN-0051
GSAPGASGGYAELEMG
7181




GERATSGSETPGT



 



RSN-0052
GSAPGGTGGYAELRPG
7182




GPIATSGSETPGT



 



RSN-0053
GSAPGEAGGYAELRAG
7183




GSAATSGSETPGT



 



RSN-0054
GSAPGPGGGYAELRLG
7184




GTIATSGSETPGT



 



RSN-0055
GSAPGSEGGYAELRIGG
7185




AAATSGSETPGT



 



RSN-0056
GSAPGTPGGYAELRSG
7186




GGIATSGSETPGT



 



RSN-0057
GSAPGASGGYAELRNG
7187




GEAATSGSETPGT



 



RSN-0058
GSAPGGTGGYAELRQG
7188




GPIATSGSETPGT



 



RSN-0059
GSAPGEAGGYAELRDG
7189




GSAATSGSETPGT



 



RSN-0060
GSAPGPGGGYAELREG
7190




GTIATSGSETPGT



 



RSN-0061
GSAPGSEGGYAELRHG
7191




GAAATSGSETPGT



 



RSN-0062
GSAPGTPGGYAELRMP
7192




GGIATSGSETPGT



 



RSN-0063
GSAPGASGGYAELRMA
7193




GEAATSGSETPGT



 



RSN-0064
GSAPGGTGGYAELRMV
7194




GPIATSGSETPGT



 



RSN-0065
GSAPGEAGGYAELRML
7195




GSAATSGSETPGT



 



RSN-0066
GSAPGPGGGYAELRMI
7196




GTIATSGSETPGT



 



RSN-0067
GSAPGSEGGYAELRMY
7197




GAIATSGSETPGT



 



RSN-0068
GSAPGTPGGYAELRMS
7198




GGAATSGSETPGT



 



RSN-0069
GSAPGASGGYAELRMN
7199




GEIATSGSETPGT



 



RSN-0070
GSAPGGTGGYAELRMQ
7200




GPAATSGSETPGT



 



RSN-0071
GSAPGANHTPAGLTGP
7201




GARATSGSETPGT



 



RSN-0072
GSAPGANTAPEGLTGPS
7202




TRATSGSETPGT



 



RSN-0073
GSAPGTGAPPGGLTGPG
7203




TRATSGSETPGT



 



RSN-0074
GSAPGANHEPSGLTEGS
7204




PRATSGSETPGT



 



RSN-0075
GSAPGANTEPPELGAGT
7205




ERATSGSETPGT



 



RSN-0076
GSAPGASGPPPGLTGPP
7206




GRATSGSETPGT



 



RSN-0077
GSAPGASGTPAPLGGEP
7207




GRATSGSETPGT



 



RSN-0078
GSAPGPAGPPEGLETEA
7208




GRATSGSETPGT



 



RSN-0079
GSAPGPTSGQGGLTGPE
7209




SRATSGSETPGT



 



RSN-0080
GSAPGSAGGAANLVRG
7210




GAIATSGSETPGT



 



RSN-0081
GSAPGTGGGAAPLVRG
7211




GGAATSGSETPGT



 



RSN-0082
GSAPGAEGGAAALVRG
7212




GEIATSGSETPGT



 



RSN-0083
GSAPGGPGGAALLVRG
7213




GPAATSGSETPGT



 



RSN-0084
GSAPGEAGGAAFLVRG
7214




GSIATSGSETPGT



 



RSN-0085
GSAPGPGGGAASLVRG
7215




GTAATSGSETPGT



 



RSN-0086
GSAPGSEGGAATLVRG
7216




GAIATSGSETPGT



 



RSN-0087
GSAPGTPGGAAGLVRG
7217




GGAATSGSETPGT



 



RSN-0088
GSAPGASGGAADLVRG
7218




GEIATSGSETPGT



 



RSN-0089
GSAPGGTGGAGNLVRG
7219




GPAATSGSETPGT



 



RSN-0090
GSAPGEAGGAPNLVRG
7220




GSIATSGSETPGT



 



RSN-0091
GSAPGPGGGAVNLVRG
7221




GTAATSGSETPGT



 



RSN-0092
GSAPGSEGGALNLVRG
7222




GAIATSGSETPGT



 



RSN-0093
GSAPGTPGGASNLVRG
7223




GGAATSGSETPGT



 



RSN-0094
GSAPGASGGATNLVRG
7224




GEIATSGSETPGT



 



RSN-0095
GSAPGGTGGAQNLVRG
7225




GPAATSGSETPGT



 



RSN-0096
GSAPGEAGGAENLVRG
7226




GSIATSGSETPGT



 



RSN-1517
GSAPEAGRSANHEPLGL
7227




VATATSGSETPGT



 



BSRS-A1-2
GSAPASGRSTNAGPSGL
7228




AGPATSGSETPGT



 



BSRS-A2-2
GSAPASGRSTNAGPQG
7229




LAGQATSGSETPGT



 



BSRS-A3-2
GSAPASGRSTNAGPPGL
7230




TGPATSGSETPGT



 



VP-1
GSAPASSRGTNAGPAG
7231




LTGPATSGSETPGT



 



RSN-1752
GSAPASSRTTNTGPSTL
7232




TGPATSGSETPGT



 



RSN-1512
GSAPAAGRSDNGTPLEL
7233




VAPATSGSETPGT



 



RSN-1517
GSAPEAGRSANHEPLGL
7234




VATATSGSETPGT



 



VP-2
GSAPASGRGTNAGPAG
7235




LTGPATSGSETPGT



 



RSN-1018
GSAPLFGRNDNHEPLEL
7236




GGGATSGSETPGT



 



RSN-1053
GSAPTAGRSDNLEPLGL
7237




VFGATSGSETPGT



 



RSN-1059
GSAPLDGRSDNFHPPEL
7238




VAGATSGSETPGT



 



RSN-1065
GSAPLEGRSDNEEPENL
7239




VAGATSGSETPGT



 



RSN-1167
GSAPLKGRSDNNAPLA
7240




LVAGATSGSETPGT



 



RSN-1201
GSAPVYSRGTNAGPHG
7241




LTGRATSGSETPGT



 



RSN-1218
GSAPANSRGTNKGFAG
7242




LIGPATSGSETPGT



 



RSN-1226
GSAPASSRLTNEAPAGL
7243




TIPATSGSETPGT



 



RSN-1254
GSAPDQSRGTNAGPEG
7244




LTDPATSGSETPGT



 



RSN-1256
GSAPESSRGTNIGQGGL
7245




TGPATSGSETPGT



 



RSN-1261
GSAPSSSRGTNQDPAGL
7246




TIPATSGSETPGT



 



RSN-1293
GSAPASSRGQNHSPMG
7247




LTGPATSGSETPGT



 



RSN-1309
GSAPAYSRGPNAGPAG
7248




LEGRATSGSETPGT



 



RSN-1326
GSAPASERGNNAGPAN
7249




LTGFATSGSETPGT



 



RSN-1345
GSAPASHRGTNPKPAIL
7250




TGPATSGSETPGT



 



RSN-1354
GSAPMSSRRTNANPAQ
7251




LTGPATSGSETPGT



 



RSN-1426
GSAPGAGRTDNHEPLE
7252




LGAAATSGSETPGT



 



RSN-1478
GSAPLAGRSENTAPLEL
7253




TAGATSGSETPGT



 



RSN-1479
GSAPLEGRPDNHEPLAL
7254




VASATSGSETPGT



 



RSN-1496
GSAPLSGRSDNEEPLAL
7255




PAGATSGSETPGT



 



RSN-1508
GSAPEAGRTDNHEPLEL
7256




SAPATSGSETPGT



 



RSN-1513
GSAPEGGRSDNHGPLEL
7257




VSGATSGSETPGT



 



RSN-1516
GSAPLSGRSDNEAPLEL
7258




EAGATSGSETPGT



 



RSN-1524
GSAPLGGRADNHEPPEL
7259




GAGATSGSETPGT



 



RSN-1622
GSAPPPSRGTNAEPAGL
7260




TGEATSGSETPGT



 



RSN-1629
GSAPASTRGENAGPAG
7261




LEAPATSGSETPGT



 



RSN-1664
GSAPESSRGTNGAPEGL
7262




TGPATSGSETPGT



 



RSN-1667
GSAPASSRATNESPAGL
7263




TGEATSGSETPGT



 



RSN-1709
GSAPASSRGENPPPGGL
7264




TGPATSGSETPGT



 



RSN-1712
GSAPAASRGTNTGPAEL
7265




TGSATSGSETPGT



 



RSN-1727
GSAPAGSRTTNAGPGG
7266




LEGPATSGSETPGT



 



RSN-1754
GSAPAPSRGENAGPATL
7267




TGAATSGSETPGT



 



RSN-1819
GSAPESGRAANTGPPTL
7268




TAPATSGSETPGT



 



RSN-1832
GSAPNPGRAANEGPPG
7269




LPGSATSGSETPGT



 



RSN-1855
GSAPESSRAANLTPPEL
7270




TGPATSGSETPGT



 



RSN-1911
GSAPASGRAANETPPGL
7271




TGAATSGSETPGT



 



RSN-1929
GSAPNSGRGENLGAPG
7272




LTGTATSGSETPGT



 



RSN-1951
GSAPTTGRAANLTPAG
7273




LTGPATSGSETPGT



 



RSN-2295
GSAPEAGRSANHTPAG
7274




LTGPATSGSETPGT



 



RSN-2298
GSAPESGRAANTTPAGL
7275




TGPATSGSETPGT



 



RSN-2038
GSAPTTGRATEAANLTP
7276




AGLTGPATSGSETPGT



 



RSN-2072
GSAPTTGRAEEAANLTP
7277




AGLTGPATSGSETPGT



 



RSN-2089
GSAPTTGRAGEAANLT
7278




PAGLTGPATSGSETPGT



 



RSN-2302
GSAPTTGRATEAANAT
7279




PAGLTGPATSGSETPGT



 



RSN-3047
GSAPTTGRAGEAEGAT
7280




SAGATGPATSGSETPGT



 



RSN-3052
GSAPTTGEAGEAANAT
7281




SAGATGPATSGSETPGT



 



RSN-3043
GSAPTTGEAGEAAGLTP
7282




AGLTGPATSGSETPGT



 



RSN-3041
GSAPTTGAAGEAANAT
7283




PAGLTGPATSGSETPGT



 



RSN-3044
GSAPTTGRAGEAAGLT
7284




PAGLTGPATSGSETPGT



 



RSN-3057
GSAPTTGRAGEAANAT
7285




SAGATGPATSGSETPGT



 



RSN-3058
GSAPTTGEAGEAAGAT
7286




SAGATGPATSGSETPGT



 



RSN-2485
GSAPESGRAANTEPPEL
7287




GAGATSGSETPGT



 



RSN-2486
GSAPESGRAANTAPEGL
7288




TGPATSGSETPGT



 



RSN-2488
GSAPEPGRAANHEPSGL
7289




TEGATSGSETPGT



 



RSN-2599
GSAPESGRAANHTGAP
7290




PGGLTGPATSGSETPGT



 



RSN-2706
GSAPTTGRTGEGANAT
7291




PGGLTGPATSGSETPGT



 



RSN-2707
GSAPRTGRSGEAANETP
7292




EGLEGPATSGSETPGT



 



RSN-2708
GSAPRTGRTGESANETP
7293




AGLGGPATSGSETPGT



 



RSN-2709
GSAPSTGRTGEPANETP
7294




AGLSGPATSGSETPGT



 



RSN-2710
GSAPTTGRAGEPANATP
7295




TGLSGPATSGSETPGT



 



RSN-2711
GSAPRTGRPGEGANAT
7296




PTGLPGPATSGSETPGT



 



RSN-2712
GSAPRTGRGGEAANAT
7297




PSGLGGPATSGSETPGT



 



RSN-2713
GSAPSTGRSGESANATP
7298




GGLGGPATSGSETPGT



 



RSN-2714
GSAPRTGRTGEEANATP
7299




AGLPGPATSGSETPGT



 



RSN-2715
GSAPATGRPGEPANTTP
7300




EGLEGPATSGSETPGT



 



RSN-2716
GSAPSTGRSGEPANATP
7301




GGLTGPATSGSETPGT



 



RSN-2717
GSAPPTGRGGEGANTTP
7302




TGLPGPATSGSETPGT



 



RSN-2718
GSAPPTGRSGEGANATP
7303




SGLTGPATSGSETPGT



 



RSN-2719
GSAPTTGRASEGANSTP
7304




APLTEPATSGSETPGT



 



RSN-2720
GSAPTYGRAAEAANTT
7305




PAGLTAPATSGSETPGT



 



RSN-2721
GSAPTTGRATEGANAT
7306




PAELTEPATSGSETPGT



 



RSN-2722
GSAPTVGRASEEANTTP
7307




ASLTGPATSGSETPGT



 



RSN-2723
GSAPTTGRAPEAANATP
7308




APLTGPATSGSETPGT



 



RSN-2724
GSAPTWGRATEPANAT
7309




PAPLTSPATSGSETPGT



 



RSN-2725
GSAPTVGRASESANATP
7310




AELTSPATSGSETPGT



 



RSN-2726
GSAPTVGRAPEGANSTP
7311




AGLTGPATSGSETPGT



 



RSN-2727
GSAPTWGRATEAPNLE
7312




PATLTTPATSGSETPGT



 



RSN-2728
GSAPTTGRATEAPNLTP
7313




APLTEPATSGSETPGT



 



RSN-2729
GSAPTQGRATEAPNLSP
7314




AALTSPATSGSETPGT



 



RSN-2730
GSAPTQGRAAEAPNLTP
7315




ATLTAPATSGSETPGT



 



RSN-2731
GSAPTSGRAPEATNLAP
7316




APLTGPATSGSETPGT



 



RSN-2732
GSAPTQGRAAEAANLT
7317




PAGLTEPATSGSETPGT



 



RSN-2733
GSAPTTGRAGSAPNLPP
7318




TGLTTPATSGSETPGT



 



RSN-2734
GSAPTTGRAGGAENLPP
7319




EGLTAPATSGSETPGT



 



RSN-2735
GSAPTTSRAGTATNLTP
7320




EGLTAPATSGSETPGT



 



RSN-2736
GSAPTTGRAGTATNLPP
7321




SGLTTPATSGSETPGT



 



RSN-2737
GSAPTTARAGEAENLSP
7322




SGLTAPATSGSETPGT



 



RSN-2738
GSAPTTGRAGGAGNLA
7323




PGGLTEPATSGSETPGT



 



RSN-2739
GSAPTTGRAGTATNLPP
7324




EGLTGPATSGSETPGT



 



RSN-2740
GSAPTTGRAGGAANLA
7325




PTGLTEPATSGSETPGT



 



RSN-2741
GSAPTTGRAGTAENLA
7326




PSGLTTPATSGSETPGT



 



RSN-2742
GSAPTTGRAGSATNLGP
7327




GGLTGPATSGSETPGT



 



RSN-2743
GSAPTTARAGGAENLT
7328




PAGLTEPATSGSETPGT



 



RSN-2744
GSAPTTARAGSAENLSP
7329




SGLTGPATSGSETPGT



 



RSN-2745
GSAPTTARAGGAGNLA
7330




PEGLTTPATSGSETPGT



 



RSN-2746
GSAPTTSRAGAAENLTP
7331




TGLTGPATSGSETPGT



 



RSN-2747
GSAPTYGRTTTPGNEPP
7332




ASLEAEATSGSETPGT



 



RSN-2748
GSAPTYSRGESGPNEPP
7333




PGLTGPATSGSETPGT



 



RSN-2749
GSAPAWGRTGASENET
7334




PAPLGGEATSGSETPGT



 



RSN-2750
GSAPRWGRAETTPNTPP
7335




EGLETEATSGSETPGT



 



RSN-2751
GSAPESGRAANHTGAE
7336




PPELGAGATSGSETPGT



 



RSN-2754
GSAPTTGRAGEAANLT
7337




PAGLTESATSGSETPGT



 



RSN-2755
GSAPTTGRAGEAANLT
7338




PAALTESATSGSETPGT



 



RSN-2756
GSAPTTGRAGEAANLT
7339




PAPLTESATSGSETPGT



 



RSN-2757
GSAPTTGRAGEAANLT
7340




PEPLTESATSGSETPGT



 



RSN-2758
GSAPTTGRAGEAANLT
7341




PAGLTGAATSGSETPGT



 



RSN-2759
GSAPTTGRAGEAANLT
7342




PEGLTGAATSGSETPGT



 



RSN-2760
GSAPTTGRAGEAANLT
7343




PEPLTGAATSGSETPGT



 



RSN-2761
GSAPTTGRAGEAANLT
7344




PAGLTEAATSGSETPGT



 



RSN-2762
GSAPTTGRAGEAANLT
7345




PEGLTEAATSGSETPGT



 



RSN-2763
GSAPTTGRAGEAANLT
7346




PAPLTEAATSGSETPGT



 



RSN-2764
GSAPTTGRAGEAANLT
7347




PEPLTEAATSGSETPGT



 



RSN-2765
GSAPTTGRAGEAANLT
7348




PEPLTGPATSGSETPGT



 



RSN-2766
GSAPTTGRAGEAANLT
7349




PAGLTGGATSGSETPGT



 



RSN-2767
GSAPTTGRAGEAANLT
7350




PEGLTGGATSGSETPGT



 



RSN-2768
GSAPTTGRAGEAANLT
7351




PEALTGGATSGSETPGT



 



RSN-2769
GSAPTTGRAGEAANLT
7352




PEPLTGGATSGSETPGT



 



RSN-2770
GSAPTTGRAGEAANLT
7353




PAGLTEGATSGSETPGT



 



RSN-2771
GSAPTTGRAGEAANLT
7354




PEGLTEGATSGSETPGT



 



RSN-2772
GSAPTTGRAGEAANLT
7355




PAPLTEGATSGSETPGT



 



RSN-2773
GSAPTTGRAGEAANLT
7356




PEPLTEGATSGSETPGT



 



RSN-3047
GSAPTTGRAGEAEGAT
7357




SAGATGPATSGSETPGT



 



RSN-2783
GSAPEAGRSAEATSAG
7358




ATGPATSGSETPGT



 



RSN-3107
GSAPSASGTYSRGESGP
7359




GSPATSGSETPGT



 



RSN-3103
GSAPSASGEAGRTDTHP
7360




GSPATSGSETPGT



 



RSN-3102
GSAPSASGEPGRAAEHP
7361




GSPATSGSETPGT



 



RSN-3119
GSAPSPAGESSRGTTIA
7362




GSPATSGSETPGT



 



RSN-3043
GSAPTTGEAGEAAGLTP
7363




AGLTGPATSGSETPGT



 



RSN-2789
GSAPEAGESAGATPAG
7364




LTGPATSGSETPGT



 



RSN-3109
GSAPSASGAPLELEAGP
7365




GSPATSGSETPGT



 



RSN-3110
GSAPSASGEPPELGAGP
7366




GSPATSGSETPGT



 



RSN-3111
GSAPSASGEPSGLTEGP
7367




GSPATSGSETPGT



 



RSN-3112
GSAPSASGTPAPLTEPP
7368




GSPATSGSETPGT



 



RSN-3113
GSAPSASGTPAELTEPP
7369




GSPATSGSETPGT



 



RSN-3114
GSAPSASGPPPGLTGPP
7370




GSPATSGSETPGT



 



RSN-3115
GSAPSASGTPAPLGGEP
7371




GSPATSGSETPGT



 



RSN-3125
GSAPSPAGAPEGLTGPA
7372




GSPATSGSETPGT



 



RSN-3126
GSAPSPAGPPEGLETEA
7373




GSPATSGSETPGT



 



RSN-3127
GSAPSPTSGQGGLTGPG
7374




SEPATSGSETPGT



 



RSN-3131
GSAPSESAPPEGLETEST
7375




EPATSGSETPGT



 



RSN-3132
GSAPSEGSEPLELGAAS
7376




ETPATSGSETPGT



 



RSN-3133
GSAPSEGSGPAGLEAPS
7377




ETPATSGSETPGT



 



RSN-3138
GSAPSEPTPPASLEAEPG
7378




SPATSGSETPGT



 



RSC-0001
GTAEAASASGGSAGGY
7379




AELRMGGAIPGSP



 



RSC-0002
GTAEAASASGGTGGGY
7380




APLRMGGGAPGSP



 



RSC-0003
GTAEAASASGGAEGGY
7381




AALRMGGEIPGSP



 



RSC-0004
GTAEAASASGGGPGGY
7382




ALLRMGGPAPGSP



 



RSC-0005
GTAEAASASGGEAGGY
7383




AFLRMGGSIPGSP



 



RSC-0006
GTAEAASASGGPGGGY
7384




ASLRMGGTAPGSP



 



RSC-0007
GTAEAASASGGSEGGY
7385




ATLRMGGAIPGSP



 



RSC-0008
GTAEAASASGGTPGGY
7386




ANLRMGGGAPGSP



 



RSC-0009
GTAEAASASGGASGGY
7387




AHLRMGGEIPGSP



 



RSC-0010
GTAEAASASGGGTGGY
7388




GELRMGGPAPGSP



 



RSC-0011
GTAEAASASGGEAGGY
7389




PELRMGGSIPGSP



 



RSC-0012
GTAEAASASGGPGGGY
7390




VELRMGGTAPGSP



 



RSC-0013
GTAEAASASGGSEGGY
7391




LELRMGGAIPGSP



 



RSC-0014
GTAEAASASGGTPGGY
7392




SELRMGGGAPGSP



 



RSC-0015
GTAEAASASGGASGGY
7393




TELRMGGEIPGSP



 



RSC-0016
GTAEAASASGGGTGGY
7394




QELRMGGPAPGSP



 



RSC-0017
GTAEAASASGGEAGGY
7395




EELRMGGSIPGSP



 



RSC-0018
GTAEAASASGGPGIGPA
7396




ELRMGGTAPGSP



 



RSC-0019
GTAEAASASGGSEIGAA
7397




ELRMGGAIPGSP



 



RSC-0020
GTAEAASASGGTPIGSA
7398




ELRMGGGAPGSP



 



RSC-0021
GTAEAASASGGASIGTA
7399




ELRMGGEIPGSP



 



RSC-0022
GTAEAASASGGGTIGN
7400




AELRMGGPAPGSP



 



RSC-0023
GTAEAASASGGEAIGQ
7401




AELRMGGSIPGSP



 



RSC-0024
GTAEAASASGGPGGPY
7402




AELRMGGTAPGSP



 



RSC-0025
GTAEAASASGGSEGAY
7403




AELRMGGAIPGSP



 



RSC-0026
GTAEAASASGGTPGVY
7404




AELRMGGGAPGSP



 



RSC-0027
GTAEAASASGGASGLY
7405




AELRMGGEIPGSP



 



RSC-0028
GTAEAASASGGGTGIY
7406




AELRMGGPAPGSP



 



RSC-0029
GTAEAASASGGEAGFY
7407




AELRMGGSIPGSP



 



RSC-0030
GTAEAASASGGPGGYY
7408




AELRMGGTAPGSP



 



RSC-0031
GTAEAASASGGSEGSY
7409




AELRMGGAIPGSP



 



RSC-0032
GTAEAASASGGTPGNY
7410




AELRMGGGAPGSP



 



RSC-0033
GTAEAASASGGASGEY
7411




AELRMGGEIPGSP



 



RSC-0034
GTAEAASASGGGTGHY
7412




AELRMGGPAPGSP



 



RSC-0035
GTAEAASASGGEAGGY
7413




AEARMGGSIPGSP



 



RSC-0036
GTAEAASASGGPGGGY
7414




AEVRMGGTAPGSP



 



RSC-0037
GTAEAASASGGSEGGY
7415




AEIRMGGAIPGSP



 



RSC-0038
GTAEAASASGGTPGGY
7416




AEFRMGGGAPGSP



 



RSC-0039
GTAEAASASGGASGGY
7417




AEYRMGGEIPGSP



 



RSC-0040
GTAEAASASGGGTGGY
7418




AESRMGGPAPGSP



 



RSC-0041
GTAEAASASGGEAGGY
7419




AETRMGGSIPGSP



 



RSC-0042
GTAEAASASGGPGGGY
7420




AELAMGGTRPGSP



 



RSC-0043
GTAEAASASGGSEGGY
7421




AELVMGGARPGSP



 



RSC-0044
GTAEAASASGGTPGGY
7422




AELLMGGGRPGSP



 



RSC-0045
GTAEAASASGGASGGY
7423




AELIMGGERPGSP



 



RSC-0046
GTAEAASASGGGTGGY
7424




AELWMGGPRPGSP



 



RSC-0047
GTAEAASASGGEAGGY
7425




AELSMGGSRPGSP



 



RSC-0048
GTAEAASASGGPGGGY
7426




AELTMGGTRPGSP



 



RSC-0049
GTAEAASASGGSEGGY
7427




AELQMGGARPGSP



 



RSC-0050
GTAEAASASGGTPGGY
7428




AELNMGGGRPGSP



 



RSC-0051
GTAEAASASGGASGGY
7429




AELEMGGERPGSP



 



RSC-0052
GTAEAASASGGGTGGY
7430




AELRPGGPIPGSP



 



RSC-0053
GTAEAASASGGEAGGY
7431




AELRAGGSAPGSP



 



RSC-0054
GTAEAASASGGPGGGY
7432




AELRLGGTIPGSP



 



RSC-0055
GTAEAASASGGSEGGY
7433




AELRIGGAAPGSP



 



RSC-0056
GTAEAASASGGTPGGY
7434




AELRSGGGIPGSP



 



RSC-0057
GTAEAASASGGASGGY
7435




AELRNGGEAPGSP



 



RSC-0058
GTAEAASASGGGTGGY
7436




AELRQGGPIPGSP



 



RSC-0059
GTAEAASASGGEAGGY
7437




AELRDGGSAPGSP



 



RSC-0060
GTAEAASASGGPGGGY
7438




AELREGGTIPGSP



 



RSC-0061
GTAEAASASGGSEGGY
7439




AELRHGGAAPGSP



 



RSC-0062
GTAEAASASGGTPGGY
7440




AELRMPGGIPGSP



 



RSC-0063
GTAEAASASGGASGGY
7441




AELRMAGEAPGSP



 



RSC-0064
GTAEAASASGGGTGGY
7442




AELRMVGPIPGSP



 



RSC-0065
GTAEAASASGGEAGGY
7443




AELRMLGSAPGSP



 



RSC-0066
GTAEAASASGGPGGGY
7444




AELRMIGTIPGSP



 



RSC-0067
GTAEAASASGGSEGGY
7445




AELRMYGAIPGSP



 



RSC-0068
GTAEAASASGGTPGGY
7446




AELRMSGGAPGSP



 



RSC-0069
GTAEAASASGGASGGY
7447




AELRMNGEIPGSP



 



RSC-0070
GTAEAASASGGGTGGY
7448




AELRMQGPAPGSP



 



RSC-0071
GTAEAASASGGANHTP
7449




AGLTGPGARPGSP



 



RSC-0072
GTAEAASASGGANTAP
7450




EGLTGPSTRPGSP



 



RSC-0073
GTAEAASASGGTGAPP
7451




GGLTGPGTRPGSP



 



RSC-0074
GTAEAASASGGANHEP
7452




SGLTEGSPRPGSP



 



RSC-0075
GTAEAASASGGANTEP
7453




PELGAGTERPGSP



 



RSC-0076
GTAEAASASGGASGPPP
7454




GLTGPPGRPGSP



 



RSC-0077
GTAEAASASGGASGTP
7455




APLGGEPGRPGSP



 



RSC-0078
GTAEAASASGGPAGPPE
7456




GLETEAGRPGSP



 



RSC-0079
GTAEAASASGGPTSGQ
7457




GGLTGPESRPGSP



 



RSC-0080
GTAEAASASGGSAGGA
7458




ANLVRGGAIPGSP



 



RSC-0081
GTAEAASASGGTGGGA
7459




APLVRGGGAPGSP



 



RSC-0082
GTAEAASASGGAEGGA
7460




AALVRGGEIPGSP



 



RSC-0083
GTAEAASASGGGPGGA
7461




ALLVRGGPAPGSP



 



RSC-0084
GTAEAASASGGEAGGA
7462




AFLVRGGSIPGSP



 



RSC-0085
GTAEAASASGGPGGGA
7463




ASLVRGGTAPGSP



 



RSC-0086
GTAEAASASGGSEGGA
7464




ATLVRGGAIPGSP



 



RSC-0087
GTAEAASASGGTPGGA
7465




AGLVRGGGAPGSP



 



RSC-0088
GTAEAASASGGASGGA
7466




ADLVRGGEIPGSP



 



RSC-0089
GTAEAASASGGGTGGA
7467




GNLVRGGPAPGSP



 



RSC-0090
GTAEAASASGGEAGGA
7468




PNLVRGGSIPGSP



 



RSC-0091
GTAEAASASGGPGGGA
7469




VNLVRGGTAPGSP



 



RSC-0092
GTAEAASASGGSEGGA
7470




LNLVRGGAIPGSP



 



RSC-0093
GTAEAASASGGTPGGA
7471




SNLVRGGGAPGSP



 



RSC-0094
GTAEAASASGGASGGA
7472




TNLVRGGEIPGSP



 



RSC-0095
GTAEAASASGGGTGGA
7473




QNLVRGGPAPGSP



 



RSC-0096
GTAEAASASGGEAGGA
7474




ENLVRGGSIPGSP



 



RSC-1517
GTAEAASASGEAGRSA
7475




NHEPLGLVATPGSP



 



BSRS-A1-3
GTAEAASASGASGRST
7476




NAGPSGLAGPPGSP



 



BSRS-A2-3
GTAEAASASGASGRST
7477




NAGPQGLAGQPGSP



 



BSRS-A3-3
GTAEAASASGASGRST
7478




NAGPPGLTGPPGSP



 



VP-1
GTAEAASASGASSRGT
7479




NAGPAGLTGPPGSP



 



RSC-1752
GTAEAASASGASSRTTN
7480




TGPSTLTGPPGSP



 



RSC-1512
GTAEAASASGAAGRSD
7481




NGTPLELVAPPGSP



 



RSC-1517
GTAEAASASGEAGRSA
7482




NHEPLGLVATPGSP



 



VP-2
GTAEAASASGASGRGT
7483




NAGPAGLTGPPGSP



 



RSC-1018
GTAEAASASGLFGRND
7484




NHEPLELGGGPGSP



 



RSC-1053
GTAEAASASGTAGRSD
7485




NLEPLGLVFGPGSP



 



RSC-1059
GTAEAASASGLDGRSD
7486




NFHPPELVAGPGSP



 



RSC-1065
GTAEAASASGLEGRSD
7487




NEEPENLVAGPGSP



 



RSC-1167
GTAEAASASGLKGRSD
7488




NNAPLALVAGPGSP



 



RSC-1201
GTAEAASASGVYSRGT
7489




NAGPHGLTGRPGSP



 



RSC-1218
GTAEAASASGANSRGT
7490




NKGFAGLIGPPGSP



 



RSC-1226
GTAEAASASGASSRLTN
7491




EAPAGLTIPPGSP



 



RSC-1254
GTAEAASASGDQSRGT
7492




NAGPEGLTDPPGSP



 



RSC-1256
GTAEAASASGESSRGTN
7493




IGQGGLTGPPGSP



 



RSC-1261
GTAEAASASGSSSRGTN
7494




QDPAGLTIPPGSP



 



RSC-1293
GTAEAASASGASSRGQ
7495




NHSPMGLTGPPGSP



 



RSC-1309
GTAEAASASGAYSRGP
7496




NAGPAGLEGRPGSP



 



RSC-1326
GTAEAASASGASERGN
7497




NAGPANLTGFPGSP



 



RSC-1345
GTAEAASASGASHRGT
7498




NPKPAILTGPPGSP



 



RSC-1354
GTAEAASASGMSSRRT
7499




NANPAQLTGPPGSP



 



RSC-1426
GTAEAASASGGAGRTD
7500




NHEPLELGAAPGSP



 



RSC-1478
GTAEAASASGLAGRSE
7501




NTAPLELTAGPGSP



 



RSC-1479
GTAEAASASGLEGRPD
7502




NHEPLALVASPGSP



 



RSC-1496
GTAEAASASGLSGRSD
7503




NEEPLALPAGPGSP



 



RSC-1508
GTAEAASASGEAGRTD
7504




NHEPLELSAPPGSP



 



RSC-1513
GTAEAASASGEGGRSD
7505




NHGPLELVSGPGSP



 



RSC-1516
GTAEAASASGLSGRSD
7506




NEAPLELEAGPGSP



 



RSC-1524
GTAEAASASGLGGRAD
7507




NHEPPELGAGPGSP



 



RSC-1622
GTAEAASASGPPSRGTN
7508




AEPAGLTGEPGSP



 



RSC-1629
GTAEAASASGASTRGE
7509




NAGPAGLEAPPGSP



 



RSC-1664
GTAEAASASGESSRGTN
7510




GAPEGLTGPPGSP



 



RSC-1667
GTAEAASASGASSRAT
7511




NESPAGLTGEPGSP



 



RSC-1709
GTAEAASASGASSRGE
7512




NPPPGGLTGPPGSP



 



RSC-1712
GTAEAASASGAASRGT
7513




NTGPAELTGSPGSP



 



RSC-1727
GTAEAASASGAGSRTT
7514




NAGPGGLEGPPGSP



 



RSC-1754
GTAEAASASGAPSRGE
7515




NAGPATLTGAPGSP



 



RSC-1819
GTAEAASASGESGRAA
7516




NTGPPTLTAPPGSP



 



RSC-1832
GTAEAASASGNPGRAA
7517




NEGPPGLPGSPGSP



 



RSC-1855
GTAEAASASGESSRAA
7518




NLTPPELTGPPGSP



 



RSC-1911
GTAEAASASGASGRAA
7519




NETPPGLTGAPGSP



 



RSC-1929
GTAEAASASGNSGRGE
7520




NLGAPGLTGTPGSP



 



RSC-1951
GTAEAASASGTTGRAA
7521




NLTPAGLTGPPGSP



 



RSC-2295
GTAEAASASGEAGRSA
7522




NHTPAGLTGPPGSP



 



RSC-2298
GTAEAASASGESGRAA
7523




NTTPAGLTGPPGSP



 



RSC-2038
GTAEAASASGTTGRAT
7524




EAANLTPAGLTGPPGSP



 



RSC-2072
GTAEAASASGTTGRAE
7525




EAANLTPAGLTGPPGSP



 



RSC-2089
GTAEAASASGTTGRAG
7526




EAANLTPAGLTGPPGSP



 



RSC-2302
GTAEAASASGTTGRAT
7527




EAANATPAGLTGPPGSP



 



RSC-3047
GTAEAASASGTTGRAG
7528




EAEGATSAGATGPPGSP



 



RSC-3052
GTAEAASASGTTGEAG
7529




EAANATSAGATGPPGSP



 



RSC-3043
GTAEAASASGTTGEAG
7530




EAAGLTPAGLTGPPGSP



 



RSC-3041
GTAEAASASGTTGAAG
7531




EAANATPAGLTGPPGSP



 



RSC-3044
GTAEAASASGTTGRAG
7532




EAAGLTPAGLTGPPGSP



 



RSC-3057
GTAEAASASGTTGRAG
7533




EAANATSAGATGPPGSP



 



RSC-3058
GTAEAASASGTTGEAG
7534




EAAGATSAGATGPPGSP



 



