B7-H3 binding proteins and uses thereof

Abstract

The invention relates to the field of disease treatment, in particular to an anti-B7-H3 antibody or an antigen binding fragment thereof, a nucleic acid molecule encoding the antibody or the antigen binding fragment, and a method for preparing the antibody or the antigen binding fragment. The anti-B7-H3 antibody or the antigen binding fragment of the anti-B7-H3 antibody has high specificity and high affinity to B7-H3. The invention further relates to the use of said antibodies or antigen-binding fragments thereof in the treatment and diagnosis of disease.

Description

B7-H3 binding protein and its use Technical Field

[0001] The present invention belongs to the field of therapeutic monoclonal antibodies, and more specifically, the present invention relates to an antibody against B7-H3; and also relates to the use of the antibody in treating and diagnosing diseases. Technical Background

[0002] B7-H3 (CD276) is a type I transmembrane protein located on human chromosome 15, with a molecular weight of 45kD-66kD. It is one of the co-stimulatory molecules of the B7 family and shares 20%-27% amino acid sequence homology with other family members. Structurally, B7-H3 has extracellular IgV / IgC tandem repeats, a transmembrane region, and an intracellular domain (similar to PD-L1). Based on the number of extracellular tandem repeat units of B7-H3, two forms have been discovered: 2Ig-B7-H3 and 4Ig-B7-H3 (with one more IgV / C repeat unit), with 4Ig-B7-H3 being considered more common. Studies have shown that matrix metalloproteinases can cleave 2Ig-B7-H3 into a serum-free form. B7-H3 mRNA is widely distributed, but positive expression has not been detected in lymphoid organs, including the spleen, lymph nodes, bone marrow, and thymus. However, B7-H3 protein is constitutively expressed in non-immune quiescent fibroblasts, endothelial cells, osteoblasts, and amniotic fluid stem cells, and inducibly expressed on activated T cells, NK cells, dendritic cells, and macrophages. In normal tissues, B7-H3 expression is negative by IHC in many tissues, but low-to-moderate expression has been detected in tissues such as the pancreas, liver, colon, stomach, placenta, skin, and adrenal gland. Studies have shown that B7-H3 mRNA is overexpressed in various tumors, including breast, colorectal, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, and thyroid cancer. Numerous studies have demonstrated that B7-H3 plays an important role in tumor progression, including promoting tumor proliferation and migration, mediating EMT in tumor cells, and influencing tumor cell metabolism. The expression of B7-H3 is regulated by oncogenic genes, and the upregulation of B7-H3 promotes tumor growth and metastasis through multiple signaling pathways.

[0003] There are currently no antibody drugs targeting B7-H3 on the market. Therefore, it is urgent and necessary to develop antibodies targeting B7-H3 with higher specificity, lower toxic side effects, better clinical efficacy, and more convenient administration methods, which will provide patients with more medication options.

[0004] Summary of the Invention

[0005] In this application, the inventors developed a high-affinity, fully human antibody with superior properties. It specifically recognizes and binds to B7-H3, does not bind or substantially binds to B7-1, B7-2, B7-H1, B7-H2, and / or B7-H4, and lacks ADCC activity, effectively avoiding side effects caused by ADCC activity. This led to the following invention.

[0006] Antibodies of the present invention

[0007] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to B7-H3, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0008] (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 2;

[0009] (b) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 3; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 4; or

[0010] (c) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and / or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and / or light chain variable region described in any one of (a) or (b), and the mutation is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids).

[0011] In certain embodiments, the substitutions are conservative substitutions.

[0012] In certain embodiments, the CDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering systems.

[0013] In certain embodiments, the B7-H3 comprises human B7-H3 and / or monkey B7-H3. In certain embodiments, the monkey is a rhesus monkey (Macaca mulatta).

[0014] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0015] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 5 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 6 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 8 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 9 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 10 or a variant thereof; or

[0016] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof; CDR-H2 of SEQ ID NO: 19 or a variant thereof; CDR-H3 of SEQ ID NO: 20 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof; CDR-L2 of SEQ ID NO: 9 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0017] Wherein, the variant described in any one of (1a) and (1b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0018] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0019] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof; CDR-H2 of SEQ ID NO: 12 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0020] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 21 or a variant thereof; CDR-H2 of SEQ ID NO: 22 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0021] Wherein, the variant described in any one of (2a) and (2b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0022] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0023] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 16 or a variant thereof; CDR-H2 of SEQ ID NO: 17 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0024] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 24 or a variant thereof; CDR-H2 of SEQ ID NO: 25 or a variant thereof; and CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0025] Wherein, the variant described in any one of (3a) and (3b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0026] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0027] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0028] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof; CDR-H2 of SEQ ID NO: 29 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0029] Wherein, the variant described in any one of (4a) and (4b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0030] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0031] (a) a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; or

[0032] (b) a VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof;

[0033] wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0034] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

[0035] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0036] In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, wherein each antibody or antigen-binding fragment may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, the N-terminal amino acid cyclized to pyroglutamic acid, or the N-terminal amino acid cyclized to pyroglutamate.

[0037] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain) (conversion of the N-terminal glutamine or glutamic acid in the heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., CHO cells) or during purification / storage.

[0038] In certain embodiments, the N-terminal glutamine of the VH comprising the sequence as shown in SEQ ID NO: 1 or 3 or its variants undergoes cyclization to form pyroglutamate or pyroglutamate; and / or the N-terminal glutamate of the VL comprising the sequence as shown in SEQ ID NO: 2 or 4 or its variants undergoes cyclization to form pyroglutamate or pyroglutamate.

[0039] In certain embodiments, the antibody or antigen-binding fragment thereof having the features of any one of (1a), (2a), (3a), (4a) or (a) above further has a feature selected from the following:

[0040] (1) binds to B7-H3 (e.g., human or monkey B7-H3) with an EC50 of less than about 100 ng / mL, e.g., less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL or less; preferably, the EC50 is measured by ELISA;

[0041] (2) binds to B7-H3 (e.g., human or monkey B7-H3) with a KD of less than about 100 nM, e.g., less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or less; preferably, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured;

[0042] (3) does not bind or does not substantially bind to B7-1, B7-2, B7-H1, B7-H2, and / or B7-H4; for example, as determined by ELISA;

[0043] (4) having CDC activity, such as inducing killing of cells expressing B7-H3 (e.g., tumor cells) through CDC;

[0044] (5) No ADCC activity;

[0045] (6) inducing B7-H3 internalization, e.g., as measured by flow cytometry;

[0046] (7) inhibiting cell (such as tumor cell) proliferation; and / or

[0047] (8) Inhibit tumor growth.

[0048] In certain embodiments, the antibody or antigen-binding fragment thereof having the features of any one of (1b), (2b), (3b), (4b) or (b) above further has a feature selected from the following:

[0049] (1) binds to B7-H3 (e.g., human or monkey B7-H3) with an EC50 of less than about 100 ng / mL, e.g., less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL or less; preferably, the EC50 is measured by ELISA;

[0050] (2) binds to B7-H3 (e.g., human or monkey B7-H3) with a KD of less than about 100 nM, e.g., less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or less; preferably, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured;

[0051] (3) does not bind or does not substantially bind to B7-1, B7-2, B7-H1, B7-H2, and / or B7-H4; for example, as determined by ELISA;

[0052] (4) having CDC activity, such as inducing killing of cells expressing B7-H3 (e.g., tumor cells) through CDC;

[0053] (5) No ADCC activity;

[0054] (6) inducing B7-H3 internalization, e.g., as measured by flow cytometry;

[0055] (7) inhibiting cell (such as tumor cell) proliferation; and / or

[0056] (8) Inhibit tumor growth.

[0057] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.

[0058] In certain embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises from or is derived from the heavy chain constant region of human immunoglobulin (such as IgG1, IgG2, IgG3 or IgG4).In certain embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises wild-type Fc district, or comprises mutated or chemically modified Fc district, which has the effector function (such as reduced ADCC activity) of change compared with wild-type Fc district.In certain exemplary embodiments, the antibody or its antigen-binding fragment of the present invention comprises the variant of human IgG1 heavy chain constant region, and the variant has the following displacement compared with the wild-type sequence from which it is derived: Leu234Ala, Leu235Ala and Gly237Ala (according to the position of EU numbering system).In such embodiments, the antibody or its antigen-binding fragment of the present invention has reduced ADCC activity. In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 30 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared thereto (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids). In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 31 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared thereto (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids).

[0059] In certain embodiments, the heavy chain constant region (CH) as shown in SEQ ID NO: 30 or 31 or a variant thereof lacks a C-terminal lysine.

[0060] In certain embodiments, the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region from or derived from a human immunoglobulin (e.g., κ or λ). In certain embodiments, the light chain of the antibody or its antigen-binding fragment comprises a sequence as shown in SEQ ID NO: 32 or a variant thereof, the variant having a conservative substitution of up to 20 amino acids compared thereto (e.g., a conservative substitution of up to 15, up to 10, or up to 5 amino acids; e.g., a conservative substitution of 1, 2, 3, 4, or 5 amino acids).

