HER3 binding proteins and uses thereof

Abstract

Provided are HER3 binding proteins and uses thereof. Specifically, provided are anti-HER3 antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding same, and methods for preparing same. Anti-HER3 antibodies or antigen-binding fragments thereof have high specificity and high affinity for HER3, and do not bind to EGFR, HER2, and HER4 of the same family. Further provided are uses of the antibodies or antigen-binding fragments thereof in the treatment and diagnosis of diseases.

Description

[Technical field]

[0001] The present invention is in the field of therapeutic monoclonal antibodies, specifically directed against HER3 and the use of said antibodies in the treatment and diagnosis of diseases. [Background technology]

[0002] Human epidermal growth factor receptor 3 (HER3, also known as Erbb3) is a receptor tyrosine protein that belongs to the epidermal growth factor receptor (EGFR) / HER subfamily of receptor protein tyrosine kinases (RTKs), which consists of EGFR (HER1 / Erbb1), HER2 / Erbb2, HER3 / Erbb3 and HER4 / Erbb4. The HER3 protein mainly consists of three domains, the extracellular domain, the transmembrane domain and the intracellular domain, and the extracellular domain contains four domains, D1, D2, D3 and D4.

[0003] EGFR and HER2 are recognized as oncogenic RTKs that promote tumorigenesis in various types of solid tumors. The major role of HER3 in tumorigenesis is to act as a scaffold protein, thereby allowing maximal induction of the PI3K / AKT pathway. HER3 contains a motif that includes a cluster of six C-terminal tyrosines that resembles the consensus PI3K / p85 binding site when phosphorylated. As a result, the upstream cancer drivers EGFR, HER2, cMET, and FGFR2 can most efficiently couple to the PI3K / AKT pathway by forming heterodimers with HER3. Therefore, it is reasonable to expect that inhibition of HER3 activity can block cancer development in various systems driven by different RTKs. In addition to promoting tumor growth under non-stress conditions, HER3 has been found to be highly involved in conferring therapeutic resistance to various targeted drugs, including EGFR tyrosine kinase inhibitors, HER2 monoclonal antibodies such as trastuzumab mAb, and small molecule inhibitors of PI3K or AKT or MEK. HER3 is therefore a promising cancer target to address the problem of cancer resistance.

[0004] At present, there is no commercially available antibody that targets HER3. Therefore, it is urgent and necessary to develop an antibody that targets HER3, which has higher specificity, lower toxicity, better clinical efficacy, and more convenient administration, and provides more drug treatment options for patients. Summary of the Invention

[0005] In the present application, the present inventors have developed a fully human antibody that can specifically recognize / bind to HER3 but cannot specifically recognize / bind to EGFR, HER2, and HER4 in the same family, and has high affinity and excellent properties, thereby providing the following invention.

[0006] Antibodies of the Invention In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to HER3, the antibody or antigen-binding fragment thereof comprising 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; (c) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 5, and / or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 6; (d) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 7, and / or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 8, or (e) CDR-H1, CDR-H2 and CDR-H3 contained in a subsequent heavy chain variable region (VH), and / or CDR-L1, CDR-L2 and CDR-L3 contained in a subsequent light chain variable region (VL), wherein the heavy chain variable region (VH) and / or the light chain variable region (VL) have at least one CDR that contains a mutation compared to the heavy chain variable region and / or light chain variable region described in any of (a) to (d), and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids).

[0007] In a specific embodiment, the heavy chain variable region (VH) and / or light chain variable region (VL) described in (e) above has at least one CDR that contains a mutation compared to the corresponding heavy chain variable region and / or light chain variable region described in any one of (a) to (d), wherein the mutation is a substitution, deletion or addition of one or several amino acids.

[0008] In certain embodiments, the substitutions are conservative substitutions.

[0009] In certain embodiments, the CDRs are according to the IMGT, Kabat, Chothia numbering system.

[0010] In certain embodiments, the HER3 comprises human HER3 and / or monkey HER3. In certain embodiments, the monkey is a rhesus monkey.

[0011] 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), the CDRs being defined according to the IMGT numbering system: (1a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 9 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 10 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 11 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 18 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 19 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 20 or a variant thereof; (1b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 24 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 25 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 26 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 67 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 68 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 69 or a variant thereof; (1c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 37 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 38 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 39 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 32 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 33 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 34 or a variant thereof; or (1d) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 46 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 47 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 53 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 33 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 54 or a variant thereof; A variant according to any one of (1a) to (1d) has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, and preferably, the substitutions are conservative substitutions.

[0012] 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: (2a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 12 or a variant thereof, CDR-H1 having the sequence set forth in SEQ ID NO: 13 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 14 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 21 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 22 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 23 or a variant thereof; (2b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 28 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 70 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 71 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 69 or a variant thereof; (2c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 40 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 41 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 42 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 35 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 34 or a variant thereof; or (2d) a heavy chain variable region (VH) comprising three CDRs, namely, CDR-H1 having the sequence set forth in SEQ ID NO: 48 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 49 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 50 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs, namely, CDR-L1 having the sequence set forth in SEQ ID NO: 72 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 54 or a variant thereof; The variant according to any one of (2a) to (2d) has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, and preferably, the substitutions are conservative substitutions.

[0013] 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: (3a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 15 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 16 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 17 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 21 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 22 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 23 or a variant thereof; (3b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 30 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 31 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 70 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 71 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 69 or a variant thereof; (3c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 43 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 44 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 42 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 35 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 34 or a variant thereof; or (3d) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 51 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 52 or a variant thereof, and CDR-H3 having the sequence set forth in SEQ ID NO: 50 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 72 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, and CDR-L3 having the sequence set forth in SEQ ID NO: 54 or a variant thereof; The variant according to any one of (3a) to (3d) has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, and preferably, the substitutions are conservative substitutions.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: (4a) VH comprising the sequence set forth in SEQ ID NO: 1 or a variant thereof, and / or VL comprising the sequence set forth in SEQ ID NO: 2 or a variant thereof; (4b) VH comprising the sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or VL comprising the sequence set forth in SEQ ID NO: 4 or a variant thereof; (4c) VH comprising the sequence set forth in SEQ ID NO: 5 or a variant thereof, and / or VL comprising the sequence set forth in SEQ ID NO: 6 or a variant thereof; or (4d) VH comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof, and / or VL comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof. A 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 several 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 said substitutions being conservative substitutions.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof having any one of features (a), (1a), (2a), (3a), (4a) in the above embodiments further has a feature selected from the following: (1) Binding to the D1 domain of HER3; (2) binding to human or monkey HER3 with an EC50 of less than about 50 ng / mL, e.g., less than about 40 ng / mL, 30 ng / mL, 20 ng / mL, 15 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 where the EC50 is measured by ELISA; (3) binds to human HER3 with a KD of less than about 50 nM, e.g., less than about 40 nM, 30 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM or less, and / or binds to monkey HER3 with a KD of less than about 100 nM, e.g., less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM or less, preferably as measured by biolayer interferometry (BLI) (e.g., ForteBio Octet®); (4) does not bind or does not substantially bind to EGFR, HER2 and / or HER4, e.g., as measured by ELISA; (5) having ADCC activity, e.g., inducing and killing HER3-expressing cells (e.g., tumor cells) via ADCC; (6) inhibiting HER3-mediated signaling, e.g., inhibiting ligand-induced AKT phosphorylation and / or inhibiting PI3K / AKT signaling; (6) inducing internalization of HER3, e.g., as determined by flow cytometry; (7) inhibiting cell proliferation, and / or (8) Inhibiting tumor growth.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof having any one of features (b), (1b), (2b), (3b), and (4b) of the above embodiments further has a feature selected from the following: (1) binding to HER3 (e.g., human or monkey HER3) with an EC50 of less than about 50 ng / mL, e.g., less than about 40 ng / mL, 30 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 or less, preferably wherein the EC50 is measured by ELISA; (2) does not bind, or does not substantially bind, to EGFR, HER2, and / or HER4, e.g., as determined by ELISA; (3) having ADCC activity, e.g., inducing and killing HER3-expressing cells (e.g., tumor cells) via ADCC; (4) inhibiting HER3-mediated signaling, e.g., inhibiting ligand-induced AKT phosphorylation and / or inhibiting PI3K / AKT signaling; (5) inducing internalization of HER3, e.g., as determined by flow cytometry; (6) inhibiting cell (e.g., tumor cell) proliferation, and / or (7) Inhibiting tumor growth.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof having any one of features (c), (1c), (2c), (3c), and (4c) in the above embodiments further has a feature selected from the following: (1) Binding to the D1 domain of HER3; (2) binding to human or monkey HER3 with an EC50 of less than about 50 ng / mL, e.g., less than about 40 ng / mL, 30 ng / mL, 20 ng / mL, 15 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL or less, preferably where the EC50 is measured by ELISA; (3) binds to human HER3 with a KD of less than about 50 nM, e.g., less than about 40 nM, 30 nM or less, and / or binds to monkey HER3 with a KD of less than about 100 nM, e.g., less than about 90 nM, 80 nM or less, preferably as measured by biolayer interferometry (BLI) (e.g., ForteBio Octet®); (4) does not bind or does not substantially bind to EGFR, HER2 and / or HER4, e.g., as measured by ELISA; (5) having ADCC activity, e.g., inducing and killing HER3-expressing cells (e.g., tumor cells) via ADCC; (6) inhibiting HER3-mediated signaling, e.g., inhibiting ligand-induced AKT phosphorylation and / or inhibiting PI3K / AKT signaling; (6) inducing internalization of HER3, e.g., as determined by flow cytometry; (7) inhibiting cell proliferation, and / or (8) Inhibiting tumor growth.

