Anti-CD100 antibodies and uses thereof

Abstract

The invention relates to an anti-CD100 antibody and a use thereof, a pharmaceutical composition or a pharmaceutical combination comprising the antibody and a use thereof. Further provided are polynucleotides and expression vectors encoding the antibodies as well as methods of producing the antibodies.

Description

Anti-CD100 antibodies and uses thereof Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to anti-CD100 antibodies and uses thereof. Background Art

[0002] CD100, also known as semaphorin 4D (SEMA4D), is a transmembrane protein belonging to the semaphorin gene family. CD100 is expressed on the cell surface as a homodimer and can be released from the cell surface by proteolysis to produce an active soluble form of CD100. CD100 is strongly expressed mainly in human lymphoid tissue, skeletal muscle, and brain (at lower levels). Its biological activity is mainly characterized in the immune system: for example, as a receptor, CD100 can enhance T cell proliferation; as a ligand, it can promote the aggregation and survival of B cells and the activation and maturation of antigen-presenting cells (dendritic cells and macrophages). Through the high-affinity receptor Plexin-B1, CD100 can inhibit the migration of monocytes and B cells.

[0003] CD100 is widely expressed in many human tumors, and its expression correlates with aggressive human disease. In the preclinical tumor microenvironment, expression of CD100 by inflammatory and tumor cells regulates the infiltration, spatial distribution, and activity of myeloid and lymphocytes. CD100 binds to Plexin receptors located on myeloid cells in the tumor microenvironment. Blocking the CD100 protein eliminates the CD100 barrier. Once the barrier is breached, inflammatory dendritic cells and proinflammatory antigen-presenting cells migrate and infiltrate the tumor. In preclinical animal models of cancer, blocking CD100 with antibodies delays tumor growth and promotes durable tumor rejection.

[0004] Some antibodies targeting CD100 have been developed in the prior art, such as Pepinemab (Vaccinex, Inc.), but there is still a need in the art for anti-CD100 antibodies that can specifically bind to CD100 and block the binding of CD100 to its Plexin receptor.

[0005] Summary of the Invention

[0006] One aspect of the present invention provides an antibody against CD100 (anti-CD100 antibody) or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically recognizes and binds to CD100.

[0007] In one embodiment, the antibody or antigen-binding fragment thereof against CD100 comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences, and wherein the HCDR1 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 1, 7, 19, 33, 41, 47 or 52 by no more than 2 amino acid additions, deletions or substitutions; the HCDR2 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 2, 8, 14, 20, 26, 42, 48 or 53 by no more than 2 amino acid additions, deletions or substitutions; and / or the HCDR3 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49 or 54 by no more than 2 amino acid additions, deletions or substitutions.

[0008] In one embodiment, the antibody or antigen-binding fragment thereof against CD100 comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences, and wherein the LCDR1 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 4, 10, 16, 36, 44, or 50 by no more than 2 amino acid additions, deletions, or substitutions; the LCDR2 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 5, 11, or 45 by no more than 2 amino acid additions, deletions, or substitutions; and / or the LCDR3 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 6, 12, 18, 24, 37, 46, 51, or 56 by no more than 2 amino acid additions, deletions, or substitutions.

[0009] In one embodiment, the heavy chain variable region comprises a HCDR1 sequence shown in SEQ ID NO: 1, 7, 13, 19, 25, 33, 41, 47, 52 or 57; a HCDR2 sequence shown in SEQ ID NO: 2, 8, 14, 20, 26, 34, 42, 48, 53 or 58; and a HCDR3 sequence shown in SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, 54, 60, 63 or 66.

[0010] In one embodiment, the light chain variable region comprises a LCDR1 sequence as shown in SEQ ID NO: 4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; a LCDR2 sequence as shown in SEQ ID NO: 5, 11, 17, 23, 28, 45, 55 or 59; and a LCDR3 sequence as shown in SEQ ID NO: 6, 12, 18, 24, 29, 32, 37, 40, 46, 51, 56, 62 or 65.

[0011] In one embodiment, the heavy chain variable region comprises a HCDR1, a HCDR2, and a HCDR3 sequence, and the light chain variable region comprises a LCDR1, a LCDR2, and a LCDR3 sequence; the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences are selected from any one of (1) to (15): (1) the HCDR1 sequence of SEQ ID NO: 1; the HCDR2 sequence of SEQ ID NO: 2; the HCDR3 sequence of SEQ ID NO: 3; the LCDR1 sequence of SEQ ID NO: 4; the LCDR2 sequence of SEQ ID NO: 5; and the LCDR3 sequence of SEQ ID NO: 6; (2) the HCDR1 sequence of SEQ ID NO: 7; the HCDR2 sequence of SEQ ID NO: 8; the HCDR3 sequence of SEQ ID NO: 9; the LCDR1 sequence of SEQ ID NO: 10; the LCDR2 sequence of SEQ ID NO: 11; and the LCDR3 sequence of SEQ ID NO: 12; (3) the HCDR1 sequence of SEQ ID NO: 13; the HCDR2 sequence of SEQ ID NO: 14; the HCDR3 sequence of SEQ ID NO: 15 NO:14; HCDR2 sequence shown in SEQ ID NO:15; HCDR3 sequence shown in SEQ ID NO:16; LCDR1 sequence shown in SEQ ID NO:17; and LCDR3 sequence shown in SEQ ID NO:18; (4) HCDR1 sequence shown in SEQ ID NO:19; HCDR2 sequence shown in SEQ ID NO:20; HCDR3 sequence shown in SEQ ID NO:21; LCDR1 sequence shown in SEQ ID NO:22; LCDR2 sequence shown in SEQ ID NO:23; and LCDR3 sequence shown in SEQ ID NO:24; (5) HCDR1 sequence shown in SEQ ID NO:25; HCDR2 sequence shown in SEQ ID NO:26; HCDR3 sequence shown in SEQ ID NO:27; LCDR1 sequence shown in SEQ ID NO:16; LCDR2 sequence shown in SEQ ID NO:28; and LCDR3 sequence shown in SEQ ID NO:29; (6) HCDR1 sequence shown in SEQ ID NO:25; HCDR2 sequence shown in SEQ ID NO:26; HCDR3 sequence shown in SEQ ID NO:27; LCDR1 sequence shown in SEQ ID NO:16; LCDR2 sequence shown in SEQ ID NO:28; and LCDR3 sequence shown in SEQ ID NO:29 HCDR3 sequence shown in SEQ ID NO:30; LCDR1 sequence shown in SEQ ID NO:31; LCDR2 sequence shown in SEQ ID NO:5; and LCDR3 sequence shown in SEQ ID NO:32; (7) HCDR1 sequence shown in SEQ ID NO:33; HCDR2 sequence shown in SEQ ID NO:34; HCDR3 sequence shown in SEQ ID NO:35;The LCDR1 sequence shown in SEQ ID NO:36; the LCDR2 sequence shown in SEQ ID NO:23; and the LCDR3 sequence shown in SEQ ID NO:37; (8) the HCDR1 sequence shown in SEQ ID NO:33; the HCDR2 sequence shown in SEQ ID NO:34; the HCDR3 sequence shown in SEQ ID NO:38; the LCDR1 sequence shown in SEQ ID NO:39; the LCDR2 sequence shown in SEQ ID NO:17; and the LCDR3 sequence shown in SEQ ID NO:40; (9) the HCDR1 sequence shown in SEQ ID NO:41; the HCDR2 sequence shown in SEQ ID NO:42; the HCDR3 sequence shown in SEQ ID NO:43; the LCDR1 sequence shown in SEQ ID NO:44; the LCDR2 sequence shown in SEQ ID NO:45; and the LCDR3 sequence shown in SEQ ID NO:46; (10) the HCDR1 sequence shown in SEQ ID NO:47; the HCDR2 sequence shown in SEQ ID NO:48; the HCDR3 sequence shown in SEQ ID NO:49; the LCDR1 sequence shown in SEQ ID NO:50; NO:11 LCDR2 sequence; and SEQ ID NO:51 LCDR3 sequence; (11) SEQ ID NO:52 HCDR1 sequence; SEQ ID NO:53 HCDR2 sequence; SEQ ID NO:54 HCDR3 sequence; SEQ ID NO:50 LCDR1 sequence; SEQ ID NO:55 LCDR2 sequence; and SEQ ID NO:56 LCDR3 sequence; (12) SEQ ID NO:57 HCDR1 sequence; SEQ ID NO:58 HCDR2 sequence; SEQ ID NO:35 HCDR3 sequence; SEQ ID NO:36 LCDR1 sequence; SEQ ID NO:59 LCDR2 sequence; and SEQ ID NO:37 LCDR3 sequence; (13) SEQ ID NO:33 HCDR1 sequence; SEQ ID NO:34 HCDR2 sequence; SEQ ID NO:60 HCDR3 sequence; SEQ ID NO:61 LCDR1 sequence; SEQ ID NO:23 LCDR2 sequence; and SEQ ID NO: LCDR3 sequence shown in NO:62; (14) HCDR1 sequence shown in SEQ ID NO:1; HCDR2 sequence shown in SEQ ID NO:2; HCDR3 sequence shown in SEQ ID NO:63; LCDR1 sequence shown in SEQ ID NO:64; LCDR2 sequence shown in SEQ ID NO:5;and the LCDR3 sequence shown in SEQ ID NO:65; (15) the HCDR1 sequence shown in SEQ ID NO:1; the HCDR2 sequence shown in SEQ ID NO:2; the HCDR3 sequence shown in SEQ ID NO:66; the LCDR1 sequence shown in SEQ ID NO:4; the LCDR2 sequence shown in SEQ ID NO:5; and the LCDR3 sequence shown in SEQ ID NO:6.

[0012] In one embodiment, the heavy chain variable region comprises 1) an amino acid sequence as set forth in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0013] In one embodiment, the light chain variable region comprises 1) an amino acid sequence as set forth in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0014] In one embodiment, the heavy chain variable region and the light chain variable region are selected from any one of (1) to (15): (1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 68; (2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 69; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 70; (3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 71; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 72; (4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 73; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74; (5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 75; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76; (6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 77; a light chain variable region comprising the amino acid sequence of SEQ ID NO: NO:78; (7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:79; a light chain variable region comprising the amino acid sequence of SEQ ID NO:80; (8) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:81; a light chain variable region comprising the amino acid sequence of SEQ ID NO:82; (9) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:83; a light chain variable region comprising the amino acid sequence of SEQ ID NO:84; (10) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:85; a light chain variable region comprising the amino acid sequence of SEQ ID NO:86; (11) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:87; a light chain variable region comprising the amino acid sequence of SEQ ID NO:88; (12) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:89; a light chain variable region comprising the amino acid sequence of SEQ ID NO:90; (13) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: (14) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 93; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 94; (15) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 95; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 96.

[0015] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising 1) an amino acid sequence as set forth in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, preferably, the addition, deletion and / or substitution does not occur in the CDR region.

[0016] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain comprising 1) an amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; 2) an amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; or 3) an amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0017] In one embodiment, the heavy chain and light chain are selected from any one of (1) to (15): (1) a heavy chain comprising the amino acid sequence of SEQ ID NO: 97; a light chain comprising the amino acid sequence of SEQ ID NO: 98; (2) a heavy chain comprising the amino acid sequence of SEQ ID NO: 99; a light chain comprising the amino acid sequence of SEQ ID NO: 100; (3) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101; a light chain comprising the amino acid sequence of SEQ ID NO: 102; (4) a heavy chain comprising the amino acid sequence of SEQ ID NO: 103; a light chain comprising the amino acid sequence of SEQ ID NO: 104; (5) a heavy chain comprising the amino acid sequence of SEQ ID NO: 105; a light chain comprising the amino acid sequence of SEQ ID NO: 106; (6) a heavy chain comprising the amino acid sequence of SEQ ID NO: 107; a light chain comprising the amino acid sequence of SEQ ID NO: 108; (7) a heavy chain comprising the amino acid sequence of SEQ ID NO: NO:109; a light chain comprising the amino acid sequence of SEQ ID NO:110; (8) a heavy chain comprising the amino acid sequence of SEQ ID NO:111; a light chain comprising the amino acid sequence of SEQ ID NO:112; (9) a heavy chain comprising the amino acid sequence of SEQ ID NO:113; a light chain comprising the amino acid sequence of SEQ ID NO:114; (10) a heavy chain comprising the amino acid sequence of SEQ ID NO:115; a light chain comprising the amino acid sequence of SEQ ID NO:116; (11) a heavy chain comprising the amino acid sequence of SEQ ID NO:117; a light chain comprising the amino acid sequence of SEQ ID NO:118; (12) a heavy chain comprising the amino acid sequence of SEQ ID NO:119; a light chain comprising the amino acid sequence of SEQ ID NO:120; (13) a heavy chain comprising the amino acid sequence of SEQ ID NO:121; a light chain comprising the amino acid sequence of SEQ ID NO: NO:122; (14) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:123; a light chain comprising the amino acid sequence shown in SEQ ID NO:124; (15) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:125; a light chain comprising the amino acid sequence shown in SEQ ID NO:126.

[0018] In another aspect, the present invention provides a pharmaceutical composition comprising the anti-CD100 antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0019] In some embodiments, the anti-CD100 antibody comprises antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5, or B13-e-2.

[0020] In another aspect, the present invention provides a pharmaceutical combination comprising the anti-CD100 antibody or antigen-binding fragment thereof of the present invention and an anti-PD-L1 antibody or antigen-binding fragment thereof.

[0021] In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof specifically recognizes and binds to PD-L1, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an immunoglobulin single variable domain;

[0022] In one embodiment, the immunoglobulin single variable domain comprises CDR1 comprising the amino acid sequence shown in SEQ ID NO: 130; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 131; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 132.

[0023] In one embodiment, the immunoglobulin single variable domain comprises: 1) the amino acid sequence of SEQ ID NO: 133; or 2) an amino acid sequence that has at least 85%, at least 90%, at least 95% or higher sequence identity to SEQ ID NO: 133.

[0024] In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc fragment of human IgG1. In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or more sequence identity to SEQ ID NO: 134.

[0025] In one embodiment, the pharmaceutical combination is a pharmaceutical composition or a kit.

[0026] In one aspect, the present invention also provides use of the antibody or antigen-binding fragment thereof, the pharmaceutical composition or the pharmaceutical combination of the present invention in the preparation of a medicament for treating cancer.

[0027] In another aspect, the present invention provides a method for treating cancer, comprising administering the antibody or antigen-binding fragment thereof, the pharmaceutical composition or the pharmaceutical combination of the present invention to a subject in need thereof.

[0028] In another aspect, the present invention relates to the antibody or antigen-binding fragment thereof, the pharmaceutical composition, or the pharmaceutical combination of the present invention for treating cancer.

[0029] In another aspect, the present invention further provides an isolated nucleic acid molecule encoding an anti-CD100 antibody or antigen-binding fragment thereof of the present invention. The present invention also relates to an expression vector comprising the nucleic acid molecule of the present invention. The present invention also relates to a host cell comprising the nucleic acid molecule or expression vector of the present invention.

[0030] In another aspect, the present invention also relates to a method for producing an anti-CD100 antibody or an antigen-binding fragment thereof, the method comprising:

[0031] a) culturing the host cell of the present invention under suitable conditions to express the anti-CD100 antibody or antigen-binding fragment thereof of the present invention; and

[0032] b) isolating the antibody or antigen-binding fragment thereof from the host cell or culture thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1 shows the ELISA test results of Pepinemab antibody and positive control 2D5 and 5D8 antibodies.

[0034] FIG2 shows the ELISA test results of antigens from different species.

[0035] FIG3 shows the FACS detection results of overexpressing cell lines of different species.

[0036] Figures 4A-4D show the FACS results of Plexin receptor-overexpressing cell lines from different species. Figure 4A shows the results for the HuPlexin-B1-HEK293 overexpressing cell line; Figure 4B shows the results for the MusPlexin-B1-HEK293 overexpressing cell line; Figure 4C shows the results for the CynoPlexin-B1-HEK293 overexpressing cell line; and Figure 4D shows the results for the HuPlexin-B2-HEK293 overexpressing cell line.

[0037] FIG5A to FIG5X show the results of ELISA testing the binding activity of candidate antibodies to antigen proteins from different species. Figures 5A-5H show the results of the binding activity of antibody molecules A14, A15, B13, H5, H12, H21, H74, H96, C-C081, C-C171 and C-B71 to the HuCD100-His antigen protein; Figures 5I-5P show the results of the binding activity of antibody molecules A14, A15, B13, H5, H12, H21, H74, H96, C-C081, C-C171 and C-B71 to the MusCD100-His antigen protein; Figures 5Q-5X show the results of the binding activity of antibody molecules A14, A15, B13, H5, H12, H21, H74, H96, C-C081, C-C171 and C-B71 to the CynoCD100-His antigen protein.

[0038] FIG6A-FIG6O show the results of FACS detection of the binding activity of candidate antibodies to overexpressing cell lines of different species. Figures 6A-6E show the results of the binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96, H12, C-C081, C-C171 and C-B71 to HuCD100-HEK293 overexpressing cell lines; Figures 6F-6J show the results of the binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96, H12, C-C081, C-C171 and C-B71 to MusCD100-HEK293 overexpressing cell lines; Figures 6K-6O show the results of the binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96, H12, C-C081, C-C171 and C-B71 to CynoCD100-HEK293 overexpressing cell lines.

[0039] Figures 7A-7H show the results of FACS testing of the binding activity of candidate antibodies to HuPBMC and Jurkat cells. Figures 7A-7D show the results of the binding activity of antibody molecules A14, A15, B13, H5, H21, H74, H96, H12, C-C081, C-C171, and C-B71 to HuPBMC cells; Figures 7E-7H show the results of the binding activity of antibody molecules A14, A15, B13, H5, H21, H74, H96, H12, C-C081, C-C171, and C-B71 to Jurkat cells.

[0040] Figures 8A-8O show the results of candidate antibodies blocking the binding of CD100 to Plexin receptors of different species. Figures 8A-8D show the results of antibody molecules A14, A15, B13, H5, H21, H74, H96, H12, C-C081, C-C171, and C-B71 blocking the binding of HuCD100 to HuPlexin-B1-HEK293 cells; Figures 8E-8I show the results of antibody molecules A14, H5, A15, B13, H21, H74, H96, H12, C-C081, C-C171, and C-B71 blocking the binding of HuCD100 to HuPlexin-B2-HEK293 cells; Figure 8J -8L shows the results of antibody molecules H5, A14, A15, B13, H12, H21, H74, H96, C-C081, C-C171 and C-B71 blocking the binding of MusCD100 to MusPlexin-B1-HEK293 cells; Figures 8M-8O show the results of antibody molecules H5, A14, A15, B13, H12, H21, H74, H96, C-C081, C-C171 and C-B71 blocking the binding of CynoCD100 to CynoPlexin-B1-HEK293 cells.

