TW202540193A - Bispecific antibodies, antibody-drug conjugates and their applications - Google Patents

Abstract

It involves bispecific antibodies, antibody-drug conjugates and their applications, specifically bispecific antibodies targeting EGFR and HER3, antibody-drug conjugates and their applications in antitumor therapy.

Description

Bispecific antibodies, antibody-drug conjugates and their applications

This disclosure is based on and asserts priority over CN application No. 202410295797.6, filed on March 14, 2024, and CN application No. 202510289027.5, filed on March 11, 2025, the contents of which are incorporated herein as a whole.

This disclosure relates to the field of targeted therapy, specifically to bispecific antibodies targeting EGFR and HER3, antibody-drug conjugates, and their applications in antitumor therapy.

The epidermal growth factor receptor (EGFR) family of ErbB receptors, including EGFR, HER2, HER3, and HER4, plays a crucial role in tumorigenesis and tumor progression (Hynes NE et al. 2005). After binding to specific ligands, these receptors can form homodimers or heterodimers, activating various downstream signaling pathways and stimulating cell division and proliferation. Dysregulation of EGFR mutations, amplification, or overexpression is closely related to adverse clinical responses and prognosis (Mishra, R. et al. 2017). Inhibitors targeting ErbB, including monoclonal antibodies and small molecule tyrosine kinase inhibitors, have been approved for the treatment of various types of cancer, such as lung cancer, breast cancer, squamous cell carcinoma of the head and neck, colon cancer, and gastric cancer (Wu, Q., et al., 2022). Despite the success of ErbB inhibitors, many patients develop 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). Overexpression of HER3 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).

Therefore, there is a need to develop a bispecific antibody that is highly specific, effective, and easy to prepare, targeting both EGFR and HER3.

This 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 heterodimer via HC-HC and HC-LC charge interactions, and then binds to a toppo isomerase 1 inhibitor (TOP1i) via a cleavable linker to form the ADC. Optimization of the EGFR and HER3 affinity of the bispecific antibody improves the ADC therapeutic window, enhances safety, and demonstrates potent antitumor efficacy in various tumor models, showing great potential as a safer and more effective ADC therapy for cancer treatment. Antibody antigen-binding domain

In a first aspect, this disclosure provides an antibody comprising a HER3 antigen-binding domain or an antigen-binding fragment thereof, said HER3 antigen-binding domain comprising a VH (heavy chain variable region) and a VL (light chain variable region), wherein the VH comprises: (1-i) CDRs contained in the heavy chain as shown in SEQ ID NO: 15; (1-ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 30, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32; or, (1-iii) an amino acid sequence as shown in SEQ ID NO: 4 or a variant thereof; and/or, the VL comprises: (1-iv) CDRs contained in the light chain as shown in SEQ ID NO: 16; (1-v) CDRs contained in the light chain as shown in SEQ ID NO: 16; LCDR1 with the amino acid sequence shown in SEQ ID NO: 34, LCDR2 with the amino acid sequence shown in SEQ ID NO: 35, and LCDR3 with 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.

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

In some embodiments, the HER3 antigen-binding domain comprises VH and VL, and according to the IMGT numbering system, the VH comprises: (1-i) CDRs contained in the heavy chain as shown in SEQ ID NO: 15; (1-ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 30, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 containing 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) CDRs contained in the light chain as shown in SEQ ID NO: 16; (1-v) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and VL containing the amino acid sequence shown in SEQ ID NO: 35. LCDR3 of the amino acid sequence shown in SEQ ID NO: 35; or, (1-vi) the amino acid sequence or a variant thereof as shown in SEQ ID NO: 5.

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

In some implementations, the substitution is a conservative substitution.

In some embodiments, the antibody or its antigen-binding fragment is single-specific.

In a second aspect, this disclosure provides an antibody comprising an EGFR antigen-binding domain or an antigen-binding fragment thereof, the EGFR antigen-binding domain comprising VH and VL, wherein the VH comprises: (2-i) CDRs contained in the heavy chain as shown in SEQ ID NO: 12 or 14; (2-ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 23, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 24 or 29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 25; or, (2-iii) an amino acid sequence as shown in SEQ ID NO: 1 or 3 or a variant thereof; and/or, the VL comprises: (2-iv) CDRs contained in the light chain as shown in SEQ ID NO: 13; (2-v) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 26, and VL containing the amino acid sequence shown in SEQ ID NO: 13; LCDR2 containing the amino acid sequence shown in SEQ ID NO: 27, and LCDR3 containing 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.

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

In some embodiments, the EGFR antigen-binding domain comprises VH and VL, and according to the IMGT numbering system, the VH comprises: (2-i) CDRs contained in the heavy chain as shown in SEQ ID NO: 12 or 14; (2-ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 23, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 24 or 29, and HCDR3 containing 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) CDRs contained in the light chain as shown in SEQ ID NO: 13; (2-v) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 25; LCDR2 containing the amino acid sequence shown in SEQ ID NO: 27, and LCDR3 containing 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.

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

In some implementations, the substitution is a conservative substitution.

In some embodiments, the antibody or its antigen-binding fragment is single-specific.

The antibody or antigen-binding fragment thereof described in either the first or second aspect of this disclosure may further contain a second antigen-binding domain, and the antibody or antigen-binding fragment thereof may be bispecific.

In some embodiments, the antibody or its antigen-binding fragment according to any of the first aspects of this disclosure further includes an EGFR antigen-binding domain. In some embodiments, the EGFR antigen-binding domain is as defined in any of the second aspects.

In some embodiments, the antibody or antigen-binding fragment thereof described in any of the second aspects of this disclosure further includes a HER3 antigen-binding domain. In some embodiments, the HER3 antigen-binding domain is as defined in any of the first aspects.

Accordingly, in a third aspect, this disclosure provides a bispecific antibody or an antigen-binding fragment thereof, said antibody comprising a first antigen-binding domain specific to HER3 and a second antigen-binding domain specific to EGFR, wherein the first antigen-binding domain comprises a first VL and a first VH, the first VL being a VL contained in the HER3 antigen-binding domain and the first VH being a VH contained in the HER3 antigen-binding domain; and/or the second antigen-binding domain comprises a second VL and a second VH, the second VL being a VL contained in the EGFR antigen-binding domain and the second VH being a VH contained in the EGFR antigen-binding domain. (Stationary region )

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.

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

In some implementations, CH is the IgG1 heavy chain stationary region.

In some embodiments, the CL is a κ or λ light chain stationary region. In some embodiments, the CL is a κ light chain stationary region.

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

In some embodiments, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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). In some embodiments, the substitution is a conservative substitution.

In some embodiments, the CL and/or the CH (e.g., CH1, Fc region) are modified (e.g., mutated) to promote dimerization (e.g., homodimerization or heterodimerization), such as promoting pairing of κCL with CH1, and/or heterodimerization of the Fc region. In some embodiments, the Fc region of the antibody contains modifications that form a knock-in-hole structure.

In some embodiments, CH comprises an amino acid sequence as shown in SEQ ID NO: 8 or 9, or a variant thereof, and/or CL comprises an amino acid sequence as shown in SEQ ID NO: 10 or 11, or a variant thereof. Bispecific antibody

The disclosed bispecific antibody can be designed to consist of two different but complementary heavy and/or light chains, forming a heterodimeric structure. Each chain contains a binding site targeting one antigen, and the structure of the bispecific antibody is stabilized through inter-chain interactions. To avoid antibody chain mismatch, various techniques known in the art can be used to ensure correct inter-chain pairing, such as the "Knob-in-Hole" method, electrostatic reversal, CrossMab technology, and Orthogonal Fab interface technology.

In some embodiments, the bispecific antibody comprises peptide chain I-A, peptide chain I-B, peptide chain I-C, and peptide chain I-D, wherein peptide chain I-A comprises a first VL and CL, peptide chain I-B comprises a first VH and CH, peptide chain I-C comprises a second VH and CH, and peptide chain I-D comprises a second VL and CL.

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

In some embodiments, adjacent domains of peptide chains I-A are connected by or without linkers, adjacent domains of peptide chains I-B are connected by or without linkers, adjacent domains of peptide chains I-C are connected by or without linkers, and/or adjacent domains of peptide chains I-D are connected by or without linkers.

In some embodiments, each of the linkers is independently selected from polypeptide linkers, such as rigid or flexible polypeptide linkers; preferably, the polypeptide linkers are glycine-rich linkers, for example, linkers having the form (GGS) _n , (GGGGS) _n , or (GGGGA) _n , where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, SEQ ID NO: 36 or 37.

In some embodiments, the bispecific antibody or its antigen-binding fragment comprises: peptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 18; peptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 17; peptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 19 or 21; and peptide chain I-D comprising the amino acid sequence shown in SEQ ID NO: 20 or 22.

In some embodiments, the bispecific antibody comprises: peptide chain I-A having the amino acid sequence shown in SEQ ID NO: 18, peptide chain I-B having the amino acid sequence shown in SEQ ID NO: 17, peptide chain I-C having the amino acid sequence shown in SEQ ID NO: 19, and peptide chain I-D having the amino acid sequence shown in SEQ ID NO: 20; or, peptide chain I-A having the amino acid sequence shown in SEQ ID NO: 18, peptide chain I-B having the amino acid sequence shown in SEQ ID NO: 17, peptide chain I-C having the amino acid sequence shown in SEQ ID NO: 21, and peptide chain I-D having the amino acid sequence shown in SEQ ID NO: 22.

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

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

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

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

In some embodiments, adjacent domains of peptide chain II-A are connected by or without linkers, and/or adjacent domains of peptide chain II-B are connected by or without linkers.