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof having any one of features (d), (1d), (2d), (3d), and (4d) of the above embodiments further has a feature selected from the following: (1) binding to the D1 and D2 domains of HER3; (2) binding to human, monkey, or mouse HER3 with an EC50 of less than about 50 ng / mL, e.g., less than about 40 ng / mL, 30 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 or less, preferably where the EC50 is measured by ELISA; (3) binds to human HER3 with a KD of less than about 20 nM, e.g., less than about 15 nM, 10 nM, 9 nM, 8 nM or less, and / or binds to monkey HER3 with a KD of less than about 20 nM, e.g., less than about 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM or less, preferably as measured by biolayer interferometry (BLI) (e.g., ForteBio Octet®); (4) does not bind or does not substantially bind to EGFR, HER2 and / or HER4, e.g., as measured by ELISA; (5) having ADCC activity, e.g., inducing and killing HER3-expressing cells (e.g., tumor cells) via ADCC; (6) inhibiting HER3-mediated signaling, e.g., inhibiting ligand-induced AKT phosphorylation and / or inhibiting PI3K / AKT signaling; (6) inducing internalization of HER3, e.g., as determined by flow cytometry; (7) inhibiting cell proliferation, and / or (8) Inhibiting tumor growth.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof according to any one of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof has a heavy chain comprising a heavy chain constant region from or derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4). In certain embodiments, the antibody or antigen-binding fragment thereof has a heavy chain comprising a wild-type Fc region or a mutated or chemically modified Fc region having altered effector function (such as enhanced ADCC activity) compared to the wild-type Fc region. In certain embodiments, the antibody or antigen-binding fragment thereof has a heavy chain comprising a sequence set forth in SEQ ID NO:63 or a variant thereof, said variant having up to 20 amino acid conservative substitutions compared thereto (e.g., up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, or 5 amino acid conservative substitutions).

[0021] In certain embodiments, the antibody or antigen-binding fragment thereof has a light chain comprising a light chain constant region from or derived from a human immunoglobulin (e.g., kappa or lambda). In certain embodiments, the antibody or antigen-binding fragment thereof has a light chain comprising the sequence set forth in SEQ ID NO:65 or a variant thereof, said variant having up to 20 amino acid conservative substitutions compared thereto (e.g., up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, or 5 amino acid conservative substitutions).

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: (1) a heavy chain comprising a VH set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) set forth in SEQ ID NO: 63, and / or a light chain comprising a VL set forth in SEQ ID NO: 2 and a light chain constant region (CL) set forth in SEQ ID NO: 65; (2) a heavy chain comprising a VH set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) set forth in SEQ ID NO: 63, and / or a light chain comprising a VL set forth in SEQ ID NO: 4 and a light chain constant region (CL) set forth in SEQ ID NO: 65; (3) a heavy chain comprising a VH set forth in SEQ ID NO: 5 and a heavy chain constant region (CH) set forth in SEQ ID NO: 63, and / or a light chain comprising a VL set forth in SEQ ID NO: 6 and a light chain constant region (CL) set forth in SEQ ID NO: 65; or (4) A heavy chain comprising a VH set forth in SEQ ID NO: 7 and a heavy chain constant region (CH) set forth in SEQ ID NO: 63, and / or a light chain comprising a VL set forth in SEQ ID NO: 8 and a light chain constant region (CL) set forth in SEQ ID NO: 65.

[0023] In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: (1) a heavy chain comprising a VH set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) set forth in SEQ ID NO: 63, and a light chain comprising a VL set forth in SEQ ID NO: 2 and a light chain constant region (CL) set forth in SEQ ID NO: 65; (2) a heavy chain comprising a VH set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) set forth in SEQ ID NO: 63, and a light chain comprising a VL set forth in SEQ ID NO: 4 and a light chain constant region (CL) set forth in SEQ ID NO: 65; (3) a heavy chain comprising a VH set forth in SEQ ID NO: 5 and a heavy chain constant region (CH) set forth in SEQ ID NO: 63, and a light chain comprising a VL set forth in SEQ ID NO: 6 and a light chain constant region (CL) set forth in SEQ ID NO: 65; or (4) A heavy chain comprising a VH set forth in SEQ ID NO: 7 and a heavy chain constant region (CH) set forth in SEQ ID NO: 63, and a light chain comprising a VL set forth in SEQ ID NO: 8 and a light chain constant region (CL) set forth in SEQ ID NO: 65.

[0024] In certain embodiments, the antibody or antigen-binding fragment thereof according to any one of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0025] In certain embodiments, the variable region of the antibody or antigen-binding fragment thereof according to any one of the above embodiments is a human variable region.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof according to any one of the above embodiments includes an ScFv, a Fab, a Fab', a (Fab')2, a Fab'-SH, an Fv fragment, a disulfide-linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof according to any one of the above embodiments carries a label. In certain embodiments, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material), or biotin.

[0028] derivatized antibodies The antibodies or antigen-binding fragments thereof of the present invention may be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to HER3 (e.g., human HER3). Thus, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent binding, etc.) to one or more other molecular moieties, e.g., another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., an avidin or polyhistidine tag) that can mediate binding of the antibody or antigen-binding fragment to another molecule.

[0029] As one type of derivative of an antibody, the present invention provides a conjugate comprising an antibody of the present invention or an antigen-binding fragment thereof and a conjugate moiety.

[0030] In certain embodiments, the conjugate moiety is selected from a detectable label. A detectable label of the present invention can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. 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, Alexa750)), acridinium esters, magnetic beads (e.g., Dynabeads®), colorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above labels. In certain embodiments, such labels can be used for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable label is selected from the group consisting of a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. In certain embodiments, such detectable labels can be attached to the antibodies or antigen-binding fragments thereof of the present invention by linkers of various lengths to reduce potential steric hindrance.

[0031] In certain embodiments, the conjugate moiety is selected from a therapeutic agent, preferably an anti-tumor agent such as a cytotoxic drug, a cytokine, a toxin or a radionuclide.

[0032] In some embodiments, the conjugate moiety is selected from substances capable of improving the biological properties of the antibody (e.g., increasing the serum half-life), for example, it may be a chemical group such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl.

[0033] The present invention provides, as one type of antibody derivative, a multispecific antibody comprising the antibody of the present invention or an antigen-binding fragment thereof.

[0034] In certain embodiments, the multispecific antibody comprises an antibody or antigen-binding fragment thereof of the invention as a first antigen-binding domain and further comprises at least one second antigen-binding domain against another target.

[0035] In certain embodiments, each antigen-binding domain of a multispecific antibody retains its original binding specificity.

[0036] In certain embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

[0037] The present invention provides a chimeric antigen receptor comprising the antibody of the present invention or an antigen-binding fragment thereof as one of the antibody derivatives. In a specific embodiment, the chimeric antigen receptor comprises the antibody of the present invention or an antigen-binding fragment thereof (e.g., ScFv) as an extracellular antigen-binding domain that specifically binds to HER3, 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 or a NK cell) that contains or expresses the chimeric antigen receptor.

[0038] Preparation of antibodies The antibody of the present invention can be prepared by various methods known in the art, for example, by genetic engineering and recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibody of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules can be inserted into an expression vector and transfected into a host cell. The transfected host cell can then be cultured under specific conditions to express the antibody of the present invention.

[0039] The antigen-binding fragments of the present invention can be obtained by hydrolysis of 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 also be produced directly by 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)). Furthermore, Fv, Fab, or F(ab')2 fragments can also be isolated directly from recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are well known to those skilled in the art.

[0040] Thus, 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. In accordance with codon degeneracy in the art, in some embodiments, the nucleotide sequence may be substituted in accordance with codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0041] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding a heavy chain variable region of an antibody, and / or a nucleic acid molecule encoding a light chain variable region of an antibody, The nucleic acid molecule encoding the heavy chain variable region of the antibody comprises: (i) the nucleotide sequence set forth in SEQ ID NO:55; (ii) a sequence substantially identical to SEQ ID NO:55 (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:55); or (iii) a degenerate sequence of (i) or (ii) above; and / or The nucleic acid molecule encoding the light chain variable region of the above-mentioned antibody comprises: (iv) a nucleotide sequence set forth in SEQ ID NO:56; (v) a sequence substantially identical to SEQ ID NO:56 (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:56); or (vi) a degenerate sequence of (iv) or (v) above.

[0042] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding a heavy chain variable region of an antibody, and / or a nucleic acid molecule encoding a light chain variable region of an antibody, The nucleic acid molecule encoding the heavy chain variable region of the antibody comprises: (i) a nucleotide sequence set forth in SEQ ID NO:57; (ii) a sequence substantially identical to SEQ ID NO:57 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:57, or a sequence having one or more nucleotide substitutions); or (iii) a degenerate sequence of (i) or (ii) above; and / or The nucleic acid molecule encoding the light chain variable region of the above-mentioned antibody comprises: (iv) a nucleotide sequence set forth in SEQ ID NO:58; (v) a sequence substantially identical to SEQ ID NO:58 (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:58); or (vi) a degenerate sequence of (iv) or (v) above.

[0043] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding a heavy chain variable region of an antibody, and / or a nucleic acid molecule encoding a light chain variable region of an antibody, The nucleic acid molecule encoding the heavy chain variable region of the antibody comprises: (i) the nucleotide sequence set forth in SEQ ID NO:59; (ii) a sequence substantially identical to SEQ ID NO:59 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:59, or a sequence having one or more nucleotide substitutions); or (iii) a degenerate sequence of (i) or (ii) above; and / or The nucleic acid molecule encoding the light chain variable region of the above-mentioned antibody comprises: (iv) a nucleotide sequence set forth in SEQ ID NO: 60; (v) a sequence substantially identical to SEQ ID NO: 60 (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: 60); or (vi) a degenerate sequence of (iv) or (v) above.

[0044] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding a heavy chain variable region of an antibody, and / or a nucleic acid molecule encoding a light chain variable region of an antibody, The nucleic acid molecule encoding the heavy chain variable region of the antibody comprises: (i) the nucleotide sequence set forth in SEQ ID NO:61; (ii) a sequence substantially identical to SEQ ID NO:61 (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:61); or (iii) a degenerate sequence of (i) or (ii) above; and / or The nucleic acid molecule encoding the light chain variable region of the above-mentioned antibody comprises: (iv) a nucleotide sequence set forth in SEQ ID NO:62; (v) a sequence substantially identical to SEQ ID NO:62 (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:62); or (vi) a degenerate sequence of (iv) or (v) above.

[0045] In another aspect of the invention, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention. In certain embodiments, a vector of the invention is, for example, a plasmid, cosmid, phage, lentivirus, etc. In certain embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject (e.g., a mammal, e.g., a human).

[0046] In a specific embodiment, the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof of the invention, and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof of the invention, the first nucleotide sequence and the second nucleotide sequence being 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 invention consists of a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0047] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used to construct chimeric antigen receptors (CARs), which comprise an extracellular antigen-binding domain (e.g., ScFv) that specifically binds to HER3, 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 comprise a nucleotide sequence encoding a chimeric antigen receptor, where the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof (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 thereof (e.g., ScFv) of an antibody of the present invention.