[0041] 9A-9C show the results of MDSC proliferation inhibition assays of candidate antibodies A14, A15, B13, H12, H21, H74, H96, H5, C-C081, C-C171, and C-B71.

[0042] Figures 10A-10B show the results of FACS analysis of the binding activity of anti-PD-L1 antibody m18 against human PD-L1-overexpressing CHO cells and PD-L1-positive HCC827 cells. Figure 10A shows the results of binding activity against human PD-L1-CHO cells; Figure 10B shows the results of binding activity against HCC827 cells.

[0043] Figures 11A-11B show the results of FACS analysis of the binding activity of anti-PD-L1 antibody m18 against cell lines overexpressing PD-L1 from different species. Figure 11A shows the binding activity against mouse PD-L1-CHO cells; Figure 11B shows the binding activity against cynomolgus monkey PD-L1-CHO cells.

[0044] FIG12 shows the results of ELISA testing of the specific binding activity of the anti-PD-L1 antibody m18 to B7-H1 and homologous proteins.

[0045] Figures 13A-13D show the results of FACS analysis of the binding activity of affinity-matured candidate antibodies H5 and B13 (H5-h-7, H5-a-2, B13-c-5, and B13-e-2) to CD100-overexpressing cell lines from different species and CD100-positive PBMCs. Figure 13A shows the results for binding activity to HuCD100-HEK293 cells; Figure 13B shows the results for binding activity to human PBMCs; Figure 13C shows the results for binding activity to MusCD100-HEK 293 cells; and Figure 13D shows the results for binding activity to CynoCD100-HEK 293 cells.

[0046] Figures 14A-14D show the results of affinity-matured antibodies H5 and B13, H5-h-7, H5-a-2, B13-c-5, and B13-e-2, blocking the binding of CD100 to Plexin receptors from different species. Figure 14A shows the results of the candidate antibodies blocking the binding of HuCD100 to HuPlexin-B1-HEK293 cells; Figure 14B shows the results of the candidate antibodies blocking the binding of HuCD100 to HuPlexin-B2-HEK293 cells; Figure 14C shows the results of the candidate antibodies blocking the binding of MusCD100 to MusPlexin-B1-HEK293 cells; and Figure 14D shows the results of the candidate antibodies blocking the binding of CynoCD100 to CynoPlexin-B1-HEK293 cells.

[0047] FIG15 shows the results of MDSC proliferation inhibition assays of the candidate antibodies H5 and affinity-matured modified antibodies H5-h-7, H5-a-2, B13-c-5, and B13-e-2.

[0048] Figures 16A and 16B show the results of tumor inhibition test: Figure 16A shows the change in tumor volume; Figure 16B shows the change in body weight. DETAILED DESCRIPTION

[0049] definition

[0050] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0051] As used herein, "at least one" or "one or more" may mean 1, 2, 3, 4, 5, 6, 7, 8 or more.

[0052] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0053] As used herein, the term "and / or" connecting multiple elements should be understood to include both individual and combined options. In other words, "and / or" includes "and" and "or." For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. More elements qualified with "and / or" are understood in a similar manner and include any one thereof and any combination thereof.

[0054] Unless otherwise indicated, any numerical value or numerical range, such as concentration or concentration range, is understood to be modified by the term "about" in any case. Thus, numerical values ​​generally include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions within the range, unless the context clearly indicates otherwise.

[0055] As used herein, "antibody" refers to an immunoglobulin or its fragment, which specifically binds to an antigenic epitope through at least one antigen binding site. Antibody encompasses antibody fragments. As used herein, the term "antibody" includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b). In preferred embodiments, the antibodies of the invention are human antibodies.

[0056] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region (e.g., one or more CDRs and / or one or more antibody binding sites) of the antibody that binds to an antigen and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments can include multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding regions), results in an antibody that immunospecifically binds to an antigen.

[0057] As used herein, "immunoglobulin single variable domain" or "single variable domain" refers to a single variable region (variable domain) with antigen binding activity. Unlike conventional antibodies, which are composed of a pair of VH and VL functional antigen binding units, single variable domains can form functional antigen binding units on their own. Single variable domains can be derived from naturally occurring light chain-free antibodies, such as the variable domain of heavy chain of heavy-chain antibodies (VHH) of camelids (such as camels and alpacas) and the single variable domain of shark's new antigen receptor (IgNAR variable single-domain, VNAR), or can be screened from full-length antibodies, such as light chain variable domains and heavy chain variable domains with antigen binding activity in human antibodies. VHH typically contains three highly variable "complementarity determining regions (CDRs)" and four relatively conserved "framework regions (FRs)", and is connected in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus.

[0058] As used herein, "single domain antibody (sdAb)" or "nanoantibody" refers to an antibody comprising a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment. Similar to the variable region of a full-length antibody, a single variable domain typically comprises CDR1, CDR2, and CDR3 that form an antigen-binding site, as well as a supporting framework region. Unlike full-length antibodies that typically comprise two heavy chains and two light chains, a single domain antibody typically comprises a single peptide chain consisting of a single variable domain, with a molecular weight of only about 15 kDa. The single variable domain can, for example, be the variable domain of a heavy-chain antibody of an alpaca (VHH), the IgNAR variable domain of a shark, or the variable domain of a human light-chain antibody.

[0059] As used herein, the terms "heavy-chain-only antibody" and "heavy-chain antibody" are used interchangeably and in their broadest sense to refer to an antibody that lacks conventional antibody light chains and comprises only one VHH and a heavy chain constant region (e.g., Fc fragment) that does not comprise CH1.

[0060] "Fc fragment" generally refers to a crystallizable fragment of a conventional antibody or heavy chain antibody after papain digestion. Generally speaking, the Fc fragment of IgG and heavy chain antibodies can include part of the hinge region, CH2 and CH3. Herein, the Fc fragment can include at least part of the hinge region (e.g., all or part of the hinge region), CH2 and CH3.

[0061] As used herein, a "full-length antibody" generally comprises two heavy chains (which may be designated as H and H') and two light chains (which may be designated as L and L'). Each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding ability, and each light chain may be a full-length light chain or any functional region. Each light chain comprises a "light chain variable region (VL)" and a "light chain constant region (CL)" from N-terminus (amino acid terminus) to C-terminus (carboxyl terminus). Each heavy chain comprises a "heavy chain variable region (VH)" and a "heavy chain constant region (CH)" from N-terminus to C-terminus. In general, the heavy chain constant region of a full-length antibody may comprise VH-CH1-hinge region (hinge) CH2-CH3 from N-terminus to C-terminus. Each heavy chain (H and H') is paired with one light chain (L and L', respectively).

[0062] Light chains are classified as κ or λ (Kappa or Lambda). Each heavy chain category can be combined with a κ or λ light chain. Typically, the light chain and the heavy chain are covalently bound to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tail" portions of the two heavy chains are bound together by covalent disulfide bonds or non-covalent linkages. In the heavy chain, the amino acids are from the N-terminus at the bifurcated end of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of some antibodies (e.g., IgG antibodies) includes two heavy chain subunits and two light chain subunits, which are covalently linked by disulfide bonds to form a "Y" structure.

[0063] Both light and heavy chains are divided into regions with structural and functional homology. The terms "constant" and "variable" are used from a functional perspective. In this regard, it should be understood that the variable region of the variable light (VL) chain or variable heavy (VH) chain portion determines antigen recognition and specificity. In contrast, the constant regions of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of the constant region domains increases as they move away from the antigen binding site or the N-terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains actually include the carboxyl termini of the heavy and light chains, respectively.

[0064] As mentioned above, the variable region (i.e., "binding domain") allows the binding molecule to selectively recognize an epitope on an antigen and specifically bind to the epitope. That is, for example, the VL domain and the VH domain of the binding molecule of an antibody, or these complementary determining regions (CDR) subgroups combine to form a variable region that determines a three-dimensional antigen binding site. More specifically, the antigen binding site is determined by three CDRs on each VH and VL chain. These six "complementary determining regions" or "CDRs" are short, non-continuous sequences of amino acids that are specifically positioned to form a binding domain as the antibody adopts its three-dimensional configuration in an aqueous environment. The remaining amino acids in the binding domain are called "framework (FR)" regions, which show smaller intermolecular differences. The binding domain formed by the positioned CDRs determines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its complementary epitope. The amino acids that make up the CDRs and framework regions, respectively, in any given heavy or light chain variable region can be identified by conventional methods (see, "Sequences of Proteins of Immunological Interest," Kabat, E. et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties). Herein, the CDRs (CDRL or LCDR) of the light chain variable region may be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region may be referred to as HCDR1, HCDR2, and HCDR3.

[0065] Unless otherwise indicated, herein, the amino acid sequences of CDRs are shown according to the AbM definition rules (the sequences in the claims of the present invention are also shown according to the AbM definition rules). However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on antibody sequence variability (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), AbM (Martin, ACR and J. Allen (2007) "Bioinformatics tools for antibody engineering," in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp. 95–118), Contact (MacCallum, RM et al., (1996) J. Mol. Biol. 262: 732-745), IMGT (Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.Although the scope of protection requested in the claims of the present invention is based on the sequences shown in the AbM definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of protection of the present invention.

[0066] Thus, when referring to antibodies defined by specific CDR sequences defined herein, the scope of said antibodies also encompasses antibodies whose variable region sequences comprise said specific CDR sequences, but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0067] As used herein, the terms "framework region" and "framework region" are used interchangeably. As used herein, the terms "framework region," "framework region," or "FR" residues refer to those amino acid residues in the antibody variable region excluding the CDR sequences as defined above.

[0068] An "Fv" fragment, consisting of a single VH and a single VL through non-covalent interactions, is generally considered the smallest antigen-binding fragment containing an antigen-binding site. However, a single variable domain (single-domain antibody) also possesses antigen-binding ability. A "single-chain Fv (scFv)" can be obtained by linking the VH and VL via a peptide linker. By introducing disulfide bonds into an Fv or scFv, a "disulfide-stabilized Fv (dsFv)" or "single-chain disulfide-stabilized Fv (scdsFv or dsscFv)" can be obtained, respectively.

[0069] As used herein, Fab fragments are antibody fragments obtained by digesting full-length immunoglobulins with papain, or fragments with the same structure, for example, synthesized by recombinant methods. It contains a complete antibody light chain (VL-CL) and an antibody heavy chain variable region and a heavy chain constant region (VH-CH1, also referred to as Fd). Single-chain "Fab (scFab)" can be obtained by connecting CL and CH1 in "Fab" with a peptide linker. "F(ab')2" is an antibody fragment resulting from digesting immunoglobulins with pepsin at pH 4.0-4.5, or a fragment with the same structure, for example, synthesized by recombinant methods. It basically contains two Fab fragments connected by a disulfide bond in the hinge region. "Fab'" is half of F(ab')2, which can be obtained by reducing the disulfide bond in the hinge region of F(ab')2.

[0070] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain and the CH2 domain. The hinge region comprises approximately 25 amino acids and is flexible, thus allowing the two N-terminal antigen binding regions to move independently.

[0071] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a sulfhydryl group that can form a disulfide bond or bridge a second sulfhydryl group.

[0072] As used herein, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species, while the constant region (which may be complete, partial, or modified) is obtained from a second species. In some embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region will be human.

[0073] As used herein, the term "humanized antibody" refers to an antibody in which a non-human antibody is modified to increase sequence homology with a human antibody. Humanized antibodies generally retain the antigen-binding ability of the non-human antibody from which they are derived and have lower immunogenicity to the human body. Humanized antibodies can be obtained by engineering any non-human species antibody or an antibody (e.g., a chimeric antibody) comprising a sequence of a non-human species. Non-human species may, for example, include mice, rats, rabbits, alpacas, sharks, or non-human primates. The technology for obtaining humanized antibodies from non-human antibodies is well known to those skilled in the art. For example, the CDR sequences of non-human antibodies (e.g., mouse antibodies) are transplanted into human antibody framework regions. In some cases, in order to maintain the antigen-binding ability and / or stability of the humanized antibody, key amino acid residues of the non-human antibody (e.g., murine antibody) framework sequence can be retained in the human antibody framework region, i.e., "back mutations" are performed (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21): 6851-6855; Neuberger et al. (1984) Nature 312: 604-608).

[0074] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human. The definition of a human antibody encompasses complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide.

[0075] As used herein, an "affinity matured" antibody comprises one or more modifications (e.g., substitutions of amino acid residues) in one or more CDRs such that the affinity matured antibody has improved affinity for the antigen compared to a parent antibody that does not comprise such modifications. Methods for affinity maturation of antibodies are known in the art, see, e.g., Marks et al., Bio / Technology 10:779-783 (1992); Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Scier et al., Gene 169:147-155 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0076] As used herein, "percent (%) sequence identity" or "sequence identity" of amino acid sequences has an art-recognized definition and refers to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). This can be determined using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0077] As used herein, an amino acid sequence that is "derived from" or "derived from" a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence. For example, an amino acid sequence derived from the heavy chain constant region of a human immunoglobulin may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the wild-type sequence of the heavy chain constant region of the human immunoglobulin from which it is derived.

[0078] "Affinity" or "binding affinity" is a measure of the strength of the non-covalent binding between an antibody and an antigen. Affinity can be determined using conventional techniques known in the art, such as biofilm interferometry (e.g., using the Octet Fortebio assay system), radioimmunoassay, surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS).

[0079] "Specific binding" generally refers to a binding molecule, such as an antibody or a fragment, variant, or derivative thereof, that binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. By this definition, a binding molecule is said to "specifically bind" to an epitope when it is easier to bind to an epitope through its antigen binding domain than to a random, unrelated epitope. The term "specificity" is used herein to qualitatively analyze the relative affinity of an antibody for binding to an epitope. For example, it can be considered that binding molecule "A" has a higher specificity for a given epitope than binding molecule "B," or it can be said that binding molecule "A" specifically binds to epitope "C" with a higher specificity than its specificity for a related epitope "D."

[0080] If a binding molecule, e.g., an antibody, or fragment, variant, or derivative thereof, preferentially binds to an epitope to the extent that it blocks binding of the reference antibody or antigen-binding fragment to the epitope, then the binding molecule, e.g., an antibody, or fragment, variant, or derivative thereof, is said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope. Competitive inhibition can be determined by any method known in the art, e.g., a competition ELISA assay. The binding molecule can be said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0081] As used herein, the term "PD-L1" refers to programmed cell death ligand 1 (PD-L1, see, e.g., Freeman et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000 Oct 2; 192 (7)). PD-L1 belongs to the B7 family. Alternative names or synonyms for PD-L1 include PDCD1L1, PDL1, B7 homolog 1 (B7-H1), surface antigen differentiation cluster 274 (cluster of differentiation 274, CD274) or B7-H, etc. The representative amino acid sequence of human PD-L1 is disclosed in NCBI accession number NP_054862.1, and the representative nucleic acid sequence encoding human PD-L1 is shown under NCBI accession number: NM_014143.4. PD-L1 is expressed in the placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found in many tumors or cancer cells. PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells, and bone marrow cells. The binding of PD-L1 to its receptor induces signal transduction to inhibit TCR-mediated activation of cytokine production and T cell proliferation. Therefore, PD-L1 plays a major role in suppressing the immune system during specific events (such as pregnancy, autoimmune diseases, tissue allografts), and is thought to allow tumors or cancer cells to bypass immune checkpoints and evade immune responses.

[0082] As used herein, the term "B7 family" refers to a class of structurally similar costimulatory factors involved in the immune process. They belong to the immunoglobulin class and are associated with T and B cell activation and immune function.

[0083] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It will be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, with deoxythymidylic acid in the DNA coding strand sequence corresponding to uridine in the RNA sequence it encodes.

[0084] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in the natural origin of nucleic acid molecules. " Isolated " nucleic acid molecules such as cDNA molecules can be substantially free of other cellular materials or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical compositions when chemosynthesis. The exemplary isolated nucleic acid molecules provided herein comprise the isolated nucleic acid molecules of the antibody or Fab provided by coding.

[0085] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and / or polypeptides.

[0086] As used herein, "vector" is a medium for introducing exogenous polynucleotides into a host cell, and when the vector is transformed into an appropriate host cell, the exogenous polynucleotides are amplified or expressed. The vector usually remains free, but can be designed to integrate a gene or part thereof into a chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes virus vectors, pox virus vectors, and baculovirus vectors, etc.

[0087] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells, such as HEK 293 cells.

[0088] Terms such as "treating" or "treating" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, alleviate, reduce the symptoms of an existing, diagnosed pathological condition or disorder, and / or arrest or slow the progression of an existing, diagnosed pathological condition or disorder. Terms such as "preventing," "preventing," "avoiding," "containment," and the like refer to preventative or prophylactic measures that prevent the progression of an undiagnosed target pathological condition or disorder. Thus, a "subject in need thereof" can include a subject already suffering from a disease; a subject susceptible to a disease; and a subject in need of prevention of a disease.

[0089] As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

[0090] The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule or other drug that is effective for "treating" a disease or condition in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; block or stop cancer cell division, reduce or block the increase in tumor size; inhibit, for example, suppress, block, prevent, stop, delay or reverse cancer cell infiltration into peripheral organs, including, for example, the spread of cancer to soft tissue and bone; inhibit, for example, suppress, block, prevent, shrink, stop, delay or reverse tumor metastasis; inhibit, for example, suppress, block, prevent, stop, delay or reverse tumor growth; alleviate to some extent one or more symptoms associated with cancer, reduce morbidity and mortality; improve quality of life; or a combination of these effects. To the extent that a drug prevents growth and / or kills existing cancer cells, it can refer to being cytostatic and / or cytotoxic.

[0091] As used herein, the term "subject" refers to a mammal, such as a human.

[0092] Antibody numbering used herein (such as B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2, m18-VHH or m18) is only used to distinguish or identify antibodies or products, and is not intended to represent that such identification is a feature of the antibody of the present invention or product. It will be understood by those skilled in the art that, for example, for the purpose of distinguishing or identifying, other antibodies or products may also use such identification, but do not refer to identical or equivalent antibodies or products. Similarly, the similar numbering or identification used in the embodiments is only for example convenience, and the antibody of the present invention or product is limited by the features described in the appended claims.

[0093] Anti-CD100 antibody or antigen-binding fragment thereof

[0094] The present invention provides an antibody against CD100 (anti-CD100 antibody) or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof specifically recognizes and binds to CD100.

