Monoclonal antibody specifically binding to hegfr and bispecific antibody

Abstract

Provided are an antibody specifically binding to hEGFR or an antigen-binding fragment thereof, and a bispecific antibody, antibody-drug conjugate and pharmaceutical composition comprising the antibody. Further provided is the use of the antibody or the antigen-binding fragment thereof or the bispecific antibody.

Description

Monoclonal antibodies and bispecific antibodies that specifically bind to hEGFR Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an antibody or antigen-binding fragment thereof that specifically binds to hEGFR, and a bispecific antibody comprising the same. Background Art

[0002] EGFR (Epidermal Growth Factor Receptor, EGFR) is a member of the human epidermal growth factor receptor (HER) family, a receptor tyrosine kinase (RTK) that includes HER1 (EGFR, ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). EGFR is a single-pass transmembrane glycoprotein primarily composed of three regions: an extracellular domain that binds to ligands, a hydrophobic transmembrane domain for receptor dimerization, and an intracellular kinase active domain. The activation process of the EGFR signaling pathway is as follows: ligand binding induces conformational changes in EGFR, followed by homodimerization or heterodimerization of EGFR with family members, and phosphorylation of the intracellular kinase domain, thereby initiating signaling cascades that ultimately affect cellular function. EGFR is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, and other cells, where it regulates cell growth, proliferation, and differentiation. Studies have shown that EGFR is abnormally activated in many epithelial tumors, such as non-small cell lung cancer, breast cancer, colorectal cancer, head and neck cancer, and glioblastoma. EGFR overexpression, gene amplification, activating mutations, overexpression of ligands, and / or loss of EGFR activity regulators can all lead to EGFR dysfunction.

[0003] EGFR tyrosine kinase inhibitors (EGFR-TKI) mainly include small molecule compounds and antibodies. The antibodies that have been marketed include Cetuximab, Nimotuzumab, Panitumumab and Necitumumab. EGFR antibodies mainly compete with endogenous ligands to bind to EGFR, inhibiting dimerization between family members to achieve the effect of blocking signal transduction. Among them, Panitumumab is the only fully humanized monoclonal antibody with a strong affinity for EGFR, reaching 10 -11In 2006, the FDA approved panitumumab for the treatment of metastatic colorectal cancer after chemotherapy failure. However, clinical practice has shown that only a small percentage of patients benefit from antibody therapy. For example, in colorectal cancer, although 65% of patients have high EGFR expression, only approximately 10% experience a delay in disease progression. Furthermore, antibody drugs like panitumumab have significant skin toxicity, likely due to their high affinity, which limits the therapeutic population. Finally, even patients who benefit can develop resistance, further reducing the effectiveness of the antibody. EGFR-TKI resistance can be categorized as both EGFR-dependent and EGFR-independent. EGFR-independent resistance primarily involves activation of bypass signaling pathways, such as amplification or upregulation of genes such as HGF, c-MET, HER2, FGFR1, and IGF1R. c-MET gene amplification is the most common, accounting for 5% to 10% of cases of acquired EGFR-TKI resistance. c-MET is also a transmembrane receptor tyrosine kinase that can bypass EGFR and activate the PI3K / AKT signaling pathway. In addition, abnormal activation of EGFR downstream signaling pathways, such as KRAS mutations activating the MAPK signaling pathway, PI3KCA mutations activating the PI3K / AKT / mTOR pathway, and PTEN deficiency activating the PI3K / AKT signaling pathway, are also important mechanisms of EGFR-independent drug resistance. In view of this, many companies have carried out research and development of antibody drugs targeting bypass-activating proteins such as c-Met and HGF. However, many drugs, such as Onartuzumab (MetMab) and Tivatinib, have failed in clinical trials due to insufficient efficacy.

[0004] Therefore, there is an urgent need to introduce new drug mechanisms and modalities, such as bispecific antibodies, to expand the treatment population, broaden the therapeutic window, and improve drug safety and efficacy. The advantage of bispecific antibody drugs is that the affinity and binding epitope of each antibody arm can be fine-tuned based on antigen expression levels, bridging multiple antigens to achieve optimal biological activity. For example, bispecific antibodies that simultaneously block EGFR and other target signaling pathways that bypass activation are promising therapeutic approaches.

[0005] Summary of the Invention

[0006] The present invention provides a modified hEGFR antibody and its antigen-binding fragment, as well as a bispecific antibody comprising the hEGFR antibody and its antigen-binding fragment. Furthermore, the present invention also provides uses of the antibody or its antigen-binding fragment or the bispecific antibody.

[0007] Specifically, the present invention relates to the following aspects:

[0008] In a first aspect, an antibody or antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor (hEGFR), wherein the antibody or antigen-binding fragment comprises:

[0009] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:3, respectively, according to the Kabat sequence numbering system, or a variant thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, according to the Kabat sequence numbering system, or a variant thereof; or

[0010] (a2) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:3, respectively, according to the Kabat sequence numbering system, or a variant thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, according to the Kabat sequence numbering system, or a variant thereof; or

[0011] (a3) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:9 and SEQ ID NO:3, respectively, according to the Kabat sequence numbering system, or a variant thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, according to the Kabat sequence numbering system, or a variant thereof; or

[0012] (a4) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:10 and SEQ ID NO:3, respectively, according to the Kabat sequence numbering system, or a variant thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, according to the Kabat sequence numbering system, or a variant thereof; or

[0013] (a5) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:11 and SEQ ID NO:3, respectively, according to the Kabat sequence numbering system, or a variant thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, according to the Kabat sequence numbering system, or a variant thereof; or

[0014] (a6) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:12, respectively, according to the Kabat sequence numbering system, or a variant thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, according to the Kabat sequence numbering system, or a variant thereof; or

[0015] (a7) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:13, respectively, according to the Kabat sequence numbering system, or a variant thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, according to the Kabat sequence numbering system, or a variant thereof; or

[0016] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, according to the Kabat sequence numbering system, or a variant thereof, and (b2) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO: 14, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, according to the Kabat sequence numbering system, or a variant thereof; or

[0017] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, according to the Kabat sequence numbering system, or a variant thereof, and (b3) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:15, respectively, according to the Kabat sequence numbering system, or a variant thereof; or

[0018] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, according to the Kabat sequence numbering system, or variants thereof, and (b4) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 16, respectively, according to the Kabat sequence numbering system, or variants thereof; or

[0019] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, according to the Kabat sequence numbering system, or variants thereof, and (b5) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 17, respectively, according to the Kabat sequence numbering system, or variants thereof;

[0020] wherein the variants of the CDRs have 3, 2 or 1 amino acid differences with the corresponding CDRs, respectively, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively.

[0021] In one embodiment, the aforementioned amino acid differences include amino acid substitutions, deletions or additions; preferably, the substitutions are conservative substitutions.

[0022] In one embodiment, the antibody or antigen-binding fragment thereof as described above, wherein the VH and VL are selected from the following groups or variants thereof:

[0023] (a) a heavy chain having a variable domain comprising an amino acid sequence selected from any one of SEQ ID NOs: 20-26 and a light chain having a variable domain comprising an amino acid sequence as shown in SEQ ID NO: 19; or

[0024] (b) a heavy chain having a variable domain comprising the amino acid sequence shown in SEQ ID NO: 18 and a light chain having a variable domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 27-30;

[0025] wherein the variants have 3, 2 or 1 amino acid differences, respectively, or are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding variable domain.

[0026] In one embodiment, the aforementioned amino acid differences include amino acid substitutions, deletions or additions; preferably, the substitutions are conservative substitutions.

[0027] In one embodiment, the antibody or antigen-binding fragment thereof as described above, wherein the antibody comprises a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a chimeric antibody, a humanized antibody or a fully human antibody; and / or the antigen-binding fragment comprises a Fab, Fab', Fv fragment, F(ab')2, Fd fragment, dAb, a complementarity determining region fragment, scFv or a single domain antibody.

[0028] In one embodiment, the antigen-binding fragment as described above is a scFv, which comprises a light chain variable region, a connecting fragment and a heavy chain variable region in sequence from N-terminus to C-terminus.

[0029] In one embodiment, the aforementioned connecting fragment is selected from (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, and n is selected from 2, 3, and 4; preferably, the connecting fragment comprises the amino acid sequence shown in SEQ ID NO:78.

[0030] In one embodiment, the antigen-binding fragment as described above is a scFv, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 32-42 or a variant thereof, wherein the variant has 3, 2 or 1 amino acid differences with the scFv or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, respectively.

[0031] In one embodiment, the aforementioned amino acid differences include amino acid substitutions, deletions or additions; preferably, the substitutions are conservative substitutions.

[0032] In one embodiment, the aforementioned scFv further comprises point mutations H44C and L100C to form a disulfide bond.

[0033] In one embodiment, the maximum effect (E max ) is lower than the maximum effect (E max A non-limiting example of the EGFR-expressing cell line is the A431 cell line.

[0034] In one embodiment, the antibody or antigen-binding fragment thereof as described above has an IC higher than 2.10 nM. 50 The value inhibits the binding of panitumumab to EGFR-expressing cell lines, such as the A431 cell line.

