Title of the Invention: Anti-LILRB1 Antibodies and Their Uses
technical field
[One]
Cross-Citation with Related Applications
[2]
This application claims the benefit of priority based on Korean Patent Application No. 10-2019-0173414 dated December 23, 2019 and Korean Patent Application No. 10-2020-0061907 dated May 22, 2020, All content disclosed in the literature is incorporated as a part of this specification.
[3]
This application relates to anti-LILRB1 antibodies and uses thereof, and specifically to anti-LILRB1 antibodies or antigen-binding fragments thereof, and uses thereof for the treatment of cancer.
[4]
background
[5]
Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1, or ILT2, CD85j, LIR-1) is an inhibitory receptor expressed on B cells, T cells, NK cells, dendritic cells, macrophages and other immune cells. to be. LILRB1 binds to classical and non-classical MHC class-I, and has a signaling mechanism that inhibits immune cell activity.
[6]
On the other hand, it is known that various cancer cells overexpress MHC class I such as HLA-G for immune evasion. When the binding of LILRB1 and MHC Class I is inhibited, it is expected to show anticancer effect by restoring the activity of suppressed immune cells.
[7]
Therefore, there is a need to develop a substance that binds to LILRB1 and inhibits LILRB1 from binding to MHC Class I and/or inhibits the interaction between LILRB1 and MHC Class I.
[8]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[9]
Provided are an antibody that binds to LILRB1 and shows anticancer activity by acting on immune cells expressing LILRB1 to regulate the activity of immune cells, and a use of the antibody for treating cancer.
[10]
One example provides an anti-LILRB1 antibody or antigen-binding fragment thereof that binds to LILRB1. The anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity of inhibiting the binding of LILRB1 to MHC Class I and/or an activity of inhibiting the interaction between LILRB1 and MHC Class I. The anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity of inhibiting immune evasion of cancer cells. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have excellent anticancer activity. The cancer may be one that expresses or overexpresses MHC Class I on the surface.
[11]
Another example provides a pharmaceutical composition for treating and/or preventing cancer, the pharmaceutical composition comprising the anti-LILRB1 antibody or antigen-binding fragment thereof.
[12]
Another example provides a pharmaceutical composition for inhibiting the binding of LILRB1 to MHC Class I and/or a pharmaceutical composition for inhibiting the interaction between LILRB1 and MHC Class I, comprising the anti-LILRB1 antibody or antigen-binding fragment thereof.
[13]
Another example provides a pharmaceutical composition for inhibiting immune evasion of cancer cells comprising the anti-LILRB1 antibody or antigen-binding fragment thereof.
[14]
means of solving the problem
[15]
One example provides an anti-LILRB1 antibody or antigen-binding fragment thereof that binds to LILRB1. The anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity of inhibiting the binding of LILRB1 to MHC Class I and/or inhibiting the interaction between LILRB1 and MHC Class I. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity of inhibiting immune evasion of cancer cells. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have excellent anticancer activity.
[16]
The anti-LILRB1 antibody or antigen-binding fragment thereof may include the following complementarity determining region (CDR):
[17]
(1) CDR definition according to Kabat numbering (Kabat, EA, Wu, TT, Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91 -3242; based on http://www.abysis.org/),
[18]
a CDR- comprising the 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; L1,
[19]
a CDR- comprising the 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; L2,
[20]
a CDR- comprising the 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; L3,
[21]
a CDR- comprising the 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; H1,
[22]
a CDR- comprising the 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; H2, and
[23]
a CDR- comprising the 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; H3
[24]
include, or
[25]
(2) Based on the CDR definition according to IMGT numbering (http://www.imgt.org/),
[26]
a CDR- comprising the 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; L1,
[27]
a CDR- comprising the 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; L2,
[28]
a CDR- comprising the 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; L3,
[29]
a CDR- comprising the 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; H1,
[30]
a CDR- comprising the 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; H2, and
[31]
a CDR- comprising the 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; H3
[32]
may include.
[33]
In one embodiment, the six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) that can be included in an anti-LILRB1 antibody or antigen-binding fragment thereof provided herein ) are illustrated in Table 1 below:
[34]
[Table 1]
CDR Amino acid sequence (N→C) (Kabat) SEQ ID NO: Amino acid sequence (N→C) (IMGT) SEQ ID NO:
E3/E3.1 CDR-L1 QGDSLRNFYAS One SLRNFY 121
CDR-L2 GKNNRPS 2 GKN 122
CDR-L3 NSRDSSGSHLTGV 3 NSRDSSGSHLTGV 3
CDR-H1 SYAMS 4 GFTFSSYA 123
CDR-H2 AISGSGGSTYYADSVKG 5 ISGSGGST 124
CDR-H3 DTYYYGSGRSNAFDI 6 ARDTYYYGSGRSNAFDI 125
B3 CDR-L1 QASQDISNYLN 7 QDISNY 126
CDR-L2 DASNLET 8 DAS 127
CDR-L3 QQYDNLP 9 QQYDNLP 9
CDR-H1 DYAM 10 GFTFDDYA 128
CDR-H2 GISWNSGSIGYADSVKG 11 ISWNSGSI 129
CDR-H3 VGDSSGWSDAFDI 12 ARVGDSSGWSDAFDI 130
A10 CDR-L1 RASQSVSSNLA 13 QSVSSSN 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 ARGGLGELDNWFDP 135
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 WISAYNGNTNYAQELQG 23 ISAYNGNT 139
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 ASQYYDGGYYMDV 145
H2 CDR-L1 QGDSLRNYYAS 31 SLRNYY 146
CDR-L2 GNNKRPS 32 GNN 147
CDR-L3 NSLDSTYNHPI 33 NSLDSTYNHPI 33
CDR-H1 SYDIH 34 GYTFSYD 148
CDR-H2 WISAYNGNTNYAQKLQG 35 ISAYNGNT 149
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 DVSRPS 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 ASQYYDGGYYMDV 160
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 ARGEIAVAQNWDYYGMDV 165
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 ASQYYDGGYYMDV 170
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 ARGWGYSSSFDY 175
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 AKDRGSYGYYYGMDV 185
B12 CDR-L1 RASQSISSYLN 79 QSISSY 186
CDR-L2 AASSLQS 80 AAS 187
CDR-L3 QQSYSTLRT 81 QQSYSTLRT 81
CDR-H1 GYYMH 82 GYTFTGYY 188
CDR-H2 WINPNSGGTNYAQKFQG 83 INNPSGGT 189
CDR-H3 AGASIVGATALDY 84 TRAGASIVGATALDY 190
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 VRGPSLNYAGYFDN 195
E12 CDR-L1 QGDSLRSYYAS 91 SLRSYY 196
CDR-L2 GKEKRPS 92 GKE 197
CDR-L3 NSRGSTTDYMV 93 NSRGSTTDYMV 93
CDR-H1 SYAM 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 GYTFSYG 203
CDR-H2 WINPNSGGTNYAQKFQG 101 INNPSGGT 204
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 WMNPNSGNSRSAQKFQG 107 MNPNSGNS 209
CDR-H3 GDYSGVVLTAALDY 108 ATGDYSGVVLTAALDY 210
E9 CDR-L1 SGSSSNIGNNYVY 109 SSNIGNNY 211
CDR-L2 RNNQRPS 110 RNN 212
CDR-L3 AAWDDSLSGWV 111 AAWDDSLSGWV 111
CDR-H1 SYGM 112 GFTFSSYG 213
CDR-H2 NIKQDGSEKYYVDSVKG 113 IKQDGSEK 214
CDR-H3 EDRIAAAGMRELDY 114 AREDRIAAAGMRELDY 215
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 ARDQLLGWDALNV 220
[35]
In one embodiment, the anti-LILRB1 antibody or antigen-binding fragment thereof, as described above,
[36]
a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, and
[37]
Heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3
[38]
may include.
[39]
In an embodiment, the anti-LILRB1 antibody or antigen-binding fragment thereof comprises:
[40]
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 a light chain variable region, and
[41]
a heavy chain variable comprising the 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 area
[42]
may include.
