Pharmaceutical combination of Anti-her3 antibody-drug conjugate and Anti-her2 antibody, and use thereof

Abstract

Provided are a pharmaceutical combination of an anti-HER3 antibody-drug conjugate and an anti-HER2 antibody, and a use of the pharmaceutical combination for treating cancer. The drug combination has good safety, has stronger anti-tumor efficacy than a single drug, and exhibits a synergistic tumor-inhibitory effect. Also provided is a kit comprising the pharmaceutical combination.

Description

Drug combinations of anti-HER3 antibody-drug conjugates and anti-HER2 antibodies and their applications Technical Field

[0001] This invention relates to the pharmaceutical field. Specifically, it relates to a pharmaceutical combination of an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or its antigen-binding fragment, said pharmaceutical combination for the treatment of cancer. Background Technology

[0002] The human epidermal growth factor receptor (HER) family (also known as the human EGFR family) is one of the four closely related type I receptor tyrosine kinase (RTK) families of cell membrane receptors and is closely related to the occurrence and development of various tumors.

[0003] The HER family mainly consists of four structurally similar receptor molecules, including HER1 (also known as epidermal growth factor receptor (EGFR), ErbB1), HER2 (also known as Neu, ErbB2), HER3 (also known as ErbB3), and HER4 (also known as ErbB4).

[0004] HER2 is a 185kDa orphan receptor without an endogenous ligand-binding domain, but it is recognized as the preferred and most catalytic binding partner for other members of the HER family. Another characteristic of HER2 is that it is constitutively activated. Clinically, HER2-targeted therapy is well-known for its role in the treatment of breast cancer. Trastuzumab, the first monoclonal antibody targeting HER2, was developed in 1990. It blocks HER2 signaling through several mechanisms, including promoting HER2 internalization and degradation, inhibiting dimerization, blocking downstream PI3K-AKT signaling, and killing tumor cells through antibody-dependent cytotoxicity (ADCC) (Klapper LN et al., Tumor-inhibitory antibodies to HER-2 / ErbB-2 may act by recruiting c-Cbl and enhancing ubiquitination of HER-2. Cancer Res, 2000. 60(13): p. 3384–3388).

[0005] HER3 lacks intrinsic tyrosine kinase activity and is often referred to as a pseudokinase. HER3 is a specific dimerizing chaperone; other HER family members, such as HER2, maximize the induction of the phosphoinositol 3-kinase (PI3K) / protein kinase B (AKT) / mTOR pathway after forming a heterodimer with HER3. Neuroregulatory proteins (NRGs) 1 and 2 are the main activating ligands of HER3, promoting heterodimerization of HER3 and HER2, leading to pathological activation of downstream signaling. Furthermore, positive HER3 expression has been reported to be associated with poor prognosis in some cancers, including breast cancer, gastric cancer, and head and neck cancer. Since HER3 directly promotes cancer signaling through PI3K / AKT / mTOR, targeting HER3 is becoming an effective strategy for treating HER3-expressing cancers; however, no specific HER3-targeting therapies have yet been approved for clinical use.

[0006] Anti-HER3 antibody-drug conjugates (anti-HER3-ADCs) are composed of a monoclonal antibody specifically targeting the HER3 antigen and a small-molecule cytotoxic drug linked by a linker. They combine the potent killing effect of traditional small-molecule chemotherapy with the tumor-targeting properties of antibody drugs. They consist of three main parts: an antibody that selectively recognizes the HER3 antigen on the surface of cancer cells, a drug payload responsible for killing cancer cells, and a linker connecting the anti-HER3 antibody and the payload. Patritumab deruxtecan (HER3-DXd; U3-1402), developed by Daiichi Sankyo, is an anti-HER3 antibody-drug conjugate covalently linked to the antibody U3-1287 (Patritumab) and a drug payload containing the topoisomerase I inhibitor exatecan (MAAA-1181a). It has shown clinical benefit in NSCLC patients with EGFR mutations; however, hematological toxicities, including thrombocytopenia and neutropenia, were the most common adverse events observed during the clinical development of Patritumab deruxtecan. PA et al., Efficacy and safety of patritumab deruxtecan(HER3-DXd) in EGFR inhibitor-resistant, EGFR-mutated non-small cell lung cancer, Cancer Discov. 2022; 12:74–89).

[0007] Furthermore, considering that the biological behavior of most tumors is not governed by a single signaling pathway, but rather by the combined action of multiple signaling pathways, drugs with different mechanisms of action can be used in combination in certain situations. However, any combination of drugs with different mechanisms of action but acting in similar areas does not necessarily produce a beneficial combination. Therefore, while there is indeed a demand for combination therapy regimens and products targeting different signaling pathways in the existing technology, given the complexity of tumorigenesis and the unpredictability of interactions between different drugs, discovering feasible combination therapy regimens and products that can deliver superior effects compared to monotherapy (reducing monotherapy dosage, improving the incidence and / or severity of adverse events (AEs) during treatment, and / or acting in a synergistic manner, etc.) remains a major challenge in the pharmaceutical field. Summary of the Invention

[0008] The present invention provides a pharmaceutical combination comprising an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or an antigen-binding fragment thereof, and the use of said combination for treating cancer.

[0009] The inventors have surprisingly discovered that the drug combination of this application can have a therapeutic effect on cancer in a synergistic manner and / or improve the incidence and / or severity of adverse events (AEs) during treatment.

[0010] Specifically, the present invention relates to the following aspects.

[0011] In a first aspect, the present invention provides a pharmaceutical combination comprising an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or an antigen-binding fragment thereof, wherein the anti-HER3 antibody drug conjugate has the structure shown in formula (I-1):

[0012] in,

[0013] Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the Ab is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment.

[0014] For example, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and Ab is not a bispecific antibody or a multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment.

[0015] Preferably, the Ab is an anti-HER3 monospecific antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0016] Preferably, the anti-HER3 monospecific antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8;

[0017] More preferably, the heavy chain amino acid sequence of the anti-HER3 monospecific antibody is shown in SEQ ID NO:9, and the light chain amino acid sequence is shown in SEQ ID NO:10;

[0018] L stands for connector unit;

[0019] -M- is selected from:

[0020] p represents the average number of drug linkages relative to each Ab molecule, and p is selected from an integer or decimal of 1 to 10, preferably an integer or decimal of 3 to 8.

[0021] In a second aspect, the present invention provides the use of the pharmaceutical combinations of the present invention in the treatment of cancer.

[0022] In a third aspect, the present invention provides a method for treating cancer, the method comprising administering, in combination, a therapeutically effective amount of the pharmaceutical combination of the present invention to an individual in need.

[0023] In a fourth aspect, the present invention provides a kit containing the drug combination of the present invention. In some embodiments, the kit is in the form of a drug dosage unit.

[0024] Other aspects and embodiments of the invention will become clear from the following detailed description. Attached Figure Description

[0025] The preferred embodiments of the invention described in the following detailed description will be better understood when read in conjunction with the accompanying drawings. The drawings show presently preferred embodiments for illustrative purposes. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0026] Figure 1 shows the expression levels of HER2 and HER3 on the surface of different breast cancer cell lines (HCC1419 cells, HCC1569 cells, HCC1954 cells, BT-483 cells, and MDA-MB-231 cells).

[0027] Figure 2 shows whether there were significant changes in the expression level of HER3 on the surface of HCC1419, HCC1569, HCC1954 and BT-483 cells after treatment with trastuzumab compared with before treatment.

[0028] Figure 3 shows whether treatment of HCC1419, HCC1569, HCC1954, BT-483, and MDA-MB-231 cells with trastuzumab and anti-HER3 antibody conjugate (i.e., DB1001) inhibited the proliferation of each tumor cell type.

[0029] Figures 4A and 4B show the activity of DB1001 in HCC1419, HCC1569, HCC1954 and BT-483 cells under co-incubation with trastuzumab.

[0030] Figure 5 shows that trastuzumab or DB1001 can partially inhibit Heregulin-B-induced HER2 and HER3 dimer formation.

[0031] Figure 6 shows that when trastuzumab and DB1001 act on cells simultaneously, the production of HER2 and HER3 dimers can be completely inhibited.

[0032] Figure 7 shows the effects of DB1001 alone, trastuzumab alone, and DB1001 and trastuzumab in combination on HER3 phosphorylation and AKT phosphorylation levels in vitro.

[0033] Figure 8 shows the effects of DB1001 alone, trastuzumab alone, and DB1001 and trastuzumab in combination on tumor volume in tumor-bearing mice.

[0034] Figure 9 shows the effects of DB1001 alone, trastuzumab alone, and DB1001 and trastuzumab in combination on the body weight of tumor-bearing mice.

[0035] Figure 10 shows the pharmacokinetics of DB1001 in the serum of tumor-bearing mice when used alone and in combination with trastuzumab.

[0036] Figure 11 shows the pharmacokinetics of DB1001 in tumor-bearing mouse tumors when used alone or in combination with trastuzumab. Detailed Implementation Plan

[0037] Before describing the invention in detail, it should be understood that the invention is not limited to the specific methods and experimental conditions described herein, as these methods and conditions can be modified. Furthermore, the terminology used herein is for illustrative purposes only and is not intended to be restrictive.

[0038] I. Definition

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below.

[0040] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 10% less than the specified numeric value and an upper limit of 10% greater than the specified numeric value.

[0041] When the term “and / or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0042] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0043] The term "antibody" is used in the broadest sense to refer to a protein containing an antigen-binding site, encompassing various structures of natural and artificial antibodies, including but not limited to complete antibodies and antigen-binding fragments of antibodies. In this invention, "anti-HER3 antibody" and "antibody that specifically binds to Her3" are used interchangeably. In this invention, the anti-HER3 antibody is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment; preferably, the anti-HER3 antibody is an anti-HER3 monospecific antibody.

