Bispecific antibody, antibody-drug conjugate, and use thereof

Abstract

Provided are a bispecific antibody, an antibody-drug conjugate, and a use thereof, specifically relating to a bispecific antibody targeting EGFR and HER3, an antibody-drug conjugate, and a use thereof against tumors.

Description

Bispecific antibodies, antibody-drug conjugates and their applications

[0001] The present disclosure is based on and claims priority to CN application No. 202410295797.6, filed on March 14, 2024, and CN application No. 202510289027.5, filed on March 11, 2025. The disclosed contents of these CN applications are hereby incorporated into the present disclosure as a whole. Technical Field

[0002] The present disclosure relates to the field of targeted therapy, and in particular to bispecific antibodies targeting EGFR and HER3, antibody-drug conjugates, and their applications in anti-tumor treatment. Background Art

[0003] The epidermal growth factor receptor (EGFR) family, ErbB, includes EGFR, HER2, HER3, and HER4, and plays a key role in tumorigenesis and progression (Hynes NE et al. 2005). Upon binding to specific ligands, these receptors can form homo- or heterodimers and activate various downstream signaling pathways, thereby stimulating cell division and proliferation. Disorders of EGFR, such as mutation, amplification, or overexpression, are closely associated with poor clinical response and prognosis (Mishra, R. et al. 2017). ErbB inhibitors, including monoclonal antibodies and small molecule tyrosine kinase inhibitors, have been approved for the treatment of various cancer types, such as lung cancer, breast cancer, head and neck squamous cell carcinoma, colon cancer, and gastric cancer (Wu, Q. et al. 2022). Despite the success of ErbB inhibitors, many patients develop drug resistance due to the emergence of alternative compensatory signaling pathways (Arteaga CL et al. 2014). For example, HER3 can activate the compensatory PI3K-AKT survival pathway, which is considered an important regulator of acquired resistance to EGFR and HER2 targeted therapies (Amin DN, et al, 2010). HER3 overexpression has been reported in a variety of cancers, such as breast cancer, ovarian cancer, lung cancer, colorectal cancer, melanoma, head and neck cancer, cervical cancer, and prostate cancer (Gandullo-Sánchez, L., et al, 2022).

[0004] Therefore, it is necessary to develop a bispecific antibody targeting both EGFR and HER3 that has good specificity, good efficacy and is easy to prepare. Summary of the Invention

[0005] The present disclosure provides a humanized anti-EGFR×HER3 IgG-like bispecific antibody and a drug conjugate (ADC). In some embodiments, the bispecific antibody forms an asymmetric 1+1 heterodimerization form through the charge interaction of HC-HC and HC-LC, and then binds to a topoisomerase 1 inhibitor (TOP1i) through a cleavable linker to form the ADC. The affinity optimization of the bispecific antibody for EGFR and HER3 can improve the ADC therapeutic window, have better safety, and show strong anti-tumor efficacy in various tumor models. It has great potential for use as a safer and more effective ADC therapeutic drug in the treatment of cancer.

[0006] Antibody

[0007] Antigen binding domain

[0008] In a first aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a HER3 antigen-binding domain comprising a VH (heavy chain variable region) and a VL (light chain variable region), wherein:

[0009] The VH comprises:

[0010] (1-i) comprising the CDRs contained in the heavy chain as shown in the amino acid sequence of SEQ ID NO: 15;

[0011] (1-ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 30, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; or,

[0012] (1-iii) the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof;

[0013] and / or,

[0014] The VL comprises:

[0015] (1-iv) comprising the CDRs contained in the light chain as shown in the amino acid sequence of SEQ ID NO: 16;

[0016] (1-v) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35; or

[0017] (1-vi) The amino acid sequence shown in SEQ ID NO: 5 or a variant thereof.

[0018] In some embodiments, the CDRs are defined by the Kabat, Chothia, AbM, or IMGT numbering systems. In some embodiments, the CDRs are defined by the IMGT numbering system.

[0019] In some embodiments, the HER3 antigen binding domain comprises a VH and a VL, as defined by the IMGT numbering system,

[0020] The VH comprises:

[0021] (1-i) comprising the CDRs contained in the heavy chain as shown in the amino acid sequence of SEQ ID NO: 15;

[0022] (1-ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 30, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; or,

[0023] (1-iii) the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof;

[0024] and / or,

[0025] The VL comprises:

[0026] (1-iv) comprising the CDRs contained in the light chain as shown in the amino acid sequence of SEQ ID NO: 16;

[0027] (1-v) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35; or

[0028] (1-vi) The amino acid sequence shown in SEQ ID NO: 5 or a variant thereof.

[0029] In some embodiments, the variant has a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared thereto.

[0030] In some embodiments, the substitutions are conservative substitutions.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof is monospecific.

[0032] In a second aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising an EGFR antigen-binding domain, wherein the EGFR antigen-binding domain comprises VH and VL, wherein:

[0033] The VH comprises:

[0034] (2-i) comprising the CDRs contained in the heavy chain as shown in the amino acid sequence of SEQ ID NO: 12 or 14;

[0035] (2-ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 24 or 29, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 25; or

[0036] (2-iii) the amino acid sequence shown in SEQ ID NO: 1 or 3 or a variant thereof;

[0037] and / or,

[0038] The VL comprises:

[0039] (2-iv) comprising the CDRs contained in the light chain as shown in the amino acid sequence of SEQ ID NO: 13;

[0040] (2-v) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 28; or,

[0041] (2-vi) the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof.

[0042] In some embodiments, the CDRs are defined by the Kabat, Chothia, AbM, or IMGT numbering systems. In some embodiments, the CDRs are defined by the IMGT numbering system.

[0043] In some embodiments, the EGFR antigen binding domain comprises a VH and a VL, as defined by the IMGT numbering system,

[0044] The VH comprises:

[0045] (2-i) comprising the CDRs contained in the heavy chain as shown in the amino acid sequence of SEQ ID NO: 12 or 14;

[0046] (2-ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 24 or 29, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 25; or

[0047] (2-iii) the amino acid sequence shown in SEQ ID NO: 1 or 3 or a variant thereof;

[0048] and / or,

[0049] The VL comprises:

[0050] (2-iv) comprising the CDRs contained in the light chain as shown in the amino acid sequence of SEQ ID NO: 13;

[0051] (2-v) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 28; or,

[0052] (2-vi) the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof.

[0053] In some embodiments, the variant has a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared thereto.

[0054] In some embodiments, the substitutions are conservative substitutions.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof is monospecific.

[0056] The antibody or antigen-binding fragment thereof according to any one of the first aspect or the second aspect of the present disclosure may further contain a second antigen-binding domain, and thus the antibody or antigen-binding fragment thereof may be bispecific.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof according to any one of the first aspects of the present disclosure further comprises an EGFR antigen-binding domain. In some embodiments, the EGFR antigen-binding domain is as defined in any one of the second aspects.

[0058] In some embodiments, the antibody or antigen-binding fragment thereof according to any one of the second aspects of the present disclosure further comprises a HER3 antigen-binding domain. In some embodiments, the HER3 antigen-binding domain is as defined in any one of the first aspects.

[0059] Accordingly, in a third aspect, the present disclosure provides a bispecific antibody or an antigen-binding fragment thereof, wherein the antibody comprises a first antigen-binding domain specific for HER3 and a second antigen-binding domain specific for EGFR, wherein:

[0060] The first antigen-binding domain comprises a first VL and a first VH, wherein the first VL is the VL comprised in the HER3 antigen-binding domain, and the first VH is the VH comprised in the HER3 antigen-binding domain; and / or

[0061] The second antigen-binding domain comprises a second VL and a second VH, wherein the second VL is the VL comprised in the EGFR antigen-binding domain, and the second VH is the VH comprised in the EGFR antigen-binding domain.

[0062] constant region

[0063] In some embodiments, the antibody described in any aspect herein is an IgG antibody. In some embodiments, the IgG antibody is an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE antibody. In some embodiments, the IgG antibody is an IgG1 antibody.

[0064] In some embodiments, the antibody further comprises a CH (heavy chain constant region) and a CL (light chain constant region).

[0065] In some embodiments, the CH is an IgG1 heavy chain constant region.

[0066] In some embodiments, the CL is a kappa or lambda light chain constant region. In some embodiments, the CL is a human kappa light chain constant region.

[0067] In some embodiments, the CH comprises the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, and / or the CL comprises the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof.

[0068] In some embodiments, the variant has a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared thereto. In some embodiments, the substitutions are conservative substitutions.

[0069] In some embodiments, the CL and / or the CH (e.g., CH1, Fc region) are altered (e.g., mutated) to promote dimerization (e.g., homodimerization or heterodimerization), for example, to promote pairing of κCL with CH1, and / or Fc region heterodimerization. In some embodiments, the Fc region of the antibody contains an alteration that forms a knob-in-hole structure.

[0070] In some embodiments, the CH comprises the amino acid sequence shown in SEQ ID NO: 8 or 9 or a variant thereof, and / or the CL comprises the amino acid sequence shown in SEQ ID NO: 10 or 11 or a variant thereof.

[0071] Bispecific antibodies

[0072] The bispecific antibodies disclosed herein can be designed to be composed of two different but complementary heavy and / or light chains to form a heterodimeric structure. Each chain contains a binding site for an antigen, and the structure of the bispecific antibody is stabilized by interchain interactions. In order to avoid mispairing of antibody chains, various methods known in the art can be used to ensure correct interchain pairing, such as the "Knob-in-Hole" method, electrostatic steering, CrossMab technology, orthogonal Fab interface technology, etc.

[0073] In some embodiments, the bispecific antibody comprises peptide chain IA, peptide chain IB, peptide chain IC and peptide chain ID, wherein the peptide chain IA comprises a first VL and CL, the peptide chain IB comprises a first VH and CH, the peptide chain IC comprises a second VH and CH, and the peptide chain ID comprises a second VL and CL.

[0074] In some embodiments, the first VL, second VL, first VH, second VH, CL, and CH are each independently as defined above.

[0075] In some embodiments, the adjacent domains of the peptide chain IA are connected by a linker or not, the adjacent domains of the peptide chain IB are connected by a linker or not, the adjacent domains of the peptide chain IC are connected by a linker or not, and / or the adjacent domains of the peptide chain ID are connected by a linker or not.

[0076] In some embodiments, each of the linkers is independently selected from a polypeptide linker, such as a rigid or flexible polypeptide linker; preferably, the polypeptide linker is a glycine-rich linker, such as (GGS) n 、(GGGGS) n or (GGGGA) n The linker shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, for example, SEQ ID NO: 36 or 37.