[0061] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0062] (1) a heavy chain comprising the VH region represented by SEQ ID NO: 1 and the heavy chain constant region (CH) represented by SEQ ID NO: 30, and a light chain comprising the VL region represented by SEQ ID NO: 2 and the light chain constant region (CL) represented by SEQ ID NO: 32;

[0063] (2) a heavy chain comprising the VH region of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 30, and a light chain comprising the VL region of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO: 32;

[0064] (3) a heavy chain comprising the VH region of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 31, and a light chain comprising the VL region of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 32;

[0065] or,

[0066] (4) A heavy chain comprising the VH represented by SEQ ID NO: 3 and the heavy chain constant region (CH) represented by SEQ ID NO: 31, and a light chain comprising the VL represented by SEQ ID NO: 4 and the light chain constant region (CL) represented by SEQ ID NO: 32.

[0067] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0068] (1) a heavy chain having the sequence shown in SEQ ID NO: 40 and a light chain having the sequence shown in SEQ ID NO: 41; or,

[0069] (2) A heavy chain having the sequence shown in SEQ ID NO:42 and a light chain having the sequence shown in SEQ ID NO:43.

[0070] In certain embodiments, the N-terminal glutamine of the heavy chain having a sequence as shown in SEQ ID NO: 40 or 42 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate; and / or the N-terminal glutamate of the light chain having a sequence as shown in SEQ ID NO: 41 or 43 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate.

[0071] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0072] In certain embodiments, the variable region of the antibody or antigen-binding fragment thereof described in any of the above embodiments is of human origin.

[0073] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is selected from ScFv, Fab, Fab', F(ab')2, Fab'-SH, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

[0074] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is labeled. In certain embodiments, the antibody or antigen-binding fragment thereof is labeled with a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0075] The present invention also provides methods for treating tumors using the antibodies or antigen-binding fragments provided herein or pharmaceutical compositions thereof.

[0076] Derivatized antibodies

[0077] The antibodies or antigen-binding fragments thereof of the present invention may be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof will not adversely affect its binding to B7-H3 (particularly human B7-H3). Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the present invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and / or a protein or polypeptide capable of mediating binding of the antibody or antigen-binding fragment to another molecule (e.g., an avidin or polyhistidine tag).

[0078] As one of the derivatives of the antibody, the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present invention and a conjugated moiety.

[0079] In certain embodiments, the coupling moiety is selected from a detectable label. The detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In certain embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme. In certain embodiments, the detectable label as described above can be linked to the antibody or antigen-binding fragment thereof of the present invention by linkers of varying lengths to reduce potential steric hindrance.

[0080] In certain embodiments, the conjugated moiety is selected from a therapeutic agent. In certain embodiments, the therapeutic agent is preferably an anti-tumor agent, such as a cytotoxic agent, a cytokine, a toxin or a radionuclide.

[0081] In certain embodiments, the conjugated moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a sugar group.

[0082] As one of the antibody derivatives, the present invention provides a multispecific antibody comprising the antibody of the present invention or an antigen-binding fragment thereof.

[0083] In certain embodiments, the multispecific antibody comprises an antibody of the present invention or an antigen-binding fragment thereof as a first antigen-binding domain and further comprises at least one second antigen-binding domain directed against another target.

[0084] In certain embodiments, each antigen-binding domain of the multispecific antibody retains its respective original binding specificity.

[0085] In certain embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0086] As one of the derivatives of the antibody, the present invention provides a chimeric antigen receptor, which includes an antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the chimeric antigen receptor includes an antibody or antigen-binding fragment thereof (e.g., ScFv) of the present invention as an extracellular antigen-binding domain that specifically binds to B7-H3, as well as a transmembrane domain and one or more intracellular T cell signaling domains. The present invention also provides a host cell (e.g., an immune cell, such as a T lymphocyte, NK cell, DC cell, macrophage) containing or expressing the chimeric antigen receptor.

[0087] Antibody preparation

[0088] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0089] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0090] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present invention, or a heavy chain variable region and / or a light chain variable region thereof. According to codon degeneracy in the art, in certain embodiments, the nucleotide sequence can be replaced according to codon degeneracy. In certain embodiments, the nucleotide sequence is codon optimized.

[0091] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence set forth in SEQ ID NO:33, (ii) a sequence substantially identical to SEQ ID NO:33 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:33), or (iii) a degenerate sequence of (i) or (ii) above; and / or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO:34, (v) a sequence substantially identical to SEQ ID NO:34 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:34), or (vi) a degenerate sequence of (iv) or (v) above.

[0092] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence set forth in SEQ ID NO:35, (ii) a sequence substantially identical to SEQ ID NO:35 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:35), or (iii) a degenerate sequence of (i) or (ii) above; and / or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO:36, (v) a sequence substantially identical to SEQ ID NO:36 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:36), or (vi) a degenerate sequence of (iv) or (v) above.

[0093] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present invention. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a lentivirus, or the like. In certain embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the present invention in a subject (e.g., a mammal, such as a human).

[0094] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0095] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used to construct a chimeric antigen receptor (CAR), which comprises an extracellular antigen binding domain (e.g., ScFv) specifically binding to B7-H3, a transmembrane domain, and one or more intracellular T cell signaling domains. In such embodiments, the isolated nucleic acid molecules of the present invention may include a nucleotide sequence encoding a chimeric antigen receptor, which further includes a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv). In certain embodiments, the isolated nucleic acid molecules of the present invention encode a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention.

[0096] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used to construct chimeric antigen receptor-modified immune cells, wherein the chimeric antigen receptor-modified immune cells comprise a chimeric antigen receptor (CAR) and an immune cell (e.g., T lymphocytes, NK cells, DC cells, macrophages).

[0097] The present invention further provides a host cell comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention. The host cell can be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44, CHO-EBNA).

[0098] In certain embodiments, the host cell of the present invention may be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule contained in the host cell may include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv). In certain embodiments, the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention.

[0099] In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0100] Therapeutic applications

[0101] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing the chimeric antigen receptor of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0102] In certain embodiments, the pharmaceutical compositions of the present invention comprise the antibodies or antigen-binding fragments thereof of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0103] In certain embodiments, the pharmaceutical composition of the present invention comprises a vector or host cell of the present invention, and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv); the host cell comprises an isolated nucleic acid molecule or vector as described above. In certain embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention. In certain embodiments, the host cell is an immune cell, such as a T cell. In certain embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0104] In certain embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent. In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity. In certain embodiments, the additional pharmaceutically active agent is selected from B7-H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutics or any combination thereof. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent are provided as independent components or as mixed components. Therefore, the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0105] In certain embodiments, the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing the chimeric antigen receptor in the pharmaceutical composition of the present invention is sufficient (e.g., in a subject):

[0106] (a) Inhibit cell (such as tumor cell) proliferation;

[0107] (b) inhibiting tumor growth;

[0108] (c) inducing and / or increasing antibody-dependent cellular cytotoxicity;

[0109] (d) inhibiting B7-H3-mediated signaling;

[0110] (e) preventing and / or treating B7-H3 mediated diseases / disorders; or

[0111] (f) Any combination of (a)-(e).

[0112] In certain embodiments, the B7-H3 mediated disease / disorder is a tumor, e.g., a tumor expressing B7-H3. In certain embodiments, the tumor is selected from breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer, or any combination thereof.

[0113] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention in the preparation of a medicament, wherein the medicament is used to inhibit cell proliferation, or prevent and / or treat and / or assist in the treatment of tumors.

[0114] In certain embodiments, the medicament is used to inhibit the proliferation of cells expressing B7-H3 (eg, tumor cells).

[0115] In another aspect, the present invention provides a method for inhibiting cell proliferation, comprising contacting the cell with an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention. In certain embodiments, the cell is a cell expressing B7-H3, such as a tumor cell.

[0116] In another aspect, the present invention provides a method for preventing and / or treating and / or adjuvant treating a tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention.

[0117] In certain embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof. In certain embodiments, the second therapy can be applied simultaneously, separately, or sequentially with the above method.

[0118] In any of the above embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention can be any tumor type. In certain embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is a B7-H3 positive tumor. In certain embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is selected from breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer or any combination thereof.

[0119] The antibodies or antigen-binding fragments thereof of the present invention, and the pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of production and storage. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the required dose of the antibody of the present invention into an appropriate solvent, and optionally, other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.

[0120] Furthermore, the antibodies or antigen-binding fragments thereof of the present invention can be presented in a pharmaceutical composition in a unit dosage form for ease of administration.

[0121] Antibody of the present invention or its Fab, pharmaceutical composition can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to intended purpose. In a preferred embodiment, antibody of the present invention or its Fab, pharmaceutical composition is given by intravenous infusion or injection.

[0122] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof of the present invention. A "prophylactically effective amount" is an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered therapies.