[0095] In some embodiments, the anti-CD100 antibody or antigen-binding fragment thereof of the present invention is a chimeric antibody, a humanized antibody, a human antibody, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide-stabilized Fv (dsFv), or a diabody. Preferably, the antibody or antigen-binding fragment thereof is a human antibody.

[0096] In one embodiment, the antibody or antigen-binding fragment thereof has at least one of the following characteristics:

[0097] 1) Having affinity activity for CD100 protein;

[0098] 2) has affinity activity for CD100-positive cells;

[0099] 3) blocking the binding of CD100 to Plexin-B1 or Plexin-B2;

[0100] 4) Inhibit MDSC cell proliferation;

[0101] 5) Inhibit tumor growth.

[0102] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can specifically bind to CD100 (eg, human CD100) and block its interaction with Plexin-B1 or Plexin-B2.

[0103] In some embodiments, the tumors targeted include but are not limited to those described below with respect to neoplastic diseases. In other embodiments, the antibodies or antigen-binding fragments thereof of the present invention can inhibit tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%.

[0104] In some embodiments, an antibody or antigen-binding fragment thereof directed against CD100 of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2, and HCDR3 sequence, and wherein the HCDR1 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 1, 7, 19, 33, 41, 47, or 52 by no more than 2 amino acid additions, deletions, or substitutions; the HCDR2 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 2, 8, 14, 20, 26, 42, 48, or 53 by no more than 2 amino acid additions, deletions, or substitutions; and / or the HCDR3 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, or 54 by no more than 2 amino acid additions, deletions, or substitutions.

[0105] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof directed against CD100, which comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences, and wherein the LCDR1 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 4, 10, 16, 36, 44, or 50 by no more than 2 amino acid additions, deletions, or substitutions; the LCDR2 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 5, 11, or 45 by no more than 2 amino acid additions, deletions, or substitutions; and / or the LCDR3 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 6, 12, 18, 24, 37, 46, 51, or 56 by no more than 2 amino acid additions, deletions, or substitutions.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof directed against CD100 of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0107] The heavy chain variable region comprises a HCDR1, a HCDR2, and a HCDR3 sequence, wherein the HCDR1 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 1, 7, 19, 33, 41, 47, or 52 by no more than 2 amino acid additions, deletions, or substitutions; the HCDR2 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 2, 8, 14, 20, 26, 42, 48, or 53 by no more than 2 amino acid additions, deletions, or substitutions; and / or the HCDR3 sequence differs in amino acid sequence from the sequence of SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, or 54 by no more than 2 amino acid additions, deletions, or substitutions; and

[0108] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences, and wherein the LCDR1 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO:4, 10, 16, 36, 44 or 50 by no more than 2 amino acids in amino acid addition, deletion or substitution; the LCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO:5, 11 or 45 by no more than 2 amino acids in amino acid addition, deletion or substitution; and / or the LCDR3 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO:6, 12, 18, 24, 37, 46, 51 or 56 by no more than 2 amino acids in amino acid addition, deletion or substitution.

[0109] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO:7 (SEQ ID NO:7; GFTFSSYSMN), wherein the amino acid at position 3 is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO:7 (SEQ ID NO:7; GFTFSSYSMN), wherein the amino acid at position 8 is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO:7 (SEQ ID NO:7; GFTFSSYSMN), wherein the amino acid at position 3 and the amino acid at position 8 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO:7 is substituted with P, and the amino acid at position 8 in SEQ ID NO:7 is substituted with E (SEQ ID NO:13; GFPFSSYEMN).

[0110] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 1; SGSFSGYYWT), wherein the amino acid at position 1 is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 1; SGSFSGYYWT), wherein the amino acid at position 10 is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 1; SGSFSGYYWT), wherein the amino acid at position 1 and the amino acid at position 10 are substituted. In one embodiment, the amino acid at position 1 in SEQ ID NO: 1 is substituted with G, and the amino acid at position 10 in SEQ ID NO: 1 is substituted with S (SEQ ID NO: 25; GGSFSGYYWS).

[0111] In one embodiment, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 33 (SEQ ID NO: 33; GGSISSSNWWS), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 33 is substituted with G (SEQ ID NO: 57; GGSISGSNWWS).

[0112] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 26 (SEQ ID NO: 26; EINHSGSTN), wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 26 is substituted with Y (SEQ ID NO: 34; EIYHSGSTN).

[0113] In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 26 (SEQ ID NO: 26; EINHSGSTN), wherein the amino acid at position 3 is substituted. In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 26 (SEQ ID NO: 26; EINHSGSTN), wherein the amino acid at position 7 is substituted. In one embodiment, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 26 (SEQ ID NO: 26; EINHSGSTN), wherein the amino acid at position 3 and the amino acid at position 7 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO: 26 is substituted with Y, and the amino acid at position 7 in SEQ ID NO: 26 is substituted with E (SEQ ID NO: 58; EIYHSGETN).

[0114] In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 35 (SEQ ID NO: 35; YDFWSGSGLDY), wherein the amino acid at position 4 is substituted. In one embodiment, the amino acid at position 4 in SEQ ID NO: 35 is substituted with E (SEQ ID NO: 60; YDFESGSGLDY).

[0115] In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 3 (SEQ ID NO: 3; GPAYYADALDGFDI), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 3 is substituted with P (SEQ ID NO: 63; GPAYYPDALDGFDI).

[0116] In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 3 (SEQ ID NO: 3; GPAYYADALDGFDI), wherein the amino acid at position 5 is substituted. In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 3 (SEQ ID NO: 3; GPAYYADALDGFDI), wherein the amino acid at position 6 is substituted. In one embodiment, the HCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 3 (SEQ ID NO: 3; GPAYYADALDGFDI), wherein the amino acid at position 5 and the amino acid at position 6 are substituted. In one embodiment, the amino acid at position 5 of SEQ ID NO: 3 is substituted with L, and the amino acid at position 6 of SEQ ID NO: 3 is substituted with P (SEQ ID NO: 66; GPAYLPDALDGFDI).

[0117] In one embodiment, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 16; SGDKLGDKYAY), wherein the amino acid at position 10 is substituted. In one embodiment, the amino acid at position 10 in SEQ ID NO: 16 is substituted with V (SEQ ID NO: 22; SGDKLGDKYVY).

[0118] In one embodiment, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 16; SGDKLGDKYAY), wherein the amino acid at position 7 is substituted. In one embodiment, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 16; SGDKLGDKYAY), wherein the amino acid at position 11 is substituted. In one embodiment, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 16; SGDKLGDKYAY), wherein the amino acid at position 7 and the amino acid at position 11 are substituted. In one embodiment, the amino acid at position 7 in SEQ ID NO: 16 is substituted with E, and the amino acid at position 11 in SEQ ID NO: 16 is substituted with F (SEQ ID NO: 31; SGDKLGEKYAF).

[0119] In one embodiment, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 16; SGDKLGDKYAY), wherein the amino acid at position 4 is substituted. In one embodiment, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 16; SGDKLGDKYAY), wherein the amino acid at position 10 is substituted. In one embodiment, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 16; SGDKLGDKYAY), wherein the amino acid at position 4 and the amino acid at position 10 are substituted. In one embodiment, the amino acid at position 4 in SEQ ID NO: 16 is substituted with R, and the amino acid at position 10 in SEQ ID NO: 16 is substituted with S (SEQ ID NO: 39; SGDRLGDKYSY).

[0120] In one embodiment, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO:36 (SEQ ID NO:36; SGDKLGDKFAS), wherein the amino acid at position 7 is substituted. In one embodiment, the amino acid at position 7 in SEQ ID NO:36 is substituted with E (SEQ ID NO:61; SGDKLGEKFAS).

[0121] In one embodiment, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO:4 (SEQ ID NO:4; SGDKLGDKYVS), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO:4 is substituted with Q (SEQ ID NO:64; SGDKLQDKYVS).

[0122] In one embodiment, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO:5 is substituted with S (SEQ ID NO:17; QDSKRPS).

[0123] In one embodiment, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO:5 is substituted with R (SEQ ID NO:23; QDRKRPS).

[0124] In one embodiment, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO:5 is substituted with A (SEQ ID NO:28; QDAKRPS).

[0125] In one embodiment, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 11 (SEQ ID NO: 11; AASSLQS), wherein the amino acid at position 1 is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 11 (SEQ ID NO: 11; AASSLQS), wherein the amino acid at position 5 is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 11 (SEQ ID NO: 11; AASSLQS), wherein the amino acid at position 1 and the amino acid at position 5 are substituted. In one embodiment, the amino acid at position 1 of SEQ ID NO: 11 is substituted with T, and the amino acid at position 5 of SEQ ID NO: 11 is substituted with V (SEQ ID NO: 55; TASSVQS).

[0126] In one embodiment, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the amino acid at position 3 is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the amino acid at position 7 is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the amino acid at position 3 and the amino acid at position 7 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO:5 is substituted with R, and the amino acid at position 7 in SEQ ID NO:5 is substituted with N (SEQ ID NO:59; QDRKRPN).

[0127] In one embodiment, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO:6 (SEQ ID NO:6; QAWDSSTKAYV), wherein the amino acid at position 8 is substituted. In one embodiment, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO:6 (SEQ ID NO:6; QAWDSSTKAYV), wherein the amino acid at position 9 is substituted. In one embodiment, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO:6 (SEQ ID NO:6; QAWDSSTKAYV), wherein the amino acid at position 8 and the amino acid at position 9 are substituted. In one embodiment, the amino acid at position 8 in SEQ ID NO:6 is substituted with A, and the amino acid at position 9 in SEQ ID NO:6 is substituted with G (SEQ ID NO:29; QAWDSSTAGYV).

[0128] In one embodiment, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; QAWDSSTV), wherein an amino acid is added between amino acid positions 7 and 8. In one embodiment, the amino acid added between amino acid positions 7 and 8 in SEQ ID NO: 18 is Y (SEQ ID NO: 32; QAWDSSTYV).

[0129] In one embodiment, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; QAWDSSTV), wherein the amino acid at position 2 is substituted. In one embodiment, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; QAWDSSTV), wherein an amino acid is added between amino acids 7 and 8. In one embodiment, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 18; QAWDSSTV), wherein the amino acid at position 2 is substituted and an amino acid is added between amino acids 7 and 8. In one embodiment, the amino acid at position 2 in SEQ ID NO: 18 is substituted with V, and the amino acid added between amino acids 7 and 8 in SEQ ID NO: 18 is A (SEQ ID NO: 40; QVWDSSTAV).

[0130] In one embodiment, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 37 (SEQ ID NO: 37; QAWDSGTVI), wherein the amino acid at position 8 is substituted. In one embodiment, the amino acid at position 8 in SEQ ID NO: 37 is substituted with A (SEQ ID NO: 62; QAWDSGTAI).

[0131] In one embodiment, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO: 6 (SEQ ID NO: 6; QAWDSSTKAYV), wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO: 6 is substituted with E (SEQ ID NO: 65; QAWDSETKAYV).

[0132] In one embodiment, the heavy chain variable region comprises a HCDR1 sequence shown in SEQ ID NO: 1, 7, 13, 19, 25, 33, 41, 47, 52 or 57; a HCDR2 sequence shown in SEQ ID NO: 2, 8, 14, 20, 26, 34, 42, 48, 53 or 58; and a HCDR3 sequence shown in SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, 54, 60, 63 or 66.

[0133] In one embodiment, the light chain variable region comprises a LCDR1 sequence as shown in SEQ ID NO: 4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; a LCDR2 sequence as shown in SEQ ID NO: 5, 11, 17, 23, 28, 45, 55 or 59; and a LCDR3 sequence as shown in SEQ ID NO: 6, 12, 18, 24, 29, 32, 37, 40, 46, 51, 56, 62 or 65.

[0134] In one embodiment, the heavy chain variable region comprises a HCDR1 sequence as shown in SEQ ID NO: 1, 7, 13, 19, 25, 33, 41, 47, 52 or 57; a HCDR2 sequence as shown in SEQ ID NO: 2, 8, 14, 20, 26, 34, 42, 48, 53 or 58; and a HCDR3 sequence as shown in SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, 54, 60, 63 or 66; and

[0135] The light chain variable region comprises a LCDR1 sequence shown in SEQ ID NO: 4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; a LCDR2 sequence shown in SEQ ID NO: 5, 11, 17, 23, 28, 45, 55 or 59; and a LCDR3 sequence shown in SEQ ID NO: 6, 12, 18, 24, 29, 32, 37, 40, 46, 51, 56, 62 or 65.

[0136] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 2, and the HCDR3 sequence of SEQ ID NO: 3. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 7, the HCDR2 sequence of SEQ ID NO: 8, and the HCDR3 sequence of SEQ ID NO: 9. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 13, the HCDR2 sequence of SEQ ID NO: 14, and the HCDR3 sequence of SEQ ID NO: 15. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 19, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 21. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 25, the HCDR2 sequence of SEQ ID NO: 26, and the HCDR3 sequence of SEQ ID NO: 27. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 25, the HCDR2 sequence of SEQ ID NO: 26, and the HCDR3 sequence of SEQ ID NO: 30. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 33, the HCDR2 sequence of SEQ ID NO: 34, and the HCDR3 sequence of SEQ ID NO: 35. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 33, the HCDR2 sequence of SEQ ID NO: 34, and the HCDR3 sequence of SEQ ID NO: 38. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 41, the HCDR2 sequence of SEQ ID NO: 42, and the HCDR3 sequence of SEQ ID NO: 43. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 47, the HCDR2 sequence of SEQ ID NO: 48, and the HCDR3 sequence of SEQ ID NO: 49. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 52, the HCDR2 sequence of SEQ ID NO: 53, and the HCDR3 sequence of SEQ ID NO: 54. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 57, the HCDR2 sequence of SEQ ID NO: 58, and the HCDR3 sequence of SEQ ID NO: 35.In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 33, the HCDR2 sequence of SEQ ID NO: 34, and the HCDR3 sequence of SEQ ID NO: 60. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 2, and the HCDR3 sequence of SEQ ID NO: 63. In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 1, the HCDR2 sequence of SEQ ID NO: 2, and the HCDR3 sequence of SEQ ID NO: 66.

[0137] In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:4, the LCDR2 sequence of SEQ ID NO:5, and the LCDR3 sequence of SEQ ID NO:6. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:10, the LCDR2 sequence of SEQ ID NO:11, and the LCDR3 sequence of SEQ ID NO:12. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:16, the LCDR2 sequence of SEQ ID NO:17, and the LCDR3 sequence of SEQ ID NO:18. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:22, the LCDR2 sequence of SEQ ID NO:23, and the LCDR3 sequence of SEQ ID NO:24. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:16, the LCDR2 sequence of SEQ ID NO:28, and the LCDR3 sequence of SEQ ID NO:29. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:31, the LCDR2 sequence of SEQ ID NO:5, and the LCDR3 sequence of SEQ ID NO:32. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:36, the LCDR2 sequence of SEQ ID NO:23, and the LCDR3 sequence of SEQ ID NO:37. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:39, the LCDR2 sequence of SEQ ID NO:17, and the LCDR3 sequence of SEQ ID NO:40. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:44, the LCDR2 sequence of SEQ ID NO:45, and the LCDR3 sequence of SEQ ID NO:46. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO:50, the LCDR2 sequence of SEQ ID NO:11, and the LCDR3 sequence of SEQ ID NO:51. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO: 50, the LCDR2 sequence of SEQ ID NO: 55, and the LCDR3 sequence of SEQ ID NO: 56. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO: 36, the LCDR2 sequence of SEQ ID NO: 59, and the LCDR3 sequence of SEQ ID NO: 37.In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO: 61, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 62. In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO: 64, the LCDR2 sequence of SEQ ID NO: 5, and the LCDR3 sequence of SEQ ID NO: 65.

[0138] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 1, the HCDR2 sequence shown in SEQ ID NO: 2, and the HCDR3 sequence shown in SEQ ID NO: 3; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO: 4, the LCDR2 sequence shown in SEQ ID NO: 5, and the LCDR3 sequence shown in SEQ ID NO: 6.

[0139] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:7, the HCDR2 sequence shown in SEQ ID NO:8 and the HCDR3 sequence shown in SEQ ID NO:9; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:10, the LCDR2 sequence shown in SEQ ID NO:11 and the LCDR3 sequence shown in SEQ ID NO:12.

[0140] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 13, the HCDR2 sequence shown in SEQ ID NO: 14, and the HCDR3 sequence shown in SEQ ID NO: 15; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO: 16, the LCDR2 sequence shown in SEQ ID NO: 17, and the LCDR3 sequence shown in SEQ ID NO: 18.

[0141] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 19, the HCDR2 sequence shown in SEQ ID NO: 20, and the HCDR3 sequence shown in SEQ ID NO: 21; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO: 22, the LCDR2 sequence shown in SEQ ID NO: 23, and the LCDR3 sequence shown in SEQ ID NO: 24.

[0142] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:25, the HCDR2 sequence shown in SEQ ID NO:26 and the HCDR3 sequence shown in SEQ ID NO:27; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:16, the LCDR2 sequence shown in SEQ ID NO:28 and the LCDR3 sequence shown in SEQ ID NO:29.

[0143] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:25, the HCDR2 sequence shown in SEQ ID NO:26, and the HCDR3 sequence shown in SEQ ID NO:30; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:31, the LCDR2 sequence shown in SEQ ID NO:5, and the LCDR3 sequence shown in SEQ ID NO:32.

[0144] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:33, the HCDR2 sequence shown in SEQ ID NO:34, and the HCDR3 sequence shown in SEQ ID NO:35; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:36, the LCDR2 sequence shown in SEQ ID NO:23, and the LCDR3 sequence shown in SEQ ID NO:37.

[0145] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:33, the HCDR2 sequence shown in SEQ ID NO:34, and the HCDR3 sequence shown in SEQ ID NO:38; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:39, the LCDR2 sequence shown in SEQ ID NO:17, and the LCDR3 sequence shown in SEQ ID NO:40.

[0146] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:41, the HCDR2 sequence shown in SEQ ID NO:42, and the HCDR3 sequence shown in SEQ ID NO:43; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:44, the LCDR2 sequence shown in SEQ ID NO:45, and the LCDR3 sequence shown in SEQ ID NO:46.

[0147] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:47, the HCDR2 sequence shown in SEQ ID NO:48 and the HCDR3 sequence shown in SEQ ID NO:49; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:50, the LCDR2 sequence shown in SEQ ID NO:11 and the LCDR3 sequence shown in SEQ ID NO:51.

[0148] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:52, the HCDR2 sequence shown in SEQ ID NO:53, and the HCDR3 sequence shown in SEQ ID NO:54; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:50, the LCDR2 sequence shown in SEQ ID NO:55, and the LCDR3 sequence shown in SEQ ID NO:56.