[0044] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions (complementarity-determining regions (CDRs) interspersed with more conserved regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions. In a given VH or VL amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of well-known schemes or combinations thereof, including, for example: the Chothia numbering scheme (Chothia et al., Canonical structures for the hypervariable regions of immunoglobulins, Journal of Molecular Biology, 196, 901-917 (1987)); and the Kabat numbering scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact (University College London); North numbering scheme (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406, 228-256 (2011)). The CDR of the anti-HER3 antibody in the anti-HER3 antibody drug conjugate of the present invention and the CDR of the anti-HER2 antibody used in combination with the anti-HER3 antibody drug conjugate can be determined according to any scheme or combination thereof in the art and human evaluation. In one embodiment, the CDR of the antibody of the present invention is a CDR sequence defined according to the Kabat numbering scheme.

[0045] Antibodies with different specificities (i.e., different binding sites against different antigens) have different core binding receptors (CDRs). Although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, and Contact methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0046] The term "antigen-binding fragment" refers to a portion or segment of a complete antibody with fewer amino acid residues than the complete antibody, capable of binding an antigen or competing with the complete antibody (i.e., the complete antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody antibodies, and single-domain antibodies (sdAb). The Fab fragment is a monovalent fragment composed of VL, VH, CL, and CH1 domains; for example, a Fab fragment can be obtained by digesting a complete antibody with papain. Furthermore, digestion of a complete antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. Fab' monomers are essentially Fab fragments with hinge regions (for a more detailed description of other antibody fragments, see: Fundamental Immunology, edited by WE. Paul, Raven Press, NY (1993)). The Fv fragment consists of the VL and VH domains of the antibody single arm. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be linked using recombinant methods via synthetic linker peptides that enable the two domains to be produced as a single protein chain, in which the VL and VH regions pair to form a single-stranded Fv. The antibody fragment can be obtained by chemical methods, recombinant DNA methods, or protease digestion.

[0047] The term "monoclonal antibody" refers to polypeptides having substantially the same amino acid sequence or derived from the same genetic source, including antibodies and antigen-binding fragments. This term also includes formulations of antibody molecules that are single-molecule components. Monoclonal antibodies exhibit single-molecule binding specificity and affinity for a specific epitope.

[0048] The term “binding site” or “antigen binding site” refers to the region in an antibody molecule that actually binds to an antigen, including the VH / VL pair, which consists of the antibody light chain variable domain (VL) and the antibody heavy chain variable domain (VH).

[0049] The term "monospecific antibody" refers to an antibody having one or more antigen-binding sites, each of which binds to the same epitope of the same antigen. The anti-HER3 antibody in the antibody-drug conjugate described in this article is an anti-HER3 monospecific antibody.

[0050] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen.

[0051] The term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, a humanized antibody comprises all or substantially all of its CDRs corresponding to those of the non-human antibody and all or substantially all of its FR regions corresponding to those of the human antibody. Optionally, a humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0052] The term "specific binding" refers to the formation of a complex between an antibody and an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, surface plasmon resonance assays and MSD assays (Estep, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2):p.270-278).

[0053] In the context of two or more nucleic acid or polypeptide sequences, the term "% identity" refers to the percentage of identity between sequences. Two sequences are "identical" if they have the same amino acid or nucleotide sequence in the compared region. When comparing and aligning within a comparison window or specified region to seek the largest correspondence measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, two sequences are "substantially identical" if they have a specified percentage of identity amino acid residues or nucleotides (i.e., 60% identity in the specified region or, when not specified, across the entire sequence, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity). Sequence identity between sequences is calculated as follows.

[0054] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0055] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. The particularly preferred set of parameters (and unless otherwise specified, a set of parameters to be used) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0056] Alternatively, the PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences or nucleotide sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0057] Additionally or alternatively, the nucleic acid and protein sequences described herein may be further used as “query sequences” to perform searches against public databases, for example, to identify other family member sequences or related sequences.

[0058] The term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker unit. In this invention, the terms "anti-HER3 antibody-drug conjugate," "anti-Her3 antibody-drug conjugate," or "anti-HER3 ADC" are used interchangeably and refer to antibody-drug conjugates that couple an anti-HER3 antibody and a cytotoxic drug molecule together via a linker, including their tautomers, mesosomes, racemates, enantiomers, diastereomers, and their pharmaceutically acceptable salts or pharmaceutically acceptable salts and solvates. In this invention, the anti-HER3 antibody in the anti-HER3 antibody-drug conjugate is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment; preferably, the anti-HER3 antibody is an anti-HER3 monospecific antibody.

[0059] In some embodiments of the present invention, the term "drug loading" refers to the average number of cytotoxic drugs loaded on each antibody, which may be called the average number of linkages, or expressed as the ratio of the amount of cytotoxic drug to the amount of antibody. The range of cytotoxic drug loading can be 0-12 linkages per ligand (Ab), for example, 1-10 cytotoxic drugs. The drug loading of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization. The average number of linkages p can be an integer or decimal from 1 to 10. For example, the average number of linkages p can be an integer or decimal from 2 to 8. For example, the average number of linkages p can be an integer or decimal from 3 to 8. For example, the average number of linkages p can be an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0060] In some embodiments of the present invention, antibody-drug conjugates refer to compounds containing the same DAR distribution. The term "drug loading" refers to the number of cytotoxic drugs loaded on each ligand, which can be called the number of links or expressed as the ratio of cytotoxic drug to antibody. The range of cytotoxic drug loading can be 0-12 links per ligand (Ab), for example, 1-10 cytotoxic drugs. The number of links p can be any integer from 1 to 10. For example, the number of links p can be any integer from 3 to 9. For example, the number of links p can be any integer from 6 to 8. For example, the number of links p can be 4, 5, 6, 7, or 8.

[0061] The term "drug combination" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) an anti-HER3 antibody-drug conjugate and (ii) an anti-HER2 antibody or its antigen-binding fragment) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of the two active agents. In some embodiments, the anti-HER2 antibody or its antigen-binding fragment and the anti-HER3 antibody-drug conjugate used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that the two active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two active agents are selected so that the combined use of the components produces an effect greater than that achieved by using either component alone in treating a disease or condition. The components may be in separate formulations, and their formulations may be the same or different.

[0062] The terms "pharmaceutically acceptable salt" and "medicinal salt" are used interchangeably and refer to salts that maintain the biological effects and properties of the anti-HER3 antibody drug conjugates of the present invention, and which are not biologically or otherwise undesirable. The anti-HER3 antibody drug conjugates of the present invention can exist in their pharmaceutically acceptable salt forms, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable, non-toxic acid addition salt refers to a salt formed by the anti-HER3 antibody drug conjugate of the present invention with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the anti-HER3 antibody drug conjugate of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N group.

[0063] The term "solvent" refers to an association formed by one or more solvent molecules with the anti-HER3 antibody drug conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0064] The terms “application” and “administration” are used interchangeably to refer to the physical introduction of the active ingredients of the pharmaceutical combination of the present invention into an individual using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration for the active ingredients of the pharmaceutical combination of the present invention include oral administration and parenteral administration, such as intravenous (e.g., infusion (also known as drip) or injection), intramuscular, subcutaneous, intraperitoneal, spinal, local, or other parenteral administration routes. The term “parenteral administration” refers to administration other than gastrointestinal administration, typically via intravenous, and non-limitingly includes intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Accordingly, the active ingredients of the pharmaceutical combination of the present invention can be formulated as injections (including infusions or injection solutions), liposomes, etc.

[0065] The term "dosage" refers to the amount of a drug that produces a therapeutic effect. Unless otherwise stated, dosage relates to the amount of the drug in its free form. If the drug is in the form of a pharmaceutically acceptable salt, the amount of the drug is increased proportionally to the amount of the drug in its free form. For example, the dosage will be stated on the product packaging or product information sheet.

[0066] The term "effective amount" refers to the amount of medicine that provides a desired biological, therapeutic, and / or preventive outcome. This outcome can be a reduction, improvement, mitigation, alleviation, and / or delay of one or more signs or symptoms of a disease, or any other desired alteration of the organism. In the context of cancer, an effective amount includes amounts sufficient to cause tumor shrinkage and / or a decrease in the tumor growth rate (e.g., inhibition of tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor development. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more doses. An effective dose of the drug can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down, and possibly prevent cancer cell infiltration into surrounding organs to a certain extent; (iv) inhibit (i.e., slow down and possibly prevent) tumor metastasis to a certain extent; (v) inhibit tumor growth; (vi) prevent or delay tumor occurrence and / or recurrence; and / or (vii) alleviate one or more cancer-related symptoms to a certain extent. In one implementation, an "effective dose" is the amount of a combination of antiHER3 antibody-drug conjugate and antiHER2 antibody that has been clinically proven to result in a significant reduction in tumor growth and / or slowed cancer progression.

[0067] The term "pharmaceutical-grade" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0068] The term "cancer" refers to a disease characterized by the rapid and uncontrolled proliferation of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Cancer includes, but is not limited to, solid tumors and hematologic malignancies. The cancers treated by the drug combination of the present invention are preferably advanced cancers, recurrent and / or refractory cancers, or cancers resistant to chemotherapy, more preferably advanced solid tumors, such as (histologically or cytologically confirmed) unresectable or metastatic advanced solid tumors.

[0069] The term "inhibition" refers to a given molecule (e.g., (i) an anti-HER3 antibody-drug conjugate and / or (ii) an anti-HER2 antibody or its antigen-binding fragment) causing a reduction in certain parameters (e.g., heterodimerization of HER2 and HER3, phosphorylation level of HER3, phosphorylation level of AKT, tumor volume). For example, the term includes inhibition of at least 5%, 10%, 20%, 30%, 40%, or more of activity. Therefore, inhibition need not be 100%.

[0070] The term "treatment" includes administering the pharmaceutical combination of the present invention to an individual in need to achieve the purpose of curing a disease or being effective in resolving or delaying the progression of a disease. When referring to a disease, the term "treatment" means alleviating the disease (i.e., slowing, stopping, or reducing the development of the disease or at least one of its clinical symptoms), preventing or delaying the onset, development, or progression of the disease.