[0123] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be a single dose, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the treatment situation.

[0124] In the present invention, the subject may be a mammal, such as a human.

[0125] Detection Application

[0126] The antibodies or antigen-binding fragments thereof of the present invention can specifically bind to B7-H3 and thus can be used to detect the presence or level of B7-H3 in a sample.

[0127] Therefore, in another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention carries a detectable label. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention. Preferably, the second antibody further comprises a detectable label.

[0128] In the present invention, the detectable label can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Particularly preferably, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, the detectable labels described above can be attached to the antibodies of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0129] In another aspect, the present invention provides a method for detecting the presence or level of B7-H3 in a sample, comprising the step of using an antibody or antigen-binding fragment thereof of the present invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention also carries a detectable label. In another preferred embodiment, the method further comprises detecting the antibody or antigen-binding fragment thereof of the present invention using a reagent with a detectable label. The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample rather than a sample from a patient).

[0130] In certain embodiments, the method comprises contacting the sample with the antibody or antigen-binding fragment thereof of the present invention under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and B7-H3, and detecting formation of the complex.

[0131] Given that B7-H3 is lowly expressed or absent in normal tissues, and is expressed or highly expressed in some cancers, tumors can be diagnosed by detecting the presence or level of B7-H3 in a sample. Therefore, in certain embodiments, the method is used to diagnose tumors, such as B7-H3-positive tumors, such as breast cancer, gastric cancer, lung cancer (such as non-small cell lung cancer), colorectal cancer, pancreatic cancer, head and neck squamous cell carcinoma, melanoma, ovarian cancer, prostate cancer, liver cancer, kidney cancer, bladder cancer, or any combination thereof.

[0132] In certain embodiments, the method comprises detecting the expression level of B7-H3 in a test sample from a subject, and comparing the expression level with a reference value (e.g., a healthy control), wherein an increase in the expression level compared to the reference value is indicative of a tumor.

[0133] In another aspect, provided is a use of the antibody or antigen-binding fragment thereof of the present invention in preparing a kit for detecting the presence or level of B7-H3 in a sample and / or diagnosing a tumor.

[0134] In another aspect, the present invention provides a diagnostic or therapeutic kit comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, or multispecific antibody as described herein, and instructions for use. The kit may also include a drug delivery device for topical administration. The drug delivery device may include a prefilled syringe or a needle-free device.

[0135] The antibodies of the present invention have high binding affinity and extremely strong specificity for B7-H3, do not bind or substantially bind to B7-1, B7-2, B7-H1, B7-H2, and / or B7-H4, and lack ADCC activity, effectively avoiding side effects caused by ADCC. The antibodies of the present invention can inhibit tumor cell proliferation and therefore have the potential to be used for the prevention and / or treatment of tumors. Furthermore, the antibodies of the present invention are fully human and can be safely administered to human subjects without triggering an immunogenic response. Therefore, the antibodies of the present invention have significant clinical value.

[0136] Abbreviations CDR Complementarity determining region of immunoglobulin variable region FR Antibody framework region: amino acid residues in antibody variable region other than CDR residues VH Antibody heavy chain variable region VL Antibody light chain variable region IgG Immunoglobulin G IMGT Based on the international Immunogenetics information system initiated by Lefranc et al. The immunoglobulin (CDR) numbering system is described in Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). Chothia The immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the location of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). The AbM CDR definition is derived from Martin's work (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). mAb monoclonal antibody EC50 concentration that produces 50% efficacy or binding IC50 concentration that produces 50% inhibition ELISA enzyme-linked immunosorbent assay PCR polymerase chain reaction HRP horseradish peroxidase K D Equilibrium dissociation constant Ka Association rate constant Kd Dissociation rate constant ADCC Antibody-dependent cell-mediated cytotoxicity CDC Complement-dependent cytotoxicity FACS Flow cytometry CDR-H1 Complementarity determining region 1 of the immunoglobulin heavy chain variable region CDR-H2 Complementarity determining region 2 of the immunoglobulin heavy chain variable region CDR-H3 Complementarity determining region 3 of the immunoglobulin heavy chain variable region CDR-L1 Complementarity determining region 1 of the immunoglobulin light chain variable region CDR-L2 Complementarity determining region 2 of the immunoglobulin light chain variable region CDR-L3 Complementarity determining region 3 of the immunoglobulin light chain variable region

[0137] definition

[0138] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, immunology, and other laboratory procedures used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0139] As used herein, the term "antibody" is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can be composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The constant domain is not directly involved in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions, known as complementarity-determining regions (CDRs), interspersed with more conserved regions known as framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The distribution of amino acids among regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0140] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0141] The term "antibody" also includes embodiments in which the heavy chain constant region comprises a C-terminal lysine, or lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized to a pyroglutamate. Thus, in a composition comprising the antibodies disclosed herein, each antibody therein may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid, or have the N-terminal amino acid cyclized to a pyroglutamate.

[0142] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0143] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably identified by the IMGT, Kabat, Chothia or AbM numbering systems.

[0144] The following general rules (published at www.bioinf.org.uk: Professor Andrew CR Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with amino acids that make up the epitope to which the antibody binds. In rare cases, these generally constant features do not appear; however, Cys residues are the most conserved feature.

[0145] V HThe entire amino acid sequence of a V is generally numbered according to Kabat, and the three CDRs within the variable region may be defined according to any of the above numbering systems. H The amino acid positions in the sequence may be numbered sequentially starting from amino acid position 1 to the end of the sequence, or according to Kabat numbering. H and V L The amino acid positions in are defined according to sequential numbering.

[0146] Amino acid positions in the heavy chain constant region can be numbered sequentially starting from amino acid position 1 and continuing to the end of the sequence, or numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu begins at position 118 and ends at position 447. Unless otherwise indicated, amino acid positions in the heavy and light chains described herein are defined according to sequential numbering.

[0147] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0148] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0149] As used herein, the term "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody and binds to the antigen bound by the intact antibody. For example, an antigen-binding fragment can be a polypeptide that is a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0150] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains." A "full-length heavy chain" or "heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, when the full-length antibody is of the IgE isotype, optionally further comprising a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" or "light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between the CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention can be derived from a single species, such as humans; they can also be chimeric antibodies or humanized antibodies. The full-length antibodies of the present invention comprise two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0151] As used herein, the term "Fd fragment" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of an intact light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains); the term "Fab'-SH" refers to a Fab fragment containing a free sulfhydryl group.

[0152] As used herein, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. An Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0153] As used herein, the term "Fc fragment" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0154] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

[0155] As used herein, the term "diabody" means an antibody whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0156] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen. In this article, those skilled in the art can use known conventional techniques to obtain an antigen-binding fragment of an antibody (e.g., an antibody provided by the present invention) from a given antibody (e.g., an antibody provided by the present invention), and screen the antigen-binding fragment of the antibody for specificity in the same manner as for the complete antibody.

[0157] As used herein, the term "multispecific antibody" refers to an antibody with multiple different antigen-binding specificities, including, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. A "bispecific antibody" refers to an antibody with two different antigen-binding specificities, which is a conjugate formed by a first antibody (or a fragment thereof) and a second antibody (or a fragment thereof) or an antibody analog through a coupling arm, and the coupling method includes but is not limited to chemical reaction, gene fusion, and enzymatic reaction. "Multispecific antibodies" include, for example, trispecific antibodies and tetraspecific antibodies. Trispecific antibodies are antibodies with three different antigen-binding specificities, and tetraspecific antibodies are antibodies with four different antigen-binding specificities.

[0158] As used herein, the terms "monoclonal antibody," "single antibody," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or an antibody fragment from a population of highly homologous antibody molecules, that is, a population of identical antibody molecules except for possible spontaneous natural mutations. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies, and these different antibodies typically recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates the characteristic of the antibody as being obtained from a population of highly homologous antibodies, and is not to be understood as requiring the antibody to be prepared by any particular method.

[0159] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it retains binding activity to the target antigen. For example, the term "chimeric antibody" may include an antibody in which the heavy and light chain variable regions are derived from a first antibody (e.g., human), while the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., mouse). For example, an antibody produced by immunizing a fully human transgenic mouse may be referred to as a chimeric antibody, which consists of a fully human variable region and a mouse constant region.

[0160] As used herein, the term "humanized antibody" refers to an antibody that can be prepared by replacing a portion of a human antibody with a portion of a non-human antibody prepared by immunizing a mammal other than a human. Typically, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). For example, a humanized antibody can be prepared by transplanting CDR sequences derived from the germline of other mammalian species onto human framework sequences.