RSC-2485
GTAEAASASGESGRAA
7535




NTEPPELGAGPGSP



 



RSC-2486
GTAEAASASGESGRAA
7536




NTAPEGLTGPPGSP



 



RSC-2488
GTAEAASASGEPGRAA
7537




NHEPSGLTEGPGSP



 



RSC-2599
GTAEAASASGESGRAA
7538




NHTGAPPGGLTGPPGSP



 



RSC-2706
GTAEAASASGTTGRTG
7539




EGANATPGGLTGPPGSP



 



RSC-2707
GTAEAASASGRTGRSG
7540




EAANETPEGLEGPPGSP



 



RSC-2708
GTAEAASASGRTGRTG
7541




ESANETPAGLGGPPGSP



 



RSC-2709
GTAEAASASGSTGRTG
7542




EPANETPAGLSGPPGSP



 



RSC-2710
GTAEAASASGTTGRAG
7543




EPANATPTGLSGPPGSP



 



RSC-2711
GTAEAASASGRTGRPG
7544




EGANATPTGLPGPPGSP



 



RSC-2712
GTAEAASASGRTGRGG
7545




EAANATPSGLGGPPGSP



 



RSC-2713
GTAEAASASGSTGRSGE
7546




SANATPGGLGGPPGSP



 



RSC-2714
GTAEAASASGRTGRTG
7547




EEANATPAGLPGPPGSP



 



RSC-2715
GTAEAASASGATGRPG
7548




EPANTTPEGLEGPPGSP



 



RSC-2716
GTAEAASASGSTGRSGE
7549




PANATPGGLTGPPGSP



 



RSC-2717
GTAEAASASGPTGRGG
7550




EGANTTPTGLPGPPGSP



 



RSC-2718
GTAEAASASGPTGRSGE
7551




GANATPSGLTGPPGSP



 



RSC-2719
GTAEAASASGTTGRAS
7552




EGANSTPAPLTEPPGSP



 



RSC-2720
GTAEAASASGTYGRAA
7553




EAANTTPAGLTAPPGSP



 



RSC-2721
GTAEAASASGTTGRAT
7554




EGANATPAELTEPPGSP



 



RSC-2722
GTAEAASASGTVGRAS
7555




EEANTTPASLTGPPGSP



 



RSC-2723
GTAEAASASGTTGRAP
7556




EAANATPAPLTGPPGSP



 



RSC-2724
GTAEAASASGTWGRAT
7557




EPANATPAPLTSPPGSP



 



RSC-2725
GTAEAASASGTVGRAS
7558




ESANATPAELTSPPGSP



 



RSC-2726
GTAEAASASGTVGRAP
7559




EGANSTPAGLTGPPGSP



 



RSC-2727
GTAEAASASGTWGRAT
7560




EAPNLEPATLTTPPGSP



 



RSC-2728
GTAEAASASGTTGRAT
7561




EAPNLTPAPLTEPPGSP



 



RSC-2729
GTAEAASASGTQGRAT
7562




EAPNLSPAALTSPPGSP



 



RSC-2730
GTAEAASASGTQGRAA
7563




EAPNLTPATLTAPPGSP



 



RSC-2731
GTAEAASASGTSGRAPE
7564




ATNLAPAPLTGPPGSP



 



RSC-2732
GTAEAASASGTQGRAA
7565




EAANLTPAGLTEPPGSP



 



RSC-2733
GTAEAASASGTTGRAG
7566




SAPNLPPTGLTTPPGSP



 



RSC-2734
GTAEAASASGTTGRAG
7567




GAENLPPEGLTAPPGSP



 



RSC-2735
GTAEAASASGTTSRAG
7568




TATNLTPEGLTAPPGSP



 



RSC-2736
GTAEAASASGTTGRAG
7569




TATNLPPSGLTTPPGSP



 



RSC-2737
GTAEAASASGTTARAG
7570




EAENLSPSGLTAPPGSP



 



RSC-2738
GTAEAASASGTTGRAG
7571




GAGNLAPGGLTEPPGSP



 



RSC-2739
GTAEAASASGTTGRAG
7572




TATNLPPEGLTGPPGSP



 



RSC-2740
GTAEAASASGTTGRAG
7573




GAANLAPTGLTEPPGSP



 



RSC-2741
GTAEAASASGTTGRAG
7574




TAENLAPSGLTTPPGSP



 



RSC-2742
GTAEAASASGTTGRAG
7575




SATNLGPGGLTGPPGSP



 



RSC-2743
GTAEAASASGTTARAG
7576




GAENLTPAGLTEPPGSP



 



RSC-2744
GTAEAASASGTTARAG
7577




SAENLSPSGLTGPPGSP



 



RSC-2745
GTAEAASASGTTARAG
7578




GAGNLAPEGLTTPPGSP



 



RSC-2746
GTAEAASASGTTSRAG
7579




AAENLTPTGLTGPPGSP



 



RSC-2747
GTAEAASASGTYGRTT
7580




TPGNEPPASLEAEPGSP



 



RSC-2748
GTAEAASASGTYSRGES
7581




GPNEPPPGLTGPPGSP



 



RSC-2749
GTAEAASASGAWGRTG
7582




ASENETPAPLGGEPGSP



 



RSC-2750
GTAEAASASGRWGRAE
7583




TTPNTPPEGLETEPGSP



 



RSC-2751
GTAEAASASGESGRAA
7584




NHTGAEPPELGAGPGSP



 



RSC-2754
GTAEAASASGTTGRAG
7585




EAANLTPAGLTESPGSP



 



RSC-2755
GTAEAASASGTTGRAG
7586




EAANLTPAALTESPGSP



 



RSC-2756
GTAEAASASGTTGRAG
7587




EAANLTPAPLTESPGSP



 



RSC-2757
GTAEAASASGTTGRAG
7588




EAANLTPEPLTESPGSP



 



RSC-2758
GTAEAASASGTTGRAG
7589




EAANLTPAGLTGAPGSP



 



RSC-2759
GTAEAASASGTTGRAG
7590




EAANLTPEGLTGAPGSP



 



RSC-2760
GTAEAASASGTTGRAG
7591




EAANLTPEPLTGAPGSP



 



RSC-2761
GTAEAASASGTTGRAG
7592




EAANLTPAGLTEAPGSP



 



RSC-2762
GTAEAASASGTTGRAG
7593




EAANLTPEGLTEAPGSP



 



RSC-2763
GTAEAASASGTTGRAG
7594




EAANLTPAPLTEAPGSP



 



RSC-2764
GTAEAASASGTTGRAG
7595




EAANLTPEPLTEAPGSP



 



RSC-2765
GTAEAASASGTTGRAG
7596




EAANLTPEPLTGPPGSP



 



RSC-2766
GTAEAASASGTTGRAG
7597




EAANLTPAGLTGGPGSP



 



RSC-2767
GTAEAASASGTTGRAG
7598




EAANLTPEGLTGGPGSP



 



RSC-2768
GTAEAASASGTTGRAG
7599




EAANLTPEALTGGPGSP



 



RSC-2769
GTAEAASASGTTGRAG
7600




EAANLTPEPLTGGPGSP



 



RSC-2770
GTAEAASASGTTGRAG
7601




EAANLTPAGLTEGPGSP



 



RSC-2771
GTAEAASASGTTGRAG
7602




EAANLTPEGLTEGPGSP



 



RSC-2772
GTAEAASASGTTGRAG
7603




EAANLTPAPLTEGPGSP



 



RSC-2773
GTAEAASASGTTGRAG
7604




EAANLTPEPLTEGPGSP



 



RSC-3047
GTAEAASASGTTGRAG
7605




EAEGATSAGATGPPGSP



 



RSC-2783
GTAEAASASGEAGRSA
7606




EATSAGATGPPGSP



 



RSC-3107
GTAEAASASGSASGTYS
7607




RGESGPGSPPGSP



 



RSC-3103
GTAEAASASGSASGEA
7608




GRTDTHPGSPPGSP



 



RSC-3102
GTAEAASASGSASGEPG
7609




RAAEHPGSPPGSP



 



RSC-3119
GTAEAASASGSPAGESS
7610




RGTTIAGSPPGSP



 



RSC-3043
GTAEAASASGTTGEAG
7611




EAAGLTPAGLTGPPGSP



 



RSC-2789
GTAEAASASGEAGESA
7612




GATPAGLTGPPGSP



 



RSC-3109
GTAEAASASGSASGAPL
7613




ELEAGPGSPPGSP



 



RSC-3110
GTAEAASASGSASGEPP
7614




ELGAGPGSPPGSP



 



RSC-3111
GTAEAASASGSASGEPS
7615




GLTEGPGSPPGSP



 



RSC-3112
GTAEAASASGSASGTPA
7616




PLTEPPGSPPGSP



 



RSC-3113
GTAEAASASGSASGTPA
7617




ELTEPPGSPPGSP



 



RSC-3114
GTAEAASASGSASGPPP
7618




GLTGPPGSPPGSP



 



RSC-3115
GTAEAASASGSASGTPA
7619




PLGGEPGSPPGSP



 



RSC-3125
GTAEAASASGSPAGAPE
7620




GLTGPAGSPPGSP



 



RSC-3126
GTAEAASASGSPAGPPE
7621




GLETEAGSPPGSP



 



RSC-3127
GTAEAASASGSPTSGQG
7622




GLTGPGSEPPGSP



 



RSC-3131
GTAEAASASGSESAPPE
7623




GLETESTEPPGSP



 



RSC-3132
GTAEAASASGSEGSEPL
7624




ELGAASETPPGSP



 



RSC-3133
GTAEAASASGSEGSGPA
7625




GLEAPSETPPGSP



 



RSC-3138
GTAEAASASGSEPTPPA
7626




SLEAEPGSPPGSP









In some embodiments, a paTCE comprises an RS1 and an RS2 that have different rates of cleavage and different cleavage efficiencies to multiple proteases for which they are substrates. As a given protease may be found in different concentrations in a tumor, compared to healthy tissues or in circulation, the disclosure provides RSs that have a higher or lower cleavage efficiency for a given protease in order to ensure that a paTCE is preferentially converted from the inactive form to the active form (i.e., by the separation and release of the binding moieties and ELNNs from the paTCE after cleavage of the RSs) when in proximity to the cancer cell or tissue and its co-localized proteases compared to the rate of cleavage of the RSs in healthy tissue or the circulation such that the released binding moieties of the TCE have a greater ability to bind to ligands in the tumor compared to the inactive form that remains in circulation. By such selective designs, the therapeutic index of the resulting compositions can be improved, resulting in reduced side effects relative to convention therapeutics that do not incorporate such site-specific activation.
In some embodiments, cleavage efficiency is the log 2 value of the ratio of the percentage of the test substrate comprising the RS cleaved to the percentage of the control substrate AC1611 cleaved when each is subjected to the protease enzyme in biochemical assays in which reaction in conducted wherein the initial substrate concentration is 6 μM, the reactions are incubated at 37° C. for 2 hours before being stopped by adding EDTA, with the amount of digestion products and uncleaved substrate analyzed by non-reducing SDS-PAGE to establish the ratio of the percentage cleaved. The cleavage efficiency may be calculated as follows:



 
  
   
    Log
     
   
   2
  
  ⁢
    
  
   
    (
    
     
      %
      ⁢
         
      Cleaved
      ⁢
         
      for
      ⁢
         
      substrate
      ⁢
         
      of
      ⁢
         
      interest
     
     
      %
      ⁢
         
      cleaved
      ⁢
         
      for
      ⁢
         
      AC
      ⁢
      1611
      ⁢
         
      in
      ⁢
         
      the
      ⁢
         
      same
      ⁢
         
      experiment
     
    
    )
   
   .
  
 



Thus, a cleavage efficiency of −1 means that the amount of test substrate cleaved was 50% compared to that of the control substrate, while a cleavage efficiency of +1 means that the amount of test substrate cleaved was 200% compared to that of the control substrate. A higher rate of cleavage by the test protease relative to the control would result in a higher cleavage efficiency, and a slower rate of cleavage by the test protease relative to the control would result in a lower cleavage efficiency. A control RS sequence AC1611 (RSR-1517), having the amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001), was established as having an appropriate baseline cleavage efficiency by the proteases legumain, MMP-2, MMP-7, MMP-9, MMP-14, uPA, and matriptase, when tested in in vitro biochemical assays for rates of cleavage by the individual proteases. By selective substitution of amino acids at individual locations in the RS peptides, libraries of RS were created and evaluated against the panel of the 7 proteases, resulting in profiles that were used to establish guidelines for appropriate amino acid substitutions in order to achieve RS with desired cleavage efficiencies. In some embodiments, in making RSs with desired cleavage efficiencies, substitutions using the hydrophilic amino acids A. E. G. P. S. and T are preferred, however other L-amino acids can be substituted at given positions in order to adjust the cleavage efficiency so long as the RSs retain at least some susceptibility to cleavage by a given protease. Conservative substitutions of amino acids in a peptide to retain or effect activity is well within the knowledge and capabilities of a person within skill in the art. In some embodiments, the disclosure provides an RS in which the RS is cleaved by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-14, uPA, or matriptase (also known as MT-SP1) with at least a 0.2 log 2, or 0.4 log 2, or 0.8 log 2, or 1.0 log, higher cleavage efficiency in an in vitro biochemical competitive assay compared to the cleavage by the same protease of a control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001). In some embodiments, the disclosure provides an RS in which the RS is cleaved by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-11, uPA, or matriptase with at least a 0.2 log 2, or 0.4 log 2, or 0.8 log 2, or 1.0 log 2 lower cleavage efficiency in an in vitro biochemical competitive assay compared to the cleavage by the same protease of a control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001). In some embodiments, the disclosure provides an RS in which the rate of cleavage of the RS by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-14, uPA, or matriptase is at least 2-fold, or at least 4-fold, or at least 8 fold, or at least 16-fold faster compared to the control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001). In some embodiments, the disclosure provides an RS in which the rate of cleavage of the RS by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-14, uPA, or matriptase is at least 2-fold, or at least 4-fold, or at least 8-fold, or at least 16-fold slower compared to the control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001).

In some embodiments, the RS comprises the amino acid sequence EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N. In some embodiments, X is S. In some embodiments, X is T. In some embodiments, X is Y. In some embodiments, X is Q. In some embodiments, X is G. In some embodiments, X is A. In some embodiments, X is V. In some embodiments, X is C. In some embodiments, X is P. In some embodiments, X is L. In some embodiments, X is I. In some embodiments, X is M. In some embodiments, X is F. In some embodiments, X is K. In some embodiments, X is R. In some embodiments, X is H. In some embodiments, X is D. In some embodiments, X is E. In some embodiments, the RS is not cleaved by legumain. In some embodiments, the RS is not cleavable by legumain in human blood, plasma, or serum.
In some embodiments, the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours. In some embodiments, the RS is cleaved by legumain less quickly or efficiently than RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 50% of the rate that legumain cleaves RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048). In some embodiments, the RS is cleaved by legumain at a rate that is less than about 25% of the rate that legumain cleaves RSR-2295. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 10% of the rate that legumain cleaves RSR-2295. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 5% of the rate that legumain cleaves RSR-2295. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 2.5% of the rate that legumain cleaves RSR-2295.
In some embodiments, the RS is cleaved by legumain at a rate that is less than about 50% of the rate that legumain cleaves RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 25% of the rate that legumain cleaves RSR-2295 in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 10% of the rate that legumain cleaves RSR-2295 in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 5% of the rate that legumain cleaves RSR-2295 in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 2.5% of the rate that legumain cleaves RSR-2295 in human plasma.
In some embodiments, the disclosure provides paTCEs comprising multiple RSs wherein each RS sequence is identified herein by the group of sequences set forth in Table 7a and the RSs are linked to each other by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, a paTCE comprises a first RS and a second RS different from the first RS wherein each RS sequence is identified herein by a sequence set forth in Table 7a and the RSs are linked to each other by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, the paTCE comprises a first RS, a second RS different from the first RS, and a third RS different from the first and the second RS wherein each sequence is identified herein by's sequence set forth in Table 7a and the first and the second and the third RS are linked to each other by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, multiple RS of the paTCE can be concatenated to form a sequence that can be cleaved by multiple proteases at different rates or efficiency of cleavage. In some embodiments, the disclosure provides a paTCE comprising an RS1 and an RS2, wherein each has a sequences set forth in Table 7a or 7b and ELNNs (e.g., an ELNN1 and ELNN2), such as those described herein, wherein the RS1 is fused between the ELNN1 and the binding moieties and the RS2 is fused between the ELNN2 and the binding moieties. In some embodiments, a paTCE is more readily cleaved in target tissues that express multiple proteases (e.g., tumor tissues), compared with healthy tissues or when in the normal circulation, with the result that the resulting fragments bearing the binding moieties would more readily penetrate the target tissue; e.g., a tumor, and have an enhanced ability to bind and link the cancer cell and the effector cell.
In some embodiments, a paTCE comprises a first release segment (RS1) positioned between a first ELNN a bispecific antibody. In some embodiments, the polypeptide further comprises a second release segment (RS2) positioned between the bispecific antibody and a second ELNN. In some embodiments, RS1 and RS2 are identical in sequence. In some embodiments, RS1 and RS2 are not identical in sequence. In some embodiments, the RS1 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence identified herein in Table 7a or 7b or a subset thereof. In some embodiments, the RS2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence identified herein in Table 7a or 7b or a subset thereof. In some embodiments, the RS1 and RS2 are each a substrate for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.
In some embodiments, the paTCE further comprises one or more reference fragments (e.g., barcode fragments) releasable from the paTCE upon digestion by the protease. In some embodiments, the one or more reference fragments is a single reference fragment that differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon digestion of the polypeptide by the protease.
Exemplary paTCEs
In some embodiments, a paTCE comprises an amino acid sequence having at least (about) 80% sequence identity to a sequence set forth in Table D (SEQ ID NOs: 1000-1007) or a subset thereof. In some embodiments, the paTCE comprises an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NOs: 1000-1007 or a subset thereof. In some embodiments, the paTCE comprises an amino acid sequence having at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NOs: 1000-1007 or a subset thereof. In some embodiments, the paTCE comprises an amino acid sequence identical to a sequence set forth in SEQ ID NOs: 1000-1007. It is specifically contemplated that the compositions of this disclosure can comprise sequence variants of the amino acid sequences set forth in Table D, such as with linker sequence(s) substituted or inserted or with purification tag sequence(s) attached thereto, so long as the variants exhibit substantially similar or same bioactivity/bioactivities and/or activation mechanism(s).







TABLE D







Exemplary amino acid sequences of polypeptides








SEQ ID 



NO
AMINO ACID SEQUENCE





1000
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGS


(AMX-525)
EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS



PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA



SHTPAGLTGPGTSESATPESDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQ



KPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLA



FGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVKPSETLS



LTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTS



KNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQEP



SLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFS



GSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPGT



SPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQ



APGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAV



YYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLTGP



ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE



PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPA



TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES



ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE



PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES



ATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE



PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA


 


1001
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS



PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA



SHTPAGLTGPGTSESATPESDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQ



KPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLA



FGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQQWGAGLLKPSETL



SLTCAVYGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDT



SKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQE



PSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF



SGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPG



TSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVR



QAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTA



VYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLT



GPATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS



TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE



PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS



ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS



TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS



ESATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS



TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA


 


1002
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS



PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA



SHTPAGLTGPGTSESATPESDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQ



KPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFDHLPL



AFGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQQWGAGLLKPSET



LSLTCAVYGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVD



TSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQ



EPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPA



RFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESG



PGTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNW



VRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTE



DTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPA



GLTGPATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP



GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP



GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP



GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP



GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP



GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP



GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP



GTSESATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE



GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA


 


1003
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS



PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA



SHTPAGLTGPGTSESATPESDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQ



KPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFDHLPL



AFGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVKPSETL



SLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDT



SKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQE



PSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF



SGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPG



TSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVR



QAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTA



VYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLT



GPATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS



TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE



PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS



ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS



TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS



ESATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS



TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA


 


1004
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS



PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA



SHTPAGLTGPGTSESATPESEIVLTQSPGTLSLSPGERATLSCQASQDISNYLNWYQQ



KPGQAPRLLIYDASNLETGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHFDHLPLA



FGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQQWGAGLLKPSETL



SLTCAVYGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDT



SKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQE



PSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF



SGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPG



TSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVR



QAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTA



VYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLT



GPATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS



TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE



PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS



ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS



TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS



ESATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS



TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA


 


1005
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS



PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA



SHTPAGLTGPGTSESATPESEIVLTQSPGTLSLSPGERATLSCQASQDISNYLNWYQQ



KPGQAPRLLIYDASNLETGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHFDHLPLA



FGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVKPSETLS



LTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTS



KNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQEP



SLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFS



GSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPGT



SPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQ



APGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAV



YYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLTGP



ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE



PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPA



TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES



ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE



PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES



ATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE



PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA


 


1006
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS



PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA



SHTPAGLTGPGTSESATPESEIVLTQSPATLSLSPGERATLSCQASQDISNYLNWYQQ



KPGQAPRLLIYDASNLETGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHFDHLPLA



FGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQQWGAGLLKPSETL



SLTCAVYGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDT



SKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQE



PSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF



SGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPG



TSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVR



QAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTA



VYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLT



GPATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS



TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE



PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS



ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS



TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS



ESATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS



TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA


 


1007
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS



PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA



SHTPAGLTGPGTSESATPESEIVLTQSPATLSLSPGERATLSCQASQDISNYLNWYQQ



KPGQAPRLLIYDASNLETGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHFDHLPLA



FGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVKPSETLS



LTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTS



KNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQEP



SLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFS



GSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPGT



SPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQ



APGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAV



YYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLTGP



ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE



PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPA



TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES



ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE



PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES



ATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE



PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA









Recombinant Production
Also provided are polynucleotides that encode any polypeptide disclosed herein and/or the reverse complements of such polynucleotides.
The disclosure herein includes an expression vector that comprises a polynucleotide sequence, such as any described in the preceding paragraph, and a regulatory sequence operably linked to the polynucleotide sequence.
The disclosure herein includes a host cell comprising an expression vector, such as described any in the preceding paragraph. In some embodiments, the host cell is a prokaryote. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is a mammalian cell.
In some embodiments, the disclosure provides methods of manufacturing the subject compositions. In some embodiments, such a method comprises culturing a host cell comprising a nucleic acid construct that encodes a polypeptide (such as a paTCE) described herein under conditions that promote the expression of the polypeptide, followed by recovery of the polypeptide using standard purification methods (e.g., column chromatography, HPLC, and the like) wherein the composition is recovered wherein at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the binding fragments of the expressed polypeptide or paTCE fusion polypeptide are correctly folded. In some embodiments of the method of making, the expressed polypeptide is recovered in which at least or at least 90%, or at least 95%, or at least 97%, or at least 99% of the polypeptide is recovered in monomeric, soluble form.
In some embodiments, the disclosure relates to methods of making a polypeptide (such as a paTCE fusion polypeptide) at high fermentation expression levels of functional protein using an E. coli or mammalian host cell, as well as providing expression vectors encoding the polypeptides useful in methods to produce the cytotoxically active polypeptide compositions at high expression levels. In some embodiments, the method comprises the steps of 1) preparing a polynucleotide encoding a polypeptide disclosed herein, 2) cloning the polynucleotide into an expression vector, which can be a plasmid or other vector under the control of appropriate transcription and translation sequences for high level protein expression in a biological system, 3) transforming an appropriate host cell with the expression vector, and 4) culturing the host cell in conventional nutrient media under conditions suitable for the expression of the polypeptide composition. Where desired, the host cell is E. coli. As used herein, the term “correctly folded” means that the antigen binding fragments component of the composition have the ability to specifically bind their target ligands (e.g., upon activation). In some embodiments, the disclosure provides a method for producing a polypeptide, the method comprising culturing in a fermentation reaction a host cell that comprises a vector encoding a polypeptide comprising the polypeptide under conditions effective to express the polypeptide product.
Pharmaceutical Composition
Disclosed herein includes a pharmaceutical composition comprising a polypeptide (such as a paTCE), such as any described herein, and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intra-arterial, intraabdominal, intraperitoneal, intravitreal, intrathecal, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for intravenous injection. In some embodiments, the pharmaceutical composition is in a liquid form or frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder to be reconstituted prior to administration.
The pharmaceutical compositions can be administered for therapy by any suitable route. In some embodiments, the dose is administered intradermally, subcutaneously, intravenously, intra-arterially, intra-abdominally, intraperitoneally, intrathecally, or intramuscularly. In some embodiments, the subject is a mouse, rat, monkey, or human. In preferred embodiments, the subject is a human.
In some embodiments, the pharmaceutical composition can be administered subcutaneously, intramuscularly, or intravenously. In some embodiments, the pharmaceutical composition is administered at a therapeutically effective amount. In some embodiments, the therapeutically effective amount results in a gain in time spent within a therapeutic window for the fusion protein compared to the corresponding TCE of the fusion protein not linked to the ELNN and administered at a comparable dose to a subject.
In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered intravenously.
In some embodiments, the composition may be supplied as a lyophilized powder or cake to be reconstituted prior to administration. In some embodiments, the composition may also be supplied in a liquid form or frozen, which can be administered directly to a subject.
Pharmaceutical Kits
In some embodiments, the present disclosure provides kits to facilitate the use of paTCEs. In some embodiments, a kit comprises (a) a first container comprising pharmaceutically effective amount of a paTCE in a lyophilized composition; and (b) a second container comprising a diluent for reconstituting the lyophilized formulation. In some embodiments, the kit further comprises instructions for storage of the kit, information regarding a cancer that is treatable with the paTCE, instructions for the reconstitution of the lyophilized formulation, and/or administration instructions.
Methods of Treatment
Disclosed herein are uses of a polypeptide, such as any described herein, in the preparation of a medicament for the treatment of a disease in a subject. In some embodiments, the particular disease to be treated will depend on the choice of the biologically active proteins. In some embodiments, the disease is cancer. Included herein are paTCE polypeptides for use in the treatment of cancer. In some cases, the cancer or tumor expresses EGFR. In some embodiments, the cancer or tumor is a solid tumor. In some embodiments, the cancer is a carcinoma, a sarcoma, or a melanoma. In some embodiments, the cancer is a carcinoma. In some embodiments, the cancer is a sarcoma. In some embodiments, the cancer is a melanoma.
EGFR is one of the most frequently altered oncogenes in solid tumors. Activation of EGFR promotes processes responsible for tumor growth and progression, including proliferation and maturation, angiogenesis, invasion, metastasis, and inhibition of apoptosis. Pathological alterations of EGFR in cancers include kinase-activating mutations in EGFR and/or over-expression of the EGFR protein. Kinase-activating mutations lead to increased tyrosine kinase activity of EGFR. Over-expression of EGFR protein can be associated with or without EGFR gene amplifications. Additionally, wild-type EGFR protein is commonly over-expressed in many types of solid cancers and is often associated with negative prognosis. Alterations of EGFR in solid cancers known in the art, for example, as described in Thomas R. and Weihua Z. Front. Oncol. 9:800 (2019) and Singal et al. Cancer Control 14 (3): 295-304 (2007), each of which is incorporated herein in its entirety. Current EGFR inhibitors, including tyrosine kinase inhibitors and monoclonal antibody inhibitors, have exhibited limited efficacies and have been challenged by innate and acquired resistance in the clinic.
In some embodiments, the cancer is associated with EGFR overexpression (e.g., relative to a non-cancerous cell of the same tissue type). In some embodiments, the cancer comprises cells that express, on average, at least 3,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; 100,000; or 200,000 EGFR proteins per cell. In some embodiments, the cancer comprises cells having one or more oncogenic mutations in an EGFR gene. In some embodiments, the cancer comprises cells having an EGFR gene amplification. In some embodiments, the cells comprise a 2 to 5-fold, 2 to 10-fold, 2 to 15-fold, 2 to 30-fold, 2 to 50-fold, 3 to 5-fold, 3 to 10-fold, 3 to 15-fold, 3 to 30-fold, 3 to 50-fold, 5 to 10-fold, 5 to 15-fold, 5 to 30-fold, or 5 to 50-fold increase in EGFR gene copy number as compared to a non-cancerous cell of the same tissue type.
In some embodiments, the cancer is lung cancer, colorectal cancer, head and neck cancer, breast cancer, pancreatic cancer, brain cancer, liver cancer, kidney cancer, ovarian cancer, prostate cancer, esophageal cancer, cervical cancer, or bladder cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple-negative breast cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the brain cancer is glioblastoma.
In some embodiments, the cancer is anaplastic and medullary thyroid cancers, appendiceal cancer, arrhenoblastoma, biliary tract carcinoma, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignancy with malignant ascites, esophageal cancer. Ewing sarcoma, fallopian tube cancer, follicular cancer, gall bladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), GE-junction cancer, genito-urinary tract cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocarcinoma, HR+ and HER2+ breast cancer, Hurthle cell cancer, Inflammatory breast cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroblastoma, neuroendocrine cancer, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary cancer, parathyroid cancer, peritoneal carcinomatosis, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, triple negative breast cancer, urothelial cancer, uterine cancer, uterine serous carcinoma, vaginal cancer, vulvar cancer, or Wilms tumor.
The present disclosure includes a method of treating a disease in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition, such as any described herein. In some embodiments, the disease is cancer. In some embodiments, the subject is a mouse, rat, monkey, or human. In some embodiments, the subject is a human.
In some embodiments, an EGFR-targeted bispecific composition of the present disclosure (such as a paTCE) may be combined with one or more checkpoint inhibitors. In some embodiments of such combination therapy, a paTCE can be combined with an antagonist of the cell surface receptor programmed cell death protein 1, also known as PD-1, and/or an antagonist of PD-L1. As used herein, the term “combination” or “combination therapy” corresponds to the administration of two or more distinct compounds (e.g., an EGFR paTCE and a checkpoint inhibitor) as part of a treatment regimen. The two or more compounds may be administered simultaneously or sequentially. The two or more compounds may be combined into a single composition prior to administration. Each compound in the combination may be separately administered as part of a defined dosing regimen.
PD-1 plays an important role in down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. Binding of the PD-1 ligands, PD-L1 and PD-L2 to the PD-1 receptor found in T cells inhibits T-cell proliferation and cytokine production. Upregulation of PD-1 ligands occurs in some tumors and signaling through this pathway can contribute to inhibition of active T-cell immune surveillance of tumors. Anti-PD-1 antibodies bind to the PD-1 receptor and block its interaction with PD-L1 and PD-L3, releasing PD-1 pathway-mediated inhibition of the immune response, including the anti-tumor immune response.
Those of skill in the art are aware of various anti-PD-1 antibodies that may be used. In some embodiments, an exemplary anti-PD-1 antibody used in combination with the compounds of the present invention is Pembrolizumab (Keytruda®). In some embodiments, the anti-PD-1 antibody used in combination with the compound described above is Nivolumab (Opdivo®). In some embodiments, the anti-PD-1 antibody used in combination with the compound described above is Pidilizumab (Medivation).
Additional PD-1 antibodies known to those of skill in the art, include AGEN-2034 (Agenus), AMP-224 (Medimmunc), BCD-100 (Biocad), BGBA-317 (Beigene), BI-754091 (Bochringer Ingelheim), CBT-501 (Genor Biopharma), CC-90006 (Celgene), cemiplimab (Regeneron Pharmaceuticals), durvalumab+MEDI-0680 (Medimmune), GLS-010 (Harbin Gloria Pharmaceuticals), IBI-308 (Eli Lilly), JNJ-3283 (Johnson & Johnson), JS-001 (Shanghai Junshi Bioscience Co.), MEDI-0680 (Medimmune), MGA-012 (MacroGenics), MGD-013 (Marcogenics), pazopanib hydrochloride+pembrolizumab (Novartis), PDR-001 (Novartis), PF-06801591 (Pfizer), SHR-1210 (Jiangsu Hengrui Medicine Co.), TSR-042 (Tesaro Inc.), LZM-009 (Livzon Pharmaceutical Group Inc) and ABBV-181 (AbbVie Inc).
In some embodiments for combination therapy of the present disclosure, the anti-PD-1 antibody is pembrolizumab (Keytruda®).
In some embodiments, the compositions of the present invention are combined with an anti-PD-L1 antibody. Exemplary such anti-PD-L1 antibodies used in the combinations of the present invention may be selected from the group consisting of Durvalumab (MedImmune LLC), Atezolizumab (Hoffmann-La Roche Ltd, Chugai Pharmaceutical Co Ltd), Avelumab (Merck KGaA), CX-072 (CytomX Therapeutics Inc), BMS-936559 (ViiV Healthcare Ltd), SHR-1316 (Jiangsu Hengrui Medicine Co Ltd), M-7824 (Merck KGaA), LY-3300054 (Eli Lilly and Co), FAZ-053 (Novartis AG), KN-035 (AlphaMab Co Ltd), CA-170 (Curis Inc), CK-301 (TG Therapeutics Inc), CS-1001 (CStone Pharmaceuticals Co Ltd), HLX-10 (Shanghai Henlius Biotech Co Ltd), MCLA-145 (Merus NV), MSB-2311 (MabSpace Biosciences (Suzhou) Co Ltd) and MEDI-4736 (Medimmune).
Other immunotherapies and checkpoint inhibitor-based therapies that may be useful in combination with the compositions of the present disclosure include CTLA4, TIGIT, OX40, and TIM3-based therapies.
In some embodiments, the disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an amount of the paTCE described herein to the subject, and a checkpoint inhibitor to the subject, wherein the cancer comprises a solid tumor, and treating the cancer comprises reducing the volume of the solid tumor.
Exemplary Embodiments
Disclosed herein further provides below non-limiting exemplary embodiments:

    
    
        1. A chimeric polypeptide comprising a bispecific antibody domain,
        wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds epidermal growth factor receptor (EGFR) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3),
        wherein the first antigen binding domain comprises:
        
            a VH domain comprising
            
                a CDR1 amino acid sequence of GGSVSSGDYYWT (SEQ ID NO: 562), a CDR2 amino acid sequence of HIYYSGNTNYNPSLKS (SEQ ID NO: 563), and a CDR3 amino acid sequence of DRVTGAFDI (SEQ ID NO: 564); and
                at least one of: a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and/or a leucine (L) residue at position 108 in FR4, wherein the FR numbering is according to Kabat; and
            
            
            a VL domain comprising
            
                a CDR1 amino acid sequence of QASQDISNYLN (SEQ ID NO: 565), a CDR2 amino acid sequence of DASNLET (SEQ ID NO: 566), a CDR3 amino acid sequence of QHFDHLPLA (SEQ ID NO: 567); and
            
            
        
        
        wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or EGFR, and wherein the protease-cleavable release segment is cleavable by at least one protease that is present in a tumor.
        2. The chimeric polypeptide of embodiment 1, wherein the VH domain comprises an asparagine (N) residue at position 76 in FR3.
        3. The chimeric polypeptide of embodiment 1 or 2, wherein the VH domain comprises alanine (A) residue at position 93 in FR3.
        4. The chimeric polypeptide of any one of embodiment 1-3, wherein the VH domain comprises a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, and an alanine (A) residue at position 93 in FR3.
        5. The chimeric polypeptide of any one of embodiments 1-4, wherein the VH domain comprises a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and a leucine (L) residue at position 108 in FR4.
        6. The chimeric polypeptide of any one of embodiment 1-5, wherein the VL domain comprises at least one of: a tyrosine (Y) residue at position 87 in FR3 and/or a glutamine (Q) residue at position 100 in FR4, wherein the FR numbering is according to Kabat.
        7. The chimeric polypeptide of embodiment 6, wherein the VL domain comprises a tyrosine (Y) residue at position 87 in FR3 and a glutamine (Q) residue at position 100 in FR4.
        8. The chimeric polypeptide of any one of embodiments 1-7, wherein:
        the VH domain comprises an amino acid sequence of QVQLQX1X2GX3GLX4KPSETLSLTCXsVX6GGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS, wherein X1 corresponds to E or Q; X2 corresponds to S or W; X3 corresponds to P or A; X4 corresponds to V or L; X5 corresponds to T or A; and X6 corresponds to S or Y (SEQ ID NO: 576); and the VL domain comprises an amino acid sequence of X1IX2X3TQSPX4XLSX6SX-GX8RXnTX10X1CQASQDISNYLNWYQQKPGX12APX13LLIYDASNLET GX14PX15RFSGSGSGTDFTX16TISX17LX18PEDX19AX20YYCQHFDHLPLAFGQGTKVEIK, wherein X1corresponds to D or E; X2 corresponds to Q or V; X3 corresponds to M or L; X4 corresponds to S, G, or A; X5 corresponds to S or T; X6 corresponds to L or A; X7 corresponds to P or V; Xx corresponds to D or E; X9 corresponds to V or A; X10 corresponds to I or L; X11 corresponds to T or S; X12 corresponds to K or Q; X13 corresponds to K or R; X14 corresponds to V or I; X15 corresponds to S, D, or A; X16 corresponds to F or L; X17 corresponds to S or R; X18 corresponds to Q or E; X19 corresponds to I or F; and X20 corresponds to Tor V (SEQ ID NO: 577).
        9. A chimeric polypeptide comprising a bispecific antibody domain,
        wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3),
        wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or EGFR, wherein the protease-cleavable release segment is not capable of being cleaved by legumain in human plasma, or wherein legumain cleaves the protease-cleavable release segment in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
        10. A chimeric polypeptide comprising a bispecific antibody domain,
        wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds epidermal growth factor receptor (EGFR) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3),
        wherein the chimeric polypeptide has a melting temperature (Tm) of greater than 62° C. and/or a thermostability ratio of greater than 0.5 at 62° C.;
        wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or EGFR, and wherein the protease-cleavable release segment is cleavable by at least one protease that is present in a tumor.
    