[0149] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 57, the HCDR2 sequence shown in SEQ ID NO: 58, and the HCDR3 sequence shown in SEQ ID NO: 35; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO: 36, the LCDR2 sequence shown in SEQ ID NO: 59, and the LCDR3 sequence shown in SEQ ID NO: 37.

[0150] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:33, the HCDR2 sequence shown in SEQ ID NO:34, and the HCDR3 sequence shown in SEQ ID NO:60; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:61, the LCDR2 sequence shown in SEQ ID NO:23, and the LCDR3 sequence shown in SEQ ID NO:62.

[0151] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 1, the HCDR2 sequence shown in SEQ ID NO: 2, and the HCDR3 sequence shown in SEQ ID NO: 63; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO: 64, the LCDR2 sequence shown in SEQ ID NO: 5, and the LCDR3 sequence shown in SEQ ID NO: 65.

[0152] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 1, the HCDR2 sequence shown in SEQ ID NO: 2, and the HCDR3 sequence shown in SEQ ID NO: 66; the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO: 4, the LCDR2 sequence shown in SEQ ID NO: 5, and the LCDR3 sequence shown in SEQ ID NO: 6.

[0153] In one embodiment, the heavy chain variable region comprises 1) an amino acid sequence as set forth in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0154] In one embodiment, the light chain variable region comprises 1) an amino acid sequence as set forth in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0155] In one embodiment, the heavy chain variable region comprises 1) an amino acid sequence as set forth in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95, preferably, the additions, deletions and / or substitutions do not occur in the CDR regions; and

[0156] The light chain variable region comprises 1) an amino acid sequence as set forth in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0157] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68.

[0158] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 69; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70.

[0159] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:71; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:72.

[0160] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 73; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 74.

[0161] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:75; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:76.

[0162] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 77; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 78.

[0163] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 79; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 80.

[0164] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:81; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:82.

[0165] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 84.

[0166] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:85; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:86.

[0167] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 87; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 88.

[0168] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 89; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90.

[0169] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:91; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:92.

[0170] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:93; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:94.

[0171] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:95; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:96.

[0172] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising 1) an amino acid sequence as set forth in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, preferably, the addition, deletion and / or substitution does not occur in the CDR region.

[0173] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain comprising 1) an amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; 2) an amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; or 3) an amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0174] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising 1) an amino acid sequence as set forth in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, preferably, the additions, deletions and / or substitutions do not occur in the CDR regions; and

[0175] The antibody or antigen-binding fragment thereof comprises a light chain comprising 1) an amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; 2) an amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; or 3) an amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0176] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:97; and the light chain comprises the amino acid sequence shown in SEQ ID NO:98.

[0177] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:99; and the light chain comprises the amino acid sequence shown in SEQ ID NO:100.

[0178] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 101; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 102.

[0179] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 103; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 104.

[0180] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 105; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 106.

[0181] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 107; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 108.

[0182] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 109; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 110.

[0183] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:111; and the light chain comprises the amino acid sequence shown in SEQ ID NO:112.

[0184] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:113; and the light chain comprises the amino acid sequence shown in SEQ ID NO:114.

[0185] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:115; and the light chain comprises the amino acid sequence shown in SEQ ID NO:116.

[0186] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:117; and the light chain comprises the amino acid sequence shown in SEQ ID NO:118.

[0187] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 119; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 120.

[0188] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 121; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 122.

[0189] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 123; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 124.

[0190] In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 125; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 126.

[0191] In one embodiment, the anti-CD100 antibody includes anti-CD100 antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5 or B13-e-2, in particular anti-CD100 antibody B13, C-C081 or B13-c-5.

[0192] Antibody B13

[0193] In one aspect, the present invention provides an antibody B13 or an antigen-binding fragment thereof directed against CD100.

[0194] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0195] The heavy chain variable region comprises:

[0196] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1,

[0197] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and

[0198] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3;

[0199] The light chain variable region comprises:

[0200] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4,

[0201] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, and

[0202] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0203] In another embodiment, the anti-CD100 antibody B13 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0204] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 67.

[0205] In another embodiment, the anti-CD100 antibody B13 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0206] wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 68.

[0207] In yet another embodiment, the anti-CD100 antibody B13 or an antigen-binding fragment thereof comprises a heavy chain,

[0208] wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 97 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 97.

[0209] In yet another embodiment, the anti-CD100 antibody B13 or antigen-binding fragment thereof comprises a light chain,

[0210] wherein the light chain comprises the amino acid sequence of SEQ ID NO: 98 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 98.

[0211] Antibody C-C081

[0212] In another aspect, the present invention provides an antibody C-C081 or an antigen-binding fragment thereof directed against CD100.

[0213] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0214] The heavy chain variable region comprises:

[0215] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 7,

[0216] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and

[0217] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 9;

[0218] The light chain variable region comprises:

[0219] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10,

[0220] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 11, and

[0221] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0222] In one embodiment, the anti-CD100 antibody C-C081 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0223] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 69.

[0224] In one embodiment, the anti-CD100 antibody C-C081 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0225] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 70.

[0226] In yet another embodiment, the anti-CD100 antibody C-C081 or an antigen-binding fragment thereof comprises a heavy chain,

[0227] wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 99 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 99.

[0228] In yet another embodiment, the anti-CD100 antibody C-C081 or an antigen-binding fragment thereof comprises a light chain,

[0229] The light chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 100.

[0230] Antibody A14

[0231] In another aspect, the present invention provides an antibody A14 or an antigen-binding fragment thereof directed against CD100.

[0232] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0233] The heavy chain variable region comprises:

[0234] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13,

[0235] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, and

[0236] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 15;

[0237] The light chain variable region comprises:

[0238] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16,

[0239] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 17, and

[0240] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:18.

[0241] In one embodiment, the anti-CD100 antibody A14 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0242] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 71.

[0243] In one embodiment, the anti-CD100 antibody A14 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0244] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 72.

[0245] In yet another embodiment, the anti-CD100 antibody A14 or an antigen-binding fragment thereof comprises a heavy chain,

[0246] The heavy chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 101.

[0247] In yet another embodiment, the anti-CD100 antibody A14 or an antigen-binding fragment thereof comprises a light chain,

[0248] The light chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 102.

[0249] Antibody A15

[0250] In another aspect, the present invention provides an antibody A15 or an antigen-binding fragment thereof directed against CD100.

[0251] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0252] The heavy chain variable region comprises:

[0253] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19,

[0254] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 20, and

[0255] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 21;

[0256] The light chain variable region comprises:

[0257] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22,

[0258] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 23, and

[0259] LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24.

[0260] In one embodiment, the anti-CD100 antibody A15 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0261] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 73.

[0262] In one embodiment, the anti-CD100 antibody A15 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0263] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 74.

[0264] In yet another embodiment, the anti-CD100 antibody A15 or an antigen-binding fragment thereof comprises a heavy chain,

[0265] The heavy chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 103.

[0266] In yet another embodiment, the anti-CD100 antibody A15 or an antigen-binding fragment thereof comprises a light chain,

[0267] The light chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 104.

[0268] Antibody H74

[0269] In another aspect, the present invention provides an antibody H74 or an antigen-binding fragment thereof directed against CD100.

[0270] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0271] The heavy chain variable region comprises:

[0272] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25,

[0273] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and

[0274] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27;

[0275] The light chain variable region comprises:

[0276] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16,

[0277] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 28, and

[0278] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:29.

[0279] In one embodiment, the anti-CD100 antibody H74 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0280] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 75 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 75.

[0281] In one embodiment, the anti-CD100 antibody H74 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0282] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 76 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 76.

[0283] In yet another embodiment, the anti-CD100 antibody H74 or an antigen-binding fragment thereof comprises a heavy chain,

[0284] The heavy chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 105.

[0285] In yet another embodiment, the anti-CD100 antibody H74 or an antigen-binding fragment thereof comprises a light chain,

[0286] wherein the light chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 106.

[0287] Antibody H96

[0288] In another aspect, the present invention provides an antibody H96 or an antigen-binding fragment thereof directed against CD100.

[0289] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0290] The heavy chain variable region comprises:

[0291] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25,

[0292] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and

[0293] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30;

[0294] The light chain variable region comprises:

[0295] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31,

[0296] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, and

[0297] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32.

[0298] In one embodiment, the anti-CD100 antibody H96 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0299] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 77.

[0300] In one embodiment, the anti-CD100 antibody H96 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0301] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 78 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 78.

[0302] In yet another embodiment, the anti-CD100 antibody H96 or an antigen-binding fragment thereof comprises a heavy chain,

[0303] wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 107.

[0304] In yet another embodiment, the anti-CD100 antibody H96 or an antigen-binding fragment thereof comprises a light chain,

[0305] wherein the light chain comprises the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 108.

[0306] Antibody H5

[0307] In another aspect, the present invention provides an antibody H5 or an antigen-binding fragment thereof directed against CD100.

[0308] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0309] The heavy chain variable region comprises:

[0310] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 33,

[0311] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and

[0312] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35;

[0313] The light chain variable region comprises:

[0314] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 36,

[0315] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 23, and

[0316] LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 37.

[0317] In one embodiment, the anti-CD100 antibody H5 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0318] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 79 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 79.

[0319] In one embodiment, the anti-CD100 antibody H5 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0320] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 80 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 80.

[0321] In yet another embodiment, the anti-CD100 antibody H5 or antigen-binding fragment thereof comprises a heavy chain,

[0322] The heavy chain comprises the amino acid sequence of SEQ ID NO: 109 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 109.

[0323] In yet another embodiment, the anti-CD100 antibody H5 or antigen-binding fragment thereof comprises a light chain,

[0324] The light chain comprises the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 110.

[0325] Antibody H12

[0326] In another aspect, the present invention provides an antibody H12 or an antigen-binding fragment thereof directed against CD100.

[0327] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0328] The heavy chain variable region comprises:

[0329] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 33,

[0330] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and

[0331] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 38;

[0332] The light chain variable region comprises:

[0333] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 39,

[0334] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 17, and

[0335] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:40.

[0336] In one embodiment, the anti-CD100 antibody H12 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0337] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 81.

[0338] In one embodiment, the anti-CD100 antibody H12 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0339] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 82.

[0340] In yet another embodiment, the anti-CD100 antibody H12 or an antigen-binding fragment thereof comprises a heavy chain,

[0341] The heavy chain comprises the amino acid sequence of SEQ ID NO: 111 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 111.

[0342] In yet another embodiment, the anti-CD100 antibody H12 or antigen-binding fragment thereof comprises a light chain,

[0343] The light chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 112.

[0344] Antibody H21

[0345] In another aspect, the present invention provides an antibody H21 or an antigen-binding fragment thereof against CD100.

[0346] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0347] The heavy chain variable region comprises:

[0348] HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41,

[0349] HCDR2 comprising the amino acid sequence shown in SEQ ID NO:42, and

[0350] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 43;

[0351] The light chain variable region comprises:

[0352] LCDR1 comprises the amino acid sequence shown in SEQ ID NO:44,

[0353] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 45, and

[0354] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:46.

[0355] In one embodiment, the anti-CD100 antibody H21 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0356] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 83.

[0357] In one embodiment, the anti-CD100 antibody H21 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0358] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 84.

[0359] In yet another embodiment, the anti-CD100 antibody H21 or an antigen-binding fragment thereof comprises a heavy chain,

[0360] The heavy chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 113.

[0361] In yet another embodiment, the anti-CD100 antibody H21 or an antigen-binding fragment thereof comprises a light chain,

[0362] The light chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 114.

[0363] Antibody C-C171

[0364] In another aspect, the present invention provides an antibody C-C171 or an antigen-binding fragment thereof directed against CD100.

[0365] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0366] The heavy chain variable region comprises:

[0367] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 47,

[0368] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 48, and

[0369] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 49;

[0370] The light chain variable region comprises:

[0371] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 50,

[0372] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 11, and

[0373] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:51.

[0374] In one embodiment, the anti-CD100 antibody C-C171 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0375] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 85.

[0376] In one embodiment, the anti-CD100 antibody C-C171 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0377] The light chain variable region comprises the amino acid sequence of SEQ ID NO:86 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:86.

[0378] In yet another embodiment, the anti-CD100 antibody C-C171 or antigen-binding fragment thereof comprises a heavy chain,

[0379] The heavy chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 115.

[0380] In yet another embodiment, the anti-CD100 antibody C-C171 or antigen-binding fragment thereof comprises a light chain,

[0381] wherein the light chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 116.

[0382] Antibody C-B71

[0383] In another aspect, the present invention provides an antibody C-B71 or an antigen-binding fragment thereof directed against CD100.

[0384] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0385] The heavy chain variable region comprises:

[0386] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 52,

[0387] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 53, and

[0388] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 54;

[0389] The light chain variable region comprises:

[0390] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 50,

[0391] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 55, and

[0392] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:56.

[0393] In one embodiment, the anti-CD100 antibody C-B71 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0394] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 87.

[0395] In one embodiment, the anti-CD100 antibody C-B71 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0396] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 88 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 88.

[0397] In yet another embodiment, the anti-CD100 antibody C-B71 or an antigen-binding fragment thereof comprises a heavy chain,

[0398] wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 117.

[0399] In yet another embodiment, the anti-CD100 antibody C-B71 or an antigen-binding fragment thereof comprises a light chain,

[0400] wherein the light chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 118.

[0401] Antibody H5-h-7

[0402] In another aspect, the present invention provides an antibody H5-h-7 or an antigen-binding fragment thereof directed against CD100.

[0403] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0404] The heavy chain variable region comprises:

[0405] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 57,

[0406] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 58, and

[0407] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35;

[0408] The light chain variable region comprises:

[0409] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 36,

[0410] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 59, and

[0411] LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 37.

[0412] In one embodiment, the anti-CD100 antibody H5-h-7 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0413] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 89 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 89.

[0414] In one embodiment, the anti-CD100 antibody H5-h-7 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0415] The light chain variable region comprises the amino acid sequence of SEQ ID NO:90 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:90.

[0416] In yet another embodiment, the anti-CD100 antibody H5-h-7 or antigen-binding fragment thereof comprises a heavy chain,

[0417] The heavy chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 119.

[0418] In yet another embodiment, the anti-CD100 antibody H5-h-7 or antigen-binding fragment thereof comprises a light chain,

[0419] wherein the light chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 120.

[0420] Antibody H5-a-2

[0421] In another aspect, the present invention provides an antibody H5-a-2 or an antigen-binding fragment thereof directed against CD100.

[0422] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0423] The heavy chain variable region comprises:

[0424] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 33,

[0425] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and

[0426] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 60;

[0427] The light chain variable region comprises:

[0428] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 61,

[0429] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 23, and

[0430] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:62.

[0431] In one embodiment, the anti-CD100 antibody H5-a-2 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0432] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 91 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 91.

[0433] In one embodiment, the anti-CD100 antibody H5-a-2 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0434] The light chain variable region comprises the amino acid sequence of SEQ ID NO:92 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:92.

[0435] In yet another embodiment, the anti-CD100 antibody H5-a-2 or antigen-binding fragment thereof comprises a heavy chain,

[0436] The heavy chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 121.

[0437] In yet another embodiment, the anti-CD100 antibody H5-a-2 or antigen-binding fragment thereof comprises a light chain,

[0438] wherein the light chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 122.

[0439] Antibody B13-c-5

[0440] In another aspect, the present invention provides an antibody B13-c-5 or an antigen-binding fragment thereof directed against CD100.

[0441] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0442] The heavy chain variable region comprises:

[0443] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1,

[0444] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and

[0445] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 63;

[0446] The light chain variable region comprises:

[0447] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 64,

[0448] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, and

[0449] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:65.

[0450] In one embodiment, the anti-CD100 antibody B13-c-5 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0451] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 93 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 93.

[0452] In one embodiment, the anti-CD100 antibody B13-c-5 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0453] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 94.

[0454] In yet another embodiment, the anti-CD100 antibody B13-c-5 or an antigen-binding fragment thereof comprises a heavy chain,

[0455] The heavy chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 123.

[0456] In yet another embodiment, the anti-CD100 antibody B13-c-5 or an antigen-binding fragment thereof comprises a light chain,

[0457] wherein the light chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 124.

[0458] Antibody B13-e-2

[0459] In another aspect, the present invention provides an antibody B13-e-2 or an antigen-binding fragment thereof directed against CD100.

[0460] The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein

[0461] The heavy chain variable region comprises:

[0462] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1,

[0463] HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and

[0464] HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 66;

[0465] The light chain variable region comprises:

[0466] LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4,

[0467] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, and

[0468] LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0469] In one embodiment, the anti-CD100 antibody B13-e-2 or an antigen-binding fragment thereof comprises a heavy chain variable region (VH),

[0470] wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 95 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 95.

[0471] In one embodiment, the anti-CD100 antibody B13-e-2 or an antigen-binding fragment thereof comprises a light chain variable region (VL),

[0472] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 96 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 96.

[0473] In yet another embodiment, the anti-CD100 antibody B13-e-2 or antigen-binding fragment thereof comprises a heavy chain,

[0474] wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 125 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 125.

[0475] In yet another embodiment, the anti-CD100 antibody B13-e-2 or antigen-binding fragment thereof comprises a light chain,

[0476] wherein the light chain comprises the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 126.

[0477] Nucleic acid molecules, vectors and host cells

[0478] In another aspect, the present invention provides a nucleic acid molecule comprising a polynucleotide sequence encoding an anti-CD100 antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the nucleic acid molecule of the present invention encodes an anti-CD100 antibody or antigen-binding fragment thereof of the present invention.

[0479] Can utilize method known in the art to obtain nucleic acid molecules of the present invention.For example, nucleic acid molecules of the present invention can be separated from phage display library, yeast display library, immune animal, immortalized cell (for example, mouse B cell hybridoma cell, EBV-mediated immortalized B cell) or chemical synthesis.Nucleic acid molecules of the present invention can be carried out codon optimization for the host cell for expression.

[0480] In yet another aspect, the present invention also provides an expression vector comprising the nucleic acid molecule of the present invention. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The nucleic acid molecule of the present invention may be present in one or more expression vectors. In one embodiment, the nucleic acid molecule of the present invention is prepared as a recombinant nucleic acid. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination technology (e.g., polymerase chain reaction (PCR) technology), etc.

[0481] The present invention also provides a host cell comprising a nucleic acid molecule or expression vector of the present invention. Nucleic acid molecules or expression vectors of the present invention can be introduced into a suitable host cell using various methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.