[0077] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises:

[0078] A peptide chain IA comprising the amino acid sequence shown in SEQ ID NO: 18;

[0079] a peptide chain IB comprising the amino acid sequence shown in SEQ ID NO: 17;

[0080] A peptide chain IC comprising the amino acid sequence shown in SEQ ID NO: 19 or 21; and

[0081] The peptide chain ID comprising the amino acid sequence shown in SEQ ID NO: 20 or 22.

[0082] In some embodiments, the bispecific antibody comprises:

[0083] Peptide chain IA having the amino acid sequence set forth in SEQ ID NO: 18, peptide chain IB having the amino acid sequence set forth in SEQ ID NO: 17, peptide chain IC having the amino acid sequence set forth in SEQ ID NO: 19, and peptide chain ID having the amino acid sequence set forth in SEQ ID NO: 20;

[0084] Alternatively, the peptide chain IA having the amino acid sequence shown in SEQ ID NO: 18, the peptide chain IB having the amino acid sequence shown in SEQ ID NO: 17, the peptide chain IC having the amino acid sequence shown in SEQ ID NO: 21, and the peptide chain ID having the amino acid sequence shown in SEQ ID NO: 22.

[0085] The bispecific antibodies disclosed herein can also be designed by fusing two single-chain variable fragments (scFv) or other antigen-binding domains to the same polypeptide chain through genetic engineering techniques. For example, the variable region of another antibody can be attached to the N-terminus of the heavy and light chains of an IgG molecule, or the scFv can be fused to the C-terminus of an antibody.

[0086] In some embodiments, the bispecific antibody comprises peptide chain II-A and peptide chain II-B, wherein the peptide chain II-A comprises a first VH, CH, a second VH, and a second VL, and the peptide chain II-B comprises a first VL and CL, or, the peptide chain II-A comprises a second VH, CH, a first VH, and a first VL, and the peptide chain II-B comprises a second VL and CL.

[0087] In some embodiments, the peptide chain II-A comprises a first VH, CH, a second VH, and a second VL from N-terminus to C-terminus, and the peptide chain II-B comprises a first VL and CL from N-terminus to C-terminus, or, the peptide chain II-A comprises a second VH, CH, a first VH, and a first VL from N-terminus to C-terminus, and the peptide chain II-B comprises a second VL and CL from N-terminus to C-terminus.

[0088] In some embodiments, the first VL, second VL, first VH, second VH, CL, and CH are each independently as defined above.

[0089] In some embodiments, adjacent domains of the peptide chain II-A are connected by a linker or not, and / or adjacent domains of the peptide chain II-B are connected by a linker or not.

[0090] In some embodiments, each of the linkers is independently selected from a polypeptide linker, such as a rigid or flexible polypeptide linker; preferably, the polypeptide linker is a glycine-rich linker, such as (GGS) n or (GGGGS) n The linker shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, for example, SEQ ID NO: 36 or 37.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0092] Peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 38; and, Peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 39; or,

[0093] a peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 40; and a peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 41.

[0094] In some embodiments, the antibody comprises:

[0095] Peptide chain II-A having the amino acid sequence shown in SEQ ID NO: 38; and, Peptide chain II-B having the amino acid sequence shown in SEQ ID NO: 39; or,

[0096] a peptide chain II-A having the amino acid sequence shown in SEQ ID NO: 40; and a peptide chain II-B having the amino acid sequence shown in SEQ ID NO: 41.

[0097] In some embodiments, the antibody comprises two peptide chains II-A. In some embodiments, the antibody comprises two peptide chains II-B. In some embodiments, the heavy chain constant regions of the two peptide chains II-A form a dimer. In some embodiments, the two peptide chains II-A dimerize via the Fc region.

[0098] The bispecific antibodies disclosed herein have at least one of the following characteristics: selective binding to EGFR / HER3 double-positive cells, capable of maintaining a binding capacity comparable to or even higher than that of the corresponding monospecific antibodies; the ability to specifically bind to EGFR / HER3 double-positive cells and mediate antibody internalization, with the internalization effect being significantly superior to that of the corresponding monospecific antibodies; and possessing enhanced tumor inhibitory effects, including EGF-EGFR blocking effects, NRG1-HER3 blocking effects, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0099] Detectable labels and detection applications

[0100] In some embodiments, the antibody is further detectably labeled.

[0101] The detectable label described herein can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In certain embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme. In certain embodiments, the detectable label as described above can be linked to the antibody or antigen-binding fragment thereof of the present disclosure by linkers of varying lengths to reduce potential steric hindrance.

[0102] The antibodies or antigen-binding fragments thereof disclosed herein can specifically bind to EGFR and / or HER3, thereby being useful for detecting the presence or level of EGFR and / or HER3 in a sample. Therefore, in another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are detectably labeled.

[0103] In another aspect, the present disclosure provides a method for detecting the presence or level of EGFR and / or HER3 in a sample, comprising the step of using the antibody or antigen-binding fragment thereof of the present disclosure.

[0104] In some embodiments, the method further comprises detecting the antibody or antigen-binding fragment thereof of the present disclosure using the detectably labeled antibody or antigen-binding fragment thereof. The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample rather than a sample from a patient).

[0105] In certain embodiments, the method comprises contacting the sample with the antibody or antigen-binding fragment thereof of the present disclosure under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and EGFR and / or HER3, and detecting formation of the complex.

[0106] In view of the significant differences in the expression levels of EGFR and / or HER3 in normal tissues and some cancers, tumors can be diagnosed by detecting the levels of EGFR and / or HER3 in samples. Therefore, in certain embodiments, the method is used to diagnose tumors, such as breast cancer, colon cancer, gastric cancer, lung cancer (e.g., lung squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), melanoma, rectal cancer, liver cancer, pancreatic cancer, glioma, ovarian cancer, bladder cancer, cervical cancer, prostate cancer and head and neck cancer. In some embodiments, the tumor is a primary or metastatic tumor.

[0107] In certain embodiments, the method comprises detecting the expression level of EGFR and / or HER3 in a test sample from a subject, and comparing the expression level with a reference value (e.g., a healthy control), wherein an increase in the expression level compared to the reference value is indicative of a tumor.

[0108] In another aspect, provided is a use of the antibody or antigen-binding fragment thereof of the present disclosure in preparing a kit for detecting the presence or level of EGFR and / or HER3 in a sample and / or diagnosing a tumor.

[0109] Nucleic acids, vectors, host cells and expression methods

[0110] In another aspect, the present disclosure provides a nucleic acid molecule encoding an antibody described in any of the preceding claims. The nucleic acid molecule can be obtained using methods known in the art, for example, by isolation from a phage display library, a yeast display library, an immune animal, an immortalized cell (e.g., a mouse B cell hybridoma, an EBV-mediated immortalized B cell), or chemical synthesis. The nucleic acid molecule can be codon-optimized for the host cell used for expression.

[0111] In another aspect, the present disclosure provides a vector comprising the nucleic acid molecule. In some embodiments, the nucleic acid sequences encoding different peptide chains of the antibody or antigen-binding fragment thereof are located in the same or different vectors. In some embodiments, the vector is a cloning vector or an expression vector.

[0112] In some embodiments, the nucleic acid molecule is prepared as a recombinant nucleic acid. In some embodiments, the nucleic acid molecule is cloned into an expression vector. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The recombinant nucleic acid comprising the nucleic acid can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination technology (such as polymerase chain reaction (PCR) technology) etc. (see Sambrook, J., E. Fritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The expression vector may also comprise a polynucleotide sequence encoding a polypeptide or protein that can promote detection and / or separation of the expressed antibody or antigen-binding fragment. Such polypeptides or proteins may include, but are not limited to, affinity tags (such as biotin, polyhistidine tags (His6) or glutathione S-transferase (GSH) tags), polypeptides comprising protease cleavage sites, and reporter proteins (such as fluorescent proteins). The nucleic acid molecule may be present in one or more vectors. In some embodiments, the expression vector is a DNA plasmid, such as a DNA plasmid for expression in bacteria, yeast, or mammalian cells. In other embodiments, the expression vector is a viral vector. In other embodiments, the expression vector is a phage vector or a phagemid vector.

[0113] In another aspect, the present disclosure provides a host cell comprising the nucleic acid molecule or vector.

[0114] In some embodiments, the host cell is used to express the antibody or antigen-binding fragment thereof.Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., Escherichia coli), eukaryotic cells (e.g., yeast, insect cells, mammalian cells).

[0115] Bacteria (e.g., E. coli BL21 (DE3)) are particularly advantageous for expressing smaller antigen-binding fragments. Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells (e.g., HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for expressing antibodies.

[0116] In another aspect, the present disclosure provides a method for producing the antibody or antigen-binding fragment thereof in a host cell, wherein the method comprises the following steps:

[0117] (1) transforming a host cell with at least one nucleic acid molecule or expression vector described herein;

[0118] (2) culturing the transformed host cell under appropriate conditions to allow expression of the nucleic acid molecule or expression vector, and

[0119] (3) Isolating and purifying the antibody or antigen-binding fragment thereof from the host cell or culture medium.

[0120] In some embodiments, the method comprises culturing the host cell under suitable conditions to allow expression of the antibody or antigen-binding fragment thereof, and collecting the antibody or antigen-binding fragment thereof from the culture medium of the host cell.

[0121] In some embodiments, the host cell further comprises a chaperone plasmid that can help improve the solubility, stability and / or folding of the antibody or antibody fragment.Techniques for isolating and purifying antibodies from host cells are well known to those skilled in the art.

[0122] Antibody-drug conjugate (ADC)

[0123] In another aspect, the present disclosure provides an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof described in any one of the foregoing items.

[0124] In some embodiments, the antibody-drug conjugate further comprises a therapeutic agent.

[0125] In some embodiments, each molecule of the antibody drug conjugate contains 1-20 therapeutic agents, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, such as at least 1, 2, 3 or 4 therapeutic agents. In some embodiments, the therapeutic agent is selected from a microtubule inhibitor and a DNA damaging drug.

[0126] In some embodiments, the tubulin inhibitor is selected from auristatins (e.g., MMAE, MMAF, or derivatives thereof), maytansines (e.g., maytansine, maytansinol, DM1, DM4, or derivatives thereof), taxanes (e.g., Taxol, Docetaxel, Cabazitaxel, or derivatives thereof), vinca alkaloids (e.g., vinblastine, vincristine, or derivatives thereof), eribulin, and colchicine, or derivatives thereof.

[0127] In some embodiments, the DNA damaging agent is selected from DNA alkylating agents (calicheamicin γ11, N-acetyl-γ11 calicheamicin, anthramycin, PBD, dukamycin or derivatives thereof), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically, camptothecin, SN-38, Dxd, irinotecan, belotecan, topotecan, PNU-159682 or derivatives thereof), doxorubicin, daunorubicin, etoposide, mitoxantrone or derivatives thereof) and amanitin or derivatives thereof.