    


11. The chimeric polypeptide of embodiment 10, wherein the Tm is determined by differential scanning fluorimetry (DSF).
12. The chimeric polypeptide of embodiment 10, wherein the thermostability ratio is determined by:

    
    
        i) incubating an input amount of a chimeric polypeptide at 62° C. for 30 minutes thereby denaturing a fraction of the input amount of chimeric polypeptide;
        ii) measuring an amount of monomeric chimeric polypeptide remaining following step i); and
        iii) dividing the amount of monomeric chimeric polypeptide by the input amount of the chimeric polypeptide to generate the thermostability ratio.
        13. The chimeric polypeptide of embodiment 12, wherein amount of monomeric chimeric polypeptide is measured by mass spectrometry.
        14. A chimeric polypeptide comprising a bispecific antibody domain,
        wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds a cancer cell antigen and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3), wherein the second antigen binding domain comprises:
        a VH domain comprising a CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), a CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and a CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and
        a VL domain comprising a CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), a CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and a CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6).
        wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or the cancer cell antigen, and wherein the protease-cleavable release segment is cleavable by at least one protease that is present in a tumor.
        15. The chimeric polypeptide of embodiment 14, wherein the second antigen binding domain comprises:
        (i) the VL domain comprising the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTN KRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLT VL (SEQ ID NO: 127); and
        (ii) the VH domain comprising the amino acid sequence of
    
    









(SEQ ID NO: 126)


EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR


 


IRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR


 


HENFGNSYVSWFAHWGQGTLVTVSS.






    
    
        16. The chimeric polypeptide of embodiment 14 or 15, wherein the cancer cell antigen is human alpha 4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, EGFR, HER2, HER3, HER4, PD-L1, prostate-specific membrane antigen (PSMA), CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUCSAC, MUC5B, MUC7, MUC16 BhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3; 9—O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, GD2, carbonicanhydrase IX, CD44v6, Sonic Hedgehog (Shh), Wue-1, plasma cell antigen 1, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, 6-transmembrane epithelial antigen of prostate (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6, desmoglein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA-125), Müellerian inhibitory substance receptor type II (MISIIR), sialylated Tn antigen (sTN), fibroblast activation antigen (FAP), endosialin (CD248), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMPs, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, or EphA2.
        17. The chimeric polypeptide of embodiment 14 or 15, wherein the cancer cell antigen is EGFR.
        18. The chimeric polypeptide of any one of embodiments 1-17, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (first antigen binding domain)-(second antigen binding domain)-(linker)-(mask polypeptide), (second antigen binding domain)-(first antigen binding domain)-(linker)-(mask polypeptide), (mask polypeptide)-(linker)-(first antigen binding domain)-(second antigen binding domain), or (mask polypeptide)-(linker)-(second antigen binding domain)-(first antigen binding domain), wherein each—is a covalent connection or a polypeptide linker.
        19. The chimeric polypeptide of any one of embodiments 1-18, wherein the mask polypeptide is an extended length non-natural polypeptide (ELNN).
        20. The chimeric polypeptide of any one of embodiments 1-19, wherein the linker further comprises a spacer.
        21. The chimeric polypeptide of any one of embodiments 1-20, wherein the protease-cleavable release segment is fused to the bispecific antibody domain via the spacer.
        22. The chimeric polypeptide of embodiment 20 or 21, wherein the spacer is characterized in that:
        
            (i) at least 90% of its amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and
            (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
        
        
        23. The chimeric polypeptide of any one of embodiments 20-22, wherein the spacer is from 9 to 14 amino acids in length.
        24. The chimeric polypeptide of any one of embodiments 20-23, wherein the spacer comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
        25. The chimeric polypeptide of any one of embodiments 20-24, wherein the amino acids of the spacer consists of A, E, G, S, P, and/or T.
        26. The chimeric polypeptide of any one of embodiments 20-25, wherein the spacer is cleavable by a non-mammalian protease.
        27. The chimeric polypeptide of embodiment 26, wherein the non-mammalian protease is Glu-C.
        28. The chimeric polypeptide of any one of embodiments 18-27, wherein the spacer comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.
        29. The chimeric polypeptide of any one of embodiments 20-28, wherein the spacer comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES (SEQ ID NO:96) or GTATPESGPG (SEQ ID NO:97).
        30. The chimeric polypeptide of any one of embodiments 1-29, wherein the protease-cleavable release segment comprises an amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
        31. The chimeric polypeptide of embodiment 30, wherein X is S.
        32. The chimeric polypeptide of any one of embodiments 1-31, comprising a first mask polypeptide joined to the first antigen binding domain via a first linker wherein the first linker comprises a first protease cleavable release segment (RS1) cleavable by at least one protease present in a tumor; and a second mask polypeptide joined to the second antigen binding domain via a second linker wherein the second linker comprises a second protease cleavable release segment (RS2) cleavable by at least one protease present in a tumor.
        33. The chimeric polypeptide of embodiment 32, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (Mask1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(Mask2), (Mask1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(Mask2), (Mask2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(Mask1), or (Mask2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(Mask1), wherein each—is, individually, a covalent bond or a polypeptide linker.
        34. The chimeric polypeptide of embodiment 32 or 33, wherein the first mask polypeptide is a first ELNN (ELNN1) and the second mask polypeptide is a second ELNN (ELNN2).
        35. The chimeric polypeptide of embodiment 34, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(ELNN2), (ELNN1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(ELNN2), (ELNN2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(ELNN1), or (ELNN2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(ELNN1), wherein each—is, individually, a covalent bond or a polypeptide linker.
        36. The chimeric polypeptide of any one of embodiments 32-35, wherein Linker1 further comprises a first spacer (Spacer1).
        37. The chimeric polypeptide of any one of embodiments 32-36, wherein Linker2 further comprises a second spacer (Spacer2).
        38. The chimeric polypeptide of embodiment 36 or 37, wherein RS1 is fused to the bispecific antibody domain via Spacer1 and/or RS2 is fused to the bispecific antibody domain via Spacer2.
        39. The chimeric polypeptide of embodiment 38, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(RS1)-(Spacer1)-(first antigen binding domain)-(second antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(Spacer1)-(second antigen binding domain)-(first antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN2)-(RS2)-(Spacer2)-(first antigen binding domain)-(second antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), or (ELNN2)-(RS2)-(Spacer2)-(second antigen binding domain)-(first antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), wherein each—is a, individually, covalent bond or a polypeptide linker.
        40. The chimeric polypeptide of any one of embodiments 36-39 wherein Spacer 1 and/or the Spacer2 is characterized in that:
        
            (iii) at least 90% of its amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and
            (iv) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
        
        
        41. The chimeric polypeptide of any one of embodiments 36-40, wherein Spacer1 and/or the Spacer2 is from 9 to 14 amino acids in length.
        42. The chimeric polypeptide of any one of embodiments 36-41, wherein Spacer1 and/or the Spacer2 comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
        43. The chimeric polypeptide of any one of embodiments 36-42, wherein the amino acids of Spacer1 and/or the Spacer2 consists of A, E, G, S, P, and/or T.
        44. The chimeric polypeptide of any one of embodiments 36-43, wherein Spacer1 and/or the Spacer2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.
        45. The chimeric polypeptide of any one of embodiments 36-44, wherein Spacer1 and/or the Spacer2 comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES (SEQ ID NO:96) or GTATPESGPG (SEQ ID NO: 97).
        46. The chimeric polypeptide of any one of embodiments 34-45, wherein the amino acid sequence of the first ELNN is between 250 amino acids and 350 amino acids in length, and wherein the amino acid sequence of the second ELNN is between 500 amino acids and 600 amino acids in length.
        47. The chimeric polypeptide of any one of embodiments 34-46, wherein the amino acid sequence of the first ELNN is 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is 582 amino acids in length.
        48. The chimeric polypeptide of any one of embodiments 32-47, wherein RS1 and/or RS2 comprises an amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
        49. The chimeric polypeptide of embodiment 48, wherein X is S.
        50. A chimeric polypeptide comprising a bispecific antibody domain,
        wherein the bispecific antibody domain comprises a first antigen binding domain that has binding specificity to a cancer cell antigen, and a second antigen binding domain that has binding specificity to an effector cell antigen expressed on an effector cell,
        wherein the chimeric polypeptide further comprises a first ELNN joined to the first antigen binding domain via a first linker comprising a first protease-cleavable release segment (RS1) positioned between the first ELNN and the first antigen binding domain such that the first ELNN is capable of reducing the binding of the first antigen binding domain to the cancer cell antigen, wherein the RS1 is cleavable by at least one protease that is present in a tumor,
        wherein the chimeric polypeptide further comprises a second ELNN joined to the second antigen binding domain via a second linker comprising second protease-cleavable release segment (RS2) positioned between the second ELNN and the second antigen binding domain such that the second ELNN is capable of reducing the binding of the first antigen binding domain to the effector cell antigen, wherein the RS2 is cleavable by at least one protease that is present in a tumor,
        wherein the first ELNN has a shorter amino acid sequence than the second ELNN, and
        wherein the cancer cell antigen is EGFR.
        51. The chimeric polypeptide of embodiment 50, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(ELNN2), (ELNN1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(ELNN2), (ELNN2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(ELNN1), or (ELNN2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(ELNN1), wherein each—is, individually, a covalent bond or a polypeptide linker.
        52. The chimeric polypeptide of embodiment 50 or 51, wherein Linker1 further comprises a first spacer (Spacer1).
        53. The chimeric polypeptide of any one of embodiments 50-52, wherein Linker2 further comprises a second spacer (Spacer2).
        54. The chimeric polypeptide of embodiment 52 or 53, wherein RS1 is fused to the bispecific antibody domain via Spacer1 and/or RS2 is fused to the bispecific antibody domain via Spacer2.
        55. The chimeric polypeptide of embodiment 54, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(RS1)-(Spacer1)-(first antigen binding domain)-(second antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(Spacer1)-(second antigen binding domain)-(first antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN2)-(RS 2)-(Spacer2)-(first antigen binding domain)-(second antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), or (ELNN2)-(RS2)-(Spacer2)-(second antigen binding domain)-(first antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), wherein each—is a, individually, covalent bond or a polypeptide linker.
        56. A chimeric polypeptide comprising a bispecific antibody domain, comprising the formulas that comprises from the N-terminal side to the C-terminal side:
        Formula 1: (Mask1)-(RS1)-(Spacer1)-(first antigen binding domain)-[antibody domain linker]-(second antigen binding domain);
        Formula 2: (first antigen binding domain)-[antibody domain linker]-(second antigen binding domain)-(Spacer2)-(RS2)-(Mask2); or
        Formula 3: (Mask1)-(RS1)-(Spacer1)-(first antigen binding domain)-[antibody domain linker]-(second antigen binding domain)-(Spacer2)-(RS2)-(Mask2),
        wherein,
        
            the first antigen binding domain has binding specificity to a cancer cell antigen;
            the second antigen binding domain has binding specificity to an effector cell antigen expressed on an effector cell;
            each—comprises, individually, a covalent connection or a polypeptide linker;
            the Mask1 is a polypeptide that is capable of reducing binding of the first antigen binding domain to its target;
            the Mask2 is a polypeptide that is capable of reducing binding of the second antigen binding domain to its target;
            if the chimeric polypeptide comprises Formula 1 then the Spacer1 consists of A, E, G, S, P, and/or T residues, if the chimeric polypeptide comprises Formula 2 then the Spacer2 consists of A, E, G, S, P, and/or T residues, and if the chimeric polypeptide comprises Formula 3 then the Spacer 1 and/or the Spacer2 consists of A, E, G, S, P, and/or T residues; and
            wherein the cancer cell antigen is EGFR.
        
        
    
    


57. The chimeric polypeptide of any one of embodiments 18-56, wherein each—is, individually, a covalent connection.
58. The chimeric polypeptide of embodiment 57, wherein each—is, individually, a covalent bond.
59. The chimeric polypeptide of embodiment 57, wherein each—is a peptide bond.
60. The chimeric polypeptide of embodiment 57, wherein each—is, individually, a polypeptide linker of no more than 5 amino acids.
61. The chimeric polypeptide of any one of embodiments 1-60, wherein the second antigen binding domain has binding specificity to human CD3 and cynomolgus monkey CD3.
62. The chimeric polypeptide of any one of embodiments 1-61, wherein the second antigen binding domain has binding specificity to human CD3.
63. The chimeric polypeptide of any one of embodiments 50-60, wherein the effector cell antigen is cluster of differentiation 3 T cell receptor (CD3).
64. The chimeric polypeptide of any one of embodiments 61-63, wherein the CD3 is CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.
65. The chimeric polypeptide of embodiment 64, wherein the CD3 is CD3 epsilon.
66. The chimeric polypeptide of any one of embodiments 33-65, wherein the Mask1 is a first ELNN and the Mask2 is a second ELNN.
67. The chimeric polypeptide of any one of embodiments 36-66, wherein the Spacer1 and/or the Spacer2 is characterized in that:

    
    
        (i) at least 90% of its amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and
        (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
    
    


68. The chimeric polypeptide of embodiment 67, wherein the Spacer1 and/or the Spacer2 is from 9 to 14 amino acids in length.
69. The chimeric polypeptide of embodiment 67 or 68, wherein the Spacer 1 and/or the Spacer2 comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
70. The chimeric polypeptide of any one of embodiments 67-69, wherein the amino acids of the Spacer 1 and/or the Spacer2 consists of A, E, G, S, P, and/or T.
71. The chimeric polypeptide of any one of embodiments 67-70, wherein the Spacer 1 and/or the Spacer2 is cleavable by a non-mammalian protease.
72. The chimeric polypeptide of embodiment 71, wherein the non-mammalian protease is Glu-C.
73. The chimeric polypeptide of any one of embodiments 67-71, wherein the Spacer 1 and/or the Spacer 2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.
74. The chimeric polypeptide of any one of embodiments 67-71, wherein the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES (SEQ ID NO:96) or GTATPESGPG (SEQ ID NO:97).
75. The chimeric polypeptide of any one of embodiments 67-74, wherein the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN.
76. The chimeric polypeptide of embodiment 75, wherein the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN.
77. The chimeric polypeptide of embodiment 75 or 76, wherein the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN.
78. The chimeric polypeptide of any one of embodiments 75-77, wherein the amino acid sequence of the first ELNN is between 250 amino acids and 350 amino acids in length, and wherein the amino acid sequence of the second ELNN is between 500 amino acids and 600 amino acids in length.
79. The chimeric polypeptide of any one of embodiments 75-78, wherein the amino acid sequence of the first ELNN is 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is 582 amino acids in length
80. The chimeric polypeptide of any one of embodiments 1-79, wherein the first antigen binding domain comprises a first antibody or an antigen-binding fragment thereof, and wherein the second antigen binding domain comprises a second antibody or an antigen-binding fragment thereof.
81. The chimeric polypeptide of any one of embodiments 1-80, wherein the first antigen binding domain is a Fab, an scFv, or an ISVD.
82. The chimeric polypeptide of any one of embodiments 1-81, wherein the second antigen binding domain is a Fab, an scFV, or an ISVD.
83. The chimeric polypeptide of embodiment 81 or 82, wherein the ISVD is a VHH domain.
84. The chimeric polypeptide of any one of embodiments 1-82, wherein the first antigen binding domain is an scFV.
85. The chimeric polypeptide of any one of embodiments 1-82, wherein the second antigen binding domain is an scFV.
86. The chimeric polypeptide of any one of embodiments 1-85, wherein there is an antibody domain linker between the first antigen binding domain and the second antigen binding domain.
87. The chimeric polypeptide of embodiment 86, wherein the antibody domain linker comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table A or B.
88. The chimeric polypeptide of embodiment 86, wherein the antibody domain linker consists of G and S amino residues.
89. The chimeric polypeptide of embodiment 88, wherein the antibody domain linker is 6-12 residues in length.
90. The chimeric polypeptide of embodiment 88 or 89, wherein the antibody domain linker comprises the amino acid sequence GGGGS (SEQ ID NO:87) or GGGGSGGGS (SEQ ID NO:125).
91. The chimeric polypeptide of any one of embodiments 1-90, wherein the first antigen binding domain and/or the second antigen binding domain comprise an scFv comprising a VL domain, a VH domain, and a linker between the VL domain and the VH domain, wherein the linker consists of A, E, G, S, P, and/or T residues.
92. The chimeric polypeptide of embodiment 91, wherein the linker is characterized in that:

    
    
        (i) at least 90% of its amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and
        (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
    
    


93. The chimeric polypeptide of embodiment 91 or 92, wherein the linker between the VL domain and the VH domain is from 25 to 35 amino acids in length.
94. The chimeric polypeptide of any one of embodiments 91-93, wherein the linker between the VL domain and the VH domain comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
95. The chimeric polypeptide of any one of embodiments 91-94, wherein the amino acids of the linker between the VL domain and the VH domain consists of A, E, G, S, P, and/or T.
96. The chimeric polypeptide of any one of embodiments 91-95, wherein the linker between the VL domain and the VH domain is cleavable by a non-mammalian protease.
97. The chimeric polypeptide of embodiment 96, wherein the non-mammalian protease is Glu-C.
98. The chimeric polypeptide of embodiment 91, wherein linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).
99. The chimeric polypeptide of any one of embodiments 1-98, wherein the second antigen binding domain comprises the following CDRs:

    
    
        a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSX1GAVTX2SNYAN (SEQ ID NO:8023), wherein X1 corresponds to T or N, and X2 corresponds to T or S;
        a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP (SEQ ID NO:4);
        a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX4NLWV (SEQ ID NO:8024), wherein X4 corresponds to S or P;
        a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX8TYAMN (SEQ ID NO:8025), wherein Xx corresponds to S or N;
        a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRX10KX11NX12YATYYADSVKX13 (SEQ ID NO:8026), wherein X10 corresponds to T or S, X11 corresponds to R or Y, X12 corresponds to D or N, and X13 corresponds to G or D;
        a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HX14NFGNSYVSWFAX15 (SEQ ID NO:8027), wherein X14 corresponds to E or G, and X15 corresponds to H or Y.
    
    


100. The chimeric polypeptide of any one of embodiments 1-99, wherein the second antigen binding domain comprises:

    
    
        a VH domain comprising a CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), a CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and a CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and
        a VL domain comprising a CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), a CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and a CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6).
        101. The chimeric polypeptide of any one of embodiments 1-100, wherein the second antigen binding domain comprises:
        a VH domain comprising an amino acid sequence of EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYA DSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126); and
        a VL domain comprising an amino acid sequence of
    
    









(SEQ ID NO: 127)


ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLI


 


GGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVF


 


GGGTKLTVL.






    
    
        102. The chimeric polypeptide of any one of embodiments 2-101, wherein the first antigen binding domain comprises the following CDRs:
        
            a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QASQDISNYLN (SEQ ID NO:565);
            a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to DASNLET (SEQ ID NO:566);
            a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QHFDHLPLA (SEQ ID NO:567);
            a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GGSVSSGDYYWT (SEQ ID NO:562);
            a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HIYYSGNTNYNPSLKS (SEQ ID NO:563); and
            a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to DRVTGAFDI (SEQ ID NO:564).
        
        
        103. The chimeric polypeptide of embodiment 102, wherein the VH domain comprises at least one of: a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and/or a leucine (L) residue at position 108 in FR4, wherein the FR numbering is according to Kabat.
        104. The chimeric polypeptide of embodiment 102 or 103, wherein the VH domain comprises an asparagine (N) residue at position 76 in FR3.
        105. The chimeric polypeptide of any one of embodiments 102-104, wherein the VH domain comprises alanine (A) residue at position 93 in FR3.
        106. The chimeric polypeptide of any one of embodiments 102-105, wherein the VH domain comprises a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, and an alanine (A) residue at position 93 in FR3.
        10−7. The chimeric polypeptide of any one of embodiments 102-106, wherein the VH domain comprises a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and a leucine (L) residue at position 108 in FR4.
        108. The chimeric polypeptide of any one of embodiments 102-10−7, wherein the VL domain comprises at least one of: a tyrosine (Y) residue at position 87 in FR3 and/or a glutamine (Q) residue at position 100 in FR4, wherein the FR numbering is according to Kabat.
        109. The chimeric polypeptide of any one of embodiments 102-108, wherein the VL domain comprises a tyrosine (Y) residue at position 87 in FR3 and a glutamine (Q) residue at position 100 in FR4.
        110. The chimeric polypeptide of any one of embodiments 102-109, wherein the first antigen binding domain comprises a VH domain comprising an amino acid sequence of SEQ ID NO: 576 and a VL domain comprising an amino acid sequence of SEQ ID NO: 577.
        111. The chimeric polypeptide of any one of embodiments 1-110, wherein the first antigen binding domain comprises:
        
            i) a VH domain comprising an amino acid sequence of SEQ ID NO: 468 and a VL domain comprising an amino acid sequence of SEQ ID NO: 469;
            ii) a VH domain comprising an amino acid sequence of SEQ ID NO: 466 and a VL domain comprising an amino acid sequence of SEQ ID NO: 467;
            iii) a VH domain comprising an amino acid sequence of SEQ ID NO: 490 and a VL domain comprising an amino acid sequence of SEQ ID NO: 491;
            iv) a VH domain comprising an amino acid sequence of SEQ ID NO: 492 and a VL domain comprising an amino acid sequence of SEQ ID NO: 493;
            v) a VH domain comprising an amino acid sequence of SEQ ID NO: 514 and a VL domain comprising an amino acid sequence of SEQ ID NO: 515;
            vi) a VH domain comprising an amino acid sequence of SEQ ID NO: 516 and a VL domain comprising an amino acid sequence of SEQ ID NO: 517;
            vii) a VH domain comprising an amino acid sequence of SEQ ID NO: 538 and a VL domain comprising an amino acid sequence of SEQ ID NO: 539; or
            viii) a VH domain comprising an amino acid sequence of SEQ ID NO: 540 and a VL domain comprising an amino acid sequence of SEQ ID NO: 541.
        
        
        112. The chimeric polypeptide of any one of embodiments 1-111, wherein the VL domain is N-terminal to the VH domain.
        113. The chimeric polypeptide of any one of embodiments 1-111, wherein the VL domain is C-terminal to the VH domain.
        114. The chimeric polypeptide of any one of embodiments 1-113, wherein the second antigen binding domain comprises a scFV comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:
    
    









(SEQ ID NO: 128)


ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLI


 


GGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVF


 


GGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQLVESGGGI


 


VQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATY


 


YADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSW


 


FAHWGQGTLVTVSS.






    
    
        115. The chimeric polypeptide of any one of embodiments 1-114, wherein the first antigen binding domain comprises a scFV comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:
    
    









(SEQ ID NO: 449)


DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD


 


ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQ


 


GTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVK


 


PSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNP


 


SLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGT


 


LVTVSS.






    
    
        116. The chimeric polypeptide of any one of embodiments 1-115, wherein the RS comprises a protease cleavage site is cleavable by at least one protease listed in Table 6.
        117. The chimeric polypeptide of any one of embodiments 1-115, wherein the RS comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 7a.
        118. The chimeric polypeptide of any one of embodiments 1-115, wherein the RS is cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.
        119. The chimeric polypeptide of any one of embodiments 1-115, wherein the RS is not cleavable by legumain.
        120. The chimeric polypeptide of embodiment 119, wherein the RS is not cleavable by legumain in human blood, plasma, or serum.
        121. The chimeric polypeptide of embodiment 119 or 120, wherein the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours.
        122. The chimeric polypeptide of any one of embodiments 119-121, wherein the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.
        123. The chimeric polypeptide of embodiment 122, wherein legumain cleaves the RS in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO: 7048) is cleaved by legumain.
        124. The chimeric polypeptide of embodiment 122, wherein legumain cleaves the RS in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO: 7048) is cleaved by legumain.
        125. The chimeric polypeptide of embodiment 122, wherein legumain cleaves the RS in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO: 7048) is cleaved by legumain.
        126. The chimeric polypeptide of embodiment 122, wherein legumain cleaves the RS in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO: 7048) is cleaved by legumain.
        127. The chimeric polypeptide of embodiment 122, wherein legumain cleaves the RS in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO: 7048) is cleaved by legumain.
        128. The chimeric polypeptide of any one of embodiments 32-115, wherein the RS1 and/or RS2 comprises protease cleavage is cleavable by at least one protease listed in Table 6.
        129. The chimeric polypeptide of any one of embodiments 32-115, wherein the RS1 and/or RS2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 7a.
        130. The chimeric polypeptide of any one of embodiments 32-115, wherein the RS1 and/or RS2 is cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.
        131. The chimeric polypeptide of any one of embodiments 32-115, wherein the RS1 and/or RS2 is not cleavable by legumain.
        132. The chimeric polypeptide of embodiment 131, wherein the RS1 and/or RS2 is not cleavable by legumain in human blood, plasma, or serum.
        133. The chimeric polypeptide of embodiment 131 or 132, wherein the RS1 and/or RS2 is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours.
        134. The chimeric polypeptide of embodiment 131 or 132, wherein the RS1 and/or RS2 is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.
        135. The chimeric polypeptide of embodiment 134, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
        136. The chimeric polypeptide of embodiment 134, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
        137. The chimeric polypeptide of embodiment 134, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
        138. The chimeric polypeptide of embodiment 134, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
        139. The chimeric polypeptide of embodiment 134, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
        140. The chimeric polypeptide of any one of embodiments 32-139, wherein the RS1 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
        141. The chimeric polypeptide of any one of embodiments 32-140, wherein the RS2 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
        142. The chimeric polypeptide of any one of embodiments 32-141, wherein RS1 and/or RS2 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSASHTPAGLTGP (SEQ ID NO: 7628).
        143. The chimeric polypeptide of any one of embodiments 32-142, wherein the RS1 and the RS2 are the same.
        144. The chimeric polypeptide of any one of embodiments 32-142, wherein the RS1 and the RS2 are different.
        145. The chimeric polypeptide of any one of embodiments 34-144, wherein the first ELNN and the second ELNN are each individually characterized in that:
        
            (i) at least 90% of each of the first ELNN's and the second ELNN's amino acids are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E), proline (P), or any combination thereof; and
            (ii) each comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
        
        
        146. The chimeric polypeptide of embodiment 145, wherein the first ELNN and the second ELNN are each individually further characterized in that:
        
            (i) each comprises at least 100 amino acid residues;
            (ii) each comprises a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acids in length, wherein the plurality of non-overlapping sequence motifs comprise a set of non-overlapping sequence motives, wherein each non-overlapping sequence motive of the set of non-overlapping sequence motifs is repeated at least two times in the ELNN.
        
        
        147. The chimeric polypeptide of embodiment 146, wherein the plurality of non-overlapping sequence motifs comprises at least one non-overlapping sequence motif that occurs only once within the ELNN.
        148. The chimeric polypeptide of embodiment 146 or 147, wherein the non-overlapping sequence motifs comprise one of or any combination of the sequence motifs listed in Table 1.
        149. The chimeric polypeptide of embodiment 146 or 147, wherein the non-overlapping sequence motifs comprise at least 2, 3, or 4 of the sequence motifs listed in Table 1.
        150. The chimeric polypeptide of embodiment 146 or 147, wherein the non-overlapping sequence motifs comprise any one of or any combination of GTSTEPSEGSAP (SEQ ID NO:189), GTSESATPESGP (SEQ ID NO:188), GSGPGTSESATP (SEQ ID NO:8028), GSEPATSGSETP (SEQ ID NO: 187), GSPAGSPTSTEE (SEQ ID NO:186), and GTSPSATPESGP (SEQ ID NO:8029).
        151. The chimeric polypeptide of any one of embodiments 145-150, wherein each of the first ELNN and the second ELNN comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
        152. The chimeric polypeptide of any one of embodiments 145-151, wherein the amino acids of each of the first ELNN and the second ELNN consists of A, E, G, S, P, and/or T.
        153. The chimeric polypeptide of any one of embodiments 145-152, wherein the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN.
        154. The chimeric polypeptide of any one of embodiments 145-152, wherein the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN.
        155. The chimeric polypeptide of any one of embodiments 145-152, wherein the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN.
        156. The chimeric polypeptide of any one of embodiments 145-152, wherein the amino acid sequence of the first ELNN is between 250 amino acids and 350 amino acids in length, and wherein the amino acid sequence of the second ELNN is between 500 amino acids and 600 amino acids in length.
        157. The chimeric polypeptide of any one of embodiments 145-152, wherein the amino acid sequence of the first ELNN is 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is 582 amino acids in length.
        158. The chimeric polypeptide of any one of embodiments 145-157, wherein the first ELNN and/or the second ELNN comprises an amino acid sequence that is at least 85% identical to an amino acid sequence listed in Table 3a or 3b.
        159. The chimeric polypeptide of any one of embodiments 145-158, wherein the first ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:
    
    









(SEQ ID NO: 8021)


ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSE


 


SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS


 


PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT


 


STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES


 


GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE


 


GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP.






    
    
        160. The chimeric polypeptide of any one of embodiments 145-159, wherein the second ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:
    
    









(SEQ ID NO: 8022)


ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATS


 


GSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG


 


SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE


 


EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG


 


SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP


 


AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE


 


PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS


 


GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE


 


SGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE


 


EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATPESGPG


 


SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS


 


TEPSEGSAPGSEPATSGSETPGTSESAGEPEA.






