Anti-LILRB1 antibody and uses thereof

The present invention relates to an anti-LILRB1 antibody having increased specificity for LILRB1, and to uses thereof. Specifically, an anti-LILRB1 antibody or an antigen-binding fragment thereof, and uses thereof in treating cancer are provided.

1. 11. An anti-LILRB1 antibody or antigen-binding fragment thereof, comprising: (1) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (2) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (3) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; (4) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 42; (5) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54; (6) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 61, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 62, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 63, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 65, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 66; (7) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 76, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 77, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 78; (8) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 85, the CDR-L2 comprising the amino acid sequence of SEQ ID NO:86, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 87, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 88, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 89, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 90; (9) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 97, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 98, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 99, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 100, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (10) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 109, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 110, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 112, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 113, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 114; (11) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 121, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 122, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 123, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 124, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 125, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 126; (12) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 133, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 134, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 135, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 136, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 137, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 138; (13) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 148, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 149, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 150; (14) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 157, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 158, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 159, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 160, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 161, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 162; (15) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 169, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 170, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 171, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 172, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 173, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 174; (16) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 181, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 182, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 183, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 184, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 185, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 186; (17) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 193, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 194, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 195, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 196, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 197, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 198; (18) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 205, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 206, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 207, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 208, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 209, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 210; or (19) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 217, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 218, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 219, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 220, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 221, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 222.
2. 22. The anti-LILRB1 antibody or antigen-binding fragment thereof of claim 1, comprising: a light chain variable region and a heavy chain variable region comprising: a) SEQ ID NOs: 7 and 9, b) SEQ ID NOs: 19 and 21, c) SEQ ID NOs: 31 and 33, d) SEQ ID NOs: 43 and 45, e) SEQ ID NOs: 55 and 57, f) SEQ ID NOs: 67 and 69, g) SEQ ID NOs: 79 and 81, h) SEQ ID NOs: 91 and 93, i) SEQ ID NOs: 103 and 105, j) SEQ ID NOs: 115 and 117, k) SEQ ID NOs: 127 and 129, l) SEQ ID NOs: 139 and 141, m) SEQ ID NOs: 151 and 153, n) SEQ ID NOs: 163 and 165, o) SEQ ID NOs: 175 and 177, p) SEQ ID NOs: 187 and 189, q) SEQ ID NOs: 199 and 201, r) SEQ ID NOs: 211 and 213, or s) SEQ ID NOs: 223 and 225, respectively.
3. 33. The anti-LILRB1 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a human IgG1 or IgG4 antibody.
4. 44. The anti-LILRB1 antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is a scFv, (scFv)2, Fab, Fab′, or F(ab′)2 of the anti-LILRB1 antibody, a fusion polypeptide comprising a scFv fused with an immunoglobulin Fc, or a fusion polypeptide comprising a scFv fused with a constant region of a light chain.
5. 55. A pharmaceutical composition for treating a cancer, comprising the anti-LILRB1 antibody or antigen-binding fragment thereof of claim 1.
6. 66. The pharmaceutical composition of claim 5, wherein the cancer has overexpression of MHC Class I.
7. 77. The pharmaceutical composition of claim 5, wherein the composition has an activity of inhibiting immune evasion of cancer cells.
8. 88. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of claim 1.
9. 99. A recombinant vector comprising the nucleic acid molecule of claim 8.
10. 1010. A recombinant cell comprising the recombinant vector of claim 9.
11. 1111. A method of preparing an anti-LILRB1 antibody or antigen-binding fragment thereof, comprising culturing the recombinant cell of claim 10.
12. 1212. A pharmaceutical composition for inhibiting immune evasion of cancer cells, comprising the anti-LILRB1 antibody or antigen-binding fragment thereof of claim 1.
13. 1313. A method for treating a cancer, comprising administering to a subject in need thereof a pharmaceutically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof of claim 1.
14. 1414. The method of claim 13, wherein the cancer has overexpression of MHC Class I.
15. 1515. A method for inhibiting immune evasion of cancer cells, comprising administering to a subject in need thereof a pharmaceutically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof of claim 1.
16. 1616. The pharmaceutical composition of claim 5, wherein the cancer is selected from the group consisting of lung cancer, peritoneal carcinoma, skin cancer, squamous cell carcinoma, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, leukemia, lymphocytic lymphoma, hepatoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma, uterine carcinoma, salivary gland tumor, renal cell carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, and bone osteosarcoma.
17. 1717. The method of claim 13, wherein the cancer is selected from the group consisting of lung cancer, peritoneal carcinoma, skin cancer, squamous cell carcinoma, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, leukemia, lymphocytic lymphoma, hepatoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma, uterine carcinoma, salivary gland tumor, renal cell carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, and bone osteosarcoma.

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 National Phase Entry Application from PCT/KR2021/009696 filed on Jul. 27, 2021, which claims the benefits of KR 10-2020-0094053 filed on Jul. 28, 2020 with the Korean Intellectual Property Office, the entire disclosure of which is herein incorporated by reference.
TECHNICAL FIELD
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on Jul. 24, 2023, is named 3570-846_ST25.txt and is 205,619 bytes in size.
The disclosure relates to an anti-LILRB1 antibody and uses thereof. More specifically, an anti-LILRB1 antibody or antigen-binding fragment thereof, and a use thereof for cancer therapy are provided.
BACKGROUND OF THE INVENTION
Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1; also known as ILT2, CD85j, or LIR-1) is an inhibitory receptor, which is expressed in cells such as B cells, T cells, NK cells, dendritic cells, macrophages, and other immune cells. LILRB1 participates in a signal transduction mechanism of inhibiting activities of immune cells by binding classical and non-classical MHC class I.
Therefore, it is required to develop a substance targeting LILRB1.
DISCLOSURE
Brief Summary of the Invention
An embodiment provides an anti-LILRB1 antibody, which binds to LILRB1, or antigen-binding fragment thereof. The anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity to inhibit immune evasion of cancer cells. Furthermore, the anti-LILRB1 antibody or antigen-binding fragment thereof may have an anti-cancer effect. The anti-cancer effect may be against a cancer cell expressing or overexpressing MHC Class I on the surface.
Another embodiment provides a pharmaceutical composition for treatment and/or prevention of a cancer, the composition comprising the anti-LILRB1 antibody or antigen-binding fragment thereof as an active ingredient. Another embodiment provides a method for treating and/or preventing cancer, comprising administering to a subject in need thereof a pharmaceutically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof. Another embodiment provides a use of the anti-LILRB1 antibody or antigen-binding fragment thereof for the treatment and/or prevention of cancer or for the manufacture of a pharmaceutical composition for the treatment and/or prevention of cancer.
Another embodiment provides a pharmaceutical composition for inhibiting immune evasion of cancer cells comprising the anti-LILRB1 antibody or antigen-binding fragment thereof. Another embodiment provides a method for inhibiting immune evasion of cancer cells, comprising administering a pharmaceutically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof to a subject in need of inhibiting immune evasion of cancer cells. Another example provides a use of the anti-LILRB1 antibody or antigen-binding fragment thereof for inhibiting immune evasion of cancer cells, or for the manufacture of a pharmaceutical composition for inhibiting immune evasion of cancer cells.
DETAILED DESCRIPTION OF THE INVENTION
An embodiment provides an anti-LILRB1 antibody, which binds to LILRB1, or antigen-binding fragment thereof. The anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity to inhibit immune evasion of cancer cells. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have an anti-cancer effect.
The anti-LILRB1 antibody or antigen-binding fragment thereof may comprise the following complementarity determining regions (CDRs):
Based on the CDR definition according to Kabat numbering (Kabat, E. A., Wu, T. T., Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242; http://www.abysis.org/),

    
    
        a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 1, 13, 25, 37, 49, 61, 73, 85, 97, 109, 121, 133, 145, 157, 169, 181, 193, 205, or 217,
        a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 2, 14, 26, 38, 50, 62, 74, 86, 98, 110, 122, 134, 146, 158, 170, 182, 194, 206, or 218,
        a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 3, 15, 27, 39, 51, 63, 75, 87, 99, 111, 123, 135, 147, 159, 171, 183, 195, 207, or 219,
        a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 4, 16, 28, 40, 52, 64, 76, 88, 100, 112, 124, 136, 148, 160, 172, 184, 196, 208 or 220,
        a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 5, 17, 29, 41, 53, 65, 77, 89, 101, 113, 125, 137, 149, 161, 173, 185, 197, 209 or 221, and
        a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 6, 18, 30, 42, 54, 66, 78, 90, 102, 114, 126, 138, 150, 162, 174, 186, 198, 210 or 222.
    
    


In a specific embodiment, combinations of 6 CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) that can be comprised in the anti-LILRB1 antibody or antigen-binding fragment thereof provided in this disclosure are illustrated in Table 1:










TABLE 1







Amino Acid Sequence
SEQ ID


Clone
CDR
 (N→C) (Kabat)
NO


















5
CDR-L1
RASQSIANYLN
1


 



CDR-L2
ATSTLQS
2


 



CDR-L3
QQSYSFPWT
3


 



CDR-H1
AYGIH
4


 



CDR-H2
WIIPLSGGAHYAQKFQG
5


 



CDR-H3
LYGWAEYFDV
6


 


6
CDR-L1
RASQSISNYLN
13


 



CDR-L2
AASTLQS
14


 



CDR-L3
QQSYSFPWT
15


 



CDR-H1
SYTIS
16


 



CDR-H2
WISPELGTSNYAQKFQG
17


 



CDR-H3
LRYGQTLYGFDI
18


 


7
CDR-L1
RASQSISNWLN
25


 



CDR-L2
GTSSLQS
26


 



CDR-L3
QQSYSFPFT
27


 



CDR-H1
SYGMH
28


 



CDR-H2
WIIPVSGGATYAQKFQG
29


 



CDR-H3
GSWAYYAEFDY
30


 


8
CDR-L1
RASQSISSYLN
37


 



CDR-L2
AASTLQS
38


 



CDR-L3
QQSYSFPYT
39


 



CDR-H1
SYGIH
40


 



CDR-H2
WIIPISGTTNYAQKFQG
41


 



CDR-H3
VGGVGLYVFDV
42


 


9
CDR-L1
RASQSISNYLN
49


 



CDR-L2
AASSLQS
50


 



CDR-L3
QQSYSFPWT
51


 



CDR-H1
SYAIH
52


 



CDR-H2
WIVPGLGVTNYAQKFQG
53


 



CDR-H3
QATLYQTEYMDV
54


 


10
CDR-L1
RASQSISNYLN
61


 



CDR-L2
AASNLQS
62


 



CDR-L3
QQSYSFPFT
63


 



CDR-H1
SHYMH
64


 



CDR-H2
WISPYLGSTNYAQKFQG
65


 



CDR-H3
DETGSTYGAFDY
66


 


