CN117866091A - anti-CD 100 antibodies and uses thereof - Google Patents

anti-CD 100 antibodies and uses thereof Download PDF

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CN117866091A
CN117866091A CN202211246863.8A CN202211246863A CN117866091A CN 117866091 A CN117866091 A CN 117866091A CN 202211246863 A CN202211246863 A CN 202211246863A CN 117866091 A CN117866091 A CN 117866091A
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seq
amino acid
sequence shown
acid sequence
antibody
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闫鑫甜
李双琦
郎国竣
方小鹏
高雪
韩洋洋
任雨琪
徐培芳
邓强强
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Sanyou Biopharmaceuticals Co Ltd
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Sanyou Biopharmaceuticals Co Ltd
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Priority to PCT/CN2023/124146 priority patent/WO2024078558A1/en
Publication of CN117866091A publication Critical patent/CN117866091A/en
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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Abstract

The invention relates to the field of biological medicine. In particular, the invention relates to anti-CD 100 antibodies and uses thereof, pharmaceutical compositions or pharmaceutical combinations comprising said antibodies and uses thereof. The invention further provides polynucleotides and expression vectors encoding the antibodies and methods of producing the antibodies.

Description

anti-CD 100 antibodies and uses thereof
Technical Field
The invention belongs to the field of biological medicine. In particular, the invention relates to anti-CD 100 antibodies and uses thereof.
Background
CD100, also known as semaphorin 4D (SEMA 4D), is a transmembrane protein belonging to the semaphorin gene family. CD100 is expressed on the cell surface as a homodimer and can be released from the cell surface by proteolysis to produce CD100 in an active soluble form. CD100 is strongly expressed mainly in human lymphoid tissues, skeletal muscles and brain (lower levels). Its biological activity is mainly characterized in the immune system: for example, CD100 can enhance T cell proliferation as a receptor; as a ligand, it promotes the aggregation and survival of B cells and the activation and maturation of antigen presenting cells (dendritic cells and macrophages). CD100 inhibits the migration of monocytes and B cells via the high affinity receptor Plexin-B1.
CD100 is widely expressed in many human tumors, whose expression is associated with human invasive disease. In the preclinical tumor microenvironment, inflammatory and tumor cell expression CD100 regulates the infiltration, spatial distribution and activity of myeloid and lymphoid cells. CD100 binds to the Plexin receptor located on myeloid cells in the tumor microenvironment. When the CD100 protein is blocked, the CD100 barrier may be eliminated. Once the barrier is breached, inflammatory dendritic cells and pro-inflammatory antigen presenting cells migrate and penetrate into the tumor. In preclinical animal models of cancer, blocking CD100 with antibodies can delay tumor growth and promote persistent tumor rejection.
Antibodies against CD100 have been developed in the prior art, for example Pepinemab (vaccine, inc.). There remains a need in the art for anti-CD 100 antibodies that specifically bind to CD100 and block the binding of CD100 to its Plexin receptor.
Disclosure of Invention
An aspect of the present invention provides an antibody (anti-CD 100 antibody) or antigen-binding fragment thereof directed against CD100, wherein the antibody or antigen-binding fragment thereof specifically recognizes and binds CD100.
In one embodiment, the antibody or antigen binding fragment thereof directed against CD100 comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences, and wherein the HCDR1 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID NO 1, 7, 19, 33, 41, 47 or 52; the HCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 2, 8, 14, 20, 26, 42, 48 or 53 by NO more than 2 amino acid additions, deletions or substitutions; and/or the HCDR3 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID No. 3, 9, 15, 21, 27, 30, 35, 38, 43, 49 or 54.
In one embodiment, the antibody or antigen binding fragment thereof directed against CD100 comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences, and wherein the LCDR1 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID No. 4, 10, 16, 36, 44 or 50; the LCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 5, 11 or 45 by NO more than 2 amino acid additions, deletions or substitutions; and/or the LCDR3 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 6, 12, 18, 24, 37, 46, 51 or 56 by NO more than 2 amino acid additions, deletions or substitutions.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth 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.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NOs 4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; 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.
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; an HCDR2 sequence shown in SEQ ID NO. 2; an HCDR3 sequence shown in SEQ ID NO. 3; an LCDR1 sequence shown in SEQ ID NO. 4; an 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; an HCDR2 sequence shown in SEQ ID NO. 8; an HCDR3 sequence shown in SEQ ID NO. 9; an LCDR1 sequence shown in SEQ ID NO. 10; an 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; an HCDR2 sequence shown in SEQ ID NO. 14; an HCDR3 sequence shown in SEQ ID NO. 15; the LCDR1 sequence shown in SEQ ID NO. 16; an LCDR2 sequence shown in SEQ ID NO. 17; and the LCDR3 sequence shown in SEQ ID NO. 18; (4) the HCDR1 sequence shown in SEQ ID NO. 19; an HCDR2 sequence shown in SEQ ID NO. 20; an HCDR3 sequence shown in SEQ ID NO. 21; the LCDR1 sequence shown in SEQ ID NO. 22; the LCDR2 sequence shown in SEQ ID NO. 23; and the LCDR3 sequence shown in SEQ ID NO. 24; (5) the HCDR1 sequence shown in SEQ ID NO. 25; the HCDR2 sequence shown in SEQ ID NO. 26; the HCDR3 sequence shown in SEQ ID NO. 27; the LCDR1 sequence shown in SEQ ID NO. 16; the LCDR2 sequence shown in SEQ ID NO. 28; and the LCDR3 sequence shown in SEQ ID NO. 29; (6) the HCDR1 sequence shown in SEQ ID NO. 25; the HCDR2 sequence shown in SEQ ID NO. 26; the HCDR3 sequence shown in SEQ ID NO. 30; the LCDR1 sequence shown in SEQ ID NO. 31; an 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; an HCDR3 sequence shown in SEQ ID NO. 35; the LCDR1 sequence shown in SEQ ID NO. 36; the LCDR2 sequence shown in SEQ ID NO. 23; and the LCDR3 sequence shown in SEQ ID NO. 37; (8) the HCDR1 sequence shown in SEQ ID NO. 33; the HCDR2 sequence shown in SEQ ID NO. 34; the HCDR3 sequence shown in SEQ ID NO. 38; the LCDR1 sequence shown in SEQ ID NO. 39; an LCDR2 sequence shown in SEQ ID NO. 17; and the LCDR3 sequence shown in SEQ ID NO. 40; (9) the HCDR1 sequence shown in SEQ ID NO. 41; an HCDR2 sequence shown in SEQ ID NO. 42; an 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; an HCDR2 sequence shown in SEQ ID NO. 48; an HCDR3 sequence shown in SEQ ID NO. 49; the LCDR1 sequence shown in SEQ ID NO. 50; an LCDR2 sequence shown in SEQ ID NO. 11; and the LCDR3 sequence shown in SEQ ID NO. 51; (11) the HCDR1 sequence shown in SEQ ID NO. 52; an HCDR2 sequence shown in SEQ ID NO. 53; an HCDR3 sequence shown in SEQ ID NO. 54; the LCDR1 sequence shown in SEQ ID NO. 50; the LCDR2 sequence shown in SEQ ID NO. 55; and the LCDR3 sequence shown in SEQ ID NO. 56; (12) the HCDR1 sequence shown in SEQ ID NO. 57; the HCDR2 sequence shown in SEQ ID NO. 58; an 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; an HCDR3 sequence shown in SEQ ID NO. 60; the LCDR1 sequence shown in SEQ ID NO. 61; the LCDR2 sequence shown in SEQ ID NO. 23; and the LCDR3 sequence shown in SEQ ID NO. 62; (14) the HCDR1 sequence shown in SEQ ID NO. 1; an 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; an LCDR2 sequence shown in SEQ ID NO. 5; and the LCDR3 sequence shown in SEQ ID NO. 65; (15) the HCDR1 sequence shown in SEQ ID NO. 1; an HCDR2 sequence shown in SEQ ID NO. 2; the HCDR3 sequence shown in SEQ ID NO. 66; an LCDR1 sequence shown in SEQ ID NO. 4; an LCDR2 sequence shown in SEQ ID NO. 5; and the LCDR3 sequence shown in SEQ ID NO. 6.
In one embodiment, the heavy chain variable region comprises 1) the amino acid sequence set forth in SEQ ID NO 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95; 2) An amino acid sequence 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to 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, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
In one embodiment, the light chain variable region comprises 1) the amino acid sequence set forth in SEQ ID NO 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96; 2) An amino acid sequence 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
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 set forth in SEQ ID NO. 67; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 68; (2) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 69; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 70; (3) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 71; a light chain variable region comprising the amino acid sequence of SEQ ID NO. 72; (4) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 73; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 74; (5) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 75; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 76; (6) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 77; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 78; (7) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 79; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 80; (8) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 81; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 82; (9) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 83; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 84; (10) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 85; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 86; (11) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 87; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 88; (12) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 89; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 90; (13) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 91; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 92; (14) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 93; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 94; (15) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 95; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 96.
In one embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain comprising 1) the amino acid sequence set forth in SEQ ID No. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; 2) An amino acid sequence 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
In one embodiment, the antibody or antigen binding fragment thereof comprises a light chain comprising 1) the amino acid sequence set forth in SEQ ID NO 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; 2) An amino acid sequence 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID No. 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
In one embodiment, the heavy and light chains are selected from any one of (1) - (15): (1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 97; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 98; (2) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 99; 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; 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; 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; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 106; (6) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 107; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 108; (7) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 109; a light chain comprising the amino acid sequence shown in SEQ ID NO. 110; (8) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 111; a light chain comprising the amino acid sequence shown in SEQ ID NO. 112; (9) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 113; a light chain comprising the amino acid sequence shown in SEQ ID NO. 114; (10) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 115; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 116; (11) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 117; a light chain comprising the amino acid sequence shown in SEQ ID NO. 118; (12) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 119; a light chain comprising the amino acid sequence shown in SEQ ID NO. 120; (13) A heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 121; a light chain comprising the amino acid sequence shown in SEQ ID NO. 122; (14) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 123; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 124; (15) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 125; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 126.
In yet another aspect, the invention provides a pharmaceutical composition comprising an anti-CD 100 antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.
In some embodiments, the anti-CD 100 antibody comprises antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-H-7, H5-a-2, B13-C-5, or B13-e-2.
In yet another aspect, the invention provides a pharmaceutical combination comprising an anti-CD 100 antibody or antigen-binding fragment thereof of the invention and an anti-PD-L1 antibody or antigen-binding fragment thereof.
In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof specifically recognizes and binds to PD-L1, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an immunoglobulin single variable domain;
in one embodiment, the immunoglobulin single variable domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO. 130; CDR2 comprising the amino acid sequence shown in SEQ ID NO. 131; and CDR3 comprising the amino acid sequence shown in SEQ ID NO. 132.
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 more sequence identity to SEQ ID NO. 133. In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc fragment of human IgG 1;
In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 134 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 134.
In one embodiment, the pharmaceutical combination is a pharmaceutical composition or kit.
In one aspect, the invention also provides the use of an antibody or antigen binding fragment thereof of the invention, a pharmaceutical composition of the invention or a pharmaceutical combination of the invention in the manufacture of a medicament for the treatment of cancer.
In another aspect, the invention provides a method of treating cancer comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof of the invention, a pharmaceutical composition of the invention, or a pharmaceutical combination of the invention.
In a further aspect, the invention relates to an antibody of the invention or an antigen binding fragment thereof, a pharmaceutical composition of the invention, or a pharmaceutical combination of the invention for use in the treatment of cancer.
In another aspect, the invention also provides an isolated nucleic acid molecule encoding an anti-CD 100 antibody or antigen-binding fragment thereof of the invention. The invention also relates to expression vectors comprising the polynucleotides of the invention. The invention also relates to host cells comprising the polynucleotides or expression vectors of the invention.
In another aspect, the invention also relates to a method of producing an anti-CD 100 antibody or antigen-binding fragment thereof, the method comprising:
a) Culturing a host cell of the invention under suitable conditions to express an anti-CD 100 antibody or antigen-binding fragment thereof of the invention; and
b) Isolating the antibody or antigen binding fragment thereof from the host cell or culture thereof.
Drawings
FIG. 1 shows ELISA detection results of Pepinetab antibodies and positive control 2D5, 5D8 antibodies.
FIG. 2 shows ELISA detection results for different species of antigens.
FIG. 3 shows the results of FACS detection of various species of overexpressing cell lines.
FIGS. 4A-4D show FACS detection results of different species of Plexin receptor overexpressing cell lines. FIG. 4A is a graph showing the results of detection of Huplexin-B1-HEK293 overexpressing cell lines; FIG. 4B is a test result of a Musplexin-B1-HEK293 overexpressing cell line; FIG. 4C shows the results of detection of Cynoplexin-B1-HEK293 overexpressing cell lines; FIG. 4D shows the results of detection of Huplexin-B2-HEK293 overexpressing cell lines.
FIGS. 5A-5X show the results of ELISA detection of candidate antibodies for binding activity to different species of antigen proteins. FIGS. 5A-5H are results of binding activity of antibody molecules A14, A15, B13, H5, H12, H21, H74, H96, C-C081, C-C171 and C-B71 to HuCD100-His antigen protein; FIGS. 5I-5P are results of binding activity of antibody molecules A14, A15, B13, H5, H12, H21, H74, H96, C-C081, C-C171 and C-B71 to MusCD100-His antigen protein; FIGS. 5Q-5X are results of binding activity of antibody molecules A14, A15, B13, H5, H12, H21, H74, H96, C-C081, C-C171 and C-B71 against CynocD100-His antigen protein.
FIGS. 6A-6O show the results of detection of candidate antibody binding activity to various species of overexpressing cell lines using FACS. FIGS. 6A-6E are results of binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96, H12, C-C081, C-C171 and C-B71 on HuCD100-HEK293 overexpressing cell lines; FIGS. 6F-6J are results of binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96, H12, C-C081, C-C171 and C-B71 on MusCD100-HEK293 overexpressing cell lines; FIGS. 6K-6O are results of binding activity of antibody molecules H5, H21, A14, A15, B13, H74, H96, H12, C-C081, C-C171 and C-B71 on CynocD100-HEK293 overexpressing cell lines.
FIGS. 7A-7H show the results of detection of candidate antibody binding activity to HuPBMC and Jurkat cells using FACS. FIGS. 7A-7D are results of binding activity of antibody molecules A14, A15, B13, H5, H21, H74, H96, H12, C-C081, C-C171 and C-B71 to HuPBMC cells; FIGS. 7E-7H are the results of binding activity of antibody molecules A14, A15, B13, H5, H21, H74, H96, H12, C-C081, C-C171 and C-B71 on Jurkat cells.
FIGS. 8A-8O show the results of candidate antibodies blocking binding of different species of CD100 to the Plexin receptor. FIGS. 8A-8D are results of blocking binding of HuCD100 to Huplexin-B1-HEK293 cells by antibody molecules A14, A15, B13, H5, H21, H74, H96, H12, C-C081, C-C171 and C-B71; FIGS. 8E-8I are results of blocking binding of HuCD100 to Huplexin-B2-HEK293 cells by antibody molecules A14, H5, A15, B13, H21, H74, H96, H12, C-C081, C-C171 and C-B71; FIGS. 8J-8L are results of blocking binding of musCD100 to MusPlexin-B1-HEK293 cells by antibody molecules H5, A14, A15, B13, H12, H21, H74, H96, C-C081, C-C171 and C-B71; FIGS. 8M-8O are the results of blocking the binding of CynogC 100 to Cynoplexin-B1-HEK293 cells by antibody molecules H5, A14, A15, B13, H12, H21, H74, H96, C-C081, C-C171 and C-B71.
FIGS. 9A-9C show 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.
FIGS. 10A-10B show the results of detection of anti-PD-L1 antibody m18 cell binding activity on overexpressing cell lines human PD-L1-CHO cells and PD-L1 positive cells HCC827 cells using FACS. FIG. 10A is a graph showing the results of binding activity to human PD-L1-CHO cells; FIG. 10B is the result of binding activity to HCC827 cells.
FIGS. 11A-11B show the results of FACS detection of binding activity of anti-PD-L1 antibody m18 against various species of overexpressing cell lines. FIG. 11A is a graph showing the results of binding activity to mouse PD-L1-CHO cells; FIG. 11B shows the results of binding activity to cynomolgus PD-L1-CHO cells.
FIG. 12 shows the results of detection of specific binding activity of the anti-PD-L1 antibody m18 to B7-H1 and cognate proteins by ELISA.
FIGS. 13A-13D show the results of detection of binding activity of candidate antibodies H5-H-7, H5-a-2, B13-c-5, B13-e-2 to different species of CD100 overexpressing cell lines and CD100 positive PBMC cells after affinity maturation of antibodies H5 and B13 using FACS. FIG. 13A is a graph showing the results of binding activity to HuCD100-HEK293 cells; FIG. 13B is a graph showing the results of cell binding activity to human PBMC; FIG. 13C is a graph showing the results of binding activity to musCD100-HEK 293 cells; FIG. 13D is a graph showing the results of binding activity to CynocD100-HEK 293 cells.
FIGS. 14A-14D show the results of antibodies H5-H-7, H5-a-2, B13-c-5, B13-e-2 blocking binding of different species of CD100 to the Plexin receptor after affinity maturation of candidate antibodies H5 and B13. FIG. 14A is a graph showing the results of candidate antibodies blocking binding of HuCD100 to Huplexin-B1-HEK293 cells; FIG. 14B is a graph showing the results of candidate antibodies blocking binding of HuCD100 to Huplexin-B2-HEK293 cells; FIG. 14C is a graph showing the results of candidate antibodies blocking binding of musCD100 to MusPlexin-B1-HEK293 cells; FIG. 14D is a graph showing the results of candidate antibodies blocking the binding of CynoCD100 to Cynoplexin-B1-HEK293 cells.
FIG. 15 shows the results of MDSC proliferation inhibition assays for antibodies H5-H-7, H5-a-2, B13-c-5, B13-e-2 after affinity maturation of candidate antibodies H5 and B13.
Fig. 16A to 16B show the detection results of the tumor suppression effect. FIG. 16A shows the results of tumor volume change; fig. 16B shows the result of weight change.