    
    
        161. The chimeric polypeptide of any one of embodiments 1-160, comprising one or more barcode fragments.
        162. The chimeric polypeptide of any one of embodiments 1-161, comprising two or more barcode fragments.
        163. The chimeric polypeptide of embodiment 161 or 162, wherein each barcode fragment is different from every other barcode fragment.
        164. The chimeric polypeptide of any one of embodiments 161-163, wherein each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that are releasable from the chimeric polypeptide upon complete digestion the chimeric polypeptide by a non-mammalian protease.
        165. The chimeric polypeptide of embodiment 164, wherein the non-mammalian protease is Glu-C.
        166. The chimeric polypeptide of any one of embodiments 1-165, comprising a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG (SEQ ID NO:8030), SGSETPGT (SEQ ID NO:8031), and GTSESATP (SEQ ID NO:8032).
        167. The chimeric polypeptide of any one of embodiments 1-165, comprising at least one of the following amino acid sequences: SGPE.SGPGXnSGPE.SGPG (SEQ ID NO:8033),
    
    











(SEQ ID NO: 8034)



SGPE.SGPGXnATPE.SGPG,



 



(SEQ ID NO: 8036)



SGPE.SGPGXnGTSE.SATP,



 



(SEQ ID NO: 8037)



SGPE.SGPGXnTTPE.SGPG,



 



(SEQ ID NO: 8038)



SGPE.SGPGXnSTPE.SGPG,



 



(SEQ ID NO: 8039)



SGPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8040)



SGPE.SGPGXnGTPE.TPGS,



 



(SEQ ID NO: 8040)



SGPE.SGPGXnGTPE.TPGS,



 



(SEQ ID NO: 8041)



SGPE.SGPGXnSGSE.TGTP,



 



(SEQ ID NO: 8042)



SGPE.SGPGXnGTPE.GSAP,



 



(SEQ ID NO: 8043)



SGPE.SGPGXnEPSE.SATP,



 



(SEQ ID NO: 8044)



ATPE.SGPGXnSGPE.SGPG,



 



(SEQ ID NO: 8045)



ATPE.SGPGXnATPE.SGPG,



 



(SEQ ID NO: 8047)



ATPE.SGPGXnGTSE.SATP,



 



(SEQ ID NO: 8049)



ATPE.SGPGXnTTPE.SGPG,



 



(SEQ ID NO: 8051)



ATPE.SGPGXnSTPE.SGPG,



 



(SEQ ID NO: 8053)



ATPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8055)



ATPE.SGPGXnGTPE.TPGS,



 



(SEQ ID NO: 8056)



ATPE.SGPGXnSGSE.TGTP,



 



(SEQ ID NO: 8057)



ATPE.SGPGXnGTPE.GSAP,



 



(SEQ ID NO: 8058)



ATPE.SGPGXnEPSE.SATP,



 



(SEQ ID NO: 8059)



GTSE.SATPXnSGPE.SGPG,



 



(SEQ ID NO: 8060)



GTSE.SATPXnATPE.SGPG,



 



(SEQ ID NO: 8061)



GTSE.SATPXnGTSE.SATP,



 



(SEQ ID NO: 8062)



GTSE.SATPXnTTPE.SGPG,



 



(SEQ ID NO: 8063)



GTSE.SATPXnSTPE.SGPG,



 



(SEQ ID NO: 8064)



GTSE.SATPXnGTPE.SGPG,



 



(SEQ ID NO: 8065)



GTSE.SATPXnGTPE.TPGS,



 



(SEQ ID NO: 8066)



GTSE.SATPXnSGSE.TGTP,



 



(SEQ ID NO: 8067)



GTSE.SATPXnGTPE.GSAP,



 



(SEQ ID NO: 8068)



GTSE.SATPXnEPSE.SATP,



 



(SEQ ID NO: 8069)



TTPE.SGPGXnSGPE.SGPG,



 



(SEQ ID NO: 8070)



TTPE.SGPGXnATPE.SGPG,



 



(SEQ ID NO: 8071)



TTPE.SGPGXnGTSE.SATP,



 



(SEQ ID NO: 8072)



TTPE.SGPGXnTTPE.SGPG,



 



(SEQ ID NO: 8074)



TTPE.SGPGXnSTPE.SGPG,



 



(SEQ ID NO: 8075)



TTPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8076)



TTPE.SGPGXnGTPE.TPGS,



 



(SEQ ID NO: 8077)



TTPE.SGPGXnSGSE.TGTP,



 



(SEQ ID NO: 8078)



TTPE.SGPGXnGTPE.GSAP,



 



(SEQ ID NO: 8079)



TTPE.SGPGXnEPSE.SATP,



 



(SEQ ID NO: 8080)



STPE.SGPGXnSGPE.SGPG,



 



(SEQ ID NO: 8081)



STPE.SGPGXnATPE.SGPG,



 



(SEQ ID NO: 8082)



STPE.SGPGXnGTSE.SATP,



 



(SEQ ID NO: 8083)



STPE.SGPGXnTTPE.SGPG,



 



(SEQ ID NO: 8084)



STPE.SGPGXnSTPE.SGPG,



 



(SEQ ID NO: 8086)



STPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8087)



STPE.SGPGXnGTPE.TPGS,



 



(SEQ ID NO: 8088)



STPE.SGPGXnSGSE.TGTP,



 



(SEQ ID NO: 8089)



STPE.SGPGXnGTPE.GSAP,



 



(SEQ ID NO: 8090)



STPE.SGPGXnEPSE.SATP,



 



(SEQ ID NO: 8091)



GTPE.SGPGXnSGPE.SGPG,



 



(SEQ ID NO: 8092)



GTPE.SGPGXnATPE.SGPG,



 



(SEQ ID NO: 8093)



GTPE.SGPGXnGTSE.SATP,



 



(SEQ ID NO: 8094)



GTPE.SGPGXnTTPE.SGPG,



 



(SEQ ID NO: 8096)



GTPE.SGPGXnSTPE.SGPG,



 



(SEQ ID NO: 8098)



GTPE.SGPGXnGTPE.SGPG,



 



(SEQ ID NO: 8100)



GTPE.SGPGXnGTPE.TPGS,



 



(SEQ ID NO: 8101)



GTPE.SGPGXnSGSE.TGTP,



 



(SEQ ID NO: 8102)



GTPE.SGPGXnGTPE.GSAP,



 



(SEQ ID NO: 8103)



GTPE.SGPGXnEPSE.SATP,



 



(SEQ ID NO: 8104)



GTPE.TPGSXnSGPE.SGPG,



 



(SEQ ID NO: 8105)



GTPE.TPGSXnATPE.SGPG,



 



(SEQ ID NO: 8106)



GTPE.TPGSXnGTSE.SATP,



 



(SEQ ID NO: 8107)



GTPE.TPGSXnTTPE.SGPG,



 



(SEQ ID NO: 8108)



GTPE.TPGSXnSTPE.SGPG,



 



(SEQ ID NO: 8109)



GTPE.TPGSXnGTPE.SGPG,



 



(SEQ ID NO: 8110)



GTPE.TPGSXnGTPE.TPGS,



 



(SEQ ID NO: 8111)



GTPE.TPGSXnSGSE.TGTP,



 



(SEQ ID NO: 8113)



GTPE.TPGSXnGTPE.GSAP,



 



(SEQ ID NO: 8114)



GTPE.TPGSXnEPSE.SATP,



 



(SEQ ID NO: 8115)



SGSE.TGTPXnSGPE.SGPG,



 



(SEQ ID NO: 8116)



SGSE.TGTPXnATPE.SGPG,



 



(SEQ ID NO: 8117)



SGSE.TGTPXnGTSE.SATP,



 



(SEQ ID NO: 8118)



SGSE.TGTPXnTTPE.SGPG,



 



(SEQ ID NO: 8119)



SGSE.TGTPXnSTPE.SGPG,



 



(SEQ ID NO: 8120)



SGSE.TGTPXnGTPE.SGPG,



 



(SEQ ID NO: 8121)



SGSE.TGTPXnGTPE.TPGS,



 



(SEQ ID NO: 8122)



SGSE.TGTPXnSGSE.TGTP,



 



(SEQ ID NO: 8123)



SGSE.TGTPXnGTPE.GSAP,



 



(SEQ ID NO: 8124)



SGSE.TGTPXnEPSE.SATP,



 



(SEQ ID NO: 8125)



GTPE.GSAPXnSGPE.SGPG,



 



(SEQ ID NO: 8126)



GTPE.GSAPXnATPE.SGPG,



 



(SEQ ID NO: 8127)



GTPE.GSAPXnGTSE.SATP,



 



(SEQ ID NO: 8128)



GTPE.GSAPXnTTPE.SGPG,



 



(SEQ ID NO: 8129)



GTPE.GSAPXnSTPE.SGPG,



 



(SEQ ID NO: 8130)



GTPE.GSAPXnGTPE.SGPG,



 



(SEQ ID NO: 8131)



GTPE.GSAPXnGTPE.TPGS,



 



(SEQ ID NO: 8132)



GTPE.GSAPXnSGSE.TGTP,



 



(SEQ ID NO: 8133)



GTPE.GSAPXnGTPE.GSAP,



 



(SEQ ID NO: 8134)



GTPE.GSAPXnEPSE.SATP,



 



(SEQ ID NO: 8136)



EPSE.SATPXnSGPE.SGPG,



 



(SEQ ID NO: 8137)



EPSE.SATPXnATPE.SGPG,



 



(SEQ ID NO: 8138)



EPSE.SATPXnGTSE.SATP,



 



(SEQ ID NO: 8139)



EPSE.SATPXnTTPE.SGPG,



 



(SEQ ID NO: 8140)



EPSE.SATPXnSTPE.SGPG,



 



(SEQ ID NO: 8141)



EPSE.SATPXnGTPE.SGPG,



 



(SEQ ID NO: 8142)



EPSE.SATPXnGTPE.TPGS,



 



(SEQ ID NO: 8143)



EPSE.SATPXnSGSE.TGTP,



 



(SEQ ID NO: 8144)



EPSE.SATPXnGTPE.GSAP,



or



 



(SEQ ID NO: 8145)



EPSE.SATPXnEPSE.SATP,






    
    
        wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50.
        168. The chimeric polypeptide of embodiment 167, comprising at least one of the following amino acid sequences: SGPE.SGPGXnATPE.SGPG (SEQ ID NO:8035), ATPE.SGPGXnGTSE.SATP (SEQ ID NO: 8048), ATPE.SGPGXnTTPE.SGPG (SEQ ID NO:8050), ATPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8052), ATPE.SGPGXnATPE.SGPG (SEQ ID NO:8046), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO: 8054), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:8054), ATPE.SGPGXnATPE.SGPG (SEQ ID NO: 8046), GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:8099), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8097), GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:8095), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8097), GTPE.TPGSXnSGSE.TGTP (SEQ ID NO:8112), GTPE.GSAPXnEPSE.SATP (SEQ ID NO: 8135), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:8054), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO: 8054), ATPE.SGPGXnATPE.SGPG (SEQ ID NO:8046), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO: 8054), TTPE.SGPGXnTTPE.SGPG (SEQ ID NO:8073), or STPE.SGPGXnSTPE.SGPG (SEQ ID NO: 8085), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 30.
        169. The chimeric polypeptide of embodiments 167 or 168, wherein n is any integer from 1 to 20.
        170. The chimeric polypeptide of any one of embodiments 167-169, wherein n is any integer from 5 to 15.
        171. The chimeric polypeptide of any one of embodiments 167-169, wherein n is any integer from 3 to 7.
        172. The chimeric polypeptide of any one of embodiments 167-169, wherein n is any integer from 5 to 10.
        173. The chimeric polypeptide of any one of embodiments 167-169, wherein n is 9.
        174. The chimeric polypeptide of any one of embodiments 167-169, wherein n is 4.
        175. The chimeric polypeptide of any one of embodiments 167-174, wherein Xn is PGTGTSAT (SEQ ID NO: 8146), PGSGPGT (SEQ ID NO:8147), PGTTPGTT (SEQ ID NO:8148), PGTPPTST (SEQ ID NO: 8149), PGTSPSAT (SEQ ID NO:8150), PGTGSAGT (SEQ ID NO:8151), PGTGGAGT (SEQ ID NO: 8152), PGTSPGAT (SEQ ID NO:8153), PGTSGSGT (SEQ ID NO:8154), PGTSSAST (SEQ ID NO: 8155), PGTGAGTT (SEQ ID NO:8156), PGTGSTST (SEQ ID NO:8157), GSEPATSG (SEQ ID NO: 8158), APGTSTEP (SEQ ID NO:8159), PGTAGSGT (SEQ ID NO:8160), PGTSSGGT (SEQ ID NO: 8161), PGTAGPAT (SEQ ID NO:8162), PGTPGTGT (SEQ ID NO:8163), PGTGGPTT (SEQ ID NO: 8164), or PGTGSGST (SEQ ID NO:8165).
        176. The chimeric polypeptide of any one of embodiments 167-174, wherein X11 is TGTS (SEQ ID NO: 8166), SGP, TTPG (SEQ ID NO:8167), TPPT (SEQ ID NO:8168), TSPS (SEQ ID NO:8169), TGSA (SEQ ID NO:8170), TGGA (SEQ ID NO:8171), TSPG (SEQ ID NO:8172), TSGS (SEQ ID NO: 8173), TSSA (SEQ ID NO:8174), TGAG (SEQ ID NO:8175), TGST (SEQ ID NO:8176), EPAT (SEQ ID NO:8177), GTST (SEQ ID NO:8178), TAGS (SEQ ID NO:8179), TSSG (SEQ ID NO: 8180), TAGP (SEQ ID NO:8181), TPGT (SEQ ID NO:8182), TGGP (SEQ ID NO:8183), or TGSG (SEQ ID NO:8184).
        177. The chimeric polypeptide of any one of embodiments 1-176, wherein neither the N-terminal amino acid nor the C-terminal amino acid of the chimeric polypeptide is included in a barcode fragment.
        178. The chimeric polypeptide of any one of embodiments 19-177, comprising an ELNN with a non-overlapping sequence motif that occurs only once within the ELNN, wherein the ELNN further comprises a barcode fragment that includes at least part of the non-overlapping sequence motif that occurs only once within the ELNN.
        179. The chimeric polypeptide of any one of embodiments 19-177, comprising a first ELNN with a first barcode fragment and a second ELNN with a second barcode fragment, wherein neither the first barcode fragment nor the second barcode fragment includes a glutamate that is immediately adjacent to another glutamate, if present, in the ELNN that contains the barcode fragment.
        180. The chimeric polypeptide of embodiment 179, wherein at least one of the barcode fragments comprises a glutamate at the C-terminus thereof.
        181. The chimeric polypeptide of embodiments 178 or 179, wherein at least one of the barcode fragments has an N-terminal amino acid that is immediately preceded by a glutamate in the chimeric polypeptide.
        182. The chimeric polypeptide of embodiment 181, wherein the glutamate that precedes the N-terminal amino acid of the barcode fragment is not immediately adjacent to another glutamate.
        183. The chimeric polypeptide of any one of embodiments 179-182, wherein at least one of the barcode fragments does not include a second glutamate at a position other than the C-terminus of the barcode fragment unless the second glutamate is immediately followed by a proline.
        184. The chimeric polypeptide of any one of embodiments 1-183, comprising a single polypeptide chain, wherein the chimeric polypeptide comprises a barcode fragment that is at a position within the polypeptide chain that is from 10 to 200 amino acids or from 10 to 125 amino acids from the N-terminus or the C-terminus of the chimeric polypeptide.
        185. The chimeric polypeptide of any one of embodiments 34-184, wherein the first ELNN is at the N-terminal side of the bispecific antibody domain, and wherein the first barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the N-terminus of the chimeric polypeptide.
        186. The chimeric polypeptide of any one of embodiments 34-184, wherein the second ELNN is at the C-terminal side of the bispecific antibody domain, and wherein the second barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.
        187. The chimeric polypeptide of any one of embodiments 161-186, wherein at least one of the barcode fragments is at least 4 amino acids in length.
        188. The chimeric polypeptide of any one of embodiments 161-187, wherein at least one of the barcode fragments is from 4 to 20, from 5 to 15, from 6 to 12, or from 7 to 10 amino acids in length.
        189. The chimeric polypeptide of embodiment 188, wherein each mask polypeptide comprises one barcode fragment that is listed in Table 2 or disclosed in Table 3a.
        190. The chimeric polypeptide of any one of embodiments 1-189, comprising a barcode fragment comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SGPGSGPGTSE (SEQ ID NO:78) or SGPGTSPSATPE (SEQ ID NO:79).
        191. The chimeric polypeptide of any one of embodiments 1-189, comprising one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGSGPGTSE (SEQ ID NO:78) and one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGTSPSATPE (SEQ ID NO:79).
        192. The chimeric polypeptide of any one of embodiments 189-191, wherein the barcode fragment consists of A, E, G, S, P, and/or T residues.
        193. The chimeric polypeptide of any one of embodiments 189-192 wherein the barcode fragment is part of a mask peptide.
        194. The chimeric polypeptide of embodiment 193, wherein the mask peptide is the first ELNN or the second ELNN.
        195. A chimeric polypeptide, comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:
    
    









(SEQ ID NO: 1000)


ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSE


 


SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS


 


PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT


 


STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES


 


GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE


 


GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA


 


SHTPAGLTGPGTSESATPESDIQMTQSPSSLSASVGDRVTITCQASQDIS


 


NYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP


 


EDIATYYCQHFDHLPLAFGQGTKVEIKSESATPESGPGTSPGATPESGPG


 


TSESATPQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPP


 


GKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA


 


VYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQEPSLTVSPGGTVT


 


LTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL


 


EGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGP


 


GTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFS


 


TYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNT


 


LYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATP


 


ESGPGEAGRSASHTPAGLTGPATPESGPGTSESATPESGPGSPAGSPTST


 


EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


 


GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT


 


STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP


 


ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP


 


SEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT


 


STEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


 


TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


 


GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS


 


PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE


 


SATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS


 


PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGE


 


PEA.






    
    
        196. The chimeric polypeptide of embodiment 195, comprising the following amino acid sequence:
    
    









(SEQ ID NO: 1000)


ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSE


 


SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS


 


PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT


 


STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES


 


GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE


 


GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA


 


SHTPAGLTGPGTSESATPESDIQMTQSPSSLSASVGDRVTITCQASQDIS


 


NYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP


 


EDIATYYCQHFDHLPLAFGQGTKVEIKSESATPESGPGTSPGATPESGPG


 


TSESATPQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPP


 


GKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA


 


VYYCARDRVTGAFDIWGQGTLVTVSSGGGGSELVVTQEPSLTVSPGGTVT


 


LTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL


 


EGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLSESATPESGP


 


GTSPGATPESGPGTSESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFS


 


TYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNT


 


LYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATP


 


ESGPGEAGRSASHTPAGLTGPATPESGPGTSESATPESGPGSPAGSPTST


 


EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


 


GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT


 


STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP


 


ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP


 


SEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT


 


STEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


 


TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


 


GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS


 


PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE


 


SATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS


 


PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGE


 


PEA.






    
    
        197. A pharmaceutical composition comprising the chimeric polypeptide of any one of embodiments 1-196 and at least one pharmaceutically acceptable excipient.
        198. The pharmaceutical composition of embodiment 197, which is in a liquid form or is frozen.
        199. The pharmaceutical composition of embodiment 197, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
        200. An injection device comprising the pharmaceutical composition of embodiment 197.
        201. The injection device of embodiment 200, which comprises a syringe.
        202. A polynucleotide sequence encoding the chimeric polypeptide of any one of embodiments 1-196.
        203. An expression vector comprising the polynucleotide sequence of embodiment 202.
        204. A host cell comprising the expression vector of embodiment 203.
        205. A method of producing the chimeric polypeptide of any one of embodiments 1-196.
        206. The method of embodiment 205, further comprising isolating the chimeric polypeptide from a host cell.
        207. A method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of the chimeric polypeptide of any one of embodiments 1-196 to the subject.
        208. The method of embodiment 207, wherein the cancer comprises a solid tumor.
        209. The method of embodiment 207 or 208, wherein the cancer is a carcinoma, a sarcoma, or a melanoma.
        210. The method of any one of embodiments 207-209, wherein the cancer expresses EGFR.
        211. The method of any one of embodiments 207-209, wherein the cancer overexpresses EGFR.
        212. The method of any one of embodiments 207-209, wherein the cancer comprises cells that express, on average, at least 3,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; 100,000; or 200,000EGFR proteins per cell.
        213. The method of any one of embodiments 207-209, wherein the cancer comprises cells having one or more oncogenic mutations in an EGFR gene.
        214. The method of any one of embodiments 207-209, wherein the cancer comprises cells having an EGFR gene amplification.
        215. The method of embodiment 214, wherein the cells comprise a 2 to 5-fold, 2 to 10-fold, 2 to 15-fold, 2 to 30-fold, 2 to 50-fold, 3 to 5-fold, 3 to 10-fold, 3 to 15-fold, 3 to 30-fold, 3 to 50-fold, 5 to 10-fold, 5 to 15-fold, 5 to 30-fold, or 5 to 50-fold increase in EGFR gene copy number as compared to a non-cancerous cell of the same tissue type.
        216. The method of any one of embodiments 207-209, wherein the cancer is lung cancer, colorectal cancer, head and neck cancer, breast cancer, pancreatic cancer, brain cancer, liver cancer, kidney cancer, ovarian cancer, prostate cancer, esophageal cancer, cervical cancer, or bladder cancer.
        217. The method of any one of embodiments 207-209, wherein the cancer is lung cancer.
        218. The method of embodiment 217, wherein the lung cancer is non-small cell lung cancer.
        219. The method of any one embodiments 207-209, wherein the cancer is colorectal cancer.
        220. The method of any one of embodiments 207-209, wherein the cancer is head and neck squamous cell carcinoma.
        221. The method of any one of embodiments 207-209, wherein the cancer is breast cancer.
        222. The method of embodiment 221, wherein the cancer is triple-negative breast cancer.
        223. The method of any one of embodiments 207-209, wherein the cancer is brain cancer.
        224. The method of embodiment 223, wherein the brain cancer is glioblastoma.
        225. The method of any one of embodiments 207-224, further comprising administering a checkpoint inhibitor to the subject.
        226. The method of embodiment 225, wherein the checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
        227. The method of embodiment 225, wherein the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
        228. The method of embodiment 225, wherein the checkpoint inhibitor is pembrolizumab or cemiplimab.
        229. An antibody or an antigen-binding fragment thereof that specifically binds EGFR, comprising:
        a VH domain comprising
        
            a CDR1 amino acid sequence of GGSVSSGDYYWT (SEQ ID NO: 562), a CDR2 amino acid sequence of HIYYSGNTNYNPSLKS (SEQ ID NO: 563), and a CDR3 amino acid sequence of DRVTGAFDI (SEQ ID NO: 564); and
            at least one of: a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine residue at position 89 in FR3, an alanine residue at position 93 in FR3, and/or a leucine residue at position 108 in FR4, wherein the FR numbering is according to Kabat; and
        
        
        a VL domain comprising
        
            a CDR1 amino acid sequence of QASQDISNYLN (SEQ ID NO: 565), a CDR2 amino acid sequence of DASNLET (SEQ ID NO: 566), a CDR3 amino acid sequence of QHFDHLPLA (SEQ ID NO: 567).
        
        
        230. The antibody or an antigen-binding fragment thereof of embodiment 229, wherein the VH domain comprises an asparagine (N) residue at position 76 in FR3.
        231. The antibody or an antigen-binding fragment thereof of embodiment 229 or 230, wherein the VH domain comprises alanine (A) residue at position 93 in FR3.
        232. The antibody or an antigen-binding fragment thereof of any one of embodiments 229-231, wherein the VH domain comprises a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, and an alanine (A) residue at position 93 in FR3.
        233. The antibody or an antigen-binding fragment thereof of any one of embodiments 229-232, wherein the VH domain comprises a proline (P) residue at position 40 in FR2, a valine (V) residue at position in position 67 in FR3, a valine (V) residue at position 71 in FR3, an asparagine (N) residue at position 76 in FR3, a valine (V) residue at position 89 in FR3, an alanine (A) residue at position 93 in FR3, and a leucine (L) residue at position 108 in FR4.
        234. The antibody or an antigen-binding fragment thereof of any one of embodiment 229-233, wherein the VL domain comprises at least one of: a tyrosine (Y) residue at position 87 in FR3 and/or a glutamine (Q) residue at position 100 in FR4, wherein the FR numbering is according to Kabat.
        235. The antibody or an antigen-binding fragment thereof of any one of embodiments 229-234, wherein the VL domain comprises a tyrosine (Y) residue at position 87 in FR3 and a glutamine (Q) residue at position 100 in FR4.
        236. The antibody or an antigen-binding fragment of any one of embodiments 229-235, comprising a VH domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 576; and a VL domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 577.
        237. The antibody of embodiment 236, comprising:
        
            i) a VH domain comprising an amino acid sequence of SEQ ID NO: 468 and a VL domain comprising an amino acid sequence of SEQ ID NO: 469;
            ii) a VH domain comprising an amino acid sequence of SEQ ID NO: 466 and a VL domain comprising an amino acid sequence of SEQ ID NO: 467;
            iii) a VH domain comprising an amino acid sequence of SEQ ID NO: 490 and a VL domain comprising an amino acid sequence of SEQ ID NO: 491;
            iv) a VH domain comprising an amino acid sequence of SEQ ID NO: 492 and a VL domain comprising an amino acid sequence of SEQ ID NO: 493;
            v) a VH domain comprising an amino acid sequence of SEQ ID NO: 514 and a VL domain comprising an amino acid sequence of SEQ ID NO: 515;
            vi) a VH domain comprising an amino acid sequence of SEQ ID NO: 516 and a VL domain comprising an amino acid sequence of SEQ ID NO: 517;
            vii) a VH domain comprising an amino acid sequence of SEQ ID NO: 538 and a VL domain comprising an amino acid sequence of SEQ ID NO: 539; or
            viii) a VH domain comprising an amino acid sequence of SEQ ID NO: 540 and a VL domain comprising an amino acid sequence of SEQ ID NO: 541.
        
        
        238. An anti-CD3 antibody or an antigen-binding fragment thereof, comprising the following CDRs:
        a VH domain comprising a CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), a CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and a CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and
        a VL domain comprising a CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO:
        1), a CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and a CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6).
        239. The anti-CD3 antibody or an antigen-binding fragment thereof of embodiment 238, wherein:
        the VL domain comprises the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTN KRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLT VL (SEQ ID NO: 127); and
        the VH domain comprises the amino acid sequence of
    
    









(SEQ ID NO: 126)


EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG


 


RIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC


 


VRHENFGNSYVSWFAHWGQGTLVTVSS.






The following are examples of compositions and evaluations of compositions of the disclosure. It is understood that various some embodiments may be practiced, given the general description provided above.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
EXAMPLES
Example 1. Improved anti-EGFR binding domains
This example demonstrates the engineering, selection, and characterization of anti-EGFR antibody fragments in a paTCE with improved properties, for example, improved thermostability.
A parental anti-EGFR scFv molecule, EGFR.2, is previously described in Internal Patent Publication No. WO/2020/264208. EGFR.2 includes the VH and VL sequences of a human anti-EGFR antibody, panitumumab. The EGFR.2 scFv molecule was determined to have low thermostability, limiting its expression and its developability in a therapeutic context.
In order to identify anti-EGFR antibody fragments with improved properties, the CDRs of panitumumab (Table 8) were grafted in a combinatorial manner into the framework regions from approved monoclonal antibody therapies (VL Table 9 and VH Table 10).







TABLE 8







Panitumumab CDRs










VL

VH



CDR
Sequence
CDR
Sequence





CDR-
QASQDISNYLN
CDR-
GGSVSSGDYYWT


L1
(SEQ ID NO: 565)
H1
(SEQ ID NO: 562)


 


CDR-
DASNLET
CDR-
HIYYSGNTNYNPSLKS


L2
(SEQ ID NO: 566)
H2
(SEQ ID NO: 563)


 


CDR-
QHFDHLPLA
CDR-
DRVTGAFDI


L3
(SEQ ID NO: 567)
H3
(SEQ ID NO: 564)
















TABLE 9







VL Framework regions











VL
VL FR1
VL FR2
VL FR3
VL FR4





Panitu-
DIQMTQSPS
WYQQKPGK
GVPSRFSGSG
FGGGTK


mumab
SLSASVGDR
APKLLIY
SGTDFTFTIS
VEIK



VTITC
(SEQ ID
SLQPEDIATY
(SEQ 



(SEQ ID
NO:
FC
ID



NO: 8209)
8210)
(SEQ ID 
NO: 





NO: 8216)
8221)


 


Donor-
DIQMTQSPS
WYQQKPGK
GVPSRFSGSG
FGQGTK


FW4
SLSASVGDR
APKLLIY
SGTDFTFTIS
VEIK*


mutation
VTITC
(SEQ ID
SLQPEDIATY
(SEQ 



(SEQ ID
NO:
FC
ID



NO: 8209)
8210) 
(SEQ ID 
NO: 





NO: 8216)
8212)


 


IGKV1-
DIQMTQSPS
WYQQKPGK
GVPSRFSGSG
FGQGTK


33
SLSASVGDR
APKLLIY
SGTDFTFTIS
VEIK*



VTITC
(SEQ ID
SLQPEDIATY
(SEQ 



(SEQ ID
NO:
YC
ID



NO: 8209) 
8210) 
(SEQ ID 
NO: 





NO: 8211)
8212)


 


IGKV1D-
DIQMTQSPS
WYQQKPGK
GVPSRFSGSG
FGQGTK


39
SLSASVGDR
APKLLIY
SGTDFTLTIS
VEIK*



VTITC
(SEQ ID
SLQPEDFATY
(SEQ 



(SEQ ID
NO:
YC
ID



NO: 8209)
8210) 
(SEQ ID 
NO: 





NO: 8217)
8212)


 


IGKV3-
EIVLTQSPG
WYQQKPGQ
GIPDRFSGSG
FGQGTK


20
TLSLSPGER
APRLLIY
SGTDFTLTIS
VEIK*



ATLSC
(SEQ ID
RLEPEDFAVY
(SEQ 



(SEQ ID 
NO: 
YC 
ID 



NO: 8213)
8215)
(SEQ ID
NO:





NO: 8218)
8212)


 


IGKV3-
EIVLTQSPA
WYQQKPGQ
GIPARFSGSG
FGQGTK


11
TLSLSPGER
APRLLIY
SGTDFTLTIS
VEIK*



ATLSC
(SEQ ID
SLEPEDFAVY
(SEQ 



(SEQ ID 
NO: 
YC 
ID 



NO: 8214)
8215)
(SEQ ID
NO:





NO: 8219)
8212)


 


IGKV1D-
DIQMTQSPS
WYQQKPGK
GVPSRFSGSG
FGQGTK


39
SLSASVGDR
APKLLIY
SGTDFTLTIS
VEIK*


(+2)
VTITC
(SEQ ID
SLQPEDFATY
(SEQ 



(SEQ ID 
NO: 
FC 
ID 



NO: 8209)
8210)
(SEQ ID
NO:





NO: 8220)
8212)


 


IGKV1-
DIQMTQSPS
WYQQKPGK
GVPSRFSGSG
FGQGTK


33
SLSASVGDR
APKLLIY
SGTDFTFTIS
VEIK*


(+2)
VTITC
(SEQ ID
SLQPEDIATY
(SEQ 



(SEQ ID 
NO: 
FC
ID 



NO: 8209)
8210)
(SEQ ID 
NO:





NO: 8216)
8212)





*Sequences from Ling et al. Front. Immunol. Vol. 9 (2018). doi.org/10.3389/fimmu.2018.00469













TABLE 10







VH Framework regions











VH
VH FR1
VH FR2
VH FR3
VH FR4





Panitu-
QVQLQESGP
WIRQSPG
RLTISIDTSK
WGQGTM


mumab
GLVKPSETL
KGLEWIG
TQFSLKLSSV
VTVSS



SLTCTVS
(SEQ ID 
TAADTAIYYC
(SEQ 



(SEQ ID
NO:
VR
ID



NO: 8206)
8233)
(SEQ ID 
NO: 





NO: 8237)
8290)


 


IGHV1-
QVQLVQSGA
WVRQAPG
RVTSTRDTSI
WGQGTL


2
EVKKPGASV
QGLEWMG
STAYMELSRL
VTVSS*



KVSCKAS
(SEQ ID 
RSDDTVVYYC
(SEQ 



(SEQ ID
NO:
AR
ID 



NO: 8223)
8234)
(SEQ ID
NO:





NO: 8238)
67)


 


IGHV1-
QVQLVQSGA
WVRQAPG
RVTMTRDTST
WGQGTL


46
EVKKPGASV
QGLEWMG
STVYMELSSL
VTVSS*



KVSCKAS
(SEQ ID 
RSEDTAVYYC
(SEQ 



(SEQ ID 
NO:
AR
ID



NO: 8223)
8234)
(SEQ ID
NO:





NO: 8239)
67)


 


IGHV1-
QVQLVQSGA
WVRQAPG
RVTITADEST
WGQGTL


69
EVKKPGSSV
QGLEWMG
STAYMELSSL
VTVSS*



KVSCKAS
(SEQ ID
RSEDTAVYYC 
(SEQ



(SEQ ID
NO:
AR
ID



NO: 8224) 
8234)
(SEQ ID
NO: 





NO: 8240)
67)


 


IGHV3-
EVQLLESGG
WVRQAPG
RFTISRDNSK
WGQGTL


23
GLVQPGGSL
KGLEWVS
NTLYLQMNSL
VTVSS*



RLSCAAS
(SEQ ID
RAEDTAVYYC
(SEQ



(SEQ ID
NO:
AK
ID



NO: 8225)
8235)
(SEQ ID
NO: 





NO: 8241)
67)


 


IGHV3-
QVQLVESGG
WVRQAPG
RFTISRDNSK
WGQGTL


30-3
GVVQPGRSL
KGLEWVA
NTLYLQMNSL
VTVSS*



RLSCAAS
(SEQ ID
RAEDTAVYYC
(SEQ



(SEQ ID
NO:
AR
ID



NO: 8226)
64)
(SEQ ID
NO: 





NO: 8242)
67)


 


IGHV3-
EVQLVESGG
WVRQAPG
RFTISRDNAK
WGQGTL


7
GLVQPGGSL
KGLEWVA
NSLYLQMNSL
VTVSS*



RLSCAAS
(SEQ ID
RAEDTAVYYC
(SEQ



(SEQ ID
NO:
AR
ID



NO: 8227)
64)
(SEQ ID
NO: 





NO: 8243)
67)


 


IGHV3-
EVQLVESGG
WVRQAPG
RFTISRDNSK
WGQGTL


66
GLVQPGGSL
KGLEWVS
NTLYLQMNSL
VTVSS*



RLSCAAS
(SEQ ID
RAEDTAVYYC
(SEQ



(SEQ ID
NO:
AR
ID



NO: 8227)
8235)
(SEQ ID
NO: 





NO: 8242)
67)


 


IGHV4-
QVQLQQWGA
WIRQPPG
RVTISVDTSK
WGQGTL


34
GLLKPSETL
KGLEWIG
NQFSLKLSSV
VTVSS*



SLTCAVY
(SEQ ID
TAADTAVYYC
(SEQ



(SEQ ID
NO:
AR
ID



NO: 8228)
8207)
(SEQ ID
NO:



 
 
NO: 8208)
67)


 


IGHV4-
QVQLQESGP
WIRQPPG
RVTISVDTSK
WGQGTL


59
GLVKPSETL
KGLEWIG
NQFSLKLSSV
VTVSS*



SLTCTVS
(SEQ ID
TAADTAVYYC
(SEQ



(SEQ ID
NO:
AR
ID 



NO: 8206)
8207)
(SEQ ID
NO:



 
 
NO: 8208)
67)


 


IGHV5-
EVQLVQSGA
WVRQMPG
QVTISADKSI
WGQGTL


51
EVKKPGESL
KGLEWMG
STAYLQWSSL
VTVSS*



KISCKGS
(SEQ ID
KASDTAMYYC
(SEQ



(SEQ ID
NO: 
AR
ID 



NO: 8230)
8236)
(SEQ ID
NO:



 

NO: 8244)
67)


 


IGHV7-
QVQLVQSGS
WVRQAPG
RFVFSLDTSV
WGQGTL


4-1
ELKKPGASV
QGLEWMG
STAYLQICSL
VTVSS*



KVSCKAS
(SEQ ID
KAEDTAVYYC 
(SEQ



(SEQ ID
NO: 
AR
ID



NO: 8231)
8234)
(SEQ ID
NO:



 

NO: 8245)
67)


 


VH1 
QVQLVQSGV
WVRQAPG
RVTLTTDSST
WGQGTL


(Ling)
EVKKPGASV
QGLEWMG*
TTAYMELKSL
VTVSS*



KVSCKAS*
(SEQ ID
QFDDTAVYYC
(SEQ



(SEQ ID
NO: 
AR
ID



NO: 8232)
8234)
(SEQ ID
NO:



 

NO: 8246)
67)


 


IGHV3-
QVQLVESGG
WVRQAPG
RLTISRDNSK
WGQGTL


30-
GVVQPGRSL
KGLEWVA
NTLYLQMNSL
VTVSS*


3(+2)
RLSCAAS
(SEQ ID 
RAEDTAVYY
(SEQ



(SEQ ID
NO:
CVR
ID



NO: 8226)
64)
(SEQ ID
NO:





NO: 8247)
67)


 


IGHV3-
EVQLVESGG
WVRQAPG
RLTISRDNAK
WGQGTL


7(+2)
GLVQPGGSL
KGLEWVA
NSLYLQMNSL
VTVSS*



RLSCAAS
(SEQ ID
RAEDTAVYY
(SEQ



(SEQ ID
NO:
CVR
ID



NO: 8227)
64)
(SEQ ID
NO: 





NO: 8248)
67)


 


IGHV1-
QVQLVQSGA
WVRQAPG
RLTITADEST
WGQGTL


69(+2)
EVKKPGSSV
QGLEWMG
STAYMELSSL
VTVSS*



KVSCKAS
(SEQ ID
RSEDTAVYY
(SEQ



(SEQ ID
NO:
CVR
ID



NO: 8224)
8234)
(SEQ ID 
NO: 





NO: 8249)
67)





*FW sequences from Ling et al. Front. Immunol. Vol. 9 (2018). Doi.org/10.3389/fimmu.2018.00469






The resulting library included approximately 56 anti-EGFR scFvs in paTCE format in combination with anti-CD3 scFv CD3.23. The paTCE library having the anti-EGFR scFvs was screened with the goal of identifying anti-EGFR antibody fragments with improved stability and expression while also exhibiting favorable binding and immunogenicity profiles (FIG. 4). Of the approximately 56 anti-EGFR containing paTCEs screened, 26 were expressed in an amount adequate to further characterize the binding and stability. The 56 constructs were expressed as a pool in a 10 L E. coli fermentation run. The protein pool was purified and screened as follows: 1) Magnetic beads coated with human EGFR were incubated with the protein pool at room temperature. The beads were washed, and the bound protein was eluted and analyzed by mass spectrometry for construct identification. 2) The protein pool was heated to 62° C. The heated sample was run through a size exclusion chromatography (SEC) column for separation of monomer from aggregated proteins. The monomer fraction was analyzed by mass spectrometry for construct identification. The screening results are provided in Table 11 and FIG. 5A (screen for thermal stability), FIG. 5B (screen for binding affinity), and FIG. 5C (thermostability ratio).








