Anti-cd100 antibodies and uses thereof

Abstract

Anti-CD100 antibodies and uses thereof, pharmaceutical compositions or medicaments comprising the antibodies or combinations thereof and uses thereof. Further provided are polynucleotides encoding the antibodies and expression vectors and methods of producing the antibodies.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine. Specifically, this invention relates to anti-CD100 antibodies and their uses. Background Technology

[0002] CD100, also known as brain signaling protein 4D (SEMA4D), is a transmembrane protein belonging to the brain signaling protein gene family. CD100 is expressed on the cell surface as a homodimer and can be released from the cell surface through proteolysis, producing an active, soluble form of CD100. CD100 is strongly expressed primarily in human lymphoid tissues, skeletal muscle, and the 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 B cell aggregation and survival, as well as 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 is associated with aggressive disease in humans. In the preclinical tumor microenvironment, inflammatory cells and tumor cells express CD100 to regulate the infiltration, spatial distribution, and activity of myeloid cells and lymphocytes. CD100 binds to the Plexin receptor on myeloid cells located in the tumor microenvironment. When the CD100 protein is blocked, the CD100 barrier is eliminated. Once the barrier is breached, inflammatory dendritic cells and pro-inflammatory antigen-presenting cells migrate and infiltrate the tumor. In preclinical animal models of cancer, blocking CD100 with antibodies slows tumor growth and promotes a durable tumor rejection response.

[0004] Several antibodies targeting CD100 have been developed in the prior art, such as Pepinemab (Vaccinex, Inc.). However, 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. Summary of the Invention

[0005] One aspect of 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.

[0006] In one embodiment, the antibody against CD100 or its antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 sequences, and wherein the HCDR1 sequence differs from the sequence shown in SEQ ID NO:1, 7, 19, 33, 41, 47, or 52 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; the HCDR2 sequence differs from the sequence shown in SEQ ID NO:2, 8, 14, 20, 26, 42, 48, or 53 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; and / or the HCDR3 sequence differs from the sequence shown in SEQ ID NO:3, 9, 15, 21, 27, 30, 35, 38, 43, 49, or 54 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions.

[0007] In one embodiment, the antibody against CD100 or its antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences, and wherein the LCDR1 sequence differs from the sequence shown in SEQ ID NO:4, 10, 16, 36, 44, or 50 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; the LCDR2 sequence differs from the sequence shown in SEQ ID NO:5, 11, or 45 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; and / or the LCDR3 sequence differs from the sequence shown in SEQ ID NO:6, 12, 18, 24, 37, 46, 51, or 56 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions.

[0008] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:1, 7, 13, 19, 25, 33, 41, 47, 52 or 57; the HCDR2 sequence shown in SEQ ID NO:2, 8, 14, 20, 26, 34, 42, 48, 53 or 58; and the HCDR3 sequence shown in SEQ ID NO:3, 9, 15, 21, 27, 30, 35, 38, 43, 49, 54, 60, 63 or 66.

[0009] In one embodiment, the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; the LCDR2 sequence shown in SEQ ID NO:5, 11, 17, 23, 28, 45, 55 or 59; and the LCDR3 sequence shown in SEQ ID NO:6, 12, 18, 24, 29, 32, 37, 40, 46, 51, 56, 62 or 65.

[0010] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 sequences, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences; the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences are selected from any one of (1)-(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) the HCDR1 sequence shown in SEQ ID NO:13; SEQ ID NO:14; HCDR2; HCDR3; HCDR1; HCDR2; HCDR3; HCDR2 ... (3) SEQ ID NO:14 HCDR2 sequence; SEQ ID NO:15 HCDR3 sequence; SEQ ID NO:16 LCDR1 sequence; SEQ ID NO:17 LCDR2 sequence; and SEQ ID NO:18 LCDR3 sequence; (4) SEQ ID NO:19 HCDR1 sequence; SEQ ID NO:20 HCDR2 sequence; SEQ ID NO:21 HCDR3 sequence; SEQ ID NO:22 LCDR1 sequence; SEQ ID NO:23 LCDR2 sequence; and SEQ ID NO:24 LCDR3 sequence; (5) SEQ ID NO:25 HCDR1 sequence; SEQ ID NO:26 HCDR2 sequence; SEQ ID NO:27 HCDR3 sequence; SEQ ID NO:16 LCDR1 sequence; SEQ ID NO:28 LCDR2 sequence; and SEQ ID NO:29 LCDR3 sequence; (6) SEQ ID NO:25 HCDR1 sequence; SEQ ID NO:26 HCDR2 sequence; SEQ ID NO:16 HCDR2 sequence; SEQ ID NO:29 HCDR3 sequence; SEQ ID NO:25 HCDR1 sequence; SEQ ID NO:26 HCDR2 sequence; SEQ ID NO:16 HCDR2 sequence; SEQ ID NO:16 HCDR2 sequence; SEQ ID NO:16 HCDR2 sequence; SEQ ID NO:17 LCDR2 sequence; and SEQ ID NO:28 LCDR2 sequence; and SEQ ID NO:29 LCDR3 sequence; The HCDR3 sequence shown in 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; and the HCDR3 sequence shown in SEQ ID NO:35;(8) 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; (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; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ ID NO:39; the LCDR1 sequence shown in SEQ ID NO:17; and the LCDR3 sequence shown in SEQ ID NO:40; (11) 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; SEQ ID NO:50; (11) The LCDR2 sequence shown in SEQ ID NO:11; and the LCDR3 sequence shown in SEQ ID NO:51; (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 HCDR3 sequence shown in SEQ ID NO:56; (14) The LCDR3 sequence shown in NO:62; 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;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.

[0011] In one embodiment, the heavy chain variable region comprises 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95; or 3 ... Compared to the amino acid sequences shown in NO:67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0012] In one embodiment, the light chain variable region comprises 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96; or 3 ... Compared to the amino acid sequences shown in NO:68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0013] In one embodiment, the heavy chain variable region and the light chain variable region are selected from any one of (1)-(15): (1) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:67; a light chain variable region comprising the amino acid sequence shown in 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 shown in SEQ ID NO:71; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:72; (4) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:73; a light chain variable region comprising the amino acid sequence shown in 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; a light 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:70; a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:71; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:72; a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:73; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:74; 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; 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:79; a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:70; a light chain variable (7) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:79; the light chain variable region containing the amino acid sequence shown in SEQ ID NO:80; (8) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:81; the light chain variable region containing the amino acid sequence shown in SEQ ID NO:82; (9) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:83; the light chain variable region containing the amino acid sequence shown in SEQ ID NO:84; (10) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:85; the light chain variable region containing the amino acid sequence shown in SEQ ID NO:86; (11) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:87; the light chain variable region containing the amino acid sequence shown in SEQ ID NO:88; (12) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:89; the light chain variable region containing the amino acid sequence shown in SEQ ID NO:90; (13) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:89; the light chain variable region containing the amino acid sequence shown in SEQ ID NO:90; (14) The amino acid sequence shown in NO:91; the light chain variable region, which contains the amino acid sequence shown in SEQ ID NO:92; the heavy chain variable region, which contains the amino acid sequence shown in SEQ ID NO:93; the light chain variable region, which contains the amino acid sequence shown in SEQ ID NO:94; the heavy chain variable region, which contains the amino acid sequence shown in SEQ ID NO:95; the light chain variable region, which contains the amino acid sequence shown in SEQ ID NO:96.

[0014] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain, the heavy chain comprising 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125; or 3 ... Compared to the amino acid sequences shown in NO:97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0015] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain comprising: 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; or 3 ... Compared to the amino acid sequences shown in NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0016] In one embodiment, the heavy chain and the light chain are selected from any one of (1)-(15): (1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:97; and a light chain comprising the amino acid sequence shown in SEQ ID NO:98; (2) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:99; and a light chain comprising the amino acid sequence shown in SEQ ID NO:100; (3) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:101; and a light chain comprising the amino acid sequence shown in SEQ ID NO:102; (4) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:103; and a light chain comprising the amino acid sequence shown in SEQ ID NO:104; (5) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:105; and a light chain comprising the amino acid sequence shown in SEQ ID NO:106; (6) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:107; and a light chain comprising the amino acid sequence shown in SEQ ID NO:108; (7) a heavy chain comprising the amino acid sequence shown in SEQ ID NO:108; and a light chain comprising the amino acid sequence shown in SEQ ID NO:109. (8) A heavy chain containing the amino acid sequence shown in SEQ ID NO:109; a light chain containing the amino acid sequence shown in SEQ ID NO:110; (9) A heavy chain containing the amino acid sequence shown in SEQ ID NO:111; a light chain containing the amino acid sequence shown in SEQ ID NO:112; (10) A heavy chain containing the amino acid sequence shown in SEQ ID NO:113; a light chain containing the amino acid sequence shown in SEQ ID NO:114; (11) A heavy chain containing the amino acid sequence shown in SEQ ID NO:115; a light chain containing the amino acid sequence shown in SEQ ID NO:116; (11) A heavy chain containing the amino acid sequence shown in SEQ ID NO:117; a light chain containing the amino acid sequence shown in SEQ ID NO:118; (12) A heavy chain containing the amino acid sequence shown in SEQ ID NO:119; a light chain containing the amino acid sequence shown in SEQ ID NO:120; (13) A heavy chain containing the amino acid sequence shown in SEQ ID NO:121; a light chain containing the amino acid sequence shown in SEQ ID NO:119; a light chain containing the amino acid sequence shown in SEQ ID NO:120; a heavy chain containing the amino acid sequence shown in SEQ ID NO:121; a light chain containing the amino acid sequence shown in SEQ ID NO:112; a heavy chain containing the amino acid sequence shown in SEQ ID NO:119; a light chain containing the amino acid sequence shown in SEQ ID NO:120; a heavy chain containing the amino acid sequence shown in SEQ ID NO:121; a light chain containing the amino acid sequence shown in SEQ ID NO:119; a heavy ... (14) The heavy chain containing the amino acid sequence shown in SEQ ID NO:123; the light chain containing the amino acid sequence shown in SEQ ID NO:124; (15) The heavy chain containing the amino acid sequence shown in SEQ ID NO:125; the light chain containing the amino acid sequence shown in SEQ ID NO:126.

[0017] In another aspect, the present invention provides a pharmaceutical composition comprising the anti-CD100 antibody of the present invention or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0018] In some embodiments, the anti-CD100 antibody includes antibodies 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.

[0019] In another aspect, the present invention provides a pharmaceutical combination comprising the anti-CD100 antibody or its antigen-binding fragment thereof and the anti-PD-L1 antibody or its antigen-binding fragment thereof.

[0020] In one embodiment, the anti-PD-L1 antibody or its antigen-binding fragment specifically recognizes and binds to PD-L1, wherein the anti-PD-L1 antibody or its antigen-binding fragment comprises an immunoglobulin monovariable domain.

[0021] In one embodiment, the immunoglobulin single variable domain comprises CDR1, which contains the amino acid sequence shown in SEQ ID NO:130; CDR2, which contains the amino acid sequence shown in SEQ ID NO:131; and CDR3, which contains the amino acid sequence shown in SEQ ID NO:132.

[0022] In one 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.

[0023] In one embodiment, the anti-PD-L1 antibody or its antigen-binding fragment further comprises an Fc fragment of human IgG1. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment 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.

[0024] In one embodiment, the drug combination is a pharmaceutical composition or a kit.

[0025] In one aspect, the present invention also provides the use of the antibody or antigen-binding fragment thereof of the present invention, the pharmaceutical composition of the present invention, or the pharmaceutical combination of the present invention in the preparation of a medicament for treating cancer.

[0026] In another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need an antibody of the present invention or an antigen-binding fragment thereof, a pharmaceutical composition of the present invention, or a pharmaceutical combination of the present invention.

[0027] In another aspect, the present invention relates to antibodies or antigen-binding fragments thereof, pharmaceutical compositions of the present invention, or pharmaceutical combinations of the present invention for the treatment of cancer.

[0028] In another aspect, the present invention also provides isolated nucleic acid molecules encoding the anti-CD100 antibody of the present invention or an antigen-binding fragment thereof. The present invention also relates to expression vectors comprising the nucleic acid molecules of the present invention. The present invention further relates to host cells comprising the nucleic acid molecules or expression vectors of the present invention.

[0029] In another aspect, the present invention also relates to a method for generating an anti-CD100 antibody or an antigen-binding fragment thereof, the method comprising:

[0030] a) Culturing the host cells of the present invention under suitable conditions to express the anti-CD100 antibody or its antigen-binding fragment as claimed in the present invention; and

[0031] b) Isolate the antibody or its antigen-binding fragment from a host cell or its culture. Attached Figure Description

[0032] Figure 1 The results of ELISA detection of Pepinemab antibody and positive controls 2D5 and 5D8 antibody are shown.

[0033] Figure 2 The results of ELISA detection for different species antigens are shown.

[0034] Figure 3 The FACS results of overexpressing cell lines from different species are shown.

[0035] Figures 4A-4D The FACS results of Plexin receptor overexpressing cell lines from different species are shown. Figure 4A The results are for the detection of HuPlexin-B1-HEK293 overexpressing cell lines; Figure 4B The results are for the detection of MusPlexin-B1-HEK293 overexpressing cell lines; Figure 4C The results are for the detection of CynoPlexin-B1-HEK293 overexpressing cell lines; Figure 4D The results are for the detection of HuPlexin-B2-HEK293 overexpressing cell lines.

[0036] Figures 5A-5X The results show the results of ELISA detection of the binding activity of candidate antibodies to antigen proteins of different species. Figures 5A-5H The results show 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; Figure 5I-5P The results show 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; Figure 5Q-5X The results show 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.

[0037] Figures 6A-6O The results of FACS detection of the binding activity of candidate antibodies to different species overexpressing cell lines are shown. Figures 6A-6E The results show the binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96, H12, C-C081, C-C171 and C-B71 on the HuCD100-HEK293 overexpressing cell line; Figure 6F-6J The results show the binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96, H12, C-C081, C-C171 and C-B71 on the MusCD100-HEK293 overexpressing cell line; Figure 6K-6O The results show the binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96, H12, C-C081, C-C171, and C-B71 to the CynoCD100-HEK293 overexpressing cell line.

[0038] Figures 7A-7H The results of FACS detection of the binding activity of candidate antibodies to HuPBMC and Jurkat cells are shown. Figures 7A-7D 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; Figure 7E-7H The results represent the binding activity of antibody molecules A14, A15, B13, H5, H21, H74, H96, H12, C-C081, C-C171, and C-B71 to Jurkat cells.

[0039] Figures 8A-8O The results show the results of candidate antibodies blocking the binding of CD100 to the Plexin receptor in different species. Figures 8A-8D The results show that antibody molecules A14, A15, B13, H5, H21, H74, H96, H12, C-C081, C-C171 and C-B71 block the binding of HuCD100 to HuPlexin-B1-HEK293 cells; Figure 8E-8IThe results show that antibody molecules A14, H5, A15, B13, H21, H74, H96, H12, C-C081, C-C171, and C-B71 block the binding of HuCD100 to HuPlexin-B2-HEK293 cells. Figure 8J-8L The results show that antibody molecules H5, A14, A15, B13, H12, H21, H74, H96, C-C081, C-C171, and C-B71 block the binding of MusCD100 to MusPlexin-B1-HEK293 cells. Figure 8M-8O The results show that antibody molecules H5, A14, A15, B13, H12, H21, H74, H96, C-C081, C-C171, and C-B71 block the binding of CynoCD100 to CynoPlexin-B1-HEK293 cells.