11
CDR-L1
RASQSISNYLN
73


 



CDR-L2
DASTLQS
74


 



CDR-L3
QQSYSFPWT
75


 



CDR-H1
SYYVH
76


 



CDR-H2
WISPYSGGTNYAQKFQG
77


 



CDR-H3
DYYVSAYGAFDY
78


 


12
CDR-L1
RASQDISNYLN
85


 



CDR-L2
ATSSLQS
86


 



CDR-L3
QQSYSFPWT
87


 



CDR-H1
SYDIH
88


 



CDR-H2
RIVPYLGVTNYAQKFQG
89


 



CDR-H3
RQSQSSVYAFDI
90


 


13
CDR-L1
RASQSISNYLN
97


 



CDR-L2
AASRLQS
98


 



CDR-L3
QQSYSFPFT
99


 



CDR-H1
GYYIH
100


 



CDR-H2
WISPSSGGTIYAQKFQG
101


 



CDR-H3
DISVRVVQAFDY
102


 


14
CDR-L1
RASQSISNYLN
109


 



CDR-L2
ATSNLQS
110


 



CDR-L3
QQSYSFPWT
111


 



CDR-H1
SYYMH
112


 



CDR-H2
WISPYLGITNYAQKFQG
113


 



CDR-H3
AGYQQAQYWFDY
114


 


15
CDR-L1
RASQSISNYLN
121


 



CDR-L2
ATSSLQS
122


 



CDR-L3
QQSYSFPYT
123


 



CDR-H1
SYAMS
124


 



CDR-H2
WIIPISGTTNYAQKFQG
125


 



CDR-H3
QHSVGSVFDY
126


 


16
CDR-L1
RASQDISSWLN
133


 



CDR-L2
AASSLQS
134


 



CDR-L3
QQSYSFPWT
135


 



CDR-H1
SYYMT
136


 



CDR-H2
GISPILGVTNYAQKFQG
137


 



CDR-H3
LLVGVSETYFDY
138


 


17
CDR-L1
RASQSISNYLN
145


 



CDR-L2
AASNMHS
146


 



CDR-L3
QQSHSFPWT
147


 



CDR-H1
TYAMS
148


 



CDR-H2
GISPTLGIANYAQKFQG
149


 



CDR-H3
VRYAGWTGYFDL
150


 


18
CDR-L1
RASQSISRWLN
157


 



CDR-L2
AASRLQS
158


 



CDR-L3
QQSESFPWT
159


 



CDR-H1
SYDIN
160


 



CDR-H2
WIIPTSGSTNYAQKFQG
161


 



CDR-H3
DSQSSYIGYFDV
162


 


19
CDR-L1
RASQSISNYLN
169


 



CDR-L2
DTSSLQS
170


 



CDR-L3
QQSYSTPYT
171


 



CDR-H1
AYGIS
172


 



CDR-H2
RIIPYLGTANYAQKFQG
173


 



CDR-H3
LSYGIGYESFDV
174


 


20
CDR-L1
RASQSISSYLN
181


 



CDR-L2
DTSTLQS
182


 



CDR-L3
QQSYSFPWT
183


 



CDR-H1
SYAMS
184


 



CDR-H2
SISSSGGSTYYADSVKG
185


 



CDR-H3
ELGGYGFSYFDY
186


 


21
CDR-L1
RASQSIRNYLN
193


 



CDR-L2
ATSSLQS
194


 



CDR-L3
QQSYSFPWT
195


 



CDR-H1
DYAMS
196


 



CDR-H2
GISGSDIYYADSVKG
197


 



CDR-H3
AVSYWSYTFDY
198


 


22
CDR-L1
RASQSIGSYLN
205


 



CDR-L2
DASTLQS
206


 



CDR-L3
QQSYSFPWT
207


 



CDR-H1
SYAMH
208


 



CDR-H2
GISSSGGTTYYADSVKG
209


 



CDR-H3
ALGVVGGTWFDY
210


 


23
CDR-L1
RASQSISNYLN
217


 



CDR-L2
DTSTLQS
218


 



CDR-L3
QQSYSFPWT
219


 



CDR-H1
DYAMH
220


 



CDR-H2
AISGSGGYTHYADSVKG
221


 



CDR-H3
SATFGVWETFDV
222









In an embodiment, the anti-LILRB1 antibody or antigen-binding fragment thereof may comprise:

    
    
        a light chain variable region comprising a CDR-L1, a CDR-L2, and CDR-L3, and
        a heavy chain variable region comprising a CDR-H1, a CDR-H2, and a CDR-H3, wherein the CDRs are as described above.
    
    


In an embodiment, the anti-LILRB1 antibody or antigen-binding fragment thereof may comprise:

    
    
        a light chain variable region comprising an amino acid sequence of SEQ ID NO: 7, 19, 31, 43, 55, 67, 79, 91, 103, 115, 127, 139, 151, 163, 175, 187, 199, 211, or 223, and
        a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 9, 21, 33, 45, 57, 69, 81, 93, 105, 117, 129, 141, 153, 165, 177, 189, 201, 213, or 225.
    
    


In a specific embodiment, combinations of a light chain variable region and a heavy chain variable region that can be comprised in the anti-LILRB1 antibody or antigen-binding fragment thereof provided in this disclosure are illustrated in Table 2:










TABLE 2






variable




Clone
region
Amino acid sequence(N→C)
SEQ ID NO







 5
light
DIQMTQSPSSLSASVGDRVTITCRASQSIANYLNWYQQK
  7



chain
PGKAPKLLIYATSTLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYGIHWVR
  9



chain
QAPGQGLEWMGWIIPLSGGAHYAQKFQGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARLYGWAEYFDVWGQGTLVT




region
VSS



 


 6
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
 19



chain
PGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVR
 21



chain
QAPGQGLEWMGWISPELGTSNYAQKFQGRVTITADEST




variable
STAYMELSSLRSEDTAVYYCARLRYGQTLYGFDIWGQGT




region
LVTVSS



 


 7
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNWLNWYQQK
 31



chain
PGKAPKLLIYGTSSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPFTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGMHWV
 33



chain
RQAPGQGLEWMGWIIPVSGGATYAQKFQGRVTITADEST




variable
STAYMELSSLRSEDTAVYYCARGSWAYYAEFDYWGQGT




region
LVTVSS



 


 8
light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK
 43



chain
PGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPYTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGIHWVR
 45



chain
QAPGQGLEWMGWIIPISGTTNYAQKFQGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARVGGVGLYVFDVWGQGTLV




region
TVSS



 


 9
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
 55



chain
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIHWVR
 57



chain
QAPGQGLEWMGWIVPGLGVTNYAQKFQGRVTITADEST




variable
STAYMELSSLRSEDTAVYYCARQATLYQTEYMDVWGQG




region
TLVTVSS



 


10
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
 67



chain
PGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPFTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHYMHWV
 69



chain
RQAPGQGLEWMGWISPYLGSTNYAQKFQGRVTITADES




variable
TSTAYMELSSLRSEDTAVYYCARDETGSTYGAFDYWGQ




region
GTLVTVSS



 


11
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
 79



chain
PGKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYVHWVR
 81



chain
QAPGQGLEWMGWISPYSGGTNYAQKFQGRVTITADEST




variable
STAYMELSSLRSEDTAVYYCARDYYVSAYGAFDYWGQG




region
TLVTVSS



 


12
light
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQK
 91



chain
PGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYDIHWVR
 93



chain
QAPGQGLEWMGRIVPYLGVTNYAQKFQGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARRQSQSSVYAFDIWGQGTL




region
VTVSS



 


13
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
103



chain
PGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPFTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSGYYIHWVR
105



chain
QAPGQGLEWMGWISPSSGGTIYAQKFQGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARDISVRVVQAFDYWGQGTL




region
VTVSS



 


14
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
115



chain
PGKAPKLLIYATSNLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYMHWV
117



chain
RQAPGQGLEWMGWISPYLGITNYAQKFQGRVTITADEST




variable
STAYMELSSLRSEDTAVYYCARAGYQQAQYWFDYWGQ




region
GTLVTVSS



 


15
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
127



chain
PGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPYTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWV
129



chain
RQAPGQGLEWMGWIIPISGTTNYAQKFQGRVTITADEST




variable
STAYMELSSLRSEDTAVYYCARQHSVGSVFDYWGQGTL




region
VTVSS



 


16
light
DIQMTQSPSSLSASVGDRVTITCRASQDISSWLNWYQQK
139



chain
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYMTWV
141



chain
RQAPGQGLEWMGGISPILGVTNYAQKFQGRVTITADEST




variable
STAYMELSSLRSEDTAVYYCARLLVGVSETYFDYWGQGT




region
LVTVSS



 


17
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
151



chain
PGKAPKLLIYAASNMHSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSHSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAMSWVR
153



chain
QAPGQGLEWMGGISPTLGIANYAQKFQGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARVRYAGWTGYFDLWGQGT




region
LVTVSS



 


18
light
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLNWYQQK
163



chain
PGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSESFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYDINWVR
165



chain
QAPGQGLEWMGWIIPTSGSTNYAQKFQGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARDSQSSYIGYFDVWGQGTL




region
VTVSS



 


19
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
175



chain
PGKAPKLLIYDTSSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSTPYTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYGISWVR
177



chain
QAPGQGLEWMGRIIPYLGTANYAQKFQGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARLSYGIGYESFDVWGQGTL




region
VTVSS



 


20
light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK
187



chain
PGKAPKLLIYDTSTLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWV
189



chain
RQAPGQGLEWMGSISSSGGSTYYADSVKGRVTITADES




variable
TSTAYMELSSLRSEDTAVYYCARELGGYGFSYFDYWGQ




region
GTLVTVSS



 


21
light
DIQMTQSPSSLSASVGDRVTITCRASQSIRNYLNWYQQK
199



chain
PGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAMSWV
201



chain
RQAPGQGLEWMGGISGSDIYYADSVKGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARAVSYWSYTFDYWGQGTLV




region
TVSS



 


22
light
DIQMTQSPSSLSASVGDRVTITCRASQSIGSYLNWYQQK
21



chain
PGKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMHWV
213



chain
RQAPGQGLEWMGGISSSGGTTYYADSVKGRVTITADES




variable
TSTAYMELSSLRSEDTAVYYCARALGVVGGTWFDYWGQ




region
GTLVTVSS



 


