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, provided are an anti-LILRB1 antibody or antigen-binding fragment thereof, and uses thereof in treating cancer.

1. 11. An anti-LILRB1 antibody or an 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: 7, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; (3) 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; (4) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (5) 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; (6) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; (7) 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; (8) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 46, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 47, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 48; (9) 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; (10) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 60; (11) 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; (12) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 68, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 69, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 71, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (13) 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; (14) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 83, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 84; (15) 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; (16) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 91, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 92, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 93, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 94, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 95, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 96; (17) 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; (18) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 103, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 104, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 105, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 106, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 107, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 108; (19) 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; or (20) the CDR-L1 comprising the amino acid sequence of SEQ ID NO: 115, the CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 117, the CDR-H1 comprising the amino acid sequence of SEQ ID NO: 118, the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 119, and the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120.
2. 22. The anti-LILRB1 antibody or an antigen-binding fragment thereof of claim 1, comprising: a light chain variable region and a heavy chain variable region comprising: a) SEQ ID NOs: 221 and 222, b) SEQ ID NOs: 223 and 224, c) SEQ ID NOs: 225 and 226, d) SEQ ID NOs: 227 and 228, e) SEQ ID NOs: 233 and 234, f) SEQ ID NOs: 235 and 236, g) SEQ ID NOs: 231 and 232, h) SEQ ID NOs: 237 and 238, i) SEQ ID NOs: 229 and 230, j) SEQ ID NOs: 239 and 240, k) SEQ ID NOs: 241 and 242, l) SEQ ID NOs: 243 and 244, m) SEQ ID NOs: 245 and 246, n) SEQ ID NOs: 247 and 248, o) SEQ ID NOs: 249 and 250, p) SEQ ID NOs: 251 and 252, q) SEQ ID NOs: 253 and 254, r) SEQ ID NOs: 255 and 256, s) SEQ ID NOs: 257 and 258, t) SEQ ID NOs: 259 and 260, or u) SEQ ID NOs: 345 and 222, respectively.
3. 33. The anti-LILRB1 antibody or an antigen-binding fragment thereof of claim 1, wherein the antibody is a human IgG1 or IgG4 antibody.
4. 44. The anti-LILRB1 antibody or an antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is a scFv, (scFv)2, Fab, Fab′, F(ab′)2, a fusion polypeptide comprising a scFv fused with an immunoglobulin Fc, or a fusion polypeptide comprising 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 an antigen-binding fragment thereof of any one of claim 1, and a pharmaceutically acceptable carrier.
6. 66. The pharmaceutical composition of claim 5, wherein the cancer is characterized by overexpression of MHC Class I.
7. 77. A nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof of claim 1.
8. 88. A recombinant vector comprising the nucleic acid molecule of claim 7.
9. 99. A recombinant cell comprising the recombinant vector of claim 8.
10. 1010. A method of preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof, comprising culturing the recombinant cell of claim 9.
11. 1111. The pharmaceutical composition of claim 6, wherein the cancer is colon adenocarcinoma, small cell lung carcinoma, breast cancer, pancreatic cancer, malignant melanoma, bone osteosarcoma, renal cell carcinoma, or gastric cancer.
12. 1212. A method for treating a cancer, comprising administering to a subject in need thereof a pharmaceutically effective amount of the anti-LILRB1 antibody or an antigen-binding fragment thereof of claim 1.
13. 1313. The method of claim 12, wherein the cancer is characterized by overexpression of MHC Class I.
14. 1414. The method of claim 13, wherein the cancer is colon adenocarcinoma, small cell lung carcinoma, breast cancer, pancreatic cancer, malignant melanoma, bone osteosarcoma, renal cell carcinoma, or gastric cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 National Phase Entry Application from PCT/KR2020/018931 filed on Dec. 22, 2020, which claims the benefits of KR 10-2019-0173414 filed on Dec. 23, 2019 and KR 10-2020-0061907 filed on May 22, 2020 with the Korean Intellectual Property Office, the entire disclosures of which are herein incorporated by reference.
TECHNICAL FIELD
The disclosure relates to an anti-LILRB1 antibody and uses thereof. More specifically, an anti-LILRB1 antibody or an antigen-binding fragment thereof, and a use thereof for cancer therapy are provided.
The present application includes a Sequence Listing filed in electronic format. The Sequence Listing is entitled “3570-819_ST25.txt” created on Dec. 21, 2022 and is 292,811 bytes in size. The information in the electronic format of the Sequence Listing is part of the present application and is incorporated herein by reference in its entirety.
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.
Meanwhile, it has been reported that various cancer cells overexpress MHC class I such as HLA-G for immune evasion. It has been expected that blocking the binding of LILRB1 to MHC Class I allows recovery of the inhibited activities of immune cells, thereby exhibiting anti-cancer effects.
Therefore, it is required to develop novel agent binding to LILRB1 and blocking the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I.
BRIEF SUMMARY OF THE INVENTION
This disclosure provides antibodies, which bind to LILRB1, act on LILRB1-expressing immune cells, regulate activities of the immune cells, and exhibit anti-cancer effects, and uses thereof for cancer therapies.
An embodiment provides an anti-LILRB1 antibody, which binds to LILRB1, or an antigen-binding fragment thereof. The anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity to block the binding of LILRB1 to MHC Class I and/or blocking the interaction between LILRB1 and MHC Class I. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity to inhibit immune evasion of cancer cells. Furthermore, the anti-LILRB1 antibody or an 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 its cell surface.
Another embodiment provides a pharmaceutical composition for treatment and/or prevention of a cancer, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
Another embodiment provides a pharmaceutical composition for inhibition of binding of LILRB1 to MHC Class I and/or blocking the interaction between LILRB1 and MHC Class I, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
Another embodiment provides a pharmaceutical composition for inhibiting immune evasion of cancer cell, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
DETAILED DESCRIPTION OF THE INVENTION
An embodiment provides an anti-LILRB1 antibody, which binds to LILRB1, or an antigen-binding fragment thereof. The anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity to block the binding of LILRB1 to MHC Class I and/or blocking the interaction between LILRB1 and MHC Class I. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity to inhibit immune evasion of cancer cells. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an anti-cancer effect.
The anti-LILRB1 antibody or an antigen-binding fragment thereof may comprise the following complementarity determining regions (CDRs):

    
    
        (1) 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, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, or 115,
        a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, or 116,
        a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, or 117,
        a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, or 118,
        a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, or 119, and
        a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, or 120; or
        (2) based on the CDR definition according to IMGT numbering (http://www.imgt.org/),
        a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 206, 211, or 216,
        a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 122, 127, 132, 137, 142, 147, 152, 157, 162, 167, 172, 177, 182, 187, 192, 197, 202, 207, 212, or 217,
        a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, or 117,
        a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 123, 128, 133, 138, 143, 148, 153, 158, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 213, or 218,
        a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 124, 129, 134, 139, 144, 149, 154, 159, 164, 169, 174, 179, 184, 189, 194, 199, 204, 209, 214, or 219, and
        a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or 220.
    
    


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 an antigen-binding fragment thereof provided in this disclosure are illustrated in Table 1:












TABLE 1







Amino Acid
SEQ
Amino Acid
SEQ




Sequence
ID
Sequence
ID



CDR
(N→C)(Kabat)
NO
(N→C)(IMGT)
NO




















E3/
CDR-L1
QGDSLRNFYAS
1
SLRNFY
121


E3.1
CDR-L2
GKNNRPS
2
GKN
122



CDR-L3
NSRDSSGSHLTGV
3
NSRDSSGSHLTG
3






V




CDR-H1
SYAMS
4
GFTFSSYA
123



CDR-H2
AISGSGGSTYYADSVKG
5
ISGSGGST
124



CDR-H3
DTYYYGSGRSNAFDI
6
ARDTYYYGSGRS
125






NAFDI



 


B3
CDR-L1
QASQDISNYLN
7
QDISNY
126



CDR-L2
DASNLET
8
DAS
127



CDR-L3
QQYDNLP
9
QQYDNLP
9



CDR-H1
DYAMH
10
GFTFDDYA
128



CDR-H2
GISWNSGSIGYADSVKG
11
ISWNSGSI
129



CDR-H3
VGDSSGWSDAFDI
12
ARVGDSSGWSD
130






AFDI



 


A10
CDR-L1
RASQSVSSNLA
13
QSVSSN
131



CDR-L2
GASTRAT
14
GAS
132



CDR-L3
QQYGSSPRMYT
15
QQYGSSPRMYT
15



CDR-H1
SYAIS
16
GGTFSSYA
133



CDR-H2
GIIPIFGTANYAQKFQG
17
IIPIFGTA
134



CDR-H3
GGLGELDNWFDP
18
ARGGLGELDNWF
135






DP



 


G1
CDR-L1
SGYKLGDRYVS
19
KLGDRY
136



CDR-L2
KDSQRPS
20
KDS
137



CDR-L3
QAWDSGTGV
21
QAWDSGTGV
21



CDR-H1
SYGIS
22
GGTFSSYG
138



CDR-H2
WISAYNGNTNYAQELQ
23
ISAYNGNT
139




G






CDR-H3
VGVAGKLDY
24
ARVGVAGKLDY
140


 


G9
CDR-L1
TGSSSDVGGYNYVS
25
SSDVGGYNY
141



CDR-L2
DVSNRPS
26
DVS
142



CDR-L3
SSYTGSSTLDVL
27
SSYTGSSTLDVL
27



CDR-H1
SYWIG
28
GYSFTSYW
143



CDR-H2
IIYPGDSDTRYSPSFQG
29
IYPGDSDT
144



CDR-H3
QYYDGGYYMDV
30
ASQYYDGGYYM
145






DV



 


H2
CDR-L1
QGDSLRNYYAS
31
SLRNYY
146



CDR-L2
GNNKRPS
32
GNN
147



CDR-L3
NSLDSTYNHPI
33
NSLDSTYNHPI
33



CDR-H1
SYDIH
34
GYTFTSYD
148



CDR-H2
WISAYNGNTNYAQKLQ
35
ISAYNGNT
149




G






CDR-H3
DGGDAFDI
36
ARDGGDAFDI
150


 


H11
CDR-L1
QGDSLRSYYAS
37
SLRSYY
151



CDR-L2
GRNNRPS
38
GRN
152



CDR-L3
KSRDSSGNHYV
39
KSRDSSGNHYV
39



CDR-H1
SYYMH
40
GYTFTSYY
153



CDR-H2
IINPSGGSTSYAQKFQG
41
INPSGGST
154



CDR-H3
DAGSSSDY
42
ARDAGSSSDY
155


 


F12
CDR-L1
AGTSSDIGDYDYVS
43
SSDIGDYDY
156



CDR-L2
DVSRRPS
44
DVS
157



CDR-L3
ASYTSSSVVV
45
ASYTSSSVVV
45



CDR-H1
SYWIG
46
GYSFTSYW
158



CDR-H2
IIYPGDSDTRYSPSFQG
47
IYPGDSDT
159



CDR-H3
QYYDGGYYMDV
48
ASQYYDGGYYM
160






DV



 


B9
CDR-L1
RASQSISRYLN
49
QSISRY
161



CDR-L2
GASSLQS
50
GAS
162



CDR-L3
QQAYGFPLT
51
QQAYGFPLT
51



CDR-H1
SYAIS
52
GGTFSSYA
163



CDR-H2
GIIPIFGTANYAQKFQG
53
IIPIFGTA
164



CDR-H3
GEIAVAQNWDYYGMDV
54
ARGEIAVAQNWD
165






YYGMDV



 


G11
CDR-L1
TGTSSDVGGYNYVS
55
SSDVGGYNY
166



CDR-L2
DVSKRPS
56
DVS
167



CDR-L3
SSYSSSSTLVV
57
SSYSSSSTLVV
57



CDR-H1
SYWIG
58
GYSFTSYW
168



CDR-H2
IIYPGDSDTRYSPSFQG
59
IYPGDSDT
169



CDR-H3
QYYDGGYYMDV
60
ASQYYDGGYYM
170






DV



 


G6
CDR-L1
QGDSLRRYYAT
61
SLRRYY
171



CDR-L2
GQNYRPS
62
GQN
172



CDR-L3
NSRDSSGNHVV
63
NSRDSSGNHVV
63



CDR-H1
SYYMH
64
GYTFTSYY
173



CDR-H2
GIIPIFGTANYAQKFQG
65
IIPIFGTA
174



CDR-H3
GWGYSSSFDY
66
ARGWGYSSSFD
175






Y



 


F11
CDR-L1
SGSSSNIGTNTVN
67
SSNIGTNT
176



CDR-L2
SNDQRPS
68
SND
177



CDR-L3
ETWDDSLKGPV
69
ETWDDSLKGPV
69



CDR-H1
SYAMS
70
GFTFSSYA
178



CDR-H2
TISGSGDSTYYADSVKG
71
ISGSGDST
179



CDR-H3
EWELGDAFDI
72
AREWELGDAFDI
180


 


D3
CDR-L1
RASQSISSYLN
73
QSISSY
181



CDR-L2
AASSLQS
74
AAS
182



CDR-L3
QQSYSTRWT
75
QQSYSTRWT
75



CDR-H1
SYAMS
76
GSTFSSYA
183



CDR-H2
AISGSGGSTYYADSVKG
77
ISGSGGST
184



CDR-H3
DRGSYGYYYGMDV
78
AKDRGSYGYYYG
185






MDV



 


B12
CDR-L1
RASQSISSYLN
79
QSISSY
186



CDR-L2
AASSLOS
80
AAS
187



CDR-L3
QQSYSTLRT
81
QQSYSTLRT
81



CDR-H1
GYYMH
82
GYTFTGYY
188



CDR-H2
WINPNSGGTNYAQKFQ
83
INPNSGGT
189




G






CDR-H3
AGASIVGATALDY
84
TRAGASIVGATAL
190






DY



 


E4
CDR-L1
TRSSGSIASNYVQ
85
SGSIASNY
191



CDR-L2
EDNQRPS
86
EDN
192



CDR-L3
QSYDTGNRNYV
87
QSYDTGNRNYV
87



CDR-H1
SYTIS
88
GGTFSSYT
193



CDR-H2
RIIPILGIANYAQKFQG
89
IIPILGIA
194



CDR-H3
GPSLNYAGYFDN
90
VRGPSLNYAGYF
195






DN



 


E12
CDR-L1
QGDSLRSYYAS
91
SLRSYY
196



CDR-L2
GKEKRPS
92
GKE
197



CDR-L3
NSRGSTTDYMV
93
NSRGSTTDYMV
93



CDR-H1
SYAMH
94
GFTFSSYA
198



CDR-H2
VISYDGSNKYYADSVKG
95
ISYDGSNK
199



CDR-H3
ERGSGMDV
96
ARERGSGMDV
200


 


D1
CDR-L1
KASQDIDDDMN
97
QDIDDD
201



CDR-L2
EASTLVP
98
EAS
202



CDR-L3
LQHDKFPYT
99
LQHDKFPYT
99



CDR-H1
SYGIS
100
GYTFTSYG
203



CDR-H2
WINPNSGGTNYAQKFQ
101
INPNSGGT
204




G






CDR-H3
RGVDEGDY
102
ASRGVDEGDY
205


 


E6
CDR-L1
TGSSGNIASNYVQ
103
SGNIASNY
206



CDR-L2
RDDQRPS
104
RDD
207



CDR-L3
QSYDSSSWV
105
QSYDSSSWV
105



CDR-H1
TYDIT
106
GYTFTTYD
208



CDR-H2
WMNPNSGNSRSAQKF
107
MNPNSGNS
209




QG






CDR-H3
GDYSGVVLTATALDY
108
ATGDYSGVVLTAT
210






ALDY



 


E9
CDR-L1
SGSSSNIGNNYVY
109
SSNIGNNY
211



CDR-L2
RNNQRPS
110
RNN
212



CDR-L3
AAWDDSLSGWV
111
AAWDDSLSGWV
111



CDR-H1
SYGMH
112
GFTFSSYG
213



CDR-H2
NIKQDGSEKYYVDSVKG
113
IKQDGSEK
214



CDR-H3
EDRIAAAGMRELDY
114
AREDRIAAAGMR
215






ELDY



 


A11
CDR-L1
RSSQSLLHSNGYNYLD
115
QSLLHSNGYNY
216



CDR-L2
LGSNRAS
116
LGS
217



CDR-L3
MQGTHWPPYT
117
MQGTHWPPYT
117



CDR-H1
SYAMT
118
GFSFTSYA
218



CDR-H2
GISSDGTTTTYADSVRG
119
ISSDGTTT
219



CDR-H3
DQLLGWDALNV
120
ARDQLLGWDALN
220






V









In an embodiment, the anti-LILRB1 antibody or an 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.
    