[0128] In some embodiments, the therapeutic agent is

[0129] In some embodiments, the antibody and therapeutic agent are connected by at least one connector. In some embodiments, each of the therapeutic agents is connected to the constant region of the antibody. In some embodiments, each of the therapeutic agents is connected to the Fc region of the antibody. In some embodiments, the therapeutic agent is connected to the Fc region of the antibody via the connector.

[0130] In some embodiments, the therapeutic agent is attached to the linker via its reactive group (eg, hydroxyl group).

[0131] In some embodiments, the linker is cleavable or non-cleavable.

[0132] In some embodiments, the cleavable linker is selected from the group consisting of a protease-sensitive, pH-sensitive, and glutathione-sensitive linker.

[0133] In some embodiments, the linker is selected from -NHCH2-, MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropionyl), Val-Cit (valine-citrulline), Val-Cit-NHCH2-, Val-Ala (valine-alanine), Val-Ala-NHCH2-, Ala-Phe (alanine-phenylalanine), Ala-Phe-NHCH2-, Gly-Gly-Phe-Gly (glycine-glycine-phenylalanine-glycine), Gly- Gly-Phe-Gly-NHCH2-, PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimidyl)-4-(pyridin-2-ylthio) pentanoate), 6-(2,5-dioxopyrrolidin-1-yl)-4-(pyridin-2-ylthio) hexanoate, 6-(2,5-dioxopyrrolidin-1-yl)-5-methyl-4-(pyridin-2-ylthio) hexanoate, SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate), and any combination thereof.

[0134] In some embodiments, the linker is selected from MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropionyl), Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Ala-Phe (alanine-phenylalanine), Gly-Gly-Phe-Gly (glycine-glycine-phenylalanine-glycine), Gly-Gly-Phe-Gly-NHCH2-, PAB (para-aminobenzyl) oxycarbonyl), SPP (5-(succinimidyl)-4-(pyridin-2-ylthio) pentanoate), 6-(2,5-dioxopyrrolidin-1-yl)-4-(pyridin-2-ylthio) hexanoate, 6-(2,5-dioxopyrrolidin-1-yl)-5-methyl-4-(pyridin-2-ylthio) hexanoate, SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), or SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate), and any combination thereof.

[0135] In some embodiments, the linker is Gly-Gly-Phe-Gly-NHCH2-.

[0136] In some embodiments, the linker is

[0137] In some embodiments, the antibody drug conjugate has the following structure:

[0138] wherein A is the antibody or antigen-binding fragment thereof described in any one of the preceding items;

[0139] p is an integer selected from 1-20, for example, an integer from 1-10 or 1-8, and further for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0140] In another aspect, the present disclosure provides an antibody drug conjugate having the following structure:

[0141] Among them, A is a bispecific antibody targeting EGFR and HER3, comprising:

[0142] Peptide chain IA having the amino acid sequence set forth in SEQ ID NO: 18, peptide chain IB having the amino acid sequence set forth in SEQ ID NO: 17, peptide chain IC having the amino acid sequence set forth in SEQ ID NO: 19, and peptide chain ID having the amino acid sequence set forth in SEQ ID NO: 20;

[0143] Alternatively, peptide chain IA having the amino acid sequence shown in SEQ ID NO: 18, peptide chain IB having the amino acid sequence shown in SEQ ID NO: 17, peptide chain IC having the amino acid sequence shown in SEQ ID NO: 21, and peptide chain ID having the amino acid sequence shown in SEQ ID NO: 22;

[0144] Alternatively, a peptide chain IA having the amino acid sequence shown in SEQ ID NO: 38; and, a peptide chain IB having the amino acid sequence shown in SEQ ID NO: 39;

[0145] Alternatively, a peptide chain IA having the amino acid sequence shown in SEQ ID NO: 40; and, a peptide chain IB having the amino acid sequence shown in SEQ ID NO: 41;

[0146] p is an integer selected from 1-20, for example, an integer from 1-10 or 1-8, and further for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0147] Pharmaceutical composition

[0148] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell or antibody-drug conjugate described in any one of the foregoing, and a pharmaceutically acceptable excipient.

[0149] The excipients may be those described in Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Non-limiting examples of suitable excipients include buffers, preservatives, binders, lubricants, disintegrants, chelating agents, surfactants, flavorings, sweeteners, coloring agents.

[0150] In some embodiments, the pharmaceutical composition contains any of the above-mentioned antibody-drug conjugates and a pharmaceutically acceptable excipient.

[0151] In some embodiments, the drug-antibody coupling ratio (DAR) of the pharmaceutical composition is 1-8, such as 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1.5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 2-2.5, 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5-7, 2.5-7.5 , 2.5-8, 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3.5-4, 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 4-4.5, 4-5, 4-5.5, 4-6, 4-6.5, 4-7, 4-7.5, 4-8, 4.5- 5, 4.5-5.5, 4.5-6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5.5-6, 5.5-6.5, 5.5-7, 5.5-7.5, 5.5-8, 6-6.5, 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-7.5, 7-8, 7.5-8.

[0152] application

[0153] In another aspect, the present disclosure provides use of any of the above antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells, antibody-drug conjugates or pharmaceutical compositions in the preparation of anti-tumor drugs.

[0154] In another aspect, the present disclosure provides the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, antibody-drug conjugate or pharmaceutical composition described in any one of the above, for use in anti-tumor treatment.

[0155] In another aspect, the present disclosure provides an anti-tumor method, comprising administering to a subject in need thereof a therapeutically effective amount of any of the above antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells, antibody-drug conjugates or pharmaceutical compositions.

[0156] In some embodiments, the tumor overexpresses EGFR and / or HER3.

[0157] In some embodiments, the tumor is selected from breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), melanoma, rectal cancer, liver cancer, pancreatic cancer, glioma, ovarian cancer, bladder cancer, cervical cancer, prostate cancer, and head and neck cancer.

[0158] In some embodiments, the tumor is a primary or metastatic tumor.

[0159] Definition of terms

[0160] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures in molecular genetics, nucleic acid chemistry, cell culture, biochemistry, cell biology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0161] As used herein, the singular forms "a", "an" and "include" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, the terms "include", "comprise", "includes", "have", "contain" or their variations are open-ended and not exclusive or exhaustive.

[0162] As used herein, the term "and / or" should be understood as a specific disclosure of each of the two specified features or combinations, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A (alone)," and "B (alone)."

[0163] As used herein, the terms "EGFR," "ErbB-1," and "HER1" are used interchangeably. They are members of the ErbB / EGFR receptor tyrosine kinase family and are widely distributed on the surfaces of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, and other cells. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation, and differentiation, and is associated with the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis. EGFR consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular kinase domain. It is activated by binding to ligands, including EGF and TGFα (transforming growth factor α). Upon activation, EGFR converts from a monomer to a dimer. Dimerization involves both the binding of two homologous receptor molecules (homodimerization) and the binding of different members of the human EGF-related receptor (HER) tyrosine kinase family (heterodimerization). EGFR dimerization can activate its intracellular kinase pathways, and receptor autophosphorylation can induce downstream phosphorylation, including the MAPK, Akt, and JNK pathways, inducing cell proliferation.

[0164] As used herein, the terms "HER3" and "ErbB-3" are used interchangeably and are 148kD transmembrane receptors of the ErbB / EGFR receptor tyrosine kinase family, but lack intrinsic kinase activity. ErbB receptors form homodimers and heterodimer complexes, which activate the ligand-dependent (ligand-independent in some cases) activation of multiple signal transduction pathways to affect the physiological functions of cells and organs. Heterodimers (e.g., ErbB2 / ErbB3) containing ErbB3 in tumor cells have been shown to be the most mitogenic and oncogenic receptor complex in the ErbB family. After binding to its physiological ligand, the ErbB3 receptor forms a dimer with other ErbB family members (mainly ErbB2). ErbB3 / ErbB2 dimerization causes ErbB3 to be transphosphorylated on the tyrosine residues included in the protein cytoplasm tail. Phosphorylation of these sites creates SH2 docking sites for SH2-containing proteins (including PI3 kinases). Therefore, heterodimeric complexes containing ErbB3 are potent activators of AKT because ErbB3 possesses six tyrosine phosphorylation sites with YXXM motifs that, when phosphorylated, serve as excellent binding sites for phosphoinositide 3-kinase (PI3K), leading to subsequent downstream activation of the AKT pathway. These six PI3K sites act as powerful amplifiers of ErbB3 signaling. Activation of this pathway further triggers several important biological processes involved in tumorigenesis, such as cell growth, migration, and survival.

[0165] As used herein, the term "inhibit" or "block" refers to any statistically significant reduction in biological activity, including complete blocking of activity. For example, "inhibit" can refer to a reduction in biological activity of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0166] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and its antigen. The binding affinity between two molecules can be expressed as K. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as a molar concentration (M). K D The smaller the value, the tighter the two molecules bind and the higher the affinity. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Bind to the antigen. K D The value can be determined by methods well known in the art, for example using surface plasmon resonance (SPR) in a BIACORE instrument.

[0167] As used herein, the terms "antibody" and "monoclonal antibody" refer to immunoglobulin molecules typically composed of two pairs of polypeptide chains, each pair comprising a light chain (LC) and a heavy chain (HC). Each chain has variable regions, referred to as the heavy chain variable region (VH) and the light chain variable region (VL). Together, the VH and VL are responsible for binding to the antigen recognized by the antibody. Mammalian immunoglobulins have five major heavy chain classes (or isotypes), which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians. Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, corresponding to the five antibody isotypes mentioned above: IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0168] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region), and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance rate of antibodies and antigen-antibody complexes, and the like. Methods for altering the effector functions of an antibody are known in the art, for example, by introducing mutations in the Fc region.

[0169] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-attached target cells and then kill the target cells by secreting cytotoxins.

[0170] The variable region of an antibody comprises a framework region (FR) and a hypervariable region (HVR), which are called "complementarity determining regions (CDR)". The CDR is primarily responsible for binding to the epitope of the antigen. VH and VL consist of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The allocation of amino acids in each region or domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The CDRs contained in the antibodies or antigen-binding fragments thereof described herein can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof are preferably identified by the Kabat, AbM, Chothia or IMGT numbering systems.

[0171] As used herein, the term "Fc region" or "Fc domain" refers to the portion of the heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In some embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0172] As used herein, the term "antigen-binding fragment" refers to a portion of an intact antibody that retains specific binding ability to an antigen, such as a fragment comprising an intact hypervariable region. Examples of antibody fragments include, but are not limited to, scFv, Fab, Fab', (Fab')2, Fd, Fv, CDR fragments, nanobodies, and disulfide-linked Fv (dsFv).

