Anti-IL-5 antibodies

Disclosed herein are fully human antibody molecules that immunospecifically bind to human IL-5. The antibody molecules can bind to human IL-5 with an equilibrium affinity constant (KD) of at least about 40 pM as determined by surface plasmon resonance.

1. 11. A human antibody molecule that immunospecifically binds to human Interleukin-5 (IL-5), wherein the antibody molecule comprises: a heavy chain complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15, and an IgG4 heavy chain comprising a S228P mutation, a M252Y mutation, a S254T mutation, a T256E mutation, and a deletion of a heavy chain C-terminal lysine residue.
2. 22. The antibody molecule of claim 1, wherein the antibody molecule comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17.
3. 33. The antibody molecule of claim 1, wherein the antibody molecule comprises the IgG4 heavy chain constant region comprising the S228P mutation, the M252Y mutation, the S254T mutation, the T256E mutation, and the deletion of the heavy chain C-terminal lysine residue, and a lambda light chain constant region.
4. 44. The antibody molecule of claim 3, wherein the antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and a light chain comprising the amino acid sequence of SEQ ID NO: 19.
5. 55. A pharmaceutical composition comprising the antibody molecule of claim 1.
6. 66. A pharmaceutical composition comprising the antibody molecule of claim 4.
7. 77. A nucleic acid molecule encoding the antibody molecule of claim 1.
8. 88. A vector comprising the nucleic acid molecule of claim 7.
9. 99. An isolated cell transformed to express the antibody molecule of claim 1.
10. 1010. A nucleic acid molecule encoding the antibody molecule of claim 4.
11. 1111. A vector comprising the nucleic acid molecule of claim 10.
12. 1212. An isolated cell transformed to express the antibody molecule of claim 4.

CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 15/848,090, filed Dec. 20, 2017 (now U.S. Pat. No. 11,111,292), which claims priority to U.S. Provisional Application No. 62/438,502, filed Dec. 23, 2016, the disclosures of each of which are hereby incorporated by reference in their entirety.


SEQUENCE LISTING
The instant application contains a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 26, 2021, is named 102085.001560_sl.txt and is 91 KB in size.
TECHNICAL FIELD
Disclosed herein are novel antibody molecules that immunospecifically bind IL-5, and uses of the disclosed antibodies.
BACKGROUND
Interleukin-5 (IL-5) is a T-helper 2 (Th2) cytokine which causes the proliferation and differentiation of both B cells and eosinophils. In B cells, IL-5 also increases immunoglobulin secretion. IL-5 is a key modulator of eosinophils, where it also regulates maturation, migration to tissues, survival, and the prevention of apoptosis.
Through two separate motifs, IL-5 binds to its specific receptor (IL5-Rα) and a signaling receptor, a common β-chain (βc) shared between Interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GMCSF). The affinity of IL-5 for IL5-Rα has been reported to be in the mid-low nM range (0.2-100 nM); this shifts into the mid-pM range (˜100 pM) in the presence of βc. IL5-Rα binds to IL-5 specifically, which then recruits βc to IL-5R.
The therapeutic potential of targeting interleukin-5 (IL-5) has been demonstrated by extensive validation in the literature and recent positive Phase III clinical data for both reslizumab and mepolizumab.
SUMMARY
Disclosed herein are human antibody molecules that immunospecifically bind to human IL-5 with an equilibrium affinity constant (KD) of at least about 40 pM as determined by surface plasmon resonance.
Also provided are antibody molecules comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR1 comprising the amino acid sequence of SEQ ID NOs: 5, 21, 24, 27, 30, 33, 36, 39, or 66, a light chain CDR2 comprising the amino acid sequence of SEQ ID NOs: 7, 42, or 45, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NOs: 15, 48, 51, 54, 57, 60, or 63.
The disclosed antibody molecules can comprise a heavy chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, or 67, wherein the variability (i.e. the at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) occurs outside of the CDR sequence.
The disclosed antibody molecules can comprise a heavy chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 18 or 20 and a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, or 68, wherein the variability (i.e. the at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) occurs outside of the CDR sequence.
Nucleic acid molecules encoding the disclosed antibody molecules, vectors comprising the nucleic acid molecules, and cells transformed to express the disclosed antibody molecules are also provided.
Also disclosed are pharmaceutical compositions comprising any of the antibody molecules disclosed herein.
Also provided are methods of treating a subject having eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis comprising administering to the subject a therapeutically effective amount of any of the herein disclosed antibody molecules, or pharmaceutical compositions comprising the same, to treat the eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis.
Use of an effective amount of any of the herein disclosed antibody molecules, or pharmaceutical compositions comprising the same, in the treatment of eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis, or eosinophilic esophagitis are also provided.
Further provided is the use of any of the herein disclosed antibody molecules, or pharmaceutical compositions comprising the same, in the manufacture of a medicament for the treatment of eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis.



BRIEF DESCRIPTION OF THE DRAWINGS
The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed antibody molecules, methods, and uses, there are shown in the drawings exemplary embodiments of the antibody molecules, methods, and uses; however, the antibody molecules, methods, and uses are not limited to the specific embodiments disclosed. In the drawings:
FIG. 1 illustrates a TF-1.6G4 assay showing that antibody variant 3A5.001 (in IgG4 format) retained an equivalent potency to the original antibody 3A5 (in IgG1 format).
FIG. 2 illustrates a TF-1.6G4 assay showing that antibody variant 3A5.040 retained an equivalent potency to the parent antibody 3A5.001.
FIG. 3 is a graphical matrix showing the position and identity of the different single amino acid substitutions that were generated on the 3A5.040 VH CDRs, aligned to the original 3A5.040 VH sequence (top sequence). The boxes contain the residues which were designated CDR residues according to the AbM nomenclature. In addition to the CDR residues, variants were made to Kabat residue numbers 93 and 94 (adjacent to HC CDR3) as described in the Example “Functional testing and characterization of antibodies.” The various sequences recited in this figure are provided in SEQ ID NO:73.
FIG. 4 is graphical matrix showing the position and identity of the different single amino acid substitutions that were generated to the 3A5.040 VL CDRs, aligned to the original 3A5.040 VL sequence (top sequence). The boxes contain the residues which were designated CDR residues according to the AbM nomenclature. The various sequences recited in this figure are provided in SEQ ID NO:74.
FIG. 5 illustrates exemplary light chain CDR consensus sequences aligned to the 3A5.040 VL sequence (top sequence). 3A5.040 VL CDR single amino acid substitution variants which showed potential improvements according to the inclusion criteria described in the Examples of (the ratio of variant kd to 3A5.040 kd≥1.5) and (the ratio of variant expression level to 3A5.040 expression level ≥0.5) were included in the consensus sequences. CDR definitions and numbering are according to AbM and Kabat nomenclature, respectively. The boxes identify the positions of CDR residues. The various sequences recited in this figure are provided in SEQ ID NO:75.
FIG. 6 illustrates a multi-concentration BIACORE™ kinetic analysis of 3A5.046 antibody binding to recombinant human IL-5 at 2.5, 1.25, 0.625, 0.313 and 0.156 μg/mL. Sensorgrams show decreasing concentrations of IL-5 in order from 2.5 μg/mL IL-5 in the top trace to 0.156 μg/mL IL-5 in the bottom trace.
FIG. 7A and FIG. 7B illustrate the proliferation of TF-1.6G4 cells in response to IL-5, and the potency of inhibition of IL-5 driven proliferation by 3A5.046. 3A5.046 was a more potent inhibitor of human IL-5 (FIG. 7A) and cynomolgus monkey IL-5 (FIG. 7B) than mepolizumab.
FIG. 8A and FIG. 8B illustrate an exemplary ELISA developed using the 3A5 antibody and a control capture antibody (R&D SYSTEMS®). The ELISA was able to detect recombinant IL-5 (FIG. 8A) and IL-5 produced from CD3/CD28/IL-33 activated primary human T-cells from 3 donors (FIG. 8B). FIG. 8B top panel: Donor 1, FIG. 8B middle panel: Donor 3; FIG. 8B bottom panel: Donor 4.
FIG. 9 illustrates the results of an experiment in which CD34+ cord blood cells differentiated into phenotypically mature eosinophils using IL-5 and other cytokines as described in the Examples. Antibody 3A5.046 was more potent than mepolizumab at inhibiting IL-5 induced eosinophil differentiation.
FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, and FIG. 10E illustrate the results of the BIACORE™ crossreactivity analysis of antibody 3A5.046 binding to human IL-5 (FIG. 10A), cynomolgus monkey IL-5 (FIG. 10B), mouse IL-5 (FIG. 0C), rat IL-5 (FIG. 10D), and guinea pig IL-5 (FIG. 10E). Double-referenced sensorgrams are shown for binding to cytokines at 1 μg/ml or 10 μg/ml.
FIG. 11A, FIG. 11B, FIG. 11C, and FIG. 11D illustrate the results of a BIACORE™ specificity analysis of antibody 3A5.046. Double-referenced sensorgrams are shown for binding to cytokines at 10 μg/ml (FIG. 11A and FIG. 11B) or 1 μg/ml (FIG. 11C and FIG. 11D).
FIG. 12 illustrates the results of Ascaris suum (A. suum) challenge in a cynomolgus monkey model of airway eosinophilia. At day 2 there was a substantial difference in lung (BALF) eosinophil numbers when comparing 3A5.046-treated and vehicle (placebo)-treated animals (p<0.01; Mann-Whitney test).
FIG. 13A and FIG. 13B illustrate the blood eosinophil response over 10 days following an A. suum challenge in cynomolgus monkeys which were pre-treated with 3A5.046 or a isotype-matched control antibody (“placebo”) one week before A. suum challenge (FIG. 13A). FIG. 13B illustrates further details of the blood eosinophil counts for animals treated with 3A5.046 antibody, up to 45 days post-challenge (52 days post-dose). Eosinophil counts remained below baseline for at least 45 days post-challenge following a single dose of 3A5.046 one week before challenge.
FIG. 14 illustrates BALF eosinophil levels in a human IL-5 knock-in (KI) rat model in response to a challenge with Alternaria alternata. This model may be used to test the potency of an anti-IL-5 antibody to modulate the numbers of BALF eosinophils following Alternaria challenge.



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The disclosed antibody molecules, methods, and uses may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed antibody molecules, methods, and uses are not limited to the specific antibody molecules, methods, and uses described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed antibody molecules, methods, and uses.
Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed antibody molecules, methods, and uses are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.
Throughout this text, the descriptions refer to antibody molecules and methods of using said antibody molecules. Where the disclosure describes or claims a feature or embodiment associated with an antibody molecule, such a feature or embodiment is equally applicable to the methods of using said antibody molecule. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of using an antibody molecule, such a feature or embodiment is equally applicable to the antibody molecule.
Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.
Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.
When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. Thus, the term “about” is used to encompass variations of ±10% or less, variations of ±5% or less, variations of ±1% or less, variations of ±0.5% or less, or variations of ±0.1% or less from the specified value.
It is to be appreciated that certain features of the disclosed antibody molecules, methods, and uses which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed antibody molecules, methods, and uses that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.
As used herein, the singular forms “a,” “an,” and “the” include the plural.
Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.
The term “comprising” is intended to include, but is not necessarily limited to, examples encompassed by the terms “consisting essentially of” and “consisting of”; similarly, the term “consisting essentially of” is intended to include, but is not necessarily limited to, examples encompassed by the term “consisting of.”
The term “antibody molecule” is meant in a broad sense and includes full length immunoglobulin molecules and antigen binding fragments thereof.
Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAQ1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (x) and lambda (k), based on the amino acid sequences of their constant domains.
“Antigen binding fragment” refers to a portion of an immunoglobulin molecule that retains the antigen binding properties of the parental full length antibody (i.e. “antigen binding fragment thereof”). Exemplary antigen binding fragments can have: heavy chain complementarity determining regions (HCDR) 1, 2, and/or 3; light chain complementarity determining regions (LCDR) 1, 2, and/or 3; a heavy chain variable region (VH); a light chain variable region (VL); and combinations thereof. Antigen binding fragments include: a Fab fragment, a monovalent fragment consisting of the VL, VH, constant light (CL), and constant heavy 1 (CH1) domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and a domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VH domain or a VL domain. VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody, described for example in Int'l Pat. Pub. Nos. WO1998/44001, WO1988/01649, WO1994/13804, and WO1992/01047. These antibody fragments are obtained using techniques well known to those of skill in the art, and the fragments are screened for utility in the same manner as are full length antibodies.
The phrase “immunospecifically binds” refers to the ability of the disclosed antibody molecules to preferentially bind to its target (IL-5 in the case of anti-IL-5 antibody molecules) without preferentially binding other molecules in a sample containing a mixed population of molecules. Antibody molecules that immunospecifically bind IL-5 are substantially free of other antibodies having different antigenic specificities (e.g., an anti-IL-5 antibody is substantially free of antibodies that specifically bind antigens other than IL-5). Antibody molecules that immunospecifically bind IL-5, however, can have cross-reactivity to other antigens, such as orthologs of human IL-5, including Macacafascicularis (cynomolgus monkey) IL-5. The antibody molecules disclosed herein are able to immunospecifically bind both naturally-produced human IL-5 and to IL-5 which is recombinantly produced in mammalian or prokaryotic cells.
An antibody variable region consists of four “framework” regions interrupted by three “antigen binding sites.” The antigen binding sites are defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDRI, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat J Exp Med 132:211-50, 1970; Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991); and (ii) “Hypervariable regions” (“HVR” or “HV”), three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) refer to the regions of the antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk Mol Biol 196:901-17, 1987). The AbM definition of CDRs is also widely used; it is a compromise between Kabat and Chothia numbering schemes and is so-called because it was used by Oxford Molecular's AbM antibody modelling software (Rees, A. R., Searle, S. M. J., Henry, A. H. and Pedersen, J. T. (1996) In Sternberg M. J. E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172). Other terms include “IMGT-CDRs” (Lefranc et al., Dev Comparat Immunol 27:55-77, 2003) and “Specificity Determining Residue Usage” (SDRU) (Almagro Mol Recognit 17:132-43, 2004). The International ImMunoGeneTics (IMGT) database (http://www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.
“Framework” or “framework sequences” are the remaining sequences of a variable region other than those defined to be antigen binding sites. Because the antigen binding sites can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigen-binding site was defined. “Human antibody,” “fully human antibody,” and like terms refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin. A human antibody comprises heavy and/or light chain variable regions that are “derived from” sequences of human origin if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci as described herein. “Human antibody” may contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to, for example, naturally occurring somatic mutations or intentional introduction of substitutions in the variable domain (framework and antigen binding sites), or constant domain. Typically, a “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., J Mol Biol 296:57-86, 2000, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, as described in, for example, Shi et al., J Mol Biol 397:385-96, 2010 and Int'l Pat. Pub. No. WO2009/085462. Antibodies in which antigen binding sites are derived from a non-human species are not included in the definition of “human antibody.”
Human antibodies, while derived from human immunoglobulin sequences, may be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties in the variable regions or the constant regions or both, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.
“Recombinant antibody” includes all antibodies that are prepared, expressed, created, or isolated by recombinant means, such as: antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below); antibodies isolated from a host cell transformed to express the antibody; antibodies isolated from a recombinant, combinatorial antibody library; and antibodies prepared, expressed, created, or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, or antibodies that are generated in vitro using Fab arm exchange.
“Monoclonal antibody” refers to a population of antibody molecules of a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes. Monoclonal antibody therefore refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well known alterations such as removal of C-terminal lysine from the antibody heavy chain. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.
“Epitope” refers to a portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar, or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule.
“Variant” refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions, or deletions. The term “mutation” as used herein is intended to mean one or more intentional substitutions which are made to a polypeptide or polynucleotide.
As used herein “90% identical to” encompasses at least 90% identical, 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical to the reference item (e.g., a biological sequence). The current specification uses the term “% identical” to describe a number of sequences. As would be understood, the term “% identical” means that in a comparison of two sequences over the specified region the two sequences have the specified number of identical residues in the same position. The level of identity may be determined using CLUSTAL W with default parameters.
“Treat,” “treatment,” and like terms refer to both therapeutic treatment and prophylactic or preventative measures, and includes reducing the severity and/or frequency of symptoms, eliminating symptoms and/or the underlying cause of the symptoms, reducing the frequency or likelihood of symptoms and/or their underlying cause, improving or remediating damage caused, directly or indirectly, by the eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis. Treatment also includes prolonging survival as compared to the expected survival of a subject not receiving treatment. Subjects to be treated include those that have the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
As used herein, “administering to the subject” and similar terms indicate a procedure by which the disclosed antibody molecules or compositions comprising the same are injected into a patient such that target cells, tissues, or segments of the body of the subject are contacted with the disclosed antibody molecules.
The phrase “therapeutically effective amount” refers to an amount of the antibody molecules, as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, biological or therapeutic results disclosed, described, or exemplified herein. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to cause a desired response in a subject. Exemplary indicators of a therapeutically effect amount include, for example, improved well-being of the patient, reduction of a disease symptom, arrested or slowed progression of disease symptoms, and/or absence of disease symptoms.
The following abbreviations are used herein: Alternaria alternata (Alternaria), Ascaris suum (A. suum); complementarity-determining region (CDR); heavy chain (HC); light chain (LC); heavy chain variable region (VH); light chain variable region (VL); surface plasmon resonance (SPR).
Antibody Molecules
Disclosed herein are human antibody molecules that immunospecifically bind to human IL-5. The human antibody molecules can immunospecifically bind to human IL-5 with an equilibrium affinity constant (KD) of at least about 40 pM as determined by surface plasmon resonance (SPR). As used herein, “of at least about 40 pM” means that the disclosed antibodies immunospecifically bind human IL-5 with a KD of less than or equal to about 40 pM. For example, the disclosed antibodies can immunospecifically bind human IL-5 with a KD of about 40 pM, about 30 pM, about 20 pM, about 10 pM, or less than about 10 pM.
The disclosed human antibody molecules can comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain consensus CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a light chain consensus CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a light chain consensus CDR3 comprising the amino acid sequence of SEQ ID NO: 3.
The light chain consensus CDR1 comprises the amino acid sequence of GX1X2X3X4X5X6KX7X8Y (SEQ ID NO: 1), wherein:

    
    
        X1 is G or K;
        X2 is N or D;
        X3 is N or H;
        X4 is I or A;
        X5 is G or D;
        X6 is S or K;
        X7 is N or H; and
        X8 is V or A.
    
    


In some embodiments, the light chain CDR1 amino acid sequence can comprise GGNNIGSKNVY (SEQ ID NO: 5). In some embodiments, the light chain CDR1 amino acid sequence can comprise GKNNIGSKNVY (SEQ ID NO: 21). In some embodiments, the light chain CDR1 amino acid sequence can comprise GGDNIGSKNVY (SEQ ID NO: 24). In some embodiments, the light chain CDR1 amino acid sequence can comprise GGNHIGSKNVY (SEQ ID NO: 27). In some embodiments, the light chain CDR1 amino acid sequence can comprise GGNNAGSKNVY (SEQ ID NO: 30). In some embodiments, the light chain CDR1 amino acid sequence can comprise GGNNIDSKNVY (SEQ ID NO: 66). In some embodiments, the light chain CDR1 amino acid sequence can comprise GGNNIGKKNVY (SEQ ID NO: 33). In some embodiments, the light chain CDR1 amino acid sequence can comprise GGNNIGSKHVY (SEQ ID NO: 36). In some embodiments, the light chain CDR1 amino acid sequence can comprise GGNNIGSKNAY (SEQ ID NO: 39).
The light chain CDR2 amino acid sequence comprises DDX8X9RPS (SEQ ID NO: 2), wherein:

    
    
        X8 is S or L; and
        X9 is D or S.
    
    


In some embodiments, the light chain CDR2 amino acid sequence can comprise DDSDRPS (SEQ ID NO: 7). In some embodiments, the light chain CDR2 amino acid sequence can comprise DDLDRPS (SEQ ID NO: 42). In some embodiments, the light chain CDR2 amino acid sequence can comprise DDSSRPS (SEQ ID NO: 45).
The light chain CDR3 amino acid sequence comprises QVWX10SSSDX11VX12 (SEQ ID NO: 3), wherein:

    
    
        X10 is D or L;
        X11 is H, S, Y, or D; and
        X12 is V, A, or W.
    
    


In some embodiments, the light chain CDR3 amino acid sequence can comprise QVWDSSSDHVV (SEQ ID NO: 15). In some embodiments, the light chain CDR3 amino acid sequence can comprise QVWLSSSDHVV (SEQ ID NO: 48). In some embodiments, the light chain CDR3 amino acid sequence can comprise QVWDSSSDSVV (SEQ ID NO: 51). In some embodiments, the light chain CDR3 amino acid sequence can comprise QVWDSSSDYVV (SEQ ID NO: 54). In some embodiments, the light chain CDR3 amino acid sequence can comprise QVWDSSSDDVV (SEQ ID NO: 57). In some embodiments, the light chain CDR3 amino acid sequence can comprise QVWDSSSDHVA (SEQ ID NO: 60). In some embodiments, the light chain CDR3 amino acid sequence can comprise QVWDSSSDHVW (SEQ ID NO: 63).
The disclosed human antibody molecules can comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR1 comprising the amino acid sequence of SEQ ID NOs: 5, 21, 24, 27, 30, 33, 36, 39, or 66, a light chain CDR2 comprising the amino acid sequence of SEQ ID NOs: 7, 42, or 45, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NOs: 15, 48, 51, 54, 57, 60, or 63. Exemplary antibody molecules comprise a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and

    
    
        a. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        b. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        c. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        d. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        e. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        f. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        g. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        h. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        i. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        j. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        k. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
        l. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 48;
        m. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 51;
        n. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 54;
        o. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 57;
        p. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 60; or
        q. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 63;
        wherein the position of the amino acid residues of the CDR is determined according to AbM.
    
    


The disclosed antibody molecules can comprise a heavy chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, or 67, wherein the variability (i.e. the at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) occurs outside of the CDR sequence. Exemplary antibody molecules are provided in Table 1 and Table 15.