[0048] 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, which consist of a chimeric antigen receptor (CAR) and an immune cell (e.g., T lymphocyte, NK cell).

[0049] In another aspect of the present invention, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention. The host cell may be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., E. coli). Suitable eukaryotic cells include, but are not limited to, NS0 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 a particular embodiment, the host cell of the present invention is a mammalian cell, such as a CHO cell (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cell).

[0050] 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 comprise a nucleotide sequence encoding a chimeric antigen receptor, which nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof (e.g., ScFv). In certain embodiments, the host cell comprises an isolated nucleic acid molecule encoding a chimeric antigen receptor comprising an antigen-binding fragment thereof (e.g., ScFv) of an antibody of the present invention.

[0051] In another aspect of the invention, there is provided a method for producing an antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from a culture of the cultured host cells.

[0052] therapeutic use Another aspect of the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a multispecific antibody, or a chimeric antigen receptor of the invention, or a host vector expressing a chimeric antigen receptor, and a pharma- ceutically acceptable carrier and / or excipient.

[0053] In certain embodiments, a pharmaceutical composition of the invention comprises an antibody or antigen-binding fragment thereof of the invention and a pharma- ceutically acceptable carrier and / or excipient.

[0054] In certain embodiments, the pharmaceutical composition of the present invention comprises a vector or host cell of the present invention and a pharma- ceutically 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, the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof (e.g., ScFv), and the host cell comprises the isolated nucleic acid molecule or vector described above. In certain embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment of an antibody of the present invention (e.g., ScFv). 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).

[0055] In certain embodiments, the pharmaceutical composition may further comprise an additional pharma- ceutical active agent. In certain embodiments, the additional pharma- ceutical active agent is a drug having antitumor activity. In certain embodiments, the additional pharma- ceutical active agent is selected from the group consisting of EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic drugs, or any combination thereof. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention and the additional pharma- ceutical active agent are provided as separate or mixed components. Thus, the antibody or antigen-binding fragment thereof of the present invention and the additional pharma- ceutical active agent can be administered simultaneously, separately, or sequentially.

[0056] 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 invention is (e.g., in a subject) (1) Inhibition of cell (e.g., tumor cell) proliferation; (2) inhibition of tumor growth; (3) Induction and / or enhancement of antibody-dependent cellular cytotoxicity, (4) inhibition of HER3-mediated signaling; (5) Prevention and / or treatment of HER3-mediated diseases / disorders, or (6) Any combination of the above (1) to (5) is effective.

[0057] In certain embodiments, the HER3-mediated disease / disorder is a tumor, e.g., a HER3-expressing tumor. In certain embodiments, the tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer (e.g., 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.

[0058] Another aspect of the invention provides the use of an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a multispecific antibody, a chimeric antigen receptor, or a host cell expressing a chimeric antigen receptor, or a pharmaceutical composition of the invention in the manufacture of a medicament for use in the inhibition of cell proliferation, or the prevention and / or treatment of a tumor, and / or as adjuvant therapy.

[0059] In certain embodiments, the medicament is used to inhibit the proliferation of HER3-expressing cells (eg, tumor cells).

[0060] Another aspect of the invention provides a method for inhibiting cell proliferation comprising contacting a cell with an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a multispecific antibody, a chimeric antigen receptor, or a host cell expressing a chimeric antigen receptor, or a pharmaceutical composition of the invention. In certain embodiments, the cell is a HER3-expressing cell, such as a tumor cell.

[0061] Another aspect of the present invention provides a method for prevention and / or treatment of a tumor in a subject, and / or adjuvant therapy, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor, or host cell expressing a chimeric antigen receptor, or pharmaceutical composition of the present invention.

[0062] In certain embodiments, the method further comprises administering to the subject a second therapy, the second therapy being selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof. In certain embodiments, the second therapy and the above method can be administered simultaneously, separately, or sequentially.

[0063] In any of the above-mentioned embodiments, the tumor associated with 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 may be any tumor type. In a particular embodiment, the tumor associated with 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 is a HER3-positive tumor. In a particular embodiment, the tumor associated with 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 is selected from the group consisting of breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, pancreatic cancer, head / neck squamous cell carcinoma, melanoma, ovarian cancer, prostate cancer, liver cancer, kidney cancer, bladder cancer, or any combination thereof.

[0064] The antibody or antigen-binding fragment thereof of the present invention and the pharmaceutical composition of the present invention can be formulated into any dosage form known in the pharmaceutical art, for example, tablets, pills, suspensions, emulsions, liquids, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injections, and concentrated solutions for injections), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention must be sterile and stable under the conditions of manufacture and storage. The preferred dosage form is an injection. Such an injection may be a sterile injection solution. For example, a sterile injection solution can be prepared by incorporating the antibody of the present invention in a required dose in a suitable solvent, optionally with other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. The sterile injection solution may also be prepared as a sterile lyophilized powder (for example, by vacuum drying or lyophilization) for ease of storage and use. Such sterile, lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0065] Further, the antibodies or antigen-binding fragments thereof of the present invention may be presented in pharmaceutical compositions in unit dosage form for ease of administration.

[0066] The antibodies or antigen-binding fragments thereof and pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, intraocular, topical, parenteral, rectal, intrathecal, intracisternal, inguinal, intravesical, specific site (e.g., powder, ointment, or drop), or nasal routes. However, for many therapeutic applications, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In some preferred embodiments, the antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present invention are administered by intravenous infusion or injection.

[0067] The pharmaceutical composition of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antigen-binding fragment thereof of the present invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent a disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention may vary depending on the severity of the disease to be treated, the general condition of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the mode of administration, and other therapies administered at the same time.

[0068] In the present invention, the dosing regimen can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dose can be pro rataly reduced or increased according to the level of exigency of the therapeutic situation.

[0069] In the present invention, the subject may be a mammal, such as a human.

[0070] Detection Applications The antibodies, or antigen-binding fragments thereof, of the present invention are capable of specifically binding to HER3 and therefore can be used to detect the presence or level of HER3 in a sample.

[0071] Thus, 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 some preferred embodiments, 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.

[0072] In the present invention, a detectable label can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such labels are suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent 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, Alexa750)), acridinium esters, magnetic beads (e.g., Dynabeads®), colorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above labels. In certain embodiments, the above-mentioned detectable labels can be attached to the antibodies of the invention by linkers of various lengths to reduce potential steric hindrance.

[0073] In another aspect, the invention provides a method of detecting the presence or level of HER3 in a sample, comprising using an antibody or antigen-binding fragment thereof of the invention. In some preferred embodiments, the antibody or antigen-binding fragment thereof of the invention further carries a detectable label. In some preferred embodiments, the method further comprises detecting the antibody or antigen-binding fragment thereof of the invention using a reagent carrying a detectable label. The method may be used for diagnostic or non-diagnostic purposes (e.g., the sample is not a patient-derived sample but is a sample of cells).

[0074] In certain embodiments, the method includes contacting the sample with an antibody or antigen-binding fragment thereof of the present invention under conditions that allow for the formation of a complex between the antibody or antigen-binding fragment thereof and HER3, and detecting the formation of a complex.

[0075] Considering that HER3 is not expressed or is low expressed in normal tissues, and is expressed or is high expressed in some cancers, tumors can be diagnosed by detecting the presence or level of HER3 in a sample.Thus, in certain embodiments, the method is used to diagnose tumors, such as HER3-positive tumors, such as breast cancer, gastric cancer, lung cancer (e.g., 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.

[0076] In certain embodiments, the method includes detecting an expression level of HER3 in a test sample from a subject and comparing the expression level to 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.

[0077] In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the present invention in the manufacture of a kit, the kit being used for detecting the presence or level of HER3 in a sample and / or for diagnosing a tumor.

[0078] In another aspect, the invention provides a diagnostic or therapeutic kit comprising an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, or a multispecific antibody of the invention, and instructions for use. The kit may further comprise an administration device for local administration. The administration device comprises a drug-loaded syringe or a needle-free device.

[0079] The antibody of the present invention has a high binding affinity to HER3 with very strong specificity, does not bind to EGFR, HER2 and HER4 of the same family, and can inhibit HER3-mediated signal transduction and inhibit cell proliferation. Therefore, the antibody of the present invention has the potential to be used for the prevention and / or treatment of tumors. Furthermore, the antibody of the present invention is a fully human antibody and can be safely administered to human subjects without causing an immunogenic response. Therefore, the antibody of the present invention has significant clinical value.

[0080] [Table 1]

[0081] definition In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the cell culture, biochemistry, nucleic acid chemistry and immunology procedures used herein are all routine procedures widely used in the corresponding fields.At the same time, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0082] 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, so long as they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule may 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 κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chains may further include a "D" region of about 3 or more amino acids. Each heavy chain is composed 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 one domain, CL. The constant domains are not directly involved in antibody-antigen binding, but exhibit 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 of the classical complement system (C1q). The VH and VL regions can also be further divided into regions of high variability (called complementarity determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) respectively form an antigen-binding site.Amino acid assignments in each region or domain can be according to 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.

[0083] As used herein, unless the context clearly indicates otherwise, reference to the term "antibody" includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0084] As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody involved in antigen binding. The exact boundaries of these amino acid residues can be determined according to various numbering systems known in the art, for example, as defined by the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272) or the IMGT numbering system (Lefranc et al. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, one skilled in the art can easily identify the CDRs defined by each numbering system. Also, the correspondence between different numbering systems is well known to those skilled in the art (e.g., Lefranc et al. Comparat. Immunol. 27: 55-77, 2003).

[0085] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined according to the IMGT, Kabat, Chothia or AbM numbering system.

[0086] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0087] The term "antibody" is not limited to any particular method for producing the antibody. It includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody may be of various isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0088] As used herein, the term "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and binds to an antigen bound by the intact antibody. For example, an antigen-binding fragment is a fragment of a full-length antibody and is a polypeptide 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 known as an "antigen-binding site." 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 (Domantis technology), domain antibodies (Ablynx technology), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005, Nat Immunol 23, 1126-1136.

[0089] 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", where a "full-length heavy chain" or "heavy chain" refers to a polypeptide chain consisting of, from the N-terminus to the C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain, and optionally, in the case of an IgE isotype, a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of, from the N-terminus to the C-terminus, VH, CH1, HR, CH2, and CH3. A "full-length light chain" or "light chain" is a polypeptide chain consisting of, from the N-terminus to the C-terminus, a light chain variable region (VL) and a light chain constant region (CL). The two pairs of full-length antibody chains are linked to each other by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be derived from a single species, such as human, and may also be a chimeric or humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites, each formed by a pair of VH and VL, and these two antigen-binding sites specifically recognize / bind the same antigen.