[0071] The term "individual" refers to both mammals and non-mammals. Mammals include any member of the mammalian class, including but not limited to: humans; non-human primates such as cattle, horses, sheep, pigs, rabbits, dogs, and cats. The term "individual" is not limited to a specific age or sex. In some implementations, an individual is a human.

[0072] The term "adverse event" (AE) is any unfavorable and generally unexpected or unwanted symptom (including abnormal laboratory findings), condition, or illness associated with the use of a medical treatment. For example, an adverse event may be associated with activation of the immune system in response to treatment or expansion of immune system cells (e.g., T cells) in response to treatment. Medical treatments may have one or more associated AEs, and these AEs may have the same or different levels of severity.

[0073] The term "overall survival" or "OS" refers to the time from when a patient first uses the investigated drug until they die from any cause.

[0074] The term “progression-free survival” or “PFS” refers to the time from when a patient first uses the investigational drug until disease progression or death from any cause.

[0075] All numerical ranges herein should be understood as disclosing every numerical value and subset thereof within that range, regardless of whether they are specifically disclosed otherwise. For example, reference to any numerical range should be considered as reference to every numerical value within that range, such as every integer within that range. This invention relates to all values ​​falling into these ranges, all smaller ranges, and upper or lower limits of numerical ranges.

[0076] Undefined technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0077] II. The drug combination of the present invention

[0078] The drug combination of the present invention comprises an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or an antigen-binding fragment thereof.

[0079] -Anti-HER3 antibody drug conjugate of the present invention

[0080] Antibody-drug conjugates (ADCs) are a technology that utilizes the specific recognition ability of antibodies on the surface of tumor cells to precisely deliver anti-tumor drugs (such as cytotoxic agents, cell inhibitors, and small-molecule chemotherapeutic agents) to tumor target cells, causing intracellular accumulation and release, thereby precisely killing tumor cells. ADCs generally consist of three parts: an antibody or antibody-like ligand, a small-molecule drug, and a linker (connector) that conjugates the antibody or antibody-like ligand to the drug. Due to their suitable molecular weight, high stability, strong targeting, and low toxicity, ADCs are considered to be among the most promising anti-tumor drugs.

[0081] The anti-HER3 antibody-drug conjugate in the drug combination of the present invention consists of three parts: an antibody that specifically binds to Her3 or its antigen-binding fragment, a small molecule drug, and a linker (connector) that conjugates the antibody and the small molecule drug together, having the structure shown in formula (I-1):

[0082] In some embodiments, the Ab in formula (I-1) is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the Ab is not a bispecific antibody or a multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment.

[0083] In some embodiments, the Ab in formula (I-1) is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and the Ab is not a bispecific antibody or a multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment.

[0084] In some embodiments, the Ab in formula (I-1) is an anti-HER3 monospecific antibody, which includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes amino acid sequences HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and the light chain variable region includes amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0085] In some embodiments, the Ab in formula (I-1) is an anti-HER3 monospecific antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7 or has at least 95%, 96%, 97%, 98%, or 99% identity with it; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8 or has at least 95%, 96%, 97%, 98%, or 99% identity with it. For example, the Ab in formula (I-1) comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:7 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:8.

[0086] In some embodiments, the Ab in formula (I-1) is an anti-HER3 monospecific antibody comprising a heavy chain sequence of SEQ ID NO:9 or having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and a light chain sequence of SEQ ID NO:10 or having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with it. For example, the Ab in formula (I-1) comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:9 and a light chain with an amino acid sequence as shown in SEQ ID NO:10.

[0087] In some embodiments, Ab in formula (I-1) is an IgG antibody; more preferably, an IgG1 antibody or an IgG4 antibody; even more preferably, an IgG1 antibody; wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, single-chain Fv, or single-chain Fab.

[0088] In some embodiments, the anti-Her3 antibody or its antigen-binding fragment described in this invention is an anti-HER3 monospecific humanized antibody or its fragment.

[0089] In some embodiments, the anti-Her3 antibody described in this invention is a monoclonal antibody.

[0090] In some implementations, L in equation (I-1) is -L a -L b-L c -,

[0091] The -L a -for

[0092] Where W is -(C(R) wa (R) wb )) wn -, Y is -(OCH2CH2) yn -O yp -, Z is -(C(R) za (R) zb )) zn ;

[0093] Where wn is 1, 2, 3 or 6,

[0094] Each methylene unit of W is independently converted by -Cyr-, -N(R) wx )C(O)-、-C(O)N(R wx - or -C(O)- substitution;

[0095] Where yn is 0, 4 or 8, and yp is 0 or 1;

[0096] Where zn is 1, 2 or 3

[0097] Each of the methylene units of Z is independently converted by -Cyr-, -N(R) zx )C(O)-、-C(O)N(R zx - or -C(O)- substitution;

[0098] -Cyr- is a 3- to 10-membered saturated subcarbonyl cycloalgide, wherein -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R. cx replace;

[0099] Each R wa R wb R za R zb R wx R zx R cx Each independently represents hydrogen, halogen, -OR r Or be R r Optional substitution of C 1-6 Aliphatic groups;

[0100] Each R r Each is independently hydrogen, halogen, or C 1-6 Aliphatic groups;

[0101] The -L b -Selected from the following groups:

[0102] The -L c -for

[0103] Where R L1 R L2 Each is independently selected from the following groups: hydrogen, halogen, -OH and C. 1-6 Aliphatic groups.

[0104] In some preferred embodiments, L in equation (I-1) is -L a -L b -L c -,

[0105] Wherein, -L a -for

[0106] The -L b -for

[0107] The -L c -for

[0108] In some preferred embodiments, L in equation (I-1) is

[0109] In some implementations, -M- in formula (I-1) is selected from:

[0110] In some embodiments, p in this invention refers to the average number of connections. For example, the average number of connections p is an integer or decimal from 2 to 8. For example, the average number of connections p is an integer or decimal from 3 to 8. For example, the average number of connections p is an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10. For example, the average number of connections p is an integer or decimal from 7 to 8.

[0111] In some embodiments, p in this invention refers to the number of connections. The number of connections p in this invention is an integer from 2 to 8. For example, the number of connections p is an integer from 3 to 8. For example, the number of connections p is an integer of 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, the number of connections p is 6, 7, or 8.

[0112] In some implementations, the structure of the anti-HER3 antibody drug conjugate is shown in formula (II-1):

[0113] in,

[0114] p is as defined in any of the embodiments described herein;

[0115] Ab is an anti-HER3 antibody or its antigen-binding fragment as defined in any of the embodiments described herein;

[0116] L 2 It can be -O- or -S-;

[0117] L 3 Selected from -C(R) 1a (R) 1b - or -C(R) 1a (R) 1b )C(R 1a (R) 1b )-;

[0118] Each R 1a or R 1b Each can be hydrogen, halogen, or C that can be optionally substituted by R. 1-6 Aliphatic groups;

[0119] Each R can be either hydrogen or halogen.

[0120] In a preferred embodiment, the anti-HER3 antibody drug conjugate has the following structural formula:

[0121] in,

[0122] p is defined as in any of the schemes described in this paper;

[0123] Ab is an anti-HER3 antibody or its antigen-binding fragment as defined in any of the embodiments described herein.

[0124] In a preferred embodiment, the anti-HER3 antibody drug conjugate has the following structural formula:

[0125] in,

[0126] p represents the average number of connections, and p is selected from an integer or decimal of 1 to 10, preferably an integer or decimal of 3 to 8, and more preferably an integer or decimal of 7 to 8;

[0127] hu3F8-2 is an anti-HER3 antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO:9 and the light chain amino acid sequence of which is shown in SEQ ID NO:10.

[0128] In a preferred embodiment, the anti-HER3 antibody drug conjugate has the following structural formula:

[0129] in,

[0130] p is the number of connections, and p is selected from an integer from 1 to 10, preferably an integer from 3 to 8, and more preferably an integer of 6, 7 and 8;

[0131] hu3F8-2 is an anti-HER3 antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO:9 and the light chain amino acid sequence of which is shown in SEQ ID NO:10.

[0132] -Anti-HER2 antibody or its antigen-binding fragment

[0133] The anti-HER2 antibody or its antigen-binding fragment in the drug combination of the present invention specifically binds to HER2 expressed on the surface of tumor cells, thereby promoting HER2 internalization and degradation, inhibiting dimerization, blocking downstream PI3K-AKT signaling, and optionally killing tumor cells through antibody-dependent cytotoxicity (ADCC), thereby exerting an anti-tumor effect.

[0134] In some embodiments, examples of anti-HER2 antibodies in the drug combinations of the present invention include trastuzumab, pertuzumab, or antigen-binding fragments thereof.

[0135] In one embodiment, the anti-HER2 antibody in the drug combination of the present invention comprises six CDRs, wherein HCDR1, HCDR2, and HCDR3 are composed of the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively, and wherein LCDR1, LCDR2, and LCDR3 are composed of the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively. The CDR sequences are CDR sequences defined according to the Kabat numbering scheme.

[0136] Preferably, the anti-HER2 antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:17 or a sequence having at least 90%, 95%, 98%, or 99% identity with it, and the light chain variable region comprises the sequence of SEQ ID NO:18 or a sequence having at least 90%, 95%, 98%, or 99% identity with it. For example, the anti-HER2 antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:18.

[0137] Preferably, the anti-HER2 antibody comprises a heavy chain sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO:19 and a light chain sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO:20. For example, the anti-HER2 antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:19 and a light chain with an amino acid sequence as shown in SEQ ID NO:20.

[0138] Preferably, the anti-HER2 antibody is trastuzumab or its antigen-binding fragment. In some embodiments, the anti-HER2 antibody is pertuzumab or its antigen-binding fragment.

[0139] - A combination of drugs containing an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or its antigen-binding fragment.

[0140] The drug combinations of the present invention are those drug combinations in which the anti-HER3 antibody drug conjugate and the anti-HER2 antibody are respectively contained as active components in different formulations and are administered simultaneously or at different times, or they may be those drug combinations in which the anti-HER3 antibody drug conjugate and the anti-HER2 antibody are contained as active components in a single formulation and are administered.