[0482] In a preferred embodiment, host cells are used to express the anti-CD100 antibodies or antigen-binding fragments thereof of the present invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for antibody expression include, but are not limited to, human cervical carcinoma cells (HeLa cells), human embryonic kidney cells (HEK cells, such as HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

[0483] The present invention also provides a method for producing the anti-CD100 antibody or antigen-binding fragment thereof of the present invention, comprising the following steps:

[0484] a) culturing the host cell of the present invention under suitable conditions to express the anti-CD100 antibody or antigen-binding fragment thereof of the present invention; and

[0485] b) isolating the antibody or antigen-binding fragment thereof from the host cell or culture thereof.

[0486] Drug combinations

[0487] In another aspect, the present invention provides a pharmaceutical combination for administering two or more therapeutic or prophylactic agents to a subject, wherein the pharmaceutical combination comprises an antibody or antigen-binding fragment thereof against CD100 and an antibody or antigen-binding fragment thereof against PD-L1.

[0488] In one embodiment, the antibody against CD100 comprises antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2, or a combination thereof.

[0489] In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof specifically recognizes and binds to PD-L1, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an immunoglobulin single variable domain.

[0490] In a preferred embodiment, the immunoglobulin single variable domain comprises:

[0491] CDR1 comprises the amino acid sequence shown in SEQ ID NO: 130,

[0492] CDR2 comprises the amino acid sequence shown in SEQ ID NO: 131, and

[0493] CDR3 comprises the amino acid sequence shown in SEQ ID NO:132.

[0494] In a preferred embodiment, the immunoglobulin single variable domain comprises: 1) the amino acid sequence shown in SEQ ID NO: 133; or 2) an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 133.

[0495] In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc fragment of human IgG1.

[0496] In a preferred embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 134.

[0497] In one embodiment, the anti-PD-L1 antibody comprises anti-PD-L1 antibody m18-VHH or m18.

[0498] Antibody m18-VHH

[0499] In one aspect, the present invention provides an antibody m18-VHH or an antigen-binding fragment thereof against PD-L1,

[0500] wherein the antibody comprises a single variable domain of an immunoglobulin, wherein

[0501] The single variable domain comprises:

[0502] CDR1 comprises the amino acid sequence shown in SEQ ID NO: 130,

[0503] CDR2 comprises the amino acid sequence shown in SEQ ID NO: 131, and

[0504] CDR3 comprises the amino acid sequence shown in SEQ ID NO:132.

[0505] In one embodiment, the anti-PD-L1 antibody m18-VHH comprises a single variable domain of an immunoglobulin,

[0506] wherein the single variable domain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 133.

[0507] Antibody m18

[0508] In another aspect, the present invention further provides an anti-PD-L1 antibody m18 or an antigen-binding fragment thereof,

[0509] wherein the antibody comprises a single variable domain of an immunoglobulin, wherein

[0510] The single variable domain comprises:

[0511] CDR1 comprises the amino acid sequence shown in SEQ ID NO: 130,

[0512] CDR2 comprises the amino acid sequence shown in SEQ ID NO: 131, and

[0513] CDR3 comprises the amino acid sequence shown in SEQ ID NO:132.

[0514] In one embodiment, the anti-PD-L1 antibody m18 comprises a single variable domain of an immunoglobulin,

[0515] wherein the single variable domain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 133.

[0516] In one embodiment, the anti-PD-L1 antibody m18 further comprises an Fc fragment of human IgG1.

[0517] The antibody m18 comprises the amino acid sequence shown in SEQ ID NO: 134 or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO: 134.

[0518] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention is a single domain antibody, a heavy chain antibody, a humanized antibody, or a chimeric antibody. Preferably, the antibody or antigen-binding fragment thereof is a human antibody.

[0519] In one embodiment, the antibody or antigen-binding fragment thereof has at least one of the following characteristics:

[0520] 1) Has affinity activity for PD-L1 positive cells;

[0521] 2) Ability to specifically bind to PD-L1 protein;

[0522] 3) Inhibit tumor growth.

[0523] In some embodiments, the tumors targeted include but are not limited to those described below with respect to neoplastic diseases. In other embodiments, the antibodies or antigen-binding fragments thereof of the present invention can inhibit tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%.

[0524] In one embodiment, the drug combination comprises an antibody against CD100 comprising antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2, or a combination thereof; and the drug combination comprises an antibody against PD-L1 comprising antibody m18-VHH or m18.

[0525] In a preferred embodiment, the drug combination comprises anti-CD100 antibodies B13, C-C081, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2, or a combination thereof and anti-PD-L1 antibodies m18-VHH or m18.

[0526] In a preferred embodiment, the pharmaceutical combination comprises the anti-CD100 antibody C-C081, B13, or B13-c-5 and the anti-PD-L1 antibody m18-VHH or m18.

[0527] In one embodiment, the pharmaceutical combination can be a pharmaceutical composition or a kit.

[0528] Pharmaceutical composition

[0529] The present invention also provides a pharmaceutical composition comprising an anti-CD100 antibody or antigenic fragment thereof of the present invention and a pharmaceutically acceptable carrier. In one embodiment, the anti-CD100 antibody comprises anti-CD100 antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2, or a combination thereof, particularly anti-CD100 antibody B13, C-C081, C-B71, H5-h-7, H5-a-2, B13-c-5, or B13-e-2. In one embodiment, the pharmaceutical composition comprises a combination of anti-CD100 antibodies B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2 and anti-PD-L1 antibodies m18 or m18-VHH. In a preferred embodiment, the pharmaceutical composition comprises a combination of anti-CD100 antibodies B13, C-C081, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2 and anti-PD-L1 antibodies m18 or m18-VHH. In a preferred embodiment, the pharmaceutical composition comprises a combination of anti-CD100 antibodies B13, C-C081, or B13-c-5 and anti-PD-L1 antibodies m18 or m18-VHH.

[0530] The pharmaceutical compositions provided herein may be in a variety of dosage forms, including but not limited to solid, semisolid, liquid, powder or lyophilized forms. For compositions comprising antibodies or antigenic fragments thereof, preferred dosage forms may generally be, for example, injection solutions and lyophilized powders.

[0531] The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or topical administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavitary), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical). Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Methods of administration can be, for example, injection or infusion.

[0532] Those skilled in the art will appreciate that the exact dosage will depend on various factors, such as the metabolic properties of the pharmaceutical composition, the duration of treatment, the excretion rate of the specific compound, the therapeutic goal, the route of administration, and the subject's condition, such as the patient's age, health, weight, sex, diet, medical history, and other factors well known in the medical field. As a general guide, the dosage range of the anti-CD100 antibodies or antigen-binding fragments thereof of the present invention is about 0.0001-100 mg / kg, more typically 0.01-20 mg / kg of the subject's body weight. For example, the dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, or 20 mg / kg body weight, or in the range of 1-20 mg / kg. Exemplary treatment regimens entail administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, once every three to six months, or with a slightly shorter initial dosing interval followed by a longer dosing interval. In one embodiment, the dosage used can be 1200 mg administered every three weeks. The administration method can be intravenous infusion.

[0533] As used herein, a "therapeutically effective dose" refers to a dose that results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of the disease, or prevents damage or disability caused by the suffering of the disease. For example, a therapeutically effective dose is used for anti-proliferative effects, preventing further tumor development, reducing tumor size, reducing tumor vascularity, reducing the number of cancer cells, inhibiting, delaying or reducing tumor and / or malignant cell growth and / or metastasis in cancer patients, and / or reducing one or more symptoms associated with the disease that can be observed. The therapeutically effective dose can vary depending on many different factors, including the mode of administration, the target site, the patient's physiological state, whether the patient is human or other animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. In certain embodiments, the patient is human, but non-human mammals, including transgenic animals, can also be treated. The therapeutic dose can be titrated to optimize safety and efficacy using conventional methods known to those skilled in the art.

[0534] A "therapeutically effective dose" of an antibody or antigen-binding fragment thereof of the present invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which inhibition can be determined in vitro by assays well known to those skilled in the art. An effective amount of an antibody or antigen-binding fragment thereof of the present invention can reduce tumor size or otherwise alleviate symptoms of a subject, such as preventing and / or treating metastasis or recurrence. Such an amount can be determined by those skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0535] treat

[0536] In yet another aspect, the present invention relates to use of the anti-CD100 antibody or antigen-binding fragment thereof, pharmaceutical composition or pharmaceutical combination of the present invention in the preparation of a medicament for treating a disease in a subject.

[0537] The present invention also relates to the anti-CD100 antibody or antigen-binding fragment thereof, pharmaceutical composition or pharmaceutical combination of the present invention, which is used for treating diseases.

[0538] The present invention also provides a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the anti-CD100 antibody or antigen-binding fragment thereof, pharmaceutical composition or pharmaceutical combination of the present invention.

[0539] In one embodiment, the disease as described above is cancer. Blockade of CD100 by the antibodies of the present invention can enhance the immune response to cancer cells in patients. CD100 is widely expressed in many human tumors, and its expression is associated with aggressive human diseases. In the preclinical tumor microenvironment, inflammatory cells and tumor cells expressing CD100 regulate the infiltration, spatial distribution and activity of myeloid cells and lymphocytes. CD100 binds to Plexin receptors located on myeloid cells in the tumor microenvironment. When the CD100 protein is blocked, the CD100 barrier can be eliminated. Once the barrier is breached, inflammatory dendritic cells and proinflammatory antigen-presenting cells migrate and infiltrate the tumor. In preclinical cancer animal models, blocking CD100 with antibodies can delay tumor growth and promote lasting tumor rejection.

[0540] As used herein, "cancer" includes but is not limited to hematologic malignancies and solid tumors. In this article, solid tumors include, for example, squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, renal cell carcinoma, mammary duct carcinoma, soft tissue sarcoma, osteosarcoma, melanoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, esophageal cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, etc., or its any combination. Hematologic malignancies include, for example, leukemia, lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, etc., or its any combination. Cancer can also be metastatic. "Metastasis" refers to the spread of cancer cells from their original site to other parts of the body.

[0541] Combination therapy

[0542] For cancer treatment, the anti-CD100 antibodies or antigen-binding fragments thereof, pharmaceutical compositions or drug combinations of the present invention can be used in combination with other treatment methods, including but not limited to surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition and palliative care.

[0543] The anti-CD100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or drug combinations of the present invention can also be administered in combination with at least one or more therapeutic agents described herein. The manner of combined administration is not limited. For example, the following therapeutic agents can all be administered at once or separately. When administered separately (using different administration regimens), they can be administered continuously without interruption or at predetermined intervals.

[0544] In certain embodiments, the anti-CD100 antibodies or antigen-binding fragments thereof, pharmaceutical compositions or drug combinations of the present invention are further administered in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutics, radioisotopes, immune checkpoint inhibitors and tumor antigen targeted drugs. Chemotherapeutics can include, for example, antimetabolites, alkylating agents, cytotoxic agents, topoisomerase inhibitors, microtubule inhibitors. Tumor antigen targeted drugs include, but are not limited to, drugs targeting tumor-associated antigens and tumor-specific antigens. Other non-limiting examples of therapeutic agents can include, for example, angiogenesis inhibitors, deacetylase (HDAC) inhibitors, Hedgehog signaling pathway blockers, mTOR inhibitors, p53 / mdm2 inhibitors, PARP inhibitors, proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib, Marizomib, Oprozomib) and tyrosine kinase inhibitors (e.g., BTK inhibitors).

[0545] In one embodiment, anti-CD100 antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2 is administered in combination with anti-PD-L1 antibody m18 or m18-VHH.

[0546] In a preferred embodiment, the anti-CD100 antibody B13, C-C081, C-B71, H5-h-7, H5-a-2, B13-c-5, or B13-e-2 is administered in combination with the anti-PD-L1 antibody m18 or m18-VHH.

[0547] In a preferred embodiment, the anti-CD100 antibody C-C081, B13, or B13-c-5 is administered in combination with the anti-PD-L1 antibody m18 or m18-VHH.

[0548] In some embodiments, the anti-CD100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or drug combinations of the present invention are administered in combination with a chemotherapeutic agent. In some embodiments, the anti-CD100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or drug combinations of the present invention are administered in combination with an immune checkpoint inhibitor. In some embodiments, the anti-CD100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or drug combinations of the present invention are administered in combination with a radioisotope. In some embodiments, the anti-CD100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or drug combinations of the present invention are administered in combination with a tumor-targeting drug.

[0549] Reagent test kit

[0550] The present invention also provides a kit comprising an anti-CD100 antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a pharmaceutical combination of the present invention, and instructions for use. The kit may also comprise a suitable container. In certain embodiments, the kit further comprises a device for administration. The kit generally includes a label indicating the intended use and / or instructions for use of the kit contents. The term "label" includes any written or recorded material provided on or with the kit, or otherwise provided with the kit.

[0551] In one embodiment, the kit comprises anti-CD100 antibodies B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2, or a combination thereof.

[0552] In one embodiment, the kit comprises anti-CD100 antibodies B13, C-C081, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2, or a combination thereof.

[0553] In one embodiment, the kit comprises anti-CD100 antibody C-C081, B13, or B13-c-5.

[0554] In one embodiment, the kit comprises an anti-CD100 antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2 in combination with an anti-PD-L1 antibody m18 or m18-VHH.

[0555] In one embodiment, the kit comprises a combination of anti-CD100 antibodies B13, C-C081, C-B71, H5-h-7, H5-a-2, B13-c-5, B13-e-2 and anti-PD-L1 antibody m18 or m18-VHH.

[0556] In one embodiment, the kit comprises a combination of anti-CD100 antibody C-C081, B13, or B13-c-5 and anti-PD-L1 antibody m18 or m18-VHH. Beneficial effects

[0557] The anti-CD100 antibody or antigen-binding fragment thereof of the present invention has at least one of the following beneficial effects: 1) having affinity activity for human, mouse or monkey CD100 protein; 2) having affinity activity for CD100-positive cells; 3) blocking the binding of CD100 to Plexin-B1 or Plexin-B2; 4) inhibiting MDSC cell proliferation; 5) inhibiting tumor growth.

[0558] The anti-CD100 antibody or antigen-binding fragment thereof of the present invention, when used in combination with an anti-PD-L1 antibody or antigen-binding fragment thereof, has at least one of the following beneficial effects: 1) inhibiting or delaying tumor growth; 2) significantly improving the response rate to single-drug therapy; 3) prolonging survival; 4) enhancing the therapeutic effect of PD-L1 tumor immunotherapy; and 5) having high safety.

[0559] Example

[0560] A further understanding of the present invention may be obtained by reference to some of the specific examples given herein, which are intended to illustrate the present invention only and are not intended to limit the scope of the present invention in any way. Obviously, various modifications and variations may be made to the present invention without departing from the spirit of the present invention and, therefore, such modifications and variations are also within the scope of the present invention. Ratios used herein include percentages and, unless otherwise specified, are by weight.

[0561] Example 1 Preparation and identification of raw materials

[0562] 1.1 Preparation and identification of anti-CD100 control antibodies

[0563] Preparation of anti-CD100 control antibody: The anti-CD100 antibody Pepinemab and Shanghai Pioneer Pharmaceutical's 2D5 and 5D8 antibodies were used as positive control antibodies. According to the sequences disclosed in WO2013148854A1 and patent WO2020011275, the coding gene sequences of Pepinemab monoclonal antibody and 2D5 and 5D8 antibodies were handed over to General Biotechnology Co., Ltd. for gene synthesis. Then, it was constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination. The constructed recombinant protein expression vectors were transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then the plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain the desired expression plasmids for Pepinemab monoclonal antibody and 2D5 and 5D8 antibodies. Through ExpiFectamine TMCHO transfection kit (Thermo Fisher, A29129) was used to transfect CHO cells with expression plasmids to express pepinemab and 2D5 and 5D8 monoclonal antibodies according to the manufacturer's instructions. Seven days after transfection, the cell culture supernatant was collected and centrifuged at 15,000 g for 10 min. The resulting supernatant was filtered through a 0.22 μm filter membrane, and the antibodies in the supernatant were affinity purified using a Protein A / G affinity chromatography column (MabSelect SuRe (Cytiva, 17543802)). The target antibody was eluted with 100 mM glycine (pH 3.0), and the eluted antibody was exchanged into PBS buffer using an ultrafiltration concentrator (Millipore, UFC901096).

[0564] Identification of anti-CD100 control antibody: The activity of the prepared positive control antibody Pepinemab (heavy chain constant region type is IgG4SP) was detected using purchased Human Semaphorin 4D / SEMA4D / CD100 Protein, Fc Tag antigen protein (Acro, CD0-H5257).

[0565] The specific method is as follows: 96-well ELISA plates were coated with purchased human CD100 protein (also known as HuCD100-Fc, 2 μg / mL, 30 μL / well) at 4°C overnight; after washing three times, the plates were blocked with 5% skim milk in PBS for 1 hour at room temperature; after washing three times, the control antibody Pepinemab was added in a gradient dilution with PBS and incubated at room temperature for 1 hour; after washing, the secondary antibodies Anti-human-IgG-Kappa-HRP (Millipore, AP502P) and Anti-human-IgG-Lambda-HRP (Millipore, AP506P) diluted in PBS (1:6000) were added and incubated at room temperature for 1 hour. The plates were then washed six times and TMB (SurModics, TMBS-1000-01) was added for color development for 5-20 minutes. After stopping the color development, the data were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190). Data were processed and graphed using Graphpad Prism.

[0566] The results are shown in Figure 1 , and the expressed control antibodies Pepinemab, 2D5, and 5D8 can all bind to CD100 protein and have normal anti-CD100 activity.

[0567] 1.2 Preparation and identification of CD100 antigen protein

[0568] Antigen protein preparation: Through genetic manipulation at the coding gene level, human Fc (SEQ ID NO: 135) or His tag was added to the C-terminus of the amino acid sequence of the 22-734th fragment of the human CD100 protein ECD region (HuCD100, Uniprot ID: Q92854), the 24-733th fragment of the mouse CD100 protein ECD region (MusCD100, Uniprot ID: O09126), and the 22-734th fragment of the cynomolgus monkey CD100 protein ECD region (CynoCD100, Uniprot ID: A0A2K5TZC9). The obtained nucleic acid sequence was constructed into the pcDNA3.4 vector and then transformed into Escherichia coli DH5α and cultured overnight at 37°C. The plasmid was then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The resulting plasmid was lysed using ExpiFectamine TM 293 transfection kit (Gibco TM , A14524) were transiently transfected into HEK293 cells ( CRL-1573 TM ). After 7 days of expression, the cell culture supernatant was collected. For proteins containing Fc tags, the cell suspension was centrifuged at high speed after the culture was completed and the supernatant was collected. The obtained supernatant was filtered through a 0.22μm filter membrane and purified by affinity chromatography using a Protein A / G column MabSelect SuRe (Cytiva, 17543802). The target protein was eluted with 100mM glycine hydrochloride (pH 3.0), concentrated, buffer exchanged, aliquoted, SDS-PAGE identified and activity detected, and then stored and frozen. Proteins containing His tags were affinity purified using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with an imidazole gradient. The eluted proteins were respectively exchanged into PBS buffer through ultrafiltration concentration tubes (Millipore, UFC901096), and finally human, mouse and monkey CD100 antigen proteins with Fc tags and His tags (HuCD100-Fc, MusCD100-Fc, CynoCD100-Fc, HuCD100-His, MusCD100-His, CynoCD100-His) were obtained.