Detailed Description
Definition of the definition
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
As used herein, "at least one" or "one or more" may mean 1, 2, 3, 4, 5, 6, 7, 8 or more.
As used herein, the terms "comprises," "comprising," "includes," "including," "having" and "containing" are open-ended, meaning the inclusion of the stated elements, steps or components, but not the exclusion of other non-recited elements, steps or components. The expression "consisting of … …" does not include any elements, steps or components not specified. The expression "consisting essentially of … …" means that the scope is limited to the specified elements, steps, or components, plus any optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of … …" and "consisting of … …" are encompassed within the meaning of the expression "comprising".
As used herein, the term "and/or" in connection with a plurality of recited elements should be understood to include both individual and combined options. In other words, "and/or" includes "and" as well as "or". For example, a and/or B include A, B and a+b. A. B and/or C include A, B, C and any combination thereof, e.g., a+ B, A + C, B +c and a+b+c. Further elements defined by "and/or" are to be understood in a similar manner and include any one of, and any combination of, these.
Any numerical value or range of numerical values, such as concentration or range of concentration, should be construed as modified by the term "about" in any event, unless otherwise indicated. Thus, a numerical value typically includes ±10% of the value. For example, a concentration of 1mg/mL includes 0.9mg/mL to 1.1mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range explicitly includes all possible subranges, all individual values within the range including integers and fractions within the range unless the context clearly indicates otherwise.
As used herein, "antibody" refers to an immunoglobulin or fragment thereof that specifically binds an epitope through at least one antigen binding site. Antibodies encompass antibody fragments. As used herein, the term "antibody" includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, fab fragments, fab 'fragments, F (ab') 2 Fragments, fv fragments, disulfide-linked Fv (dsFv), fd fragments, fd' fragments, single chain Fv (scFv), single chain Fab (scFab), diabodies, anti-idiotype (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin class (e.g., igG, igM, igD, igE, igA and IgY), any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass (e.g., igG2a and IgG2 b). In a preferred embodiment, the antibody of the invention is a human antibody.
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 that comprises at least the antigen-binding fragmentA portion of the variable region of the antibody (e.g., one or more CDRs and/or one or more antibody binding sites) and thus retains binding specificity as well as at least a portion of the specific binding capacity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds the same antigen as an antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2 Single chain Fv (scFv), fv, dsFv, diabodies, fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., methods in Molecular Biology, vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); chapter 1; p 3-25, kipriyanov). The fragments may comprise multiple strands linked together, for example by disulfide bonds and/or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacement of the corresponding region), obtains antibodies that immunospecifically bind to an antigen.
As used herein, "immunoglobulin single variable domain" or "single variable domain" refers to a single variable region (variable domain) having antigen binding activity. Unlike conventional antibodies, which consist of a pair of VH and VL functional antigen-binding units, a single variable domain can alone form a functional antigen-binding unit. The single variable domains may be derived from naturally occurring light chain-free antibodies, such as the variable domains (variable domain of heavy chain of heavy-chain antibodies, VHH) of heavy chain antibodies of camelids (e.g., camels and alpacas) and the single variable domains (IgNAR variable single-domain, VNAR) of the shark's neoantigen receptor, or may be selected from full length antibodies, such as the light chain variable domains and heavy chain variable domains of human antibodies that have antigen binding activity. VHHs typically comprise three highly variable "Complementarity Determining Regions (CDRs)" and four relatively conserved "Framework Regions (FRs)" and are connected in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus.
As used herein, "single domain antibody (sdAb)" or "nanobody" refers to an antibody that comprises a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment. Like the variable regions of full length antibodies, single variable domains typically comprise CDR1, CDR2, and CDR3, which form the antigen binding site, and a framework region that serves as a support. Unlike full length antibodies, which typically comprise two heavy and two light chains, single domain antibodies typically comprise a single peptide chain consisting of a single variable domain, with a molecular weight of only about 15 kDa. The single variable domain may be, for example, a variable domain of a heavy chain antibody of alpaca (variable domain of heavy-chain antibody, VHH), a variable domain of a IgNAR of shark or a variable domain of a human light chain antibody.
As used herein, the terms "Heavy-chain-only antibody" and "Heavy-chain antibody" are used interchangeably and are presented in their broadest sense to refer to antibodies lacking the light chain of a conventional antibody that comprise only one VHH and do not comprise the Heavy chain constant region of CH1 (e.g., an Fc fragment).
"Fc fragment" generally refers to a crystallizable fragment of a conventional antibody or heavy chain antibody that has been digested with papain. In general, the Fc fragment of IgG and heavy chain antibodies may comprise part of the hinge region, CH2 and CH3. In this context, the Fc fragment may comprise at least part of the hinge region (e.g., all or part of the hinge region), CH2 and CH3.
As used herein, a "full length antibody" generally comprises two heavy chains (which may be labeled H and H ') and two light chains (which may be labeled L and L'). Wherein each heavy chain may be a full length immunoglobulin heavy chain or any functional region thereof that retains antigen binding capacity, and each light chain may be a full length light chain or any functional region. Each light chain comprises a "light chain variable region (VL)" and a "light chain constant region (CL)" from the N-terminus (amino acid terminus) to the C-terminus (carboxy terminus). Each heavy chain comprises, from the N-terminus to the C-terminus, a "heavy chain variable region (VH)" and a "heavy chain constant region (CH)". In general, the heavy chain constant region of a full length antibody may comprise the VH-CH 1-hinge region (range) CH2-CH3 from the N-terminus to the C-terminus. Each heavy chain (H and H ') is paired with a light chain (L and L', respectively).
Light chains are classified as either Kappa or Lambda (Kappa or Lambda). Each heavy chain class may be associated with either a kappa or lambda light chain. Typically, the light and heavy chains are covalently bound to each other, and when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the "tail" portions of the two heavy chains are bound together by covalent disulfide bonds or non-covalent linkages. In the heavy chain, the amino acids range from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of some antibodies (e.g., igG antibodies) includes two heavy chain subunits and two light chain subunits, which are covalently linked by disulfide bonds to form a "Y" structure.
Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used from a functional perspective. In this regard, it is understood that the variable region of the Variable Light (VL) chain or Variable Heavy (VH) chain portion determines antigen recognition and specificity. In contrast, the constant regions of the light Chain (CL) and heavy chain (CH 1, CH2 or CH 3) confer biological properties such as secretion, trans-placental movement, fc receptor binding, complement fixation, and the like. According to convention, the numbering of the constant region domains increases away from the antigen binding site or the N-terminus of the antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chains, respectively.
As described above, the variable region (i.e., the "binding domain") allows the binding molecule to selectively recognize an epitope on an antigen and specifically bind the epitope. That is, for example, the VL domain and VH domain of a binding molecule of an antibody, or a subset of these Complementarity Determining Regions (CDRs), combine to form a variable region that defines a three-dimensional antigen binding site. More specifically, the antigen binding site is defined by three CDRs on each VH and VL chain. These total six "complementarity determining regions" or "CDRs" are non-contiguous short sequences of amino acids that are specifically positioned to form a binding domain as the antibody adopts its three-dimensional configuration in an aqueous environment. The remaining amino acids in the binding domain are referred to as the "Framework (FR)" region, exhibiting minor intermolecular differences. The binding domain formed by the localized CDRs defines a surface complementary to an epitope on the immunoreactive antigen. Such complementary surfaces promote non-covalent binding of the antibody to its complementary epitope. The amino acids of the CDR and framework regions, respectively, that make up any given heavy or light chain variable region can be identified by conventional methods (see, "immunohot protein sequence (Sequences of Proteins of Immunological Interest)", kabat, e.et al, U.S. department of health and public service (U.S. device 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 entirety). Herein, CDRs of the light chain variable region (CDRL or LCDR) may be referred to as LCDR1, LCDR2 and LCDR3, and CDRs of the heavy chain variable region (CDRH or HCDR) may be referred to as HCDR1, HCDR2 and HCDR3.
In the present invention, the amino acid sequences of the CDRs are all shown according to the AbM definition rules (the sequences shown according to the AbM definition rules are also in the claims of the present invention). However, it is well known to those skilled in the art that CDRs of an antibody can be defined in a variety of ways, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (see, e.g., chothia, c.et Al, nature,342,877-883 (1989); and Al-Lazikani, B.et Al, J.mol.biol.,273,927-948 (1997)), kabat based on antibody sequence variability (see, e.g., kabat, E.A.et Al. (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. device of Health and Human Services, NIH Publication No. 91-3242), abM (Martin, A.C. R.and J.Allen (2007) "Bioinformatics tools for antibody engineering," in S.Dben (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)), GT, the International ImMunoGeneTics Information System Cold Spring Harb protc.; and Lefranc, M-P.et Al., (6)), and the clustering of which is based on the mass of the crystal structure of the neighbor (s.J.Biol., J.J.Lev. J.6), J.J.Lev. 6) is defined by the nearest neighbor (J.J.LbL.Verlag, pp.95-118). It will be appreciated by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present invention. Although the scope of the claimed invention is based on the sequences shown by the AbM definition rules, the amino acid sequences corresponding to the definition rules according to other CDRs should also fall within the scope of the claimed invention.
Thus, when referring to defining antibodies with a particular CDR sequence as defined herein, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the particular CDR sequence, but whose purported CDR boundaries differ from the particular CDR boundaries defined herein by the application of different protocols (e.g., different assignment system rules or combinations).
As used herein, the terms "framework region" and "framework region" are used interchangeably. As used herein, the term "framework region", "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR sequences as defined above.
Generally, an "Fv" fragment consisting of one VH and one VL by non-covalent interactions is considered to be the smallest antigen-binding fragment that contains an antigen-binding site. But single variable domains (single domain antibodies) also have antigen binding capacity. "Single chain Fv (scFv)" may be obtained by linking VH and VL via a peptide linker. "disulfide stabilized Fv (dsFv)" or "single chain disulfide stabilized Fv (scdsFv or dsscFv)", respectively, can be obtained by introducing disulfide bonds into Fv or scFv.
As used herein, fab fragments are antibody fragments obtained by digestion of full length immunoglobulins with papain, or fragments of the same structure synthetically produced, e.g., by recombinant methods. It comprises one complete antibody light chain (VL-CL) and antibody heavy chain variable region and one heavy chain constant region (VH-CH 1, also called Fd). A single chain "Fab (scFab)" can be obtained by ligating CL and CH1 in "Fab" with a peptide linker. "F (ab') 2 "is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at a pH of 4.0-4.5, or a fragment having the same structure synthetically produced, for example, by recombinant means. Which essentially comprises the steps ofDisulfide-linked two Fab fragments of the chain region. "Fab '" is F (ab') 2 Is capable of reducing F (ab') 2 Disulfide bonds of the hinge region are obtained.
The term "hinge region" as used herein includes the portion of the heavy chain molecule that connects the CH1 domain and the CH2 domain. The hinge region comprises about 25 amino acids and is flexible, thus allowing the two N-terminal antigen binding regions to move independently.
The term "disulfide" as used herein includes covalent bonds formed between two sulfur atoms. The amino acid cysteine comprises a sulfhydryl group that may form a disulfide bond or bridge a second sulfhydryl group.
The term "chimeric antibody" as used herein refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species, while the constant region (which may be intact, a portion or modified) is obtained from a second species. In some embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.
As used herein, the term "humanized antibody" refers to an antibody in which a non-human antibody is modified to increase sequence homology with a human antibody. Humanized antibodies generally retain the antigen binding capacity of the non-human antibody from which they are derived and have low immunogenicity to humans. Humanized antibodies may be obtained by antibody engineering any non-human species antibody or antibodies (e.g., chimeric antibodies) comprising sequences derived from a non-human species therein. The 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, CDR sequences of a non-human antibody (e.g., a murine antibody) are grafted into human antibody framework regions. In some cases, to preserve the antigen binding capacity and/or stability of a humanized antibody, critical amino acid residues of a non-human antibody (e.g., murine antibody) framework sequence may be retained in the human antibody framework region, i.e., subjected to "back-mutations" (see, e.g., morrison et al (1984) Proc. Natl. Acad. Sci.81 (21): 6851-6855;Neuberger et al (1984) Nature 312:604-608).
As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human. The definition of human antibody encompasses whole or full length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.
As used herein, an "affinity matured" antibody comprises one or more modifications (e.g., substitutions of amino acid residues) in one or more CDRs such that the affinity of the affinity matured antibody for the antigen is improved as compared to a parent antibody that does not comprise such modifications. Methods of affinity maturation of antibodies are known in the art, see, e.g., marks et al, bio/Technology 10:779-783 (1992); barbas et al, proc.Nat.Acad.Sci.USA91:3809-3813 (1994); scier et al, gene 169:147-155 (1995); and Hawkins et al, J.mol. Biol.226:889-896 (1992).
As used herein, "percent (%) sequence identity" of amino acid sequences, sequence identity "has art-recognized definitions that refer to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or by a known algorithm). The determination may be made using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, clustal Omega and FASTA software.
Herein, an amino acid sequence "derived from" or "derived from" a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence. For example, an amino acid sequence derived from a heavy chain constant region of a human immunoglobulin may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the wild-type sequence of the heavy chain constant region of the human immunoglobulin from which it is derived.
"affinity" or "binding affinity" is used to measure the strength of the interaction between an antibody and an antigen through non-covalent interactions. Affinity can be determined using conventional techniques known in the art, such as biofilm interference techniques (e.g., octet Fortebio detection systems can be employed), radioimmunoassay, surface plasmon resonance, enzyme-linked immunoassay (ELISA), or flow cytometry (FACS), among others.
"specific binding" generally means that a binding molecule, such as an antibody or fragment, variant or derivative thereof, binds an epitope through its antigen binding domain, and that the 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 the epitope through its antigen binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualitatively analyze the relative affinity of an antibody for binding to an epitope. For example, binding molecule "a" may be considered to have a higher specificity for a given epitope than binding molecule "B", or may be said to bind epitope "C" with a higher specificity than it has for the relevant epitope "D".
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 the reference antibody or antigen-binding fragment to the epitope, it can be said that the binding molecule, e.g., antibody or fragment, variant or derivative thereof, competitively inhibits the binding of the reference antibody or antigen-binding fragment to a given epitope. Competitive inhibition may be determined by any method known in the art, for example, a competition ELISA assay. It can be said that the binding molecule competitively inhibits the binding of the reference antibody or antigen binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60% or at least 50%.
The term "PD-L1" as used herein refers to a programmed cell death ligand 1 (PD-L1, see, e.g., freeman et al energy of the PD-1immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.J Exp Med.2000Oct 2;192 (7)). PD-L1 belongs to the B7 family. Alternative names or synonyms for PD-L1 include PDCD1L1, PDL1, B7 homolog (B7 homolog 1, B7-H1), surface antigen cluster 274 (cluster of differentiation, CD 274), or B7-H, among others. A representative amino acid sequence of human PD-L1 is disclosed in NCBI accession number NP-054862.1, and a representative nucleic acid sequence encoding human PD-L1 is shown in NCBI accession number: nm_ 014143.4. PD-L1 is expressed in placenta, spleen, lymph node, thymus, heart, fetal liver, and is also found in many tumor 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. Binding of PD-L1 and its receptor induces signal transduction to inhibit TCR-mediated cytokine production and activation of T cell proliferation. Thus, PD-L1 inhibits a major role in the immune system during specific events (e.g., pregnancy, autoimmune disease, tissue allograft) and is thought to allow tumor or cancer cells to bypass immune checkpoints and evade immune responses.
As used herein, the term "B7 family" refers to a class of structurally similar co-stimulatory factors in the course of an organism's immunity. It belongs to the class of immunoglobulins, which are involved in the activation of T, B cells and in the immunization of the body.
As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably to refer to the ordering of nucleotides in a polynucleotide. It will be appreciated by those skilled in the art that the coding strand (sense strand) of DNA can be considered to have the same nucleotide sequence as the RNA it encodes, with deoxythymidylate in the sequence of the coding strand of DNA corresponding to uridylate in the sequence of the RNA it encodes.
As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, 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 the provided antibodies or antigen binding fragments.
As used herein, the term "expression" includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides.
As used herein, a "vector" is a vehicle for introducing an exogenous polynucleotide into a host cell, which exogenous polynucleotide is amplified or expressed when the vector is transformed into an appropriate host cell. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, poxviral vectors, and baculovirus vectors, among others.
As used herein, a "host cell" is a cell that is used to receive, hold, replicate, and amplify a vector. Host cells may also be used to express the polypeptides encoded by the vectors. When the host cell is divided, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell may be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, heLa cells, HEK cells such as HEK 293 cells.
Terms such as "treating" or "alleviating" refer to therapeutic measures that cure, slow, reduce symptoms of, and/or intercept or slow the progression of an existing pathological condition or disorder that has been diagnosed. Terms such as "preventing," "defending," "avoiding," "suppressing," and the like refer to a prophylactic or preventative measure that prevents the progress of an undiagnosed target pathological condition or disorder. Thus, a "subject in need thereof" may include a subject already with a disease; subjects prone to disease; and a subject in need of disease prevention.
As used herein, "therapeutic effect" refers to the effect resulting from treatment of an individual that alters, generally improves or ameliorates symptoms of, or cures a disease or condition.
The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other drug that is effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; blocking or stopping cancer cell division, reducing or blocking tumor size increase; inhibiting, e.g., suppressing, blocking, preventing, stopping, delaying or reversing infiltration of cancer cells into peripheral organs, including, e.g., spread of cancer to soft tissues and bones; inhibition, e.g., suppression, retardation, prevention, shrinkage, cessation, delay, or reversal of tumor metastasis; inhibition, e.g., suppression, retardation, prevention, cessation, delay, or reversal of tumor growth; to some extent, one or more symptoms associated with cancer are alleviated, reducing morbidity and mortality; improving the quality of life; or a combination of these effects. To the extent that the drug prevents growth and/or kills existing cancer cells, it may refer to inhibition of cell growth and/or cytotoxicity.
As used herein, the term "subject" refers to a mammal, such as a human.