[0141] In some embodiments, the present invention provides a drug combination in which the anti-HER3 antibody drug conjugate and the anti-HER2 antibody are contained as active ingredients in different formulations for simultaneous or sequential administration.

[0142] In some embodiments, the present invention provides a drug combination in which the anti-HER3 antibody drug conjugate and the anti-HER2 antibody are contained as active ingredients in the same formulation for simultaneous administration.

[0143] In some embodiments, the anti-HER2 antibody or its antigen-binding fragment of the present invention specifically binds to HER2 expressed on the surface of tumor cells, thereby promoting HER2 internalization and degradation, inhibiting dimerization, blocking downstream PI3K-AKT signaling, and optionally killing tumor cells via antibody-dependent cytotoxicity (ADCC).

[0144] In some embodiments, the pharmaceutical combination of the present invention comprises an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or an antigen-binding fragment thereof, wherein the anti-HER3 antibody drug conjugate is as defined in “II. Use of the anti-HER3 antibody drug conjugate of the present invention” below, “Anti-HER3 antibody drug conjugate of the present invention”, and the anti-HER2 antibody or an antigen-binding fragment thereof is, for example, trastuzumab, pertuzumab, or an antigen-binding fragment thereof.

[0145] In one embodiment, the anti-HER2 antibody comprises six CDRs, wherein HCDR1, HCDR2, and HCDR3 are composed of the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively, and wherein LCDR1, LCDR2, and LCDR3 are composed of the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively. The CDR sequences are defined according to the Kabat numbering scheme.

[0146] Preferably, the anti-HER2 antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:17 or a sequence having at least 90%, 95%, 98%, or 99% identity with it, and the light chain variable region comprises the sequence of SEQ ID NO:18 or a sequence having at least 90%, 95%, 98%, or 99% identity with it. For example, the anti-HER2 antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:18.

[0147] Preferably, the anti-HER2 antibody comprises a heavy chain sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO:19 and a light chain sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO:20. For example, the anti-HER2 antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:19 and a light chain with an amino acid sequence as shown in SEQ ID NO:20.

[0148] Preferably, the anti-HER2 antibody is trastuzumab or its antigen-binding fragment.

[0149] In some embodiments, the pharmaceutical combination of the present invention comprises an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or an antigen-binding fragment thereof, wherein the anti-HER3 antibody drug conjugate has the structure shown in formula (II-1):

[0150] in,

[0151] Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the Ab is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment.

[0152] For example, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and Ab is not a bispecific antibody or a multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment.

[0153] Preferably, the Ab is an anti-HER3 antibody hu3F8-2, whose heavy chain amino acid sequence is shown in SEQ ID NO:9 and whose light chain amino acid sequence is shown in SEQ ID NO:10.

[0154] L 2 It can be -O- or -S-;

[0155] L 3 Selected from -C(R) 1a (R) 1b - or -C(R) 1a (R) 1b )C(R 1a (R) 1b )-;

[0156] Each R 1a or R 1b Each can be hydrogen, halogen, or C that can be optionally substituted by R. 1-6 Aliphatic groups;

[0157] Each R can be either hydrogen or halogen.

[0158] p is the average number of connections as defined in any of the above schemes;

[0159] The anti-HER2 antibody or its antigen-binding fragment is the anti-HER2 antibody or its antigen-binding fragment as defined in any of the above schemes.

[0160] In some embodiments, the pharmaceutical combination of the present invention comprises an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or an antigen-binding fragment thereof, wherein the anti-HER3 antibody drug conjugate has the following structural formula:

[0161] in,

[0162] Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the Ab is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment.

[0163] For example, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and Ab is not a bispecific antibody or a multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment.

[0164] Preferably, the Ab is an anti-HER3 antibody hu3F8-2, whose heavy chain amino acid sequence is shown in SEQ ID NO:9 and whose light chain amino acid sequence is shown in SEQ ID NO:10.

[0165] p is the average number of connections as defined in any of the above schemes;

[0166] The anti-HER2 antibody or its antigen-binding fragment is the anti-HER2 antibody or its antigen-binding fragment as defined in any of the above schemes.

[0167] In some embodiments, the pharmaceutical combination of the present invention comprises an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or an antigen-binding fragment thereof, wherein the anti-HER3 antibody drug conjugate has the following structural formula:

[0168] in,

[0169] hu3F8-2 is an anti-HER3 antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO:9 and the light chain amino acid sequence of which is shown in SEQ ID NO:10.

[0170] p represents the average number of connections, and p is selected from an integer or decimal of 1 to 10, preferably an integer or decimal of 3 to 8, and more preferably an integer or decimal of 7 to 8;

[0171] The anti-HER2 antibody or its antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:18. For example, the anti-HER2 antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:19, and a light chain with an amino acid sequence as shown in SEQ ID NO:20.

[0172] III. Uses of the pharmaceutical combination of the present invention and treatment methods using the pharmaceutical combination of the present invention.

[0173] The present invention provides the aforementioned pharmaceutical combinations of the present invention for treating the severity of at least one symptom or indication of cancer in an individual or for inhibiting the growth of cancer cells.

[0174] The present invention provides the aforementioned anti-HER3 antibody drug conjugate and anti-HER2 antibody, or the aforementioned drug combination of the present invention, for the treatment of cancer.

[0175] This invention provides a method for treating cancer, comprising administering, to an individual in need, a therapeutically effective amount of the aforementioned anti-HER3 antibody-drug conjugate and anti-HER2 antibody, or administering the aforementioned drug combination of this invention. This invention also provides the use of the aforementioned drug combination of this invention in the preparation of a medicament for treating cancer.

[0176] This invention provides the use of the aforementioned anti-HER3 antibody drug conjugate and anti-HER2 antibody in the preparation of a medicament for treating cancer.

[0177] This invention provides the use of the aforementioned anti-HER3 antibody drug conjugate in the preparation of a drug for the combined treatment of cancer with an anti-HER2 antibody.

[0178] This invention provides the use of the aforementioned antiHER2 antibody in the preparation of a medicament for the combined treatment of cancer with an antiHER3 antibody drug conjugate.

[0179] The present invention provides an anti-HER3 antibody drug conjugate for the treatment of cancer, which is used in combination with an anti-HER2 antibody, said anti-HER2 antibody and anti-HER3 antibody drug conjugate as defined in any embodiment of the present invention.

[0180] The present invention provides an anti-HER2 antibody for treating cancer, which is used in combination with an anti-HER3 antibody-drug conjugate, said anti-HER2 antibody and anti-HER3 antibody-drug conjugate as defined in any embodiment of the present invention.

[0181] The pharmaceutical combination of the present invention is administered to individuals exhibiting at least the cancer-related biomarkers HER2 and / or HER3. For example, a therapeutically effective amount of the pharmaceutical combination of the present invention is administered to an individual with cancer expressing HER3 and / or HER2. For example, the cancer described in the present invention is a cancer that is positive for HER2 and / or HER3 expression.

[0182] The cancers described in this invention include solid tumors and hematologic malignancies, such as breast cancer.

[0183] In some embodiments, the cancer described in this invention is advanced cancer, refractory cancer, and / or cancer resistant to chemotherapy, more preferably advanced solid tumor, histologically or cytologically confirmed unresectable or metastatic advanced solid tumor. For example, the cancer is advanced recurrent or metastatic cancer (advanced malignancy), such as brain metastases from breast cancer.

[0184] In some embodiments, the cancer described in this invention is an advanced / unresectable or metastatic cancer.

[0185] In some embodiments, the cancer described in this invention is HER3-positive breast cancer.

[0186] In some embodiments, the cancer described in this invention is a cancer that has been previously treated with HER2-targeted therapy, for example, a cancer that has been previously treated with trastuzumab (Enhertu) or other HER2-targeting topoisomerase 1 inhibitor ADCs.

[0187] In some preferred embodiments, the cancer described in this invention is breast cancer that has been previously treated with HER2-targeted therapy, for example, breast cancer that has been previously treated with trastuzumab (Enhertu) or other HER2-targeting topoisomerase 1 inhibitor ADCs; preferably, the HER2-targeted therapy is HER2-targeting topoisomerase 1 inhibitor ADC therapy.

[0188] In some embodiments, the cancer described in this invention is HER2-positive breast cancer. In some embodiments, the cancer described in this invention is HER2-positive and HER3-positive breast cancer. For example, the HER2-positive breast cancer is a breast cancer that scores 3+ for HER2 expression in immunohistochemical IHC (IHC 3+); or the HER2-positive breast cancer is a breast cancer that scores 2+ for HER2 expression in immunohistochemical IHC and is confirmed as positive for HER2 expression in in situ hybridization ISH (IHC 2+ and ISH+).

[0189] In some embodiments, the cancer described in this invention is breast cancer with low HER2 expression. In some embodiments, the cancer described in this invention is breast cancer with low HER2 expression and positive HER3 expression. The low HER2 expression breast cancer is breast cancer that receives a HER2 expression score of 2+ in immunohistochemistry and is negative for HER2 expression in in situ hybridization (IHC 2+ and ISH-); or the low HER2 expression breast cancer is breast cancer that receives a HER2 expression score of 1+ in immunohistochemistry (IHC 1+); or the low HER2 expression breast cancer is breast cancer that receives a HER2 expression score >0 and <1+ in immunohistochemistry.

[0190] In some embodiments, the cancer described in this invention is breast cancer with ultra-low HER2 expression. In some embodiments, the cancer described in this invention is breast cancer with ultra-low HER2 expression and HER3 positive expression. The ultra-low HER2 expression breast cancer is breast cancer with an immunohistochemical HER2 expression score of 0 and membrane staining (IHC 0 with membrane staining). In some embodiments, the cancer described in this invention is triple-negative breast cancer. In some embodiments, the breast cancer is selected from HER3 positive triple-negative breast cancer (TNBC).