TABLE 1







Antibody chain/domain composition summary











Antibody
VH protein/
VL protein/
HC protein/
LC protein/


ID
(SEQ ID)
(SEQ ID)
(SEQ ID)
(SEQ ID)





3A5
3A5 VH
3A5 VL
3A5 HC
3A5 LC



(SEQ ID NO: 10)
(SEQ ID NO: 11)
(SEQ ID NO: 12)
(SEQ ID NO: 13)


 


3A5.001
3A5 VH
3A5 VL
3A5.001 HC
3A5 LC



(SEQ ID NO: 10)
(SEQ ID NO: 11)
(SEQ ID NO: 14)
(SEQ ID NO: 13)


 


3A5.040
3A5.040 VH
3A5.040 VL
3A5.040 HC
3A5.040 LC



(SEQ ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 18)
(SEQ ID NO: 19)


 


3A5.046
3A5.040 VH
3A5.040 VL
3A5.046 HC
3A5.040 LC



(SEQ ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 20)
(SEQ ID NO: 19)


 


3A5.063
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
G25K
(SEQ ID NO: 18)
G25K




(SEQ ID NO: 22)

(SEQ ID NO: 23)


 


3A5.070
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
N26D
(SEQ ID NO: 18)
N26D




(SEQ ID NO: 25)

(SEQ ID NO: 26)


 


3A5.082
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
N27H
(SEQ ID NO: 18)
N27H




(SEQ ID NO: 28)

(SEQ ID NO: 29)


 


3A5.084
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
I28A
(SEQ ID NO: 18)
I28A




(SEQ ID NO: 31)

(SEQ ID NO: 32)


 


3A5.097
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
G29D
(SEQ ID NO: 18)
G29D




(SEQ ID NO: 67)

(SEQ ID NO: 68)


 


3A5.107
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
S30K
(SEQ ID NO: 18)
S30K




(SEQ ID NO: 34)

(SEQ ID NO: 35)


 


3A5.125
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
N32H
(SEQ ID NO: 18)
N32H




(SEQ ID NO: 37)

(SEQ ID NO: 38)


 


3A5.127
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
V33A
(SEQ ID NO: 18)
V33A




(SEQ ID NO: 40)

(SEQ ID NO: 41)


 


3A5.161
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
S52L
(SEQ ID NO: 18)
S52L




(SEQ ID NO: 43)

(SEQ ID NO: 44)


 


3A5.169
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
D53S
(SEQ ID NO: 18)
D53S




(SEQ ID NO: 46)

(SEQ ID NO: 47)


 


3A5.232
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
D92L
(SEQ ID NO: 18)
D92L




(SEQ ID NO: 49)

(SEQ ID NO: 50)


 


3A5.276*
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
H95bS
(SEQ ID NO: 18)
H95bS




(SEQ ID NO: 52)

(SEQ ID NO: 53)


 


3A5.278*
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
H95bY
(SEQ ID NO: 18)
H95bY




(SEQ ID NO: 55)

(SEQ ID NO: 56)


 


3A5.279*
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
H95bD
(SEQ ID NO: 18)
H95bD




(SEQ ID NO: 58)

(SEQ ID NO: 59)


 


3A5.294
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
V97A
(SEQ ID NO: 18)
V97A




(SEQ ID NO: 61)

(SEQ ID NO: 62)


 


3A5.302
3A5.040 VH
3A5.040 VL +
3A5.040 HC
3A5.040 LC +



(SEQ ID NO: 16)
V97W
(SEQ ID NO: 18)
V97W




(SEQ ID NO: 64)

(SEQ ID NO: 65)





*The lower case “b” in each of these sequences refers to the Kabat numbering for the CDR position. Kabat numbering allows for CDRs of variable sizes, by using alphanumeric numbering to denote amino acid insertions at certain positions. In these CDR sequences, additional amino acids were present, which were numbered as positions 95a and 95b (corresponding to Kabat positions 95A and 95B respectively). Thus, for antibody 3A5.276, for example, H95bS indicates a histidine (“H”) to serine (“S”) mutation at position 95B according to Kabat number relative to the 3A5.040 VL chain. The lower case notation is therefore used here to distinguish the Kabat number, separate from the mutation at the indicated Kabat position.






In some embodiments, the disclosed antibody molecules can comprise a heavy chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16 and

    
    
        a. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17;
        b. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22;
        c. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25;
        d. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28;
        e. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31;
        f. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34;
        g. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37;
        h. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40;
        i. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43;
        j. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46;
        k. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49;
        l. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52;
        m. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55;
        n. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 58;
        o. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 61;
        p. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64; or
        q. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67,
        wherein the variability (i.e. the at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) occurs outside of the CDR sequence.
    
    


The disclosed antibody molecules can comprise one or more mutations, deletions, or insertions, in the framework and/or constant regions. In some embodiments, an IgG4 antibody molecule can comprise a S228P mutation. S228 is located in the hinge region of the IgG4 antibody molecule. Mutation of the serine (“S”) to a proline (“P”) serves to stabilize the hinge of the IgG4 and prevent Fab arm exchange in vitro and in vivo. In some embodiments, the antibody molecules can comprise one or more modifications which increase the in vivo half life of the antibody molecules. For instance in certain embodiments the antibody can comprise a M252Y mutation, a S254T mutation, and a T256E mutation (collectively referred to as the “YTE” mutation). M252, S254, and T256 are located in in the CH2 domain of the heavy chain. Mutation of these residues to tyrosine (“Y”), threonine (“T”), and glutamate (“E”), respectively, protects the antibody molecules from lysosomal degradation, thereby enhancing the serum half-life of the antibody molecules. Based on the example of other antibodies, it is contemplated that the introduction of the YTE mutation in an anti-IL-5 antibody may provide sufficient extension of serum half life to allow for administration regimes with 3 months or longer inter-dosing intervals. In some embodiments, the antibody molecules can comprise a deletion of the heavy chain C-terminal lysine residue. Deletion of the heavy chain C-terminal lysine residue reduces heterogeneity of the antibody molecules when produced by mammalian cells. In some embodiments, the antibody molecules can comprise a combination of mutations, deletions, or insertions. For example, in some aspects, the disclosed antibody molecules can comprise a S228P mutation and a deletion of a heavy chain C-terminal lysine residue. The disclosed antibodies comprising a heavy chain sequence of SEQ ID NO: 18, for example, comprise a S228P mutation and a deletion of a heavy chain C-terminal lysine residue. In some aspects, the disclosed antibody molecule can comprise a S228P mutation, a M252Y mutation, a S254T mutation, a T256E mutation, and a deletion of a heavy chain C-terminal lysine residue. The 3A5.046 antibody, for example, which comprises a heavy chain of SEQ ID NO: 20, comprises a S228P mutation, a M252Y mutation, a S254T mutation, a T256E mutation, and a deletion of a heavy chain C-terminal lysine residue.
The antibody molecule can comprise an IgG1 or IgG4 heavy chain constant region and a lambda light chain constant region. In some embodiments, the antibody molecule comprises an IgG1 heavy chain constant region and a lambda light chain constant region (antibody 3A5, for example). In some embodiments, the antibody molecule comprises an IgG4 heavy chain constant region and a lambda light chain constant region.
The disclosed antibody molecules can comprise a heavy chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 18 or 20 and a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, or 68, wherein the variability (i.e. the at least 90% identity) occurs outside of the CDR sequence. Exemplary antibody molecules are provided in Table 1 and Table 15. In some embodiments, the antibody molecules can comprise a heavy chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18 and

    
    
        a. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19;
        b. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23;
        c. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26;
        d. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29;
        e. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32;
        f. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35;
        g. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 38;
        h. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41;
        i. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44;
        j. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47;
        k. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50;
        l. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53;
        m. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56;
        n. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 59;
        o. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62;
        p. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65; or
        q. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 68,
        wherein the variability (i.e. the at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) occurs outside of the CDR sequence.
    
    


The disclosed antibody molecules can comprise a heavy chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20 and a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19, wherein the variability (i.e. the at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) occurs outside of the CDR sequence.
In some embodiments, the antibody molecules are full length antibody molecules (with or without a deletion of the heavy chain C-terminal lysine residue). In other embodiments, the antibody molecules are antigen binding fragments. Suitable antibody binding fragments include, but are not limited to, a Fab fragment, a Fab2 fragment, or a single chain antibody.
The antibody molecules can have one or more of the following properties:

    
    
        a. binds to human IL-5 with an equilibrium affinity constant (KD) of at least about 40 pM as determined by surface plasmon resonance;
        b. reduces binding of IL-5 to the IL-5 receptor;
        c. has a serum half-life of at least about 20 days; or
        d. binds human and cynomolgus monkey IL-5 but not mouse, rat, or guinea pig IL-5.
    
    


Pharmaceutical compositions comprising any of the disclosed antibody molecules are also provided.
Also provided are nucleic acid molecules encoding any of the disclosed antibody molecules and vectors comprising the disclosed nucleic acid molecules.
Cells transformed to express any of the disclosed antibody molecules are further provided.
Methods and Uses
The disclosed antibody molecules, or pharmaceutical compositions comprising the same, can be used to treat eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis and eosinophilic esophagitis. Any of the antibody molecule characteristics disclosed herein apply equally to the antibodies used in the disclosed methods and uses.
Disclosed herein are methods of treating a subject having eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis comprising administering to the subject a therapeutically effective amount of any of the antibody molecules disclosed herein, or pharmaceutical compositions comprising the same, to treat the eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis.
The use of an effective amount of any of the disclosed antibody molecules, or pharmaceutical compositions comprising the same, in the treatment of eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis is also provided.
Also provided is the use of any of the disclosed antibody molecules, or pharmaceutical compositions comprising the same, in the manufacture of a medicament for the treatment of eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis.
EXAMPLES
The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.
Generation of Anti-Human-IL-5 Antibodies
Anti-human-IL-5 antibodies were obtained from transgenic rats (OMT) with human V-genes cloned into their genomes and which produce antibodies with human V-domains and rat Fc domains. Briefly, the transgenic rats were genetically immunized with DNA encoding IL-5 four times over 21 days (on days 0, 7, 14, 21) and boosted with recombinant human IL-5 at day 28 of the immunization protocol. Serum antibody titres were determined at days 0 and 38 of the immunization protocol by an ELISA assay using recombinant human IL-5. Briefly, sera from each animal were diluted in PBS 1% BSA and were tested using ELISA plates coated with 1 μg/ml human IL-5, or BSA as a control. A goat anti-rat IgG R-phycoerythrin conjugate (SOUTHERN BIOTECH®, #3030-09) was used at 10 μg/ml as a secondary antibody. Specific animals were chosen for hybridoma fusion based on these serum titres.
To generate hybridomas which produce monoclonal antibodies to human IL-5, splenocytes and/or lymph node cells from immunized animals were isolated and fused to P3X63Ag8.653 non-secreting mouse myeloma cells (ATCC®, CRL-1580). Cells were plated at approximately 1×105 cells/mL in flat bottom microtiter plates, followed by a two week incubation in selective medium which included 10% fetal clone serum and 1×HAT (SIGMA). Hybridomas were expanded by serial passage through four media changes in 96-well plates (96-well stages 1 to 4), then into T25 and finally T75 flasks.
The supernatants from hybridoma clones were assayed during the hybridoma expansion process, initially in a whole-cell ELISA using cells transfected with GPI-anchored human IL-5 and then ELISA using a recombinant human IL-5 ELISA assay (the latter as described above). Hybridomas which survived the scale-up process to T75 stage and which gave signals above a given threshold for binding in both of these assays were frozen as cell pellets, for cloning and sequencing of Ig v-domains. Approximately 20 hybridomas producing chimeric IgGs were selected for cloning following these screening steps.
The human v-domains from the candidate chimeric IgGs were isolated by generation of cDNA from hybridoma cell pellets, PCR amplification of v-domains, subcloning, and DNA sequencing. A total of approximately 35 heavy and light chain combinations were obtained from the sequencing of these hybridomas. All antibodies were cloned into a mammalian expression vector and transiently transfected into HEK-293 cells. Antibodies were purified using standard Protein A purification protocols.
Functional Testing and Characterization of Antibodies
Selected antibodies in human IgG1 format were first assayed for specificity in a human IL-5 ELISA. Briefly, antibodies were diluted in PBS 0.1% BSA and were tested using ELISA plates coated with 1 μg/ml human IL-5, or an irrelevant control protein. A horseradish peroxidase-conjugated Anti-IgG antibody was used as a secondary antibody.
Antibodies that showed immunospecific binding to human IL-5 were ranked for potency in a human IL-5-dependent cell proliferation assay using human TF-1.6G4 (a derivative of the human erythroleukemic cell line TF-1) cells. The TF-1.6G4 cell line was subcloned and selected for enhanced surface expression of IL-5Rα and consistent proliferative response to human IL-5. The TF-1.6G4 cell line was maintained in culture following standard conditions used for the TF-1 human erythroleukemic cell line (ATCC®: CRL-2003). Briefly, dilutions of each antibody were incubated in the presence of 45 pM human IL-5 and 5×104 TF-1.6G4 cells per well, incubated for 48 h, and cell proliferation was determined using the CELLTITER-GLO® Luminescent Cell Viability Assay (PROMEGA®, WI). All proliferation and inhibition curves were fitted using a three- or four-parameter dose-response model in GraphPad Prism®6 (Version 6.04) software. Table 2 summarizes these results.







TABLE 2







Summary of screening and characterization


of an initial test panel in IgG1 format.













Equilibrium
Inhibits
Specificity


Test
IC50 (pM)
Affinity
IL-5Rα
confirmed


Antibody
in TF1.6G4
(KD)
binding
by ELISA





3A5
 35.07
36 pM
Yes
Yes


1A3
 55.5
18 pM
Yes
Yes


2B4
 56.75
42 pM
Yes
Yes


6G10
 72.34
61 pM
Yes
Yes


1E8
 72.45
28 pM
Yes
Yes


3G4
 80.92
41 pM
Yes
Yes


6C9
 81.89
41 pM
Yes
Yes


5H1
 86.42
36 pM
Yes
Yes


1H10
 93.26
18 pM
Yes
Yes


5H11
109.3
36 pM
No
Yes


5G9
184.4
52 pM
Yes
Yes


1B2
214.4
45 pM
Yes
Yes









In Table 2, the results of tests were ranked in order of TF1.6G4 assay potency. The initial test panel was selected based upon potency, affinity, IL-5Rα inhibition and sequence liabilities (immunogenicity and developability).
BIACORE™ assays were used to determine the affinity of test antibodies to recombinant human IL-5 and their potency in inhibiting IL-5Rα binding to human IL-5. Table 2 summarizes these results. Binding affinity of test antibodies to human IL-5 (KD; FIG. 6) was determined on a BIACORE™ T200 system (GE HEALTHCARE®) by coating a BIACORE™ Series S Sensor Chip Protein A (GE HEALTHCARE®) with selected purified IgG antibody to a capture level of 75 RU, then recombinant human IL-5 was injected at 60 μL/min across a 7-step two-fold serial dilution range, starting at 1 μg/mL. All experiments were run using HBS-EP+ buffer (GE HEALTHCARER). The resulting sensorgrams were double-referenced (test flowcell values subtracted from control (Protein A surface with no coated antibody) flowcell values and also a buffer blank). Binding constants were determined by fitting a 1:1 Langmuir binding model to double-referenced sensorgrams.
To determine whether each test antibody inhibited the binding of IL-5 to IL-5Rα, either a BIACORE™ T200 or a BIACORE™ 3000 system (GE HEALTHCARE®) was used. A BIACORE™ CM5 Sensor Chip was first derivatized with a Fab capture kit (GE HEALTHCARER) in accordance with the manufacturer's instructions on two adjacent (test and control) flowcells. This surface was used to capture each purified IgG test antibody on a single test flowcell. Recombinant human IL-5 at 5 μg/mL or a buffer blank was then injected across both test and control flowcells, to saturate the test flowcell surface and control for non-specific association of IL-5, respectively. A second injection of purified IgG test antibody at 10 μg/mL or a buffer blank was performed on the test flowcell to block free IL-5 binding sites and control for dissociation of the IgG test antibody from the Fab capture antibody, respectively.
Subsequent injection of IL-5Rα-Fc (R&D SYSTEMS®) at either 5 or 20 μg/mL or a buffer blank across both flowcells was used to determine whether the IgG test antibody blocked the interaction between IL-5 and IL-5Rα or to control for dissociation of IgG antibody from the Fab capture antibody during this step. Antibodies which inhibited the binding of IL-5 to IL-5Rα showed a markedly reduced signal upon injection of IL-5Rα (Table 2). A triple reference subtraction method was used to analyze data in the manner detailed above. All data was exported from BIACORE™ evaluation software and subtracted in Excel (MICROSOFT®) software. All experiments were run using HBS-EP+ buffer (GE HEALTHCARE®).
A smaller panel of test antibodies (3A5, 5H11 and 2B4 in Table 2) was chosen on the basis of these results. The antibodies were reformatted as human IgG4 and tested in the same assays. The IgG4 version of antibody 3A5 (originally an IgG1) was designated as 3A5.001. This fully-human antibody was demonstrated to have equivalent potency to the original test 3A5 in IgG1 format (FIG. 1).
A variant of antibody 3A5.001 (designated 3A5.040) was prepared with specific amino acid substitutions (VH: S[68]T, N[82A]S; VL: S[2]Y, I[3]V, Y[92]D, wherein the residues in square brackets represent the Kabat positions) introduced in the V-domain regions to remove predicted T-cell epitopes. Antibody 3A5.040 was demonstrated to have equivalent potency to its parental antibody, 3A5.001 (FIG. 2) in the TF-1.6G4 assay.
CDR Scanning of Antibody 3A5.040
Generation of antibody 3A5.040 variants—Single-mutant variants of antibody 3A5.040 were made by substituting one of a group of nine representative amino acids—A, S, L, Y, D, Q, K, H, W—at each amino acid position in the light chain CDR1 (CDR-L1), the light chain CDR2 (CDR-L2), the heavy chain CDR1 (CDR-H1) and the heavy chain CDR2 (CDR-H2) (as defined by AbM nomenclature). Antibody variants were also made by substituting one of a group of ten representative amino acids—A, S, L, Y, D, Q, K, H, W, P—at each CDR amino acid position in the light chain CDR3 (CDR-L3), heavy chain CDR3 (CDR-H3), and at Kabat positions 93 and 94 in the variable heavy chain. A complete list of all single-mutant antibody variants generated is shown in FIG. 3 (variable heavy chain) and FIG. 4 (variable light chain), respectively.
Construction of Vectors Expressing Antibodies—Variable region variants were generated by back-translation of amino acid sequences into DNA sequences which were subsequently synthesized de novo by assembly of synthetic oligonucleotides. Variable heavy (VH) variants were subcloned into a mammalian expression vector containing a human constant region to produce full-length antibody heavy chains (human IgG4 heavy chain CHI, hinge, CH2, and CH3 domains). Similarly, variable light (VL) variants were subcloned into a mammalian expression vector containing a human lambda light chain constant region to produce full-length antibody lambda chains.
Expression of antibody variants—Antibodies were produced by co-transfecting separate expression vectors encoding antibody heavy chains and light chains into EXPI293® cells (LIFE TECHNOLOGIES®, Carlsbad, CA). Each single-mutant chain was paired with a parental chain for protein expression in the EXP1293® system. For each 20 mL transfection, 3.6×107 cells were required in 20 mL of EXP1293® Expression Medium. On the day prior to transfection, cells were seeded at a density of 0.9×106 viable cells/mL and incubated overnight at 37° C. in a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 200 rpm. On the day of transfection, the cell number and viability were determined using an automated cell counter. Only cultures with >98% viable cells were used. For each 20 mL transfection, lipid-DNA complexes were prepared by diluting 10 μg of heavy chain DNA and 10 μg of light chain DNA in OPTI-MEM® (LIFE TECHNOLOGIES®, Carlsbad, CA) I Reduced Serum Medium (Cat. no. 31985-062) to a total volume of 1.0 mL. 54 μL of EXPIFECTAMINE® 293 Reagent (LIFE TECHNOLOGIES®, Carlsbad, CA) was diluted in OPTI-MEM® I medium to a total volume of 1.0 mL. Both vials were mixed gently and incubated for 5 minutes at room temperature. Following incubation, the diluted DNA was mixed with the diluted EXPIFECTAMINE® 293 Reagent and the DNA-EXPIFECTAMINE® 293 Reagent mixture and incubated a further 20 minutes at room temperature to allow the formation of DNA-EXPIFECTAMINE® 293 Reagent complexes. Following incubation, 2 mL of DNA-EXPIFECTAMINE® 293 Reagent complex was added to each 50 mL bioreactor tube (TPP TECHNO PLASTIC PRODUCTS® AG). 2 mL of OPTI-MEM® (LIFE TECHNOLOGIES®, Carlsbad, CA) I medium was added to the negative control tube instead of DNA-EXPIFECTAMINE® 293 Reagent complex. The cells were incubated in a 37° C. incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 200 rpm. Approximately 16-18 hours post-transfection, 100 μL of EXPIFECTAMINE® 293 Transfection Enhancer 1 and 1.0 mL of EXPIFECTAMINE® 293 Transfection Enhancer 2 were added to each bioreactor. Supernatants were harvested at approximately 48 hours post-transfection.
Purification of antibody variants—Each antibody variant was expressed in EXP1293® cells in either 20 or 100 mL of cell culture. Cultures were spun down in 50 mL falcon tubes at 3000×g for 20 minutes, and supernatants were filtered using a 0.22 μm filter. Supernatants were purified using a GILSON® ASPEC GX274 robot. Briefly, SPE cartridges (AGILENT®, 12131014) packed with 1.2 mL MABSELECT SURE® protein A resin (GE HEALTHCARE®) were pre-equilibrated with 3 column volumes of 1×PBS. Supernatant was run over the columns followed by a 4 mL 1×PBS wash. Each column was washed with 9 mL of 1 M citric acid, pH 2.9. Antibodies were eluted with 2 mL 0.1 M citric acid, pH 2.9. Antibodies were desalted into Sorensens PBS (5 mM KH2PO4, 3 mM Na2HPO4·2H2O, 145.4 mM NaCl (pH˜5.8)) using PD-10 columns (GE HEALTHCARE®).
Determination of 3A5.040 variant antibody titre in supernatant by BIACORE™—Supernatants containing antibody from expression in EXPI293® cells were analyzed using a Protein A series S chip on the BIACORE™ T200 to determine their titre and rank them with respect to the parental antibody 3A5.040. Each supernatant sample was diluted with running buffer (1×HBS-EP+, 350 mM NaCl) and captured by injection at 60 μL/min onto flow cell (FC) 2. The resulting capture level in response units (RUs) were measured on FC 2-1 by subtracting a report point 5 sec after cycle start from one 5 sec after injection. The surface was regenerated by injecting 50 mM NaOH for 12 sec at a flow rate of 60 μL/min onto FC 1 and 2 every 4 cycles. After each regeneration, the surface and sensorgram was stabilized for 120 sec by injection of running buffer. Multiple batches were run due to the large number of antibodies that were screened in this manner.
The capture levels (in BIACORE™ response units “RU”) obtained for each supernatant sample were adjusted by multiplying the values obtained for each 30 sec injection by the supernatant dilution factor, enabling comparison of capture levels between supernatants diluted to varying degrees across different experimental runs. The relative antibody expression level (titre) of each mutant was compared to a batch-specific 3A5.040 supernatant, (Table 3) using the following formula:

(adjusted 3A5.040 reference RU for 30 sec injection/adjusted variant antibody RU for 30 sec injection)×100=proportional titre of parental antibody(% 3A5.040 titre)   Formula 1

Variant antibodies with a higher titre than parent 3A5.040 antibody had a “% 3A5.040 titre” value >100% and those with a lower titre, a value <100%. This enabled the identification of variant antibodies that might have improved expression over the parental antibody, 3A5.040. The titres of some variants were not determined by BIACORE™ analysis of supernatant samples, but were expressed and purified as above and their purified yields determined by spectrophotometric analysis (A280), then compared to parental 3A5.040 antibody (Table 5).
Determination of 3A5.040 variant antibody IL-5-binding kinetics by BIACORE™—Supernatants containing antibody from expression in EXPI293® cells or purified antibodies were analyzed using a Protein A series S chip on a BIACORE™ T200 system (GE HEALTHCARE®) to determine their binding affinity for recombinant human IL-5 and rank them with respect to the parental antibody 3A5.040.
Each antibody was diluted in running buffer (1×HBS-EP+, 350 mM NaCl) and captured at 60 μL/min to approximately 50 RU on FC 2. The surface and sensorgram was then stabilized for 120 sec by injection of running buffer. Recombinant human IL-5 at 5 μg/mL or running buffer was injected onto FC 1 and 2 at a flow rate of 60 μL/min for 70 sec. The IL-5 was allowed to dissociate in running buffer for 300 sec. The surface was regenerated by injecting 50 mM NaOH for 12 sec at a flow rate of 60 μL/min onto FC 1 and 2. Buffer was injected to further clean up drift for 60 sec at a flow rate of 60 μL/min onto FC 1 and 2. The surface was stabilized for 300 sec with running buffer over FC 1 and 2. Supernatant containing the parental 3A5.040 antibody and that had been transfected with each batch was run approximately every 25 cycles throughout each run. A purified sample of 3A5.040 and an IgG4 lambda isotype control was run with every batch as a measure of inter-assay variability. Multiple batches were run due to the large number of antibodies that were screened in this manner.
Data was double-referenced (flow cell 2 was subtracted from flow cell 1 and a buffer blank) and sensorgrams fitted to a 1:1 Langmuir binding model with BIACORE™ Evaluation software. A kd (off-rate) value was calculated for each 3A5.040 supernatant sample throughout the run and these were averaged to give a reference kd, with the exception of run 2.1, which only had one 3A5.040 supernatant sample (Table 3 and Tables 6-11). The calculated kd value for each supernatant antibody was compared against the 3A5.040 average reference kd using the following formula:

(3A5.040 reference kd/variant antibody kd)×100=proportional kd of parental antibody(% 3A5.040 kd)   Formula 2

Variant antibodies with a lower kd (slower off-rate) than parent 3A5.040 antibody had a “% 3A5.040 kd” value >100% and those with a higher kd (faster off-rate) a value <100%. This enabled the identification of variant antibodies that might have improved binding kinetics for IL-5 and therefore improved function over the parental antibody, 3A5.040. For variants where only purified protein was made (Table 6), the BIACORE™ kinetic analysis was performed in triplicate as above using these purified antibodies and the average values from each triplicate analysis used to perform the proportional kd calculation, as above (Table 4 and Tables 8-11).







TABLE 3







Determination of supematant 3A5.040 variant antibody titre and IL-5 binding kinetics by Biacore


























%
%


Run
Antibody
Replicate
Amino acid
Capture



Rmax
3A5.040
3A5.04


#
ID
Number
Substitution
level
ka (1/Ms)
kd (1/s)
KD (M)
(RU)
kd
0 titre




















1.1
3A5.040
1
Wild type
51.6
7.20E+05
1.15E−04
1.60E−10
16.3




1.1
3A5.040
2
Wild type
51.6
7.27E+05
8.96E−05
1.23E−10
16.6




1.1
3A5.040
3
Wild type
51.8
7.18E+05
9.21E−05
1.28E−10
16.7




1.1
3A5.040
4
Wild type
52
6.96E+05
9.95E−05
1.43E−10
16.8




1.1
3A5.040
Average
Wild type
51.75
7.15E+05
9.91E−05
1.39E−10
16.6
100
100


1.1
IgG4 


54.4
N/A
N/A
N/A
0.7
N/A
N/A



Isotype











1.1
3A5.040

Wild type
56.4
7.17E+05
1.06E−04
1.48E−10
17.8
93.0




(Purified)











1.1
3A5.304

VH G26A
54.6
7.41E+05
7.73E−05
1.04E−10
17.1
128
49


1.1
3A5.305

VH G26S
53.1
7.36E+05
1.09E−04
1.48E−10
16.7
91
59


1.1
3A5.306

VH G26L
49.1
7.49E+05
8.74E−05
1.17E−10
15.6
113
15


1.1
3A5.308

VH G26D
52.5
6.75E+05
8.57E−05
1.27E−10
16.4
116
61


1.1
3A5.309

VH G26Q
55.1
7.47E+05
9.23E−05
1.24E−10
17.4
107
86


1.1
3A5.310

VH G26K
51.8
7.36E+05
1.01E−04
1.37E−10
16.4
98
100


1.1
3A5.311

VH G26H
50.9
7.60E+05
1.11E−04
1.46E−10
16.3
89
85


1.1
3A5.312

VH G26W
56.1
8.54E+05
1.21E−04
1.41E−10
18
82
107


1.1
3A5.313

VH G27A
54.5
6.97E+05
1.17E−04
1.68E−10
17.3
85
99


1.1
3A5.314

VH G27S
50.9
7.16E+05
9.81E−05
1.37E−10
16.2
101
95


1.1
3A5.315

VH G27L
54.3
7.01E+05
1.31E−04
1.87E−10
17.1
76
118


1.1
3A5.316

VH G27Y
56
6.66E+05
8.56E−04
1.29E−09
16.9
12
80


1.1
3A5.317

VH G27D
57.4
6.56E+05
1.16E−04
1.76E−10
17.8
85
73


1.1
3A5.318

VH G27Q
55.2
7.31E+05
1.49E−04
2.04E−10
17.4
67
82


1.1
3A5.319

VH G27K
54.8
7.70E+05
2.04E−04
2.65E−10
17.5
49
95


1.1
3A5.320

VH G27H
54.4
7.12E+05
2.43E−04
3.42E−10
16.9
41
71


1.1
3A5.322

VH S28A
58
7.49E+05
1.05E−04
1.41E−10
18
94
62


1.1
3A5.323

VH S28L
53.8
7.18E+05
9.30E−05
1.30E−10
17
107
70


1.1
3A5.324

VH S28Y
54.8
8.13E+05
8.60E−05
1.06E−10
17
115
51


1.1
3A5.325

VH S28D
54.9
6.03E+05
9.16E−05
1.52E−10
17
108
68


1.1
3A5.326

VH S28Q
56.9
6.78E+05
9.97E−05
1.47E−10
17.7
99
77


1.1
3A5.327

VH S28K
53.7
7.22E+05
1.15E−04
1.59E−10
16.9
86
84


1.1
3A5.328

VH S28H
54
7.82E+05
1.23E−04
1.57E−10
16.9
81
71


1.1
3A5.329

VH S28W
55
8.33E+05
1.36E−04
1.63E−10
17
73
41


1.1
3A5.330

VH I29A
56.3
6.54E+05
1.69E−04
2.58E−10
16.8
59
30


1.1
3A5.331

VH I29S
53.3
5.87E+05
2.04E−04
3.48E−10
15.7
49
28


1.1
3A5.332

VH I29L
55.3
8.26E+05
1.16E−04
1.41E−10
17.5
85
75


1.1
3A5.333

VH I29Y
55.4
5.44E+05
9.31E−04
1.71E−09
15.7
11
24


1.1
3A5.334

VH I29D
52.8
3.53E+06
5.37E−03
1.52E−09
11.9
2
21


1.1
3A5.336

VH I29K
53.7
6.17E+05
6.75E−04
1.10E−09
14.8
15
29


1.1
3A5.338

VH I29W
56.6
1.31E+06
2.34E−03
1.78E−09
14.9
4
38


1.1
3A5.340

VH S3OL
48.7
6.51E+05
1.47E−04
2.25E−10
15.6
67
153


1.1
3A5.341

VH S30Y
54.3
7.54E+05
1.37E−04
1.82E−10
17.3
72
103


1.1
3A5.342

VH S3OD
54.9
6.17E+05
1.23E−04
2.00E−10
17.4
81
103


1.1
3A5.347

VH N31A
53.8
7.53E+05
6.75E−04
8.97E−10
17.3
15
118


1.1
3A5.348

VH N31S
54.6
7.82E+05
5.03E−04
6.44E−10
17.7
20
107


1.1
3A5.350

VH N31Y
56.2
9.01E+05
8.80E−04
9.76E−10
17.6
11
NE


1.1
3A5.351

VH N31D
59.8
6.40E+05
2.03E−04
3.17E−10
19.2
49
75


1.1
3A5.352

VH N31Q
54.8
6.93E+05
4.21E−04
6.07E−10
17.7
24
156


1.1
3A5.353

VH N31K
55.5
6.85E+05
7.54E−04
1.10E−09
17.7
13
170


1.1
3A5.354

VH N31H
53.7
7.63E+05
1.32E−04
1.73E−10
17.5
75
143


1.1
3A5.356

VH G32A
52.3
5.25E+05
3.02E−04
5.76E−10
16.4
33
179


1.1
3A5.357

VH G32S
49.2
6.02E+05
1.23E−03
2.04E−09
15.3
8
153


1.1
3A5.495

VH S94K
55
6.76E+05
2.91E−04
4.31E−10
17.6
34
127


1.1
3A5.497

VH S94W
56.8
1.67E+06
2.92E−03
1.75E−09
12.5
3
38


1.1
3A5.498

VH S94P
55.5
4.45E+05
1.17E−04
2.63E−10
16
85
17


1.1
3A5.500

VH L95S
2.7




NE
NE


1.1
3A5.504

VH L95K
51.7
1.53E+06
3.12E−03
2.03E−09
11.3
3
67


1.1
3A5.505

VH L95H
52.9
5.59E+05
6.66E−04
1.19E−09
16.3
15
88


1.1
3A5.507

VH L95P
54.7




NB
40


1.1
3A5.509

VH G96S
54.4
5.94E+05
1.92E−04
3.23E−10
17
52
88


1.1
3A5.510

VH G96L
54
4.11E+06
7.90E−03
1.92E−09
8.5
1
63


1.1
3A5.513

VH G96Q
54.8
4.45E+05
5.13E−04
1.15E−09
16.5
19
64


1.1
3A5.514

VH G96K
54.9
5.14E+06
6.84E−03
1.33E−09
11.4
1
147


1.1
3A5.515

VH G96H
53.2
4.37E+05
1.20E−03
2.74E−09
15.3
8
117


1.1
3A5.516

VH G96W
53
1.31E+06
1.40E−02
1.07E−08
6.7
1
55


1.1
3A5.517

VH G96P
47.8
1.35E+05
5.04E−04
3.73E−09
11.7
20
15


1.1
3A5.518

VH N97A
52
5.35E+05
1.72E−04
3.21E−10
16.3
58
95


1.1
3A5.520

VH N97L
54.5
4.73E+05
1.11E−04
2.35E−10
16
89
96


1.1
3A5.521

VH N97Y
54.7
5.05E+05
3.34E−04
6.62E−10
16.1
30
75


1.1
3A5.523

VH N97Q
53.7
5.06E+05
9.79E−05
1.94E−10
16.6
101
127


1.1
3A5.524

VH N97K
52.5
2.89E+05
1.37E−04
4.75E−10
15.1
72
167


1.1
3A5.525

VH N97H
53.1
4.11E+05
2.14E−04
5.20E−10
15
46
113


1.1
3A5.526

VH N97W
53.7
5.28E+05
5.26E−04
9.95E−10
15.5
19
56


1.1
3A5.527

VH N97P
55.1
4.21E+05
4.38E−04
1.04E−09
15.5
23
68


1.1
3A5.528

VH W98A
57.6
7.26E+05
1.87E−04
2.57E−10
18.4
53
114


1.1
3A5.529

VH W98S
53.9
6.23E+05
2.36E−04
3.78E−10
16.8
42
83


1.1
3A5.530

VH W98L
54.3
3.38E+05
1.72E−04
5.08E−10
14.9
58
38


1.1
3A5.531

VH W98Y
56.3
7.15E+05
1.12E−04
1.56E−10
18.1
88
143


1.1
3A5.532

VH W98D
56.4
2.17E+05
8.87E−05
4.09E−10
14.6
112
42


1.1
3A5.533

VH W98Q
53.8
5.01E+05
1.54E−04
3.08E−10
15.8
64
51


1.1
3A5.534

VH W98K
53.4
3.46E+05
8.25E−04
2.38E−09
11.5
12
73


1.1
3A5.535

VH W98H
53.8
6.13E+05
1.45E−04
2.37E−10
17
68
81


1.1
3A5.536

VH W98P
56.1
9.97E+04
8.17E−04
8.20E−09
14.2
12
31


1.1
3A5.537

VH F99A
54.2
1.54E+05
4.90E−04
3.18E−09
13.7
20
37


1.1
3A5.538

VH F99S
53.3
1.18E+05
1.02E−03
8.67E−09
9.9
10
40


1.1
3A5.539

VH F99L
54.2
7.05E+05
1.15E−04
1.63E−10
16.7
86
55


1.1
3A5.540

VH F99Y
55.6
5.27E+05
9.13E−05
1.73E−10
14.9
109
41


1.1
3A5.541

VH F99D
53.9
1.46E+05
1.11E−02
7.56E−08
4.4
1
32


1.1
3A5.542

VH F99Q
53.8
9.93E+04
1.00E−03
1.01E−08
13
10
35


1.1
3A5.543

VH F99K
54.6




NB
38


1.1
3A5.544

VH F99H
54.8
2.05E+05
9.50E−05
4.64E−10
14.5
104
41


1.1
3A5.545

VH F99W
54.5
5.35E+04
4.09E−04
7.66E−09
6
24
29


1.1
3A5.546

VH F99P
53.1




NB
43


1.1
3A5.547

VH D101A
55.6
1.68E+05
1.40E−04
8.37E−10
14.7
71
69


1.1
3A5.548

VH D101S
55.2
2.38E+05
1.11E−04
4.67E−10
15.3
89
78


1.1
3A5.549

VH D101L
53.6
5.94E+05
4.76E−04
8.01E−10
16.3
21
43


1.1
3A5.551

VH D101Q
54.5
5.93E+05
2.19E−04
3.69E−10
17.1
45
78


1.2
3A5.040
1
Wild type
50.3
7.40E+05
1.33E−04
1.80E−10
16.3




1.2
3A5.040
2
Wild type
50.3
7.14E+05
8.22E−05
1.15E−10
16.1




1.2
3A5.040
3
Wild type
50
7.28E+05
1.01E−04
1.39E−10
16.1




1.2
3A5.040
4
Wild type
50.2
7.32E+05
1.10E−04
1.50E−10
16.2




1.2
3A5.040
5
Wild type
50.1
7.27E+05
9.36E−05
1.29E−10
16.1




1.2
3A5.040
Average
Wild type
50.2
7.28E+05
1.04E−04
1.43E−10
16.2
100
100


1.2
IgG4


50.8
N/A
N/A
N/A
0
N/A




Isotype











1.2
3A5.040

Wild type
52.6
7.35E+05
1.11E−04
1.51E−10
16.6
94




(Purified)











1.2
3A5.553

VH D101H
51.9
4.41E+05
1.63E−04
3.69E−10
15.5
64
66


1.2
3A5.555

VH D101P
51.3
7.54E+03
8.00E−04
1.06E−07
23.6
13
39


1.2
3A5.556

VH Y102A
50.6
6.60E+05
8.94E−05
1.35E−10
16.4
116
106


1.2
3A5.557

VH Y102S
53.5
6.85E+05
1.11E−04
1.61E−10
17.2
94
135


1.2
3A5.558

VH Y102L
53.2
7.40E+05
1.14E−04
1.54E−10
16.8
91
51


1.2
3A5.559

VH Y102D
52.8
7.03E+05
1.41E−04
2.01E−10
16.7
74
Si


1.2
3A5.560

VH Y102Q
52.6
7.14E+05
1.08E−04
1.51E−10
16.9
96
87


1.2
3A5.561

VH Y102K
55.9
7.23E+05
8.01E−05
1.11E−10
17.7
130
90


1.2
3A5.562

VH Y102H
50.4
7.22E+05
1.00E−04
1.39E−10
16.4
104
132


1.2
3A5.563

VH Y102W
50.9
7.51E+05
1.16E−04
1.54E−10
16.8
90
117


1.2
3A5.564

VH Y102P
53
6.44E+05
1.29E−04
2.01E−10
15
81
32


2.1
3A5.040
1
Wild type
68.5
7.13E+05
1.16E−04
1.63E−10
22.6




2.1
3A5.040
Average
Wild type
68.5
7.13E+05
1.16E−04
1.63E−10
22.6
100
100


2.1
IgG4


60.7
N/A
N/A
N/A
1.6
N/A




Isotype











2.1
3A5.040

Wild type
75.6
7.37E+05
1.05E−04
1.42E−10
24.3
110




(Purified)











2.1
3A5.048

VL G24A
69
7.16E+05
9.18E−05
1.28E−10
22.7
126
104


2.1
3A5.049

VL G24S
72.5
6.98E+05
1.03E−04
1.47E−10
23.7
113
104


2.1
3A5.050

VL G24L
73.8
6.79E+05
8.13E−05
1.20E−10
23.9
143
103


2.1
3A5.051

VL G24Y
61.7
6.40E+05
1.12E−04
1.75E−10
20.3
104
91


2.1
3A5.052

VL G24D
66.7
7.16E+05
9.71E−05
1.36E−10
21.9
119
104


2.1
3A5.053

VL G24Q
72.6
6.99E+05
9.84E−05
1.41E−10
23.6
118
95


2.1
3A5.054

VL G24K
74.2
7.12E+05
9.59E−05
1.35E−10
24.1
121
98


2.1
3A5.055

VL G24H
60.9
6.90E+05
9.17E−05
1.33E−10
20.2
126
99


2.1
3A5.056

VL G24W
64.4
6.41E+05
8.06E−05
1.26E−10
21.1
144
111


2.1
3A5.057

VL G25A
69.9
7.04E+05
8.75E−05
1.24E−10
22.9
133
109


2.1
3A5.059

VL G25L
63.1
7.16E+05
1.04E−04
1.45E−10
21
112
83


2.1
3A5.060

VL G25Y
67.7
7.05E+05
8.55E−05
1.21E−10
22.2
136
96


2.1
3A5.061

VL G25D
71.9
7.26E+05
1.11E−04
1.53E−10
23.4
105
94


2.1
3A5.062

VL G25Q
73.2
6.52E+05
9.24E−05
1.42E−10
23.5
126
94


2.1
3A5.064

VL G25H
60.4
6.90E+05
9.21E−05
1.33E−10
20.1
126
108


2.1
3A5.065

VL G25W
70.8
7.37E+05
8.97E−05
1.22E−10
23.1
129
81


2.1
3A5.066

VL N26A
76.8
7.27E+05
8.75E−05
1.20E−10
24.7
133
77


2.1
3A5.069

VL N26Y
71.2
6.79E+05
8.73E−05
1.28E−10
22.9
133
100


2.1
3A5.070

VL N26D
62.7
7.22E+05
7.87E−05
1.09E−10
20.7
147
95


2.1
3A5.071

VL N26Q
67.4
7.05E+05
1.08E−04
1.53E−10
22
107
95


2.1
3A5.072

VL N26K
69.7
7.04E+05
1.17E−04
1.66E−10
22.7
99
99


2.1
3A5.073

VL N26H
74.8
7.15E+05
1.11E−04
1.55E−10
24
105
95


2.1
3A5.074

VL N26W
62.6
6.41E+05
9.82E−05
1.53E−10
20.3
118
87


2.1
3A5.075

VL N27A
62.7
6.55E+05
9.29E−05
1.42E−10
20.7
125
120


2.1
3A5.076

VL N27S
71.7
7.04E+05
1.09E−04
1.55E−10
23.2
106
93


2.1
3A5.077

VL N27L
71.7
6.73E+05
8.98E−05
1.33E−10
23.4
129
102


2.1
3A5.078

VL N27Y
61.5
6.16E+05
1.02E−04
1.65E−10
20.3
114
103


2.1
3A5.080

VL N27Q
68.3
6.52E+05
1.07E−04
1.64E−10
22.1
108
91


2.1
3A5.081

VL N27K
71.5
6.89E+05
8.84E−05
1.28E−10
23.2
131
96


2.1
3A5.082

VL N27H
74.7
7.06E+05
7.85E−05
1.11E−10
24.1
148
90


2.1
3A5.084

VL I28A
62.1
6.71E+05
6.20E−05
9.24E−11
20.3
187
91


2.1
3A5.085

VL I28S
91.9
6.37E+05
1.16E−04
1.82E−10
31.3
100
47


2.1
3A5.086

VL I28L
81.5
6.02E+05
1.04E−04
1.73E−10
27.2
112
62


2.1
3A5.087

VL I28Y
73.1
7.07E+05
8.52E−05
1.20E−10
23.8
136
93


2.1
3A5.088

VL I28D
80.3
5.56E+05
9.83E−05
1.77E−10
27.9
118
40


2.1
3A5.089

VL I28Q
79.8
5.80E+05
1.03E−04
1.77E−10
27.8
113
54


2.1
3A5.093

VL G29A
68.4
5.42E+05
1.10E−04
2.04E−10
22.6
105
59


2.1
3A5.094

VL G29S
67.6
6.98E+05
9.31E−05
1.33E−10
21.9
119
93


2.2
3A5.040
1
Wild type
52.4
7.77E+05
1.05E−04
1.35E−10
17.9




2.2
3A5.040
2
Wild type
52.7
7.89E+05
1.09E−04
1.38E−10
17.9




2.2
3A5.040
3
Wild type
52.7
7.72E+05
1.37E−04
1.77E−10
18.1




2.2
3A5.040
Average
Wild type
52.6
7.79E+05
1.17E−04
1.50E−10
17.9
100
100


2.2
IgG4


56.6
N/A
N/A
N/A
0.5
N/A




Isotype











2.2
3A5.040

Wild type
54.9
7.93E+05
1.06E−04
1.33E−10
18.7
110




(Purified)