23
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQK
223



chain
PGKAPKLLIYDTSTLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSFPWTFGQGTKVEIK




region





heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAMHWV
225



chain
RQAPGQGLEWMGAISGSGGYTHYADSVKGRVTITADES




variable
TSTAYMELSSLRSEDTAVYYCARSATFGVWETFDVWGQ




region
GTLVTVSS









In this disclosure, an antibody (for example, CDR, variable region, or heavy chain/light chain) “comprising a specific amino acid sequence or consisting of a specific amino acid sequence” refers to all cases which the amino acid sequence is essentially included, and/or an insignificant mutation (for example, substitution, deletion, and/or addition of amino acid residue(s)) that does not affect antibody activity is introduced into the amino acid sequence.
The anti-LILRB1 antibody or antigen-binding fragment thereof provided in this disclosure may have a binding affinity (KD) to LILRB1 (for example, human LILRB1) of 10 mM or less, 5 mM or less, 1 mM or less, 0.5 mM or less, 0.2 mM or less, 0.15 mM or less, for example, 0.001 nM to 10 mM, 0.005 nM to 10 mM, 0.01 nM to 10 mM, 0.05 nM to 10 mM, 0.1 nM to 10 mM, 0.5 nM to 10 mM, 1 nM to 10 mM, 0.001 nM to 5 mM, 0.005 nM to 5 mM, 0.01 nM to 5 mM, 0.05 nM to 5 mM, 0.1 nM to 5 mM, 0.5 nM to 5 mM, 1 nM to 5 mM, 0.001 nM to 1 mM, 0.005 nM to 1 mM, 0.01 nM to 1 mM, 0.05 nM to 1 mM, 0.1 nM to 1 mM, 0.5 nM to 1 mM, 1 nM to 1 mM, 0.001 nM to 0.5 mM, 0.005 nM to 0.5 mM, 0.01 nM to 0.5 mM, 0.05 nM to 0.5 mM, 0.1 nM to 0.5 mM, 0.5 nM to 0.5 mM, 1 nM to 0.5 mM, 0.001 nM to 0.2 mM, 0.005 nM to 0.2 mM, 0.01 nM to 0.2 mM, 0.05 nM to 0.2 mM, 0.1 nM to 0.2 mM, 0.5 nM to 0.2 mM, 1 nM to 0.2 mM, 0.001 nM to 0.15 mM, 0.005 nM to 0.15 mM, 0.01 nM to 0.15 mM, 0.05 nM to 0.15 mM, 0.1 nM to 0.15 mM, 0.5 nM to 0.15 mM, or 1 nM to 0.15 mM, when measured by surface plasmon resonance (SPR).
Another embodiment provides a pharmaceutical composition comprising the anti-LILRB1 antibody or antigen-binding fragment thereof as an active ingredient. For example, the pharmaceutical composition may be a pharmaceutical composition for treating and/or preventing a cancer. In an embodiment, the pharmaceutical composition may have an activity to inhibit immune evasion of a cancer cell. The cancer cell may be a cell expressing or overexpressing MHC Class I on cell surface.
Another embodiment provides a method of treating and/or preventing a cancer, comprising administering (orally or parenterally) a pharmaceutically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof to a subject (e.g., a mammal including human) in need of treating and/or preventing the cancer.
The methods provided in this disclosure may further comprise a step of identifying the subject in need of treating and/or preventing the cancer, and/or inhibiting immune evasion of the cancer cell, prior to the step of administering.
Another embodiment provides the use of the anti-LILRB1 antibody or antigen-binding fragment thereof for the treatment and/or prevention of cancer or for use in the manufacture of a pharmaceutical composition for the treatment and/or prevention of cancer. Another example provides the use of the anti-LILRB1 antibody or antigen-binding fragment thereof for inhibiting immune evasion of cancer cells or the use in the preparation of a pharmaceutical composition for inhibiting immune evasion of cancer cells.
Another embodiment provides a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of CDR (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, a combination of CDR-L1, CDR-L2, and CDR-L3, or a combination of CDR-H1, CDR-H2, and CDR-H3), a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3; a light chain comprising the light chain variable region, and a heavy chain comprising the heavy chain variable region, of the anti-LILRB1 antibody described above.
Another embodiment provides a recombinant vector comprising the nucleic acid molecule. In an embodiment, the recombinant vector may include the light chain variable region or light chain, and the heavy chain variable region or heavy chain (e.g., in two separate vectors), respectively, or (e.g., in one vector) together. The recombinant vector may be an expression vector for expressing the light chain variable region or light chain and the heavy chain variable region or heavy chain in an appropriate cell.
Another embodiment provides a recombinant cell comprising the nucleic acid molecule or the recombinant vector.
Another embodiment provides a method for producing an anti-LILRB1 antibody or antigen-binding fragment thereof, comprising expressing the antibody in the recombinant cell.
As described herein, the antigen-binding fragment of an anti-LILRB1 antibody may refer to a fragment which is derived from an anti-LILRB1 antibody and retain antigen (LILRB1) binding affinity of the antibody. In an embodiment, the antigen-binding fragment may be a polypeptide comprising the 6 CDRs of an anti-LILRB1 antibody as described above, and, for example, may be scFv, scFv-Fc, scFv-Ck (kappa constant region), scFv-CA (lambda constant region), (scFv)2, Fab, Fab′, or a F(ab′)2, but not be limited thereto.
The anti-LILRB1 antibody or antigen-binding fragment thereof may have a regulatory activity, for example, an antagonistic or agonistic activity, on LILRB1 protein. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity of inhibiting immune evasion of a cancer cell. Furthermore, the anti-LILRB1 antibody or antigen-binding fragment thereof may have an anti-cancer effect.
A protein LILRB1, which is an antigen of an anti-LILRB1 antibody or antigen-binding fragment thereof provided in this disclosure, may be derived from mammal. For example, LILRB1 as an antigen may be a human LILRB1 (e.g., GenBank accession numbers NP_001265328.2, NP_001265327.2, NP_001075108.2, NP_001075107.2, NP_001075106.2, NP_006660.4, NM_001081637.2, NM_001081638.3, NM_001081639.3, NM_001278398.2, NM_001278399.2, etc.), but not be limited thereto.
MHC Class I may be one of classes of major histocompatibility complex (MHC) molecules. In an embodiment, the MHC Class I may be a human MHC Class I and may be at least one selected from the group consisting of HLA (human leukocyte antigen)-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, but not be limited thereto.
As described herein, the term “antibody” may refer to a protein that specifically binds to a specific antigen, and may be a protein produced by stimulation of an antigen in the immune system, or a protein produced by chemical synthesis or recombinant production, with no specific limitation. The antibody may be non-naturally occurring, for example, produced by recombinant or synthetic production. The antibody may be an animal antibody (e.g., a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal or polyclonal antibody.
In the anti-LILRB1 antibody or antigen-binding fragment thereof provided herein, the portion, except for the heavy-chain CDR and light-chain CDR portions or the heavy-chain variable and light-chain variable regions as defined above, may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like), and, for example, derived from the framework portions, and/or light-chain constant region and/or heavy-chain constant region. In an embodiment, the anti-LILRB1 antibody provided in this disclosure may be an antibody in a form of human IgG, for example, IgG1, IgG2, IgG3, or IgG4, but not be limited thereto.
An intact antibody (e.g., IgG type) has a structure with two full-length light chains and two full-length heavy chains, in which each light chain is linked to a corresponding heavy chain via a disulfide bond. The constant region of an antibody is divided into a heavy-chain constant region and a light-chain constant region. The heavy-chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, and has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1) or alpha2 (α2) as its subclass. The light chain constant region is of either a kappa (κ) or lambda (λ) type.
As used herein, the term “heavy chain” may be intended to encompass a full-length heavy chains and fragments thereof, wherein the full-length heavy chain may comprise a variable region VH including amino acid sequences sufficient to provide specificity to antigens, three constant regions CH1, CH2, and CH3, and a hinge. The term “light chain” may be intended to encompass full-length light chains and fragments thereof, wherein the full-length light chain may comprise a variable region VL including amino acid sequences sufficient to provide specificity to antigens, and a constant region CL.
The term “complementarity determining region (CDR)” may refer to a portion that confers antigen-binding specificity in a variable region of an antibody, and may refer to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contacting residues that play an important role in the binding of an antibody to its antigen or an epitope of the antigen. As used herein, the terms “specifically binding” and “specifically recognizing” may have the same general meaning as known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological reaction.
In this disclosure, unless differently stated, the term “antibody” may encompass may be understood to include an antigen-binding fragment of an antibody having antigen-binding ability.
The term “antigen-binding fragment” used herein may refer to a polypeptide in any type, which comprises a portion (e.g., 6 CDRs as described herein) capable of binding to an antigen, and, for example, may be scFv, (scFv)2, scFv-Fc, Fab, Fab′, or F(ab′)2, but is not limited thereto. In addition, as described above, the antigen-binding fragment may be scFv, a fusion polypeptide wherein scFv is fused with a Fc region of an immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.) or a constant region (e.g., kappa or lambda).
Among the antigen-binding fragments, Fab has a structure having variable regions of light and heavy chains, a constant region of a light chain and a first constant region (CH1) of a heavy chain, and has one antigen-binding site.
Fab′ is different from Fab in that Fab′ comprises a hinge region having at least one cysteine residue at the C-terminal of CH1.
F(ab′)2 antibody is formed through disulfide bridging of the cysteine residues in the hinge region of Fab′.
Fv is a minimal antibody fragment composed of only a heavy chain variable region and a light chain variable region. Recombination techniques of generating an Fv fragment are widely known in the art.
Two-chain Fv comprises a heavy chain variable region and a light chain variable region which are linked to each other by a non-covalent bond. Single-chain Fv generally comprises a heavy-chain variable region and a light-chain variable region which are linked to each other by a covalent bond via a peptide linker or directly linked at the C-terminals to have a dimer structure like two-chain Fv.
The antigen-binding fragments may be obtained using protease (for example, Fab may be obtained by restrictively cleaving a whole antibody with papain, and an F(ab′)2 fragment may be obtained by cleaving with pepsin), or may be prepared by using a genetic recombination technique.
The term “hinge region” may refer to a region between CH1 and CH2 domains within heavy chain of an antibody, which functions to provide flexibility for the antigen-binding site in the antibody.
The anti-LILRB1 antibody may be a monoclonal or polyclonal antibody and, for example, a monoclonal antibody. A monoclonal antibody can be prepared using a method widely known in the art, for example, using a phage display technique. Alternatively, the anti-LILRB1 antibody may be constructed in the form of a mouse-derived monoclonal antibody by a conventional method.
Meanwhile, individual monoclonal antibodies can be screened using a typical ELISA (Enzyme-Linked ImmunoSorbent Assay) format, based on the binding potential against LILRB1. Inhibitory activities can be verified through functional analysis such as competitive ELISA for verifying the molecular interaction of binding assemblies or functional analysis such as a cell-based assay. Then, with regard to monoclonal antibody members selected on the basis of their strong inhibitory activities, their affinities (Kd values) to LILRB1 may be each verified.
The finally selected antibodies can be prepared and used as humanized antibodies as well as human immunoglobulin antibodies in which the remaining parts except for the antigen-binding portion are humanized. Methods for producing humanized antibodies are well known in the art.
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, in addition to the active ingredient (the anti-LILRB1 antibody or antigen-binding fragment thereof). The pharmaceutically acceptable carrier may be anyone selected from those commonly used for the formulation of antibodies. For example, the pharmaceutically acceptable carrier may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. The pharmaceutical composition may further comprise one or more selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, preservative, and the like, which can be commonly used for manufacturing pharmaceutical composition.