[0569] Antigen identification: The prepared antigens (HuCD100-Fc, MusCD100-Fc, CynoCD100-Fc, HuCD100-His, MusCD100-His, CynoCD100-His) were detected using the qualified antibody Pepinemab (IgG4SP) obtained in Example 1.1.

[0570] The specific method is as follows: ELISA plates were coated with 2 μg / mL of antigen overnight at 4°C. A commercially available antigen protein, Human Semaphorin 4D / SEMA4D / CD100 Protein, and Fc Tag antigen protein (Acro, CD0-H5257) were used as positive controls. After washing three times, the plates were blocked with 5% skim milk in PBS for 1 hour at room temperature. After washing three times, the plate was incubated with pepinemab antibody serially diluted in PBS for 1 hour at room temperature. After washing, the plate was incubated with secondary antibodies, Anti-human IgG-Kappa-HRP (Millipore, AP502P) or Anti-human IgG-Lambda-HRP (Millipore, AP506P), diluted 1:6000 in PBS, for 1 hour at room temperature. The plate was washed six times, and then TMB was added for color development for 5-20 minutes. The color reaction was terminated, and the data were read at OD450 using a microplate reader. Data were processed and plotted using GraphPad Prism.

[0571] The results are shown in FIG2 . The affinity activity of the antibody Pepinemab for binding to the homemade antigen was comparable to that for binding to the purchased CD100 antigen protein, wherein the two HuCD100-Fcs were, in order, the homemade HuCD100 antigen and the commercially purchased HuCD100 antigen.

[0572] Example 2 Construction and identification of overexpression cell lines

[0573] 2.1 Construction and identification of CD100-overexpressing cell lines

[0574] Construction of HEK293 cell lines overexpressing CD100 (hereinafter referred to as HuCD100-HEK293, MusCD100-HEK293, CynoCD100-HEK293): The coding nucleic acid sequences of full-length human CD100 protein HuCD100 (Uniprot ID: Q92854), full-length mouse CD100 protein MusCD100 (Uniprot ID: O09126), and full-length cynomolgus monkey CD100 protein CynoCD100 (Uniprot ID: A0A2K5TZC9) were constructed into pLVX-puro plasmid (Clontech, catalog number 632164). Then, the obtained plasmids were electroporated into HEK293 cells ( CRL-1573 TMAfter electroporation, the cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) and without antibiotics. The cells were then transferred to 10×10 cm cell culture dishes and cultured for 48 hours. 4 The cells were distributed into 96-well cell culture plates at a density of 10 cells / well, and puromycin with a final concentration of 2 μg / mL was added as a screening pressure. After about 2 weeks, the cell lines that formed clones were picked for identification.

[0575] Flow cytometric identification of HuCD100-HEK293, MusCD100-HEK293, and CynoCD100-HEK293 cells: Cells of the above cell lines in the logarithmic growth phase were trypsinized and plated into 96-well plates. After washing with FACS buffer (1× PBS buffer containing 2% FBS by volume), the primary antibody (Pepinemab) diluted in PBS was added and incubated at 4°C for 30 minutes. After washing, the prepared fluorescent secondary antibody anti-human IgG Fc (abcam, 98596) was added and incubated at 4°C for 30 minutes. Finally, the cells were detected by flow cytometry (Beckman, CytoFLEXAOO-1-1102).

[0576] The detection results are shown in FIG3 , which show that HuCD100-HEK293, MusCD100-HEK293, and CynoCD100-HEK293 cell lines highly express human, mouse, and monkey CD100 on their surfaces, respectively.

[0577] 2.2 Construction and identification of cell lines overexpressing HuPlexin-B1, MusPlexin-B1, CynoPlexin-B1, and HuPlexin-B2

[0578] Construction of HEK293 cell lines overexpressing Plexin-B1 and Plexin-B2 (hereinafter referred to as HuPlexin-B1-HEK293, MusPlexin-B1-HEK293, CynoPlexin-B1-HEK293, and HuPlexin-B2-HEK293): full-length human Plexin-B1 protein HuPlexin-B1 (Uniprot ID: O43157), full-length mouse Plexin-B1 protein MusPlexin-B1 (Uniprot ID: Q8CJH3), full-length cynomolgus monkey Plexin-B1 protein CynoPlexin-B1 (Uniprot ID: A0A1D5QMB8), full-length human Plexin-B2 protein HuPlexin-B2 (Uniprot ID: The coding nucleic acid sequence of the gene encoding SEQ ID NO: 015031 was constructed into the pLVX-puro plasmid (Clontech, Cat. No. 632164). The construction method was similar to that in Example 2.1.

[0579] Flow cytometry identification of HuPlexin-B1-HEK293, MusPlexin-B1-HEK293, CynoPlexin-B1-HEK293, and HuPlexin-B2-HEK293 cells: The cells of the above-mentioned cell lines in the logarithmic growth phase were digested and plated into 96-well plates. After washing with FACS buffer (1×PBS buffer containing 2% by volume of FBS), antigen proteins (biotin-labeled in Example 1. 2. Prepare HuCD100-Fc, MusCD100-Fc, and CynoCD100-Fc to obtain HuCD100-Fc-Biotin, MusCD100-Fc-Biotin, and CynoCD100-Fc-Biotin (for biotin labeling methods, see the instructions for the Roche Biotin Protein Labeling Kit, Catalog Number: 11418165001), and incubate at 4°C for 30 min. After washing, add the prepared eBioscience Streptavidin PE (Invitrogen, 2265658, 1:300) and incubate at 4°C for 30 min. Finally, detect by flow cytometry (Beckman, CytoFLEXAOO-1-1102).

[0580] The detection results are shown in Figures 4A-4D, where Figure 4A is a schematic diagram of the results of identifying the HuPlexin-B1-HEK293 cell line using human CD100 protein, Figure 4B is a schematic diagram of the results of identifying the MusPlexin-B1-HEK293 cell line using mouse CD100 protein, Figure 4C is a schematic diagram of the results of identifying the CynoPlexin-B1-HEK293 cell line using monkey CD100 protein, and Figure 4D is a schematic diagram of the results of identifying the HuPlexin-B2-HEK293 cell line using human CD100 protein. The results show that the HuPlexin-B1-HEK293, MusPlexin-B1-HEK293, CynoPlexin-B1-HEK293, and HuPlexin-B2-HEK293 cell lines highly express human, mouse, and monkey Plexin-B1, as well as human Plexin-B2, on their surfaces, respectively.

[0581] Example 3 Construction and screening of human phage-displayed recombinant antibody library

[0582] In this example, an antibody gene phage display library was constructed, and the library was screened using the antigen proteins HuCD100-Fc, MusCD100-Fc, CynoCD100-Fc, HuCD100-His, MusCD100-His, and CynoCD100-His prepared in Example 1.2 as screening antigens to obtain multiple antibody molecules that specifically bind to human CD100.

[0583] 3.1 Construction of a human antibody gene library

[0584] Peripheral blood mononuclear cells (PBMC) of normal human blood were separated using Ficoll-Paque density gradient separation solution (purchased from GE, catalog number: 17144003S), and total RNA was extracted from the isolated PBMC cells using a conventional method. The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa, catalog number: 6210A) according to the manufacturer's instructions. Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers were designed at the front end of the V region of the heavy chain and light chain and the rear end of the first constant region, and the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody were obtained after PCR. The fragments containing the light chain variable region and heavy chain variable region of the antibody were amplified by fusion PCR. The PCR product and the phage display vector were digested, recovered, and ligated. The ligated product was recovered using a recovery kit (Omega, catalog number: D6492-02) (Li Xiaolin, Construction and Preliminary Screening of a Large-Capacity Non-Immune Human Fab Phage Antibody Library, Master's Thesis, Peking Union Medical College, June 2007). Finally, the cells were transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) using an electroporator (Bio-Rad, MicroPulser), and the transformed E. coli SS320 bacterial solution was spread on ampicillin-resistant 2-YT solid plates (solid plates were prepared with 1.5% tryptone, 1% yeast extract, 0.5% NaCl, and 1.5% agar, at a mass volume of g / mL). By gradient dilution plating, the library capacity was measured to be 3×10 11 cfu, i.e. 3×10 11 An antibody gene library containing 100 antibody genes (for library capacity calculation, see Example 2.2 in CN112250763A ). VSCM13 helper phage (purchased from Stratagene) was used to package the library to obtain an antibody gene phage display library (for preparation of the antibody gene phage display library, see Example 2.3 in CN112250763A ).

[0585] 3.2 Screening of Antibody Gene Phage Display Library

[0586] 3.2.1 Screening of Antibody Gene Phage Display Library by Magnetic Bead Method

[0587] Magnetic bead screening is to biotin-label the antigen protein and then bind it to magnetic beads coupled with streptavidin. The antigen-bound magnetic beads and the antibody gene phage display library are incubated, washed, and eluted in an elutriation process. Usually 3-4 rounds of elutriation are performed, whereby specific monoclonal antibodies against the antigen can be enriched in large quantities. In this embodiment, biotin-labeled antigen proteins HuCD100-Fc, CynoCD100-Fc, HuCD100-His, and MusCD100-His are used for phage display library screening. After 3 rounds of elutriation, a primary screening of monoclonal antibody Fab against human CD100 is performed. The specific method is described in Example 2.4.1 in CN112250763A.

[0588] 3.2.2 Screening of Antibody Gene Phage Display Library by Immunotube Method

[0589] Immunotube screening involves coating the antigen proteins HuCD100-Fc, CynoCD100-Fc, HuCD100-His, or MusCD100-His on the highly adsorbable surface of an immunotube. The phage-displayed antibody library is then added to the immunotube and incubated with the adsorbed antigen proteins, followed by washing and elution. After two to four rounds of panning, antigen-specific monoclonal antibody Fabs are ultimately enriched. In this example, monoclonal antibody Fabs targeting human CD100 were enriched after three rounds of panning. For specific methods, refer to Example 2.4.2 in CN112250763A.

[0590] 3.3 Selection of monoclonal clones

[0591] The phage pool eluted in each round was tested by ELISA to evaluate the enrichment effect, and 10 clones were randomly selected from the phage pool in each round of screening for sequence analysis. The enrichment effect and the repeatability ratio of the measured sequences were comprehensively analyzed to select the appropriate round for single clone selection.

[0592] The ELISA monoclonal primary screening used the antigen proteins HuCD100-His, MusCD100-His, and CynoCD100-His. The antibody Fabs binding to HuCD100-His, MusCD100-His, and CynoCD100-His obtained in the primary screening were prepared into Fab lysates, and then detected and verified by flow cytometry (FACS) using the overexpression cells HuCD100-HEK293, MusCD100-HEK293, and CynoCD100-HEK293 prepared in Example 2.1. A total of 11 antibody Fab molecules that specifically bind to human CD100 were screened, and the 11 obtained antibody Fabs were named according to the corresponding clone numbers (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, and C-B71). The amino acid sequences of the obtained antibody variable regions are shown in Table 1 , and the CDR sequences were determined using the AbM CDR definition method.

[0593] Table 1. Amino acid sequences of the variable regions of 11 anti-CD100 antibodies (SEQ ID NO:)

[0594] Example 4 Antibody Construction, Expression and Purification

[0595] 4.1 Plasmid construction

[0596] The VH coding sequence from the Fab sequences of the screened monoclonal antibodies A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, and C-B71 was ligated with the coding sequence of the heavy chain constant region of human IgG4SP (SEQ ID NO: 127) to obtain the fully human heavy chain coding sequence of the antibody. The VL coding sequence from the Fab sequence was ligated with the coding sequence of the human light chain constant region (CL) of either the kappa type (SEQ ID NO: 128) or the lambda type (SEQ ID NO: 129) to obtain the fully human light chain coding sequence of the antibody. The coding sequences for the heavy and light chains of the antibodies were separately inserted into the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), transformed into Escherichia coli DH5α, and cultured overnight at 37°C. Plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody plasmids for eukaryotic expression.

[0597] 4.2 Antibody Expression and Purification

[0598] Follow ExpiCHO TMExpression System USER GUIDE instructions, using the ExpiCHO transient expression system (Thermo Fisher, A29133) kit, the full-length sequence of the antibody obtained above was expressed. The specific method is as follows: on the day of transfection, confirm that the CHO cell density is 7×10 6 to 1×10 7 The cell viability was >98%. Fresh ExpiCHO expression medium pre-warmed at 37°C was used to adjust the cells to a final concentration of 6×10 6 cells / mL. Use OptiPRO pre-cooled at 4°C TM The target plasmid was diluted in SFM (1 μg of plasmid was added to 1 mL of the culture medium) and OptiPRO TM Dilute ExpiFectamine in SFM TM CHO reagent, and then mix the two in equal volumes and gently pipette to mix to prepare ExpiFectamine TM Incubate the CHO / plasmid DNA mixture at room temperature for 1-5 minutes, then slowly add it to the prepared cell suspension while gently shaking. Finally, place the mixture in a cell culture shaker and incubate at 37°C, 8% CO2.

[0599] 18-22 hours after transfection, add ExpiCHO to the cell culture medium TM Enhancer Reagent and ExpiCHO TM Feed reagent, place the shake flask in a 32°C shaker and continue to culture under 5% CO2 conditions. On the 5th day after transfection, add the same volume of ExpiCHO TM Feed reagent, slowly add while gently mixing the cell suspension. 7 days after transfection, collect the cell culture supernatant expressing the target antibody protein and centrifuge at 15000g for 10 minutes. The resulting supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403), and then the target antibody protein was eluted with 100mM sodium acetate (pH 3.0), followed by neutralization with 1M Tris-HCl, and finally the resulting antibody protein was exchanged into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096).

[0600] Example 5: Detection of the physicochemical properties of antibodies

[0601] In this example, the relative molecular weight and purity of the candidate antibodies were detected by SDS-PAGE and SEC-HPLC.

[0602] 5.1 Antibody SDS-PAGE Identification

[0603] Preparation of non-reducing solution: Add 1 μg of each obtained antibody and the quality control IPI (ipilimumab) to 5× SDS loading buffer and 40 mM iodoacetamide. Heat in a 75°C dry bath for 10 minutes. After cooling the mixture to room temperature, centrifuge at 12,000 rpm for 5 minutes and collect the supernatant.

[0604] Preparation of reducing solution: Add 2 μg of each obtained antibody and the quality control product IPI to 5× SDS loading buffer and 5 mM DTT, and heat in a dry bath at 100°C for 10 minutes. After cooling the mixture to room temperature, centrifuge at 12,000 rpm for 5 minutes and collect the supernatant.

[0605] The supernatant was added to a 4-15% Bis-tris gradient gel (GenScript) for gel electrophoresis and stained with Coomassie Brilliant Blue to visualize the protein bands. The protein gel with the visualized protein bands was scanned using an EPSON V550 color scanner (the gel was destained with a destaining solution until the background was transparent). The purity of the reduced and non-reduced bands was calculated using ImageJ using peak area normalization.

[0606] The experimental results showed that the bands of each antibody in the non-reducing gel were around 150kD, and the bands in the reducing gel were around 55kD and 25kD, which were consistent with the expected size. The purity of all candidate antibodies tested by reducing gel was greater than 95% (Table 2).

[0607] 5.2 SEC-HPLC Identification of Monomeric Antibody Purity

[0608] Material preparation: Mobile phase: 150 mmol / L phosphate buffer, pH 7.4; dilute each antibody and quality control product IPI to 0.5 mg / mL with mobile phase solution.

[0609] Experimental Methods: An Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph was used with an XBridge BEH column (SEC 3.5 μm, 7.8 mm ID x 30 cm). A Waters flow rate of 0.8 mL / min and a 20 μL injection volume were used. The VWD detector wavelengths were set at 280 nm and 214 nm. A blank solution, an IPI quality control solution, and an antibody sample solution were injected sequentially. The percentages of high-molecular-weight polymers, antibody monomers, and low-molecular-weight species in the samples were calculated using the area normalization method.

[0610] The results are shown in Table 2. The monomer purity of all candidate antibodies was greater than 89% (Table 2).

[0611] Table 2. Expression levels and physicochemical properties of candidate antibodies

[0612] Example 6 Antigen Binding Activity Detection of Antibodies

[0613] In this example, the binding of the expressed candidate antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, C-B71) to the CD100 antigen proteins HuCD100-His, MusCD100-His, and CynoCD100-His was detected based on the ELISA method, and the expressed antibodies (A14, A15, H74 , H96, H5, H12, H21, B13, C-C081, C-C171, C-B71) and CD100-overexpressing cells HuCD100-HEK293, MusCD100-HEK293, CynoCD100-HEK293 cells, human peripheral blood mononuclear cells naturally expressing human CD100 (hereinafter referred to as HuPBMC cells) and human T lymphocytic leukemia cells Jurkat cells.

[0614] 6.1 ELISA-based detection of antibody binding ability to antigen protein CD100-His

[0615] A 96-well ELISA plate (30 μL / well) was coated with 2 μg / mL of HuCD100-His, MusCD100-His, or CynoCD100-His overnight at 4°C. The next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three more times with PBST, a serial dilution of each antibody (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002 μg / mL) and the positive control antibody Pepinemab were added and incubated for 1 hour. The plate was then washed three times with PBST before the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After incubation, the plate was washed six times with PBST and color was developed with TMB (SurModics, TMBS-1000-01). According to the color development results, 2 M stop solution was added to terminate the reaction, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0616] The results are shown in Figures 5A-5X and Table 3: For the antigen protein HuCD100-His (Figures 5A-5H), the binding activities of antibody molecules A14, A15, B13, H5, H21, H74 and C-B71 to the antigen protein HuCD100-His were better than those of the positive control antibody, and the binding activities of antibody molecules H12, H96, C-C081 and C-C171 to the antigen protein HuCD100-His were comparable to those of the positive control antibody; for the antigen protein MusCD100-His (Figures 5I-5P), except for the antibody molecule C-C081, the binding activities of antibody molecules H12, H96, C-C081 and C-C171 to the antigen protein MusCD100-His The binding activity of is was weaker than that of the positive control antibody, and the binding activities of antibody molecules H12 and H96 to the antigen protein MusCD100-His were comparable to those of the positive control antibody. The binding activities of the other antibody molecules to the antigen protein MusCD100-His were better than those of the positive control antibody. For the antigen protein CynoCD100-His (Figures 5Q-5X), the binding activities of antibody molecules H12 and H96 to the antigen protein CynoCD100-His were comparable to those of the positive control antibody. The binding activities of the other antibody molecules to the antigen protein CynoCD100-His were better than those of the positive control antibody.