In some embodiments, each of the linkers is independently selected from polypeptide linkers, such as rigid or flexible polypeptide linkers; preferably, the polypeptide linkers are glycine-rich linkers, for example, linkers having the form (GGS) _n or (GGGGS) _n , where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, for example, SEQ ID NO: 36 or 37.

In some embodiments, the antibody or its antigen-binding fragment comprises: a peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 38; and a peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 39; or, 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.

In some embodiments, the antibody comprises: a peptide chain II-A having the amino acid sequence shown in SEQ ID NO: 38; and a peptide chain II-B having the amino acid sequence shown in SEQ ID NO: 39; or, 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.

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.

The bispecific antibody disclosed herein possesses at least one of the following characteristics: selective binding to EGFR/HER3 double-positive cells, maintaining a binding capacity comparable to or even higher than that of corresponding single-specific antibodies; specifically binding to EGFR/HER3 double-positive cells and mediating antibody internalization, with an internalization effect significantly superior to that of corresponding single-specific antibodies; and enhanced tumor suppressor activity, including EGF-EGFR blockade, NRG1-HER3 blockade, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or complement-dependent cytotoxicity (CDC) activity. Detectable markers and detection applications are also available .

In some embodiments, the antibody also carries a detectable marker.

The detectable markers described herein can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, basic phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., ^3H , ^125I , ^35S , ^14C , or ^32P ), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa). 750), acridine ester compounds, magnetic beads (e.g., Dynabeads®), pyrometric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioisotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the disclosed antibody or its antigen-binding fragment via linkers of different lengths to reduce potential steric hindrance.

The disclosed antibody or its antigen-binding fragment can specifically bind to EGFR and/or HER3, thereby enabling the detection of the presence or level of EGFR and/or HER3 in a sample. Therefore, in another aspect, this disclosure provides a kit comprising the disclosed antibody or its antigen-binding fragment. In some embodiments, the disclosed antibody or its antigen-binding fragment carries a detectable label.

In another aspect, this disclosure provides a method for detecting the presence or level of EGFR and/or HER3 in a sample, which includes steps using an antibody or an antigen-binding fragment of the present disclosure.

In some embodiments, the method further includes using the antibody or antigen-binding fragment thereof bearing the detectable marker to detect the disclosed antibody or antigen-binding fragment thereof. The method may be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, rather than a sample from a patient).

In some embodiments, the method includes contacting the sample with the antibody or antigen-binding fragment of the present disclosure, and detecting the formation of the complex, under conditions that allow the antibody or its antigen-binding fragment to form a complex with EGFR and/or HER3.

Given the significant differences in EGFR and/or HER3 expression levels between normal tissues and some cancers, tumors can be diagnosed by detecting the levels of EGFR and/or HER3 in a sample. Therefore, in some embodiments, the method is used to diagnose tumors such as breast cancer, colon cancer, gastric cancer, lung cancer (e.g., 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.

In some embodiments, the method includes detecting the expression levels of EGFR and/or HER3 in a test sample from a subject, and comparing the expression levels to a reference value (e.g., a healthy control), wherein an increase in the expression level compared to the reference value is an indicator of tumor.

In another aspect, the use of the antibodies or antigen-binding fragments thereof disclosed herein in the preparation of kits for detecting the presence or level of EGFR and/or HER3 in a sample and/or diagnosing tumors is provided. Nucleic acids, vectors, host cells, and expression methods are also included.

In another aspect, this disclosure provides a nucleic acid molecule that encodes any of the antibodies described above. The nucleic acid molecule can be obtained using methods known in the art, such as isolation from phage display libraries, yeast display libraries, immunized animals, immortalized cells (e.g., mouse B-cell hybridoma cells, EBV-mediated immortalized B cells), or chemical synthesis. The nucleic acid molecule can be codon-optimized for the host cells used for expression.

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

In some embodiments, the nucleic acid molecule is prepared as a recombinant nucleic acid. In some embodiments, the nucleic acid molecule is selectively implanted into an expression vector. The expression vector may further contain additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The recombinant nucleic acid containing the nucleic acid can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR)), etc. (see Sambrook, J., EF 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 contain a polynucleotide sequence encoding a polypeptide or protein that facilitates the detection and/or isolation of the expressed antibody or antigen-binding fragment. Such peptides or proteins may include, but are not limited to, affinity tags (e.g., biotin, polyhistamine tags (His ₆ ), or glutathione S-transferase (GSH) tags), peptides containing protease cleavage sites, and reporter proteins (e.g., 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 still other embodiments, the expression vector is a phage vector or a phage particle vector.

In another aspect, this disclosure provides a host cell comprising the aforementioned nucleic acid molecule or vector.

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

Bacteria (e.g., Escherichia coli BL21(DE3)) are particularly favorable 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 antibody expression.

In another aspect, this disclosure provides a method for generating the antibody or an antigen-binding fragment thereof in a host cell, wherein the method comprises the steps of: (1) transforming the host cell with at least one of the nucleic acid molecules or expression vectors described herein; (2) culturing the transformed host cell under suitable conditions to allow expression of the nucleic acid molecule or expression vector; and (3) isolating and purifying the antibody or an antigen-binding fragment thereof from the host cell or culture medium.

In some embodiments, the method includes culturing the host cells under suitable conditions to allow expression of the antibody or its antigen-binding fragment, and collecting the antibody or its antigen-binding fragment from the culture medium of the host cells.

In some implementations, the host cell also contains a companion plasmid, which 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. Antibody-drug conjugates (ADCs)

In another aspect, this disclosure provides an antibody-drug conjugate comprising any of the antibodies described above or an antigen-binding fragment thereof.

In some embodiments, the antibody-drug conjugate further includes a therapeutic agent.

In some embodiments, each molecule of the antibody-drug conjugate contains 1-20 of the therapeutic agents, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, at least 1, 2, 3, or 4 therapeutic agents. In some embodiments, the therapeutic agents are selected from microtubule inhibitors and DNA-damaging drugs.

In some embodiments, the tubulin inhibitor is selected from olistatin compounds (e.g., MMAE, MMAF, or derivatives thereof), maytansine compounds (e.g., maytansine, maytanol, DM1, DM4, or derivatives thereof), taxanes (e.g., taxol, docetaxel, carbazitaxel, or derivatives thereof), vinca alkaloids (e.g., vinca alkaloid, vincristine, or derivatives thereof), eribulin, and colchicine, or derivatives thereof.

In some embodiments, the DNA damaging agent is selected from DNA alkylating agents (cachicin γ1l, N-acetylated γ1I cachicin, ampicillin, PBD, ducachicin or derivatives thereof), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically, camptothecin, SN-38, Dxd, irinotecan, belotecone, topoisomerone, PNU-159682 or derivatives thereof), amycin, daunorubicin, etoposide, mitoxantrone or derivatives thereof), and muscarinic acid or derivatives thereof.

In some implementation schemes, the treatment agent is .

In some embodiments, the antibody and the treatment are linked by at least one linker. In some embodiments, each of the treatments is linked to the constant region of the antibody. In some embodiments, each of the treatments is linked to the Fc region of the antibody. In some embodiments, the treatment is linked to the Fc region of the antibody via the linker.

In some embodiments, the treatment agent is linked to the linker via its reactive groups (e.g., hydroxyl groups).

In some embodiments, the connector may be cuttable or incuttable.

In some embodiments, the cleavable linker is selected from protease-sensitive, pH-sensitive, and glutathione-sensitive linkers.

In some embodiments, the linker is selected from _-NHCH2- , MC (6-maleiminohexyl), MCC (maleiminomethylcyclohexane-1-carboxylate), MP (maleiminopropyl), Val-Cit (citrulline), Val-Cit-NHCH2-, Val-Ala (citrulline), 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-(succinimino)-4-(pyridin-2-ylthio)valerate), 6-(2,5-dioxopyrrolidone-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidone-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate), SIAB (N-succinimino-(4-iodoacetylated)aminobenzoate) and any combination thereof.

In some embodiments, the linker is selected from MC (6-maleiminohexyl), MCC (maleiminomethylcyclohexane-1-carboxylate), MP (maleiminopropyl), Val-Cit (citrulline), Val-Ala (villiine-alanine), Ala-Phe (alanine-phenylalanine), Gly-Gly-Phe-Gly (glycine-glycine-phenylalanine-glycine), and Gly-Gly-Phe-Gly-NHCH _2. - PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimino)-4-(pyridin-2-ylthio)valerate), 6-(2,5-dioxopyrrolidone-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidone-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate) or SIAB (N-succinimino-(4-iodoacetylated)aminobenzoate) and any combination thereof.

In some embodiments, the connector is Gly-Gly-Phe-Gly-NHCH _2- .

In some embodiments, the connector is .

In some embodiments, the antibody-drug conjugate has the following structure: Wherein, A is any of the antibodies or antigen-binding fragments described above; p is an integer selected from 1 to 20, for example, an integer from 1 to 10 or 1 to 8, or for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In another aspect, this disclosure provides an antibody-drug conjugate having the following structure: Wherein, A is a bispecific antibody targeting EGFR and HER3, comprising: a peptide chain IA having the amino acid sequence shown in SEQ ID NO: 18, a peptide chain IB having the amino acid sequence shown in SEQ ID NO: 17, a peptide chain IC having the amino acid sequence shown in SEQ ID NO: 19, and a peptide chain ID having the amino acid sequence shown in SEQ ID NO: 20; or, a peptide chain IA having the amino acid sequence shown in SEQ ID NO: 18, a peptide chain IB having the amino acid sequence shown in SEQ ID NO: 17, a peptide chain IC having the amino acid sequence shown in SEQ ID NO: 21, and a peptide chain ID having the amino acid sequence shown in SEQ ID NO: 22; or, 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; or, a peptide chain having the amino acid sequence shown in SEQ ID NO: 18; or, a peptide chain having the amino acid sequence shown in SEQ ID NO: 19 ... Peptide chain IA having the amino acid sequence shown in SEQ ID NO: 40; and 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. Pharmaceutical composition.