[0035] In another aspect, the present invention also provides a multispecific antibody comprising multiple different antigen-binding domains, wherein the first antigen-binding domain comprises the aforementioned antibody or antigen-binding fragment thereof.

[0036] On the other hand, the present invention also provides a bispecific antibody containing two different antigen-binding domains, wherein the first antigen-binding domain comprises the above-mentioned antibody or its antigen-binding fragment, and the second antigen-binding domain comprises an antibody or its antigen-binding fragment that specifically binds to a tumor-associated antigen (TAA).

[0037] In one embodiment, the tumor-associated antigen as described above is selected from one of the following groups: c-MET, AFP, ALK, BAGE protein, BIRC5, BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20, CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFRvIII, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-ra f, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein, MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16, MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA, RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Tang-Novo antigen, TRP-1, TRP-2, tyrosinase, and urokinin-3.

[0038] In one embodiment, the second antigen binding domain as described above specifically binds c-MET.

[0039] In one embodiment, the second antigen binding domain that specifically binds to c-MET as described above comprises:

[0040] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively, according to the Kabat sequence numbering system, or variants thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences of SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, respectively, according to the Kabat sequence numbering system, or variants thereof; or

[0041] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively, according to the Kabat sequence numbering system, or variants thereof, and (b2) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences of SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:49, respectively, according to the Kabat sequence numbering system, or variants thereof; or

[0042] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively, according to the Kabat sequence numbering system, or variants thereof, and (b3) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences of SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:50, respectively, according to the Kabat sequence numbering system, or variants thereof; or

[0043] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively, according to the Kabat sequence numbering system, or variants thereof, and (b4) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences of SEQ ID NO:51, SEQ ID NO:47 and SEQ ID NO:48, respectively, according to the Kabat sequence numbering system, or variants thereof; or

[0044] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively, according to the Kabat sequence numbering system, or variants thereof, and (b5) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences of SEQ ID NO:52, SEQ ID NO:47 and SEQ ID NO:48, respectively, according to the Kabat sequence numbering system, or variants thereof; or

[0045] (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3, comprising the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively, according to the Kabat sequence numbering system, or variants thereof, and (b6) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3, comprising the amino acid sequences of SEQ ID NO:53, SEQ ID NO:47 and SEQ ID NO:48, respectively, according to the Kabat sequence numbering system, or variants thereof;

[0046] wherein the variants of the variant CDRs have 3, 2 or 1 amino acid differences, respectively, or are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding CDRs, respectively.

[0047] In one embodiment, the aforementioned amino acid differences include amino acid substitutions, deletions or additions; preferably, the substitutions are conservative substitutions.

[0048] In one embodiment, the second antigen binding domain that specifically binds to c-MET as described above has:

[0049] 1) a heavy chain comprising a variable domain having the amino acid sequence shown in SEQ ID NO: 54, or a variant thereof; and

[0050] 2) a light chain comprising a variable domain selected from the group consisting of an amino acid sequence shown in any one of SEQ ID NOs: 55-60, or a variant thereof;

[0051] wherein the variants have 3, 2 or 1 amino acid differences, respectively, or are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding variable domain.

[0052] In one embodiment, the aforementioned amino acid differences include amino acid substitutions, deletions or additions; preferably, the substitutions are conservative substitutions.

[0053] In one embodiment, the second antigen binding domain as described above further comprises a first heavy chain constant region (CH1) and a light chain constant region (CL).

[0054] In one embodiment, the CH1 as described above comprises the amino acid sequence shown in SEQ ID NO: 75, and the CL comprises the amino acid sequence shown in SEQ ID NO: 76 or 77.

[0055] In one embodiment, the second antigen-binding domain as described above consists of a light chain-heavy chain pair, the light chain sequence comprises the amino acid sequence shown in any one of SEQ ID NOs: 63-67, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 61.

[0056] In one embodiment, the second antigen binding domain as described above comprises a Fab, Fab', Fv fragment, F(ab')2, Fd fragment, dAb, complementarity determining region fragment, scFv or single domain antibody format.

[0057] In one embodiment, the bispecific antibody as described above further comprises an Fc domain.

[0058] In one embodiment, the Fc domain as described above is selected from IgA Fc fragment, IgG Fc fragment, IgM Fc fragment, IgD Fc fragment, IgE Fc fragment; the IgG Fc fragment is preferably IgG1 Fc fragment, IgG2 Fc fragment, IgG3 Fc fragment or IgG4 Fc fragment; the Fc domain is preferably IgG1 Fc fragment, whose amino acid sequence is shown in SEQ ID NO:79.

[0059] In one embodiment, the aforementioned Fc domain further comprises an amino acid substitution that promotes pairing between the first subunit and the second subunit of the Fc domain.

[0060] In one embodiment, the first Fc domain subunit as described above comprises the following amino acid substitutions: Y349C / T366S / L368A / Y407V, and the second Fc domain subunit comprises the following amino acid substitutions: S354C / T366W.

[0061] In one embodiment, the N-terminus of the Fc domain as described above is fused to the C-terminus of the first antigen-binding domain, and / or the N-terminus of the Fc domain is fused to the C-terminus of the heavy chain of the second antigen-binding domain.

[0062] In one embodiment, the bispecific antibody as described above is selected from the following group or variants thereof:

[0063] (a) a first antigen-binding domain comprising HCDR1-3 of the sequences shown in SEQ ID NOs: 1-3 and LCDR1-3 of the sequences shown in SEQ ID NOs: 4-6; or

[0064] (b) a first antigen-binding domain comprising HCDR1-3 of the sequences shown in SEQ ID NOs: 8, 2, and 3 and LCDR1-3 of the sequences shown in SEQ ID NOs: 4-6; or

[0065] (c) a first antigen-binding domain comprising HCDR1-3 of the sequences shown in SEQ ID NOs: 1-3 and LCDR1-3 of the sequences shown in SEQ ID NOs: 4, 5, and 15; or

[0066] (d) a first antigen-binding domain comprising HCDR1-3 of SEQ ID NOs: 7, 2, and 3 and LCDR1-3 of SEQ ID NOs: 4, 5, and 15; or

[0067] (e) a first antigen-binding domain comprising HCDR1-3 of the sequences shown in SEQ ID NOs: 1, 10, and 3 and LCDR1-3 of the sequences shown in SEQ ID NOs: 4-6; or

[0068] (f) a first antigen-binding domain comprising HCDR1-3 of the sequences shown in SEQ ID NOs: 1, 2, and 12 and LCDR1-3 of the sequences shown in SEQ ID NOs: 4-6;

[0069] and a second antigen-binding domain comprising HCDR1-3 of the sequences shown in SEQ ID NOs: 43-45 and LCDR1-3 of the sequences shown in SEQ ID NOs: 46-48;

[0070] wherein the variants have 3, 2 or 1 amino acid differences, respectively, or are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding CDRs, respectively.

[0071] In one embodiment, the aforementioned amino acid differences include amino acid substitutions, deletions or additions; preferably, the substitutions are conservative substitutions.

[0072] In one embodiment, the bispecific antibody as described above is selected from the following group or variants thereof:

[0073] (a) a first antigen-binding domain comprising a VH sequence as shown in SEQ ID NO: 18 and a VL sequence as shown in SEQ ID NO: 19; or

[0074] (b) a first antigen-binding domain comprising a VH sequence as shown in SEQ ID NO: 21 and a VL sequence as shown in SEQ ID NO: 19; or

[0075] (c) a first antigen-binding domain comprising a VH sequence as shown in SEQ ID NO: 18 and a VL sequence as shown in SEQ ID NO: 28; or

[0076] (d) a first antigen-binding domain comprising a VH sequence as shown in SEQ ID NO: 20 and a VL sequence as shown in SEQ ID NO: 28; or

[0077] (e) a first antigen-binding domain comprising a VH sequence as shown in SEQ ID NO: 23 and a VL sequence as shown in SEQ ID NO: 19; or

[0078] (f) a first antigen-binding domain comprising a VH sequence as shown in SEQ ID NO: 25 and a VL sequence as shown in SEQ ID NO: 19;

[0079] and a second antigen-binding domain comprising a VH having a sequence as shown in SEQ ID NO: 54 and a VL having a sequence as shown in SEQ ID NO: 55;

[0080] wherein the variants have 3, 2 or 1 amino acid differences, respectively, or are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding variable domain.

[0081] In one embodiment, the aforementioned amino acid differences include amino acid substitutions, deletions or additions; preferably, the substitutions are conservative substitutions.

[0082] In one embodiment, the bispecific antibody as described above is composed of a first heavy chain comprising a first antigen-binding domain, a second heavy chain comprising a second antigen-binding domain, and a first light chain; wherein the first heavy chain is selected from the amino acid sequence shown in any one of SEQ ID NOs: 69-74 or a variant thereof, the second heavy chain is the sequence shown in SEQ ID NO: 68 or a variant thereof, and the first light chain is the sequence shown in SEQ ID NO: 62 or a variant thereof;

[0083] wherein the variants have 3, 2 or 1 amino acid differences, respectively, or are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding sequence, respectively.