    


More specifically, the anti-LILRB1 antibody or an antigen-binding fragment thereof may comprise:

    
    
        a light chain variable region comprising an amino acid sequence of SEQ ID NO: 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, or 345, and
        a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, or 260.
    
    


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 an antigen-binding fragment thereof provided in this disclosure are illustrated in Table 2:










TABLE 2








SEQ



variable

ID



region
Amino acid sequence(N→C)
NO







E3
light
SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKS
221



chain
GQAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTITGAQ




variable
AEDEADYYCNSRDSSGSHLTGVFGGGTKVTVLGQPAAA




region





heavy
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
222



chain
QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKN




variable
TLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAFDIWGQ




region
GTLVTVSS



 


B3
light
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQK
223



chain
PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQ




variable
PEDIATYYCQQYDNLPFGGGTKVDIKRTAAA




region





heavy
EVQLLESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVR
224



chain
QAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKN




variable
TLYLQMNSLRAEDTAVYYCARVGDSSGWSDAFDIWGQG




region
TMVTVSS



 


A10
light
DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQK
225



chain
PGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS




variable
EDFAVYYCQQYGSSPRMYTFGQGTKVDIKRTAAA




region





heavy
QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR
226



chain
QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSIST




variable
AYMELSSLRSEDTAVYYCARGGLGELDNWFDPWGQGTL




region
VTVSS



 


G1
light
SYELTQPPSLSVSPGQTASITCSGYKLGDRYVSWYQQKT
227



chain
GQSPVVVIYKDSQRPSGVPERFSGSNSGNTATLTISGTQ




variable
AMDEADYYCQAWDSGTGVFGGGTKLTVLGQPAAA




region





heavy
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVR
228



chain
QAPGQGLEWMGWISAYNGNTNYAQELQGRVTMTTDTS




variable
TSTAYMELRSLRSDDTAVYYCARVGVAGKLDYWGQGTLV




region
TVSS



 


G9
light
QSALTQPASVSGSPGQSITISCTGSSSDVGGYNYVSWYQ
233



chain
QHPGKAPKLMIYDVSNRPSGVSDRFSGSKSGNMASLTIS




variable
GLQAEDEADYYCSSYTGSSTLDVLFGGGTKLTVLGQPAA




region
A




heavy
QVQLVQPGAEVKKPGESLKISCKGSGYSFTSYWIGWVR
234



chain
QMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS




variable
TAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGT




region
LVTVSS



 


H2
light
SYELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKP
235



chain
GQAPILVISGNNKRPSGIPDRFSGSSSGDTASLTISGAQA




variable
EDEADYYCNSLDSTYNHPIFGGGTKVTVLGQPAAA




region





heavy
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIHWVR
236



chain
QATGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTST




variable
STAYMELRSLRSDDTAVYYCARDGGDAFDIWGQGTLVTV




region
SS



 


H11
light
SYELTQDPAASVALGQTVRITCQGDSLRSYYASWYQQKP
231



chain
GQAPVVVIYGRNNRPSGIPDRFSGSSSGDTASLTITGAQ




variable
AEDEADYYCKSRDSSGNHYVFGTGTKLTVLGQPAAA




region





heavy
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVR
232



chain
QAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTST




variable
STVYMELSSLRSEDTAVYYCARDAGSSSDYWGRGTLVT




region
VSS



 


F12
light
QSVLTQPASVSGSPGQSITISCAGTSSDIGDYDYVSWYQ
237



chain
QHPGKTPKLMIYDVSRRPSGVPDRFSGSKSGNTASLTIS




variable
GLQTEDEADYYCASYTSSSVVVFGGGTKLTVLGQPAAA




region





heavy
QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVR
238



chain
QMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS




variable
TAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGT




region
LVTVSS



 


B9
light
DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQK
229



chain
PGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYHCQQAYGFPLTLGGGTKVEIKRTAAA




region





heavy
QVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVR
230



chain
QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARGEIAVAQNWDYYGMDVWG




region
QGTLVTVSS



 


G11
light
QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWY
239



chain
QQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTI




variable
SGLQAEDEADYYCSSYSSSSTLVVFGGGTKLTVLGQPAA




region
A




heavy
QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVR
240



chain
QMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS




variable
TAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGT




region
LVTVSS



 


G6
light
SYELTQDPAVSVALGQTVTITCQGDSLRRYYATWYQQKP
241



chain
GQAPVLVIYGQNYRPSGIPDRFSGSNSGTTASLTITGAQA




variable
EDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPAAA




region





heavy
EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVR
242



chain
QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTST




variable
AYMELSSLRSEDTAVYYCARGWGYSSSFDYWGQGTTVT




region
VSS



 


F11
light
QSVLTQPPSTSGTPGQTFSIFCSGSSSNIGTNTVNWYQQ
243



chain
LPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAISGL




variable
QSEDEADYYCETWDDSLKGPVFGGGTKVTVLGQPAAA




region





heavy
EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYAMSWVR
244



chain
RAPGKGLEWVSTISGSGDSTYYADSVKGRFTISRDNSKN




variable
TLYLQMNNLRAEDTAVYYCAREWELGDAFDIWGRGTLVT




region
VSS



 


D3
light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK
245



chain
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSTRWTFGQGTKVEIKRTAAA




region





heavy
EVQLLESGGGVVQPGRSLRLSCAASGSTFSSYAMSWVR
246



chain
QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKN




variable
TLYLQMNSLRAEDTAVYYCAKDRGSYGYYYGMDVWGQ




region
GTMVTVSS



 


B12
light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK
247



chain
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ




variable
PEDFATYYCQQSYSTLRTFGQGTKVEIKRTAAA




region





heavy
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV
248



chain
RQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTITADES




variable
TSTAYMELSSLRSEDTAVYYCTRAGASIVGATALDYWGQ




region
GTLVTVSS



 


E4
light
NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQ
249



chain
RPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTIS




variable
GLKTEDEADYYCQSYDTGNRNYVFGTGTQLTVLGQPAA




region
A




heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVR
250



chain
QAPGQGLEWMGRIIPILGIANYAQKFQGRVTMTRDMSTD




variable
TAYMELSSLTYDDTAVYFCVRGPSLNYAGYFDNWGQGT




region
LVTVSS



 


E12
light
SYELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKS
251



chain
GQAPVLVIYGKEKRPSGIPDRFSGSSSGNTASLTITGARA




variable
EDEADYYCNSRGSTTDYMVFGGGTQLTVLGQPAAA




region





heavy
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVR
252



chain
QAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKN




variable
TLYLQMNSLRAEDTAVYYCARERGSGMDVWGQGTLVTV




region
SS



 


D1
light
ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQK
253



chain
PGEAAISIIQEASTLVPGIPPRFSGSGYGTDFTLTINNIESE




variable
DAAYYFCLQHDKFPYTFGQGTKLEIKRTAAA




region





heavy
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVR
254



chain
QAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTS




variable
ISTAYMELSRLRSDDTAVYYCASRGVDEGDYWGQGTMV




region
TVSS



 


E6
light
NFMLTQPHSVSESPGKTVTLSCTGSSGNIASNYVQWYQ
255



chain
HRPGSAPTTVIYRDDQRPSGVPDRFSGSIDSSSNSASLTI




variable
SGLRPEDEADYYCQSYDSSSWVFGGGTKLTVLGQPAAA




region





heavy
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYDITWVR
256



chain
QAPGQGLEWMGWMNPNSGNSRSAQKFQGRVSMTSDS




variable
SISTAYMELSSLRSEDTAVYYCATGDYSGVVLTATALDY




region
WGQGTLVTVSS



 


E9
light
QSELTQLPSASETPGQRVTISCSGSSSNIGNNYVYWYQQ
257



chain
LPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGL




variable
RSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPAAA




region





heavy
QVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMHWV
258



chain
RQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNA




variable
KNTLYLQMNSLRAEDTAVYYCAREDRIAAAGMRELDYW




region
GQGTLVTVSS



 


A11
light
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLD
259



chain
WYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFT




variable
LKISRVEAEDVGVYYCMQGTHWPPYTFGQGTKVEIKRTA




region
AA




heavy
EVQLLESGGGLEQPGGFLRLSCAASGFSFTSYAMTWVR
260



chain
QAPGKGLEWVSGISSDGTTTTYADSVRGRFTISRDNAKN




variable
TVYLQMNSLRDEDTAVYYCARDQLLGWDALNVWGQGT




region
MVTVSS



 


E3.1
light
SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKS
345



chain
GQAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTITGAQ




variable
AEDEADYYCNSRDSSGSHLTGVFGGGTKVTVL




region





heavy
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
222



chain
QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKN




variable
TLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAFDIWGQ




region
GTLVTVSS









In this disclosure, the expression “an antibody or an antigen-binding fragment (for example, CDR, variable region, or heavy chain/light chain) comprising, consists of, or represented by a certain amino acid sequence” may refer to an antigen-binding fragment that consists essentially of (1) the certain amino acid sequence or (2) an amino acid sequence wherein an insignificant mutation (for example, substitution, deletion, and/or addition of an amino acid residue(s); leading to no impact on the activity of the antibody) is introduced in the amino acid sequence (1).
The anti-LILRB1 antibody or an 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 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 an 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. The pharmaceutical composition may have an activity to inhibit the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I. The cancer may be a cancer associated with the interaction between LILRB1 and MHC Class I. 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 composition for blocking the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
Another embodiment provides a composition for inhibiting immune evasion of a cancer cell, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
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 an antigen-binding fragment thereof to a subject (e.g., a mammal including human) in need of treating and/or preventing the cancer.
Another embodiment provides a method of blocking the binding of LILRB1 to MHC Class I and/or a method of blocking the interaction between LILRB1 and MHC Class I, comprising administering (orally or parenterally) a pharmaceutically effective amount of the anti-LILRB1 antibody or an antigen-binding fragment thereof to a subject (e.g., a mammal including human) in need of inhibiting the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I.
Another embodiment provides a method of inhibiting immune evasion of a cancer cell, comprising administering (orally or parenterally) a pharmaceutically effective amount of the anti-LILRB1 antibody or an antigen-binding fragment thereof to a subject (e.g., a mammal including human) in need of inhibiting immune evasion of the cancer cell.
The methods provided in this disclosure may further comprise a step of identifying the subject in need of treating and/or preventing the cancer, inhibiting the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I, and/or inhibiting immune evasion of the cancer cell, prior to the step of administering.
Another embodiment provides a nucleic acid molecule (polynucleotide) 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 comprise a nucleic acid molecule encoding the light chain variable region or light chain, and a nucleic acid molecule encoding the heavy chain variable region or heavy chain, respectively (e.g., in two separate vectors) or all together (e.g., in one vector). The recombinant vector may be used as an expression vector.
Another embodiment provides a recombinant cell comprising the nucleic acid molecule or the recombinant vector.
Another embodiment provides a method of preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof, comprising expressing the nucleic acid molecule in a cell. The step of expressing the nucleic acid molecule may comprise culturing 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 an 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-Cλ (lambda constant region), (scFv)2, Fab, Fab′, or a F(ab′)2, but not be limited thereto. In an embodiment, the antigen-binding fragment may be scFv, a fusion polypeptide (scFv-Fc) 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 fusion polypeptide (scFv-Ck or scFv-Cλ) wherein scFv is fused with a constant region (e.g., kappa or lambda) of a light chain.
The anti-LILRB1 antibody or an 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 an antigen-binding fragment thereof may have an activity of blocking the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity of inhibiting immune evasion of a cancer cell. Furthermore, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an anti-cancer effect.
A protein LILRB1, which is an antigen of an anti-LILRB1 antibody or an 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 AAH15731.1 (SEQ ID NO: 348), 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 an 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 comprises 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 not only an intact antibody but also an antigen-binding fragment of the antibody possessing an antigen-binding capability.
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 includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region CH1.
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 pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, in addition to the active ingredient (the anti-LILRB1 antibody or an 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 an 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 anti-LILRB1 antibody or an antigen-binding fragment thereof may be comprised in the pharmaceutical composition or administered to a subject in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” may refer to an amount of an active ingredient (the antibody or fragment thereof) at which the active ingredient can exert desired effects (e.g., anti-cancer effect). The pharmaceutically effective amount may be prescribed in a variety of ways, depending on various factors, such as age, body weight, gender, pathologic conditions, diets, excretion speed, and/or reaction sensitivity of a subject, formulation types, administration time, administration interval, administration route, administration manner, and the like. For example, anti-LILRB1 antibody or an 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 compared to that of a normal cell or a cancer cell which is non-responsive or resistant to the immunotherapy, for example, T-cell (e.g., cytotoxic 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 an 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 an 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 an 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 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 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 nucleic acid molecule or 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 CMV 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.
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 an 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 an 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 B3 according to an example.
FIG. 3 is a sensorgram showing the results of SPR assay for anti-LILRB1 antibody E3 according to an example.
FIG. 4a is a graph showing binding ability of anti-LILRB1 antibody A10 according to an example to a human natural killer cell, KHYG-1; FIG. 4b is a graph showing binding ability of anti-LILRB1 antibody E3 according to an example to a human natural killer cell, KHYG-1; and FIG. 4c is a graph showing binding ability of human IgG4 isotype control antibody to a human natural killer cell, KHYG-1.
FIG. 5 is a graph showing the level of binding of recombinant LILRB1-Fc proteins to HLA-G overexpressing cell surface measured by iQue screener, when treated with anti-LILRB1 antibodies according to an example and human IgG4 isotype control antibody, respectively.
FIG. 6 is a graph showing in vivo antitumor effects of anti-LILRB1 antibody E3 and B3 according to an example.
FIGS. 7a to 7d are flow cytometry diagrams of binding of anti-LILRB1 antibody E3.1 according to an example to cells expressing various members of human LILR family.
FIGS. 8a to 8d are flow cytometry diagrams of binding of anti-LILRB1 antibody H11 according to an example to cells expressing various members of human LILR family.
FIG. 9 shows graphs showing release level of granzyme B in a human natural killer cell, KHYG-1, when treated with anti-LILRB1 antibody E3.1 or H11 according to an example, comparing with that in the cell treated with a control antibody (human IgG4 isotype).
FIG. 10 shows graphs showing release level of perforin in a human natural killer cell, KHYG-1, when treated with anti-LILRB1 antibody E3.1 or H11 according to an example, comparing with that in the cell treated with a control antibody (human IgG4 isotype).
FIG. 11 is a graph showing results of luciferase reporter assay for evaluating ability of anti-LILRB1 antibody E3.1 or H11 according to an example to block LILRB1 signal pathway.
FIG. 12 is a graph showing in vivo anti-tumor effects of anti-LILRB1 antibody E3.1 and H11 according to an example.