[0040] Figures 9A-9C The results of MDSC proliferation inhibition assays for candidate antibodies A14, A15, B13, H12, H21, H74, H96, H5, C-C081, C-C171, and C-B71 are shown.

[0041] Figures 10A-10B The results show the results of FACS detection of the binding activity of anti-PD-L1 antibody m18 on overexpressing cell lines human PD-L1-CHO cells and PD-L1 positive cells HCC827 cells. Figure 10A Results of binding activity against human PD-L1-CHO cells; Figure 10B The results show the binding activity against HCC827 cells.

[0042] Figures 11A-11B The results of FACS detection of the binding activity of anti-PD-L1 antibody m18 to different species overexpressing cell lines are shown. Figure 11A Results of binding activity against mouse PD-L1-CHO cells; Figure 11B The results show the binding activity of PD-L1-CHO cells in cynomolgus monkeys.

[0043] Figure 12 The results show the results of detecting the specific binding activity of anti-PD-L1 antibody m18 to B7-H1 and its homologous proteins using ELISA.

[0044] Figures 13A-13D The results show the binding activity of the modified antibodies H5-h-7, H5-a-2, B13-c-5, and B13-e-2 to CD100-overexpressing cell lines and CD100-positive PBMCs, as determined by FACS, for candidate antibodies H5 and B13. Figure 13A Results of binding activity against HuCD100-HEK293 cells; Figure 13B Results of binding activity to human PBMC cells; Figure 13C Results of binding activity against MusCD100-HEK 293 cells; Figure 13D Results of binding activity against CynoCD100-HEK 293 cells.

[0045] Figures 14A-14D The results show that the modified antibodies H5-h-7, H5-a-2, B13-c-5, and B13-e-2, after affinity maturation of candidate antibody H5 and B13, blocked the binding of CD100 to Plexin receptor in different species. Figure 14A The results show that the candidate antibody blocked the binding of HuCD100 to HuPlexin-B1-HEK293 cells; Figure 14B The results show that the candidate antibody blocked the binding of HuCD100 to HuPlexin-B2-HEK293 cells; Figure 14C The results show that the candidate antibody blocked the binding of MusCD100 to MusPlexin-B1-HEK293 cells; Figure 14D The results show the effect of the candidate antibody blocking the binding of CynoCD100 to CynoPlexin-B1-HEK293 cells.

[0046] Figure 15 The results of MDSC assays for proliferation inhibition of candidate antibody H5 and the modified antibodies H5-h-7, H5-a-2, B13-c-5, and B13-e-2 after affinity maturation are shown.

[0047] Figures 16A-16B The results of the tumor suppression effect test are shown. Figure 16A Results of tumor volume changes; Figure 16B This represents the results of weight change. Detailed Implementation

[0048] definition

[0049] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0050] As used herein, “at least one species” or “one or more species” can mean 1, 2, 3, 4, 5, 6, 7, 8 or more species.

[0051] As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “essentially composed 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 essential and novel nature of the claimed subject matter. It should be understood that the expressions “essentially composed of” and “composed of” are encompassed within the meaning of the expression “comprising.”

[0052] As used herein, the connecting term “and / or” between multiple referred elements should be understood to include both individual and combined options. In other words, “and / or” includes both “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. Further elements qualified by “and / or” are understood in a similar manner and include any one of them and any combination thereof.

[0053] Unless otherwise stated, any numerical value or range, such as concentration or concentration range, shall in any case be understood to be modified by the term “about”. Thus, numerical values ​​typically 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 that range, including integers and fractions within that range, unless the context clearly indicates otherwise.

[0054] As used herein, “antibody” refers to an immunoglobulin or a fragment thereof that 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, recombinant 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), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described 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 a preferred embodiment, the antibody of the present invention is a human antibody.

[0055] 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 antibody’s variable region (e.g., one or more CDRs and / or one or more antibody-binding sites) that binds to an antigen, and thus retains binding specificity as well as at least a portion of the full-length antibody’s specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant 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, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise 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, upon insertion into an antibody framework (e.g., by replacing the corresponding region), acquires an antibody that specifically binds to the antigen.

[0056] 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 consist of a pair of VH and VL to form a functional antigen-binding unit, a single variable domain can form a functional antigen-binding unit independently. Single variable domains can be derived from naturally occurring light-chain-free antibodies, such as the variable domain of heavy chain of heavy-chain antibody (VHH) from camels (e.g., camels and alpacas) and the single variable domain of shark neoantigen receptors (IgNAR variable single-domain, VNAR). They can also be obtained through screening full-length antibodies, such as the light chain variable domain and heavy chain variable domain with antigen-binding activity in human antibodies. A VHH typically contains three highly variable "complementarity-determining regions (CDRs)" and four relatively conserved "framework regions (FRs)," linked from the N-terminus to the C-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0057] As used herein, "single-domain antibody (sdAb)" or "nanobody" refers to an antibody containing 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 contains CDR1, CDR2, and CDR3, which form the antigen-binding site, as well as a supporting framework region. Unlike full-length antibodies, which typically contain two heavy chains and two light chains, single-domain antibodies usually consist of a single peptide chain composed of a single variable domain, with a molecular weight of only about 15 kDa. Examples of single variable domains include the variable domain of a heavy-chain antibody (VHH) from alpacas, the variable domain of IgNAR from sharks, or the variable domain of a human light-chain antibody.

[0058] As used herein, the terms “heavy-chain-only antibody” and “heavy-chain antibody” are used interchangeably and exist in their broadest sense, referring to an antibody that lacks the conventional antibody light chain and contains only a VHH and a heavy chain constant region (e.g., Fc fragment) that does not contain CH1.

[0059] "Fc fragment" generally refers to a crystallizable fragment of a conventional antibody or heavy chain antibody after papain digestion. Typically, the Fc fragment of IgG and heavy chain antibodies may contain a partial hinge region, CH2, and CH3. In this article, the Fc fragment may contain at least a partial hinge region (e.g., all or part of the hinge region), CH2, and CH3.

[0060] As used herein, a “full-length antibody” typically comprises two heavy chains (which may be labeled H and H') and two light chains (which may be labeled 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 contains a “light chain variable region (VL)” and a “light chain constant region (CL)” from the N-terminus to the C-terminus. Each heavy chain contains a “heavy chain variable region (VH)” and a “heavy chain constant region (CH)” from the N-terminus to the C-terminus. Generally, the heavy chain constant region of a full-length antibody may contain a VH-CH1-hinge region CH2-CH3 from the N-terminus to the C-terminus. Each heavy chain (H and H') pairs with one light chain (L and L', respectively).

[0061] Light chains are classified as κ or λ (Kappa or Lambda). Each heavy chain class can bind to a κ or λ light chain. Typically, light and heavy chains are covalently linked, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tail" portions of the two heavy chains are linked together via covalent disulfide bonds or non-covalent connections. In the heavy chain, amino acids extend from the N-terminus at the bifurcation end of the Y-configuration to the C-terminus at the base of each chain. The basic structure of some antibodies (e.g., IgG antibodies) comprises two heavy chain subunits and two light chain subunits covalently linked by disulfide bonds to form a "Y" structure.

[0062] 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 regions of the variable light (VL) chain or variable heavy (VH) chain portion determine antigen recognition and specificity. Conversely, the constant regions of the light chain (CL) and heavy chain (CH1, CH2, or CH3) endow biological properties such as secretion, transplacental movement, Fc receptor binding, complement binding, etc. By convention, the numbering of constant region domains increases with distance 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 comprise the carboxyl termini of the heavy and light chains, respectively.

[0063] As described above, variable regions (i.e., “binding domains”) allow binding molecules to selectively recognize and specifically bind to epitopes on antigens. That is, for example, the VL and VH domains of an antibody binding molecule, or combinations of these complementarity-determining region (CDR) subgroups, form variable regions that define three-dimensional antigen-binding sites. More specifically, the antigen-binding site is defined by three CDRs on each VH and VL chain. These six “complementarity-determining regions” or “CDRs” are discontinuous short sequences of amino acids that are specifically localized to form binding domains as the antibody adopts its three-dimensional conformation in an aqueous environment. The remaining amino acids in the binding domains are called “framework (FR)” regions, exhibiting smaller intermolecular differences. The binding domains formed by the localized CDRs define a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its complementary epitope. The amino acids constituting the CDRs and framework regions of 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 full text). In this paper, 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.

[0064] Unless otherwise specified, the amino acid sequence of CDR is shown in accordance with the AbM definition rules (the sequence in the claims of this invention is also shown in accordance with the AbM definition rules). However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (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)) based on the antibody's three-dimensional structure and the topology of the CDR ring; Kabat (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242) based on antibody sequence variability; and 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, R.M 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. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementarity-determining region” for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described above by means of the known schemes described in this invention.Although the scope of protection sought in the claims of this invention is based on the sequence defined by the AbM definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0065] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0066] As used herein, the terms “frame region” and “architecture region” are used interchangeably. As used herein, the terms “frame region,” “architecture region,” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR sequence as defined above.

[0067] The "Fv" fragment, consisting of a VH and a VL domain connected by non-covalent interactions, is generally considered the smallest antigen-binding fragment containing an antigen-binding site. However, single variable domain (single-domain antibody) also possesses antigen-binding capability. A "single-chain Fv (scFv)" can be obtained by linking VH and VL domains via peptide linkers. Introducing disulfide bonds into Fv or scFv yields either a "disulfide-stable Fv (dsFv)" or a "single-chain disulfide-stable Fv (scdsFv or dsscFv)," respectively.

[0068] As used herein, the Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment with the same structure synthesized, for example, through recombinant methods. It contains a complete antibody light chain (VL-CL), a variable region of the antibody heavy chain, and a constant region of the heavy chain (VH-CH1, also known as Fd). Linking the CL and CH1 regions of “Fab” with peptide linkers yields a single-chain “Fab(scFab)”. “F(ab')2” is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at pH 4.0–4.5, or a fragment with the same structure synthesized, for example, through recombinant methods. It essentially consists of two Fab fragments linked by disulfide bonds in their hinge regions. “Fab'” is half of F(ab')2 and can be obtained by reducing the disulfide bonds in the hinge region of F(ab')2.

[0069] As used in this article, the term "hinge region" refers to the heavy chain portion of the molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 amino acids and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.

[0070] As used in this article, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge a second thiol group.

[0071] As used herein, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is derived from or derived from a first species, while the constant region (which may be complete, partial, or modified) is derived from a second species. In some embodiments, the target binding region or site will be of a non-human origin (e.g., mouse or primate) and the constant region will be human.

[0072] As used herein, the term "humanized antibody" refers to a non-human antibody modified to increase sequence homology with human antibodies. Humanized antibodies typically retain the antigen-binding ability of their derived non-human antibody and exhibit low immunogenicity in humans. Humanized antibodies can be obtained by antibody engineering of any non-human species antibody or antibody containing sequences derived from a non-human species (e.g., chimeric antibodies). Non-human species may include, for example, mice, rats, rabbits, alpacas, sharks, or non-human primates. Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art. For example, the CDR sequence of a non-human antibody (e.g., a mouse antibody) can be transplanted into the framework region of a human antibody. In some cases, in order to maintain the antigen-binding ability and / or stability of humanized antibodies, key amino acid residues of the framework sequence of non-human antibodies (e.g., murine antibodies) can be retained in the human antibody framework region, i.e., "reverse mutation" can be performed (see, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21):6851-6855; Neuberger et al. (1984) Nature 312:604-608).

[0073] 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 that has an amino acid sequence corresponding to that of a human-produced antibody. The definition of a human antibody encompasses full-length or complete antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide.

[0074] As used herein, “affinity-matured” antibodies contain one or more modifications (e.g., substitution of amino acid residues) in one or more CDRs such that the affinity-matured antibody exhibits improved affinity for the antigen compared to the parent antibody without such modifications. Methods for affinity maturation of antibodies are known in the art, see, for example, 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).

[0075] As used herein, the term "percentage (%) sequence identity" or "sequence identity" has a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a known algorithm). This can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0076] In this document, amino acid sequences “derived from” or “from” a reference amino acid sequence are partially or wholly identical or homologous to the reference amino acid sequence. For example, an amino acid sequence derived from the heavy chain constant region of 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 from which the heavy chain constant region of human immunoglobulin from which it is derived.

[0077] "Affinity" or "binding affinity" measures the strength of the binding between an antibody and an antigen through non-covalent interactions. Affinity can be determined using conventional techniques known in the art, such as biomembrane interference techniques (e.g., using the Octet Fortebio detection system), radioimmunoassay, surface plasmon resonance assay, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS).

[0078] "Specific binding" generally refers to a binding molecule, such as an antibody or its fragments, variants, or derivatives, binding 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 binds to it more readily through its antigen-binding domain than to a random, unrelated epitope. In this paper, the term "specificity" is used to qualitatively analyze the relative affinity of an antibody for a given epitope. For example, binding molecule "A" can be considered to have higher specificity for a given epitope than binding molecule "B," or it can be said that binding molecule "A" binds to epitope "C" with higher specificity than its specificity for related epitope "D."

[0079] If a binding molecule, such as an antibody or a fragment, variant, or derivative thereof, preferentially binds to an epitope to a degree that blocks the binding of a reference antibody or antigen-binding fragment to that epitope, then it can be said that the binding molecule, for example, the antibody or a fragment, variant, or derivative thereof, competitively inhibits the 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, for example, a competitive ELISA assay. It can be said that the binding molecule competitively inhibits at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% of the binding of the reference antibody or antigen-binding fragment to a given epitope.

[0080] As used herein, the term “PD-L1” refers to programmed cell death ligand 1 (PD-L1, see, for example, Freeman et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 familymember 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), 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, and 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 and its receptor induces signal transduction to inhibit TCR-mediated 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, and tissue allogeneic grafts) and is thought to allow tumors or cancer cells to bypass immune checkpoints and evade immune responses.

[0081] As used in this article, the term "B7 family" refers to a class of structurally similar co-stimulatory factors in the body's immune process. They belong to the immunoglobulin class and are involved in the activation of T and B cells and the body's immunity.

[0082] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to polymers of deoxyribonucleotides (DNA) or polymers of ribonucleotides (RNA). The terms "polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the sequence of nucleotides in a polynucleotide. Those skilled in the art will understand that the DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, with the deoxythymidine nucleotide in the DNA coding strand sequence corresponding to the uridine nucleotide in its encoded RNA sequence.

[0083] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0084] As used herein, the term “expression” includes the transcription and / or translation of nucleotide sequences. Therefore, expression can involve the production of transcripts and / or peptides.

[0085] As used herein, a "vector" is a medium used to introduce exogenous polynucleotides into host cells, whereby the exogenous polynucleotides are amplified or expressed when the vector is transformed into a suitable host cell. Vectors typically remain free, but can be designed to integrate genes or portions thereof into the chromosome of the genome. As used herein, the definition of a vector encompasses plasmids, linearized plasmids, viral vectors, granules, phage vectors, phage particles, 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), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, poxvirus vectors, and baculovirus vectors, etc.

[0086] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0087] Terms such as “treatment,” “curing,” “with treatment,” “relief,” or “with relief” refer to therapeutic measures that cure, alleviate, or reduce the symptoms of an existing diagnosed pathological condition or disorder, and / or halt or slow the progression of an existing diagnosed pathological condition or disorder. Terms such as “prevention,” “defense,” “avoidance,” or “containment” refer to preventive or preventative measures that prevent the progression of an undiagnosed target pathological condition or disorder. Therefore, “subjects in need” may include subjects who already have the disease; subjects who are susceptible to the disease; and subjects who need to prevent the disease.