2.2
3A5.095

VL G29L
55.4
7.17E+05
9.08E−05
1.27E−10
18.7
122
75


2.2
3A5.096

VL G29Y
56.7
6.92E+05
1.33E−04
1.92E−10
19.2
83
58


2.2
3A5.097

VL G29D
56.2
7.31E+05
7.19E−05
9.84E−11
18.7
154
85


2.2
3A5.099

VL G29K
49.9
7.18E+05
9.08E−05
1.26E−10
17.2
122
71


2.2
3A5.100

VL G29H
52
7.38E+05
8.65E−05
1.17E−10
17.7
128
100


2.2
3A5.101

VL G29W
55.8
7.59E+05
9.40E−05
1.24E−10
18.7
118
114


2.2
3A5.102

VL S30A
56.3
6.85E+05
1.09E−04
1.58E−10
18.7
102
84


2.2
3A5.103

VL S3OL
49.5
7.87E+05
9.94E−05
1.26E−10
16.9
112
126


2.2
3A5.104

VL S30Y
54.7
8.17E+05
1.08E−04
1.33E−10
18.5
103
93


2.2
3A5.105

VL S3OD
56.4
8.20E+05
8.91E−05
1.09E−10
18.8
125
119


2.2
3A5.106

VL S30Q
58.3
7.22E+05
9.10E−05
1.26E−10
19.7
122
78


2.2
3A5.107

VL S3OK
50.6
7.85E+05
7.34E−05
9.34E−11
17.3
151
102


2.2
3A5.113

VL K31Y
53.8
8.35E+05
8.73E−05
1.05E−10
18.4
127
99


2.2
3A5.114

VL K31D
54.5
4.89E+05
4.73E−04
9.65E−10
17.9
23
105


2.2
3A5.115

VL K31Q
57.9
4.65E+05
4.60E−04
9.91E−10
19
24
85


2.2
3A5.116

VL K31H
49.5
5.52E+05
2.46E−04
4.46E−10
16.7
45
104


2.2
3A5.117

VL K31W
52.7
5.34E+05
1.33E−04
2.49E−10
17.7
83
125


2.2
3A5.118

VL N32A
55.8
5.07E+05
6.39E−04
1.26E−09
18.3
17
67


2.2
3A5.121

VL N32Y
56.1
9.57E+05
9.68E−05
1.01E−10
19.1
115
91


2.2
3A5.122

VL N32D
49.8
5.68E+05
1.25E−04
2.20E−10
15.8
89
115


2.2
3A5.123

VL N32Q
53.9
8.85E+05
1.25E−04
1.41E−10
18.6
89
102


2.2
3A5.125

VL N32H
56.4
9.01E+05
7.73E−05
8.58E−11
19.2
144
90


2.2
3A5.126

VL N32W
56.6
6.35E+05
9.26E−05
1.46E−10
18.8
120
79


2.2
3A5.127

VL V33A
49.1
7.15E+05
8.05E−05
1.13E−10
16.6
138
74


2.2
3A5.128

VL V33S
51.5
2.85E+05
9.65E−05
3.38E−10
13.7
115
126


2.2
3A5.130

VL V33Y
56.1
7.41E+05
9.16E−05
1.24E−10
19
121
68


2.2
3A5.131

VL V33D
52.4
7.40E+05
9.67E−05
1.31E−10
19.3
115
40


2.2
3A5.132

VL V33Q
48.7
6.61E+05
1.38E−04
2.09E−10
16.7
80
50


2.2
3A5.135

VL V33W
48.4
5.26E+05
1.04E−04
1.99E−10
18.8
107
24


2.2
3A5.137

VL Y34S
48.3
6.96E+05
9.88E−05
1.42E−10
19
112
32


2.2
3A5.138

VL Y34L
49.4
5.94E+05
2.16E−04
3.64E−10
16.6
51
56


2.2
3A5.139

VL Y34D
52.8
4.63E+05
4.90E−04
1.06E−09
13.1
23
68


2.2
3A5.140

VL Y34Q
54.4
7.01E+05
5.40E−04
7.71E−10
18.2
21
55


2.2
3A5.141

VL Y34K
56
8.16E+05
3.72E−04
4.56E−10
18.8
30
64


2.2
3A5.142

VL Y34H
48.8
6.71E+05
5.00E−04
7.46E−10
16.3
22
82


2.2
3A5.143

VL Y34W
53.5
6.98E+05
2.19E−03
3.13E−09
9.7
5
58


2.2
3A5.165

VL S52K
52.6
5.32E+05
9.30E−04
1.75E−09
16.6
12
167


2.2
3A5.168

VL D53A
57.2
1.20E+06
2.04E−03
1.71E−09
17.1
5
51


2.2
3A5.172

VL D53Q
50.3
7.13E+05
1.08E−04
1.51E−10
17.1
103
97


2.2
3A5.174

VL D53H
53
7.45E+05
1.23E−04
1.65E−10
17.7
90
120


2.2
3A5.186

VL P55S
55.8
7.97E+05
1.08E−04
1.35E−10
18.6
103
108


2.2
3A5.199

VL S56K
57.3
7.71E+05
1.18E−04
1.54E−10
18.8
94
104


2.2
3A5.201

VL S56W
49.6
7.41E+05
1.24E−04
1.67E−10
17.1
90
92


2.2
3A5.210

VL Q89P
53.5
3.80E+05
4.78E−04
1.26E−09
18
25
72


2.2
3A5.211

VL V90A
55.7
7.65E+05
1.06E−04
1.39E−10
18.8
105
78


3.1
3A5.040
1
Wild type
53.3
7.72E+05
1.32E−04
1.71E−10
18.2




3.1
3A5.040
2
Wild type
53.4
7.76E+05
1.15E−04
1.48E−10
18.1




3.1
3A5.040
3
Wild type
53.3
7.73E+05
1.03E−04
1.34E−10
18.1




3.1
3A5.040
4
Wild type
54
7.69E+05
1.14E−04
1.49E−10
18.5




3.1
3A5.040
5
Wild type
53.8
7.84E+05
1.20E−04
1.53E−10
18.4




3.1
3A5.040
Average
Wild type
53.6
7.75E+05
1.17E−04
1.51E−10
18.2
100
100


3.1
IgG4


56
N/A
N/A
N/A
0.7
N/A




Isotype











3.1
3A5.040

Wild type
52.5
8.07E+05
1.40E−04
1.74E−10
17.9
83




(Purified)











3.1
3A5.213

VL V9OL
53.7
7.49E+05
1.55E−04
2.06E−10
18.2
75
58


3.1
3A5.214

VL V90Y
54
6.50E+05
2.92E−04
4.49E−10
18
40
85


3.1
3A5.215

VL V9OD
52.9
5.54E+05
4.19E−04
7.56E−10
19.9
28
27


3.1
3A5.216

VL V90Q
48.6
5.93E+05
1.88E−04
3.18E−10
16.3
62
74


3.1
3A5.217

VL V9OK
44.3
6.34E+05
8.30E−05
1.31E−10
17
141
48


3.1
3A5.218

VL V9OH
47.5
5.90E+05
2.99E−04
5.07E−10
17.6
39
60


3.1
3A5.219

VL V9OW
53.4
5.69E+05
6.64E−04
1.17E−09
17.9
18
77


3.1
3A5.220

VL V9OP
47.7
2.72E+05
7.61E−03
2.79E−08
1.7
2
76


3.1
3A5.221

VL W91A
51.8
3.05E+05
7.08E−03
2.32E−08
2.4
2
79


3.1
3A5.223

VL W91L
53.6
2.59E+06
2.55E−02
9.83E−09
7.7
0
98


3.1
3A5.224

VL W91Y
45.1
8.82E+05
9.03E−04
1.02E−09
15.8
13
64


3.1
3A5.226

VL W91Q
51.1
5.38E+06
3.79E−02
7.05E−09
6.8
0
75


3.1
3A5.227

VL W91K
51.8




NB
71


3.1
3A5.228

VL W91H
50.9
1.53E+06
7.24E−03
4.74E−09
9
2
62


3.1
3A5.229

VL W91P
39.4
4.02E+05
7.40E−03
1.84E−08
1.4
2
36


3.1
3A5.230

VL D92A
51.2
5.63E+05
9.94E−05
1.77E−10
16.5
118
113


3.1
3A5.231

VL D92S
53.2
7.67E+05
9.46E−05
1.23E−10
17.5
124
113


3.1
3A5.232

VL D92L
54.3
9.17E+05
7.59E−05
8.27E−11
18
154
130


3.1
3A5.233

VL D92Y
47.7
1.11E+06
1.10E−04
9.92E−11
16.4
106
72


3.1
3A5.234

VL D92Q
50.9
5.25E+05
1.39E−04
2.64E−10
15.7
84
194


3.1
3A5.235

VL D92K
54.7
7.63E+05
8.84E−05
1.16E−10
18
132
92


3.1
3A5.236

VL D92H
54.3
7.93E+05
1.09E−04
1.37E−10
17.6
107
122


3.1
3A5.237

VL D92W
0.5




NE
NE


3.1
3A5.238

VL D92P
46.8
5.21E+05
1.43E−04
2.75E−10
16.1
82
63


3.1
3A5.239

VL S93A
52.2
3.12E+06
3.65E−03
1.17E−09
15.9
3
112


3.1
3A5.240

VL S93L
54.8
2.39E+07
2.45E−01
1.03E−08
6.5
0
124


3.1
3A5.241

VL S93Y
50.1
6.20E+05
7.48E−03
1.21E−08
2
2
118


3.1
3A5.242

VL S93D
51.6
5.18E+05
1.68E−02
3.25E−08
2.9
1
73


3.1
3A5.243

VL S93Q
54.4
1.90E+06
1.51E−02
7.95E−09
8.2
1
111


3.1
3A5.244

VL S93K
55.5




NB
162


3.1
3A5.245

VL S93H
49.7
2.92E+06
2.23E−02
7.64E−09
7.3
1
133


3.1
3A5.246

VL S93W
52.8
3.27E+05
4.33E−03
1.33E−08
0.8
3
115


3.1
3A5.247

VL S93P
53.8
6.94E+05
5.33E−02
7.68E−08
1.7
0
112


3.1
3A5.248

VL S94A
56.1
8.15E+05
1.22E−04
1.50E−10
19.1
96
101


3.1
3A5.249

VL S94L
49.7
7.81E+05
5.00E−04
6.41E−10
17.1
23
90


3.1
3A5.253

VL S94K
53
6.74E+05
1.48E−04
2.20E−10
17.9
79
109


3.1
3A5.254

VL S94H
53.7
6.24E+05
1.51E−04
2.42E−10
18.1
77
104


3.1
3A5.256

VL S94P
49.7
6.97E+05
1.27E−04
1.82E−10
16.7
92
139


3.1
3A5.257

VL S95A
52.1
7.34E+05
1.19E−04
1.62E−10
17.7
98
99


3.1
3A5.258

VL S95L
54
7.73E+05
1.13E−04
1.47E−10
18.3
104
99


3.1
3A5.259

VL S95Y
55.7
8.35E+05
1.07E−04
1.28E−10
18.7
109
83


3.1
3A5.260

VL S95D
49.2
6.07E+05
1.25E−04
2.07E−10
16.5
94
109


3.1
3A5.261

VL S95Q
48.6
7.87E+05
9.19E−05
1.17E−10
17.8
127
62


3.1
3A5.262

VL S95K
53.1
7.99E+05
1.01E−04
1.26E−10
18.2
116
118


3.1
3A5.263

VL S95H
55.4
7.83E+05
1.00E−04
1.28E−10
18.7
117
101


3.1
3A5.264

VL S95W
49
7.95E+05
1.27E−04
1.60E−10
16.5
92
87


3.1
3A5.265

VL S95P
52.7
6.45E+05
5.55E−03
8.61E−09
7.1
2
147


3.1
3A5.266

VL D95aA
53.7
5.78E+06
8.07E−03
1.40E−09
10.4
1
109


3.1
3A5.267

VL D95aS
55.3
4.53E+05
1.21E−03
2.68E−09
16.7
10
101


3.1
3A5.268

VL D95aL
48.5
9.19E+06
1.35E−02
1.47E−09
8.7
1
71


3.1
3A5.269

VL D95aY
51.8
1.77E+06
6.74E−03
3.80E−09
8
2
95


3.1
3A5.270

VL D95aQ
53.6
5.26E+06
7.11E−03
1.35E−09
11.4
2
110


3.1
3A5.271

VL D95aK
53.8
3.92E+05
3.83E−02
9.77E−08
2
0
70


3.1
3A5.272

VL D95aH
49.7
2.56E+06
4.90E−03
1.92E−09
10.1
2
188


3.1
3A5.273

VL D95aW
53.1
1.27E+06
1.47E−02
1.15E−08
7.1
1
94


3.1
3A5.274

VL D95aP
54.6
1.17E+06
3.05E−03
2.61E−09
9.8
4
150


3.1
3A5.276

VL H95bS
52.1
8.47E+05
7.33E−05
8.66E−11
19.6
160
61


3.1
3A5.281

VL H95bK
45.5
7.08E+05
9.58E−05
1.35E−10
16.3
122
64


3.1
3A5.307

VH G26Y
48.1
8.73E+05
1.02E−04
1.17E−10
18.3
115
60


3.1
3A5.321

VH G27W
55
7.64E+05
5.59E−04
7.31E−10
18.4
21
97


3.1
3A5.335

VH I29Q
37.9
5.64E+05
3.90E−04
6.92E−10
14.9
30
37


3.1
3A5.343

VH S30Q
51.9
7.55E+05
1.06E−04
1.40E−10
17.7
110
121


3.1
3A5.344

VH S3OK
53.9
7.41E+05
1.39E−04
1.88E−10
18
84
156


3.1
3A5.345

VH S3OH
54.8
7.34E+05
1.46E−04
1.99E−10
18.7
80
77


3.1
3A5.349

VH N31L
47.1
7.69E+05
7.13E−04
9.27E−10
16.3
16
69


3.1
3A5.355

VH N31W
45.3
8.52E+05
4.80E−04
5.63E−10
17.3
24
59


3.1
3A5.359

VH G32Y
53.7
3.28E+06
4.30E−03
1.31E−09
15.5
3
90


3.1
3A5.360

VH G32D
54.2
1.05E+06
6.78E−03
6.44E−09
8.1
2
105


3.1
3A5.361

VH G32Q
49.5
3.98E+06
6.22E−03
1.56E−09
11.8
2
133


3.1
3A5.362

VH G32K
52.6
6.07E+05
2.20E−03
3.62E−09
7.6
5
171


3.1
3A5.363

VH G32H
55.4
1.81E+06
3.01E−03
1.66E−09
15.3
4
114


3.1
3A5.364

VH G32W
52.3
1.46E+07
1.27E−02
8.65E−10
13.5
1
66


3.1
3A5.365

VH G33A
48.7
6.97E+05
9.24E−05
1.33E−10
16.6
127
116


3.1
3A5.366

VH G33S
53
8.56E+05
2.68E−03
3.14E−09
8.3
4
117


3.1
3A5.367

VH G33L
54.2
8.03E+05
3.05E−04
3.79E−10
17.9
38
82


3.1
3A5.368

VH G33Y
55.4
3.97E+06
4.08E−03
1.03E−09
13.4
3
107


3.2
3A5.040
1
Wild type
54.7
6.75E+05
9.46E−05
1.40E−10
19




3.2
3A5.040
2
Wild type
55.2
6.82E+05
7.26E−05
1.07E−10
19




3.2
3A5.040
3
Wild type
55.7
7.17E+05
9.30E−05
1.30E−10
19.3




3.2
3A5.040
4
Wild type
55.3
7.08E+05
8.82E−05
1.25E−10
19.1




3.2
3A5.040
5
Wild type
55.2
6.95E+05
1.05E−04
1.51E−10
19.2




3.2
3A5.040
Average
Wild type
55.2
6.95E+05
9.07E−05
1.31E−10
19.1
100
100


3.2
IgG4


55.8
N/A
N/A
N/A
0
N/A




Isotype











3.2
3A5.040

Wild type
54.5
7.12E+05
8.25E−05
1.16E−10
1.85E+01
110




(Purified)