Hereafter, 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 scFv antibodies. As an antigen, human LILRB1-His (Cat. No. 8989-T2) and human LILRB1-Fc (Cat. No. 2017-T2) (RnD systems) were used respectively. Each antigen was conjugated with biotin by EZ-Link Sulfo-NHS-Biotin kit (ThermoFisher Scientific) for use.
The phage display screening was performed using total 4-types of LILRB1 antigens (LILRB1-His, LILRB1-Fc, LILRB1-His-Biotin, and LILRB1-Fc-Biotin) through solid-phase screening and solution-phase screening. Additional screenings were performed by gradually decreasing the concentration of the used antigen, competitively eluting with control antibodies against LILRB1, conducting negative selection to Fc when LILRB1-Fc is used as an antigen, etc. The selected products were confirmed for their binding to the antigen through polyclonal phage ELISA.
1.2. Screening and Analysis of Monoclonal Soluble scFvs
Genes encoding the scFvs, which were verified to bind the antigen in Example 1.1, were amplified by PCR to prepare expression vectors. For each selection, a certain number of transformants were transferred to a 96 well culture plate for screening. Antibodies in a scFv form were expressed using Autoinduction media (Studier, F. W. (2005) Protein Expression and Purification 41, 207-34) and then analyzed for their binding to the antigen by performing DELFIA immune assay (PerkinElmer). In addition, after allowing a certain amount of each scFv antibody to be captured on the surface, DELFIA for the antigen was performed to determine the ranking for antigen-antibody binding affinity.
1.3. Conversion of the Screened scFvs into IgG Antibodies
Among the clones which were confirmed to bind to the antigen in Example 1.2, a total of 376 clones were selected, and the DNA sequences of genes encoding the selected scFvs were analyzed by a general DNA sequencing to remove duplicate clones. In addition, a total of 93 clones were selected based on the ranking of the antigen-antibody binding affinity determined in Example 1.2. Genes encoding a heavy chain variable region (VH) and a light chain variable region (VL) were respectively amplified by PCR from each of the genes encoding the selected scFvs, and inserted into an expression vector (pTRIOZ-hIgG4, InvivoGen; alternatively, any one of vectors comprising CMV promoter or CMV/CHO beta-actin fusion promoter (KR10-1038126B1) and genes encoding human IgG4 heavy chain constant region and kappa or lambda light chain constant region can be used), wherein the expression vector was designed for encoding a human IgG4 antibody (IgG4 Fc: SEQ ID NO: 341, Kappa constant region: SEQ ID NO: 342, Lambda constant region: SEQ ID NO: 343). The DNA sequence of the expression vector was confirmed by sequencing.
1.4. Preparation of Selected Antibodies
The vectors constructed in Example 1.3 were purified using Plasmid Plus Maxi kit (Qiagen). The purified vectors were used for expressing antibodies in ExpiCHO-S™ cells or Expi293™ cells.
In particular, the vectors constructed in Example 1.3 were transfected into ExpiCHO-S™ cells (Gibco) (1.5×108 cells/Culture Volume 25 mL) by adding 80 μL of ExpiFectamine™ CHO reagent (Thermo Fisher). One day post-transfection, 150 μL of ExpiCHO™ Enhancer (Thermo Fisher) and 4 mL of ExpiCHO™ Feed (Thermo Fisher) were added to the culture. On day 5, 4 mL of ExpiCHO™ Feed was added to the culture. The transfected cells were cultured under the conditions of 32° C. and 5% CO2 for 7-11 days in total.
In addition, the vectors constructed in Example 1.3 were transfected into Expi293F™ cells (Gibco) (3×108 cells/Culture Volume 100 mL) by adding 320 μL of ExpiFectamine™ 293 Reagent (Gibco) according to manufacturer's protocol. One day post-transfection, ExpiFectamine™ 293 Enhancer 1 (Thermo Fisher), ExpiFectamine™ Enhancer 2 (Thermo Fisher), and glucose were added in the amount of 0.6 mL per Culture Volume 100 mL, 6 mL per Culture Volume 100 mL, and 3.6 g per 1 liter, respectively. The transfected cells were cultured under the conditions of 36.5° C. and 5% CO2 for 5 days in total. The cultured cells of two types were respectively centrifuged at 4000 rpm at 4° C. for 20 minutes, and then, filtrated using 0.22 um 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 1 mL column (GE healthcare) at the flow rate of 1 mL/min,, and the column was washed with 20 column volumes (CV) of 1×PBS. Then, elutionbuffer (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 (4-20% or 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.
Among 93 antibodies, gel electrophoresis images for antibodies A10, B3, E3, G1, G9 and H2 were representatively shown in FIG. 1. As shown in FIG. 1, the production of antibodies having disulfide bond was confirmed.
1.5. Analysis of Binding Affinity of the Selected Antibodies
The binding affinities of the 93 antibodies, which were selected in Example 1.3, 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-1000-508). The antigen, human LILRB1 protein (LILRB1-His, RnD systems Cat. No. 8989-T2) was injected thereto in 4˜9 different concentrations from 3.13 nM to 1600 nM at the flow rate of 30 μL/min to determine ka and kd values as shown in Table 3 and calculate KD value therefrom.
Among the 93 antibodies, 20 antibodies showing excellent binding affinities (KD values) were selected and summarized in Table 3. Among them, SPR sensorgrams for antibody B3 showing the LILRB1 binding affinity (KD) of about 99.8 nM and for antibody E3 showing the LILRB1 binding affinity (KD) of about 101.2 nM are shown in FIGS. 2 and 3, respectively (FIG. 2: SPR sensorgram for B3, FIG. 3: SPR sensorgram for E3):







TABLE 3







Antigen Binding Affinities (KD) of Anti-


LILRB1 antibodies to human LILRB1










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













A10
0.504
76.5
152


A11
0.001801
9.814
5448


B3
0.149
14.87
99.8


B9
0.09324
6.16
66.1


B12
1.84
14.42
7.84


D1
1.165
57.44
49.33


D3
0.0311
5.58
180


E3
0.3460
35.00
101.2


E4
0.1065
7.73
72.55


E6
0.2679
16.27
60.73


E9
0.105
10.48
99.86


E12
2.331
102.6
44.01


F11
2.72
6.15
2.26


F12
2.811
9.731
3.462


G1
4.33
14.19
3.28


G6
2.58
152.4
59.06


G9
1.43
4.36
3.05


G11
0.454
20.53
45.23


H2
5.865
95
16.20


H11
2.962
22.57
7.621









1.6. Sequence Analysis of the Selected Antibodies
In the 20 antibodies which are analyzed for antigen binding affinity in Example 1.5, amino acid sequences of the CDRs defined according to Kabat numbering, light chain variable region, heavy chain variable region, light chain, and heavy chain, and nucleic acid sequence encoding the light chain variable region and the heavy chain variable region were analyzed by general amino acid sequencing and DNA sequencing methods and summarized in Tables 4-23:







TABLE 4







Antibody clone E3










Amino acid sequence (N→C) / 
SEQ ID



Nucleic acid sequence (5′→3′)
NO












CDR-L1
QGDSLRNFYAS
1


 


CDR-L2
GKNNRPS
2


 


CDR-L3
NSRDSSGSHLTGV
3


 


CDR-H1
SYAMS
4


 


CDR-H2
AISGSGGSTYYADSVKG
5


 


CDR-H3
DTYYYGSGRSNAFDI
6


 


light
SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKSG
221


chain
QAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTITGAQAE



variable
DEADYYCNSRDSSGSHLTGVFGGGTKVTVLGQPAAA



region




 


light
TCCTATGAGCTGACTCAGGACCCTGCTGTGTCTGTGGC
261


chain
CTTGGGACAGACAGTCAGGATCACATGCCAGGGAGACA



variable
GCCTCAGAAACTTTTATGCAAGCTGGTACCAGCAGAAGT



region
CAGGACAGGCCCCAGTTCTTGTCATGTATGGTAAAAACA



coding
ACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCC



gene
ACCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGC




TCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCG




GGACAGCAGTGGTAGCCATTTGACGGGCGTATTCGGCG




GAGGGACCAAGGTCACCGTCCTAGGTCAGCCCGCGGC




CGCA



 


heavy
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
222


chain
QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKN



variable
TLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAFDIWGQG



region
TLVTVSS



 


heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAC
262


chain
AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC



variable
TGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCG



region
CCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT



coding
ATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCC



gene
GTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAA




GAATACGCTGTATCTGCAAATGATTAGCCTGAGAGCTGA




GGACACGGCTGTGTATTACTGTGCGAGAGATACGTATTA




CTATGGTTCGGGGAGAAGTAATGCTTTTGATATATGGGG




CCAGGGAACCCTGGTCACCGTCTCGAGT



 


light
SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKSG
301


chain
QAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTITGAQAE



(Lambda)
DEADYYCNSRDSSGSHLTGVFGGGTKVTVLGQPAAAPSV




TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV




KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ




VTHEGSTVEKTVAPTECS



 


heavy
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
302


chain
QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKN




TLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAFDIWGQG




TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF




PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS




SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP




EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE




VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ




DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL




PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN




YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH




EALHNHYTQKSLSLSLGK
















TABLE 5







Antibody clone B3










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
QASQDISNYLN
7


 


CDR-L2
DASNLET
8


 


CDR-L3
QQYDNLP
9


 


CDR-H1
DYAMH
10


 


CDR-H2
GISWNSGSIGYADSVKG
11


 


CDR-H3
VGDSSGWSDAFDI
12


 


light chain
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQK
223


variable
PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSL



region
QPEDIATYYCQQYDNLPFGGGTKVDIKRTAAA



 


light chain
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
263


variable
TCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGT



region
CAGGACATTAGCAACTATTTGAATTGGTATCAGCAGAAA



coding
CCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCC



gene
AATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGT




GGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTG




CAGCCTGAAGATATTGCAACATATTACTGTCAACAGTAT




GATAATCTCCCTTTCGGCGGAGGGACCAAAGTGGATATC




AAACGTACCGCGGCCGCA



 


heavy chain
EVQLLESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ
224


variable
APGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNT



region
LYLQMNSLRAEDTAVYYCARVGDSSGWSDAFDIWGQGTM




VTVSS



 


heavy chain
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG
264


variable
CCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



region
TTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAA



coding
GCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGT



gene
TGGAATAGTGGTAGCATAGGCTACGCAGACTCCGTGAAG




GGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG




CTGTATCTTCAAATGAACAGTCTGAGAGCCGAGGACACG




GCCGTGTATTACTGTGCGAGAGTTGGGGATAGCAGTGGC




TGGTCCGATGCTTTTGATATCTGGGGCCAAGGGACAATG




GTCACCGTCTCGAGT



 


light chain
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQK
303


(Kappa)
PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSL




QPEDIATYYCQQYDNLPFGGGTKVDIKRTAAAPSVFIFP




PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN




SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVT




HQGLSSPVTKSFNRGEC



 


heavy chain
EVQLLESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ
304



APGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNT




LYLQMNSLRAEDTAVYYCARVGDSSGWSDAFDIWGQGTM




VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP




EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS




SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP




EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP




EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH




QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY




TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE




NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV




MHEALHNHYTQKSLSLSLGK
















TABLE 6







Antibody clone A10










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
RASQSVSSNLA
13


 


CDR-L2
GASTRAT
14


 


CDR-L3
QQYGSSPRMYT
15


 


CDR-H1
SYAIS
16


 


CDR-H2
GIIPIFGTANYAQKFQG
17


 


CDR-H3
GGLGELDNWFDP
18


 


light chain
DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQK
225


variable
PGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSL



region
QSEDFAVYYCQQYGSSPRMYTFGQGTKVDIKRTAAA



 


light chain
GATATTGTGATGACACAGTCTCCAGCCACCCTGTCTGTG
265


variable
TCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT



region
CAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAA



coding
CCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC



gene
ACCAGGGCCACCGGTATCCCAGCCAGGTTCAGTGGCAGT




GGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTG




CAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTAT




GGTAGCTCACCTCGGATGTACACTTTTGGCCAGGGGACC




AAAGTGGATATCAAACGTACCGCGGCCGCA



 


heavy chain
QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ
226


variable
APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSIST



region
AYMELSSLRSEDTAVYYCARGGLGELDNWFDPWGQGTLV




TVSS



 


heavy chain
CAAATGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAG
266


variable
CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGA



region
GGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAG



coding
GCCCCTGGACAAGGGCTTGAGTGGATGGGTGGGATCATC



gene
CCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAG




GGCAGAGTCACGATTACCGCGGACAAATCCATCAGCACA




GCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACG




GCCGTGTATTACTGTGCGAGAGGCGGCCTCGGGGAGTTG




GACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTC




ACCGTCTCGAGT



 


light chain
DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQK
305


(Kappa)
PGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSL




QSEDFAVYYCQQYGSSPRMYTFGQGTKVDIKRTAAAPSV




FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL




QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYA




CEVTHQGLSSPVTKSFNRGEC



 


heavy chain
QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ
306



APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSIST




AYMELSSLRSEDTAVYYCARGGLGELDNWFDPWGQGTLV




TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE




PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS




SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE




FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE




VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ




DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT




LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN




NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM




HEALHNHYTQKSLSLSLGK
















TABLE 7







Antibody clone G1










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
SGYKLGDRYVS
19


 