[0084] In one embodiment, the aforementioned amino acid differences include amino acid substitutions, deletions or additions; preferably, the substitutions are conservative substitutions.

[0085] In one embodiment, the bispecific antibody described above can bind to EGFR and c-MET simultaneously with an EC lower than 10 nM. 50 The value was combined with EGFR and c-MET co-expressing cell lines, such as H1975 or H441 cell lines.

[0086] In one embodiment, the bispecific antibody described above has an EC higher than 50 nM. 50 The value is combined with EGFR high-expressing cell lines, such as A431 cell line or keratinocytes.

[0087] In one embodiment, the aforementioned bispecific antibody is preferentially internalized by a cell line that co-expresses EGFR and c-MET and a cell line that overexpresses EGFR. For example, when H1975 cells and keratinocytes are co-present, the bispecific antibody is preferentially internalized by the H1975 cells. This demonstrates that the bispecific antibody exhibits significant selectivity in in vitro cytotoxicity assays.

[0088] In another aspect, the present invention also provides a nucleic acid molecule encoding the aforementioned antibody specifically binding to human epidermal growth factor receptor (hEGFR) or its antigen-binding fragment or the aforementioned bispecific antibody.

[0089] In another aspect, the present invention also provides an expression vector comprising the nucleic acid molecule as described above.

[0090] In another aspect, the present invention also provides a host cell containing the nucleic acid molecule or the expression vector as described above, or expressing the antibody or antigen-binding fragment thereof as described above, the multispecific antibody as described above, or the bispecific antibody as described above.

[0091] On the other hand, the present invention also provides an antibody-drug conjugate (ADC), which is composed of an antibody, a linker, and a payload, wherein the antibody is an antibody or an antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor (hEGFR) as described above, or a multispecific antibody as described above, or a bispecific antibody as described above.

[0092] On the other hand, the present invention also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor (hEGFR) as described above, or a multispecific antibody as described above, or a bispecific antibody as described above, or an antibody-drug conjugate as described above, and at least one pharmaceutically acceptable carrier and / or excipient.

[0093] On the other hand, the present invention also provides a method for inhibiting tumor cell growth and / or killing the tumor cells, which comprises contacting the tumor cells with an effective amount of the antibody or antigen-binding fragment thereof as described above, or the multispecific antibody as described above, or the bispecific antibody as described above, or the antibody-drug conjugate as described above, or the pharmaceutical composition as described above.

[0094] On the other hand, the present invention also provides the use of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned multispecific antibody, the above-mentioned bispecific antibody, the above-mentioned antibody-drug conjugate, or the above-mentioned pharmaceutical composition in the preparation of tumor therapeutic drugs.

[0095] In one embodiment, according to the above use, the tumor is selected from pancreatic cancer, melanoma, glioblastoma, head and neck cancer, prostate cancer, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, renal oncocytoma, renal transitional cell carcinoma, urothelial carcinoma, adenocarcinoma or small cell carcinoma.

[0096] In another aspect, the present invention also provides a method for treating a tumor in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof as described above, or the multispecific antibody or bispecific antibody as described above, or the antibody-drug conjugate as described above, or the pharmaceutical composition as described above.

[0097] In one embodiment, the tumor is selected from pancreatic cancer, melanoma, glioblastoma, head and neck cancer, prostate cancer, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, renal oncocytoma, renal transitional cell carcinoma, urothelial carcinoma, adenocarcinoma, or small cell carcinoma.

[0098] In one embodiment, the subject is a mammal, preferably a human.

[0099] In one embodiment, the method further comprises the simultaneous administration of other drugs with anti-tumor activity, including but not limited to alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.

[0100] In one embodiment, the method further comprises administering an additional anti-tumor therapy including, but not limited to, surgery, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0101] The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to hEGFR, as well as a bispecific antibody, antibody-drug conjugate, and pharmaceutical composition comprising the antibody. Furthermore, the present application also provides applications of the antibody or antigen-binding fragment thereof or the bispecific antibody. Compared with wild-type antibodies, the bispecific antibodies provided by the present invention can promote the endocytosis and degradation of EGFR and c-MET antigens and inhibit the conduction of EGFR downstream signals. It should be particularly noted that the bispecific antibodies provided by the present invention have selective activity against cell lines with different EGFR and c-MET expression levels, and have good activity immunity against human keratinocytes with high EGFR expression but low c-MET expression. They can overcome the problems of skin side effects and ocular toxicity caused by common clinical EGFR-targeted drugs, and have potential clinical safety advantages. Therefore, the bispecific antibodies provided by the present application have great clinical value.

[0102] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0103] In the present invention, unless otherwise indicated, the terms involved herein have conventional meanings understood by those skilled in the art. Where a term has two or more definitions, as used and / or accepted in the art, the definitions of the terms used herein are intended to include all of them. The procedures used herein in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA are conventional procedures widely used in the corresponding fields. For a better understanding of the present invention, definitions and explanations of the relevant terms are provided below.

[0104] Those of ordinary skill in the art will appreciate that the CDR district of an antibody is responsible for the binding specificity of the antibody to the antigen. In the case of known antibody heavy chain and light chain variable region sequences, there are currently several methods for determining the CDR district of an antibody, including Kabat, IMGT, Chothia, and AbM numbering systems. However, the application of the definition of the CDR of an antibody or its variants will fall within the scope of the term defined and used herein. If the variable region amino acid sequence of the given antibody is given, those skilled in the art can determine specific CDRs generally without relying on any experimental data outside the sequence itself.

[0105] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules, including monoclonal or polyclonal human, humanized, composite and chimeric antibodies, as well as antibody fragments. Therefore, the term "antibody" includes any protein or peptide containing a specific molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of this include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or its ligand-binding portion, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein. In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art. The immunoglobulin molecules or antibody molecules of the present application can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0106] As used herein, "antibody fragments" or "antigen-binding fragments" include, but are not limited to, F(ab')2, Fab', Fab, Fv, Fd, dAb, Fab / c, complementarity determining region (CDR) fragments, single-chain Fvs (ScFv), disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv), diabodies, disulfide-stabilized diabodies (ds-Diabodies), ScFv multimers (e.g., ScFv dimers, ScFv trimers), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, nanobodies, single domain antibodies (sdAbs), domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. Regardless of the structure, antigen-binding fragments include any polypeptide or polypeptide complex that is capable of binding to the same antigen as the parent antibody or parent antibody fragment. As used herein, "antibody fragments" include aptamers, aptamer enantiomers (spiegelmers), and diabodies. As used herein, "antibody fragments" also include any synthetic or genetically modified proteins that, like antibodies, can bind to a specific antigen to form a complex. Typically, an antibody fragment has at least about 50 consecutive amino acids of an antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0107] As used herein, "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0108] As used herein, "Fv" 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 the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0109] As used herein, "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0110] As used herein, "single-chain variable fragment" or "ScFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In certain aspects, these regions are connected by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and also contain serine or threonine for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, and vice versa. The protein retains the properties of the original immunoglobulin, except that the constant region has been removed and a linker has been introduced. ScFv molecules are known in the art. In some cases, a disulfide bond can also be present between the VH and VL of the scFv.

[0111] As used herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody. Single-domain antibodies are also known as nanobodies.

[0112] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0113] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0114] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0115] As used herein, "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen. In certain embodiments, the term "chimeric antibody" may include an antibody in which the heavy and light chain variable regions of the antibody are derived from a first antibody, and the heavy and light chain constant regions of the antibody are derived from a second antibody.

[0116] As used herein, "bispecific antibody" or "bi-antibody" refers to a conjugate formed by a first antibody (or fragment thereof) and a second antibody (or fragment thereof) or antibody analog via a coupling arm, wherein the coupling method includes but is not limited to chemical reaction, gene fusion, and enzymatic reaction. Bispecific antibodies can be linked or produced by various methods, for example, see the method of Songsivilai et al. (Clin. Exp. Immunol., 79: 315-321 (1990)), and the method of Kostelny et al. (J. Immunol., 148: 1547-1553 (1992)).

[0117] The antigen-binding domain of the antibodies of the present invention is Fab, or ScFv, or a non-covalent pairing of a heavy chain variable region (VH) and a light chain variable region (VL) (Fv). Any of the above antibodies or polypeptides may also include additional polypeptides, such as a signal peptide at the N-terminus of the antibody for directing secretion, or a 6×His tag for purification. The present invention encompasses not only intact antibodies but also immunologically active antibody fragments or fusion proteins formed by antibodies with other sequences.

[0118] The present invention also includes fragments, variants, derivatives and analogs of the antibodies. The antibodies, antigen-binding fragments, variants or derivatives of the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, etc.), human antibodies, animal-derived antibodies, humanized antibodies, primatized antibodies, or chimeric antibodies, CDR-grafted and / or modified antibodies, single-chain antibodies (e.g., ScFv), double-chain antibodies, epitope-binding fragments, such as Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (ScFv), single-chain antibodies, disulfide-linked Fv (dsFv), fragments comprising a VL domain or a VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. The antibody fragments, antigen-binding fragments, derivatives or analogs of the present invention can also be polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted.