[43]
Combinations of light chain variable regions and heavy chain variable regions that can be included in the anti-LILRB1 antibody or antigen-binding fragment thereof provided herein are exemplified in Table 2 below:
[44]
[Table 2]
variable region Amino acid sequence (N→C) SEQ ID NO:
E3 light chain variable region SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKSGQAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTITGAQAEDEADYYCNSRDSSGSHLTGVFGGGTKVTVLGQPAAA 221
heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAFDIWGQGTLVTVSS 222
B3 light chain variable region DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPFGGGTKVDIKRTAAA 223
heavy chain variable region EVQLLESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGDSSGWSDAFDIWGQGTMVTVSS 224
A10 light chain variable region DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSSPRMYTFGQGTKVDIKRTAAA 225
heavy chain variable region QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSISTAYMELSSLRSEDTAVYYCARGGLGELDNWFDPWGQGTLVTVSS 226
G1 light chain variable region SYELTQPPSLSVSPGQTASITCSGYKLGDRYVSWYQQKTGQSPVVVIYKDSQRPSGVPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSGTGVFGGGTKLTVLGQPAAA 227
heavy chain variable region EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQELQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVGVAGKLDYWGQGTLVTVSS 228
G9 light chain variable region QSALTQPASVSGSPGQSITISCTGSSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSDRFSGSKSGNMASLTISGLQAEDEADYYCSSYTGSSTLDVLFGGGTKLTVLGQPAAA 233
heavy chain variable region QVQLVQPGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVTVSS 234
H2 light chain variable region SYELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQAPILVISGNNKRPSGIPDRFSGSSSGDTASLTISGAQAEDEADYYCNSLDSTYNHPIFGGGTKVTVLGQPAAA 235
heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIHWVRQATGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGGDAFDIWGQGTLVTVSS 236
H11 light chain variable region SYELTQDPAASVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVVVIYGRNNRPSGIPDRFSGSSSGDTASLTITGAQAEDEADYYCKSRDSSGNHYVFGTGTKLTVLGQPAAA 231
heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDAGSSSDYWGRGTLVTVSS 232
F12 light chain variable region QSVLTQPASVSGSPGQSITISCAGTSSDIGDYDYVSWYQQHPGKTPKLMIYDVSRRPSGVPDRFSGSKSGNTASLTISGLQTEDEADYYCASYTSSSVVVFGGGTKLTVLGQPAAA 237
heavy chain variable region QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVTVSS 238
B9 light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYHCQQAYGFPLTLGGGTKVEIKRTAAA 229
heavy chain variable region QVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGEIAVAQNWDYYGMDVWGQGTLVTVSS 230
G11 light chain variable region QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYSSSSTLVVFGGGTKLTVLGQPAAA 239
heavy chain variable region QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVTVSS 240
G6 light chain variable region SYELTQDPAVSVALGQTVTITCQGDSLRRYYATWYQQKPGQAPVLVIYGQNYRPSGIPDRFSGSNSGTTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPAAA 241
heavy chain variable region EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGWGYSSSFDYWGQGTTVTVSS 242
F11 light chain variable region QSVLTQPPSTSGTPGQTFSIFCSGSSSNIGTNTVNWYQQLPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCETWDDSLKGPVFGGGTKVTVLGQPAAA 243
heavy chain variable region EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYAMSWVRRAPGKGLEWVSTISGSGDSTYYADSVKGRFTISRDNSKNTLYLQMNNLRAEDTAVYYCAREWELGDAFDIWGRGTLVTVSS 244
D3 light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTRWTFGQGTKVEIKRTAAA 245
heavy chain variable region EVQLLESGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGSYGYYYGMDVWGQGTMVTVSS 246
B12 light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTLRTFGQGTKVEIKRTAAA 247
heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTRAGASIVGATALDYWGQGTLVTVSS 248
E4 light chain variable region NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDTGNRNYVFGTGTQLTVLGQPAAA 249
heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTMTRDMSTDTAYMELSSLTYDDTAVYFCVRGPSLNYAGYFDNWGQGTLVTVSS 250
E12 light chain variable region SYELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKSGQAPVLVIYGKEKRPSGIPDRFSGSSSGNTASLTITGARAEDEADYYCNSRGSTTDYMVFGGGTQLTVLGQPAAA 251
heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERGSGMDVWGQGTLVTVSS 252
D1 light chain variable region ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQKPGEAAISIIQEASTLVPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQHDKFPYTFGQGTKLEIKRTAAA 253
heavy chain variable region EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASRGVDEGDYWGQGTMVTVSS 254
E6 light chain variable region NFMLTQPHSVSESPGKTVTLSCTGSSGNIASNYVQWYQHRPGSAPTTVIYRDDQRPSGVPDRFSGSIDSSSNSASLTISGLRPEDEADYYCQSYDSSSWVFGGGTKLTVLGQPAAA 255
heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYDITWVRQAPGQGLEWMGWMNPNSGNSRSAQKFQGRVSMTSDSSISTAYMELSSLRSEDTAVYYCATGDYSGVVLTATALDYWGQGTLVTVSS 256
E9 light chain variable region QSELTQLPSASETPGQRVTISCSGSSSNIGNNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPAAA 257
heavy chain variable region QVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAREDRIAAAGMRELDYWGQGTLVTVSS 258
A11 light chain variable region DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPYTFGQGTKVEIKRTAAA 259
heavy chain variable region EVQLLESGGGLEQPGGFLRLSCAASGFSFTSYAMTWVRQAPGKGLEWVSGISSDGTTTTYADSVRGRFTISRDNAKNTVYLQMNSLRDEDTAVYYCARDQLLGWDALNVWGQGTMVTVSS 260
E3.1 light chain variable region SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKSGQAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTITGAQAEDEADYYCNSRDSSGSHLTGVFGGGTKVTVL 345
heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAFDIWGQGTLVTVSS 222
[45]
In the present specification, an antibody or antigen-binding fragment thereof (eg, CDR, variable region, or heavy chain / light chain) "comprises a specific amino acid sequence or is expressed or consists of a specific amino acid sequence" means essentially comprising the amino acid sequence case, and a case in which meaningless mutations (eg, substitution, deletion, and/or addition of amino acid residues) that do not affect antibody activity are introduced into the amino acid sequence.
[46]
An anti-LILRB1 antibody or antigen-binding fragment thereof provided herein has a binding affinity (K D ) for LILRB1 (eg, human LILRB1).) may be, for example, 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, based on the case measured by surface plasmon resonance (SPR), 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 , 1nM to 1mM, 0.001nM to 0.5mM, 0.005nM to 0.5mM, 0.01nM to 0.5mM, 0.05nM to 0.5mM, 0.1nM to 0.5mM, 0.5nM to 0.5mM, 1nM to 0.5mM, 0.001nM 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.
[47]
Another example provides a pharmaceutical composition comprising the anti-LILRB1 antibody or antigen-binding fragment thereof as an active ingredient. For example, the pharmaceutical composition may be a pharmaceutical composition for the treatment and/or prevention of cancer. The pharmaceutical composition may have an activity of inhibiting 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 an interaction between LILRB1 and MHC Class I. In one example, the pharmaceutical composition may have an activity to inhibit immune evasion of cancer cells. The cancer cell may be one that expresses or overexpresses MHC Class I on the surface.
[48]
Another example provides a composition for inhibiting binding of LILRB1 to MHC Class I and/or a composition for inhibiting interaction between LILRB1 and MHC Class I, comprising the anti-LILRB1 antibody or antigen-binding fragment thereof as an active ingredient.
[49]
Another example provides a composition for inhibiting immune evasion of cancer cells comprising the anti-LILRB1 antibody or antigen-binding fragment thereof as an active ingredient.
[50]
Another example is the step of administering (oral or parenteral administration) a pharmaceutically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof to a subject in need of treatment and/or prevention of cancer (eg, a mammal including a human) It provides a method for treating and/or preventing cancer, comprising a.
[51]
Another example is to administer a pharmaceutically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof to a subject in need of binding LILRB1 to MHC Class I and/or inhibiting the interaction between LILRB1 and MHC Class I (e.g., mammals including humans ) to (oral or parenteral administration), providing a method for inhibiting the binding of LILRB1 to MHC Class I and/or a method for inhibiting the interaction between LILRB1 and MHC Class I.
[52]
Another example includes the step of administering (oral or parenteral administration) a pharmaceutically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof to a subject in need of inhibiting immune evasion of cancer cells (eg, mammals including humans) It provides a method for inhibiting immune evasion of cancer cells.
[53]
The method provided herein requires inhibition of binding of LILRB1 to MHC Class I and/or inhibition of interaction between LILRB1 and MHC Class I in a subject in need of treatment and/or prevention of cancer, prior to the administering step It may further comprise the step of identifying a subject in need, and/or a subject in need of inhibition of immune evasion of cancer cells.
[54]
Other examples include the CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, or CDR-L1, CDR-L2, and CDR-L3 combination, 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 it provides a nucleic acid molecule (polynucleotide) encoding one or more polypeptides selected from the group consisting of a heavy chain comprising the heavy chain variable region.
[55]
Another example provides a recombinant vector comprising the nucleic acid molecule. In one embodiment, the recombinant vector comprises the light chain variable region or light chain encoding nucleic acid molecule and the heavy chain variable region or heavy chain encoding nucleic acid molecule, respectively (eg, in two vectors) or together (eg, in one vector) may be doing The recombinant vector may be used as an expression vector.
[56]
Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector.
[57]
Another example provides a method for producing an anti-LILRB1 antibody or antigen-binding fragment thereof, comprising expressing the nucleic acid molecule in a cell. The step of expressing the nucleic acid molecule may include culturing the recombinant cell.
[58]
As used herein, the antigen-binding fragment of the anti-LILRB1 antibody refers to a fragment derived from the anti-LILRB1 antibody and having binding affinity to the antigen (LILRB1), and the six CDRs of the anti-LILRB1 antibody. Any polypeptide comprising 2 , but is not limited thereto. In one embodiment, the antigen-binding fragment comprises an scFv, or a fusion polypeptide ( scFv-Fc) or a fusion polypeptide (scFv-Ck or scFv-Cλ) fused to the constant region of the light chain (eg, kappa or lambda).
[59]
The anti-LILRB1 antibody or antigen-binding fragment thereof may have a regulatory action on the LILRB1 protein, for example, antagonism or agonism. The anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity of inhibiting 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 antigen-binding fragment thereof may have an activity of inhibiting immune evasion of cancer cells. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have excellent anticancer activity.
[60]
LILRB1 acting as an antigen of the antibody or antigen-binding fragment provided herein may be of mammalian origin, for example, human-derived LILRB1 (eg, 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 is not limited thereto.
[61]
MHC Class I described herein is a class of major histocompatibility complex (MHC) molecules. In one embodiment, the MHC Class I may be of human origin, and selected from the group consisting of human leukocyte antigen (HLA)-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. It may be one or more, but is not limited thereto.
[62]
As used herein, the term "antibody" refers 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 chemically synthesized or recombinantly prepared protein thereof, The type is not particularly limited. The antibody may be non-naturally produced, eg, recombinantly or synthetically produced. The antibody may be an animal antibody (eg, a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal antibody or a polyclonal antibody.