CDR-L2
KDSQRPS
20


 


CDR-L3
QAWDSGTGV
21


 


CDR-H1
SYGIS
22


 


CDR-H2
WISAYNGNTNYAQELQG
23


 


CDR-H3
VGVAGKLDY
24


 


light chain
SYELTQPPSLSVSPGQTASITCSGYKLGDRYVSWYQQKT
227


variable
GQSPVVVIYKDSQRPSGVPERFSGSNSGNTATLTISGTQ



region
AMDEADYYCQAWDSGTGVFGGGTKLTVLGQPAAA



 


light chain
TCCTATGAGCTGACTCAGCCACCCTCACTGTCCGTGTCC
267


variable
CCAGGACAGACAGCCAGCATCACCTGCTCAGGATATAAA



region
CTGGGAGATAGATATGTTTCCTGGTATCAGCAGAAGACA



coding
GGCCAGTCCCCTGTGGTGGTCATCTATAAAGATAGCCAG



gene
CGGCCCTCAGGGGTCCCTGAACGATTCTCTGGCTCCAAC




TCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAG




GCTATGGATGAGGCTGACTATTACTGTCAGGCGTGGGAC




AGCGGCACTGGGGTATTCGGCGGAGGGACCAAGCTGACC




GTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQ
228


variable
APGQGLEWMGWISAYNGNTNYAQELQGRVTMTTDTSTST



region
AYMELRSLRSDDTAVYYCARVGVAGKLDYWGQGTLVTVS




S



 


heavy chain
GAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAG
268


variable
CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGA



region
GGCACCTTCAGCAGCTATGGTATCAGCTGGGTGCGACAG



coding
GCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGC



gene
GCTTACAATGGTAACACAAACTATGCACAGGAGCTCCAG




GGCAGAGTCACCATGACCACAGACACATCCACGAGCACA




GCCTATATGGAGCTGAGGAGCCTGAGATCTGACGACACG




GCCGTGTATTACTGTGCGAGAGTAGGGGTGGCTGGTAAA




CTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCG




AGT



 


light chain
SYELTQPPSLSVSPGQTASITCSGYKLGDRYVSWYQQKT
307


(Lambda)
GQSPVVVIYKDSQRPSGVPERFSGSNSGNTATLTISGTQ




AMDEADYYCQAWDSGTGVFGGGTKLTVLGQPAAAPSVTL




FPPSSEELQANKATLVCLISDFYPGAVTVAWKEDSSPVK




AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV




THEGSTVEKTVAPTECS



 


heavy chain
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQ
308



APGQGLEWMGWISAYNGNTNYAQELQGRVTMTTDTSTST




AYMELRSLRSDDTAVYYCARVGVAGKLDYWGQGTLVTVS




SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT




VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG




TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG




GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF




NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL




NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP




SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK




TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA




LHNHYTQKSLSLSLGK
















TABLE 8







Antibody clone G9










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
TGSSSDVGGYNYVS
25


 


CDR-L2
DVSNRPS
26


 


CDR-L3
SSYTGSSTLDVL
27


 


CDR-H1
SYWIG
28


 


CDR-H2
IIYPGDSDTRYSPSFQG
29


 


CDR-H3
QYYDGGYYMDV
30


 


light chain
QSALTQPASVSGSPGQSITISCTGSSSDVGGYNYVSWYQ
233


variable
QHPGKAPKLMIYDVSNRPSGVSDRFSGSKSGNMASLTIS



region
GLQAEDEADYYCSSYTGSSTLDVLFGGGTKLTVLGQPAA




A



 


light chain
CAGTCTGCGCTGACTCAGCCTGCCTCCGTGTCTGGGTCT
269


variable
CCTGGACAGTCGATCACCATCTCCTGCACTGGAAGCAGC



region
AGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAG



coding
CAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGAT



gene
GTCAGTAATCGGCCCTCAGGGGTTTCTGATCGCTTCTCT




GGCTCCAAGTCTGGCAACATGGCCTCCCTGACCATCTCT




GGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGC




TCATATACAGGAAGCAGCACTCTCGACGTGCTATTCGGC




GGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCC




GCA



 


heavy chain
QVQLVQPGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQ
234


variable
MPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIST



region
AYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVT




VSS



 


heavy chain
CAGGTGCAGCTGGTGCAGCCTGGAGCAGAGGTGAAAAAG
270


variable
CCGGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGA



region
TACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAG



coding
ATGCCCGGGAAGGGCCTGGAGTGGATGGGGATCATCTAT



gene
CCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAA




GGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACC




GCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACC




GCCATGTATTACTGTGCGAGTCAATATTACGATGGGGGT




TACTACATGGACGTCTGGGGCCAGGGAACCCTGGTCACC




GTCTCGAGT



 


light chain
QSALTQPASVSGSPGQSITISCTGSSSDVGGYNYVSWYQ
309


(Lambda)
QHPGKAPKLMIYDVSNRPSGVSDRFSGSKSGNMASLTIS




GLQAEDEADYYCSSYTGSSTLDVLFGGGTKLTVLGQPAA




APSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA




DSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHR




SYSCQVTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVQPGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQ
310



MPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIST




AYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVT




VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP




VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS




LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF




LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV




QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD




WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL




PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN




YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH




EALHNHYTQKSLSLSLGK
















TABLE 9







Antibody clone H2










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
QGDSLRNYYAS
31


 


CDR-L2
GNNKRPS
32


 


CDR-L3
NSLDSTYNHPI
33


 


CDR-H1
SYDIH
34


 


CDR-H2
WISAYNGNTNYAQKLQG
35


 


CDR-H3
DGGDAFDI
36


 


light chain
SYELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKP
235


variable
GQAPILVISGNNKRPSGIPDRFSGSSSGDTASLTISGAQ



region
AEDEADYYCNSLDSTYNHPIFGGGTKVTVLGQPAAA



 


light chain
TCCTATGAGCTGACTCAGGACCCTGCTGTGTCGGTGGCC
271


variable
TTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGC



region
CTCAGAAACTATTATGCAAGCTGGTACCAGCAGAAGCCA



coding
GGACAGGCCCCTATTCTTGTCATCTCTGGTAACAACAAA



gene
CGGCCCTCGGGGATCCCAGACCGATTCTCTGGCTCCAGC




TCAGGAGACACAGCTTCCTTGACCATCTCTGGGGCTCAG




GCGGAAGATGAGGCTGACTATTACTGTAACTCCCTAGAC




AGCACTTATAACCATCCGATATTCGGCGGAGGGACCAAG




GTCACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIHWVRQ
236


variable
ATGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTST



region
AYMELRSLRSDDTAVYYCARDGGDAFDIWGQGTLVTVSS



 


heavy chain
CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAAG
272


variable
CCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGA



region
TACACCTTCACCAGTTATGATATCCACTGGGTGCGACAG



coding
GCCACTGGACAAGGGCTTGAGTGGATGGGATGGATCAGC



gene
GCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAG




GGCAGAGTCACCATGACCACAGACACATCCACGAGCACA




GCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACG




GCCGTGTATTACTGTGCGAGAGATGGGGGTGATGCTTTT




GATATCTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT



 


light chain
SYELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKP
311


(Lambda)
GQAPILVISGNNKRPSGIPDRFSGSSSGDTASLTISGAQ




AEDEADYYCNSLDSTYNHPIFGGGTKVTVLGQPAAAPSV




TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP




VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC




QVTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIHWVRQ
312



ATGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTST




AYMELRSLRSDDTAVYYCARDGGDAFDIWGQGTLVTVSS




ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV




SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT




KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG




PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN




WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN




GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS




QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT




TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL




HNHYTQKSLSLSLGK
















TABLE 10







Antibody clone H11










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
QGDSLRSYYAS
37


 


CDR-L2
GRNNRPS
38


 


CDR-L3
KSRDSSGNHYV
39


 


CDR-H1
SYYMH
40


 


CDR-H2
IINPSGGSTSYAQKFQG
41


 


CDR-H3
DAGSSSDY
42


 


light chain
SYELTQDPAASVALGQTVRITCQGDSLRSYYASWYQQKP
231


variable
GQAPVVVIYGRNNRPSGIPDRFSGSSSGDTASLTITGAQ



region
AEDEADYYCKSRDSSGNHYVFGTGTKLTVLGQPAAA



 


light chain
TCCTATGAGCTGACTCAGGACCCTGCTGCGTCTGTGGCC
273


variable
TTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGC



region
CTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCA



coding
GGACAGGCCCCTGTAGTTGTCATCTATGGTAGAAACAAC



gene
CGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGC




TCAGGAGACACAGCTTCCTTGACCATCACTGGGGCTCAG




GCGGAAGATGAGGCTGACTATTACTGTAAGTCCCGGGAC




AGCAGTGGTAACCATTATGTCTTCGGAACTGGGACCAAG




CTGACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQ
232


variable
APGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTST



region
VYMELSSLRSEDTAVYYCARDAGSSSDYWGRGTLVTVSS



 


heavy chain
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAG
274


variable
CCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGA



region
TACACCTTCACCAGCTACTATATGCACTGGGTGCGACAG



coding
GCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAAC



gene
CCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAG




GGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACA




GTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACG




GCCGTGTATTACTGTGCGAGAGATGCCGGCAGCTCGTCC




GATTACTGGGGCCGTGGCACCCTGGTCACCGTCTCGAGT



 


light chain
SYELTQDPAASVALGQTVRITCQGDSLRSYYASWYQQKP
313


(Lambda)
GQAPVVVIYGRNNRPSGIPDRFSGSSSGDTASLTITGAQ




AEDEADYYCKSRDSSGNHYVFGTGTKLTVLGQPAAAPSV




TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP




VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC




QVTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQ
314



APGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTST




VYMELSSLRSEDTAVYYCARDAGSSSDYWGRGTLVTVSS




ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV




SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT




KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG




PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN




WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN




GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS




QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT




TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL




HNHYTQKSLSLSLGK
















TABLE 11







Antibody clone F12










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
AGTSSDIGDYDYVS
43


 


CDR-L2
DVSRRPS
44


 


CDR-L3
ASYTSSSVVV
45


 


CDR-H1
SYWIG
46


 


CDR-H2
IIYPGDSDTRYSPSFQG
47


 


CDR-H3
QYYDGGYYMDV
48


 


light chain
QSVLTQPASVSGSPGQSITISCAGTSSDIGDYDYVSWYQ
237


variable
QHPGKTPKLMIYDVSRRPSGVPDRFSGSKSGNTASLTIS



region
GLQTEDEADYYCASYTSSSVVVFGGGTKLTVLGQPAAA



 


light chain
CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCT
275


variable
CCTGGACAGTCGATCACCATCTCCTGCGCTGGAACCAGC



region
AGTGACATTGGTGATTATGACTATGTCTCCTGGTACCAA



coding
CAGCACCCAGGCAAGACTCCCAAACTCATGATTTATGAT



gene
GTCAGTAGGCGGCCCTCAGGGGTCCCTGATCGCTTCTCT




GGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCT




GGGCTCCAGACTGAGGACGAGGCTGATTATTACTGCGCC




TCATATACAAGCAGCAGCGTCGTGGTCTTCGGCGGAGGG




ACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQ
238


variable
MPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIST



region
AYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVT




VSS



 


heavy chain
CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAG
276


variable
CCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGA



region
TACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAG



coding
ATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTAT



gene
CCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAA




GGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACC




GCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACC




GCCATGTATTACTGTGCGAGTCAATATTACGATGGGGGT




TACTACATGGACGTCTGGGGCCAGGGCACCCTGGTCACC




GTCTCGAGT



 


light chain
QSVLTQPASVSGSPGQSITISCAGTSSDIGDYDYVSWYQ
315


(Lambda)
QHPGKTPKLMIYDVSRRPSGVPDRFSGSKSGNTASLTIS




GLQTEDEADYYCASYTSSSVVVFGGGTKLTVLGQPAAAP




SVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS




SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSY




SCQVTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQ
316



MPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIST




AYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVT




VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP




VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS




LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF




LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV




QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD




WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL




PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN




YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH




EALHNHYTQKSLSLSLGK
















TABLE 12







Antibody clone B9










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
RASQSISRYLN
49


 


CDR-L2
GASSLQS
50


 


CDR-L3
QQAYGFPLT
51


 


CDR-H1
SYAIS
52


 


CDR-H2
GIIPIFGTANYAQKFQG
53


 


CDR-H3
GEIAVAQNWDYYGMDV
54


 


light chain
DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQK
229


variable
PGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSL



region
QPEDFATYHCQQAYGFPLTLGGGTKVEIKRTAAA



 


light chain
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
277


variable
TCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT



region
CAGAGCATTAGCAGGTATTTAAATTGGTATCAGCAGAAA



coding
CCAGGGAAAGCCCCCAAGCTCCTGATCTATGGTGCATCC



gene
AGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGT




GGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG




CAGCCTGAAGATTTCGCAACTTACCATTGTCAACAGGCT




TACGGTTTCCCCCTCACTCTCGGGGGAGGGACCAAGGTG




GAGATCAAACGTACCGCGGCCGCA



 


heavy chain
QVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ
230


variable
APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTST



region
AYMELSSLRSEDTAVYYCARGEIAVAQNWDYYGMDVWGQ




GTLVTVSS



 


heavy chain
CAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAG
278


variable
CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGA



region
GGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAG



coding
GCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATC



gene
CCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAG




GGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACA




GCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACG




GCCGTGTATTACTGTGCGAGAGGGGAAATAGCAGTGGCT




CAAAACTGGGACTACTACGGTATGGACGTCTGGGGCCAG




GGCACCCTGGTCACCGTCTCGAGT



 


light chain
DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQK
317


(Kappa)
PGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSL




QPEDFATYHCQQAYGFPLTLGGGTKVEIKRTAAAPSVFI




FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS




GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACE




VTHQGLSSPVTKSFNRGEC



 


heavy chain
QVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ
318



APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTST




AYMELSSLRSEDTAVYYCARGEIAVAQNWDYYGMDVWGQ




GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD




YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT




VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC




PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ




EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT




VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP




QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG




QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS




CSVMHEALHNHYTQKSLSLSLGK
















TABLE 13







Antibody clone G11










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
TGTSSDVGGYNYVS
55


 


CDR-L2
DVSKRPS
56


 


CDR-L3
SSYSSSSTLVV
57


 


CDR-H1
SYWIG
58


 


CDR-H2
IIYPGDSDTRYSPSFQG
59


 