[0088] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0089] The term "therapeuticly effective amount" refers to the amount of an antibody, peptide, polynucleotide, small organic molecule, or other drug that is effective for the disease or condition in the "treated" subject or mammal. In the case of cancer, a therapeutically effective amount of drug may reduce the number of cancer cells; inhibit or stop cancer cell division, reduce or stop tumor size growth; inhibit, for example, suppress, block, prevent, stop, delay, or reverse cancer cell infiltration into surrounding organs, including, for example, cancer spread to soft tissues 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 one or more cancer-related symptoms to some extent, reduce morbidity and mortality; improve quality of life; or a combination of these effects. In terms of the extent to which a drug prevents the growth of and / or kills existing cancer cells, it may refer to inhibition of cell growth and / or cytotoxicity.

[0090] As used in this article, the term "subject" refers to a mammal, such as a human.

[0091] The antibody designations 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) are for identification purposes only and are not intended to represent a characteristic of the antibodies or products of this invention. Those skilled in the art will understand that other antibodies or products may also use such designations for identification purposes, but these do not necessarily refer to the same or equivalent antibodies or products. Similarly, similar designations used in the examples are merely for illustrative purposes, and the antibodies or products of this invention are defined by the features described in the appended claims.

[0092] Anti-CD100 antibody or its antigen-binding fragment

[0093] 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.

[0094] In some embodiments, the anti-CD100 antibody or its antigen-binding fragment of the present invention is a chimeric antibody, a humanized antibody, a human antibody, scFv, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv (dsFv), or a biantibody. Preferably, the antibody or its antigen-binding fragment is a human antibody.

[0095] In one embodiment, the antibody or its antigen-binding fragment has at least one of the following characteristics:

[0096] 1) It has affinity activity for CD100 protein;

[0097] 2) It has affinity activity for CD100 positive cells;

[0098] 3) Block the binding of CD100 to Plexin-B1 or Plexin-B2;

[0099] 4) Inhibits MDSC cell proliferation;

[0100] 5) Inhibits tumor growth.

[0101] In some embodiments, the antibody or antigen-binding fragment of the present invention is capable of specifically binding to CD100 (such as human CD100) and blocking its interaction with Plexin-B1 or Plexin-B2.

[0102] In some embodiments, the targeted tumors include, but are not limited to, those described below with respect to neoplastic diseases. In other embodiments, the antibody or antigen-binding fragment of the present invention is capable of inhibiting 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%.

[0103] In some embodiments, the antibody against CD100 of the present invention or its antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 sequences, and wherein the HCDR1 sequence differs from the sequence shown in SEQ ID NO:1, 7, 19, 33, 41, 47, or 52 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; the HCDR2 sequence differs from the sequence shown in SEQ ID NO:2, 8, 14, 20, 26, 42, 48, or 53 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; and / or the HCDR3 sequence differs from the sequence shown in SEQ ID NO:3, 9, 15, 21, 27, 30, 35, 38, 43, 49, or 54 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions.

[0104] In some embodiments, the antibody against CD100 of the present invention or its antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences, and wherein the LCDR1 sequence differs from the sequence shown in SEQ ID NO:4, 10, 16, 36, 44, or 50 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; the LCDR2 sequence differs from the sequence shown in SEQ ID NO:5, 11, or 45 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; and / or the LCDR3 sequence differs from the sequence shown in SEQ ID NO:6, 12, 18, 24, 37, 46, 51, or 56 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions.

[0105] In some embodiments, the antibody against CD100 of the present invention, or its antigen-binding fragment, comprises a heavy chain variable region and a light chain variable region, wherein

[0106] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1 sequence differs from the sequence shown in SEQ ID NO: 1, 7, 19, 33, 41, 47, or 52 by no more than two amino acid additions, deletions, or substitutions; the HCDR2 sequence differs from the sequence shown in SEQ ID NO: 2, 8, 14, 20, 26, 42, 48, or 53 by no more than two amino acid additions, deletions, or substitutions; and / or the HCDR3 sequence differs from the sequence shown in SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, or 54 by no more than two amino acid additions, deletions, or substitutions; and

[0107] The light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence differs from the sequence shown in SEQ ID NO:4, 10, 16, 36, 44, or 50 by no more than two amino acid additions, deletions, or substitutions; the LCDR2 sequence differs from the sequence shown in SEQ ID NO:5, 11, or 45 by no more than two amino acid additions, deletions, or substitutions; and / or the LCDR3 sequence differs from the sequence shown in SEQ ID NO:6, 12, 18, 24, 37, 46, 51, or 56 by no more than two amino acid additions, deletions, or substitutions.

[0108] In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:7 (SEQ ID NO:7; GFTFSSYSMN), wherein the 3rd amino acid is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:7 (SEQ ID NO:7; GFTFSSYSMN), wherein the 8th amino acid is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:7 (SEQ ID NO:7; GFTFSSYSMN), wherein the 3rd and 8th amino acids are substituted. In one embodiment, the 3rd amino acid in SEQ ID NO:7 is substituted with P, and the 8th amino acid in SEQ ID NO:7 is substituted with E (SEQ ID NO:13; GFPFSSYEMN).

[0109] In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:1 (SEQ ID NO:1; SGSFSGYYWT), wherein the first amino acid is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:1 (SEQ ID NO:1; SGSFSGYYWT), wherein the tenth amino acid is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:1 (SEQ ID NO:1; SGSFSGYYWT), wherein the first and tenth amino acids are substituted. In one embodiment, the first amino acid in SEQ ID NO:1 is substituted with G, and the tenth amino acid in SEQ ID NO:1 is substituted with S (SEQ ID NO:25; GGSFSGYYWS).

[0110] In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:33 (SEQ ID NO:33; GGSISSSNWWS), wherein the 6th amino acid is substituted. In another embodiment, the 6th amino acid in SEQ ID NO:33 is substituted with G (SEQ ID NO:57; GGSISGSNWWS).

[0111] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:26 (SEQ ID NO:26; EINHSGSTN), wherein the third amino acid is substituted. In another embodiment, the third amino acid in SEQ ID NO:26 is substituted with Y (SEQ ID NO:34; EIYHSGSTN).

[0112] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:26 (SEQ ID NO:26; EINHSGSTN), wherein the third amino acid is substituted. In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:26 (SEQ ID NO:26; EINHSGSTN), wherein the seventh amino acid is substituted. In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:26 (SEQ ID NO:26; EINHSGSTN), wherein the third and seventh amino acids are substituted. In one embodiment, the third amino acid in SEQ ID NO:26 is substituted with Y, and the seventh amino acid in SEQ ID NO:26 is substituted with E (SEQ ID NO:58; EIYHSGETN).

[0113] In one embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:35 (SEQ ID NO:35; YDFWSGSGLDY), wherein the fourth amino acid is substituted. In another embodiment, the fourth amino acid in SEQ ID NO:35 is substituted with E (SEQ ID NO:60; YDFESGSGLDY).

[0114] In one embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:3 (SEQ ID NO:3; GPAYYADALDGFDI), wherein the 6th amino acid is substituted. In another embodiment, the 6th amino acid in SEQ ID NO:3 is substituted with P (SEQ ID NO:63; GPAYYPDALDGFDI).

[0115] In one embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:3 (SEQ ID NO:3; GPAYYADALDGFDI), wherein the 5th amino acid is substituted. In another embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:3 (SEQ ID NO:3; GPAYYADALDGFDI), wherein the 6th amino acid is substituted. In yet another embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:3 (SEQ ID NO:3; GPAYYADALDGFDI), wherein the 5th and 6th amino acids are substituted. In yet another embodiment, the 5th amino acid in SEQ ID NO:3 is substituted with L, and the 6th amino acid in SEQ ID NO:3 is substituted with P (SEQ ID NO:66; GPAYLPDALDGFDI).

[0116] In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:16 (SEQ ID NO:16; SGDKLGDKYAY), wherein the 10th amino acid is substituted. In another embodiment, the 10th amino acid in SEQ ID NO:16 is substituted with V (SEQ ID NO:22; SGDKLGDKYVY).

[0117] In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:16 (SEQ ID NO:16; SGDKLGDKYAY), wherein the 7th amino acid is substituted. In another embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:16 (SEQ ID NO:16; SGDKLGDKYAY), wherein the 11th amino acid is substituted. In yet another embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:16 (SEQ ID NO:16; SGDKLGDKYAY), wherein the 7th and 11th amino acids are substituted. In one embodiment, the 7th amino acid in SEQ ID NO:16 is substituted with E, and the 11th amino acid in SEQ ID NO:16 is substituted with F (SEQ ID NO:31; SGDKLGEKYAF).

[0118] In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:16 (SEQ ID NO:16; SGDKLGDKYAY), wherein the 4th amino acid is substituted. In another embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:16 (SEQ ID NO:16; SGDKLGDKYAY), wherein the 10th amino acid is substituted. In yet another embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:16 (SEQ ID NO:16; SGDKLGDKYAY), wherein the 4th and 10th amino acids are substituted. In one embodiment, the 4th amino acid in SEQ ID NO:16 is substituted with R, and the 10th amino acid in SEQ ID NO:16 is substituted with S (SEQ ID NO:39; SGDRLGDKYSY).

[0119] In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:36 (SEQ ID NO:36; SGDKLGDKFAS), wherein the 7th amino acid is substituted. In another embodiment, the 7th amino acid in SEQ ID NO:36 is substituted with E (SEQ ID NO:61; SGDKLGEKFAS).

[0120] In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO:4 (SEQ ID NO:4; SGDKLGDKYVS), wherein the 6th amino acid is substituted. In another embodiment, the 6th amino acid in SEQ ID NO:4 is substituted with Q (SEQ ID NO:64; SGDKLQDKYVS).

[0121] In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the third amino acid is substituted. In another embodiment, the third amino acid in SEQ ID NO:5 is substituted with S (SEQ ID NO:17; QDSKRPS).

[0122] In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the third amino acid is substituted. In another embodiment, the third amino acid in SEQ ID NO:5 is substituted with R (SEQ ID NO:23; QDRKRPS).

[0123] In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the third amino acid is substituted. In another embodiment, the third amino acid in SEQ ID NO:5 is substituted with A (SEQ ID NO:28; QDAKRPS).

[0124] In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:11 (SEQ ID NO:11; AASSLQS), wherein the first amino acid is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:11 (SEQ ID NO:11; AASSLQS), wherein the fifth amino acid is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:11 (SEQ ID NO:11; AASSLQS), wherein the first and fifth amino acids are substituted. In one embodiment, the first amino acid in SEQ ID NO:11 is substituted with T, and the fifth amino acid in SEQ ID NO:11 is substituted with V (SEQ ID NO:55; TASSVQS).

[0125] In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the third amino acid is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the seventh amino acid is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:5 (SEQ ID NO:5; QDNKRPS), wherein the third and seventh amino acids are substituted. In one embodiment, the third amino acid in SEQ ID NO:5 is substituted with R, and the seventh amino acid in SEQ ID NO:5 is substituted with N (SEQ ID NO:59; QDRKRPN).

[0126] In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:6 (SEQ ID NO:6; QAWDSSTKAYV), wherein the 8th amino acid is substituted. In another embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:6 (SEQ ID NO:6; QAWDSSTKAYV), wherein the 9th amino acid is substituted. In yet another embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:6 (SEQ ID NO:6; QAWDSSTKAYV), wherein the 8th and 9th amino acids are substituted. In one embodiment, the 8th amino acid in SEQ ID NO:6 is substituted with A, and the 9th amino acid in SEQ ID NO:6 is substituted with G (SEQ ID NO:29; QAWDSSTAGYV).

[0127] In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:18 (SEQ ID NO:18; QAWDSSTV), wherein one amino acid is added between the 7th and 8th amino acids. In one embodiment, the amino acid added between the 7th and 8th amino acids in SEQ ID NO:18 is Y (SEQ ID NO:32; QAWDSSTYV).

[0128] In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:18 (SEQ ID NO:18; QAWDSSTV), wherein the second amino acid is substituted. In another embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:18 (SEQ ID NO:18; QAWDSSTV), wherein one amino acid is added between the seventh and eighth amino acids. In yet another embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:18 (SEQ ID NO:18; QAWDSSTV), wherein the second amino acid is substituted, and one amino acid is added between the seventh and eighth amino acids. In one embodiment, the second amino acid in SEQ ID NO:18 is substituted with V, and the amino acid added between the seventh and eighth amino acids in SEQ ID NO:18 is A (SEQ ID NO:40; QVWDSSTAV).

[0129] In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:37 (SEQ ID NO:37; QAWDSGTVI), wherein the 8th amino acid is substituted. In another embodiment, the 8th amino acid in SEQ ID NO:37 is substituted with A (SEQ ID NO:62; QAWDSGTAI).

[0130] In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO:6 (SEQ ID NO:6; QAWDSSTKAYV), wherein the 6th amino acid is substituted. In another embodiment, the 6th amino acid in SEQ ID NO:6 is substituted with E (SEQ ID NO:65; QAWDSETKAYV).

[0131] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:1, 7, 13, 19, 25, 33, 41, 47, 52 or 57; the HCDR2 sequence shown in SEQ ID NO:2, 8, 14, 20, 26, 34, 42, 48, 53 or 58; and the HCDR3 sequence shown in SEQ ID NO:3, 9, 15, 21, 27, 30, 35, 38, 43, 49, 54, 60, 63 or 66.

[0132] In one embodiment, the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; the LCDR2 sequence shown in SEQ ID NO:5, 11, 17, 23, 28, 45, 55 or 59; and the LCDR3 sequence shown in SEQ ID NO:6, 12, 18, 24, 29, 32, 37, 40, 46, 51, 56, 62 or 65.

[0133] In one embodiment, the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 1, 7, 13, 19, 25, 33, 41, 47, 52 or 57; the HCDR2 sequence shown in SEQ ID NO: 2, 8, 14, 20, 26, 34, 42, 48, 53 or 58; and the HCDR3 sequence shown in SEQ ID NO: 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, 54, 60, 63 or 66; and

[0134] The light chain variable region includes the LCDR1 sequence shown in SEQ ID NO:4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; the LCDR2 sequence shown in SEQ ID NO:5, 11, 17, 23, 28, 45, 55 or 59; and the LCDR3 sequence shown in SEQ ID NO:6, 12, 18, 24, 29, 32, 37, 40, 46, 51, 56, 62 or 65.

[0135] 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. In another 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.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. 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. In another 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.

[0136] In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In another embodiment, 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.In one embodiment, 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. In another embodiment, 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[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: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.

[0143] 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.

[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: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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[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: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.

[0152] In one embodiment, the heavy chain variable region comprises 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95; or 3 ... Compared to the amino acid sequences shown in NO:67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0153] In one embodiment, the light chain variable region comprises 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96; or 3 ... Compared to the amino acid sequences shown in NO:68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0154] In one embodiment, the heavy chain variable region comprises 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95; or 3 ... Compared to the amino acid sequences shown in NO:67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95, the amino acid sequences have one or more amino acid substitutions, additions, and / or deletions, preferably, the additions, deletions, and / or substitutions do not occur in the CDR region; and

[0155] The light chain variable region comprises: 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 96; or 3 ... Compared to the amino acid sequences shown in NO:68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0156] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:67; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:68.

[0157] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:69; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:70.

[0158] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:71; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:72.

[0159] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:73; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:74.

[0160] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:75; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:76.

[0161] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:77; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:78.

[0162] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:79; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:80.

[0163] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:81; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:82.

[0164] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:83; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:84.

[0165] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:85; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:86.

[0166] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:87; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:88.

[0167] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:89; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:90.

[0168] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:91; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:92.

[0169] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:93; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:94.

[0170] In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:95; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:96.