[63]
In the anti-LILRB1 antibody or antigen-binding fragment thereof provided herein, the regions other than the heavy and light chain CDR regions, or heavy and light chain variable regions, as defined above, are of all subtypes of immunoglobulins (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.), e.g., the framework regions of immunoglobulins of all subtypes, and/or light chain constant regions and/or heavy chain constant regions. It may be from the realm. In one embodiment, the anti-LILRB1 antibody provided herein may be a human IgG-type antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody, but is not limited thereto.
[64]
A complete antibody (eg, IgG type) has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has a gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) type, subclass gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) or alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.
[65]
The term "heavy chain" refers to a variable region domain V H comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains C H1 , C H2 and C H3 and a hinge ( hinge) is interpreted as meaning including all of the full-length heavy chain and fragments thereof. In addition, the term "light chain" refers to both a full-length light chain comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and fragments thereof . interpreted as including
[66]
The term “complementarity determining region (CDR)” refers to a region that provides antigen-binding specificity among variable regions of an antibody, and refers to the amino acid sequence of the hypervariable region of the heavy and light chains of an immunoglobulin. . The heavy and light chains may each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for the antibody to bind antigen or epitope. Meanwhile, in the present specification, the terms, "specifically binding" or "specifically recognized" have the same meaning as commonly known to those skilled in the art, and the antigen and the antibody specifically interact to produce an immunological reaction. means that
[67]
In the present specification, an antibody, unless otherwise specified, may be understood to include an antigen-binding fragment of an antibody having antigen-binding ability as well as a complete antibody.
[68]
The term "antigen-binding fragment" refers to any form of a polypeptide comprising an antigen-binding portion (eg, six CDRs as defined herein). For example, the antibody may be scFv, (scFv) 2 , scFvFc, Fab, Fab' or F(ab') 2 , but is not limited thereto. In addition, as described above, the antigen-binding fragment is an scFv, or the scFv is an immunoglobulin (eg, IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.) Fc region or light chain constant It may be a fusion polypeptide fused to a region (eg, kappa or lambda).
[69]
Among the antigen-binding fragments, Fab has a structure having variable regions of a light chain and a heavy chain, a constant region of a light chain, and a first constant region of a heavy chain (C H1 ).
[70]
Fab' differs from Fab in that it has a hinge region comprising one or more cysteine ​​residues at the C-terminus of the heavy chain C H1 domain.
[71]
The F(ab') 2 antibody is produced by forming a disulfide bond with a cysteine ​​residue in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombinant technique for generating an Fv fragment is well known in the art.
[72]
In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in a single-chain Fv (single-chain Fv), the heavy chain variable region and the single chain variable region are generally shared through a peptide linker. Since they are linked by a bond or are linked directly at the C-terminus, they can form a dimer-like structure like a double-stranded Fv.
[73]
The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, by restriction digestion of the whole antibody with papain, Fab can be obtained, and when digested with pepsin, F(ab') 2 fragment can be obtained), It can be produced through genetic recombination technology.
[74]
The term “hinge region” refers to a region included in the heavy chain of an antibody, which exists between the CH1 and CH2 regions, and functions to provide flexibility of the antigen-binding site in the antibody.
[75]
The anti-LILRB1 antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared by methods well known in the art. For example, it may be manufactured using a phage display technique. Alternatively, the anti-LILRB1 antibody may be prepared as a mouse-derived monoclonal antibody by a conventional method.
[76]
Meanwhile, individual monoclonal antibodies can be screened based on their ability to bind to LILRB1 using a typical ELISA (Enzyme-Linked ImmunoSorbent Assay) format. Inhibitory activity can be assayed for binding agents through functional assays such as competitive ELISA (Competitive ELISA) or functional assays such as cell-based assays for assaying molecular interactions. Then, the respective affinity (Kd values) for LILRB1 can be assayed for selected monoclonal antibody members based on their strong inhibitory activity.
[77]
The pharmaceutical composition provided herein may further include a pharmaceutically acceptable carrier, in addition to the active ingredient (anti-LILRB1 antibody or antigen-binding fragment thereof). The pharmaceutically acceptable carriers are those commonly used in drug formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose. , polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, may be at least one selected from the group consisting of mineral oil, etc., but limited thereto it's not going to be The pharmaceutical composition may further include at least one selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, which are commonly used in the manufacture of pharmaceutical compositions.
[78]
The effective amount of the pharmaceutical composition or the antibody or antigen-binding fragment thereof may be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, intranasal administration, intrapulmonary administration, rectal administration, or local administration at the lesion site may be administered. Since the protein or peptide is digested upon oral administration, oral compositions may be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device capable of transporting the active agent to a target cell (eg, a cancer cell).
[79]
The anti-LILRB1 antibody or antigen-binding fragment thereof may be included in the pharmaceutical composition or administered to a patient in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” may mean an amount of the active ingredient (anti-LILRB1 antibody or antigen-binding fragment thereof) capable of exerting a desired effect (eg, anticancer effect). The pharmaceutically effective amount may be prescribed in various ways depending on factors such as the patient's age, weight, sex, pathological condition, food, excretion rate, reaction sensitivity, formulation method, administration time, administration interval, administration route, administration method, etc. . For example, the daily dose of the anti-LILRB1 antibody or antigen-binding fragment thereof is 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, but is not limited thereto. The daily dosage is formulated as one preparation in unit dosage form, or
[80]
The pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in oil or aqueous medium, or may be formulated in the form of an extract, powder, powder, granule, tablet or capsule, and a dispersant or stabilizer for formulation. may additionally include.
[81]
A patient to which the present invention is applied may be a mammal including humans and primates including monkeys, and rodents including mice and rats.
[82]
The cancer may be a solid cancer or a blood cancer, but is not limited to lung cancer (eg, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cancer, etc.), peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, anus Peripheral cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, leukemia (eg chronic or acute leukemia), lymphoma, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, Liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cell cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, osteosarcoma, etc. It may be at least one selected from the group consisting of. The cancer may be a primary cancer or a metastatic cancer. The cancer may be one in which MHC Class I is expressed or overexpressed on the surface, for example, colon adenocarcinoma, small cell lung carcinoma, breast cancer, pancreatic cancer, malignant melanoma ), bone osteosarcoma, renal cell carcinoma, and gastric cancer. The overexpression of MHC Class I is a normal cell or cancer cell that does not show an anticancer effect (eg, is not responsive to or resistant to the immunotherapy) to immunotherapy, such as T-cell (eg, cytotoxic T-cell) mediated immunotherapy. Compared to , it may mean that the antibody is overexpressed in the target cancer cells.
[83]
In the present specification, the treatment of cancer may refer to any anti-cancer action that prevents, alleviates, or improves symptoms of cancer, such as inhibiting proliferation of cancer cells, killing cancer cells, inhibiting metastasis, and the like, or partially or completely annihilating cancer.
[84]
The anti-LILRB1 antibody or antigen-binding fragment thereof provided herein may be used in combination with other drugs, such as at least one selected from the group consisting of commonly used immunotherapeutic agents, anticancer agents, cytotoxic agents, and the like. Accordingly, an example includes (1) an anti-LILRB1 antibody or antigen-binding fragment thereof, and (2) one or more drugs selected from the group consisting of immunotherapeutic agents, anticancer agents, cytotoxic agents, etc. or a pharmaceutical composition for concomitant administration for treatment. Another example is one or more selected from the group consisting of 1) an anti-LILRB1 antibody or antigen-binding fragment thereof, and (2) an immunotherapeutic agent, an anticancer agent, a cytotoxic agent, etc. to a patient in need of prevention and/or treatment of cancer A method for preventing and/or treating cancer, comprising administering a drug, is provided. The immunotherapeutic agent, anticancer agent, and cytotoxic agent are generally used for cancer treatment, and/or include all drugs having cytotoxic activity, proteins such as antibodies, nucleic acid molecules such as siRNA, and/or paclitaxel, docetaxel At least one may be selected from among small molecule compounds such as, but not limited thereto.
[85]
In another example, 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) of the above-described anti-LILRB1 antibody , or a combination thereof), or a combination thereof; Or a polypeptide molecule comprising a heavy chain variable region, a light chain variable region, or a combination thereof is provided. The polypeptide molecule can be used for antibody production as a precursor of an antibody, and can be included as a component of a protein scaffold (eg, a peptibody) having a structure similar to that of an antibody, a bispecific antibody, or a multispecific antibody. In another example, the polypeptide molecule can be used as a target (antigen) recognition moiety, a secreted antibody, or a cell therapeutic agent designed to secrete the anti-LILRB1 antibody in a target cell therapy agent such as CAR-T.
[86]
Another example 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 an anti-LILRB1 antibody.
[87]
Another example 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 an anti-LILRB1 antibody.
[88]
Another example is a nucleic acid molecule encoding a heavy chain complementarity determining region, heavy chain variable region or heavy chain of the anti-LILRB1 antibody and a nucleic acid molecule encoding a light chain complementarity determining region, light chain variable region or light chain of the anti-LILRB1 antibody in one vector Recombinant vectors contained together or contained in separate vectors are provided.
[89]
Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector.
[90]
The term “vector” refers to a means for expressing a gene of interest in a host cell. Viral vectors such as, for example, plasmid vectors, cosmid vectors and bacteriophage vectors, lentiviral vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors are included. Vectors that can be used as the recombinant vector include plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14). , pGEX series, pET series, and pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1 and M13, etc.) or viruses (eg, SV40, etc.).
[91]
In the recombinant vector, the nucleic acid molecule may be operably linked to a promoter. The term “operatively linked” refers to a functional linkage between a nucleotide expression control sequence (eg, a promoter sequence) and another nucleotide sequence. Such regulatory sequences may be "operatively linked" to control the transcription and/or translation of other nucleotide sequences.