CDR-H3
QYYDGGYYMDV
60


 


light chain
QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQ
239


variable
QHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTIS



region
GLQAEDEADYYCSSYSSSSTLVVFGGGTKLTVLGQPAAA



 


light chain
CAGTCTGCGCTGACTCAGCCTCGCTCAGTGTCCGGGTCT
279


variable
CCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGC



region
AGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAA



coding
CAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAT



gene
GTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCT




GGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCT




GGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGC




TCATATTCAAGCAGCAGCACTCTCGTGGTTTTCGGCGGA




GGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQ
240


variable
MPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIST



region
AYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVT




VSS



 


heavy chain
CAGGTCCAGCTGGTACAGTCTGGAGCAGAGGTGAAAAAG
280


variable
CCGGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGA



region
TACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAG



coding
ATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTAT



gene
CCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAA




GGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACC




GCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACC




GCCATGTATTACTGTGCGAGTCAATATTACGATGGGGGT




TACTACATGGACGTCTGGGGCCAGGGAACCCTGGTCACC




GTCTCGAGT



 


light chain
QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQ
319


(Lambda)
QHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTIS




GLQAEDEADYYCSSYSSSSTLVVFGGGTKLTVLGQPAAA




PSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD




SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRS




YSCQVTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQ
320



MPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIST




AYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVT




VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP




VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS




LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF




LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV




QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD




WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL




PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN




YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH




EALHNHYTQKSLSLSLGK
















TABLE 14







Antibody clone G6










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
QGDSLRRYYAT
61


 


CDR-L2
GQNYRPS
62


 


CDR-L3
NSRDSSGNHVV
63


 


CDR-H1
SYYMH
64


 


CDR-H2
GIIPIFGTANYAQKFQG
65


 


CDR-H3
GWGYSSSFDY
66


 


light chain
SYELTQDPAVSVALGQTVTITCQGDSLRRYYATWYQQKP
241


variable
GQAPVLVIYGQNYRPSGIPDRFSGSNSGTTASLTITGAQ



region
AEDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPAAA



 


light
TCCTATGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCC
281


chain
TTGGGACAGACAGTCACGATCACATGCCAAGGAGACAGC



variable
CTCAGAAGGTATTATGCAACCTGGTACCAGCAGAAGCCA



region
GGACAGGCCCCTGTCCTTGTCATCTATGGTCAAAACTAC



coding
CGGCCCTCGGGGATCCCAGACCGATTCTCTGGCTCCAAC



gene
TCAGGAACCACAGCTTCCTTGACCATCACTGGGGCTCAG




GCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGAC




AGCAGTGGTAACCATGTGGTATTCGGCGGAGGGACCAAG




CTGACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQ
242


variable
APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTST



region
AYMELSSLRSEDTAVYYCARGWGYSSSFDYWGQGTTVTV




SS



 


heavy chain
GAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAG
282


variable
CCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGA



region
TACACCTTCACCAGCTACTATATGCACTGGGTGCGACAG



coding
GCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATC



gene
CCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAG




GGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACA




GCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACG




GCCGTGTACTACTGTGCGAGAGGGTGGGGGTATAGCAGC




TCGTTTGACTACTGGGGGCAAGGGACCACGGTCACCGTC




TCGAGT



 


light chain
SYELTQDPAVSVALGQTVTITCQGDSLRRYYATWYQQKP
321


(Lambda)
GQAPVLVIYGQNYRPSGIPDRFSGSNSGTTASLTITGAQ




AEDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPAAAPSV




TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP




VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC




QVTHEGSTVEKTVAPTECS



 


heavy chain
EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQ
322



APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTST




AYMELSSLRSEDTAVYYCARGWGYSSSFDYWGQGTTVTV




SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV




TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL




GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFL




GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ




FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW




LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP




PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY




KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE




ALHNHYTQKSLSLSLGK
















TABLE 15







Antibody clone F11










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
SGSSSNIGTNTVN
67


 


CDR-L2
SNDQRPS
68


 


CDR-L3
ETWDDSLKGPV
69


 


CDR-H1
SYAMS
70


 


CDR-H2
TISGSGDSTYYADSVKG
71


 


CDR-H3
EWELGDAFDI
72


 


light chain
QSVLTQPPSTSGTPGQTFSIFCSGSSSNIGTNTVNWYQQ
243


variable
LPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAISG



region
LQSEDEADYYCETWDDSLKGPVFGGGTKVTVLGQPAAA



 


light chain
CAGTCTGTGCTGACTCAGCCACCCTCAACGTCTGGGACC
283


variable
CCCGGGCAGACGTTCTCCATTTTTTGTTCTGGAAGCAGT



region
TCGAACATCGGAACTAATACTGTTAATTGGTACCAGCAG



coding
CTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAAT



gene
GATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGC




TCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGG




CTCCAGTCTGAGGATGAGGCTGATTATTACTGTGAAACA




TGGGATGACAGCCTGAAAGGCCCGGTGTTCGGCGGGGGG




ACCAAGGTCACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYAMSWVRR
244


variable
APGKGLEWVSTISGSGDSTYYADSVKGRFTISRDNSKNT



region
LYLQMNNLRAEDTAVYYCAREWELGDAFDIWGRGTLVTV




SS



 


heavy chain
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAG
284


variable
CCTGGGGGGTCCCTGAAACTCTCCTGTGCAGCGTCTGGA



region
TTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCGG



coding
GCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGT



gene
GGTAGTGGTGATAGCACATACTACGCAGACTCCGTGAAG




GGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACG




CTGTATCTGCAAATGAACAACCTGAGAGCCGAGGACACG




GCCGTATATTACTGTGCGAGAGAATGGGAACTAGGCGAT




GCTTTTGATATCTGGGGCCGTGGCACCCTGGTCACCGTC




TCGAGT



 


light chain
QSVLTQPPSTSGTPGQTFSIFCSGSSSNIGTNTVNWYQQ
323


(Lambda)
LPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAISG




LQSEDEADYYCETWDDSLKGPVFGGGTKVTVLGQPAAAP




SVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS




SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSY




SCQVTHEGSTVEKTVAPTECS



 


heavy chain
EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYAMSWVRR
324



APGKGLEWVSTISGSGDSTYYADSVKGRFTISRDNSKNT




LYLQMNNLRAEDTAVYYCAREWELGDAFDIWGRGTLVTV




SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV




TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL




GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFL




GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ




FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW




LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP




PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY




KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE




ALHNHYTQKSLSLSLGK
















TABLE 16







Antibody clone D3










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
RASQSISSYLN
73


 


CDR-L2
AASSLQS
74


 


CDR-L3
QQSYSTRWT
75


 


CDR-H1
SYAMS
76


 


CDR-H2
AISGSGGSTYYADSVKG
77


 


CDR-H3
DRGSYGYYYGMDV
78


 


light chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK
245


variable
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL



region
QPEDFATYYCQQSYSTRWTFGQGTKVEIKRTAAA



 


light chain
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
285


variable
TCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT



region
CAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA



coding
CCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCC



gene
AGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGT




GGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG




CAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGT




TACAGTACCCGGTGGACGTTCGGCCAAGGGACCAAGGTG




GAAATCAAACGTACCGCGGCCGCA



 


heavy chain
EVQLLESGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQ
246


variable
APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT



region
LYLQMNSLRAEDTAVYYCAKDRGSYGYYYGMDVWGQGTM




VTVSS



 


heavy chain
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAG
286


variable
CCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



region
TCCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAG



coding
GCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGT



gene
GGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAG




GGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACG




CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACG




GCCGTATATTACTGTGCGAAAGACAGAGGCAGCTATGGT




TACTACTACGGTATGGACGTCTGGGGCCAAGGGACAATG




GTCACCGTCTCGAGT



 


light chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK
325


(Kappa)
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL




QPEDFATYYCQQSYSTRWTFGQGTKVEIKRTAAAPSVTL




FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK




AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV




THEGSTVEKTVAPTECS



 


heavy chain
EVQLLESGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQ
326



APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT




LYLQMNSLRAEDTAVYYCAKDRGSYGYYYGMDVWGQGTM




VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP




EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS




SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP




EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP




EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH




QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY




TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE




NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV




MHEALHNHYTQKSLSLSLGK
















TABLE 17







Antibody clone B12










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
RASQSISSYLN
79


 


CDR-L2
AASSLQS
80


 


CDR-L3
QQSYSTLRT
81


 


CDR-H1
GYYMH
82


 


CDR-H2
WINPNSGGTNYAQKFQG
83


 


CDR-H3
AGASIVGATALDY
84


 


light chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK
247


variable
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL



region
QPEDFATYYCQQSYSTLRTFGQGTKVEIKRTAAA



 


light chain
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCA
287


variable
TCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT



region
CAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA



coding
CCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCC



gene
AGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGT




GGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG




CAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGT




TACAGTACCCTCCGGACGTTCGGCCAAGGGACCAAGGTG




GAGATCAAACGTACCGCGGCCGCA



 


heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ
248


variable
APGQGLEWMGWINPNSGGTNYAQKFQGRVTITADESTST



region
AYMELSSLRSEDTAVYYCTRAGASIVGATALDYWGQGTL




VTVSS



 


heavy chain
CAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAG
288


variable
CCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGA



region
TACACCTTCACCGGCTACTATATGCACTGGGTGCGACAG



coding
GCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAAC



gene
CCTAACAGTGGTGGCACAAACTACGCACAGAAGTTCCAG




GGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACA




GCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACG




GCCGTGTATTACTGTACGAGAGCCGGTGCTTCTATAGTG




GGAGCTACCGCGCTTGACTACTGGGGCCAGGGAACCCTG




GTCACCGTCTCGAGT



 


light chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK
327


(Kappa)
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL




QPEDFATYYCQQSYSTLRTFGQGTKVEIKRTAAAPSVFI




FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS




GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACE




VTHQGLSSPVTKSFNRGEC



 


heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ
328



APGQGLEWMGWINPNSGGTNYAQKFQGRVTITADESTST




AYMELSSLRSEDTAVYYCTRAGASIVGATALDYWGQGTL




VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP




EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS




SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP




EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP




EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH




QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY




TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE




NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV




MHEALHNHYTQKSLSLSLGK
















TABLE 18







Antibody clone E4










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
TRSSGSIASNYVQ
85


 


CDR-L2
EDNQRPS
86


 


CDR-L3
QSYDTGNRNYV
87


 


CDR-H1
SYTIS
88


 


CDR-H2
RIIPILGIANYAQKFQG
89


 


CDR-H3
GPSLNYAGYFDN
90


 


light chain
NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQR
249


variable
PGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISG



region
LKTEDEADYYCQSYDTGNRNYVFGTGTQLTVLGQPAAA



 


light chain
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTC
289


variable
CGGGAAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGG



region
CAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGC



coding
CCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACC



gene
AAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCAT




CGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGA




CTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTT




ATGATACCGGCAATCGGAATTATGTCTTCGGAACTGGGAC




CCAGCTCACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQA
250


variable
PGQGLEWMGRIIPILGIANYAQKFQGRVTMTRDMSTDTAY



region
MELSSLTYDDTAVYFCVRGPSLNYAGYFDNWGQGTLVTVS




S



 


heavy chain
CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGC
290


variable
CTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGG



region
CACCTTCAGCAGCTATACTATCAGCTGGGTGCGACAGGCC



coding
CCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCTA



gene
TCCTTGGTATAGCAAACTACGCACAGAAGTTCCAGGGCAG




AGTCACCATGACCAGGGACATGTCCACAGACACAGCCTAC




ATGGAGTTGAGCAGCCTGACATATGATGACACGGCCGTAT




ATTTTTGTGTGAGAGGCCCTAGTCTTAATTATGCCGGCTA




TTTTGACAACTGGGGCCAGGGCACCCTGGTCACCGTCTCG




AGT



 


light chain
NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQR
329


(Lambda)
PGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISG




LKTEDEADYYCQSYDTGNRNYVFGTGTQLTVLGQPAAAPS




VTLFPPSSEEIQANKATLVCLISDFYPGAVTVAWKADSSP




VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ




VTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQA
330



PGQGLEWMGRIIPILGIANYAQKFQGRVTMTRDMSTDTAY




MELSSLTYDDTAVYFCVRGPSLNYAGYFDNWGQGTLVTVS




SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV




SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK




TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPS




VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV




DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY




KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT




KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD




SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK




SLSLSLGK
















TABLE 19







Antibody clone E12










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
QGDSLRSYYAS
91


 


CDR-L2
GKEKRPS
92


 


CDR-L3
NSRGSTTDYMV
93


 


CDR-H1
SYAMH
94


 


CDR-H2
VISYDGSNKYYADSVKG
95


 


CDR-H3
ERGSGMDV
96


 


light chain
SYELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKS
251


variable
GQAPVLVIYGKEKRPSGIPDRFSGSSSGNTASLTITGAR



region
AEDEADYYCNSRGSTTDYMVFGGGTQLTVLGQPAAA



 


light chain
TCCTATGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCC
291


variable
TTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGC



region
CTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGTCA



coding
GGACAGGCCCCTGTACTTGTCATCTATGGTAAAGAAAAG



gene
CGCCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGC




TCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCGG




GGGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGGC




AGCACTACTGACTATATGGTGTTCGGGGGGGGGACCCAG




CTCACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQ
252


variable
APGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNT



region
LYLQMNSLRAEDTAVYYCARERGSGMDVWGQGTLVTVSS



 


heavy chain
CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAG
292


variable
CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA



region
TTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAG



coding
GCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCA



gene
TATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAG




GGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACG




CTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACG




GCTGTGTATTACTGTGCGAGAGAACGGGGAAGTGGTATG




GACGTCTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT



 


light chain
SYELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKS
331


(Lambda)
GQAPVLVIYGKEKRPSGIPDRFSGSSSGNTASLTITGAR




AEDEADYYCNSRGSTTDYMVFGGGTQLTVLGQPAAAPSV




TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP




VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC




QVTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQ
332



APGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNT




LYLQMNSLRAEDTAVYYCARERGSGMDVWGQGTLVTVSS




ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV




SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT




KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG




PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN




WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN




GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS




QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT




TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL




HNHYTQKSLSLSLGK
















TABLE 20







Antibody clone D1










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
KASQDIDDDMN
97


 


CDR-L2
EASTLVP
98


 


CDR-L3
LQHDKFPYT
99


 


CDR-H1
SYGIS
100


 


CDR-H2
WINPNSGGTNYAQKFQG
101


 