The pharmaceutical composition, or the antibody or antigen-binding fragment thereof may be administered orally or parenterally in a pharmaceutically effective amount. The parenteral administration may be intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, intranasal administration, intrapulmonary administration, rectal administration or intralesional local administration. Since proteins or peptides are digested when administered orally, the active ingredient in the compositions for oral administration may be coated or formulated to prevent digestion in stomach. In addition, the antibody or the compositions may be administered using an optional device that enables the active ingredient to be delivered to target cells (e.g., cancer cells).
The content of the anti-LILRB1 antibody or antigen-binding fragment thereof or the dosage of the anti-LILRB1 antibody or antigen-binding fragment thereof in the pharmaceutical composition may be prescribed in a variety of ways, depending on various factors, such as the formulation method, administration method, age, weight, sex, pathology, food, administration time of the patient., administration interval, administration route, excretion rate, response sensitivity, etc. For example, anti-LILRB1 antibody or antigen-binding fragment thereof may be administered at the amount of 0.005 ug/kg to 1000 mg/kg, 0.005 ug/kg to 500 mg/kg, 0.005 ug/kg to 250 mg/kg, 0.005 ug/kg to 100 mg/kg, 0.005 ug/kg to 75 mg/kg, 0.005 ug/kg to 50 mg/kg, 0.01 ug/kg to 1000 mg/kg, 0.01 ug/kg to 500 mg/kg, 0.01 ug/kg to 250 mg/kg, 0.01 ug/kg to 100 mg/kg, 0.01 ug/kg to 75 mg/kg, 0.01 ug/kg to 50 mg/kg, 0.05 ug/kg to 1000 mg/kg, 0.05 ug/kg to 500 mg/kg, 0.05 ug/kg to 250 mg/kg, 0.05 ug/kg to 100 mg/kg, 0.05 ug/kg to 75 mg/kg, or 0.05 ug/kg to 50 mg/kg per day, but not be limited thereto. The daily dosage may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container.
The pharmaceutical compositions may be formulated into a form of a solution in oil or an aqueous medium, a suspension, syrup, an emulsifying solution, an extract, powder, granules, a tablet, or a capsule, and may further comprise a dispersing or a stabilizing agent for the formulation.
The subject, to whom the antibody, pharmaceutical composition, or method provided in this disclosure is applied, may be selected from mammals including a mammal including primates such as humans and monkeys, rodents such as rats and mice, and the like.
The cancer may be a solid cancer or blood cancer. The cancer may be, but not limited to, one or more selected from the group consisting of lung cancer (e.g., squamous cell carcinoma of the lung, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung), peritoneal carcinoma, skin cancer, squamous cell carcinoma, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, leukemia (e.g., chronic or acute leukemia), lymphocytic lymphoma, hepatoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, renal cell carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, bone osteosarcoma, and the like. The cancer may be a primary cancer or a metastatic cancer. The cancer may be a cancer characterized by expression or overexpression of MHC Class I on a surface of cancer cell, and, for example, may be colon adenocarcinoma, small cell lung carcinoma, breast cancer, pancreatic cancer, malignant melanoma, bone osteosarcoma, renal cell carcinoma, or gastric cancer. The overexpression of MHC Class I may refer to an overexpression in cancer cells to which the antibody is applied as compared to normal cells. In one embodiment, the cancer may be a cancer that does not show anticancer effect (resistant) to T-cell mediated immunotherapy.
As used herein, the term “treatment of cancer” may refer to all anti-cancer actions that prevent, alleviate or ameliorate the symptoms of cancer, or partially or completely remove a cancer, such as, cancer cell death, inhibition of cancer cell proliferation, inhibition of cancer metastasis, and the like.
The anti-LILRB1 antibody or antigen-binding fragment thereof provided in this disclosure may be co-administered with another drug, for example, at least one selected from the group consisting of conventionally used agents for immunotherapy, anti-cancer agents, cytotoxic agents, and the like. Accordingly, an embodiment provides a pharmaceutical composition of combined administration for treating and/or preventing a cancer, comprising (1) an anti-LILRB1 antibody or antigen-binding fragment thereof, and (2) at least one selected from the group consisting of agents for immunotherapy, anti-cancer agents, cytotoxic agents, and the like. Another embodiment provides a method of treating and/or preventing a cancer, comprising administering (1) an anti-LILRB1 antibody or antigen-binding fragment thereof, and (2) at least one selected from the group consisting of agents for immunotherapy, anti-cancer agents, cytotoxic agents, and the like, to a subject in need of treating and/or preventing the cancer. The agents for immunotherapy, anti-cancer agents, and cytotoxic agents may include any drugs which are conventionally used for cancer therapy, and/or have cytotoxic activity, and for example, they may be at least one selected from the group consisting of proteins such as cell therapeutics, antibodies, nucleic acid molecules such as siRNA, and/or small molecular chemicals such as paclitaxel, docetaxel, and the like, but not limited thereto.
Another embodiment provides a polypeptide molecule comprising a heavy chain complementarity determining region (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), a light chain complementarity determining region (CDR-L1, CDR-L2, CDR-L3, or a combination thereof), a combination thereof; or heavy chain variable region, light chain variable region, or a combination thereof, of the anti-LILRB1 antibody as described above. The polypeptide molecule may be used in preparing an antibody as a precursor of antibody, or comprised in a protein scaffold having an antibody-like structure (e.g., peptibody), a bispecific antibody, or a multispecific antibody, as a component thereof. In another embodiment, the polypeptide molecule comprising a heavy chain complementarity determining region (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), a light chain complementarity determining region (CDR-L1, CDR-L2, CDR-L3, or a combination thereof), a combination thereof; or heavy chain variable region, light chain variable region, or a combination thereof, of the anti-LILRB1 antibody as described above may be used as a target (antigen) recognition domain or a secreted antibody, in cell therapeutics for target therapy, such as CAR-T. In another embodiment, the polypeptide molecule may be used for constructing anti-LILRB1 antibody-secreting cells as cell therapeutics.
Another embodiment provides a nucleic acid molecule encoding a heavy chain complementarity determining region (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), a heavy chain variable region, or a heavy chain, of the anti-LILRB1 antibody.
Another embodiment provides a nucleic acid molecule encoding a light chain complementarity determining region (CDR-L1, CDR-L2, CDR-L3, or a combination thereof), a light chain variable region, or a light chain, of the anti-LILRB1 antibody.
Another embodiment provides a recombinant vector comprising a nucleic acid molecule encoding a heavy chain variable region or a heavy chain of the anti-LILRB1 antibody, and a light chain variable region or a light chain of the anti-LILRB1 antibody, respectively in two separate vectors or all together in one vector.
Another embodiment provides a recombinant cell comprising the recombinant vector.
The term “vector” refers to a means for expressing a target gene in a host cell, as exemplified by a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, a lentivirus vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The recombinant vector may be constructed from or by manipulating a plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), a phage (for example, λgt4λB, λ-Charon, λΔz1, M13, etc.), or a virus vector (for example, SV40, etc.), which is commonly used in the art.
In the recombinant vector, the nucleic acid molecule may be operatively linked to a promoter. The term “operatively linked” is intended to pertain to a functional linkage between a nucleotide sequence of interest and an expression regulatory sequence (for example, a promoter sequence). When being “operatively linked”, the regulatory element can control the transcription and/or translation of a polynucleotide of interest.
The recombinant vector may be constructed typically as a cloning vector or an expression vector. For recombinant expression vectors, a vector generally available in the relevant art for expressing a foreign protein in plant, animal, or microbial cells may be employed. Various methods well known in the art may be used for the construction of recombinant vectors.
For use in hosts, such as prokaryotic or eukaryotic cells, the recombinant vector may be constructed accordingly. For example, when a vector is constructed as an expression vector for use in a prokaryotic host, the vector typically includes a strong promoter for transcription (e.g., a pLλ promoter, a trp promoter, a lac promoter, a tac promoter, a T7 promoter, etc.), a ribosomal binding site for initiating translation, and transcriptional/translational termination sequences. On the other hand, an expression vector for use in a eukaryotic host includes an origin of replication operable in a eukaryotic cell, such as an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, and a BBV origin of replication, but is not limited thereto. In addition, the expression vector typically includes a promoter derived from genomes of mammalian cells (for example, metallothionein promoter) or from mammalian viruses (for example, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, etc.), and a polyadenylation sequence as a transcription termination sequence.
Another embodiment provides a recombinant cell comprising the recombinant vector.
The recombinant cell may be prepared by introducing the recombinant vector into a suitable host cell. As long as it allows the sequential cloning and expression of the recombinant vector in a stable manner, any host cell known in the art may be employed in the present disclosure. Examples of the prokaryotic host cell available for the present disclosure may be selected from E. coli such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus spp. such as Bacillus subtilis and Bacillus thuringiensis, and enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species. Eukaryotic host cells that may be used for transformation may selected from, but are not limited to, Saccharomyces cerevisiae, insect cells, and animal cells, such as Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK, etc.
The nucleic acid molecule or a recombinant vector carrying the same may be introduced (transfected) into a host cell using a method well known in the relevant art. For example, this transfection may be carried out using a CaCl2) or electroporation method when the host cell is prokaryotic. For eukaryotic host cells, the genetic introduction may be achieved using, but not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, or particle bombardment.
To select a transformed host cell, advantage may be taken of a phenotype associated with a selection marker according to methods well known in the art. For example, when the selection marker is a gene conferring resistance to a certain antibiotic, the host cells may be grown in the presence of the antibiotic in a medium to select a transformant of interest.
Another embodiment provides a method of preparing the anti-LILRB1 antibody or antigen-binding fragment thereof, comprising expressing the nucleic acid molecule or a recombinant vector in a host cell. The step of expressing may be conducted by culturing the recombinant cell comprising the nucleic acid molecule (for example, in a recombinant vector) under a condition allowing the expression of the nucleic acid molecule. The method may further comprise isolating and/or purifying the antibody or its fragment from the cell culture, after the step of expressing or culturing.
Advantageous Effects
The anti-LILRB1 antibody or antigen-binding fragment thereof provided in this disclosure can have high anti-cancer effect by inhibiting the immune evasion mechanism of cancer cells, allowing that the immune cells can exhibit their anti-cancer effect.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows electrophoresis images showing the results of SDS-PAGE gel analysis for anti-LILRB1 antibodies purified in an example.
FIG. 2 is a sensorgram showing the results of SPR (surface plasmon resonance) assay for anti-LILRB1 antibody No. 13 according to an example.
FIG. 3 shows graphs showing the binding ability of anti-LILRB1 antibodies No. 8, No. 10, No. 11, No. 13, and No. 18 to LILRB1 overexpressing CHO cells according to an example.
FIG. 4 is a graph showing the IncuCyte S3-analyzed results of cell killing activity using HLA-G overexpressing HEK293 cells and natural killer cells KHYG-1, where those cells were treated with anti-LILRB1 antibodies (antibodies No. 10, No. 11 and No. 13) or human IgG4 isotype control antibody (negative control) according to an example.
FIG. 5 is a graph showing in vivo anti-tumor effects of anti-LILRB1 antibodies No. 10, No. 11, and No. 13 according to an example.