[0161] As used herein, the term "fully human antibody" refers to an antibody comprising only sequences derived from human germline immunoglobulin sequences. Those skilled in the art will appreciate that a fully human antibody does not exclude the inclusion of amino acid residues not encoded by human germline immunoglobulin sequences, such as mutations introduced by random or in vitro site-specific mutagenesis or by in vivo somatic mutation. Fully human antibodies can be prepared by using transgenic mice (XenoMouse (Chemical Biology (2000), vol.7, issue 8, p.R185-6), HuMAb-Mouse (Infection and Immunity (2002), vol.70, issue 2, p.612-9), TC mouse (Biotechnology and Genetics Enginnering Revew (2002), vol.19, p.73-82), and KM mouse (Cloning Stem Cells (2002), vol.4, issue 1, p.91-102)), in which human immunoglobulin genes are transferred, and the target antibody can be mass-produced by isolating antibody-producing lymphocytes from the mouse to hybridomas. Fully human antibodies can also be prepared by phage display methods (FEBS Letter (1998), vol.441, p.20-24). In this method, by using a phage that integrates human antibody genes into a circular single-stranded DNA, fully human antibodies can be expressed on the phage surface in the form of a fusion with the coat protein of the phage. Fully human antibodies can also be prepared by using transgenic mice that have transferred human immunoglobulin variable region genes. The mice can produce chimeric antibodies consisting of fully human variable regions and mouse constant regions, and then fully human variable regions can be connected to human immunoglobulin constant regions using methods known in the art to produce fully human antibodies. For example, the DNA encoding VH can be operably connected to another DNA molecule encoding the heavy chain constant region to obtain full-length heavy chain genes. The sequence of human heavy chain constant region genes is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region.For example, the DNA encoding VL is operably linked to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and a Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0162] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0163] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured in terms of the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC). 50 )express.

[0164] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant, KD (see Davies et al., Annual Rev Biochem, 1990;59:439-473). KD, kon, and kdis values ​​can be measured using any valid method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method). Surface plasmon resonance techniques (e.g., Biacore) or Kinexa can also be used to measure the dissociation constant.

[0165] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0166] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide, which comprises an expression regulatory sequence operably linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by host cells or in vitro expression systems. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0167] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, NSO cells, Vero cells, Hela cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 or CHO-EBNA cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0168] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0169] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0170] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0171] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0172] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or entirely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival (if not receiving treatment), extending the survival period.

[0173] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a tumor, or is at risk of suffering from the above-mentioned disease.

[0174] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0175] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region), and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance rate of antibodies and antigen-antibody complexes, and the like. Methods for altering the effector functions of an antibody are known in the art, for example, by introducing mutations in the Fc region.

[0176] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-attached target cells and then kill the target cells by secreting cytotoxins.

[0177] As used herein, the term "complement-dependent cytotoxicity" refers to the cytotoxic effect in which complement participates, i.e., the classical complement pathway is activated by the binding of specific antibodies to corresponding antigens on the cell membrane surface to form a complex, and the resulting membrane attack complex exerts a lytic effect on target cells.

[0178] As used herein, the term "antibody-mediated internalization" refers to the phenomenon that an antibody crosses the cell membrane after binding to a cell surface antigen. Internalization includes antibody-mediated receptor (eg, HER3) internalization.

[0179] As used herein, combination therapy includes the use of an anti-HER3 antibody or antigen-binding fragment thereof encompassed by the invention in combination with one or more additional active therapeutic agents of a second therapy (e.g., a chemotherapeutic agent) or other prophylactic or therapeutic modality (e.g., radiation therapy).

[0180] In such combination therapies, various active agents often have different complementary mechanisms of action, and combination therapies may result in synergistic effects. Combination therapies include therapeutic agents that affect immune responses (e.g., enhance or activate responses) and therapeutic agents that affect (e.g., inhibit or kill) tumor / cancer cells. Combination therapies can reduce the likelihood of drug-resistant cancer cells occurring. Combination therapies can allow one or more of the reagents in the reagent to be dosed to reduce or eliminate adverse effects associated with one or more of the reagents. Such combination therapies can have a synergistic therapeutic or preventive effect on potential diseases, disorders, or conditions.

[0181] As used herein, "combination" includes therapies that can be administered separately, such as separately formulated for individual administration (e.g., provided in a kit), and therapies that can be administered together in a single formulation (i.e., a "co-formulation"). In certain embodiments, the anti-B7-H3 antibodies or antigen-binding fragments thereof of the present invention can be administered sequentially. In other embodiments, the anti-B7-H3 antibodies or antigen-binding fragments thereof can be administered simultaneously. The anti-B7-H3 antibodies or antigen-binding fragments thereof of the present invention can be used in combination with at least one other (active) agent in any manner.

[0182] In this article, B7-H3 positivity was obtained by immunohistochemistry and staining intensity evaluation by professional clinical pathologists.

[0183] The terms "cancer" and "tumor" are used interchangeably to refer to a broad category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors, or cells that invade neighboring tissues and may spread to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0184] The term "hematological malignancy" includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Hematological malignancies also include leukemias, such as secondary leukemia or acute lymphoblastic leukemia. Hematological malignancies also include myeloma (e.g., multiple myeloma) and other hematological and / or B-cell or T-cell related cancers.

[0185] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only intended to illustrate the present invention and are not intended to limit the scope of the invention. Based on the following detailed description of the drawings and preferred embodiments, the various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0186] Figure 1A: Binding activity assay of anti-human B7-H3 fully human antibody 2# to human B7-H3-4Ig protein

[0187] Figure 1B: Binding activity assay of anti-human B7-H3 fully human antibody 2# to monkey B7-H3-4Ig protein

[0188] Figure 2: Binding activity assay of anti-human B7-H3 fully human antibody to human B7-H3-4Ig protein

[0189] Figure 3A: Binding activity assay of anti-human B7-H3 fully human antibody to human colon cancer cells HT29

[0190] Figure 3B: Binding activity assay of anti-human B7-H3 fully human antibody to human gastric cancer cells NCI-N87

[0191] Figure 3C: Binding activity assay of anti-human B7-H3 fully human antibody to human breast squamous carcinoma cells HCC1806

[0192] Figure 3D: Binding activity assay of anti-human B7-H3 fully human antibody to human non-small cell lung cancer cells HCC827

[0193] Figure 3E: Binding activity assay of anti-human B7-H3 fully human antibody and CHO-S-human B7-H3-4Ig

[0194] Figure 3F: Binding activity assay of anti-human B7-H3 fully human antibody and CHO-S-human B7-H3-2Ig

[0195] Figure 4A: Binding activity assay of anti-human B7-H3 fully human antibody to CHO-S-monkey B7-H3

[0196] Figure 4B: Binding activity assay of anti-human B7-H3 fully human antibody to CHO-S-rat B7-H3

[0197] Figure 5: ADCC activity detection of anti-human B7-H3 fully human antibody

[0198] Figure 6: CDC activity assay of anti-human B7-H3 fully human antibody

[0199] Figure 7A: Endocytosis activity assay of anti-human B7-H3 fully human antibody and human gastric cancer cell line NCI-N87

[0200] Figure 7B: Endocytosis activity assay of anti-human B7-H3 fully human antibody in human breast squamous carcinoma cells HCC1806 Figure 7C: Endocytosis activity assay of anti-human B7-H3 fully human antibody in human non-small cell lung cancer cells HCC827 Figure 8: Epitope competition assay of anti-human B7-H3 fully human antibody

[0201] Figure 9: Specificity detection of anti-human B7-H3 fully human antibody

[0202] Sequence information

[0203] The information of the sequences involved in the present invention is described in the following table: DETAILED DESCRIPTION

[0204] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0205] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0206] Example 1: Preparation of B7-H3 antigen and control antibody proteins

[0207] 1.1 Preparation of B7-H3 antigen

[0208] The full-length sequences of human B7-H3-2Ig (NCBI: NP_001316557.1), human B7-H3-4Ig (Uniprot: Q5ZPR3), monkey B7-H3-4Ig (XP_015308534.1), and rat B7-H3 (Uniprot: Q7TPB4) were synthesized at GenScript and constructed into the pLVX vector. After plasmid extraction, viruses were packaged and used to construct overexpression cell lines. The extracellular segment nucleic acid sequences of human 4Ig, 2Ig, monkey antigen, and rat 2Ig were constructed into the pTT5 vector, and a 6-histidine tag was introduced at the C-terminus of the extracellular segment. The recombinant plasmids were extracted and transiently transfected into HEK293-EBNA cells for transient expression after sequencing. After 6 days, the cell supernatant was collected and purified to obtain human B7-H3-4Ig-His, B7-H3-2Ig-His proteins, monkey B7-H3-4Ig-His proteins, and rat B7-H3-2Ig-His proteins.

[0209] 1.2 B7-H3 control antibody expression

[0210] The B7-H3 control antibody is derived from patent CN 103687945 A. After codon optimization by GenScript, the antibody heavy and light chain nucleotide sequences were synthesized and cloned into the pTT5 vector. After plasmid extraction, the pTT5 plasmids corresponding to the antibody heavy and light chain nucleotides were simultaneously transfected into CHO-S cells. The cell supernatant was collected by centrifugation and purified using Protein A (MabSelect SuRe, GE) to obtain the control antibody protein DS7300.