TABLE 11







Screening of anti-EGFR scFvs in a paTCE















AC#







Thermo-


(with





Amount
Thermal
stability


CD3.23)
EGFR
CDR Donor
VL FW
VH FW
Expression
Bound
Stability
ratio





AC2884
EGFR.36
Panitumumab
IGKV1-33
IGHV4-34
Low
High
High
0.76


AC2885
EGFR.37
Panitumumab
IGKV1-33
IGHV4-59
Med
High
High
0.78


AC2896
EGFR.48
Panitumumab
IGKV1D-39
IGHV4-34
Med
Med
High
0.76


AC2897
EGFR.49
Panitumumab
IGKV1D-39
IGHV4-59
Med
Med
High
0.76


AC2908
EGFR.60
Panitumumab
IGKV3-20
IGHV4-34
High
Med
High
0.77


AC2909
EGFR.61
Panitumumab
IGKV3-20
IGHV4-59
High
High
High
0.94


AC2920
EGFR.72
Panitumumab
IGKV3-11
IGHV4-34
Med
Med
High
0.80


AC2921
EGFR.73
Panitumumab
IGKV3-11
IGHV4-59
High
High
High
0.86


AC2876
EGFR.2
Panitumumab
Panitumumab
Panitumumab
Low
High
Low
0.31



(parent)









AC2879
EGFR.31
Panitumumab
IGKV1-33
IGHV1-69
Low
Low
Low
0.13


AC2887
EGFR.39
Panitumumab
IGKV1-33
IGHV7-4-1
Low
Low
Low
0.00


AC2888
EGFR.40
Panitumumab
IGKV1-33
VH1 (Ling)
Low
Low
Low
0.00


AC2890
EGFR.42
Panitumumab
IGKV1D-39
IGHV1-46
High
Low
Low
0.10


AC2891
EGFR.43
Panitumumab
IGKV1D-39
IGHV1-69
Med
Low
Low
0.24


AC2895
EGFR.47
Panitumumab
IGKV1D-39
IGHV3-66
Low
Low
Low
0.16


AC2900
EGFR.52
Panitumumab
IGKV1D-39
VH1 (Ling)
Low
Low
Low
0.37


AC2903
EGFR.55
Panitumumab
IGKV3-20
IGHV1-69
High
Low
Low
0.18


AC2905
EGFR.57
Panitumumab
IGKV3-20
IGHV3-30-3
Med
Low
Low
0.14


AC2906
EGFR.58
Panitumumab
IGKV3-20
IGHV3-7
High
Low
Low
0.22


AC2907
EGFR.59
Panitumumab
IGKV3-20
IGHV3-66
Low
Low
Low
0.11


AC2914
EGFR.66
Panitumumab
IGKV3-11
IGHV1-46
High
Low
Low
0.04


AC2915
EGFR.67
Panitumumab
IGKV3-11
IGHV1-69
High
Low
Low
0.04


AC2918
EGFR.70
Panitumumab
IGKV3-11
IGHV3-7
Med
Low
Low
0.37


AC2919
EGFR.71
Panitumumab
IGKV3-11
IGHV3-66
Med
Low
Low
0.12


AC2922
EGFR.74
Panitumumab
IGKV3-11
IGHV5-51
Med
Low
Low
0.04


AC2931
EGFR.87
Panitumumab
Panitumumab +
Panitumumab +
Low
High
Low
0.05





FRW4mut
FRW4mut






AC2877
EGFR.29
Panitumumab
IGKV1-33
IGHV1-2
Low
n.d.
n.d.
n.d.


AC2878
EGFR.30
Panitumumab
IGKV1-33
IGHV1-46
Low
n.d.
n.d.
n.d.


AC2880
EGFR.32
Panitumumab
IGKV1-33
IGHV3-23
Low
n.d.
n.d.
n.d.


AC2881
EGFR.33
Panitumumab
IGKV1-33
IGHV3-30-3
Low
n.d.
n.d.
n.d.


AC2882
EGFR.34
Panitumumab
IGKV1-33
IGHV3-7
Low
n.d.
n.d.
n.d.


AC2883
EGFR.35
Panitumumab
IGKV1-33
IGHV3-66
Low
n.d.
n.d.
n.d.


AC2886
EGFR.38
Panitumumab
IGKV1-33
IGHV5-51
Low
n.d.
n.d.
n.d.


AC2889
EGFR.41
Panitumumab
IGKV1D-39
IGHV1-2
Low
n.d.
n.d.
n.d.


AC2892
EGFR.44
Panitumumab
IGKV1D-39
IGHV3-23
Low
n.d
n.d.
n.d.


AC2893
EGFR.45
Panitumumab
IGKV1D-39
IGHV3-30-3
Low
n.d.
n.d.
n.d.


AC2894
EGFR.46
Panitumumab
IGKV1D-39
IGHV3-7
Low
n.d.
n.d.
n.d.


AC2898
EGFR.50
Panitumumab
IGKV1D-39
IGHV5-51
Low
n.d.
n.d.
n.d.


AC2899
EGFR.51
Panitumumab
IGKV1D-39
IGHV7-4-1
Low
n.d.
n.d.
n.d.


AC2901
EGFR.53
Panitumumab
IGKV3-20
IGHV1-2
Low
n.d.
n.d.
n.d.


AC2902
EGFR.54
Panitumumab
IGKV3-20
IGHV1-46
Low
n.d.
n.d.
n.d.


AC2904
EGFR.56
Panitumumab
IGKV3-20
IGHV3-23
Low
n.d.
n.d.
n.d.


AC2910
EGFR.62
Panitumumab
IGKV3-20
IGHV5-51
Low
n.d.
n.d.
n.d.


AC2911
EGFR.63
Panitumumab
IGKV3-20
IGHV7-4-1
Low
n.d.
n.d.
n.d.


AC2912
EGFR.64
Panitumumab
IGKV3-20
VH1 (Ling)
Low
n.d.
n.d.
n.d.


AC2913
EGFR.65
Panitumumab
IGKV3-11
IGHV1-2
Low
n.d.
n.d.
n.d.


AC2916
EGFR.68
Panitumumab
IGKV3-11
IGHV3-23
Low
n.d.
n.d.
n.d.


AC2917
EGFR.69
Panitumumab
IGKV3-11
IGHV3-30-3
Low
n.d.
n.d.
n.d.


AC2923
EGFR.75
Panitumumab
IGKV3-11
IGHV7-4-1
Low
n.d.
n.d.
n.d.


AC2924
EGFR.76
Panitumumab
IGKV3-11
VH1 (Ling)
Low
n.d.
n.d.
n.d.


AC2925
EGFR.81
Panitumumab
IGKV1D-
IGHV3-30-
Low
n.d.
n.d.
n.d.





39(+2)
3(+2)






AC2926
EGFR.82
Panitumumab
IGKV1D-
IGHV3-7(+2)
Low
n.d.
n.d.
n.d.





39(+2)







AC2927
EGFR.83
Panitumumab
IGKV1D-
IGHV1-
Low
n.d.
n.d.
n.d.





39(+2)
69(+2)






AC2928
EGFR.84
Panitumumab
IGKV1-33
IGHV3-30-
Low
n.d.
n.d.
n.d.





(+2)
3(+2)






AC2929
EGFR.85
Panitumumab
IGKV1-33
IGHV3-7(+2)
Low
n.d.
n.d.
n.d.





(+2)







AC2930
EGFR.86
Panitumumab
IGKV1-33
IGHV1-
Low
n.d.
n.d.
n.d.





(+2)
69(+2)





n.d. = no data






Anti-EGFR variants in a paTCE format together with CD3.23 were co-expressed and purified as a pool. The pool was subjected to various temperatures for 30 minutes (unheated, heated at 58° C., and heated at 62° C.) to induce denaturation and therefore aggregation. The pool was subsequently placed on ice. The thermostable, monomeric variants which survived the heated conditions were separated from the aggregated variants using anion exchange chromatography. The unheated condition and heated monomeric fractions were run on LCMS to determine individual abundance of each monomeric variant as compared to the input. To analyze the data and select hits: the abundance of each variant in the heated monomeric fraction at 62° C. was divided by its abundance in the unheated, control sample (input).
The thermostability ratio above and in FIG. 5C shows the amount of thermostable monomeric protein remaining at 62° C. divided by the input amount. A thermostability ratio value of less than 0.5 suggests that less than 50% protein remains monomeric at 62° C. (e.g., has formed denatured aggregates) and therefore the melting Temperature™ of the protein is less than 62° C. By contrast, a thermostability ratio of more than 0.5 suggests that more than 50% protein remains monomeric at 62° C. and therefore the Tm of the protein in greater than 62° C. FIG. 5C shows the thermostability ratio of the paTCE including EGFR.2/CD3.23 is 0.3 at 62° C., suggesting a Tm of less than 62° C. By contrast, each of the thermostable anti-EGFR variants in combination with CD3.23 has a thermostability ratio of greater than 0.5 at 62° C., suggesting that each of EGFR.36/CD3.23, EGFR.37/CD3.23, EGFR.48/CD3.23, EGFR.49/CD3.23, EGFR.60/CD3.23, EGFR.61/CD3.23, EGFR.72/CD3.23, and EGFR.73/CD3.23 have a Tm of greater than 62° C.
The VH and VL amino acid sequences of the parent anti-EGFR scFv, EGFR.2, and selected thermostable variants are provided in Table 12 (VL), Table 13 (VH). For screening purposes, the anti-EGFR scFv format was VL-linker-VH, with the linker having that amino acid sequence of GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 84).







TABLE 12







Sequences of select anti-EGFR scFvs: VL








anti-



EGFR VL
Amino acid sequence





EGFR.2
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWY


(parent)
QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT



FTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK



(SEQ ID NO: 451)


 


EGFR.36
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWY



QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT



FTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIK



(SEQ ID NO: 469)


 


EGFR.37
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWY



QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT



FTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIK



(SEQ ID NO: 469)


 


EGFR.48
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWY



QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT



LTISSLQPEDFATYYCQHFDHLPLAFGQGTKVEIK



(SEQ ID NO: 477)


 


EGFR.49
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWY



QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT



LTISSLQPEDFATYYCQHFDHLPLAFGQGTKVEIK



(SEQ ID NO: 477)


 


EGFR.60
EIVLTQSPGTLSLSPGERATLSCQASQDISNYLNWY



QQKPGQAPRLLIYDASNLETGIPDRFSGSGSGTDFT



LTISRLEPEDFAVYYCQHFDHLPLAFGQGTKVEIK



(SEQ ID NO: 501)


 


EGFR.61
EIVLTQSPGTLSLSPGERATLSCQASQDISNYLNWY



QQKPGQAPRLLIYDASNLETGIPDRFSGSGSGTDFT



LTISRLEPEDFAVYYCQHFDHLPLAFGQGTKVEIK



(SEQ ID NO: 501)


 


EGFR.72
EIVLTQSPATLSLSPGERATLSCQASQDISNYLNWY



QQKPGQAPRLLIYDASNLETGIPARFSGSGSGTDFT



LTISSLEPEDFAVYYCQHFDHLPLAFGQGTKVEIK



(SEQ ID NO: 525)


 


EGFR.73
EIVLTQSPATLSLSPGERATLSCQASQDISNYLNWY



QQKPGQAPRLLIYDASNLETGIPARFSGSGSGTDFT



LTISSLEPEDFAVYYCQHFDHLPLAFGQGTKVEIK



(SEQ ID NO: 525)
















TABLE 13







Sequences of select anti-EGFR scFvs: VH








anti-



EGFR VH
Amino acid sequence





EGFR.2
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYW


(parent)
TWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTIS



IDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDI



WGQGTMVTVSS



(SEQ ID NO: 450)


 


EGFR.36
QVQLQQWGAGLLKPSETLSLTCAVYGGSVSSGDYYW



TWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTIS



VDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDI



WGQGTLVTVSS



(SEQ ID NO: 466)


 


EGFR.37
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYW



TWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTIS



VDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDI



WGQGTLVTVSS



(SEQ ID NO: 468)


 


EGFR.48
QVQLQQWGAGLLKPSETLSLTCAVYGGSVSSGDYYW



TWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTIS



VDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDI



WGQGTLVTVSS



(SEQ ID NO: 466)


 


EGFR.49
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYW



TWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTIS



VDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDI



WGQGTLVTVSS



(SEQ ID NO: 468)


 


EGFR.60
QVQLQQWGAGLLKPSETLSLTCAVYGGSVSSGDYYW



TWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTIS



VDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDI



WGQGTLVTVSS



(SEQ ID NO: 466)


 


EGFR.61
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYW



TWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTIS



VDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDI



WGQGTLVTVSS



(SEQ ID NO: 468)


 


EGFR.72
QVQLQQWGAGLLKPSETLSLTCAVYGGSVSSGDYYW



TWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTIS



VDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDI



WGQGTLVTVSS



(SEQ ID NO: 466)


 


EGFR.73
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYW



TWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTIS



VDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDI



WGQGTLVTVSS



(SEQ ID NO: 468)









An alignment of the VH and VL of parental EGFR.2 and selected thermostable variants is provided below (CDRs underlined; differences relative to EGFR.2 highlighted).












VL alignment









EGFR.2
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPS
 60


 


EGFR.36
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPS
 60


 


EGFR.37
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPS
 60


 


EGFR.48
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPS
 60


 


EGFR.49
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPS
 60


 


EGFR.60
EIVLTQSPGTLSLSPGERATLSCQASQDISNYLNWYQQKPGQAPRLLIYDASNLETGIPD
 60


 


EGFR.61
EIVLTQSPGTLSLSPGERATLSCQASQDISNYLNWYQQKPGQAPRLLIYDASNLETGIPD
 60


 


EGFR.72
EIVLTQSPATLSLSPGERATLSCQASQDISNYLNWYQQKPGQAPRLLIYDASNLETGIPA
 60


 


EGFR.73
EIVLTQSPATLSLSPGERATLSCQASQDISNYLNWYQQKPGQAPRLLIYDASNLETGIPA
 60



:* :****.:** * *:*.*::*******************:**:************:*



 


EGFR.2
RFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK  
107



(SEQ ID NO: 451)



 


EGFR.36
RFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIK  
107



(SEQ ID NO: 453)



 


EGFR.37
RFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIK  
107



(SEQ ID NO: 453)



 


EGFR.48
RFSGSGSGTDFTLTISSLQPEDFATYYCQHFDHLPLAFGQGTKVEIK  
107



(SEQ ID NO: 477)



 


EGFR.49
RFSGSGSGTDFTLTISSLQPEDFATYYCQHFDHLPLAFGQGTKVEIK  
107



(SEQ ID NO: 477)



 


EGFR.60
RFSGSGSGTDFTLTISRLEPEDFAVYYCQHFDHLPLAFGQGTKVEIK  
107



(SEQ ID NO: 501)



 


EGFR.61
RFSGSGSGTDFTLTISRLEPEDFAVYYCQHFDHLPLAFGQGTKVEIK  
107



(SEQ ID NO: 501)



 


EGFR.72
RFSGSGSGTDFTLTISSLEPEDFAVYYCQHFDHLPLAFGQGTKVEIK  
107



(SEQ ID NO: 525)



 


EGFR.73
RFSGSGSGTDFTLTISSLEPEDFAVYYCQHFDHLPLAFGQGTKVEIK  
107



(SEQ ID NO: 525)




************:*** *:***:*.*:************ *******





Bold: VL mutations relative to EGFR.2


Bold, double underline. VL mutations conserved in thermostable variants






The VL sequences of the thermostable variants included the VL framework regions of IGKV1-33, IGKV1D-39, IGKV3-20, or IGKV3-11 (each with VL FW4 from Ling). Two conserved mutations (F87Y and G100Q, shown in bold in the VL alignment above, numbering according to Kabat) were identified that are present in each of the thermostable variants and which are not present in the donor EGFR.2 VL.












VH alignment









EGFR.2
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTN
 60


 


EGFR.36
QVQLQQWGAGLLKPSETLSLTCAVYGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN
 60


 


EGFR.37
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN
 60


 


EGFR.48
QVQLQQWGAGLLKPSETLSLTCAVYGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN
 60


 


EGFR.49
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN
 60


 


EGFR.60
QVQLQQWGAGLLKPSETLSLTCAVYGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN
 60


 


EGFR.61
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN
 60


 


EGFR.72
QVQLQQWGAGLLKPSETLSLTCAVYGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN
 60


 


EGFR.73
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTN
 60



*****: * **:**********:* **************** ******************



 


EGFR. 2
YNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS
119


 
(SEQ ID NO: 450)



 


EGFR.36
YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS
119



(SEQ ID NO: 466)



 


EGFR.37
YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS
119


 
(SEQ ID NO: 468)



 


EGFR.48
YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS
119


 
(SEQ ID NO: 466)



 


EGFR.49
YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS
119


 
(SEQ ID NO: 468)



 


EGFR.60
YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS
119



(SEQ ID NO: 466)



 


EGFR.61
YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS
119



(SEQ ID NO: 468)



 


EGFR.72
YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS
119



(SEQ ID NO: 466)



 


EGFR.73
YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS
119



(SEQ ID NO: 468)




********:***:****.***************:***.***************:*****





Bold: VH mutations relative to EGFR.2


Bold, double underline. VH mutations conserved in thermostable variants






Each of the VH sequences of the thermostable variants included the VH FW regions of either IGHV4-34 or IGHV4-59 (each with VH FW4 from Ling). Seven conserved mutations (S40P, L67V, 171V. T76N, 189V. V93A, and M108L; shown in bold in the VH alignment above; numbering according to Kabat) were identified that are present in each of the thermostable variants and which are not present in the donor EGFR.2 VH.
To determine the structural basis for the observed improved thermostability in the anti-EGFR variants, SWISS MODEL was used to simulate the EGFR.2 Fab, using an existing structure of Panitumumab (PDB 5SX4). The same template was used to generate a model of EGFR.2 having a T76N mutation in VH FW3 and thermostable variant EGFR.61 which, like all the other improved thermostable variants, includes the T76N mutation in VH FW3. SWISS MODEL Deep View was used to determine molecular energy using GROMOS. Individual residues and their hydrogen bond partners were analyzed. Residues that resulted in a significant change in energy were analyzed in PyMol. The T76N mutation in the VH FW3 appears to reduce electrostatic clash resulting in a favorable change in free energy. Addition of the T76N mutation resulted in free energy change of −175 (relative to EGFR.2 without the mutation), with a negative change in free energy being predictive of increased stability. As shown in FIG. 5D, the asparagine mutation at position 76 results in a formation of a hydrogen bond with a valine residue at position 29 in the VH CDR1, which may account, at least in part, for the increased stability and decreased binding affinity observed in the thermostable variants.
Based on the screening data, anti-EGFR scFvs were selected for further characterization as individual constructs. Each construct was expressed in small-scale and purified. Thermal stability was determined by Differential Scanning Fluorimetry. Melting temperature (Tm) determined by Differential Scanning Fluorimetry experiments described herein were performed either in duplicate or triplicate using GloMelt™ dye and QuantStudio5™. GloMelt™ dye undergoes fluorescence enhancement upon binding to hydrophobic regions of denatured proteins. Therefore, the dye can be used to detect protein unfolding or measure thermal stability. GloMelt™ dye is optimized for detection in the SYBR® Green channel of qPCR instruments. The Differential Scanning Fluorimetry experiments were performed in 96-well plates with 10 μg protein/reaction (equal to 0.5 mg/mL final protein concentration), reaction buffer, and GloMelt™ 10× dye according to manufacturer's instructions. Fluorescence of 96-well plates was read and melt curve plots were generated in the QuantStudio5TM qPCR system. Binding affinity was analyzed with bio-layer interferometry at room temperature with human EGFR as the antigen. The potency of each construct was determined by in vitro cytotoxicity in an HT-29 cell line with an Effector to Target (E: T) ratio of 5 to 1.
The results are provided in Table 14. The loss of potency observed corresponds to the loss in affinity for the anti-EGFR scFvs observed during screening and confirmed here. This result is favorable since decreasing the potency of the paTCE can result in a safer molecule. EGFR.2 was previously shown to cause CRS toxicity, so the lower potency molecules discovered here are desired (see also Example 3).







TABLE 14







Characterization of anti-EGFR scFvs in a paTCE














anti-
Anti-

EGFR

EC50HT-



EGFR
CD3
Tm
KD
CD3 KD
29


AC#
scFV
scFv
(° C.)*
(nM)
(nM)
(pM)
















AC1955
EGFR.2
CD3.9
n.d.
0.72
99
2-4


AC2885
EGFR.37
CD3.23
66.7
2.3
203
20-29


AC2897
EGFR.49
CD3.23
66.9
7.2
approx. 203
n.d.


AC2908
EGFR.60
CD3.23
66.6
7.3
approx. 203
94


AC2909
EGFR.61
CD3.23
67.0
2.4
approx. 203
34-40


AC2920
EGFR.72
CD3.23
67.0
4.4
approx. 203
85


AC2921
EGFR.73
CD3.23
66.9
2.45
approx. 203
43-44





*Thermostability determined for uTCE; after unmasking by proteolytic cleavage of N- and C-terminal ELNNs






EGFR.37 was selected for further characterization. The thermostability of AMX-525 (EGFR.37 in combination with CD3.318) was compared to the thermostability of the parent anti-EGFR paTCE described in Internal Patent Publication No. WO/2020/264208 (EGRF-XPAT gen1; construct ID pJB0169) which includes EGFR.2 in combination with CD3.9. Thermal stability was determined by Differential Scanning Fluorimetry. The results in Table 15 demonstrate that AMX-525 (EGFR.37/CD3.318) is more stable than the EGFR-XPAT gen1 (EGFR.2/CD3.9). Further comparisons of AMX-525 and EGFR-XPAT gen1 are also provided in Example 3.







TABLE 15







Characterization of anti-EGFR scFvs in a paTCE











anti-EGFR
Anti-CD3
Tm


AC#
scFV
scFv
(° C.)*













AC1955 (EGFR-XPAT gen1)
EGFR.2
CD3.9
59.9


AC4230 (AMX-525)
EGFR.37
CD3.318
68.3





*Thermostability determined for masked paTCE






Example 2. Improved Anti-CD3 Binding Domains
CD3 scFv paTCE arm optimization was conducted to reduce molecule immunogenicity and improve stability, while maintaining binding affinity with CD3 close to the affinity observed for the CD3.23 parental molecule. Putative T cell epitope (PTE) scores were calculated based on a proprietary computer prediction program, where a lower PTE score is predictive of decreased immunogenicity.
To achieve this, Pool 1 was created, which included 74 paTCE molecules, each containing an anti-PSMA VHH and one of the 74 CD3.23 mutation variants. The amino acid sequences of each of the 74 CD3.23 mutation variants are provided in Table 18. Single mutations were chosen based on analyses including CD3.23 PTE score analysis (using internal PTE algorithm v12) and structural analysis. Structural considerations included: possible contact disruption, anticipated steric clashes, side chain charge maintenance and possible pockets filling. Stability and affinity of the individually expressed molecules in the form of crude lysate was evaluated by Octet (ForteBio).
Based on the results of the Pool 1 screening, mutations that did not disrupt paTCE molecule affinity and stability were taken further to evaluate as combinations in Pool 2. Pool 2 consisted of paTCE molecules each containing an anti-PSMA VHH and one of 64 CD3.23 mutation combination variants. The amino acid sequences of each of the 64 CD3.23 mutation combination variants are provided in Table 19. Stability and affinity of the individually expressed molecules in the form of crude lysate was evaluated by Octet. The four most stable paTCE molecules from Pool 2 were additionally expressed in a larger volume (2.5 L) and purified. The binding of these anti-CD3 molecules (CD3.227, CD3.228, CD3.229 and CD3.230) to human and cynomolgus CD3 was measured by Octet and the Tm was measured by Differential Scanning Fluorimetry. All variants were paired with an anti-PSMA VHH. Values are reported below in Table 16. Based on these data that included an additional PTE score evaluation using internal PTE algorithm v22 (FIG. 6), CD3.228 was chosen from Pool 2 over the other leads in Table 19.







TABLE 16







Binding affinities, melting temperatures, and PTE values for select CD3 antibodies from Pool 2




















kon
kdiss
PTE



Anti-CD3
KD,
kon (1/Ms),
kdiss (1/s),
KD,
(1/Ms),
(1/s),
score
Tm


scFv
huCD3e-Fc
huCD3e-Fc
huCD3e-Fc
cyCD3e-Fc
cyCD3e-Fc
cyCD3e-Fc
(v22)
(° C.)





CD3.227
 57 nM
3.42E+05
1.96E−02
 80 nM
3.15E+05
2.53E−02
10
63.71


CD3.228
 69 nM
3.17E+05
2.17E−02
 80 nM
3.34E+05
2.66E−02
10
64.45


CD3.229
162 nM
3.21E+05
5.20E−02
193 nM
3.17E+05
6.11E−02
15
63.46


CD3.230
195 nM
3.25E+05
6.33E−02
216 nM
3.36E+05
7.26E−02
15
63.71


CD3.23
131 nM
2.20E+05
2.89E−02
130 nM
2.40E+05
3.12E−02
73
62.62









PTE score analysis of the top molecules from Pool 2 (CD3.228, CD3.229 and CD3.230) was performed using PTE algorithm v12. Lowering PTE score mutations were chosen to address potential immunogenicity of two peptide clusters in VH and one peptide cluster in VL. Stability enhancing mutations from Pool 1 (L67D and G68E) were incorporated in the Pool 3 design. Mutations, that potentially detune CD3 binding affinity, were also tested: mutations in CDR-H3 (N100A or S100A) and mutations that detuned CD3 binding as demonstrated in Pool 1 (W47D, V48G, K52bP, A56T, Y58T, Y59D, Y59W). In total-69 new combinations of mutations in the context of single CD3 domain having a 144 amino acid C-terminal ELNN mask, a 144 amino acid N-terminal ELNN mask, and without a tumor binder were evaluated for anti-CD3 binding affinity, stability, and immunogenicity risk. Based on the expression, binding, and stability data two anti-CD3 domains (CD3.295 and CD3.318) were expressed in a larger volume and purified. The binding of these anti-CD3 molecules to human CD3 was measured by Octet and the stability was measured by Differential Scanning Fluorimetry. Values are reported below in Table 17. The CD3.318 scFv was chosen to be combined with anti-EGFR for AMX-525 because of its low immunogenicity risk as determined by internal PTE algorithm v22 (FIG. 6) and increased stability relative to either CD3.23 or CD3.295.







TABLE 17







Binding affinities, stability, and PTE values


for select CD3 antibodies from Pool 3













KD
PTE score
% remaining


AC#
Anti-CD3 scFv
(nM)
(v22)
stability














AC3796
CD3.292*
3.89
10
n.d.


AC3799
CD3.295
3.11
3
73.85%


AC3822
CD3.318
5.38
4
85.02%


AC3768
CD3.23
4.67
n.d.
66.11%





*Clone CD3.292 is identical to CD3.228 but produced as a 144/144 masked scFv without tumor binder


n.d. = no data






An alignment of parental CD3.8, CD3.9, and CD3.23 and selected CD3.228 and CD3.318 VL and VH molecules with differences highlighted is provided below. CD3.8 and CD3.9 are humanized versions of the SP34 monoclonal mouse antibody. CD3.23 has 8 mutations compared to CD3.9 and has an estimated 2-4 fold lower affinity vs CD3.9 based on ELISA, Octet, and cell binding data. CD3.228 has 8 mutations compared to CD3.23 and 16 mutations compared to CD3.9. CD3.228 has increased stability and lower immunogenicity risk compared to CD3.23. CD3.318 has increased stability and lower immunogenicity risk as compared to CD3.23.












>CD3.8_VL


QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARF


SGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 358)


 


>CD3.9_VL


ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFS


GSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 359)


 


>CD3.23_VL


ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFS


GSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361)


 


>CD3.228_VL


ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFS


GSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361)


 


>CD3.318_VL


ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFS


GSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127)


 









CD3.8_VL
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGV
 60


 


CD3.9_VL
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGT
 60


 


CD3.23_VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGT
 60


 


CD3.228_VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGT
 60


 


CD3.318_VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGT
 60



: ******************** **.****:****************************.



 


CD3.8_VL
PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKHTVL
109



(SEQ ID NO: 358)



 


CD3.9_VL
PARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKHTVL
109



(SEQ ID NO: 108)



 


CD3.23_VL
PARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKHTVL
109



(SEQ ID NO: 101)



 


CD3.228_VL
PARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKHTVL
109



(SEQ ID NO: 101)



 


CD.318_VL
PARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKHTVL
109



(SEQ ID NO: 127)




********* *********.****** ******* **************








Bold: mutations relative to CD3.8


 


>CD3.8_VH


EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYY


ADSVKGRFTISRDDSKNTLYLQMNSLREADTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS


(SEQ ID NO: 308)


 


>CD3.9_VH


EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYY


ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS


S (SEQ ID NO: 309)


 


>CD3.23_VH


EVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA


DSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS


(SEQ ID NO: 102)


 


>CD3.228_VH


EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYA


DSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS


(SEQ ID NO: 311)


 


>CD3.318_VH


EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYA


DSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS


(SEQ ID NO: 126)


 









CD3.8_VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYAT 
 60


 


CD3.9_VH
EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYAT 
 60


 


CD3.23_VH
EVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYAT 
 60


 


CD3.228_VH
EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYAT 
 60


 


CD3.318_VH
EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYAT 
 60



****:*****:*******:**********.******************.***:* *:***



 


CD3.8_VH
YYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL 
120


 


CD3.9_VH
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL 
120


 


CD3.23_VH
YYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTL 
120


 


CD3.228_VH
YYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTL 
120


 


CD3.318_VH
YYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTL 
120



*******.************ *****.*::*********** ***********:******



 


CD3.8_VH
VTVSS
125 



(SEQ ID NO: 308)



 


CD3.9_VH
VTVSS
125 



(SEQ ID NO: 109)



 


CD3.23_VH
VTVSS
125 



(SEQ ID NO: 102)



 


CD3.228_VH
VTVSS
125 



(SEQ ID NO: 311)



 


CD3.318_VH
VTVSS
125 



(SEQ ID NO: 126)




*****





Bold: mutations relative to CD3.8


Bold, underlined: PTE removal mutations


Bold, double underlined: mutations relative to CD3.8 and PTE removal mutations













TABLE 18





Pool 1 CD3.23 Mutation Variants





















VL

VH




SEQ

SEQ




ID

ID


AC
VL sequence
NO:
VH sequence
NO:





AC3364
ELVVTQEPSLTVSPGGTVTLTCRSS
834
EVQLLESGGGIVQPGGSLKLSCAASGF
835



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3366
ELVVTQEPSLTVSPGGTVTLTCRSS
836
EVQLLESGGGIVQPGGSLKLSCAASGF
837



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3367
ELVVTQEPSLTVSPGGTVTLTCRSS
838
EVQLLESGGGIVQPGGSLKLSCAASGF
839



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEDVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3368
ELVVTQEPSLTVSPGGTVTLTCRSS
840
EVQLLESGGGIVQPGGSLKLSCAASGF
841



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEEVARIRS




GLIGGTNKRAPGTPARFSGSLLGG

KYNNYATYYADSVKDRFTISRDDSKN




KAALTLSGVQPEDEAVYYCALWY

TVYLQMNNLKTEDTAVYYCVRHENF




PNLWVFGGGTKLTVL

GNSYVSWFAHWGQGTLVTVSS



 


AC3369
ELVVTQEPSLTVSPGGTVTLTCRSS
842
EVQLLESGGGIVQPGGSLKLSCAASGF
843



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWAARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3370
ELVVTQEPSLTVSPGGTVTLTCRSS
844
EVQLLESGGGIVQPGGSLKLSCAASGF
845



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWEARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3371
ELVVTQEPSLTVSPGGTVTLTCRSS
846
EVQLLESGGGIVQPGGSLKLSCAASGF
847



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWGARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3372
ELVVTQEPSLTVSPGGTVTLTCRSS
848
EVQLLESGGGIVQPGGSLKLSCAASGF
849



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWSARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3373
ELVVTQEPSLTVSPGGTVTLTCRSS
850
EVQLLESGGGIVQPGGSLKLSCAASGF
851



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWTARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3374
ELVVTQEPSLTVSPGGTVTLTCRSS
852
EVQLLESGGGIVQPGGSLKLSCAASGF
853



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWWARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3375
ELVVTQEPSLTVSPGGTVTLTCRSS
854
EVQLLESGGGIVQPGGSLKLSCAASGF
855



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVDRIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3376
ELVVTQEPSLTVSPGGTVTLTCRSS
856
EVQLLESGGGIVQPGGSLKLSCAASGF
857



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVERIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3377
ELVVTQEPSLTVSPGGTVTLTCRSS
858
EVQLLESGGGIVQPGGSLKLSCAASGF
859



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVGRIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3378
ELVVTQEPSLTVSPGGTVTLTCRSS
860
EVQLLESGGGIVQPGGSLKLSCAASGF
861



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVAQIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3379
ELVVTQEPSLTVSPGGTVTLTCRSS
862
EVQLLESGGGIVQPGGSLKLSCAASGF
863



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVAGIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3380
ELVVTQEPSLTVSPGGTVTLTCRSS
864
EVQLLESGGGIVQPGGSLKLSCAASGF
865



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVAHIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3381
ELVVTQEPSLTVSPGGTVTLTCRSS
866
EVQLLESGGGIVQPGGSLKLSCAASGF
867



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVAPIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3382
ELVVTQEPSLTVSPGGTVTLTCRSS
868
EVQLLESGGGIVQPGGSLKLSCAASGF
869



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVAWI




GLIGGTNKRAPGTPARFSGSLLGG

RSKYNNYATYYADSVKDRFTISRDDS




KAALTLSGVQPEDEAVYYCALWY

KNTVYLQMNNLKTEDTAVYYCVRHE




PNLWVFGGGTKLTVL

NFGNSYVSWFAHWGQGTLVTVSS



 


AC3383
ELVVTQEPSLTVSPGGTVTLTCRSS
870
EVQLLESGGGIVQPGGSLKLSCAASGF
871



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARA




GLIGGTNKRAPGTPARFSGSLLGG

RSKYNNYATYYADSVKDRFTISRDDS




KAALTLSGVQPEDEAVYYCALWY

KNTVYLQMNNLKTEDTAVYYCVRHE




PNLWVFGGGTKLTVL

NFGNSYVSWFAHWGQGTLVTVSS



 


AC3384
ELVVTQEPSLTVSPGGTVTLTCRSS
872
EVQLLESGGGIVQPGGSLKLSCAASGF
873



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARG




GLIGGTNKRAPGTPARFSGSLLGG

RSKYNNYATYYADSVKDRFTISRDDS




KAALTLSGVQPEDEAVYYCALWY

KNTVYLQMNNLKTEDTAVYYCVRHE




PNLWVFGGGTKLTVL

NFGNSYVSWFAHWGQGTLVTVSS



 