[0119] Amino acid numbering follows the Kabat numbering system. "Kabat numbering" refers to the numbering system described by Kabat et al., as described in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0120] Antibodies can be modified to improve heterodimer pairing efficiency. For example, in certain aspects, the Fc fragment of the heavy chain of the monovalent unit and / or the Fc fragment of the fusion peptide can contain one or more substitutions compared to a wild-type antibody fragment that form a knob-in-hole pair. Knob-in-hole configurations are known in the art.

[0121] As used herein, "identity" refers to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, a gap can be introduced in the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0122] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0123] As used herein, "variant", in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by the introduction of amino acid residue substitutions, deletions or additions. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limiting, a polypeptide can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0124] As used herein, "conservative substitutions" means amino acid substitutions that do not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art.

[0125] The “E max " or "maximum effect" means that when the drug dose or concentration is increased within a certain range, the intensity of its effect increases accordingly. However, when the effect reaches its maximum, further increasing the dose or concentration will no longer increase the effect. This limit of pharmacological effect is called the maximum effect. max It reflects the intrinsic activity of the drug and is the ability of the drug to produce an effect.

[0126] The “EC 50 " or "half-effective concentration" refers to the drug dose that causes 50% of the maximum effect. It reflects the affinity of the drug to the target.

[0127] As used herein, "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0128] As used herein, "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0129] It should be noted that the definition of an entity without a clear number should refer to one or more (kinds) of the entity; for example, "multifunctional antibody" should be understood to mean one or more (kinds) of multifunctional antibodies. Similarly, the terms "one or more" and "at least one" without a clear number of definitions are used interchangeably herein.

[0130] As used herein, a "pharmaceutical composition" is a product comprising one or more active ingredients (e.g., antibodies, small molecule drugs) in optionally specific amounts, as well as any product produced directly or indirectly by combining one or more active ingredients in optionally specific amounts. The different active ingredients in the pharmaceutical composition can be administered independently in separate formulations, including administration simultaneously or at different time points to enhance synergy. In the present disclosure, "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0131] As used herein, "pharmaceutically acceptable carriers and / or excipients" refer to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, and are well known in the art.

[0132] As used herein, "treat" refers to both therapeutic treatment and prophylactic or preventative measures, in which an undesirable physiological change or disease, such as cancer, is prevented or slowed (mitigated) in a subject. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (e.g., preventing it from worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether or not detectable. "Treatment" may also refer to prolonging survival compared to the expected survival if not receiving treatment. Conditions in need of treatment include those already having the condition or symptom as well as those susceptible to having the condition or symptom or those in which the condition or symptom is to be prevented.

[0133] As used herein, "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domestic animals, farm animals, zoos, sports farms, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, rats, horses, cattle, cows, primates (e.g., humans, monkeys such as cynomolgus monkeys, macaques, baboons, and chimpanzees, etc.), and the like.

[0134] As used herein, an "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactic amount is an amount sufficient to prevent, arrest, or delay the onset of the disease; a therapeutic amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the method of administration of the drug, and any other concurrently administered therapies.

[0135] As described herein, the antigen-binding polypeptides, variants or derivatives of the present application can be used in certain treatments and diagnostic methods related to cancer or infectious diseases. The application also relates to antibody-based treatments, which include administering the bispecific antibodies of the present application to patients, such as animals, mammals and humans, for the treatment of one or more diseases or conditions described herein. The therapeutic drugs of the present application include, but are not limited to, antibodies of the present application (including their variants and derivatives as described herein) and nucleic acids or polynucleotides encoding antibodies of the present application (including their variants and derivatives as described herein). The antibodies of the present application can also be used to treat, suppress or prevent diseases, disorders or conditions, including malignant diseases, disorders, or conditions related to such diseases or disorders, such as diseases related to immune responses. In some embodiments, the antibodies of the present invention can be used as immunosuppressants. In some embodiments, the antibodies of the present invention can be used to treat autoimmune diseases. The antigen-binding polypeptides of the present application, their variants or derivatives are used to suppress the growth, development and / or metastasis of cancer, particularly those listed above or listed in the following paragraphs.

[0136] The antibodies of the present application or their variants or derivatives can be used to treat, prevent, diagnose and / or predict other diseases or conditions associated with increased cell survival, including but not limited to cancer or tumors, including the development and / or metastasis of malignant tumors, and related diseases (such as malignant ascites, malignant pleural effusion, malignant effusion), such as EGFR-positive tumors.

[0137] The method of administering antibody, its variant or derivative includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral route.The antibody or composition can be administered by any convenient route, for example, by infusion or bolus injection, absorbed by epithelium or mucosa and skin inner layer (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents.Therefore, the pharmaceutical composition containing antibody of the present application can be administered orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneally, topically (such as through powder, ointment, drops or transdermal patch), buccal administration or as oral or nasal spray.Term " parenteral " used herein refers to the mode of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.Administration can be systemic or topical. It may also be desirable to administer the antigen-binding polypeptides or compositions of the present invention locally to the area in need of treatment, which can be achieved by, for example, but not limited to, local infusion during surgery, topical application, such as in conjunction with a postoperative wound dressing, by injection, by catheter, by suppository, or by implant, wherein the implant is a porous, non-porous, or gel-like material, including membranes or fibers. Preferably, when administering the proteins (including antibodies) of the present invention, care must be taken to use materials that do not absorb the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1: Schematic diagram of an exemplary anti-EGFR and c-METs bispecific antibody. Figure 1A shows the "F(ab')2 fragment" format expressed in a cell-free system. Figure 1B shows the structural format of a bispecific antibody expressed in a eukaryotic system.

[0139] Figure 2: Determination of the binding activity of EGFR mutant antibodies to A431 cells.

[0140] Figure 3: Flow cytometry analysis of the competitive binding activity between EGFR mutant antibodies and parental antibodies.

[0141] FIG4 : Flow cytometry detection of the binding activity of bispecific F(ab')2 fragments to H1975 cell line.

[0142] FIG5 : Flow cytometry detection of the binding activity of bispecific F(ab')2 fragments to H441 cell lines.

[0143] FIG6 : Flow cytometry detection of the binding activity of bispecific F(ab')2 fragments to A431 cell lines.

[0144] Figure 7: ELISA bridging assay to detect the simultaneous binding ability of bispecific F(ab')2 fragments to EGFR and c-MET.

[0145] FIG8 : Fluorescence labeling method is used to detect the activity of bispecific F(ab')2 fragments in inducing target endocytosis in H1975 cell line.

[0146] FIG9 : Fluorescence labeling method is used to detect the activity of bispecific F(ab')2 fragments in inducing target endocytosis in H441 cell lines.

[0147] FIG10 : Fluorescence labeling method is used to detect the activity of bispecific F(ab')2 fragments in inducing target endocytosis in A431 cell lines.

[0148] Figure 11: Flow cytometry analysis of the affinity activity of the bispecific antibodies against H1975, H441, and A431 cell lines. Figures 11A-B are for the H1975 cell line, Figure 11C is for the H441 cell line, and Figure 11D is for the A431 cell line.

[0149] Figure 12: Fluorescence labeling method was used to detect the activity of bispecific antibodies in inducing target endocytosis in different tumor cell lines.

[0150] Figure 13: Fluorescence labeling method was used to detect the activity of the dual antibody in inducing endocytosis in human primary keratinocyte cell lines.

[0151] Figure 14: αFc-MMAE toxin secondary antibody assay to detect the activity of dual antibodies in inducing target endocytosis in different tumor cell lines.

[0152] Figure 15: DT3C toxin killing assay to detect the activity of bispecific antibodies in inducing target endocytosis in different tumor cell lines.

[0153] Figure 16: ELISA method was used to detect the activity of the dual antibody in reducing the expression of EGFR and c-MET proteins in H1975 cells.

[0154] Figure 17: Western blot detection of the inhibitory activity of the dual antibody on downstream pERK and pAKT signals in EGF-activated H1975 cells.

[0155] Figure 18: Detection of the selective killing activity of the bispecific antibodies against A431 cells and H1975 cells in mixed culture. DETAILED DESCRIPTION

[0156] The methods and applications of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or replacements can also be made, all of which should be deemed to fall within the scope of protection of the present invention. Unless otherwise specified, the experiments and methods described in the embodiments are basically carried out according to conventional methods well known in the art and described in various references. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0157] Example 1: Affinity modification by antibody sequence mutation

[0158] The structure of the panitumumab:EGFR complex (PDB:5XS4) was analyzed, and the PISA program was used to calculate the amino acids involved in the antigen-antibody interaction. The amino acid residues on the panitumumab CDR involved in EGFR binding were counted. The amino acid residues that provide hydrogen bonds, salt bridges, and other interactions were defined as key amino acid residues. Mutating these key amino acid residues to alanine resulted in antibodies with reduced affinity, as detailed in Table 1. Similarly, the structure of the onartuzumab:c-MET complex (PDB:4K3J) was analyzed, and the PISA program was used to calculate the key amino acid residues involved in the antigen-antibody interaction. Based on these statistical results, several antibodies with varying affinities were designed, as detailed in Table 2.