Antibody numbering as used herein (e.g., 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 m 18) is used only to distinguish or identify antibodies or products and is not intended to indicate that such identification is characteristic of antibodies or products of the invention. It will be appreciated by those skilled in the art that other antibodies or products are equally possible to use such a label, for example for the purpose of distinguishing or identifying, but not referring to the same or equivalent antibody or product. Similarly, like numbers or designations used in the examples are also for convenience only and the antibodies or products of the invention are defined by the features described in the appended claims.
anti-CD 100 antibodies or antigen-binding fragments thereof
The present invention provides an antibody (anti-CD 100 antibody) or antigen-binding fragment thereof directed against CD100, wherein the antibody or antigen-binding fragment thereof specifically recognizes and binds CD100.
In some embodiments, the anti-CD 100 antibodies or antigen-binding fragments thereof of the invention are chimeric, humanized, human, scFv, fab, fab ', F (ab') 2 Fv fragments, disulfide stabilized Fv (dsFv) or diabodies. Preferably, the antibody or antigen binding fragment thereof is a human antibody.
In one embodiment, the antibody or antigen binding fragment thereof has at least one of the following characteristics:
1) Has affinity activity to CD100 protein;
2) Has affinity activity to CD100 positive cells;
3) Blocking binding of CD100 to Plexin-B1 or Plexin-B2;
4) Inhibit MDSC cell proliferation;
5) Inhibit tumor growth.
In some embodiments, the antibodies or antigen binding fragments thereof of the invention are capable of specifically binding to CD100 (e.g., human CD 100) and blocking its interaction with Plexin-B1 or Plexin-B2.
In some embodiments, the tumors targeted include, but are not limited to, those described below with respect to neoplastic disease. In other embodiments, the antibodies or antigen binding fragments thereof of the invention are 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%, more preferably at least about 80%.
In some embodiments, an antibody or antigen binding fragment thereof directed against CD100 of the invention comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences, and wherein the HCDR1 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID No. 1, 7, 19, 33, 41, 47 or 52; the HCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 2, 8, 14, 20, 26, 42, 48 or 53 by NO more than 2 amino acid additions, deletions or substitutions; and/or the HCDR3 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID No. 3, 9, 15, 21, 27, 30, 35, 38, 43, 49 or 54.
In some embodiments, an antibody or antigen-binding fragment thereof directed against CD100 of the invention comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences, and wherein the LCDR1 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID No. 4, 10, 16, 36, 44 or 50; the LCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 5, 11 or 45 by NO more than 2 amino acid additions, deletions or substitutions; and/or the LCDR3 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 6, 12, 18, 24, 37, 46, 51 or 56 by NO more than 2 amino acid additions, deletions or substitutions.
In some embodiments, an antibody or antigen-binding fragment thereof of the invention directed against CD100 comprises a heavy chain variable region and a light chain variable region, wherein
The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences and wherein the HCDR1 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID No. 1, 7, 19, 33, 41, 47 or 52; the HCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 2, 8, 14, 20, 26, 42, 48 or 53 by NO more than 2 amino acid additions, deletions or substitutions; and/or the HCDR3 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID NO 3, 9, 15, 21, 27, 30, 35, 38, 43, 49 or 54; and
The light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences and wherein the LCDR1 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID No. 4, 10, 16, 36, 44 or 50; the LCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 5, 11 or 45 by NO more than 2 amino acid additions, deletions or substitutions; and/or the LCDR3 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 6, 12, 18, 24, 37, 46, 51 or 56 by NO more than 2 amino acid additions, deletions or substitutions.
In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 7 (SEQ ID NO. 7; GFTFSSYSMN) wherein amino acid position 3 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 amino acid at position 8 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 amino acid 3 and amino acid 8 are substituted. In one embodiment, the amino acid at position 3 of SEQ ID NO. 7 is substituted with P and the amino acid at position 8 of SEQ ID NO. 7 is substituted with E (SEQ ID NO. 13; GFPFSSYEMN).
In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 1 (SEQ ID NO. 1; SGSFSGYYWT) wherein amino acid 1 is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 1 (SEQ ID NO. 1; SGSFSGYYYWT) wherein amino acid 10 is substituted. In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 1 (SEQ ID NO. 1; SGSFSGYYYWT) wherein amino acid 1 and amino acid 10 are substituted. In one embodiment, the amino acid at position 1 of SEQ ID NO. 1 is substituted with G and the amino acid at position 10 of SEQ ID NO. 1 is substituted with S (SEQ ID NO. 25; GGSFSGYYWS).
In one embodiment, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 33 (SEQ ID NO. 33; GGSISSSNWWS) wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 of SEQ ID NO. 33 is substituted with G (SEQ ID NO. 57; GGSISGSNSWWS).
In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 26 (SEQ ID NO. 26; EINHSGSTN) wherein amino acid 3 is substituted. In one embodiment, amino acid 3 of SEQ ID NO. 26 is substituted with Y (SEQ ID NO. 34; EIYHSGSTN).
In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 26 (SEQ ID NO. 26; EINHSGSTN) wherein amino acid 3 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 amino acid 7 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 amino acid 3 and amino acid 7 are substituted. In one embodiment, the amino acid at position 3 of SEQ ID NO. 26 is substituted with Y and the amino acid at position 7 of SEQ ID NO. 26 is substituted with E (SEQ ID NO. 58; EIYHSGETN).
In one embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 35 (SEQ ID NO. 35; YDFWSGSGLDY), wherein amino acid 4 is substituted. In one embodiment, the amino acid at position 4 of SEQ ID NO. 35 is substituted with E (SEQ ID NO. 60; YDFESGSGGLDY).
In one embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 3 (SEQ ID NO. 3; GPAYYADALDGFDI) wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 of SEQ ID NO. 3 is substituted with P (SEQ ID NO. 63; GPAYPDADGFDI).
In one embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 3 (SEQ ID NO. 3; GPAYYADALDGFDI) wherein the amino acid at position 5 is substituted. In one embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 3 (SEQ ID NO. 3; GPAYYADALDGFDI) wherein the amino acid at position 6 is substituted. In one embodiment, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 3 (SEQ ID NO. 3; GPAYYADALDGFDI) wherein amino acid 5 and amino acid 6 are substituted. In one embodiment, the 5 th amino acid in SEQ ID NO. 3 is substituted with L and the 6 th amino acid in SEQ ID NO. 3 is substituted with P (SEQ ID NO. 66; GPAYLPDALDGFDI).
In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 16 (SEQ ID NO. 16; SGDKLGDKYAY) wherein amino acid 10 is substituted. In one embodiment, amino acid 10 of SEQ ID NO. 16 is substituted with V (SEQ ID NO. 22; SGDKLGDKYVY).
In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 16 (SEQ ID NO. 16; SGDKLGDKYAY) wherein amino acid 7 is substituted. In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 16 (SEQ ID NO. 16; SGDKLGDKYAY) wherein amino acid 11 is substituted. In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 16 (SEQ ID NO. 16; SGDKLGDKYAY) wherein amino acid 7 and amino acid 11 are substituted. In one embodiment, the 7 th amino acid of SEQ ID NO. 16 is substituted with E and the 11 th amino acid of SEQ ID NO. 16 is substituted with F (SEQ ID NO. 31; SGDKGLGEKYAF).
In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 16 (SEQ ID NO. 16; SGDKLGDKYAY) wherein amino acid 4 is substituted. In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 16 (SEQ ID NO. 16; SGDKLGDKYAY) wherein amino acid 10 is substituted. In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 16 (SEQ ID NO. 16; SGDKLGDKYAY) wherein amino acid 4 and amino acid 10 are substituted. In one embodiment, the amino acid at position 4 of SEQ ID NO. 16 is substituted with R and the amino acid at position 10 of SEQ ID NO. 16 is substituted with S (SEQ ID NO. 39; SGDRLGDKYSY).
In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 36 (SEQ ID NO. 36; SGDKLGDKFAS) with amino acid 7 substituted. In one embodiment, the amino acid at position 7 of SEQ ID NO. 36 is substituted with E (SEQ ID NO. 61; SGDKLGEKFAS).
In one embodiment, the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO. 4 (SEQ ID NO. 4; SGDKLGDKYVS) with the amino acid at position 6 being substituted. In one embodiment, the amino acid at position 6 in SEQ ID NO. 4 is substituted with Q (SEQ ID NO. 64; SGDKLQDKYVS).
In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 5 (SEQ ID NO. 5; QDNKRRS) wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 of SEQ ID NO. 5 is substituted with S (SEQ ID NO. 17; QDSKRUS).
In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 5 (SEQ ID NO. 5; QDNKRRS) wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 of SEQ ID NO. 5 is substituted with R (SEQ ID NO. 23; QDRKRUS).
In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 5 (SEQ ID NO. 5; QDNKRRS) wherein the amino acid at position 3 is substituted. In one embodiment, the amino acid at position 3 of SEQ ID NO. 5 is substituted with A (SEQ ID NO. 28; QDAKRUS).
In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 11 (SEQ ID NO. 11; AASSLQS) wherein amino acid 1 is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 11 (SEQ ID NO. 11; AASSLQS) with the amino acid at position 5 being substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 11 (SEQ ID NO. 11; AASSLQS) wherein amino acid 1 and amino acid 5 are substituted. In one embodiment, the amino acid at position 1 of SEQ ID NO. 11 is substituted with T and the amino acid at position 5 of SEQ ID NO. 11 is substituted with V (SEQ ID NO. 55; TASSVQS).
In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 5 (SEQ ID NO. 5; QDNKRRS) wherein the amino acid at position 3 is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 5 (SEQ ID NO. 5; QDNKRRS) wherein amino acid 7 is substituted. In one embodiment, the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO. 5 (SEQ ID NO. 5; QDNKRRS) wherein amino acid 3 and amino acid 7 are substituted. In one embodiment, the amino acid at position 3 in SEQ ID NO. 5 is substituted with R and the amino acid at position 7 in SEQ ID NO. 5 is substituted with N (SEQ ID NO. 59; QDRKRN).
In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 6 (SEQ ID NO. 6; QAWDSSTKAYV) with the amino acid at position 8 being substituted. In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 6 (SEQ ID NO. 6; QAWDSSTKAYV) with the amino acid at position 9 being substituted. In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 6 (SEQ ID NO. 6; QAWDSSTKAYV) wherein amino acids 8 and 9 are substituted. In one embodiment, the amino acid at position 8 of SEQ ID NO. 6 is substituted with A and the amino acid at position 9 of SEQ ID NO. 6 is substituted with G (SEQ ID NO. 29; QAWDSSTAGYV).
In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 18 (SEQ ID NO. 18; QAWDSSTV) with the addition of 1 amino acid between amino acid 7 and amino acid 8. In one embodiment, the amino acid added between amino acid 7 and amino acid 8 in SEQ ID NO. 18 is Y (SEQ ID NO. 32; QAWDSSTYV).
In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown as SEQ ID NO. 18 (SEQ ID NO. 18; QAWDSSTV) with the amino acid at position 2 being substituted. In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 18 (SEQ ID NO. 18; QAWDSSTV) with the addition of 1 amino acid between amino acid 7 and amino acid 8. In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 18 (SEQ ID NO. 18; QAWDSSTV) wherein amino acid 2 is substituted and 1 amino acid is added between amino acid 7 and amino acid 8. In one embodiment, the amino acid at position 2 of SEQ ID NO. 18 is substituted with V and the amino acid added between amino acid at positions 7 and 8 of SEQ ID NO. 18 is A (SEQ ID NO. 40; QVWLDSTAV).
In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 37 (SEQ ID NO. 37; QAWDSGTVI) with the amino acid at position 8 being substituted. In one embodiment, amino acid 8 of SEQ ID NO. 37 is substituted with A (SEQ ID NO. 62; QAWDSGTAI).
In one embodiment, the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO. 6 (SEQ ID NO. 6; QAWDSSTKAYV) wherein the amino acid at position 6 is substituted. In one embodiment, the amino acid at position 6 of SEQ ID NO. 6 is substituted with E (SEQ ID NO. 65; QAWDSETKAYV).
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth 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.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NOs 4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; 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.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth 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 of SEQ ID NO 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, 54, 60, 63 or 66; and
the light chain variable region comprises the LCDR1 sequence of SEQ ID NOs 4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; 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.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 1, the HCDR2 sequence set forth in SEQ ID NO. 2 and the HCDR3 sequence set forth in SEQ ID NO. 3.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 7, the HCDR2 sequence set forth in SEQ ID NO. 8 and the HCDR3 sequence set forth in SEQ ID NO. 9.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 13, the HCDR2 sequence set forth in SEQ ID NO. 14 and the HCDR3 sequence set forth 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 set forth in SEQ ID NO. 25, the HCDR2 sequence set forth in SEQ ID NO. 26 and the HCDR3 sequence set forth in SEQ ID NO. 27.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 25, the HCDR2 sequence set forth in SEQ ID NO. 26 and the HCDR3 sequence set forth in SEQ ID NO. 30.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 33, the HCDR2 sequence set forth in SEQ ID NO. 34 and the HCDR3 sequence set forth in SEQ ID NO. 35.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 33, the HCDR2 sequence set forth in SEQ ID NO. 34 and the HCDR3 sequence set forth in SEQ ID NO. 38.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 41, the HCDR2 sequence of SEQ ID NO. 42 and the HCDR3 sequence of SEQ ID NO. 43.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 47, the HCDR2 sequence set forth in SEQ ID NO. 48 and the HCDR3 sequence set forth in SEQ ID NO. 49.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 52, the HCDR2 sequence of SEQ ID NO. 53 and the HCDR3 sequence of SEQ ID NO. 54.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO:57, the HCDR2 sequence set forth in SEQ ID NO:58 and the HCDR3 sequence set forth in SEQ ID NO: 35.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 33, the HCDR2 sequence set forth in SEQ ID NO. 34 and the HCDR3 sequence set forth in SEQ ID NO. 60.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 1, the HCDR2 sequence set forth in SEQ ID NO. 2 and the HCDR3 sequence set forth in SEQ ID NO. 63.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 1, the HCDR2 sequence set forth in SEQ ID NO. 2 and the HCDR3 sequence set forth in SEQ ID NO. 66.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 4, the LCDR2 sequence of SEQ ID NO. 5 and the LCDR3 sequence of SEQ ID NO. 6.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 10, the LCDR2 sequence of SEQ ID NO. 11 and the LCDR3 sequence of SEQ ID NO. 12.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 16, the LCDR2 sequence of SEQ ID NO. 17 and the LCDR3 sequence of SEQ ID NO. 18.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 22, the LCDR2 sequence of SEQ ID NO. 23 and the LCDR3 sequence of SEQ ID NO. 24.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 16, the LCDR2 sequence of SEQ ID NO. 28 and the LCDR3 sequence of SEQ ID NO. 29.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 31, the LCDR2 sequence of SEQ ID NO. 5 and the LCDR3 sequence of SEQ ID NO. 32.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 36, the LCDR2 sequence of SEQ ID NO. 23 and the LCDR3 sequence of SEQ ID NO. 37.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 39, the LCDR2 sequence of SEQ ID NO. 17 and the LCDR3 sequence of SEQ ID NO. 40.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 44, the LCDR2 sequence of SEQ ID NO. 45 and the LCDR3 sequence of SEQ ID NO. 46.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 50, the LCDR2 sequence of SEQ ID NO. 11 and the LCDR3 sequence of SEQ ID NO. 51.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 50, the LCDR2 sequence of SEQ ID NO. 55 and the LCDR3 sequence of SEQ ID NO. 56.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 36, the LCDR2 sequence of SEQ ID NO. 59 and the LCDR3 sequence of SEQ ID NO. 37.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 61, the LCDR2 sequence of SEQ ID NO. 23 and the LCDR3 sequence of SEQ ID NO. 62.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 64, the LCDR2 sequence of SEQ ID NO. 5 and the LCDR3 sequence of SEQ ID NO. 65.
In one embodiment, the light chain variable region comprises the LCDR1 sequence of SEQ ID NO. 4, the LCDR2 sequence of SEQ ID NO. 5 and the LCDR3 sequence of SEQ ID NO. 6.
In one embodiment, the 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 an LCDR1 sequence shown as SEQ ID NO. 4, an LCDR2 sequence shown as SEQ ID NO. 5 and an LCDR3 sequence shown as SEQ ID NO. 6.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 7, the HCDR2 sequence of SEQ ID NO. 8 and the HCDR3 sequence of SEQ ID NO. 9; the light chain variable region comprises an LCDR1 sequence shown as SEQ ID NO. 10, an LCDR2 sequence shown as SEQ ID NO. 11 and an LCDR3 sequence shown as SEQ ID NO. 12.
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.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 19, the HCDR2 sequence of SEQ ID NO. 20 and the HCDR3 sequence of SEQ ID NO. 21; 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 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.
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.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 33, the HCDR2 sequence set forth in SEQ ID NO. 34 and the HCDR3 sequence set forth 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.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 33, the HCDR2 sequence set forth in SEQ ID NO. 34 and the HCDR3 sequence set forth 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.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 41, the HCDR2 sequence of SEQ ID NO. 42 and the HCDR3 sequence of SEQ ID NO. 43; the light chain variable region comprises the LCDR1 sequence shown as SEQ ID NO. 44, the LCDR2 sequence shown as SEQ ID NO. 45 and the LCDR3 sequence shown as SEQ ID NO. 46.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 47, the HCDR2 sequence set forth in SEQ ID NO. 48 and the HCDR3 sequence set forth in SEQ ID NO. 49; the light chain variable region comprises an LCDR1 sequence shown as SEQ ID NO. 50, an LCDR2 sequence shown as SEQ ID NO. 11 and an LCDR3 sequence shown as SEQ ID NO. 51.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 52, the HCDR2 sequence of SEQ ID NO. 53 and the HCDR3 sequence of SEQ ID NO. 54; the light chain variable region comprises the LCDR1 sequence shown as SEQ ID NO. 50, the LCDR2 sequence shown as SEQ ID NO. 55 and the LCDR3 sequence shown as SEQ ID NO. 56.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 57, the HCDR2 sequence of SEQ ID NO. 58 and the HCDR3 sequence of SEQ ID NO. 35; 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 heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 33, the HCDR2 sequence set forth in SEQ ID NO. 34 and the HCDR3 sequence set forth 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.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO. 1, the HCDR2 sequence of SEQ ID NO. 2 and the HCDR3 sequence of SEQ ID NO. 63; the light chain variable region comprises the LCDR1 sequence shown as SEQ ID NO. 64, the LCDR2 sequence shown as SEQ ID NO. 5 and the LCDR3 sequence shown as SEQ ID NO. 65.