Hereinafter, the present invention will be described in detail by examples.
The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.
EXAMPLE 1: PREPARATION OF HUMAN ANTIBODIES AGAINST LILRB1
1.1. Selection of Human Antibodies Against LILRB1 Using Phage Display
In order to select antibodies that specifically recognize human LILRB1, a phage display screening was performed using a library composed of human Fab antibodies. Phage panning was performed up to 4 rounds using human LILRB1-Fc (Cat. No. 2017-T2) (RnD systems) as an antigen. Additionally, since the antigen is in the form of Fc fusion, Fc control panning to remove the Fc binder in the panning step was also performed. The selected products were confirmed for their binding to the antigen through polyclonal phage ELISA.
1.2. Monoclonal Phage ELISA
Monoclonal phage ELISA was performed to select a clone that specifically binds to an antigen among the phage obtained through panning in Example 1.1. For the antigen of Example 1.1, an absorbance (A450 nm) cut-off of 0.4 or more was determined to confirm a positive clone, and the sequence of the corresponding gene was analyzed. In order to confirm the specificity of the antigen, the purified phage ELISA of the unique Fab clone for the antigen was performed to obtain the EC50 (pfu) value.
1.3. Monoclonal Soluble Fab Analysis
Among the 47 unique clones that bind to the antigen obtained through panning in Example 1.2, genes encoding the Fab of the top 19 clones based on EC50 in the phage specificity ELISA were amplified by PCR to produce expression vectors. After the antibody was expressed using TB media, soluble protein was obtained through periplasmic extraction. After purification through affinity chromatography, ELISA was performed to confirm binding to antigen.
EXAMPLE 2: CONVERSION OF SELECTED ANTIBODIES TO IGG
For the genes selected from the Fab-type phage display library in Example 1.3, genes corresponding to each heavy chain variable region (VH) and light chain variable region (VL) were amplified by PCR. In the case of some clones with low expression level, the genes of the light chain variable region (VL) were amplified in the same manner by PCR, and the gene sequences corresponding to the heavy chain variable region (VH) were generated by grafting CDRs into the sequence corresponding to the framework region (FR) of the clone with high expression level. The constructed heavy chain variable region (VH) and light chain variable region (VL) gene sequences were inserted into an expression vector (pCB-LIR-mAB, other than that, vectors including CMV promoter, or CMV/CHO beta-actin fusion promoter (KR10-1038126B1) and genes encoding human IgG4 heavy chain constant region and kappa light chain constant region can be used) designed to encode a human antibody in the form of IgG4 (IgG4 Fc: SEQ ID NO: 229, Kappa constant region: SEQ ID NO: 230). The DNA sequence of the prepared expression vector was confirmed through sequencing.
The amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, light chain variable region, heavy chain variable region, light chain, and heavy chain of the 19 selected antibodies, and the nucleic acid sequences of the light chain variable region gene and the heavy chain variable region genes are shown in Tables 3 to 21 below. A clone number was assigned to each clone. Hereinafter, only simple clone numbers are indicated.










TABLE 3







Amino acid sequence (N→C) or Nucleic acid



clone
region
sequence (5′→3′)
SEQ ID NO


















5
CDR-L1
RASQSIANYLN
 1


 



CDR-L2
ATSTLQS
 2


 



CDR-L3
QQSYSFPWT
 3


 



CDR-H1
AYGIH
 4


 



CDR-H2
WIIPLSGGAHYAQKFQG
 5


 



CDR-H3
LYGWAEYFDV
 6


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSIANYLNWYQQKP
 7



chain
GKAPKLLIYATSTLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
 8



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA 




variable
GCCAGTCTATCGCAAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAACT




coding
TCCACTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




gene
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG





CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYGIHWVRQ
 9



chain
APGQGLEWMGWIIPLSGGAHYAQKFQGRVTITADESTSTA




variable
YMELSSLRSEDTAVYYCARLYGWAEYFDVWGQGTLVTVS




region
S



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
10



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTGCATACGGTATCCATTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGG




coding
ATTATCCCACTGTCTGGTGGTGCACATTATGCACAAAAAT




gene
TCCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGACTGTACGGTTG





GGCAGAATACTTCGATGTTTGGGGTCAGGGTACTCTGG





TTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSIANYLNWYQQKP
11



chain
GKAPKLLIYATSTLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYGIHWVRQ
12



chain
APGQGLEWMGWIIPLSGGAHYAQKFQGRVTITADESTSTA





YMELSSLRSEDTAVYYCARLYGWAEYFDVWGQGTLVTVS





SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV





SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK





TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP





SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY





VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK





EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE





MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV





LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY





TQKSLSLSLG




















TABLE 4







Amino acid sequence (N→C) or Nucleic acid



clone
region
sequence (5′→3′)
SEQ ID NO


















6
CDR-L1
RASQSISNYLN
13


 



CDR-L2
AASTLQS
14


 



CDR-L3
QQSYSFPWT
15


 



CDR-H1
SYTIS
16


 



CDR-H2
WISPELGTSNYAQKFQG
17


 



CDR-H3
LRYGQTLYGFDI
18


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
19



chain
GKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
20



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCA




coding
TCCACTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




gene
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG





CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQ
21



chain
APGQGLEWMGWISPELGTSNYAQKFQGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARLRYGQTLYGFDIWGQGTLVT




region
VSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
22



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACACCATTTCTTGGGTGCGG




region
CAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGGA




coding
TTTCTCCAGAACTGGGTACCTCTAACTATGCACAAAAATT




gene
CCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGACTGCGTTACGG





TCAGACTCTGTACGGTTTCGATATCTGGGGTCAGGGTAC





TCTGGTTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
23



chain
GKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQ
24



chain
APGQGLEWMGWISPELGTSNYAQKFQGRVTITADESTST





AYMELSSLRSEDTAVYYCARLRYGQTLYGFDIWGQGTLVT





VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV





TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG





TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAG





GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN





WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN





GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ





EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP





PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN





HYTQKSLSLSLG



















TABLE 5







Amino acid sequence (N→C) or Nucleic acid



clone
region
sequence (5′→3′)
SEQ ID NO


















7
CDR-L1
RASQSISNWLN
25


 



CDR-L2
GTSSLQS
26


 



CDR-L3
QQSYSFPFT
27


 



CDR-H1
SYGMH
28


 



CDR-H2
WIIPVSGGATYAQKFQG
29


 



CDR-H3
GSWAYYAEFDY
30


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNWLNWYQQK
31



chain
PGKAPKLLIYGTSSLQSGVPSRFSGSGSGTDFTLTISSLQP




variable
EDFATYYCQQSYSFPFTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
32



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTGGCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGGTACTT




coding
CCTCTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGGC




gene
AGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGC





CTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAAT





CTTACTCTTTTCCGTTTACGTTCGGGCAGGGAACTAAAG





TGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGMHWVR
33



chain
QAPGQGLEWMGWIIPVSGGATYAQKFQGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARGSWAYYAEFDYWGQGTLVT




region
VSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
34



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACGGTATGCATTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGG




coding
ATTATCCCAGTTTCTGGTGGTGCAACCTATGCACAAAAAT




gene
TCCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGGTTCTTGGGC





ATACTACGCTGAATTCGATTACTGGGGTCAGGGCACTTT





AGTGACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNWLNWYQQK
35



chain
PGKAPKLLIYGTSSLQSGVPSRFSGSGSGTDFTLTISSLQP




(Kappa)
EDFATYYCQQSYSFPFTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGMHWVR
36



chain
QAPGQGLEWMGWIIPVSGGATYAQKFQGRVTITADESTST





AYMELSSLRSEDTAVYYCARGSWAYYAEFDYWGQGTLVT





VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV





TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG





TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAG





GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN





WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN





GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ





EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP





PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN





HYTQKSLSLSLG




















TABLE 6







Amino acid sequence (N→C) or Nucleic acid



clone
region
sequence (5′→3′)
SEQ ID NO


















8
CDR-L1
RASQSISSYLN
37


 



CDR-L2
AASTLQS
38


 



CDR-L3
QQSYSFPYT
39


 



CDR-H1
SYGIH
40


 



CDR-H2
WIIPISGTTNYAQKFQG
41


 



CDR-H3
VGGVGLYVFDV
42


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP
43



chain
GKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPYTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
44



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTTCTTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCA




coding
TCCACTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




gene
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG





CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTTACTCTTTTCCGTACACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGIHWVRQ
45



chain
APGQGLEWMGWIIPISGTTNYAQKFQGRVTITADESTSTAY




variable
MELSSLRSEDTAVYYCARVGGVGLYVFDVWGQGTLVTVS




region
S



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
46



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACGGTATCCATTGGGTGCGG




region
CAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGGA




coding
TTATCCCAATCTCTGGTACCACCAACTATGCACAAAAATT




gene
CCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGTTGGTGGTGT





TGGTCTGTACGTTTTCGATGTTTGGGGTCAGGGTACTCT





GGTTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP
47



chain
GKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPYTFGQGTKVEIKRTVAAPSVFIFPPSD





EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE





SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL





SSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGIHWVRQ
48



chain
APGQGLEWMGWIIPISGTTNYAQKFQGRVTITADESTSTAY





MELSSLRSEDTAVYYCARVGGVGLYVFDVWGQGTLVTVS





SASTKGPSVFPLAPCSRSTSESTAALGQLVKDYFPEPVTV





SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK





TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP





SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY





VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK





EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE





MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV





LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY





TQKSLSLSLG



















TABLE 7







Amino acid sequence (N→C) or Nucleic acid



clone
region
sequence (5′→3′)
SEQ ID NO


















9
CDR-L1
RASQSISNYLN
49


 



CDR-L2
AASSLQS
50


 



CDR-L3
QQSYSFPWT
51


 



CDR-H1
SYAIH
52


 



CDR-H2
WIVPGLGVTNYAQKFQG
53


 