[0090] As used herein, the term "Fd fragment" refers to an antibody fragment consisting of the VH and CH1 domains, the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)), the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains, the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by disulfide bonds in the hinge region, the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment, and consisting of an intact light chain and an Fd fragment (consisting of the VH and CH1 domains) of the heavy chain, and the term "Fab'-SH" refers to a Fab fragment containing a free sulfhydryl group.

[0091] As used herein, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to an antibody. However, even a variable region (e.g., an Fd fragment that contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although perhaps with a lower affinity than the complete binding site.

[0092] As used herein, the term "Fc fragment" refers to an antibody fragment formed by linking the second and third constant regions of a first heavy chain of an antibody to the second and third constant regions of a second heavy chain via disulfide bonds. The Fc fragment of an antibody has a variety of different functions that are not involved in antigen binding.

[0093] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, where the VL and VH are linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg 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 a repeated GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, as well as variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described in 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.

[0094] As used herein, the term "diabody" refers to a diabody whose VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, so that the domains pair with the complementary domains of another chain and form 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)).

[0095] Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and / or competes with the full-length antibody in specific binding to the antigen. Herein, one 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, and screen the antigen-binding fragment of the antibody for specificity in the same manner as an intact antibody.

[0096] As used herein, the term "multispecific antibody" refers to an antibody having multiple different antigen-binding specificities, including, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. A "bispecific antibody" refers to an antibody having two different antigen-binding specificities, which is a conjugate formed from a first antibody (or a fragment thereof) and a second antibody (or a fragment thereof) or an antibody mimic via conjugation arms, and conjugation methods include, but are not limited to, chemical reaction, gene fusion, and enzyme catalysis. A "multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies, where a trispecific antibody refers to an antibody having three different antigen-binding specificities and a tetraspecific antibody refers to an antibody having four different antigen-binding specificities.

[0097] As used herein, the terms "monoclonal antibody", "McAb" and "mAb" can be used interchangeably with the same meaning and refer to an antibody or antibody fragment from a highly homogenous group of antibody molecules, i.e., a population of antibody molecules that are identical except for natural mutations that may occur naturally. A monoclonal antibody is highly specific to a single epitope on an antigen. In comparison with a monoclonal antibody, a polyclonal antibody usually contains at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Furthermore, the modifier "monoclonal" merely indicates that the antibody is characterized as being obtained from a highly homogenous antibody population and cannot be construed as requiring any particular method for preparing the antibody.

[0098] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light or / and heavy chain is derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass) and another portion of the light or / and heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), yet still retains binding activity to a target antigen. For example, the term "chimeric antibody" can include an antibody whose heavy and light chain variable regions are derived from a first antibody (e.g., a human antibody) and whose heavy and light chain constant regions are derived from a second antibody (e.g., a mouse antibody). For example, an antibody produced by immunizing a fully human transgenic mouse can be referred to as a chimeric antibody, which consists of a fully human variable region and a mouse constant region.

[0099] 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 a portion of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR region (e.g., FR region of the variable region and / or constant region) is derived from a human immunoglobulin (acceptor antibody). For example, a humanized antibody can be prepared by grafting CDR sequences derived from the germline of another mammalian species onto human framework sequences.

[0100] As used herein, the term "fully human antibody" refers to an antibody that contains only immunoglobulin sequences derived from human germline. Those skilled in the art will understand that a fully human antibody does not exclude the inclusion of amino acid residues that are not encoded by human germline immunoglobulin sequences, such as mutations introduced by random or in vitro site-specific mutation or by in vivo somatic mutation. Fully human antibodies can be produced using transgenic mice into which human immunoglobulin genes have been introduced (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 Engineering Reviews (2002), vol. 19, p. 73-82), KM mouse (Cloning Stem Cells (2002), vol. 4, issue 1, p. 91-102)), and the antibody of interest can be produced in large quantities by isolating antibody-producing lymphocytes from the mouse and forming hybridomas. Fully human antibodies can also be produced by phage display (FEBS Letter (1998), vol. 441, p. 20-24). In this method, a phage containing a human antibody gene integrated into a circular single-stranded DNA can be used to express a fully human antibody on the surface of the phage in a form fused to the coat protein of the phage. Fully human antibodies can also be produced using transgenic mice introduced with genes for human immunoglobulin variable regions, and the mice can produce chimeric antibodies consisting of fully human variable regions and mouse constant regions, which can then be linked to human immunoglobulin constant regions using methods known in the art to produce fully human antibodies. For example, a DNA encoding VH can be operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene.Gene sequences of human heavy chain constant regions are known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment 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 can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but generally is preferably an IgG1 or IgG4 constant region. For example, the DNA encoding the VL can be operably linked to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (as well as a Fab light chain gene). Gene sequences of human light chain constant regions are known in the art (see, for example, Kabat, EA et al. (1991) J. Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0101] As used herein, the term "variant" in the context of a polypeptide (e.g., in the case of a polypeptide) refers to a polypeptide or peptide that contains an altered amino acid sequence introduced by substitution, deletion, or addition of amino acid residues. In some cases, the term "variant" also refers to a modified polypeptide or peptide (by covalently attaching any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be made 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. Furthermore, a variant has a similar, identical, or improved function compared to the polypeptide or peptide from which it is derived.

[0102] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as, for example, the reaction between an antibody and its target. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (KD) or the half maximal effective concentration (EC 50 )

[0103] 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 k) and the "dissociation rate constant" (k or k) can be calculated based on the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k / k is equal to the dissociation constant K (see Davies et al., Annual Rev Biochem, 1990:16:1799-473). Any valid method can be used to measure the K, k and k values. In certain embodiments, bioluminescence interference techniques (e.g., ForteBio Octet techniques) can be used to measure the dissociation constant. Additionally, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0104] As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. If the vector allows for the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector can 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, yeast artificial chromosomes (YACs), artificial chromosomes such as 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 (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may also contain an origin of replication.

[0105] Expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. Generally, in cloning vectors, this sequence allows the vector to replicate independently of the host chromosomal DNA, and includes an origin of replication or an autonomously replicating sequence. As used herein, the term "expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be supplied by the host cell or by an in vitro expression system. 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).

[0106] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as E. 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, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0107] As used herein, the term "identity" refers to the degree of correspondence between two polypeptides or two nucleic acids. If the two sequences being compared have the same monomer subunit of a base or amino acid at a particular position (e.g., two DNA molecules each have an adenine at a particular position, or two polypeptides each have a lysine at a particular position), the two molecules are identical at that site. The percent identity between two sequences is a function of the number of identical positions shared by the two sequences to the total number of positions being compared x 100. For example, if 6 out of 10 positions of two sequences are identical, then these two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions are identical). Generally, the comparison of two sequences is performed in a manner that produces maximum identity. Such alignments can be performed using computer programs such as the Align program (DNAStar, Inc.), which is based on the method of Needleman et al. (J. Mol. Biol. 48:443-453, 1970). 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)) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap extension penalty of 12, and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) as incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and extension weights of 1, 2, 3, 4, 5, or 6.

[0108] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide that contains that 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 the replacement of an amino acid residue with an amino acid residue that has a similar side chain, e.g., an amino acid residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has chemical properties including similar size, shape, charge, ability to form covalent 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, 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 a 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)).

[0109] The twenty conventional amino acids referred to herein are written according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" are used interchangeably with the same meaning. Also, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0110] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, osmotic pressure maintaining agents, absorption retarding agents, and preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffered saline. Surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants such as Tween-80. Ionic strength enhancing agents include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, trichloro-t-butanol, phenol, sorbic acid, and the like. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, etc. Absorption retarding agents include, but are not limited to, monostearate salts, gelatin, etc. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, trichloro-t-butanol, phenol, sorbic acid, etc. Stabilizers have the meanings commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or their degradation products (e.g., lactalbumin hydrolysate), etc.

[0111] As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease or disorder or condition (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, delay or slowing of disease progression, improvement or remission of the disease state, and alleviation of symptoms, whether detectable or undetectable. In addition, "treatment" also refers to prolonging survival compared to the expected survival in the absence of treatment.

[0112] As used herein, the term "subject" refers to a mammal, such as, for example, a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) has a tumor or is at risk of having the above-mentioned disease.

[0113] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a tumor), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. It is entirely within the capabilities of a person skilled in the art to determine such an effective amount. For example, an effective amount for therapeutic use depends on the severity of the disease to be treated, the general condition of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the mode of administration, and other therapies administered at the same time.

[0114] As used herein, the term "effector function" refers to the biological activity attributable to an antibody Fc region (either a native sequence Fc region or an amino acid sequence variant Fc region), which 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 phagocytosis (ADCP), down-regulation 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 antibody effector functions are known in the art and are performed, for example, by introducing mutations into the Fc region.

[0115] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which cytotoxic effector cells specifically bind to antigen-bearing target cells via Ig binding to Fc receptors (FcR) displayed on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages) and then kill the target cells by secreting cytotoxins.

[0116] As used herein, the term "antibody-mediated internalization" refers to the phenomenon in which an antibody crosses the cell membrane after binding to an antigen on the cell surface. Internalization includes antibody-mediated internalization of receptors (e.g., HER3).

[0117] As used herein, combination therapy includes the use of an anti-HER3 antibody or antigen-binding fragment thereof encompassed by the present invention in combination with one or more additional active therapeutic agents of a second therapy (e.g., a chemotherapeutic agent) or other modalities of prevention or treatment (e.g., radiation therapy).

[0118] In such combination therapy, various active agents often have different complementary mechanisms of action, and combination therapy can provide synergistic effects. Combination therapy includes therapeutic agents that affect (e.g., enhance or activate) immune responses and therapeutic agents that affect (e.g., inhibit or kill) tumor / cancer cells. Combination therapy can reduce the likelihood of developing drug-resistant cancer cells. Combination therapy can allow for a reduction in the dose of one or more agents to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapy can have synergistic therapeutic or preventive effects against an underlying disease, disorder, or condition.