In one embodiment, the heavy chain variable region comprises the HCDR1 sequence set forth in SEQ ID NO. 1, the HCDR2 sequence set forth in SEQ ID NO. 2 and the HCDR3 sequence set forth in SEQ ID NO. 66; the light chain variable region comprises an LCDR1 sequence shown as SEQ ID NO. 4, an LCDR2 sequence shown as SEQ ID NO. 5 and an LCDR3 sequence shown as SEQ ID NO. 6.
In one embodiment, the heavy chain variable region comprises 1) the amino acid sequence set forth in SEQ ID NO 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95; 2) An amino acid sequence 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to 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, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
In one embodiment, the light chain variable region comprises 1) the amino acid sequence set forth in SEQ ID NO 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96; 2) An amino acid sequence 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
In one embodiment, the heavy chain variable region comprises 1) the amino acid sequence set forth in SEQ ID NO 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95; 2) An amino acid sequence 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95, preferably the additions, deletions and/or substitutions do not occur in the CDR regions; and
the light chain variable region comprises 1) the amino acid sequence set forth in SEQ ID NOs 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 67; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 68.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 69; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 70.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 71; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 72.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 73; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 74.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 75; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 76.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 77; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 78.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 79; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 80.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 81; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 82.
In one embodiment, the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 83; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 84.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 85; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 86.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 87; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 88.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 89; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 90.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 91; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 92.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 93; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 94.
In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 95; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 96.
In one embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain comprising 1) the amino acid sequence set forth in SEQ ID No. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; 2) An amino acid sequence 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
In one embodiment, the antibody or antigen binding fragment thereof comprises a light chain comprising 1) the amino acid sequence set forth in SEQ ID NO 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, or 126; 2) An amino acid sequence 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID No. 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
In one embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain comprising 1) the amino acid sequence set forth in SEQ ID No. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; 2) An amino acid sequence 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, preferably the additions, deletions and/or substitutions do not occur in the CDR regions; and
the antibody or antigen binding fragment thereof comprises a light chain comprising 1) the amino acid sequence set forth in SEQ ID NOs 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID No. 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 97; the light chain comprises an amino acid sequence shown in SEQ ID NO. 98.
In one embodiment, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO. 99; the light chain comprises an amino acid sequence shown in SEQ ID NO. 100.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 101; the light chain comprises the amino acid sequence shown in SEQ ID NO. 102.
In one embodiment, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO. 103; the light chain comprises the amino acid sequence shown in SEQ ID NO. 104.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 105; the light chain comprises the amino acid sequence shown in SEQ ID NO. 106.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 107; the light chain comprises the amino acid sequence shown in SEQ ID NO. 108.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 109; the light chain comprises the amino acid sequence shown in SEQ ID NO. 110.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 111; the light chain comprises the amino acid sequence shown in SEQ ID NO. 112.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 113; the light chain comprises the amino acid sequence shown in SEQ ID NO. 114.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 115; the light chain comprises the amino acid sequence shown in SEQ ID NO. 116.
In one embodiment, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO. 117; the light chain comprises the amino acid sequence shown in SEQ ID NO. 118.
In one embodiment, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO. 119; the light chain comprises an amino acid sequence shown as SEQ ID NO. 120.
In one embodiment, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO. 121; the light chain comprises the amino acid sequence shown in SEQ ID NO. 122.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 123; the light chain comprises the amino acid sequence shown in SEQ ID NO. 124.
In one embodiment, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 125; the light chain comprises the amino acid sequence shown in SEQ ID NO. 126.
In one embodiment, the anti-CD 100 antibody comprises anti-CD 100 antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-H-7, H5-a-2, B13-C-5 or B13-e-2, particularly anti-CD 100 antibody B13 or C-C081.
Antibody B13
In one aspect, the invention provides an antibody B13 or antigen binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 1,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO. 2, and
h CDR3 comprising the amino acid sequence set forth in SEQ ID No. 3;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 4,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 5, and
l CDR3 comprising the amino acid sequence shown in SEQ ID NO. 6.
In another embodiment, the anti-CD 100 antibody B13 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 67.
In another embodiment, the anti-CD 100 antibody B13 or antigen-binding fragment thereof comprises a light chain variable region (VL),
wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 68 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 68.
In yet another embodiment, the anti-CD 100 antibody B13 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 97.
In yet another embodiment, the anti-CD 100 antibody B13 or antigen-binding fragment thereof comprises a light chain,
wherein 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 more sequence identity to SEQ ID NO. 98.
Antibody C-C081
In another aspect, the invention provides an antibody C-C081 or antigen-binding fragment thereof to CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 7,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO. 8, and
h CDR3 comprising the amino acid sequence set forth in SEQ ID No. 9;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 10,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 11, and
L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 12.
In one embodiment, the anti-CD 100 antibody C-C081 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 69 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 69.
In one embodiment, the anti-CD 100 antibody C-C081 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises 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 more sequence identity to SEQ ID NO. 70.
In yet another embodiment, the anti-CD 100 antibody C-C081 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 99.
In yet another embodiment, the anti-CD 100 antibody C-C081 or antigen-binding fragment thereof comprises a light chain,
wherein 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 more sequence identity to SEQ ID NO. 100.
Antibody A14
In another aspect, the invention provides an antibody A14 or antigen-binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 13,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO. 14, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 15;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 16,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO:17, and
l CDR3 comprising the amino acid sequence shown in SEQ ID NO. 18.
In one embodiment, the anti-CD 100 antibody A14 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 71.
In one embodiment, the anti-CD 100 antibody A14 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises 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 more sequence identity to SEQ ID NO. 72.
In yet another embodiment, the anti-CD 100 antibody A14 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 101.
In yet another embodiment, the anti-CD 100 antibody A14 or antigen-binding fragment thereof comprises a light chain,
wherein 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 more sequence identity to SEQ ID NO. 102.
Antibody A15
In another aspect, the invention provides an antibody A15 or antigen binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 19,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO:20, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 21;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 22,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, and
L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 24.
In one embodiment, the anti-CD 100 antibody A15 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 73 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 73.
In one embodiment, the anti-CD 100 antibody A15 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises 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 more sequence identity to SEQ ID NO. 74.
In yet another embodiment, the anti-CD 100 antibody A15 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 103.
In yet another embodiment, the anti-CD 100 antibody A15 or antigen-binding fragment thereof comprises a light chain,
wherein 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 more sequence identity to SEQ ID NO. 104.
Antibody H74
In another aspect, the invention provides an antibody H74 or antigen binding fragment thereof to CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 25,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO. 26, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 27;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 16,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 28, and
l CDR3 comprising the amino acid sequence shown in SEQ ID NO. 29.
In one embodiment, the anti-CD 100 antibody H74 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 75 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 75.
In one embodiment, the anti-CD 100 antibody H74 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 76 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 76.
In yet another embodiment, the anti-CD 100 antibody H74 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 105.
In yet another embodiment, the anti-CD 100 antibody H74 or antigen-binding fragment thereof comprises a light chain,
wherein 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 more sequence identity to SEQ ID NO. 106.
Antibody H96
In another aspect, the invention provides an antibody H96 or antigen binding fragment thereof to CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 25,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO. 26, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 30;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 31,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 5, and
L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 32.
In one embodiment, the anti-CD 100 antibody H96 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 77.
In one embodiment, the anti-CD 100 antibody H96 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 78 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 78.
In yet another embodiment, the anti-CD 100 antibody H96 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 107.
In yet another embodiment, the anti-CD 100 antibody H96 or antigen-binding fragment thereof comprises a light chain,
wherein the light chain comprises the amino acid sequence shown as SEQ ID NO. 108 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 108.
Antibody H5
In another aspect, the invention provides an antibody H5 or antigen binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 33,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO 34, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 35;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 36,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, and
l CDR3 comprising the amino acid sequence shown in SEQ ID NO. 37.
In one embodiment, the anti-CD 100 antibody H5 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 79.
In one embodiment, the anti-CD 100 antibody H5 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 80 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 80.
In yet another embodiment, the anti-CD 100 antibody H5 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 109.
In yet another embodiment, the anti-CD 100 antibody H5 or antigen-binding fragment thereof comprises a light chain,
wherein the light chain comprises the amino acid sequence shown as SEQ ID NO. 110 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 110.
Antibody H12
In another aspect, the invention provides an antibody H12 or antigen binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 33,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO 34, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 38;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO 39,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO:17, and
L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 40.
In one embodiment, the anti-CD 100 antibody H12 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 81.
In one embodiment, the anti-CD 100 antibody H12 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 82 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 82.
In yet another embodiment, the anti-CD 100 antibody H12 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 111.
In yet another embodiment, the anti-CD 100 antibody H12 or antigen-binding fragment thereof comprises a light chain,
wherein the light chain comprises the amino acid sequence shown as SEQ ID NO. 112 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 112.
Antibody H21
In another aspect, the invention provides an antibody H21 or antigen binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 41,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO. 42, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 43;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown as SEQ ID NO. 44,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 45, and
l CDR3 comprising the amino acid sequence shown in SEQ ID NO. 46.
In one embodiment, the anti-CD 100 antibody H21 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 83 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 83.
In one embodiment, the anti-CD 100 antibody H21 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 84 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 84.
In yet another embodiment, the anti-CD 100 antibody H21 or antigen-binding fragment thereof comprises a heavy chain,
wherein the heavy chain comprises the amino acid sequence shown as SEQ ID NO. 113 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 113.
In yet another embodiment, the anti-CD 100 antibody H21 or antigen-binding fragment thereof comprises a light chain,
wherein the light chain comprises the amino acid sequence shown as SEQ ID NO. 114 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 114.
Antibody C-C171
In another aspect, the invention provides an antibody C-C171 or antigen binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 47,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO:48, and
h CDR3 comprising the amino acid sequence set forth in SEQ ID No. 49;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown as SEQ ID NO. 50,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 11, and
L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 51.
In one embodiment, the anti-CD 100 antibody C-C171 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 85 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 85.
In one embodiment, the anti-CD 100 antibody C-C171 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 86 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 86.
In yet another embodiment, the anti-CD 100 antibody C-C171 or antigen-binding fragment thereof comprises a heavy chain,
wherein the heavy chain comprises the amino acid sequence shown as SEQ ID NO. 115 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 115.
In yet another embodiment, the anti-CD 100 antibody C-C171 or antigen-binding fragment thereof comprises a light chain,
wherein 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 more sequence identity to SEQ ID NO. 116.
Antibody C-B71
In another aspect, the invention provides an antibody C-B71 or antigen binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 52,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO:53, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 54;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown as SEQ ID NO. 50,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO:55, and
l CDR3 comprising the amino acid sequence shown in SEQ ID NO: 56.
In one embodiment, the anti-CD 100 antibody C-B71 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 87.
In one embodiment, the anti-CD 100 antibody C-B71 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 88 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 88.
In yet another embodiment, the anti-CD 100 antibody C-B71 or antigen-binding fragment thereof comprises a heavy chain,
wherein the heavy chain comprises the amino acid sequence shown as SEQ ID NO. 117 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 117.
In yet another embodiment, the anti-CD 100 antibody C-B71 or antigen-binding fragment thereof comprises a light chain,
wherein 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 more sequence identity to SEQ ID NO. 118.
Antibody H5-H-7
In another aspect, the invention provides an antibody H5-H-7 or antigen binding fragment thereof to CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO:57,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO:58, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 35;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 36,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO:59, and
L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 37.
In one embodiment, the anti-CD 100 antibody H5-H-7 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 89.
In one embodiment, the anti-CD 100 antibody H5-H-7 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 90 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 90.
In yet another embodiment, the anti-CD 100 antibody H5-H-7 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 119.
In yet another embodiment, the anti-CD 100 antibody H5-H-7 or antigen-binding fragment thereof comprises a light chain,
wherein the light chain comprises the amino acid sequence shown as SEQ ID NO. 120 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 120.
Antibody H5-a-2
In another aspect, the invention provides an antibody H5-a-2 or antigen binding fragment thereof to CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 33,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO 34, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 60;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 61,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, and
l CDR3 comprising the amino acid sequence shown in SEQ ID NO. 62.
In one embodiment, the anti-CD 100 antibody H5-a-2 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 91.
In one embodiment, the anti-CD 100 antibody H5-a-2 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 92 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 92.
In yet another embodiment, the anti-CD 100 antibody H5-a-2 or antigen-binding fragment thereof comprises a heavy chain,
wherein 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 more sequence identity to SEQ ID NO. 121.
In yet another embodiment, the anti-CD 100 antibody H5-a-2 or antigen-binding fragment thereof comprises a light chain,
wherein 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 more sequence identity to SEQ ID NO. 122.
Antibody B13-c-5
In another aspect, the invention provides an antibody B13-c-5 or antigen binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 1,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO. 2, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 63;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 64,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 5, and
L CDR3 comprising the amino acid sequence shown in SEQ ID NO. 65.
In one embodiment, the anti-CD 100 antibody B13-c-5 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 93.
In one embodiment, the anti-CD 100 antibody B13-c-5 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 94 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 94.
In yet another embodiment, the anti-CD 100 antibody B13-c-5 or antigen-binding fragment thereof comprises a heavy chain,
wherein the heavy chain comprises the amino acid sequence shown as SEQ ID NO. 123 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 123.
In yet another embodiment, the anti-CD 100 antibody B13-c-5 or antigen-binding fragment thereof comprises a light chain,
wherein the light chain comprises the amino acid sequence shown as SEQ ID NO. 124 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 124.
Antibody B13-e-2
In another aspect, the invention provides an antibody B13-e-2 or antigen binding fragment thereof directed against CD100,
wherein the antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein
The heavy chain variable region comprises:
h CDR1 comprising the amino acid sequence shown in SEQ ID NO. 1,
h CDR2 comprising the amino acid sequence shown in SEQ ID NO. 2, and
h CDR3 comprising the amino acid sequence shown in SEQ ID NO. 66;
the light chain variable region comprises:
l CDR1 comprising the amino acid sequence shown in SEQ ID NO. 4,
l CDR2 comprising the amino acid sequence shown in SEQ ID NO. 5, and
l CDR3 comprising the amino acid sequence shown in SEQ ID NO. 6.
In one embodiment, the anti-CD 100 antibody B13-e-2 or antigen-binding fragment thereof comprises a heavy chain variable region (VH),
wherein 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 more sequence identity to SEQ ID NO. 95.
In one embodiment, the anti-CD 100 antibody B13-e-2 or antigen-binding fragment thereof comprises a light chain variable region (VL),
the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 96 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 96.
In yet another embodiment, the anti-CD 100 antibody B13-e-2 or antigen-binding fragment thereof comprises a heavy chain,
wherein the heavy chain comprises the amino acid sequence shown as SEQ ID NO. 125 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 125.
In yet another embodiment, the anti-CD 100 antibody B13-e-2 or antigen-binding fragment thereof comprises a light chain,
wherein the light chain comprises the amino acid sequence shown as SEQ ID NO. 126 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 126.
Polynucleotides, vectors and host cells
In another aspect, the invention provides a polynucleotide comprising a polynucleotide sequence encoding an anti-CD 100 antibody or antigen-binding fragment thereof of the invention.
The polynucleotides of the invention may be obtained using methods known in the art. For example, polynucleotides of the invention may be isolated from phage display libraries, yeast display libraries, immune animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemical synthesis. The polynucleotides of the invention may be codon optimized for the host cell used for expression.
In yet another aspect, the invention also provides an expression vector comprising a polynucleotide of the invention. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. Polynucleotides of the invention may be present in one or more expression vectors. In one embodiment, the polynucleotides of the 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), and the like.
The invention also provides a host cell comprising a polynucleotide or expression vector of the invention. The polynucleotides or expression vectors of the invention may be introduced into a suitable host cell using a variety of methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection, among others.
In a preferred embodiment, the host cell is used to express an anti-CD 100 antibody or antigen-binding fragment thereof of the invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., E.coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for antibody expression include, but are not limited to, external human cervical cancer cells (HeLa cells), human embryonic kidney cells (HEK cells, e.g., HEK 293 cells), chinese Hamster Ovary (CHO) cells, and other mammalian cells suitable for expression of antibodies.
The invention also provides a method of producing an anti-CD 100 antibody or antigen-binding fragment thereof of the invention comprising the steps of:
a) Culturing a host cell of the invention under suitable conditions to express an anti-CD 100 antibody or antigen-binding fragment thereof of the invention; and
b) Isolating the antibody or antigen binding fragment thereof from the host cell or culture thereof.
Pharmaceutical combination
In another aspect, the invention provides a pharmaceutical combination, two or more therapeutic or prophylactic agents may be administered to a subject by administering the pharmaceutical combination. The pharmaceutical combination comprises an antibody or antigen-binding fragment thereof directed against CD100 and an antibody or antigen-binding fragment thereof directed against PD-L1.
In one embodiment, the antibodies to CD100 include 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.
In one embodiment, the antibody or antigen-binding fragment thereof directed against PD-L1 specifically recognizes and binds PD-L1, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an immunoglobulin single variable domain.
In a preferred embodiment, the immunoglobulin single variable domain comprises:
CDR1 comprising the amino acid sequence shown in SEQ ID NO. 130,
CDR2 comprising the amino acid sequence shown in SEQ ID NO. 131, and
CDR3 comprising the amino acid sequence shown in SEQ ID NO. 132.
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 more sequence identity to SEQ ID NO. 133.
In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc fragment of human IgG 1.
In a preferred embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 134 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 134.
In one embodiment, the anti-PD-L1 antibody comprises an anti-PD-L1 antibody m18-VHH or m18.
Antibody m18-VHH
In one aspect, the invention provides an antibody m18-VHH or antigen-binding fragment thereof to PD-L1,
wherein the antibody comprises a single variable domain of an immunoglobulin, wherein
The single variable domain comprises:
CDR1 comprising the amino acid sequence shown in SEQ ID NO. 130,
CDR2 comprising the amino acid sequence shown in SEQ ID NO. 131, and
CDR3 comprising the amino acid sequence shown in SEQ ID NO. 132.