CDR-H3
RGVDEGDY
102


 


light chain
ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQK
253


variable
PGEAAISIIQEASTLVPGIPPRFSGSGYGTDFTLTINNI



region
ESEDAAYYFCLQHDKFPYTFGQGTKLEIKRTAAA



 


light chain
GAAACGACACTCACGCAGTCTCCAGCATTCATGTCAGCG
293


variable
ACTCCAGGAGACAAAGTCAACATCTCCTGCAAAGCCAGC



region
CAAGACATTGATGATGATATGAACTGGTACCAACAGAAA



coding
CCAGGAGAAGCTGCTATTTCCATTATTCAAGAAGCTAGT



gene
ACTCTCGTTCCTGGAATCCCACCTCGATTCAGTGGCAGC




GGGTATGGAACAGATTTTACCCTCACAATTAATAACATA




GAATCTGAGGATGCTGCATATTACTTCTGTCTACAACAT




GATAAGTTCCCGTACACTTTTGGCCAGGGGACCAAGCTG




GAGATCAAACGTACCGCGGCCGCA



 


heavy chain
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQ
254


variable
APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSIST



region
AYMELSRLRSDDTAVYYCASRGVDEGDYWGQGTMVTVSS



 


heavy chain
GAAGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAG
294


variable
CCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGT



region
TACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAG



coding
GCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAAC



gene
CCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAG




GGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACA




GCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACG




GCCGTGTATTACTGTGCGAGTCGGGGGGTTGATGAGGGG




GACTACTGGGGCCAAGGGACAATGGTCACCGTCTCGAGT



 


light chain
ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQK
333


(Kappa)
PGEAAISIIQEASTLVPGIPPRFSGSGYGTDFTLTINNI




ESEDAAYYFCLQHDKFPYTFGQGTKLEIKRTAAAPSVFI




FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS




GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACE




VTHQGLSSPVTKSFNRGEC



 


heavy chain
EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQ
334



APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSIST




AYMELSRLRSDDTAVYYCASRGVDEGDYWGQGTMVTVSS




ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV




SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT




KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG




PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN




WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN




GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS




QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT




TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL




HNHYTQKSLSLSLGK
















TABLE 21







Antibody clone E6










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO





CDR-L1
TGSSGNIASNYVQ
103


 


CDR-L2
RDDQRPS
104


 


CDR-L3
QSYDSSSWV
105


 


CDR-H1
TYDIT
106


 


CDR-H2
WMNPNSGNSRSAQKFQG
107


 


CDR-H3
GDYSGVVLTATALDY
108


 


light chain
NFMLTQPHSVSESPGKTVTLSCTGSSGNIASNYVQWYQH
255


variable
RPGSAPTTVIYRDDQRPSGVPDRFSGSIDSSSNSASLTI



region
SGLRPEDEADYYCQSYDSSSWVFGGGTKLTVLGQPAAA



 


light chain
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCT
295


variable
CCGGGGAAGACGGTTACCCTCTCCTGCACCGGCAGCAGC



region
GGCAACATTGCCAGTAACTATGTGCAGTGGTACCAGCAC



coding
CGCCCGGGCAGTGCCCCCACCACTGTGATCTACCGGGAT



gene
GACCAAAGACCCTCTGGAGTCCCTGATCGCTTCTCTGGC




TCCATCGACAGTTCATCCAACTCTGCCTCCCTCACGATC




TCTGGACTGAGGCCTGAGGACGAGGCTGACTATTACTGT




CAGTCTTATGATAGCAGCTCTTGGGTGTTCGGCGGAGGG




ACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYDITWVRQ
256


variable
APGQGLEWMGWMNPNSGNSRSAQKFQGRVSMTSDSSIST



region
AYMELSSLRSEDTAVYYCATGDYSGVVLTATALDYWGQG




TLVTVSS



 


heavy chain
CAGGTCCAGCTTGTGCAGTCTGGAGCAGAGGTGAAGAAG
296


variable
CCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGA



region
TACACCTTCACCACTTATGATATCACCTGGGTGCGACAG



coding
GCCCCTGGACAAGGCCTTGAGTGGATGGGATGGATGAAC



gene
CCGAACAGTGGTAACTCACGCTCTGCACAGAAGTTCCAG




GGCAGAGTCAGCATGACCAGTGACTCCTCCATAAGCACA




GCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACG




GCCGTGTATTACTGTGCAACAGGAGACTACTCGGGTGTG




GTACTAACTGCAACAGCACTTGACTACTGGGGCCAGGGA




ACCCTGGTCACCGTCTCGAGT



 


light chain
NFMLTQPHSVSESPGKTVTLSCTGSSGNIASNYVQWYQH
335


(Lambda)
RPGSAPTTVIYRDDQRPSGVPDRFSGSIDSSSNSASLTI




SGLRPEDEADYYCQSYDSSSWVFGGGTKLTVLGQPAAAP




SVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS




SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSY




SCQVTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYDITWVRQ
336



APGQGLEWMGWMNPNSGNSRSAQKFQGRVSMTSDSSIST




AYMELSSLRSEDTAVYYCATGDYSGVVLTATALDYWGQG




TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY




FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV




PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP




APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE




DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV




LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ




VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ




PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC




SVMHEALHNHYTQKSLSLSLGK
















TABLE 22







Antibody clone E9










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO





CDR-L1
SGSSSNIGNNYVY
109


 


CDR-L2
RNNQRPS
110


 


CDR-L3
AAWDDSLSGWV
111


 


CDR-H1
SYGMH
112


 


CDR-H2
NIKQDGSEKYYVDSVKG
113


 


CDR-H3
EDRIAAAGMRELDY
114


 


light chain
QSELTQLPSASETPGQRVTISCSGSSSNIGNNYVYWYQQ
257


variable
LPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISG



region
LRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPAAA



 


light chain
CAGTCTGAGCTGACTCAGCTACCCTCAGCGTCTGAGACC
297


variable
CCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGC



region
TCCAACATCGGAAATAATTATGTATACTGGTACCAGCAA



coding
CTCCCCGGAACGGCCCCCAAACTCCTCATCTATAGGAAT



gene
AATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGC




TCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGG




CTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCA




TGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGG




ACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA



 


heavy chain
QVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMHWVRQ
258


variable
APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNT



region
LYLQMNSLRAEDTAVYYCAREDRIAAAGMRELDYWGQGT




LVTVSS



 


heavy chain
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAG
298


variable
CCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGA



region
TTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAG



coding
GCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAG



gene
CAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAG




GGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACG




CTGTATCTCCAAATGAACAGCCTGAGAGCTGAGGACACG




GCTGTGTATTACTGTGCGAGAGAGGACCGTATAGCAGCA




GCTGGGATGCGGGAGTTGGACTACTGGGGCCAGGGCACC




CTGGTCACCGTCTCGAGT



 


light chain
QSELTQLPSASETPGQRVTISCSGSSSNIGNNYVYWYQQ
337


(Lambda)
LPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISG




LRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPAAAP




SVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS




SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSY




SCQVTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMHWVRQ
338



APGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNT




LYLQMNSLRAEDTAVYYCAREDRIAAAGMRELDYWGQGT




LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF




PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP




SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA




PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED




PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL




HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV




YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP




ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS




VMHEALHNHYTQKSLSLSLGK
















TABLE 23







Antibody clone A11










Amino acid sequence (N→C)/




Nucleic acid sequence (5′→3′)
SEQ ID NO





CDR-L1
RSSQSLLHSNGYNYLD
115


 


CDR-L2
LGSNRAS
116


 


CDR-L3
MQGTHWPPYT
117


 


CDR-H1
SYAMT
118


 


CDR-H2
GISSDGTTTTYADSVRG
119


 


CDR-H3
DQLLGWDALNV
120


 


light chain
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLD
259


variable
WYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTL



region
KISRVEAEDVGVYYCMQGTHWPPYTFGQGTKVEIKRTAA




A



 


light chain
GATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTC
299


variable
ACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT



region
CAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGAT



coding
TGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTG



gene
ATCTATTTGGGTTCTAACCGGGCCTCCGGGGTCCCTGAC




AGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTG




AAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTAT




TACTGCATGCAAGGTACACACTGGCCTCCGTACACCTTT




GGCCAGGGGACCAAGGTGGAGATCAAACGTACCGCGGCC




GCA



 


heavy chain
EVQLLESGGGLEQPGGFLRLSCAASGFSFTSYAMTWVRQ
260


variable
APGKGLEWVSGISSDGTTTTYADSVRGRFTISRDNAKNT



region
VYLQMNSLRDEDTAVYYCARDQLLGWDALNVWGQGTMVT




VSS



 


heavy chain
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGAACAG
300


variable
CCTGGGGGGTTCCTGAGACTCTCCTGTGCAGCCTCTGGA



region
TTCTCCTTTACCAGCTACGCCATGACCTGGGTCCGCCAG



coding
GCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGT



gene
AGTGATGGGACCACTACAACCTACGCGGACTCCGTGAGG




GGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACACG




GTGTATCTCCAAATGAACAGTCTGAGAGACGAGGACACG




GCTGTGTATTATTGTGCAAGAGATCAATTGTTGGGCTGG




GATGCTCTGAATGTCTGGGGCCAAGGGACAATGGTCACC




GTCTCGAGT



 


light chain
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLD
339


(Kappa)
WYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTL




KISRVEAEDVGVYYCMQGTHWPPYTFGQGTKVEIKRTAA




APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV




DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH




KLYACEVTHQGLSSPVTKSFNRGEC



 


heavy chain
EVQLLESGGGLEQPGGFLRLSCAASGFSFTSYAMTWVRQ
340



APGKGLEWVSGISSDGTTTTYADSVRGRFTISRDNAKNT




VYLQMNSLRDEDTAVYYCARDQLLGWDALNVWGQGTMVT




VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP




VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS




LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF




LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV




QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD




WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL




PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN




YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH




EALHNHYTQKSLSLSLGK









Example 2: Assay of In Vitro Biological Activities of the Selected Antibodies
2.1. Natural Killer Cell (NK Cell) Surface Binding Assay
In order to test whether or not 93 antibodies selected in Example 1.4 bind LILRB1 expressed on surface of immune cells, natural killer cell (NK cell) surface binding assay was performed. A human NK cell, KHYG-1 cell (JCRB) was cultured in RPMI 1640 medium (Gibco) supplemented with 10% (w/v) of FBS (Gibco) and 100 U/mL of interleukin-2 (Novartis). KHYG-1 cells were added to a U-bottom 96-well tissue culture plate (BD Falcon) at the amount of 5×104 cells/well. Each of the selected antibodies was added to the well to the final concentration of 50 μg/mL per well and incubated at 4° C. for 1 hour.
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, the cells were treated with streptavidin PE (BD Pharmigen) and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were resuspended and subjected to analysis using iQue screener (Sartorius).
Among the obtained results, the results for antibodies A10, E3, E4, F12, G1, G9, G11, H2 and H11 are representatively compared with that of human IgG4 isotype (control), which are shown in Table 24. The flow cytometry diagrams for A10, E3 and human IgG4 isotype (control) are shown in FIGS. 4a (A10), 4b (E3), and 4c (isotype IgG4), respectively:










TABLE 24







Mean Fluorescence




Intensity
% of population 2


















human IgG4 isotype
142917.2
2.95


control


A10
222660.2
28.68


E3
268702.2
40.22


E4
272295.5
43.25


F12
262012.7
38.02


G1
321051.7
56.23


G9
263079.3
41.32


G11
262771.9
40.11


H2
238570.9
29.00


H11
244818.2
32.59









As shown in Table 24 and FIGS. 4a˜4c, the tested antibodies show higher level of binding to human NK cells (surface), compared to that of human IgG4 isotype control antibody.
2.2. Analysis of Inhibition of LILRB1 Binding to HLA-G by the Selected Antibodies
In order to test whether or not the antibodies selected in Example 1.5 exert an inhibitory effect on binding of LILRB1 to its ligand, HLA-G, the degree of blocking by the selected antibodies was analyzed.
For this purpose, JEG-3 cells (ATCC cat #HTB-36), which show high expression level of HLA-G, were used. JEG-3 cells were cultured in MEM medium (Gibco) supplemented with 10% (v/v) of FBS (Gibco) and 1% (v/v) of pen-strep (Gibco). The JEG-3 cells were added to U-bottom 96-well tissue culture plate (BD Falcon) at the amount of 5×104 cells/well. The well plate was washed with 1×PBS buffer. Each of the antibodies selected in Example 1.5 (A10, E3, F12, G1, G9, H2 and H11) and LILRB1-Fc (RnD systems) were mixed in FACS buffer (1×PBS+1% BSA+1 mM EDTA) to the final concentrations of 10 μg/mL and 5 μg/mL, respectively. The cells were treated with 100 μL of the mixture solution per well and incubated on ice for 2 hours. An anti-LILRB1 antibody (clone HP-F1, Abcam) as a positive control and an anti-lysozyme IgG4 antibody (clone D1.3) as a negative control were treated in the same manner. After washing with FACS buffer twice, the cells were treated with PE-anti-hulgG-Fc antibody (Biolegend, 10 μg/mL) and incubated on ice for one hour. After washing with FACS buffer twice, the cells were resuspended in 100 μL of the same buffer and subjected to analysis using iQue screener (Sartorius).
The obtained results are shown in FIG. 5. As shown in FIG. 5, all the tested antibodies A10, E3, F12, G1, G9, H2 and H11 effectively inhibit the binding of LILRB1-Fc to HLA-G-overexpressing cell line.
2.3. Assay of Cancer Cell Lysis by NK Cells
In order to test whether or not the selected antibodies 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 addeded to 96-well tissue culture plate (BD Falcon) at the amount of 2×104 cells/well (4×104 cells/mL, total volume 50 μL). The cells were treated with each antibody (Table 25) to the final concentration of 20 μg/mL per well, and left 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 (which were prepared by transduction of HEK293 cells (American Typo Culture Collection) with lentivirus constructed for expressing HLA-G) were stained 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 1×104 cells/well (2×104 cells/mL, total volume 50 μL). 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. Red area confluence indicating the density of live HLA-G-overexpressing HEK293 cells was measured, and cell viability was calculated. The obtained cell viabilities are shown in Table 25 (wherein the cell viabilities are shown as a relative value to that of control antibody (cell viability of IgG4 isotype-treated well=1)):



 
  

  
   
    Relative
    ⁢
       
    cell
    ⁢
       
    viability
    ⁢
       
    
     (
     
      
       IgG
       ⁢
       4
       ⁢
          
       Isotype
      
      =
      1
     
     )
    
   
   =
   
    
     Normalized
     ⁢
        
     red
     ⁢
        
     area
     ⁢
        
     confluence
     ⁢
        
     value
     ⁢
        
     of
     ⁢
        
     antibody
    
    
     Normalized
     ⁢
        
     red
     ⁢
        
     area
     ⁢
        
     confluence
     ⁢
        
     value
     ⁢
        
     of
     ⁢
        
     IgG
     ⁢
     4
     ⁢
        
     Isotype
    
   
  
 