[0171] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain, the heavy chain comprising 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125; or 3 ... Compared to the amino acid sequences shown in NO:97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0172] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain comprising: 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; or 3 ... Compared to the amino acid sequences shown in NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0173] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain, the heavy chain comprising 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125; or 3 ... Compared to the amino acid sequences shown in NO:97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125, the amino acid sequences having one or more amino acid substitutions, additions, and / or deletions, preferably, the additions, deletions, and / or substitutions do not occur in the CDR region; and

[0174] The antibody or its antigen-binding fragment comprises a light chain comprising: 1) an amino acid sequence shown 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 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 with the amino acid sequence shown in SEQ ID NO: 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; or 3 ... Compared to the amino acid sequences shown in NO:98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, the amino acid sequences have one or more amino acid substitutions, additions and / or deletions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] In one embodiment, the anti-CD100 antibody includes 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, or B13-e-2, particularly anti-CD100 antibodies B13, C-C081, or B13-c-5.

[0191] Antibody B13

[0192] In one aspect, the present invention provides an antibody B13 or its antigen-binding fragment against CD100.

[0193] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0194] The heavy chain variable region includes:

[0195] HCDR1 contains the amino acid sequence shown in SEQ ID NO:1.

[0196] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:2, and

[0197] HCDR3 contains the amino acid sequence shown in SEQ ID NO:3;

[0198] The light chain variable region includes:

[0199] LCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0200] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:5, and

[0201] LCDR3 contains the amino acid sequence shown in SEQ ID NO:6.

[0202] In another embodiment, the anti-CD100 antibody B13 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0203] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0204] In another embodiment, the anti-CD100 antibody B13 or its antigen-binding fragment includes a light chain variable region (VL).

[0205] The light chain variable region comprises the amino acid sequence shown in 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.

[0206] In yet another embodiment, the anti-CD100 antibody B13 or its antigen-binding fragment contains a heavy chain.

[0207] The heavy chain comprises the amino acid sequence shown in 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.

[0208] In yet another embodiment, the anti-CD100 antibody B13 or its antigen-binding fragment comprises a light chain.

[0209] The light chain comprises the amino acid sequence shown in 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.

[0210] Antibody C-C081

[0211] In another aspect, the present invention provides an antibody C-C081 or an antigen-binding fragment thereof targeting CD100.

[0212] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0213] The heavy chain variable region includes:

[0214] HCDR1 contains the amino acid sequence shown in SEQ ID NO:7.

[0215] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:8, and

[0216] HCDR3 contains the amino acid sequence shown in SEQ ID NO:9;

[0217] The light chain variable region includes:

[0218] LCDR1 contains the amino acid sequence shown in SEQ ID NO:10.

[0219] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:11, and

[0220] LCDR3 contains the amino acid sequence shown in SEQ ID NO:12.

[0221] In one embodiment, the anti-CD100 antibody C-C081 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0222] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0223] In one embodiment, the anti-CD100 antibody C-C081 or its antigen-binding fragment includes a light chain variable region (VL).

[0224] The light chain variable region contains the amino acid sequence shown in 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.

[0225] In yet another embodiment, the anti-CD100 antibody C-C081 or its antigen-binding fragment contains a heavy chain.

[0226] The heavy chain comprises the amino acid sequence shown in 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.

[0227] In yet another embodiment, the anti-CD100 antibody C-C081 or its antigen-binding fragment comprises a light chain.

[0228] The light chain comprises the amino acid sequence shown in 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.

[0229] Antibody A14

[0230] In another aspect, the present invention provides an antibody A14 or an antigen-binding fragment thereof targeting CD100.

[0231] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0232] The heavy chain variable region includes:

[0233] HCDR1 contains the amino acid sequence shown in SEQ ID NO:13.

[0234] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:14, and

[0235] HCDR3 contains the amino acid sequence shown in SEQ ID NO:15;

[0236] The light chain variable region includes:

[0237] LCDR1 contains the amino acid sequence shown in SEQ ID NO:16.

[0238] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:17, and

[0239] LCDR3 contains the amino acid sequence shown in SEQ ID NO:18.

[0240] In one embodiment, the anti-CD100 antibody A14 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0241] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0242] In one embodiment, the anti-CD100 antibody A14 or its antigen-binding fragment includes a light chain variable region (VL).

[0243] The light chain variable region contains the amino acid sequence shown in 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.

[0244] In yet another embodiment, the anti-CD100 antibody A14 or its antigen-binding fragment contains a heavy chain.

[0245] The heavy chain comprises the amino acid sequence shown in 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.

[0246] In yet another embodiment, anti-CD100 antibody A14 or its antigen-binding fragment comprises a light chain.

[0247] The light chain comprises the amino acid sequence shown in 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.

[0248] Antibody A15

[0249] In another aspect, the present invention provides an antibody A15 or an antigen-binding fragment thereof targeting CD100.

[0250] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0251] The heavy chain variable region includes:

[0252] HCDR1 contains the amino acid sequence shown in SEQ ID NO:19.

[0253] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:20, and

[0254] HCDR3 contains the amino acid sequence shown in SEQ ID NO:21;

[0255] The light chain variable region includes:

[0256] LCDR1 contains the amino acid sequence shown in SEQ ID NO:22.

[0257] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:23, and

[0258] LCDR3 contains the amino acid sequence shown in SEQ ID NO:24.

[0259] In one embodiment, the anti-CD100 antibody A15 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0260] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0261] In one embodiment, the anti-CD100 antibody A15 or its antigen-binding fragment includes a light chain variable region (VL).

[0262] The light chain variable region contains the amino acid sequence shown in 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.

[0263] In yet another embodiment, the anti-CD100 antibody A15 or its antigen-binding fragment contains a heavy chain.

[0264] The heavy chain comprises the amino acid sequence shown in 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.

[0265] In yet another embodiment, anti-CD100 antibody A15 or its antigen-binding fragment comprises a light chain.

[0266] The light chain comprises the amino acid sequence shown in 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.

[0267] Antibody H74

[0268] In another aspect, the present invention provides an antibody H74 or an antigen-binding fragment thereof targeting CD100.

[0269] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0270] The heavy chain variable region includes:

[0271] HCDR1, which contains the amino acid sequence shown in SEQ ID NO:25,

[0272] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:26, and

[0273] HCDR3 contains the amino acid sequence shown in SEQ ID NO:27;

[0274] The light chain variable region includes:

[0275] LCDR1 contains the amino acid sequence shown in SEQ ID NO:16.

[0276] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:28, and

[0277] LCDR3 contains the amino acid sequence shown in SEQ ID NO:29.

[0278] In one embodiment, the anti-CD100 antibody H74 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0279] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0280] In one embodiment, the anti-CD100 antibody H74 or its antigen-binding fragment includes a light chain variable region (VL).

[0281] The light chain variable region contains the amino acid sequence shown in 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.

[0282] In yet another embodiment, the anti-CD100 antibody H74 or its antigen-binding fragment contains a heavy chain.

[0283] The heavy chain comprises the amino acid sequence shown in 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.

[0284] In yet another embodiment, the anti-CD100 antibody H74 or its antigen-binding fragment comprises a light chain.

[0285] The light chain comprises the amino acid sequence shown in 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.

[0286] Antibody H96

[0287] In another aspect, the present invention provides an antibody H96 or an antigen-binding fragment thereof targeting CD100.

[0288] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0289] The heavy chain variable region includes:

[0290] HCDR1, which contains the amino acid sequence shown in SEQ ID NO:25,

[0291] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:26, and

[0292] HCDR3 contains the amino acid sequence shown in SEQ ID NO:30;

[0293] The light chain variable region includes:

[0294] LCDR1 contains the amino acid sequence shown in SEQ ID NO:31.

[0295] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:5, and

[0296] LCDR3 contains the amino acid sequence shown in SEQ ID NO:32.

[0297] In one embodiment, the anti-CD100 antibody H96 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0298] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0299] In one embodiment, the anti-CD100 antibody H96 or its antigen-binding fragment includes a light chain variable region (VL).

[0300] The light chain variable region contains the amino acid sequence shown in 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.

[0301] In yet another embodiment, the anti-CD100 antibody H96 or its antigen-binding fragment contains a heavy chain.

[0302] The heavy chain comprises the amino acid sequence shown in 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.

[0303] In yet another embodiment, the anti-CD100 antibody H96 or its antigen-binding fragment comprises a light chain.

[0304] The light chain comprises the amino acid sequence shown in 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.

[0305] Antibody H5

[0306] In another aspect, the present invention provides an antibody H5 or an antigen-binding fragment thereof targeting CD100.

[0307] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0308] The heavy chain variable region includes:

[0309] HCDR1 contains the amino acid sequence shown in SEQ ID NO:33.

[0310] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:34, and

[0311] HCDR3 contains the amino acid sequence shown in SEQ ID NO:35;

[0312] The light chain variable region includes:

[0313] LCDR1 contains the amino acid sequence shown in SEQ ID NO:36.

[0314] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:23, and

[0315] LCDR3 contains the amino acid sequence shown in SEQ ID NO:37.

[0316] In one embodiment, the anti-CD100 antibody H5 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0317] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0318] In one embodiment, the anti-CD100 antibody H5 or its antigen-binding fragment includes a light chain variable region (VL).

[0319] The light chain variable region contains the amino acid sequence shown in 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.

[0320] In yet another embodiment, the anti-CD100 antibody H5 or its antigen-binding fragment contains a heavy chain.

[0321] The heavy chain comprises the amino acid sequence shown in 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.

[0322] In yet another embodiment, the anti-CD100 antibody H5 or its antigen-binding fragment comprises a light chain.

[0323] The light chain comprises the amino acid sequence shown in 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.

[0324] Antibody H12

[0325] In another aspect, the present invention provides an antibody H12 or an antigen-binding fragment thereof targeting CD100.

[0326] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0327] The heavy chain variable region includes:

[0328] HCDR1 contains the amino acid sequence shown in SEQ ID NO:33.

[0329] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:34, and

[0330] HCDR3 contains the amino acid sequence shown in SEQ ID NO:38;

[0331] The light chain variable region includes:

[0332] LCDR1 contains the amino acid sequence shown in SEQ ID NO:39.

[0333] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:17, and

[0334] LCDR3 contains the amino acid sequence shown in SEQ ID NO:40.

[0335] In one embodiment, the anti-CD100 antibody H12 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0336] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0337] In one embodiment, the anti-CD100 antibody H12 or its antigen-binding fragment includes a light chain variable region (VL).

[0338] The light chain variable region contains the amino acid sequence shown in 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.

[0339] In yet another embodiment, the anti-CD100 antibody H12 or its antigen-binding fragment contains a heavy chain.

[0340] The heavy chain comprises the amino acid sequence shown in 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.

[0341] In yet another embodiment, the anti-CD100 antibody H12 or its antigen-binding fragment comprises a light chain.

[0342] The light chain comprises the amino acid sequence shown in 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.

[0343] Antibody H21

[0344] In another aspect, the present invention provides an antibody H21 or an antigen-binding fragment thereof targeting CD100.

[0345] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0346] The heavy chain variable region includes:

[0347] HCDR1, which contains the amino acid sequence shown in SEQ ID NO:41,

[0348] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:42, and

[0349] HCDR3 contains the amino acid sequence shown in SEQ ID NO:43;

[0350] The light chain variable region includes:

[0351] LCDR1 contains the amino acid sequence shown in SEQ ID NO:44.

[0352] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:45, and

[0353] LCDR3 contains the amino acid sequence shown in SEQ ID NO:46.

[0354] In one embodiment, the anti-CD100 antibody H21 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0355] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0356] In one embodiment, the anti-CD100 antibody H21 or its antigen-binding fragment includes a light chain variable region (VL).

[0357] The light chain variable region contains the amino acid sequence shown in 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.

[0358] In yet another embodiment, the anti-CD100 antibody H21 or its antigen-binding fragment contains a heavy chain.

[0359] The heavy chain comprises the amino acid sequence shown in 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.

[0360] In yet another embodiment, the anti-CD100 antibody H21 or its antigen-binding fragment comprises a light chain.

[0361] The light chain comprises the amino acid sequence shown in 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.

[0362] Antibody C-C171

[0363] In another aspect, the present invention provides an antibody C-C171 or an antigen-binding fragment thereof targeting CD100.

[0364] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0365] The heavy chain variable region includes:

[0366] HCDR1 contains the amino acid sequence shown in SEQ ID NO:47.

[0367] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:48, and

[0368] HCDR3 contains the amino acid sequence shown in SEQ ID NO:49;

[0369] The light chain variable region includes:

[0370] LCDR1 contains the amino acid sequence shown in SEQ ID NO:50.

[0371] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:11, and

[0372] LCDR3 contains the amino acid sequence shown in SEQ ID NO:51.

[0373] In one embodiment, the anti-CD100 antibody C-C171 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0374] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0375] In one embodiment, the anti-CD100 antibody C-C171 or its antigen-binding fragment includes a light chain variable region (VL).

[0376] The light chain variable region contains the amino acid sequence shown in 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.

[0377] In yet another embodiment, the anti-CD100 antibody C-C171 or its antigen-binding fragment contains a heavy chain.

[0378] The heavy chain comprises the amino acid sequence shown in 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.

[0379] In yet another embodiment, the anti-CD100 antibody C-C171 or its antigen-binding fragment comprises a light chain.

[0380] The light chain comprises the amino acid sequence shown in 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.

[0381] Antibody C-B71

[0382] In another aspect, the present invention provides an antibody C-B71 or an antigen-binding fragment thereof targeting CD100.

[0383] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0384] The heavy chain variable region includes:

[0385] HCDR1 contains the amino acid sequence shown in SEQ ID NO:52.

[0386] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:53, and

[0387] HCDR3 contains the amino acid sequence shown in SEQ ID NO:54;

[0388] The light chain variable region includes:

[0389] LCDR1 contains the amino acid sequence shown in SEQ ID NO:50.

[0390] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:55, and

[0391] LCDR3 contains the amino acid sequence shown in SEQ ID NO:56.

[0392] In one embodiment, the anti-CD100 antibody C-B71 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0393] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0394] In one embodiment, the anti-CD100 antibody C-B71 or its antigen-binding fragment includes a light chain variable region (VL).

[0395] The light chain variable region contains the amino acid sequence shown in 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.

[0396] In yet another embodiment, the anti-CD100 antibody C-B71 or its antigen-binding fragment contains a heavy chain.

[0397] The heavy chain comprises the amino acid sequence shown in 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.

[0398] In yet another embodiment, the anti-CD100 antibody C-B71 or its antigen-binding fragment comprises a light chain.

[0399] The light chain comprises the amino acid sequence shown in 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.

[0400] Antibody H5-h-7

[0401] In another aspect, the present invention provides an antibody H5-h-7 or an antigen-binding fragment thereof targeting CD100.

[0402] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0403] The heavy chain variable region includes:

[0404] HCDR1 contains the amino acid sequence shown in SEQ ID NO:57.

[0405] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:58, and

[0406] HCDR3 contains the amino acid sequence shown in SEQ ID NO:35;

[0407] The light chain variable region includes:

[0408] LCDR1 contains the amino acid sequence shown in SEQ ID NO:36.

[0409] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:59, and

[0410] LCDR3 contains the amino acid sequence shown in SEQ ID NO:37.

[0411] In one embodiment, the anti-CD100 antibody H5-h-7 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0412] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0413] In one embodiment, the anti-CD100 antibody H5-h-7 or its antigen-binding fragment includes a light chain variable region (VL).

[0414] The light chain variable region contains the amino acid sequence shown in 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.

[0415] In yet another embodiment, the anti-CD100 antibody H5-h-7 or its antigen-binding fragment contains a heavy chain.

[0416] The heavy chain comprises the amino acid sequence shown in 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.