In another aspect, this disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any of the preceding claims, a nucleic acid molecule, a vector, a host cell or antibody-drug conjugate, and a pharmaceutically acceptable excipient.

The excipient may be an excipient described in the 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, and colorants.

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

In some embodiments, the drug-antibody conjugation ratio (DAR) of the drug combination 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. Application

In another aspect, this disclosure provides the use of any of the antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells, antibody-drug conjugates or drug compositions described in any of the preceding claims in the preparation of antitumor drugs.

In another aspect, this disclosure provides antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells, antibody-drug conjugates or drug combinations described in any of the preceding claims for use in antitumor purposes.

In another aspect, this disclosure provides an antitumor method comprising administering to a subject in need a therapeutically effective amount of any of the preceding claims an antibody or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate, or a drug combination.

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

In some embodiments, the tumor is selected from breast cancer, colon cancer, stomach cancer, lung cancer (e.g., 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. Terminology Definitions

In this document, unless otherwise stated, scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the procedures used in this document, such as molecular genetics, nucleic acid chemistry, cell culture, biochemistry, and cell biology, are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

As used herein, the singular forms “a kind” and “an” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the terms “including,” “containing,” “having,” “containing,” or their variations are open-ended, not exclusive or exhaustive.

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

As used in this article, the terms "EGFR," "ErbB-1," and "HER1" are used interchangeably. EGFR is a member of the ErbB/EGFR receptor tyrosine kinase family, widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, and keratinocytes. The EGFR signaling pathway plays a crucial 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 includes 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 transforms from a monomer into a dimer. Dimerization includes both the binding of two identical receptor molecules (homogeneous dimerization) and the binding of different members of the human EGF-associated receptor (HER) tyrosine kinase family (heterogeneous dimerization). EGFR dimerization can activate its intracellular kinase pathways, and receptor autophosphorylation can lead to downstream phosphorylation, including the MAPK, Akt, and JNK pathways, inducing cell proliferation.

As used herein, the terms “HER3” and “ErbB-3” are used interchangeably. HER3 is a 148kD transmembrane receptor of the ErbB/EGFR receptor tyrosine kinase family, but lacks intrinsic kinase activity. ErbB receptors form homodimeric and heterodimeric complexes, influencing cellular and organ physiology by mediating ligand-dependent (and in some cases ligand-independent) activation of multiple signaling pathways. Heterodimers containing ErbB3 in tumor cells (e.g., ErbB2/ErbB3) have been shown to be the most mitogenic and oncogenic receptor complexes in the ErbB family. Upon binding to its physiological ligand, the ErbB3 receptor forms a dimer with other ErbB family members, primarily ErbB2. ErbB3/ErbB2 dimerization leads to transphosphorylation of tyrosine residues contained in the cytoplasmic tail of ErbB3. Phosphorylation at these sites creates SH2 binding sites for SH2-containing proteins, including PI3 kinase. Therefore, the ErbB3-containing heterodimeric complex is a potent activator of AKT because ErbB3 possesses six tyrosine phosphorylation sites with YXXM motifs, which, upon phosphorylation, serve as preferred binding sites for phosphatidylinositol 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.

As used herein, the terms “inhibition” or “blockage” refer to any statistically significant reduction in biological activity, including complete blockage of activity. For example, “inhibition” can refer to a reduction in biological activity of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., the binding molecule and the target molecule), such as the reaction between an antibody and its target antigen. The binding affinity between two molecules can be described by the _KD value. The _KD value is the dissociation constant obtained by the ratio of kd (the dissociation rate of the specific binding molecule-target molecule interaction; also known as koff) to ka (the binding rate of the specific binding molecule-target molecule interaction; also known as kon), or kd/ka expressed as molar concentration (M). The smaller the _KD value, the tighter the binding between the two molecules and the higher the affinity. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to the antigen with a _KD value 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 less. The _KD value can be determined by methods well known in the art, for example, by measuring it in a BIACORE instrument using surface plasma resonance (SPR).

As used herein, the terms “antibody” and “monoclonal antibody” refer to immunoglobulin molecules that are typically composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Each chain has a variable region, called 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. There are five major classes (or isotypes) of heavy chains in mammalian immunoglobulins, 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 a primary antibody produced by birds and reptiles, 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 κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, corresponding to the five isotypes of the aforementioned antibodies: IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids; the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a variable region (VH) and a constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain does not directly participate in the binding of antibodies to antigens, but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

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

As used herein, the term “antibody-dependent cell-mediated cytotoxicity (ADCC)” refers to a form of cytotoxicity in which Ig binds specifically to antigen-attached target cells by binding to Fc receptors (FcRs) present on cytotoxic cells (such as natural killer (NK) cells, neutrophils, or macrophages), and then kills the target cells by secreting cytotoxins.

The variable region of an antibody comprises a framework region (FR) and hypervariable regions (HVR), collectively known as the complementarity determining region (CDR). The CDR is primarily responsible for binding to epitopes of the antigen. VH and VL are composed of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus to the carboxyl terminus. The allocation of amino acids in various regions or structural domains can follow the definitions in 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 described herein can be determined according to various numbering systems known in the art. In some embodiments, the CDRs contained in the antibodies or antigen-binding fragments are preferably determined using the Kabat, AbM, Chothia, or IMGT numbering systems.

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

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

As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH₂ _- VL-linker-VH-COOH or _NH₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variations thereof. For example, a connector with the amino acid sequence (GGGGS) ₄ can be used, but variations thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other connectors available for use in this 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 exist between the VH and VL of the scFv.

As used herein, the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains; the term "F(ab') ₂ fragment" refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on hinge regions; the term "Fab'fragment" refers to the fragment obtained by reducing the disulfide bonds of the two heavy chain fragments in the F(ab') ₂ fragment, consisting of a complete light and heavy chain Fd fragment (consisting of VH and CH1 domains); and the term "Fd" refers to an antibody fragment consisting of VH and CH1 domains.

As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, referring 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 bound by a full-length antibody. Single-domain antibodies are also known as nanobodies.

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

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

As used in this article, the term "conservative variant" refers to a protein containing a conserved amino acid substitution that does not substantially affect or reduce the protein's affinity.

Conserved amino acid substitutions of functionally similar amino acids are well known to those skilled in the art. The following six groups of amino acids are considered to be examples of conserved substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) aspartamide (N), glutamylamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), volcanic acid (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

As used in this article, amino acids may be represented by their commonly known three-letter symbols or by single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The specific abbreviations are as follows: alanine (Ala; A), aspartic acid (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamylamine (Gln; Q), glycine (Gly; G), histamine (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 valence (Val; V). Similarly, nucleotides are represented by their universally accepted single-letter codes.

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

Knob-in-hole techniques are described, for example, in 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, this method involves introducing a protrusion (“knob”) at the interface of a first polypeptide and a corresponding cavity (“mortise”) at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and inhibit homodimer formation. 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). Compensating cavities of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). The protrusions and cavities can be prepared by altering the nucleic acids encoding the polypeptide, for example, through site-specific mutagenesis or peptide synthesis. In a specific embodiment, the pestle modification comprises an amino acid substitution of T366W in one of the two subunits of the Fc domain, while the mortar modification comprises an amino acid substitution of T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit of the Fc domain containing the pestle modification further comprises an amino acid substitution of S354C, while the subunit of the Fc domain containing the mortar modification further comprises an amino acid substitution of Y349C. The introduction of these two cysteine residues results in the formation of disulfide bridges between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). The amino acid residues in the Fc region are numbered 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, the term "subunit" of the Fc domain refers to one of the two polypeptides forming the dimer Fc domain, namely the polypeptide containing the C-terminal constant region of the immunoglobulin heavy chain, which is capable of stable autoreplication. For example, the subunits of the IgG Fc domain contain the IgG CH2 constant domain and the IgG CH3 constant domain.

In alternative embodiments, modifications that promote heterodimerization of two distinct polypeptide chains include modifications that mediate electrostatic reversal effects, such as those described in WO 2009/089004. Typically, this method involves substituting one or more amino acid residues at the interface of the two polypeptide chains with charged amino acid residues, making homodimerization electrostatically unfavorable but heterodimerization electrostatically favorable.

As used herein, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, which may typically include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

As used in this article, the term "isolated" means that a substance (such as a nucleic acid molecule or polypeptide) is isolated from its source or environment, that is, it essentially does not contain any other components.

As used herein, the term "vector" is a medium used to introduce foreign nucleic acids into host cells, whereby the foreign nucleic acids are amplified or expressed when the vector is transformed into a suitable host cell. Vectors typically remain free, but can be designed to integrate genes or portions thereof into chromosomes of the genome. In this paper, the definition of vectors encompasses plasmids, linearized plasmids, viral vectors, granules, phage vectors, phage particles, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc.

As used herein, the term "expression vector" refers to a vector capable of expressing DNA operatively linked to regulatory sequences (such as promoters, ribosome-binding sites) that influence DNA expression. Regulatory sequences may include promoter and terminator sequences, and optionally may include replication origins, selection markers, enhancers, polyadenylation signals, etc. Expression vectors may be plasmids, phage vectors, recombinant viruses, or other vectors that, when introduced into a suitable host cell, result in the expression of selected DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and/or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

As used herein, the term "host cell" refers to the cell used to receive, maintain, replicate, or amplify a vector. Host cells can also be used to express nucleic acids or polypeptides encoded by the vector. Host cells can be eukaryotic or prokaryotic cells.