TABLE 25





Antibody
Relative cell viability (IgG4 isotype = 1)
















human IgG4 Isotype control
1.00


A10
0.70


B9
0.81


D3
0.83


E1
0.82


E3
0.64


F12
0.81


G1
0.64


G6
0.78


G9
0.77


G11
0.82


H2
0.78


H11
0.60









As shown in Table 25, all the tested antibodies including A10, B9, D3, E1, E3, F12, G1, G6, G9, G11, H2 and H11 increase cell death of HLA-G-overexpressing HEK293 cells by KHYG-1, compared to that of human IgG4 isotype control antibody.
Example 3: Assay of In Vivo Biological Activities of the Selected Antibodies
Among the antibodies selected in Example 1.5, two antibodies (E3 and B3) were tested for their in vivo anti-cancer efficacies. For this purpose, it was tested whether or not administration of the two antibodies reduces tumor size where the tumor was generated by engrafting human colorectal carcinoma cells (Bioware Brite Cell Line HCT116 Red-Fluc colorectal carcinoma cells (PerkinElmer)) and THP-1 derived macrophages 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). Hereinafter, the processes are described in detail:
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).
Measurement of Anti-Cancer Efficacy in Mouse Model
5-week old female CIEA NOG mice [NOG immunodeficient mouse] (Central Institute for Experimental Animals, Japan) were subcutaneously injected with a mixture of 3×106 cells of HCT116 Red-Fluc colorectal carcinoma cells, 3×106 cells of THP-1 derived macrophages and each of two test antibodies (E3 or B3 antibody; 20 μg per mouse). From the 4th day after tumor grafting, the antibody was administered to the mouse model at the dosage of 5 mg/kg by intraperitoneal injection twice a week. Then, the size (mm3) of the grafted tumor was measured and shown in FIG. 6. As shown in FIG. 6, all the tested antibodies, particularly antibody E3, exhibit statistically significant effect of inhibiting tumor growth in mouse models grafted with HCT116 colon cancer cells and THP-1 derived macrophages.
Example 4: Preparation of Anti-LILRB1 Antibody (E3.1)
The nucleic acid sequence encoding the full-length heavy chain (SEQ ID NO: 302) of antibody E3, which was confirmed to have particularly significant effect in Example 3, was amplified by PCR. The nucleic acid sequence encoding the region from Ser1 to Leu110 of the light chain variable region (VL) (SEQ ID NO: 221) of antibody E3 was amplified by PCR and ligated to a nucleic acid sequence encoding the lambda constant region (Lambda CL.1, SEQ ID NO: 344) to amplify the nucleic acid sequence encoding lambda light chain by PCR. The amplified sequences were inserted into an expression vector (pTRIOZ-hIgG4, InvivoGen; alternatively, any one of vectors comprising CMV promoter or CMV/CHO beta-actin fusion promoter (KR10-1038126B1) and genes encoding human IgG4 heavy chain constant region and lambda light chain constant region, can be used), wherein the expression vector was designed for encoding a human IgG4 antibody. The DNA sequence of the expression vector was confirmed by sequencing.
An antibody (E3.1) was prepared using the constructed expression vector referring to Example 1.4, and the sequence of the antibody was analyzed referring to Example 1.6 and summarized in Table 26:







TABLE 26







Antibody clone E3.1










amino acid sequence (N→C)/




nucleic acid sequence (5′→3′)
SEQ ID NO












CDR-L1
QGDSLRNFYAS
1


 


CDR-L2
GKNNRPS
2


 


CDR-L3
NSRDSSGSHLTGV
3


 


CDR-H1
SYAMS
4


 


CDR-H2
AISGSGGSTYYADSVKG
5


 


CDR-H3
DTYYYGSGRSNAFDI
6


 


light chain
SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQ
345


variable
KSGQAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTI



region
TGAQAEDEADYYCNSRDSSGSHLTGVFGGGTKVTVL



 


light chain
TCCTATGAGCTGACTCAGGACCCTGCTGTGTCTGTGG
346


variable
CCTTGGGACAGACAGTCAGGATCACATGCCAGGGAGA



region
CAGCCTCAGAAACTTTTATGCAAGCTGGTACCAGCAG



coding
AAGTCAGGACAGGCCCCAGTTCTTGTCATGTATGGTA



gene
AAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTC




TGGCTCCACCTCAGGAAACACAGCTTCCTTGACCATC




ACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACT




GTAACTCCCGGGACAGCAGTGGTAGCCATTTGACGGG




CGTATTCGGCGGAGGGACCAAGGTCACCGTCCTA



 


heavy chain
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWV
222


variable
RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN



region
SKNTLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAF




DIWGQGTLVTVSS



 


heavy chain
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAC
262


variable
AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC



region
TGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTC



coding
CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAG



gene
CTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGA




CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT




TCCAAGAATACGCTGTATCTGCAAATGATTAGCCTGA




GAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGA




TACGTATTACTATGGTTCGGGGAGAAGTAATGCTTTT




GATATATGGGGCCAGGGAACCCTGGTCACCGTCTCGA




GT



 


light chain
SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQ
347


(Lambda)
KSGQAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTI




TGAQAEDEADYYCNSRDSSGSHLTGVFGGGTKVTVLG




QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAV




TVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLT




PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS



 


heavy chain
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWV
302



RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN




SKNTLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAF




DIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA




LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG




LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV




ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR




TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR




EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP




SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT




CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS




FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS




LSLSLGK









Example 5: Generation of Human LILR-Overexpressing Cell Lines
The nucleic acid sequences encoding the full-length human LILR family proteins (Table 27) were amplified by PCR, and each of the amplified sequences was inserted into an expression vector (pTRIOZ-hIgG4, InvivoGen; alternatively, any one of vectors comprising CMV promoter or CMV/CHO beta-actin fusion promoter (KR10-1038126B1) and genes encoding human IgG4 heavy chain constant region and lambda light chain constant region, can be used). The DNA sequence of the expression vector was confirmed by sequencing. The constructed vector was transfected into CHO cells, to generate 11 stable cell lines overexpressing each LILR protein on its surface.











TABLE 27






Genbank

SEQ




Accession

ID



Protein
No.
amino acid sequence (N→C)
NO
Antibody







LILRB1
AAH15731
MTPILTVLICLGLSLGPRTHVQAGHL
348
Human




PKPTLWAEPGSVITQGSPVTLRCQGG

LILRB1




QETQEYRLYREKKTAPWITRIPQELV

antibody




KKGQFPIPSITWEHAGRYRCYYGSDT

(ab185796,




AGRSESSDPLELVVTGAYIKPTLSAQ

Abcam)




PSPVVNSGGNVTLQCDSQVAFDGFIL






CKEGEDEHPQCLNSQPHARGSSRAIF






SVGPVSPSRRWWYRCYAYDSNSPYEW






SLPSDLLELLVLGVSKKPSLSVQPGP






IVAPEETLTLQCGSDAGYNRFVLYKD






GERDFLQLAGAQPQAGLSQANFTLGP






VSRSYGGQYRCYGAHNLSSEWSAPSD






PLDILIAGQFYDRVSLSVQPGPTVAS






GENVTLLCQSQGWMQTFLLTKEGAAD






DPWRLRSTYQSQKYQAEFPMGPVTSA






HAGTYRCYGSQSSKPYLLTHPSDPLE






LVVSGPSGGPSSPTTGPTSTSGPEDQ






PLTPTGSDPQSGLGRHLGVVIGILVA






VILLLLLLLLLFLILRHRRQGKHWTS






TQRKADFQHPAGAVGPEPTDRGLQWR






SSPAADAQEENLYAAVKHTQPEDGVE






MDTRSPHDEDPQAVTYAEVKHSRPRR






EMASPPSPLSGEFLDTKDRQAEEDRQ






MDTEAAASEAPQDVTYAQLHSLTLRR






KATEPPPSQEGPSPAVPSIYATLAIH




 


LILRB2
AAH36827
MTPIVTVLICLGLSLGPRTHVQTGTI
349
Human




PKPTLWAEPDSVITQGSPVTLSCQGS

LILRB2/




LEAQEYRLYREKKSASWITRIRPELV

CD85d/




KNGQFHIPSITWEHTGRYGCQYYSRA

ILT4




RWSELSDPLVLVMTGAYPKPTLSAQP

antibody




SPVVTSGGRVTLQCESQVAFGGFILC

(MAB2078,




KEGEDEHPQCLNSQPHARGSSRAIFS

R&D




VGPVSPNRRWSHRCYGYDLNSPYVWS

Systems)




SPSDLLELLVPGVSKKPSLSVQPGPV






VAPGESLTLQCVSDVGYDRFVLYKEG






ERDLRQLPGRQPQAGLSQANFTLGPV






SRSYGGQYRCYGAYNLSSEWSAPSDP






LDILITGQIHGTPFISVQPGPTVASG






ENVTLLCQSWRQFHTFLLTKAGAADA






PLRLRSIHEYPKYQAEFPMSPVTSAH






AGTYRCYGSLNSDPYLLSHPSEPLEL






VVSGPSMGSSPPPTGPISTPAGPEDQ






PLTPTGSDPQSGLGRHLGVVIGILVA






VVLLLLLLLLLFLILRHRRQGKHWTS






TQRKADFQHPAGAVGPEPTDRGLQWR






SSPAADAQEENLYAAVKDTQPEDGVE






MDTRAAASEAPQDVTYAQLHSLTLRR






KATEPPPSQEGEPPAEPSIYATLAIH




 


LILRB3
XP_006726377
MTPALTALLCLGLSLGPRTRVQAGPF
350
Human




PKPTLWAEPGSVISWGSPVTIWCQGS

LILRB3/




LEAQEYRLDKEGSPEPLDRNNPLEPK

CD85a/




NKARFSIPSMTEHHAGRYRCHYYSSA

ILT5




GWSEPSDPLELVMTGFYNKPTLSALP

antibody




SPVVASGGNMTLRCGSQKGYHHFVLM

(MAB1806,




KEGEHQLPRTLDSQQLHSGGFQALFP

R&D




VGPVNPSHRWRFTCYYYYMNTPQVWS

Systems)




HPSDPLEILPSGVSRKPSLLTLQGPV






LAPGQSLTLQCGSDVGYDRFVLYKEG






ERDFLQRPGQQPQAGLSQANFTLGPV






SPSHGGQYRCYGAHNLSSEWSAPSDP






LNILMAGQIYDTVSLSAQPGPTVASG






ENVTLLCQSWWQFDTFLLTKEGAAHP






PLRLRSMYGAHKYQAEFPMSPVTSAH






AGTYRCYGSYSSNPHLLSFPSEPLEL






MVSGHSGGSSLPPTGPPSTPGLGRYL






EVLIGVSVAFVLLLFLLLFLLLRRQR






HSKHRTSDQRKTDFQRPAGAAETEPK






DRGLLRRSSPAADVQEENLYAAVKDT






QSEDRVELDSQSPHDEDPQAVTYAPV






KHSSPRREMASPPSSLSGEFLDTKDR






QVEEDRQMDTEAAASEASQDVTYAQL






HSLTLRRKATEPPPSQEGEPPAEPSI






YATLAIH




 


LILRB4
NP_001265355
MIPTFTALLCLGLSLGPRTHMQAGPL
351
Human




PKPTLWAEPGSVISWGNSVTIWCQGT

LILRB4/




LEAREYRLDKEESPAPWDRQNPLEPK

CD85k/




NKARFSIPSMTEDYAGRYRCYYRSPV

ILT3




GWSQPSDPLELVMTGAYSKPTLSALP

antibody




SPLVTSGKSVTLLCQSRSPMDTFLLI

(MAB24251,




KERAAHPLLHLRSEHGAQQHQAEFPM

R&D




SPVTSVHGGTYRCFSSHGFSHYLLSH

Systems)




PSDPLELIVSGSLEGPRPSPTRSVST






AAGPEDQPLMPTGSVPHSGLRRHWEV






LIGVLVVSILLLSLLLFLLLQHWRQG






KHRTLAQRQADFORPPGAAEPEPKDG






GLQRRSSPAADVQGENFCAAVKNTQP






EDGVEMDTRQSPHDEDPQAVTYAKVK






HSRPRREMASPPSPLSGEFLDTKDRQ






AEEDRQMDTEAAASEAPQDVTYARLH






SFTLRQKATEPPPSQEGASPAEPSVY






ATLAIH




 


LILRB5
NP_006831
MTLTLSVLICLGLSVGPRTCVQAGTL
352
Human




PKPTLWAEPASVIARGKPVTLWCQGP

LILRB5/




LETEEYRLDKEGLPWARKRONPLEPG

CD85c/




AKAKFHIPSTVYDSAGRYRCYYETPA

LIR-8




GWSEPSDPLELVATGFYAEPTLLALP

antibody




SPVVASGGNVTLQCDTLDGLLTFVLV

(MAB3065,




EEEQKLPRTLYSQKLPKGPSQALFPV

R&D




GPVTPSCRWRFRCYYYYRKNPQVWSN

Systems)




PSDLLEILVPGVSRKPSLLIPQGSVV






ARGGSLTLQCRSDVGYDIFVLYKEGE






HDLVQGSGQQPQAGLSQANFTLGPVS






RSHGGQYRCYGAHNLSPRWSAPSDPL






DILIAGLIPDIPALSVQPGPKVASGE






NVTLLCQSWHQIDTFFLTKEGAAHPP






LCLKSKYQSYRHQAEFSMSPVTSAQG






GTYRCYSAIRSYPYLLSSPSYPQELV






VSGPSGDPSLSPTGSTPTPGPEDQPL






TPTGLDPQSGLGRHLGVVTGVSVAFV






LLLFLLLFLLLRHRHQSKHRTSAHFY






RPAGAAGPEPKDQGLQKRASPVADIQ






EEILNAAVKDTQPKDGVEMDARAAAS






EAPQDVTYAQLHSLTLRREATEPPPS






QEREPPAEPSIYAPLAIH




 


LILRA1
NP_006854
MTPIVTVLICLRLSLGPRTHVQAGTL
353
Human




PKPTLWAEPGSVITQGSPVTLWCQGI

LILRA1/




LETQEYRLYREKKTAPWITRIPQEIV

LILRB1




KKGQFPIPSITWEHTGRYRCFYGSHT

antibody




AGWSEPSDPLELVVTGAYIKPTLSAL

(MAB30851,




PSPVVTSGGNVTLHCVSQVAFGSFIL

R&D




CKEGEDEHPQCLNSQPRTHGWSRAIF

Systems)




SVGPVSPSRRWSYRCYAYDSNSPHVW






SLPSDLLELLVLGVSKKPSLSVQPGP






IVAPGESLTLQCVSDVSYDRFVLYKE






GERDFLQLPGPQPQAGLSQANFTLGP






VSRSYGGQYRCSGAYNLSSEWSAPSD






PLDILIAGQFRGRPFISVHPGPTVAS






GENVTLLCQSWGPFHTFLLTKAGAAD






APLRLRSIHEYPKYQAEFPMSPVTSA






HSGTYRCYGSLSSNPYLLSHPSDSLE






LMVSGAAETLSPPQNKSDSKAGAANT






LSPSQNKTASHPQDYTVENLIRMGIA






GLVLVVLGILLFEAQHSQRSL




 