[0159] Table 1 List of EGFR mutation antibodies

[0160] Table 2 List of c-MET mutant antibodies

[0161] Example 2: Preparation of antibodies

[0162] The eukaryotic expression of the antibody used the common protein expression pcDNA3.4 / CHO-K1 system, and the expression plasmid was synthesized by Taizhou Baiying Biotechnology Co., Ltd.

[0163] In a 1 L shake flask, 0.6 × 10 6 Cells were inoculated into 250 ml of CHO cells at a density of 3.5-4 × 10 cells / mL and cultured until the cell density reached 3.5-4 × 10 6 When the cell culture medium reaches 1000 rpm for 5 minutes at room temperature, the cell culture is harvested by centrifugation at 1000 rpm for 5 minutes. The supernatant is discarded and the cells are resuspended in fresh, pre-warmed culture medium. The cells are then placed on a cell shaker for transfection. 500 μg of the target protein expression plasmid is diluted in 5 mL of culture medium, and 3 mg of PEI is diluted in 5 mL of culture medium. The mixture is mixed thoroughly and allowed to stand at room temperature for 5 minutes. The plasmid / PEI mixture is then added directly to the cells to be transfected. Supplemental culture medium and feed are added to each flask 4 hours after transfection. After 24 hours, valproic acid is added to each flask to a final concentration of 3.8 mM. After 72 hours, glucose is added to each flask to a final concentration of 4 g / L. The supernatant is collected 5-6 days after transfection. The supernatant is purified using Protein A to obtain purified antibodies for subsequent detection.

[0164] Example 3: Affinity activity of EGFR mutant antibodies on antigen-expressing cells

[0165] Flow cytometry was used to detect the binding of antibodies to target cells expressing antigens. Target cells in the logarithmic growth phase were selected, collected, washed with PBS, centrifuged, and resuspended in 2% FBS PBS. 1×10 5 Plate cells and centrifuge at 400xg for 2 minutes. Add different concentrations of the antibody to be tested, incubate at 4°C for 1 hour, wash with 2% FBS PBS, and centrifuge at 400g for 2 minutes. Add 100μL of secondary antibody with a fluorescent group, incubate at 4°C for 30 minutes, wash with 2% FBS PBS, and then load onto the BD FACSLyric TM Detection.

[0166] Figure 2 used A431 cell line (purchased from ATCC, CRL-1555 TM ), the results showed that some mutant antibodies activated E max lower than the parental antibody.

[0167] Table 3 Affinity data of some mutant antibodies to A431 cells

[0168] Example 4: Detection of competitive binding activity between mutated antibodies and parental antibodies

[0169] The affinity of EGFR antibodies changes after point mutation, but it does not affect the affinity epitope of the antibody to the antigen. The competitive binding ability between mutant antibodies can be detected by flow cytometry.

[0170] A431 cells in the logarithmic growth phase (purchased from ATCC, CRL-1555) were selected. TM ), collect cells, wash with PBS, centrifuge, and resuspend cells in 2% FBS / PBS. Use 100 μL of 1×10 5 Cells were plated and centrifuged at 400xg for 2 minutes. Cells were stimulated with different concentrations of mutant antibodies combined with a fixed concentration of competitive antibodies, incubated in a 4°C refrigerator for 1 hour, washed with 2% FBS / PBS, and centrifuged at 400g for 2 minutes. 100 μL of secondary antibody PE anti-His Tag Antibody with a fluorescent group (purchased from biolegend, 362603) was added, incubated in a 4°C refrigerator for 30 minutes, washed with 2% FBS / PBS, and loaded onto a BD FACSLyric TM Detection.

[0171] FIG3 shows that the competitive binding activity of antibodies with different point mutations to the parent antibody is lower than that of the parent antibody, and the competitive binding activity levels are consistent with the affinity levels shown in FIG2 .

[0172] Table 4 Competitive binding activity of mutant antibodies against parent antibodies on A431 cells

[0173] Example 5: Expression of bispecific F(ab')2 fragments in a cell-free system

[0174] Thaw an appropriate amount of cell-free extract to room temperature, add 50 mM iodoacetamide, and incubate for 30 minutes. Add 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (1 mM tyrosine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, 2-10 mg / mL plasmid DNA template, 10 mM GSH, and 5 mM GSSG. After reacting at 180 rpm and 25°C for 16 hours, centrifuge the supernatant and purify the F(ab')2 protein using an automated purification system, according to the method described in Cell-free gene expression. Nat Rev Methods Primers 1, 49 (2021). F(ab')2 antibody numbers and corresponding mutation numbers are shown in Table 3.

[0175] Table 5 F(ab')2 antibody combinations

[0176] Example 6: Detection of the affinity activity of bispecific F(ab')2 fragments for antigen-expressing cells

[0177] The affinity activity of the bispecific F(ab')2 fragment for antigen-expressing cells was detected using a method consistent with that described in Example 3. The results are shown in Figures 4-6.

[0178] Figure 4, using the H1975 cell line (purchased from ATCC, CRL-5908), shows that the affinity of OLM7 with HM2, HM3, and Panitumumab for F(ab')2 is lower than that of the same group. Figure 5, using the H441 cell line (ATCC, CRM-HTB-174), shows that the affinity of OLM7 with HM2, HM3, and HM4 for F(ab')2 is lower than that of the same group. Figure 6, using the A431 cell line, shows that the affinity of groups A, B, F, and G is lower than that of the control group D. Figures 4-6 all used a fluorescently labeled secondary antibody, PE anti-His Tag Antibody (purchased from biolegend, 362603).

[0179] Example 7: Activity of bispecific F(ab')2 fragments binding to two antigenic epitopes simultaneously

[0180] To develop a bispecific antibody that simultaneously targets two different antigens on the same cell, the bispecific antibody must have the ability to bind to two different antigens simultaneously. The enzyme-linked immunosorbent assay was used to detect the activity of the bispecific F(ab')2 fragment in binding to both EGFR and c-MET antigens simultaneously.

[0181] Using an enzyme-linked immunosorbent assay (ELISA), 96-well plates were coated with 1 μg / mL c-MET protein (purchased from Nearshore Protein, CS57). The sample to be tested was diluted serially and added to the 96-well plate. After complete incubation, 0.25 μg / mL EGFR protein (purchased from SinoBiological, 10001-H27H-B) was added to bind to the other antibody site. Peroxidase-Streptavidin (purchased from Jackson, AB_2337238) was then added at a 1:2000 dilution and incubated. Finally, TMB was used for color development. After the reaction was terminated, the sample was measured at 450 nm, and the results were plotted and analyzed graphically.

[0182] The results in FIG7 show that the F(ab')2 antibodies have the ability to bind to two antigens simultaneously.

[0183] Example 8: Detection of the activity of bispecific F(ab')2 fragments in mediating cell surface antigen endocytosis

[0184] Antibody endocytosis activity can be monitored by labeling the antibody with a tracer. CypHer5E is a pH-sensitive cyanine dye that emits red fluorescence. It is non-fluorescent at alkaline pH but fluorescent at acidic pH, with a pKa of 7.3. CypHer5E mono-NHS ester is suitable for labeling monoclonal antibodies and other proteins. CypHer5E-labeled secondary antibodies are used to investigate primary antibody-mediated target internalization. When the CypHer5E secondary antibody enters the acidic endosome from the neutral cell surface, the time-dependent internalization activity can be reflected by measuring the intensity of the fluorescence signal.

[0185] The internalization of the antibody on the cells was detected using an Incucyte instrument (purchased from Sartorius). Cells in the logarithmic growth phase were collected, washed with PBS, centrifuged, and resuspended in 10% FBSDMEM medium. 80 μL of 1×10 4 Plate cells and place in a 37°C ± 2°C, 5% CO2 constant temperature incubator for 21 hours. Dye preparation: Dilute AffiniPure Fab Fragment Goat Anti-Human IgG (purchased from Jackson, 109-007-008) to 1 mg / mL, calculate and add the required CypHer5E Mono NHS Ester (purchased from Cytiva, PA15405) based on the molar ratio of secondary antibody: dye = 1:20, mix and incubate at room temperature in the dark for 1 hour. Use Slide-A-Lyzer TM After dialysis using mini dialysis cups (Thermo Scientific, 88404), the concentration was determined. A molar ratio of primary antibody to dye-containing secondary antibody (1:3) was added to the first well of the dilution plate. The plate was incubated at 37°C for 30 minutes to form a complex. The complex was then serially diluted and added to the cell plate at a concentration of 20 μL / well. The cells were cultured in Incucyte and the fluorescence intensity of the cells was measured after 24 hours.

[0186] Figure 8 uses the H1975 cell line, and the results show that the maximum endocytic activity of antibodies in groups C and E is comparable to that of wild-type antibody group D, while groups A, B, and F are weaker than the control group D.

[0187] Figure 9 uses the H441 cell line, and the results show that group A exhibits superior endocytic activity.