As used herein, the terms “subject,” “patient,” or “individual” include both mammals and non-mammalians. Mammals can be any member of the class Mammalia, including but not limited to humans; non-human primates such as chimpanzees, apes, or other monkeys; livestock such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs (or canines), and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; and so on. Non-mammalians can include birds, fish, etc. In some protocols, subjects can be mammals. In some protocols, subjects can be humans. In some cases, humans can be adults. In some cases, humans can be children. In some cases, humans can be 0-17 years of age. In some cases, humans can be 18-130 years of age. In some cases, the subject may be male. In some cases, the subject may be female. In some cases, the subject has been diagnosed with or is suspected of having a certain disease. In some cases, the disease is cancer. The subject may be a patient or an individual. In some cases, the terms subject, patient, or individual may be used interchangeably.

As used herein, the terms “treatment,” “management,” “improvement,” or “relief” include relieving or reducing the symptoms of a disease, inhibiting the disease (e.g., preventing its progression), alleviating the disease, causing the disease to subside, relieving the symptoms caused by the disease, or stopping the symptoms of the disease. The terms “treatment,” “management,” “improvement,” or “relief” may further include obtaining therapeutic benefits. Therapeutic benefits may refer to the eradication of the treated disease. Additionally, therapeutic benefits may also be achieved by eradicating one or more physiological symptoms associated with the treated disease, resulting in observable improvement in the subject, although in some implementation protocols the subject may still suffer from the underlying disease.

As used herein, the terms "effective dose" and "therapeutic effective dose" refer to an adequate amount of medication administered that will at least partially relieve the symptoms of the treated disease. Dosage regimens can be adjusted to provide the optimal desired response. For example, a single bolus injection may be administered, or several fractions may be given over time, or the dose may be reduced or increased proportionally as needed. It should be noted that dose values can vary depending on the type and severity of the disease to be alleviated and may include single or multiple doses. To further understand, for any given individual, the specific dosing regimen should be adjusted over time based on individual needs and the drug's instructions or the clinician's professional judgment. Generally, the effective dose is approximately 0.0001 to approximately 50 mg per kg of body weight per day, for example, approximately 0.01 to approximately 10 mg/kg/day (single or divided doses). For a 70 kg person, this would total approximately 0.007 mg/day to approximately 3500 mg/day, for example, approximately 0.7 mg/day to approximately 700 mg/day. In some cases, dose levels not exceeding the lower limit of the aforementioned range may be sufficient, while in others, larger doses may be used without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses administered throughout the day. Beneficial Effects of the Invention

This disclosure provides a humanized bispecific antibody against EGFR and HER3 and its antibody-drug conjugate, wherein the antibody exhibits higher binding activity against EGFR and HER3-duplicated cancer cells. The antibody-drug conjugate is obtained by heterodimerization under HC-HC and HC-LC charge, followed by binding to a toppo isomerase 1 inhibitor (TOP1i) via a cleavable linker. Optimization of the antibody's affinity for EGFR and HER3 improves the therapeutic window of the antibody-drug conjugate, enhances its safety profile, and demonstrates potent antitumor efficacy in various tumor models, potentially serving as a safer and more effective ADC for cancer treatment.

The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially. Example 1: Selection and expression of EGFR×HER3 bispecific antibodies and preparation of antibody-drug conjugates 1. Selection and expression of antibodies 1.1. Expression and purification of antibodies

The antibody of this invention was expressed by transfection using HEK293 cells as the vector. The cell density was adjusted to 2.5 × ^10⁶ cells/ml one day before transfection, and diluted to 3.0 × ^10⁶ cells/ml the next day before transfection. MEM medium was used as the transfection buffer, and the antibody plasmid was transfected into HEK293 cells at a PEI:plasmid ratio of 3:1. After 5 days of culture, the cell supernatant was collected. The target protein was purified using a Protein A column. First, 10 column volumes of PBS were added to the column to equilibrate it. Then, the cell supernatant was added to the gravity column, allowing it to flow through by gravity. Unbound impurities were then washed away with 20 column volumes of PBS. Finally, the target protein was obtained by elution with 3-5 column volumes of elution buffer (0.1M sodium citrate, pH 3.2), and collected using neutralization buffer (1M Tris, pH 8.54). 1.2. Cell Line Selection and Construction

The vectors pCHO2.0-GS-Puro-H1-L1, containing both the heavy and light chain genes of the anti-EGFR antibody, and pCHO2.0-GS-Puro-H2-L2, containing both the heavy and light chain genes of the anti-HER3 antibody, were co-transfected into CHOS-ADP-FUT8-KO host cells using electroporation. High-yielding minipools were obtained through pressure selection using Puromycin and MSX. Following a round of limited dilution and single-strain identification, high-yielding and stable selected cell lines were obtained. Cell culture...

Using Dynamis AGT Medium as the culture medium, the inoculation density was (1.0±0.2) × ^10⁶ cells/ml. On days 3, 5, 7, 9, and 11, 5.0±0.5% (w/w) of the initial culture weight of Cell Boost 7a powder supplement (purchased from Cytiva, product name SH31026.02, batch number MF29430725A) and 0.5±0.05% (w/w) of the initial culture weight of Cell Boost 7b powder supplement (purchased from Cytiva, product name SH31027.02, batch number MF29442466B) were added, respectively. Dissolved oxygen was set at 40%, and the initial culture temperature was 36.5℃, decreasing to 33.0℃ on day 4. Daily, based on glucose concentration monitoring results, 300 g/kg glucose concentrate was added to maintain a glucose concentration of 6.0 g/L in the cell slurry, except on the day of harvest. Culture was terminated on day 14 or when cell viability fell below 80%. During culture, cell density and viability were monitored using Vicell (Beckman Biotechnology). From day 7 onwards, antibody production was monitored daily using Cedex (Roche Biotechnology). Quality assessment of products from stable cell lines was conducted.

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℃, flow rate: 0.5 ml/min, detection time: 30 min; TSKgel G3000SWXL column. The heavy-light chain pairing of the protein was determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). The instruments used were a Vanquish UHPLC (Thermo) liquid chromatography system, a Q Exactive (Thermo) mass spectrometer, and a Waters ACQUITY UPLC BEH C4 column, 2.1 mm × 100 mm. Take 50 μg of sample, dilute with ultrapure water to 25 μl, centrifuge, transfer 20 μl of sample to a sample vial, inject 5 μl, and analyze the complete molecular weight using LC-MS. The chromatographic conditions were: column temperature: 80℃; 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). The mass spectrometry parameters were: ESI ion source: ion transfer tube temperature 320℃, voltage 3.8 kV, gas flow rate 36 L/min; mode: positive ion Full MS; resolution: 17500; scan range: 600-4000 m/z.

1.3. In this embodiment, five EGFR×HER3 bispecific antibodies, two anti-EGFR monoclonal antibodies and one HER3 monoclonal antibody were constructed, namely: Anti-EGFR-01×HER3: composed of four polypeptide chains, the structural diagram of which is shown in Figure 1.

Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 17, which includes 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 introducing the CH3 Knob mutation and the CH1/CL preferred mutation CH SET1 (SEQ ID NO: 8).

Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 18, which includes the light chain variable region amino acid sequence of the anti-HER3 monoclonal antibody Patritumab (SEQ ID NO: 5), and the introduction of the CH1/CL-preferred mutation CL SET1 (SEQ ID NO: 10) into the human κ light chain constant region (CL) amino acid sequence at the C-terminus of the VL amino acid sequence.

Peptide chain #3 has the amino acid sequence shown in SEQ ID NO: 19, which includes 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 introducing the CH3 Hole mutation and the CH1/CL preferred mutation CH SET2 (SEQ ID NO: 9).

Peptide chain #4 has the amino acid sequence shown in SEQ ID NO: 20, which includes the light chain variable region amino acid sequence of the anti-EGFR monoclonal antibody Zalutumumab (SEQ ID NO: 2), and the introduction of the CH1/CL-preferred mutation CL SET2 (SEQ ID NO: 11) into the human κ light chain constant region (CL) amino acid sequence at the C-terminus of the VL amino acid sequence.

Anti-EGFR-03×HER3: Composed of 4 polypeptide chains, its structural diagram is shown in Figure 1.

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

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

Peptide chain #3 has the amino acid sequence shown in SEQ ID NO: 21, which includes the heavy chain variable region amino acid sequence of the anti-EGFR monoclonal antibody EGFR-03 (SEQ ID NO: 3) and the human IgG1 amino acid sequence introducing the CH3 Hole mutation and the CH1/CL preferred mutation CH SET2 (SEQ ID NO: 9).

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

Anti-EGFR-01×HER3(2+2): Composed of two polypeptide chains, the structural diagram of which is shown in Figure 1.

Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 38, which includes 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). The N-terminus of the heavy chain variable region amino acid sequence (SEQ ID NO: 4) of the anti-HER3 monoclonal antibody Patritumab is linked to the C-terminus of Fc via a flexible peptide consisting of 10 amino acid residues (G4A)2 (SEQ ID NO: 36), and the N-terminus of the light chain variable region amino acid sequence (SEQ ID NO: 5) of the anti-HER3 monoclonal antibody Patritumab is linked to the C-terminus of the heavy chain variable region amino acid sequence of Patritumab via a flexible peptide consisting of 20 amino acid residues (G4S)4 (SEQ ID NO: 37).

Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 39, which includes 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 at the C-terminus of the VL amino acid sequence (SEQ ID NO: 6).