[0119] As used herein, "combination" includes therapies that may be administered separately, e.g., the therapies may be formulated separately for separate administration (e.g., provided as a kit) or may be administered together as a single formulation (i.e., "co-formulation"). In certain embodiments, the anti-HER3 antibodies or antigen-binding fragments thereof of the present invention may be administered sequentially. In other embodiments, the anti-HER3 antibodies or antigen-binding fragments thereof may be administered simultaneously. The antibodies or antigen-binding fragments thereof of the present invention may be used in any combination with at least one additional (active) agent.

[0120] In this context, HER3 positivity is obtained by immunohistochemistry and evaluation of staining intensity by an expert clinical pathologist.

[0121] The terms "cancer" and "tumor" are used interchangeably and refer to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can result in the formation of malignant tumors or cells that can invade adjacent tissues and metastasize to distant sites in the body via the lymphatic system or bloodstream. Cancer includes benign or malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.

[0122] The term "hematological tumors" includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as splenic 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 tumors also include leukemias, such as secondary leukemia or acute lymphocytic leukemia. Hematological tumors also include myelomas (e.g., multiple myeloma) and other hematological and / or B-cell or T-cell related cancers.

[0123] As used herein, the terms "about" or "approximately," when applied to a numerical variable, generally mean that the variable has a value within experimental error (e.g., within a 95% confidence interval of the mean) or within ±10% of the particular value or within a broader range.

[0124] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments. [Brief description of the drawings]

[0125] [Figure 1A] 1 shows the determination of the binding activity of anti-human HER3 fully human antibodies to human HER3 protein.

[0126] [Figure 1B] 1 shows the determination of the binding activity of anti-human HER3 fully human antibodies to monkey HER3 protein.

[0127] [Figure 1C] 1 shows the determination of the binding activity of anti-human HER3 fully human antibodies to rat HER3 protein.

[0128] [Figure 2A]1 shows the determination of the binding activity of anti-human HER3 fully human antibodies to human breast cancer cells T47D.

[0129] [Figure 2B] 1 shows the determination of the binding activity of anti-human HER3 fully human antibodies to human gastric cancer cells NCI-N87.

[0130] [Figure 2C] 1 shows the determination of the binding activity of anti-human HER3 fully human antibodies to human non-small cell lung cancer cells NCI-H358.

[0131] [Diagram 3] 1 shows detection of ADCC activity of an anti-human HER3 fully human antibody.

[0132] [Figure 4] 1 shows detection of inhibition of AKT phosphorylation by anti-human HER3 fully human antibody.

[0133] [Figure 5A] 1 shows detection of the internalization activity of an anti-human HER3 fully human antibody against human breast cancer cells MDA-MB-453.

[0134] [Figure 5B] 1 shows detection of the internalization activity of an anti-human HER3 fully human antibody against human colorectal cancer cells WIDR.

[0135] [Figure 5C] 1 shows detection of the internalization activity of an anti-human HER3 fully human antibody against human non-small cell lung cancer cells NCI-H358.

[0136] [Figure 5D] 1 shows detection of the internalization activity of an anti-human HER3 fully human antibody against human pancreatic cancer cells HPAF-II.

[0137] [Figure 6] 1 shows detection of the binding domain of an anti-human HER3 fully human antibody.

[0138] [Figure 7A] ELISA competitive binding assay between 100H7D3-hIgG, 47A3E3-hIgG and control antibody U1-59.

[0139] [Figure 7B] ELISA competitive binding assay between 47A3E3-hIgG, 100H7D3-hIgG and 22B6D2-hIgG.

[0140] [Figure 7C] ELISA competitive binding assay between 100H7D3-hIgG and 47A3E3-hIgG.

[0141] [Figure 8] 1 shows a FACS-detected competitive binding assay between 22B6D2-hIgG, 47A3E3-hIgG, 100H7D3-hIgG and the control antibody U1-59.

[0142] [Figure 9] 1 shows detection of binding specificity of anti-human HER3 fully human antibody.

[0143] Sequence information The sequence information encompassed by the present invention is set forth in Table 1 below. [Table 2-1] [Table 2-2] EXAMPLES

[0144] The invention will now be described with reference to the following examples which are intended to illustrate, but not limit, the invention.

[0145] Unless otherwise specified, the molecular biology experimental methods and immunodetection methods used in the present invention are basically performed 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., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will understand that these examples are illustrative of the present invention and are not intended to limit the scope of the claims of the present invention.

[0146] Example 1: Preparation of HER3 antigen and control antibody 1.1 Preparation of Her3 antigen The extracellular sequences of human HER3 (UniProt: P21860) and monkey HER3 (UniProt: G7N7B1) were synthesized by GenScript, and six histidines were introduced into the C-terminus to construct them on pTT5 vector. The recombinant plasmids were extracted and then verified by sequencing, and then transiently transfected into HEK293 cells for transient expression. The supernatants were collected on the 6th day and purified to obtain human HER3 protein and monkey HER3 protein, respectively.

[0147] 1.2 Expression of HER3 control antibodies The HER3 control antibody was derived from U1-59 of patent CN200680049887. The nucleotide sequences of the heavy and light chains of the antibody after codon optimization by GenScript were synthesized and cloned into pTT5 vectors, respectively, and then the pcDNA3.4 plasmids corresponding to the nucleotides of the heavy and light chains of the antibody were transfected simultaneously into CHOS cells (Thermo). After collecting the cell supernatant by centrifugation, the supernatant was purified with Protein A (MabSelect SuRe, GE) to obtain the control antibody.

[0148] Example 2: Preparation of anti-human HER3 monoclonal antibody 2.1 Immunization of mice Harbor Biomed's fully human transgenic mouse H2L2 (the antibody produced by this mouse is a chimeric antibody consisting of a fully human variable region and a mouse constant region) was used to immunize with the human HER3 protein prepared in 1.1. Freund's complete adjuvant, Adjuvant system (Sigma) was used for immunization by footpad, subcutaneous, tail base, or intraperitoneal injection, or Titermax Gold Adjuvant (Sigma), Imject Alum (Thermo) was used for immunization by footpad injection once every 2-3 days. During the immunization period, serum titers of anti-HER3 antibodies were monitored every 2 weeks by ELISA. Furthermore, mouse hybridomas with optimal titers were generated using protocols 2.2 and 2.3 below.

[0149] 2.2 Hybridoma fusion and screening 1 μg / mL human HER3 protein was coated on ELISA plates (BIOFIL) at 100 μL / well overnight at 4° C., and after washing once with 300 μL PBST (0.2% Tween-20), 100 μl 2% BSA in PBS was added to each well and incubated at 37° C. for 1 hour, and 20 μl hybridoma supernatant was added directly to the ELISA plate and incubated at 37° C. for 2 hours. After discarding the solution, the ELISA plate was mechanically washed three times with 320 μl PBST (0.2% Tween-20) per well. The ELISA plate was dried, and then 100 μL (1:5000) diluted HRP-conjugated goat anti-rat IgG (Thermo Fisher) was added to each well and incubated at 37° C. for 1 hour. The solution was discarded, and the ELISA plate was washed mechanically five times with 320 μl of PBST (0.2% Tween-20) per well. After drying the ELISA plate, 100 μL of TMB (InnoReagents) was added to each well to develop the color reaction in the dark, and then 50 μL of 2N H2SO4 was added to stop the color reaction, and the absorbance at 450 nm was read on a microplate reader. The clones with OD450 nm values ​​5 times higher than the negative control were identified as positive clones. The positive clones were then subjected to ELISA for cross-reactivity against monkey HER3 protein (see 2.1) and detection of cell binding with human breast cancer T47D cells to select the best clones for subcloning. The subclones were screened in the same manner, and the best subclones were selected to purify the chimeric antibodies.

[0150] Example 3: Evaluation of anti-human HER3 chimeric antibodies 3.1 Evaluation of binding activity of anti-human HER3 chimeric antibodies All favorable monoclones were transferred to a 6-well plate for serum-free culture, and then affinity purification was performed using Protein A beads with 4 to 6 ml of culture supernatant. The antibody protein bound to the Protein A beads was eluted with 1 M glycine solution (pH 2.5), and the eluted antibody protein was neutralized with 1 M Tris solution. The antibody protein concentration was quantified by Nanodrop, and candidate evaluation was performed.

[0151] Determination of ELISA affinity of HER3 chimeric antibody: Human and monkey HER3 protein were diluted to 1 μg / ml in CBS coating solution, added to ELISA plate at 100 ng / well, and left at 4 ° C overnight. The next day, the ELISA plate was washed once with PBS, then 100 μl of 2% BSA was added to each well and incubated at 37 ° C for 2 hours. The purified chimeric antibody and the control antibody were serially diluted with 2% BSA respectively (starting from 10 μg / ml, 8 concentration points with a 3-fold gradient), the blocking solution was removed, then 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 320 μl of PBST (0.2% Tween-20) per well, and after drying the plate, 100 μl of HRP-conjugated goat anti-rat IgG (Thermo) was added to each well and incubated at 37 ° C for 1 hour. The plates were washed five times with PBST (0.2% Tween-20). After drying the plates, 100 μl of TMB (CoWin Biotech) colorimetric substrate was added to each well and reacted at room temperature for 3-5 minutes, 50 μl of 2N H2SO4 was added to each well to stop the colorimetric reaction, and the absorbance values ​​at OD450nm were read on a microplate reader. The raw data was imported into GraphPad Prism 6 software and nonlinear curve fitting was performed to calculate the EC50 values ​​of binding of each antibody to human HER3 and monkey HER3. As shown in Table 2 below, all the screened antibodies were able to bind to human and monkey HER3 proteins. [Table 3]

[0152] 3.2 Obtaining the sequence of anti-human HER3 chimeric antibody After culturing the hybridoma cells until they reached about 8000 cells, the cells were lysed and the first strand cDNA was synthesized using a cDNA reverse transcription kit (Thermo Fisher). The VH and VK genes were amplified from the cDNA by PCR using primers, and the PCR products were purified by a DNA purification kit (Qiagen) and ligated into a TOPO vector (Thermo Fisher Sci). About 12 clones were taken in each ligation reaction for sequencing. The sequences were analyzed by abYsis. The variable region sequences and CDR sequences of the obtained anti-human HER3 antibodies are shown in Table 3-1 and Table 3-2. [Table 4] [Table 5]

[0153] Example 4: Evaluation of anti-human HER3 fully human antibodies 4.1 Construction of fully human anti-HER3 antibodies The amino acid sequences of the light chain variable regions of 22B6D2, 47A3E3 and 100H7D3 were linked to the amino acid sequence of the kappa constant region of the human light chain (SEQ ID NO: 65), and the amino acid sequences of their heavy chain variable regions were linked to the amino acid sequence of the constant region of the human IgG1 heavy chain (SEQ ID NO: 63), respectively, to construct HER3 fully human antibodies, 22B6D2-hIgG, 47A3E3-hIgG and 100H7D3-hIgG. The cDNA synthesized by codon optimization was linked to the plasmid pTT5 (GenScript). The pTT5 plasmids corresponding to the heavy and light chain nucleotides of the fully human antibodies were co-transfected into CHO-S cells, the supernatant was collected by centrifugation, and the recombinant antibody in the supernatant was purified using Protein A (MabSelect SuRe, GE) to obtain an anti-human HER3 fully human antibody.