In one embodiment, the anti-PD-L1 antibody m18-VHH comprises a single variable domain of an immunoglobulin,
wherein 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 more sequence identity to SEQ ID NO. 133.
Antibody m18
In a further aspect, the invention also provides an antibody m18 or antigen binding fragment thereof directed against PD-L1,
wherein the antibody comprises a single variable domain of an immunoglobulin, wherein
The single variable domain comprises:
CDR1 comprising the amino acid sequence shown in SEQ ID NO. 130,
CDR2 comprising the amino acid sequence shown in SEQ ID NO. 131, and
CDR3 comprising the amino acid sequence shown in SEQ ID NO. 132.
In one embodiment, the anti-PD-L1 antibody m18 comprises a single variable domain of an immunoglobulin,
wherein 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 more sequence identity to SEQ ID NO. 133.
In one embodiment, the anti-PD-L1 antibody m18 further comprises an Fc fragment of human IgG1,
The antibody m18 comprises the amino acid sequence shown as SEQ ID NO. 134 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 134.
In some embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof of the invention are single domain antibodies, heavy chain antibodies, humanized antibodies, or chimeric antibodies. Preferably, the antibody or antigen binding fragment thereof is a human antibody.
In one embodiment, the antibody or antigen binding fragment thereof has at least one of the following characteristics:
1) Has affinity activity to PD-L1 positive cells;
2) Capable of specifically binding to PD-L1 protein;
3) Inhibit tumor growth.
In some embodiments, the tumors targeted include, but are not limited to, those described below with respect to neoplastic disease. In other embodiments, the antibodies or antigen binding fragments thereof of the invention are 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%, more preferably at least about 80%.
In one embodiment, the pharmaceutical combination comprises an antibody against CD100 comprising antibodies B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-H-7, H5-a-2, B13-C-5, B13-e-2 or a combination thereof; and the pharmaceutical combination comprises an antibody against PD-L1 comprising the antibody m18-VHH or m18.
In a preferred embodiment, the pharmaceutical combination comprises the anti-CD 100 antibody B13, C-C081, C-B71, H5-H-7, H5-a-2, B13-C-5, B13-e-2 or a combination thereof and the anti-PD-L1 antibody m18-VHH or m18.
In a preferred embodiment, the pharmaceutical combination comprises the anti-CD 100 antibody C-C081, B13 or B13-C-5 and the anti-PD-L1 antibody m18-VHH or m18.
In one embodiment, the pharmaceutical combination may be a pharmaceutical composition or a kit.
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising an anti-CD 100 antibody or antigen fragment thereof of the invention, and a pharmaceutically acceptable carrier. In one embodiment, the anti-CD 100 antibodies described above include anti-CD 100 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-CD 100 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-CD 100 antibodies B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-H-7, H5-a-2, B13-C-5, B13-e-2 and anti-PD-L1 antibodies m18 or m 18-VHH. In a preferred embodiment, the pharmaceutical composition comprises a combination of anti-CD 100 antibody B13, C-C081, C-B71, H5-H-7, H5-a-2, B13-C-5, B13-e-2 and anti-PD-L1 antibody m18 or m 18-VHH. In a preferred embodiment, the pharmaceutical composition comprises a combination of the anti-CD 100 antibody B13, C-C081 or B13-C-5 with the anti-PD-L1 antibody m18 or m 18-VHH.
The pharmaceutical compositions provided herein may be in a variety of dosage forms including, but not limited to, solid, semi-solid, liquid, powder, or lyophilized forms. For compositions comprising antibodies or antigen fragments thereof, preferred dosage forms may generally be, for example, injectable solutions and lyophilized powders.
The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or topical administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavity), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical). Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion). The method of administration may be, for example, injection or infusion.
It will be appreciated by those skilled in the art that the exact dosage administered will depend on various factors such as the pharmacokinetic properties of the pharmaceutical composition, the duration of the treatment, the rate of excretion of the particular compound, the purpose of the treatment, the route of administration and the condition of the subject, such as the age, health, weight, sex, diet, medical history of the patient, and other factors well known in the medical arts. As a general guideline, the anti-CD 100 antibodies or antigen-binding fragments thereof of the invention are administered in a dosage range of about 0.0001-100mg/kg, more typically 0.01-20mg/kg of subject body weight. For example, the dosage may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight or 20mg/kg body weight, or in the range of 1-20 mg/kg. Exemplary treatment regimens require weekly dosing, biweekly dosing, tricyclically dosing, weekly dosing, monthly dosing, 3 months dosing, 3-6 months dosing, or slightly shorter initial dosing intervals followed by longer post dosing intervals. In one embodiment, the dosage used may be 1200mg administered every three weeks. The mode of administration may be intravenous drip.
As used herein, a "therapeutically effective dose" refers to a dose that causes a decrease in the severity of symptoms of a disease, an increase in the frequency and duration of the disease asymptomatic phase, or prevents damage or disability due to the suffering of the disease. For example, for antiproliferative effects, preventing further tumor progression, reducing tumor size, reducing tumor blood vessels, reducing the number of cancer cells, inhibiting, delaying or reducing tumor and/or malignant cell growth and/or metastasis in a cancer patient, and/or reducing the therapeutically effective dose at which one or more symptoms associated with the disease can be observed. The therapeutically effective dose may vary depending on a number of different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is a human or other animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. In certain embodiments, the patient is a human, but non-human mammals, including transgenic animals, may also be treated. The therapeutic dose may be titrated to optimize safety and efficacy using conventional methods known to those skilled in the art.
The "therapeutically effective dose" of an antibody or antigen-binding fragment thereof of the invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, it may be assessed by examining the ability to inhibit cell growth, which inhibition may be determined in vitro by experiments well known to those skilled in the art. An effective amount of an antibody or antigen-binding fragment thereof of the invention is capable of reducing tumor size or otherwise alleviating a symptom in a subject, such as preventing and/or treating metastasis or recurrence. Such amounts may be determined by one of skill in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.
Treatment of
In a further aspect, the invention relates to the use of an anti-CD 100 antibody or antigen-binding fragment thereof, a pharmaceutical composition or a pharmaceutical combination of the invention in the manufacture of a medicament for treating a disease in a subject.
The invention also relates to an anti-CD 100 antibody or antigen-binding fragment thereof, a pharmaceutical composition or a pharmaceutical combination of the invention for use in the treatment of a disease.
The invention also provides a method of treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD 100 antibody or antigen-binding fragment thereof, a pharmaceutical composition or a pharmaceutical combination of the invention.
In one embodiment, the disease as described above is cancer. Blocking CD100 by the antibodies of the invention may enhance the immune response to cancer cells in a patient. CD100 is widely expressed in many human tumors, whose expression is associated with human invasive disease. In the preclinical tumor microenvironment, inflammatory and tumor cell expression CD100 regulates the infiltration, spatial distribution and activity of myeloid and lymphoid cells. CD100 binds to the Plexin receptor located on myeloid cells in the tumor microenvironment. When the CD100 protein is blocked, the CD100 barrier may be eliminated. Once the barrier is breached, inflammatory dendritic cells and pro-inflammatory antigen presenting cells migrate and penetrate into the tumor. In preclinical animal models of cancer, blocking CD100 with antibodies can delay tumor growth and promote persistent tumor rejection.
As used herein, "cancer" includes, but is not limited to, hematological tumors and solid tumors. Herein, 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 carcinoma, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma, renal cancer, hepatic carcinoma, prostate cancer, vulval cancer, thyroid cancer, head and neck cancer, and the like, or any combination thereof. Hematological neoplasms include, for example, leukemia, lymphoma, myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma, and the like, or any combination thereof. The cancer may also be metastatic cancer. "metastasis" refers to the spread of cancer cells from their original site to other parts of the body.
Combination therapy
For cancer treatment, the anti-CD 100 antibodies or antigen-binding fragments, pharmaceutical compositions, or combinations thereof of the invention may be used in combination with other therapeutic methods, including, but not limited to: surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibition and palliative therapy.
The anti-CD 100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or pharmaceutical combinations of the invention may also be administered in combination with at least one or more of the therapeutic agents described herein. The mode of administration of the combination is not limited. For example, the therapeutic agents described below may be administered all at once or separately. When administered separately (in the case of using mutually different administration regimens), they can be administered continuously without interruption or at predetermined intervals.
In certain embodiments, the anti-CD 100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or pharmaceutical combinations of the invention are further administered in combination with one or more therapeutic agents selected from the group consisting of: chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors and tumor antigen targeting drugs. Chemotherapeutic agents may include, for example: antimetabolites, alkylating agents, cytotoxic agents, topoisomerase inhibitors, 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, oprozomib) and tyrosine kinase inhibitors (e.g., BTK inhibitors).
In one embodiment, the anti-CD 100 antibody B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-H-7, H5-a-2, B13-C-5, B13-e-2 is administered in combination with the anti-PD-L1 antibody m18 or m 18-VHH.
In a preferred embodiment, the anti-CD 100 antibody B13, C-C081, C-B71, H5-H-7, H5-a-2, B13-C-5 or B13-e-2 is administered in combination with the anti-PD-L1 antibody m18 or m 18-VHH.
In a preferred embodiment, the anti-CD 100 antibody C-C081, B13 or B13-C-5 is administered in combination with the anti-PD-L1 antibody m18 or m 18-VHH.
In some embodiments, the anti-CD 100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or combinations of the invention are administered in combination with a chemotherapeutic agent. In some embodiments, the anti-CD 100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or combinations of the invention are administered in combination with an immune checkpoint inhibitor. In some embodiments, an anti-CD 100 antibody or antigen-binding fragment thereof, pharmaceutical composition or pharmaceutical combination of the invention is administered in combination with a radioisotope. In some embodiments, the anti-CD 100 antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or combinations of the invention are administered in combination with a tumor-targeting drug.
Kit for detecting a substance in a sample
The invention also provides a kit comprising an anti-CD 100 antibody or antigen-binding fragment thereof, a pharmaceutical composition or a pharmaceutical combination of the invention, and instructions for use. The kit may also comprise a suitable container. In certain embodiments, the kit further comprises a device for administering the drug. Kits generally include a label that indicates 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 or otherwise with the kit.
In one embodiment, the kit comprises anti-CD 100 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.
In one embodiment, the kit comprises anti-CD 100 antibodies B13, C-C081, C-B71, H5-H-7, H5-a-2, B13-C-5, B13-e-2, or combinations thereof.
In one embodiment, the kit comprises anti-CD 100 antibody C-C081, B13 or B13-C-5.
In one embodiment, the kit comprises a combination of anti-CD 100 antibodies B13, C-C081, A14, A15, H74, H96, H5, H12, H21, C-C171, C-B71, H5-H-7, H5-a-2, B13-C-5, B13-e-2 and anti-PD-L1 antibodies m18 or m 18-VHH.
In one embodiment, the kit comprises a combination of anti-CD 100 antibody B13, C-C081, C-B71, H5-H-7, H5-a-2, B13-C-5, B13-e-2 and anti-PD-L1 antibody m18 or m 18-VHH.
In one embodiment, the kit comprises a combination of the anti-CD 100 antibody C-C081, B13 or B13-C-5 with the anti-PD-L1 antibody m18 or m 18-VHH.
Advantageous effects
The anti-CD 100 antibodies or antigen binding fragments thereof of the invention have at least one of the following beneficial effects: 1) Has affinity activity to human, mouse or monkey CD100 protein; 2) Has affinity activity to CD100 positive cells; 3) Blocking binding of CD100 to Plexin-B1 or Plexin-B2; 4) Inhibit MDSC cell proliferation; 5) Inhibit tumor growth.
The anti-CD 100 antibodies or antigen-binding fragments thereof of the invention in combination with an anti-PD-L1 antibody or antigen-binding fragment thereof have at least one of the following beneficial effects: 1) Inhibit or delay tumor growth; 2) The response rate to single medication is obviously improved; 3) Prolonging the life span; 4) Enhancing the therapeutic effect of PD-L1 tumor immunotherapy; 5) The safety is high.
Examples
A further understanding of the present invention may be obtained by reference to the specific examples which are set forth to illustrate, but are not intended to limit the scope of the present invention. It will be apparent that various modifications and variations can be made to the present invention without departing from the spirit thereof, and therefore, such modifications and variations are also within the scope of the present application as hereinafter claimed. The proportions used herein include percentages, by weight unless otherwise indicated.
Example 1 raw material preparation and identification
1.1 preparation and identification of anti-CD 100 control antibodies
anti-CD 100 control antibody preparation: anti-CD 100 antibody Pepinetab and 2D5 of Shanghai developer pharmaceutical industry,The 5D8 antibody served as a positive control antibody. The coding gene sequences of Pepinemab and 2D5, 5D8 antibodies were submitted to gene synthesis by the company of general biotechnology, inc, according to the sequences disclosed in WO2013148854A1 and patent WO 2020011275. Then constructed to eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination. The constructed recombinant protein expression vectors are respectively transformed into escherichia coli DH5 alpha, cultured overnight at 37 ℃, and then subjected to plasmid extraction by utilizing an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain the expected expression plasmids for expressing the Pepinetab monoclonal antibody and the 2D5 and 5D8 antibodies. By means of an Expifectamine TM CHO transfection kit (Thermo Fisher, a 29129), CHO cells were transfected with the expression plasmid to express Pepinemab and 2D5, 5D8 mab according to the manufacturer's instructions. Cell culture supernatants were collected 7 days after transfection and centrifuged at 15000g for 10min. The resulting supernatant was filtered through a 0.22 μm filter, and the antibodies in the supernatant were affinity purified using a Protein A/G affinity column (MabSelect SuRe (Cytiva, 17543802)). The antibody of interest was eluted with 100mM glycinate (pH 3.0) and the eluted antibody was exchanged through ultrafiltration concentrate tube (Millipore, UFC 901096) into PBS buffer.
Identification of anti-CD 100 control antibody: the activity of the positive control antibody Pepinetab (heavy chain constant region type IgG4 SP) prepared was tested with the purchased Human Semaphorin 4D/SEMA4D/CD100 Protein, fc Tag antigen Protein (Acro, CD 0-H5257).
The specific method comprises the following steps: the 96-well ELISA plate was coated with the commercially available human CD100 protein (also known as HuCD100-Fc, 2. Mu.g/mL, 30. Mu.L/well) at 4℃overnight; after washing the plate 3 times, 5% skim milk prepared with PBS was used for sealing for 1 hour at room temperature; after washing the plate 3 times, adding a control antibody Pepinetab diluted by PBS gradient, and incubating for 1 hour at room temperature; after washing the plates, secondary Anti-human-IgG-Kappa-HRP (Millipore, AP 502P) diluted with PBS (1:6000) and Anti-human-IgG-Lambda-HRP (Millipore, AP 506P) were added, and after incubation for 1 hour at room temperature, TMB (SurModics, TMBS-1000-01) was added to the plates 6 times for development of color for 5-20min. After termination of the development, the data were read at OD450 with a microplate reader (Molecular Devices, spectromax 190). Data were processed and plotted using Graphpad prism.
As a result, as shown in FIG. 1, the expressed control antibodies Pepinemab, 2D5 and 5D8 all bind to CD100 protein and have normal anti-CD 100 activity.
1.2CD100 antigen protein preparation and identification
Antigen protein preparation: by genetic manipulation at the level of the coding gene, the C-terminal of the amino acid sequence of the 22-734 fragment of the ECD region of human CD100 protein (HuCD 100, uniprot ID: Q92854), the 24-733 fragment of the ECD region of mouse CD100 protein (musCD 100, uniprot ID: O09126), the 22-734 fragment of the ECD region of cynomolgus monkey CD100 protein (CynocD 100, uniprot ID: A0A2K5TZC 9) was added with a human Fc (SEQ ID NO: 135) or His tag, respectively. The obtained nucleic acid sequences were respectively constructed into pcDNA3.4 vectors, then transformed into E.coli DH 5. Alpha. And cultured overnight at 37℃after which plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The resulting plasmid was subjected to an Expiectamine TM 293 transfection kit (Gibco) TM A 14524) is transiently transferred to HEK293 cellsCRL-1573 TM ) Is a kind of medium. After 7 days of expression, cell culture supernatants were harvested. After the completion of the culture, the cell suspension was centrifuged at a high speed to collect the supernatant, and the supernatant was filtered through a 0.22 μm filter, and then purified by affinity chromatography using a Protein A/G column MabSelect SuRe (Cytiva, 17543802). The target protein is eluted by using 100mM glycine hydrochloride (pH 3.0), concentrated, replaced by buffer solution, split charging, SDS-PAGE identification and activity detection, and then stored in a warehouse for freezing. His-tagged proteins were affinity purified using Ni Smart Beads 6FF (Hemsl and Biotechnology Co., ltd., SA 036050) and then the target proteins were eluted with an imidazole gradient. The eluted proteins were subjected to ultrafiltration and concentration tube (Millipore, UFC 901096) exchange to PBS buffer solution, and finally human and mouse monkey CD100 antigen proteins (HuCD 100-Fc, musCD100-Fc, cynocD100-Fc, huCD100-His, musCD100-His, and CynocD 100-His) with Fc tags and His tags were obtained.
Antigen identification: the antigen (HuCD 100-Fc, musCD100-Fc, cynocD100-Fc, huCD100-His, musCD100-His, cynocD 100-His) obtained was tested with the quality-acceptable antibody Pepinemab (IgG 4 SP) obtained in example 1.1.
The specific method comprises the following steps: ELISA plates were coated with 2. Mu.g/mL antigen at 4℃overnight, respectively, using the purchased antigen Protein Human Semaphorin 4D/SEMA4D/CD100 Protein, fc Tag antigen Protein (Acro, CD 0-H5257) as positive control; after washing the plate 3 times, 5% skim milk prepared with PBS was used for sealing for 1 hour at room temperature; after washing the plate 3 times, adding antibody Pepinetab diluted by PBS gradient, and incubating for 1 hour at room temperature; after washing the plates, secondary Anti-human-IgG-Kappa-HRP (Millipore, AP 502P) or Anti-human-IgG-Lambda-HRP (Millipore, AP 506P) diluted with PBS (1:6000) was added, incubated for 1 hour at room temperature, washed 6 times and then developed with TMB for 5-20min. The chromogenic reaction was terminated and the data read with a microplate reader at OD 450. Data were processed and plotted using Graphpad prism.