Anti-HER3×EGFR-01(2+2): Composed of two polypeptide chains, the structural diagram of which is shown in Figure 1.

Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 40, which includes 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). The N-terminus of the heavy chain variable region amino acid sequence (SEQ ID NO: 1) of the anti-EGFR monoclonal antibody Zalutuzumab is linked to the C-terminus of Fc via a flexible peptide consisting of 10 amino acid residues (G4A)2 (SEQ ID NO: 36), and the N-terminus of the light chain variable region amino acid sequence (SEQ ID NO: 2) of the anti-EGFR monoclonal antibody Zalutuzumab is linked to the C-terminus of the heavy chain variable region amino acid sequence of Zalutuzumab via a flexible peptide consisting of 20 amino acid residues (G4S)4 (SEQ ID NO: 37).

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 κ light chain constant region (CL) amino acid sequence at the C-terminus of the VL amino acid sequence (SEQ ID NO: 6).

Anti-EGFR(Cet)×HER3(Syst)(2+2)(Patent Publication No.: WO2016106157A1): Composed of two polypeptide chains, the structural diagram of which is shown in Figure 1.

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

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

Anti-EGFR-01: Composed of 4 polypeptide chains, its structural diagram is shown in Figure 1.

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

The light chain has the amino acid sequence shown in SEQ ID NO: 13, which includes 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 at the C-terminus of the VL amino acid sequence (SEQ ID NO: 6).

Anti-EGFR-03: Composed of 4 polypeptide chains, its structural diagram is shown in Figure 1.

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

The light chain has the amino acid sequence shown in SEQ ID NO: 13, which includes 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 at the C-terminus of the VL amino acid sequence (SEQ ID NO: 6).

Anti-HER3: It consists of 4 polypeptide chains, and its structural diagram is shown in Figure 1.

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

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

Anti-HER3, Anti-EGFR-01, and Anti-EGFR-03 sequence information (SEQ ID NO:) antibody VH VL Definition method HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 CH CL HC LC Anti-HER3 4 5 IMGT 30 31 32 33 34 35 7 6 15 16 Anti-EGFR-01 1 2 twenty three twenty four 25 26 27 28 12 13 Anti-EGFR-03 3 2 twenty three 29 25 26 27 28 14 13

Anti-EGFR-01×HER3 and Anti-EGFR-03×HER3 sequence information (SEQ ID NO:) antibody peptide chain VH VL CH CL HC LC Anti-EGFR-01×HER3 Peptide chain #1 (heavy chain 1) 4 / 8 / 17 / Peptide Chain #2 (Light Chain 1) / 5 / 10 / 18 Peptide chain #3 (heavy chain 2) 1 / 9 / 19 / Peptide Chain #4 (Light Chain 2) / 2 / 11 / 20 Anti-EGFR-03×HER3 Peptide chain #1 (heavy chain 1) 4 / 8 / 17 / Peptide Chain #2 (Light Chain 1) / 5 / 10 / 18 Peptide chain #3 (heavy chain 2) 3 / 9 / twenty one / Peptide Chain #4 (Light Chain 2) / 2 / 11 / twenty two 2. Preparation of antibody-drug conjugates

In this embodiment, EGFR×HER3 bispecific antibody-drug conjugates (Anti-EGFR-01×HER3-ADC-1 and Anti-EGFR-03×HER3-ADC-1, structural diagrams are 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: 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, and the antibody concentration was adjusted to approximately 5 mg/mL with PBS (Cytiva; pH 7.4). Add 0.1 M EDTA (Thermo, 0.001 mL) and 0.1 M TCEP (Sigma) aqueous solution (0.013 mL, equivalent to 20.0 equivalents per molecule of antibody) to the antibody. Incubate the resulting solution in a 37 °C water bath for 2 h to reduce the disulfide bonds between antibody chains. Subsequently, dimethyl monoxide (Sigma, 0.05 mL) and a 15 mM Deruxtecan conjugate (MCE, catalog number HY-13631E) solution (0.069 mL, equivalent to 16.0 equivalences per molecule of antibody) prepared with dimethyl monoxide were added to the above solution at room temperature. The coupling reaction was carried out at room temperature for 1 h. Then, 100 mM acetylcysteine (MCE, catalog number HY-B0215/CS-2160) aqueous solution (0.022 mL, equivalent to 32.0 equivalences per molecule of antibody) was added. The mixture was stirred at room temperature for 20 min, and then the coupling reaction was terminated. The obtained ADC mixture was replaced with a 30 K concentration tube (Amicon Ultra-4) (the replacement volume was 10 times that of the reaction solution), and finally replaced with Lite 6.0 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 replacement 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 this invention met the requirements, with a DAR value of approximately 8. Example 2. Binding of EGFR×HER3 bispecific antibody to human EGFR/HER3 cells.

In this experiment, expanded LS180 cells (self-expressing EGFR and HER3) were digested with 0.25% EDTA trypsin, centrifuged, and the supernatant was discarded. The cell pellet was resuspended in medium, counted, and then the cell density was adjusted to 2× ^10⁶ cells/ml with 2% BSA solution. Jurkat-EGFR cells (overexpressing EGFR) and CHOS-HER3 cells (overexpressing HER3) were used as suspension cells. An appropriate amount of cells was taken, counted, and the cell density was adjusted to 2× ^10⁶ cells/ml with 2% BSA solution. The cell suspension was added at 100 μl/well to a 96-well flow cytometry plate and centrifuged for later use. Gradual-diluted antibodies were added at 100 μl/well to the cell-bearing 96-well flow cytometry plates and incubated at 4°C for 60 min. After washing twice with PBS, 100 μl/well of Goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1000-fold with 2% BSA solution was added 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.

The experimental results are shown in Figure 3. The Anti-EGFR-01×HER3 bispecific antibody preferentially binds to EGFR/HER3 double-positive cells, and its binding to EGFR or HER3 single-positive cells is weaker, significantly weaker than that of Anti-EGFR-01 or Anti-HER3 monoclonal antibodies. Furthermore, although the Anti-EGFR-03×HER3 bispecific antibody further reduced its binding to EGFR single-positive cells, it still maintained good binding in double-positive cells. Bispecific antibodies in the “2+2” configuration exhibit comparable binding levels to their corresponding monoclonal antibodies in double-positive cells. However, for monopositive cells, the binding of Anti-EGFR-01×HER3(2+2) and Anti-HER3×EGFR-01(2+2) bispecific antibodies to HER3 or EGFR monopositive cells is significantly weaker than that of their corresponding monoclonal antibodies because their anti-HER3 or anti-EGFR antibodies are designed as scFv at the C-terminus of the Fc. However, their binding to EGFR and HER3 double-positive cells maintains comparable binding capacity to their corresponding monoclonal antibodies. Example 3. EGFR×HER3 bispecific antibodies block EGF-EGFR interaction.

In this experiment, HEK293T-hEGFR (overexpressing human EGFR) cells were expanded and cultured to a cell density of 2 × ^10⁶ cells/ml. 100 μl/well was added to each well of a 96-well flow cytometry plate and centrifuged. Serially diluted antibodies were added at 100 μl/well to the same 96-well flow cytometry plates containing the cells and incubated at 4°C for 60 min. After washing once with PBS, 100 μl/well of Bio-EGF diluted with 2% BSA solution was added and incubated at 4°C for 60 min. After washing twice with PBS, 100 μl/well of Streptavidin (BD Pharmingen, 554061) diluted 1000-fold with 2% BSA solution was added and incubated at 4°C for 60 min. The cells were washed twice with PBS, and then resuspended in PBS at a rate of 100 μl/well. The cells were then analyzed and the corresponding MFI was calculated using a CytoFlex (Beckman) flow cytometer.

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 EGF-EGFR interaction. In EGFR monopositive cells, the Anti-EGFR-01×HER3 bispecific antibody significantly blocked 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 EGFR, the Anti-EGFR-03×HER3 bispecific antibody did not show significant EGF-EGFR blocking activity in EGFR monopositive cells. Furthermore, the "2+2" bispecific antibody exhibits a stronger blocking effect against EGF-EGFR, with its blocking activity comparable to that of the corresponding anti-EGFR monoclonal antibody. Example 4. EGFR×HER3 bispecific antibody blocks EGF-EGFR signal transduction.

In this experiment, HEK293T-hEGFR-STAT3 reporter gene (overexpressing human EGFR and STAT3) cells were adjusted to a cell density of 2.5 × 10⁶ cells/ml, and 40 μl/well was added to each well of a 96-well plate for later use. Serially diluted antibodies were added at 40 μl/well to the cell-containing 96-well plates and incubated for 60 min. Then, EGF solution diluted in complete medium was added at 40 μl/well, and the plates were incubated for 16 h. Finally, the chemiluminescence signal was collected using a microplate reader after colorimetric development using a Bio-Glo luciferase assay system (Promega, G7940).

The experimental results are shown in Figure 4B. This study used a reporter gene approach to verify the blocking effect of the Anti-EGFR×HER3 bispecific antibody on the EGF-EGFR signaling pathway. In EGFR monopositive cells, the Anti-EGFR-01×HER3 bispecific antibody significantly blocked 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 EGFR, the Anti-EGFR-03×HER3 bispecific antibody did not show significant EGF-EGFR blocking activity in EGFR monopositive cells. Furthermore, the "2+2" bispecific antibody exhibits a stronger blocking effect against EGF-EGFR, with its blocking activity comparable to that of the corresponding anti-EGFR monoclonal antibody. Example 5. EGFR×HER3 bispecific antibody blocks the interaction between NRG1 and HER3.