3.2
3A5.369

VH G33D
52.4
9.85E+05
2.46E−03
2.50E−09
9.4
4
277


3.2
3A5.370

VH G33Q
54.5
5.33E+05
1.07E−03
2.00E−09
17.2
8
279


3.2
3A5.371

VH G33K
58.5
2.08E+06
2.02E−03
9.72E−10
14.1
4
350


3.2
3A5.372

VH G33H
51.5
1.45E+06
2.67E−03
1.83E−09
14.4
3
273


3.2
3A5.373

VH G33W
52.4
6.71E+05
1.32E−03
1.96E−09
15.9
7
172


3.2
3A5.374

VH Y34A
56.4
5.91E+05
1.27E−04
2.16E−10
19.2
71
162


3.2
3A5.375

VH Y34S
56.4
6.23E+05
1.24E−04
2.00E−10
19.1
73
159


3.2
3A5.376

VH Y34L
50
5.79E+05
1.60E−04
2.77E−10
17.2
57
160


3.2
3A5.377

VH Y34D
54.7
5.40E+05
6.04E−04
1.12E−09
18.1
15
103


3.2
3A5.378

VH Y34Q
54.4
5.98E+05
1.51E−04
2.53E−10
18.5
60
165


3.2
3A5.379

VH Y34K
56.6
6.17E+05
4.91E−04
7.96E−10
19.1
18
407


3.2
3A5.380

VH Y34H
49.8
6.40E+05
7.06E−04
1.10E−09
16.7
13
223


3.2
3A5.382

VH Y35A
52.8
2.48E+05
2.46E−03
9.91E−09
0.9
4
289


3.2
3A5.383

VH Y35S
55.8
3.34E+05
8.50E−03
2.54E−08
1.9
1
129


3.2
3A5.384

VH Y35L
57
5.98E+05
1.42E−03
2.37E−09
17.5
6
190


3.2
3A5.385

VH Y35D
48.5
2.75E+06
6.13E−03
2.23E−09
8.1
1
87


3.2
3A5.386

VH Y35Q
51.8
1.18E+07
2.72E−01
2.31E−08
5.4
0
127


3.2
3A5.387

VH Y35K
56.2




NB
382


3.2
3A5.388

VH Y35H
58
3.40E+05
9.25E−03
2.72E−08
3.3
1
195


3.2
3A5.389

VH Y35W
50
3.65E+06
5.41E−03
1.48E−09
8.8
2
225


3.2
3A5.390

VH W35aA
47
6.84E+05
1.74E−03
2.55E−09
15.4
5
37


3.2
3A5.392

VH W35aL
55
4.59E+05
1.14E−03
2.49E−09
17.1
8
100


3.2
3A5.393

VH W35aY
49.1
6.34E+05
1.87E−04
2.94E−10
16.7
49
104


3.2
3A5.394

VH W35aD
0.7




NE
NE


3.2
3A5.395

VH W35aQ
50.4
5.46E+05
8.47E−04
1.55E−09
18.8
11
34


3.2
3A5.397

VH W35aH
39.6
5.07E+05
2.44E−04
4.82E−10
15.8
37
53


3.2
3A5.399

VH S35bL
46
1.08E+05
2.86E−04
2.64E−09
14.1
32
39


3.2
3A5.400

VH S35bY
51.7
4.43E+05
7.06E−03
1.59E−08
1.8
1
88


3.2
3A5.402

VH S35bQ
38.8
1.84E+05
5.15E−04
2.80E−09
13.6
18
45


3.2
3A5.404

VH S35bH
48.8
6.18E+05
1.88E−03
3.05E−09
13.3
5
75


3.2
3A5.405

VH S35bW
49.6
2.93E+05
7.19E−03
2.45E−08
0.7
1
50


3.2
3A5.406

VH Y50A
50.6
4.79E+05
5.88E−04
1.23E−09
16.1
15
268


3.2
3A5.407

VH Y5OS
56.1
4.95E+05
3.64E−04
7.34E−10
17.5
25
327


3.2
3A5.408

VH Y5OL
57
4.31E+05
3.22E−04
7.46E−10
17.3
28
122


3.2
3A5.409

VH Y5OD
56.9
7.63E+05
1.45E−03
1.90E−09
16
6
169


3.2
3A5.410

VH Y50Q
50.9
5.95E+05
3.69E−04
6.20E−10
17.1
25
241


3.2
3A5.411

VH Y5OK
53.5
5.51E+05
4.47E−04
8.11E−10
17.5
20
135


3.2
3A5.412

VH Y5OH
55.5
6.72E+05
7.88E−04
1.17E−09
18.2
12
300


3.2
3A5.413

VH Y5OW
57.2
6.67E+05
1.61E−03
2.41E−09
16.9
6
131


3.2
3A5.414

VH I51A
51.9
7.29E+05
1.13E−04
1.56E−10
17.7
80
244


3.2
3A5.415

VH I51S
54.2
7.38E+05
1.15E−04
1.56E−10
18.6
79
130


3.2
3A5.416

VH I51L
54.6
6.62E+05
1.39E−04
2.10E−10
19
65
87


3.2
3A5.417

VH I51Y
57.2
5.42E+05
7.77E−03
1.43E−08
7.9
1
106


3.2
3A5.418

VH I51D
40.4
5.15E+05
6.36E−04
1.23E−09
15.8
14
48


3.2
3A5.419

VH I51Q
51.6
6.26E+05
5.02E−04
8.01E−10
17.7
18
95


3.2
3A5.420

VH I51K
53.3
7.42E+05
1.16E−03
1.57E−09
17.3
8
124


3.2
3A5.421

VH I51H
54.1
7.46E+06
8.34E−03
1.12E−09
13.4
1
93


3.2
3A5.422

VH I51W
49.7
3.18E+05
3.31E−03
1.04E−08
0.9
3
139


3.2
3A5.423

VH Y52A
54.9
1.49E+07
1.08E−02
7.23E−10
11.8
1
304


3.2
3A5.424

VH Y52S
57.3
6.79E+05
1.29E−03
1.89E−09
11.7
7
325


3.2
3A5.425

VH Y52L
59.4
1.90E+06
8.83E−03
4.66E−09
10.1
1
209


3.2
3A5.426

VH Y52D
51.7




NB
333


3.2
3A5.427

VH Y52Q
55.5
1.86E+06
8.86E−03
4.76E−09
9.2
1
264


3.2
3A5.428

VH Y52K
58.4




NB
348


3.2
3A5.429

VH Y52H
61.5
5.62E+05
2.91E−04
5.17E−10
20.6
31
286


3.2
3A5.430

VH Y52W
48.6
6.79E+05
1.26E−03
1.86E−09
15.9
7
102


3.2
3A5.431

VH Y53A
53
6.79E+05
5.21E−04
7.67E−10
17.9
17
249


3.2
3A5.432

VH Y53S
55
6.94E+05
1.57E−04
2.26E−10
18.8
58
226


3.2
3A5.433

VH Y53L
57.3
6.02E+05
4.43E−04
7.36E−10
19.1
20
102


3.2
3A5.434

VH Y53D
50.8
4.80E+05
5.94E−04
1.24E−09
16.6
15
182


3.2
3A5.435

VH Y53Q
53.4
6.27E+05
4.41E−04
7.03E−10
17.9
21
206


3.2
3A5.436

VH Y53K
56.7
6.17E+05
9.61E−04
1.56E−09
18.1
9
232


3.2
3A5.437

VH Y53H
57
6.71E+05
1.91E−04
2.85E−10
19.1
47
206


3.2
3A5.438

VH Y53W
49.2
6.91E+05
2.35E−04
3.40E−10
16.8
39
152


3.2
3A5.439

VH S54A
53.1
6.98E+05
1.54E−04
2.20E−10
18.1
59
112


3.2
3A5.440

VH S54L
53.4
9.17E+06
8.95E−03
9.76E−10
13.6
1
158


3.2
3A5.441

VH S54Y
55
1.57E+06
2.17E−03
1.38E−09
16.5
4
92


3.2
3A5.442

VH S54D
51.4
8.06E+05
2.02E−03
2.50E−09
14.4
4
200


3.2
3A5.443

VH S54Q
54
6.04E+05
1.07E−03
1.77E−09
17.6
8
150


3.2
3A5.444

VH S54K
55.6
2.65E+06
3.55E−03
1.34E−09
15.5
3
201


3.2
3A5.445

VH S54H
57.5
7.10E+05
4.59E−04
6.46E−10
19.3
20
97


3.2
3A5.446

VH S54W
46.7
7.24E+05
8.68E−04
1.20E−09
14.8
10
112


3.2
3A5.447

VH G55A
45.7
6.76E+05
1.05E−04
1.55E−10
17.1
86
68


3.2
3A5.448

VH G55S
48.4
7.30E+05
1.24E−04
1.70E−10
18
73
72


3.2
3A5.449

VH G55L
47.7
6.14E+05
6.30E−04
1.03E−09
17.7
14
66


3.2
3A5.450

VH G55Y
38.6
6.88E+05
7.06E−04
1.03E−09
14.2
13
49


4.1
3A5.040
1
Wild type
82.7
1.00E+06
1.16E−04
1.16E−10
24.9




4.1
3A5.040
2
Wild type
82.7
1.02E+06
1.07E−04
1.04E−10
25.1




4.1
3A5.040
3
Wild type
85.2
1.03E+06
1.12E−04
1.09E−10
25.1




4.1
3A5.040
4
Wild type
85.1
1.01E+06
1.06E−04
1.05E−10
25.3




4.1
3A5.040
5
Wild type
83.5
9.91E+05
9.29E−05
9.37E−11
25.3




4.1
3A5.040
Average
Wild type
83.84
1.01E+06
1.07E−04
1.06E−10
25.14
100
100


4.1
IgG4


61.4
N/A
N/A
N/A
0
N/A




Isotype











4.1
3A5.040

Wild type
84.2
1.06E+06
1.09E−04
1.03E−10
23.8
98




(Purified)











4.1
3A5.133

VL V33K
52.7
7.83E+05
1.34E−04
1.71E−10
15.9
80
15


4.1
3A5.222

VL W91S
62.8
1.38E+06
2.85E−03
2.06E−09
12.5
4
14


4.1
3A5.391

VH W35aS
68.4
6.35E+05
1.19E−03
1.87E−09
15.6
9
22


4.1
3A5.396

VH W35aK
65.9
7.21E+05
2.08E−03
2.88E−09
9.4
5
26


4.1
3A5.401

VH S35bD
53.1
5.96E+05
1.19E−04
2.01E−10
14.5
90
16


4.1
3A5.403

VH S35bK
53.1
1.83E+05
1.23E−02
6.68E−08
2.8
1
12


4.1
3A5.275

VL H95bA
69.7
5.47E+05
1.62E−04
2.95E−10
18
66
24


4.1
3A5.282

VL H95bW
77.5
7.18E+05
1.07E−04
1.49E−10
21.1
100
152


4.1
3A5.283

VL H95bP
63.4
5.90E+05
4.22E−02
7.15E−08
3.6
0
154


4.1
3A5.285

VL V96S
74.4
9.02E+05
2.01E−04
2.23E−10
19.9
53
21


4.1
3A5.287

VL V96Y
92.4
7.33E+06
1.50E−03
2.05E−10
17.6
7
62


4.1
3A5.288

VL V96D
68.8
7.32E+05
1.30E−03
1.78E−09
16.8
8
9.5


4.1
3A5.289

VL V96Q
77.1
1.62E+06
6.54E−04
4.03E−10
20.4
16
25


4.1
3A5.290

VL V96K
64.6
8.08E+05
2.32E−03
2.87E−09
13.1
5
15


4.1
3A5.291

VL V96H
5.2




NB
45


4.1
3A5.292

VL V96W
66.4
3.83E+07
2.01E−02
5.26E−10
10.6
1
19


4.1
3A5.293

VL V96P
81.5
8.54E+05
5.22E−04
6.11E−10
23.4
20
106


4.1
3A5.110

VL K31A
74
7.01E+05
1.86E−04
2.65E−10
21.1
58
122


4.1
3A5.111

VL K31S
79.2
7.10E+05
1.36E−04
1.92E−10
23.6
79
109


4.1
3A5.112

VL K31L
79.9
6.30E+05
3.91E−04
6.21E−10
22.3
27
125


4.1
3A5.134

VL V33H
5.8




NB
52


4.1
3A5.136

VL Y34A
59.9
1.05E+06
3.45E−04
3.28E−10
17.2
31
170


4.1
3A5.206

VL Q89D
71.6
5.68E+05
8.76E−05
1.54E−10
18.6
122
12


4.1
3A5.225

VL W91D
114.2
6.31E+05
1.05E−02
1.66E−08
3.7
1
74


4.1
3A5.250

VL S94Y
74.6
8.14E+05
3.48E−04
4.27E−10
20.3
31
147


4.1
3A5.252

VL S94Q
63.4
8.25E+05
1.65E−04
2.00E−10
18
65
136


4.2
3A5.040
1
Wild type
50.4
6.96E+05
1.03E−04
1.48E−10
16.2




4.2
3A5.040
2
Wild type
50.5
7.10E+05
1.06E−04
1.49E−10
16




4.2
3A5.040
Average
Wild type
50.4
7.03E+05
1.05E−04
1.49E−10
16.1
100
100


4.2
IgG4


52.5
N/A
N/A
N/A
N/A





Isotype











4.2
3A5.040

Wild type
48.7
7.21E+05
6.63E−05
9.20E−11
16.5
150




(Purified)











4.2
3A5.451

VH G55D
33.1
4.54E+05
2.50E−04
5.50E−10
9.9
42
39


4.2
3A5.452

VH G55Q
37.2
6.09E+05
1.94E−04
3.18E−10
11.7
54
42


4.2
3A5.453

VH G55K
43.8
5.49E+05
2.07E−04
3.76E−10
12.4
50
51


4.2
3A5.454

VH G55H
38.2
5.92E+05
1.81E−04
3.05E−10
11.7
58
41


4.2
3A5.455

VH G55W
31.2
6.17E+05
6.65E−04
1.08E−09
10
16
33


4.2
3A5.456

VH S56A
49.7
6.44E+05
1.18E−04
1.84E−10
15.5
89
104


4.2
3A5.457

VH S56L
51.1
5.49E+05
8.63E−05
1.57E−10
15.6
121
101


4.2
3A5.458

VH S56Y
47.6
5.44E+05
1.92E−04
3.53E−10
14.2
54
85


4.2
3A5.459

VH S56D
49
5.05E+05
4.57E−04
9.06E−10
15
23
84


4.2
3A5.460

VH S56Q
50.2
5.90E+05
9.69E−05
1.64E−10
15.8
108
106


4.2
3A5.461

VH S56K
52.9
6.11E+05
7.84E−05
1.28E−10
16.6
133
119


4.2
3A5.463

VH S56W
47.7
4.72E+05
2.85E−04
6.03E−10
13.9
37
98


4.2
3A5.464

VH T57A
50
7.50E+3005
1.26E−04
1.67E−10
16.1
83
104


4.2
3A5.465

VH T57S
48.5
7.29E+05
1.26E−04
1.73E−10
15.6
83
69


4.2
3A5.467

VH T57Y
44
7.66E+05
9.50E−05
1.24E−10
13.8
110
49


4.2
3A5.468

VH T57D
42.5
7.02E+05
9.89E−05
1.41E−10
14
106
46


4.2
3A5.469

VH T57Q
45.1
7.24E+05
1.07E−04
1.48E−10
14.2
98
58


4.2
3A5.470

VH T57K
46.5
7.28E+05
1.21E−04
1.66E−10
14.6
86
61


4.2
3A5.471

VH T57H
43.8
7.34E+05
1.09E−04
1.49E−10
13.9
96
50


4.2
3A5.472

VH T57W
39.2
7.57E+05
1.34E−04
1.77E−10
13.3
78
40


4.2
3A5.473

VH Y58A
48.1
1.56E+06
2.86E−03
1.83E−09
13
4
95


4.2
3A5.474

VH Y58S
50.7
2.76E+06
3.84E−03
1.39E−09
13.7
3
101


4.2
3A5.475

VH Y58L
48.6
5.58E+06
5.88E−03
1.05E−09
8.3
2
70


4.2
3A5.476

VH Y58D
42.4
1.89E+07
1.68E−02
8.88E−10
7.3
1
47


4.2
3A5.477

VH Y58Q
46.6
6.62E+05
1.33E−03
2.02E−09
13.9
8
75


4.2
3A5.478

VH Y58K
49.2
5.75E+05
1.31E−03
2.28E−09
14.6
8
102


4.2
3A5.479

VH Y58H
50.6
6.68E+05
5.75E−04
8.62E−10
15.8
18
91


4.2
3A5.480

VH Y58W
48.1
6.58E+05
5.53E−04
8.41E−10
14.7
19
79


4.2
3A5.481

VH A93S
45.3
7.18E+05
9.31E−05
1.30E−10
13.9
112
58


4.2
3A5.482

VH A93L
13
2.55E+05
2.02E−04
7.90E−10
5.2
52
17


4.2
3A5.484

VH A93D
0.5




NE
NE


4.2
3A5.485

VH A93Q
40.4
6.48E+05
1.00E−04
1.54E−10
12.7
105
44


4.2
3A5.486

VH A93K
17




NE
NE


4.2
3A5.487

VH A93H
28.8




NE
NE


4.2
3A5.489

VH A93P
6.5




NE
NE


4.2
3A5.491

VH S94L
37.4
5.33E+05
8.11E−04
1.52E−09
12.5
13
40


4.2
3A5.492

VH S94Y
30.3
3.27E+05
1.10E−03
3.37E−09
9.7
10
30


4.2
3A5.493

VH S94D
4.6




NE
NE


4.2
3A5.494

VH S94Q
42
6.74E+05
6.37E−04
9.45E−10
13.6
16
49


4.2
3A5.496

VH S94H
47.1
2.19E+06
4.62E−03
2.11E−09
6.5
2
72


4.2
3A5.501

VH L95Y
49
4.17E+06
5.88E−03
1.41E−09
12.4
2
96


4.2
3A5.502

VH L95D
21.2




NB
22


4.2
3A5.506

VH L95W
46.4
6.20E+06
7.36E−03
1.19E−09
8.4
1
68


4.2
3A5.511

VH G96Y
45.5




NB
59


4.2
3A5.519

VH N97S
48
5.61E+05
2.27E−04
4.04E−10
14.9
46
80


4.2
3A5.552

VH D101K
47.1
6.10E+05
3.63E−04
5.95E−10
14.6
29
67


4.2
3A5.358

VH G32L
48.4
2.01E+06
3.83E−03
1.91E−09
12.3
3
75


4.2
3A5.381

VH Y34W
42.4
7.09E+05
2.18E−04
3.07E−10
14
48
47


4.2
3A5.398

VH S35bA
47
6.82E+05
1.02E−04
1.50E−10
14.7
102
88


4.2
3A5.488

VH A93W




14.2
NB
17


4.2
3A5.490

VH S94A
49.6
6.78E+05
8.70E−05
1.28E−10
15.1
120
104


4.2
3A5.499

VH L95A
43.4
3.09E+06
7.48E−03
2.42E−09
10.4
1
51


4.2
3A5.508

VH G96A
47.1
5.77E+05
1.25E−04
2.17E−10
14.8
84
75


4.2
3A5.522

VH N97D
49
1.00E+06
1.92E−04
1.91E−10
15.5
54
91


4.2
3A5.554

VH D101W
32.7
1.38E+05
6.18E−04
4.47E−09
8.3
17
33


4.2
3A5.144

VL D50A
47.1
5.29E+05
1.67E−04
3.15E−10
14.5
63
77


4.2
3A5.145

VL DSOS
48.2
5.74E+05
1.05E−04
1.83E−10
15.1
100
90


4.2
3A5.146

VL D5OL
48
5.92E+05
2.59E−04
4.38E−10
15
40
86


4.2
3A5.147

VL D5OY
48
5.36E+05
1.95E−04
3.63E−10
15
54
78


4.2
3A5.148

VL D50Q
47
5.99E+05
1.93E−04
3.22E−10
15
54
70


4.2
3A5.149

VL D5OK
45.8
6.00E+05
1.61E−04
2.68E−10
13.2
65
74


4.2
3A5.150

VL D5OH
48.2
4.07E+05
1.68E−04
4.14E−10
14.3
62
95


4.2
3A5.151

VL D5OW
48
5.36E+05
2.35E−04
4.38E−10
15
44
74


4.2
3A5.152

VL D51A
47
6.34E+05
1.34E−04
2.11E−10
14.6
78
88


4.2
3A5.153

VL D51S
46.1
8.53E+05
1.60E−04
1.87E−10
14.1
65
72


4.2
3A5.154

VL D51L
47.8
6.22E+05
1.63E−04
2.63E−10
14.3
64
83


4.2
3A5.155

VL D51Y
48.1
6.14E+05
1.78E−04
2.90E−10
13.7
59
75


4.2
3A5.156

VL D51Q
47.8
6.48E+05
1.15E−04
1.77E−10
15
91
85


4.2
3A5.157

VL D51K
45.2
5.85E+05
1.20E−04
2.05E−10
13.2
87
68


4.2
3A5.158

VL D51H
48.3
6.15E+05
1.82E−04
2.95E−10
14.7
57
86


4.2
3A5.159

VL D51W
45.7
4.93E+05
1.96E−04
3.98E−10
14.1
53
57


4.2
3A5.160

VL S52A
47.7
6.61E+05
1.11E−04
1.68E−10
15.2
94
80


4.2
3A5.161

VL S52L
47.8
6.87E+05
5.74E−05
8.36E−11
15
182
106


4.2
3A5.162

VL S52Y
49
7.26E+05
9.91E−05
1.36E−10
15.8
105
114


5.1
3A5.040
1
Wild type
49.9
6.56E+05
1.22E−04
1.86E−10
14.5




5.1
3A5.040
2
Wild type
48.5
6.43E+05
1.12E−04
1.75E−10
14.4




5.1
3A5.040
3
Wild type
48.1
6.67E+05
1.25E−04
1.87E−10
13.7




5.1
3A5.040
4
Wild type
50.4
6.63E+05
1.17E−04
1.77E−10
14.4




5.1
3A5.040
Average
Wild type
49.2
6.57E+05
1.19E−04
1.81E−10
14.3
100
100


5.1
IgG4


51.2
N/A
N/A
N/A
0.1
N/A




Isotype











5.1
3A5.040

Wild type
48.9
7.07E+05
1.08E−04
1.53E−10
15.6
110




(Purified)