[0211] Example 2: Preparation of anti-human B7-H3 chimeric antibody

[0212] 2.1 Mouse immunization

[0213] The fully human transgenic mouse H2L2 (the antibody produced by this mouse is a chimeric antibody composed of the variable region of a fully human antibody and the constant region of rat origin) of Hebo Pharmaceuticals was used for immunization with the human B7-H3-4Ig-His protein prepared in 1.1. Freund's complete adjuvant, Adjuvant system (Sigma) were used for footpad, subcutaneous, tail root, intraperitoneal immunization or Titermax Gold Adjuvant (Sigma), Imject Alum (Thermo), with footpad immunization every 2-3 days. During the immunization period, the serum titer of the anti-B7-H3 antibody was monitored every two weeks by ELISA. And the following 2.2 protocol was used to generate mouse hybridomas with the best titer.

[0214] 2.2 Hybridoma fusion screening

[0215] 1 μg / ml human B7-H3-4Ig-His protein, 100 μl / well, was coated onto an ELISA plate (BIOFIL) overnight at 4°C. The plate was washed once with 300 μl of PBST (0.2% Tween-20), and 100 μl of 2% BSA in PBS was added to each well. The plate was incubated at 37°C for 1 hour. Twenty μl of hybridoma supernatant was then added directly to the ELISA plate and incubated at 37°C for 2 hours. The solution was discarded, and the plate was machine-washed three times with PBST (0.2% Tween-20), 320 μl per well. The plate was drained, and 100 μl (1:5000) of HRP-conjugated Goat anti-Rat IgG (Thermo Fisher) diluted in PBST was added to each well and incubated at 37°C for 1 hour. Discard the solution and wash the plate five times with PBST (0.2% Tween-20), using a 320 μl buffer per well. Drain the plate dry and add 100 μl of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) to each well for color development in the dark. 50 μl of 2M H₂SO₄ was added to terminate the color development reaction. The absorbance at 450 nm was read on a microplate reader. Clones with an OD 450 nm greater than five times that of the negative control were identified as positive clones. Positive clones were then subjected to monkey B7-H3-4Ig-His protein cross-ELISA (see 2.1) and binding assays with human breast cancer MCF-7 cells. The best clones were selected for subcloning. Subclones were screened using similar methods, and the best subclones were selected for purification of chimeric antibodies.

[0216] Example 3: Evaluation of anti-human B7-H3 chimeric antibodies

[0217] 3.1 Evaluation of Binding Activity of Anti-Human B7-H3 Chimeric Antibodies

[0218] All preferred monoclonal clones were transferred to T25 square flasks for serum-free culture. 4-6 ml of culture supernatant was affinity purified using Protein-A beads. The antibody protein bound to the Protein-A beads was eluted with a 1M Glycine solution at a pH of 2.5. After elution, the antibody protein was neutralized with a 1M Tris solution. The antibody protein concentration was quantified using Nanodrop and used for candidate evaluation.

[0219] B7-H3 chimeric antibody affinity assay: Human and monkey B7-H3-4Ig-His proteins were diluted to 1 μg / ml and 100 ng / well in CBS coating buffer and incubated overnight at 4°C. The next day, the ELISA plate was washed once with PBS. 100 μl of 2% BSA was then added to each well and incubated at 37°C for 2 hours. The purified chimeric antibody and control antibody were serially diluted in 2% BSA, starting at 10 μg / ml and progressing through 8 concentrations in a 3-fold gradient. The blocking buffer was removed, and the diluted antibody solution was added to the ELISA plate and incubated at 37°C for 2 hours. The plate was washed three times with PBST (0.2% Tween-20), using 320 μl per well. After the plate was drained, 100 μl of HRP-conjugated Goat anti-Rat IgG (Thermo) was added to each well and incubated at 37°C for 1 hour. The plate was washed five times with PBST (0.2% Tween-20). After drying the plate, 100 μl of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) chromogenic substrate was added to each well. After incubation at room temperature for 3-5 minutes, 50 μl of 2M H2SO4 was added to each well to terminate the color reaction. The absorbance at OD450 nm was read using a microplate reader. The raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting. The EC values ​​for each antibody binding to human B7-H3-4Ig-His and monkey B7-H3-4Ig-His were calculated. 50 As shown in Table 1 below, the screened antibodies can all bind to human and monkey B7-H3-4Ig-His proteins.

[0220] The affinity of anti-human B7-H3 fully human antibody to human breast cancer MCF-7 cells was detected by flow cytometry (Beckman, model Cytoflex). Adherent cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution, counted, and the cell density was adjusted to 4.0×10 6 / ml, washed twice with 1% BSA, resuspended in 1% BSA solution, and 50 μl of cell suspension was added to each well of a 96-well pointed bottom plate (cell number 2×10 5 1% BSA was used to dilute the candidate antibody to a final concentration of 20 μg / ml, followed by a 3-fold serial dilution for a total of 10 dilution points. 50 μl of the diluted antibody was added to the conical bottom plate containing cells and incubated at 4°C for 60 min. The cells were washed twice with 1% BSA, and 50 μl of the diluted secondary antibody was added to each well, mixed, and incubated at 4°C for 30 min. The cells were washed twice with 1% BSA and resuspended in 200 μl of 1% BSA for flow cytometry analysis. Data processing: Median PE values ​​were exported and imported into GraphPad Prism 6 software to calculate EC values. 50 , the results are shown in Table 1.

[0221] Table 1: Protein binding and tumor cell binding ability detection of chimeric antibodies

[0222] 3.2 Anti-human B7-H3 chimeric antibody sequence extraction

[0223] Hybridoma cells were cultured to approximately 8,000 cells, lysed, and first-strand cDNA was synthesized using a cDNA reverse transcription kit (Thermo Fisher). Primers were used to amplify the VH and VK genes from the cDNA using PCR. The PCR products were purified using a DNA purification kit (Qiagen) and ligated into a TOPO vector (Thermo Fisher Scientific). Approximately 12 clones were selected for sequencing from each ligation reaction. Sequences were analyzed using IMGT. The resulting anti-human B7-H3 antibody variable region and CDR sequences are shown in Table 2.

[0224] Table 2: Anti-human B7-H3 antibody variable region and CDR amino acid sequences

[0225] Example 4: Evaluation of fully human anti-human B7-H3 antibodies

[0226] 4.1 Expression of fully human anti-human B7-H3 antibody

[0227] The heavy chain variable region amino acid sequence of 20G11G6 was linked to the human IgG1 heavy chain constant region amino acid sequence (SEQ ID NO: 30), and the light chain variable region amino acid sequence was linked to the human light chain κ constant region amino acid sequence (SEQ ID NO: 32) to construct B7-H3 fully human antibody 2#, whose heavy chain amino acid sequence and light chain amino acid sequence are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. The cDNA was synthesized by codon optimization and ligated into the plasmid pTT5 (commissioned to GenScript). The pTT5 plasmids corresponding to the fully human antibody heavy chain and light chain nucleotides were simultaneously transfected into CHO-EBNA cells, and the supernatant was collected by centrifugation. The recombinant antibody in the supernatant was purified using Protein A (MabSelect SuRe, GE) to obtain the anti-human B7-H3 fully human antibody 2# protein (hereinafter referred to as "2# antibody").

[0228] 4.2 Anti-human B7-H3 fully human antibody protein binding assay

[0229] To detect the affinity of anti-human B7-H3 fully human antibody to human B7-H3-4Ig-his and monkey B7-H3-4Ig-his, human and monkey B7-H3-4Ig-his proteins were diluted to 1 μg / ml with CBS coating buffer, and 100 μl / well was coated on a 96-well ELISA plate and incubated at 4°C overnight; the ELISA plate was washed once with PBS, the plate was drained, and then 100 μl of 2% BSA was added to each well. After incubation at 37°C for 2 hours, the blocking solution was removed; the human antibody and control antibody DS7300 were serially diluted with 2% BSA, starting at 10 μg / ml, with a 3-fold gradient and 11 concentration points. 100 μl was added to each well of the ELISA plate and incubated at 37°C for 2 hours; the plate was washed three times with PBST (0.2% Tween-20), the plate was drained, and 100 μl of HRP Goat Anti-Human IgG (H+L) (Jackson), incubated at 37°C for 1 hour. The plate was washed five times with PBST (0.2% Tween-20). After the plate was drained, 100 μl of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) colorimetric substrate was added to each well. After incubation at room temperature for 3-5 minutes, 50 μl of 2M H2SO4 was added to each well to terminate the colorimetric reaction. The OD was read on a microplate reader. 450nm The raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting and calculation of EC 50 As shown in Figures 1A-B and Table 3, antibody #2 has good binding affinity to human and monkey B7-H3-4Ig.