The results are shown in FIG. 2, where the affinity of the antibody Pepinetab to self-made antigen is comparable to the affinity to the purchased CD100 antigen protein, with two HuCD100-Fc sequences, self-made HuCD100 antigen and commercially available HuCD100 antigen, respectively.
EXAMPLE 2 construction and identification of overexpressing cell lines
2.1 construction and identification of over-expressed CD100 cell lines
Construction of HEK293 cell lines overexpressing CD100 (hereinafter abbreviated as HuCD100-HEK293, mus CD100-HEK293, cynocD100-HEK 293): the nucleic acid sequences encoding 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 CynogCD 100 (Uniprot ID: A0A2K5TZC 9) were each constructed on the pLVX-puro plasmid (Clontech, cat No. 632164). The resulting plasmid was then electrotransformed into HEK293 cells by electrotransformation apparatus (Invitrogen, neonTM Transfection System, MP 922947) CRL-1573 TM ) Is a kind of medium. After electrotransformation, the resulting cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume and no antibiotics, respectively. The cells were then transferred to 10X 10cmThe cells were cultured in a cell culture dish for 48 hours, followed by an average of 10 4 Cell/well density cells were sub-packed into 96 well cell culture plates, puromycin at a final concentration of 2 μg/mL was added as screening pressure, and cell lines forming clones were picked up for about 2 weeks for identification.
Flow identification of HuCD100-HEK293, musCD100-HEK293, cynocD100-HEK293 cells: cells of the above cell line in the logarithmic growth phase were digested with pancreatin and plated into 96-well plates, washed with FACS buffer (1 x PBS buffer containing 2% FBS by volume), and then incubated with primary antibody (pepinemia) diluted in PBS gradient at 4 ℃ for 30min; after washing, adding the prepared fluorescent secondary antibody anti human IgG Fc (abcam, 98596) and incubating for 30min at 4 ℃; finally, detection was performed by flow cytometry (Beckman, cytoFLEXAOO-1-1102).
The results of the detection are shown in FIG. 3, and show that the HuCD100-HEK293, musCD100-HEK293 and CynocD100-HEK293 cell lines express human, mouse and monkey CD100 on the surface.
2.2 construction and identification of cell lines overexpressing Huplexin-B1, musplexin-B1, cynoplexin-B1, huplexin-B2
Construction of HEK293 cell lines overexpressing Plexin-B1 and Plexin-B2 (hereinafter referred to as Huplexin-B1-HEK293, musplexin-B1-HEK293, cynoplexin-B1-HEK293, huplexin-B2-HEK 293): the coding nucleic acid sequences of the full length human Plexin-B1 protein Huplexin-B1 (Uniprot ID: O43157), the full length mouse Plexin-B1 protein Musplexin-B1 (Uniprot ID: Q8CJH 3), the full length cynomolgus monkey Plexin-B1 protein Cynoplexin-B1 (Uniprot ID: A0A1D5QMB 8), the full length human Plexin-B2 protein Huplexin-B2 (Uniprot ID: O15031) were constructed on the pLVX-puro plasmid (Clontech, accession number 632164). The construction is described in example 2.1.
Flow identification of Huplexin-B1-HEK293, musplexin-B1-HEK293, cynoplexin-B1-HEK293, huplexin-B2-HEK293 cells: cells of the above cell line in the logarithmic growth phase were digested and plated into 96-well plates, washed with FACS buffer (1 XPBS buffer containing 2% by volume of FBS), then added with antigen proteins (HuCD 100-Fc, musCD100-Fc, cynocD100-Fc prepared in example 1.2 by Biotin labeling to obtain HuCD100-Fc-Biotin, musCD100-Fc-Biotin, cynoCD100-Fc-Biotin, biotin labeling method see instructions of Biotin protein labeling kit from Roche Co., cat., cat# 11418165001) and incubated at 4℃for 30min; after washing, formulated eBioscience Streptavidin PE (Invitrogen, 2265658, 1:300) was added and incubated at 4℃for 30min; finally, detection was performed by flow cytometry (Beckman, cytoFLEXAOO-1-1102).
The results of the assay are shown in FIGS. 4A-4D, wherein FIG. 4A is a schematic diagram showing the results of identifying Huplexin-B1-HEK293 cell lines with human CD100 protein, FIG. 4B is a schematic diagram showing the results of identifying Musplexin-B1-HEK293 cell lines with murine CD100 protein, FIG. 4C is a schematic diagram showing the results of identifying Cynoplexin-B1-HEK293 cell lines with monkey CD100 protein, and FIG. 4D is a schematic diagram showing the results of identifying Huplexin-B2-HEK293 cell lines with human CD100 protein. The results show that the Huplexin-B1-HEK293, musplexin-B1-HEK293, cynoplexin-B1-HEK293, huplexin-B2-HEK293 cell lines were surface highly expressed in human, murine, monkey Plexin-B1 and human Plexin-B2, respectively.
EXAMPLE 3 construction and screening of a humanized phage display recombinant antibody library
In this example, an antibody gene phage display library was constructed and screened using the antigen proteins HuCD100-Fc, musCD100-Fc, cynocD100-Fc, huCD100-His, musCD100-His, and CynocD100-His prepared in example 1.2 as screening antigens to obtain a plurality of antibody molecules having specific binding to human CD 100.
3.1 construction of Gene libraries of human antibodies
Peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC) of normal human blood were isolated from Ficoll-Paque density gradient (available from GE company under the catalog number 17144003S) and total RNA was extracted from the isolated PBMC cells by conventional methods. The total RNA extracted was reverse transcribed into cDNA using a reverse transcription kit (available from TaKaRa, cat# 6210A) according to the manufacturer's instructions. Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers are respectively designed at the front end of the V region and the rear end of the first constant region of the heavy chain and the light chain, and the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody are obtained after PCR. By fusion PCR method The fragment containing the light chain variable region and the heavy chain variable region of the antibody was amplified, the PCR product and the phage display vector were digested, recovered and ligated, and the ligation product was recovered by a recovery kit (Omega, catalog number: D6492-02) (Li Xiaolin, construction of a large-capacity non-immune humanized Fab phage antibody library, preliminary screening, university of Kyoto medical science, china, university of Shuoshi, 6 months of 2007). Finally, the mixture was transformed into competent E.coli SS320 (Lucigen, MC 1061F) by an electrotransfer apparatus (Bio-Rad, micropulser), and the transformed E.coli SS320 bacterial liquid was spread on an ampicillin-resistant 2-YT solid plate (solid plate was prepared from 1.5% tryptone, 1% yeast extract, 0.5% NaCl,1.5% agar, in mass volume g/mL). The library capacity was determined to be 3X 10 by gradient dilution plating 11 cfu, i.e. 3X 10 11 Antibody gene libraries of individual antibody genes (for methods of calculation of the reservoir capacity see example 2.2 in CN112250763 a). Packaging with VSCM13 helper phage (from Stratagene) resulted in an antibody gene phage display library (preparation of antibody gene phage display library is referred to in example 2.3 of CN 112250763A).
3.2 screening of antibody Gene phage display libraries
3.2.1 screening of antibody Gene phage display library by magnetic bead method
The magnetic bead screening is to carry out biotin labeling on antigen protein, then combine the antigen protein with magnetic beads coupled with streptavidin, and carry out incubation, washing and elution on the antigen-combined magnetic beads and an antibody gene phage display library. Typically, 3-4 rounds of panning are experienced, whereby specific monoclonal antibodies directed against the antigen can be enriched in large amounts. In this example, the biotin-labeled antigen proteins HuCD100-Fc, cynocD100-Fc, huCD100-His, musCD100-His were used for phage display library screening, and after 3 rounds of panning, monoclonal antibody Fab primary screening against human CD100 was performed, for specific procedures as described in example 2.4.1 of CN 112250763A.
3.2.2 screening of antibody Gene phage display library by Immunotube method
The immune tube screening is a panning process in which antigen proteins HuCD100-Fc, cynoCD100-Fc, huCD100-His, musCD100-His are coated on the surface of an immune tube having high adsorption power, and phage display antibody libraries are added to the immune tube and incubated, washed and eluted with the antigen proteins adsorbed on the surface of the immune tube. After 2-4 rounds of panning, the specific monoclonal antibody Fab against the antigen was eventually enriched. In this example, monoclonal antibody Fab against human CD100 was enriched after 3 rounds of panning, for specific procedures reference example 2.4.2 in CN112250763 a.
3.3 selection of monoclonal
ELISA detection is carried out on phage pools eluted in each round to evaluate enrichment effect, 10 clones are randomly selected from phage pools screened in each round to carry out sequence analysis, comprehensive analysis is combined with enrichment effect and the proportion of repeatability of the sequenced sequences, and proper rounds are selected to carry out monoclonal selection.
ELISA monoclonal screening Using the antigen proteins HuCD100-His, musCD100-His, cynocD100-His, the antibody Fab obtained from the screening, which bound HuCD100-His, musCD100-His, cynocD100-His, was prepared as a Fab lysate, and then examined by flow cytometry (FACS) using the overexpressing cells HuCD100-HEK293, musCD100-HEK293, cynocD100-HEK293 prepared in example 2.1, 11 antibody Fab molecules specifically binding to human CD100 were screened and the 11 obtained antibody Fab was named (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, C-B71) respectively with the corresponding clone numbers. The amino acid sequences of the variable regions of the obtained antibodies are shown in Table 1, and the CDR sequences are determined by defining the CDRs by using AbM.
TABLE 1.11 amino acid sequence of the variable region of anti-CD 100 antibody (SEQ ID NO:)
EXAMPLE 4 construction, expression and purification of antibodies
4.1 plasmid construction
The VH coding sequence in the Fab sequence of the monoclonal A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171 and C-B71 obtained by screening is connected with the coding sequence of the heavy chain constant region (SEQ ID NO: 127) of the human IgG4SP to obtain the heavy chain coding sequence of the fully human antibody, and the VL coding sequence in the Fab sequence is connected with the Kappa type (SEQ ID NO: 128) or Lambda type (SEQ ID NO: 129) coding sequence of the human light chain constant region (CL) to obtain the light chain coding sequence of the fully human antibody. The coding sequences for the heavy and light chains of the antibodies were inserted into eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), respectively, and transformed into E.coli DH 5. Alpha. And incubated overnight at 37 ℃. Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody plasmids for eukaryotic expression.
4.2 expression and purification of antibodies
According to the method of ExpiCHO TM Expression System USER GUIDE the full-length sequence of the antibody obtained above was expressed by using an expcho transient expression system (Thermo Fisher, a 29133) kit, as follows: on the day of transfection, CHO cell density was confirmed to be 7X 10 6 Up to 1X 10 7 Cell viability about living cells/mL>98% at this time, the cells were adjusted to a final concentration of 6X 10 using fresh ExpiCHO expression medium pre-warmed at 37 ℃ 6 Individual cells/mL. OptiPRO pre-cooled at 4deg.C TM SFM dilution of plasmid of interest (1. Mu.g plasmid was added to 1mL of the medium) with OptiPRO TM SFM dilution of Expifectamine TM CHO reagent, mixing the two with equal volume, and gently blowing to obtain the product TM CHO/plasmid DNA cocktail. Incubating the mixture at room temperature for 1-5min, slowly adding into the prepared cell suspension while gently shaking, and placing into a cell culture shaker at 37deg.C and 8% CO 2 Culturing under the condition.
After 18-22h of transfection, expiCHO was added to the cell culture broth TM Enhance reagent and ExpiCHO TM Feed reagent, shake flask placed in shaker at 32deg.C and 5% CO 2 Culturing was continued under the conditions. On day 5 post transfection, the same volume of ExpiCHO was added TM Feed reagent, slowly added while gently mixing the cell suspension. Cell culture supernatant expressing the target antibody protein was collected 7 days after transfection, 15000g of the supernatant was isolatedHeart for 10min. The resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), then eluted with 100mM sodium acetate (pH 3.0), then neutralized with 1M Tris-HCl, and finally the resulting antibody protein was changed to PBS buffer by ultrafiltration of a concentrate tube (Millipore, UFC 901096).
Example 5 physicochemical Property detection of antibodies
In this example, SDS-PAGE and SEC-HPLC were used to detect the relative molecular weight and purity of the candidate antibodies.
5.1 SDS-PAGE identification of antibodies
Preparation of non-reducing solution: mu.g of each of the obtained antibodies and the quality control IPI (Ipilimumab ) were added to 5 XSDS loading buffer and 40mM iodoacetamide, respectively, and heated in a dry bath at 75℃for 10min. After cooling the mixture to room temperature, the supernatant was collected by centrifugation at 12000rpm for 5 min.
Preparation of a reduction solution: mu.g of each of the obtained antibodies and the quality control IPI were added to 5 XSDS loading buffer and 5mM DTT, respectively, and heated in a dry bath at 100℃for 10 minutes. After cooling the mixture to room temperature, the supernatant was collected by centrifugation at 12000rpm for 5 min.
The supernatant was gel-electrophoresed with Bis-tris 4-15% gradient gel (gold srey) and the protein bands were visualized by coomassie brilliant blue staining. Protein gels with chromogenic protein bands (decolorized gel with a decolorizing solution to its background transparent) were scanned using EPSON V550 color scanner and reduced and non-reduced band purity was calculated by ImageJ according to peak area normalization.
Experimental results show that the band of the non-reducing gel of each antibody is about 150kD, and the band of the reducing gel is about 55kD and about 25kD, which accords with the expected size. All candidate antibodies detected by the reduction gel were greater than 95% pure (table 2).
5.2SEC-HPLC to identify monomer purity of antibodies
Material preparation: mobile phase: 150mmol/L phosphate buffer, pH 7.4; each antibody was diluted with mobile phase solution to 0.5mg/mL of IPI and quality control, respectively.
The experimental method comprises the following steps: the column was XBridge BEH (SEC 3.5 μm,7.8mm I.D.×30 cm) using an Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph, the Waters flow rate was set at 0.8mL/min, the sample injection volume was 20. Mu.L, and the VWD detector wavelengths were 280nm and 214nm. And sequentially feeding a blank solution, an IPI quality control product solution and an antibody sample solution. The percentages of high molecular polymers, antibody monomers and low molecular substances in the sample are calculated according to an area normalization method.
The results are shown in Table 2, with monomer purities of more than 89% for all candidate antibodies (Table 2).
TABLE 2 expression level and physicochemical Properties of candidate antibodies
Antibody name Species of genus Expression level (μg/mL) SDS-PAGE(％) SEC-HPLC(％)
A14 Human body 139.00 >95.0 92.90
A15 Human body 209.00 >95.0 90.63
H74 Human body 114.00 >95.0 89.40
H96 Human body 170.00 >95.0 99.21
H5 Human body 88.60 >95.0 94.69
H12 Human body 93.50 >95.0 97.07
H21 Human body 219.00 >95.0 98.66
B13 Human body 135.00 >95.0 95.66
C-C081 Human body 107.00 >95.0 97.79
C-C171 Human body 65.40 >95.0 100.00
C-B71 Human body 122.0 >95.0 95.02
Example 6 detection of antigen binding Activity of antibodies
In this example, binding of the expressed candidate antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, C-B71) to the CD100 antigen proteins HuCD100-His, musCD100-His, cynocD100-His was examined based on ELISA, and binding capacity of the expressed antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, C-B71) to CD100 overexpressing cells HuCD100-HEK293, musCD100-HEK293, cynocD100-HEK293 cells, and human peripheral blood mononuclear cells (hereinafter abbreviated as HuPBMC cells) and human T lymphocyte leukemia cell Jurkat cells were examined based on FACS.
6.1 detection of the binding Capacity of antibodies to the antigen protein CD100-His based on ELISA
96-well ELISA plates (30. Mu.L/well) were coated with 2. Mu.g/mL of HuCD100-His, musCD100-His, cynocD100-His, overnight at 4 ℃. The next day, the well plates were washed 3 times with PBST and blocked with 5% skimmed milk for 2h. After washing the plates 3 times with PBST, each antibody was added in gradient dilutions (3.0, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014, 0.0002. Mu.g/mL) and incubated for 1h with the positive control antibody Pepinemab. The plates were then washed 3 times with PBST and secondary antibody, goat-anti-human Fc-HRP (abcam, ab 97225), was added and incubated for 1h. After incubation, the plates were washed 6 times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was stopped by adding 2M stop solution, and the absorbance was read at OD450 by a microplate reader (Molecular Devices, specterMax 190).
The results are shown in FIGS. 5A-5X and Table 3: for the antigen protein HuCD100-His (FIGS. 5A-5H), the binding activity of the antibody molecules A14, A15, B13, H5, H21, H74 and C-B71 to the antigen protein HuCD100-His was superior to that of the positive control antibody, and the binding activity of the antibody molecules H12, H96, C-C081 and C-C171 to the antigen protein HuCD100-His was comparable to that of the positive control antibody; for the antigen protein MusCD100-His (fig. 5I-5P), except that the binding activity of antibody molecule C-C081 to the antigen protein MusCD100-His was weaker than that of the positive control antibody and that of antibody molecules H12 and H96 to the antigen protein MusCD100-His were comparable to the positive control antibody, the binding activity of the remaining antibody molecules to the antigen protein MusCD100-His was superior to that of the positive control antibody; for the antigen protein CynocD100-His (FIGS. 5Q-5X), the binding activity of the antibody molecules H12 and H96 to the antigen protein CynocD100-His was superior to that of the positive control antibody, except that the binding activity of the antibody molecules H12 and H96 to the antigen protein CynocD100-His was comparable to that of the positive control antibody.
TABLE 3 antigen binding Activity of candidate antibodies
6.2 detection of antibody binding Capacity to CD100-HEK293 cells based on FACS
In this example, three kinds of cells, huCD100-HEK293, musCD100-HEK293 and CynocD100-HEK293, which were human CD 100-overexpressing cells, were used to evaluate the binding activity of the antibodies.