[0173] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

[0174] As used herein, the term "Fab fragment" means an antibody fragment consisting of 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 a complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain; the term "Fd" means an antibody fragment consisting of the VH and CH1 domains.

[0175] As used herein, the term "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.

[0176] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly 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 called nanobodies.

[0177] 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.

[0178] 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 of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and screened for specificity in the same manner as for intact antibodies.

[0179] As used herein, the term "conservative variant" refers to a protein containing conservative amino acid substitutions that do not substantially affect or reduce the affinity of the protein.

[0180] Conservative amino acid substitutions of functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0181] 1) Alanine (A), serine (S), threonine (T);

[0182] 2) Aspartic acid (D), glutamic acid (E);

[0183] 3) Asparagine (N), glutamine (Q);

[0184] 4) Arginine (R), Lysine (K);

[0185] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0186] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0187] As used herein, amino acids may be represented by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Specific abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

[0188] As used herein, the term "identity" refers to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0189] Knob-in-hole technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be positioned in the cavity to promote the formation of heterodimers and hinder the formation of homodimers. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity having the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis. In a specific embodiment, the knob is modified to include the amino acid substitution T366W in one of the two subunits of the Fc domain, while the hole is modified to include the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit comprising the knob-modified Fc domain additionally includes the amino acid substitution S354C, while the subunit comprising the hole-modified Fc domain additionally includes the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). The numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain, which polypeptide is capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 constant domain and an IgG CH3 constant domain.

[0190] In an alternative embodiment, modifications that promote heterodimerization of two non-identical polypeptide chains comprise modifications that mediate an electrostatic steering effect, such as described in WO 2009 / 089004. Typically, this approach involves replacing one or more amino acid residues at the interface of the two polypeptide chains with charged amino acid residues such that homodimer formation becomes electrostatically unfavorable but heterodimerization becomes electrostatically favorable.

[0191] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, and generally can include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0192] As used herein, the term "isolated" refers to a substance (eg, a nucleic acid molecule or a polypeptide) that is separated from its source or environment, ie, does not substantially contain any other components.

[0193] As used herein, the term "vector" is a medium for importing exogenous nucleic acid into a host cell, and when the vector is transformed into an appropriate host cell, the exogenous nucleic acid is amplified or expressed. The vector usually remains free, but can be designed to integrate a gene or part thereof into a chromosome of the genome. In this article, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc.

[0194] As used herein, the term "expression vector" refers to a vector capable of expressing DNA, and the DNA is operably connected to a regulatory sequence (such as a promoter, a ribosome binding site) that can affect expression of the DNA. Regulatory sequences can include promoter and terminator sequences, and optionally can include an origin of replication, a selective marker, an enhancer, a polyadenylation signal, etc. The expression vector can be a plasmid, a phage vector, a recombinant virus, or other vectors that, when introduced into a suitable host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, and are included in reproducible expression vectors in eukaryotic cells and / or prokaryotic cells and keep free expression vectors or are integrated into the expression vector of the host cell genome.

[0195] As used herein, the term "host cell" is a cell that is used to receive, maintain, replicate, or amplify a vector. A host cell can also be used to express a nucleic acid or a polypeptide encoded by the vector. The host cell can be a eukaryotic cell or a prokaryotic cell.

[0196] As used herein, the terms "subject," "patient," or "individual" include mammals and non-mammals. A mammal can be any member of the class Mammalia, including but not limited to humans; non-human primates such as chimpanzees, apes, or other monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs (or canines), and cats; laboratory animals including rodents such as rats, mice, and guinea pigs; and the like. Non-mammals can include birds, fish, and the like. In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. In some cases, the human can be an adult. In some cases, the human can be a child. In some cases, the human can be 0-17 years old. In some cases, the human can be 18-130 years old. In some cases, the subject can be male. In some cases, the subject can be female. In some cases, the subject is diagnosed with or suspected of having a disease. In some cases, the disease is cancer. The subject can be a patient or an individual. In some cases, subject, patient, or individual are used interchangeably.

[0197] As used herein, the terms "treat," "treat," "improve," or "alleviate" include alleviating or relieving the symptoms of a disease, inhibiting a disease, such as arresting the development of a disease, relieving a disease, causing regression of a disease, alleviating symptoms caused by a disease, or stopping the symptoms of a disease. The terms "treat," "treat," "improve," or "alleviate" may further include obtaining a therapeutic benefit. A therapeutic benefit may refer to the eradication of the disease being treated. Additionally, a therapeutic benefit may also be achieved by eradicating one or more physiological symptoms associated with the disease being treated, resulting in an observable improvement in the subject, although in some embodiments, the subject may still be suffering from the underlying disease.

[0198] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to a sufficient amount of the drug administered that will at least partially alleviate the symptoms of the disease being treated. The dosage regimen can be adjusted to provide the best desired response. For example, a single bolus injection can be administered, or several divided doses can be administered over time, or the dosage can be proportionally reduced or increased according to the treatment situation. It should be noted that the dosage value can vary with the type and severity of the disease to be alleviated, and can include single or multiple doses. It is further understood that for any particular individual, a specific dosage regimen should be adjusted over time according to individual needs and drug instructions or clinical physician professional judgment. In general, the effective dose is about 0.0001 to about 50 mg per kg body weight per day, for example, about 0.01 to about 10 mg / kg / day (single or divided administration). For a 70 kg person, this would add up to about 0.007 mg / day to about 3500 mg / day, for example, about 0.7 mg / day to about 700 mg / day. In some cases, dosage levels no higher than the lower limit of the aforementioned range may be sufficient, while in other cases, larger doses may still be employed without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses to be administered throughout the day.

[0199] Advantageous Effects of the Invention

[0200] The present disclosure provides a humanized anti-EGFR and HER3 bispecific antibody and its antibody-drug conjugate, which has higher binding activity against EGFR and HER3 dual-expressing cancer cells. Under the action of HC-HC and HC-LC charges, heterodimerization is carried out, and then it is combined with a topoisomerase 1 inhibitor (TOP1i) through a cleavable linker to obtain an antibody-drug conjugate. The antibody's affinity optimization for EGFR and HER3 can improve the therapeutic window of the antibody-drug conjugate, have better safety, and show strong anti-tumor efficacy in various tumor models. It may be used as a safer and more effective ADC therapeutic drug for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0201] FIG1 shows a schematic structural diagram of the antibody of the present invention.

[0202] FIG2 shows a schematic structural diagram of the antibody-drug conjugate (ADC-1) of the present invention.

[0203] FIG3 shows the test results of the cell binding activity of the antibodies of the present invention, expressed as EC50 (nM).

[0204] FIG4 shows the test results of the blocking activity of the antibodies of the present invention on EGF-EGFR interaction and signal transduction, expressed as IC50 (nM).

[0205] FIG5 shows the test results of the blocking activity of the antibodies of the present invention on the NRG1-HER3 interaction, expressed as IC50 (nM).

[0206] FIG6 shows the test results of the internalization effect mediated by the antibodies of the present invention.

[0207] FIG7 shows the test results of the antibody drug conjugate (ADC-1) of the present invention inhibiting cell proliferation.

[0208] FIG8 shows the results of studying the tumor inhibitory activity of the antibody drug conjugate of the present invention (ADC-1) in NOD SCID model mice subcutaneously inoculated with MKN45 cells.

[0209] FIG9 shows the results of studying the tumor inhibitory activity of the antibody drug conjugate of the present invention (ADC-1) in a tumor model in which A375 was subcutaneously inoculated in Balb / c Nude mice.

[0210] FIG10 shows the results of studying the tumor inhibitory activity of the antibody drug conjugate of the present invention (ADC-1) in a tumor model in which NSG mice were subcutaneously inoculated with MKN45. DETAILED DESCRIPTION

[0211] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0212] Example 1 Cloning and Expression of EGFR×HER3 Bispecific Antibody and Preparation of Antibody Drug Conjugate

[0213] 1. Antibody Cloning and Expression

[0214] 1.1. Antibody Expression and Purification

[0215] The antibody of the present invention was transfected and expressed using HEK293 cells as carrier cells. The cell density was adjusted to 2.5×10 6 cells / ml, diluted to 3.0×10 cells / ml for transfection the next day 6cells / ml. Using MEM medium as the transfection buffer, the antibody plasmid was transferred into HEK293 cells at a ratio of PEI: plasmid = 3:1. After culturing for 5 days, the cell supernatant was collected. The target protein was purified using a Protein A filler column. First, 10 column volumes of PBS were added to the filler column to balance the column. Then, the above cell supernatant was added to the gravity column and gravity flowed through. Unbound foreign proteins were then washed away with 20 column volumes of PBS. Finally, the target protein was eluted with 3-5 column volumes of elution buffer (0.1M sodium citrate, pH3.2) and the protein was collected with neutralization buffer (1M Tris, pH8.54).

[0216] 1.2. Cell line screening

[0217] Cell line construction

[0218] The vectors pCHO2.0-GS-Puro-H1-L1 containing the heavy and light chain genes of the anti-EGFR antibody and pCHO2.0-GS-Puro-H2-L2 containing the heavy and light chain genes of the anti-HER3 antibody were co-transfected into the host cells CHOS-ADP-FUT8-KO by electroporation. The cells were screened under pressure using Puromycin and MSX screening pressure to obtain a high-yield minipool. Then, a high-yield and stable clonal cell line was obtained through a round of limiting dilution and monoclonal identification.

[0219] Cell culture

[0220] Dynamis AGT Medium was used as the basal medium, and the inoculation density was (1.0±0.2)×10 6cells / ml. Cell boost 7a powder supplement (available from Cytiva, product name SH31026.02, production batch number MF29430725A) was added at 5.0±0.5% (w / w) of the initial culture weight on days 3, 5, 7, 9, and 11, respectively. Cell boost 7b powder supplement (available from Cytiva, product name SH31027.02, production batch number MF29442466B) was added at 0.5±0.05% (w / w) of the initial culture weight. The dissolved oxygen level was maintained at 40%, and the initial culture temperature was 36.5°C, which was lowered to 33.0°C on day 4. Based on glucose concentration measurements, 300 g / kg of glucose concentrate was added daily to maintain a glucose concentration of 6.0 g / L in the cell sap, except on the day of harvest. Cultures were terminated on day 14 or when cell viability fell below 80%. During the culture process, the cell density and viability were detected using Vicell (Beckman Company), and starting from the 7th day, the antibody production was detected every day using Cedex (Roche Company).