[0417] In yet another embodiment, the anti-CD100 antibody H5-h-7 or its antigen-binding fragment contains a light chain.

[0418] The light chain comprises the amino acid sequence shown in 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.

[0419] Antibody H5-a-2

[0420] In another aspect, the present invention provides an antibody H5-a-2 or an antigen-binding fragment thereof targeting CD100.

[0421] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0422] The heavy chain variable region includes:

[0423] HCDR1 contains the amino acid sequence shown in SEQ ID NO:33.

[0424] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:34, and

[0425] HCDR3 contains the amino acid sequence shown in SEQ ID NO:60;

[0426] The light chain variable region includes:

[0427] LCDR1 contains the amino acid sequence shown in SEQ ID NO:61.

[0428] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:23, and

[0429] LCDR3 contains the amino acid sequence shown in SEQ ID NO:62.

[0430] In one embodiment, the anti-CD100 antibody H5-a-2 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0431] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0432] In one embodiment, the anti-CD100 antibody H5-a-2 or its antigen-binding fragment includes a light chain variable region (VL).

[0433] The light chain variable region contains the amino acid sequence shown in 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.

[0434] In yet another embodiment, the anti-CD100 antibody H5-a-2 or its antigen-binding fragment contains a heavy chain.

[0435] The heavy chain comprises the amino acid sequence shown in 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.

[0436] In yet another embodiment, the anti-CD100 antibody H5-a-2 or its antigen-binding fragment comprises a light chain.

[0437] The light chain comprises the amino acid sequence shown in 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.

[0438] Antibody B13-c-5

[0439] In another aspect, the present invention provides an antibody B13-c-5 or an antigen-binding fragment thereof targeting CD100.

[0440] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0441] The heavy chain variable region includes:

[0442] HCDR1 contains the amino acid sequence shown in SEQ ID NO:1.

[0443] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:2, and

[0444] HCDR3 contains the amino acid sequence shown in SEQ ID NO:63;

[0445] The light chain variable region includes:

[0446] LCDR1 contains the amino acid sequence shown in SEQ ID NO:64.

[0447] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:5, and

[0448] LCDR3 contains the amino acid sequence shown in SEQ ID NO:65.

[0449] In one embodiment, the anti-CD100 antibody B13-c-5 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0450] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0451] In one embodiment, the anti-CD100 antibody B13-c-5 or its antigen-binding fragment includes a light chain variable region (VL).

[0452] The light chain variable region contains the amino acid sequence shown in 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.

[0453] In yet another embodiment, the anti-CD100 antibody B13-c-5 or its antigen-binding fragment contains a heavy chain.

[0454] The heavy chain comprises the amino acid sequence shown in 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.

[0455] In yet another embodiment, the anti-CD100 antibody B13-c-5 or its antigen-binding fragment comprises a light chain.

[0456] The light chain comprises the amino acid sequence shown in 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.

[0457] Antibody B13-e-2

[0458] In another aspect, the present invention provides an antibody B13-e-2 or its antigen-binding fragment against CD100.

[0459] The antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein

[0460] The heavy chain variable region includes:

[0461] HCDR1 contains the amino acid sequence shown in SEQ ID NO:1.

[0462] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:2, and

[0463] HCDR3 contains the amino acid sequence shown in SEQ ID NO:66;

[0464] The light chain variable region includes:

[0465] LCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0466] LCDR2, which contains the amino acid sequence shown in SEQ ID NO:5, and

[0467] LCDR3 contains the amino acid sequence shown in SEQ ID NO:6.

[0468] In one embodiment, the anti-CD100 antibody B13-e-2 or its antigen-binding fragment includes a heavy chain variable region (VH).

[0469] The heavy chain variable region comprises the amino acid sequence shown in 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.

[0470] In one embodiment, the anti-CD100 antibody B13-e-2 or its antigen-binding fragment includes a light chain variable region (VL).

[0471] The light chain variable region contains the amino acid sequence shown in 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.

[0472] In yet another embodiment, the anti-CD100 antibody B13-e-2 or its antigen-binding fragment contains a heavy chain.

[0473] The heavy chain comprises the amino acid sequence shown in 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.

[0474] In yet another embodiment, the anti-CD100 antibody B13-e-2 or its antigen-binding fragment comprises a light chain.

[0475] The light chain comprises the amino acid sequence shown in 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.

[0476] Nucleic acid molecules, vectors, and host cells

[0477] In another aspect, the present invention provides a nucleic acid molecule comprising a polynucleotide sequence encoding the anti-CD100 antibody of the present invention or an antigen-binding fragment thereof. In some embodiments, the nucleic acid molecule of the present invention encodes the anti-CD100 antibody of the present invention or an antigen-binding fragment thereof.

[0478] The nucleic acid molecules of the present invention can be obtained using methods known in the art. For example, the nucleic acid molecules of the present invention can be isolated from phage display libraries, yeast display libraries, immunized animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemically synthesized. The nucleic acid molecules of the present invention can be codon-optimized for the host cells used for expression.

[0479] In another aspect, the present invention also provides expression vectors comprising the nucleic acid molecules of the present invention. The expression vectors may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The nucleic acid molecules of the present invention may be present in one or more expression vectors. In one embodiment, the nucleic acid molecules of the present invention are prepared as recombinant nucleic acids. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR) techniques), etc.

[0480] This invention also provides a host cell containing the nucleic acid molecule or expression vector of this invention. The nucleic acid molecule or expression vector of this invention can be introduced into a suitable host cell using various methods known in the art. These methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.

[0481] In a preferred embodiment, the host cell is used to express the anti-CD100 antibody of the present invention or its antigen-binding fragment. 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). Suitable mammalian host cells for antibody expression include, but are not limited to, exogenous human cervical cancer 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.

[0482] The present invention also provides a method for generating the anti-CD100 antibody or its antigen-binding fragment of the present invention, comprising the following steps:

[0483] a) Culturing the host cells of the present invention under suitable conditions to express the anti-CD100 antibody or its antigen-binding fragment of the present invention; and

[0484] b) Isolate the antibody or its antigen-binding fragment from a host cell or its culture.

[0485] Drug combination

[0486] In another aspect, the present invention provides a drug combination that can be used to administer two or more therapeutic or prophylactic agents to a subject. The drug combination comprises an antibody against CD100 or an antigen-binding fragment thereof and an antibody against PD-L1 or an antigen-binding fragment thereof.

[0487] In one embodiment, the antibody against CD100 includes 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 combinations thereof.

[0488] In one embodiment, the antibody against PD-L1 or its antigen-binding fragment specifically recognizes and binds to PD-L1, wherein the anti-PD-L1 antibody or its antigen-binding fragment comprises an immunoglobulin monovariable domain.

[0489] In a preferred embodiment, the immunoglobulin monovariable domain comprises:

[0490] CDR1 contains the amino acid sequence shown in SEQ ID NO:130.

[0491] CDR2, which contains the amino acid sequence shown in SEQ ID NO:131, and

[0492] CDR3 contains the amino acid sequence shown in SEQ ID NO:132.

[0493] 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.

[0494] In one embodiment, the anti-PD-L1 antibody or its antigen-binding fragment further comprises an Fc fragment of human IgG1.

[0495] In a preferred embodiment, the anti-PD-L1 antibody or its antigen-binding fragment 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.

[0496] In one embodiment, the anti-PD-L1 antibody includes anti-PD-L1 antibody m18-VHH or m18.

[0497] Antibody m18-VHH

[0498] In one aspect, the present invention provides an antibody m18-VHH or its antigen-binding fragment targeting PD-L1.

[0499] The antibody described herein comprises a single variable domain of an immunoglobulin, wherein

[0500] The single variable structural domain includes:

[0501] CDR1 contains the amino acid sequence shown in SEQ ID NO:130.

[0502] CDR2, which contains the amino acid sequence shown in SEQ ID NO:131, and

[0503] CDR3 contains the amino acid sequence shown in SEQ ID NO:132.

[0504] In one embodiment, the anti-PD-L1 antibody m18-VHH contains a single variable domain of an immunoglobulin.

[0505] The single variable domain comprises the amino acid sequence shown in 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.

[0506] Antibody m18

[0507] In another aspect, the present invention also provides an antibody m18 or an antigen-binding fragment thereof targeting anti-PD-L1.

[0508] The antibody described herein comprises a single variable domain of an immunoglobulin, wherein

[0509] The single variable structural domain includes:

[0510] CDR1 contains the amino acid sequence shown in SEQ ID NO:130.

[0511] CDR2, which contains the amino acid sequence shown in SEQ ID NO:131, and

[0512] CDR3 contains the amino acid sequence shown in SEQ ID NO:132.

[0513] In one embodiment, the anti-PD-L1 antibody m18 comprises a single variable domain of an immunoglobulin.

[0514] The single variable domain comprises the amino acid sequence shown in 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.

[0515] In one embodiment, the anti-PD-L1 antibody m18 further comprises an Fc fragment of human IgG1.

[0516] 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.

[0517] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment of the present invention is a single-domain antibody, a heavy-chain antibody, a humanized antibody, or a chimeric antibody. Preferably, the antibody or its antigen-binding fragment is a human antibody.

[0518] In one embodiment, the antibody or its antigen-binding fragment has at least one of the following characteristics:

[0519] 1) It has affinity activity for PD-L1 positive cells;

[0520] 2) It can specifically bind to the PD-L1 protein;

[0521] 3) Inhibit tumor growth.

[0522] In some embodiments, the targeted tumors include, but are not limited to, those described below with respect to neoplastic diseases. In other embodiments, the antibody or antigen-binding fragment of the present invention is capable of inhibiting 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%.

[0523] In one embodiment, the drug combination comprises an antibody against CD100, which includes 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 combinations thereof; and the drug combination comprises an antibody against PD-L1, which includes antibody m18-VHH or m18.

[0524] In a preferred embodiment, the drug combination comprises anti-CD100 antibody 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 antibody m18-VHH or m18.

[0525] In a preferred embodiment, the drug combination comprises an anti-CD100 antibody C-C081, B13 or B13-c-5 and an anti-PD-L1 antibody m18-VHH or m18.

[0526] In one embodiment, the drug combination may be a pharmaceutical composition or a kit.

[0527] Pharmaceutical Composition

[0528] The present invention also provides a pharmaceutical composition comprising the anti-CD100 antibody of the present invention or an antigen fragment thereof, and a pharmaceutically acceptable carrier. In one embodiment, the anti-CD100 antibody 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 combinations thereof, particularly anti-CD100 antibodies 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 antibody 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 antibody m18 or m18-VHH. In another preferred embodiment, the pharmaceutical composition comprises a combination of anti-CD100 antibodies B13, C-C081, or B13-c-5 and anti-PD-L1 antibody m18 or m18-VHH.

[0529] The pharmaceutical compositions described herein can be in various dosage forms, including but not limited to solid, semi-solid, liquid, powder, or lyophilized forms. For compositions containing antibodies or antigen fragments thereof, preferred dosage forms are typically, for example, injection solutions and lyophilized powders.

[0530] The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, such as systemic or local administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavitary), local (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or local). Preferably, the pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Administration methods may include, for example, injection or infusion.

[0531] Those skilled in the art will understand that the exact dosage will depend on various factors, such as the pharmacokinetic properties of the pharmaceutical composition, the duration of treatment, the excretion rate of a particular compound, the therapeutic purpose, the route of administration, and the condition of the subject, such as the patient's age, health status, weight, sex, diet, medical history, and other factors known in the medical field. As a general guideline, the dosage range for the anti-CD100 antibody or its antigen-binding fragment of the present invention is about 0.0001-100 mg / kg, more typically 0.01-20 mg / kg of subject weight. For example, the dosage may 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 may require weekly dosing, every two weeks, every three weeks, every four weeks, monthly, every three months, every three to six months, or a slightly shorter initial dosing interval followed by a longer dosing interval. In one embodiment, the dosage used may be 1200 mg every three weeks. The administration method can be intravenous infusion.

[0532] The term "therapeutic effective dose" as used herein refers to a dose that reduces the severity of disease symptoms, increases the frequency and duration of asymptomatic periods, or prevents damage or disability caused by disease-related suffering. For example, a therapeutically effective dose may be used for antiproliferative effects, preventing further tumor development, reducing tumor size, reducing tumor angiogenesis, 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 observable as associated with the disease. Therapeutic effective doses can vary depending on many different factors, including the route of administration, target site, patient's physiological state, whether the patient is human or other animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. In some embodiments, the patient is human, but non-human mammals, including transgenic animals, may also be treated. Therapeutic doses can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0533] Therapeutic doses of the antibody or antigen-binding fragment of the present invention preferably inhibit cell 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 animal model systems used to predict the efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro using experiments known to those skilled in the art. Effective doses of the antibody or antigen-binding fragment of the present invention can reduce tumor size or otherwise alleviate symptoms in the subject, such as prevention and / or treatment of metastasis or recurrence. Those skilled in the art can determine this dose 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 chosen.

[0534] treat

[0535] In another aspect, the present invention relates to the use of the anti-CD100 antibody of the present invention or its antigen-binding fragment, pharmaceutical composition or combination thereof in the preparation of a medicament for treating a disease in a subject.

[0536] This invention also relates to the anti-CD100 antibody or its antigen-binding fragment, pharmaceutical composition or combination thereof, for the treatment of diseases.

[0537] The present invention also provides a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-CD100 antibody of the present invention or its antigen-binding fragment, pharmaceutical composition or combination of pharmaceuticals.

[0538] In one embodiment, the disease described above is cancer. Blocking CD100 with the antibody of the present invention can enhance the immune response against cancer cells in patients. CD100 is widely expressed in many human tumors, and its expression is associated with aggressive human disease. In the preclinical tumor microenvironment, inflammatory cells and tumor cells express CD100, regulating the infiltration, spatial distribution, and activity of myeloid cells and lymphocytes. CD100 binds to the Plexin receptor located on myeloid cells in the tumor microenvironment. When the CD100 protein is blocked, the CD100 barrier is eliminated. Once the barrier is breached, inflammatory dendritic cells and pro-inflammatory antigen-presenting cells migrate and infiltrate into the tumor. In preclinical animal models of cancer, blocking CD100 with antibodies can delay tumor growth and promote a durable tumor rejection response.

[0539] As used herein, “cancer” includes, but is not limited to, hematologic malignancies and solid tumors. In this context, solid tumors include, for example, squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, renal cell carcinoma, ductal carcinoma of the breast, 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 carcinoma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatocellular carcinoma, 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 any combination thereof. Hematologic malignancies include, for example, leukemia, lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, etc., or any combination thereof. Cancer can also be metastatic. "Metastasis" refers to the spread of cancer cells from their original site to other parts of the body.

[0540] Combination therapy

[0541] For cancer treatment, the anti-CD100 antibody or its antigen-binding fragment, pharmaceutical composition or combination of drugs 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 treatment.

[0542] The anti-CD100 antibody or its antigen-binding fragment, pharmaceutical composition, or combination thereof of the present invention can also be administered in combination with at least one or more of the therapeutic agents described herein. There are no limitations on the manner of administration. For example, all of the following therapeutic agents can be administered at once or separately. When administered separately (using different administration regimens), they can be administered continuously without interruption or at predetermined intervals.

[0543] In some embodiments, the anti-CD100 antibody or its antigen-binding fragment, pharmaceutical composition, or combination of pharmaceuticals of the present invention is further administered in combination with one or more therapeutic agents selected from: chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen-targeting drugs. Chemootherapeutic agents may include, for example, antimetabolites, alkylating agents, cytotoxic agents, topoisomerase inhibitors, and microtubule inhibitors. Tumor antigen-targeting drugs include, but are not limited to, drugs that target tumor-associated antigens and tumor-specific antigens. Other non-limiting examples of therapeutic agents may 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, oprazomib), and tyrosine kinase inhibitors (e.g., BTK inhibitors).