5.1
3A5.337

VH I29H
48.1
4.79E+05
1.12E−03
2.34E−09
13.7
11
24


5.1
3A5.339

VH S30A
50.7
6.79E+05
1.12E−04
1.65E−10
16.3
106
6


5.1
3A5.346

VH S3OW
53.4
6.42E+05
1.32E−04
2.06E−10
16.7
90
69


5.1
3A5.058

VL G25S
47.6
6.93E+05
1.08E−04
1.56E−10
15.7
110
43


5.1
3A5.090

VL I28K
49.1
5.73E+05
1.04E−04
1.81E−10
16.2
114
51


5.1
3A5.092

VL I28W
47
6.13E+05
1.30E−04
2.12E−10
15.5
92
35


5.1
3A5.212

VL V9OS
46.1
5.71E+05
3.11E−04
5.45E−10
14.6
38
4.8


5.1
3A5.251

VL S94D
52.9
2.77E+05
4.98E−04
1.79E−09
15.5
24
7


5.1
3A5.255

VL S94W
48.5
5.54E+05
2.86E−04
5.16E−10
14.7
42
19


5.1
3A5.299

VL V97Q
48.5
5.86E+05
9.90E−05
1.69E−10
15.5
120
71


5.1
3A5.462

VH S56H
50
6.35E+05
1.16E−04
1.83E−10
15.9
103
41


5.1
3A5.466

VH T57L
44.9
6.39E+05
1.02E−04
1.59E−10
14.2
117
14


5.1
3A5.483

VH A93Y
38.2




NB
8


5.1
3A5.503

VH L95Q
48.5
4.04E+05
6.18E−04
1.53E−09
14.4
19
18


5.1
3A5.163

VL S52D
49.3
6.65E+05
1.01E−04
1.52E−10
16.4
118
177


5.1
3A5.164

VL S52Q
49.6
6.57E+05
1.17E−04
1.78E−10
15.8
102
9.8


5.1
3A5.166

VL S52H
51.6
6.71E+05
1.11E−04
1.66E−10
16.9
107
167


5.1
3A5.167

VL S52W
52.3
7.19E+05
1.11E−04
1.55E−10
17.2
107
166


5.1
3A5.169

VL D53S
50.6
6.38E+05
6.35E−05
9.97E−11
16.5
187
151


5.1
3A5.170

VL D53L
48.8
6.87E+05
9.50E−05
1.38E−10
15.8
125
143


5.1
3A5.171

VL D53Y
51
6.79E+05
1.09E−04
1.60E−10
16.1
109
208


5.1
3A5.173

VL D53K
52.6
6.35E+05
1.09E−04
1.71E−10
16.7
109
175


5.1
3A5.175

VL D53W
50
5.88E+05
9.74E−05
1.66E−10
15.7
122
178


5.1
3A5.176

VL R54A
50.4
6.51E+05
9.75E−05
1.50E−10
16.8
122
138


5.1
3A5.177

VL R54S
50.1
6.45E+05
1.11E−04
1.72E−10
16.8
107
131


5.1
3A5.178

VL R54L
51.5
6.60E+05
1.01E−04
1.53E−10
17
118
135


5.1
3A5.179

VL R54Y
49.2
6.66E+05
1.24E−04
1.86E−10
16.4
96
138


5.1
3A5.180

VL R54D
49.5
6.79E+05
9.89E−05
1.46E−10
16.6
120
128


5.1
3A5.181

VL R54Q
51.3
6.58E+05
9.55E−05
1.45E−10
17
125
148


5.1
3A5.182

VL R54K
51.5
6.50E+05
1.17E−04
1.79E−10
17
102
165


5.1
3A5.183

VL R54H
49.1
6.47E+05
8.44E−05
1.30E−10
16.3
141
137


5.1
3A5.184

VL R54W
50
6.14E+05
1.22E−04
1.99E−10
15.8
98
7.5


5.1
3A5.185

VL P55A
51.9
6.87E+05
1.34E−04
1.95E−10
16.5
89
8.9


5.1
3A5.187

VL P55L
49.6
7.00E+05
9.58E−05
1.37E−10
16.3
124
153


5.1
3A5.188

VL P55Y
49.2
6.61E+05
1.19E−04
1.80E−10
16.4
100
127


5.1
3A5.189

VL P55D
51.1
6.48E+05
9.21E−05
1.42E−10
16.7
129
155


5.1
3A5.190

VL P55Q
48.8
6.55E+05
1.08E−04
1.64E−10
16.4
110
151


5.1
3A5.191

VL P55K
49.3
6.38E+05
1.05E−04
1.64E−10
16.3
113
137


5.1
3A5.192

VL P55H
51.5
6.51E+05
1.16E−04
1.78E−10
16.7
103
176


5.1
3A5.193

VL P55W
51.5
6.41E+05
8.80E−05
1.37E−10
16.8
135
160


5.1
3A5.194

VL S56A
49.8
6.41E+05
1.06E−04
1.65E−10
16.1
112
143


5.1
3A5.195

VL S56L
49.6
6.85E+05
1.06E−04
1.54E−10
16.3
112
131


5.1
3A5.196

VL S56Y
50.2
6.89E+05
1.14E−04
1.66E−10
16.5
104
123


5.1
3A5.197

VL S56D
50.1
6.87E+05
1.19E−04
1.73E−10
16.5
100
153


5.1
3A5.198

VL S56Q
49.6
6.86E+05
8.85E−05
1.29E−10
16.4
134
131


5.1
3A5.200

VL S56H
49.3
6.83E+05
1.15E−04
1.68E−10
16.3
103
138


5.1
3A5.207

VL Q89K
49.9
6.84E+05
1.27E−04
1.86E−10
16.4
94
136


5.1
3A5.209

VL Q89W
48.6
5.57E+05
1.27E−04
2.28E−10
15.7
94
72


5.1
3A5.278

VL H95bY
49.9
7.65E+05
7.90E−05
1.03E−10
16.3
151
139


5.1
3A5.279

VL H95bD
49.6
7.01E+05
7.84E−05
1.12E−10
16.5
152
210


5.1
3A5.280

VL H95bQ
50.3
7.47E+05
1.51E−04
2.02E−10
16.6
79
133


5.1
3A5.284

VL V96A
48.2
7.24E+05
1.88E−04
2.60E−10
15.9
63
65


5.1
3A5.286

VL V96L
48.4
6.50E+05
1.94E−04
2.98E−10
15.9
61
79


5.1
3A5.294

VL V97A
48.6
6.27E+05
7.68E−05
1.23E−10
16
155
133


5.1
3A5.295

VL V97S
48.6
6.12E+05
1.07E−04
1.74E−10
15.9
111
110


5.1
3A5.296

VL V97L
48
6.71E+05
9.60E−05
1.43E−10
15.9
124
95


5.1
3A5.297

VL V97Y
48.4
6.72E+05
1.07E−04
1.60E−10
16.2
111
90


5.1
3A5.298

VL V97D
47.7
5.94E+05
1.30E−04
2.18E−10
15.8
92
96


5.1
3A5.300

VL V97K
50.1
5.88E+05
1.18E−04
2.01E−10
15.8
101
4.7


5.1
3A5.301

VL V97H
49.4
6.34E+05
8.79E−05
1.39E−10
16.1
135
168


5.1
3A5.063

VL G25K
49
6.90E+05
7.21E−05
1.05E−10
16.2
165
90


5.1
3A5.067

VL N26S
49
6.52E+05
1.10E−04
1.69E−10
16
108
162


5.1
3A5.083

VL N27W
49.6
6.03E+05
1.29E−04
2.14E−10
16.4
92
148


5.1
3A5.091

VL I28H
47.7
5.71E+05
1.30E−04
2.27E−10
15.7
92
75


5.1
3A5.098

VL G29Q
49.7
6.53E+05
1.05E−04
1.61E−10
16.2
113
139


5.1
3A5.108

VL S3OH
49.4
6.95E+05
1.02E−04
1.46E−10
16.2
117
145


5.1
3A5.109

VL S3OW
48
6.77E+05
1.35E−04
2.00E−10
15.5
88
112


5.1
3A5.119

VL N32S
48.6
8.35E+05
1.08E−04
1.29E−10
16.3
110
98


5.1
3A5.120

VL N32L
49.2
8.72E+05
1.03E−04
1.18E−10
16.4
116
119


5.1
3A5.129

VL V33L
48.8
5.80E+05
1.42E−04
2.45E−10
15.8
84
99


5.1
3A5.277

VL H95bL
50.4
6.79E+05
1.40E−04
2.06E−10
16.6
85
180


5.1
3A5.302

VL V97W
47.7
7.18E+05
7.59E−05
1.06E−10
15.9
157
73


5.1
3A5.303

VL V97P
48.7
6.99E+05
1.21E−04
1.73E−10
16.2
98
21





This table contains a summary of the calculated kinetic and titre data for each antibody supernatant tested Variant antibodies with a lower kd (slower off-rate) than parent 3A5.040 antibody have a “% 3A5.040 kd”value >100% and those with a higher kd (faster off-rate) a value <100%. Variant antibodies with a higher titre than parent 3A5.040 antibody have a “% 3A5.040 titre”value >100% and those with a lower titre, a value <100%. N/A, non-applicable. NB, No binding. NE, No expression.













TABLE 4







Determination of purified 3A5.040 variant antibody IL-5 binding kinetics by BIACORE ™

























%


Run


Amino acid
Capture



Rmax
3A5.040


#
Antibody ID
Replicates
Substitution
level
ka (1/Ms)
kd (Vs)
KD (M)
(RU)
kd



















6
3A5.040 (batch 6)
1
Wild type
48.6
1.44E+06
2.32E−04
1.61E−10
16



6
3A5.040 (batch 6)
2
Wild type
49.4
1.44E+06
2.14E−04
1.48E−10
15.7



6
3A5.040 (batch 6)
3
Wild type
49.2
1.36E+06
2.61E−04
1.93E−10
16



6
3A5.040 (batch 6)
4
Wild type
49.2
1.36E+06
2.46E−04
1.80E−10
16



6
3A5.040 (batch 6)
5
Wild type
49.3
1.47E+06
2.52E−04
1.71E−10
16.1



6
3A5.040 (batch 6)
6
Wild type
49.2
1.45E+06
2.13E−04
1.47E−10
16



6
3A5.040 (batch 6)
7
Wild type
49.2
1.41E+06
2.29E−04
1.63E−10
16.1



6
3A5.040 (batch 6)
8
Wild type
50.9
1.40E+06
2.45E−04
1.75E−10
16.8



6
3A5.040 (batch 6)
Average
Wild type
49.3
1.42E+06
2.37E−04
1.67E−10
16.1
100


6
IgG4 iso
1
48.8
N/A
N/A
N/A
0




6
IgG4 iso
2
48.9
N/A
N/A
N/A
0




6
IgG4 iso
3
48.8
N/A
N/A
N/A
0




6
IgG4 Isotype
Average
49.8
N/A
N/A
N/A
0
N/A



6
3A5.040 (Purified)
1
Wild type
49.6
1.43E+06
2.39E−04
1.68E−10
16.1



6
3A5.040 (Purified)
2
Wild type
49.8
1.48E+06
2.85E−04
1.93E−10
16.2



6
3A5.040 (Purified)
3
Wild type
49.7
1.45E+06
2.32E−04
1.60E−10
16.1



6
3A5.040 (Purified)
Average
Wild type
49.7
1.45E+06
2.52E−04
1.74E−10
16.1
94


6
3A5.237
1
VL_D92W
47.9
1.57E+06
2.67E−04
1.70E−10
15.5



6
3A5.237
2
VL_D92W
48.7
1.55E+06
2.58E−04
1.66E−10
15.6



6
3A5.237
3
VL_D92W
48.5
1.52E+06
2.12E−04
1.40E−10
15.7



6
3A5.237
Average
VL_D92W
48.3
1.55E+06
2.46E−04
1.59E−10
15.6
96


6
3A5.161
1
VL_S52L
49.4
1.47E+06
2.62E−04
1.78E−10
16.2



6
3A5.161
2
VL_S52L
49.9
1.43E+06
2.41E−04
1.69E−10
16.2



6
3A5.161
3
VL_S52L
49.9
1.40E+06
2.19E−04
1.57E−10
16.4



6
3A5.161
Average
VL_S52L
49.7
1.43E+06
2.41E−04
1.68E−10
16.2
99


6
3A5.169
1
VL_D53S
48.9
1.23E+06
2.43E−04
1.98E−10
15.6



6
3A5.169
2
VL_D53S
49.5
1.26E+06
2.44E−04
1.93E−10
15.9



6
3A5.169
3
VL_D53S
49.7
1.23E+06
2.46E−04
1.99E−10
16.1



6
3A5.169
Average
VL_D535
49.3
1.24E+06
2.44E−04
1.97E−10
15.8
97


6
3A5.183
1
VL_R54H
47.9
1.44E+06
2.32E−04
1.61E−10
15.6



6
3A5.183
2
VL_R54H
48.2
1.44E+06
2.50E−04
1.74E−10
15.8



6
3A5.183
3
VL_R54H
48.4
1.39E+06
2.04E−04
1.47E−10
15.8



6
3A5.183
Average
VL_R54H
48.1
1.42E+06
2.29E−04
1.61E−10
15.7
104


6
3A5.193
1
VL_P55W
48.5
1.32E+06
2.32E−04
1.76E−10
15.7



6
3A5.193
2
VL_P55W
48.3
1.34E+06
2.51E−04
1.88E−10
15.3



6
3A5.193
3
VL_P55W
48.7
1.29E+06
2.57E−04
1.99E−10
15.6



6
3A5.193
Average
VL_P55W
48.5
1.32E+06
2.47E−04
1.88E−10
15.5
96


6
3A5.198
1
VL_S56Q
49.1
1.42E+06
2.43E−04
1.71E−10
16



6
3A5.198
2
VL_S56Q
49
1.49E+06
2.90E−04
1.95E−10
15.7



6
3A5.198
3
VL_S56Q
49.3
1.42E+06
2.38E−04
1.68E−10
15.9



6
3A5.198
Average
VL_S56Q
49.1
1.44E+06
2.57E−04
1.78E−10
15.8
92


6
3A5.278
1
VL_H95bY
48.3
1.55E+06
2.53E−04
1.63E−10
15.9



6
3A5.278
2
VL_H95bY
48.2
1.62E+06
2.43E−04
1.49E−10
15.7



6
3A5.278
3
VL_H95bY
48.5
1.57E+06
2.14E−04
1.36E−10
15.8



6
3A5.278
Average
VL_H95bY
48.3
1.58E+06
2.37E−04
1.49E−10
15.8
100


6
3A5.279
1
VL_H95bD
50
1.58E+06
2.50E−04
1.58E−10
16.3



6
3A5.279
2
VL_H95bD
49.7
1.61E+06
2.42E−04
1.50E−10
16.1



6
3A5.279
3
VL_H95bD
50
1.61E+06
2.43E−04
1.51E−10
16.4



6
3A5.279
Average
VL_H95bD
49.9
1.60E+06
2.45E−04
1.53E−10
16.2
97


6
3A5.294
1
VL_V97A
49.1
1.31E+06
2.54E−04
1.94E−10
15.9



6
3A5.294
2
VL_V97A
48.6
1.31E+06
2.13E−04
1.62E−10
15.7



6
3A5.294
3
VL_V97A
48.6
1.37E+06
2.15E−04
1.57E−10
15.6



6
3A5.294
Average
VL_V97A
48.7
1.33E+06
2.27E−04
1.71E−10
15.7
104


6
3A5.301
1
VL_V97H
48.4
1.51E+06
2.76E−04
1.83E−10
15.8



6
3A5.301
2
VL_V97H
48.1
1.58E+06
1.06E−04
6.71E−11
15.6



6
3A5.301
3
VL_V97H
47.8
1.60E+06
2.45E−04
1.53E−10
15.4



6
3A5.301
Average
VL_V97H
48.1
1.56E+06
2.09E−04
1.34E−10
15.6
114


6
3A5.063
1
VL_G25K
48
1.37E+06
2.61E−04
1.90E−10
15.7



6
3A5.063
2
VL_G25K
48.1
1.36E+06
2.70E−04
1.99E−10
15.5



6
3A5.063
3
VL_G25K
47.8
1.42E+06
2.50E−04
1.76E−10
15.3



6
3A5.063
Average
VL_G25K
47.9
1.38E+06
2.60E−04
1.88E−10
15.5
91


6
3A5.302
1
VL_V97W
48.9
1.65E+06
2.35E−04
1.42E−10
16



6
3A5.302
2
VL_V97W
48.9
1.69E+06
2.35E−04
1.39E−10
15.8



6
3A5.302
3
VL_V97W
48.5
1.66E+06
2.40E−04
1.45E−10
15.7



6
3A5.302
Average
VL_V97W
48.7
1.67E+06
2.37E−04
1.42E−10
15.8
100


6
3A5.202
1
VL_Q89A
48.9
1.09E+06
2.49E−04
2.29E−10
15.8



6
3A5.202
2
VL Q89A
48.7
1.07E+06
1.89E−04
1.77E−10
15.6



6
3A5.202
3
VL Q89A
48.8
1.09E+06
2.44E−04
2.25E−10
15.7



6
3A5.202
Average
VL_Q89A
48.8
1.08E+06
2.27E−04
2.10E−10
15.7
104


6
3A5.203
1
VL Q89S
49
1.28E+06
2.82E−04
2.20E−10
15.9



6
3A5.203
2
VL Q89S
48.6
1.28E+06
2.26E−04
1.76E−10
15.6



6
3A5.203
3
VL Q89S
48.9
1.27E+06
2.36E−04
1.86E−10
15.7



6
3A5.203
Average
VL_Q895
48.8
1.28E+06
2.48E−04
1.94E−10
15.7
96


6
3A5.204
1
VL Q89L
49.9
5.01E+05
2.20E−04
4.38E−10
14.4



6
3A5.204
2
VL Q89L
49.9
5.02E+05
1.88E−04
3.74E−10
14.2



6
3A5.204
3
VL Q89L
49.9
4.95E+05
2.11E−04
4.26E−10
14.3



6
3A5.204
Average
VL_Q89L
49.9
4.99E+05
2.06E−04
4.13E−10
14.3
115


6
3A5.205
1
VL Q89Y
48.4
9.87E+05
2.42E−04
2.45E−10
15.5



6
3A5.205
2
VL Q89Y
48.6
1.04E+06
2.37E−04
2.28E−10
15.6



6
3A5.205
3
VL Q89Y
48.5
1.05E+06
2.60E−04
2.47E−10
15.6



6
3A5.205
Average
VL_Q89Y
48.5
1.03E+06
2.46E−04
2.40E−10
15.5
96


6
3A5.208
1
VL_Q89H
51
9.41E+05
2.53E−04
2.68E−10
16



6
3A5.208
2
VL_Q89H
51.1
9.45E+05
2.52E−04
2.66E−10
16.1



6
3A5.208
3
VL_Q89H
51
9.54E+05
2.88E−04
3.02E−10
16



6
3A5.208
Average
VL_Q89H
51
9.47E+05
2.64E−04
2.79E−10
16
90


6
3A5.550
1
VH_D101Y
48.3
1.16E+06
4.40E−04
3.81E−10
15.5



6
3A5.550
2
VH_D101Y
48.5
1.17E+06
4.54E−04
3.87E−10
15.7



6
3A5.550
3
VH_D101Y
48.3
1.19E+06
4.86E−04
4.10E−10
15.5



6
3A5.550
Average
VH_D101Y
48.3
1.17E+06
4.60E−04
3.93E−10
15.5
51


6
3A5.512
1
VH_G96D
50.4
1.70E+07
8.63E−03
5.07E−10
13.7



6
3A5.512
2
VH_G96D
50.7
1.88E+07
9.87E−03
5.26E−10
13.8



6
3A5.512
3
VH_G96D
50.5
1.28E+06
1.52E−03
1.19E−09
12.5



6
3A5.512
Average
VH_G96D
50.5
1.24E+07
6.67E−03
7.41E−10
13.3
3.5


6
3A5.124
1
VL_N32K
50.9
9.94E+05
2.46E−04
2.47E−10
16.3



6
3A5.124
2
VL_N32K
51.2
1.05E+06
2.62E−04
2.49E−10
16.5



6
3A5.124
3
VL_N32K
50.7
1.02E+06
2.28E−04
2.24E−10
16.3



6
3A5.124
Average
VL_N32K
50.9
1.02E+06
2.45E−04
2.40E−10
16.3
97


7
3A5.040 (batch 7)
1
Wild type
51.2
1.37E+06
2.30E−04
1.68E−10
16.4



7
3A5.040 (batch 7)
2
Wild type
51.6
1.43E+06
2.15E−04
1.50E−10
16.6



7
3A5.040 (batch 7)
3
Wild type
51.6
1.42E+06
2.84E−04
2.00E−10
16.6



7
3A5.040 (batch 7)
Average
Wild type
51.4
1.41E+06
2.43E−04
1.73E−10
16.5
100


7
IgG4 Isotype
1
48.8
N/A
N/A
N/A
0




7
IgG4 Isotype
2
48.9
N/A
N/A
N/A
0




7
IgG4 Isotype
3
48.8
N/A
N/A
N/A
0




7
IgG4 Isotype
Average
49.8
N/A
N/A
N/A
0
N/A



7
3A5.040 (Purified)
1
Wild type
49.6
1.43E+06
2.39E−04
1.68E−10
16.1



7
3A5.040 (Purified)
2
Wild type
49.8
1.48E+06
2.85E−04
1.93E−10
16.2



7
3A5.040 (Purified)
3
Wild type
49.7
1.45E+06
2.32E−04
1.60E−10
16.1



7
3A5.040 (Purified)
Average
Wild type
49.7
1.45E+06
2.52E−04
1.74E−10
16.1
94


7
3A5.068
1
VL_N26L
52.3
1.29E+06
2.68E−04
2.07E−10
16.8



7
3A5.068
2
VL_N26L
53
1.39E+06
2.08E−04
1.50E−10
17.1



7
3A5.068
3
VL_N26L
53.1
1.43E+06
2.69E−04
1.88E−10
17.2



7
3A5.068
Average
VL_N26L
52.8
1.37E+06
2.48E−04
1.82E−10
17
98


7
3A5.079
1
VL_N27D
49.3
1.22E+06
2.29E−04
1.88E−10
16.1



7
3A5.079
2
VL_N27D
50
1.29E+06
2.39E−04
1.85E−10
16.4



7
3A5.079
3
VL_N27D
49.9
1.25E+06
2.63E−04
2.09E−10
16.2



7
3A5.079
Average
VL_N27D
49.7
1.25E+06
2.44E−04
1.94E−10
16.2
100





This table contains a summary of the calculated kinetic data for each purified antibody tested. Variant antibodies with a lower kd (slower off-rate) than parent 3A5.040 antibody have a “% 3A5.040 kd” value >100% and those with a higher kd (faster off-rate) a value <100%. Runs 6 and 7 were transfected separately but run in the same BIACORE ™ batch, and therefore use the same isotype and 3A5.040 (purified) controls. N/A: non-applicable.













TABLE 5







Purified protein expression yield for selected


3A5.040 antibody variants














Amino acid
% parent 3A5.040



Run #
Antibody ID
Substitution
purified yield







6
3A5.040
Parental sequence
100



6
3A5.237
VL_D92W
 89



6
3A5.161
VL_S52L
160



6
3A5.169
VL_D53S
173



6
3A5.183
VL_R54H
172



6
3A5.193
VL_P55W
187



6
3A5.198
VL_S56Q
205



6
3A5.278
VL_H95bY
210



6
3A5.279
VL_H95bD
143



6
3A5.294
VL_V97A
140



6
3A5.301
VL_V97H
151



6
3A5.063
VL_G25K
110



6
3A5.302
VL_V97W
156



6
3A5.202
VL_Q89A
 64



6
3A5.203
VL_Q89S
129



6
3A5.204
VL_Q89L
 61



6
3A5.205
VL_Q89Y
 41



6
3A5.208
VL_Q89H
 54



6
3A5.550
VH_D101Y
 88



6
3A5.512
VH_G96D
 72



6
3A5.124
VL_N32K
128



7
3A5.040
Parental Sequence
100



7
3A5.068
VL_N26L
 96



7
3A5.079
VL_N27D
 99

















TABLE 6





VH CDR1 Scanning Matrix

















VH CDR1















26
27
28
29

30
31









sc














G
G
S
I
S
N






















A
0.5 
1.3
1 
0.8
0.6 
0.9
0.3 
0.6
0.1 
1.1
1.2 
0.1


S
0.6 
0.9
1 
1


0.3 
0.5


1.1 
0.2


L
0.2 
1.1
1.2 
0.8
0.7 
1.1
0.8 
0.9
1.5 
0.7
0.7 
0.2


Y
0.6 
1.1
0.8 
0.1
0.5 
1.2
0.2 
0.1
1 
0.7
0.6 
0.1


D
0.6 
1.2
0.7 
0.9
0.7 
1.1
0.2 
0
1 
0.8
0.8
0.5


Q
0.9 
1.1
0.8 
0.7
0.8 
1
0.4 
0.3
1.2 
1.1
1.6 
0.2


K
1 
1
1 
0.5
0.8 
0.9
0.3 
0.1
1.6 
0.8
1.7 
0.1


H
0.9 
0.9
0.7 
0.4
0.7 
0.8
0.2 
0.1
0.8 
0.8
1.4 
0.8


W
1.1 
0.8
1 
0.2
0.4 
0.7
0.4 
0
0.7 
0.9
0.6 
0.2


P












VH CDR1














32
33
34
35
35A
35B









sc














G
G
Y
Y
W
S






















A
1.8 
0.3
1.2 
1.3
1.6 
0.7
2.9
0
0.4 
0.1
0.9 
1


S
1.5 
0.1
1.2
0
1.6 
0.7
1.3
0
0.2 
0.1




L
0.8 
0
0.8 
0.4
1.6 
0.6
1.9
0.1
1 
0.1
0.4 
0.3


Y
0.9 
0
1.1 
0




1 
0.5
0.9
0


D
1.1 
0
2.8 
0
1 
0.2
0.9
0
NE 
NE
0.2 
0.9


Q
1.3 
0.1
2.8 
0.1
1.7 
0.6
1.3
0
0.3 
0.1
0.5 
0.2


K
1.7 
0
3.5 
0
4.1 
0.2
3.8
NB
0.3 
0.1
0.1 
0


H
1.1 
0
2.7 
0
2.2 
0.1
2
0
0.5 
0.4
0.8 
0


W
0.7 
0
1.7 
0.1
0.5 
0.5
2.3
0


0.5 
0


P





Summary of difference in 3A5.040 variant antibody titre and kd for variants of VH CDR1 (AbM definition).


Kabat amino acid numbering and parent 3A5.040 amino acids are given in the top row.


Mutated residue identities are listed in the left column.


The leftmost value in each cell is the proportional titre of each variant vs parent 3A5.040 (>1 = higher titre, <1 = lower titre), while the right value is the proportional kd (>1 = lower kd, <1 = higher kd) of each variant vs parent 3A5.040.


Variants not made are blank.


NB, no binding.


NE, no expression. (VH CDR1 sequence disclosed as residues 26-37 of SEQ ID NO: 73).