[92]
The recombinant vector is typically constructed as a vector for cloning or a vector for expression. The expression vector may be a conventional vector used to express a foreign protein in plants, animals or microorganisms in the art. The recombinant vector can be constructed through various methods known in the art.
[93]
The recombinant vector may be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and a prokaryotic cell is a host, a strong promoter capable of propagating transcription (eg, pL λ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. In the case of a eukaryotic cell as a host, the origin of replication operating in the eukaryotic cell contained in the vector includes the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, and the BBV origin of replication. It is not limited. In addition, a promoter derived from the genome of a mammalian cell (eg, a metallotionine promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, etc.) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.
[94]
The recombinant cell may be obtained by introducing the recombinant vector into an appropriate host cell. As the host cell, any host cell known in the art may be used as a cell capable of stably and continuously cloning or expressing the recombinant vector, and as a prokaryotic cell, for example, E. coli JM109, E. coli E. coli such as BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus sp. strains such as Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium Um, there are enterobacteriaceae and strains such as Serratia marcescens and various Pseudomonas species, and in the case of transformation into eukaryotic cells, as a host cell, yeast ( Saccharomyces cerevisiae), insect cells, plant 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 cell lines, etc. may be used, but is not limited thereto.
[95]
For the delivery (introduction) of the nucleic acid molecule or a recombinant vector containing the same into a host cell, a delivery method well known in the art may be used. The transport method, for example, when the host cell is a prokaryotic cell, CaCl 2 method or electroporation method, etc. can be used, and when the host cell is a eukaryotic cell, microinjection method, calcium phosphate precipitation method, electroporation method, Liposome-mediated transfection and gene bombardment may be used, but are not limited thereto.
[96]
The method of selecting the transformed host cell can be easily carried out according to a method well known in the art using the phenotype expressed by the selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.
[97]
Another example provides a method for producing an anti-LILRB1 antibody or antigen-binding fragment thereof, comprising expressing the nucleic acid molecule or a recombinant vector containing the same in a host cell. The expressing step may be performed by culturing a recombinant cell containing the nucleic acid molecule (eg, contained in a recombinant vector) under conditions permissive for expression of the nucleic acid molecule. The production method may include the step of isolating and/or purifying the antibody or antigen-binding fragment from the culture medium after the step of expressing or culturing.
[98]
Effects of the Invention
[99]
The anti-LILRB1 antibody or antigen-binding fragment thereof provided herein can exhibit excellent anticancer activity by inhibiting the immune evasion mechanism of cancer cells so that the anticancer efficacy of immune cells can be well exerted without being inhibited.
[100]
Brief description of the drawing
[101]
1 is an electrophoresis photograph showing the results of SDS-PAGE gel analysis of the purified anti-LILRB1 antibody in one embodiment.
[102]
2 shows a surface plasmon resonance (SPR) sensorgram result for anti-LILRB1 antibody B3 according to an embodiment.
[103]
3 shows SPR sensorgram results for anti-LILRB1 antibody E3 according to an embodiment.
[104]
Figure 4a is a graph showing the binding ability of the anti-LILRB1 antibody A10 according to an embodiment to KHYG-1 cells, which are human natural killer cells, Figure 4b is a human natural killer cell of the anti-LILRB1 antibody E3 according to an embodiment It is a graph showing the binding ability to KHYG-1 cells, Figure 4c is a graph showing the binding ability of the human IgG4 isotype control antibody to KHYG-1 cells, which are human natural killer cells.
[105]
5 is a graph showing the results of analyzing the binding degree of the recombinant LILRB1-Fc protein to the HLA-G overexpressing cell surface with the iQue screener when the anti-LILRB1 antibody and the human IgG4 isotype control antibody according to an embodiment are treated, respectively. .
[106]
6 is a graph showing the in vivo antitumor effect of anti-LILRB1 antibodies E3 and B3 according to an embodiment.
[107]
7A to 7D are flow cytometry diagrams showing that the anti-LILRB1 antibody E3.1 according to an embodiment binds to various human LILR family overexpressing cells.
[108]
8A to 8D are flow cytometry diagrams showing that the anti-LILRB1 antibody H11 according to an embodiment binds to various human LILR family overexpressing cells.
[109]
9 is a graph showing the amount of granzyme B secretion in KHYG-1 cells, which are human natural killer cells, compared with a control antibody (human IgG4 isotype) upon treatment with anti-LILRB1 antibody E3.1 or H11 according to an embodiment; .
[110]
10 is a graph showing the amount of perforin secretion from KHYG-1 cells, which are human natural killer cells, when treated with anti-LILRB1 antibody E3.1 or H11 according to an embodiment in comparison with a control antibody (human IgG4 isotype).
[111]
11 is a graph showing the results of a luciferase reporter assay for evaluating the LILRB1 signaling inhibitory ability of the anti-LILRB1 antibody E3.1 or H11 according to an embodiment.
[112]
12 is a graph showing the in vivo antitumor effect of the anti-LILRB1 antibody E3.1 or H11 according to an embodiment.
[113]
Modes for carrying out the invention
[114]
Hereinafter, the present invention will be described in more detail by way of examples, but this is merely illustrative and not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the embodiments described below can be modified without departing from the essential gist of the invention.
[115]
[116]
Example 1: Production of human antibodies to LILRB1
[117]
1.1. Selection of human antibodies to LILRB1 using phage display
[118]
In order to select an antibody that specifically recognizes human LILRB1, a phage display selection method was performed using a library consisting of human scFv antibodies. As antigens, human LILRB1-His (Cat. No. 8989-T2) and human LILRB1-Fc (Cat. No. 2017-T2) manufactured by RnD Systems were used, respectively. In addition, each antigen was conjugated with biotin using the EZ-Link Sulfo-NHS-Biotin kit (ThermoFisher Scientific).
[119]
Phage display screening uses a total of four types of LILRB1 antigens (LILRB1-His, LILRB1-Fc, LILRB1-His-Biotin, LILRB1-Fc-Biotin) through solid-phase and solution-phase screening methods. was performed. Additional selection was performed by gradually reducing the concentration of the antigen used, eluting competitively using a control antibody against LILRB1, or performing negative selection for Fc when LILRB1-Fc was used as an antigen. Binding to the selected product was confirmed by polyclonal phage ELISA.
[120]
[121]
1.2. Monoclonal soluble scFv screening and analysis
[122]
An expression vector was prepared by amplifying the genes encoding the scFvs whose antigen binding was confirmed in Example 1.1 by PCR. For screening for each selection group, a certain number of transformants were transferred to a 96-well culture plate. After expressing the antibody in scFv form using autoinduction media (Studier, FW (2005) Protein Expression and Purification 41, 207-34), DELFIA immune assay (PerkinElmer) was performed to confirm binding to the antigen. In addition, after allowing a certain amount of scFv antibody to be coated on the surface, DELFIA was performed on the antigen to determine the ranking for antigen-antibody binding ability.
[123]
[124]
1.3. Conversion of selected scFv antibodies to IgG antibodies
[125]
A total of 376 clones were selected from among the clones whose antigen binding was confirmed in Example 1.2, and the nucleic acid sequence of the selected scFv-encoding gene was analyzed by a general DNA sequencing method to remove duplicate clones. In addition, a total of 93 clones were selected based on the ranking for antigen-antibody binding ability determined in Example 1.2. Sequences corresponding to each heavy chain variable region (VH) and light chain variable region (VL) from the selected scFv-encoding gene were amplified by PCR, and an IgG4 human antibody (IgG4 Fc: SEQ ID NO: 341, Kappa constant region: SEQ ID NO: 342, Lambda constant region: SEQ ID NO: 343) of human IgG4 with an expression vector (pTRIOZ-hIgG4; InvivoGen, in addition, CMV promoter, or CMV/CHO beta-actin fusion promoter (KR10-1038126B1)) Any of the vectors containing the heavy chain constant region and the constant region sequence of kappa or lambda light chain may be used). The expression vector has its DNA sequence confirmed through sequencing.
[126]
[127]
1.4. Preparation of Selected Antibodies
[128]
The vector constructed in Example 1.3 was purified using the Plasmid Plus Maxi kit (Qiagen). The purified vector was used for antibody expression using ExpiCHO-S TM cells or Expi293 TM cells.
[129]
Specifically, the vector constructed in Example 1.3 was transfected by adding 80 μl of ExpiFectamine TM CHO reagent (Thermo Fisher) to ExpiCHO-S TM cells (Gibco) (1.5 x 10 8 cells/Culture Volume 25 mL) ( transfection). One day after transfection, 150 μl of ExpiCHO TM Enhancer (Thermo Fisher) and 4 mL of ExpiCHO TM Feed (Thermo Fisher) were added. On day 5, 4 mL of ExpiCHO ™ Feed was added. The transfected cells were cultured for a total of 7 to 11 days under conditions of 32° C. and 5% CO 2 .
[130]
In addition, the vector constructed in Example 1.3 was transfected by adding 320 μl of ExpiFectamine TM 293 Reagent (Gibco) to Expi293F TM cells (Gibco) (3 x 10 8 cells/Culture Volume 100 mL) according to the manufacturer's instructions. did. One day after transfection, 0.6 mL of ExpiFectamine TM 293 Enhancer 1 (Thermo Fisher) per 100 mL of Culture Volume, 6 mL of ExpiFectamine TM Enhancer 2 (Thermo Fisher) per 100 mL of Culture Volume, and 3.6 g of glucose per liter were added. . The transfected cells were cultured for a total of 5 days under 36.5° C., 5% CO 2 conditions.