[0617] Table 3. Antigen binding activity of candidate antibodies

[0618] 6.2 FACS-based detection of antibody binding ability to CD100-HEK293 cells

[0619] In this example, three types of human CD100-overexpressing cells, HuCD100-HEK293, MusCD100-HEK293, and CynoCD100-HEK293, were used to evaluate the binding activity of the antibody.

[0620] The specific method is as follows: HuCD100-HEK293, MusCD100-HEK293, and CynoCD100-HEK293 cells in the logarithmic growth phase were prepared into single-cell suspensions, and the density was adjusted to 1×10 6100 μL of the suspension was added to each well of a 96-well plate. The cells were centrifuged at 300 g at 4°C, and the supernatant was removed. A serial dilution of each antibody (20.0, 6.67, 2.22, 0.74, 0.25, 0.08, 0.027, and 0.0027 μg / mL) and the positive control antibody Pepinemab were added to the corresponding wells, mixed, and incubated at 4°C for 30 min. The incubated cell mixture was washed three times, and 100 μL of a 1:300 dilution of the secondary antibody Goat F(ab')2 Anti-Human IgG-Fc(PE) (abcam, ab98596) was added. The cells were incubated at 4°C in the dark for 30 min, washed three times, and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0621] The results are shown in Figures 6A-6O and Table 4: For the overexpression cell line HuCD100-HEK293 (Figures 6A-6E), except for the antibody molecules C-C081, C-C171 and C-B71, whose binding activity to HuCD100-HEK293 was comparable to that of the positive control antibody, the binding activity of the remaining antibody molecules to HuCD100-HEK293 was better than that of the positive control antibody; for MusCD100-HEK293 (Figures 6F-6J), all antibody molecules had good mouse cross-reactivity and The binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96 and H12 to MusCD100-HEK293 was better than that of the positive control antibody; for CynoCD100-HEK293 ( Figures 6K-6O ), all antibody molecules showed good monkey cross-reactivity, among which H5, H21, A14, A15, B13, H74, H96, H12 and C-C081 had better binding activity to CynoCD100-HEK293 than the positive control antibody.

[0622] Table 4. Cell binding activity of candidate antibodies

[0623] 6.3 FACS-based detection of antibody binding to HuPBMC and Jurkat cells

[0624] In this example, HuPBMC and Jurkat cells were used to evaluate the binding activity of the antibody.

[0625] The specific method is as follows: HuPBMC (AllCells, catalog number PB004F-C (Y1246)) and Jurkat cells (ATCC, TIB-152) in the logarithmic growth phase were prepared into single-cell suspensions, and the density was adjusted to 1×10 6100 μL of the suspension was added to each well of a 96-well plate. The cells were centrifuged at 300 g at 4°C, and the supernatant was removed. Serial dilutions of each antibody (5.00, 0.50, 0.17, 0.056, 0.019, 0.006, 0.002, and 0.0002 μg / mL) and the positive control antibody Pepinemab were added to the corresponding wells, mixed, and incubated at 4°C for 30 min. The incubated cell mixture was washed three times, and 100 μL of a 1:300 dilution of the secondary antibody Goat F(ab')2 Anti-Human IgG-Fc(PE) (abcam, ab98596) was added. The cells were incubated at 4°C in the dark for 30 min, washed three times, and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0626] The results are shown in Figures 7A-7H and Table 5: Except for antibody molecules C-C081, C-C171 and C-B71, the binding activities of the remaining antibody molecules to HuPBMC cells (Figures 7A-7D) and Jurkat cells (Figures 7E-7H) were better than those of the positive control antibodies.

[0627] Table 5. Cell binding activity of candidate antibodies

[0628] Example 7 Detection of blocking activity of antibodies

[0629] In this example, the blocking activity of candidate antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, C-B71) was evaluated using four cell lines: HuPlexin-B1-HEK293, MusPlexin-B1-HEK293, CynoPlexin-B1-HEK293, and HuPlexin-B2-HEK293.

[0630] The specific method is as follows: cultured HuPlexin-B1-HEK293, MusPlexin-B1-HEK293, CynoPlexin-B1-HEK293, and HuPlexin-B2-HEK293 cells were collected and centrifuged at 300 g to remove the supernatant. The cells were resuspended in the prepared FACS buffer, counted, and the cell suspension density was adjusted to 1 × 10 6cells / mL; HuPlexin-B1-HEK293, MusPlexin-B1-HEK293, CynoPlexin-B1-HEK293, and HuPlexin-B2-HEK293 cells were added to a 96-well plate at 100 μL per well and centrifuged at 300 g to remove the supernatant; each antibody with a gradient dilution (20.0, 2.00, 0.67, 0.22, 0.074, 0.025, 0.008, 0.0008 μg / mL) and the positive control antibody Pepinemab were added to the corresponding wells of the 96-well plate, the cells were resuspended and incubated at 4°C for 30 minutes; the diluted antibodies were respectively stained with the biotinylated antigen protein HuCD1 Mix 100 μL of HuCD100-Fc dilution (0.5 μg / mL), CynoCD100-Fc dilution (0.3 μg / mL), or MusCD100-Fc dilution (3 μg / mL) (HuCD100-Fc, MusCD100-Fc, and CynoCD100-Fc prepared in Example 1.2 were obtained by biotinylation to obtain HuCD100-Fc-Biotin, MusCD100-Fc-Biotin, and CynoCD100-Fc-Biotin. For biotinylation methods, refer to the instructions for the Roche Biotin Protein Labeling Kit, Catalog No. 11418165001). Add the mixture to the cells and incubate for 1 hour. Then wash the cells three times. The cells were resuspended and incubated at 4°C for 30 min; the incubated cell mixture was washed three times, and then PE-labeled streptavidin (eBioscience, 12-4317-87) was added and incubated at 4°C for 30 min; the incubated cell mixture was washed three times, and then FACS buffer was added to the wells, 200 μL per well, and the cells were resuspended and detected by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0631] The results are shown in Figures 8A-8O: All candidate antibodies can effectively block the binding of HuCD100 antigen protein to HuPlexin-B1-HEK293 and HuPlexin-B2-HEK293 cells, the binding of MusCD100 antigen protein to MusPlexin-B1-HEK293 cells, and the binding of CynoCD100 antigen protein to CynoPlexin-B1-HEK293 cells. Among them, the antibody molecules A14, A15, B13, C-C081 and C-C171 had better activities in blocking the binding of HuCD100 antigen protein to HuPlexin-B1-HEK293 than the positive control antibody; the antibody molecules A15, B13, H74, H96, H12 and C-C081 had better activities in blocking the binding of HuCD100 antigen protein to HuPlexin-B2-HEK293 than the positive control antibody; the antibody molecules H12, H74, H96 and C-C081 had better activities in blocking the binding of MusCD100 antigen protein to MusPlexin-B1-HEK293 than the positive control antibody; the antibody molecules A15, B13, H12, H96, C-C081, C-C171 and C-B71 had better activities in blocking the binding of CynoCD100 antigen protein to CynoPlexin-B1-HEK293 than the positive control antibody.

[0632] Example 8 MDSC proliferation inhibition assay

[0633] This example evaluates the in vitro pharmacodynamics of candidate antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-B71) by examining their ability to inhibit the proliferation of myeloid-derived suppressor cells (MDSCs).

[0634] CD33+ cells were isolated from fresh PBMCs using CD33 magnetic beads (Miltenyi Biotech). After the isolation, the cell density was adjusted to 1 × 10 6 / mL for later use. The antibody was serially diluted with 1640 complete medium to 100.0, 33.33, 11.11, 3.704, 1.235, and 0.412 μg / mL. The HuCD100-His antigen protein was diluted with 1640 complete medium to 400.0 μg / mL. The antibody dilution solution and the HuCD100-His antigen protein dilution solution were evenly mixed in a 1:1 ratio and incubated at room temperature for 30 minutes. Take a 96-well cell culture plate and add 100 μL of a 1×10 6 / mL CD33 + Cells (1×10 per well5 Cells were collected from 96-well plates (100 μL of a mixture of antibodies and HuCD100-His antigen protein, totaling 200 μL). The cell culture plates were incubated in a 37°C cell culture incubator for 72 h. The cells were then transferred to 96-well U-shaped plates and washed twice with FACS buffer. PE-anti-human CD33 (Biolegend, 303404), FITC-anti-human HLA-DR (Biolegend, 307604), and APC anti-human CD11b Antibody (Biolegend, 301310) were diluted 1:100 in FACS buffer and 100 μL of the mixture was added to each well. The plates were incubated at 4°C for 30 min. The plates were washed twice with FACS buffer. Flow cytometry was performed (Beckman, CytoFLEX AOO-1-1102).

[0635] The experimental results are shown in Figures 9A-9C and Table 6: All candidate antibodies and positive control antibodies can effectively neutralize the induction effect of CD100 on MDSC cell populations. Among them, antibody molecules A14, A15, B13, H12, H21, H74 and H96 have better inhibitory effects on MDSC proliferation than positive control antibodies 2D5 and 5D8, and antibody molecules C-C081 and C-B71 have significantly better inhibitory effects on MDSC proliferation than the control antibody Pepinemab molecule.

[0636] Table 6. Anti-CD100 antibodies inhibit MDSC activity

[0637] Example 9 Preparation of anti-PD-L1 antibodies

[0638] In this example, affinity maturation of the antibody NB22D-21-huVH2 (see, for example, CN112745391A) was performed to improve antibody affinity and other biological activities. Affinity maturation was performed using M13 phage display technology. Codon-based primers (a single codon consisting of NNK during primer synthesis) were used to introduce mutations in the CDR regions. Four phage display libraries were constructed: Library 1, which contained single-point mutations, contained CDR1+CDR2+CDR3 mutations; Library 2, Library 3, and Library 4, which contained double-point mutations. Library 2 contained double-point mutations in CDR1+CDR3, Library 3 contained double-point mutations in CDR2+CDR3, and Library 4 contained double-point mutations in CDR1+CDR2.

[0639] Specific library construction method: First, synthesize primers containing point mutations (Jinweizhi Biotechnology Co., Ltd.); secondly, use the coding sequence of the antibody to be modified (hereinafter referred to as the parent antibody) NB22D-21-huVH as a PCR amplification template to amplify the sequence containing the mutation in the CDR region, and combine the fragments containing different CDR mutations by the bridge PCR method. Then, the point mutation antibody is connected to the phage display vector by double enzyme digestion (HindⅢ and NotⅠ) and double sticky end connection, and finally, the antibody sequence with the mutation site is transferred into Escherichia coli SS320 by electroporation. For the specific process of library capacity calculation, phage library preparation and library screening, please refer to Example 3. The obtained antibody is named m18-VHH. The variable region amino acid sequence of the obtained antibody is shown in Table 7, and the CDR sequence is determined by using the AbM method to define CDR.

[0640] Table 7. Amino acid sequences of anti-PD-L1 antibody variable regions (SEQ ID NO:

[0641] Example 10 Production and expression of antibody m18

[0642] A fusion expression vector was constructed by linking the C-terminus of the VHH gene sequence to the N-terminus of the human IgG1 Fc segment gene sequence to fuse the antibody m18-VHH and the human IgG1 Fc segment (SEQ ID NO: 135). The fusion expression vector plasmid was transformed into ExpiCHO cells and induced for expression to obtain a VHH-Fc chimeric antibody protein fused to the Fc segment (SEQ ID NO: 134). The VHH-Fc antibody is hereinafter designated as antibody m18.

[0643] The antibody expression was performed using the ExpiCHO transient expression system. TM Expression medium (Gibco, A29100-01) and Gibco TM ExpiFectamine TM CHO transfection kit (Gibco, A29129). For specific methods, see Example 4.

[0644] Example 11 Antigen Binding Activity Detection of Antibody m18

[0645] In this example, the binding ability of the VHH-Fc antibody to PD-L1-overexpressing human PD-L1-CHO cells, human non-small cell lung cancer cell line HCC827 cells, mouse PD-L1-CHO cells, and cynomolgus monkey PD-L1-CHO cells was detected based on the FACS method. The sources or preparation methods of the above cell lines can be found in CN112745391A.

[0646] 11.1 FACS-based detection of antibody binding to human PD-L1-CHO cells

[0647] In this example, human PD-L1-CHO cells were used to evaluate the binding activity of the antibody to human PD-L1 overexpressing cells.

[0648] For specific methods, please refer to Example 5 in CN112745391A, wherein the positive control antibody used is Avelumab (the preparation method is described in patent WO2013079174).

[0649] The results are shown in Figure 10A , and the binding activity of antibody m18 to human PD-L1-CHO cells was superior to that of the parent antibody NB22D-21-huVH2 and the positive control antibody Avelumab.

[0650] 11.2 FACS-based detection of antibody binding to human non-small cell lung cancer cell line HCC827 cells

[0651] In this example, the binding activity of the antibody to the PD-L1 protein on human tumor cells was evaluated using the human non-small cell lung cancer cell line HCC827 cells (ATCC: CRL-2868).

[0652] For specific methods, please refer to Example 8 in CN112745391A, wherein the positive control antibody used is Avelumab (the preparation method is described in patent WO2013079174).

[0653] The results are shown in FIG10B . The binding activity of antibody m18 to the human non-small cell lung cancer cell line HCC827 cells was comparable to that of the parent antibody NB22D-21-huVH2 and the positive control antibody Avelumab.

[0654] 11.3 FACS-based detection of antibody binding to mouse PD-L1-CHO cells and cynomolgus monkey PD-L1-CHO cells

[0655] In this example, mouse PD-L1-CHO cells and cynomolgus monkey PD-L1-CHO cells were used to evaluate cross-binding activity with monkey and mouse PD-L1. For specific methods, see Example 9 in CN112745391A, where the positive control antibody used was avelumab (preparation method described in patent WO2013079174).

[0656] The results are shown in Figures 11A and 11B . Antibody m18 has good binding activity to mouse PD-L1-CHO cells, while the parent antibody and the positive control antibody do not bind to mouse PD-L1-CHO cells ( Figure 11A ). It can be expected that the antibody molecule m18 can be used in animal model experiments in Balb / C mice. The binding activity of antibody m18 to cynomolgus monkey PD-L1-CHO cells is better than that of the positive control antibody and is comparable to that of the parent antibody ( Figure 11B ).

[0657] Example 12 Specific Detection of Antibody m18 Binding to PD-L1

[0658] In this example, the binding activity of the VHH-Fc antibody to other B7 family proteins was detected by ELISA to evaluate the specificity of antibody m18 for the PD-L1 protein. For specific methods, see Example 10 of Chinese invention patent application CN112745391A.

[0659] The results are shown in Table 8 and Figure 12. Antibody m18 has no binding activity to B7 family molecules other than B7-H1 (i.e., PD-L1), and only has binding activity to B7-H1. This binding specificity is consistent with that of the parent antibody.

[0660] Table 8. Specificity of candidate molecules binding to PD-L1 protein +: indicates that binding activity was detected; N / A: indicates that binding activity was not detected.

[0661] Example 13 Affinity maturation of anti-CD100 antibody molecules

[0662] 13.1 Affinity Maturation Library Design and Construction

[0663] Antibodies B13 and H5 underwent affinity maturation to improve affinity and biological activity. Affinity maturation was performed using M13 phage display technology. Codon-based primers (a single codon consisting of NNK during primer synthesis) were used to introduce mutations into the CDR regions. Four phage display libraries were constructed: Library 1 and Library 2 contained single-point mutagenesis (CDRL1+CDRL3+CDRH3 combined mutations, Library 1 contained CDRL2+CDRH1+CDRH2 combined mutations); Library 3 and Library 4 contained double-point saturation mutagenesis (CDRL3 combined mutations, Library 4 contained CDRH3 combined mutations). The specific library construction method is as follows: First, primers containing point mutations are synthesized (Genwizhi Biotechnology Co., Ltd.); second, sequences containing mutations in the CDR regions are amplified using the antibodies to be modified, B13 and H5, as PCR amplification templates. Fragments containing different CDR mutations are combined using the bridge PCR method. The point mutation antibodies are then linked to a phage display vector via double enzyme digestion (Hind III and Not I) and double sticky end ligation. Finally, the antibody sequences containing the mutation sites are transferred into Escherichia coli SS320 by electroporation. The specific procedures for library capacity calculation and phage library preparation are shown in Example 3.

[0664] 13.2 Screening of Affinity Maturation Libraries

[0665] The specific procedures for library screening are described in Example 3. After library screening, initial screening, affinity ranking, and sequence analysis, 74 positive clones were obtained for H5 and 57 for B13, respectively, for affinity ranking. Combining the affinity ranking and sequence analysis data, 20 candidate molecules were selected from the H5 and 28 from the B13 for construction, expression, and functional screening.

[0666] 13.3 Preparation of Affinity Maturation Candidate Molecules, Evaluation of Physicochemical Properties, and Testing of Binding Activity and Blocking Effects

[0667] The specific methods for the preparation, physicochemical property testing, affinity activity testing, blocking activity testing, and MDSC proliferation inhibition assay of the 48 candidate antibodies are shown in Examples 4-8.

[0668] 13.4 Selection of candidate molecules for affinity maturation

[0669] Based on the results of antibody physicochemical properties, affinity activity, and blocking activity testing, two affinity-matured anti-C100 antibodies, B13-c-5 and B13-e-2, were selected for B13. Two affinity-matured anti-C100 antibodies, H5-h-7 and H5-a-2, were selected for H5. The resulting antibody variable region amino acid sequences are shown in Table 9. CDR sequences were determined using the AbM CDR definition method.

[0670] The expression levels and in vitro physicochemical property test results are shown in Table 10. The purity of the antibodies was determined by SDS-PAGE, and the purity of the modified antibodies was preferably greater than 95%. The monomer purity of the antibodies was determined by SEC-HPLC, and the monomer purity of the modified antibodies was preferably greater than 98%.

[0671] The results of FACS-based affinity activity assays are shown in Figures 13A-13D. Figure 13A shows the results of binding activity to HuCD100-HEK293 cells. Figure 13B shows the results of binding activity to human PBMC cells. Figure 13C shows the results of binding activity to MusCD100-HEK 293 cells. Figure 13D shows the results of binding activity to CynoCD100-HEK 293 cells. Among them, the affinity activities of the modified antibodies B13-c-5, B13-e-2, H5-h-7 and H5-a-2 for human CD100-overexpressing cells (HuCD100-HEK293) and CD100-positive human PBMC cells were significantly better than those of the positive control antibody Pepinemab, and were superior to their corresponding parent antibodies; and the affinity activities of the modified antibodies for mouse CD100-overexpressing cells (MusCD100-HEK 293) and crab-eating macaque CD100-overexpressing cells (CynoCD100-HEK 293) were significantly better than those of the positive control antibody Pepinemab, and were superior to their corresponding parent antibodies.