[0230] Table 3: Anti-human B7-H3 fully human antibody protein binding test results

[0231] 4.3 Determination of the isoelectric point (PI) of recombinant fully human antibodies

[0232] The isoelectric point (PI) of candidate antibodies was determined using isoelectric focusing. The method is briefly described as follows: Analysis was performed using a ProteinSimple Maurice Isoelectric Focusing System in conjunction with a capillary cartridge. The antibody was diluted with water, and the detection system used a pH gradient formed using ampholytes 3-10 (GE) at a final concentration of 4%. The isoelectric points of the antibodies are shown in Table 4.

[0233] The results showed that the isoelectric point of antibody 2# was relatively high, about 9.3, so the sequence of antibody 2# needed to be modified to lower the isoelectric point.

[0234] Table 4: Isoelectric point determination results of anti-human B7-H3 fully human antibody

[0235] 4.4 Sequence modification of fully human anti-human B7-H3 antibody:

[0236] To lower the isoelectric point of fully human anti-human B7-H3 antibody 2# and remove PTM risk sites while retaining its activity, the heavy chain variable region sequence of antibody 2# was modified in multiple locations. The heavy chain constant region of the antibody was also modified (L234A, L235A, G237A) to eliminate ADCC activity. The amino acid sequence of the mutated human IgG1 heavy chain constant region is SEQ ID NO: 31. The light chain variable region sequence of antibody 2# was modified in multiple locations to lower the isoelectric point, while the amino acid sequence of the light chain kappa constant region was not modified (SEQ ID NO: 32). The designed antibody was named 2#8890. The variable region sequence and CDR sequence of anti-human B7-H3 antibody 2#8890 are shown in Table 5. The heavy chain amino acid sequence and light chain amino acid sequence of 2#8890 are shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0237] Table 5: Anti-human B7-H3 antibody 2#8890 variable region and CDR amino acid sequences

[0238] 4.5 Expression of fully human anti-human B7-H3 antibody

[0239] The cDNA sequence of 2#8890 was synthesized through codon optimization and ligated into plasmid pTT5 (commissioned to GenScript). The pTT5 plasmids corresponding to the nucleotide sequence of the fully human antibody heavy and light chains were simultaneously transfected into CHO-EBNA cells, and the supernatant was collected by centrifugation. The recombinant antibody in the supernatant was purified using Protein A (MabSelect SuRe, GE) to obtain a fully human anti-human B7-H3 antibody.

[0240] According to the isoelectric point determination method described in 4.3, the isoelectric point of antibody 2#8890 was measured again, and its isoelectric point was reduced to 8.4, successfully lowering the isoelectric point of the anti-human B7-H3 fully human antibody.

[0241] 4.6 Anti-human B7-H3 fully human antibody protein binding assay

[0242] To test the affinity of the fully human anti-human B7-H3 antibody to human B7-H3-4Ig-his, human B7-H3-4Ig-his protein was diluted to 1 μg / ml with CBS coating buffer, and 100 μl / well was coated on a 96-well ELISA plate and incubated at 4°C overnight. The plate was washed once with PBS, drained, and then 100 μl of 2% BSA was added to each well. The plate was incubated at 37°C for 2 hours, and the blocking solution was removed. The human antibody and control antibody were serially diluted in 2% BSA, starting at 10 μg / ml, and the gradient was 4-fold with 8 concentration points. 100 μl was added to each well of the ELISA plate and incubated at 37°C for 2 hours. The plate was washed three times with PBST (0.2% Tween-20). After draining, 100 μl of HRP Goat Anti-Human IgG (H+L) (Jackson) was added to each well and incubated at 37°C for 1 hour. The plate was washed five times with PBST (0.2% Tween-20). After the plate was dried, 100 μl of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) colorimetric substrate was added to each well. After reacting at room temperature for 3-5 min, 50 μl of 2M H2SO4 was added to each well to terminate the colorimetric reaction. The OD value was read using a microplate reader. 450nm The raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting and calculation of EC 50 As shown in Table 6 and Figure 2, 2#8890 has a good binding affinity to B7-H3-4Ig.

[0243] Table 6: Binding results of anti-human B7-H3 fully human antibody to human B7-H3-4Ig-His protein

[0244] 4.7 Dynamic affinity testing of fully human anti-human B7-H3 antibodies

[0245] The dynamic affinity of the anti-human B7-H3 fully human antibody to human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his, and monkey B7-H3-his proteins was tested using ForteBio (Pall Life Sciences). The specific method is as follows: the test antibody is diluted to 5 μg / ml in PBST (0.02% Tween-20), and human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his, and monkey B7-H3-his proteins are gradiently diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, and 0 nM. The test antibodies are then captured in PBST (0.02% Tween-20) solution using a Protein A Sensor (Pall Life Sciences), each of which is captured for 60 s. The antibodies then bind to the four proteins for 60 s, followed by dissociation for 180 s. The results are analyzed in Data Analysis 11.0 software using a 1:1 global fitting mode to obtain affinity constants. The results are shown in Table 7, which show that 2#8890 binds strongly to human and monkey B7-H3, but does not bind to rat B7-H3.

[0246] Table 7: Dynamic affinity test results of anti-human B7-H3 fully human antibody

[0247] 4.8 Cellular affinity and species cross-species testing of fully human anti-human B7-H3 antibodies

[0248] A flow cytometer (Beckman, model Cytoflex) was used to detect the affinity of the anti-human B7-H3 fully human antibody to human colon cancer cells HT29 (Cell Bank of the Chinese Academy of Sciences), human gastric cancer cells NCI-N87 (ATCC), human breast squamous cancer cells HCC1806 (ATCC) and human non-small cell lung cancer cells HCC827 (ATCC); the affinity of the anti-human B7-H3 fully human antibody to CHO-S-human B7-H3-4Ig and CHO-S-human B7-H3-2Ig was detected; and the species cross-species of the anti-human B7-H3 fully human antibody was detected: affinity to CHO-S-rat B7-H3 and CHO-S-monkey B7-H3.

[0249] The adherent cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution, counted, and the cell density was adjusted to 4.0 × 10 6 / ml, washed twice with 1% BSA, resuspended in 1% BSA solution, and 50 μl of cell suspension was added to each well of a 96-well pointed bottom plate (cell number 2×105 The candidate antibody and negative control antibody hIgG1 were diluted in 1% BSA to a final concentration of 10 μg / ml, followed by a 3-fold serial dilution to a total of 11 concentration points. 50 μl of the diluted antibody was added to the conical bottom plate containing cells and incubated at 4°C for 60 min. The cells were washed twice with 1% BSA, and 50 μl of the diluted secondary antibody was added to each well, mixed, and incubated at 4°C for 30 min. The cells were washed twice with 1% BSA and resuspended in 200 μl of 1% BSA for flow cytometry analysis. Data processing: Median PE values ​​were exported and imported into GraphPad Prism 6 software to calculate EC values. 50 .

[0250] The affinity results of the fully human anti-human B7-H3 antibodies for HT29, NCI-N87, HCC1806, and HCC827 tumor cells are shown in Figures 3A, 3B, 3C, and 3D, respectively, and Table 8. 2#8890 exhibited stronger affinity for these tumor cells than DS7300. The affinity results of the fully human anti-human B7-H3 antibodies for CHO-S-human B7-H3-4Ig and CHO-S-human B7-H3-2Ig cells are shown in Figures 3E and 3F, and Table 9. 2#8890 and DS7300 exhibited comparable affinity for these two overexpressing cell lines. The affinity results of the fully human anti-human B7-H3 antibody for CHO-S-monkey B7-H3 and CHO-S-rat B7-H3 cells are shown in Figures 4A and 4B and Table 10. The results showed that 2#8890 bound to monkey B7-H3 overexpressing cells but not to rat B7-H3 overexpressing cells.

[0251] Table 8: Anti-human B7-H3 fully human antibodies and HT29, NCI-N87, HCC1806 and HCC827

[0252] Cell affinity measurement results

[0253] Table 9: Anti-human B7-H3 fully human antibodies and CHO-S-human B7-H3-4Ig, CHO-S-human B7-H3-2Ig cell affinity

[0254] Strength measurement results

[0255] Table 10: Affinity determination results of anti-human B7-H3 fully human antibodies to CHO-S-rat B7-H3 and CHO-S-monkey B7-H3 cells

[0256] 4.9 ADCC activity detection of fully human anti-human B7-H3 antibody

[0257] Effector cells, Jurkat-NFAT / luciferase-CD16a (engineered Jurkat cells stably expressing CD16a and a luciferase reporter gene regulated by an NFAT response element, obtained by lentiviral packaging, infection, and pressure screening), activate luciferase expression in the presence of both antibodies and target cells. Using One-Glo reagent (Promega) as a substrate, a fluorescent signal is emitted. The specific method is as follows: Jurkat-NFAT / luciferase-CD16a and CHO-S-human B7-H3-4Ig cells (engineered CHO-S cells stably expressing human B7-H3-4Ig, obtained by lentiviral packaging, infection, and pressure screening) are collected by centrifugation and resuspended in RPMI1640 + 1% FBS medium. The density is adjusted to 2.0 × 10 6 / ml and 1.0×10 6 / ml, 50 μl / well was added to a 96-well plate; the antibody was diluted in RPMI 1640 + 1% FBS medium (starting at 10 μg / ml, 3-fold dilution, 11 concentration points), 50 μl / well was added to a 96-well plate, and incubated at 37°C for 5 h; 20 μl One-glo (Promega) detection reagent was added, and the fluorescence signal value was read using a microplate reader after shaking and mixing. The results were imported into Graph Prism 6 software to calculate EC 50 The results are shown in Figure 5 and Table 11. 2#8890 has no ADCC activity, while DS7300 retains ADCC activity and EC 50 It is 108ng / ml.