[0188] Figure 10, using the A431 cell line, shows that the maximum endocytic activity of antibodies in groups A and F is much lower than that in the wild-type group. The EGFR protein expression level in the A431 cell line is much higher than that of c-MET protein, indicating that the corresponding EGFR mutations in groups A and F have potential safety advantages.

[0189] Example 9: Expression of bispecific antibodies in a eukaryotic expression system

[0190] Eukaryotic expression of bispecific antibodies was performed using the common protein expression system pcDNA3.4 / CHO-K1. Expression plasmids were synthesized by Taizhou Baiying Biotechnology Co., Ltd. Bispecific antibodies for subsequent testing were prepared using the method described in Example 2. Antibody serial numbers and corresponding mutation sites are shown in the table below.

[0191] Table 6 List of eukaryotic expression bispecific antibodies

[0192] Example 10: Affinity activity of bispecific antibodies for antigen-expressing cells

[0193] The affinity activity of the bispecific antibody for antigen-expressing cells was detected using a method consistent with that described in Example 3. The results are shown in FIG11 .

[0194] Figures 11A-B were obtained using the H1975 cell line, Figure 11C was obtained using the H441 cell line, and Figure 11D was obtained using the A431 cell line. Figure 11 used the same fluorescent secondary antibody (purchased from Invitrogen, 12-4998-82). Figure 11 shows that SVAB38 and SVAB39 have strong affinity for H1975 cells, similar to that of wild-type SVAB14, but have lower affinity for A431 cells.

[0195] Table 7 Affinity EC of bispecific antibodies to H1975, H441 and A431 cells 50 contrast

[0196] Example 11: Detection of bispecific antibody-mediated reduction of cell surface antigens

[0197] The endocytic activity of an antibody can be detected by either labeling the antibody or by expressing the antibody-carrying toxin in killing cells. This example uses different endocytic activity detection schemes to detect bispecific antibody-mediated endocytosis.

[0198] A) Fluorescence endocytosis assay

[0199] The same fluorescence endocytosis method as in Example 8 was used to detect the reduction of cell surface antigens mediated by the bispecific antibody.

[0200] The results in Figure 12 show that in H1975 and H441 cell lines, the endocytic activity of bispecific antibodies synthesized from EGFR antibody sequences with different mutations is similar, while the results in the A431 cell line show that endocytic activity is related to affinity, and lower EGFR affinity can improve potential safety.

[0201] Figure 13 uses primary human epidermal keratinocytes HEKs (ATCC, PCS-200-011), and the results show that the antibody SVAB38 with reduced EGFR affinity has significantly reduced endocytic activity on these cells compared to the EGFR wild-type bispecific antibody SVAB14.

[0202] B) Toxin secondary antibody method

[0203] Monomethyl auristatin E (MMAE) is a cytotoxin that can kill cancer cells. MMAE is an analog of dolastatin 10. Microtubules, composed of α-tubulin and β-tubulin, are the primary components of the cytoskeleton. Tubulin inhibitors kill tumor cells by disrupting their cytoskeletal structure and interfering with mitosis. MMAE-labeled secondary antibodies can bind to primary antibodies with endocytic activity, enter cells, and kill tumor cells. The killing effect is assessed by measuring the number of remaining viable cells. Therefore, MMAE secondary antibodies are often used to test the ability of primary antibodies to induce endocytosis of cell membrane antigens.

[0204] The activity of antibody-mediated cell surface antigen endocytosis was detected by cell proliferation inhibition. Cells were seeded in 96-well cell plates. After the cells adhered to the wall, MMAE-labeled anti-Fc secondary antibody (purchased from MORADEC, AH-202AE) was pre-incubated with the sample to be tested at a molar ratio of 6:1 at 37°C in serum-free medium for 30 minutes. Then, a series of gradient dilutions were performed and added to the cell culture plate for incubation for a period of time. CTG was added to detect fluorescence, and the cell killing rate was converted according to the fluorescence intensity.

[0205] The results in Figure 14 show that SVAB14 is a bispecific antibody composed of wild-type sequences and mediates EC on H1975 and H441 cells. 50 The lowest endocytic activity, however, A431 cell line also showed EC 50 The lowest endocytosis activity suggests that while the strongest in vitro efficacy may also lead to greater safety risks. In contrast, the SVAB38 molecule has slightly weaker endocytosis activity than the wild type in 1975 and H441 cells, but significantly reduces endocytosis in the A431 cell line.

[0206] C) DT3C method

[0207] DT3C is a recombinant protein produced through genetic recombination technology. It consists of a diphtheria toxin (DT) without a receptor binding domain and the C1, C2, and C3 (3C) domains of Streptococcus protein G. After the mAb-DT3C conjugate complex recognizes and binds to a cell surface antigen, the mAb-DT3C-antigen complex is internalized. DT3C is then cleaved by cytoplasmic furin, releasing the DT3C catalytic domain into the cytoplasm. The DT3C catalytic domain leads to ADP-ribosylation of elongation factor (EF)-2, subsequently causing cytotoxicity by inhibiting protein translation.

[0208] The activity of antibody-mediated cell surface antigen endocytosis was detected by cell proliferation inhibition. Cells were seeded in 96-well cell plates. After the cells adhered, DT3C (purchased from Huamei Biotechnology, CSB-EP360556CQR1) and the sample to be tested were pre-incubated at 37°C for 30 minutes in a serum-free medium at a molar ratio of 4:1. Then, a series of gradient dilutions were performed and added to the cell culture plate for incubation for a period of time. CTG was added to detect fluorescence, and the fluorescence intensity was converted into cell killing rate.

[0209] The results in Figure 15 show that in the A431 cell line with high EGFR expression, the endocytic activity of SVAB38 is significantly lower than that of the wild-type SVAB14 bispecific antibody molecule, which has potential safety advantages.

[0210] Example 12: Detection of Antibody-Mediated Reduction of Cell Surface Antigens

[0211] The binding of antibodies to specific surface antigen targets mediates the endocytosis of surface antigens, which are then degraded through the lysosomal pathway. In order to detect the activity of antibody-mediated antigen reduction, the ELISA method is used to detect the antigen content. The specific experimental method is as follows:

[0212] Sample preparation: 2x10 cells were seeded in 6-well cell plates. 5 Cells / well A549 cells (purchased from ATCC, CCL-185) were cultured overnight, and 30 μg / mL of antibody sample was added and placed in an incubator overnight. Cell lysis buffer was added and the supernatant was collected by centrifugation.

[0213] Detection of EGFR protein: 96-well coated plates were coated with 0.25 μg / mL panitumumab (purchased from Bio-Tech, B21796801). 100 ng / mL EGFR protein (purchased from Kaixia Bio, EGF-HM201) was serially diluted and the test sample diluted several times was added to the 96-well coated plates. After complete incubation, 0.25 μg / mL EGFR-biotin antibody (purchased from SinoBiological, 10001-MM08T-B) was added. Peroxidase-Streptavidin (purchased from Jackson, AB_2337238) was then added at a 1:2000 dilution and incubated. Finally, TMB was used for color development. After the reaction was terminated, the sample was measured at 450 nm. A standard curve with a good linear relationship was constructed for the EGFR protein group. After the equation was obtained, the OD value of the sample group was substituted into the converted concentration.

[0214] Detection of c-MET: 2 μg / mL c-MET antibody (purchased from Sino Biological, 10692-R261) was coated on a 96-well plate. 100 ng / mL c-MET protein (purchased from Nearshore Protein, CS57) was serially diluted and added to the 96-well plate along with several times diluted test samples. After complete incubation, 1 μg / mL c-MET antibody was added, followed by a 1:5000 dilution of Fab-HRP (purchased from GenScript, C5592HI060-4 / P9HI001) and incubation. Finally, TMB was used for color development. After the reaction was terminated, the sample was measured at 450 nm. A standard curve with a good linear relationship was constructed for the c-MET protein group. The resulting equation was substituted into the sample OD value to calculate the concentration.

[0215] The results in FIG16 show that the SVAB38 molecule has the activity of promoting the degradation of EGFR and c-MET antigens compared with the negative control antibody.

[0216] Example 13: Detecting the inhibitory activity of antibodies on downstream signals of EGF-activated target cells

[0217] Epidermal growth factor (EGF) is one of the major ligands discovered for EGFR. When the EGF ligand binds to the extracellular receptor, EGFR dimerization triggers activation of the intracellular domain, forming a tyrosine kinase. Specifically, the intracellular TK domain reacts biochemically with ATP, converting ATP to ADP. TK phosphorylation activates EGFR enzyme activity. Activated EGFR transmits proliferative and anti-apoptotic signals to the cell nucleus through multiple downstream signaling pathways, including PI3K-AKT-mTOR and Ras-Raf-MEK-ERK1 / 2, thereby controlling cell growth and division. Antibodies can inhibit EGF-induced downstream signaling by competitively binding to the extracellular EGF-binding epitope of EGFR. Activation signals are often indicated by phosphorylation levels of AKT and ERK.