CDR-H3
QATLYQTEYMDV
54


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
55



chain
GKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
56



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCA




coding
TCCTCTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




gene
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG





CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIHWVRQ
57



chain
APGQGLEWMGWIVPGLGVTNYAQKFQGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARQATLYQTEYMDVWGQGTLV




region
TVSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
58



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACGCAATCCATTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGG




coding
ATTGTTCCAGGTCTGGGTGTTACCAACTATGCACAAAAA




gene
TTCCAAGGCCGCGTAACTATTACCGCCGACGAATCAACC





TCCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGA





AGACACGGCCGTCTATTATTGCGCCAGACAGGCAACTC





TGTACCAGACTGAATACATGGATGTTTGGGGTCAGGGTA





CTCTGGTTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
59



chain
GKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIHWVRQ
60



chain
APGQGLEWMGWIVPGLGVTNYAQKFQGRVTITADESTST





AYMELSSLRSEDTAVYYCARQATLYQTEYMDVWGQGTLV





TVSSASTKGPSVFPLAPCSRSTSESTAALGQLVKDYFPEP





VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL





GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAA





GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF





NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL





NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS





QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT





TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL





HNHYTQKSLSLSLG



















TABLE 8







Amino acid sequence (N→C) or Nucleic acid



clone
region
sequence (5′→3′)
SEQ ID NO


















10
CDR-L1
RASQSISNYLN
61


 



CDR-L2
AASNLQS
62


 



CDR-L3
QQSYSFPFT
63


 



CDR-H1
SHYMH
64


 



CDR-H2
WISPYLGSTNYAQKFQG
65


 



CDR-H3
DETGSTYGAFDY
66


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
67



chain
GKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPFTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
68



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCA




coding
TCCAATCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGGC




gene
AGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGC





CTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAAT





CTTACTCTTTTCCGTTTACGTTCGGGCAGGGAACTAAAG





TGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHYMHWVR
69



chain
QAPGQGLEWMGWISPYLGSTNYAQKFQGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARDETGSTYGAFDYWGQGTL




region
VTVSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
70



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTCATTACATGCATTGGGTGCGG




region
CAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGGA




coding
TTTCTCCATACCTGGGTTCTACCAACTATGCACAAAAATT




gene
CCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGATGAAACTGGT





TCTACTTACGGTGCATTCGATTACTGGGGTCAGGGTACT





CTGGTTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
71



chain
GKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPFTFGQGTKVEIKRTVAAPSVFIFPPSD





EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE





SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL





SSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSHYMHWVR
72



chain
QAPGQGLEWMGWISPYLGSTNYAQKFQGRVTITADESTS





TAYMELSSLRSEDTAVYYCARDETGSTYGAFDYWGQGTL





VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE





PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS





LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEA





AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ





FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW





LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP





SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK





TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA





LHNHYTQKSLSLSLG



















TABLE 9







Amino acid sequence (N→C) or Nucleic acid



clone
region
sequence (5′→3′)
SEQ ID NO


















11
CDR-L1
RASQSISNYLN
73


 



CDR-L2
DASTLQS
74


 



CDR-L3
QQSYSFPWT
75


 



CDR-H1
SYYVH
76


 



CDR-H2
WISPYSGGTNYAQKFQG
77


 



CDR-H3
DYYVSAYGAFDY
78


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
79



chain
GKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
80



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGATGCAT




coding
CCACTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGGC




gene
AGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGC





CTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAAT





CTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAAA





GTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYVHWVR
81



chain
QAPGQGLEWMGWISPYSGGTNYAQKFQGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARDYYVSAYGAFDYWGQGTL




region
VTVSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
82



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACTACGTTCATTGGGTGCGG




region
CAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGGA




coding
TTTCTCCATACTCTGGTGGTACCAACTATGCACAAAAATT




gene
CCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGATTACTACGTT





TCTGCATACGGTGCATTCGATTACTGGGGTCAGGGTACT





CTGGTTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
83



chain
GKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYVHWVR
84



chain
QAPGQGLEWMGWISPYSGGTNYAQKFQGRVTITADESTS





TAYMELSSLRSEDTAVYYCARDYYVSAYGAFDYWGQGTL





VTVSSASTKGPSVFPLAPCSRSTSESTAALGQLVKDYFPE





PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS





LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEA





AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ





FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW





LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP





SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK





TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA





LHNHYTQKSLSLSLG



















TABLE 10







Amino acid sequence (N→C) or Nucleic acid



clone
region
sequence (5′→3′)
SEQ ID NO


















12
CDR-L1
RASQDISNYLN
85


 



CDR-L2
ATSSLQS
86


 



CDR-L3
QQSYSFPWT
87


 



CDR-H1
SYDIH
88


 



CDR-H2
RIVPYLGVTNYAQKFQG
89


 



CDR-H3
RQSQSSVYAFDI
90


 



light
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKP
91



chain
GKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
92



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGGATATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAACT




coding
TCCTCTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




gene
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG





CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYDIHWVRQ
93



chain
APGQGLEWMGRIVPYLGVTNYAQKFQGRVTITADESTSTA




variable
YMELSSLRSEDTAVYYCARRQSQSSVYAFDIWGQGTLVTV




region
SS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
94



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACGATATCCATTGGGTGCGG




region
CAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGCGTA




coding
TTGTTCCATACCTGGGTGTTACCAACTATGCACAAAAATT




gene
CCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGACGTCAGTCTCA





GTCTTCTGTTTACGCATTCGATATCTGGGGTCAGGGCAC





TTTAGTGACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKP
95



chain
GKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYDIHWVRQ
96



chain
APGQGLEWMGRIVPYLGVTNYAQKFQGRVTITADESTSTA





YMELSSLRSEDTAVYYCARRQSQSSVYAFDIWGQGTLVTV





SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT





VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT





KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGG





PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW





YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG





KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE





EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP





VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH





YTQKSLSLSLG



















TABLE 11







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO


















13
CDR-L1
RASQSISNYLN
97


 



CDR-L2
AASRLQS
98


 



CDR-L3
QQSYSFPFT
99


 



CDR-H1
GYYIH
100


 



CDR-H2
WISPSSGGTIYAQKFQG
101


 



CDR-H3
DISVRVVQAFDY
102


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
103



chain
GKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQP




variable
EDFATYYCQQSYSFPFTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
104



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCA




coding
TCCCGTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




gene
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG





CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTTACTCTTTTCCGTTTACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSGYYIHWVRQ
105



chain
APGQGLEWMGWISPSSGGTIYAQKFQGRVTITADESTSTA




variable
YMELSSLRSEDTAVYYCARDISVRVVQAFDYWGQGTLVTV




region
SS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
106



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTGGTTACTACATCCATTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGG




coding
ATTTCTCCATCTTCTGGTGGTACCATCTATGCACAAAAAT




gene
TCCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGATATCTCTGTT





CGTGTTGTTCAGGCATTCGATTACTGGGGTCAGGGTACT





CTGGTTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
107



chain
GKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPFTFGQGTKVEIKRTVAAPSVFIFPPSD





EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE





SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL





SSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSGYYIHWVRQ
108



chain
APGQGLEWMGWISPSSGGTIYAQKFQGRVTITADESTSTA





YMELSSLRSEDTAVYYCARDISVRVVQAFDYWGQGTLVTV





SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT





VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT





KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGG





PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW





YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG





KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE





EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP





VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH





YTQKSLSLSLG



















TABLE 12







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







14
CDR-L1
RASQSISNYLN
109


 



CDR-L2
ATSNLQS
110


 



CDR-L3
QQSYSFPWT
111


 



CDR-H1
SYYMH
112


 



CDR-H2
WISPYLGITNYAQKFQG
113


 



CDR-H3
AGYQQAQYWFDY
114


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
115



chain
GKAPKLLIYATSNLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
116



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAACT




coding
TCCAATCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGGC




gene
AGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGC





CTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAAT





CTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAAA





GTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYMHWVR
117



chain
QAPGQGLEWMGWISPYLGITNYAQKFQGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARAGYQQAQYWFDYWGQGTL




region
VTVSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
118



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACTACATGCATTGGGTGCGG




region
CAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGGA




coding
TTTCTCCATACCTGGGTATCACCAACTATGCACAAAAATT




gene
CCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGCAGGTTACCA





GCAGGCACAGTACTGGTTCGATTACTGGGGTCAGGGCA





CTTTAGTGACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
119



chain
GKAPKLLIYATSNLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYMHWVR
120



chain
QAPGQGLEWMGWISPYLGITNYAQKFQGRVTITADESTST





AYMELSSLRSEDTAVYYCARAGYQQAQYWFDYWGQGTL





VTVSSASTKGPSVFPLAPCSRSTSESTAALGQLVKDYFPE





PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS





LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEA





AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ





FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW





LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP





SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK





TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA





LHNHYTQKSLSLSLG




















TABLE 13







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







15
CDR-L1
RASQSISNYLN
121


 



CDR-L2
ATSSLQS
122


 



CDR-L3
QQSYSFPYT
123


 



CDR-H1
SYAMS
124


 



CDR-H2
WIIPISGTTNYAQKFQG
125


 



CDR-H3
QHSVGSVFDY
126


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
127



chain
GKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPYTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
128



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAACT




coding
TCCTCTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




gene
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG





CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTTACTCTTTTCCGTACACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVR
129



chain
QAPGQGLEWMGWIIPISGTTNYAQKFQGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARQHSVGSVFDYWGQGTLVTV




region
SS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
130



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACGCAATGTCTTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGG




coding
ATTATCCCAATCTCTGGTACCACCAACTATGCACAAAAAT




gene
TCCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGACAGCATTCTGTT





GGTTCTGTTTTCGATTACTGGGGTCAGGGTACTCTGGTT





ACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
131



chain
GKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPYTFGQGTKVEIKRTVAAPSVFIFPPSD





EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE





SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL





SSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVR
132



chain
QAPGQGLEWMGWIIPISGTTNYAQKFQGRVTITADESTST





AYMELSSLRSEDTAVYYCARQHSVGSVFDYWGQGTLVTV





SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT





VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT





KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGG





PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW





YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG





KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE





EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP





VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH





YTQKSLSLSLG



















TABLE 14







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







16
CDR-L1
RASQDISSWLN
133


 



CDR-L2
AASSLQS
134


 



CDR-L3
QQSYSFPWT
135


 



CDR-H1
SYYMT
136


 



CDR-H2
GISPILGVTNYAQKFQG
137


 



