Antibody-drug conjugate targeting her3

Abstract

The present invention relates to an antibody-drug conjugate targeting HER3, a pharmaceutical composition containing same, a use thereof for treating or preventing diseases or conditions related to HER3 activity, especially HER3 positive tumors, and a use thereof for preparing a drug for treating or preventing diseases or conditions related to HER3 activity, especially HER3 positive tumors.

Description

Antibody-drug conjugates targeting HER3 Technical Field

[0001] The present disclosure relates to an antibody-drug conjugate (ADC) targeting HER3, a pharmaceutical composition comprising the ADC, the use of the ADC for treating or preventing diseases or conditions associated with HER3 activity, particularly HER3-positive tumors, and the use of the ADC for preparing a medicament for treating or preventing diseases or conditions associated with HER3 activity, particularly HER3-positive tumors. Background Art

[0002] HER3 (encoded by ErbB3) is a member of the HER protein receptor family. Initially thought to lack kinase activity, recent studies have shown that HER3 can bind ATP and promote phosphorylation of its intracellular domain, but its kinase activity is relatively weak. Studies have shown that HER3 plays a crucial role in HER2 / HER3 heterodimerization and subsequent activation of the PI3K / AKT cascade. Consequently, HER3 has garnered extensive attention and research in recent years.

[0003] HER3 is overexpressed in various cancers, including gastric, breast, colorectal, and lung cancers, and HER3 expression is associated with poor survival after surgery. Studies have shown that MET amplification may induce resistance to EGFR-targeted therapy by maintaining HER3-mediated PI3K / AKT signaling. Furthermore, HER3 is not only overexpressed in various cancers but also has the potential to mediate resistance to targeted therapies, making it a promising and attractive therapeutic target.

[0004] Patritumab is an IgG1 HER3 monoclonal antibody with extremely strong cellular internalization. Daiichi Sankyo leverages the potent endocytosis of HER3 to combine patritumab with a cleavable tetrapeptide linker and a topoisomerase I inhibitor (DXd) to create a novel HER3-DXd (U3-1402) ADC molecule with a DAR of 8. Clinical study data for U3-1402 demonstrate that HER3-DXd exhibits anti-tumor activity not only in breast and lung cancer but also in patients with tumors harboring multiple EGFR resistance mechanisms (including EGFR C797S mutation, MET amplification, HER2 mutation, BRAF fusion, and PIK3CA mutation), with clinically observed long-lasting efficacy.

[0005] Although the above-mentioned drugs have appeared in the prior art, there is still a widespread demand for therapeutic drugs that can target HER3 and be used to treat HER3-positive tumors, especially antibody-drug conjugates with high affinity, good specificity, low immunogenicity risk, strong anti-tumor activity and / or better bystander effect.

[0006] Summary of the Invention

[0007] The present disclosure is based on the HER3-targeting antibody-drug conjugates developed by the inventors, which exhibit enhanced anti-tumor activity and a better bystander effect, as well as improved pharmacodynamics and PK performance compared to existing technologies. Therefore, the HER3-targeting antibody-drug conjugates disclosed herein are particularly suitable for treating or preventing diseases or conditions associated with HER3 activity, particularly HER3-positive tumors.

[0008] In one aspect, the present disclosure provides an antibody-drug conjugate having formula (I), a stereoisomer or a pharmaceutically acceptable salt or solvate thereof:

[0009] Ab-(LD) n (Ⅰ)

[0010] in,

[0011] Ab is an antibody or antigen-binding fragment thereof that binds to HER3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6,

[0012] L is a linker having the structure

[0013] :-ZE-NH-CH2-,

[0014] Wherein Z is connected to Ab, -CH2- is connected to D;

[0015] Z is selected from

[0016] where m a1 and m a2 independently selected from integers from 0 to 20;

[0017] m is an integer selected from 1 to 10,

[0018] The carbonyl group on the right end of Z is covalently linked to E;

[0019] E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted with one or more groups selected from C 1-6 an alkyl group and a polyol group, wherein the N-terminus of the peptide residue is covalently linked to Z,

[0020] D is a cytotoxic compound having the formula -QL 2 -L 1 -D 1 structure,

[0021] wherein Q is -O- or -S-;

[0022] L 1 Does not exist or -(C1-C 10 alkylene)-;

[0023] L 2 Does not exist, *-(C1-C 10 Alkylene)-C(O)N(R 5 )-or*-(C1-C 10 Alkylene)-N(R 5 )C(O)-; wherein * indicates that the terminus is covalently linked to Q; and R 5 is H or C1-C6 alkyl,

[0024] Among them D 1 It has the structure shown in formula (D-1):

[0025] where R 1 Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;

[0026] R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and-SR 4 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 ; or R 2 and R 3 Together they form -O(CH2) n1 O- or -O(CF2) n1 O-, where n1 is 1 or 2;

[0027] R 4 is selected from H or C1-C4 alkyl; and

[0028] n represents DAR, and n is a natural number selected from 1-15.