[0221] Stable cell line product quality identification

[0222] The antibody was purified using a one-step affinity purification method, and the protein purity was determined by HPLC. The HPLC method was as follows: mobile phase: 150 mM Na₂HPO₄·12H₂O, pH 7.0. Chromatographic conditions: detection wavelength: 280 nm, column temperature: 25°C, flow rate: 0.5 ml / min, detection time: 30 min, using a TSKgel G3000SWXL column. High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to determine the heavy and light chain pairing of the protein. The instrument used was a Vanquish UHPLC system (Thermo), a Q Exactive mass spectrometer (Thermo), and a Waters ACQUITY UPLC BEH C4 column, 2.1 mm × 100 mm. A 50 μg sample was diluted to 25 μl with ultrapure water. After centrifugation, 20 μl of the sample was transferred to an injection vial, and 5 μl was injected. The intact molecular weight was analyzed by LC-MS. Chromatographic conditions were: column temperature: 80°C; UV detection wavelength: 280 nm; flow rate: 0.3 mL / min; mobile phase A: aqueous solution (containing 0.1% formic acid); mobile phase B: acetonitrile solution (containing 0.1% formic acid). Mass spectrometry parameters were: ESI ion source: ion transfer tube temperature 320°C, voltage 3.8 kV, gas flow rate 36 L / min; mode: positive ion Full MS; resolution: 17500; scan range: 600-4000 m / z.

[0223] 1.3. In this example, five EGFR×HER3 bispecific antibodies, two anti-EGFR monoclonal antibodies, and one HER3 monoclonal antibody were constructed, namely:

[0224] Anti-EGFR-01×HER3: It is composed of 4 polypeptide chains, and its structural diagram is shown in Figure 1.

[0225] Peptide chain #1 has the amino acid sequence shown in SEQ ID NO:17, which comprises the heavy chain variable region amino acid sequence of the anti-HER3 monoclonal antibody Patritumab (SEQ ID NO:4) and the human IgG1 amino acid sequence introduced with a CH3Knob mutation and a CH1 / CL-preferring mutation CH SET1 (SEQ ID NO:8).

[0226] Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:18, which comprises the light chain variable region amino acid sequence of the anti-HER3 monoclonal antibody Patritumab (SEQ ID NO:5), and a human κ light chain constant region (CL) amino acid sequence introduced with a CH1 / CL preferential mutation CL SET1 (SEQ ID NO:10) at the C-terminus of the VL amino acid sequence.

[0227] Peptide chain #3 has the amino acid sequence shown in SEQ ID NO:19, which comprises the heavy chain variable region amino acid sequence of the anti-EGFR monoclonal antibody Zalutumumab (SEQ ID NO:1) and the human IgG1 amino acid sequence introduced with a CH3 Hole mutation and a CH1 / CL-preferring mutation CH SET2 (SEQ ID NO:9).

[0228] Peptide chain #4 has the amino acid sequence shown in SEQ ID NO:20, which comprises the light chain variable region amino acid sequence of the anti-EGFR monoclonal antibody Zalutumumab (SEQ ID NO:2), and a human κ light chain constant region (CL) amino acid sequence introduced with a CH1 / CL preferential mutation CL SET2 (SEQ ID NO:11) at the C-terminus of the VL amino acid sequence.

[0229] Anti-EGFR-03×HER3: It is composed of 4 polypeptide chains, and its structural diagram is shown in Figure 1.

[0230] Peptide chain #1 has the amino acid sequence shown in SEQ ID NO:17.

[0231] Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:18.

[0232] Peptide chain #3 has the amino acid sequence shown in SEQ ID NO:21, which comprises the amino acid sequence of the heavy chain variable region of the anti-EGFR monoclonal antibody EGFR-03 (SEQ ID NO:3) and the human IgG1 amino acid sequence introduced with a CH3 Hole mutation and a CH1 / CL-preferring mutation CH SET2 (SEQ ID NO:9).

[0233] Peptide chain #4 has the amino acid sequence shown in SEQ ID NO:22, which comprises the light chain variable region amino acid sequence of the anti-EGFR monoclonal antibody EGFR-03 (SEQ ID NO:2), and the human κ light chain constant region (CL) amino acid sequence introduced with the CH1 / CL preferential mutation CL SET2 (SEQ ID NO:11) at the C-terminus of the VL amino acid sequence.

[0234] Anti-EGFR-01×HER3(2+2): It is composed of two polypeptide chains, and its structural diagram is shown in Figure 1.

[0235] Peptide chain #1 has the amino acid sequence set forth in SEQ ID NO:38, and comprises the amino acid sequence of the heavy chain variable region of the anti-EGFR monoclonal antibody Zalutumumab (SEQ ID NO:1) and the amino acid sequence of the heavy chain constant region of human IgG1 (SEQ ID NO:7). The N-terminus of the heavy chain variable region of the anti-HER3 monoclonal antibody Patritumab (SEQ ID NO:4) is linked to the C-terminus of the Fc via a flexible peptide of 10 amino acid residues (G4A)2 (SEQ ID NO:36), and the N-terminus of the light chain variable region of the anti-HER3 monoclonal antibody Patritumab (SEQ ID NO:5) is linked to the C-terminus of the heavy chain variable region of Patritumab via a flexible peptide of 20 amino acid residues (G4S)4 (SEQ ID NO:37).

[0236] Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:39, which comprises the light chain variable region amino acid sequence of the anti-EGFR monoclonal antibody zalutuzumab (SEQ ID NO:2), and the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO:6) at the C-terminus of the VL amino acid sequence.

[0237] Anti-HER3×EGFR-01(2+2): It is composed of two polypeptide chains, and its structural diagram is shown in Figure 1.

[0238] Peptide chain #1 has the amino acid sequence set forth in SEQ ID NO:40 and comprises the amino acid sequence of the heavy chain variable region of the anti-HER3 monoclonal antibody patritumab (SEQ ID NO:4) and the amino acid sequence of the heavy chain constant region of human IgG1 (SEQ ID NO:7). The N-terminus of the heavy chain variable region of the anti-EGFR monoclonal antibody zalutuzumab (SEQ ID NO:1) is linked to the C-terminus of the Fc via a flexible peptide of 10 amino acid residues (G4A)2 (SEQ ID NO:36), and the N-terminus of the light chain variable region of the anti-EGFR monoclonal antibody zalutuzumab (SEQ ID NO:2) is linked to the C-terminus of the heavy chain variable region of zalutuzumab via a flexible peptide of 20 amino acid residues (G4S)4 (SEQ ID NO:37).

[0239] Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 41. It comprises the light chain variable region amino acid sequence of the anti-HER3 monoclonal antibody patritumab (SEQ ID NO: 5), and the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 6) at the C-terminus of the VL amino acid sequence.

[0240] Anti-EGFR (Cet) × HER3 (Syst) (2+2) (Patent Publication No.: WO2016106157A1): consists of two polypeptide chains, and its structural schematic is shown in Figure 1.

[0241] Peptide chain #1 has the amino acid sequence shown in SEQ ID NO:42.

[0242] Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:43.

[0243] Anti-EGFR-01: It is composed of 4 polypeptide chains, and its structural diagram is shown in Figure 1.

[0244] The heavy chain has the amino acid sequence shown in SEQ ID NO: 12, which comprises the heavy chain variable region amino acid sequence of the anti-EGFR monoclonal antibody Zalutumumab (SEQ ID NO: 1) and the human IgG1 heavy chain constant region amino acid sequence (SEQ ID NO: 7).

[0245] The light chain has the amino acid sequence shown in SEQ ID NO: 13, which comprises the light chain variable region amino acid sequence of the anti-EGFR monoclonal antibody Zalutuzumab (SEQ ID NO: 2), and the human κ light chain constant region (CL) amino acid sequence (SEQ ID NO: 6) at the C-terminus of the VL amino acid sequence.

[0246] Anti-EGFR-03: It is composed of four polypeptide chains, and its structural diagram is shown in Figure 1.

[0247] The heavy chain has the amino acid sequence shown in SEQ ID NO: 14, which comprises the amino acid sequence of the heavy chain variable region of the anti-EGFR monoclonal antibody EGFR-03 (SEQ ID NO: 3) and the amino acid sequence of the human IgG1 heavy chain constant region (SEQ ID NO: 7).

[0248] The light chain has the amino acid sequence shown in SEQ ID NO: 13, which comprises the light chain variable region amino acid sequence of the anti-EGFR monoclonal antibody EGFR-03 (SEQ ID NO: 2) and the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 6) at the C-terminus of the VL amino acid sequence.

[0249] Anti-HER3: It is composed of four polypeptide chains, and its structural diagram is shown in Figure 1.

[0250] The heavy chain has the amino acid sequence shown in SEQ ID NO: 15, which comprises the heavy chain variable region amino acid sequence of the anti-HER3 monoclonal antibody patritumab (SEQ ID NO: 4) and the human IgG1 heavy chain constant region amino acid sequence (SEQ ID NO: 7).

[0251] The light chain has the amino acid sequence shown in SEQ ID NO: 16, which comprises the light chain variable region amino acid sequence of the anti-HER3 monoclonal antibody patritumab (SEQ ID NO: 5), and the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 6) at the C-terminus of the VL amino acid sequence.

[0252] 2. Preparation of Antibody-Drug Conjugates

[0253] In this example, EGFR × HER3 bispecific antibody-drug conjugates (Anti-EGFR-01 × HER3-ADC-1 and Anti-EGFR-03 × HER3-ADC-1, structures shown in Figure 2), EGFR monoclonal antibody-drug conjugates (Anti-EGFR-01-ADC-1 and Anti-EGFR-03-ADC-1), HER3 monoclonal antibody-drug conjugate (Anti-HER3-ADC-1), and Isotype Ctrl-ADC-1 were also constructed:

[0254] 5 mg of antibodies (Anti-EGFR-01×HER3, Anti-EGFR-03×HER3, Anti-EGFR-01, Anti-EGFR-03, Anti-HER3 and Isotype Ctrl) were placed at the bottom of a 2 mL centrifuge tube. The antibody concentration was adjusted to approximately 5 mg / mL (1 mL) with PBS (Cytiva; pH 7.4). 0.1 M EDTA (Thermo, 0.001 mL) and 0.1 M TCEP (Sigma) aqueous solution (0.013 mL, 20.0 equivalents per antibody molecule) were added, and the resulting solution was incubated in a 37°C water bath for 2 h to reduce the disulfide bonds between the antibody chains. Subsequently, dimethyl sulfoxide (Sigma, 0.05 mL) and a 15 mM solution of Deruxtecan conjugate (MCE, Product No. HY-13631E) (0.069 mL, 16.0 equivalents per antibody molecule) prepared in dimethyl sulfoxide were added to the above solution at room temperature. The coupling reaction was allowed to proceed at room temperature for 1 hour. Next, a 100 mM aqueous solution of acetylcysteine ​​(MCE, Product No. HY-B0215 / CS-2160) (0.022 mL, 32.0 equivalents per antibody molecule) was added and mixed at room temperature for 20 minutes before terminating the coupling reaction. The resulting ADC mixture was exchanged with a 30K concentrator tube (Amicon Ultra-4) (the replacement volume was 10 times that of the reaction solution) and finally exchanged into Lite6.0 solution (10 mM citric acid, 200 mM sucrose, 40 mM NaCl, 0.02 g EDTA, pH 6.0). The purity of the ADC-1 sample after the liquid exchange was determined by HPLC-SEC, and the DAR value was determined by LC-MS. The test results showed that the purity and DAR value of the ADC-1 sample of the present invention met the requirements, and the DAR value was approximately 8.