In this experiment, HEK293T-hHER3 (overexpressing human HER3) cells were expanded to a cell density of 2 × ^10⁶ cells/ml and added 100 μl/well to a 96-well flow cytometry plate for centrifugation. Serially diluted antibodies were added 100 μl/well to the cell-containing 96-well flow cytometry plates and incubated at 4°C for 60 min. After washing once with PBS, 100 μl/well of Bio-NRG1 diluted with 2% BSA solution was added and incubated at 4°C for 60 min. After washing twice with PBS, 100 μl/well of Streptavidin (BD Pharmingen, 554061) diluted 1000-fold with 2% BSA solution was added and incubated at 4°C for 60 min. The cells were washed twice with PBS, and then resuspended in PBS at a rate of 100 μl/well. The cells were then analyzed and the corresponding MFI was calculated using a CytoFlex (Beckman) flow cytometer.

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 monopositive cells, the Anti-EGFR-01×HER3 and Anti-EGFR-03×HER3 bispecific antibodies significantly blocked the binding of NRG1 and HER3, but their blocking activity was significantly weaker than that of the corresponding Anti-HER3 monoclonal antibody. Furthermore, the "2+2" bispecific antibody showed a stronger blocking effect on NRG1-HER3, with blocking activity comparable to that of the corresponding Anti-HER3 monoclonal antibody. Example 6. EGFR×HER3 Bispecific Antibody Induces Internalization Effect

In this experiment, expanded LS180 and MKN45 cells (self-expressing human EGFR and HER3) were adjusted to a cell density of 4 × ^10⁶ cells/ml using culture medium, and 50 μl/well was added to each well of a 96-well flow cytometry plate. 50 μl/well of serially diluted bispecific antibody (pre-incubated with a pH-labeled secondary antibody for 1 h) was added to each well, and the cells were incubated for 2 hours. After washing twice with PBS, the cells were resuspended in PBS at 100 μl/well. The cells were then analyzed using a CytoFlex (Beckman) flow cytometer, and the corresponding MFI was calculated.

The experimental results are shown in Figure 6. The Anti-EGFR-01×HER3 bispecific antibody can specifically bind to EGFR/HER3 double-positive cells and mediate antibody internalization. Its internalization effect is significantly better than that of the corresponding Anti-EGFR-01 and Anti-HER3 monoclonal antibodies. Furthermore, Anti-EGFR-01×HER3 is also superior to or no less effective than the "2+2" form of bispecific antibody and the internalization mediated by Anti-EGFR-03×HER3. Example 7. Inhibition of cell proliferation by EGFR×HER3 bispecific antibody-drug conjugate (ADC-1).

In this experiment, expanded LS180 and MKN45 cells (self-expressing EGFR and HER3) were digested with 0.25% EDTA trypsin, centrifuged, and resuspended. After washing once with medium, the cell density was adjusted to 6.25 × ^10⁴ cells/ml, and 80 μl/well was added to each well of a 96-well plate for later use. Serially diluted antibodies were added at 80 μl/well to the cell-containing 96-well plates and incubated in a cell culture incubator for 3–5 days. Finally, the chemiluminescence signal was collected using a microplate reader after colorimetric development with the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7572) kit.

The experimental results are shown in Figure 7. The Anti-EGFR-01xHER3-ADC-1 and Anti-EGFR-03xHER3-ADC-1 bispecific antibody-drug conjugates of this invention can significantly inhibit the growth and proliferation of LS180 and MKN45 cells, and their inhibitory effect is comparable to that of the corresponding monoclonal antibody-drug conjugates. Example 8. Study on tumor inhibitory activity of EGFR×HER3 bispecific antibody-drug conjugate (ADC-1)

This experiment serves as a proof-of-concept study of the antitumor activity of the EGFR×HER3 bispecific antibody-drug conjugate (ADC-1) of this invention in a NOD SCID mouse tumor model induced by subcutaneous inoculation with MKN45.

First, a mouse model bearing tumor was established by subcutaneous inoculation with MKN45 cells. When the tumor volume reached approximately 150 ^mm³ , the mice were divided into groups and treated with intraperitoneal injections of PBS (G1), Isotype Ctrl-ADC-1 (G2), Anti-EGFR-01×HER3 (G3), and Anti-EGFR-01×HER3-ADC-1 (G4) (all groups received equal molar doses). The tumor volume and body weight of each group were monitored every 2-3 days for 2-3 weeks. The dosage and administration method are shown in Table 1.

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 this invention (TGI: 94.12%) exhibits stronger antitumor activity and can significantly inhibit tumor growth. Table 1: Dosing regimen for the tumor inhibitory activity study of Anti-EGFR-01×HER3-ADC-1 Group Dosage Number of doses G1:PBS N/A Q2d×2 G2:Isotype Ctrl-ADC-1 3 mg/kg Q2d×2 G3: Anti-EGFR-01×HER3 3 mg/kg Q2d×2 G4: Anti-EGFR-01×HER3-ADC-1 3 mg/kg Q2d×2 Example 9. Study on tumor inhibitory activity of EGFR×HER3 bispecific antibody-drug conjugate (ADC-1)

This experiment serves as a proof-of-concept study of the antitumor activity of the EGFR×HER3 bispecific antibody-drug conjugate (ADC-1) of this invention in a tumor model in which Balb/c Nude mice are subcutaneously inoculated with A375.

First, a mouse model bearing tumor was established by subcutaneous inoculation with A375 cells. When the tumor volume reached approximately 200 ^mm³ , the mice were divided into groups and treated with intraperitoneal injections of PBS (G1), Isotype Ctrl-ADC-1 (G2), Anti-EGFR-01×HER3 (G3), and Anti-EGFR-01×HER3-ADC-1 (G4) (all groups received equal molar doses). The tumor volume and body weight of each group were monitored every 2-3 days for 2-3 weeks. The dosage and administration method are shown in Table 2.

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 (TGI: 100%) of this invention has stronger antitumor activity and can significantly inhibit tumor growth. Table 2: Dosing regimen for the tumor inhibitory activity study of EGFR×HER3-ADC Group Dosage Number of doses G1:PBS N/A 1 G2:Isotype Ctrl-ADC-1 2 mg/kg 1 G3: Anti-EGFR-01×HER3 2 mg/kg 1 G4: Anti-EGFR-01×HER3-ADC-1 2 mg/kg 1 Example 10. Study on tumor inhibitory activity of EGFR×HER3 bispecific antibody-drug conjugate (ADC-1)

This experiment serves as a proof-of-concept study to demonstrate the antitumor activity of the EGFR×HER3 bispecific antibody-drug conjugate (ADC-1) of this invention in a tumor model in which NSG mice are subcutaneously inoculated with MKN45.

First, a tumor-bearing mouse model was established by subcutaneous inoculation with MKN45 cells. When the tumor volume reached approximately 100 ^mm³ , the mice were divided into groups and administered intraperitoneal injections of the following: G1: PBS; G2: 3 mg/kg Isotype Ctrl-ADC-1; G3: 3 mg/kg Anti-EGFR-01-ADC-1; G4: 3 mg/kg Anti-EGFR-03-ADC-1; G5: 3 mg/kg Anti-HER3-ADC-1; G6: Enhertu (Daiichi-Sankyo AstraZeneca, batch number: 389085); G7: 3 mg/kg Anti-EGFR-01×HER3-ADC-1; G8: 3 mg/kg Anti-EGFR-03×HER3-ADC-1; and G9: 4 mg/kg Anti-EGFR-03×HER3-ADC-1. Mice were treated with Anti-EGFR(Cet)×HER3(Syst)(2+2)-ADC-1 at a dose of mg/kg (equal molar doses in all groups). Tumor volume and body weight changes were monitored in each group at a frequency of 2-3 days per session for 2-3 weeks. Dosage and administration method are shown in Table 3.

The experimental results are shown in Figure 10. The antitumor 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 antitumor 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%). Table 3: Dosing regimens for the tumor inhibitory activity study of EGFR×HER3-ADC. Group Dosage Number of doses G1:PBS N/A Q2d×4 G2:Isotype Ctrl-ADC-1 3 mg/kg Q2d×4 G3: Anti-EGFR-01-ADC-1 3 mg/kg Q2d×4 G4: Anti-EGFR-03-ADC-1 3 mg/kg Q2d×4 G5: Anti-HER3-ADC-1 3 mg/kg Q2d×4 G6:Enhertu 3 mg/kg Q2d×4 G7: Anti-EGFR-01×HER3-ADC-1 3 mg/kg Q2d×4 G8: Anti-EGFR-03×HER3-ADC-1 3 mg/kg Q2d×4 G9:Anti-EGFR(Cet)×HER3(Syst)(2+2)-ADC-1 3 mg/kg Q2d×4

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such modifications are within the scope of protection of the present invention. The full scope of the present invention is given by the appended patent claims and any equivalents thereof.

References 1. Hynes, NE and HA Lane, ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer, 2005. 5(5): p. 341-54.2. Mishra, R., AB Hanker, and JT Garrett, Genomic alterations of ERBB receptors in cancer: clinical implications. Oncotarget, 2017. 8(69): p. 114371-114392.3. Wu, Q., et al., Small-molecule inhibitors, immune checkpoint inhibitors, and more: FDA-approved novel therapeutic drugs for solid tumors from 1991 to 2021. J Hematol Oncol, 2022. 15(1): p. 143.4. Arteaga, CL and JA Engelman, ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics. Cancer Cell, 2014. 25(3): p. 282-303.5. Amin, DN, MR Campbell, and MM 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. 944-50.6. 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.