[0258] Table 11: ADCC activity of anti-human B7-H3 fully human antibodies

[0259] 4.10 CDC activity assay of fully human anti-human B7-H3 antibody

[0260] To detect the CDC (complement-dependent cytotoxicity) effect and strength of human B7-H3 fully human antibody, target cells CHO-S-B7-H3-4Ig were collected and counted, and the cell density was adjusted to 2.5×10 5 40 μl of cell suspension was added to each well of a 96-well plate (the number of cells was 1×10 41 vial of freeze-dried guinea pig serum complement was added to 1 ml of DMEM basal medium to reconstitute the complement concentration at 100%. 950 μl of 100% complement was added to 950 μl of DMEM basal medium and mixed thoroughly. The complement concentration was 50%. 20 μl of 50% guinea pig serum complement was added to the 96-well plate to make the final complement concentration in the well 10%. 40 μl of the test antibody dilution was added to each well. The final concentration of the test antibody was 100,000 ng / ml, diluted 3-fold, and 11 concentrations. The 96-well plate was placed in a 37°C, CO2 constant temperature incubator and incubated for 2 hours. After incubation, 50 μl / well of Cell Counting luminescent substrate solution was added to the 96-well plate. After vortexing and mixing, the fluorescence signal value was read using a microplate reader. The results were imported into Graph Prism 6 software to calculate the EC 50 The results are shown in Figure 6 and Table 12, indicating that 2#8890 has CDC activity.

[0261] Table 12: CDC activity of anti-human B7-H3 fully human antibodies

[0262] 4.11 Endocytic activity detection of fully human anti-human B7-H3 antibody

[0263] The endocytic activity of the anti-human B7-H3 fully human antibody in human gastric cancer cells NCI-N87 (ATCC), human breast squamous carcinoma cells HCC1806 (ATCC), and human non-small cell lung cancer cells HCC827 (ATCC) was detected using a flow cytometer (Thermo, model Attune NxT).

[0264] The adherent cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution and counted, and the cell density was adjusted to 1×10 5 100 μl of cell suspension was added to each well of a 96-well plate (the number of cells was 1×10 4100 μg / well) and incubate the 96-well plate in a 37°C, CO2 incubator for 24 h. Remove the 96-well plate, discard the culture medium, and add 50 μl of fresh complete culture medium to each well. Use complete culture medium to dilute the test antibody and negative control antibody hIgG1, starting with a final concentration of 1.2 μg / ml, and perform a 5-fold serial dilution for a total of 6 concentration points. Use complete culture medium to dilute 300 μg / ml of PHrodo reagent (Thermo, Cat#Z25612) to 12 μg / ml (the final concentration of PHrodo is 3 μg / ml). The pHrodo reagent was mixed evenly in a 1:1 ratio (30 μl:30 μl) and incubated at room temperature in the dark for 30 minutes. 50 μl of the test antibody and pHrodo reagent mixture was added to a 96-well plate and incubated at 37°C, 5% CO2 for 24 hours. The 96-well plate was removed, the culture medium was aspirated, and the plate was washed once with sterile PBS. 100 μl of Trypsin-EDTA (0.25%) was added to each well to digest the cells, and then 100 μl of complete culture medium was added to neutralize the cells. After pipetting and dispersing the cells in the wells, the cells were detected by FACS. Data processing: Median APC values ​​were exported and then imported into GraphPad Prism 6 software to calculate EC. 50 The results are shown in Figures 7A, 7B, 7C and Table 13. The endocytic activity of 2#8890 is stronger than that of DS7300-.

[0265] Table 13: Endocytic activity assay of anti-human B7-H3 fully human antibody

[0266] 4.12 Antigen Binding Epitope Analysis of Fully Human Antibody Against Human B7-H3

[0267] Collect the suspended CHO-S-human B7-H3-4Ig cells, count them and adjust the cell density to 4.0×10 6 / ml, washed twice with 1% BSA, resuspended in 1% BSA solution, and 50 μl of cell suspension was added to each well of a 96-well pointed bottom plate (cell number 2×10 5100 μg / well); Biotin-2#8890 was diluted to 80 ng / ml (final concentration 20 ng / ml) in 1% BSA, and 25 μl was added to the conical-bottom plate containing cells. DS7300 and 2#8890 were diluted in 1% BSA in a 3-fold gradient starting at 10 μg / ml, for a total of 6 concentration points. 25 μl of each was added to the 96-well plate. The cells, the antibody concentration gradient, and the constant concentration of biotin-labeled antibody were mixed and incubated at 4°C for 60 min. The cells were washed twice with 1% BSA, and 50 μl of diluted PE-streptavidin secondary antibody (Biolegend) was added to each well and incubated at 4°C for 30 min. The cells were washed twice with 1% BSA and resuspended in 200 μl of PBS for flow cytometry analysis. Data processing: Median PE values ​​were exported and imported into GraphPad Prism 6 software for nonlinear curve fitting (four parameters). The results are shown in FIG8 , and 2#8890 does not compete with the DS7300 epitope.

[0268] 4.13 Specificity Detection of Anti-Human B7-H3 Fully Human Antibody

[0269] To investigate the binding specificity of the anti-human B7-H3 fully human antibody to human B7-H3, the ELISA method was used to detect the binding activity of the anti-human B7-H3 fully human antibody to human B7-1-his (purchased from nearshore), B7-2-his (purchased from nearshore), B7-H1-mFc (self-constructed), B7-H2-his (purchased from nearshore), B7-H3-4Ig-his and B7-H4-hFc (purchased from nearshore). Human B7-1-his, B7-2-his, B7-H1-mFc, B7-H2-his, B7-H3-4Ig-his and B7-H4-hFc proteins were diluted to 1 μg / ml with CBS coating solution, and 100 μl / well was coated on a 96-well ELISA plate and incubated at 4°C overnight; the ELISA plate was washed once with PBS, the plate was drained, and then 100 μl of 2% BSA was added to each well. After incubation at 37°C for 2 hours, the blocking solution was removed; human antibodies 2#8890, 2#8890-biotin and control antibodies DS7300, DS7300-biotin were serially diluted with 2% BSA, starting at 10 μg / ml, with a 4-fold gradient and 8 concentration points. For human B7-1-his, B7-2-his, B7-H2-his, and B7-H3-4Ig-his, 100 μl of 2#8890 and DS7300 were added to each well, respectively. For human B7-H1-mFc and B7-H4-hFc, 100 μl of 2#8890-biotin and DS7300-biotin were added to each well, respectively, and incubated at 37°C for 2 hours. The plates were washed three times with PBST (0.2% Tween-20). After the plates were dried, 100 μl of 1:10,000 diluted HRP Goat Anti-Human IgG (H+L) (Jackson) was added to each well for human B7-1-his, B7-2-his, B7-H2-his, and B7-H3-4Ig-his. For human B7-H1-mFc and B7-H4-hFc, 100 μl of 1:10,000 diluted peroxidase-conjugated streptavidin secondary antibody (Jackson) was added to each well and incubated at 37°C for 1 hour. The plate was washed five times with PBST (0.2% Tween-20). After the plate was dried, 100 μl of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) colorimetric substrate was added to each well. After reacting at room temperature for 3-5 min, 50 μl of 2M H2SO4 was added to each well to terminate the colorimetric reaction. The OD value was read using a microplate reader. 450nm The raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting and calculation of EC 50As shown in Table 14 and Figure 9, 2#8890 has specific binding to B7-H3-4Ig, and 2#8890 does not bind to B7-1, B7-2, B7-H1, B7-H2, and B7-H4.