[0255] Example 2. Binding of EGFR×HER3 Bispecific Antibody to Human EGFR / HER3 Cells

[0256] In this experiment, the expanded cultured LS180 cells (which express EGFR and HER3) were digested with 0.25% EDTA trypsin and centrifuged to remove the supernatant. The cell pellet was resuspended in culture medium and counted. The cell density was then adjusted to 2×10 cells using 2% BSA solution. 6 cells / ml; Jurkat-EGFR cells (overexpressing EGFR) and CHOS-HER3 cells (overexpressing HER3) were suspension cells. An appropriate amount of cells was counted and the cell density was adjusted to 2×10 cells / ml using 2% BSA solution. 6 cells / ml. The cell suspension was added to a 96-well flow cytometry plate at 100 μl / well and centrifuged for later use. The gradient diluted antibody was added to the above 96-well flow cytometry plate with cells at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, 1000-fold diluted Goat anti-human IgG-Fc (PE) (Abcam, ab98596) with 2% BSA solution was added at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, the cells were resuspended in PBS at 100 μl / well and detected on a CytoFlex (Beckman) flow cytometer and the corresponding mean fluorescence intensity (MFI) was calculated.

[0257] The experimental results, shown in Figure 3, show that the Anti-EGFR-01 × HER3 bispecific antibody preferentially binds to EGFR / HER3 double-positive cells, while its binding to EGFR or HER3 single-positive cells is weaker, significantly weaker than that of the Anti-EGFR-01 or Anti-HER3 monoclonal antibodies. Furthermore, although the Anti-EGFR-03 × HER3 bispecific antibody further reduces its binding to EGFR single-positive cells, it still maintains strong binding to double-positive cells. The "2+2" format of bispecific antibodies has a binding level comparable to that of the corresponding monoclonal antibodies in double-positive cells. However, for single-positive cells, the binding of Anti-EGFR-01×HER3(2+2) and Anti-HER3×EGFR-01(2+2) bispecific antibodies to HER3 or EGFR single-positive cells, respectively, is significantly weakened compared to the corresponding monoclonal antibodies because their anti-HER3 or anti-EGFR is designed in the form of scFv at the C-terminus of the Fc. However, their binding to EGFR and HER3 double-positive cells, respectively, maintains a binding ability comparable to that of the corresponding monoclonal antibodies.

[0258] Example 3. EGFR×HER3 bispecific antibody blocks EGF-EGFR interaction

[0259] In this experiment, the cell density of HEK293T-hEGFR (overexpressing human EGFR) cells was adjusted to 2×106 Cells were diluted 100 μl / well into a 96-well flow cytometry plate and centrifuged for later use. The serially diluted antibodies were added to the 96-well flow cytometry plate containing cells at 100 μl / well and incubated at 4°C for 60 min. The cells were washed once with PBS, and Bio-EGF diluted in 2% BSA solution was added at 100 μl / well and incubated at 4°C for 60 min. The cells were washed twice with PBS, and Streptavidin (BD Pharmingen, 554061) diluted 1000-fold in 2% BSA solution was added at 100 μl / well and incubated at 4°C for 60 min. The cells were washed twice with PBS, and finally, 100 μl / well of PBS was added to resuspend the cells. The cells were detected on a CytoFlex (Beckman) flow cytometer and the corresponding MFI was calculated.

[0260] The experimental results are shown in Figure 4A. This study used flow cytometry to verify the inhibitory effect of the Anti-EGFR×HER3 bispecific antibody on the EGF-EGFR interaction. On EGFR single-positive cells, the Anti-EGFR-01×HER3 bispecific antibody was able to significantly block the binding of EGF and EGFR, but its blocking activity was significantly weaker than that of the corresponding Anti-EGFR-01 monoclonal antibody. Due to its weaker affinity for binding to EGFR, the Anti-EGFR-03×HER3 bispecific antibody had no significant EGF-EGFR blocking effect on EGFR single-positive cells. In addition, the "2+2" bispecific antibody had a stronger blocking effect on EGF-EGFR, and its blocking activity was comparable to that of the corresponding Anti-EGFR monoclonal antibody.

[0261] Example 4. EGFR×HER3 bispecific antibody blocks EGF-EGFR signal transduction

[0262] In this experiment, the cell density of HEK293T-hEGFR-STAT3 reporter gene (overexpressing human EGFR and STAT3) cells was adjusted to 2.5×10 6 Cells / ml, 40 μl / well was added to a 96-well plate and set aside. The serially diluted antibody was added to the 96-well plate containing cells at 40 μl / well and incubated in a cell culture incubator for 60 minutes. Then, 40 μl / well of EGF solution diluted in complete culture medium was added and the cells were incubated in a cell culture incubator for 16 hours. Finally, the color was developed using the Bio-Glo luciferase assay system (Promega, G7940) and the chemiluminescent signal was collected using a microplate reader.

[0263] The experimental results are shown in Figure 4B. This study used a reporter gene method to verify the blocking effect of the Anti-EGFR×HER3 bispecific antibody on the EGF-EGFR signaling pathway. On EGFR single-positive cells, the Anti-EGFR-01×HER3 bispecific antibody was able to significantly block EGF and EGFR signaling, but its blocking activity was significantly weaker than that of the corresponding Anti-EGFR-01 monoclonal antibody. Due to its weaker affinity for binding to EGFR, the Anti-EGFR-03×HER3 bispecific antibody had no significant EGF-EGFR blocking effect on EGFR single-positive cells. In addition, the "2+2" bispecific antibody had a stronger blocking effect on EGF-EGFR, and its blocking activity was comparable to that of the corresponding Anti-EGFR monoclonal antibody.

[0264] Example 5. EGFR×HER3 bispecific antibody blocks NRG1-HER3 interaction

[0265] In this experiment, the cell density of HEK293T-hHER3 (overexpressing human HER3) cells was adjusted to 2×10 6 cells / ml, 100 μl / well was added to a 96-well flow cytometry plate and centrifuged for later use. The gradient diluted antibody was added to the above 96-well flow cytometry plate with cells at 100 μl / well and incubated at 4°C for 60 min. Wash once with PBS, add 100 μl / well of Bio-NRG1 diluted with 2% BSA solution, and incubate at 4°C for 60 min. Wash twice with PBS, add 1000-fold diluted Streptavidin (BD Pharmingen, 554061) with 2% BSA solution, and incubate at 4°C for 60 min. Wash twice with PBS, and finally resuspend the cells in PBS at 100 μl / well. Detect on a CytoFlex (Beckman) flow cytometer and calculate the corresponding MFI.

[0266] The experimental results are shown in Figure 5. This study used flow cytometry to verify the inhibitory effect of the Anti-EGFR×HER3 bispecific antibody on the NRG1-HER3 interaction. On HER3 single-positive cells, the Anti-EGFR-01×HER3 and Anti-EGFR-03×HER3 bispecific antibodies were able to significantly block the binding of NRG1 and HER3, but their blocking activity was significantly weaker than that of the corresponding Anti-HER3 monoclonal antibody. In addition, the "2+2" bispecific antibody had a stronger blocking effect on NRG1-HER3, and its blocking activity was comparable to that of the corresponding Anti-HER3 monoclonal antibody.

[0267] Example 6. EGFR×HER3 bispecific antibody induced internalization effect

[0268] In this experiment, the cell density of LS180 and MKN45 cells (which express human EGFR and HER3) was adjusted to 4×10 6 Cells were added to a 96-well flow cytometer at a concentration of 50 μl / well. Serially diluted bispecific antibodies (prepared with a pH dye-labeled secondary antibody for 1 hour) were added to the cells at a concentration of 50 μl / well and incubated in an incubator for 2 hours. After washing twice with PBS, cells were resuspended in 100 μl / well of PBS and analyzed on a CytoFlex (Beckman) flow cytometer, and the corresponding MFI was calculated.

[0269] The experimental results, shown in Figure 6, show that the Anti-EGFR-01 × HER3 bispecific antibody specifically binds to EGFR / HER3 double-positive cells and mediates antibody internalization, with internalization significantly superior to that of the corresponding Anti-EGFR-01 and Anti-HER3 monoclonal antibodies. Furthermore, the internalization mediated by Anti-EGFR-01 × HER3 is superior to or comparable to that of the "2+2" bispecific antibody and Anti-EGFR-03 × HER3.

[0270] Example 7. EGFR×HER3 bispecific antibody-drug conjugate (ADC-1) inhibits cell proliferation

[0271] In this experiment, the expanded cultured LS180 and MKN45 cells (which express EGFR and HER3) were digested with 0.25% EDTA trypsin and resuspended by centrifugation. After washing once with culture medium, the cell density was adjusted to 6.25×10 4 cells / ml, 80μl / well was added to the 96-well plate and set aside. The gradient diluted antibody was added to the 96-well plate with cells at 80μl / well and placed in a cell culture incubator for 3-5 days. The chemiluminescent signal was collected using a microplate reader after color development using the Luminescent Cell Viability Assay (Promega, G7572) kit.

[0272] As shown in Figure 7, the Anti-EGFR-01xHER3-ADC-1 and Anti-EGFR-03xHER3-ADC-1 bispecific antibody-drug conjugates of the present invention can significantly inhibit the growth and proliferation of LS180 and MKN45 cells, and their proliferation inhibition effects are comparable to those of the corresponding monoclonal antibody-drug conjugates.

[0273] Example 8. Study on tumor inhibitory activity of EGFR×HER3 bispecific antibody-drug conjugate (ADC-1)

[0274] This experiment was conducted to verify the concept of the anti-tumor activity of the EGFR×HER3 bispecific antibody-drug conjugate (ADC-1) of the present invention in a tumor model in which MKN45 was subcutaneously inoculated in NOD SCID mice.