[0544] In one implementation scheme, 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, and B13-e-2 are administered in combination with anti-PD-L1 antibody m18 or m18-VHH.

[0545] In a preferred embodiment, anti-CD100 antibodies B13, C-C081, C-B71, H5-h-7, H5-a-2, B13-c-5, or B13-e-2 are administered in combination with anti-PD-L1 antibody m18 or m18-VHH.

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

[0547] In some embodiments, the anti-CD100 antibody or its antigen-binding fragment, pharmaceutical composition, or combination of drugs of the present invention is administered in combination with a chemotherapeutic agent. In some embodiments, the anti-CD100 antibody or its antigen-binding fragment, pharmaceutical composition, or combination of drugs of the present invention is administered in combination with an immune checkpoint inhibitor. In some embodiments, the anti-CD100 antibody or its antigen-binding fragment, pharmaceutical composition, or combination of drugs of the present invention is administered in combination with a radioisotope. In some embodiments, the anti-CD100 antibody or its antigen-binding fragment, pharmaceutical composition, or combination of drugs of the present invention is administered in combination with a tumor-targeting drug.

[0548] Reagent test kit

[0549] The present invention also provides a kit comprising the anti-CD100 antibody of the present invention or an antigen-binding fragment thereof, a pharmaceutical composition or combination of pharmaceuticals, and instructions for use. The kit may also comprise a suitable container. In some embodiments, the kit further comprises a device for administration. The kit generally includes a label indicating the intended use and / or method of use of the kit contents. The term “label” includes any written or recorded material provided on or with the kit or otherwise accompanied by the kit.

[0550] 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 combinations thereof.

[0551] 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 combinations thereof.

[0552] In one embodiment, the kit contains anti-CD100 antibody C-C081, B13, or B13-c-5.

[0553] In one embodiment, the kit 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 antibody m18 or m18-VHH.

[0554] 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.

[0555] 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.

[0556] Beneficial effects

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

[0558] The combination of the anti-CD100 antibody or its antigen-binding fragment with the anti-PD-L1 antibody or its antigen-binding fragment of the present invention 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) high safety.

[0559] Example

[0560] A further understanding of the invention can be obtained by referring to the specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of the invention in any way. Obviously, many modifications and variations can be made to the invention without departing from its spirit, and therefore, such modifications and variations are also within the scope of protection claimed herein. All proportions used herein include percentages, and unless otherwise specified, are by weight.

[0561] Example 1: Raw Material Preparation and Identification

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

[0563] Preparation of anti-CD100 control antibodies: Pepinemab, an anti-CD100 antibody, and 2D5 and 5D8 antibodies from Shanghai Pioneer Pharmaceuticals were used as positive control antibodies. The coding gene sequences for Pepinemab monoclonal antibody and 2D5 and 5D8 antibodies were synthesized by General Biotechnology Co., Ltd. according to the sequences disclosed in WO2013148854A1 and patent WO2020011275. Then, the eukaryotic expression vector pcDNA3.4 (Invitrogen) was constructed using homologous recombination. The constructed recombinant protein expression vector was transformed into E. coli DH5α and cultured overnight at 37°C. Plasmid extraction was then performed 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. The expression plasmids were analyzed using ExpiFectamine. TMThe CHO transfection kit (Thermo Fisher, A29129) was used to transfect CHO cells with the expression plasmid to express Pepinemab and 2D5 and 5D8 monoclonal antibodies, following the manufacturer's instructions. Seven days after transfection, the cell culture supernatant was collected and centrifuged at 15000g for 10 min. The resulting supernatant was filtered through a 0.22 μm filter and the antibodies in the supernatant were 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 transferred to PBS buffer via 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 IgG4SP) was detected using purchased Human Semaphorin 4D / SEMA4D / CD100Protein,Fc Tag antigen protein (Acro, CD0-H5257).

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

[0566] The results are as follows Figure 1 As shown, 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, a human Fc (SEQ ID NO: 135) or His tag was added to the C-terminus of the amino acid sequences of the following fragments: human CD100 protein ECD region (22-734, HuCD100, Uniprot ID: Q92854), mouse CD100 protein ECD region (24-733, MusCD100, Uniprot ID: O09126), and cynomolgus monkey CD100 protein ECD region (22-734, CynoCD100, Uniprot ID: A0A2K5TZC9). The obtained nucleic acid sequences were constructed into the pcDNA3.4 vector, transformed into *E. coli* DH5α, and cultured overnight at 37°C. Plasmids were then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The obtained plasmids were analyzed using ExpiFectamine. TM 293 Transfection Kit (Gibco) TM A14524) was transiently transfected into HEK293 cells. CRL-1573 TM After 7 days of expression, the cell culture supernatant was collected. For proteins containing the Fc tag, the cell suspension was centrifuged at high speed after culture and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter and purified by affinity chromatography using a Protein A / G column (MabSelectSuRe, Cytiva, 17543802). The target protein was eluted with 100 mM glycine hydrochloride (pH 3.0), concentrated, buffer-replaced, aliquoted, identified by SDS-PAGE, and its activity was tested before being stored frozen. Proteins containing the His tag were purified by affinity chromatography using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), followed by elution with an imidazole gradient. The eluted proteins were transferred to PBS buffer via ultrafiltration concentrators (Millipore, UFC901096) to obtain human, mouse, and monkey CD100 antigen proteins (HuCD100-Fc, MusCD100-Fc, CynoCD100-Fc, HuCD100-His, MusCD100-His, and CynoCD100-His) tagged with Fc and His tags, respectively.

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

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

[0571] The results are as follows Figure 2 As shown, the affinity activity of the antibody Pepinemab for the self-made antigen is comparable to that for the commercially available CD100 antigen protein. The two HuCD100-Fc antigens are, in order, the self-made HuCD100 antigen and the commercially available 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, and CynoCD100-HEK293): The coding nucleic acid sequences of the full-length human CD100 protein HuCD100 (Uniprot ID: Q92854), the full-length mouse CD100 protein MusCD100 (Uniprot ID: O09126), and the full-length cynomolgus monkey CD100 protein CynoCD100 (Uniprot ID: A0A2K5TZC9) were constructed into the pLVX-puro plasmid (Clontech, catalog number 632164). Then, the obtained plasmids were electroporated into HEK293 cells using an Invitrogen, Neon™ Transfection System, MP922947. CRL-1573 TMAfter electroporation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) and free of antibiotics. The cells were then cultured in 10×10 cm cell culture dishes for 48 hours, followed by inoculation at an average rate of 10... 4 Cells were aliquoted into 96-well cell culture plates at a density of 1 cell / well, and puromycin was added to a final concentration of 2 μg / mL as a selection pressure. Cell lines that formed clones were picked for identification after about 2 weeks.

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

[0576] Test results as follows Figure 3 As shown, the results indicate that the 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), and full-length human Plexin-B2 protein HuPlexin-B2 (Uniprot ID: A0A1D5QMB8). The encoding nucleic acid sequence of ID: O15031 was constructed onto the pLVX-puro plasmid (Clontech, catalog number 632164). The construction method is as described in Example 2.1.

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

[0580] Test results as follows Figures 4A-4D As shown, where Figure 4A This is a schematic diagram showing the results of identifying the HuPlexin-B1-HEK293 cell line using human CD100 protein. Figure 4B This is a schematic diagram illustrating the results of identifying the MusPlexin-B1-HEK293 cell line using mouse CD100 protein. Figure 4C This is a schematic diagram illustrating the results of identifying the CynoPlexin-B1-HEK293 cell line using monkey CD100 protein. Figure 4D This is a schematic diagram showing 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, respectively.

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

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

[0583] 3.1 Construction of a gene library of human antibodies

[0584] Peripheral blood mononuclear cells (PBMCs) from normal human blood were isolated using Ficoll-Paque density gradient separation buffer (GE, catalog number: 17144003S). Total RNA was extracted from the isolated PBMCs using standard methods. The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, catalog number: 6210A) according to the manufacturer's instructions. Based on the sequence similarity of the heavy and light chain germline genes, degenerate primers were designed at the front end of the V region and the back end of the first constant region of the heavy and light chains, respectively. PCR yielded the antibody's heavy chain variable region gene fragment and light chain variable region gene fragment. Fragments containing antibody-containing light and heavy chain variable regions were amplified using fusion PCR. The PCR product and phage display vector were then digested, recovered, and ligated. The ligation 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 transformed E. coli SS320 (Lucigen, MC1061F) was transformed into competent E. coli SS320 using an electroporator (Bio-Rad, MicroPulser). The transformed E. coli SS320 culture was then plated onto ampicillin-resistant 2-YT solid plates (the solid plates were prepared from 1.5% tryptone, 1% yeast extract, 0.5% NaCl, and 1.5% agar, at a mass / volume ratio of g / mL). The library size was determined to be 3 × 10⁻⁶ cells / mL through serial dilution plating. 11 CFU, i.e., 3 × 10 11 An antibody gene library of 1 antibody gene was prepared (the library size calculation method is described in Example 2.2 of CN112250763A). The library was packaged using VSCM13 helper phage (purchased from Stratagene) to obtain an antibody gene phage display library (the preparation of the antibody gene phage display library is described in Example 2.3 of CN112250763A).

[0585] 3.2 Screening of antibody gene phage display libraries

[0586] 3.2.1 Screening of antibody gene phage display libraries using magnetic bead method

[0587] Magnetic bead screening involves biotin-labeling antigen proteins and then binding them to magnetic beads conjugated with streptavidin. The process involves incubating, washing, and eluting the antigen-bound magnetic beads and antibody gene phage display libraries. Typically, 3-4 rounds of screening are performed, resulting in a large enrichment of antigen-specific monoclonal antibodies. In this example, biotin-labeled antigen proteins HuCD100-Fc, CynoCD100-Fc, HuCD100-His, and MusCD100-His were used for phage display library screening. After three rounds of screening, a preliminary screening for monoclonal antibodies against human CD100 (Fab) was conducted, following the specific method described in Example 2.4.1 of CN112250763A.

[0588] 3.2.2 Screening of antibody gene phage display libraries using the immunotube method

[0589] Immunotube screening involves coating the antigen proteins HuCD100-Fc, CynoCD100-Fc, HuCD100-His, and MusCD100-His onto the surface of an immunotube with high adsorption capacity. A phage display antibody library is added to the immunotube, and the protein adsorbed on the immunotube surface is incubated, washed, and eluted. After 2-4 rounds of panning, specific monoclonal antibodies (Fab) against the antigen are finally enriched. In this example, after 3 rounds of panning, monoclonal antibody Fab against human CD100 was enriched. The specific method is described in Example 2.4.2 of CN112250763A.

[0590] 3.3 Selection of Monoclonal Cells

[0591] The enrichment effect was evaluated by ELISA detection of the phage pools eluted in each round. Ten clones were randomly selected from each round of phage pools for sequence analysis. The enrichment effect and the reproducibility ratio of the measured sequences were combined to select an appropriate round for single clone selection.

[0592] The ELISA monoclonal screening used the antigen proteins HuCD100-His, MusCD100-His, and CynoCD100-His. The antibodies Fab that bound HuCD100-His, MusCD100-His, and CynoCD100-His obtained from the initial screening were prepared into Fab lysis buffers. Then, the overexpressing cells HuCD100-HEK293, MusCD100-HEK293, and CynoCD100-HEK293 prepared in Example 2.1 were used for detection and verification by flow cytometry (FACS). A total of 11 antibody Fab molecules that specifically bind to human CD100 were screened. The 11 antibody Fab strains obtained were named with their corresponding clone numbers (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, C-B71). The amino acid sequence of the variable region of the obtained antibody is shown in Table 1. The CDR sequence was determined using the AbM definition method.

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

[0594]

[0595] Example 4 Antibody Construction, Expression and Purification

[0596] 4.1 Plasmid Construction

[0597] The VH coding sequences in the Fab sequences of the selected monoclonal antibodies A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, and C-B71 were ligated to the coding sequence of the heavy chain constant region (SEQ ID NO: 127) of human IgG4SP to obtain the heavy chain coding sequence of the fully human antibody. The VL coding sequences in the Fab sequences were ligated to the coding sequences of the Kappa type (SEQ ID NO: 128) or Lambda type (SEQ ID NO: 129) of the human light chain constant region (CL) to obtain the light chain coding sequence of the fully human antibody. The antibody heavy and light chain coding sequences were inserted into the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), transformed into *E. 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.

[0598] 4.2 Antibody Expression and Purification

[0599] According to ExpiCHO TMFollowing the user manual for the Expression System, the full-length antibody sequence obtained above was expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133) kit. The specific method is as follows: On the day of transfection, confirm that the CHO cell density is 7 × 10⁶ cells / year. 6 Up to 1×10 7 With approximately 100 viable cells / mL and a cell viability >98%, the cells were adjusted to a final concentration of 6 × 10⁶ cells / mL using fresh ExpiCHO expression medium pre-warmed to 37°C. 6 Cells / mL. OptiPRO pre-cooled to 4°C TM SFM dilution of the target plasmid (add 1 μg plasmid to 1 mL of the culture medium described above), and simultaneous use of OptiPRO TM SFM diluted ExpiFectamine TM CHO reagent, then mix the two in equal volumes and gently blow to mix thoroughly to prepare ExpiFectamine. TM CHO / plasmid DNA mixture. Incubate the 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 and 8% CO2.

[0600] 18-22 hours after transfection, add ExpiCHO to the cell culture medium. TM Enhancer reagent and ExpiCHO TM Feed the reagent, and incubate the shake flask at 32°C on a shaker with 5% CO2. On day 5 post-transfection, add the same volume of ExpiCHO. TM Feed the reagent slowly while gently mixing the cell suspension. Seven days after transfection, collect the cell culture supernatant expressing the target antibody protein and centrifuge at 15000g for 10 min. The obtained supernatant is affinity purified using MabSelect SuRe LX (GE, 17547403), then the target antibody protein is eluted with 100mM sodium acetate (pH 3.0), followed by neutralization with 1M Tris-HCl. Finally, the obtained antibody protein is transferred to PBS buffer via ultrafiltration concentrator (Millipore, UFC901096).

[0601] Example 5: Detection of the physicochemical properties of the antibody

[0602] In this embodiment, the relative molecular weight and purity of the candidate antibody were detected using SDS-PAGE and SEC-HPLC.

[0603] 5.1 Antibody SDS-PAGE Identification

[0604] 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, respectively, and heat in a dry bath at 75 °C for 10 min. After cooling the mixture to room temperature, centrifuge at 12000 rpm for 5 min and collect the supernatant.

[0605] Preparation of reducing solution: Add 2 μg of each obtained antibody and the quality control IPI to 5×SDS loading buffer and 5 mM DTT, respectively, and heat in a dry bath at 100 °C for 10 min. After cooling the mixture to room temperature, centrifuge at 12000 rpm for 5 min and collect the supernatant.

[0606] The supernatant was added to a Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis and the protein bands were stained with Coomassie Brilliant Blue. The protein gels with stained protein bands were scanned using an EPSON V550 color scanner (the gels were destained until the background was transparent). The purity of reduced and non-reduced bands was calculated using ImageJ according to the peak area normalization method.

[0607] Experimental results showed that the bands of each antibody on the non-reducing gel were around 150 kDa, while the bands on the reducing gel were around 55 kDa and 25 kDa, respectively, which were in line with expectations. All candidate antibodies detected by reducing gel assay showed a purity greater than 95% (Table 2).