[131]
The two cultured cells were centrifuged at 4000 rpm at 4° C. for 20 minutes, and then filtered using a 0.22 um bottle-top filter system (Corning). The recovered culture was purified using AKTA Pure L (GE healthcare). A Hitrap MabSelectSure 1mL column (GE healthcare) was mounted on AKTA Pure L, and the culture medium was flowed at a flow rate of 1 mL/min, followed by washing with 1X PBS for 20 column volumes (CV). Elution buffer (0.1 M sodium citrate pH 3.4 buffer) was flowed to elute the target protein. The eluate was concentrated using an Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge, and then the buffer was exchanged with 1xPBS buffer.
[132]
The purified antibody sample was diluted with 1X PBS to prepare about 1 mg/mL. After mixing 10 μl of Reducing Loading Buffer (3X) or Non-reducing Loading Buffer (3X) with 20 μl of the purified antibody sample, it was left in a heating bath at 95° C. for 2 minutes, then taken out and cooled. After SDS-PAGE Gradient Gel (4-20% or 4-12%) was mounted on the electrophoresis device, 10 μg of sample per well was injected and the gel was developed. For molecular weight analysis of the sample, Precision Plus Protein TM Dual Color Standards (BIO-RAD) was injected into a separate well. After staining and destaining the gel with Coomassie staining solution, a photograph of the gel was taken.
[133]
Among these 93 antibodies, gel electrophoresis images of A10, B3, E3, G1, G9 and H2 antibodies are shown in FIG. 1 as representative. As shown in Figure 1, it can be confirmed that the production of an antibody having a disulfide bond.
[134]
[135]
1.5. Binding affinity analysis of selected antibodies
[136]
The affinity of the 93 antibodies selected in Example 1.3 to the LILRB1 antigen was measured using a Biacore T200 (GE healthcare). An anti-human IgG (Fc) antibody ( GE healthcare, Cat. No. BR-1008-39, final concentration of 25 ug/mL) was flowed at 5 μl/min for 360 seconds to fix at about 5000-7000 RU. The antigen human LILRB1 protein (LILRB1-His, RnD systems Cat. No. 8989-T2) was injected at a rate of 30 μl/min at 4-9 different concentrations within a concentration range from 3.13 nM to 1600 nM, As shown in the table below, k a and k d values ​​were obtained, and the K D value was calculated therefrom.
[137]
Among the 93 types of antibodies, 20 types of antibodies exhibiting excellent binding affinity (K D value) were selected, and the results are shown in Table 3 below. Among them, B3 and The SPR sensorgram of E3, which showed a binding force of about 101.2 nM to the LILRB1 antigen, is shown in FIGS. 2 and 3, respectively (FIG. 2: SPR sensorgram for B3, FIG. 3: SPR sensorgram for E3)
[138]
[Table 3] Antigen binding activity of KD LILRB1 antibody ( )
Clone name k a (x 10 5 ) (1/Ms) k d (x 10 -4 ) (1/s) K D (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
[139]
[140]
1.6. Sequence analysis of selected antibodies
[141]
The amino acid sequences of the CDRs, light chain variable regions, heavy chain variable regions, light chains, and heavy chains and the light chain variable regions and The nucleic acid sequence of the coding gene of the heavy chain variable region was analyzed by general amino acid sequencing and DNA sequencing methods, and summarized in Tables 4 to 23 below:
[142]
[Table 4] Antibody clone E3
Amino acid sequence (N→C) / Nucleic acid sequence (5'→3') SEQ ID NO
CDR-L1 QGDSLRNFYAS One
CDR-L2 GKNNRPS 2
CDR-L3 NSRDSSGSHLTGV 3
CDR-H1 SYAMS 4
CDR-H2 AISGSGGSTYYADSVKG 5
CDR-H3 DTYYYGSGRSNAFDI 6
light chain variable region SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKSGQAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTITGAQAEDEADYYCNSRDSSGSHLTGVFGGGTKVTVLGQPAAA 221
light chain variable region gene TCCTATGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAGGGAGACAGCCTCAGAAACTTTTATGCAAGCTGGTACCAGCAGAAGTCAGGACAGGCCCCAGTTCTTGTCATGTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCACCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAGCCATTTGACGGGCGTATTCGGCGGAGGGACCAAGGTCACCGTCCTAGGTCAGCCCGCGGCCGCA 261
heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAFDIWGQGTLVTVSS 222
heavy chain variable region gene CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTATCTGCAAATGATTAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATACGTATTACTATGGTTCGGGGAGAAGTAATGCTTTTGATATATGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 262
light chain (Lambda) SYELTQDPAVSVALGQTVRITCQGDSLRNFYASWYQQKSGQAPVLVMYGKNNRPSGIPDRFSGSTSGNTASLTITGAQAEDEADYYCNSRDSSGSHLTGVFGGGTKVTVLGQPAAAPSVTLFPPSSNELQANKATLVCLISDFYPGAVTSSKATLVCLISDFYPGAVTSQAPVLVMYGKSNRPSGIPDRFSGSTSGNTASLTITGAQAEDEADYYCNSRDSSGSHLTGVFGGGTKVTVLGQPAAAPSVTLFPPSSNELQANKATLVCLISDFYPGAVTSSKATLVCLISDFYPGAVTVAWKADSSPVKTECKSKSKS 301
heavy chain QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMISLRAEDTAVYYCARDTYYYGSGRSNAFDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 302
[143]
[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 DYAM 10
CDR-H2 GISWNSGSIGYADSVKG 11
CDR-H3 VGDSSGWSDAFDI 12
light chain variable region DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPFGGGTKVDIKRTAAA 223
light chain variable region gene GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTGAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTTTCGGCGGAGGGACCAAAGTGGATATCAAACGTACCGCGGCCGCA 263
heavy chain variable region EVQLLESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGDSSGWSDAFDIWGQGTMVTVSS 224
heavy chain variable region gene GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGTTGGGGATAGCAGTGGCTGGTCCGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGT 264
light chain (Kappa) DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPFGGGTKVDIKRTAAAPSVFIFPPSDEQLKSGTASVTYVCLLNNFYPREAKVQQKPGKVDNALQSSKHSKSSQESTLNNFYPREAKVQWKVKVDNALQSSKHSKSSQYDNLPFGGGTKVDIKRTAAAPSVFIFPPSD 303
heavy chain EVQLLESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGDSSGWSDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 304
[144]
[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 variable region DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSSPRMYTFGQGTKVDIKRTAAA 225
light chain variable region gene GATATTGTGATGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACCGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATGGTAGCTCACCTCGGATGTACACTTTTGGCCAGGGGACCAAAGTGGATATCAAACGTACCGCGGCCGCA 265
heavy chain variable region QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSISTAYMELSSLRSEDTAVYYCARGGLGELDNWFDPWGQGTLVTVSS 226
heavy chain variable region gene CAAATGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCATCAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGCGGCCTCGGGGAGTTGGACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 266
light chain (Kappa) DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSSPRMYTFGQGTKVDIKRTAAAPSVFIFPPSDEQLKSGTASVTKSLECLLNNFYPREAKVQWQTLSSFHQGLQSSEKNSQLACESSHQKATEQSSEKNSK 305
heavy chain QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSISTAYMELSSLRSEDTAVYYCARGGLGELDNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 306
[145]
[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 variable region SYELTQPPSLSVSPGQTASITCSGYKLGDRYVSWYQQKTGQSPVVVIYKDSQRPSGVPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSGTGVFGGGTKLTVLGQPAAA 227
light chain variable region gene TCCTATGAGCTGACTCAGCCACCCTCACTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACCTGCTCAGGATATAAACTGGGAGATAGATATGTTTCCTGGTATCAGCAGAAGACAGGCCAGTCCCCTGTGGTGGTCATCTATAAAGATAGCCAGCGGCCCTCAGGGGTCCCTGAACGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTATTACTGTCAGGCGTGGGACAGCGGCACTGGGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA 267
heavy chain variable region EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQELQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVGVAGKLDYWGQGTLVTVSS 228
heavy chain variable region gene GAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGGAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTATATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGTAGGGGTGGCTGGTAAACTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 268
light chain (Lambda) SYELTQPPSLSVSPGQTASITCSGYKLGDRYVSWYQQKTGQSPVVVIYKDSQRPSGVPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSGTGVFGGGTKLTVLGQPAAAPSVTLFPPSSEELQANKYATLVCLISDFYPGAVTVLTSKATLVCLISDFYPGAVTVSLTQPEDSSPVKAGSYS 307
heavy chain EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQELQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVGVAGKLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 308
[146]
[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 variable region QSALTQPASVSGSPGQSITISCTGSSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSDRFSGSKSGNMASLTISGLQAEDEADYYCSSYTGSSTLDVLFGGGTKLTVLGQPAAA 233
light chain variable region gene CAGTCTGCGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAAGCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAGCAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACATGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAGGAAGCAGCACTCTCGACGTGCTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA 269
heavy chain variable region QVQLVQPGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVTVSS 234
heavy chain variable region gene CAGGTGCAGCTGGTGCAGCCTGGAGCAGAGGTGAAAAAGCCGGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAGGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCAATATTACGATGGGGGTTACTACATGGACGTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 270
light chain (Lambda) QSALTQPASVSGSPGQSITISCTGSSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSDRFSGSKSGNMASLTISGLQAEDEADYYCSSYTGSSTLDVLFGGGTKLTVLGQPAAAPSVTLFPPSSEELQANKYATLVCLISDFYPGAVTVLTSKAPTHQQWKAGSYS 309
heavy chain QVQLVQPGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 310
[147]
[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 variable region SYELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQAPILVISGNNKRPSGIPDRFSGSSSGDTASLTISGAQAEDEADYYCNSLDSTYNHPIFGGGTKVTVLGQPAAA 235