[0275] First, a tumor-bearing mouse model was established by subcutaneously inoculating MKN45 cells. 3 The mice were divided into groups at approximately 4 dpi and treated with intraperitoneal injection of G1: PBS, G2: 3 mg / kg Isotype Ctrl-ADC-1, G3: 3 mg / kg Anti-EGFR-01×HER3, and G4: 3 mg / kg Anti-EGFR-01×HER3-ADC-1 (all groups received equal molar doses). The changes in tumor volume and body weight of the mice in each group were monitored every 2-3 days for 2-3 weeks. The dosage and method of administration are shown in Table 1.

[0276] The experimental results are shown in Figure 8. Compared with Anti-EGFR-01×HER3 naked antibody (TGI: 12.90%) and Isotype Ctrl-ADC-1 (TGI: 11.05%), the Anti-EGFR-01×HER3-ADC-1 of the present invention (TGI: 94.12%) has stronger anti-tumor activity and can significantly inhibit tumor growth.

[0277] Table 1: Dosing regimen for the tumor inhibitory activity study of Anti-EGFR-01×HER3-ADC-1

[0278] Example 9. Study on tumor inhibitory activity of EGFR×HER3 bispecific antibody-drug conjugate (ADC-1)

[0279] This experiment was conducted to verify the anti-tumor activity of the EGFR×HER3 bispecific antibody-drug conjugate (ADC-1) of the present invention in a Balb / c Nude mouse subcutaneous inoculation A375 tumor model.

[0280] First, a tumor-bearing mouse model was established by subcutaneously inoculating A375 cells. 3The mice were divided into groups at approximately 4 dpi and treated with intraperitoneal injection of G1: PBS, G2: 2 mg / kg of Isotype Ctrl-ADC-1, G3: 2 mg / kg of Anti-EGFR-01×HER3, and G4: 2 mg / kg of Anti-EGFR-01×HER3-ADC-1 (all groups received equal molar doses). The changes in tumor volume and body weight of the mice in each group were monitored every 2-3 days for 2-3 weeks. The dosage and method of administration are shown in Table 2.

[0281] The experimental results are shown in Figure 9. Compared with Anti-EGFR-01×HER3 naked antibody (TGI: 0%) and Isotype Ctrl-ADC-1 (TGI: 0%), the Anti-EGFR-01×HER3-ADC-1 of the present invention (TGI: 100%) has stronger anti-tumor activity and can significantly inhibit tumor growth.

[0282] Table 2: Dosing regimens for tumor inhibitory activity studies of EGFR×HER3-ADCs

[0283] Example 10. Study on tumor inhibitory activity of EGFR×HER3 bispecific antibody-drug conjugate (ADC-1)

[0284] This experiment was conducted to verify the concept of the anti-tumor activity of the EGFR×HER3 bispecific antibody-drug conjugate (ADC-1) of the present invention in a tumor model in which MKN45 was subcutaneously inoculated in NSG mice.

[0285] First, a tumor-bearing mouse model was established by subcutaneously inoculating MKN45 cells. 3The mice were divided into groups at 14:00 pm and intraperitoneally injected with G1: PBS, G2: 3 mg / kg of Isotype Ctrl-ADC-1, G3: 3 mg / kg of Anti-EGFR-01-ADC-1, G4: 3 mg / kg of Anti-EGFR-03-ADC-1, G5: 3 mg / kg of Anti-HER3-ADC-1, G6: Enhertu (Daiichi-Sankyo AstraZeneca, batch number: 389085), G7: 3 mg / kg of Anti-EGFR-01×HER3-ADC-1, G8: 3 mg / kg of Anti-EGFR-03×HER3-ADC-1, and G9: 4 mg / kg of Anti-EGFR(Cet)×HER3(Syst)(2+2)-ADC-1 (all groups were treated with equal molar doses). The changes in tumor volume and body weight of mice in each group were monitored at a frequency of 2-3 days / time for 2 to 3 weeks. The dosage and method of administration are shown in Table 3.

[0286] As shown in Figure 10 , the anti-tumor activity of the bispecific antibody-drug conjugates Anti-EGFR-01×HER3-ADC-1 (TGI: 77.83%) and Anti-EGFR-03×HER3-ADC-1 (TGI: 71.21%) was significantly stronger than that of the corresponding monoclonal antibody-drug conjugates (Anti-EGFR-01-ADC-1 (TGI: 70.84%), Anti-EGFR-03-ADC-1 (TGI: 57.55%), and Anti-HER3-ADC-1 (TGI: 56.28%). Furthermore, the anti-tumor activity of Anti-EGFR-01×HER3-ADC-1 was superior to that of Anti-EGFR-03×HER3-ADC-1 and Anti-EGFR(Cet)×HER3(Syst)(2+2)-ADC-1 (TGI: 54.09%).

[0287] Table 3: Dosing regimens for tumor inhibitory activity studies of EGFR×HER3-ADCs

[0288] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

[0289] References

[0290] 1.Hynes,N.E.and H.A.Lane,ERBB receptors and cancer:the complexity of targeted inhibitors.Nat Rev Cancer,2005.5(5):p.341-54.

[0291] 2.Mishra,R.,A.B.Hanker,and J.T.Garrett,Genomic alterations of ERBB receptors in cancer:clinical implications.Oncotarget,2017.8(69):p.114371-114392.

[0292] 3.Wu,Q.,et al.,Small-molecule inhibitors,immune checkpoint inhibitors,and more:FDA-approved novel therapeutic drugs for solid tumors from 1991to 2021.J Hematol Oncol,2022.15(1):p.143.

[0293] 4.Arteaga,C.L.and J.A.Engelman,ERBB receptors:from oncogene discovery to basic science to mechanism-based cancer therapeutics.Cancer Cell,2014.25(3):p.282-303.

[0294] 5.Amin,D.N.,M.R.Campbell,and M.M.Moasser,The role of HER3,the unpretentious member of the HER family,in cancer biology and cancer therapeutics.Semin Cell Dev Biol,2010.21(9):p.944-50.

[0295] 6. Gandullo-Sanchez, L., A. Ocana, and A. Pandiella, HER3 in cancer: from the bench to the bedside. J Exp Clin Cancer Res, 2022. 41(1): p. 310.

[0296] Sequence information

Claims

1. An antibody or antigen-binding fragment thereof comprising a HER3 antigen-binding domain, wherein the HER3 antigen-binding domain comprises a VH (heavy chain variable region) and a VL (light chain variable region), wherein: The VH comprises: (1-i) comprising the CDRs contained in the heavy chain as shown in the amino acid sequence of SEQ ID NO: 15; (1-ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 30, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; or, (1-iii) the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof; and / or, The VL comprises: (1-iv) comprising the CDRs contained in the light chain as shown in the amino acid sequence of SEQ ID NO: 16; (1-v) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35; or (1-vi) the amino acid sequence shown in SEQ ID NO: 5 or a variant thereof; Preferably, the CDRs are defined by the Kabat, Chothia, AbM or IMGT numbering systems; Preferably, the variant has a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions.

2. An antibody or antigen-binding fragment thereof comprising an EGFR antigen-binding domain, wherein the EGFR antigen-binding domain comprises VH and VL, wherein: The VH comprises: (2-i) comprising the CDRs contained in the heavy chain as shown in the amino acid sequence of SEQ ID NO: 12 or 14; (2-ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 24 or 29, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 25; or (2-iii) the amino acid sequence shown in SEQ ID NO: 1 or 3 or a variant thereof; and / or, The VL comprises: (2-iv) comprising the CDRs contained in the light chain as shown in the amino acid sequence of SEQ ID NO: 13; (2-v) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 28; or, (2-vi) the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof; Preferably, the CDRs are defined by the Kabat, Chothia, AbM or IMGT numbering systems; Preferably, the variant has a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is monospecific. The antibody or antigen-binding fragment thereof of claim 1 , which is bispecific.

5. The antibody or antigen-binding fragment thereof of claim 4, further comprising an EGFR antigen-binding domain, wherein the EGFR antigen-binding domain is as defined in claim 2. The antibody or antigen-binding fragment thereof of claim 2 , which is bispecific.

7. The antibody or antigen-binding fragment thereof of claim 6, further comprising a HER3 antigen-binding domain, wherein the HER3 antigen-binding domain is as defined in claim 1.

8. A bispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain specific for HER3 and a second antigen-binding domain specific for EGFR, wherein: The first antigen binding domain comprises a first VL and a first VH, wherein the first VL is the VL defined in claim 1 and the first VH is the VH defined in claim 1; and / or The second antigen-binding domain comprises a second VL and a second VH, wherein the second VL is the VL defined in claim 2 and the second VH is the VH defined in claim 2.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is an IgG antibody; preferably, the IgG antibody is an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD or IgE antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, further comprising a CH (heavy chain constant region) and a CL (light chain constant region); Preferably, the CH is an IgG1 heavy chain constant region; Preferably, the CL is a kappa or lambda light chain constant region; Preferably, the CH comprises the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, and / or the CL comprises the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof; Preferably, the variant has a sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions; Preferably, the CL and / or the CH (e.g., CH1, Fc region) are altered (e.g., mutated) to promote dimerization (e.g., homodimerization or heterodimerization, such as promoting pairing of κ CL with CH1, and / or heterodimerization of the Fc region); Preferably, the CH comprises the amino acid sequence shown in SEQ ID NO: 8 or 9 or a variant thereof, and / or the CL comprises the amino acid sequence shown in SEQ ID NO: 10 or 11 or a variant thereof.

11. The antibody or antigen-binding fragment thereof according to any one of claims 4 to 10, wherein the antibody comprises peptide chain IA, peptide chain IB, peptide chain IC and peptide chain ID, wherein: The peptide chain IA comprises a first VL and CL, the peptide chain IB comprises a first VH and CH, the peptide chain IC comprises a second VH and CH, and the peptide chain ID comprises a second VL and CL.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the adjacent domains of the peptide chain IA are connected by or without a linker, the adjacent domains of the peptide chain IB are connected by or without a linker, the adjacent domains of the peptide chain IC are connected by or without a linker, and / or the adjacent domains of the peptide chain ID are connected by or without a linker; Preferably, each of the linkers is independently selected from a polypeptide linker, such as a rigid or flexible polypeptide linker; preferably, the polypeptide linker is a glycine-rich linker, such as (GGS) n 、(GGGGS) n or (GGGGA) n The linker shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, for example, SEQ ID NO: 36 or 37.