[0191] There are no particular limitations on the methods used to score HER2 expression levels by immunohistochemistry or to determine HER2 expression positivity or negativity by in situ hybridization, as long as they are methods recognized by those skilled in the art. For example, the method may include the method for breast cancer detection described in ASCO 2018. In some embodiments, the drug combination of the present invention exhibits significantly superior antitumor effects compared to the single administration of the antiHER3 antibody-drug conjugate.

[0192] In some embodiments, the drug combinations of the present invention exhibit synergistic antitumor effects.

[0193] The drug combination of the present invention can be administered to individuals who have been treated with one or more prior therapies but subsequently relapse or metastasize.

[0194] The anti-HER3 antibody-drug conjugate in the drug combination of the present invention can be administered to individuals in need at one or more doses. For example, in the case of multiple doses, the next dose can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the previous dose.

[0195] The anti-HER2 antibody or its antigen-binding fragment in the drug combination of the present invention can be administered to individuals in need at one or more doses. For example, in the case of multiple doses, the next dose may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the previous dose.

[0196] The pharmaceutical combination of the present invention can be any dosage form known to those skilled in the art, such as tablets, capsules, granules, syrups, powders, lozenges, capsules, flat capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, and injections. The anti-HER3 antibody drug conjugate and the anti-HER2 antibody or its antigen-binding fragment can each be in separate dosage forms, which can be different or the same. The anti-HER3 antibody drug conjugate can be an intravenous dosage form or an intraperitoneal injection. The anti-HER2 antibody or its antigen-binding fragment can be an intravenous dosage form or an intraperitoneal injection.

[0197] The drug combination of the present invention may be in the form of a drug dosage unit, such as a single drug dosage unit.

[0198] The anti-HER3 antibody drug conjugate and the anti-HER2 antibody or its antigen-binding fragment in the drug combination of the present invention can be administered separately, simultaneously or sequentially.

[0199] In some embodiments, administration of at least one cycle of the drug combination of the present invention results in an increased, preferably synergistically increased, progression-free survival (PFS) or overall survival (OS) compared to patients receiving monotherapy with an anti-HER3 antibody-drug conjugate or monotherapy with an anti-HER2 antibody or its antigen-binding fragment. In some embodiments, administration of at least one cycle of the drug combination of the present invention results in an increased PFS of at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, or longer compared to patients receiving monotherapy with an anti-HER3 antibody-drug conjugate or monotherapy with an anti-HER2 antibody or its antigen-binding fragment. In some implementations, compared with monotherapy of an anti-HER3 antibody drug conjugate or monotherapy of an anti-HER2 antibody or its antigen-binding fragment, administration of at least one cycle of the drug combination of the present invention resulted in an increase in overall survival (OS) of at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, or longer.

[0200] Compared to monotherapy with an anti-HER3 antibody-drug conjugate or monotherapy with an anti-HER2 antibody or its antigen-binding fragment, the drug combinations of the present invention result in an increased, preferably synergistic, increase in the inhibition of tumor growth. In some embodiments, compared to monotherapy with an anti-HER3 antibody-drug conjugate or monotherapy with an anti-HER2 antibody or its antigen-binding fragment, the drug combinations of the present invention result in an inhibition of tumor growth of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some embodiments, administration of the drug combinations of the present invention results in increased tumor regression, tumor shrinkage, and / or disappearance. In some embodiments, the drug combinations of the present invention increase the duration of survival of individuals compared to untreated individuals or to monotherapy with an anti-HER3 antibody drug conjugate or monotherapy with an anti-HER2 antibody or its antigen-binding fragment, for example, by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months. In some embodiments, the drug combinations of the present invention may increase progression-free survival or overall survival.

[0201] In some embodiments, administration of the pharmaceutical combination of the present invention to an individual with cancer results in the complete disappearance of the tumor (“complete response”). In some embodiments, administration of the pharmaceutical combination of the present invention to an individual with cancer results in a reduction of tumor cells or tumor size by at least 30% or more (“partial response”). The reduction in tumor size can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytological, histological, or molecular genetic analysis.

[0202] In some embodiments, the pharmaceutical combinations of the present invention can reduce adverse events, such as hematological toxicities, non-hematological toxicities, or other toxic reactions, caused by administration of anti-HER2 antibodies or their antigen-binding fragments and / or anti-HER3 antibody drug conjugates.

[0203] IV. The medicine box of the present invention

[0204] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0205] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0206] - A first container containing a pharmaceutical composition for administration, the pharmaceutical composition comprising an anti-HER3 antibody-drug conjugate;

[0207] - A second container containing a pharmaceutical composition for administration, the pharmaceutical composition comprising an anti-HER2 antibody or an antigen-binding fragment thereof.

[0208] The various embodiments / technical solutions and features described in this invention should be understood as being arbitrarily combined with each other, and all solutions obtained by such combinations are included within the scope of this invention, just as these solutions obtained by such combinations are specifically and individually listed herein, unless the context clearly indicates otherwise.

[0209] Example

[0210] The following examples provide a complete disclosure and description of how to prepare the compositions of the present invention and how to use them to those skilled in the art, and are not intended to limit the scope of the invention as understood by the inventors.

[0211] Example 1. Preparation of anti-HER3 antibody drug conjugate

[0212] According to the method described in PCT Publication No. WO 2023 / 143365, an anti-HER3 antibody-drug conjugate (also referred to as "anti-HER3 ADC") DB1001 was prepared by linking an anti-HER3 antibody hu3F8-2 (corresponding to the heavy chain amino acid sequence shown in WO 2023 / 143365 as shown in SEQ ID NO:13 and the light chain amino acid sequence shown in SEQ ID NO:12) with a linker-cytotoxic agent.

[0213] Specifically, the reducing agent and protecting agent were prepared using ultrapure water as follows: 2 mg / ml TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo) and 100 mmol / L sodium ethylenediaminetetraacetate (manufacturer: Sigma). 150 mg of the 7.4 mg / ml anti-HER3 antibody hu3F8-2 was placed in a 50 ml centrifuge tube, and 50 mM sodium phosphate buffer was added to dilute the antibody concentration to 5 mg / ml at pH 7.5. 100 mmol / L sodium ethylenediaminetetraacetate was added at 5% of the total reaction volume, and the mixture was shaken and mixed. Then, 2 mg / ml TCEP was added for antibody reduction. The molar ratio of TCEP to antibody was 6.0:1. After shaking and mixing, the mixture was placed in a refrigerated constant-temperature mixer at 37°C for 2 h. A 10 mg / mL solution of linker-payload in dimethyl sulfoxide (DMSO) (manufacturer: Sinopharm Group) was prepared. The DMSO was slowly added at a drug-to-antibody molar ratio of 21:1, and the mixture was shaken and stirred. The mixture was then reacted in a refrigerated constant-temperature mixer at 4°C for 1 hour. The sample was replaced with storage buffer using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). The sample was first ultrafiltered three times with 30 mM histidine acetate buffer containing 10% dimethyl sulfoxide at pH 5.5, followed by three ultrafiltrations with 30 mM histidine acetate buffer without DMSO at pH 5.5. The resulting ultrafiltration concentration yielded the anti-HER3 antibody-drug conjugate DB1001 at a concentration of 20.56 mg / mL, with a yield of 64%. Purity and DAR value were determined using size exclusion chromatography and hydrophobic chromatography. Structure of the linker-cytotoxin (linker-payload):

[0214] The structure of the anti-HER3 antibody-drug conjugate DB1001:

[0215] The purity of the anti-HER3 antibody-drug conjugate DB1001 was found to be 99.02%, and the DAR value p was 7.53.

[0216] Example 2. Detection of HER2 and HER3 expression levels on the surface of different breast cancer cells using flow cytometry.

[0217] In this embodiment, the expression levels of HER2 and HER3 on the surface of different breast cancer cells were detected by flow cytometry, with the aim of selecting cell lines with different HER2 and HER3 expression levels for subsequent experiments.

[0218] Experimental methods

[0219] Cell culture: HCC1419, HCC1569, HCC1954, BT-483, and MDA-MB-231 cells were cultured in vitro according to the conditions in Table 1. Cell culture was conducted at 37°C in a 5% CO2 incubator. Cells were collected for subsequent experiments when cell saturation reached 80%-90%.

[0220] Table 1. Cell lines and culture methods

[0221] Flow cytometry was used to detect the expression levels of Her2 and Her3 on the surface of different breast cancer cells: Cells were treated with trypsin and counted to ensure cell viability was above 90.0%. 2 × 10⁻⁶ cells were added to each well of a 96-well U-plate. 5 Cells were washed twice with FACS staining buffer (PBS + 1% FBS), centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. Then, 100 μL of anti-HER3 antibody hu3F8-2 (i.e., the antibody unit in DB1001 used as an ADC) or isotype IgG1 (final concentration 100 nM) was added and incubated at 4°C for 60 minutes; or 100 μL of anti-HER2 antibody trastuzumab or isotype IgG1 (final concentration 100 nM) was added and incubated at 4°C for 60 minutes. After incubation, cells were washed twice with FACS staining buffer, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. PE anti-human IgG Fc Antibody (Biolegend410708) (1:500 dilution) was added to each well and incubated at 4°C for 40 minutes in the dark. After incubation, the cells were washed twice with FACS staining buffer, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 200 μL of FACS staining buffer and then analyzed by flow cytometry (BD Fortessa X20).

[0222] result

[0223] Figure 1 shows that Her2 is expressed on the surface of HCC1419, HCC1569, and HCC1954 cells, and HER3 is expressed on the surface of HCC1419, HCC1569, and BT-483 cells, while no Her2 or Her3 expression is detected on the surface of MDA-MB-231 cells. The highest Her2 expression level was observed on the surface of HCC1419 cells, followed by HCC1569 and HCC1954 cells. Furthermore, the highest Her3 expression level was observed on the surface of HCC1569 cells, followed by HCC1419 and BT-483 cells.

[0224] Example 3. Flow cytometry was used to detect the effect of trastuzumab treatment on the expression level of HER3 on the cell surface of different breast cancer cells.