[0270] Table 14: Specificity test results of anti-human B7-H3 fully human antibody

[0271] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to B7-H3, wherein: The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 2; (b) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 3; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 4; or (c) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and / or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and / or light chain variable region described in any one of (a) or (b), and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution; Preferably, the CDRs are defined according to the IMGT, Kabat, Chothia or AbM numbering systems.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 5 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 6 or a variant thereof; and CDR-H3 having a sequence of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 8 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 9 or a variant thereof; and CDR-L3 having a sequence of SEQ ID NO: 10 or a variant thereof; (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof; CDR-H2 of SEQ ID NO: 19 or a variant thereof; CDR-H3 of SEQ ID NO: 20 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof; CDR-H2 of SEQ ID NO: 19 or a variant thereof; CDR-H3 of SEQ ID NO: 20 or a variant thereof; and / or or a variant thereof; CDR-L1 having a sequence of SEQ ID NO: 9 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 10 or a variant thereof; or, (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof; CDR-H2 of SEQ ID NO: 12 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof; (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 21 or a variant thereof; CDR-H2 of SEQ ID NO: 22 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 16 or a variant thereof; CDR-H2 of SEQ ID NO: 17 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof; (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 24 or a variant thereof; CDR-H2 of SEQ ID NO: 25 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are numbered according to the AbM system righteous: (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof; (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof; CDR-H2 of SEQ ID NO: 29 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof; Wherein, the variant described in any one of (1a), (1b), (2a), (2b), (3a), (3b), (4a), and (4b) has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises: (a) a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; or (b) a VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof; wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The antibody or antigen-binding fragment thereof further comprises a constant region from or derived from a human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region from or derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4); preferably, the antibody or antigen-binding fragment thereof comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region having altered effector function compared to the wild-type Fc region; Preferably, the antibody or antigen-binding fragment thereof comprises a variant of the human IgG1 heavy chain constant region having the following substitutions compared to the wild-type sequence from which it is derived: Leu234Ala, Leu235Ala and Gly237Ala (positions according to the EU numbering system); Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region from or derived from a human immunoglobulin (eg, κ or λ); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 30, or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 30 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 31 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 31 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions); Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO: 32, or a variant thereof having up to 20 conservative amino acid substitutions compared to SEQ ID NO: 32 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions); More preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 30 and a light chain constant region (CL) as shown in SEQ ID NO: 32; or, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 31 and a light chain constant region (CL) as shown in SEQ ID NO:

32.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein The heavy chain constant region (CH) as shown in SEQ ID NO: 30 or 31 or a variant thereof lacks a C-terminal lysine.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising the VH region represented by SEQ ID NO: 1 and the heavy chain constant region (CH) represented by SEQ ID NO: 30, and a light chain comprising the VL region represented by SEQ ID NO: 2 and the light chain constant region (CL) represented by SEQ ID NO: 32; (2) a heavy chain comprising the VH region of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 30, and a light chain comprising the VL region of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO: 32; (3) a heavy chain comprising the VH represented by SEQ ID NO: 1 and the heavy chain constant region (CH) represented by SEQ ID NO: 31, and a light chain comprising the VL represented by SEQ ID NO: 2 and the light chain constant region (CL) represented by SEQ ID NO: 32; or, (4) A heavy chain comprising the VH represented by SEQ ID NO: 3 and the heavy chain constant region (CH) represented by SEQ ID NO: 31, and a light chain comprising the VL represented by SEQ ID NO: 4 and the light chain constant region (CL) represented by SEQ ID NO:

32.

8. The antibody or antigen-binding fragment thereof according to claim 3 or 7, wherein The N-terminal glutamine of the VH comprising the sequence as shown in SEQ ID NO: 1 or 3 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate salt; and / or The N-terminal glutamic acid of the VL comprising the sequence as shown in SEQ ID NO: 2 or 4 or a variant thereof undergoes cyclization to form pyroglutamic acid or pyroglutamate salt.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain having the sequence shown in SEQ ID NO: 40 and a light chain having the sequence shown in SEQ ID NO: 41; or, (2) A heavy chain having the sequence shown in SEQ ID NO:42 and a light chain having the sequence shown in SEQ ID NO:

43.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein The N-terminal glutamine of the heavy chain having a sequence as shown in SEQ ID NO: 40 or 42 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate salt; and / or The N-terminal glutamic acid of the light chain having the sequence as shown in SEQ ID NO: 41 or 43 or a variant thereof undergoes cyclization to form pyroglutamic acid or pyroglutamate salt.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein: The antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', Fab'-SH, F(ab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein The antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof is detectably labeled, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or biotin.

13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof has a characteristic selected from the group consisting of: (1) binds to B7-H3 (e.g., human or monkey B7-H3) with an EC50 of less than about 100 ng / mL, e.g., less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL or less; preferably, the EC50 is measured by ELISA; (2) binds to B7-H3 (e.g., human or monkey B7-H3) with a KD of less than about 100 nM, e.g., less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or less; preferably, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured; (3) does not bind or does not substantially bind to B7-1, B7-2, B7-H1, B7-H2, and / or B7-H4; for example, as determined by ELISA; (4) having CDC activity, such as inducing killing of cells expressing B7-H3 (e.g., tumor cells) through CDC; (5) No ADCC activity; (6) inducing B7-H3 internalization, e.g., as measured by flow cytometry; (7) inhibiting cell (such as tumor cell) proliferation; and / or (8) Inhibit tumor growth.

14. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, the heavy chain and / or light chain thereof, or the heavy chain variable region and / or light chain variable region thereof according to any one of claims 1 to 13.

15. The isolated nucleic acid molecule of claim 14, comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein: (a) the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO:33, (ii) a sequence substantially identical to SEQ ID NO:33 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:33), or (iii) a degenerate sequence of (i) or (ii) above; and / or, the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of SEQ ID NO:34, (v) a sequence substantially identical to SEQ ID NO:34 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:34), or (vi) a degenerate sequence of (iv) or (v) above; or (b) the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO:35, (ii) a sequence substantially identical to SEQ ID NO:35 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:35), or (iii) a degenerate sequence of (i) or (ii) above; and / or, the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of SEQ ID NO:36, (v) a sequence substantially identical to SEQ ID NO:36 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:36), or (vi) a degenerate sequence of (iv) or (v) above.

16. A vector comprising the nucleic acid molecule according to any one of claims 14 to 15; preferably, the vector is a cloning vector or an expression vector.

17. A host cell comprising the nucleic acid molecule according to any one of claims 14 to 15 or the vector according to claim 13.

18. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, comprising culturing the host cell according to claim 14 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell.

19. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and a conjugated moiety connected thereto; Preferably, the conjugated moiety is selected from a detectable label (such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme) or a therapeutic agent (such as a cytotoxic agent, a cytokine, a toxin or a radionuclide).

20. A multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13; Preferably, the multispecific antibody comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 as a first antigen-binding domain, and further comprises at least one second antigen-binding domain directed against another target; Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

21. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof (e.g., ScFv) of any one of claims 1-13, a transmembrane domain, and one or more intracellular T cell signaling domains.

22. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20, or the chimeric antigen receptor according to claim 21 or a host cell expressing the chimeric antigen receptor, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from the group consisting of: a B7-H3 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic agent, or any combination thereof; Preferably, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as mixed components.

23. A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 13, or the isolated nucleic acid molecule of claim 14 or 15, or the vector of claim 16, or the host cell of claim 17, or the conjugate of claim 19, or the multispecific antibody of claim 20, or the chimeric antigen receptor or a host cell expressing the chimeric antigen receptor of claim 21, or the pharmaceutical composition of claim 22, and optionally instructions for use and / or an administration device.

24. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20, or the chimeric antigen receptor or a host cell expressing the chimeric antigen receptor according to claim 21, or the pharmaceutical composition according to claim 22 in the preparation of a medicament for inhibiting cell proliferation (e.g., cells expressing B7-H3, such as tumor cells) or preventing and / or treating and / or adjuvanting the treatment of tumors; Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, e.g., simultaneously, separately, or sequentially; Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from: B7-H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapy drugs or any combination thereof.

25. The use according to claim 24, wherein the tumor is a B7-H3 positive tumor; Preferably, the tumor is selected from breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer or any combination thereof.

26. A method for inhibiting cell proliferation, comprising contacting the cell with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20, or the chimeric antigen receptor according to claim 21 or a host cell expressing the chimeric antigen receptor, or the pharmaceutical composition according to claim 22; Preferably, the cell is a cell expressing B7-H3, such as a tumor cell.

27. A method for preventing and / or treating and / or adjuvant treating a tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20, or the chimeric antigen receptor according to claim 21 or a host cell expressing the chimeric antigen receptor, or the pharmaceutical composition according to claim 22.

28. The method of claim 27, further comprising administering to the subject a second therapy selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and any combination thereof; Optionally, the second therapy may be applied simultaneously, separately or sequentially with the method of claim 27 .

29. The method of claim 27 or 28, wherein The tumor is a B7-H3 positive tumor; Preferably, the tumor is selected from breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer or any combination thereof.

30. A method for detecting the presence or level of B7-H3 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 13 under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and B7-H3, and detecting formation of the complex; Preferably, the method is used to diagnose a tumor, such as a B7-H3 positive tumor, such as breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer or any combination thereof; Preferably, the method comprises detecting the expression level of B7-H3 in a test sample from a subject, and comparing the expression level with a reference value, wherein an increase in the expression level compared to the reference value is indicative of a tumor.

31. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20 in the preparation of a detection kit for detecting the presence or level of B7-H3 in a sample and / or diagnosing a tumor; Preferably, the tumor is a B7-H3 positive tumor; Preferably, the tumor is selected from breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer or any combination thereof.

Images