Sequence information SEQ ID NO: describe sequence 1 Anti-EGFR-01 mAb heavy chain variable region EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS 2 Anti-EGFR-01 mAb/Anti-EGFR-03 mAb Light Chain Variable Region DIQLTQSPSSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK 3 Anti-EGFR-03 mAb heavy chain variable region EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIEDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS 4 Anti-HER3 mAb heavy chain variable region EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS 5 Anti-HER3 mAb light chain variable zone DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIK 6 Human κ light chain constant region (CL) amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 7 Human IgG1 heavy chain constant region amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 8 The CH1/CL-preferred mutation in human IgG1 Fc amino acid sequence CH SET1 (introduces Knob mutation) ASTKGPSVFPLAPSSKSTSGGTAALGCQVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 9 The CH1/CL-preferred mutation in human IgG1 Fc amino acid sequence CH SET2 (introduces Hole mutation) ASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 10 The human κ light chain constant region (CL) amino acid sequence of the CH1/CL-preferred mutation. RTVAAPSVFIFPPSDEQLKSGRASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSRLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 11 CH1/CL-preferred mutant human κ light chain constant region (CL) amino acid sequence CL SET2 RTVAAPSVFIFPPSDEELKSGTASVQCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSELTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 12 Anti-EGFR-01 mAb heavy chain EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13 Anti-EGFR-01 mAb / Anti-EGFR-03 mAb Light Chain DIQLTQSPSSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 14 Anti-EGFR-03 mAb heavy chain EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIEDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 15 Anti-HER3 mAb heavy chain EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 16 Anti-HER3 mAb Light Chain DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 17 Anti-EGFR-01×HER3/Anti-EGFR-03×HER3 Heavy Chain 1 EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGT LVTVSSASTKGPSVFPLPSSTSGGTAALGCQVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 18 Anti-EGFR-01×HER3/Anti-EGFR-03×HER3 Light Chain 1 DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGRASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSRLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 19 Anti-EGFR-01×HER3 Heavy Chain 2 EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFD YWGQGTLVTVSSASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 20 Anti-EGFR-01×HER3 Light Chain 2 DIQLTQSPSSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEELKSGTASVQCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSELTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC twenty one Anti-EGFR-03×HER3 heavy chain 2 EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIEDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFD YWGQGTLVTVSSASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG twenty two Anti-EGFR-03×HER3 Light Chain 2 DIQLTQSPSSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEELKSGTASVQCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSELTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC twenty three Anti-EGFR-01 mAb/ Anti-EGFR-03 mAb VH CDR1 GFTFSTYG twenty four Anti-EGFR-01 mAb VH CDR2 IWDDGSYK 25 Anti-EGFR-01 mAb/ Anti-EGFR-03 mAb VH CDR3 ARDGITMVRGVMKDYFDY 26 Anti-EGFR-01 mAb/ Anti-EGFR-03 mAb VL CDR1 QDISSA 27 Anti-EGFR-01 mAb/ Anti-EGFR-03 mAb VL CDR2 DAS 28 Anti-EGFR-01 mAb/ Anti-EGFR-03 mAb VL CDR3 QQFNSYPLT 29 Anti-EGFR-03 mAb VH CDR2 IEDDGSYK 30 Anti-HER3 mAb VH CDR1 GGSFSGYY 31 Anti-HER3 mAb VH CDR2 INHSGST 32 Anti-HER3 mAb VH CDR3 ARDKWTWYFDL 33 Anti-HER3 mAb VL CDR1 QSVLYSSSNRNY 34 Anti-HER3 mAb VL CDR2 WAS 35 Anti-HER3 mAb VL CDR3 QQYYSTPRT 36 (G4A)2 GGGGAGGGGA 37 (G4S)4 GGGGSGGGGSGGGGSGGGGS 38 Anti-EGFR-01×HER3(2+2) heavy chain 39 Anti-EGFR-01×HER3(2+2) Light Chain DIQLTQSPSSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 40 Anti-HER3×EGFR-01(2+2) heavy chain 41 Anti-HER3×EGFR-01(2+2) Light Chain DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 42 Anti-EGFR(Cet)×HER3(Syst)(2+2) heavy chain QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQS PGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFF KMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLQESGGGLVKPGGSLRLSC AASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYY CSSYGSSSTH VIFGGGTKVTVL 43 Anti-EGFR (Cet) × HER3 (Syst) (2+2) Light Chain DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

(without)

Figure 1 shows a schematic diagram of the structure of the antibody of the present invention. Figure 2 shows a schematic diagram of the structure of the antibody-drug conjugate (ADC-1) of the present invention. Figure 3 shows the results of the cell-binding activity test of the antibody of the present invention, expressed as EC50 (nM). Figure 4 shows the results of the test of the blocking activity of the antibody of the present invention against EGF-EGFR interaction and signal transduction, expressed as IC50 (nM). Figure 5 shows the results of the test of the blocking activity of the antibody of the present invention against NRG1-HER3 interaction, expressed as IC50 (nM). Figure 6 shows the results of the test of the internalization effect mediated by the antibody of the present invention. Figure 7 shows the results of the test of the inhibition of cell proliferation by the antibody-drug conjugate (ADC-1) of the present invention. Figure 8 shows the results of investigating the tumor-inhibiting activity of the antibody-drug conjugate (ADC-1) of the present invention in NOD SCID model mice subcutaneously inoculated with MKN45 cells. Figure 9 shows the results of investigating the tumor-inhibiting activity of the antibody-drug conjugate (ADC-1) of the present invention in a Balb/c Nude mouse tumor model subcutaneously inoculated with A375. Figure 10 shows the results of investigating the tumor-inhibiting activity of the antibody-drug conjugate (ADC-1) of the present invention in an NSG mouse tumor model subcutaneously inoculated with MKN45.

TW202540193A_114109649_SEQL.xml

(without)

Claims (35)