[0225] In this embodiment, flow cytometry was used to detect the effect of trastuzumab treatment on the expression level of HER3 on the cell surface of different breast cancer cells.

[0226] Experimental methods

[0227] Cell culture: HCC1419, HCC1569, HCC1954, and BT-483 cells were cultured in vitro according to the conditions in Table 1. Cell culture was conducted at 37°C in a 5% CO2 incubator. When cell saturation reached 80%-90%, cells were collected for subsequent plate-coating experiments.

[0228] Cell treatment: Cells were treated with trypsin and counted to ensure a cell viability of over 90.0%. 2 × 10⁻⁶ cells were added to each well of a 96-well U-plate. 5 Add 100 μL of trastuzumab (final concentration: 100 nM) to the corresponding well of the 96-well plate and incubate for 24 hours in a cell culture incubator.

[0229] Flow cytometry assay: After incubation, cells were washed twice with FACS staining buffer (PBS + 1% FBS), centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. Then, 100 μL of hu3F8-2 or isotype IgG1 (final concentration 100 nM) was added and incubated at 4°C for 60 minutes. After incubation, cells were washed twice with FACS staining buffer, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. PE anti-human IgG Fc Antibody (Biolegend 410708) (1:500 dilution) was added to each well, and the cells were incubated at 4°C for 40 minutes in the dark. After incubation, cells were washed twice with FACS staining buffer, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in 200 μL of FACS staining buffer and analyzed using a flow cytometer (BD Fortessa X20).

[0230] result

[0231] Figure 2 shows that before and after trastuzumab treatment of HCC1419, HCC1569, HCC1954 and BT-483 cells, under the conditions of this experiment, there was no significant change in the expression level of HER3 on the cell surface.

[0232] Example 4. Evaluation of the synergistic effect of DB1001 and trastuzumab in inhibiting tumor cell proliferation by cell viability assay.

[0233] In this embodiment, the synergistic effect of DB1001 and trastuzumab on inhibiting the proliferation of HCC1419, HCC1569, HCC1954, BT-483 and MDA-MB-231 cells was evaluated by cell viability assay.

[0234] Experimental methods

[0235] Cell culture: HCC1419, HCC1569, HCC1954, BT-483, and MDA-MB-231 cells were cultured in vitro according to the conditions in Table 1. Cell culture was conducted at 37°C in a 5% CO2 incubator. When cell saturation reached 80%-90%, cells were collected for subsequent plate-coating experiments.

[0236] Cell plating: Stain cells with trypan blue and count viable cells, adjusting the cell concentration to an appropriate level. Add 90 μL of cell suspension to each well of the culture plate, and add cell-free culture medium to the blank control wells. Incubate the culture plate overnight in an incubator.

[0237] Preparation of working solution of test substance: The test substance stock solution was diluted from high concentration to low concentration in cell culture medium to prepare a 10-fold working solution of test substance.

[0238] Cells were treated with the drug: 10 μL of a 10-fold dilution of the test substance working solution was added to the cell culture plate (final drug concentration: 100 nM). 10 μL of cell culture medium mixture was added to both the solvent control and blank control. Each condition was applied in duplicate. The 96-well cell culture plate was then returned to the incubator and cultured for 7 days.

[0239] CellTiter-Glo luminescence assay for cell viability: Follow the steps outlined in the instructions for the Promega CellTiter-Glo luminescence assay kit (Promega-G7573).

[0240] 1) Melt the CellTiter-Glo buffer and let it reach room temperature.

[0241] 2) Allow the CellTiter-Glo substrate to reach room temperature.

[0242] 3) Add CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate, thereby preparing the CellTiter-Glo working solution.

[0243] 4) Slowly vortex the CellTiter-Glo working solution to fully dissolve the CellTiter-Glo substrate.

[0244] 5) Remove the 96-well cell plate that has been cultured in the incubator for 7 days and let it stand for 30 minutes to equilibrate to room temperature.

[0245] 6) Add 50 μL (equivalent to half the volume of cell culture medium in each well) of CellTiter-Glo working solution to each well of the cell culture plate. Wrap the cell plate with aluminum foil to protect it from light.

[0246] 7) Shake the culture plate on a track shaker for 2 minutes to induce cell lysis.

[0247] 8) Place the culture plate at room temperature for 10 minutes to stabilize the luminescence signal.

[0248] 9) Detect the light emission signal on the 2104 EnVision reader.

[0249] Data Analysis: The inhibition rate (IR) of the test compound was calculated using the following formula:

[0250] IR (%) = (1 – (RLU compound – RLU blank control) / (RLU solvent control – RLU blank control)) * 100%.

[0251] Calculate the inhibition rate of compounds at different concentrations in Excel. Calculate the combination index (CI) using Compusyn (1.0). The CI value has the following meanings: CI < 0.9: synergistic effect; 0.9 < CI < 1.1: additive effect; CI > 1.1: antagonistic effect.

[0252] Results

[0253] Culture HCC1419 cells, HCC1569 cells, HCC1954 cells, BT-483 cells, and MDA-MB-231 cells alone with 100 nM trastuzumab, 100 nM DB1001, or in combination with 100 nM trastuzumab and 100 nM DB1001 for 7 days, and detect cell proliferation by CellTiter-Glo luminescence cell assay. Figure 3 shows that trastuzumab and DB1001 both show the effect of inhibiting cell proliferation on HCC1419 cells and HCC1569 cells. Since CI < 0.9, it indicates that the combination of trastuzumab and DB1001 has a synergistic effect on inhibiting cell proliferation in the above two types of cells. In addition, trastuzumab and DB1001 do not show a synergistic effect on HCC1954 cells, BT-483 cells, and MDA-MB-231 cells with low or no expression of HER3 or HER2.

[0254] Example 5. Detection of the in vitro activity of the internalization of DB1001 by tumor cells under trastuzumab treatment conditions based on a live cell imaging-based internalization method

[0255] In this example, a live cell imaging-based internalization method was used to detect the in vitro activity of the internalization of DB1001 by tumor cells through the antibody on DB1001 under trastuzumab treatment conditions.

[0256] Experimental method

[0257] Cell culture: HCC1419 cells, HCC1569 cells, HCC1954 cells, and BT-483 cells were cultured in vitro according to the conditions in Table 1. Cell culture was carried out in a 37 °C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were collected for subsequent experimental plating.

[0258] Dye-labeled DB1001: Dilute the test compound DB1001 or the control isotype IgG1 to 40 nM, and mix it thoroughly with the Fabfluor-pH antibody-labeled dye at a volume ratio of 1:3, and incubate at 37 °C for 30 min before use in the experiment.

[0259] In vitro activity assay for ADC internalization: Cells were seeded into 96-well plates at a density of 10,000 cells per well and cultured overnight. Four groups were prepared: allotype IgG1-dye group, DB1001-dye group, DB1001-dye + allotype IgG1 group, and DB1001-dye + trastuzumab group. Dye-labeled allotype IgG1 control / sample working solution was transferred to the corresponding wells of the experimental plate. The concentration of DB1001 was 40 nM, and the concentration of trastuzumab was 40 nM. The experimental plate was transferred to the Incucyte live cell analyzer, and the appropriate scanning and imaging program was set up. Images were acquired using the Incucyte live cell analyzer system. The analytical results are expressed as: total fluorescence signal intensity (RCU × μm). 2 / Image). If a dose-response curve needs to be fitted for multiple concentration points, the fitted curve can be directly exported using Incucyte software or analyzed using GraphPad Prism 6 software.

[0260] result

[0261] Figures 4A and 4B show that, compared with incubation of DB1001 alone or incubation of DB1001 + isotype IgG1, co-incubation of trastuzumab with DB1001 exhibited higher antibody-mediated internalization activity on HCC1419, HCC1569, and HCC1954 cells. In Figure 4, ** indicates P < 0.01, indicating a statistically significant difference.

[0262] Example 6. Inhibition of Heregulin-B-induced HER2 and HER3 dimer formation by trastuzumab and DB1001

[0263] In this embodiment, Heregulin-B, acting as a ligand for HER3, specifically binds to HER3, inducing the formation of a dimer between HER3 and HER2 and activating downstream signaling pathways. This experiment was conducted using... The kinase-dimerization assay was used to detect the inhibitory effect of trastuzumab and DB1001 on the formation of Heregulin-B-induced HER3 and HER2 dimers.

[0264] Experimental methods

[0265] Cell culture: thawing cryopreserved cells (Eurofins Discovery) cell lines were cultured at 37°C in a 5% CO2 incubator. When cell saturation reached 80%-90%, cells were collected and transferred to 384-well cell culture plates at a volume of 20 μL per well.

[0266] The Heregulin-B stimulation conditions for cells were determined as follows: Heregulin-B was serially diluted 3-fold starting from 1 μg / ml. 5 μL of Heregulin-B, prepared to a final stimulation concentration of 5-fold, was added to a 384-well cell culture plate and cultured for 16 hours. Then, 12.5 μL of PathHunter Detection reagent cocktail was added, and the plate was incubated at room temperature for one hour. The chemiluminescence signal was then read using a PerkinElmer Envision microplate reader. Based on the concentration-luminescence value dose curve, the optimal EC80 concentration for Heregulin-B was determined to be 0.0048 μg / ml.

[0267] To determine the inhibitory effect of the test substance on the formation of HER3 and HER2 dimers: Trastuzumab or DB1001 was serially diluted 3-fold starting from 1 μM. The antibody was then diluted 6-fold to the final concentration. 5 μL of 0.0048 μg / ml Heregulin-B was added to each cell well, and the cells were incubated at room temperature for 1 hour. Then, 5 μL of the antibody was added. The cells were incubated at 37°C, 5% CO2 for 16 hours. 15 μL of PathHunter Detection reagent cocktail was added, and the cells were incubated at room temperature for 1 hour. The chemiluminescence signal was then read using a PerkinElmer Envision microplate reader.