light chain variable region gene TCCTATGAGCTGACTCAGGACCCTGCTGTGTCGGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAACTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTATTCTTGTCATCTCTGGTAACAACAAACGGCCCTCGGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAGACACAGCTTCCTTGACCATCTCTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCTAGACAGCACTTATAACCATCCGATATTCGGCGGAGGGACCAAGGTCACCGTCCTAGGTCAGCCCGCGGCCGCA 271
heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIHWVRQATGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGGDAFDIWGQGTLVTVSS 236
heavy chain variable region gene CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATATCCACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATGGGGGTGATGCTTTTGATATCTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 272
light chain (Lambda) SYELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQAPILVISGNNKRPSGIPDRFSGSSSGDTASLTISGAQAEDEADYYCNSLDSTYNHPIFGGGTKVTVLGQPAAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTAPTECVSKQSNKAGVETTPTECQSNKSKAGVETTAPTECS 311
heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIHWVRQATGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGGDAFDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 312
[148]
[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 variable region SYELTQDPAASVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVVVIYGRNNRPSGIPDRFSGSSSGDTASLTITGAQAEDEADYYCKSRDSSGNHYVFGTGTKLTVLGQPAAA 231
light chain variable region gene TCCTATGAGCTGACTCAGGACCCTGCTGCGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTAGTTGTCATCTATGGTAGAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAGACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAAGTCCCGGGACAGCAGTGGTAACCATTATGTCTTCGGAACTGGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA 273
heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDAGSSSDYWGRGTLVTVSS 232
heavy chain variable region gene CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATGCCGGCAGCTCGTCCGATTACTGGGGCCGTGGCACCCTGGTCACCGTCTCGAGT 274
light chain (Lambda) SYELTQDPAASVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVVVIYGRNNRPSGIPDRFSGSSSGDTASLTITGAQAEDEADYYCKSRDSSGNHYVFGTGTKLTVLGQPAAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKPGQAPVVVIYGRNNRPSGIPDRFSGSSSGDTASLTITGAQAEDEADYYCKSRDSSGNHYVFGTGTKLTVLGQPAAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWLTKADSSPKSVKAGVETTQTPSKQSSNKAGVETTQTPSKQSSNKAGVETTQ 313
heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDAGSSSDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 314
[149]
[Table 11] Antibody clone F12
Amino acid sequence (N→C) / Nucleic acid sequence (5'→3') SEQ ID NO
CDR-L1 AGTSSDIGDYDYVS 43
CDR-L2 DVSRPS 44
CDR-L3 ASYTSSSVVV 45
CDR-H1 SYWIG 46
CDR-H2 IIYPGDSDTRYSPSFQG 47
CDR-H3 QYYDGGYYMDV 48
light chain variable region QSVLTQPASVSGSPGQSITISCAGTSSDIGDYDYVSWYQQHPGKTPKLMIYDVSRRPSGVPDRFSGSKSGNTASLTISGLQTEDEADYYCASYTSSSVVVFGGGTKLTVLGQPAAA 237
light chain variable region gene CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCGCTGGAACCAGCAGTGACATTGGTGATTATGACTATGTCTCCTGGTACCAACAGCACCCAGGCAAGACTCCCAAACTCATGATTTATGATGTCAGTAGGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGACTGAGGACGAGGCTGATTATTACTGCGCCTCATATACAAGCAGCAGCGTCGTGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA 275
heavy chain variable region QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVTVSS 238
heavy chain variable region gene CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCAATATTACGATGGGGGTTACTACATGGACGTCTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGT 276
light chain (Lambda) QSVLTQPASVSGSPGQSITISCAGTSSDIGDYDYVSWYQQHPGKTPKLMIYDVSRRPSGVPDRFSGSKSGNTASLTISGLQTEDEADYYCASYTSSSVVVFGGGTKLTVLGQPAAAPSVTLFPPSSEELQANKATYDYVSWYQQQHPGKTPKLMIYDVSRRPSGVPDRFSGSKSGNTASLTISGLQTEDEADYYCASYTSSSVVVFGGGTKLTVLGQPAAAPSVTLFPPSSEELQANKATSKATLVCLISDFYPGAVTVAWKADSSPVKAGVEKASTSAPSKTVQWYPGAVTVAWKADSSPVKAGVEKA 315
heavy chain QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 316
[150]
[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 variable region DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYHCQQAYGFPLTLGGGTKVEIKRTAAA 229
light chain variable region gene GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGGTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCCAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGATTTCGCAACTTACCATTGTCAACAGGCTTACGGTTTCCCCCTCACTCTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACCGCGGCCGCA 277
heavy chain variable region QVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGEIAVAQNWDYYGMDVWGQGTLVTVSS 230
heavy chain variable region gene CAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGAAATAGCAGTGGCTCAAAACTGGGACTACTACGGTATGGACGTCTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGT 278
light chain (Kappa) DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYHCQQAYGFPLTLGGGTKVEIKRTAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQSQLNNFYPREAKVQSSALNFLNFYPREAKHSSKSSVKVKATEQSSEKHSS 317
heavy chain QVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGEIAVAQNWDYYGMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 318
[151]
[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 variable region QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYSSSSTLVVFGGGTKLTVLGQPAAA 239
light chain variable region gene CAGTCTGCGCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATTCAAGCAGCAGCACTCTCGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA 279
heavy chain variable region QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVTVSS 240
heavy chain variable region gene CAGGTCCAGCTGGTACAGTCTGGAGCAGAGGTGAAAAAGCCGGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCAATATTACGATGGGGGTTACTACATGGACGTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 280
light chain (Lambda) QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYSSSSTLVVFGGGTKLTVLGQPAAAPSVTLFPPSSEELQANKATYLSCLISDFYPGAVTVAWKATVCLISDFYPGAVTVAWKADSSPVKAGVETTCAPTHQWKAGVECKS 319
heavy chain QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASQYYDGGYYMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 320
[152]
[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 variable region SYELTQDPAVSVALGQTVTITCQGDSLRRYYATWYQQKPGQAPVLVIYGQNYRPSGIPDRFSGSNSGTTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPAAA 241
light chain variable region gene TCCTATGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCACGATCACATGCCAAGGAGACAGCCTCAGAAGGTATTATGCAACCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTCCTTGTCATCTATGGTCAAAACTACCGGCCCTCGGGGATCCCAGACCGATTCTCTGGCTCCAACTCAGGAACCACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA 281
heavy chain variable region EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGWGYSSSFDYWGQGTTVTVSS 242
heavy chain variable region gene GAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTACTACTGTGCGAGAGGGTGGGGGTATAGCAGCTCGTTTGACTACTGGGGGCAAGGGACCACGGTCACCGTCTCGAGT 282
light chain (Lambda) SYELTQDPAVSVALGQTVTITCQGDSLRRYYATWYQQKPGQAPVLVIYGQNYRPSGIPDRFSGSNSGTTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPAAAPSVTLFPPSSEELQANKATYLSCLISDFYPGAVTVAWKPGQAPVLVIYGQNYRPSGIPDRFSGSNSGTTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPAAAPSVTLFPPSSEELQANKATYLSCLISDFYPGAVTVAWLTKADSSPKSVKAGVETTQTPSKQSSNKAGVETTQTPSKSEGSNKAGVETTQ 321
heavy chain EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGWGYSSSFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 322
[153]
[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 variable region QSVLTQPPSTSGTPGQTFSIFCSGSSSNIGTNTVNWYQQLPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCETWDDSLKGPVFGGGTKVTVLGQPAAA 243
light chain variable region gene CAGTCTGTGCTGACTCAGCCACCCTCAACGTCTGGGACCCCCGGGCAGACGTTCTCCATTTTTTGTTCTGGAAGCAGTTCGAACATCGGAACTAATACTGTTAATTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAATGATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGAAACATGGGATGACAGCCTGAAAGGCCCGGTGTTCGGCGGGGGGACCAAGGTCACCGTCCTAGGTCAGCCCGCGGCCGCA 283
heavy chain variable region EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYAMSWVRRAPGKGLEWVSTISGSGDSTYYADSVKGRFTISRDNSKNTLYLQMNNLRAEDTAVYYCAREWELGDAFDIWGRGTLVTVSS 244
heavy chain variable region gene GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAAACTCTCCTGTGCAGCGTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCGGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGTGGTAGTGGTGATAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAACCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGAATGGGAACTAGGCGATGCTTTTGATATCTGGGGCCGTGGCACCCTGGTCACCGTCTCGAGT 284
light chain (Lambda) QSVLTQPPSTSGTPGQTFSIFCSGSSSNIGTNTVNWYQQLPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCETWDDSLKGPVFGGGTKVTVLGQPAAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCETWDDSLKGPVFGGGTKVTVLGQPAAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKATLVCLISDFYPGAVTVAWKADSSPKSVKAGVETTQTSAPSKSNKYSYSEKTVA 323
heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYAMSWVRRAPGKGLEWVSTISGSGDSTYYADSVKGRFTISRDNSKNTLYLQMNNLRAEDTAVYYCAREWELGDAFDIWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 324
[154]
[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 variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTRWTFGQGTKVEIKRTAAA 245
light chain variable region gene GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCGGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACCGCGGCCGCA 285
heavy chain variable region EVQLLESGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGSYGYYYGMDVWGQGTMVTVSS 246
heavy chain variable region gene GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATCCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACAGAGGCAGCTATGGTTACTACTACGGTATGGACGTCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGT 286