[0218] Western blotting was used to detect the downstream signaling pathways of target cells. H1975 cells were cultured at 4x10 5 Cells were plated in 6-well plates at a density of 1 cell / well. After attachment, the cells were replaced with serum-free medium and cultured overnight. They were then pretreated with 30 μg / ml of the test antibody for 1 hour and stimulated with 100 ng / ml EGF (SinoBiological, 10605-H01H) for 15 minutes. The medium was removed, and the cells were washed once with pre-chilled PBS. 200 μl of lysis buffer (Thermo Fisher Scientific, FNN0011) containing protease inhibitors (Thermo, 78425) and phosphatase inhibitors (Roche, 04906845001) was added. After incubation on ice for 30 minutes, the cell lysate was collected using a cell scraper. The cells were then centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was transferred to a fresh 1.5 ml EP tube for later use. The protein concentration of the supernatant was determined using a BCA kit (Beyotime, P0010S), followed by incubation in a metal bath with 2x SDS-PAGE loading buffer for 5 minutes. Electrophoresis was performed on an 8% precast gel (GenScript, M00657) and then transferred to a PVDF membrane (Thermo, IB31002). The membrane was blocked with 5% BSA-TBST blocking buffer for 1 hour and then blocked overnight at 4°C with primary antibodies (CST, anti-AKT-rabbit 9272, anti-P-AKT (Ser473)-rabbit 9271, p44 / 42 MAPK (Erk1 / 2) antibody 9102, Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) antibody 9101, Vinculin Rabbit mAb 13901). The membrane was washed three times with TBST (5 minutes each) and incubated with an HRP-conjugated secondary antibody (GenScript, A01827) at room temperature for 1 hour. The membrane was then washed three times with TBST (5 minutes each). Finally, a developer (purchased from Merck Millipore, WBKLS0500) was added for color development.

[0219] The results in Figure 17 show that SVAB38 can significantly reduce the phosphorylation levels of downstream AKT and ERK compared to the negative control antibody.

[0220] Example 14: Detection of Selective Killing of Keratinocytes by Different Antibodies

[0221] The most common side effects of EGFR-targeted drugs are skin reactions, including rash, dry skin, and paronychia. EGFR is not only a tumor target but is also widely expressed in the human body, particularly in epithelial cells, which are associated with these skin side effects. To test whether antibodies specifically target tumor tissue rather than normal tissue, A431 cells were selected as a surrogate for normal cells due to their similar antigen expression characteristics to epithelial cells, and H1975 cells were selected as tumor target cells.

[0222] Take A431 and H1975 cells in the logarithmic growth phase, collect the cells, wash with PBS, centrifuge, and resuspend the cells in complete medium. TM A431 cells were labeled with the Far Infrared Cell Proliferation Kit (Thermo, C34564-180T); TM H1975 cells were labeled with CFSE cell proliferation kit (Thermo, C34554-180T) and then mixed at a cell density of 1:1. 1900 μL of 2 × 10 5 Cells were plated and cultured overnight in a 37°C ± 2°C, 5% CO2 incubator. The next day, 100 μl of antibodies and secondary antibodies at different concentrations were added to treat the cells. After 3-5 days of treatment, the cells were collected, washed with PBS, centrifuged, and resuspended in 2% FBS / PBS before loading onto a BD FACSLyric analyzer. TM Detection.

[0223] The flow cytometry results in Figure 18 show that compared with the wild-type control antibody SVAB14, the SVAB38 antibody has obvious killing selectivity for killing H1975 cells.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor (hEGFR), wherein the antibody or antigen-binding fragment comprises: (a1) A heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:2, and SEQ ID NO:3 or variants thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or variants thereof; or (a2) A heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:2, and SEQ ID NO:3 or variants thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or variants thereof; or (a3) A heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:9, and SEQ ID NO:3 or variants thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or variants thereof; or (a4) A heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:10, and SEQ ID NO:3 or variants thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or variants thereof; or (a5) A heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:11 and The amino acid sequence shown in SEQ ID NO:3 or a variant thereof, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or variants thereof according to the Kabat sequence numbering system; or (a6) a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:12 or variants thereof according to the Kabat sequence numbering system, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or variants thereof according to the Kabat sequence numbering system; or (a7) a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:13 or variants thereof according to the Kabat sequence numbering system, and (b1) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or variants thereof according to the Kabat sequence numbering system; or (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 or variants thereof according to the Kabat sequence numbering system, and (b2) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:5, and SEQ ID NO:6 or variants thereof according to the Kabat sequence numbering system; or (a1) a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 or variants thereof according to the Kabat sequence numbering system, and (b3) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:15 or variants thereof according to the Kabat sequence numbering system; or (a1) A variable heavy chain (VH) comprising HCDR1, HCDR2 and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 or variants thereof according to the Kabat sequence numbering system, and (b4) a variable light chain (VL) comprising LCDR1, LCDR2 and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:16 or variants thereof according to the Kabat sequence numbering system; or (a1) A variable heavy chain (VH) comprising HCDR1, HCDR2 and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 or variants thereof according to the Kabat sequence numbering system, and (b5) a variable light chain (VL) comprising LCDR1, LCDR2 and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:17 or variants thereof according to the Kabat sequence numbering system; Wherein the variants of the CDRs have 3, 2 or 1 amino acid differences respectively from the corresponding CDRs or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH and VL are selected from the following groups or variants thereof: (a) A heavy chain having a variable domain comprising the amino acid sequence shown in any one of SEQ ID NO:20-26 and a light chain having a variable domain comprising the amino acid sequence shown in SEQ ID NO:19; or (b) A heavy chain having a variable domain comprising the amino acid sequence shown in SEQ ID NO:18 and a light chain having a variable domain comprising the amino acid sequence shown in any one of SEQ ID NO:27-30; Wherein the variants have 3, 2 or 1 amino acid differences respectively from the corresponding variable domains or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity respectively.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, wherein the antibody comprises a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a chimeric antibody, a humanized antibody or a fully human antibody; and / or, the antigen-binding fragment comprises Fab, Fab’, Fv fragment, F(ab’)2, Fd fragment, dAb, complementarity-determining region fragment, scFv or single-domain antibody.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, wherein the antigen-binding fragment is scFv, which sequentially comprises a variable light chain, a linker fragment and a variable heavy chain from the N-terminus to the C-terminus.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the linker fragment is selected from (GS) n , (GGS) n , (GGGS) n , (GGGGS) n , and n is selected from 2, 3, and 4.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the linker fragment comprises the amino acid sequence shown in SEQ ID NO:

78.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, wherein the antigen-binding fragment is a scFv, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 32-42 or a variant thereof, wherein the variant has 3, 2 or 1 amino acid differences from the scFv or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the scFv, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the scFv further comprises point mutations H44C and L100C.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the maximum effect caused by the binding of the antibody or antigen-binding fragment thereof to the EGFR-expressing cell line is more than 10% lower than the maximum effect caused by the binding of the parental antibody to the EGFR-expressing cell line.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof inhibits the binding of panitumumab to a cell line expressing EGFR with an IC 50 value higher than 2.10 nM.

11. A multispecific antibody, which contains multiple different antigen-binding domains, wherein the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-10.

12. A bispecific antibody, which contains two different antigen-binding domains, wherein the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-10, and the second antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to a tumor-associated antigen (TAA).

13. The bispecific antibody according to claim 12, wherein the tumor-associated antigen is selected from one of the following groups: c-MET, AFP, ALK, BAGE protein, BIRC5, BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20, CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFRvIII, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein, GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein, MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16, MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA, RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thomsen-Friedenreich antigen, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

14. The bispecific antibody according to claim 12, wherein the second antigen-binding domain specifically binds to c-MET.

15. The bispecific antibody according to claim 14, wherein the second antigen-binding domain that specifically binds to c-MET comprises: (a1) a heavy-chain variable region (VH) comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45 or variants thereof according to the Kabat sequence numbering system, and (b1) a light-chain variable region (VL) comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48 or variants thereof according to the Kabat sequence numbering system; or (a1) A heavy chain variable region (VH) comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45 or variants thereof according to the Kabat sequence numbering system, and (b2) a light chain variable region (VL) comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:49 or variants thereof according to the Kabat sequence numbering system; or (a1) A heavy chain variable region (VH) comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45 or variants thereof according to the Kabat sequence numbering system, and (b3) a light chain variable region (VL) comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:50 or variants thereof according to the Kabat sequence numbering system; or (a1) A heavy chain variable region (VH) comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45 or variants thereof according to the Kabat sequence numbering system, and (b4) a light chain variable region (VL) comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:51, SEQ ID NO:47, and SEQ ID NO:48 or variants thereof according to the Kabat sequence numbering system; or (a1) A heavy chain variable region (VH) comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45 or variants thereof according to the Kabat sequence numbering system, and (b5) a light chain variable region (VL) comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:52, SEQ ID NO:47, and SEQ ID NO:48 or variants thereof according to the Kabat sequence numbering system; or (a1) A heavy chain variable region (VH) comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45 or variants thereof according to the Kabat sequence numbering system, and (b5) a light chain variable region (VL) comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, which respectively comprise the amino acid sequences shown in SEQ ID NO:52, SEQ ID NO:47, and SEQ ID NO:48 or variants thereof according to the Kabat sequence numbering system; or (a1) A heavy chain variable region (VH) comprising an HCDR1 region, an HCDR2 region, and an HCDR3 region, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45 or variants thereof, and (b6) a light chain variable region (VL) comprising an LCDR1 region, an LCDR2 region, and an LCDR3 region, which, according to the Kabat sequence numbering system, respectively comprise the amino acid sequences shown in SEQ ID NO:53, SEQ ID NO:47, and SEQ ID NO:48 or variants thereof; Wherein the variants of the variant CDRs have 3, 2, or 1 amino acid differences respectively from the corresponding CDRs or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity respectively.