[0154] 4.2 Detection of binding activity of anti-human HER3 fully human antibodies To detect the affinity of anti-human HER3 fully human antibodies to human, monkey and rat HER3 proteins (UniProt: Q62799), human, monkey and rat HER3 proteins were diluted to 1 μg / ml in CBS coating solution, coated on a 96-well ELISA plate with 100 μl / well, and left to stand overnight at 4°C. The ELISA plate was washed once with PBS and dried, and then 100 μl of 2% BSA was added to each well and incubated at 37°C for 2 hours. After incubation, the blocking solution was removed, and the human and control antibodies were serially diluted with 2% BSA (starting from 5 μg / ml, 11 concentration points in a 3-fold gradient), added to the ELISA plate at 100 μl per well, and incubated at 37° C. for 2 hours. The plate was washed three times with PBST (0.2% Tween-20) and dried, after which 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) and dried, and then 100 μl of TMB (InnoReagents) color substrate was added to each well and reacted at room temperature for 3 to 5 minutes, and then 50 μl of 2N H2SO4 was added to each well to stop the color reaction, and the absorbance value at OD450nm was read with a microplate reader. The raw data was imported into GraphPad Prism 6 software for nonlinear curve fitting to calculate EC50 values. The results are shown in Figure 1A, Figure 1B, Figure 1C and Table 4. 22B6D2-hIgG, 47A3E3-hIgG, and 100H7D3-hIgG all had good binding affinity to human and monkey proteins, and 22B6D2-hIgG and 47A3E3-hIgG essentially did not bind to rat proteins, while 100H7D3-hIgG had good binding affinity to rat proteins. [Table 6]

[0155] 4.3 Detection of dynamic affinity of anti-human HER3 fully human antibodies The dynamic affinity of the candidate antibodies, 22B6D2-hIgG, 47A3E3-hIgG, 100H7D3-hIgG and U1-59, to human and monkey HER3-His proteins was detected by ForteBio (Pall life sciences). The specific method is as follows. The antibody to be tested was diluted to 5 μg / ml in PBST (0.02% Tween-20), human HER3-His protein was serially diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, 0 nM, and monkey HER3-His protein was serially diluted to 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 0 nM. The antibody to be tested was then captured by a Protein A sensor (Pall life sciences) in PBST (0.02% Tween-20) solution for 60 seconds, then allowed to bind to the human or monkey HER3-His protein for 60 seconds, and then allowed to dissociate for 180 seconds. The measurement results were input into Data Analysis 11.0 software using 1:1 mode and global fitting, and the results were analyzed to obtain the association rate, dissociation rate and affinity constant. The results are shown in Table 5. [Table 7]

[0156] 4.4 Detection of cellular affinity of anti-human HER3 fully human antibodies The affinity of anti-human HER3 fully human antibodies to human breast cancer cells T47D (Kangnuotai), human gastric cancer cells NCI-N87 (ATCC), and human non-small cell lung cancer cells NCI-H358 (Nanjing Cobioer) was detected by a flow cytometer (Beckman, model Cytoflex).

[0157] Adherent cells were digested with StemPro Accutase (Gibco) solution, counted, appropriate amount of cells were harvested, washed twice with 1xPBS, resuspended in 1% BSA solution, then cells were transferred to 96-well V-bottom plate at 50μl per well, candidate antibodies were diluted in 1% BSA (starting from 10μg / ml or 15μg / ml or 5μg / ml or 1μg / ml, diluted in a 2-fold or 2.5-fold gradient), then 50μl of diluted antibodies were added to the V-bottom plate containing cells, incubated at 4℃ for 40 minutes, cells were washed twice with PBS, then 50μl of diluted secondary antibody was added to each well, mixed well, incubated at 4℃ for 30 minutes, cells were washed twice with PBS, then cells were resuspended in 400μl of PBS and detected by flow cytometry. Data processing: PE median values ​​were exported and then imported into GraphPad Prism 6 software to calculate EC50 values. The results are shown in Figures 2A, 2B, and 2C and Table 6. The EC50 values ​​of 22B6D2-hIgG, 47A3E3-hIgG6, and 100H7D3-hIgG were smaller than that of U1-59, indicating that their affinity for cells was higher than that of U1-59, with 22B6D2-hIgG having the highest affinity. [Table 8]

[0158] 4.5 Detection of ADCC activity of anti-human HER3 fully human antibodies The effector cells Jurkat-NFAT / luciferase-CD16a (genetically engineered Jurkat cells stably expressing CD16a and a luciferase reporter gene regulated by NFAT response elements obtained by lentivirus packaging, infection, and stress screening) can activate the expression of luciferase in the presence of antibodies and target cells, and can emit a fluorescent signal by using ONE-Glo reagent (Promega) as a substrate. The specific method is as follows. Jurkat-NFAT / luciferase-CD16a and CHO-S-hHER3 cells (genetically engineered CHO-S cells stably expressing human HER3 obtained by lentivirus packaging, infection, and stress screening) were collected by centrifugation, resuspended in RPMI 1640 + 1% FBS medium, and each was diluted with 2.0 × 10 6 / ml and 1.0 × 10 6 The antibody was diluted in RPMI 1640+1% FBS medium (starting from 10 μg / mL, 11 concentration points with 3-fold dilutions) and added to a 96-well plate at 50 μL / well, incubated at 37°C for 5 hours, then 20 μL of One-glo (Promega) detection reagent was added, shaken to mix, the fluorescence signal value was read by a microplate reader, and the results were imported into Graph Prism 6 software to calculate the EC50 value. The results are shown in Figure 3 and Table 7. The ADCC activity of 22B6D2-hIgG was significantly better than that of U1-59, with a 1.86-fold enhancement, while those of 47A3E3-hIgG and 100H7D3-hIgG were comparable to that of U1-59. [Table 9]

[0159] 4.6 Inhibition of ligand-induced AKT phosphorylation by fully human anti-HER3 antibodies Human breast cancer cells MCF-7 (ATCC) were digested with Tryple and counted, seeded at 30000 cells / well / 100μl in 96-well plates with growth medium, incubated overnight at 37℃, 5%CO2, starved by removing growth medium and replacing with 100μl / well serum-free medium, and removing medium after 24 hours. Antibodies were serially diluted in serum-free medium (11 concentration points in a 3-fold gradient starting from 30μg / ml) and added to the plates at 50μl / well and incubated in a 37℃ incubator for 1 hour. 40ng / ml NRG1 protein (Sino Biological) was added at 50μl / well and the cells were returned to a 37℃ incubator for stimulation for 20 minutes. After removing the liquid, 150μl / well of 4% paraformaldehyde was added for fixation at room temperature for 30 minutes. After removing the liquid, 150μl / well of absolute methanol was added for fixation at room temperature for 20 minutes. After removing the liquid, the cells were washed five times with PBST (0.2% Tween-20) and 200μl / well of 1% BSA was added for blocking at room temperature for 2 hours. Then, Akt-P antibody (Cell Signaling Technology) diluted 1:1000 in 1% BSA was added at 50 μl / well, and placed in a refrigerator at 4 °C overnight. The primary antibody was removed, and after washing three times with PBST (0.2% Tween-20), HRP goat anti-rabbit secondary antibody diluted 1:3000 in 1% BSA was added at 50 μl / well, and incubated at room temperature for 1 hour. The secondary antibody was removed, and washed five times with PBST (0.2% Tween-20), and an equal volume of HRP ECL Solution A and Solution B were mixed and equilibrated, and added at 100 μl / well, and the luminescence value was read by a microplate reader. The raw data was imported into Graph Prism 6 to calculate the EC50 value. The results are shown in Figure 4 and Table 8. 22B6D2-hIgG could effectively inhibit ligand-induced AKT phosphorylation. [Table 10]

[0160] 4.7 Detection of internalization activity of anti-human HER3 fully human antibody The internalization activity of anti-human HER3 fully human antibodies against human breast cancer cells MDA-MB-453 (Kangnuotai), human colorectal cancer cells WIDR (Nanjing Cobioer), human non-small cell lung cancer cells NCI-H358 (Nanjing Cobioer), and human pancreatic cancer cells HPAF-II (Nanjing Cobioer) was detected by flow cytometry (Beckman, model Cytoflex).