TABLE 7







VH CDR2 Scanning Matrix









VH CDR2

















50
51
52
53
54
55
56
57
58


sc
Y
I
Y
Y
S
G
S
T
Y




























A
2.7
0.2
2.4
0.8
3
0
2.5
0.2
1.1
0.6
0.7
0.9
1
0.9
1
0.8
1
0


S
3.3
0.2
1.3
0.8
3.3
0.1
2.3
0.6


0.7
0.7


0.7
0.8
1
0


L
1.2
0.3
0.9
0.7
2.1
0
1
0.2
1.6
0
0.7
0.1
1
1.2
0.1
1.2
0.7
0


Y


1.1
0




0.9
0
0.5
0.1
0.9
0.5
0.5
1.1




D
1.7
0.1
0.5
0.1
3.3
NB
1.8
0.2
2
0
0.4
0.4
0.8
0.2
0.5
1.1
0.5
0


Q
2.4
0.2
1
0.2
2.6
0
2.1
0.2
1.5
0.1
0.4
0.5
1.1
1.1
0.6
1
0.8
0.1


K
1.4
0.2
1.2
0.1
3.5
NB
2.3
0.1
2
0
0.5
0.5
1.2
1.3
0.6
0.9
1
0.1


H
3
0.1
0.9
0
2.9
0.3
2.1
0.5
1
0.2
0.4
0.6
0.4
1
0.5
1
0.9
0.2


W
1.3
0.1
1.4
0
1
0.1
1.5
0.4
1.1
0.1
0.3
0.2
1
0.4
0.4
0.8
0.8
0.2


P























Summary of difference in 3A5.040 variant antibody titre and kd for variants of VH CDR2 (AbM definition). Kabat amino acid numbering and parent 3A5.040 amino acids are given in the top row. Mutated residue identities are listed in the left column. The leftmost value in each cell is the proportional titre of each variant vs parent 3A5.040 (>1 = higher titre, <1 = lower titre), while the right value is the proportional kd (>1 = lower kd, <1 = higher kd) of each variant vs parent 3A5.040. Variants not made are blank. NB, no binding. NE, no expression. (VH CDR2 sequence disclosed as residues 52-60 of SEQ ID NO: 73).













TABLE 8







VH CDR3 Scanning Matrix

















VH CDR3

















93
94
95
96
97
98
99
101
102


sc
A
S
L
G
N
W
F
D
Y




























A


1
1.2
0.5
0
0.8
0.8
1
0.6
1.1
0.5
0.4
0.2
0.7
0.7
1.1
1.2


S
0.6
1.1


NE 
NE
0.9
0.5
0.8
0.5
0.8
0.4
0.4
0.1
0.8
0.9
1.4
0.9


L
0.2
0.5
0.4
0.1


0.6
0
1
0.9
0.4
0.6
0.6
0.9
0.4
0.2
0.5
0.9


Y
0.1
NB
0.3
0.1
1
0
0.6
NB
0.8
0.3
1.4
0.9
0.4
1.1
0.9
0.5




D
NE
NE
NE
NE
0.2
NB
0.7
0
0.9
0.5
0.4
1.1
0.3
0


0.5
0.7


Q
0.4
1
0.5
0.2
0.2
0.2
0.6
0.2
1.3
1
0.5
0.6
0.4
0.1
0.8
0.5
0.9
1


K
0.2
NB
1.3
0.3
0.7
0
1.5
0
1.7
0.7
0.7
0.1
0.4 
NB
0.7
0.3
0.9
1.3


H
0.3
NB
0.7
0
0.9
0.1
1.2
0.1
1.1
0.5
0.8
0.7
0.4
1
0.7
0.6
1.3
1


W
0.2
NB
0.4
0.0
0.7
0
0.6
0
0.6
0.2
0.3
0.2


0.3
0.2
1.2
0.9


P
0.1
NB
0.2
0.8
0.4
NB
0.2
0.2
0.7
0.2
0.3
0.1
0.4 
NB
0.4
0.1
0.3
0.8





Summary of difference in 3A5.040 variant antibody titre and kd for variants of VH CDR3 (AbM definition). and Kabat amino acid positions 93 and 94. Kabat amino acid numbering and parent 3A5.040 amino acids are given in the top row. Mutated residue identities are listed in the left column. The leftmost value in each cell is the proportional titre of each variant vs parent 3A5.040 (>1 = higher titre, <1 = lower titre), while the right value is the proportional kd (>1 = lower kd, <1 = higher kd) of each variant vs parent 3A5.040. Variants not made are blank. NB, no binding. NE, no expression. (VH CDR3 sequence disclosed as residues 98-106 of SEQ ID NO: 73).













TABLE 9





VL CDR1 Scanning Matrix

















VL CDR1














24
25
26
27
28
29









sc














G
G
N
N
I
G






















A
1 
1.3
1.1 
1.3
0.8 
1.3
1.2
1.2
0.9 
1.9
0.6 
1.1


S
1
1.1
0.4 
1.1
1.6 
1.1
0.9
1.1
0.5 
1
0.9 
1.2


L
1 
1.4
0.8
1.1
1 
1
1
1.3
0.6 
1.1
0.8 
1.3


Y
0.9 
1
1 
1.4
1 
1.3
1
1.1
0.9 
1.4
0.6 
0.9


D
1 
1.2
0.9 
1
1 
1.5
1
1
0.4 
1.2
0.9 
1.6


Q
1 
1.2
0.9 
1.3
1 
1.1
0.9
1.1
0.5 
1.1
1.4 
1.1


K
1 
1.2
0.9 
1.7
1 
1
1
1.3
0.5 
1.1
0.7 
1.3


H
1 
1.3
1.1 
1.3
1 
1
0.9
1.5
0.8 
0.9
1 
1.4


W
1.1 
1.4
0.8 
1.3
0.9 
1.2
1.5
0.9
0.4 
0.9
1.1 
1.2


P












VL CDR1













30
31
32
33
34









sc













S
K
N
V
Y




















A
0.8 
1.1
1.2
0.6
0.7 
0.2
0.7 
1.5
1.7
0.3


S


1.1 
0.8
1 
1.1
1.3 
1.2
0.3 
1.2


L
1.3 
1.2
1.3 
0.3
1.2 
1.2
1 
0.8
0.6 
0.5


Y
0.9 
1.1
1 
1.3
0.9
1.2
0.7 
1.3




D
1.2 
1.3
1.1 
0.2
1.2 
0.9
0.4 
1.2
0.7 
0.2


Q
0.8 
1.3
0.9 
0.3
1
0.9
0.5 
0.8
0.6 
0.2


K
1 
1.6


1.3 
1
0.1 
1
0.6 
0.3


H
1.5 
1.2
1 
0.5
0.9
1.5
0.5 
NB
0.8 
0.2


W
1.1 
0.9
1.3 
0.9
0.8
1.3
0.2 
1.1
0.6 
0.1


P





Summary of difference in 3A5.040 variant antibody titre and kd for variants of VL CDRI (AbM definition).


Kabat amino acid numbering and parent 3A5.040 amino acids are given in the top row.


Mutated residue identities are listed in the left column. The leftmost value in each cell is the proportional titre of each variant vs parent 3A5.040 (>1 = higher titre, <1 = lower titre), while the right value is the proportional kd (>1 = lower kd, <1 = higher kd) of each variant vs parent 3A5.040.


Variants not made are blank.


NB, no binding.


NE, no expression. (VL CDR1 sequence disclosed as residues 23-33 of SEQ ID NO: 74).













TABLE 10







VL CDR2 Scanning Matrix









VL CDR2















50
51
52
53
54
55
56


sc
D
D
S
D
R
P
S
























A
0.8
0.6
0.9
0.8
0.8
0.9
0.5
0.1
1.4
1.2
0.1
0.9
1.4
1.1


S
0.9
1
0.7
0.7


1.5
1.9
1.3
1.1
1.1
1




L
0.9
0.4
0.8
0.6
1.1
1.8
1.4
1.3
1.4
1.2
1.5
1.2
1.3
1.1


Y
0.8
0.5
0.8
0.6
1.1
1.1
2.1
1.1
1.4
1.0
1.3
1
1.2
1


D




1.8
1.2


1.3
1.2
1.6
1.3
1.5
1


Q
0.7
0.5
0.9
0.9
0.1
1
1
1
1.5
1.2
1.5
1.1
1.3
1.3


K
0.7
0.6
0.7
0.9
1.7
0.1
1.8
1.1
1.7
1.0
1.4
1.1
1
0.9


H
1
0.6
0.9
0.6
1.7
1.1
1.2
0.9
1.4
1.4
1.8
1
1.4
1


W
0.7
0.4
0.6
0.5
1.7
1.1
1.8
1.2
0.1
1.0
1.6
1.4
0.9
0.9


P



















Summary of difference in 3A5.040 variant antibody titre and kd for variants of VL CDR2 (AbM definition). Kabat amino acid numbering and parent 3A5.040 amino acids are given in the top row. Mutated residue identities are listed in the left column. The leftmost value in each cell is the proportional titre of each variant vs parent 3A5.040 (>1 = higher titre, <1 = lower titre), while the right value is the proportional kd (>1 = lower kd, <1 = higher kd) of each variant vs parent 3A5.040. Variants not made are blank. NB, no binding. NE, no expression. (VL CDR2 sequence disclosed as residues 49-55 of SEQ ID NO: 74).













TABLE 11





VL CDR3 Scanning Matrix

















VL CDR3














89
90
91
92
93
94









sc














Q
V
W
D
S
S






















A
0.6
1
0.8
1
0.8
0
1.1
1.2
1.1
0
1
1


S
1.3
1
0
0.4
1
0
1.1
1.2






L
0.6
1.2
0.6
0.8
1
0
1.3
1.5
1.2
0
0.9
0.2


Y
0.4
1
0.9
0.4
0.6
0.1
0.7
1.1
1.2
0
1.5
0.3


D
0.1
1.2
0.3
0.3
0.7
0


0.7
0
0.1
0.2


Q


0.8
0.6
0.8
0
1.9
0.8
1.1
0
1.4
0.6


K
1.4
0.9
0.5
1.4
0.7
NB
0.9
1.3
1.6
NB
1.1
0.8


H
0.5
0.9
0.6
0.4
0.6
0
1.2
1.1
1.3
0
1
0.8


W
0.7
0.9
0.8
0.2


NE
NE
1.2
0
0.2
0.4


P
0.7
0.2
0.8
0
0.4
0
0.6
0.8
1.1
0
1.4
0.9












VL CDR3













95
95A
95B
96
97









sc













S
D
H
V
V





















1
1
1.1
0
0.2
0.7
0.7 
0.6
1.3
1.5





1
0.1
0.6
1.6
0.2 
0.5
1.1 
1.1



1
1
0.7
0
1.8
0.9
0.8 
0.6
1 
1.2



0.8
1.1
1
0
1.4
1.5
0.6 
0.1
0.9
1.1



1.1
0.9


2.1
1.5
0.1 
0.1
1 
0.9



0.6
1.3
1.1
0
1.3
0.8
0.3 
0.2
0.7 
1.2



1.2
1.2
0.7
0
0.6
1.2
0.2 
0
0
1



1
1.2
1.9
0


0.5 
NB
1.7 
1.4



0.9
0.9
0.9
0
1.5
1
0.2
0
0.7
1.6



1.5
0
1.5
0
1.5
0
1.1 
0.2
0.2
1





Summary of difference in 3A5.040 variant antibody titre and kd for variants of VL CDR3 (AbM definition).


Kabat amino acid numbering and parent 3A5.040 amino acids are given in the top row.


Mutated residue identities are listed in the left column.


The leftmost value in each cell is the proportional titre of each variant vs parent 3A5.040 (>1 = higher titre, <1 = lower titre), while the right value is the proportional kd (>1 = lower kd, <1 = higher kd) of each variant vs parent 3A5.040.


Variants not made are blank.


NB, no binding.


NE, no expression. (VL CDR3 sequence disclosed as residues 88-98 of SEQ ID NO: 74).






Expression Vector Construction

Nucleotide sequence can influence gene expression and subsequent protein expression level. Several different nucleotide sequences were examined for optimal expression of 3A5.046. These sequences are summarized in the following table:







TABLE 12







Nucleotide sequence










Gene Name
SEQ ID Nos:







3A5.046 VH
SEQ ID NO: 69



3A5.046 VL
SEQ ID NO: 71



3A5.046 HC constant region
SEQ ID NO: 70



3A5.046 LC constant region
SEQ ID NO: 72










Measurement of the Affinity of Antibody 3A5.046 for Human Recombinant IL-5 by SPR

The affinity of antibody 3A5.046 for recombinant human IL-5 was determined using a BIACORE™ T200 system (GE HEALTHCARE®). Briefly, a commercial mouse anti-human IgG antibody (THERMO SCIENTIFIC®) was coupled to two adjacent (control and test) flowcells on a BIACORE™ CM5 chip (GE HEALTHCARE®) according to the manufacturer's recommendations. The 3A5.046 antibody was captured at a low density (approximately 125 RU) on the test flowcell surface and dilutions of recombinant human IL-5 or a buffer blank were injected over both test and control flowcells, at 30 μL/min (FIG. 6, Table 13).
The resulting sensorgrams were double-referenced (test flowcell values subtracted from control (anti-human IgG-coupled surface with no coated antibody) flowcell values and also a buffer blank). Subtracted sensorgrams from injections of human IL-5 at 2.5, 1.25, 0.625, 0.313, and 0.156 μg/mL were fitted to a 1:1 Langmuir binding model using BIACORE™ Evaluation software to determine binding constants from duplicate assays (Table 13).







TABLE 13







Kinetic constants from a multi-concentration BIACORE ™ kinetic


analysis of 3A5.046 antibody binding to recombinant


human IL-5 at 2.5, 1.25, 0.625, 0.313 and 0.156 μg/mL












Antibody
Experiment
ka (1/Ms)
kd (1/s)
KD (pM)
Chi2 (RU2)





3A5.046
Repeat 1
8.16E+05
1.58E−05
19.4
0.0269



Repeat 2
8.49E+05
1.84E−05
21.7
0.0296









These results demonstrate that antibody 3A5.046 has a high affinity for recombinant human IL-5.
Potency of 3A5.046 Compared to Mepolizumab in the TF-1.6G4 Cell Line Proliferation Assay
3A5.046 and mepolizumab were run in the TF-1.6G4 cell line proliferation assay. 3A5.046 and mepolizumab inhibited human IL-5 proliferation with a mean IC50 of 13.8 pM and 534 pM respectively (See Table 14 and FIG. 7A). Both antibodies demonstrated inhibition of cynomolgus monkey IL-5 on the TF1.6G4 cell line (FIG. 7B), with a mean IC50 for 3A5.046 of 43.8 pM and for mepolizumab of 584 pM.











TABLE 14







Number of
Mean
Std.


Antibody
Agonist
Observations
(pM)
Dev.







Mepolizumab
Cynomolgus
4
584.0982
129.9019



IL-5






Human IL-5
4
534.2803
150.2546


3A5.046
Cynomolgus
8
 43.80916
 12.62095



IL-5






Human IL-5
8
 13.83667
  4.219783









Detection of Native IL5 from Primary Cells Via ELISA Using Anti-IL-5 Antibodies of the Invention

Antibody 3A5 was used as a capture reagent in a sandwich ELISA. Paired with a commercially available detection antibody (TRFk5R; R&D SYSTEMS®, #MAB405) quantification of recombinant IL-5 levels in spiked buffer samples could be determined (FIG. 8). This method was then applied to the detection of native IL-5 secreted by primary cells. CD4+ T-cells from four donors were stimulated to secrete IL-5. The IL-5 levels were then determined using the sandwich ELISA with antibody 3A5 as the capture reagent. Three out of four donor T-cells responded to T-cell stimulation and had detectable IL-5 levels in the cell culture supernatant (FIG. 8). Similar studies were performed on human IL-5 secreted from primary human donor CD4+ T-cells using antibody 3A5.046 as a capture agent, and the results obtained were consistent with the results obtained with antibody 3A5 (data not shown).
Anti-IL-5 Antibody Inhibition of Eosinophil Differentiation
A survival assay using CD34+ cord blood cells was used to assess the capacity of anti-IL-5 antibodies to inhibit the IL-5 dependent differentiation of bone marrow progenitors to eosinophils and to assess the capacity of anti-IL-5 antibodies to inhibit phenotypically mature eosinophil survival. In IL-5 treated control cells, phenotypically mature eosinophils were the predominant population at day 28 (FIG. 9, shaded region in graph). Antibody 3A5.046 was able to inhibit the differentiation of CD34+ cord blood cells, incubated with IL-5 for 28-days, into eosinophils. Antibody 3A5.046 was a more potent inhibitor of IL-5 mediated eosinophil differentiation than mepolizumab (FIG. 9).
X-Ray Crystallography Analysis
The molecular structure of the complex between antibody 3A5.046 Fab and recombinant human IL-5 is investigated by X-ray crystallography. To prepare for X-ray crystallography experiments, 3A5.046 is digested by papain, which cleaves the intact antibody in the hinge region, separating the Fc and Fab domains. The 3A5.046 Fab is purified by standard Protein A affinity chromatography and complexed with recombinant non-glycosylated human IL-5. The 3A5.046 Fab:human IL-5 complex is purified for crystallization experiments using standard size-exclusion chromatography. The complex is set up in variable concentrations, into commercially available sparse-matrix crystallization screens for broad sampling of crystallization conditions. Optimisations of any ‘hit’ conditions that provide crystals of the complex from the sparse-matrix screens are performed to improve upon crystal morphology and crystal diffraction as required.
Crystals of the 3A5.046 Fab:human IL-5 complex are harvested, flash-frozen in liquid nitrogen and transported to a synchrotron facility for diffraction tests and data collection. Crystal diffraction testing is performed at a synchrotron, which houses high energy X-ray micro beam lines for producing high-resolution diffraction. In this way, X-ray diffraction data on crystals of 3A5.046 Fab:human IL-5 complex is collected. An initial structure model of the complex may be obtained using standard molecular replacement techniques with published molecular structures of IL-5 and Fab as templates. The final structure of the 3A5.046 Fab and human IL-5 complex may be obtained by iterative refinement of the structure model, until the errors of fitting are better than 30% (final Rfree of 0.3) and chemical bonding parameters (e.g. the distribution of bond lengths and angles, stereochemistry and hydrogen bonds) are within acceptable limits.
Characterisation of Antibody 3A5.046 Binding to IL-5 from Non-Human Model Species
Antibody 3A5.046 was tested by BIACORE™ assay using a T200 system (GE HEALTHCARE®) for binding to recombinant cytokines human IL-5, rat IL-5, mouse IL-5, and guinea pig IL-5. This panel of non-human IL-5s was chosen to represent major animal model species used in preclinical development. An isotype control antibody (IgG4) and antibody 3A5.046 were immobilized on flowcells 1 and 2 of a Series S CM5 chip (GE HEALTHCARE®), respectively, using standard amine coupling methods. Cytokines were diluted to 1 and 10 μg/ml in HBS-EP+ running buffer (GE HEALTHCARE®) and injected across flowcells 1 and 2. Buffer-only injections across flowcells 1 and 2 were included for double-referencing. Data analysis was performed using BIACORE™ T200 evaluation software. The resulting sensorgrams were double-referenced (test flowcell values subtracted from control flowcell values and also a buffer blank).
Antibody 3A5.046 was found to bind to human IL-5 and cynomolgus monkey IL-5, but not to mouse IL-5, rat IL-5, or guinea pig IL-5 (FIG. 10A-FIG. 10E). This indicated that 3A5.046 could be used in cynomolgus macaque preclinical models.
Antibody 3A5.046 does not Bind to Other Human Cytokines Closely Related to IL-5
Antibody 3A5.046 was tested by BIACORE™ assay using a T200 system (GE HEALTHCARE®) for binding to recombinant human cytokines IL-2, IL-3, IL-4, IL-5, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and growth hormone (GH). This representative panel of closely-related human cytokines was chosen based on their structural and functional similarity to human IL-5. An isotype control antibody (IgG4) and antibody 3A5.046 were immobilized on flowcells 1 and 2 of a Series S CM5 chip (GE HEALTHCARE®), respectively, using standard amine coupling methods. Cytokines were diluted to 1 and 10 μg/ml in HBS-EP+ running buffer (GE HEALTHCARE®) and injected across flowcells 1 and 2. Buffer-only injections across flowcells 1 and 2 were included for double-referencing. Data analysis was performed using BIACORE™ T200 evaluation software. The resulting sensorgrams were double-referenced (test flowcell values subtracted from control flowcell values and also a buffer blank).
Antibody 3A5.046 was found to bind only to human IL-5 and not human cytokines IL-2, IL-3, IL-4, GM-CSF, M-CSF, or GH (FIG. 11A-FIG. 11D), thus demonstrating high specificity of 3A5.046 for human IL-5.
Evaluation of Antibody 3A5.046 in an Ascaris suum-Induced Cynomolgus Monkey Airways Eosinophilia Model
Antibody 3A5.046 was tested using the Ascaris suum cynomolgus model of acute airways eosinophilia as described in Hart, T. K., et al., Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys. J Allergy Clin Immunol, 2001. 108(2): p. 250-7.
Sixteen female cynomolgus macaques that had been previously sensitized to Ascaris suum were assessed for level of retained sensitivity by intradermal challenge with A. suum. Fourteen of these animals were selected for participation in the study based on the skin wheal response to challenge and randomized into two groups of n=7 per group. Two weeks after intradermal skin wheal testing, baseline blood and bronchoalveolar lavage fluid (BALF) samples were collected (Day 8). About 24 hours after baseline sample collection, animals were treated with either placebo (comprising aqueous buffer pH 6.3 with 200 mM arginine and 25 mM histidine) or antibody 3A5.046 (10 mg/kg intravenously, in aqueous buffer pH 6.3 with 200 mM arginine and 25 mM histidine) in a blinded manner (Day 7). One week after treatment with placebo or antibody a second intradermal A. suum challenge was performed and an A. suum inhalation challenge was given to all animals by delivery of an equal amount of A. suum extract (Greer Laboratories Inc.; 5 mg/ml in sterile water) administered via PARI® LC nebulizer (Day 0). Skin wheal diameter was measured 15 minutes after challenge. Two days afterA. suum inhalation challenge, blood and BALF samples were collected. Additional blood samples for haematology endpoints and PK analysis were collected weekly starting on Day 10 until Day 45.
There was no significant difference in the skin wheal responses between the first and second intradermal A. suum challenges. All animals showed an increase in eosinophils in the BALF as a result of A. suum challenge but in animals treated with antibody 3A5.046, this increase was negligible. There were significantly fewer eosinophils in the BALF of animals treated with antibody 3A5.046, compared to placebo-treated animals 48 hours after A. suum challenge (p<0.01; Mann-Whitney test; FIG. 12).
A. suum challenge also led to significantly increased blood eosinophils over baseline in placebo-treated animals to day 10 post-challenge (FIG. 13A). Treatment with antibody 3A5.046 led to a significant decrease in blood eosinophils to levels below baseline (FIG. 13A), and this depression in blood eosinophil numbers was maintained over a period of at least 52 days after initial dose (FIG. 13B).
In summary, treatment with 3A5.046 significantly reduced A. suum-induced airways eosinophilia 48 hours after challenge, and significantly decreased blood eosinophils, to below baseline levels, for at least 52 days following a single intravenous (IV) 10 mg/kg dose.
Investigation of Antibody Function in a Humanized IL-5 Rat Model of Airways Eosinophilia
Alternaria alternata (Alternaria) is a fungal allergen that is known to trigger asthma in humans and can cause eosinophilia and asthma-like symptoms in rodents. When challenged with Alternaria, humanized IL-5 knock-in rats (animals in which the rat IL-5 has been replaced by human IL-5) show elevated eosinophil levels in bronchoalveolar lavage fluid (BALF) from lungs (FIG. 14).
A single intratracheal instillation of Alternaria suspension is administered to humanized IL-5 rats, and blood and BALF samples are collected 2, 3 or 4 days after Alternaria administration. Typically in this model eosinophils are significantly increased in BALF samples and may be either increased or unchanged in blood samples. Test antibodies are administered prior to induction of eosinophilia with Altemaria and assessed for their ability to reduce eosinophil numbers in BALF and blood.
Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.
The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in its entirety.