[0672] The results of FACS-based blocking activity detection are shown in Figures 14A to 14D. The modified antibodies can effectively block the binding of HuCD100 antigen protein to HuPlexin-B1-HEK293 and HuPlexin-B2-HEK293 cells, the binding of MusCD100 antigen protein to MusPlexin-B1-HEK293 cells, and the binding of CynoCD100 antigen protein to CynoPlexin-B1-HEK293 cells. Among them, the modified antibody had better activity in blocking the binding of HuCD100 antigen protein to HuPlexin-B1-HEK293 cells than the positive control antibody and its corresponding parent antibody (Figure 14A); the modified antibody had better activity in blocking the binding of HuCD100 antigen protein to HuPlexin-B2-HEK293 cells (Figure 14B) and blocking the binding of MusCD100 antigen protein to HuPlexin-B1-HEK293 cells (Figure 14C) than the positive control antibody but weaker than the corresponding parent antibody; H5-h-7 and H5-a-2 had better activity in blocking the binding of CynoCD100 antigen protein to CynoPlexin-B1-HEK293 cells than the positive control antibody and the parent antibody H5, and B13-c-5 and B13-e-2 had better activity in blocking the binding of CynoCD100 antigen protein to CynoPlexin-B1-HEK293 cells than the positive control antibody but weaker than the parent antibody B13 (Figure 14D).

[0673] The results of the MDSC proliferation inhibition assay are shown in Figure 15 and Table 11. The results show that the anti-CD100 antibodies H5-h-7, H5-a-2, H5, B13, B13-c-5, and B13-e-2, as well as the control antibody Pepinemab, can effectively neutralize the induction of MDSC cell populations by CD100. Among them, the 100 μg / mL H5-h-7, H5-a-2, H5, B13, B13-c-5, and B13-e-2 showed significantly higher MDSC inhibition activity than the control antibody Pepinemab.

[0674] Table 9. Amino acid sequences of the variable regions of four affinity-matured anti-CD100 antibodies (SEQ ID NO:)

[0675] Table 10. Expression levels and in vitro physicochemical properties of four affinity-matured anti-CD100 antibodies

[0676] Table 11. Anti-CD100 antibodies inhibit MDSC activity

[0677] Example 14: Evaluation of drug efficacy in CT-26 animal model

[0678] This example tested the tumor inhibitory effects of seven anti-CD100 antibodies (antibodies B13, C-C081, C-B71, H5-h-7, H5-a-2, B13-c-5, and B13-e-2) in combination with anti-PD-L1 antibodies in animals. The tumor cells used were CT-26 (Shanghai Cell Bank, Chinese Academy of Sciences, catalog number TCM37), and the antibody Pepinemab was used as a positive control.

[0679] The specific method is as follows: 6-8 weeks old female Balb / C mice weighing about 20 g (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were used, and 5×10 5 CT-26 cells were randomly divided into groups and cages two days after tumor loading. There were 8 tumor-bearing nude mice in each group, for a total of 10 groups, including PBS negative control group, candidate antibody combination group (antibody B13 + antibody m18, antibody C-C081 + antibody m18, antibody C-B71 + antibody m18, antibody H5-h-7 + antibody m18, antibody H5-a-2 + antibody m18, antibody B13-c-5 + antibody m18, antibody B13-e-2 + antibody m18) and positive or reference control antibody group (Pepinemab + antibody m18, antibody m18). The dosage of anti-CD100 antibody was 50 mpk, and the dosage of anti-PD-L1 antibody was 5 mpk. The administration method was intraperitoneal injection, 2 times a week and the tumor volume was measured 2 times, for a total of 8 times / 4 weeks (BIW*4). The body weight of the mice was measured at the same time point. Tumor volume (V) calculation method: V = L × W 2 = / 2 (where L is the longest tumor diameter and W is the shortest tumor diameter). One week after the end of administration, the mice were euthanized and tumor tissues were obtained. Tumor volume and mouse body weight changes were analyzed and tumor inhibition rates were calculated. Tumor inhibition rate data are detailed in Table 12.

[0680] The results are shown in Figures 16A and 16B and Table 12. In the combination group of anti-CD100 antibodies and anti-PD-L1 antibodies, the combination of anti-CD100 antibodies (particularly antibodies C-C081, B13, or B13-c-5) and anti-PD-L1 antibody m18 can further synergistically inhibit or delay tumor growth compared to the anti-PD-L1 antibody m18 alone.

[0681] This indicates that the combination of anti-CD100 antibodies (particularly antibodies C-C081, B13, or B13-c-5) and the anti-PD-L1 antibody m18 can significantly improve the response rate of mice vaccinated with CT-26 to m18 antibody alone and prolong survival, suggesting that such a combination can enhance the therapeutic effect of PD-L1 tumor immunotherapy. At the same time, after administration, the weight of the mice increased, and there was no significant difference in the weight of the mice between the experimental and control groups. This shows that the antibodies did not cause significant toxic side effects in mice and were safe.

[0682] Table 12. Tumor inhibition rate (TGI%) of the combined use of candidate antibodies and antibody m18 in animal experiments

[0683] It will be clear to those skilled in the art that many modifications and variations of the present invention may be made without departing from its spirit and scope. The specific embodiments described herein are provided by way of example only and are not intended to be limiting in any way. The true scope and spirit of the present invention are shown by the appended claims, and the description and examples are merely exemplary.

[0684] Sequence Listing

[0685] B13

[0686] C-C081

[0687] A14

[0688] A15

[0689] H74

[0690] H96

[0691] H5

[0692] H12

[0693] H21

[0694] C-C171

[0695] C-B71

[0696] H5-h-7

[0697] H5-a-2

[0698] B13-c-5

[0699] B13-e-2

[0700] m18

Claims

1. An antibody or antigen-binding fragment thereof directed against CD100, comprising a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region comprises a HCDR1, a HCDR2, and a HCDR3 sequence, wherein the HCDR1 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO: 1, 7, 19, 33, 41, 47, or 52 by no more than 2 amino acid additions, deletions, or substitutions; the HCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO: 2, 8, 14, 20, 26, 42, 48, or 53 by no more than 2 amino acid additions, deletions, or substitutions; and / or the HCDR3 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, or 54 by no more than 2 amino acid additions, deletions, or substitutions; and / or The light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences, and wherein the LCDR1 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO:4, 10, 16, 36, 44 or 50 by no more than 2 amino acids in amino acid addition, deletion or substitution; the LCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO:5, 11 or 45 by no more than 2 amino acids in amino acid addition, deletion or substitution; and / or the LCDR3 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO:6, 12, 18, 24, 37, 46, 51 or 56 by no more than 2 amino acids in amino acid addition, deletion or substitution.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein The heavy chain variable region comprises a HCDR1 sequence as shown in SEQ ID NO: 1, 7, 13, 19, 25, 33, 41, 47, 52 or 57; a HCDR2 sequence as shown in SEQ ID NO: 2, 8, 14, 20, 26, 34, 42, 48, 53 or 58; and a HCDR3 sequence as shown in SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, 54, 60, 63 or 66; and / or The light chain variable region comprises a LCDR1 sequence shown in SEQ ID NO: 4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; a LCDR2 sequence shown in SEQ ID NO: 5, 11, 17, 23, 28, 45, 55 or 59; and a LCDR3 sequence shown in SEQ ID NO: 6, 12, 18, 24, 29, 32, 37, 40, 46, 51, 56, 62 or 65.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 sequences, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences; and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences are selected from any one of (1) to (15): (1) the HCDR1 sequence shown in SEQ ID NO: 1; the HCDR2 sequence shown in SEQ ID NO: 2; the HCDR3 sequence shown in SEQ ID NO: 3; the LCDR1 sequence shown in SEQ ID NO: 4; the LCDR2 sequence shown in SEQ ID NO: 5; and the LCDR3 sequence shown in SEQ ID NO: 6; (2) the HCDR1 sequence shown in SEQ ID NO: 7; the HCDR2 sequence shown in SEQ ID NO: 8; the HCDR3 sequence shown in SEQ ID NO: 9; the LCDR1 sequence shown in SEQ ID NO: 10; the LCDR2 sequence shown in SEQ ID NO: 11; and the LCDR3 sequence shown in SEQ ID NO: 12; (3) HCDR1 sequence shown in SEQ ID NO: 13; HCDR2 sequence shown in SEQ ID NO: 14; HCDR3 sequence shown in SEQ ID NO: 15; LCDR1 sequence shown in SEQ ID NO: 16; LCDR2 sequence; and LCDR3 sequence shown in SEQ ID NO: 18; (4) the HCDR1 sequence shown in SEQ ID NO: 19; the HCDR2 sequence shown in SEQ ID NO: 20; the HCDR3 sequence shown in SEQ ID NO: 21; the LCDR1 sequence shown in SEQ ID NO: 22; the LCDR2 sequence shown in SEQ ID NO: 23; and the LCDR3 sequence shown in SEQ ID NO: 24; (5) the HCDR1 sequence shown in SEQ ID NO: 25; the HCDR2 sequence shown in SEQ ID NO: 26; the HCDR3 sequence shown in SEQ ID NO: 27; the LCDR1 sequence shown in SEQ ID NO: 16; the LCDR2 sequence shown in SEQ ID NO: 28; and the LCDR3 sequence shown in SEQ ID NO: 29; (6) the HCDR1 sequence shown in SEQ ID NO:25; the HCDR2 sequence shown in SEQ ID NO:26; the HCDR3 sequence shown in SEQ ID NO:30; the LCDR1 sequence shown in SEQ ID NO:31; the LCDR2 sequence shown in SEQ ID NO:5; and the LCDR3 sequence shown in SEQ ID NO:32; (7) the HCDR1 sequence shown in SEQ ID NO:33; the HCDR2 sequence shown in SEQ ID NO:34; the HCDR3 sequence shown in SEQ ID NO:35; the LCDR1 sequence shown in SEQ ID NO:36; the LCDR2 sequence shown in SEQ ID NO:23; and the LCDR3 sequence shown in SEQ ID NO:37; (8) the HCDR1 sequence shown in SEQ ID NO:33; the HCDR2 sequence shown in SEQ ID NO:34; the HCDR3 sequence shown in SEQ ID NO:38; the LCDR1 sequence shown in SEQ ID NO:39; the LCDR2 sequence shown in SEQ ID NO:17; and the LCDR3 sequence shown in SEQ ID NO:40; (9) the HCDR1 sequence shown in SEQ ID NO:41; the HCDR2 sequence shown in SEQ ID NO:42; the HCDR3 sequence shown in SEQ ID NO:43; the LCDR1 sequence shown in SEQ ID NO:44; the LCDR2 sequence shown in SEQ ID NO:45; and the LCDR3 sequence shown in SEQ ID NO:46; (10) the HCDR1 sequence shown in SEQ ID NO:47; the HCDR2 sequence shown in SEQ ID NO:48; the HCDR3 sequence shown in SEQ ID NO:49; the LCDR1 sequence shown in SEQ ID NO:50; the LCDR2 sequence shown in SEQ ID NO:11; and the LCDR3 sequence shown in SEQ ID NO:51; (11) the HCDR1 sequence shown in SEQ ID NO: 52; the HCDR2 sequence shown in SEQ ID NO: 53; the HCDR3 sequence shown in SEQ ID NO: 54; the LCDR1 sequence shown in SEQ ID NO: 50; the LCDR2 sequence shown in SEQ ID NO: 55; and the LCDR3 sequence shown in SEQ ID NO: 56; (12) the HCDR1 sequence shown in SEQ ID NO:57; the HCDR2 sequence shown in SEQ ID NO:58; the HCDR3 sequence shown in SEQ ID NO:35; the LCDR1 sequence shown in SEQ ID NO:36; the LCDR2 sequence shown in SEQ ID NO:59; and the LCDR3 sequence shown in SEQ ID NO:37; (13) the HCDR1 sequence shown in SEQ ID NO:33; the HCDR2 sequence shown in SEQ ID NO:34; the HCDR3 sequence shown in SEQ ID NO:60; the LCDR1 sequence shown in SEQ ID NO:61; the LCDR2 sequence shown in SEQ ID NO:23; and the LCDR3 sequence shown in SEQ ID NO:62; (14) the HCDR1 sequence shown in SEQ ID NO: 1; the HCDR2 sequence shown in SEQ ID NO: 2; the HCDR3 sequence shown in SEQ ID NO: 63; the LCDR1 sequence shown in SEQ ID NO: 64; the LCDR2 sequence shown in SEQ ID NO: 5; and the LCDR3 sequence shown in SEQ ID NO: 65; (15) The HCDR1 sequence shown in SEQ ID NO: 1; the HCDR2 sequence shown in SEQ ID NO: 2; the HCDR3 sequence shown in SEQ ID NO: 66; the LCDR1 sequence shown in SEQ ID NO: 4; the LCDR2 sequence shown in SEQ ID NO: 5; and the LCDR3 sequence shown in SEQ ID NO:

6.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The heavy chain variable region comprises 1) an amino acid sequence as set forth in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95, preferably, the additions, deletions and / or substitutions do not occur in the CDR regions; and / or The light chain variable region comprises 1) an amino acid sequence as set forth in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain variable region and the light chain variable region are selected from any one of (1) to (15): (1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 67; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 68; (2) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 70; (3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 71; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 72; (4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 73; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74; (5) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 75; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 76; (6) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 77; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 78; (7) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 79; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 80; (8) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82; (9) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 83; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 84; (10) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 85; a light chain variable region comprising SEQ The amino acid sequence shown in ID NO:86; (11) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88; (12) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 89; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 90; (13) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 91; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 92; (14) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 93; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 94; (15) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 95; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

96.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain comprising 1) an amino acid sequence as set forth in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; 2) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; or 3) an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, preferably, the additions, deletions and / or substitutions do not occur in the CDR regions; and / or The antibody or antigen-binding fragment thereof comprises a light chain comprising 1) an amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; 2) an amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; or 3) an amino acid sequence as set forth in SEQ ID NO: An amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the heavy chain and light chain are selected from any one of (1) to (15): (1) a heavy chain comprising the amino acid sequence of SEQ ID NO: 97; a light chain comprising the amino acid sequence of SEQ ID NO: 98; (2) a heavy chain comprising the amino acid sequence of SEQ ID NO: 99; a light chain comprising the amino acid sequence of SEQ ID NO: 100; (3) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101; a light chain comprising the amino acid sequence of SEQ ID NO: 102; (4) a heavy chain comprising the amino acid sequence of SEQ ID NO: 103; a light chain comprising the amino acid sequence of SEQ ID NO: 104; (5) a heavy chain comprising the amino acid sequence of SEQ ID NO: 105; a light chain comprising the amino acid sequence of SEQ ID NO: 106; (6) a heavy chain comprising the amino acid sequence of SEQ ID NO: 107; a light chain comprising the amino acid sequence of SEQ ID NO: 108; (7) a heavy chain comprising the amino acid sequence of SEQ ID NO: 109; a light chain comprising the amino acid sequence of SEQ ID NO: 110; (8) a heavy chain comprising the amino acid sequence of SEQ ID NO: 111; a light chain comprising the amino acid sequence of SEQ ID NO: 112; (9) a heavy chain comprising the amino acid sequence of SEQ ID NO: 113; a light chain comprising the amino acid sequence of SEQ ID NO: 114; (10) a heavy chain comprising the amino acid sequence of SEQ ID NO: 115; a light chain comprising the amino acid sequence of SEQ ID NO: 116; (11) a heavy chain comprising the amino acid sequence of SEQ ID NO: 117; a light chain comprising the amino acid sequence of SEQ ID NO: 118; (12) a heavy chain comprising the amino acid sequence of SEQ ID NO: 119; a light chain comprising the amino acid sequence of SEQ ID NO: 120; (13) a heavy chain comprising the amino acid sequence of SEQ ID NO: 121; a light chain comprising the amino acid sequence of SEQ ID NO: 122; (14) a heavy chain comprising the amino acid sequence of SEQ ID NO: 123; a light chain comprising the amino acid sequence of SEQ ID NO: 124; (15) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 125; and a light chain comprising the amino acid sequence shown in SEQ ID NO:

126.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, which is a chimeric antibody, a humanized antibody, a human antibody, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide-stabilized Fv (dsFv) or a diabody; preferably, the antibody or antigen-binding fragment thereof is a human antibody.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof has at least one of the following characteristics: 1) Having affinity activity for CD100 protein; 2) has affinity activity for CD100-positive cells; 3) blocking the binding of CD100 to Plexin-B1 or Plexin-B2; 4) Inhibit MDSC cell proliferation; 5) Inhibit tumor growth.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. A pharmaceutical combination comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and an anti-PD-L1 antibody or antigen-binding fragment thereof.

12. The pharmaceutical combination of claim 11, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof specifically recognizes and binds to PD-L1, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an immunoglobulin single variable domain; Preferably, the immunoglobulin single variable domain comprises: CDR1 comprises the amino acid sequence shown in SEQ ID NO: 130, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 131, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:

132. More preferably, the immunoglobulin single variable domain comprises: 1) the amino acid sequence shown in SEQ ID NO: 133; or 2) an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:

133.

13. The pharmaceutical combination of claim 12, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc fragment of human IgG1; Preferably, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 134, or an amino acid sequence having at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:

134.

14. The pharmaceutical combination according to any one of claims 11 to 13, which is a pharmaceutical composition or a kit.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the pharmaceutical composition according to claim 10, or the pharmaceutical combination according to any one of claims 11 to 14 in the preparation of a medicament for treating cancer; preferably, the cancer is a hematological tumor or a solid tumor.

16. The method of claim 15, wherein The solid tumor comprises squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, renal cell carcinoma, mammary duct carcinoma, soft tissue sarcoma, osteosarcoma, melanoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, esophageal cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer or head and neck cancer; The blood tumor includes leukemia, lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma or multiple myeloma.

17. The use of claim 16, wherein the drug is used in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen targeting drugs.

18. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9.

19. An expression vector comprising the nucleic acid molecule of claim 18.

20. A host cell comprising the nucleic acid molecule of claim 18 or the expression vector of claim 19.

21. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the method comprising: a) culturing the host cell of claim 20 under suitable conditions to express the antibody or antigen-binding fragment thereof of any one of claims 1 to 9; as well as b) isolating the antibody or antigen-binding fragment thereof from the host cell or culture thereof.

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