LILRA2
AAH17412
MTPILTVLICLGLSLGPRTHVQAGHL
354
Human




PKPTLWAEPGSVIIQGSPVTLRCQGS

LILRA2/




LQAEEYHLYRENKSASWVRRIQEPGK

CD85h/




NGQFPIPSITWEHAGRYHCQYYSHNH

ILT1




SSEYSDPLELVVTGAYSKPTLSALPS

antibody




PVVTLGGNVTLQCVSQVAFDGFILCK

(MAB6364,




EGEDEHPQRLNSHSHARGWSWAIFSV

R&D




GPVSPSRRWSYRCYAYDSNSPYVWSL

Systems)




PSDLLELLVPGVSKKPSLSVQPGPMV






APGESLTLQCVSDVGYDRFVLYKEGE






RDFLQRPGWQPQAGLSQANFTLGPVS






PSHGGQYRCYSAHNLSSEWSAPSDPL






DILITGQFYDRPSLSVQPVPTVAPGK






NVTLLCQSRGQFHTFLLTKEGAGHPP






LHLRSEHQAQQNQAEFRMGPVTSAHV






GTYRCYSSLSSNPYLLSLPSDPLELV






VSASLGQHPQDYTVENLIRMGVAGLV






LVVLGILLFEAQHSQRSLQDAAGR




 


LILRA3
AAH28208
MTSILTVLICLGLSLDPRTHVQAGPL
355
Human




PKPTLWAEPGSVITQGSPVTLRCQGS

LILRA3/




LETQEYHLYREKKTALWITRIPQELV

CD85e




KKGQFPILSITWEHAGRYCCIYGSHT

antibody




VGLSESSDPLELVVTGAYSKPTLSAL

(PA5-47349,




PSPVVTSGGNVTIQCDSQVAFDGFIL

Invitrogen)




CKEGEDEHPQCLNSHSHARGSSRAIF






SVGPVSPSRRWSYRCYGYDSRAPYVW






SLPSDLLGLLVPGVSKKPSLSVQPGP






VVAPGEKLTFQCGSDAGYDRFVLYKE






WGRDFLQRPGRQPQAGLSQANFTLGP






VSRSYGGQYTCSGAYNLSSEWSAPSD






PLDILITGQIRARPFLSVRPGPTVAS






GENVTLLCQSQGGMHTFLLTKEGAAD






SPLRLKSKRQSHKYQAEFPMSPVTSA






HAGTYRCYGSLSSNPYLLTHPSDPLE






LVVSGAAETLSPPQNKSDSKAGE




 


LILRA4
NP_036408
MTLILTSLLFFGLSLGPRTRVQAENL
356
CD85g




PKPILWAEPGPVITWHNPVTIWCQGT

(ILT7)




LEAQGYRLDKEGNSMSRHILKTLESE

antibody




NKVKLSIPSMMWEHAGRYHCYYQSPA

(16-5179-82,




GWSEPSDPLELVVTAYSRPTLSALPS

Invitrogen)




PVVTSGVNVTLRCASRLGLGRFTLIE






EGDHRLSWTLNSHQHNHGKFQALFPM






GPLTFSNRGTFRCYGYENNTPYVWSE






PSDPLQLLVSGVSRKPSLLTLQGPVV






TPGENLTLQCGSDVGYIRYTLYKEGA






DGLPQRPGRQPQAGLSQANFTLSPVS






RSYGGQYRCYGAHNVSSEWSAPSDPL






DILIAGQISDRPSLSVQPGPTVTSGE






KVTLLCQSWDPMFTFLLTKEGAAHPP






LRLRSMYGAHKYQAEFPMSPVTSAHA






GTYRCYGSRSSNPYLLSHPSEPLELV






VSGATETLNPAQKKSDSKTAPHLQDY






TVENLIRMGVAGLVLLFLGILLFEAQ






HSQRSPPRCSQEANSRKDNAPFRVVE






PWEQI




 


LILRA5
NP_067073
MAPWSHPSAQLQPVGGDAVSPALMVL
357
Human




LCLGLSLGPRTHVQAGNLSKATLWAE

LILRA5/




PGSVISRGNSVTIRCQGTLEAQEYRL

CD85f




VKEGSPEPWDTQNPLEPKNKARFSIP

antibody




SMTEHHAGRYRCYYYSPAGWSEPSDP

(MAB6754,




LELVVTGFYNKPTLSALPSPVVTSGE

R&D




NVTLQCGSRLRFDRFILTEEGDHKLS

Systems)




WTLDSQLTPSGQFQALFPVGPVTPSH






RWMLRCYGSRRHILQVWSEPSDLLEI






PVSGAADNLSPSQNKSDSGTASHLQD






YAVENLIRMGMAGLILVVLGILIFQD






WHSQRSPQAAAGR




 


LILRA6
NP_001347096
MTPALTALLCLGLSLGPRTRVQAGPF
358
Human




PKPTLWAEPGSVISWGSPVTIWCQGS

LILRA6/




LEAQEYQLDKEGSPEPLDRNNPLEPK

CD85b




NKARFSIPSMTQHHAGRYRCHYYSSA

antibody




GWSEPSDPLELVMTGFYNKPTLSALP

(MAB86




SPVVASGGNMTLRCGSQKGYHHFVLM

56, R&D




KEGEHQLPRTLDSQQLHSGGFQALFP

Systems)




VGPVTPSHRWRFTCYYYYTNTPRVWS






HPSDPLEILPSGVSRKPSLLTLQGPV






LAPGQSLTLQCGSDVGYDRFVLYKEG






ERDFLQRPGQQPQAGLSQANFTLGPV






SPSHGGQYRCYGAHNLSSEWSAPSDP






LNILMAGQIYDTVSLSAQPGPTVASG






ENVTLLCQSRGYFDTFLLTKEGAAHP






PLRLRSMYGAHKYQAEFPMSPVTSAH






AGTYRCYGSYSSNPHLLSFPSEPLEL






MVSGHSGGSSLPPTGPPSTPASHAKD






YTVENLIRMGMAGLVLVFLGILLFEA






QHSQRNPQDAAGR









Example 6: Determination of EC50 for Binding of the Selected Antibodies to LILRB1 Overexpressing Cell Surface
In order to determine EC50 values of the antibodies prepared in Examples 1 and 4 for binding to human LILRB1-overexpressing cell lines, a cell surface binding assay was performed. Representing the prepared antibodies, EC50 values of E3.1 and H11 antibodies were measured. The CHO cells prepared in Example 5, which overexpress LILRB1 on surface, were added to U-bottom 96-well tissue culture plate (BD Falcon) at the amount of 1×105 cells/well. Threefold serial dilutions of E3.1 and H11 antibodies were prepared starting from the final concentrations of 600 ug/mL and 27 ug/mL, respectively. The cells were treated with each of the diluted antibodies and incubated at 4° C. for 60 minutes. After washing with FACS buffer, the cells were treated with anti-human Fc-biotin antibody (Invitrogen) and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were treated with streptavidin (BD Pharmigen) labeled with PE fluorescence and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were resuspended and subjected to analysis using iQue screener (Sartorius). EC50 values were calculated using nonlinear regression formula of GraphPad Prism software, and the obtained results are shown in Table 28:












TABLE 28








E3.1

H11









EC50 (nM)
7.154

0.376










Example 7: Assessment of Cross-Reactivity of the Selected Antibodies to Human LILR Family-Overexpressing Cell Lines
In order to confirm whether or not the selected antibodies bind to human LILR family proteins other than LILRB1, a cell surface binding assay was performed. The CHO cells (prepared in Example 5) expressing each of various LILR family proteins on surface were added to U-bottom 96-well tissue culture plate (BD Falcon) at the amount of 1×105 cells/well. The cells in each well were treated with the selected antibody in the final concentration of 20 ug/mL and incubated at 4° C. for 60 minutes. After washing with FACS buffer, the cells were treated with anti-human Fc-biotin antibody (Invitrogen) and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were treated with streptavidin (BD Pharmigen) labeled with PE or FITC fluorescence and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were resuspended and subjected to analysis using iQue screener (Sartorius). The cells treated with each LILR protein specific antibody (Table 27) were used as a positive control, and the cells treated with human IgG4 isotype control antibody (Biolegend) were used as a negative control.
The results obtained for antibody E3.1 are shown in FIGS. 7a to 7d (E3.1: red; LILR-specific antibody: blue; Isotype (hIgG4) control: gray), and the results obtained for antibody H11 are shown in FIGS. 8a to 8d (H11: red; LILR-specific antibody: blue; Isotype (hIgG4) control: gray). As shown in FIGS. 7a to 7d and FIGS. 8a to 8d, the E3.1 and H11 antibodies do not bind at all or hardly bind to LILRs other than LILRB1. These results indicate that the antibodies provided by the examples have binding abilities specifically to LILRB1.
Example 8: Measurement of Release of Granzyme B and Perforin by Enzyme-Linked Immune Absorbent Spot (ELISPOT) Assay
In order to confirm whether the E3.1 and H11 antibodies increase the level of cytotoxicity of NK cells, an enzyme-linked immune absorbent spot (ELISPOT) assay was performed. The level of cytotoxicity was determined by the release of cytotoxic granules, granzyme B and perforin, in NK cells.
5×103 cells of LILRB1-expressing KHYG-1 cell lines (JCRB) and 5×103 cells of HLA-G-overexpressing K562 cells (which were prepared by transduction of K562 cells (American Type Culture Collection) with lentivirus constructed for expressing HLA-G) were co-cultured in U-bottom 96-well tissue culture plate. Each antibody (E3.1, H11 or human IgG4 isotype control antibody) was added thereto with final concentration of 50 ug/mL per each well and left incubated at 37° C. for 30 minutes. The co-cultured cells were transferred onto 96 well plates (Immunospot, Cat. HGZBPFN-2M) (PVDF membrane) for ELISPOT, which were coated with anti-perforin antibody and anti-granzyme B antibody, respectively, and further incubated at 37° C. for 8 hours. The PVDF membranes were washed with a washing solution (0.05% tween 20 in PBS), then treated with anti-granzyme B-HRP and anti-perforin-biotin antibodies. Then, detection processes were performed according to the manufacturer's protocol. The PVDF membranes were dried at room temperature for 24 hours, and the number of spots for granzyme B and perforin were counted by ELISPOT analyzer (Immunospot).
The results are shown in FIG. 9 (granzyme b; Gzmb) and FIG. 10 (perforin; Prf), respectively (Y-axis indicates the total number of spots). As shown in FIG. 9 and FIG. 10, the release levels of both of granzyme B and perforin are significantly increased in E3.1 or H11 antibody-treated group, as compared with the human IgG4 isotype control antibody-treated group. Unpaired T-test was performed, and all experiments were performed three times under the same conditions for the reliability of the experiment, and the results are shown as average values.
Example 9: Preparation of Chimeric GHI/75 Antibody
In order to see if antibodies provided by the examples show higher efficacy compared to pre-existing antibodies, a chimeric GHI/75 antibody comprising a variable region of GHI/75 antibody (Biolegend, cat #333721), which is a mouse-derived anti-human LILRB1 antibody, and a constant region of human antibody was prepared.
More specifically, the amino acid sequence of the GHI/75 antibody was analyzed through peptide mapping, and a vector, in which the nucleic acid sequence corresponding to the variable region (VH and VL domain) of a human IgG4 antibody was replaced by the nucleic acid sequence corresponding to the variable region (VH and VL domain) of mouse GHI/75 antibody, was prepared. In the vector, the region corresponding to upper hinge of human IgG4 was substituted with the nucleic acid sequence corresponding to the amino acid sequence (EPKSCDKTHT; SEQ ID NO: 359) of human IgG1 upper hinge. The vector was expressed as described in Example 1.4, and the obtained antibody was purified and used as a comparative antibody in examples below.
Example 10: Measurement of Inhibitory Effect of the Selected Antibodies on LILRB1 Signaling Using IL-2 Promoter Luciferase Assay
In order to confirm whether the antibodies prepared in Examples 1 and 4 inhibit signaling by LILRB1, a luciferase reporter assay was performed. Among the antibodies prepared in Examples 1 and 4, the assay was performed representatively for E3.1 and H11 antibodies, and the chimeric GHI/75 antibody prepared in Example 9 was used for comparison. Jurkat cells expressing LILRB1 and interleukin 2 (IL-2) promoter luciferase (which were prepared by inserting IL-2 promoter luciferase vector (Promega) into Jurkat cell line (American Type Culture Collection) followed by transduction with lentivirus constructed for expressing LILRB1) and HLA-G-overexpressing K562 cells were used. Ninety six-well plates were coated with anti-CD3 antibody (Biolegend) by incubating with the antibody overnight at 4° C. On the next day, Jurkat cells expressing LILRB1 and IL-2 promoter luciferase were added to U-bottom 96-well plates at the amount of 1×105 cells/well, and the plates were treated with each antibody (E3.1, H11, chimeric GHI/75 or human IgG4 isotype (control)) to the final concentration of 20 ug/mL and incubated at 37° C. for one hour. HLA-G-overexpressing K562 cells (1×105 cells/well) were added to the plates and incubated at 37° C. for 30 minutes. The obtained suspension was transferred to the plate coated with anti-CD3 antibody, and anti-CD28 antibody (Biolegend) was added thereto to the final concentration of 10 ug/mL. The plate was incubated at 37° C. for 6 hours. Steady-Glo® solution (Promega) was added to each well, and the luminescence intensity was recorded using a luminometer (Envision, PerkinElmer).
The results are shown in FIG. 11. As shown in FIGS. 11, E3.1 and H11 antibodies provided in examples exhibit considerably increased LILRB1 signaling inhibitory activity compared to human IgG4 isotype control antibody and the chimeric GHI/75 control antibody.
Example 11: Analysis of Anti-Cancer Effect of Selected Antibodies in Mouse Model
For analysis of anti-cancer effects of selected antibodies, referring to Example 3, a mixture of 3×106 cells of HCT116 Red-Fluc colorectal carcinoma cells, 3×106 cells of THP-1 derived macrophages and an antibody (20 μg/mouse) was subcutaneously injected to 5-week old female CIEA NOG mice (NOG immunodeficient mouse; Central Institute for Experimental Animals, Japan) The antibody used was E3.1 or H11 antibody, and human IgG4 isotype was used as a control antibody for comparison. From the 4th day after grafting tumor cells, the antibody was administered to the mouse model at the dosage of 5 mg/kg by intraperitoneal injection twice a week, and the tumor volume was measured and shown in FIG. 12. As shown in FIGS. 12, E3.1 and H11 antibodies exhibit significant effect of inhibiting tumor growth in mouse models grafted with HCT116 colon cancer cells and THP-1 derived macrophages compared to the control antibody.