TABLE 15







Sequences










Protein chain
Sequence
Protein Chain
Sequence





Consensus LCDR1
GX1X2X3X4X5X6KX7X8Y





SEQ ID NO: 1




 


Consensus LCDR2
DDX8X9RPS





SEQ ID NO: 2




 


Consensus LCDR3
QVWX10SSSDX11VX12





SEQ ID NO: 3




 


Antibody 3A5





3A5 HCDR1
GGSISNGGYYWS
3A5 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5 HCDR2
YIYYSGSTY
3A5 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5 HCDR3
LGNWFDY
3A5 LCDR3
QVWYSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 9


 








3A5 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCASL



GNWFDYWGQGTLVTVSS



SEQ ID NO: 10


 


3A5 VL
SSILTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHDD



SDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWYSSSDHVVFGG



GTKLTVLG



SEQ ID NO: 11


 


3A5 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCASL



GNWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY



FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC



NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT



LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST



YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV



YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL



DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG



SEQ ID NO: 12


 


3A5 LC
SSILTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHDD



SDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWYSSSDHVVFGG



GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK



ADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG



STVEKTVAPTECS



SEQ ID NO: 13


 










Antibody 3A5.001





3A5.001 HCDR1
GGSISNGGYYWS
3A5.001 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.001 HCDR2
YIYYSGSTY
3A5.001 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.001 HCDR3
LGNWFDY
3A5.001 LCDR3
QVWYSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 9


 








3A5.001 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCASL



GNWFDYWGQGTLVTVSS



SEQ ID NO: 10


 


3A5.001 VL
SSILTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHDD



SDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWYSSSDHVVFGG



GTKLTVLG



SEQ ID NO: 11


 


3A5.001 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVSISVDTSKNQFSLKLNSVTAADTAVYYCASL



GNWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF



PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 14


 


3A5.001 LC
SSILTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHDD



SDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWYSSSDHVVFGG



GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK



ADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG



STVEKTVAPTECS



SEQ ID NO: 13


 










Antibody 3A5.040





3A5.040 HCDR1
GGSISNGGYYWS
3A5.040 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.040 HCDR2
YIYYSGSTY
3A5.040 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.040 HCDR3
LGNWFDY
3A5.040 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.040 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.040 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD



DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 17


 


3A5.040 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.040 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD



DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 19


 










Antibody 3A5.046





3A5.046 HCDR1
GGSISNGGYYWS
3A5.046 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.046 HCDR2
YIYYSGSTY
3A5.046 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.046 HCDR3
LGNWFDY
3A5.046 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.046 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.046 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD



DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 17


 


3A5.046 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLYITR



EPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 20


 


3A5.046 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD



DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 19


 










Antibody 3A5.063





3A5.063 HCDR1
GGSISNGGYYWS
3A5.063 LCDR1
GKNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 21


 


3A5.063 HCDR2
YIYYSGSTY
3A5.063 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.063 HCDR3
LGNWFDY
3A5.063 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.063 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.063 VL
SYVLTQPPSVSVAPGQTARITCGKNNIGSKNVYWYQQKPGQAPVLVVHD


G25K
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 22


 


3A5.063 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.063 LC
SYVLTQPPSVSVAPGQTARITCGKNNIGSKNVYWYQQKPGQAPVLVVHD


G25K
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 23


 










Antibody 3A5.070





3A5.070 HCDR1
GGSISNGGYYWS
3A5.070 LCDR1
GGDNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 24


 


3A5.070 HCDR2
YIYYSGSTY
3A5.070 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.070 HCDR3
LGNWFDY
3A5.070 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.070 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.070 VL
SYVLTQPPSVSVAPGQTARITCGGDNIGSKNVYWYQQKPGQAPVLVVHD


N26D
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 25


 


3A5.070 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.070 LC
SYVLTQPPSVSVAPGQTARITCGGDNIGSKNVYWYQQKPGQAPVLVVHD


N26D
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 26


 










Antibody 3A5.082





3A5.082 HCDR1
GGSISNGGYYWS
3A5.082 LCDR1
GGNHIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 27


 


3A5.082 HCDR2
YIYYSGSTY
3A5.082 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.082 HCDR3
LGNWFDY
3A5.082 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.082 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.082 VL
SYVLTQPPSVSVAPGQTARITCGGNHIGSKNVYWYQQKPGQAPVLVVHD


N27H
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 28


 


3A5.082 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.082 LC
SYVLTQPPSVSVAPGQTARITCGGNHIGSKNVYWYQQKPGQAPVLVVHD


N27H
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 29


 










Antibody 3A5.084





3A5.084 HCDR1
GGSISNGGYYWS
3A5.084 LCDR1
GGNNAGSKNVY



SEQ ID NO: 4

SEQ ID NO: 30


 


3A5.084 HCDR2
YIYYSGSTY
3A5.084 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.084 HCDR3
LGNWFDY
3A5.084 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.084 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.084 VL
SYVLTQPPSVSVAPGQTARITCGGNNAGSKNVYWYQQKPGQAPVLVVH


I28A
DDSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVF



GGGTKLTVLG



SEQ ID NO: 31


 


3A5.084 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.084 LC
SYVLTQPPSVSVAPGQTARITCGGNNAGSKNVYWYQQKPGQAPVLVVH


I28A
DDSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVF



GGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA



WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT



HEGSTVEKTVAPTECS



SEQ ID NO: 32


 










Antibody 3A5.107





3A5.107 HCDR1
GGSISNGGYYWS
3A5.107 LCDR1
GGNNIGKKNVY



SEQ ID NO: 4

SEQ ID NO: 33


 


3A5.107 HCDR2
YIYYSGSTY
3A5.107 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.107 HCDR3
LGNWFDY
3A5.107 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.107 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.107 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGKKNVYWYQQKPGQAPVLVVHD


S30K
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 34


 


3A5.107 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.107 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGKKNVYWYQQKPGQAPVLVVHD


S30K
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 35


 










Antibody 3A5.125





3A5.125 HCDR1
GGSISNGGYYWS
3A5.125 LCDR1
GGNNIGSKHVY



SEQ ID NO: 4

SEQ ID NO: 36


 


3A5.125 HCDR2
YIYYSGSTY
3A5.125 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.125 HCDR3
LGNWFDY
3A5.125 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.125 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.125 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKHVYWYQQKPGQAPVLVVHD


N32H
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 37


 


3A5.125 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.125 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKHVYWYQQKPGQAPVLVVHD


N32H
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 38


 










Antibody 3A5.127





3A5.127 HCDR1
GGSISNGGYYWS
3A5.127 LCDR1
GGNNIGSKNAY



SEQ ID NO: 4

SEQ ID NO: 39


 


3A5.127 HCDR2
YIYYSGSTY
3A5.127 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.127 HCDR3
LGNWFDY
3A5.127 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.127 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.127 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNAYWYQQKPGQAPVLVVHD


V33A
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 40


 


3A5.127 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.127 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNAYWYQQKPGQAPVLVVHD


V33A
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 41


 










Antibody 3A5.161





3A5.161 HCDR1
GGSISNGGYYWS
3A5.161 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.161 HCDR2
YIYYSGSTY
3A5.161 LCDR2
DDLDRPS



SEQ ID NO: 6

SEQ ID NO: 42


 


3A5.161 HCDR3
LGNWFDY
3A5.161 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.161 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.161 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


S52L
DLDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVF



GGGTKLTVLG



SEQ ID NO: 43


 


3A5.161 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.161 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


S52L
DLDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVF



GGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA



WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT



HEGSTVEKTVAPTECS



SEQ ID NO: 44


 










Antibody 3A5.169





3A5.169 HCDR1
GGSISNGGYYWS
3A5.169 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.169 HCDR2
YIYYSGSTY
3A5.169 LCDR2
DDSSRPS



SEQ ID NO: 6

SEQ ID NO: 45


 


3A5.169 HCDR3
LGNWFDY
3A5.169 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.169 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.169 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


D53S
DSSRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 46


 


3A5.169 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.169 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


D53S
DSSRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 47


 










Antibody 3A5.232





3A5.232 HCDR1
GGSISNGGYYWS
3A5.232 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.232 HCDR2
YIYYSGSTY
3A5.232 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.232 HCDR3
LGNWFDY
3A5.232 LCDR3
QVWLSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 48


 








3A5.232 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.232 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


D92L
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWLSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 49


 


3A5.232 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.232 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


D92L
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWLSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 50


 










Antibody 3A5.276





3A5.276 HCDR1
GGSISNGGYYWS
3A5.276 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.276 HCDR2
YIYYSGSTY
3A5.276 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.276 HCDR3
LGNWFDY
3A5.276 LCDR3
QVWDSSSDSVV



SEQ ID NO: 8

SEQ ID NO: 51


 








3A5.276 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.276 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


H95bS
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDSVVFG



GGTKLTVLG



SEQ ID NO: 52


 


3A5.276 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.276 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


H95bS
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDSVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 53


 










Antibody 3A5.278





3A5.278 HCDR1
GGSISNGGYYWS
3A5.278 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.278 HCDR2
YIYYSGSTY
3A5.278 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.278 HCDR3
LGNWFDY
3A5.278 LCDR3
QVWDSSSDYVV



SEQ ID NO: 8

SEQ ID NO: 54


 








3A5.278 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.278 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


H95bY
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDYVVFG



GGTKLTVLG



SEQ ID NO: 55


 


3A5.278 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.278 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


H95bY
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDYVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 56


 










Antibody 3A5.279





3A5.279 HCDR1
GGSISNGGYYWS
3A5.279 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.279 HCDR2
YIYYSGSTY
3A5.279 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.279 HCDR3
LGNWFDY
3A5.279 LCDR3
QVWDSSSDDVV



SEQ ID NO: 8

SEQ ID NO: 57


 








3A5.279 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.279 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


H95bD
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDDVVFG



GGTKLTVLG



SEQ ID NO: 58


 


3A5.279 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.279 LC
SYVLTQPP SVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


H95bD
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDDVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 59


 










Antibody 3A5.294





3A5.294 HCDR1
GGSISNGGYYWS
3A5.294 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.294 HCDR2
YIYYSGSTY
3A5.294 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.294 HCDR3
LGNWFDY
3A5.294 LCDR3
QVWDSSSDHVA



SEQ ID NO: 8

SEQ ID NO: 60


 








3A5.294 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.294 VL
SYVLTQPP SVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


V97A
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVAFG



GGTKLTVLG



SEQ ID NO: 61


 


3A5.294 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.294 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


V97A
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVAFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 62


 










Antibody 3A5.302





3A5.302 HCDR1
GGSISNGGYYWS
3A5.302 LCDR1
GGNNIGSKNVY



SEQ ID NO: 4

SEQ ID NO: 5


 


3A5.302 HCDR2
YIYYSGSTY
3A5.302 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.302 HCDR3
LGNWFDY
3A5.302 LCDR3
QVWDSSSDHVW



SEQ ID NO: 8

SEQ ID NO: 63


 








3A5.302 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.302 VL
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


V97W
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVWF



GGGTKLTVLG



SEQ ID NO: 64


 


3A5.302 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.302 LC
SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVYWYQQKPGQAPVLVVHD


V97W
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVWF



GGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA



WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT



HEGSTVEKTVAPTECS



SEQ ID NO: 65


 










3A5.097





3A5.097 HCDR1
GGSISNGGYYWS
3A5.097 LCDR1
GGNNIDSKNVY



SEQ ID NO: 4

SEQ ID NO: 66


 


3A5.097 HCDR2
YIYYSGSTY
3A5.097 LCDR2
DDSDRPS



SEQ ID NO: 6

SEQ ID NO: 7


 


3A5.097 HCDR3
LGNWFDY
3A5.097 LCDR3
QVWDSSSDHVV



SEQ ID NO: 8

SEQ ID NO: 15


 








3A5.097 VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSS



SEQ ID NO: 16


 


3A5.097 VL
SYVLTQPPSVSVAPGQTARITCGGNNIDSKNVYWYQQKPGQAPVLVVHD


G29D
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLG



SEQ ID NO: 67


 


3A5.097 HC
QVQLQESGPGLVKPSQTLSLTCTVSGGSISNGGYYWSWIRQHPGKGLEWI



GYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASLG



NWFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP



EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN



VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV



SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP



SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS



FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG



SEQ ID NO: 18


 


3A5.097 LC
SYVLTQPPSVSVAPGQTARITCGGNNIDSKNVYWYQQKPGQAPVLVVHD


G29D
DSDRPSGIPERFSGSNSGNTATLTISRVEVGDEADYSCQVWDSSSDHVVFG



GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW



KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE



GSTVEKTVAPTECS



SEQ ID NO: 68


 


3A5.046 VH
CAGGTGCAGCTGCAGGAATCTGGCCCTGGCCTGGTCAAGCCCAGCCA


nucleotide 
GACCCTGAGCCTGACCTGTACCGTGTCCGGCGGCAGCATCAGCAACGG


sequence
CGGCTACTACTGGTCCTGGATCAGACAGCACCCCGGCAAGGGCCTGG


(synthetic)
AATGGATCGGCTACATCTACTACAGCGGCAGCACCTACTACAACCCCA



GCCTGAAGTCCAGAGTGACCATCAGCGTGGACACCAGCAAGAACCAG



TTCAGCCTGAAGCTGAGCAGCGTGACAGCCGCCGACACCGCCGTGTAC



TACTGCGCCAGCCTGGGCAATTGGTTCGACTACTGGGGCCAGGGCACC



CTCGTGACAGTGTCCTCA



SEQ ID NO: 69


 


3A5.046 human HC
GCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCTTGTAGCAGA


constant
AGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTGAAAGACTA


region
CTTCCCCGAGCCCGTCACCGTGTCCTGGAACAGCGGAGCCCTGACCAG


nucleotide
CGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACAG


sequence
CCTGAGCAGCGTGGTGACAGTGCCCTCCAGCAGCCTGGGCACCAAGA


(synthetic)
CCTACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGAC



AAGCGGGTGGAATCTAAGTACGGCCCACCCTGCCCCCCCTGCCCTGCC



CCTGAATTTCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCAAAGCCCA



AGGACACCCTGTATATCACTCGGGAGCCCGAAGTGACCTGCGTGGTGG



TGGACGTGTCCCAGGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGG



ACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACA



GTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCA



GGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGG



GCCTGCCCAGCTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAG



CCCCGCGAGCCTCAGGTGTACACACTGCCCCCCAGCCAGGAAGAGAT



GACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCC



CAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACA



ACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCC



TGTACTCCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAAGGCAAC



GTCTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACC



CAGAAGTCCCTGAGCCTGAGCCTGGGC



SEQ ID NO: 70


 


3A5.046 VL
AGCTACGTGCTGACCCAGCCTCCTAGCGTGTCCGTGGCCCCTGGCCAG


nucleotide
ACCGCCAGAATCACCTGTGGCGGCAACAACATCGGCAGCAAGAACGT


sequence
GTACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCGTGCTGGTGGTGCA


(synthetic)
CGACGACAGCGACAGACCCAGCGGCATCCCCGAGCGGTTCAGCGGCA



GCAACAGCGGCAATACCGCCACCCTGACCATCAGCCGGGTGGAAGTG



GGCGACGAGGCCGACTACAGCTGCCAGGTCTGGGACAGCAGCAGCGA



CCACGTGGTGTTCGGCGGAGGCACCAAGCTGACCGTCCTAGGT



SEQ ID NO: 71


 


3A5.046 human LC
CAGCCCAAGGCCGCTCCCAGCGTGACCCTGTTCCCCCCAAGCAGCGAG


constant
GAACTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTC


region
TACCCTGGGGCCGTGACCGTGGCCTGGAAGGCCGATAGCAGCCCTGTG


nucleotide
AAGGCCGGCGTGGAAACCACCACCCCCTCCAAGCAGAGCAACAACAA


sequence
ATACGCCGCCAGCAGCTACCTGTCCCTGACCCCCGAGCAGTGGAAGTC


(synthetic)
CCACCGGTCCTACAGCTGCCAGGTGACACACGAGGGCAGCACCGTGG



AAAAGACCGTGGCCCCCACCGAGTGCAGC



SEQ ID NO: 72









EMBODIMENTS
The following list of embodiments is intended to complement, rather than displace or supersede, the previous descriptions.

    
    
        Embodiment 1. A human antibody molecule that immunospecifically binds to human IL-5 with an equilibrium affinity constant (KD) of at least about 40 pM as determined by surface plasmon resonance.
        Embodiment 2. A human antibody molecule that immunospecifically binds to human IL-5, the antibody molecule comprising:
        
            a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR1 comprising the amino acid sequence of SEQ ID NOs: 5, 21, 24, 27, 30, 33, 36, 39, or 66, a light chain CDR2 comprising the amino acid sequence of SEQ ID NOs: 7, 42, or 45, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NOs: 15, 48, 51, 54, 57, 60, or 63.
        
        
        Embodiment 3. The antibody molecule of embodiment 1 or 2, wherein the antibody molecule comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and
        
            a. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            b. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            c. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            d. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            e. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            f. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            g. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            h. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            i. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 66, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            j. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            k. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
            l. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 48;
            m. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 51;
            n. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 54;
            o. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 57;
            p. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 60; or
            q. a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 63;
            
                wherein the position of the amino acid residues of the CDR is determined according to AbM.
            
            
        
        
        Embodiment 4. The antibody molecule of embodiment 3, wherein the antibody molecule comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15.
        Embodiment 5. The antibody molecule of any one of the previous embodiments, wherein the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16 and
        
            a. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17;
            b. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22;
            c. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25;
            d. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28;
            e. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31;
            f. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34;
            g. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37;
            h. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40;
            i. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43;
            j. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46;
            k. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49;
            l. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52;
            m. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55;
            n. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 58;
            o. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 61;
            p. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 64; or
            q. a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67.
        
        
        Embodiment 6. The antibody molecule of any one of the previous embodiments, wherein the antibody molecule comprises a heavy chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.
        Embodiment 7. The antibody molecule of any one of the previous embodiments, wherein the antibody molecule comprises:
        
            a. a S228P mutation;
            b. a M252Y mutation, a S254T mutation, and a T256E mutation;
            c. a deletion of a heavy chain C-terminal lysine residue; or
            d. any combination of a to c.
        
        
        Embodiment 8. The antibody molecule of embodiment 7, wherein the antibody molecule comprises a S228P mutation and a deletion of a heavy chain C-terminal lysine residue.
        Embodiment 9. The antibody molecule of embodiment 7, wherein the antibody molecule comprises a S228P mutation, a M252Y mutation, a S254T mutation, a T256E mutation, and a deletion of a heavy chain C-terminal lysine residue.
        Embodiment 10. The antibody molecule of any one of the previous embodiments, wherein the antibody molecule comprises an IgG4 heavy chain constant region and a lambda light chain constant region.
        Embodiment 11. The antibody molecule of any one of the previous embodiments, wherein the antibody molecule comprises a heavy chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18 and
        
            a. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19;
            b. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23;
            c. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26;
            d. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29;
            e. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32;
            f. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35;
            g. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 38;
            h. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41;
            i. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44;
            j. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47;
            k. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50;
            l. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 53;
            m. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56;
            n. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 59;
            o. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 62;
            p. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 65; or
            q. a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 68.
        
        
        Embodiment 12. The antibody molecule of any one of embodiments 1-10, wherein the antibody molecule comprises a heavy chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
        
            the amino acid sequence of SEQ ID NO: 20 and a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19.
        
        
        Embodiment 13. The antibody molecule of embodiment 2 or embodiment 3, wherein the antibody molecule is a Fab fragment, a Fab2 fragment, or a single chain antibody.
        Embodiment 14. The antibody molecule of any one of the previous embodiments, wherein the antibody molecule has one or more of the following properties:
        
            a. reduces binding of IL-5 to the IL-5 receptor;
            b. has a serum half-life of at least about 20 days; or
            c. binds human and cynomolgus monkey IL-5 but not mouse, rat, or guinea pig IL-5.
        
        
        Embodiment 15. The antibody molecule of embodiment 2, wherein the antibody molecule binds to human IL-5 with an equilibrium affinity constant (KD) of at least about 40 pM as determined by surface plasmon resonance.
        Embodiment 16. A pharmaceutical composition comprising the antibody molecule of any one of embodiments 1 to 15.
        Embodiment 17. A nucleic acid molecule encoding the antibody molecule of any one of embodiments 1 to 15.
        Embodiment 18. A vector comprising the nucleic acid molecule of embodiment 17.
        Embodiment 19. A cell transformed to express the antibody molecule of any one of embodiments 1 to 15.
        Embodiment 20. A method of treating a subject having eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis, or eosinophilic esophagitis comprising:
        
            administering to the subject a therapeutically effective amount of the antibody molecule of any one of embodiments 1 to 15 or the pharmaceutical composition of embodiment 16 to treat the eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis.
        
        
        Embodiment 21. Use of an effective amount of the antibody molecule of any one of embodiments 1 to 15 or the pharmaceutical composition of embodiment 16 in the treatment of eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis, or eosinophilic esophagitis.
        Embodiment 22. Use of the antibody molecule of any one of embodiments 1 to 15 or the pharmaceutical composition of embodiment 16 in the manufacture of a medicament for the treatment of eosinophilic asthma, hypereosinophilic syndrome, nasal polyposis with eosinophilic involvement, eosinophilic granulomatosis with polyangiitis, atopic dermatitis or eosinophilic esophagitis.