AC3385
ELVVTQEPSLTVSPGGTVTLTCRSS
874
EVQLLESGGGIVQPGGSLKLSCAASGF
875



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVART




GLIGGTNKRAPGTPARFSGSLLGG

RSKYNNYATYYADSVKDRFTISRDDS




KAALTLSGVQPEDEAVYYCALWY

KNTVYLQMNNLKTEDTAVYYCVRHE




PNLWVFGGGTKLTVL

NFGNSYVSWFAHWGQGTLVTVSS



 


AC3386
ELVVTQEPSLTVSPGGTVTLTCRSS
876
EVQLLESGGGIVQPGGSLKLSCAASGF
877



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIN




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3387
ELVVTQEPSLTVSPGGTVTLTCRSS
878
EVQLLESGGGIVQPGGSLKLSCAASGF
879



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARID




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3388
ELVVTQEPSLTVSPGGTVTLTCRSS
880
EVQLLESGGGIVQPGGSLKLSCAASGF
881



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIE




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3389
ELVVTQEPSLTVSPGGTVTLTCRSS
882
EVQLLESGGGIVQPGGSLKLSCAASGF
883



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIQ




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3390
ELVVTQEPSLTVSPGGTVTLTCRSS
884
EVQLLESGGGIVQPGGSLKLSCAASGF
885



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIG




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3391
ELVVTQEPSLTVSPGGTVTLTCRSS
886
EVQLLESGGGIVQPGGSLKLSCAASGF
887



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIH




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3392
ELVVTQEPSLTVSPGGTVTLTCRSS
888
EVQLLESGGGIVQPGGSLKLSCAASGF
889



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARI




GLIGGTNKRAPGTPARFSGSLLGG

WSKYNNYATYYADSVKDRFTISRDDS




KAALTLSGVQPEDEAVYYCALWY

KNTVYLQMNNLKTEDTAVYYCVRHE




PNLWVFGGGTKLTVL

NFGNSYVSWFAHWGQGTLVTVSS



 


AC3393
ELVVTQEPSLTVSPGGTVTLTCRSS
890
EVQLLESGGGIVQPGGSLKLSCAASGF
891



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

NKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3394
ELVVTQEPSLTVSPGGTVTLTCRSS
892
EVQLLESGGGIVQPGGSLKLSCAASGF
893



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

DKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3395
ELVVTQEPSLTVSPGGTVTLTCRSS
894
EVQLLESGGGIVQPGGSLKLSCAASGF
895



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

EKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3396
ELVVTQEPSLTVSPGGTVTLTCRSS
896
EVQLLESGGGIVQPGGSLKLSCAASGF
897



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

TKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3397
ELVVTQEPSLTVSPGGTVTLTCRSS
898
EVQLLESGGGIVQPGGSLKLSCAASGF
899



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SPYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3398
ELVVTQEPSLTVSPGGTVTLTCRSS
900
EVQLLESGGGIVQPGGSLKLSCAASGF
901



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKANNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3399
ELVVTQEPSLTVSPGGTVTLTCRSS
902
EVQLLESGGGIVQPGGSLKLSCAASGF
903



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKRNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3400
ELVVTQEPSLTVSPGGTVTLTCRSS
904
EVQLLESGGGIVQPGGSLKLSCAASGF
905



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKGNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3401
ELVVTQEPSLTVSPGGTVTLTCRSS
906
EVQLLESGGGIVQPGGSLKLSCAASGF
907



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKKNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3402
ELVVTQEPSLTVSPGGTVTLTCRSS
908
EVQLLESGGGIVQPGGSLKLSCAASGF
909



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKPNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3403
ELVVTQEPSLTVSPGGTVTLTCRSS
910
EVQLLESGGGIVQPGGSLKLSCAASGF
911



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKINNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3404
ELVVTQEPSLTVSPGGTVTLTCRSS
912
EVQLLESGGGIVQPGGSLKLSCAASGF
913



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKWNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3405
ELVVTQEPSLTVSPGGTVTLTCRSS
914
EVQLLESGGGIVQPGGSLKLSCAASGF
915



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYDNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3406
ELVVTQEPSLTVSPGGTVTLTCRSS
916
EVQLLESGGGIVQPGGSLKLSCAASGF
917



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYENYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3407
ELVVTQEPSLTVSPGGTVTLTCRSS
918
EVQLLESGGGIVQPGGSLKLSCAASGF
919



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNDYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3408
ELVVTQEPSLTVSPGGTVTLTCRSS
920
EVQLLESGGGIVQPGGSLKLSCAASGF
921



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNEYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3409
ELVVTQEPSLTVSPGGTVTLTCRSS
922
EVQLLESGGGIVQPGGSLKLSCAASGF
923



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNGATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3410
ELVVTQEPSLTVSPGGTVTLTCRSS
924
EVQLLESGGGIVQPGGSLKLSCAASGF
925



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNFATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3411
ELVVTQEPSLTVSPGGTVTLTCRSS
926
EVQLLESGGGIVQPGGSLKLSCAASGF
927



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNWATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3412
ELVVTQEPSLTVSPGGTVTLTCRSS
928
EVQLLESGGGIVQPGGSLKLSCAASGF
929



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYGTYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3413
ELVVTQEPSLTVSPGGTVTLTCRSS
930
EVQLLESGGGIVQPGGSLKLSCAASGF
931



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYTTYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3414
ELVVTQEPSLTVSPGGTVTLTCRSS
932
EVQLLESGGGIVQPGGSLKLSCAASGF
933



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATDYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3415
ELVVTQEPSLTVSPGGTVTLTCRSS
934
EVQLLESGGGIVQPGGSLKLSCAASGF
935



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATEYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3416
ELVVTQEPSLTVSPGGTVTLTCRSS
936
EVQLLESGGGIVQPGGSLKLSCAASGF
937



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATTYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3417
ELVVTQEPSLTVSPGGTVTLTCRSS
938
EVQLLESGGGIVQPGGSLKLSCAASGF
939



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYDADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3418
ELVVTQEPSLTVSPGGTVTLTCRSS
940
EVQLLESGGGIVQPGGSLKLSCAASGF
941



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYEADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3419
ELVVTQEPSLTVSPGGTVTLTCRSS
942
EVQLLESGGGIVQPGGSLKLSCAASGF
943



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYQADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3420
ELVVTQEPSLTVSPGGTVTLTCRSS
944
EVQLLESGGGIVQPGGSLKLSCAASGF
945



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYGADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3421
ELVVTQEPSLTVSPGGTVTLTCRSS
946
EVQLLESGGGIVQPGGSLKLSCAASGF
947



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYWADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3422
ELVVTQEPSLTVSPGGTVTLTCRSS
948
EVQLLESGGGIVQPGGSLKLSCAASGF
949



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYKDSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3423
ELVVTQEPSLTVSPGGTVTLTCRSS
950
EVQLLESGGGIVQPGGSLKLSCAASGF
951



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYPDSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3424
ELVVTQEPSLTVSPGGTVTLTCRSS
952
EVQLLESGGGIVQPGGSLKLSCAASGF
953



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKGRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3425
ELVVTQEPSLTVSPGGTVTLTCRSS
954
EVQLLESGGGIVQPGGSLKLSCAASGF
955



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVDLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3426
ELVVTQEPSLTVSPGGTVTLTCRSS
956
EVQLLESGGGIVQPGGSLKLSCAASGF
957



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVGLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3427
ELVVTQEPSLTVSPGGTVTLTCRSS
958
EVQLLESGGGIVQPGGSLKLSCAASGF
959



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVSLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3428
ELVVTQEPSLTVSPGGTVTLTCRSS
960
EVQLLESGGGIVQPGGSLKLSCAASGF
961



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNELKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3429
ELVVTQEPSLTVSPGGTVTLTCRSS
962
EVQLLESGGGIVQPGGSLKLSCAASGF
963



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNQLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3430
ELVVTQEPSLTVSPGGTVTLTCRSS
964
EVQLLESGGGIVQPGGSLKLSCAASGF
965



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNSLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3431
ELVVTQEPSLTVSPGGTVTLTCRSS
966
EVQLLESGGGIVQPGGSLKLSCAASGF
967



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNYLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3432
ELVVTQEPSLTVSPGGTVTLTCRSS
968
EVQLLESGGGIVQPGGSLKLSCAASGF
969



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVGRIR




GLIGGTNKRAPGTPARFSGSLLGG

SKYNNGATYYADSVKGRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNSLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3433
ELVVTQEPSLTVSPGGTVTLTCRSS
970
EVQLLESGGGIVQPGGSLKLSCAASGF
971



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLQGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3434
ELVVTQEPSLTVSPGGTVTLTCRSS
972
EVQLLESGGGIVQPGGSLKLSCAASGF
973



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLLEG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3435
ELVVTQEPSLTVSPGGTVTLTCRSS
974
EVQLLESGGGIVQPGGSLKLSCAASGF
975



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSLDGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3436
ELVVTQEPSLTVSPGGTVTLTCRSS
976
EVQLLESGGGIVQPGGSLKLSCAASGF
977



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSSLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3437
ELVVTQEPSLTVSPGGTVTLTCRSS
978
EVQLLESGGGIVQPGGSLKLSCAASGF
979



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSKLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3438
ELVVTQEPSLTVSPGGTVTLTCRSS
980
EVQLLESGGGIVQPGGSLKLSCAASGF
981



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSNLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS



 


AC3439
ELVVTQEPSLTVSPGGTVTLTCRSS
982
EVQLLESGGGIVQPGGSLKLSCAASGF
983



NGAVTSSNYANWVQQKPGQAPR

TFNTYAMNWVRQAPGKGLEWVARIR




GLIGGTNKRAPGTPARFSGSTLGG

SKYNNYATYYADSVKDRFTISRDDSK




KAALTLSGVQPEDEAVYYCALWY

NTVYLQMNNLKTEDTAVYYCVRHEN




PNLWVFGGGTKLTVL

FGNSYVSWFAHWGQGTLVTVSS




















Relative 








Expression








(1-lowest








expression,








5-highest








expression -








evaluated by

%





PTE
PEG gel

Remaining



CD3.23

score
electro-
Primary KD
after


AC
domain
Mutation
v12
phoresis)
(nM)
heating





AC3364
CD3.23-
WT
50
4
    8.5
56.90%



L7







 


AC3366
CD3.23-
WT
50
3
   14.0
28.73%



D







 


AC3367
CD3.38
W47D
41
4
   40.2
ND


 


AC3368
CD3.39
W47E
42
4
   18.1
 0


 


AC3369
CD3.40
V48A
38
4
    8.3
 0


 


AC3370
CD3.41
V48E
38
4
  164.8
ND


 


AC3371
CD3.42
V48G
38
4
   71.5
ND


 


AC3372
CD3.43
V48S
38
4
   11.2
 0


 


AC3373
CD3.44
V48T
38
4
    5.7
 0


 


AC3374
CD3.45
V48W
40
4
34270.0
ND


 


AC3375
CD3.46
A49D
48
4
Weak binding
ND


 


AC3376
CD3.47
A49E
46
4
No binding
ND


 


AC3377
CD3.48
A49G
45
4
    4.5
79.06%


 


AC3378
CD3.49
R50Q
39
4
No binding
ND


 


AC3379
CD3.50
R50G
39
4
No binding
ND


 


AC3380
CD3.51
R50H
41
4
No binding
ND


 


AC3381
CD3.52
R50P
39
4
No binding
ND


 


AC3382
CD3.53
R50W
39
2
No binding
ND


 


AC3383
CD3.54
I51A
45
2
 1182.0
ND


 


AC3384
CD3.55
I51G
42
2
Weak binding
ND


 


AC3385
CD3.56
I51T
44
2
  424.6
ND


 


AC3386
CD3.57
R52N
39
2
No binding
ND


 


AC3387
CD3.58
R52D
35
2
No binding
ND


 


AC3388
CD3.59
R52E
35
2
No binding
ND


 


AC3389
CD3.60
R52Q
48
2
  434.4
ND


 


AC3390
CD3.61
R52G
41
2
No binding
ND


 


AC3391
CD3.62
R52H
50
2
No binding
ND


 


AC3392
CD3.63
R52W
42
2
No binding
ND


 


AC3393
CD3.64
S52aN
48
2
Weak binding
ND


 


AC3394
CD3.65
S52aD
42
4
No binding
ND


 


AC3395
CD3.66
S52aE
42
4
No binding
ND


 


AC3396
CD3.67
S52aT
49
4
    4.8
60.06%


 


AC3397
CD3.68
K52bP
45
4
   51.0
ND


 


AC3398
CD3.69
Y52cA
37
4
   11.5
14.99%


 


AC3399
CD3.70
Y52cR
38
4
    3.8
56.68%


 


AC3400
CD3.71
Y52cG
36
4
   20.1
 0


 


AC3401
CD3.72
Y52cK
40
4
    5.1
60.72%


 


AC3402
CD3.73
Y52cP
36
4
   33.1
ND


 


AC3403
CD3.74
Y52cT
36
4
   11.1
35.59%


 


AC3404
CD3.75
Y52cW
48
4
   10.5
15.25%


 


AC3405
CD3.76
N53D
34
4
No binding
ND


 


AC3406
CD3.77
N53E
34
4
  574.6
ND


 


AC3407
CD3.78
N54D
37
4
    7.4
61.45%


 


AC3408
CD3.79
N54E
42
4
    8.3
43.27%


 


AC3409
CD3.80
Y55G
34
4
   11.3
 0


 


AC3410
CD3.81
Y55F
44
4
    6.1
23.50%


 


AC3411
CD3.82
Y55W
38
4
    7.8
 6.79%


 


AC3412
CD3.83
A56G
49
4
    8.2
 9.14%


 


AC3413
CD3.84
A56T
49
4
   10.7
26.45%


 


AC3414
CD3.85
Y58D
35
4
  938.6
ND


 


AC3415
CD3.86
Y58E
35
4
  183.4
ND


 


AC3416
CD3.87
Y58T
35
4
   17.9
26.86%


 


AC3417
CD3.88
Y59D
42
4
   63.2
ND


 


AC3418
CD3.89
Y59E
42
4
    9.7
 0


 


AC3419
CD3.90
Y59Q
42
4
    7.2
 0


 


AC3420
CD3.91
Y59G
42
4
    8.3
 0


 


AC3421
CD3.92
Y59W
42
4
   37.2
ND


 


AC3422
CD3.93
A60K
37
4
    8.0
 0


 


AC3423
CD3.94
A60P
35
4
    8.2
 0


 


AC3424
CD3.95
D65G
46
4
    5.4
47.80%


 


AC3425
CD3.96
Y79D
31
4
    9.8
 0


 


AC3426
CD3.97
Y79G
31
2
  121.1
ND


 


AC3427
CD3.98
Y79S
31
4
    9.6
 0


 


AC3428
CD3.99
N82bE
39
4
    5.9
39.70%


 


AC3429
CD3.100
N82bQ
40
4
    7.1
18.12%


 


AC3430
CD3.101
N82bS
32
4
    4.8
 4.22%


 


AC3431
CD3.102
N82bY
46
4
    5.2
 1.66%


 


AC3432
CD3.103
A49G,
 8
4
   11.4
 0




Y52cG,








D65G,








N82bS






 


AC3433
CD3.104
L67Q
55
4
    4.6
59.68%


 


AC3434
CD3.105
G68E
54
4
    4.9
70.99%


 


AC3435
CD3.106
L67D
50
4
    6.1
43.75%


 


AC3436
CD3.107
L66S
50
4
    7.3
 0


 


AC3437
CD3.108
L66K
50
4
    3.2
 0


 


AC3438
CD3.109
L66N
50
4
    8.3
 0


 


AC3439
CD3.110
L66T
50
4
    8.9
 0
















TABLE 19





Pool 2 CD3.23 Mutation Combination Variants





















VL






SEQ ID

VH


AC
VL sequence
NO:
VH sequence
SEQ ID NO:





AC3632
ELVVTQEPSLTVSPGGTVTLTCRSS
700
EVQLLESGGGIVQPGGSLRLSCAAS
701



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3633
ELVVTQEPSLTVSPGGTVTLTCRSS
702
EVQLVESGGGIVQPGGSLRLSCAAS
703



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3634
ELVVTQEPSLTVSPGGTVTLTCRSS
704
EVQLLESGGGIVQPGGSLRLSCAAS
705



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKINNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3635
ELVVTQEPSLTVSPGGTVTLTCRSS
706
EVQLVESGGGIVQPGGSLRLSCAAS
707



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKINNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3636
ELVVTQEPSLTVSPGGTVTLTCRSS
708
EVQLLESGGGIVQPGGSLRLSCAAS
709



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3637
ELVVTQEPSLTVSPGGTVTLTCRSS
710
EVQLVESGGGIVQPGGSLRLSCAAS
711



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3638
ELVVTQEPSLTVSPGGTVTLTCRSS
712
EVQLLESGGGIVQPGGSLRLSCAAS
713



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNNYATTYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3639
ELVVTQEPSLTVSPGGTVTLTCRSS
714
EVQLVESGGGIVQPGGSLRLSCAAS
715



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNNYATTYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3640
ELVVTQEPSLTVSPGGTVTLTCRSS
716
EVQLVESGGGIVQPGGSLRLSCAAS
717



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3641
ELVVTQEPSLTVSPGGTVTLTCRSS
718
EVQLVESGGGIVQPGGSLRLSCAAS
719



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3642
ELVVTQEPSLTVSPGGTVTLTCRSS
720
EVQLVESGGGIVQPGGSLRLSCAAS
721



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKINNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3643
ELVVTQEPSLTVSPGGTVTLTCRSS
722
EVQLVESGGGIVQPGGSLRLSCAAS
723



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKTNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3644
ELVVTQEPSLTVSPGGTVTLTCRSS
724
EVQLVESGGGIVQPGGSLRLSCAAS
725



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3645
ELVVTQEPSLTVSPGGTVTLTCRSS
726
EVQLVESGGGIVQPGGSLRLSCAAS
727



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3646
ELVVTQEPSLTVSPGGTVTLTCRSS
728
EVQLVESGGGIVQPGGSLRLSCAAS
729



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3647
ELVVTQEPSLTVSPGGTVTLTCRSS
730
EVQLVESGGGIVQPGGSLRLSCAAS
731



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKTNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3648
ELVVTQEPSLTVSPGGTVTLTCRSS
732
EVQLVESGGGIVQPGGSLRLSCAAS
733



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3649
ELVVTQEPSLTVSPGGTVTLTCRSS
734
EVQLVESGGGIVQPGGSLRLSCAAS
735



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNNYATTYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3650
ELVVTQEPSLTVSPGGTVTLTCRSS
736
EVQLVESGGGIVQPGGSLRLSCAAS
737



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3651
ELVVTQEPSLTVSPGGTVTLTCRSS
738
EVQLVESGGGIVQPGGSLRLSCAAS
739



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3652
ELVVTQEPSLTVSPGGTVTLTCRSS
740
EVQLVESGGGIVQPGGSLRLSCAAS
741



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKTNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3653
ELVVTQEPSLTVSPGGTVTLTCRSS
742
EVQLVESGGGIVQPGGSLRLSCAAS
743



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKTNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3654
ELVVTQEPSLTVSPGGTVTLTCRSS
744
EVQLVESGGGIVQPGGSLRLSCAAS
745



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3655
ELVVTQEPSLTVSPGGTVTLTCRSS
746
EVQLVESGGGIVQPGGSLRLSCAAS
747



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3656
ELVVTQEPSLTVSPGGTVTLTCRSS
748
EVQLVESGGGIVQPGGSLRLSCAAS
749



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNNYATTYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3657
ELVVTQEPSLTVSPGGTVTLTCRSS
750
EVQLVESGGGIVQPGGSLRLSCAAS
751



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNNYATTYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3658
ELVVTQEPSLTVSPGGTVTLTCRSS
752
EVQLVESGGGIVQPGGSLRLSCAAS
753



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3659
ELVVTQEPSLTVSPGGTVTLTCRSS
754
EVQLVESGGGIVQPGGSLRLSCAAS
755



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3660
ELVVTQEPSLTVSPGGTVTLTCRSS
756
EVQLVESGGGIVQPGGSLRLSCAAS
757



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKTNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3661
ELVVTQEPSLTVSPGGTVTLTCRSS
758
EVQLVESGGGIVQPGGSLRLSCAAS
759



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKTNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3662
ELVVTQEPSLTVSPGGTVTLTCRSS
760
EVQLVESGGGIVQPGGSLRLSCAAS
761



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3663
ELVVTQEPSLTVSPGGTVTLTCRSS
762
EVQLVESGGGIVQPGGSLRLSCAAS
763



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3664
ELVVTQEPSLTVSPGGTVTLTCRSS
764
EVQLVESGGGIVQPGGSLRLSCAAS
765



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNNYATTYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3665
ELVVTQEPSLTVSPGGTVTLTCRSS
766
EVQLVESGGGIVQPGGSLRLSCAAS
767



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNNYATTYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3666
ELVVTQEPSLTVSPGGTVTLTCRSS
768
EVQLVESGGGIVQPGGSLRLSCAAS
769



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNEYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3667
ELVVTQEPSLTVSPGGTVTLTCRSS
770
EVQLVESGGGIVQPGGSLRLSCAAS
771



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNNGATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3668
ELVVTQEPSLTVSPGGTVTLTCRSS
772
EVQLVESGGGIVQPGGSLRLSCAAS
773



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNNGATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3669
ELVVTQEPSLTVSPGGTVTLTCRSS
774
EVQLVESGGGIVQPGGSLRLSCAAS
775



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRTKYNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3670
ELVVTQEPSLTVSPGGTVTLTCRSS
776
EVQLVESGGGIVQPGGSLRLSCAAS
777



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRSKRNNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTVYLQMNSLKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3671
ELVVTQEPSLTVSPGGTVTLTCRSS
778
EVQLVESGGGIVQPGGSLRLSCAAS
779



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRSKKNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



AC3672
ELVVTQEPSLTVSPGGTVTLTCRSS
780
EVQLVESGGGIVQPGGSLRLSCAAS
781



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRSKINNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTVYLQMNSLKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3673
ELVVTQEPSLTVSPGGTVTLTCRSS
782
EVQLVESGGGIVQPGGSLRLSCAAS
783



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNDYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTLYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3674
ELVVTQEPSLTVSPGGTVTLTCRSS
784
EVQLVESGGGIVQPGGSLRLSCAAS
785



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNEYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTLYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3675
ELVVTQEPSLTVSPGGTVTLTCRSS
786
EVQLVESGGGIVQPGGSLRLSCAAS
787



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNNGATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTLYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3676
ELVVTQEPSLTVSPGGTVTLTCRSS
788
EVQLVESGGGIVQPGGSLRLSCAAS
789



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNNGATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTLYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3677
ELVVTQEPSLTVSPGGTVTLTCRSS
790
EVQLVESGGGIVQPGGSLRLSCAAS
791



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRTKYNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTLYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3678
ELVVTQEPSLTVSPGGTVTLTCRSS
792
EVQLVESGGGIVQPGGSLRLSCAAS
793



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRSKRNNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTLYLQMNSLKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3679
ELVVTQEPSLTVSPGGTVTLTCRSS
794
EVQLVESGGGIVQPGGSLRLSCAAS
795



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRSKKNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTLYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3680
ELVVTQEPSLTVSPGGTVTLTCRSS
796
EVQLVESGGGIVQPGGSLRLSCAAS
797



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRSKINNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTLYLQMNSLKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3681
ELVVTQEPSLTVSPGGTVTLTCRSS
798
EVQLVESGGGIVQPGGSLRLSCAAS
799



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3682
ELVVTQEPSLTVSPGGTVTLTCRSS
800
EVQLVESGGGIVQPGGSLRLSCAAS
801



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKRNNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTVYLQMNSLKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3683
ELVVTQEPSLTVSPGGTVTLTCRSS
802
EVQLVESGGGIVQPGGSLRLSCAAS
803



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKKNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3684
ELVVTQEPSLTVSPGGTVTLTCRSS
804
EVQLVESGGGIVQPGGSLRLSCAAS
805



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKINNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTVYLQMNSLKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3685
ELVVTQEPSLTVSPGGTVTLTCRSS
806
EVQLVESGGGIVQPGGSLRLSCAAS
807



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3686
ELVVTQEPSLTVSPGGTVTLTCRSS
808
EVQLVESGGGIVQPGGSLRLSCAAS
809



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKRNNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTVYLQMNELKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3687
ELVVTQEPSLTVSPGGTVTLTCRSS
810
EVQLVESGGGIVQPGGSLRLSCAAS
811



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKKNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNELKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3688
ELVVTQEPSLTVSPGGTVTLTCRSS
812
EVQLVESGGGIVQPGGSLRLSCAAS
813



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKTNNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTVYLQMNELKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3689
ELVVTQEPSLTVSPGGTVTLTCRSS
814
EVQLVESGGGIVQPGGSLRLSCAAS
815



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKYNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTLYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3690
ELVVTQEPSLTVSPGGTVTLTCRSS
816
EVQLVESGGGIVQPGGSLRLSCAAS
817



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKRNNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTLYLQMNSLKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3691
ELVVTQEPSLTVSPGGTVTLTCRSS
818
EVQLVESGGGIVQPGGSLRLSCAAS
819



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKKNNYATTYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTLYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3692
ELVVTQEPSLTVSPGGTVTLTCRSS
820
EVQLVESGGGIVQPGGSLRLSCAAS
821



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKINNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTLYLQMNSLKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3693
ELVVTQEPSLTVSPGGTVTLTCRSS
822
EVQLVESGGGIVQPGGSLRLSCAAS
823



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKRNNYATTYADSVKDRFTISR




KAALTLSGVQPEDEAVYYCALWY

DDSKNTLYLQMNELKTEDTAVYYC




PNLWVFGGGTKLTVL

VRHENFGNSYVSWFAHWGQGTLVT






VSS



 


AC3694
ELVVTQEPSLTVSPGGTVTLTCRSS
824
EVQLVESGGGIVQPGGSLKLSCAAS
825



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3695
ELVVTQEPSLTVSPGGTVTLTCRSS
826
EVQLVESGGGIVQPGGSLKLSCAAS
827



NGAVTSSNYANWVQQKPGQAPR

GFTFSTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRTKRNNYATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3471
ELVVTQEPSLTVSPGGTVTLTCRSS
828
EVQLLESGGGIVQPGGSLKLSCAAS
829



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRSKYNNYATYYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNNLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC3432
ELVVTQEPSLTVSPGGTVTLTCRSS
830
EVQLLESGGGIVQPGGSLKLSCAAS
831



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

GRIRSKYNNGATYYADSVKGRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNSLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS



 


AC2717
ELVVTQEPSLTVSPGGTVTLTCRSS
832
EVQLLESGGGIVQPGGSLKLSCAAS
833



NGAVTSSNYANWVQQKPGQAPR

GFTFNTYAMNWVRQAPGKGLEWV




GLIGGTNKRAPGTPARFSGSLLGG

ARIRSKYNNYATYYADSVKDRFTIS




KAALTLSGVQPEDEAVYYCALWY

RDDSKNTVYLQMNNLKTEDTAVYY




PNLWVFGGGTKLTVL

CVRHENFGNSYVSWFAHWGQGTLV






TVSS























Primary
%


















PTE
PTE


Screen
Remaining



CD3.23

score
score
Expression
Expression
huCD3e
of


AC
domain
Mutation
v12
v22
Level**
Ratio***
(nM)
stability





AC3632
CD3.201
K19R,
9
18
3050.0
1.2
8.6
55.24%




A49G,










Y52cR,










D65G,










N82bS








 


AC3633
CD3.202
L5V,
9
18
2220.0
0.9
8.4
60.79%




K19R,










A49G,










Y52cR,










D65G,










N82bS








 


AC3634
CD3.203
K19R,
10
19
ND
ND
ND
ND




A49G,










Y52cT,










D65G,










N82bS








 


AC3635
CD3.204
L5V,
10
19
1870.0
0.8
7.2
36.92%




K19R,










A49G,










Y52cT,










D65G,










N82bS








 


AC3636
CD3.205
K19R,
10
19
3000.0
1.2
7.2
58.50%




A49G,










N54D,










D65G,










N82bS








 


AC3637
CD3.206
L5V,
10
19
1450.0
0.6
10.3
28.98%




K19R,










A49G,










N54D,










D65G,










N82bS








 


AC3638
CD3.207
K19R,
12
21
2420.0
1.0
15.6
1.01%




A49G,










Y58T,










D65G,










N82bS








 


AC3639
CD3.208
L5V,
12
21
2400.0
1.0
16.8
24.61%




K19R,










A49G,










Y58T,










D65G,










N82bS








 










AC3640
CD3.209
L5V,
16
25
2280.0
0.9
5.6
37.31%




K19R,










A49G,










Y52cR,










D65G,










N82bE








 


AC3641
CD3.210
L5V,
16
25
2120.0
0.9
5.7
69.25%




K19R,










N30S,










A49G,










Y52cR,










D65G,










N82bE








 


AC3642
CD3.211
L5V,
17
26
2610.0
1.1
9.5
29.37%




K19R,










A49G,










Y52cT,










D65G,










N82bE








 


AC3643
CD3.212
L5V,
17
26
890.0
0.4
35.6
9.81%




K19R,










N30S,










A49G,










Y52cT,










D65G,










N82bE








 


AC3644
CD3.213
L5V,
17
26
3280.0
1.2
6.6
57.91%




K19R,










A49G,










N54D,










D65G,










N82bE








 


AC3645
CD3.214
L5V,
17
26
3420.0
1.3
6.6
67.42%




K19R,










N30S,










A49G,










N54D,










D65G,










N82bE








 


AC3646
CD3.215
L5V,
9
18
2020.0
0.8
9.7
54.85%




K19R,










N30S,










A49G,










Y52cR,










D65G,










N82bS








 


AC3647
CD3.216
L5V,
10
19
2100.0
0.8
11.2
61.45%




K19R,










N30S,










A49G,










Y52cT










D65G,










N82bS








 


AC3648
CD3.217
L5V,
10
19
1040.0
0.4
10.7
31.11%




K19R,










N30S,










A49G,










N54D,










D65G,










N82bS








 


AC3649
CD3.218
L5V,
12
21
800.0
0.3
52.6
3.83%




K19R,










N30S,










A49G,










Y58T,










D65G,










N82bS








 


AC3650
CD3.219
L5V,
10
17
2010.0
0.8
4.7
57.93%




K19R,










A49G,










S52aT,










Y52cR,










D65G,










N82bE








 


AC3651
CD3.220
L5V,
10
17
1950.0
0.7
4.8
63.19%




K19R,










N30S,










A49G,










S52aT,










Y52cR,










D65G,










N82bE








 


AC3652
CD3.221
L5V,
15
22
2600.0
1.0
11.7
5.17%




K19R,










A49G,










S52aT,










Y52cT










D65G,










N82bE








 


AC3653
CD3.222
L5V,
15
22
2440.0
0.9
10.8
33.59%




K19R,










N30S,










A49G,










S52aT,










Y52cT










D65G,










N82bE








 


AC3654
CD3.223
L5V,
17
24
2890.0
1.1
6.0
68.99%




K19R,










A49G,










S52aT,










N54D,










D65G,










N82bE








 


AC3655
CD3.224
L5V,
17
24
2530.0
1.0
6.4
52.55%




K19R,










N30S,










A49G,










S52aT,










N54D,










D65G,










N82bE








 


AC3656
CD3.225
L5V,
19
26
3110.0
1.0
19.5
37.24%




K19R,










A49G,










S52aT,










Y58T,










D65G,










N82bE








 


AC3657
CD3.226
L5V,
19
26
1320.0
0.4
50.9
8.94%




K19R,










N30S,










A49G,










S52aT,










Y58T,










D65G,










N82bE








 


AC3658
CD3.227
L5V,
3
10
2850.0
1.0
8.2
62.96%




K19R,










A49G,










S52aT,










Y52cR,










D65G,










N82bS








 


AC3659
CD3.228
L5V,
3
10
2750.0
0.9
6.2
80.86%




K19R,










N30S,










A49G,










S52aT,










Y52cR,










D65G,










N82bS








 


AC3660
CD3.229
L5V,
8
15
3310.0
1.1
11.9
51.28%




K19R,










A49G,










S52aT,










Y52cT,










D65G,










N82bS








 


AC3661
CD3.230
L5V,
8
15
2740.0
0.9
17.0
62.00%




K19R,










N30S,










A49G,










S52aT,










Y52cT,










D65G,










N82bS








 


AC3662
CD3.231
L5V,
10
17
3590.0
1.2
5.1
54.69%




K19R,










A49G,










S52aT,










N54D,










D65G,










N82bS








 


AC3663
CD3.232
L5V,
10
17
3890.0
1.3
5.1
74.10%




K19R,










N30S,










A49G,










S52aT,










N54D,










D65G,










N82bS








 


AC3664
CD3.233
L5V,
12
19
3310.0
1.1
24.9
15.01%




K19R,










A49G,










S52aT,










Y58T,










D65G,










N82bS








 


AC3665
CD3.234
L5V,
12
19
3310.0
1.1
17.4
13.49%




K19R,










N30S,










A49G,










S52aT,










Y58T










D65G,










N82bS








 


AC3666
CD3.235
L5V,
14
23
3620.0
1.2
6.5
63.15%




K19R,










A49G,










N54E,










D65G,










N82bS








 


AC3667
CD3.236
L5V,
8
17
3180.0
1.1
21.9
0.65%




K19R,










A49G,










D65G,










N82bS








 


AC3668
CD3.237
L5V,
15
24
690.0
0.3
22.2
0




K19R,










A49G,










D65G,










N82bE








 


AC3669
CD3.238
L5V,
16
23
1680.0
0.7
23.1
−5.55%




K19R,










S52aT,










Y58T,










N82bS








 


AC3670
CD3.239
L5V,
14
21
1590.0
0.7
29.9
−12.81%




K19R,










Y52cR,










Y58T,










N82bS








 


AC3671
CD3.240
L5V,
16
23
1790.0
0.7
16.5
−25.28%




K19R,










Y52cK,










Y58T










N82bS








 


AC3672
CD3.241
L5V
14
21
2280.0
0.9
92.6
−183.07%




K19R,










Y52cT,










Y58T,










N82bS








 


AC3673
CD3.242
L5V,
9
18
2620.0
1.1
7.7
34.70%




K19R,










A49G,










N54D,










D65G,










V78L,










N82bS








 


AC3674
CD3.243
L5V,
13
22
ND
ND
ND
ND




K19R,










A49G,










N54E,










D65G,










V78L,










N82bS








 


AC3675
CD3.244
L5V,
7
16
740.0
0.3
43.6
−43.16%




K19R,










A49G,










D65G,










V78L,










N82bS








 


AC3676
CD3.245
L5V,
17
26
2490.0
1.0
19.3
−65.26%




K19R,










A49G,










D65G,










V78L,










N82bE








 


AC3677
CD3.246
L5V,
15
22
1970.0
0.8
35.2
−0.10%




K19R,










S52aT,










Y58T,










V78L,










N82bS








 


AC3678
CD3.247
L5V,
13
20
1880.0
0.8
28.9
−59.35%




K19R,










Y52cR,










Y58T,










V78L,










N82bS








 


AC3679
CD3.248
L5V,
15
22
1700.0
0.7
49.2
−25.51%




K19R,










Y52cK,










Y58T,










V78L,










N82bS








 


AC3680
CD3.249
L5V,
13
20
3180.0
1.0
128.0
−193.59%




K19R,










Y52cT,










Y58T.