An antibody comprising a HER3 antigen-binding domain or an antigen-binding fragment thereof, said HER3 antigen-binding domain comprising a VH (heavy chain variable region) and a VL (light chain variable region), wherein the VH comprises: (1-i) CDRs contained in the heavy chain as shown in SEQ ID NO: 15; (1-ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 30, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32; or, (1-iii) an amino acid sequence as shown in SEQ ID NO: 4 or a variant thereof; and/or, the VL comprises: (1-iv) CDRs contained in the light chain as shown in SEQ ID NO: 16; (1-v) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 4; or, (1-iii) an amino acid sequence as shown in SEQ ID NO: 4; or, (1-iv) a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 16; or, (1-v) a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 3 ...2; or, (1-iv) a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 33; or, (1-iv) a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 32; or, (1-iv) a light chain variable region 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 system; preferably, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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); preferably, the substitution is a conservative substitution. An antibody comprising an EGFR antigen-binding domain or an antigen-binding fragment thereof, said EGFR antigen-binding domain comprising VH and VL, wherein the VH comprises: (2-i) CDRs contained in the heavy chain as shown in SEQ ID NO: 12 or 14; (2-ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 23, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 24 or 29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 25; or, (2-iii) an amino acid sequence as shown in SEQ ID NO: 1 or 3 or a variant thereof; and/or, the VL comprises: (2-iv) CDRs contained in the light chain as shown in SEQ ID NO: 13; (2-v) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 13; LCDR2 containing the amino acid sequence shown in SEQ ID NO: 27, and LCDR3 containing 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 system; preferably, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to it; preferably, the substitution is a conservative substitution. The antibody or antigen-binding fragment thereof described in claim 1 or 2 is single-specific. The antibody or antigen-binding fragment thereof described in claim 1 is bispecific. The antibody or antigen-binding fragment thereof as described in claim 4, the antibody further comprising an EGFR antigen-binding domain as defined in claim 2. The antibody or antigen-binding fragment thereof described in claim 2 is bispecific. The antibody or antigen-binding fragment thereof as described in claim 6, the antibody further comprising a HER3 antigen-binding domain as defined in claim 1. A bispecific antibody or an antigen-binding fragment thereof, said antibody comprising a first antigen-binding domain specific to HER3 and a second antigen-binding domain specific to EGFR, wherein the first antigen-binding domain comprises a first VL and a first VH, the first VL being a VL as defined in claim 1 and the first VH being a VH as defined in claim 1; and/or the second antigen-binding domain comprises a second VL and a second VH, the second VL being a VL as defined in claim 2 and the second VH being a VH as defined in claim 2. The antibody or antigen-binding fragment thereof as described in any one of claims 1-8 is an IgG antibody; preferably, the IgG antibody is an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD or IgE antibody. The antibody or antigen-binding fragment thereof as described in any one of claims 1-9, the antibody further comprising CH (heavy chain constant region) and CL (light chain constant region); preferably, CH is an IgG1 heavy chain constant region; preferably, CL is a κ or λ light chain constant region; preferably, CH comprises an amino acid sequence as shown in SEQ ID NO: 7 or a variant thereof, and/or, CL comprises an amino acid sequence as shown in SEQ ID NO: 7. The amino acid sequence shown in 6 or a variant thereof; preferably, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the variant; preferably, the substitution is a conservative substitution; preferably, the CL and/or the CH (e.g., CH1, Fc region) are modified (e.g., mutated) to promote dimerization (e.g., homodimerization or heterodimerization, such as promoting pairing of κCL with CH1, and/or Fc region heterodimerization); preferably, the CH comprises as in SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 8 or 9 or a variant thereof, and/or the CL contains the amino acid sequence shown in SEQ ID NO: 10 or 11 or a variant thereof. An antibody or antigen-binding fragment thereof as described in any of claims 4-10, said antibody comprising peptide chain I-A, peptide chain I-B, peptide chain I-C and peptide chain I-D, wherein peptide chain I-A comprises a first VL and CL, peptide chain I-B comprises a first VH and CH, peptide chain I-C comprises a second VH and CH, and peptide chain I-D comprises a second VL and CL. The antibody or antigen-binding fragment thereof as described in claim 11, wherein adjacent domains of the peptide chain IA are linked by or without linkers, adjacent domains of the peptide chain IB are linked by or without linkers, adjacent domains of the peptide chain IC are linked by or without linkers, and/or adjacent domains of the peptide chain ID are linked by or without linkers; preferably, each linker is independently selected from polypeptide linkers, such as rigid or flexible polypeptide linkers; preferably, the polypeptide linkers are glycine-rich linkers, for example, linkers having the form (GGS) _n , (GGGGS) _n , or (GGGGA) _n , where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, SEQ ID NO: 36 or 37. The antibody or antigen-binding fragment thereof as described in claim 11 or 12 comprises: peptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 18; peptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 17; peptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 19 or 21; and peptide chain I-D comprising the amino acid sequence shown in SEQ ID NO: 20 or 22; preferably, the antibody comprises: peptide chain I-A having the amino acid sequence shown in SEQ ID NO: 18, peptide chain I-B having the amino acid sequence shown in SEQ ID NO: 17, peptide chain I-C having the amino acid sequence shown in SEQ ID NO: 19, and peptide chain I-D having the amino acid sequence shown in SEQ ID NO: 20; or, peptide chain I-A having the amino acid sequence shown in SEQ ID NO: 18, peptide chain I-B having the amino acid sequence shown in SEQ ID NO: 17, peptide chain I-C having the amino acid sequence shown in SEQ ID NO: 19, and peptide chain I-D having the amino acid sequence shown in SEQ ID NO: 20; or peptide chain I-A having the amino acid sequence shown in SEQ ID NO: 18, peptide chain I-B having the amino acid sequence shown in SEQ ID NO: 19, and peptide chain I-D having the amino acid sequence shown in SEQ ID NO: 20. Peptide chains I-B with the amino acid sequence shown in SEQ ID NO: 21, peptide chains I-C with the amino acid sequence shown in SEQ ID NO: 22, and peptide chains I-D with the amino acid sequence shown in SEQ ID NO: 22. An antibody or antigen-binding fragment thereof as described in any of claims 4-10, the antibody comprising peptide chain II-A and peptide chain II-B, wherein peptide chain II-A comprises a first VH, CH, a second VH and a second VL, and peptide chain II-B comprises a first VL and CL; or, peptide chain II-A comprises a second VH, CH, a first VH and a first VL, and peptide chain II-B comprises a second VL and CL. As described in claim 14, the peptide chain II-A comprises a first VH, 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 CL from the N-terminus to the C-terminus; or, the peptide chain II-A comprises a second VH, 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 CL from the N-terminus to the C-terminus. The antibody or antigen-binding fragment thereof as described in claim 14 or 15, wherein adjacent domains of peptide chain II-A are linked by or without linkers, and/or adjacent domains of peptide chain II-B are linked by or without linkers; preferably, each linker is independently selected from polypeptide linkers, such as rigid or flexible polypeptide linkers; preferably, the polypeptide linkers are glycine-rich linkers, for example, linkers having the form (GGS) _n or (GGGGS) _n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, SEQ ID NO: 36 or 37. An antibody or antigen-binding fragment thereof as described in any one of claims 14-16 comprises: a peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 38; and a peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 39; or, 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; preferably, the antibody comprises: a peptide chain II-A having the amino acid sequence shown in SEQ ID NO: 38; and a peptide chain II-B having the amino acid sequence shown in SEQ ID NO: 39; or, 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. The peptide chain II-B of the amino acid sequence shown in 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. An antibody or antigen-binding fragment thereof as described in any of claims 1-17, wherein the antibody further carries a detectable marker, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. An isolated nucleic acid molecule that encodes an antibody or an antigen-binding fragment thereof as described in any one of claims 1-18. A vector comprising the nucleic acid molecule of claim 19; preferably, nucleic acid sequences encoding different peptide chains of the antibody or its antigen-binding fragment are located in the same or different vectors; preferably, the vector is a selection vector or an expression vector. A host cell comprising the nucleic acid molecule described in claim 19 or the vector described in claim 20. A method for preparing an antibody or antigen-binding fragment thereof as described in any one of claims 1-18, wherein the method comprises culturing the host cells of 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 cells. An antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof as described in any one of claims 1-18. The antibody-drug conjugate as claimed in claim 23 further comprises therapeutic agents, for example, containing 1 to 20 of the therapeutic agents per molecule of the antibody-drug conjugate, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The antibody-drug conjugate as described in claim 24, wherein the therapeutic agent is selected from microtubule inhibitors and DNA damage agents; preferably, the microtubule inhibitor is selected from olistatin compounds (e.g., MMAE, MMAF or derivatives thereof), maytansine compounds (e.g., maytansine, maytanol, DM1, DM4 or derivatives thereof), taxanes (e.g., taxol, docetaxel, carbazitaxel or derivatives thereof), vinca alkaloids (e.g., vinca alkaloid, vincristine or derivatives thereof), and ergotamine. Bulolin or its derivatives and colchicine or its derivatives; preferably, the DNA damaging agent is selected from DNA alkylating agents (cachicin γ11, N-acetyl-γ1I cachicin, ampicillin, PBD, ducacicin or its derivatives), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically, camptothecin, SN-38, Dxd, irinotecan, beloteticon, topothecin, PNU-159682 or its derivatives), amycin, daunorubicin, etoposide, mitoxantrone or its derivatives) and senna or its derivatives; preferably, the treatment agent is . The antibody-drug conjugate as described in claim 24 or 25, wherein the antibody and the treatment are linked by at least one linker; preferably, the linker may be 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-maleiminohexyl), MCC (maleiminomethylcyclohexane-1-carboxylate), MP (maleiminopropyl), Val-Cit (virucine-citrulline), Val-Cit- _NHCH2- , Val-Ala (virucine-alanine), Val-Ala- _NHCH2- , Ala-Phe (alanine-phenylalanine), Ala-Phe- _NHCH2- - Gly-Gly-Phe-Gly (glycine-glycine-phenylalanine-glycine), Gly-Gly-Phe-Gly-NHCH ₂ - PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimidyl)-4-(pyridin-2-ylthio)valerate), 6-(2,5-dioxopyrrolidone-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidone-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimidyl-4-(N-maleiminomethyl)cyclohexane-1-carboxylate), SIAB (N-succinimidyl-(4-iodoacetylated)aminobenzoate) and any combination thereof; preferably, the linker is . The antibody-drug conjugate as described in any of claims 23-26 has the following structure: Wherein, A is the antibody or antigen-binding fragment thereof described in any of claims 1-18; p is an integer selected from 1-20, for example, an integer from 1-10 or 1-8, or for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Antibody-drug conjugates have the following structure: Wherein, A is a bispecific antibody targeting EGFR and HER3, comprising: a peptide chain IA having the amino acid sequence shown in SEQ ID NO: 18, a peptide chain IB having the amino acid sequence shown in SEQ ID NO: 17, a peptide chain IC having the amino acid sequence shown in SEQ ID NO: 19, and a peptide chain ID having the amino acid sequence shown in SEQ ID NO: 20; or, a peptide chain IA having the amino acid sequence shown in SEQ ID NO: 18, a peptide chain IB having the amino acid sequence shown in SEQ ID NO: 17, a peptide chain IC having the amino acid sequence shown in SEQ ID NO: 21, and a peptide chain ID having the amino acid sequence shown in SEQ ID NO: 22; or, 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; or, a peptide chain having the amino acid sequence shown in SEQ ID NO: 18; or, a peptide chain having the amino acid sequence shown in SEQ ID NO: 19 ... Peptide chain IA having the amino acid sequence shown in SEQ ID NO: 40; and peptide chain IB having the amino acid sequence shown in SEQ ID NO: 41; p is an integer selected from 1 to 20, for example, an integer from 1 to 10 or 1 to 8, and for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-18, a nucleic acid molecule as described in claim 19, a vector as described in claim 20, a host cell as described in claim 21, or an antibody-drug conjugate as described in any one of claims 23-28, and a pharmaceutically acceptable excipient; preferably, the drug-antibody conjugation 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. Use of an antibody or antigen-binding fragment thereof as described in any one of claims 1-18, a nucleic acid molecule as described in claim 19, a vector as described in claim 20, a host cell as described in claim 21, an antibody-drug conjugate as described in any one of claims 23-28, or a drug composition as described in claim 29 in the preparation of an antitumor 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 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. The antibody or antigen-binding fragment thereof described in any one of claims 1-18, the nucleic acid molecule described in claim 19, the vector described in claim 20, the host cell described in claim 21, or the antibody-drug conjugate described in any one of claims 23-28, or the drug combination described in claim 29, is used for antitumor purposes; 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 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. An antitumor method comprising administering to a subject in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-18, a nucleic acid molecule as described in claim 19, a vector as described in claim 20, a host cell as described in claim 21, or an antibody-drug conjugate as described in any one of claims 23-28, or a drug combination as described in claim 29; preferably, the 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 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. A diagnostic or therapeutic kit comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-18, and optionally instructions for use and/or a delivery device. 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, under conditions allowing the formation of a complex between the antibody or antigen-binding fragment thereof described in any one of claims 1-18 and EGFR and/or HER3, and detecting the formation of the complex; preferably, the method is used for diagnosing tumors, such as breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell carcinoma, small cell lung cancer). 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 includes 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 an indicator of tumor. The use of an antibody or antigen-binding fragment thereof as described in any of claims 1-18 in the preparation of a reagent 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 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.