13. The antibody or antigen-binding fragment thereof according to claim 11 or 12, comprising: A peptide chain IA comprising the amino acid sequence shown in SEQ ID NO: 18; a peptide chain IB comprising the amino acid sequence shown in SEQ ID NO: 17; A peptide chain IC comprising the amino acid sequence shown in SEQ ID NO: 19 or 21; and A peptide chain ID comprising the amino acid sequence shown in SEQ ID NO: 20 or 22; Preferably, the antibody comprises: Peptide chain IA having the amino acid sequence set forth in SEQ ID NO: 18, peptide chain IB having the amino acid sequence set forth in SEQ ID NO: 17, peptide chain IC having the amino acid sequence set forth in SEQ ID NO: 19, and peptide chain ID having the amino acid sequence set forth in SEQ ID NO: 20; Alternatively, the peptide chain IA having the amino acid sequence shown in SEQ ID NO: 18, the peptide chain IB having the amino acid sequence shown in SEQ ID NO: 17, the peptide chain IC having the amino acid sequence shown in SEQ ID NO: 21, and the peptide chain ID having the amino acid sequence shown in SEQ ID NO:

22.

14. The antibody or antigen-binding fragment thereof according to any one of claims 4 to 10, wherein the antibody comprises peptide chain II-A and peptide chain II-B, wherein: The peptide chain II-A comprises a first VH, a CH, a second VH and a second VL, and the peptide chain II-B comprises a first VL and a CL. Alternatively, the peptide chain II-A comprises a second VH, a CH, a first VH and a first VL, and the peptide chain II-B comprises a second VL and a CL.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the peptide chain II-A comprises a first VH, a CH, a second VH, and a second VL from the N-terminus to the C-terminus, and the peptide chain II-B comprises a first VL and a CL from the N-terminus to the C-terminus; or, the peptide chain II-A comprises a second VH, a CH, a first VH, and a first VL from the N-terminus to the C-terminus, and the peptide chain II-B comprises a second VL and a CL from the N-terminus to the C-terminus.

16. The antibody or antigen-binding fragment thereof according to claim 14 or 15, wherein the adjacent domains of the peptide chain II-A are connected by a linker or not, and / or the adjacent domains of the peptide chain II-B are connected by a linker or not; Preferably, each of the linkers is independently selected from a polypeptide linker, such as a rigid or flexible polypeptide linker; preferably, the polypeptide linker is a glycine-rich linker, such as (GGS) n or (GGGGS) n The linker shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, for example, SEQ ID NO: 36 or 37.

17. The antibody or antigen-binding fragment thereof according to any one of claims 14 to 16, comprising: Peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 38; and, Peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 39; or, Peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO:40; and, peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO:41; Preferably, the antibody comprises: Peptide chain II-A having the amino acid sequence shown in SEQ ID NO: 38; and, Peptide chain II-B having the amino acid sequence shown in SEQ ID NO: 39; or, Peptide chain II-A having the amino acid sequence shown in SEQ ID NO:40; and, peptide chain II-B having the amino acid sequence shown in SEQ ID NO:41; Preferably, the antibody comprises two peptide chains II-A and two peptide chains II-B; preferably, the heavy chain constant regions of the two peptide chains II-A form a dimer.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein The antibody can also be detectably labeled, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.

19. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-18.

20. A vector comprising the nucleic acid molecule of claim 19; Preferably, the nucleic acid sequences encoding different peptide chains of the antibody or antigen-binding fragment thereof are located in the same or different vectors; Preferably, the vector is a cloning vector or an expression vector.

21. A host cell comprising the nucleic acid molecule of claim 19 or the vector of claim 20.

22. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the method comprises culturing the host cell according to claim 21 under suitable conditions to allow expression of the antibody or antigen-binding fragment thereof, and collecting the antibody or antigen-binding fragment thereof from the culture medium of the host cell.

23. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18.

24. The antibody drug conjugate of claim 23, further comprising a therapeutic agent, such as 1-20 therapeutic agents per molecule of the antibody drug conjugate, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

25. The antibody drug conjugate of claim 24, wherein the therapeutic agent is selected from the group consisting of a microtubule inhibitor and a DNA damaging agent; Preferably, the tubulin inhibitor is selected from auristatins (e.g., MMAE, MMAF or derivatives thereof), maytansines (e.g., maytansine, maytansinol, DM1, DM4 or derivatives thereof), taxanes (e.g., Taxol, Docetaxel, Cabazitaxel or derivatives thereof), vinca alkaloids (e.g., vinblastine, vincristine or derivatives thereof), eribulin or derivatives thereof, and colchicine or derivatives thereof; Preferably, the DNA damaging agent is selected from DNA alkylating agents (calicheamicin γ1l, N-acetyl-γ1I calicheamicin, anthramycin, PBD, dukamycin or derivatives thereof), DNA topoisomerase inhibitors (for example, camptothecin compounds (specifically, camptothecin, SN-38, Dxd, irinotecan, belotecan, topotecan, PNU-159682 or derivatives thereof), doxorubicin, daunorubicin, etoposide, mitoxantrone or derivatives thereof) and amanitin or derivatives thereof; Preferably, the therapeutic agent is 26. The antibody-drug conjugate of claim 24 or 25, wherein the antibody and therapeutic agent are linked via at least one linker; Preferably, the linker is cleavable or non-cleavable; Preferably, the cleavable linker is selected from protease-sensitive, pH-sensitive and glutathione-sensitive linkers; Preferably, the linker is selected from MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropionyl), Val-Cit (valine-citrulline), Val-Cit-NHCH2-, Val-Ala (valine-alanine), Val-Ala-NHCH2-, Ala-Phe (alanine-phenylalanine), Ala-Phe-NHCH2-, Gly-Gly-Phe-Gly (glycine-glycine-phenylalanine-glycine), Gly-Gly-Phe -Gly-NHCH2-, PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimidyl)-4-(pyridin-2-ylthio) pentanoate), 6-(2,5-dioxopyrrolidin-1-yl)-4-(pyridin-2-ylthio) hexanoate, 6-(2,5-dioxopyrrolidin-1-yl)-5-methyl-4-(pyridin-2-ylthio) hexanoate, SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate), and any combination thereof; Preferably, the linker is 27. The antibody drug conjugate of any one of claims 23 to 26, having the following structure: in, A is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18; p is an integer selected from 1-20, for example, an integer from 1-10 or 1-8, and further for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

28. An antibody-drug conjugate having the following structure: in, A is a bispecific antibody targeting EGFR and HER3, comprising: Peptide chain IA having the amino acid sequence set forth in SEQ ID NO: 18, peptide chain IB having the amino acid sequence set forth in SEQ ID NO: 17, peptide chain IC having the amino acid sequence set forth in SEQ ID NO: 19, and peptide chain ID having the amino acid sequence set forth in SEQ ID NO: 20; Alternatively, peptide chain IA having the amino acid sequence shown in SEQ ID NO: 18, peptide chain IB having the amino acid sequence shown in SEQ ID NO: 17, peptide chain IC having the amino acid sequence shown in SEQ ID NO: 21, and peptide chain ID having the amino acid sequence shown in SEQ ID NO: 22; Alternatively, a peptide chain IA having the amino acid sequence shown in SEQ ID NO: 38; and, a peptide chain IB having the amino acid sequence shown in SEQ ID NO: 39; Alternatively, a peptide chain IA having the amino acid sequence shown in SEQ ID NO: 40; and, a peptide chain IB having the amino acid sequence shown in SEQ ID NO: 41; p is an integer selected from 1-20, for example, an integer from 1-10 or 1-8, and further for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

29. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, the nucleic acid molecule according to claim 19, the vector according to claim 20, the host cell according to claim 21, or the antibody-drug conjugate according to any one of claims 23 to 28, and a pharmaceutically acceptable excipient; Preferably, the drug-antibody coupling ratio (DAR) of the pharmaceutical composition is 1-8, for example, 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5- 6, 1.5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 2-2.5, 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5-7, 2.5-7.5, 2. 5-8, 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3.5-4, 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 4-4.5, 4-5, 4-5.5, 4-6, 4-6.5, 4-7, 4-7.5, 4-8, 4.5-5, 4.5-5.5, 4.5-6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5.5-6, 5.5-6.5, 5.5-7, 5.5-7.5, 5.5-8, 6-6.5, 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-7.5, 7-8, 7.5-8.

30. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, the nucleic acid molecule according to claim 19, the vector according to claim 20, the host cell according to claim 21, the antibody-drug conjugate according to any one of claims 23 to 28, or the pharmaceutical composition according to claim 29 in the preparation of an anti-tumor drug; Preferably, the tumor overexpresses EGFR and / or HER3; Preferably, the tumor is selected from breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), melanoma, rectal cancer, liver cancer, pancreatic cancer, glioma, ovarian cancer, bladder cancer, cervical cancer, prostate cancer and head and neck cancer; Preferably, the tumor is a primary or metastatic tumor.

31. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, the nucleic acid molecule according to claim 19, the vector according to claim 20, the host cell according to claim 21, the antibody-drug conjugate according to any one of claims 23 to 28, or the pharmaceutical composition according to claim 29, for use in anti-tumor treatment; Preferably, the tumor overexpresses EGFR and / or HER3; Preferably, the tumor is selected from breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), melanoma, rectal cancer, liver cancer, pancreatic cancer, glioma, ovarian cancer, bladder cancer, cervical cancer, prostate cancer and head and neck cancer; Preferably, the tumor is a primary or metastatic tumor.

32. An anti-tumor method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, the nucleic acid molecule according to claim 19, the vector according to claim 20, the host cell according to claim 21, the antibody-drug conjugate according to any one of claims 23 to 28, or the pharmaceutical composition according to claim 29; Preferably, the tumor overexpresses EGFR and / or HER3; Preferably, the tumor is selected from breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), melanoma, rectal cancer, liver cancer, pancreatic cancer, glioma, ovarian cancer, bladder cancer, cervical cancer, prostate cancer and head and neck cancer; Preferably, the tumor is a primary or metastatic tumor.

33. A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, and optionally instructions for use and / or a drug delivery device.

34. A method for detecting the presence or level of EGFR and / or HER3 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and EGFR and / or HER3, and detecting formation of the complex; Preferably, the method is used to diagnose tumors, such as breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell lung carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), melanoma, rectal cancer, liver cancer, pancreatic cancer, glioma, ovarian cancer, bladder cancer, cervical cancer, prostate cancer and head and neck cancer; Preferably, the tumor is a primary or metastatic tumor; Preferably, the method comprises detecting the expression level of EGFR and / or HER3 in a test sample from a subject, and comparing the expression level with a reference value (e.g., a healthy control), wherein an increase in the expression level compared to the reference value is indicative of a tumor.

35. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 in the preparation of a kit for detecting the presence or level of EGFR and / or HER3 in a sample and / or diagnosing a tumor; Preferably, the tumor is selected from breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), melanoma, rectal cancer, liver cancer, pancreatic cancer, glioma, ovarian cancer, bladder cancer, cervical cancer, prostate cancer and head and neck cancer; Preferably, the tumor is a primary or metastatic tumor.

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