[0161] Adherent cells were digested with StemPro™ Accutase (Gibco) solution, counted, and an appropriate amount of cells was harvested, washed twice with 1xPBS, and resuspended in 1% BSA solution; cells were then transferred to a 96-well V-bottom plate at 50 μl / well; candidate antibodies were diluted in 1% BSA (starting at 10 μg / ml or 5 μg / ml, diluted in a 3-fold or 2.5-fold gradient); 50 μl of diluted antibodies were then added to the V-bottom plate containing cells and incubated at 4° C. for 40 minutes; cells were washed twice with PBS, followed by addition of 50 μl of diluted secondary antibody anti-human F(ab')2 Alexa Fluor 660 to each well, mixed well, and incubated at 4° C. for 30 minutes; cells were washed twice with PBS, centrifuged at 500 g for 3 minutes, and then resuspended in 150 μl of complete medium and incubated at 4° C. and 37° C. for 4 hours, respectively. The medium was removed by centrifugation at 500g for 3 minutes, and each well was resuspended in 100μl of acid (150mM NaCl, 0.1M glycine, adjusted to pH 2.0 with HCl), washed for 2 minutes, neutralized by adding 60μl of alkali (3g / L Tris base, 9g / L NaCl, adjusted to pH 13 with NaOH) and mixing, and the supernatant was removed by centrifugation, resuspended in PBS, and then detected. The APC median was exported and then imported into GraphPad Prism 6 software to calculate the EC50. The results are shown in Figure 5A, Figure 5B, Figure 5C, Figure 5D, and Table 9. The internalization activity of 22B6D2-hIgG was stronger than that of U1-59. [Table 11]

[0162] 4.8 Detection of binding domains of anti-human HER3 fully human antibodies Domain proteins of human HER3 D1+D2 (aa20-329), human HER3 D2 (aa185-329), human HER3 D3+D4 (aa330-643), and human HER3 D4 (aa496-643) were diluted with CBS coating solution to a final concentration of 1 μg / ml, coated on a 96-well ELISA plate at 100 μl / well, left to stand overnight at 4°C, washed once with PBS, added 100 μl of 2% BSA to each well, blocked at 37°C for 2 hours, removed the blocking solution, and diluted biotin-labeled antibodies with 2% BSA to 5 μg / mL, added to the ELISA plate at 100 μl / well, and then incubated at 4°C for 2 hours. After incubation at 7°C for 2 hours and washing three times with PBST (0.2% Tween-20), 100 μl of diluted peroxidase-conjugated streptavidin (Proteintech) was added to the corresponding wells and incubated at 37°C for 1 hour. The plate was then washed five times with PBST (0.2% Tween-20), dried, and 100 μl of TMB (InnoReagents) color substrate was added to each well and reacted at room temperature for 3 to 5 minutes. 50 μl of 2N H2SO4 was added to each well to stop the color reaction, and the absorbance value at OD450nm was read with a microplate reader. The raw data was imported into GraphPad Prism 6 software to generate histograms. The results are shown in Table 6. U1-59, 22B6D2-hIgG, and 47A3E3-hIgG mainly bound to the D1 domain of HER3, while 100H7D3-hIgG bound to both the D1 and D2 domains of HER3.

[0163] 4.9 Analysis of antigen-epitope binding by fully human anti-human HER3 antibodies 4.9.1 Elisa detection Human HER3 protein was diluted to 1 μg / ml in CBS coating solution, coated on a 96-well ELISA plate at 100 μl / well, and left to stand overnight at 4°C. The plate was washed once with PBS, 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, and the human antibody and control antibody were serially diluted with 2% BSA (starting from 30 μg / ml, 11 concentration points with a 3-fold gradient) and added to the ELISA plate at 50 μl / well. Then, biotin-labeled U1-59 (U1-59-Bio) was diluted to 50 ng / mL with 2% BSA, biotin-labeled 22B6D2-hIgG (22B6D2-hIgG-Bio) was diluted to 3 ng / mL with 2% BSA, and biotin-labeled 47A3E3-hIgG (47A3E3-hIgG-Bio) was diluted to 15 ng / mL with 2% BSA, and added to the corresponding wells at 50 μl / well. The serially diluted antibodies and biotin-labeled antibodies were thoroughly mixed and incubated at room temperature for 2 hours. The plate was washed three times with PBST (0.2% Tween-20), dried, 100 μl of diluted peroxidase-conjugated streptavidin secondary antibody (Proteintech) was added to each well and incubated at room temperature for 1 h, the plate was washed five times with PBST (0.2% Tween-20), dried, 100 μl of TMB (InnoReagents) color substrate was added to each well and incubated at room temperature for 3 to 5 min, 50 μl of 2N H2SO4 was added to each well to stop the color reaction, and the absorbance value at OD450nm was read with a microplate reader. The raw data was imported into GraphPad Prism 6 software and nonlinear curve fitting (4 parameters) was performed. The results are shown in Figure 7A, Figure 7B, and Figure 7C. The three antibodies, 22B6D2-hIgG, 47A3E3-hIgG and 100H7D3-hIgG, did not compete with each other, and the epitope of 47A3E3-hIgG was close to the epitope of U1-59.

[0164] 4.9.2 FACS detection Adherent cells were digested with StemPro™ Accutase (Gibco) solution, counted, and an appropriate amount of cells was harvested, washed once with 1x PBS, and resuspended in 1% BSA, then cells were transferred to a 96-well V-bottom plate at 50 μl per well, and candidate antibodies were diluted in 1% BSA (starting at 45 μg / ml, a total of 11 concentration points in a 3-fold gradient), and then 50 μl of diluted antibodies were added to the V-bottom plate containing cells, 22B6D2-hIgG-Bio at 75 ng / ml was diluted to a final concentration of 25 ng / ml, and 100H7D3-hIgG-Bio at 600 ng / ml was diluted to a final concentration of 25 ng / ml. After diluting the antibody to a final concentration of 200ng / ml and diluting U1-59-Bio at 750ng / ml to a final concentration of 250ng / ml), 50μl of the diluted antibody was added to the corresponding wells, and the added cells, serially diluted antibodies and a fixed concentration of biotin-labeled antibodies were mixed and incubated at 4℃ for 40 minutes, the cells were washed twice with PBS, and 50μl of the diluted PE streptavidin secondary antibody (Biolegend) was added to each well and incubated at 4℃ for 30 minutes, the cells were washed twice with PBS, and finally resuspended in 400μl PBS for detection by flow cytometry. Data processing: The PE median value was exported and then imported into GraphPad Prism 6 software for nonlinear curve fitting (4 parameters). The results are shown in Table 8. The three antibodies, 22B6D2-hIgG, 47A3E3-hIgG and 100H7D3-hIgG, did not compete with each other, and the epitope of 47A3E3-hIgG was close to the epitope of U1-59.

[0165] 4.10 Detection of specificity of anti-human HER3 fully human antibodies To detect binding of anti-human HER3 fully human antibodies to human EGFR (UniProt: P00533), human HER2 (UniProt: P04626), human HER3 (UniProt: P21860) and human HER4 (UniProt: Q15303) in the same family, human EGFR, human HER2, human HER3 and human HER4 proteins were diluted to 1 μg / ml in CBS coating solution and coated on a 96-well ELISA plate with 100 μl / well and left at 4°C overnight. The ELISA plate was washed once with PBS and dried, after which 100 μl of 2% BSA was added to each well and incubated for 37 h. After incubation at 37°C for 2 hours, the blocking solution was removed, and the human antibodies, positive control antibodies against human EGFR and positive control antibodies against human HER2 were each diluted to 10 μg / ml in 2% BSA, and the h.HER4 positive control protein Bio-NRG1-His was also diluted to 100 μg / ml, and then they were added to the ELISA plate at 100 μl / well and incubated at 37°C for 2 hours, the plate was washed three times with PBST (0.2% Tween-20), dried, and 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) and dried, and 100 μl of TMB (InnoReagents) color substrate was added to each well and reacted at room temperature for 3 to 5 minutes. Then, 50 μl of 2N H2SO4 was added to each well to stop the color reaction, and the absorbance value at OD450nm was read by a microplate reader. The raw data was imported into GraphPad Prism 6 software to create a histogram. The results are shown in Table 9. 22B6D2-hIgG, 47A3E3-hIgG and 100H7D3-hIgG all specifically bound to HER3 protein and essentially did not bind to other proteins of the same family.

[0166] Although the specific implementations of the present application have been described in detail, it is understood that those skilled in the art can make various modifications and substitutions to those details according to all the teachings disclosed, and all these modifications are within the scope of protection of the present application. The full scope of the present application is defined by the appended claims and all equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to HER3, comprising 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; (c) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 5, and / or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 6; (d) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 7, and / or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 8, or (e) comprising CDR-H1, CDR-H2 and CDR-H3 in a subsequent heavy chain variable region (VH) and / or CDR-L1, CDR-L2 and CDR-L3 in a subsequent light chain variable region (VL), wherein the heavy chain variable region (VH) and / or the light chain variable region (VL) have at least one CDR that contains a mutation compared to the corresponding heavy chain variable region and / or light chain variable region according to any one of (a) to (d), respectively, wherein the mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of one, two or three amino acids), preferably the substitution is a conservative substitution; Preferably, the CDRs are defined according to the IMGT, Kabat or Chothia numbering systems.

2. the antibody or antigen-binding fragment thereof (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system: (1a) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 9 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 10 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 11 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 18 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 19 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 20 or a variant thereof, (1b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 24 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 25 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 26 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 67 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 68 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 69 or a variant thereof, (1c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 37 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 38 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 39 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 32 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 33 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 34 or a variant thereof, or (1d) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 46 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 47 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 53 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 33 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 54 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 Kabat numbering system: (2a) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 12 or a variant thereof, CDR-H1 having the sequence set forth in SEQ ID NO: 13 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 14 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 21 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, (2b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 28 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 29 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 70 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 71 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 69 or a variant thereof; (2c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 40 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 41 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 42 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 35 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 34 or a variant thereof, or (2d) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 48 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 49 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 50 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 72 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 54 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 by the Chothia numbering system: (3a) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 15 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 16 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 17 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 21 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 22 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 23 or a variant thereof, (3b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence described in SEQ ID NO: 30 or a variant thereof, CDR-H2 having the sequence described in SEQ ID NO: 31 or a variant thereof, CDR-H3 having the sequence described in SEQ ID NO: 29 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence described in SEQ ID NO: 70 or a variant thereof, CDR-L2 having the sequence described in SEQ ID NO: 71 or a variant thereof, CDR-L3 having the sequence described in SEQ ID NO: 69 or a variant thereof, (3c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 43 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 44 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 42 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 35 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 34 or a variant thereof, or (3d) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 51 or a variant thereof, CDR-H2 having the sequence set forth in SEQ ID NO: 52 or a variant thereof, CDR-H3 having the sequence set forth in SEQ ID NO: 50 or a variant thereof, and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 72 or a variant thereof, CDR-L2 having the sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-L3 having the sequence set forth in SEQ ID NO: 54 or a variant thereof, The antibody or antigen-binding fragment thereof according to claim 1, wherein the variant according to any one of (1a) to (1d), (2a) to (2d), and (3a) to (3d) has one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, and preferably the substitutions are conservative substitutions.