16. The bispecific antibody according to claim 14, wherein the second antigen-binding domain that specifically binds to c-MET has: 1) A heavy chain with a variable domain comprising the amino acid sequence shown in SEQ ID NO:54 or a variant thereof; and 2) A light chain with a variable domain comprising the amino acid sequence selected from any one of SEQ ID NO:55 - 60 or a variant thereof; Wherein the variants have 3, 2, or 1 amino acid differences respectively from the corresponding variable domains or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity respectively.

17. The bispecific antibody according to any one of claims 14 - 16, wherein the second antigen-binding domain further comprises a first heavy chain constant region (CH1) and a light chain constant region (CL).

18. The bispecific antibody according to claim 17, wherein the CH1 comprises the amino acid sequence shown in SEQ ID NO:75, and the CL comprises the amino acid sequence shown in SEQ ID NO:76 or 77.

19. The bispecific antibody according to any one of claims 14 - 16, wherein the second antigen-binding domain consists of a light chain-heavy chain pair, the light chain sequence comprises the amino acid sequence selected from any one of SEQ ID NO:63 - 67, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:

61.

20. The bispecific antibody according to any one of claims 14 - 19, wherein the second antigen-binding domain is in the form of Fab, Fab’, Fv fragment, F(ab’)2, Fd fragment, dAb, complementarity-determining region fragment, scFv, or single-domain antibody.

21. The bispecific antibody according to claim 12, wherein the bispecific antibody further comprises an Fc domain.

22. The bispecific antibody according to claim 21, wherein the Fc domain is selected from IgA Fc fragment, IgG Fc fragment, IgM Fc fragment, IgD Fc fragment, IgE Fc fragment.

23. The bispecific antibody according to claim 22, wherein the Fc domain is selected from an IgG1 Fc fragment, an IgG2 Fc fragment, an IgG3 Fc fragment, and an IgG4 Fc fragment.

24. The bispecific antibody according to claim 22, wherein the Fc domain is an IgG1 Fc fragment, and its amino acid sequence is as shown in SEQ ID NO:

79.

25. The bispecific antibody according to any one of claims 21-24, wherein the Fc domain further comprises an amino acid substitution that promotes the pairing of the first and second subunits of the Fc domain.

26. The bispecific antibody according to claim 25, wherein the first subunit of the Fc domain comprises the following amino acid substitutions: Y349C / T366S / L368A / Y407V, and the second subunit of the Fc domain comprises the following amino acid substitutions: S354C / T366W.

27. The bispecific antibody according to any one of claims 12-26, which is selected from the following group or its variants: (a) a first antigen-binding domain comprising HCDR1-3 with sequences as shown in SEQ ID NO:1-3 and LCDR1-3 with sequences as shown in SEQ ID NO:4-6; or (b) a first antigen-binding domain comprising HCDR1-3 with sequences as shown in SEQ ID NO:8, 2, and 3 and LCDR1-3 with sequences as shown in SEQ ID NO:4-6; or (c) a first antigen-binding domain comprising HCDR1-3 with sequences as shown in SEQ ID NO:1-3 and LCDR1-3 with sequences as shown in SEQ ID NO:4, 5, 15; or (d) a first antigen-binding domain comprising HCDR1-3 with sequences as shown in SEQ ID NO:7, 2, 3 and LCDR1-3 with sequences as shown in SEQ ID NO:4, 5, 15; or (e) a first antigen-binding domain comprising HCDR1-3 with sequences as shown in SEQ ID NO:1, 10, and 3 and LCDR1-3 with sequences as shown in SEQ ID NO:4-6; or (f) a first antigen-binding domain comprising HCDR1-3 with sequences as shown in SEQ ID NO:1, 2, and 12 and LCDR1-3 with sequences as shown in SEQ ID NO:4-6; and a second antigen-binding domain comprising HCDR1-3 with sequences as shown in SEQ ID NO:43-45 and LCDR1-3 with sequences as shown in SEQ ID NO:46-48; wherein the variants have 3, 2, or 1 amino acid differences from the corresponding CDRs respectively or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity respectively.

28. The bispecific antibody according to any one of claims 12-26, which is selected from the following group or its variants: (a) a first antigen-binding domain comprising a VH having the sequence shown in SEQ ID NO:18 and a VL having the sequence shown in SEQ ID NO:19; or (b) a first antigen-binding domain comprising a VH having the sequence shown in SEQ ID NO:21 and a VL having the sequence shown in SEQ ID NO:19; or (c) a first antigen-binding domain comprising a VH having the sequence shown in SEQ ID NO:18 and a VL having the sequence shown in SEQ ID NO:28; or (d) a first antigen-binding domain comprising a VH having the sequence shown in SEQ ID NO:20 and a VL having the sequence shown in SEQ ID NO:28; or (e) a first antigen-binding domain comprising a VH having the sequence shown in SEQ ID NO:23 and a VL having the sequence shown in SEQ ID NO:19; or (f) a first antigen-binding domain comprising a VH having the sequence shown in SEQ ID NO:25 and a VL having the sequence shown in SEQ ID NO:19; and a second antigen-binding domain comprising a VH having the sequence shown in SEQ ID NO:54 and a VL having the sequence shown in SEQ ID NO:55; wherein the variant has 3, 2 or 1 amino acid differences or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the corresponding variable domain, respectively.

29. The bispecific antibody according to any one of claims 12-26, which consists of a first heavy chain comprising a first antigen-binding domain, a second heavy chain comprising a second antigen-binding domain, and a first light chain; wherein, The first heavy chain is selected from the amino acid sequences shown in any of SEQ ID NO:69-74 or a variant thereof, the second heavy chain is the sequence shown in SEQ ID NO:68 or a variant thereof, and the first light chain is the sequence shown in SEQ ID NO:62 or a variant thereof; wherein the variant has 3, 2 or 1 amino acid differences or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the corresponding sequence, respectively.

30. The bispecific antibody according to any one of claims 12-29, wherein the bispecific antibody can simultaneously bind to EGFR and c-MET and binds to the EGFR and c-MET co-expressing cell line with an EC 50 value lower than 10 nM.

31. The bispecific antibody according to any one of claims 12-29, wherein the bispecific antibody binds to the EGFR-highly expressing cell line with an EC 50 value higher than 50 nM.

32. The bispecific antibody according to any one of claims 12-29, wherein the bispecific antibody is preferentially internalized by EGFR and c-MET co-expressing cell lines in the simultaneous presence of EGFR and c-MET co-expressing cell lines and EGFR highly expressing cell lines.

33. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-10, or the multispecific antibody according to claim 11, or the bispecific antibody according to any one of claims 12-32.

34. An expression vector containing the nucleic acid molecule according to claim 33.

35. A host cell containing the nucleic acid molecule according to claim 33 or the expression vector according to claim 34, or expressing the antibody or antigen-binding fragment thereof according to any one of claims 1-10, or the multispecific antibody according to claim 11, or the bispecific antibody according to any one of claims 12-32.

36. An antibody-drug conjugate (ADC) composed of an antibody, a linker, and a payload, wherein the antibody is the antibody according to any one of claims 1-10 or an antigen-binding fragment thereof, or the multispecific antibody according to claim 11, or the bispecific antibody according to any one of claims 12-32.

37. A pharmaceutical composition containing the antibody according to any one of claims 1-10 or an antigen-binding fragment thereof, or the multispecific antibody according to claim 11, or the bispecific antibody according to any one of claims 12-32, or the antibody-drug conjugate according to claim 36, and at least one pharmaceutically acceptable carrier and / or excipient.

38. A method for inhibiting the growth and / or killing of tumor cells, which comprises contacting the tumor cells with an effective amount of the antibody according to any one of claims 1-10 or an antigen-binding fragment thereof, or the multispecific antibody according to claim 11, or the bispecific antibody according to any one of claims 12-32, or the antibody-drug conjugate according to claim 36, or the pharmaceutical composition according to claim 37.

39. Use of the antibody according to any one of claims 1-10 or an antigen-binding fragment thereof, or the multispecific antibody according to claim 11, or the bispecific antibody according to any one of claims 12-32, or the antibody-drug conjugate according to claim 36, or the pharmaceutical composition according to claim 37 in the preparation of a tumor therapeutic drug.

40. The use according to claim 39, wherein the tumor is selected from pancreatic cancer, melanoma, glioblastoma, head and neck cancer, prostate cancer, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, renal oncocytoma, renal transitional cell carcinoma, urothelial carcinoma, adenocarcinoma or small cell carcinoma.

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