[0268] The inhibitory effect of trastuzumab and DB1001 combined on the formation of HER3 and HER2 dimers was determined: Trastuzumab and DB1001 mixed antibody solutions were prepared at a final concentration of 6x according to the concentrations corresponding to those in Table 2. 5 μL of 0.0048 μg / ml Heregulin-B was added to each cell well, and after incubation at room temperature for 1 hour, 5 μL of anti-HER2 antibody and / or ADC mixture was added. The cells were incubated at 37°C, 5% CO2 for 16 hours. 15 μL of PathHunter Detection reagent cocktail was added, and after incubation at room temperature for 1 hour, the chemiluminescence signal value was read using a PerkinElmer Envision microplate reader.

[0269] Table 2 Conditions for combined treatment with trastuzumab and DB1001

[0270] result

[0271] Figure 5 and Table 3 show that trastuzumab or DB1001 can partially inhibit Heregulin-B-induced HER2 and HER3 dimer formation. Figure 6 shows that when trastuzumab and DB1001 act on cells simultaneously, the production of HER2 and HER3 dimers can be completely inhibited.

[0272] Table 3. Inhibition rates of trastuzumab or DB1001 on Heregulin-B-induced HER2 and HER3 dimers

[0273] Example 7. Trastuzumab and DB1001 inhibit HER3 phosphorylation and AKT phosphorylation levels in cells.

[0274] In this embodiment, Heregulin-B, as a ligand for HER3, specifically binds to HER3 and induces HER3 phosphorylation and the phosphorylation of downstream effector protein AKT. This experiment uses Western blotting to study the inhibitory effects of DB1001 and trastuzumab on Heregulin-B-induced HER3 phosphorylation and AKT phosphorylation in HCC1569 and HCC1419 cells.

[0275] Experimental methods

[0276] Cell culture: HCC1569 and HCC1419 cells were cultured in vitro according to the conditions in Table 1. Cell culture was conducted at 37°C in a 5% CO2 incubator. When cell saturation reached 80%-90%, cells were collected for subsequent plate-coating experiments.

[0277] Cell treatment: HCC1569 and HCC1419 cells were collected, stained with trypan blue, and viable cells were counted. The cell concentration was adjusted to an appropriate level. 2 mL of cell culture medium (without FBS) was transferred to a 6-well plate and cultured overnight. The next day, the culture medium was removed, and 1.5 mL of fresh, pre-chilled cell culture medium containing 100 nM DB1001, 100 nM trastuzumab, or 100 nM DB1001 + 100 nM trastuzumab (final concentration of the analyte: 100 nM) was added. The cells were incubated at 4°C for 0.5 h, and then 0.5 mL of Heregulin-B was added to the cell culture medium (final concentration: 0.5 nM).

[0278] Cell lysis buffer preparation: After cells were treated with the analyte for 0.5 h, they were washed twice with PBS. 500 μL of pre-chilled RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphorylase inhibitor) was added to each culture plate (six-well plate), and the plates were incubated on ice for 30 min, mixing several times during incubation. Cell debris was removed by centrifugation at approximately 14,000 rpm for 10 min at 4 °C. The supernatant was transferred to a new tube for protein concentration determination and subsequent experiments.

[0279] Protein concentration determination: Protein quantification was performed using a BCA quantitative kit. Based on the quantification results, protein samples were prepared for loading, with the protein concentration standardized to 1-2 μg / μL. LDS loading buffer (4X) (Thermo Scientific, NP0008) and sample reducing agent (10X) (Thermo Scientific, NP0009) were added, and the samples were heated at 100°C for 10 minutes. Subsequent immunoblotting was then performed.

[0280] Immunoblot assay: After thawing, the sample was loaded into an SDS-PAGE gel, 10 μL per well (the loading volume depends on the antibody titer). Electrophoresis was performed at 80 V for 30 minutes, followed by 120 V for 90 minutes. The membrane was transferred using the iBlot2 transfer kit and transfer apparatus, running the P3 transfer program for 7 minutes. After transfer, the membrane was cut to the molecular weight of the protein to be detected. The membrane was washed three times with 1x TBST, 5 minutes each time, at room temperature with shaking. The membrane was then blocked in blocking buffer (5% skim milk prepared with 1x TBST) at room temperature for 1 hour. The membrane was washed three times with 1x TBST, 5 minutes each time. Appropriate dilutions of the primary antibody (see Table 4 below) (diluted with 5% bovine serum albumin prepared with 1x TBST) were added, and the membrane was incubated overnight at 4°C with gentle shaking. The membrane was washed three times with 1x TBST, 10 minutes each time. Add the appropriate dilution of the second antibody (Goat anti-Rabbit IgG-HRP, Thermo Fisher, #31462) and incubate at room temperature for 1 hour. Wash the membrane three times with 1x TBST for 10 minutes each time. Add the HRP substrate from the West Femto Ultrasensitive Chemiluminescence Kit (Thermo Scientific, 34096) to the membrane. Detect the chemiluminescence and photograph it using a Bio-Rad ChemiDoc™ xrs instrument. The results are shown in Figure 7. In Figure 7, NRG1 is an abbreviation for NeuReGulin 1 (neural regulatory protein 1), which is synonymous with Heregulin-B and can be used interchangeably in this article.

[0281] Table 4. Antibody Sources

[0282] result

[0283] Figure 7 shows that DB1001 can partially inhibit HER3 phosphorylation and AKT phosphorylation. Trastuzumab has a partial inhibitory effect on HER3 phosphorylation but no inhibitory effect on AKT phosphorylation. When DB1001 and trastuzumab act together on cells, they also have a partial inhibitory effect on HER3 phosphorylation or AKT phosphorylation.

[0284] Example 8. In a subcutaneous xenograft NOD / SCID female mouse model of human breast cancer HCC1569 cell line, trastuzumab and DB1001 synergistically inhibited tumor growth.

[0285] In this embodiment, the synergistic effect of trastuzumab and DB1001 on tumor inhibition in vivo was tested using a human breast cancer HCC1569 model.

[0286] Experimental methods

[0287] Cell culture: HCC1569 cells were cultured in vitro according to the conditions in Table 1. Cell culture was carried out at 37°C in a 5% CO2 incubator.

[0288] Test drugs and materials:

[0289] G01: Blank control group (negative control group): PBS

[0290] G02:DB1001 (Alone Treatment Group 1): 0.3 mg / kg

[0291] G03:DB1001 (Alone Treatment Group 2): 1 mg / kg

[0292] G04: Trastuzumab (monotherapy group): 20 mg / kg

[0293] G05: DB1001 0.3mg / kg + Trastuzumab 20mg / kg (Combination Therapy Group 1)

[0294] G06: DB1001 1mg / kg + Trastuzumab 20mg / kg (Combination therapy group 2)

[0295] Preparation method: All samples were prepared by diluting with PBS.

[0296] Laboratory animals:

[0297] 8-10 week old NOD / SCID mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0298] Animal inoculation and tumor measurement:

[0299] The experimental mice were subcutaneously inoculated with 2×10⁻⁶ mice at the right anterior scapula. 6 HCC1569 cells were resuspended in a 1:1 mixture of PBS and Matrigel (0.1 ml / cell). Tumor growth was observed until the tumor reached an average volume of 100-150 mm. 3 Mice were randomly assigned to groups of 6 mice each, based on tumor size and body weight. The day of administration was defined as day 0.

[0300] The drugs were administered via tail vein injection. DB1001 was given once every two weeks, and trastuzumab was given once a week. The experiment ended after 35 days of administration. Tumor volume and mouse weight were measured twice a week, and the data were recorded. At the end of the experiment, the mice were euthanized, and the tumor inhibition rate (TGI%) was calculated (TGI = (1 - (Ti - T0) / (Vi - V0))). Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the negative control group on day i of administration; V0: mean tumor volume in the negative control group on day 0 of administration.

[0301] Data Analysis:

[0302] Data were statistically analyzed using Excel 2016: mean values ​​were calculated as averages (avg); SD values ​​were calculated as standard deviations (STDEV); and p-values ​​for intergroup differences were calculated as tsets (TTEST).

[0303] The experimental results are shown in Table 5 and Figure 8. At the end of the experiment, the average tumor volume in the blank control group was 2042.99 mm. 3 The efficacy comparisons among the groups are shown in Table 6.

[0304] in conclusion

[0305] As shown in Figure 8, DB1001 exhibited an inhibitory effect on tumor growth at a dose of 1 mg / kg. Trastuzumab alone at a dose of 20 mg / kg had no inhibitory effect on tumors. Complete tumor clearance was achieved with the combined treatment of DB1001 and trastuzumab. DB1001 and trastuzumab demonstrated a synergistic effect in tumor suppression. In this experiment, mice in the solvent group, DB1001 monotherapy group, trastuzumab monotherapy group, and DB1001 and trastuzumab combination therapy group showed no significant weight loss (Figure 9).

[0306] Table 5. Efficacy results at the experimental endpoint (i.e., day 35).

[0307] Table 6. Comparative analysis of drug efficacy among groups

[0308] Note: Because the Bartlett test value is <0.05 and the Kruskal-Walis test value is <0.05, the Conover test is used to compare all groups pairwise; the p-value is calculated from the absolute tumor volume, and italicized and bold text indicates a significant difference between the two groups. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0309] Example 9. Pharmacokinetic determination of DB1001 in a female mouse model of subcutaneous xenograft of human breast cancer HCC1569 cell line into NOD / SCID.

[0310] In this embodiment, the human breast cancer HCC1569 model was used to test the ADC pharmacokinetics of DB1001 in the tumor body and peripheral blood under DB1001 treatment alone and trastuzumab + DB1001 combination therapy.

[0311] Experimental methods

[0312] Cell culture: HCC1569 cells were cultured in vitro according to the conditions in Table 1. Cell culture was carried out at 37°C in a 5% CO2 incubator.