3. the antibody or antigen-binding fragment thereof (4a) VH comprising the sequence set forth in SEQ ID NO: 1 or a variant thereof, and / or VL comprising the sequence set forth in SEQ ID NO: 2 or a variant thereof; (4b) VH comprising the sequence set forth in SEQ ID NO: 3 or a variant thereof, and / or VL comprising the sequence set forth in SEQ ID NO: 4 or a variant thereof; (4c) VH comprising the sequence set forth in SEQ ID NO: 5 or a variant thereof, and / or VL comprising the sequence set forth in SEQ ID NO: 6 or a variant thereof; or (4d) VH comprising a sequence as set forth in SEQ ID NO: 7 or a variant thereof, and / or VL comprising a sequence as set forth in SEQ ID NO: 8 or a variant thereof; 3. The antibody or antigen-binding fragment thereof of claim 1 or 2, 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 several amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived, preferably wherein 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 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), and preferably, the antibody or antigen-binding fragment thereof comprises a wild-type Fc region or a mutated or chemically modified Fc region having an altered effector function compared to the wild-type Fc region; Preferably, the antibody or antigen-binding fragment thereof has a light chain comprising a light chain constant region from or derived from a human immunoglobulin (e.g., kappa or lambda), Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) according to SEQ ID NO: 63 or a variant thereof, wherein the variant has up to 20 amino acid conservative substitutions (e.g. up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g. 1, 2, 3, 4 or 5 amino acid conservative substitutions) compared to SEQ ID NO: 63; Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) according to SEQ ID NO: 65 or a variant thereof, wherein the variant has up to 20 amino acid conservative substitutions (e.g. up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g. 1, 2, 3, 4 or 5 amino acid conservative substitutions) compared to SEQ ID NO: 65; More preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 63 and a light chain constant region (CL) set forth in SEQ ID NO:

65.

6. the antibody or antigen-binding fragment thereof (1) A heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 63, and / or a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO: 65; (2) A heavy chain comprising a VH as set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 63, and / or a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 65; (3) A heavy chain comprising a VH as set forth in SEQ ID NO: 5 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 63, and / or a light chain comprising a VL as set forth in SEQ ID NO: 6 and a light chain constant region (CL) as set forth in SEQ ID NO: 65; or (4) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising a heavy chain comprising a VH set forth in SEQ ID NO: 7 and a heavy chain constant region (CH) set forth in SEQ ID NO: 63, and / or a light chain comprising a VL set forth in SEQ ID NO: 8 and a light chain constant region (CL) set forth in SEQ ID NO:

65.

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 is selected from the group consisting of ScFv, Fab, Fab', Fab'-SH, (Fab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

8. 8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof carries a label, preferably the antibody or antigen-binding fragment thereof carries 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.

9. the antibody or antigen-binding fragment thereof (1) binding to HER3 (e.g., human or monkey HER3) 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 wherein the EC50 is determined by ELISA; (2) binding to HER3 (e.g., human or monkey HER3) 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 as determined by biolayer interferometry (BLI) (e.g., ForteBio Octet®); (3) does not bind or does not substantially bind to EGFR, HER2, and / or HER4, e.g., as determined by ELISA; (4) having ADCC activity, for example, inducing and killing HER3-expressing cells (e.g., tumor cells) via ADCC; (5) inhibiting HER3-mediated signaling, for example, inhibiting ligand-induced AKT phosphorylation and / or inhibiting PI3K / AKT signaling; (6) inducing HER3 internalization, e.g., as determined by flow cytometry; (7) inhibiting cell (e.g., tumor cell) proliferation, and / or (8) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, having a characteristic selected from the group consisting of: (1) inhibiting tumor growth.

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, its heavy chain and / or light chain, or its heavy chain variable region and / or light chain variable region according to any one of claims 1 to 9.

11. 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; (5a) the nucleic acid molecule encoding the antibody heavy chain variable region comprises (i) the nucleotide sequence set forth in SEQ ID NO:55, (ii) a sequence substantially identical to SEQ ID NO:55 (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:55), or (iii) a degenerate sequence of (i) or (ii); and / or the nucleic acid molecule encoding the antibody light chain variable region comprises (iv) the nucleotide sequence set forth in SEQ ID NO:56, (v) a sequence substantially identical to SEQ ID NO:56 (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:56), or (vi) a degenerate sequence of (iv) or (v); or (5b) the nucleic acid molecule encoding the antibody heavy chain variable region comprises (i) the nucleotide sequence set forth in SEQ ID NO:57, (ii) a sequence substantially identical to SEQ ID NO:57 (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:57), or (iii) a degenerate sequence of (i) or (ii); and / or the nucleic acid molecule encoding the antibody light chain variable region comprises (iv) the nucleotide sequence set forth in SEQ ID NO:58, (v) a sequence substantially identical to SEQ ID NO:58 (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:58), or (vi) a degenerate sequence of (iv) or (v); or (5c) the nucleic acid molecule encoding the antibody heavy chain variable region comprises (i) the nucleotide sequence set forth in SEQ ID NO:59, (ii) a sequence substantially identical to SEQ ID NO:59 (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:59), or (iii) a degenerate sequence of (i) or (ii), and / or the nucleic acid molecule encoding the antibody light chain variable region comprises (iv) the nucleotide sequence set forth in SEQ ID NO:60, (v) a sequence substantially identical to SEQ ID NO:60 (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:60), or (vi) a degenerate sequence of (iv) or (v); or (5d) The isolated nucleic acid molecule of claim 10, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises (i) the nucleotide sequence set forth in SEQ ID NO:61, (ii) a sequence substantially identical to SEQ ID NO:61 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity compared to SEQ ID NO:61, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii), and / or the nucleic acid molecule encoding the antibody light chain variable region comprises (iv) the nucleotide sequence set forth in SEQ ID NO:62, (v) a sequence substantially identical to SEQ ID NO:62 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity compared to SEQ ID NO:62, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v).

12. A vector comprising a nucleic acid molecule according to claim 10 or 11, preferably said vector being a cloning vector or an expression vector.

13. A host cell comprising a nucleic acid molecule according to claim 10 or 11 or a vector according to claim 12.

14. 13. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising culturing a host cell according to claim 13 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cells.

15. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a conjugate moiety linked thereto, Preferably, the conjugate moiety is selected from the group consisting of a detectable label (e.g., a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme) or a therapeutic agent (e.g., a cytotoxic agent, a cytokine, a toxin or a radionuclide).

16. A multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, Preferably, said multispecific antibody comprises an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 as a first antigen-binding domain and further comprises at least one second antigen-binding domain against another target, Preferably, said multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

17. A chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof (e.g., ScFv) according to any one of claims 1 to 9, a transmembrane domain, and one or more intracellular T cell signaling domains.

18. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or an isolated nucleic acid molecule according to claim 10 or 11, or a vector according to claim 12, or a host cell according to claim 13, or a conjugate according to claim 15, or a multispecific antibody according to claim 16, or a chimeric antigen receptor according to claim 17, or a host cell expressing said chimeric antigen receptor, and a pharma- ceutically acceptable carrier and / or excipient. Preferably, the pharmaceutical composition further comprises an additional pharma- ceutical active agent, Preferably, said additional pharma- ceutically active agent is a drug having antitumor activity; Preferably, said additional pharma- ceutically active agent is selected from the group consisting of 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 pharmaceutical composition, wherein said antibody or antigen-binding fragment thereof and said additional pharma- ceutically active agent are provided as separate or mixed components.

19. 19. A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the isolated nucleic acid molecule according to claim 10 or 11, or the vector according to claim 12, or the host cell according to claim 13, or the conjugate according to claim 15, or the multispecific antibody according to claim 16, or the chimeric antigen receptor according to claim 17, and or a host cell expressing said chimeric antigen receptor, or the pharmaceutical composition according to claim 18, optionally with instructions for use and / or an administration device.

20. 19. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or an isolated nucleic acid molecule according to claim 10 or 11, or a vector according to claim 12, or a host cell according to claim 13, or a conjugate according to claim 15, or a multispecific antibody according to claim 16, or a chimeric antigen receptor according to claim 17, or a host cell expressing said chimeric antigen receptor, or a pharmaceutical composition according to claim 18, in the manufacture of a medicament for inhibiting the proliferation of a cell (e.g. a cell expressing HER3 such as a tumor cell) or for the prevention and / or treatment and / or adjuvant treatment of a tumor, comprising: Preferably, the antibody or antigen-binding fragment thereof, the isolated nucleic acid molecule, the vector, the host cell, the conjugate, the multispecific antibody, or the pharmaceutical composition is administered in combination with an additional pharma- ceutically active agent, IMGT, Kabat, or Chothia numbering systems, e.g., simultaneously, separately, or sequentially; Preferably, said additional pharma- ceutically active agent is a drug having antitumor activity; Preferably, the additional pharma- ceutically active agent is selected from the group consisting of 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.

21. the tumor is a HER3 positive tumor; 21. The use according to claim 20, wherein the tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer (e.g. 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.

22. A method for inhibiting cell proliferation, comprising contacting said cell with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or an isolated nucleic acid molecule according to claim 10 or 11, or a vector according to claim 12, or a host cell according to claim 13, or a conjugate according to claim 15, or a multispecific antibody according to claim 16, or a chimeric antigen receptor according to claim 17, or a host cell expressing said chimeric antigen receptor, or a pharmaceutical composition according to claim 18, Preferably, the cell is a HER3 expressing cell, such as a tumor cell.

23. 19. A method for the prevention and / or treatment and / or adjuvant treatment of a tumor in a subject, comprising administering to said subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the isolated nucleic acid molecule according to claim 10 or 11, or the vector according to claim 12, or the host cell according to claim 13, or the conjugate according to claim 15, or the multispecific antibody according to claim 16, or the chimeric antigen receptor according to claim 17, or a host cell expressing said chimeric antigen receptor, or the pharmaceutical composition according to claim 18.

24. administering a second therapy to the subject, wherein the second therapy is selected from the group consisting of surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; 24. The method of claim 23, wherein optionally the second therapy may be applied simultaneously, separately, or sequentially with the method of claim 23.

25. the tumor is a HER3 positive tumor; 25. The method of claim 23 or 24, wherein the tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer (e.g. 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.

26. 10. A method for detecting the presence or level of HER3 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 under conditions allowing the formation of a complex between the antibody or antigen-binding fragment thereof and HER3, and detecting the formation of the complex; Preferably, the method is used for the diagnosis of a tumor, e.g. a HER3 positive tumor, such as breast cancer, gastric cancer, lung cancer (e.g. 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; Preferably, the method comprises detecting an expression level of HER3 in a test sample from the 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.

27. 17. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or an isolated nucleic acid molecule according to claim 10 or 11, or a vector according to claim 12, or a host cell according to claim 13, or a conjugate according to claim 15, or a multispecific antibody according to claim 16, in the manufacture of a kit, wherein said kit is used for detecting the presence or level of HER3 in a sample and / or for diagnosing a tumor, Preferably, the tumor is a HER3 positive tumor; Preferably, the tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer (e.g. 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.

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