CDR-H3
LLVGVSETYFDY
138


 



light
DIQMTQSPSSLSASVGDRVTITCRASQDISSWLNWYQQK
139



chain
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP




variable
EDFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
140



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGGATATCTCTTCTTGGCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCA




coding
TCCTCTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




gene
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG





CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYMTWVR
141



chain
QAPGQGLEWMGGISPILGVTNYAQKFQGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARLLVGVSETYFDYWGQGTLVT




region
VSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
142



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACTACATGACCTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGGGT




coding
ATTTCTCCAATCCTGGGTGTTACCAACTATGCACAAAAAT




gene
TCCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGACTGCTGGTTGG





TGTTTCTGAAACTTACTTCGATTACTGGGGTCAGGGTAC





TCTGGTTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQDISSWLNWYQQK
143



chain
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP




(Kappa)
EDFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYMTWVR
144



chain
QAPGQGLEWMGGISPILGVTNYAQKFQGRVTITADESTST





AYMELSSLRSEDTAVYYCARLLVGVSETYFDYWGQGTLVT





VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV





TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG





TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAG





GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN





WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN





GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ





EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP





PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN





HYTQKSLSLSLG



















TABLE 15







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







17
CDR-L1
RASQSISNYLN
145


 



CDR-L2
AASNMHS
146


 



CDR-L3
QQSHSFPWT
147


 



CDR-H1
TYAMS
148


 



CDR-H2
GISPTLGIANYAQKFQG
149


 



CDR-H3
VRYAGWTGYFDL
150


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
151



chain
GKAPKLLIYAASNMHSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSHSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
152



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCA




coding
TCCAATATGCACTCTGGTGTACCGTCCCGTTTCTCTGGC




gene
AGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGC





CTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAAT





CTCACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAAA





GTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAMSWVR
153



chain
QAPGQGLEWMGGISPTLGIANYAQKFQGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARVRYAGWTGYFDLWGQGTLV




region
TVSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
154



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTACCTACGCAATGTCTTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGGGT




coding
ATTTCTCCAACCCTGGGTATCGCAAACTATGCACAAAAAT




gene
TCCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGTTCGTTACGCA





GGTTGGACTGGTTACTTCGATCTGTGGGGTCAGGGTAC





TCTGGTTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
155



chain
GKAPKLLIYAASNMHSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSHSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAMSWVR
156



chain
QAPGQGLEWMGGISPTLGIANYAQKFQGRVTITADESTST





AYMELSSLRSEDTAVYYCARVRYAGWTGYFDLWGQGTLV





TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP





VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL





GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAA





GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF





NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL





NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS





QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT





TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL





HNHYTQKSLSLSLG



















TABLE 16







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







18
CDR-L1
RASQSISRWLN
157


 



CDR-L2
AASRLQS
158


 



CDR-L3
QQSESFPWT
159


 



CDR-H1
SYDIN
160


 



CDR-H2
WIIPTSGSTNYAQKFQG
161


 



CDR-H3
DSQSSYIGYFDV
162


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLNWYQQKP
163



chain
GKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSESFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
164



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTCGTTGGCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCA




coding
TCCCGTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




gene
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG





CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTGAATCTTTTCCGTGGACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYDINWVRQ
165



chain
APGQGLEWMGWIIPTSGSTNYAQKFQGRVTITADESTSTA




variable
YMELSSLRSEDTAVYYCARDSQSSYIGYFDVWGQGTLVTV




region
SS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
166



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTACGATATCAACTGGGTGCGG




region
CAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTGGA




coding
TTATCCCAACCTCTGGTTCTACCAACTATGCACAAAAATT




gene
CCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGATTCTCAGTCT





TCTTACATCGGTTACTTCGATGTTTGGGGTCAGGGTACT





CTGGTTACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLNWYQQKP
167



chain
GKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSESFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYDINWVRQ
168



chain
APGQGLEWMGWIIPTSGSTNYAQKFQGRVTITADESTSTA





YMELSSLRSEDTAVYYCARDSQSSYIGYFDVWGQGTLVTV





SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT





VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT





KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGG





PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW





YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG





KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE





EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP





VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH





YTQKSLSLSLG



















TABLE 17







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







19
CDR-L1
RASQSISNYLN
169


 



CDR-L2
DTSSLQS
170


 



CDR-L3
QQSYSTPYT
171


 



CDR-H1
AYGIS
172


 



CDR-H2
RIIPYLGTANYAQKFQG
173


 



CDR-H3
LSYGIGYESFDV
174


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
175



chain
GKAPKLLIYDTSSLQSGVPSRFSGSGSGTDFTLTISSLQP




variable
EDFATYYCQQSYSTPYTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
176



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGATACTT




coding
CCTCTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGGC




gene
AGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGC





CTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAAT





CTTACTCTACTCCGTACACGTTCGGGCAGGGAACTAAAG





TGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYGISWVRQ
177



chain
APGQGLEWMGRIIPYLGTANYAQKFQGRVTITADESTSTAY




variable
MELSSLRSEDTAVYYCARLSYGIGYESFDVWGQGTLVTVS




region
S



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
178



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTGCATACGGTATCTCTTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGCGT




coding
ATTATCCCATACCTGGGTACCGCAAACTATGCACAAAAAT




gene
TCCAAGGCCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGACTGTCTTACGGT





ATCGGTTACGAATCTTTCGATGTTTGGGGTCAGGGCACT





TTAGTGACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
179



chain
GKAPKLLIYDTSSLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSTPYTFGQGTKVEIKRTVAAPSVFIFPPSD





EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE





SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL





SSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYGISWVRQ
180



chain
APGQGLEWMGRIIPYLGTANYAQKFQGRVTITADESTSTAY





MELSSLRSEDTAVYYCARLSYGIGYESFDVWGQGTLVTVS





SASTKGPSVFPLAPCSRSTSESTAALGQLVKDYFPEPVTV





SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK





TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP





SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY





VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK





EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE





MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV





LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY





TQKSLSLSLG



















TABLE 18







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







20
CDR-L1
RASQSISSYLN
181


 



CDR-L2
DTSTLQS
182


 



CDR-L3
QQSYSFPWT
183


 



CDR-H1
SYAMS
184


 



CDR-H2
SISSSGGSTYYADSVKG
185


 



CDR-H3
ELGGYGFSYFDY
186


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP
187



chain
GKAPKLLIYDTSTLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
188



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTTCTTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGATACTT




coding
CCACTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGGC




gene
AGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGC





CTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAAT





CTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAAA





GTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVR
189



chain
QAPGQGLEWMGSISSSGGSTYYADSVKGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARELGGYGFSYFDYWGQGTLV




region
TVSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
190



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTATGCAATGTCTTGGGTGCGG




region
CAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGTCTA




coding
TCTCTTCTTCTGGTGGTTCTACTTACTATGCCGATTCAGT




gene
GAAGGGTCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGAACTGGGTGG





TTACGGTTTCTCTTACTTCGATTACTGGGGTCAGGGCAC





TTTAGTGACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP
191



chain
GKAPKLLIYDTSTLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMSWVR
192



chain
QAPGQGLEWMGSISSSGGSTYYADSVKGRVTITADESTST





AYMELSSLRSEDTAVYYCARELGGYGFSYFDYWGQGTLV





TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP





VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL





GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAA





GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF





NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL





NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS





QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT





TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL





HNHYTQKSLSLSLG



















TABLE 19







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







21
CDR-L1
RASQSIRNYLN
193


 



CDR-L2
ATSSLQS
194


 



CDR-L3
QQSYSFPWT
195


 



CDR-H1
DYAMS
196


 



CDR-H2
GISGSDIYYADSVKG
197


 



CDR-H3
AVSYWSYTFDY
198


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSIRNYLNWYQQKP
199



chain
GKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
200



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCCGTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAACT




coding
TCCTCTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGG




y
CAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAG




gene
CCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA





ATCTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAA





AGTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAMSWVR
201



chain
QAPGQGLEWMGGISGSDIYYADSVKGRVTITADESTSTAY




variable
MELSSLRSEDTAVYYCARAVSYWSYTFDYWGQGTLVTVS




region
S



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
202



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTGATTATGCAATGTCTTGGGTGCGG




region
CAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGGGTA




coding
TCTCTGGTTCTGATATCTACTATGCCGATTCAGTGAAGG




gene
GTCGCGTAACTATTACCGCCGACGAATCAACCTCCACCG





CCTACATGGAACTCAGCTCTCTGAGGTCAGAAGACACG





GCCGTCTATTATTGCGCCAGAGCAGTTTCTTACTGGTCT





TACACTTTTGATTACTGGGGTCAGGGCACTTTAGTGACC





GTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSIRNYLNWYQQKP
203



chain
GKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAMSWVR
204



chain
QAPGQGLEWMGGISGSDIYYADSVKGRVTITADESTSTAY





MELSSLRSEDTAVYYCARAVSYWSYTFDYWGQGTLVTVS





SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV





SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK





TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGP





SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY





VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK





EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE





MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV





LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY





TQKSLSLSLG



















TABLE 20







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







22
CDR-L1
RASQSIGSYLN
205


 



CDR-L2
DASTLQS
206


 



CDR-L3
QQSYSFPWT
207


 



CDR-H1
SYAMH
208


 



CDR-H2
GISSSGGTTYYADSVKG
209


 



CDR-H3
ALGVVGGTWFDY
210


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSIGSYLNWYQQKP
211



chain
GKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
212



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCGGTTCTTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGATGCAT




coding
CCACTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGGC




gene
AGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGC





CTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAAT





CTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAAA





GTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMHWVR
213



chain
QAPGQGLEWMGGISSSGGTTYYADSVKGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARALGVVGGTWFDYWGQGTL




region
VTVSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
214



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTTCTTATGCAATGCACTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGGGT




coding
ATCTCTTCTTCTGGTGGTACTACTTACTATGCCGATTCAG




gene
TGAAGGGTCGCGTAACTATTACCGCCGACGAATCAACCT





CCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAA





GACACGGCCGTCTATTATTGCGCCAGAGCACTGGGTGT





TGTTGGTGGTACTTGGTTCGATTACTGGGGTCAGGGCA





CTTTAGTGACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSIGSYLNWYQQKP
215



chain
GKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMHWVR
216



chain
QAPGQGLEWMGGISSSGGTTYYADSVKGRVTITADESTS





TAYMELSSLRSEDTAVYYCARALGVVGGTWFDYWGQGTL





VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE





PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS





LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEA





AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ





FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW





LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP





SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK





TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA





LHNHYTQKSLSLSLG



















TABLE 21







Amino acid sequence (N→C)
SEQ




or Nucleic acid sequence
ID


clone
region
(5′→3′)
NO







23
CDR-L1
RASQSISNYLN
217


 



CDR-L2
DTSTLQS
218


 



CDR-L3
QQSYSFPWT
219


 



CDR-H1
DYAMH
220


 



CDR-H2
AISGSGGYTHYADSVKG
221


 