[0313] Test drugs and materials:

[0314] DB1001 (monotherapy group 1): 1 mg / kg

[0315] DB1001 (monotherapy group 2): 3 mg / kg

[0316] DB1001 1mg / kg + trastuzumab 20mg / kg (combination therapy group 1)

[0317] DB1001 3mg / kg + trastuzumab 20mg / kg (combination therapy group 2)

[0318] Preparation method: All samples were prepared by diluting with PBS.

[0319] Laboratory animals:

[0320] 8-10 week old NOD / SCID mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0321] Animal inoculation and sample collection:

[0322] The experimental mice were subcutaneously inoculated with 2×10⁻⁶ mice at the right anterior scapula. 6 HCC1569 cells were resuspended in a 1:1 mixture of PBS and Matrigel (0.1 ml / cell). Tumor growth was observed until the tumor reached an average volume of 150 mm. 3 Mice were randomly divided into groups of 3 mice each, based on tumor size and body weight. Administration was via tail vein injection. Mice were euthanized at 15 minutes, 3 hours, 24 hours, 48 ​​hours, and 96 hours post-administration. Serum was collected, tumor weight was measured, and tumor samples were quick-frozen.

[0323] PK test:

[0324] DB1001 concentration was determined using an ELISA method. ELISA plates (96 wells) (Thermo, 446469) were coated overnight at 2–8°C with coating buffer (0.05 M CBS) containing anti-DB1001 antibody (obtained through in-house screening). After overnight coating at 4°C, the plates were washed with washing buffer (PBS, 0.05% Tween 20, pH 7.4) and treated with blocking buffer (PBS, 0.05% Tween 20, 0.2% I-Block, pH 7.4) for 2 h, followed by incubation with standards and samples diluted in blocking buffer for 2 h. The ELISA plates were washed four times with washing buffer and then incubated for 1 h with diluted HRP-goat anti-human IgG polyclonal antibody (H+L) (monkey ads) (Bethyl, A80-319P) buffer. After four washes, the bound HRP conjugates were detected using tetramethylbenzidine (TMB) peroxidase substrate. The enzymatic reaction was stopped by adding 1 M sulfuric acid after 15 minutes. Absorbance was measured at 450 nm using a Molecular Devices (SpectraMax M3) microplate reader, with a reference wavelength of 630 nm. The limit of quantitation (LLOQ) was 100 ng / mL.

[0325] result

[0326] In serum, there was no significant difference in the pharmacokinetics of DB1001 between treatment with DB1001 alone and in combination therapy with trastuzumab + DB1001 (Figure 10). In tumors, DB1001 was better enriched in the trastuzumab + DB1001 combination group compared to the DB1001 alone group, resulting in higher concentrations of ADC molecules in the tumor (Figure 11).

[0327] Exemplary sequence

[0328] Although some representative embodiments and details have been shown for illustrative purposes, it will be apparent to those skilled in the art that various changes and modifications can be made to them without departing from the scope of the subject matter. In this respect, the scope of the invention is defined only by the following claims.

Claims

1. A drug combination comprising an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or an antigen-binding fragment thereof, wherein the anti-HER3 antibody drug conjugate has the structure shown in formula (I-1): in, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the Ab is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. For example, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and Ab is not a bispecific antibody or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. Preferably, the Ab is an anti-HER3 monospecific antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. Preferably, the anti-HER3 monospecific antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; More preferably, the heavy chain amino acid sequence of the anti-HER3 monospecific antibody is shown in SEQ ID NO:9, and the light chain amino acid sequence is shown in SEQ ID NO:10; L is -L a -L b -L c -, The -L a -for Where W is -(C(R) wa (R) wb )) wn -, Y is -(OCH2CH2) yn -O yp -, Z is -(C(R) za (R) zb )) zn ; Where wn is 1, 2, 3 or 6, Each methylene unit of W is independently converted by -Cyr-, -N(R) wx )C(O)-、-C(O)N(R wx - or -C(O)- substitution; Where yn is 0, 4 or 8, and yp is 0 or 1; Where zn is 1, 2 or 3 Each of the methylene units of Z is independently converted by -Cyr-, -N(R) zx )C(O)-、-C(O)N(R zx - or -C(O)- substitution; -Cyr- is a 3- to 10-membered saturated subcarbonyl cycloalgide, wherein -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R. cx replace; Each R wa R wb R za R zb R wx R zx R cx Each independently represents hydrogen, halogen, -OR r Or be R r Optional substitution of C 1-6 Aliphatic groups; Each R r Each is independently hydrogen, halogen, or C 1-6 Aliphatic groups; Preferably, the -L a -for The -L b -Selected from the following groups: Preferably, the -L b -for The -L c -for Where R L1 R L2 Each is independently selected from the following groups: hydrogen, halogen, -OH and C. 1-6 Aliphatic groups; Preferably, the -L c -for Most preferably, L is -M- is selected from: p represents the average number of drug connections relative to each Ab molecule, and p is selected from an integer or decimal from 1 to 10; for example, the average number of connections p is an integer or decimal from 2 to 8; for example, the average number of connections p is an integer or decimal from 3 to 8; for example, the average number of connections p is an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10; for example, the average number of connections p is an integer or decimal from 7 to 8. Alternatively, p represents the number of drug linkages relative to each Ab molecule, for example, the number of linkages p is an integer from 2 to 8; for example, the number of linkages p is an integer from 3 to 8; for example, the number of linkages p is an integer of 2, 3, 4, 5, 6, 7, 8, 9 or 10; for example, the number of linkages p is 6, 7 or 8; For example, the antibody-drug conjugate has the following structural formula: in, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the Ab is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. For example, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and Ab is not a bispecific antibody or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. Preferably, the Ab is an anti-HER3 monospecific antibody, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:8; preferably, the heavy chain amino acid sequence is shown in SEQ ID NO:9, and the light chain amino acid sequence is shown in SEQ ID NO:

10. p is the number of connections, and p is selected from an integer from 1 to 10, preferably an integer from 3 to 8, and more preferably an integer of 6, 7 and 8.

2. The drug combination according to claim 1, wherein the anti-HER2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16 respectively; Preferably, the anti-HER2 antibody or its antigen-binding fragment comprises The heavy chain variable region and the light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:17 or has at least 95%, 96%, 97%, 98% or 99% identity with it; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:18 or has at least 95%, 96%, 97%, 98% or 99% identity with it; More preferably, the anti-HER2 antibody contains SEQ ID NO:19 or a heavy chain sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity with it, and SEQ ID NO:20 or a light chain sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity with it; For example, the anti-HER2 antibody or its antigen-binding fragment is trastuzumab, pertuzumab, or its antigen-binding fragment.

3. The drug combination according to claim 1 or 2, wherein the anti-HER3 antibody drug conjugate has the following structural formula: in, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the Ab is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. For example, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and Ab is not a bispecific antibody or a multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. Preferably, the Ab is an anti-HER3 monospecific antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. Preferably, the anti-HER3 monospecific antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; More preferably, the heavy chain amino acid sequence of the anti-HER3 monospecific antibody is as shown in SEQ ID NO:9, and the light chain amino acid sequence is as shown in SEQ ID NO:10; p is an integer or decimal of 2 to 8; for example, the average number of links p is an integer or decimal of 3 to 8; for example, the average number of links p is an integer or decimal of 7 to 8. The anti-HER2 antibody or its antigen-binding fragment as defined in claim 2, preferably, the anti-HER2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively; and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; more preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:18; for example, the anti-HER2 antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:19, and a light chain with an amino acid sequence as shown in SEQ ID NO:20; for example, the anti-HER2 antibody is trastuzumab.

4. Use of the pharmaceutical combination according to any one of claims 1 to 3 in the preparation of a cancer treatment drug, for example, the cancer being an advanced / metastatic solid tumor, such as breast cancer.

5. The use according to claim 4, wherein the cancer is a Her2-positive cancer; preferably a Her2-positive breast cancer.

6. The use according to claim 4, wherein the cancer is a cancer with low Her2 expression; preferably, breast cancer with low Her2 expression.

7. The use according to claim 4, wherein the cancer is a cancer with ultra-low Her2 expression; preferably, breast cancer with ultra-low Her2 expression.

8. The use according to claim 4, wherein the cancer is breast cancer that has been previously treated with HER2-targeted therapy; preferably, the HER2-targeted therapy is HER2-targeting topoisomerase 1 inhibitor ADC therapy.

9. The use according to any one of claims 4-8, wherein the cancer is a Her3-positive cancer.

10. The use according to claims 4-9, wherein the cancer is advanced / unresectable or metastatic cancer.

11. A method of treating cancer, comprising administering to an individual in need a therapeutically effective amount of the drug combination according to any one of claims 1-3, in, The anti-HER3 antibody-drug conjugate has the following structural formula: in, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the Ab is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. For example, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and Ab is not a bispecific antibody or a multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. Preferably, the Ab is an anti-HER3 monospecific antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. Preferably, the anti-HER3 monospecific antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; More preferably, the heavy chain amino acid sequence of the anti-HER3 monospecific antibody is shown in SEQ ID NO:9, and the light chain amino acid sequence is shown in SEQ ID NO:10; p is an integer or decimal between 2 and 8.

12. Use of an anti-HER3 antibody drug conjugate and an anti-HER2 antibody or its antigen-binding fragment in combination in the preparation of a medicament for treating cancer, wherein the anti-HER3 antibody drug conjugate has the following structural formula: in, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the Ab is not a bispecific or multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. For example, Ab is an anti-HER3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; and Ab is not a bispecific antibody or a multispecific antibody containing an anti-EGFR antibody or its antigen-binding fragment. Preferably, the Ab is an anti-HER3 monospecific antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. Preferably, the anti-HER3 monospecific antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; More preferably, the heavy chain amino acid sequence of the anti-HER3 monospecific antibody is shown in SEQ ID NO:9, and the light chain amino acid sequence is shown in SEQ ID NO:10; p is an integer or decimal between 2 and 8.

13. The pharmaceutical combination according to any one of claims 1 to 3, for the treatment of cancer.

14. A kit comprising the drug combination according to any one of claims 1 to 3, preferably the kit being in the form of a drug dosage unit.

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