V78L,










N82bS








 


AC3681
CD3.250
L5V,
12
19
2950.0
0.9
31.0
5.62%




K19R,










A49G,










S52aT,










Y58T,










N82bS








 


AC3682
CD3.251
L5V,
8
17
2730.0
0.9
32.8
11.12%




K19R,










A49G,










Y52cR,










Y58T,










N82bS








 


AC3683
CD3.252
L5V,
9
19
2280.0
0.7
22.9
−13.59%




K19R,










A49G,










Y52cK,










Y58T,










N82bS








 


AC3684
CD3.253
L5V,
10
19
2900.0
0.9
79.9
−105.16%




K19R,










A49G,










Y52cT,










Y58T,










N82bS








 


AC3685
CD3.254
L5V,
19
26
2650.0
0.8
32.3
24.18%




K19R,










A49G,










S52aT,










Y58T,










N82bE








 


AC3686
CD3.255
L5V,
15
24
2110.0
0.7
22.4
32.74%




K19R,










A49G,










Y52cR,










Y58T,










N82bE








AC3687
CD3.256
L5V,
16
26
ND
ND
ND
ND




K19R,










A49G










Y52cK,










Y58T,










N82bE








 


AC3688
CD3.257
L5V,
17
26
2970.0
0.9
159.0
−92.57%




K19R,










A49G,










Y52cT,










Y58T,










N82bE








 


AC3689
CD3.258
L5V,
11
18
2840.0
0.9
38.0
22.13%




K19R,










A49G,










S52aT,










Y58T,










V78L,










N82bS








 


AC3690
CD3.259
L5V,
7
16
2540.0
0.8
29.4
14.38%




K19R,










A49G,










Y52cR,










Y58T,










V78L,










N82bS








 


AC3691
CD3.260
L5V,
8
18
2730.0
0.9
45.6
27.07%




K19R,










A49G,










Y52cK,










Y58T,










V78L,










N82bS








 


AC3692
CD3.261
L5V,
9
18
2200.0
0.9
97.0
−122.00%




K19R,










A49G,










Y52cT,










Y58T,










V78L,










N82bS








 


AC3693
CD3.262
L5V,
17
26
2100.0
0.9
25.4
−4.58%




K19R,










A49G,










Y52cR,










Y58T,










V78L,










N82bE








 


AC3694
CD3.263
L5V,
17
26
2050.0
0.8
7.2
53.06%




A49G,










S52aT,










Y52cR,










D65G,










N82bS








 


AC3695
CD3.264
L5V,
17
26
2500.0
1.0
4.5
55.56%




N30S,










A49G,










S52aT,










Y52cR,










D65G,










N82bS








 


AC3471
CD3.23
WT
50
73
2738.0
1.0
13.5
63.73%


 


AC3432*
CD3.103
A49G,
8
33
3843.3
1.3
13.5
21.44%




Y52cG,










D65G,










N82bS








 


AC2717*
CD3.23
WT
50
73
3723.3
1.3
12.9
73.42%





*AC3432 and AC2717 paired with anti-PSMA VHH variant.


**These values are arbitrary reads from the Octet data. A higher number means more protein is presented.


***These values are ratios compared to expression level of CD3.23. Higher ration means higher expression level compared to expression of CD3.23.













TABLE 20





Pool 3 CD3 scFv Variants





















SEQ

SEQ




ID

ID


AC
VL sequence
NO:
VH sequence
NO:





AC3796
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3797
ELVVTQEPSLTVSPGGTVTLTCRSSN
8250
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSALGGKAA

GKGLEWVGRIRTKRNNYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTV




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3798
ELVVTQEPSLTVSPGGTVTLTCRSSN
8251
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSGLGGKAA

GKGLEWVGRIRTKRNNYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTV




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3799
ELVVTQEPSLTVSPGGTVTLTCRSSN
8252
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLNGGKAA

GKGLEWVGRIRTKRNNYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTV




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3800
ELVVTQEPSLTVSPGGTVTLTCRSSN
8253
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLEGGKAAL

GKGLEWVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3801
ELVVTQEPSLTVSPGGTVTLTCRSSN
8254
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLGGGKAA

GKGLEWVGRIRTKRNNYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTV




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3802
ELVVTQEPSLTVSPGGTVTLTCRSSN
974
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLDGGKAA

GKGLEWVGRIRTKRNNYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTV




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3803
ELVVTQEPSLTVSPGGTVTLTCRSSN
8255
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAGL

GKGLEWVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3804
ELVVTQEPSLTVSPGGTVTLTCRSSN
127
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLEGKAAL

GKGLEWVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3805
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8256



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNDYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTA




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3806
ELVVTQEPSLTVSPGGTVTLTCRSSN
8250
EVQLVESGGGIVQPGGSLRLS
8256



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSALGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTA




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3807
ELVVTQEPSLTVSPGGTVTLTCRSSN
8251
EVQLVESGGGIVQPGGSLRLS
8256



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSGLGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTA




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3808
ELVVTQEPSLTVSPGGTVTLTCRSSN
8252
EVQLVESGGGIVQPGGSLRLS
8256



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLNGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTA




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3809
ELVVTQEPSLTVSPGGTVTLTCRSSN
8253
EVQLVESGGGIVQPGGSLRLS
8256



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLEGGKAAL

GKGLEWVGRIRTKRNDYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTA




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3810
ELVVTQEPSLTVSPGGTVTLTCRSSN
8254
EVQLVESGGGIVQPGGSLRLS
8256



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLGGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTA




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3811
ELVVTQEPSLTVSPGGTVTLTCRSSN
974
EVQLVESGGGIVQPGGSLRLS
8256



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLDGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTA




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3812
ELVVTQEPSLTVSPGGTVTLTCRSSN
8255
EVQLVESGGGIVQPGGSLRLS
8256



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAGL

GKGLEWVGRIRTKRNDYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTA




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3813
ELVVTQEPSLTVSPGGTVTLTCRSSN
127
EVQLVESGGGIVQPGGSLRLS
8256



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLEGKAAL

GKGLEWVGRIRTKRNDYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTA




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3814
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
126



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNDYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTLY




FGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3815
ELVVTQEPSLTVSPGGTVTLTCRSSN
8250
EVQLVESGGGIVQPGGSLRLS
126



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSALGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTLY




VFGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3816
ELVVTQEPSLTVSPGGTVTLTCRSSN
8251
EVQLVESGGGIVQPGGSLRLS
126



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSGLGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTLY




VFGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3817
ELVVTQEPSLTVSPGGTVTLTCRSSN
8252
EVQLVESGGGIVQPGGSLRLS
126



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLNGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTLY




VFGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3818
ELVVTQEPSLTVSPGGTVTLTCRSSN
8253
EVQLVESGGGIVQPGGSLRLS
126



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLEGGKAAL

GKGLEWVGRIRTKRNDYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTLY




FGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3819
ELVVTQEPSLTVSPGGTVTLTCRSSN
8254
EVQLVESGGGIVQPGGSLRLS
1261



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLGGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTLY




VFGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3820
ELVVTQEPSLTVSPGGTVTLTCRSSN
974
EVQLVESGGGIVQPGGSLRLS
26



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLDGGKAA

GKGLEWVGRIRTKRNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTLY




VFGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3821
ELVVTQEPSLTVSPGGTVTLTCRSSN
8255
EVQLVESGGGIVQPGGSLRLS
126



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAGL

GKGLEWVGRIRTKRNDYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTLY




FGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3822
ELVVTQEPSLTVSPGGTVTLTCRSSN
127
EVQLVESGGGIVQPGGSLRLS
126



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLEGKAAL

GKGLEWVGRIRTKRNDYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTLY




FGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3823
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
757



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLGGKAAL

PGKGLEWVGRIRTKTNNYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNT




FGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3824
ELVVTQEPSLTVSPGGTVTLTCRSSN
8250
EVQLVESGGGIVQPGGSLRL
757



GAVTSSNYANWVQQKPGQAPRGLI

SCAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSALGGKAA

PGKGLEWVGRIRTKTNNYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3825
ELVVTQEPSLTVSPGGTVTLTCRSSN
8251
EVQLVESGGGIVQPGGSLRLS
757



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSGLGGKAA

PGKGLEWVGRIRTKTNNYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3826
ELVVTQEPSLTVSPGGTVTLTCRSSN
8252
EVQLVESGGGIVQPGGSLRLS
757



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLNGGKAA

PGKGLEWVGRIRTKTNNYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3827
ELVVTQEPSLTVSPGGTVTLTCRSSN
8253
EVQLVESGGGIVQPGGSLRLS
757



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLEGGKAAL

PGKGLEWVGRIRTKTNNYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNT




FGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3828
ELVVTQEPSLTVSPGGTVTLTCRSSN
8254
EVQLVESGGGIVQPGGSLRLS
757



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLGGGKAA

PGKGLEWVGRIRTKTNNYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3829
ELVVTQEPSLTVSPGGTVTLTCRSSN
974
EVQLVESGGGIVQPGGSLRLS
757



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLDGGKAA

PGKGLEWVGRIRTKTNNYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3830
ELVVTQEPSLTVSPGGTVTLTCRSSN
8255
EVQLVESGGGIVQPGGSLRL
757



GAVTSSNYANWVQQKPGQAPRGLI

SCAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLGGKAGL

PGKGLEWVGRIRTKTNNYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNT




FGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3831
ELVVTQEPSLTVSPGGTVTLTCRSSN
127
EVQLVESGGGIVQPGGSLRLS
757



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLEGKAAL

PGKGLEWVGRIRTKTNNYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNT




FGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3832
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8257



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLGGKAAL

PGKGLEWVGRIRTKTNDYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNT




FGGGTKLTVL

AYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3833
ELVVTQEPSLTVSPGGTVTLTCRSSN
8250
EVQLVESGGGIVQPGGSLRLS
8257



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSALGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

AYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3834
ELVVTQEPSLTVSPGGTVTLTCRSSN
8251
EVQLVESGGGIVQPGGSLRLS
8257



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSGLGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

AYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3835
ELVVTQEPSLTVSPGGTVTLTCRSSN
8252
EVQLVESGGGIVQPGGSLRLS
8257



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLNGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

AYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3836
ELVVTQEPSLTVSPGGTVTLTCRSSN
8253
EVQLVESGGGIVQPGGSLRLS
8257



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLEGGKAAL

PGKGLEWVGRIRTKTNDYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNT




FGGGTKLTVL

AYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3837
ELVVTQEPSLTVSPGGTVTLTCRSSN
8254
EVQLVESGGGIVQPGGSLRLS
8257



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLGGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

AYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3838
ELVVTQEPSLTVSPGGTVTLTCRSSN
974
EVQLVESGGGIVQPGGSLRLS
8257



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLDGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNT




VFGGGTKLTVL

AYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3839
ELVVTQEPSLTVSPGGTVTLTCRSSN
8255
EVQLVESGGGIVQPGGSLRLS
8257



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLGGKAGL

PGKGLEWVGRIRTKTNDYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNT




FGGGTKLTVL

AYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3840
ELVVTQEPSLTVSPGGTVTLTCRSSN
127
EVQLVESGGGIVQPGGSLRLS
8257



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLEGKAAL

PGKGLEWVGRIRTKTNDYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNT




FGGGTKLTVL

AYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3841
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8258



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLGGKAAL

PGKGLEWVGRIRTKTNDYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNTL




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3842
ELVVTQEPSLTVSPGGTVTLTCRSSN
8250
EVQLVESGGGIVQPGGSLRLS
8258



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSALGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNTL




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3843
ELVVTQEPSLTVSPGGTVTLTCRSSN
8251
EVQLVESGGGIVQPGGSLRLS
8258



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSGLGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNTL




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3844
ELVVTQEPSLTVSPGGTVTLTCRSSN
8252
EVQLVESGGGIVQPGGSLRLS
8258



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLNGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNTL




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3845
ELVVTQEPSLTVSPGGTVTLTCRSSN
8253
EVQLVESGGGIVQPGGSLRLS
8258



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLEGGKAAL

PGKGLEWVGRIRTKTNDYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNTL




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3846
ELVVTQEPSLTVSPGGTVTLTCRSSN
8254
EVQLVESGGGIVQPGGSLRLS
8258



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLGGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNTL




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3847
ELVVTQEPSLTVSPGGTVTLTCRSSN
974
EVQLVESGGGIVQPGGSLRLS
8258



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLDGGKAA

PGKGLEWVGRIRTKTNDYAT




LTLSGVQPEDEAVYYCALWYPNLW

YYADSVKGRFTISRDDSKNTL




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3848
ELVVTQEPSLTVSPGGTVTLTCRSSN
8255
EVQLVESGGGIVQPGGSLRLS
8258



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLGGKAGL

PGKGLEWVGRIRTKTNDYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNTL




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3849
ELVVTQEPSLTVSPGGTVTLTCRSSN
127
EVQLVESGGGIVQPGGSLRLS
8258



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLEGKAAL

PGKGLEWVGRIRTKTNDYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNTL




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3850
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
759



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKTNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3857
ELVVTQEPSLTVSPGGTVTLTCRSSN
8255
EVQLVESGGGIVQPGGSLRLS
759



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAGL

GKGLEWVGRIRTKTNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3860
ELVVTQEPSLTVSPGGTVTLTCRSSN
8250
EVQLVESGGGIVQPGGSLRLS
8259



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSALGGKAA

GKGLEWVGRIRTKTNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTA




VFGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3867
ELVVTQEPSLTVSPGGTVTLTCRSSN
127
EVQLVESGGGIVQPGGSLRLS
8259



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLEGKAAL

GKGLEWVGRIRTKTNdYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTaY




FGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3869
ELVVTQEPSLTVSPGGTVTLTCRSSN
8250
EVQLVESGGGIVQPGGSLRLS
8260



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSALGGKAA

GKGLEWVGRIRTKTNDYATY




LTLSGVQPEDEAVYYCALWYPNLW

YADSVKGRFTISRDDSKNTLY




VFGGGTKLTVL

LQMNSLKTEDTAVYYCVRHE






NFGNSYVSWFAHWGQGTLV






TVSS



 


AC3877
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8261



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEDVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3878
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8262



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWGGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3879
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8263



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTPRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3880
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8264



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKPNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3881
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8265



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNNYATT




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3882
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8266



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

DADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3883
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8267



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

WADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3884
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8268



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNNYTTY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3885
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8269



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGASYVSWFAHWGQGT






LVTVSS



 


AC3886
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
8270



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNAYVSWFAHWGQGT






LVTVSS



 


AC3768
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLLESGGGIVQPGGSLKLS
833



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLGGKAAL

PGKGLEWVARIRSKYNNYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKDRFTISRDDSKNT




FGGGTKLTVL

VYLQMNNLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC2885
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLLESGGGIVQPGGSLKLS
833



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLGGKAAL

PGKGLEWVARIRSKYNNYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKDRFTISRDDSKNT




FGGGTKLTVL

VYLQMNNLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS



 


AC3659
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
755



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFSTYAMNWVRQAP




GGTNKRAPGTPARFSGSLLGGKAAL

GKGLEWVGRIRTKRNNYATY




TLSGVQPEDEAVYYCALWYPNLWV

YADSVKGRFTISRDDSKNTV




FGGGTKLTVL

YLQMNSLKTEDTAVYYCVR






HENFGNSYVSWFAHWGQGT






LVTVSS



 


AC3660
ELVVTQEPSLTVSPGGTVTLTCRSSN
832
EVQLVESGGGIVQPGGSLRLS
757



GAVTSSNYANWVQQKPGQAPRGLI

CAASGFTFNTYAMNWVRQA




GGTNKRAPGTPARFSGSLLGGKAAL

PGKGLEWVGRIRTKTNNYAT




TLSGVQPEDEAVYYCALWYPNLWV

YYADSVKGRFTISRDDSKNT




FGGGTKLTVL

VYLQMNSLKTEDTAVYYCV






RHENFGNSYVSWFAHWGQG






TLVTVSS






















Binding
%
Binding,




PTE

%
KD
Remaining
KD



CD3.23
score
Binding,
Remaining
(nM)
Stability
(nM)


AC#
domain
v22
KD (nM)
Stability*
R2
R2**
R3





 


AC3796
CD3.292
10
3.89
ND
4.1
81%
3.0


 


AC3797
CD3.293
3
3.80
61.59%
3.72
ND
ND


 


AC3798
CD3.294
3
3.05
63.93%
5.75
ND
ND


 


AC3799
CD3.295
3
3.11
73.85%
3.70
78.71%
ND


 


AC3800
CD3.296
3
4.74
72.95%
17.01
ND
ND


 


AC3801
CD3.297
3
3.92
19.10%
21.18
78.71%
ND


 


AC3802
CD3.298
3
4.07
73.43%
8.00
83.80%
ND


 


AC3803
CD3.299
3
ND
ND
7.964
ND
ND


 


AC3804
CD3.300
7
36.56
ND
ND
ND
ND


 


AC3805
CD3.301
7
5.53
ND
ND
84%
4.4


 


AC3806
CD3.302
0
5.09
38.94%
ND
48.59%
ND


 


AC3807
CD3.303
0
6.08
ND
ND
54.57%
ND


 


AC3808
CD3.304
0
4.31
51.07%
ND
ND
ND


 


AC3809
CD3.305
0
6.28
ND
ND
69%
4.6


 


AC3810
CD3.306
0
5.09
54.79%
ND
67.27%
ND


 


AC3811
CD3.307
0
6.35
ND
ND
63%
4.8


 


AC3812
CD3.308
0
5.56
35.06%
ND
58.92%
ND


 


AC3813
CD3.309
4
5.82
67.83%
ND
85%
5.4


 


AC3814
CD3.310
7
4.10
ND
ND
ND
ND


 


AC3815
CD3.311
0
4.53
55.16%
ND
ND
ND


 


AC3816
CD3.312
0
4.12
53.29%
ND
ND
ND


 


AC3817
CD3.313
0
3.86
34.23%
ND
ND
ND


 


AC3818
CD3.314
0
4.02
5.76%
ND
ND
ND


 


AC3819
CD3.315
0
3.40
54.09%
ND
ND
ND


 


AC3820
CD3.316
0
3.99
50.35%
ND
ND
ND


 


AC3821
CD3.317
0
4.10
1.15%
ND
ND
ND


 


AC3822
CD3.318
4
5.38
85.02%
5.7
61.81%
ND


 


AC3823
CD3.319
15
ND
ND
ND
ND
ND


 


AC3824
CD3.320
8
10.94
ND
ND
ND
ND


 


AC3825
CD3.321
8
10.03
ND
ND
ND
ND


 


AC3826
CD3.322
8
7.00
ND
ND
ND
ND


 


AC3827
CD3.323
8
ND
ND
ND
ND
ND


 


AC3828
CD3.324
8
7.21
ND
ND
ND
ND


 


AC3829
CD3.325
8
10.07
ND
ND
ND
ND


 


AC3830
CD3.326
8
6.65
ND
ND
ND
ND


 


AC3831
CD3.327
12
ND
ND
ND
ND
ND


 


AC3832
CD3.328
14
ND
ND
ND
ND
ND


 


AC3833
CD3.329
7
ND
ND
ND
ND
ND


 


AC3834
CD3.330
7
23.35
ND
ND
ND
ND


 


AC3835
CD3.331
7
35.72
ND
ND
ND
ND


 


AC3836
CD3.332
7
30.57
ND
ND
ND
ND


 


AC3837
CD3.333
7
17.88
ND
ND
ND
ND


 


AC3838
CD3.334
7
30.69
ND
ND
ND
ND


 


AC3839
CD3.335
7
24.48
ND
ND
ND
ND


 


AC3840
CD3.336
11
32.65
ND
ND
ND
ND


 


AC3841
CD3.337
14
12.18
ND
ND
ND
ND


 


AC3842
CD3.338
7
17.02
ND
ND
ND
ND


 


AC3843
CD3.339
7
15.37
ND
ND
ND
ND


 


AC3844
CD3.340
7
10.58
ND
ND
ND
ND


 


AC3845
CD3.341
7
15.75
ND
ND
ND
ND


 


AC3846
CD3.342
7
9.99
ND
ND
ND
ND


 


AC3847
CD3.343
7
14.01
ND
ND
ND
ND


 


AC3848
CD3.344
7
14.17
ND
ND
ND
ND


 


AC3849
CD3.345
11
18.01
ND
ND
ND
ND


 


AC3850
CD3.346
15
9.83
ND
10.8
75.67%
ND


 


AC3857
CD3.353
8
12.27
ND
ND
ND
ND


 


AC3860
CD3.356
7
29.42
ND
ND
68%
19.7


 


AC3867
CD3.363
11
62.74
ND
ND

ND


 


AC3869
CD3.365
7
18.77
ND
ND
52.21%
ND


 


AC3877
CD3.373
10
8.92
ND
ND
51.39%
ND


 


AC3878
CD3.374
11
ND
ND
17.0
44.73%
ND


 


AC3879
CD3.375
8
ND
ND
ND
ND
ND


 


AC3880
CD3.376
8
12.95
ND
ND
57.88%
ND


 


AC3881
CD3.377
8
10.66
ND
ND
69%
10.4


 


AC3882
CD3.378
8
3.74
65.49%
ND
ND
ND


 


AC3883
CD3.379
9
4.19
46.17%
ND
ND
ND


 


AC3884
CD3.380
8
4.54
71.07%
ND
ND
ND


 


AC3885
CD3.381
10
ND
ND
ND
75%
4.2


 


AC3886
CD3.382
10
ND
ND
21.2
70.38%
ND


 


AC3768
CD3.23
73
4.67
66%
8.0
64%
5.6



control








 


AC2885
CD3.23
73
15.41
ND
ND
ND
ND


 


AC3659
CD3.228
10
5.14
68.89%
ND
ND
ND


 


AC3660
CD3.229
15
13.61
ND
ND
ND
ND





Only antibodies AC2885, AC3659, and AC3660 were paired with an anti-tumor antibody to form a paTCE.


All other CD3 scFv antibodies were unpaired.


*5 min at 60° C. in lysis buffer vs huCD3e-mFc


**5 min at 62° C. in lysis buffer (Triton-free) vs huCD3e-mFc






Example 3. Selection of Anti-EGFR and Anti-CD3 paTCE
paTCEs including the improved anti-EGFR binding sequence of EGFR.37 and anti-CD3 binding sequences selected from Example 2 were produced and tested. The anti-CD3 domains tested with EGFR.37 were chosen due to low PTE score and increased thermal stability. Binding to human CD3 was determined by Bio-Layer Interferometry at room temperature with CD3& as the antigen. Stability was determined by Differential Scanning Fluorimetry. In vitro cytotoxicity with unmasked uTCE was determined in an HT-29 cell line with an Effector to Target (E: T) ratio of 5 to 1. Predicted immunogenicity was determined by a proprietary v25 PTE algorithm. The results are shown in Table 21.







TABLE 21







Anti-CD3 domains with EGFR.37

















PTE
PTE






EC50 
Score
Score
Combined


Anti-
Anti-
Tm
HT-29
v25
v25
PTE Score


EGFR
CD3
(° C.)
(pM)
CD3
EGFR
v25
















EGFR.2
CD3.9
n.d.
14
100
75
175


EGFR.37
CD3.23
68.65
38
112
60
172



CD3.228
70.63
n.d.
44
60
104



CD3.295
69.84
n.d.
52
60
112



CD3.318
69.79
64
35
60
95





n.d. = no data






EGFR.37 was selected for at least its improved thermostability and expression as compared to EGFR.2 (see Example 1). CD3.318 was selected for at least its decreased PTE score as compared to any of CD3.23, CD3.228, CD3.295. A paTCE having the combination of EGFR.37/CD3.318 exhibited increased stability, decreased predicted immunogenicity, and decreased potency as compared to prior paTCEs.
Importantly, the paTCE including the combination of EGFR.37/CD3.318 demonstrated a lower potency (increase EC50 in cytotoxicity assay) as compared to a previously described paTCE including EGFR.2/CD3.9 (referred to above as EGRF-XPAT gen1). This is meaningful as decreased potency relative to EGRF-XPAT gen1 is desirable as it is expected to attenuate cytokine release syndrome (CRS) and T cell activation, resulting in a greater therapeutic safety window. Preliminary studies in cynomolgus monkeys suggest that the maximum tolerated dose for EGFR-XPAT gen1 under the conditions tested was 1 mg/kg at which point symptoms of CRS were observed. By contrast, the maximum tolerated dose under the same conditions for EGFR.37/CD3.318 (in AMX-525) was increased to 4.5 mg/kg and the observed dose-limiting toxicities suggested that they were no longer the result of CRS. This lends further evidence that the loss of potency observed for the EGFR.37/CD3.318 combination may result in a greater therapeutic index.
In view of the above, the combination of EGFR.37/CD3.318 was selected to be incorporated into AMX-525.
Example 4. Design of Barcoded ELNNs by Minimal Mutations in ELNNs
ELNN polypeptide sequences can optionally contain a barcode fragment releasable from the polypeptide upon digestion by a protease. A barcode fragment may be, e.g., (1) a portion of the ELNN that includes at least part of a (non-recurring, non-overlapping) sequence motif that occurs only once within the ELNN; and (2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide containing them (e.g., a paTCE) upon complete digestion of the polypeptide by a protease. The term “barcode fragment” (“barcode,” or “barcode sequence”) can refer to either the portion of the ELNN cleavably fused within the polypeptide, or the resulting peptide fragment released from the polypeptide. Previous barcode sequences (see, e.g., PCT International Patent Publication No. WO2021/263058, the entire content of which is incorporated herein by reference) were designed with the intention of creating unique barcode polypeptide sequences with as minimal mutations in the original ELNN sequence as possible. However, such barcode sequences required 1000 μg/mL of Glu-C and an overnight digest to release them from peptides containing them, such as paTCEs. The barcode polypeptide sequences described in this Example were designed and tested to perform against a second criteria: That the barcode polypeptide is releasable from the ELNN polypeptide rapidly (in approximately two hours vs an overnight digest) by a low concentration of protease (less than 30 μg/mL protease); in addition to the criteria of introducing the fewest mutations to the original ELNN sequence as possible.
In order to determine which peptide sequences were most favorably cleaved by Glu-C protease in a two-hour protease digest, a library of approximately 1000 peptides was constructed with each peptide containing a different cleavage sequence for the protease Glu-C. Equimolar concentrations of these Glu-C site-containing peptides were tested in a 2-hour digest against a range of Glu-C protease concentrations from 0.05 μg/mL to 1000 μg/mL of protease. After digestion the peptides were analyzed by liquid chromatography mass spectrometry. The Glu-C cleavage site sequences that were cleaved by the lowest concentrations of protease were cataloged. From this list of the fastest sequences, a select few were selected that were most compatible with ELNN polypeptides. These sequences were then implemented to flank new “Generation 2” barcode sequences.
A selection of Generation 2 barcode sequences was cloned into ELNN sequences and their performance as barcode peptides was tested by Glu-C digestion and subsequent liquid chromatography mass spectrometry analyses. Successful barcode sequences from this experiment had 3 criteria: 1.) The barcode peptide was fully releasable from the ELNN polypeptide in a 2-hour digest by a concentration of 40 μg/mL of protease. 2.) The barcode peptide was not cleaved or otherwise degraded by much higher concentrations of protease, and 3.) The barcode peptide that met conditions 1 and 2 contained the fewest mutations from the original ELNN polypeptide sequence. Table 22 provides examples of successful Generation 2 barcode sequences according to the criteria of the aforementioned selection process.







TABLE 22





Exemplary Generation 2 Barcode Sequences
















Gen 2 Barcode 01
SGPE.SGPGTGTSATPE.SGPG (SEQ ID



NO: 8271)


 


Gen 2 Barcode 02
ATPE.SGPGSGPGTSE.SATP (SEQ ID



NO: 8272)


 


Gen 2 Barcode 03
ATPE.SGPGTTPGTTPE.SGPG (SEQ ID



NO: 8273)


 


Gen 2 Barcode 04
ATPE.SGPGTPPTSTPE.SGPG (SEQ ID



NO: 8274)


 


Gen 2 Barcode 05
ATPE.SGPGTSPSATPE.SGPG (SEQ ID



NO: 8275)


 


Gen 2 Barcode 06
ATPE.SGPGTGSAGTPE.SGPG (SEQ



ID NO: 8276)


 


Gen 2 Barcode 07
ATPE.SGPGTGGAGTPE.SGPG (SEQ



ID NO: 8277)


 


Gen 2 Barcode 08
ATPE.SGPGTSPGATPE.SGPG (SEQ ID



NO: 8278)


 


Gen 2 Barcode 09
GTPE.SGPGTSGSGTPE.SGPG (SEQ ID



NO: 8279)


 


Gen 2 Barcode 10
GTPE.SGPGTSSASTPE.SGPG (SEQ ID



NO: 8280)


 


Gen 2 Barcode 11
GTPE.SGPGTGAGTTPE.SGPG (SEQ



ID NO: 8281)


 


Gen 2 Barcode 12
GTPE.SGPGTGSTSTPE.SGPG (SEQ ID



NO: 8282)


 


Gen 2 Barcode 13
GTPE.TPGSEPATSGSE.TGTP (SEQ ID



NO: 8283)


 


Gen 2 Barcode 14
GTPE.GSAPGTSTEPSE.SATP (SEQ ID



NO: 8284)


 


Gen 2 Barcode 15
ATPE.SGPGTAGSGTPE.SGPG (SEQ



ID NO: 8285)


 


Gen 2 Barcode 16
ATPE.SGPGTSSGGTPE.SGPG (SEQ ID



NO: 8286)


 


Gen 2 Barcode 17
ATPE.SGPGTAGPATPE.SGPG (SEQ



ID NO: 8287)


 


Gen 2 Barcode 18
ATPE.SGPGTPGTGTPE.SGPG (SEQ



ID NO: 8288)


 


Gen 2 Barcode 19
TTPE.SGPGTGGPTTPE.SGPG (SEQ ID



NO: 8289)


 


Gen 2 Barcode 20
STPE.SGPGTGSGSTPE.SGPG (SEQ ID



NO: 8222)









Example 5. Release Site Engineering
Incubation of a paTCE comprising RSR-2295 in human plasma showed some cleavage that, though not high, was unexpected. Further investigation revealed that the cleavage was surprisingly due to legumain, which has previously believed to be specifically present in tumor tissues. Additionally, it was initially believed that legumain cleavage provided meaningful levels of paTCE activation in tumor tissues.
A new release site was designed to avoid cleavage by legumain, resulting in RSR-3213. Surprisingly, a paTCE containing RSR-3213 release sequences was cleaved less in plasma but at comparable amounts to a corresponding paTCE comprising RSR-2295 release sequences in multiple tumor types (including gastric carcinoma (NCI-N87), colorectal adenocarcinoma (HT-29), colon carcinoma (HT-55) tumors). Thus, paTCEs comprising RSR-3213 have enhanced specificity for tumor tissues without a significant loss of activation in tumor tissues.
In Vitro Digest:
In vitro digest assays were performed to demonstrate that RSR-3213 is cleaved by MMP and ST14/matriptase, but not legumain. Two EpCAM-targeting paTCE (EpCAM-paTCE) molecules (one of having RSR-2295 on both sides of the TCE, and the other having RSR-3213 on both sides of the TCE) flanking the TCE core were digested with 5-fold dilutions of MMP9, legumain, or ST14/matriptase. Similar banding patterns were observed for both MMP9 and matriptase, suggesting the mutation of the legumain cleavage site did not affect cleavability of the MMP and serine protease cleavage sites. uTCE was observed for the paTCE containing RSR-2295 after digestion with legumain, indicating cleavage at the protease cleavable linker by legumain. uTCE was not observed for the paTCE containing RSR-3213 after digestion with legumain, indicating the mutation successfully prevented cleavage at the protease cleavable linker by legumain (FIG. 7A and FIG. 7B).
Plasma Stability—In Vivo Cleavability
Fluorescently labeled variants of an EpCAM-paTCE containing either RSR-2295 or RSR-3213 were labeled with Sulfo-Cy5.5 or Sulfo-Cy7.5. Opposite colors were co-injected into mice containing NCI-N87, HT-29, or HT-55 xenograft tumors. 48 hours after injection, tumors were harvested, homogenized, and protein extracts were analyzed by SDS-PAGE and LI-COR. Relative abundances for paTCE, 1×-C, 1×-N, and uTCE were quantified. No significant differences were observed in uTCE and 1×-C between the two protease cleavable linkers. paTCE containing RSR-2295 showed a small but statistically significant increase (average 2.19% more) in 1×-N than the corresponding paTCE containing RSR-3213. (FIG. 8A and FIG. 8B).
The observed cleavability in vivo from tumor homogenates was also determined from 3 different mouse tumor models. The % abundance for metabolites 1×-C, 1×-N, and uTCE was measured with results depicted in FIG. 8C. Finally, FIG. 8D depicts the % of total for paTCE plus the 3 metabolites (1×-N, 1×-C, and uTCE) when employing RSR-2295 or RSR-3213.
Overall, these data suggest that differences between in vivo cleavability of RSR-2295 and RSR-3213 are minor across 3 different tumor models.
Tumor Uptake:
Tumor uptake between EpCAM-paTCEs containing either RSR-2295 or RSR-3213 were compared using the ratio of calculated concentrations of total drug (paTCE, 1×-C, 1×-N, and uTCE).
While differences in tumor uptake were observed across 3 different tumor models, no significant differences were observed between RSR-2295 and RSR-3213 within each model. This indicates that the changes to the protease cleavable linkers between RSR-2295 and RSR-3213 do not affect tumor uptake of paTCE (FIG. 9).
Example 6. AMX-525, an Exemplary EGFR-Targeting Protease-Activated TCE
This example provides data relating to an exemplary paTCE, referred to as AMX-525. AMX-525 comprises the amino acid sequence set forth as SEQ ID NO: 1000. The annotated amino acid sequence for AMX-525 is provided below in Table 23:














SEQ ID




Component
NO:
Sequence
Annotation







X294(K)
8021
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESA
Barcode




TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS
underlined




EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS





ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP





GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSG





SETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE





PSEGSAPGSEPATSGSETPGTSESATP



 


RSR-3213
7628
EAGRSASHTPAGLTGP
—


 


Spacer
  96
GTSESATPES
—


 


EGFR.37 VL
 469
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGK
CDRs




APKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATY
underlined




YCQHFDHLPLAFGQGTKVEIK



 


Linker
  81
SESATPESGPGTSPGATPESGPGTSESATP
Barcode





underlined


 


EGFR.37 VH
 468
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQP
CDRs




PGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLS
underlined




SVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS



 


Linker
  87
GGGGS
—


 


hCD3.318-
 127
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQK
CDRs


VL

PGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPE
underlined




DEAVYYCALWYPNLWVFGGGTKLTVL



 


Linker
  81
SESATPESGPGTSPGATPESGPGTSESATP
Barcode





underlined


 


hCD3.318-
 126
EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAP
CDRs


VH

GKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLY
underlined




LQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVT





VSS



 


Spacer
  97
GTATPESGPG
—


 


RSR-3213
7628
EAGRSASHTPAGLTGP
—


 