CDR-H3
SATFGVWETFDV
222


 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
223



chain
GKAPKLLIYDTSTLQSGVPSRFSGSGSGTDFTLTISSLQPE




variable
DFATYYCQQSYSFPWTFGQGTKVEIK




region




 



light
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGC
224



chain
TAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTA




variable
GCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCAGA




region
AACCGGGCAAGGCGCCAAAATTGCTGATTTACGATACTT




coding
CCACTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGGC




gene
AGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGC





CTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAAT





CTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAAA





GTGGAAATTAAA



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAMHWVR
225



chain
QAPGQGLEWMGAISGSGGYTHYADSVKGRVTITADESTS




variable
TAYMELSSLRSEDTAVYYCARSATFGVWETFDVWGQGTL




region
VTVSS



 



heavy
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGA
226



chain
AGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCA




variable
GGCGGGACTTTTAGTGATTATGCAATGCACTGGGTGCG




region
GCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGGGCA




coding
ATCTCTGGTTCTGGTGGTTACACTCACTATGCCGATTCA




gene
GTGAAGGGTCGCGTAACTATTACCGCCGACGAATCAAC





CTCCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAG





AAGACACGGCCGTCTATTATTGCGCCAGATCTGCAACTT





TCGGTGTTTGGGAAACTTTCGATGTTTGGGGTCAGGGC





ACTTTAGTGACCGTCTCATCG



 



light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKP
227



chain
GKAPKLLIYDTSTLQSGVPSRFSGSGSGTDFTLTISSLQPE




(Kappa)
DFATYYCQQSYSFPWTFGQGTKVEIKRTVAAPSVFIFPPS





DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ





ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG





LSSPVTKSFNRGEC



 



heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAMHWVR
228



chain
QAPGQGLEWMGAISGSGGYTHYADSVKGRVTITADESTS





TAYMELSSLRSEDTAVYYCARSATFGVWETFDVWGQGTL





VTVSSASTKGPSVFPLAPCSRSTSESTAALGQLVKDYFPE





PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS





LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEA





AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ





FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW





LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP





SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK





TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA





LHNHYTQKSLSLSLG









EXAMPLE 3. PREPARATION OF SELECTED ANTIBODIES
The vectors constructed in Example 2 were prepared using the Plasmid Plus Maxi kit (Qiagen). These vectors were used for expressing antibodies in ExpiCHO-S™ cells. The vectors were transfected into ExpiCHO-S™ cells (Gibco) (1.2×109 cells/Culture Volume 200 mL) by adding 640 μL of ExpiFectamine™ CHO reagent (Thermo Fisher). One day post-transfection, the cells were incubated in ExpiCHO™ Expression Media (Thermo Fisher) under the condition of 32° C. and 5% CO2 for 7 to 11 days in total. On Day 1, 1200 μL of ExpiCHO™ Enhancer (Thermo Fisher) and 48 mL of ExpiCHO™ Feed (Thermo Fisher) were added to the culture.
The cultured cells were centrifuged at 3500 rpm at 4° C. for 20 minutes, and then, filtrated using 0.22 μm bottle-top filter system (Corning). The culture supernatant was harvested and purified using AKTA Pure L (GE healthcare). The culture supernatant was loaded into AKTA Pure L equipped with Hitrap MabSelectSure 5 mL column (GE healthcare) at the flow rate of 5 mL/min, and the column was washed with column volumes (CV) of 1×PBS. Then, elution buffer (0.1 M sodium citrate pH 3.4 buffer) was loaded to the column, to elute a protein of interest. The eluate was concentrated using Amicon Ultra Filter Device (MWCO 10K, Merck), centrifuged and subjected to buffer exchange with 1×PBS buffer.
The purified antibody samples were diluted with 1×PBS, to make the final concentration about 1 mg/mL. Ten (10) μL of Reducing Loading Buffer (3×) or Non-reducing Loading Buffer (3×) and 20 μL of the purified antibody sample were mixed and left in 95° C. heating bath for 2 minutes, and then, brought out and cooled. The sample was injected into SDS-PAGE Gradient Gel (4-12%) equipped on an electrophoresis device at the amount of 10 μg per well and developed on the gel. In order to analyze molecular weight of the sample, Precision Plus Protein™ Dual Color Standards (BIO-RAD) was injected to another separate well. The gel was stained with Coomassie staining solution and destained to obtain gel images (FIG. 1).
EXAMPLE 4. ANALYSIS OF BINDING AFFINITY OF THE SELECTED ANTIBODIES
The binding affinities of the 19 antibodies, which were selected in Example 3, to the antigen, LILRB1, were measured using Biacore T200 (GE healthcare). An anti-human IgG (Fc) antibody (GE healthcare, Cat. No. BR-1008-39, final concentration of 25 μg/mL) was flowed at the flow rate of 5 μL/min for 360 seconds to be immobilized at 5000-7000 RU on Series S Sensor Chip CM5 (GE healthcare, Cat. No. BR-1005-30) using Amine Coupling Kit (GE healthcare, Cat. No. BR-1000-50). The antigen, human LILRB1 protein (LILRB1-His, RnD systems Cat. No. 8989-T2) was injected thereto in 5 different concentrations from 25 nM to 400 nM at the flow rate of 30 μL/min to determine ka and kd values as shown in Table 22 and calculate KD value therefrom. Antibody No. 10 showed a binding affinity (KD) of about 24.13 nM to the LILRB1 antigen, and antibody No. 13 showed a binding affinity (KD) of about 30.27 nM to the LILRB1 antigen (Table 22). The sensorgram results for the antibody No. 13 are shown in FIG. 2.







TABLE 22







Antigen Binding Affinities (KD) of LILRB1 antibodies










Clone number
ka (× 105) (1/Ms)
kd (× 10−4) (1/s)
KD (nM)













8
0.6166
46.37
75.2


10
0.1233
2.977
24.13


11
0.08662
1.061
12.25


13
0.9729
2.945
30.27


14
1.621
663.1
409.1


16
1.157
96.35
83.3


18
1.439
6.221
4.32


22
0.6826
340.8
499.3









EXAMPLE 5. ASSAY OF IN VITRO BIOLOGICAL ACTIVITIES OF THE SELECTED ANTIBODIES
5.1. Cell Surface Binding Assay
In order to test whether or not the antibodies selected in Example 4 bind LILRB1 expressed on surface of cells, cell surface binding assay was performed. CHO cells overexpressing LILRB1 were cultured in Chemical Defined Medium, and added to a U-bottomed 96-well tissue culture plate (BD Falcon) to 2×105 cells/well. Each of the selected antibodies was added to the well to the final concentration of 10 μg/mL per well, and incubated at 4° C. for 30 minutes. In order to see the level of LILRB1-specific binding of the selected antibodies, a human IgG4 isotype control antibody (Biolegend) was treated in the same manner. After washing with FACS buffer, the cells were treated with an anti-human Fc-biotin antibody (life technologies) and incubated at 4° C. for 1 hour. After washing with FACS buffer, streptavidin PE (BD Pharmigen) was added to each well and left at 4° C. for 30 minutes. After washing with FACS buffer, it was suspended and analyzed by iQue screener (Sartorius). As shown in FIG. 3, the No. 8, No. 10, No. 11, No. 13, and No. 18 antibodies showed a higher level of binding than that of the human IgG4 isotype control antibody.
5.2. Analysis of Increased Cancer Cell Killing Ability by Natural Killer (NK) Cells
In order to determine whether the antibodies selected in Example 4 increase the degree of cancer cell lysis by NK cells, the cell death rate of HLA-G-overexpressing HEK293 cell by NK cell KHYG-1 was analyzed. KHYG-1 cells (JCRB) were added to 96-well tissue culture plate (BD Falcon) at the amount of 4×105 cells/well (2×104 cells/mL). The selected antibody was added to the well to the final concentration of 10 μg/mL and incubated at 37° C. for one hour. As a negative control, a human IgG4 isotype control antibody (Biolegend) was treated in the same manner. HLA-G-overexpressing HEK293 cells were stained in a separate tube with IncuCyte CytoLight Rapid Red Reagent (Sartorius) according to the manufacturer's protocol. After one hour, the HLA-G-overexpressing HEK293 cells were added to the plate at the amount of 4×105 cells/well (2×104 cells/mL). The plate was placed in IncuCyte S3 (Sartorius) equipped in an incubator under the condition of 37° C. and 5% CO2, and images thereof were taken for 72 hours.
For comparison of the efficacy of each antibody, the normalized red area confluence value of the isotype control was converted to 1 to obtain the relative cell viability (Isotype=1) as shown in Equation 1 below.

Relative cell viability=[Normalized red area confluence value of antibody]/[Normalized red area confluence value of Isotype]  [Equation 1]

The obtained results are shown in FIG. 4. In FIG. 4, it can be interpreted that the lower the relative cell viability, the higher the NK cell mediated cytotoxicity by the anti-LILRB1 mAb. As shown in FIG. 4, all of the tested antibodies (antibody No. 10, No. 11, and No. 13) increased cell death of HLA-G-overexpressing HEK293 cells compared to human IgG4 isotype control. These results show that the antibody provided in the present invention exhibits high cytotoxicity against cancer cells.
EXAMPLE 6: ASSAY OF IN VIVO BIOLOGICAL ACTIVITIES OF THE TEST ANTIBODIES
Three antibodies (antibody No. 10, No. 11, and No. 13) whose binding ability to the antigen was confirmed in Example 3 were tested for their in vivo anti-cancer efficacies. For this purpose, it was tested whether or not the administration of three types of antibodies reduces the size of tumor where the tumor was generated by engrafting human colorectal carcinoma cells (Bioware Brite Cell Line HCT116 Red-Fluc colorectal cancer cells (PerkinElmer)) and THP-1 derived macrophage to the mice. As a negative control, human colon cancer xenograft mice prepared as above were treated with a human IgG1 isotype control antibody (BioXcell, Cat. No. BP0297).
6.1. Preparation of THP-1 Derived Macrophages
The THP-1 derived macrophages used above were prepared by differentiating THP-1 cells (ATCC) with 150 nM phorbol 12-myristate 13-acetate (PMA, Sigma), 20 ng/mL of interferon gamma (Peprotech) and 10 pg/mL of lipopolysaccharide (LPS, Sigma).
6.2. Measurement of Anti-Cancer Efficacy in Mouse Model
Five (5)-week-old female CIEA NOG Mouse (NOG immunodeficient mouse, Central Institute of Experimental Animals, Japan) were subcutaneously injected with a mixture of 3×106 cells of HCT116 Red-Fluc colorectal cancer cells, 3×106 cells of THP-1 derived macrophages and each of three antibodies (20 μg per mouse). From the 4th day after cell transplantation, each antibody was administered twice a week at the dosage of 5 mg/kg by intraperitoneal injection.
The change in tumor volume according to the administration of the antibody was measured and shown in FIG. 5. As shown in FIG. 5, all the tested antibodies (antibody No. 10, No. 11, and No. 13) showed statistically significant effect of inhibiting tumor growth in the mouse model transplanted with HCT116 colorectal cancer cells and THP-1 derived macrophages.