[0608] 5.2 SEC-HPLC was used to identify the monomer purity of the antibody.

[0609] Materials preparation: Mobile phase: 150 mmol / L phosphate buffer, pH 7.4; dilute each antibody and the quality control IPI to 0.5 mg / mL with the mobile phase solution.

[0610] Experimental Methods: An Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph was used. The chromatographic column was an XBridge BEH (SEC 3.5μm, 7.8mm ID×30cm). The Waters flow rate was set to 0.8mL / min, the injection volume was 20μL, and the VWD detector wavelengths were 280nm and 214nm. Blank solution, IPI control solution, and antibody sample solution were injected sequentially. The percentages of high molecular weight polymers, antibody monomers, and low molecular weight substances in the samples were calculated using the area normalization method.

[0611] The results are shown in Table 2. The monomer purity of all candidate antibodies was greater than 89% (Table 2).

[0612] Table 2. Expression levels and physicochemical properties of candidate antibodies

[0613]

[0614] Example 6: Detection of Antibody Antigen Binding Activity

[0615] In this embodiment, the binding of expressed candidate antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, C-B71) to CD100 antigen proteins HuCD100-His, MusCD100-His, and CynoCD100-His was detected using the ELISA method, and the binding of expressed antibodies (A14, A15, H74) to CD100 antigen proteins HuCD100-His, MusCD100-His, and CynoCD100-His was detected using the FACS method. The binding ability of H96, H5, H12, H21, B13, C-C081, C-C171, and C-B71 to CD100 overexpressing cells HuCD100-HEK293, MusCD100-HEK293, CynoCD100-HEK293, as well as human peripheral blood mononuclear cells naturally expressing human CD100 (hereinafter referred to as HuPBMC cells) and human T lymphocyte leukemia cells Jurkat cells.

[0616] 6.1 ELISA-based detection of antibody binding affinity to antigen protein CD100-His

[0617] 96-well ELISA plates were coated with 2 μg / mL HuCD100-His, MusCD100-His, and CynoCD100-His (30 μL / well) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 5% skim milk for 2 h. After washing three more times with PBST, serially diluted (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002 μg / mL) of each antibody and the positive control antibody Pepinemab were added and incubated for 1 h. After washing three times with PBST, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 h. After incubation, the plates were washed six times with PBST and then developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was terminated by adding 2M stop solution, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0618] The results are as follows Figure 5A-5X As shown in Table 3: For the antigen protein HuCD100-His ( Figures 5A-5HAntibody molecules A14, A15, B13, H5, H21, H74, and C-B71 showed superior binding activity to the antigen protein HuCD100-His compared to the positive control antibody, while antibody molecules H12, H96, C-C081, and C-C171 exhibited comparable binding activity to the antigen protein HuCD100-His compared to the positive control antibody. For the antigen protein MusCD100-His... Figure 5I-5P Except for antibody molecule C-C081, whose binding activity to the antigen protein MusCD100-His was weaker than that of the positive control antibody, and antibody molecules H12 and H96, whose binding activity to the antigen protein MusCD100-His was comparable to that of the positive control antibody, the binding activity of the other antibody molecules to the antigen protein MusCD100-His was superior to that of the positive control antibody; for the antigen protein CynoCD100-His ( Figure 5Q-5X Except for antibody molecules H12 and H96, whose binding activity with the antigen protein CynoCD100-His was comparable to that of the positive control antibody, the binding activity of the other antibody molecules with the antigen protein CynoCD100-His was superior to that of the positive control antibody.

[0619] Table 3. Antigen-binding activity of candidate antibodies

[0620]

[0621] 6.2 Detection of antibody binding ability to CD100-HEK293 cells based on FACS

[0622] In this embodiment, the antibody binding activity was evaluated using three types of human CD100 overexpressing cells: HuCD100-HEK293, MusCD100-HEK293, and CynoCD100-HEK293.

[0623] 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 1×10⁻⁶. 6Cells / mL were added to 100 μL per well of a 96-well plate, centrifuged at 300 g at 4 °C, and the supernatant was removed. Gradual dilutions (20.0, 6.67, 2.22, 0.74, 0.25, 0.08, 0.027, 0.0027 μg / mL) of each antibody and the positive control antibody Pepinemab were added to the corresponding wells, mixed, and incubated at 4 °C for 30 min. After washing the incubated cell mixture three times, 100 μL of a 1:300 dilution of the secondary antibody Goat F(ab')2 Anti-Human IgG-Fc(PE) (abcam, ab98596) was added, and the mixture was incubated at 4 °C in the dark for 30 min. After washing three times, the cells were analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0624] The results are as follows Figure 6A-6O As shown in Table 4: For the overexpression cell line HuCD100-HEK293 ( Figures 6A-6E Except for antibody molecules C-C081, C-C171, and C-B71, whose binding activity against HuCD100-HEK293 was comparable to that of the positive control antibody, the binding activity of the other antibody molecules against HuCD100-HEK293 was superior to that of the positive control antibody; for MusCD100-HEK293 ( Figure 6F-6J All antibody molecules exhibited good mouse cross-activity, and antibody molecules H5, H21, A14, A15, B13, H74, H96, and H12 showed superior binding activity to MusCD100-HEK293 compared to the positive control antibody; for CynoCD100-HEK293 ( Figure 6K-6O All antibody molecules exhibited good cross-activity with monkeys, with H5, H21, A14, A15, B13, H74, H96, H12, and C-C081 showing superior binding activity to CynoCD100-HEK293 compared to the positive control antibody.

[0625] Table 4. Cell-binding activity of candidate antibodies

[0626]

[0627] 6.3 Detection of antibody binding ability to HuPBMCs and Jurkat cells based on FACS

[0628] In this embodiment, the binding activity of antibodies was evaluated using both HuPBMC and Jurkat cells.

[0629] The specific method is as follows: Log-phase HuPBMCs (AllCells, catalog number PB004F-C(Y1246)) and Jurkat cells (ATCC, TIB-152) were prepared into single-cell suspensions, and the density was adjusted to 1×10⁻⁶. 6 Cells / mL were added to 100 μL per well of a 96-well plate, centrifuged at 300 g at 4 °C, and the supernatant was removed. Gradual dilutions (5.00, 0.50, 0.17, 0.056, 0.019, 0.006, 0.002, 0.0002 μg / mL) of each antibody and the positive control antibody Pepinemab were added to the corresponding wells, mixed, and incubated at 4 °C for 30 min. After washing the incubated cell mixture three times, 100 μL of a 1:300 dilution of the secondary antibody Goat F(ab')2 Anti-Human IgG-Fc(PE) (abcam, ab98596) was added, and the mixture was incubated at 4 °C in the dark for 30 min. After washing three times, the cells were analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0630] The results are as follows Figures 7A-7H As shown in Table 5: Except for antibody molecules C-C081, C-C171, and C-B71, the remaining antibody molecules are effective against HubBMC cells ( Figures 7A-7D ) and Jurkat cells ( Figure 7E-7H The binding activity of the antibodies was superior to that of the positive control antibody.

[0631] Table 5. Cell-binding activity of candidate antibodies

[0632]

[0633]

[0634] Example 7: Detection of Antibody Blocking Activity

[0635] In this embodiment, the blocking activity of four cell types—HuPlexin-B1-HEK293, MusPlexin-B1-HEK293, CynoPlexin-B1-HEK293, and HuPlexin-B2-HEK293—against candidate antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, and C-B71) was evaluated.

[0636] The specific method is as follows: Collect cultured HuPlexin-B1-HEK293, MusPlexin-B1-HEK293, CynoPlexin-B1-HEK293, and HuPlexin-B2-HEK293 cells, centrifuge at 300g to remove the supernatant, resuspend the cells in prepared FACS buffer, count them, and adjust the cell suspension density to 1×10⁻⁶. 6 Cells / mL; HuPlexin-B1-HEK293, MusPlexin-B1-HEK293, CynoPlexin-B1-HEK293, and HuPlexin-B2-HEK293 cells were added to 96-well plates at 100 μL per well, and centrifuged at 300g to remove the supernatant; various serially diluted (20.0, 2.00, 0.67, 0.22, 0.074, 0.025, 0.008, 0.0008 μg / mL) antibodies and the positive control antibody Pepinemab were added to the corresponding wells of the 96-well plates, respectively. After resuspending the cells, they were incubated at 4℃ for 30 min; the diluted antibodies were then reacted with the biotinylated antigen protein HuCD1. 100 μL of HuCD100-Fc dilution buffer (0.5 μg / mL), CynoCD100-Fc dilution buffer (0.3 μg / mL), or MusCD100-Fc dilution buffer (3 μg / mL) (HuCD100-Fc-Biotin, MusCD100-Fc-Biotin, and CynoCD100-Fc-Biotin were prepared by biotin labeling in Example 1.2; the biotin labeling method is described in the instructions for the Roche Biotinylate Labeling Kit, catalog number: 11418165001) was mixed and added to the cells, and incubated for 1 h. The cell mixture was then washed three times. Resuspend the cells and incubate them at 4°C for 30 min. Wash the incubated cell mixture three times, then add PE-labeled streptavidin (eBioscience, 12-4317-87) and incubate at 4°C for 30 min. Wash the incubated cell mixture three times, then add 200 μL of FACS buffer to each well to resuspend the cells and analyze them by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0637] The results are as follows Figure 8A-8OAs shown, 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, antibody molecules A14, A15, B13, C-C081, and C-C171 showed superior activity in blocking the binding of HuCD100 antigen protein to HuPlexin-B1-HEK293 compared to the positive control antibody; antibody molecules A15, B13, H74, H96, H12, and C-C081 showed superior activity in blocking the binding of HuCD100 antigen protein to HuPlexin-B2-HEK293 compared to the positive control antibody; antibody molecules H12, H74, H96, and C-C081 showed superior activity in blocking the binding of MusCD100 antigen protein to MusPlexin-B1-HEK293 compared to the positive control antibody; and antibody molecules A15, B13, H12, H96, C-C081, C-C171, and C-B71 showed superior activity in blocking the binding of CynoCD100 antigen protein to CynoPlexin-B1-HEK293 compared to the positive control antibody.

[0638] Example 8: MDSC proliferation inhibition assay

[0639] This embodiment evaluates the in vitro pharmacological efficacy of candidate antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-B71) by examining their inhibitory effect on the proliferation of myeloid-derived suppressor cells (MDSCs).

[0640] CD33+ cells were sorted from fresh PBMCs using CD33 sorting magnetic beads (Miltenyi Biotech). After sorting, the cell density was adjusted to 1×10⁻⁶. 6 / mL for later use. Serially dilute the antibody with 1640 complete medium to 100.0, 33.33, 11.11, 3.704, 1.235, and 0.412 μg / mL. Dilute the HuCD100-His antigen protein with 1640 complete medium to 400.0 μg / mL. Mix the antibody dilution with the HuCD100-His antigen protein dilution 1:1 and incubate at room temperature for 30 min. Add 100 μL of a 1×10⁻⁶ mcg solution to each well of a 96-well cell culture plate. 6 / mL of CD33 + Cells (1×10⁶ per well) 5Cells were collected from each well, and then 100 μL of antibody-HuCD100-His antigen protein mixture was added to each well, for a total of 200 μL. The cell culture plate was incubated at 37°C for 72 h. Cells from the sample wells were then transferred to 96-well U-shaped plates. The plates were washed twice with FACS buffer. Three direct-labeled antibodies—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 with FACS buffer, and 100 μL of a mixture of the three direct-labeled antibodies was added to each well. The plates were incubated at 4°C for 30 min. The plates were washed twice with FACS buffer. Detection was performed using a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0641] Experimental results are as follows Figures 9A-9C As shown in Table 6, all candidate antibodies and positive control antibodies effectively neutralized the induction of MDSC cell population by CD100. Among them, antibody molecules A14, A15, B13, H12, H21, H74 and H96 showed better inhibitory effects on MDSC proliferation than positive control antibodies 2D5 and 5D8. Antibody molecules C-C081 and C-B71 showed significantly better inhibitory effects on MDSC proliferation than the control antibody Pepinemab.

[0642] Table 6. Anti-CD100 antibody inhibits MDSC activity

[0643]

[0644] Example 9: Preparation of anti-PD-L1 antibody

[0645] This embodiment describes affinity maturation modification of the antibody NB22D-21-huVH2 (see, for example, CN112745391A) to enhance antibody affinity and other biological activities. Affinity maturation modification is based on M13 phage display technology. Codon-based primers (in primer synthesis, a single codon is composed of NNK) are used to introduce mutations in the CDR region, constructing four phage display libraries: Library 1 contains a single-point combination mutation (CDR1+CDR2+CDR3 combination mutation); Libraries 2, 3, and 4 contain two-point combination mutations (CDR1+CDR3, CDR2+CDR3, and CDR1+CDR2).

[0646] The specific library construction method is as follows: First, primers containing point mutations were synthesized (Genewiz Biotechnology Co., Ltd.); second, using the coding sequence of the antibody to be modified (hereinafter referred to as the parent antibody) NB22D-21-huVH as a PCR amplification template, sequences containing mutations in the CDR region were amplified. Fragments containing different CDR mutations were combined using bridging PCR. Then, the point-mutant antibody was ligated into the phage display vector by double enzyme digestion (HindIII and NotI) and double-end ligation. Finally, the antibody sequence with the mutation site was transformed into *E. coli* SS320 by electroporation. The specific procedures for library capacity calculation, phage library preparation, and library screening are detailed in Example 3. The obtained antibody was named m18-VHH. The amino acid sequence of the variable region of the obtained antibody is shown in Table 7. The CDR sequence was determined using the AbM definition method.

[0647] Table 7. Amino acid sequence of the variable region of anti-PD-L1 antibody (SEQ ID NO:)

[0648]

[0649] Example 10: Production and Expression of Antibody m18

[0650] 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 fragment gene sequence to fuse antibody m18-VHH and human IgG1 Fc fragment (SEQ ID NO:135). The plasmid of this fusion expression vector was transformed into ExpiCHO cells and induced to express the protein to obtain the VHH-Fc chimeric antibody protein fused with the Fc fragment (SEQ ID NO:134). The VHH-Fc antibody will be named antibody m18 below.

[0651] Antibody expression was performed using the ExpiCHO transient expression system, employing ExpiCHO... TM Expression medium (Gibco, A29100-01) and Gibco TM ExpiFectamine TM CHO transfection kit (Gibco, A29129). See Example 4 for specific methods.

[0652] Example 11: Detection of antigen-binding activity of antibody m18

[0653] In this embodiment, the binding ability of 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 using the FACS method. The source or preparation method of the above cell lines can be found in CN112745391A.

[0654] 11.1 Detection of antibody binding ability to human PD-L1-CHO cells based on FACS

[0655] In this embodiment, human PD-L1-CHO cells were used to evaluate the binding activity of the antibody to human PD-L1 overexpressing cells.

[0656] For specific methods, please refer to Example 5 in CN112745391A, where the positive control antibody used is Avelumab (the preparation method is described in patent WO2013079174).

[0657] The results are as follows Figure 10A As shown, the binding activity of antibody m18 to human PD-L1-CHO cells is superior to that of the parent antibody NB22D-21-huVH2 and the positive control antibody Avelumab.

[0658] 11.2 Detection of antibody binding ability to human non-small cell lung cancer cell line HCC827 based on FACS

[0659] In this embodiment, 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 (ATCC:CRL-2868).

[0660] For specific methods, please refer to Example 8 in CN112745391A, where the positive control antibody used is Avelumab (the preparation method is described in patent WO2013079174).

[0661] The results are as follows Figure 10B As shown, the binding activity of antibody m18 to human non-small cell lung cancer cell line HCC827 is comparable to that of the parent antibody NB22D-21-huVH2 and the positive control antibody Avelumab.