The specific method comprises the following steps: preparing HuCD100-HEK293, musCD100-HEK293 and CynocD100-HEK293 cells in logarithmic growth phase into single cell suspension with density of 1×10 6 Each cell/mL was added to a 96-well plate at 100. Mu.L per well, centrifuged at 4℃at 300g and the supernatant removed. Adding each gradient dilution to the corresponding wellThe individual antibodies (20.0, 6.67, 2.22, 0.74, 0.25, 0.08, 0.027, 0.0027. Mu.g/mL) and the positive control antibody Pepinetab were mixed and incubated at 4℃for 30min. After washing the incubated cell mixture 3 times, 100. Mu.L of secondary antibody-Goat F (ab') 2Anti-Human IgG-Fc (PE) (abcam, ab 98596) diluted 1:300 was added, incubated at 4℃for 30min in the absence of light, and after washing 3 times, detected by flow cytometry (Beckman, cytoFLEX AOO-1-1102).
The results are shown in FIGS. 6A-6O and Table 4: for the overexpressing cell line HuCD100-HEK293 (FIGS. 6A-6E), the binding activity of the remaining antibody molecules to HuCD100-HEK293 was superior to that of the positive control antibody except that the binding activities of antibody molecules C-C081, C-C171 and C-B71 to HuCD100-HEK293 were comparable to those of the positive control antibody; for MusCD100-HEK293 (fig. 6F-6J), all antibody molecules had good murine cross-activity, and the binding activity of antibody molecules H5, H21, a14, a15, B13, H74, H96 and H12 to MusCD100-HEK293 was superior to positive control antibodies; for CynocD100-HEK293 (FIG. 6K-6O), all antibody molecules showed good monkey cross-activity, with H5, H21, A14, A15, B13, H74, H96, H12 and C-C081 binding activities to CynocD100-HEK293 being superior to positive control antibodies.
TABLE 4 candidate antibody cell binding Activity
6.3 detection of antibody binding Capacity to HuPBMC and Jurkat cells based on FACS
In this example, binding activity of antibodies was evaluated using two types of HuPBMC and Jurkat cells, respectively.
The specific method comprises the following steps: huPBMC (AllCells, cat. No. PB004F-C (Y1246)) in logarithmic growth phase and Jurkat cells (ATCC, TIB-152) were prepared as single cell suspensions with a density of 1X 10 6 Each cell/mL was added to a 96-well plate at 100. Mu.L per well, centrifuged at 4℃at 300g and the supernatant removed. Adding each antibody of each gradient dilution (5.00, 0.50, 0.17, 0.056, 0.019, 0.006, 0.002, 0.0002. Mu.g/mL) and positive control antibody Pepinemab to corresponding wells, mixing well and adding to the mixture at a concentration of 4Incubating at a temperature of 30min. Washing the incubated cell mixture 3 times, and adding 100 mu L of secondary antibody coat F (ab') diluted by 1:300 2 Anti-Human IgG-Fc (PE) (abcam, ab 98596), incubated at 4deg.C for 30min in the absence of light, washed 3 times and then detected by flow cytometry (Beckman, cytoFLEX AOO-1-1102).
The results are shown in FIGS. 7A-7H and Table 5: the binding activity of the remaining antibody molecules, except antibody molecules C-C081, C-C171 and C-B71, was superior to that of the positive control antibodies for both HuPBMC cells (FIGS. 7A-7D) and Jurkat cells (FIGS. 7E-7H).
TABLE 5 candidate antibody cell binding Activity
Example 7 detection of blocking Activity of antibodies
In this example, the blocking activity of candidate antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-C171, C-B71) was evaluated using four types of cells, huplexin-B1-HEK293, musplexin-B1-HEK293, cynoplexin-B1-HEK293, huplexin-B2-HEK293, respectively.
The specific method comprises the following steps: cultured Huplexin-B1-HEK293, musplexin-B1-HEK293, cynoplexin-B1-HEK293, huplexin-B2-HEK293 cells were collected, the supernatant was removed by centrifugation at 300g, the cells were resuspended in the formulated FACS buffer, counted and the cell suspension density was adjusted to 1X 10 6 Individual cells/mL; huplexin-B1-HEK293, musplexin-B1-HEK293, cynoplexin-B1-HEK293, huplexin-B2-HEK293 cells were added to 96-well plates at 100. Mu.L per well and the supernatant was removed by centrifugation at 300 g; each antibody of each gradient dilution (20.0, 2.00, 0.67, 0.22, 0.074, 0.025, 0.008, 0.0008 μg/mL) and the positive control antibody Pepinemab were added to each corresponding well of the 96-well plate, and the cells were incubated at 4 ℃ for 30min after being resuspended; the diluted antibodies were respectively diluted with HuCD100-Fc dilution (0.5. Mu.g/mL), cynocCD 100-Fc dilution (0.3. Mu.g/mL) or musCD100-Fc dilution (3. Mu.g/mL) 100. Mu.L (HuCD 100-Fc, musCD100-Fc, hunocCD 100-Fc prepared by the Biotin labeling example 1.2, huCD100-Fc-Biotin, musCD100-Fc-Biotin, humocD 100-Fc-Biotin), CynocD100-Fc-Biotin, biotin labeling methods see Roche, inc. Biotin labeling kit Specification, cat: 11418165001 A) and adding the mixture to the cells and incubating for 1h. The cell mixture was then washed 3 times. Resuspension the cells and incubating the cells at 4 ℃ for 30min; washing the incubated cell mixture 3 times, adding PE-labeled streptavidin (eBioscience, 12-4317-87), and incubating at 4deg.C for 30min; the incubated cell mixture was washed 3 times, then FACS buffer was added to the wells, 200. Mu.L per well, and the cells were resuspended and detected by flow cytometry (Beckman, cytoFLEX AOO-1-1102).
The results are shown in FIGS. 8A-8O: all candidate antibodies can effectively block the binding of HuCD100 antigen protein and Huplexin-B1-HEK293 cells, the binding of musCD100 antigen protein and MusPlexin-B1-HEK293 cells and the binding of CynogCD 100 antigen protein and Cynoplexin-B1-HEK293 cells. Wherein the antibody molecules A14, A15, B13, C-C081 and C-C171 have better activity of blocking the binding of HuCD100 antigen protein to Huplexin-B1-HEK293 than the positive control antibody; antibody molecules a15, B13, H74, H96, H12 and C-C081 blocked the binding of HuCD100 antigen protein to HuPlexin-B2-HEK293 with better activity than positive control antibodies; antibody molecules H12, H74, H96 and C-C081 have better activity in blocking the binding of the musCD100 antigen protein to MusPlexin-B1-HEK293 than the positive control antibody; antibody molecules A15, B13, H12, H96, C-C081, C-C171 and C-B71 blocked the binding of the CynoCD100 antigen protein to the Cynoplexin-B1-HEK293 with better activity than the positive control antibody.
Example 8MDSC proliferation inhibition assay
This example evaluates the in vitro pharmacodynamic function of candidate antibodies by examining the inhibition of proliferation of Myeloid-derived suppressor cells (Myeloid-derived suppressor cells, MDSC) by candidate antibodies (A14, A15, H74, H96, H5, H12, H21, B13, C-C081, C-B71).
Cd33+ cells were sorted from fresh PBMCs using CD33 sorting beads (Miltenyi Biotech) and after sorting was completed, the cell density was adjusted to 1 x 10 6 prepare/mL for use. The antibodies were diluted in gradient to 100.0, 33.33, 11.11, 3.70 with 1640 complete medium4. 1.235, 0.412. Mu.g/mL. HuCD100-His antigen protein was diluted to 400.0. Mu.g/mL with 1640 complete medium. The antibody diluent and HuCD100-His antigen protein diluent are uniformly mixed in a ratio of 1:1, and incubated for 30min at room temperature. 96-well cell culture plates were prepared and 100. Mu.L of 1X 10 density was added to each well 6 CD 33/mL + Cells (1X 10 per well) 5 Cells) and then a further 100uL of antibody in combination with HuCD100-His antigen protein was added per well, totaling 200 uL. After placing the cell culture plate in a 37 ℃ cell incubator for 72 hours, transferring the sample cells to a 96-well U-shaped plate, washing the plate for 2 times by using FACS buffer, diluting three direct standard antibodies of PE-anti-human CD33 (Biolegend, 303404), FITC-anti-human HLA-DR (Biolegend, 307604) and APC anti-human CD11b anti-body (Biolegend, 301310) with FACS buffer at a ratio of 1:100, adding 100 mu L of a mixed solution of the three direct standard antibodies to each well, and incubating for 30 minutes at 4 ℃. The FACS buffer was washed 2 times. Detection was by flow cytometry (Beckman, cytoFLEX AOO-1-1102).
The experimental results are shown in FIGS. 9A-9C and Table 6: all candidate antibodies and positive control antibodies can effectively neutralize the induction of CD100 on MDSC cell populations, wherein the inhibition effect of antibody molecules A14, A15, B13, H12, H21, H74 and H96 on MDSC proliferation is better than that of positive control antibodies 2D5 and 5D8, and the inhibition effect of antibody molecules C-C081 and C-B71 on MDSC proliferation is obviously better than that of control antibody Pepinetab molecules.
TABLE 6 anti-CD 100 antibodies inhibit MDSC Activity
Example 9 preparation of anti-PD-L1 antibodies
This example demonstrates the affinity maturation engineering of antibody NB22D-21-huVH2 (see, e.g., CN112745391 a) for improved antibody affinity and other biological activities. Affinity maturation was based on the M13 phage display technique, using codon-based (codon-based) primers (single codons consisting of NNK during primer synthesis) to introduce CDR region mutations, 4 phage display libraries were constructed: library 1 and library 2 are single point combinatorial mutations, library 1 is cdrl1+cdrl3+cdrh3 combinatorial mutation, and library 2 is cdrl2+cdrl1+cdrh2 combinatorial mutation; library 3 and library 4 are double point saturation mutations, library 3 is a double point saturation mutation of CDRL3, and library 4 is a double point saturation mutation of CDRH 3.
The specific method for building the library comprises the following steps: first, a primer containing a point mutation (Jin Weizhi Biotech Co., ltd.) was synthesized; secondly, respectively taking the coding sequence of an antibody to be modified (hereinafter referred to as a maternal antibody) NB22D-21-huVH as a PCR amplification template, amplifying a sequence with mutation in a CDR region, combining fragments with different CDR mutations by a bridging PCR method, then connecting the point mutation antibody into a phage display carrier by double digestion (HindIII and Not I) and double sticky end connection, and finally transferring the antibody sequence with mutation sites into escherichia coli SS320 by electrotransformation. For specific procedures of calculation of library capacity, phage library preparation and library screening see example 3. The obtained antibody was designated as m18-VHH. The amino acid sequences of the variable regions of the obtained antibodies are shown in Table 7, and the CDR sequences are determined by defining the CDRs by using AbM.
TABLE 7 amino acid sequence of the variable region of anti-PD-L1 antibody (SEQ ID NO:)
Antibody name HCDR1 HCDR2 HCDR3 VHH
m18-VHH 130 131 132 133
Example 10 production and expression of antibody m18
A fusion expression vector was constructed by ligating the C-terminus of the VHH gene sequence to the N-terminus of the human IgG1 Fc segment gene sequence to fuse the antibody m18-VHH and the human IgG1 Fc segment (SEQ ID NO: 135), and the fusion expression vector plasmid was transformed into an ExpiCHO cell to induce expression, to obtain a VHH-Fc chimeric antibody protein (SEQ ID NO: 134) fused with the Fc segment, which was designated as antibody m18 hereinafter.
The antibody is expressed by using an ExpiCHO transient expression system, and the ExpiCHO transient expression system is used TM Expression Medium (Gibco, A29100-01) and Gibco TM ExpiFectamine TM CHO transfection kit (Gibco, a 29129). See example 4 for specific methods.
Example 11 antigen binding Activity detection of antibody m18
In this example, the binding capacity of VHH-Fc antibodies to PD-L1 overexpressing cells human PD-L1-CHO cells, human non-small cell lung cancer cell lines HCC827 cells, mouse PD-L1-CHO cells and cynomolgus PD-L1-CHO cells was examined based on the FACS method, the sources of the above cell lines or methods of preparation are described in CN112745391A.
11.1 detection of antibody binding Capacity to human PD-L1-CHO cells based on FACS
In this example, the binding activity of an antibody to a human PD-L1-overexpressing cell was evaluated using a human PD-L1-CHO cell.
For a specific method see example 5 in CN112745391a, wherein the positive control antibody used is Avelumab (preparation method described in patent WO 2013079174).
As shown in FIG. 10A, the binding activity of antibody m18 to human PD-L1-CHO cells was superior to that of the parent antibody NB22D-21-huVH2 and the positive control antibody Avelumab.
11.2 detection of antibody binding Capacity to human non-Small cell Lung cancer cell line HCC827 cells based on FACS
In this example, the binding activity of the antibody to the PD-L1 protein on human tumor cells was evaluated using the human non-small cell lung cancer cell line HCC827 cell (ATCC: CRL-2868).
For a specific method see example 8 in CN112745391a, wherein the positive control antibody used is Avelumab (preparation method described in patent WO 2013079174).
As shown in FIG. 10B, the binding activity of antibody m18 to the cells of the human non-small cell lung cancer cell line HCC827 was comparable to that of the maternal antibody NB22D-21-huVH2 and the positive control antibody Avelumab.
11.3 detection of binding ability of antibodies to mouse PD-L1-CHO cells and cynomolgus monkey PD-L1-CHO cells based on FACS
In this example, the activity of cross-binding to monkey and mouse PD-L1 was evaluated using mouse PD-L1-CHO cells and cynomolgus monkey PD-L1-CHO cells. For a specific method see example 9 in CN112745391a, wherein the positive control antibody used is Avelumab (preparation method described in patent WO 2013079174).
As shown in fig. 11A and 11B, antibody m18 had good binding activity to mouse PD-L1-CHO cells, whereas the maternal antibody and the positive control antibody did not bind to mouse PD-L1-CHO cells (fig. 11A), and it was expected that antibody molecule m18 could be used in animal model experiments of Balb/C breeds of mice; the binding activity of antibody m18 to cynomolgus PD-L1-CHO cells was superior to the positive control antibody and comparable to the maternal antibody (fig. 11B).
Example 12 specific detection of antibody m18 binding to PD-L1
This example uses ELISA to test the binding activity of VHH-Fc antibodies to other proteins of the B7 family to evaluate the specificity of antibody m18 for PD-L1 proteins. For a specific method see example 10 in chinese invention patent application CN112745391 a.
As shown in Table 8 and FIG. 12, antibody m18 had no binding activity to B7 family molecules other than B7-H1 (i.e., PD-L1) and only B7-H1, and this binding specificity was consistent with that of the parent antibody.
TABLE 8 specificity of candidate molecule binding to PD-L1 protein
Cloning/proteins B7-H1 B7-H2 B7-H3 B7-H4 B7-DC
m18 + N/A N/A N/A N/A
+: indicating detection of binding activity; N/A: indicating that no binding activity was detected.
Example 13 affinity maturation engineering of anti-CD 100 antibody molecules
13.1 affinity maturation library design and construction
Antibodies B13, H5 were engineered for affinity maturation for improved affinity and biological activity. Affinity maturation was based on the M13 phage display technique, using codon-based primers (single codons consisting of NNK during primer synthesis) to introduce CDR region mutations, 4 phage display libraries were constructed: library 1 and library 2 are single point combinatorial mutations, library 1 is cdrl1+cdrl3+cdrh3 combinatorial mutation, and library 2 is cdrl2+cdrl1+cdrh2 combinatorial mutation; library 3 and library 4 are double point saturation mutations, library 3 is a double point saturation mutation of CDRL3, and library 4 is a double point saturation mutation of CDRH 3. The specific method for building the library comprises the following steps: first, a primer containing a point mutation (Jin Weizhi Biotech Co., ltd.) was synthesized; secondly, using antibodies B13 and H5 to be modified as PCR amplification templates, amplifying a CDR region containing mutated sequences, combining fragments containing different CDR mutations by a bridging PCR method, then connecting the point mutated antibodies into phage display vectors by double digestion (HindIII and Not I) and double sticky end connection, and finally transferring the antibody sequences with mutation sites into escherichia coli SS320 by electrotransformation. The calculation of the storage capacity and the preparation of phage library are described in example 3.
13.2 screening of affinity maturation libraries
For specific procedures for screening libraries see example 3. After performing the sea selection, preliminary screening and affinity sequencing and sequence analysis of the library, a total of 74 positive clones of H5 and 57 positive clones of B13 were obtained, and affinity sequencing was performed, respectively. Binding affinity sequencing and sequence analysis data, finally, 20 candidate molecules are selected from H5 molecules, and 28 candidate molecules are selected from B13 molecules for construction, expression and function screening.
13.3 preparation of affinity maturation modification candidate molecule, physicochemical property evaluation, binding Activity and blocking Effect detection
Specific methods for the preparation of 48 candidate antibodies, physicochemical property assays, affinity activity assays, and blocking activity assays, and assays for MDSC proliferation inhibition assays are described in examples 4-8.
13.4 affinity maturation engineering candidate molecule selection
According to the detection results of physicochemical properties, affinity activity and blocking activity of the antibodies, 2 anti-C100 affinity matured antibodies B13-C-5 and B13-e-2 were selected for B13. H5 2 anti-C100 affinity matured antibodies H5-H-7 and H5-a-2 were selected. The amino acid sequences of the variable regions of the obtained antibodies are shown in Table 9, and the CDR sequences are determined by defining the CDRs by using AbM.
The results of the expression level and in vitro physicochemical property are shown in Table 10, and SDS-PAGE identifies the purity of the antibody, and the purity of the modified antibody is preferably more than 95%. SEC-HPLC identifies the monomer purity of the antibody, preferably engineered antibodies are all greater than 98%.
The results of FACS-based affinity assay are shown in FIGS. 13A-13D, and FIG. 13A shows the results of binding activity to HuCD100-HEK293 cells. FIG. 13B is a graph showing the results of cell binding activity to human PBMC. FIG. 13C is a graph showing the results of binding activity to musCD100-HEK 293 cells. FIG. 13D is a graph showing the results of binding activity to CynocD100-HEK 293 cells. Wherein the affinity activity of the engineered antibodies B13-c-5, B13-e-2, H5-H-7 and H5-a-2 to human CD100 overexpressing cells (HuCD 100-HEK 293) and to CD100 positive human PBMC cells are significantly better than the positive control antibodies Pepinetab and are each better than their corresponding parent antibodies; and the affinity activity of the engineered antibody to mouse CD100 over-expressing cells (MusCD 100-HEK 293) and cynomolgus monkey CD100 over-expressing cells (CynoCD 100-HEK 293) is obviously better than that of the positive control antibody Pepinetab and better than that of the corresponding maternal antibody.