light chain (Kappa) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTRWTFGQGTKVEIKRTAAAPSVTLFPPSSEELQANKATSKLSCLISDFYPGAVTVAWKADSSPVKAGVETCSAPSKSLISDFYPGAVTVAWKADSSPVKAGVEDFATYYCQQSYSTRWTFGQGTKVEIKRTAAAPSVTLFPPSSEELQANKATSKLSCLISDFYPGAVTVAWKADSSPVKAGVECKA 325
heavy chain EVQLLESGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGSYGYYYGMDVWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 326
[155]
[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 variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTLRTFGQGTKVEIKRTAAA 247
light chain variable region gene GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCTCCGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAACGTACCGCGGCCGCA 287
heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTRAGASIVGATALDYWGQGTLVTVSS 248
heavy chain variable region gene CAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTACGAGAGCCGGTGCTTCTATAGTGGGAGCTACCGCGCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 288
light chain (Kappa) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTLRTFGQGTKVEIKRTAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVKPSTLNFYPREAKHKNSQKVKAVTKVEKG 327
heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTRAGASIVGATALDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 328
[156]
[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 variable region NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDTGNRNYVFGTGTQLTVLGQPAAA 249
light chain variable region gene AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGAAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATACCGGCAATCGGAATTATGTCTTCGGAACTGGGACCCAGCTCACCGTCCTAGGTCAGCCCGCGGCCGCA 289
heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTMTRDMSTDTAYMELSSLTYDDTAVYFCVRGPSLNYAGYFDNWGQGTLVTVSS 250
heavy chain variable region gene CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATACTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCTATCCTTGGTATAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACATGTCCACAGACACAGCCTACATGGAGTTGAGCAGCCTGACATATGATGACACGGCCGTATATTTTTGTGTGAGAGGCCCTAGTCTTAATTATGCCGGCTATTTTGACAACTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGT 290
light chain (Lambda) NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDTGNRNYVFGTGTQLTVLGQPAAAPSVTLFPPSSEEIQANKAPSKATLVCLISDFYPGAVTVLSTTVCLISDFYPGAVTVAWKADSSPVKAGEKS 329
heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTMTRDMSTDTAYMELSSLTYDDTAVYFCVRGPSLNYAGYFDNWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 330
[157]
[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 SYAM 94
CDR-H2 VISYDGSNKYYADSVKG 95
CDR-H3 ERGSGMDV 96
light chain variable region SYELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKSGQAPVLVIYGKEKRPSGIPDRFSGSSSGNTASLTITGARAEDEADYYCNSRGSTTDYMVFGGGTQLTVLGQPAAA 251
light chain variable region gene TCCTATGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGTCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAGAAAAGCGCCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCGGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGGCAGCACTACTGACTATATGGTGTTCGGCGGGGGGACCCAGCTCACCGTCCTAGGTCAGCCCGCGGCCGCA 291
heavy chain variable region QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERGSGMDVWGQGTLVTVSS 252
heavy chain variable region gene CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAACGGGGAAGTGGTATGGACGTCTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 292
light chain (Lambda) SYELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKSGQAPVLVIYGKEKRPSGIPDRFSGSSSGNTASLTITGARAEDEADYYCNSRGSTTDYMVFGGGTQLTVLGQPAAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPKSKAGVETTAPTECVCLISDFYPGAVTVAWKADSSPKSKAGVETTAPTECSQSNKSKAGVETTAPTEC 331
heavy chain QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERGSGMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 332
[158]
[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 variable region ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQKPGEAAISIIQEASTLVPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQHDKFPYTFGQGTKLEIKRTAAA 253
light chain variable region gene GAAACGACACTCACGCAGTCTCCAGCATTCATGTCAGCGACTCCAGGAGACAAAGTCAACATCTCCTGCAAAGCCAGCCAAGACATTGATGATGATATGAACTGGTACCAACAGAAACCAGGAGAAGCTGCTATTTCCATTATTCAAGAAGCTAGTACTCTCGTTCCTGGAATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCTACAACATGATAAGTTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACCGCGGCCGCA 293
heavy chain variable region EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASRGVDEGDYWGQGTMVTVSS 254
heavy chain variable region gene GAAGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGTCGGGGGGTTGATGAGGGGGACTACTGGGGCCAAGGGACAATGGTCACCGTCTCGAGT 294
light chain (Kappa) ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQKPGEAAISIIQEASTLVPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQHDKFPYTFGQGTKLEIKRTAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQKPGEAAISIIQEASTLVPGIPPRFSGSGYGTKLEIKRTAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQQWKVHQTEQDSSGNSQGTLSSFHQGLDYQSSKNSQ 333
heavy chain EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASRGVDEGDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 334
[159]
[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 GDYSGVVLTAALDY 108
light chain variable region NFMLTQPHSVSESPGKTVTLSCTGSSGNIASNYVQWYQHRPGSAPTTVIYRDDQRPSGVPDRFSGSIDSSSNSASLTISGLRPEDEADYYCQSYDSSSWVFGGGTKLTVLGQPAAA 255
light chain variable region gene AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTTACCCTCTCCTGCACCGGCAGCAGCGGCAACATTGCCAGTAACTATGTGCAGTGGTACCAGCACCGCCCGGGCAGTGCCCCCACCACTGTGATCTACCGGGATGACCAAAGACCCTCTGGAGTCCCTGATCGCTTCTCTGGCTCCATCGACAGTTCATCCAACTCTGCCTCCCTCACGATCTCTGGACTGAGGCCTGAGGACGAGGCTGACTATTACTGTCAGTCTTATGATAGCAGCTCTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA 295
heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYDITWVRQAPGQGLEWMGWMNPNSGNSRSAQKFQGRVSMTSDSSISTAYMELSSLRSEDTAVYYCATGDYSGVVLTATALDYWGQGTLVTVSS 256
heavy chain variable region gene CAGGTCCAGCTTGTGCAGTCTGGAGCAGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCACTTATGATATCACCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGATGGATGAACCCGAACAGTGGTAACTCACGCTCTGCACAGAAGTTCCAGGGCAGAGTCAGCATGACCAGTGACTCCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCAACAGGAGACTACTCGGGTGTGGTACTAACTGCAACAGCACTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 296
light chain (Lambda) NFMLTQPHSVSESPGKTVTLSCTGSSGNIASNYVQWYQHRPGSAPTTVIYRDDQRPSGVPDRFSGSIDSSSNSASLTISGLRPEDEADYYCQSYDSSSWVFGGGTKLTVLGQPAAAPSVTLFPPSSEELQANKATLSTVCLISDFYPGAVTVAWKADSSPKSKAGVETTAPSTVS 335
heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYDITWVRQAPGQGLEWMGWMNPNSGNSRSAQKFQGRVSMTSDSSISTAYMELSSLRSEDTAVYYCATGDYSGVVLTATALDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 336
[160]
[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 SYGM 112
CDR-H2 NIKQDGSEKYYVDSVKG 113
CDR-H3 EDRIAAAGMRELDY 114
light chain variable region QSELTQLPSASETPGQRVTISCSGSSSNIGNNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPAAA 257
light chain variable region gene CAGTCTGAGCTGACTCAGCTACCCTCAGCGTCTGAGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAATAATTATGTATACTGGTACCAGCAACTCCCCGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCGCGGCCGCA 297
heavy chain variable region QVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAREDRIAAAGMRELDYWGQGTLVTVSS 258
heavy chain variable region gene CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTCCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGACCGTATAGCAGCAGCTGGGATGCGGGAGTTGGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGT 298
light chain (Lambda) QSELTQLPSASETPGQRVTISCSGSSSNIGNNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPAAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTAPTECQSNSPVKAGVETTAPTECVSKQSNKSKAGVETTAPTECS 337
heavy chain QVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAREDRIAAAGMRELDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 338
[161]
[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 variable region DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPYTFGQGTKVEIKRTAAA 259
light chain variable region gene GATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAACCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCTCCGTACACCTTTGGCCAGGGGACCAAGGTGGAGATCAAACGTACCGCGGCCGCA 299
heavy chain variable region EVQLLESGGGLEQPGGFLRLSCAASGFSFTSYAMTWVRQAPGKGLEWVSGISSDGTTTTYADSVRGRFTISRDNAKNTVYLQMNSLRDEDTAVYYCARDQLLGWDALNVWGQGTMVTVSS 260
heavy chain variable region gene GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGAACAGCCTGGGGGGTTCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTTACCAGCTACGCCATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTAGTGATGGGACCACTACAACCTACGCGGACTCCGTGAGGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCCAAATGAACAGTCTGAGAGACGAGGACACGGCTGTGTATTATTGTGCAAGAGATCAATTGTTGGGCTGGGATGCTCTGAATGTCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGT 300
light chain (Kappa) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPYTFGQGTKVEIKRTAAAPSVFIFPPSTKPVGLQDSSGTASVVCLLSSTLPVYPREAKDSSFLYSTLNNFYPREAKVQWKVTSG 339
heavy chain EVQLLESGGGLEQPGGFLRLSCAASGFSFTSYAMTWVRQAPGKGLEWVSGISSDGTTTTYADSVRGRFTISRDNAKNTVYLQMNSLRDEDTAVYYCARDQLLGWDALNVWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 340
[162]
[163]
Example 2: Analysis of In Vitro Biological Activity of Selected Antibodies
[164]
2.1. NK cell surface binding assay
[165]
In order to confirm whether the 93 antibodies selected in Example 1.4 also bind to LILRB1 expressed on the surface of immune cells, a natural killer cell surface binding assay (NK cell surface binding assay) was performed. Human natural killer cells, KHYG-1 cells (JCRB), were cultured in RPMI 1640 medium (Gibco) containing 10% (w/v) FBS (Gibco) and 100 U/mL interleukin-2 (Novartis). KHYG-1 cells were aliquoted in a U-bottom 96-well tissue culture plate (BD Falcon) to 5x10 4 cells/well. A test antibody having a final concentration of 50 μg/mL was added to each well, and placed at 4° C. for 1 hour.