X582(F)
8022
ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG
Barcode




SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
underlined




APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS





EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE





PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP





GTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT





STEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES





ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG





TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES





GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS





PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS





PSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE





GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGE





PEA









Method of Production of AMX-525

Methods for producing paTCEs proteins are known in the art, e.g., as described in PCT International Patent Publication No. WO2017/040344. For example, paTCE was expressed in E. coli, which was transformed with an expression vector encoding the paTCE and grown in fermentation. Fermentation cultures were grown with animal-free complex medium at 37° C. and temperature shifted to 26° C. before phosphate depletion, which triggered induction (PhoA). Target protein was partitioned into the periplasm via an N-terminal secretory leader sequence, which was cleaved during translocation. During collection, fermentation whole broth was centrifuged to pellet the product-containing cells, which were retained and frozen at ≤−70° C. The frozen cell pellet was resuspended and, once homogenous, the resuspension was mechanically lysed. The chilled flocculate was centrifuged (12,000 RCF, 10° C., 30 min) and the supernatant was decanted and retained, while the pellet was discarded. The following day, centrifugation was performed again (12,000 RCF, 10° C., 30 min) and the supernatant was decanted, submicron filtered and purified via a chromatographic process comprising an Anion Exchange (AEX) chromatography step. paTCEproteins and their derivatives were prepared as aqueous solutions and stored frozen at ≤−70° C. and, after thawing, at temperatures between 2° C. and 8° C.
An exemplary nucleotide sequence for the production of AMX-525 is provided below:








(SEQ ID NO: 8229)



GCATCTTCGGCGACGCCGGAAAGCGGTCCGGGTACGTCCACCGAACCGAGCGAGGGTAGCG



 


CTCCGGGCACCAGCGAGTCCGCGACCCCGGAAAGCGGTCCGGGTAGCGGTCCGGGCACCTC


 


CGAGAGCGCGACCCCGGGCACCTCTGAATCAGCCACCCCGGAGTCTGGCCCAGGTAGCGAG


 


CCGGCAACCTCTGGCAGCGAAACCCCGGGCACCAGCGAATCCGCGACGCCAGAGAGCGGTC


 


CGGGCACCTCTACGGAGCCTAGCGAGGGCTCAGCACCAGGTAGCCCTGCAGGTTCCCCGACG


 


TCAACCGAGGAAGGTACAAGCGAAAGCGCCACCCCTGAGTCGGGCCCTGGCAGCGAACCGG


 


CAACTAGCGGCAGCGAGACTCCGGGTACCAGCGAGTCTGCTACGCCAGAGAGCGGCCCAGG


 


TTCGCCAGCGGGTTCGCCGACTAGCACGGAGGAGGGCAGCCCAGCGGGTAGCCCTACCAGC


 


ACTGAAGAGGGTACGTCCACCGAACCGAGCGAAGGTAGCGCACCAGGTACCTCCGAGTCTG


 


CCACCCCTGAATCCGGTCCAGGTACCAGCGAATCAGCCACCCCGGAGTCGGGTCCAGGTACG


 


AGCGAATCTGCTACCCCGGAATCCGGCCCAGGCAGCGAACCTGCTACTAGCGGCAGCGAAA


 


CGCCGGGCAGCGAACCTGCCACGTCAGGCAGCGAGACGCCGGGTTCCCCTGCAGGCTCCCC


 


GACCAGCACTGAGGAGGGCACCTCCACCGAACCATCAGAAGGTAGCGCGCCTGGTACGTCA


 


ACCGAACCTTCCGAGGgcaGCGCACCGGGTTCAGAACCAGCTACGTCTGGGAGCGAGACCCC


 


GGGCACCTCCGAGTCGGCGACCCCGGAGGCAGGTCGTTCTGCTAGCCATACCCCTGCAGGGT


 


TAACTGGCCCCGGAACTTCAGAAAGTGCTACACCCGAGTCTGACATCCAGATGACCCAGAGC


 


CCGAGCAGCCTGAGCGCGAGCGTGGGTGATCGTGTTACCATTACCTGCCAGGCCTCCCAGGA


 


CATTTCTAACTATCTGAACTGGTACCAGCAAAAGCCGGGTAAAGCGCCGAAGCTGCTGATCT


 


ATGATGCTTCTAACCTGGAAACGGGTGTTCCGAGCCGTTTCAGCGGTAGCGGCAGCGGTACC


 


GACTTCACCTTTACCATCAGCAGCCTGCAGCCGGAGGATATTGCGACCTACTATTGCCAGCA


 


TTTTGATCATCTGCCGCTGGCGTTCGGTCAGGGCACCAAGGTGGAGATCAAATCGGAATCAG


 


CGACACCTGAATCTGGCCCTGGTACAAGTCCCGGCGCAACGCCCGAATCGGGTCCGGGGAC


 


GAGTGAATCTGCGACACCGCAGGTGCAACTGCAGGAGAGCGGTCCGGGCCTGGTTAAGCCG


 


AGCGAAACCCTGAGCCTGACCTGCACCGTGAGCGGTGGCAGCGTGAGCAGCGGCGATTATT


 


ATTGGACCTGGATTCGTCAGCCGCCGGGCAAGGGTCTGGAGTGGATTGGTCATATTTATTATT


 


CCGGTAACACCAACTACAACCCGAGCCTGAAAAGCCGTGTGACCATCAGCGTTGACACCAG


 


CAAGAACCAGTTCAGCCTGAAACTGAGCAGCGTGACCGCGGCGGATACCGCGGTTTACTATT


 


GCGCGCGTGATCGTGTGACCGGCGCGTTCGACATTTGGGGTCAGGGCACCCTGGTGACGGTT


 


AGCAGCGGTGGTGGCGGCAGCGAGTTAGTTGTGACCCAAGAGCCGAGCCTGACCGTTAGCC


 


CGGGTGGTACGGTCACCCTGACGTGCCGTAGCAGCAACGGTGCGGTCACGAGCAGCAACTA


 


TGCCAATTGGGTCCAGCAGAAACCGGGTCAAGCACCGCGTGGCCTGATCGGCGGCACCAAT


 


AAACGTGCCCCGGGTACTCCTGCGCGTTTCTCCGGTAGCCTGCTGGAAGGCAAAGCCGCTCT


 


GACCCTGAGCGGTGTCCAGCCGGAAGATGAAGCGGTGTACTACTGCGCGCTGTGGTATCCGA


 


ATCTGTGGGTTTTTGGCGGCGGTACCAAGCTGACCGTATTGAGCGAGAGCGCAACGCCAGAG


 


AGCGGTCCAGGCACCAGCCCAGGTGCCACCCCTGAGAGCGGCCCAGGTACTTCTGAGAGCG


 


CCACTCCGGAGGTCCAACTGGTGGAGTCTGGTGGTGGCATTGTTCAACCGGGTGGCTCGTTG


 


CGCCTGAGCTGTGCAGCTAGCGGCTTTACCTTCAGCACCTATGCGATGAATTGGGTTCGTCA


 


GGCACCGGGTAAGGGCCTGGAATGGGTGGGCCGTATCCGCACCAAGCGCAACGATTACGCG


 


ACCTACTACGCGGATAGCGTTAAAGGCCGCTTCACGATTAGCCGTGACGATTCCAAGAATAC


 


GCTGTATCTGCAAATGAACAGCCTGAAAACCGAAGATACCGCGGTGTATTACTGTGTGCGCC


 


ACGAAAATTTCGGCAACAGCTACGTGAGCTGGTTTGCACATTGGGGTCAGGGCACCCTGGTT


 


ACGGTGAGCTCCGGTACAGCTACTCCAGAATCAGGACCCGGGGAAGCTGGAAGAAGCGCCT


 


CACACACACCAGCTGGACTTACAGGCCCGGCTACTCCCGAAAGTGGGCCAGGAACATCAGA


 


GTCCGCGACCCCGGAAAGCGGTCCGGGTTCTCCAGCTGGCAGCCCGACCTCCACTGAAGAAG


 


GCACCTCTGAGTCTGCTACCCCTGAATCTGGTCCTGGCTCCGAACCTGCTACCTCTGGTTCCG


 


AAACTCCAGGTACCTCGGAATCTGCGACTCCGGAATCTGGCCCGGGCACGAGCACGGAGCC


 


GTCTGAGGGTAGCgcaccAGGTACCAGCACTGAGCCTTCTGAGGGCTCTGCACCGGGTACCTCC


 


ACGGAACCTTCGGAAGGTTCTGCGCCGGGTACCTCCACTGAGCCATCCGAGGGTTCAGCACC


 


AGGTACTAGCACGGAACCGTCCGAGGGCTCTGCACCAGGTACGAGCACCGAACCGTCGGAG


 


GGTAGCGCTCCAGGTAGCCCAGCGGGCTCTCCGACAAGCACCGAAGAAGGCACCAGCACCG


 


AGCCGTCCGAAGGTTCCGCACCAGGTACAAGCGAGAGCGCGACTCCTGAATCTGGTCCGGGT


 


AGCGAGCCTGCAACCAGCGGTTCTGAGACGCCGGGCACTTCCGAATCTGCGACCCCGGAGTC


 


CGGTCCAGGTTCAGAGCCGGCGACGAGCGGTTCGGAAACGCCGGGTACGTCTGAATCAGCC


 


ACGCCGGAGTCTGGTCCGGGTACCTCGACCGAACCAAGCGAAGGTTCGGCACCGGGTACTA


 


GCGAGAGCGCAACCCCTGAAAGCGGTCCGGGCAGCCCGGCAGGTTCTCCAACCAGCACCGA


 


AGAAGGTTCCCCTGCTGGTAGCCCGACCTCTACGGAGGAAGGTAGCCCTGCAGGTTCCCCAA


 


CTTCTACTGAGGAAGGTACTTCTGAGTCCGCTACCCCAGAAAGCGGTCCTGGTACCTCCACT


 


GAACCGTCTGAAGGCTCTGCACCAGGCACTTCTGAGTCTGCTACTCCAGAAAGCGGCCCAGG


 


TTCTGAACCAGCAACTTCTGGCTCTGAGACTCCAGGCACTTCTGAGTCCGCAACGCCTGAAT


 


CCGGTCCTGGTTCTGAACCAGCTACTTCCGGCAGCGAAACCCCAGGTACCTCTGAGTCTGCG


 


ACTCCAGAGTCTGGTCCTGGTACTTCCACTGAGCCTAGCGAGGGTTCCGCACCAGGTTCTCC


 


GGCTGGTAGCCCGACCAGCACGGAGGAGGGTACGTCTGAATCTGCAACGCCGGAATCGGGC


 


CCAGGTTCGGAGCCTGCAACGTCTGGCAGCGAAACCCCGGGTACCTCCGAATCTGCTACACC


 


GGAAAGCGGTCCTGGCAGCCCTGCTGGTTCTCCAACCTCTACCGAGGAGGGTTCACCGGCAG


 


GTAGCCCGACTAGCACTGAAGAAGGTACTAGCACGGAGCCGAGCGAGGGTAGTGCTCCGGG


 


TACGAGCGAGAGCGCAACGCCAGAGAGCGGTCCAGGCACCAGCGAATCGGCCACCCCTGAG


 


AGCGGCCCAGGTACTTCACCCTCTGCTACGCCGGAAAGCGGTCCGGGTTCCGAGCCGGCGAC


 


CAGCGGCTCCGAGACTCCGGGTTCGGAGCCGGCGACCTCCGGCTCGGAAACCCCGGGTAGC


 


CCGGCTGGTTCTCCGACCAGCACTGAGGAAGGCACCAGCACCGAACCAAGCGAGGGCAGCG


 


CGCCAGGTACGAGCACCGAACCGAGCGAGGGTTCAGCCCCTGGCTCTGAGCCGGCGACGTC


 


TGGCTCCGAAACCCCGGGCACCAGCGAGAGCGCTGGTGAACCGGAAGCG






In Vitro Binding Kinetics and Affinity

Kinetic studies were performed by surface plasmon resonance by BIAcore at 37° C. to determine binding KD of AMX-525 and its metabolites to human and cynomolgus monkey EGFR and CD3. The results are provided in Table 24 and show that the KD of AMX-525 is greater than either of the singly masked metabolites (1×-N or 1×-C) which is in turn greater that the unmasked metabolite (uTCE). Accordingly, masking decreases binding to EGFR and CD3. Additionally, biding to human and cynomolgus monkey EGFR and CD3 occurs with similar affinity.







TABLE 24







Binding kinetics and affinity of AMX-525 and metabolites












Human EGFR
Human CD3
Cyno EGFR
Cyno CD3




















KD
ka
kd
KD
ka
kd
KD
ka
kd
KD
ka
kd



(nM)
(M−1 s−1)
(s−1)
(nM)
(M−1 s−1)
(s−1)
(nM)
(M−1 s−1)
(s−1)
(nM)
(M−1 s−1)
(s−1)





AMX-525
0.736
1.04E+6
 7.7E−4
57
3.1E+6
0.18
1.048
 9.1E+5
1.0E−3
167
1.1E+6
0.18


AMX-525
0.205
2.03E+6
4.17E−4
36
4.0E+6
0.16
0.25
1.81E+6
4.5E−4
47
3.2E+6
0.15


(1x-N)














AMX-525
0.184
 2.0E+6
 3.6E−4
32
3.6E+6
0.114
0.238
2.12E+6
5.1E−4
43
2.1E+6
0.09


(1x-C)














AMX-525
0.034
6.45E+6
 2.3E−4
29.2
4.4E+6
0.128
0.042
 4.1E+6
1.7E−4
28
5.0E+6
0.14


(uTCE)









In Vitro Cytotoxicity

In vitro cytotoxicity of AMX-525 was determined in the following cancer cell lines: HT-29 (Colorectal, 22K EGFR/cell), MDA-MB-231 (Breast, 75K EGFR/cell), or A-431 (Epidermal; 590K EGFR/cell) with an Effector to Target (E: T) ratio of 5 to 1. The results are provided Table 25. The results show that the potency is as follows: uTCE >1×-N ≅1×-C >AMX-525>NoClvSite. Therefore, the results demonstrate that AMX-525 exhibits reduced potency compared to unmasked TCE and that two masks provide more protection than one. Unmasked AMX-525 induced potent cytotoxicity at about 3-10 μM in the cell lines tested. Cytotoxicity curves for exemplary donor are provided in FIG. 10A (HT-29), FIG. 10B (MDA-MB-231), and FIG. 10C (A-431).







TABLE 25







Cytotoxicity of AMX-525 and metabolites in cancer cell lines









EC50 (nM) - Geometric mean of donors










AMX-525
HT-29
MDA-MB-231
A-431





uTCE
  0.0096
 0.0029
 0.0032


AMX-525
>300   
1.0 
0.030


AMX-525(1x-C)
1.8
0.042
0.013


AMX-525(1x-N)
2.0
0.056
0.010


AMX-525(NoClvSite)
>300   
>3.0  
8.5 


Fold protection
>30,000x   
345x   
9.4x 


AMX-525/uTCE





Fold protection
>30,000X    
>1000x    
2700x    


AMX-525(NoClvSite)/





uTCE









In Vitro Cytokine Induction

The supernatants of HT-29 cells after cytotoxic reactions were harvested and in vitro cytokine induction assays were performed. The induction of IFNγ (FIG. 11A), TNFα (FIG. 11B), IL-6 (FIG. 11C), IL-10 (FIG. 11D) from a representative HT-29 donor are shown. In general, AMX-525 (uTCE) potently induced cytokine upregulation while AMX-525 and single-masked metabolites (1×-N and 1×-C) protected against cytokine induction (in line with cytotoxicity results). Two masks (AMX-525 and AMX-525 NoClvSite) generally protected better than single masked molecules.
In Vitro T Cell Activation
To evaluate the activity of T cells, AMX-525 or its metabolites were co-cultured with healthy human PBMCs together with HT-29 cells. Human PBMCs were incubated with titrations of AMX-525 or metabolites in the presence of HT-29 cells at 37° C. (PMBC: HT-29 cells at 5:1). After 72 hours, PBMCs were analyzed by flow cytometric analysis. Specifically, CD4 and CD8 T cells were interrogated for CD69, CD25, and PD-1 expression. Results for a representative donor are depicted in FIG. 12. The results show that unmasked AMX-525 (uTCE) resulted in T cell activation as determined by CD69, CD25, and PD-1 upregulation. Additionally, fully masked AMX-525 protected against T cell activation relative to AMX-525 (uTCE). AMX-525 intermediates (1X—C, 1X—N) maintained protection relative to AMX-525 (uTCE); albeit to a lesser extent (in general) than AMX-525.
Cytokine Release Assay
In vitro assessment of cytokine release is a predictive indicator for cytokine release syndrome (CRS). Overall, neither AMX-525 nor AMX-525 (uTCE) elicited significant cytokine induction in the cytokine release assay with human PBMC (Table 26). Stimulation of IL-6 (data not shown) was observed in the soluble treated groups and determined to be an artifact of the experiment based on corresponding non-human primate data and the lack of upregulation of other cytokines, which would be expected for a CRS response.







TABLE 26







Cytokine release assay










AMX-525
AMX-525 (uTCE)












Soluble
Wet-coated
Soluble
Wet-coated


Analyte
treatment
treatment
treatment
treatment





IL-2
−
−
−
−


IL-4
−
−
−
−


IL-10
+
−
−
−


TNFα
+
−
−
−


IFNγ
−
−
−
−





− No stimulation


+ moderate stimulation (at highest concentrations tested or in single donor)






In Vitro Plasma Stability

The in vitro plasma stability of AMX-525 was determined. Fluorescently labeled AMX-525 was spiked into various plasma samples at 200 nM. The samples were healthy human donors, human cancer donors (8 pancreatic, 2 head and neck, 4 ovarian), healthy cynomolgus monkeys, healthy mice, and tumor-bearing mice (HT-29-implanted CDX). The plasma samples were incubated at 37° C. for up to 7 days. The relative levels of AMX-525 and cleavage product was quantified by SDS-PAGE and LI-COR detection after 3 & 7 days, with the 7-day timepoint shown in FIG. 13. The results show that plasma stability of AMX-525 and its metabolites is similar between humans, monkeys, and mice. Accumulation of fully cleaved and intermediate products was observed over time. Overall, AMX-525 is relatively stable in healthy and cancer patient plasma.
In Vivo Efficacy in EGFR-Expressing Tumor Bearing Mice
AMX-525 was evaluated in three in vivo cell line-derived mouse xenograft models which include MDA-MB-231 (Breast; EGFR receptor density: 75K/cell), HT-29 (Colorectal; EGFR receptor density: 22K/cell), and LoVo (Colorectal; EGFR receptor density: 28K/cell).
The in vivo efficacy of AMX-525, AMX-525-NoClvSite, and AMX-525 (uTCE) was evaluated in the human PBMC-engrafted HT-29 human colorectal tumor model in nonobese diabetic (NOD).Cg-Prkdcscid 112rgtmlwjl/SzJ (NSG) mice.
Mice bearing HT-29 tumors were randomized into 7 groups of 6 mice each and administered vehicle diluent (no PBMC), vehicle diluent (PBMC), 0.5 mg/kg AMX-525, 1 mg/kg AMX-525, 3 mg/kg AMX-525, 3 mg/kg AMX-525-NoClvSite, and 0.3 mg/kg AMX-525 (uTCE) by weekly bolus IV. Experimental design and results summary are shown in Table 27. Tumor growth curves between treatment initiation (Day 4) and study termination (Day 24) are shown in FIG. 14.
All test articles were well tolerated by the experimental animals, as evidenced by the similar average body weight change (% BW) in the range of 6.3-14.5% across all experimental groups.
AMX-525 treatment promoted anti-tumor activity at all dose levels evaluated when compared with the applicable PBMC-engrafted control, Group 2. At Day 24, the end of the study, AMX-525 at a dose level of 3 mg/kg QW showed TGI of 75% (p<0.0001), while the intermediate (1.5 mg/kg QW) and lowest (0.5 mg/kg QW) dose levels showed TGIs of 73% (p=0.0001) and 65% (p=0.0001), respectively. The AMX-525-NoClvSite group showed non-statistically significant TGI of 15% (p=0.885). The protease-activatable AMX-525 exhibited a greater anti-tumor effect at a dose of 0.5 mg/kg (65% TGI) than that observed with AMX-500-NoClvSite at the 6 times greater QW dose of 3 mg/kg (15% TGI), indicating that the ELNN masks of AMX-525 may be removed in the tumor micro-environment, releasing the potent unmasked TCE.







TABLE 27







Study Design and Results Summary, HT-29










Study Design




















Dose
Dosing

Dosing
Day 24 Results



















Level
Volume

Frequency and


Tumor


Group
N
Treatment
(mg/kg)
(mL/kg)
Route
Duration
±BWa
TGIb
Regression c (#/n)





1
6
Vehicle diluent,
—
10
IV
QW × 3 weeks
 6.3%
—
0/6




no PBMC









2
6
Vehicle diluent,
—
10
IV
QW × 3 weeks
12.7%
—
0/6




PBMC









3
6
AMX-500
0.5
10
IV
QW × 3 weeks
11.4%
 65%
0/6


4
6
AMX-500
1.0
10
IV
QW × 3 weeks
11.2%
 73%
0/6


5
6
AMX-500
3.0
10
IV
QW × 3 weeks
 8.0%
 75%
1/6


6
6
AMX-
3.0
10
IV
QW × 3 weeks
11.3%
 15%
0/6




500-NoClvSite









7
6
AMX-500(uTCE)
0.3
10
IV
QW × 3 weeks
14.5%
108%
5/6





Abbreviations: BW, body weight; IV, intravenous; NA, not applicable; PBMC, peripheral blood mononuclear cells; QW, one time a week; TGI, tumor growth inhibition.


a±BW % = percent body weight change compared with body weight at the start of treatment


bTGI (%) = (Vc-Vt)/(Vc-Vo) × 100, where Vc and Vt are the mean tumor volume of the control and treated groups at the end of the study (respectively) and Vo is the mean tumor volume of the control group at the start of dosing. TGI was calculated versus Group 2 (Vehicle diluent, PBMC).


c Tumor regression was defined as tumor volume at study end (Day 24), which is less than the starting tumor volume prior to dosing.






The in vivo efficacy of AMX-525, AMX-525-NoClvSite, and AMX-525 (uTCE) was evaluated in the human PBMC-engrafted LoVo human colorectal tumor model in nonobese diabetic (NOD).Cg-Prkdcscid 12rgtmlWjl/SzJ (NSG) mice.
Mice bearing LoVo tumors were randomized into 7 groups of 8 mice each and administered vehicle diluent (no PBMC), vehicle diluent (PBMC), 0.5 mg/kg AMX-525, 1 mg/kg AMX-525, 3 mg/kg AMX-525, 3 mg/kg AMX-525-NoClvSite, and 0.35 mg/kg AMX-525 (uTCE) by weekly bolus IV. Experimental design and results summary are shown in Table 28. Tumor growth curves between treatment initiation (Day 5) and study termination (Day 27) are shown in FIG. 15.
All test articles were well tolerated by the experimental animals, as evidenced by the similar average body weight loss (BWL) in the range of 1.1-6.4% across all experimental groups engrafted with PBMCs.
AMX-525 treatment promoted anti-tumor activity at all dose levels evaluated when compared with the applicable PBMC-engrafted control, Group 2. At Day 27, the end of the study, AMX-525 displayed dose-dependent TGI with the highest dose level of 3 mg/kg QW showing TGI of 95% (p<0.0001), while the intermediate (1.5 mg/kg QW) and lowest (0.5 mg/kg QW) dose levels showed TGIs of 75% (p=0.0001) and 36% (p=0.0001), respectively. At Day 27, AMX-525 treatment at the highest tested dose of 3 mg/kg QW had similar TGI (95% TGI) as the enzymatically cleaved and activated AMX-525 (uTCE) (94% TGI) using a 0.35 mg/kg QW dose. The protease-activatable AMX-525 exhibited a greater anti-tumor effect at a dose of 0.5 mg/kg (64% TGI) than that observed with AMX-500-NoClvSite at a QW dose of 3 mg/kg (52% TGI), indicating that the ELNN masks of AMX-500 may be removed in the tumor micro-environment, releasing the potent unmasked TCE.
Immunohistochemistry (IHC) was performed on tumor tissue from the LoVo xenograft mouse model at days 2, 6, and 9. IHC shows that AMX-525 recruits CD8+ T cells to LoVo xenograft tumor in a dose and time dependent manner (FIG. 16). Likewise, AMX-525 recruits CD4+T cells to LoVo xenograft tumor in a dose and time dependent manner (FIG. 17). Additionally, PD-L1 upregulation on tumor and immune cells was observed in AMX-525 treated tumors (FIG. 18).







TABLE 28







Study Design and Results Summary, LoVo










Study Design


















Dose
Dosing

Dosing
Day 27 Results



















Level
Volume

Frequency and


Tumor


Group
N
Treatment
(mg/kg)
(mL/kg)
Route
Duration
±BWa
TGIb
Regression c (#/n)





1
8
Vehicle diluent,
—
10
IV
QW × 3 weeks
−2.7%

8/0




no PBMC









2
8
Vehicle diluent,
—
10
IV
QW × 3 weeks
 1.1%

0/8




PBMC









3
8
AMX-500
0.5
10
IV
QW × 3 weeks
 5.4%
 36%
0/8


4
8
AMX-500
1.5
10
IV
QW × 3 weeks
 3.5%
 75%
1/8


5
8
AMX-500
3.0
10
IV
QW × 3 weeks
 6.4%
 95%
3/8


6
8
AMX-
3.0
10
IV
QW × 3 weeks
 1.9%
−50%
0/8




500-NoClvSite









7
8
AMX-500(uTCE)
0.35
10
IV
QW × 3 weeks
 5.4%
 94%
3/8





Abbreviations: BW, body weight; IV, intravenous; NA, not applicable; PBMC, peripheral blood mononuclear cells; QW, one time a week; TGI, tumor growth inhibition.


a#BW % = percent body weight change compared with body weight at the start of treatment


bTGI (%) = (Vc-Vt)/(Vc-Vo) × 100, where Vc and Vt are the mean tumor volume of the control and treated groups at the end of the study (respectively) and Vo is the mean tumor volume of the control group at the start of dosing. TGI was calculated versus Group 2 (Vehicle diluent, PBMC).


c Tumor regression was defined as tumor volume at study end (Day 27), which is less than the starting tumor volume prior to dosing.






The in vivo efficacy of AMX-525, AMX-525-NoClvSite, and AMX-525 (uTCE) was evaluated in the human PBMC-engrafted MDA-MB-231 human breast tumor model in nonobese diabetic (NOD).Cg-Prkdcscid I12rgtmlWjl/SzJ (NSG) mice.
Mice bearing MDA-MB-231 tumors were randomized into 6 groups of 8 mice each and administered vehicle diluent (no PBMC), vehicle diluent (PBMC), 0.1 mg/kg AMX-525, 0.5 mg/kg AMX-525, 2 mg/kg AMX-525, and 0.1 mg/kg AMX-525 (uTCE) by weekly bolus IV. Experimental design and results summary are shown in Table 29. Tumor growth curves between treatment initiation (Day 5) and study termination (Day 27) are shown in FIG. 19.
All test articles were well tolerated by the experimental animals, as evidenced by the similar 0.5-5.5% average body weight gain in the range of 0.5-5.5% across all experimental groups.
AMX-525 treatment promoted anti-tumor activity at all dose levels evaluated when compared with the applicable PBMC-engrafted control, Group 2. At Day 36, the end of the study, AMX-525 displayed dose-dependent TGI with the highest dose level of 2 mg/kg QW showing TGI of 114% (p<0.0001), while the intermediate (0.5 mg/kg QW) and lowest (0.1 mg/kg QW) dose levels showed TGIs of 91% (p=0.0001) and 50% (p=0.0001), respectively. At Day 27, AMX-525 treatment at the highest tested dose of 3 mg/kg QW had similar TGI (114% TGI) as the enzymatically cleaved and activated AMX-525 (uTCE) (106% TGI) using a 0.1 mg/kg QW dose.







TABLE 29







Study Design and Results Summary, MDA-MB-231










Study Design


















Dose
Dosing

Dosing
Day 36 Results



















Level
Volume

Frequency and


Tumor


Group
N
Treatment
(mg/kg)
(mL/kg)
Route
Duration
±BWa
TGIb
Regression c (#/n)





1
8
Vehicle diluent,
—
10
IV
QW × 3 weeks
2.1%

0/8




no PBMC









2
8
Vehicle diluent,
—
10
IV
QW × 3 weeks
2.0%

0/8




PBMC









3
8
AMX-500
0.1
10
IV
QW × 3 weeks
5.5%
 50%
0/8


4
8
AMX-500
0.5
10
IV
QW × 3 weeks
4.3%
 91%
1/8


5
8
AMX-500
2
10
IV
QW × 3 weeks
2.3%
114%
5/8


6
8
AMX-500(uTCE)
0.1
10
IV
QW × 3 weeks
0.5%
106%
5/8





Abbreviations: BW, body weight; IV, intravenous; NA, not applicable; PBMC, peripheral blood mononuclear cells; QW, one time a week; TGI, tumor growth inhibition.


aBW % = percent body weight change compared with body weight at the start of treatment


bTGI (%) = (Vc-Vt)/(Vc-Vo) × 100, where Vc and Vt are the mean tumor volume of the control and treated groups at the end of the study (respectively) and Vo is the mean tumor volume of the control group at the start of dosing. TGI was calculated versus Group 2 (Vehicle diluent, PBMC).


c Tumor regression was defined as tumor volume at study end (Day 36), which is less than the starting tumor volume prior to dosing.






In Vivo Tumor Distribution and Tumor Cleavage

The tumor tissue distribution and masking polypeptide cleavage of AMX-525 was determined. Tumor-bearing mice were administered fluorescently labeled AMX-525. Five patient-derived xenograft models of non-small cell lung cancer were evaluated (n=2 mice per model) and select healthy tissues (tumor, heart, lung, liver) and plasma was collected 48 hours post-administration. A control paTCE was spiked in during homogenization of tissues. Relative abundance of AMX-525 and cleavage products were quantified by SDS-PAGE and LI-COR detection. AMX-525 distributed to healthy tissue and xenografted tumor within 48 hours after administration. As shown in Table 30, AMX-525 cleavage intermediates (1X—N and 1X—C) and fully unmasked AMX-525 (uTCE) were detected in the NSCLC tumor xenograft. By contrast, minimal cleavage of AMX-525 was observed in plasma or healthy tissue.







TABLE 30







In vivo cleavage (% relative abundance)












AMX-525
1X-N
1X-C
uTCE
















Tumor
56%
6.1%
4.4%
34%



Heart
99%
BLQ
BLQ
BLQ



Lung
99%
BLQ
BLQ
BLQ



Liver
99%
BLQ
BLQ
BLQ



Plasma
97%
1.4%
1.4%
BLQ





BLQ = below level of quantification; noted if ≥50% of animals are BLQ






Combination with PD-1 and PD-L1 Inhibitors

As described above, PD-1 was upregulated on CD4+ and CD8+ T cells following treatment with AMX-525 in an in vitro T cell activation assay (FIG. 12). Also as described above, in a LoVo mouse xenograft tumor model, PD-L1 was upregulated on tumor and immune cells in AMX-525 treated tumors (FIG. 18). This suggests administering an EGFR-targeted paTCE such as AMX-525 with an anti-PD-1 or anti-PD-L1 inhibitor.
In Vivo Efficacy in Mice-Pembrolizumab Combination
In addition, the in vivo efficacy of AMX-525 or AMX-525-NoClvSite in combination with an anti-PD-1 antibody, pembrolizumab, was evaluated in the human peripheral blood mononuclear (PBMC)-engrafted SK—OV-3 human ovarian tumor model in NOD.Cg-Prkdcscid H2-K1tlBpe H2-Ab1emlMvw H2-D1tmlBpe 112rgtmlWjl/SzJ (NSG-MHC I/II DKO) mice.
Mice were inoculated subcutaneously with 5×106SK—OV-3 tumor cells (Day 0). On Day 18, randomization was performed using a tumor volume-stratified randomization method and were engrafted with 1×107PBMCs. Mice were administered either vehicle diluent (no PBMCs), vehicle diluent (with PBMCs), 0.01 mg/kg AMX-525, 0.1 mg/kg AMX-525, 0.5 mg/kg AMX-525, 2 mg/kg AMX-525, 2 mg/kg AMX-525-NoClvSite, 10 mg/kg pembrolizumab, or both 0.1 mg/kg AMX-525 and 10 mg/kg pembrolizumab for 2 weeks. AMX-525 and AMX-525-NoClvSite were administered once weekly via bolus intravenous (IV) lateral tail vein injection. Pembrolizumab was administered twice weekly via bolus intraperitoneal (IP) injection. Experimental design and results summary are shown in Table 31Table. Tumor growth curves between treatment initiation (Day 19) and study termination (Day 29) of AMX-525 (FIG. 20) and in combination with pembrolizumab (FIG. 21) are provided. All test agents were well tolerated by test animals, as shown by the body weight gain (BWG) across all groups.
At Day 29, AMX-525 treatment promoted anti-tumor activity at all dose levels with tumor growth inhibitions (TGIs) in the range of 27.8% to 58.2% when compared with the applicable PBMC-engrafted control, Group 2. AMX-525-NoClvSite did not exhibit anti-tumor activity, suggesting AMX-525's TGI is cleavage dependent. AMX-525 and pembrolizumab have greater anti-tumor activity when combined (Group 9:67.9% TGI) than single-agent 0.1 mg/kg AMX-525 (Group 4:27.8% TGI) and single-agent pembrolizumab (Group 8:30.4% TGI).







TABLE 31







Study Design and Results Summary








Study Design





















Dosing
Day 29 Results


















Dose Level
Dosing Volume

Frequency,
BWG
TGIª


Group
N
Treatment
(mg/kg)
(mL/kg)
Route
Duration
(%)
(%)





1
8
Vehicle diluent,
NA
10
Bolus
QW × 2
9.8
NA




no PBMCs


IV
weeks




2
8
Vehicle diluent,
NA
10
Bolus
QW × 2
2.9
NA




PBMCs


IV
weeks




3
8
AMX-525
0.01
10
Bolus
QW × 2
6.4
34.6







IV
weeks




4
8
AMX-525
0.1
10
Bolus
QW × 2
7.5
27.8







IV
weeks




5
8
AMX-525
0.5
10
Bolus
QW × 2
4.2
52.4







IV
weeks




6
8
AMX-525
2
10
Bolus
QW × 2
9.1
58.2







IV
weeks




7
8
AMX-525-
2
10
Bolus
QW × 2
3.6
−16.5




NoClvSite


IV
weeks




8
8
Pembrolizumab
10
10
Bolus
BIW × 2
8.4
30.4







IP
weeks




9
8
AMX-525 and
0.1 and 10
10 and 10
Bolus
QW × 2
7.1
67.9




pembrolizumab


IV and
weeks and









IP
BIW × 2










weeks





Abbreviations: BIW, twice weekly; BWG, body weight gain compared with body weight at the start of treatment; IP, intraperitoneal; IV, intravenous; NA, not applicable; PBMC, peripheral blood mononuclear cell; QW, once per week; TGI, tumor growth inhibition.


aTGI (%) = Vc-Vt)/(Vc-Vo) × 100, where Vc and Vt are the mean tumor volume of the control and treated groups at the end of the study (respectively) and Vo is the mean tumor volume of the control group at the start of dosing. TGI was calculated vs Group 2: vehicle diluent, PBMCs.






While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.