[0662] 11.3 Detection of antibody binding ability to mouse PD-L1-CHO cells and cynomolgus monkey PD-L1-CHO cells based on FACS

[0663] In this embodiment, mouse PD-L1-CHO cells and cynomolgus monkey PD-L1-CHO cells were used to evaluate the cross-binding activity with monkey and mouse PD-L1. For specific methods, please refer to Example 9 in CN112745391A, where the positive control antibody used is Avelumab (the preparation method is described in patent WO2013079174).

[0664] The results are as follows Figure 11A and Figure 11BAs shown, antibody m18 exhibits good binding activity to mouse PD-L1-CHO cells, while the maternal antibody and positive control antibody do not bind to mouse PD-L1-CHO cells. Figure 11A It can be predicted that antibody molecule m18 can be used in animal model experiments of Balb / C breed mice; the binding activity of antibody m18 to cynomolgus monkey PD-L1-CHO cells is superior to that of the positive control antibody and comparable to that of the maternal antibody. Figure 11B ).

[0665] Example 12 Specificity detection of antibody m18 binding to PD-L1

[0666] In this embodiment, the binding activity of the VHH-Fc antibody to other proteins in the B7 family was detected by ELISA to evaluate the specificity of antibody m18 for PD-L1 protein. For specific methods, please refer to Example 10 in Chinese Invention Patent Application CN112745391A.

[0667] The results are shown in Table 8 and Figure 12 As shown, antibody m18 has no binding activity against B7 family molecules other than B7-H1 (i.e., PD-L1), but only against B7-H1. This binding specificity is consistent with that of the parent antibody.

[0668] Table 8. Specificity of candidate molecules binding to PD-L1 protein

[0669]

[0670] +: indicates detected binding activity; N / A: indicates no detected binding activity.

[0671] Example 13: Modification of Anti-CD100 Antibody Molecular Affinity

[0672] 13.1 Design and Construction of an Affinity-Oriented Mature Library

[0673] Affinity maturation was performed on antibodies B13 and H5 to improve their affinity and biological activity. The affinity maturation was based on M13 phage display technology, using codon-based primers (in primer synthesis, each codon is composed of NNK) to introduce mutations in the CDR region, constructing four phage display libraries: Libraries 1 and 2 contained single-point combination mutations, with Library 1 containing a CDRL1+CDRL3+CDRH3 combination mutation and Library 2 containing a CDRL2+CDRH1+CDRH2 combination mutation; Libraries 3 and 4 contained double-point saturation mutations, with Library 3 containing a double-point saturation mutation of CDRL3 and Library 4 containing a double-point saturation mutation of CDRH3. The specific library construction method is as follows: First, primers containing point mutations (Genewiz Biotechnology Co., Ltd.) are synthesized; second, using the antibodies B13 and H5 to be modified as PCR amplification templates, sequences containing mutations in the CDR region are amplified. Fragments containing different CDR mutations are combined using bridging PCR. Then, the point mutation antibodies are ligated into the phage display vector via double enzyme digestion (HindIII and NotI) and double-end ligation. Finally, the antibody sequence with the mutation site is transformed into *E. coli* SS320 via electroporation. For the specific process of library capacity calculation and phage library preparation, please refer to Example 3.

[0674] 13.2 Screening of Affinity Mature Libraries

[0675] The specific procedures for library screening are detailed in Example 3. After initial screening, affinity sequencing, and sequence analysis, 74 positive clones for H5 and 57 positive clones for B13 were obtained, and affinity sequencing was performed on each. Combining the affinity sequencing and sequence analysis data, 20 candidate molecules were selected from H5 and 28 candidate molecules were selected from B13 for construction, expression, and functional screening.

[0676] 13.3 Preparation of candidate molecules for affinity maturation modification, evaluation of physicochemical properties, and detection of binding activity and blocking effect

[0677] For the specific methods of preparing the 48 candidate antibodies, detecting their physicochemical properties, affinity activity, blocking activity, and MDSC proliferation inhibition assay, please refer to Examples 4-8.

[0678] 13.4 Selection of candidate molecules for affinity maturation modification

[0679] Based on the physicochemical properties, affinity activity, and blocking activity of the antibodies, two mature antibodies with anti-C100 affinity, B13-c-5 and B13-e-2, were selected for B13. Two mature antibodies with anti-C100 affinity, H5-h-7 and H5-a-2, were selected for H5. The amino acid sequences of the variable regions of the obtained antibodies are shown in Table 9. The CDR sequence was determined using the AbM method for defining CDRs.

[0680] The expression levels and in vitro physicochemical properties were measured, as shown in Table 10. SDS-PAGE was used to identify the antibody purity; the purity of the preferred modified antibodies was greater than 95%. SEC-HPLC was used to identify the monomeric purity of the antibody; the monomeric purity of the preferred modified antibodies was greater than 98%.

[0681] FACS-based affinity activity detection results are as follows: Figures 13A-13D As shown, Figure 13A The results show the binding activity against HuCD100-HEK293 cells. Figure 13B The results show the binding activity against human PBMC cells. Figure 13C The results show the binding activity against MusCD100-HEK 293 cells. Figure 13D The results show the binding activity against CynoCD100-HEK 293 cells. The modified antibodies B13-c-5, B13-e-2, H5-h-7, and H5-a-2 showed significantly better affinity for human CD100-overexpressing cells (HuCD100-HEK293) and CD100-positive human PBMCs than the positive control antibody Pepinemab, and were also superior to their respective parent antibodies. Furthermore, the modified antibodies showed significantly better affinity for mouse CD100-overexpressing cells (MusCD100-HEK 293) and cynomolgus monkey CD100-overexpressing cells (CynoCD100-HEK 293) than the positive control antibody Pepinemab, and were also superior to their respective parent antibodies.

[0682] The results of FACS-based blocking activity detection are as follows: Figures 14A-14D As shown, the modified antibodies effectively blocked 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 these, the modified antibodies showed superior activity in blocking the binding of HuCD100 antigen protein to HuPlexin-B1-HEK293 cells compared to the positive control antibody and its corresponding maternal antibody. Figure 14A The modified antibody blocked the binding of HuCD100 antigen protein to HuPlexin-B2-HEK293 cells. Figure 14B ), blocking the binding of MusCD100 antigen protein to HuPlexin-B1-HEK293 cells ( Figure 14CThe activity of H5-h-7 and H5-a-2 in blocking the binding of CynoCD100 antigen protein to CynoPlexin-B1-HEK293 cells was superior to that of the positive control antibody and the maternal antibody H5. The activity of B13-c-5 and B13-e-2 in blocking the binding of CynoCD100 antigen protein to CynoPlexin-B1-HEK293 cells was superior to that of the positive control antibody but weaker than that of the maternal antibody B13. Figure 14D ).

[0683] MDSC proliferation inhibition detection results are as follows Figure 15 As shown in Table 11, the results indicate that anti-CD100 antibodies H5-h-7, H5-a-2, H5, B13, B13-c-5, B13-e-2, and the control antibody Pepinemab can effectively neutralize the induction of MDSC cell population by CD100. Among them, the inhibitory activity of MDSCs by H5-h-7, H5-a-2, H5, B13, B13-c-5, and B13-e-2 at 100 μg / mL was significantly higher than that of the control antibody Pepinemab.

[0684] Table 9. Amino acid sequences of the variable regions of four affinity-modified anti-CD100 antibodies (SEQ ID NO:)

[0685]

[0686] Table 10. Expression levels and in vitro physicochemical properties of anti-CD100 antibodies from four affinity-modified strains.

[0687]

[0688] Table 11. Anti-CD100 antibody inhibits MDSC activity

[0689]

[0690] Example 14: Pharmacological Evaluation of the CT-26 Animal Model

[0691] This embodiment tested the tumor-suppressing 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 an anti-PD-L1 antibody 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.

[0692] The specific method is as follows: Female Balb / C mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 6-8 weeks and weighing approximately 20g were used. Each mouse was subcutaneously injected 5×10⁵ mmol / L into one side. 5 Two days after tumor formation, CT-26 cells were randomly assigned to 10 groups of 8 tumor-bearing nude mice. These groups included a PBS negative control group, candidate antibody combination therapy groups (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 groups (Pepinemab + antibody m18, antibody m18). The anti-CD100 antibody was administered at a dose of 50 mpk, and the anti-PD-L1 antibody at a dose of 5 mpk, via intraperitoneal injection. Administration was twice weekly, with tumor volume measured twice, for a total of 8 administrations / 4 weeks (BIW*4). Mouse body weight was measured at the same time points. Tumor volume (V) was calculated as: V = L × W 2 / 2 (where L is the longest tumor diameter and W is the shortest tumor diameter). One week after drug administration, mice were euthanized, and tumor tissue was collected. Tumor volume and mouse weight changes were analyzed to calculate the tumor inhibition rate. Tumor inhibition rate data are detailed in Table 12.

[0693] The results are as follows Figure 16A , 16B As shown in Table 12, in the combination of anti-CD100 antibody and anti-PD-L1 antibody, the combination of anti-CD100 antibody (especially antibody C-C081, B13 or B13-c-5) and anti-PD-L1 antibody m18 can further synergistically inhibit or delay tumor growth compared with anti-PD-L1 antibody m18 alone.

[0694] This indicates that the combined use of anti-CD100 antibodies (especially antibodies C-C081, B13, or B13-c-5) and anti-PD-L1 antibody m18 significantly improved the response rate of CT-26-inoculated mice to m18 antibody monotherapy and prolonged their survival, suggesting that such combined use can enhance the therapeutic effect of PD-L1 tumor immunotherapy. Simultaneously, mouse body weight increased after administration, and there was no significant difference in body weight between the experimental groups and the control group. This indicates that the antibodies did not produce significant toxic side effects in mice and are safe.

[0695] Table 12. Tumor inhibition rate (TGI%) in animal studies of the combination of candidate antibodies and antibody m18.

[0696]

[0697] Those skilled in the art will recognize that many modifications and variations can be made to the invention without departing from its spirit and scope. The specific embodiments described herein are provided by way of example only and are not intended to limit the scope in any way. The true scope and spirit of the invention are shown in the appended claims, and the description and embodiments are merely exemplary.

[0698] sequence list

[0699] B13

[0700]

[0701]

[0702] C-C081

[0703]

[0704] A14

[0705]

[0706]

[0707] A15

[0708]

[0709] H74

[0710]

[0711]

[0712] H96

[0713]

[0714] H5

[0715]

[0716] H12

[0717]

[0718]

[0719] H21

[0720]

[0721] C-C171

[0722]

[0723]

[0724] C-B71

[0725]

[0726] H5-h-7

[0727]

[0728]

[0729] H5-a-2

[0730]

[0731] B13-c-5

[0732]

[0733] B13-e-2

[0734]

[0735]

[0736] m18

[0737]

Claims

1. An antibody against CD100 or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3; the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from any one of (1) - (3): (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: 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; (3) 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.

2. The antibody of claim 1 or its antigen-binding fragment, wherein... The heavy chain variable region comprises 1) the amino acid sequence shown in SEQ ID NO: 67, 93, or 95; 2) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 67, 93, or 95; or 3) an amino acid sequence having one or more amino acid substitutions, additions, and / or deletions compared to the amino acid sequence shown in SEQ ID NO: 67, 93, or 95; and / or The light chain variable region comprises 1) the amino acid sequence shown in SEQ ID NO: 68, 94 or 96; 2) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 68, 94 or 96; or 3) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in SEQ ID NO: 68, 94 or 96.

3. The antibody of claim 2 or its antigen-binding fragment, wherein the heavy chain variable region and the light chain variable region are selected from any one of (1)-(3): (1) Heavy chain variable region, which contains the amino acid sequence shown in SEQ ID NO:67; light chain variable region, which contains the amino acid sequence shown in SEQ ID NO:68; (2) Heavy chain variable region, which contains the amino acid sequence shown in SEQ ID NO:93; light chain variable region, which contains the amino acid sequence shown in SEQ ID NO:94; (3) Heavy chain variable region, which contains the amino acid sequence shown in SEQ ID NO:95; light chain variable region, which contains the amino acid sequence shown in SEQ ID NO:

96.

4. The antibody of claim 1 or its antigen-binding fragment, wherein... The antibody or its antigen-binding fragment comprises a heavy chain, the heavy chain comprising 1) the amino acid sequence shown in SEQ ID NO: 97, 123 or 125; 2) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 97, 123 or 125; or 3) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in SEQ ID NO: 97, 123 or 125; and / or The antibody or its antigen-binding fragment comprises a light chain, the light chain comprising 1) the amino acid sequence shown in SEQ ID NO: 98, 124 or 126; 2) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 98, 124 or 126; or 3) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to the amino acid sequence shown in SEQ ID NO: 98, 124 or 126.

5. The antibody of claim 4 or an antigen-binding fragment thereof, wherein the heavy chain and light chain are selected from any one of (1) - (3): (1) The heavy chain, comprising the amino acid sequence shown in SEQ ID NO:97; and the light chain, comprising the amino acid sequence shown in SEQ ID NO:

98. (2) The heavy chain, comprising the amino acid sequence shown in SEQ ID NO:123; and the light chain, comprising the amino acid sequence shown in SEQ ID NO:124; (3) The heavy chain, which contains the amino acid sequence shown in SEQ ID NO:125; and the light chain, which contains the amino acid sequence shown in SEQ ID NO:

126.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein it is a chimeric antibody, a humanized antibody, a human antibody, scFv, Fab, Fab', F(ab')2 or Fv fragment.

7. The antibody of claim 6 or its antigen-binding fragment, wherein, The antibody or its antigen-binding fragment is a human antibody.

8. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof of any one of claims 1-7 and a pharmaceutically acceptable carrier.

9. A pharmaceutical combination comprising an antibody or antigen-binding fragment thereof of any one of claims 1-7 and an anti-PD-L1 antibody or antigen-binding fragment thereof.

10. The pharmaceutical combination of claim 9, wherein the anti-PD-L1 antibody or its antigen-binding fragment specifically recognizes and binds to PD-L1, wherein the anti-PD-L1 antibody or its antigen-binding fragment comprises an immunoglobulin monovariable domain.

11. The pharmaceutical combination of claim 10, wherein, The immunoglobulin single variable domain includes: CDR1, which consists of the amino acid sequence shown in SEQ ID NO:130, CDR2, which consists of the amino acid sequence shown in SEQ ID NO:131, and CDR3 consists of the amino acid sequence shown in SEQ ID NO:

132.

12. The pharmaceutical combination of claim 10, wherein 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% sequence identity with SEQ ID NO:

133.

13. The pharmaceutical combination of claim 10, wherein the anti-PD-L1 antibody or its antigen-binding fragment further comprises an Fc fragment of human IgG1.

14. The pharmaceutical combination of claim 13, wherein the anti-PD-L1 antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:134 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

134.

15. The pharmaceutical combination of any one of claims 9-14, wherein it is a pharmaceutical composition or a kit.

16. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7, the pharmaceutical composition of claim 8, or the pharmaceutical combination of any one of claims 9-15 in the preparation of a medicament for treating colorectal cancer.

17. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of any one of claims 1-7.

18. An expression vector comprising the nucleic acid molecule of claim 17.

19. A host cell comprising the nucleic acid molecule of claim 17 or the expression vector of claim 18.

20. A method for generating an antibody or antigen-binding fragment thereof according to any one of claims 1-7, the method comprising: a) Culture the host cells of claim 19 under suitable conditions to express the antibody or antigen-binding fragment of any one of claims 1-7; as well as b) Isolate the antibody or its antigen-binding fragment from a host cell or its culture.

Images