[166]
In order to show specific binding to LILRB1, a human IgG4 isotype control antibody (Biolegend) was also treated in the same manner. After washing with FACS buffer, anti-human Fc-biotin antibody (life technologies) was added to each well, and placed at 4°C for 1 hour. After washing with FACS buffer, streptavidin PE (BD Pharmigen) was added to each well and left at 4°C for 30 minutes. After washing with FACS buffer, it was suspended and analyzed by iQue screener (Sartorius).
[167]
Among the results obtained, the results of A10, E3, E4, F12, G1, G9, G11, H2, and H11 are compared with the human IgG4 isotype (control) and shown in Table 24, and the A10, E3 and human IgG4 isotypes ( control) are shown in FIGS. 4a (A10), 4b (E3), and 4c (isotype IgG4), respectively:
[168]
[Table 24]
Mean Fluorescence Intensity % of population 2
human IgG4 isotype control 142917.2 2.95
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
[169]
As shown in Table 24 and Figures 4a to 4c, the tested antibodies showed a high degree of binding to human natural killer cells (surface) compared to the human IgG4 isotype control antibody.
[170]
[171]
2.2. Analysis of LILRB1/HLA-G binding inhibition ability of the selected antibody
[172]
In order to determine whether the antibodies selected in Example 1.5 inhibit the binding of LILRB1 and its ligand, HLA-G, LILRB1/HLA-G binding inhibition analysis was performed.
[173]
For this purpose, JEG-3 (ATCC cat# HTB-36) known to overexpress HLA-G was used. JEG-3 was cultured in MEM medium (Gibco) containing 10% (v/v) FBS (Gibco) and 1% (v/v) pen-strep (Gibco). JEG-3 cells were aliquoted in a U-bottom 96-well tissue culture plate (BD Falcon) to 5x10 4 cells/well. The well plate was washed with 1X PBS buffer. Each of the test antibodies (A10, E3, F12, G1, G9, H2, H11) and LILRB1-Fc (RnD systems) selected in Example 1.5 were finalized in FACS buffer (1X PBS + 1% BSA + 1 mM EDTA). The cells were mixed at a concentration of 10 μg/mL and 5 μg/mL, treated with 100 μl per well, and placed 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 prepared in the same manner as above. After washing twice with FACS buffer, PE-anti-huIgG-Fc antibody (Biolegend, 10 μg/mL) was added to each well, and placed on ice for 1 hour. After washing twice with FACS buffer, it was suspended in 100 μl of the same buffer and analyzed by iQue screener (Sartorius).
[174]
The obtained results are shown in FIG. 5 . As shown in FIG. 5 , all of the tested antibodies A10, E3, F12, G1, G9, H2, and H11 effectively inhibited the binding of LILRB1-Fc to HLA-G overexpressing cell lines.
[175]
[176]
2.3. Analysis of cancer cell killing ability by natural killer cells
[177]
To determine whether the selected antibodies increase the ability to kill cancer cells by natural killer cells, the apoptosis rate of HLA-G overexpressing HEK293 cells by natural killer cells KHYG-1 was analyzed. KHYG-1 cells (JCRB) were aliquoted in a 96-well tissue culture plate (BD Falcon) to 2x10 4 cells/well (4x10 4 cells/mL, total volume 50 μl). The test antibody (Table 25) was added to a final concentration of 20 μg/mL per well, and placed at 37° C. for 1 hour.
[178]
As a negative control, a human IgG4 isotype control antibody (Biolegend) was also treated in the same manner.
[179]
HLA-G overexpressing HEK293 cells (HLA-G overexpressing cell line was prepared by transducing HEK293 cells (American Type Culture Collection) with a lentivirus designed to express HLA-G1) using IncuCyte CytoLight Rapid Red Reagent (Sartorius) Stained according to the instructions of After 1 hour, HLA-G overexpressing HEK293 cells were added to the plate so as to become 1×10 4 cells/well (2 ×10 4 cells/mL, total volume 50 μl). The plate was placed in an IncuCyte S3 (Sartorius) installed in an incubator at 37 ° C., 5% CO 2 and images were taken for 72 hours, and the red area confluence indicating the density of live HLA-G overexpressing HEK293 was measured to determine the cell viability. Cell viability is shown in Table 25 below (represented as relative values ​​with the control antibody equal to 1):
[180]

[181]
[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
[182]
As shown in Table 25, test antibodies A10, B9, D3, E1, E3, F12, G1, G6, G9, G11, H2, H11 overexpressed HLA-G by KHYG-1 HEK293 compared to human IgG4 isotype control. increased cell death.
[183]
[184]
Example 3: In vivo biological activity analysis of the selected antibody
[185]
Among the antibodies selected in Example 1.5, it was confirmed in vivo whether the anticancer efficacy of the two test antibodies (E3, B3) was improved. To this end, Bioware Brite Cell Line HCT116 Red-Fluc colorectal cancer cells (PerkinElmer) and THP-1 derived macrophage (THP-1 derived macrophage), and in vivo ( In vivo), it was confirmed whether the size of the tumor was reduced by the administration of the two types of antibodies. As a negative control, a xenograft colorectal cancer mouse animal model in which human IgG1 isotype control antibody (BioXcell, Cat. No. BP0297) was administered in the same manner as above was prepared. Hereinafter, the above process is described in more detail:

Claims
[Claim 1]
An anti-LILRB1 antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, CDR-L1 comprising the amino acid sequence of 67, 73, 79, 85, 91, 97, 103, 109, or 115, SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, a CDR-L2 comprising the amino acid sequence of 62, 68, 74, 80, 86, 92, 98, 104, 110, or 116, SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, a CDR-L3 comprising the amino acid sequence of 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, or 117, SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, a CDR-H1 comprising the amino acid sequence of 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, or 118, SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, a CDR-H2 comprising the amino acid sequence of 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, or 119, and SEQ ID NOs: 6, 12, 18, 24, 30, 36 , 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, or a CDR-H3 comprising an amino acid sequence of 120 (the CDRs are defined based on Kabat numbering) .
[Claim 2]
The anti-LILRB1 antibody or antigen-binding fragment thereof according to claim 1, comprising: (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2; A CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR comprising the amino acid sequence of SEQ ID NO: 6 -H3; (2) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence of SEQ ID NO: 10 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; (3) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, comprising the amino acid sequence of SEQ ID NO: 16 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (4) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, comprising the amino acid sequence of SEQ ID NO: 22 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (5) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, CDR- comprising the amino acid sequence of SEQ ID NO: 29 H2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; (6) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33, and the amino acid sequence of SEQ ID NO: 34 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; (7) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39, and the amino acid sequence of SEQ ID NO: 40 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 42; (8) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45, and the amino acid sequence of SEQ ID NO: 46 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 48; (9) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54; (10) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57, comprising the amino acid sequence of SEQ ID NO: 58 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 60; (11) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 61, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 62, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 63, comprising the amino acid sequence of SEQ ID NO: 64 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 66; (12) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 68, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 69, comprising the amino acid sequence of SEQ ID NO: 70 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 71, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72; (13) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75, comprising the amino acid sequence of SEQ ID NO: 76 CDR-H1 comprising the amino acid sequence of SEQ ID NO: 77, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 78; (14) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81, comprising the amino acid sequence of SEQ ID NO: 82 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 83, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 84; (15) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 85, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 86, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 87, comprising the amino acid sequence of SEQ ID NO: 88 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 89, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 90; (16) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 91, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 92, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 93, comprising the amino acid sequence of SEQ ID NO: 94 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 95, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 96; (17) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 97, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 98, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 99, comprising the amino acid sequence of SEQ ID NO: 100 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 101, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (18) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 103; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 104, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 105, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 106, CDR- comprising the amino acid sequence of SEQ ID NO: 107 H2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:108; (19) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 109, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 110, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, comprising the amino acid sequence of SEQ ID NO: 112 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 113, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 114; or (20) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 115, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 117, the amino acid sequence of SEQ ID NO: 118 A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 119, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120. a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 113, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 114; or (20) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 115, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 117, the amino acid sequence of SEQ ID NO: 118 A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 119, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120. a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 113, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 114; or (20) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 115, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 117, the amino acid sequence of SEQ ID NO: 118 A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 119, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120.
[Claim 3]
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 a light chain variable region comprising the amino acid sequence of , or a heavy chain variable region comprising an amino acid sequence of 260, an anti-LILRB1 antibody or antigen-binding fragment thereof.
[Claim 4]
The anti-LILRB1 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a human IgG1 or IgG4 antibody.
[Claim 5]
The method of claim 1, wherein the antigen-binding fragment is scFv, (scFv) 2 , Fab, Fab', F(ab') 2 , a fusion polypeptide in which scFv is fused with an Fc of immunoglobulin, or scFv is a constant region of a light chain An anti-LILRB1 antibody or antigen-binding fragment thereof, which is a fusion polypeptide fused to
[Claim 6]
A pharmaceutical composition for preventing or treating cancer, comprising the anti-LILRB1 antibody or antigen-binding fragment thereof of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.
[Claim 7]
The pharmaceutical composition according to claim 6, wherein the cancer has MHC Class I overexpression.
[Claim 8]
A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-5.
[Claim 9]
A recombinant vector comprising the nucleic acid molecule of claim 8 .
[Claim 10]
A recombinant cell comprising the nucleic acid molecule of claim 8 or a recombinant vector comprising the same.
[Claim 11]
A method for producing an anti-LILRB1 antibody or antigen-binding fragment thereof, comprising the step of culturing the recombinant cell of claim 10 .