As shown in FIGS. 14A-14D, the modified antibodies can effectively block the binding of HuCD100 antigen protein to Huplexin-B1-HEK293 cells and Huplexin-B2-HEK293 cells, the binding of musCD100 antigen protein to MusPlexin-B1-HEK293 cells and the binding of CynocD100 antigen protein to Cynogplexin-B1-HEK 293 cells. Wherein the activity of the engineered antibody to block binding of HuCD100 antigen protein to HuPlexin-B1-HEK293 cells is superior to that of the positive control antibody and its corresponding maternal antibody (fig. 14A); the activity of the engineered antibodies to block binding of HuCD100 antigen protein to Huplexin-B2-HEK293 cells (FIG. 14B) and to block binding of musCD100 antigen protein to Huplexin-B1-HEK293 cells (FIG. 14C) was superior to that of the positive control antibody but weaker than that of the corresponding maternal antibody; the activity of H5-H-7 and H5-a-2 blocking the binding of the CynoCD100 antigen protein to the Cynoplexin-B1-HEK293 cells was superior to that of the positive control antibody and the maternal antibodies H5, B13-c-5 and B13-e-2 blocking the binding of the CynoCD100 antigen protein to the Cynoplexin-B1-HEK293 cells but was weaker than that of the positive control antibody B13 (FIG. 14D).
The results of MDSC proliferation inhibition assays are shown in FIG. 15 and Table 11, and demonstrate that anti-CD 100 antibodies H5-H-7, H5-a-2, H5, B13-c-5, B13-e-2, and control antibody Pepinetab are effective in neutralizing the induction of CD100 on MDSC cell populations. Wherein, the activity of H5-H-7, H5-a-2, H5, B13-c-5 and B13-e-2 for inhibiting MDSC of 100 mug/mL is obviously higher than that of a control antibody Pepinetab.
TABLE 9.4 amino acid sequence of variable region of affinity matured engineered anti-CD 100 antibody (SEQ ID NO:)
TABLE 10.4 affinity maturation engineered anti-CD 100 antibody expression levels and in vitro physicochemical Properties
Antibody name Species of genus Expression level (μg/mL) SDS-PAGE(％) SEC-HPLC(％)
H5-h-7 Human body 167.00 >95.0 98.96
H5-a-2 Human body 194.00 >95.0 100.00
B13-c-5 Human body 401.00 >95.0 98.41
B13-e-2 Human body 283.00 >95.0 98.43
Pepinemab Human body 35.4 >95.0 98.96
TABLE 11 anti-CD 100 antibodies inhibit MDSC Activity
EXAMPLE 14 evaluation of efficacy of CT-26 animal models
In this example, the tumor-inhibiting effect of 7 anti-CD 100 antibodies (antibodies B13, C-C081, C-B71, H5-H-7, H5-a-2, B13-C-5, B13-e-2) in combination with an anti-PD-L1 antibody in animals was examined, and the tumor cell used was CT-26 (Shanghai cell Bank, cat# TCM 37) and Pepinetab was used as a positive control.
The specific method comprises the following steps: female Balb/C mice (Peking Violet laboratory animal technologies Co., ltd.) of about 20g, 6-8 weeks old, each injected subcutaneously on one side 5X 10 5 CT-26 cells were randomly plated with fraction cages after two days of tumor bearing. Each group comprises 10 tumor-bearing nude mice, including PBS negative control group and candidate antibody combined administration group (antibody B13+antibody m18, antibody C-C081+antibody m18, antibody C-B71+antibody m18, and antibody H5-H-7+ antibody m18, antibody H5-a-2+ antibody m18, antibody B13-c-5+ antibody m18, antibody B13-e-2+ antibody m 18) and a positive or reference control antibody group (Pepinemab+ antibody m18, antibody m 18). Wherein the doses of anti-CD 100 antibody are 50mpk and anti-PD-L1 antibody are 5mpk, and the administration mode is intraperitoneal injection, 2 times per week and 2 tumor volumes are measured, and the administration is 8 times per 4 weeks (BIW 4). Tumor volume (V) calculation mode: v=l×w 2 2 (where L is the longest of the tumor diameters and W is the shortest of the tumor diameters). Mice were euthanized 1 week after the end of dosing, tumor tissue was removed and tumor weights were measured. Tumor volume, tumor weight and mouse weight change data were analyzed to calculate tumor inhibition rate. Tumor inhibition data are detailed in table 12.
As shown in fig. 16A, 16B and table 12, in the group of anti-CD 100 antibodies combined with anti-PD-L1 antibodies, the anti-CD 100 antibodies (particularly antibodies C-C081, B13 or B13-C-5) combined with anti-PD-L1 antibody m18 were able to further synergistically inhibit or retard tumor growth compared to the anti-PD-L1 antibody m18 alone.
This demonstrates that the combination of anti-CD 100 antibody (particularly antibody C-C081, B13 or B13-C-5) with anti-PD-L1 antibody m18 antibody can significantly increase the response rate of CT-26 vaccinated mice to m18 antibody single administration, prolong the survival period, and suggest that such combination can enhance the therapeutic effect of PD-L1 tumor immunotherapy. Meanwhile, after administration, the weight of the mice is increased, and compared with the control group, the weight of the mice in each experimental group is not significantly different. This indicates that the antibody has no obvious toxic or side effect on mice and has safety.
TABLE 12 tumor rejection (TGI%) in animal experiments with candidate antibody and antibody m18 combination
It will be apparent to those skilled in the art that many modifications and variations of the present invention can be made without departing from its spirit and scope. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way. The true scope and spirit of the invention is indicated by the following claims, which are exemplary only.
Sequence listing
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

Claims (21)

1. An antibody or antigen-binding fragment thereof directed against CD100 comprising a heavy chain variable region and a light chain variable region, wherein
The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences and wherein the HCDR1 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID No. 1, 7, 19, 33, 41, 47 or 52; the HCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 2, 8, 14, 20, 26, 42, 48 or 53 by NO more than 2 amino acid additions, deletions or substitutions; and/or the HCDR3 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID NO 3, 9, 15, 21, 27, 30, 35, 38, 43, 49 or 54; and/or
The light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences and wherein the LCDR1 sequence differs in amino acid sequence by NO more than 2 amino acid additions, deletions or substitutions from the sequence shown in SEQ ID No. 4, 10, 16, 36, 44 or 50; the LCDR2 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 5, 11 or 45 by NO more than 2 amino acid additions, deletions or substitutions; and/or the LCDR3 sequence differs in amino acid sequence from the sequence shown in SEQ ID NO. 6, 12, 18, 24, 37, 46, 51 or 56 by NO more than 2 amino acid additions, deletions or substitutions.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein
The heavy chain variable region comprises the HCDR1 sequence of 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 of SEQ ID NO 3, 9, 15, 21, 27, 30, 35, 38, 43, 49, 54, 60, 63 or 66; and/or
The light chain variable region comprises the LCDR1 sequence of SEQ ID NOs 4, 10, 16, 22, 31, 36, 39, 44, 50, 61 or 64; 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.
3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences; the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences are selected from any one of (1) - (15):
(1) An HCDR1 sequence shown in SEQ ID NO. 1; an HCDR2 sequence shown in SEQ ID NO. 2; an HCDR3 sequence shown in SEQ ID NO. 3; an LCDR1 sequence shown in SEQ ID NO. 4; an LCDR2 sequence shown in SEQ ID NO. 5; and the LCDR3 sequence shown in SEQ ID NO. 6;
(2) An HCDR1 sequence shown in SEQ ID NO. 7; an HCDR2 sequence shown in SEQ ID NO. 8; an HCDR3 sequence shown in SEQ ID NO. 9; an LCDR1 sequence shown in SEQ ID NO. 10; an LCDR2 sequence shown in SEQ ID NO. 11; and the LCDR3 sequence shown in SEQ ID NO. 12;
(3) An HCDR1 sequence shown in SEQ ID NO. 13; an HCDR2 sequence shown in SEQ ID NO. 14; an HCDR3 sequence shown in SEQ ID NO. 15; the LCDR1 sequence shown in SEQ ID NO. 16; an LCDR2 sequence shown in SEQ ID NO. 17; and the LCDR3 sequence shown in SEQ ID NO. 18;
(4) An HCDR1 sequence shown in SEQ ID NO. 19; an HCDR2 sequence shown in SEQ ID NO. 20; an HCDR3 sequence shown in SEQ ID NO. 21; the LCDR1 sequence shown in SEQ ID NO. 22; the LCDR2 sequence shown in SEQ ID NO. 23; and the LCDR3 sequence shown in SEQ ID NO. 24;
(5) The HCDR1 sequence shown in SEQ ID NO. 25; the HCDR2 sequence shown in SEQ ID NO. 26; the HCDR3 sequence shown in SEQ ID NO. 27; the LCDR1 sequence shown in SEQ ID NO. 16; the LCDR2 sequence shown in SEQ ID NO. 28; and the LCDR3 sequence shown in SEQ ID NO. 29;
(6) The HCDR1 sequence shown in SEQ ID NO. 25; the HCDR2 sequence shown in SEQ ID NO. 26; the HCDR3 sequence shown in SEQ ID NO. 30; the LCDR1 sequence shown in SEQ ID NO. 31; an 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; an HCDR3 sequence shown in SEQ ID NO. 35; the LCDR1 sequence shown in SEQ ID NO. 36; the LCDR2 sequence shown in SEQ ID NO. 23; and the LCDR3 sequence shown in SEQ ID NO. 37;
(8) The HCDR1 sequence shown in SEQ ID NO. 33; the HCDR2 sequence shown in SEQ ID NO. 34; the HCDR3 sequence shown in SEQ ID NO. 38; the LCDR1 sequence shown in SEQ ID NO. 39; an LCDR2 sequence shown in SEQ ID NO. 17; and the LCDR3 sequence shown in SEQ ID NO. 40;
(9) The HCDR1 sequence shown in SEQ ID NO. 41; an HCDR2 sequence shown in SEQ ID NO. 42; an 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) An HCDR1 sequence shown in SEQ ID NO. 47; an HCDR2 sequence shown in SEQ ID NO. 48; an HCDR3 sequence shown in SEQ ID NO. 49; the LCDR1 sequence shown in SEQ ID NO. 50; an LCDR2 sequence shown in SEQ ID NO. 11; and the LCDR3 sequence shown in SEQ ID NO. 51;
(11) The HCDR1 sequence shown in SEQ ID NO. 52; an HCDR2 sequence shown in SEQ ID NO. 53; an HCDR3 sequence shown in SEQ ID NO. 54; the LCDR1 sequence shown in SEQ ID NO. 50; the LCDR2 sequence shown in SEQ ID NO. 55; and the LCDR3 sequence shown in SEQ ID NO. 56;
(12) The HCDR1 sequence shown in SEQ ID NO. 57; the HCDR2 sequence shown in SEQ ID NO. 58; an 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; an HCDR3 sequence shown in SEQ ID NO. 60; the LCDR1 sequence shown in SEQ ID NO. 61; the LCDR2 sequence shown in SEQ ID NO. 23; and the LCDR3 sequence shown in SEQ ID NO. 62;
(14) An HCDR1 sequence shown in SEQ ID NO. 1; an 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; an LCDR2 sequence shown in SEQ ID NO. 5; and the LCDR3 sequence shown in SEQ ID NO. 65;
(15) An HCDR1 sequence shown in SEQ ID NO. 1; an HCDR2 sequence shown in SEQ ID NO. 2; the HCDR3 sequence shown in SEQ ID NO. 66; an LCDR1 sequence shown in SEQ ID NO. 4; an LCDR2 sequence shown in SEQ ID NO. 5; and the LCDR3 sequence shown in SEQ ID NO. 6.
4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein
The heavy chain variable region comprises 1) the amino acid sequence set forth in SEQ ID NOs 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95, preferably the additions, deletions and/or substitutions do not occur in the CDR regions; and/or
The light chain variable region comprises 1) the amino acid sequence set forth in SEQ ID NOs 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 or 96, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
5. The antibody or antigen-binding fragment thereof of claim 4, wherein 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 set forth in SEQ ID NO. 67; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 68;
(2) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 69; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 70;
(3) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 71; a light chain variable region comprising the amino acid sequence of SEQ ID NO. 72;
(4) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 73; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 74;
(5) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 75; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 76;
(6) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 77; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 78;
(7) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 79; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 80;
(8) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 81; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 82;
(9) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 83; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 84;
(10) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 85; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 86;
(11) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 87; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 88;
(12) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 89; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 90;
(13) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 91; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 92;
(14) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 93; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 94;
(15) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 95; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 96.
6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein
The antibody or antigen binding fragment thereof comprises a heavy chain comprising 1) an amino acid sequence set forth in SEQ ID No. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; 2) An amino acid sequence 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%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, or 125; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO. 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 or 125, preferably the additions, deletions and/or substitutions do not occur in the CDR regions; and/or
The antibody or antigen binding fragment thereof comprises a light chain comprising 1) the amino acid sequence set forth in SEQ ID NOs 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%, at least 99% sequence identity to an amino acid sequence set forth in SEQ ID No. 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126; or 3) an amino acid sequence having one or more amino acid substitutions, additions and/or deletions compared to the amino acid sequence set forth in SEQ ID NO 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 or 126, preferably the additions, deletions and/or substitutions do not occur in the CDR regions.
7. The antibody or antigen-binding fragment thereof of claim 6, wherein the heavy and light chains are selected from any one of (1) - (15):
(1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 97; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 98;
(2) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 99; 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; 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; 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; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 106;
(6) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 107; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 108;
(7) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 109; a light chain comprising the amino acid sequence shown in SEQ ID NO. 110;
(8) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 111; a light chain comprising the amino acid sequence shown in SEQ ID NO. 112;
(9) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 113; a light chain comprising the amino acid sequence shown in SEQ ID NO. 114;
(10) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 115; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 116;
(11) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 117; a light chain comprising the amino acid sequence shown in SEQ ID NO. 118;
(12) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 119; a light chain comprising the amino acid sequence shown in SEQ ID NO. 120;
(13) A heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 121; a light chain comprising the amino acid sequence shown in SEQ ID NO. 122;
(14) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 123; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 124;
(15) A heavy chain comprising the amino acid sequence shown in SEQ ID NO. 125; a light chain comprising the amino acid sequence set forth in SEQ ID NO. 126.
8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, which 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 diabody; preferably, the antibody or antigen binding fragment thereof is a human antibody.
9. The antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof has at least one of the following characteristics:
1) Has affinity activity to CD100 protein;
2) Has affinity activity to CD100 positive cells;
3) Blocking binding of CD100 to Plexin-B1 or Plexin-B2;
4) Inhibit MDSC cell proliferation;
5) Inhibit tumor growth.
10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9 and a pharmaceutically acceptable carrier.
11. A pharmaceutical combination comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9 and an anti-PD-L1 antibody or antigen-binding fragment thereof.
12. The pharmaceutical combination of claim 11, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof specifically recognizes and binds to PD-L1, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an immunoglobulin single variable domain;
preferably, the immunoglobulin single variable domain comprises:
CDR1 comprising the amino acid sequence shown in SEQ ID NO. 130,
CDR2 comprising the amino acid sequence shown in SEQ ID NO. 131, and
CDR3 comprising the amino acid sequence shown in SEQ ID NO. 132.
More preferably, the immunoglobulin single variable domain comprises: 1) The amino acid sequence shown in SEQ ID NO. 133; or 2) an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 133.
13. The pharmaceutical combination of claim 12, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises an Fc fragment of human IgG 1;
preferably, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 134 or an amino acid sequence having at least 85%, at least 90%, at least 95% or more sequence identity to SEQ ID NO. 134.
14. The pharmaceutical combination according to any one of claims 11-13, which is a pharmaceutical composition or a kit.
15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-9, the pharmaceutical composition of claim 10 or the pharmaceutical combination of any one of claims 11-14 in the manufacture of a medicament for the treatment of cancer; preferably, the cancer is a hematological tumor or a solid tumor.
16. The use according to claim 15, wherein
The solid tumor comprises 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 carcinoma, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, esophageal cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, or head and neck cancer;
The hematological neoplasm comprises leukemia, lymphoma, myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma or multiple myeloma.
17. The use of claim 16, wherein the medicament is used in combination with one or more therapeutic agents selected from the group consisting of: chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors and tumor antigen targeting drugs.
18. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9.
19. An expression vector comprising the nucleic acid molecule of claim 18.
20. A host cell comprising the nucleic acid molecule of claim 18 or the expression vector of claim 19.
21. A method of producing the antibody or antigen-binding fragment thereof of any one of claims 1-9, the method comprising:
a) Culturing the host cell of claim 20 under suitable conditions to express the antibody or antigen-binding fragment thereof of any one of claims 1-9; and
b) Isolating the antibody or antigen binding fragment thereof from the host cell or culture thereof.
CN202211246863.8A 2022-10-12 2022-10-12 anti-CD 100 antibodies and uses thereof Pending CN117866091A (en)

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EP1442749A1 (en) * 2003-01-31 2004-08-04 Institut National De La Sante Et De La Recherche Medicale (Inserm) Use of anti-CD100 antibodies for the treatment and the diagnosis of inflammatory disorder affecting the central nervous system
GB0520644D0 (en) * 2005-10-11 2005-11-16 Domantis Ltd Antibody polypeptide library screening and selected antibody polypeptides
US7919594B2 (en) * 2007-02-14 2011-04-05 Vaccinex, Inc. Human anti-CD100 antibodies
AU2010245668B2 (en) * 2009-05-08 2016-07-14 Vaccinex, Inc. Anti-CD100 antibodies and methods for using the same
CN110713537B (en) * 2018-07-13 2023-04-21 江苏怀瑜药业有限公司 SEMA4D antibody and preparation method and application thereof

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