[0029] In some embodiments of the present disclosure, the Ab comprises a heavy chain variable region, the heavy chain variable region

[0030] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7; or

[0031] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7; or

[0032] (iii) comprises or consists of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 7, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions.

[0033] In some embodiments of the present disclosure, the Ab comprises a light chain variable region, the light chain variable region

[0034] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; or

[0035] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 9; or

[0036] (iii) comprises or consists of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 9, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions.

[0037] In some embodiments of the present disclosure, the Ab further comprises a heavy chain constant region, the heavy chain constant region

[0038] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11; or

[0039] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11; or

[0040] (iii) comprising an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 11, or consisting of said amino acid sequence.

[0041] In some embodiments of the present disclosure, the Ab further comprises a light chain constant region, the light chain constant region

[0042] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12; or

[0043] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12; or

[0044] (iii) comprising an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 12, or consisting of said amino acid sequence.

[0045] In some embodiments of the present disclosure, the Ab comprises a heavy chain and / or a light chain, wherein

[0046] Heavy chain

[0047] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13; or

[0048] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 13; or

[0049] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 13; and / or

[0050] light chain

[0051] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14; or

[0052] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 14; or

[0053] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 14.

[0054] In some embodiments of the present disclosure, the Ab is a whole antibody. In some embodiments of the present disclosure, the Ab is a multispecific antibody such as a bispecific antibody.

[0055] In some embodiments of the present disclosure, the Ab is selected from an antigen-binding fragment Fv, Fab, Fab', or F(ab')2; a single-chain antibody scFv; or a single-domain antibody VHH.

[0056] In some embodiments of the present disclosure, the linker is attached to the Ab via a cysteine ​​residue side chain.

[0057] In some embodiments of the present disclosure, the antibody-drug conjugate has the following structure:

[0058] wherein Ab is as defined herein, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0059] In some embodiments of the disclosure, the antibody-drug conjugate has an average DAR of 2-10, 4-9, or 7.5-8.5.

[0060] In one aspect, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate described in the present disclosure, its stereoisomers or pharmaceutically acceptable salts or solvates, and a pharmaceutically acceptable carrier or excipient.

[0061] In one aspect, the present disclosure provides use of the antibody-drug conjugate, stereoisomers, or pharmaceutically acceptable salts or solvates thereof described herein in the preparation of a medicament for treating or preventing a disease or condition associated with HER3 activity.

[0062] In some embodiments of the present disclosure, the disease or disorder associated with HER3 activity is a HER3-positive tumor.

[0063] In some embodiments of the present disclosure, the HER3-positive tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer, and bladder cancer.

[0064] In one aspect, the present disclosure provides the antibody-drug conjugate of the present disclosure, its stereoisomers, or pharmaceutically acceptable salts or solvates for use in treating or preventing diseases or conditions associated with HER3 activity.

[0065] In some embodiments of the present disclosure, the disease or disorder associated with HER3 activity is a HER3-positive tumor.

[0066] In some embodiments of the present disclosure, the HER3-positive tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer, and bladder cancer.

[0067] In one aspect, the present disclosure provides a method for treating or preventing a disease or condition associated with HER3 activity, comprising administering to a subject an effective amount of an antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0068] In some embodiments of the present disclosure, the disease or disorder associated with HER3 activity is a HER3-positive tumor.

[0069] In some embodiments of the present disclosure, the HER3-positive tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer, and bladder cancer.

[0070] In some embodiments of the present disclosure, the method further comprises co-administering one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, and immunomodulators.

[0071] In some embodiments of the present disclosure, the antibody-drug conjugate, its stereoisomers or pharmaceutically acceptable salts or solvates and one or more therapeutic agents are administered simultaneously or sequentially in any order.

[0072] In some embodiments of the present disclosure, the method further comprises administering to the patient one or more treatment modalities selected from radiation therapy or surgery.

[0073] Compared with the prior art, the HER3-targeting antibody-drug conjugate provided by the present disclosure has the following advantages:

[0074] 1) The HER3-targeting antibody-drug conjugates of the present invention release a small molecule drug moiety, MB1, that is more active in vivo than the prior art DXd, and in vitro and in vivo efficacy data demonstrate superior anti-tumor activity;

[0075] 2) The small molecule drug portion MB3 in the HER3-targeting antibody-drug conjugate of the present invention is inherently hydrophilic, but after intracellular cleavage, it is more likely to release the lipophilic free small molecule drug MB1, resulting in a stronger bystander effect.

[0076] 3) The HER3-targeting antibody-drug conjugates of the present invention demonstrated excellent results in pathological experiments, including high in vivo drug exposure, good PK, and good efficacy;

[0077] 4) The HER3-targeting antibody-drug conjugate of the present invention has shown high drug safety and clinical treatment safety in toxicology experiments, and can be administered at a higher drug dose. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG1 shows the results of different HER3-ADCs killing SW620 cells in vitro.

[0079] FIG2 shows the results of different HER3-ADC killing of MCF-7 cells in vitro.

[0080] FIG3 shows the in vitro efficacy evaluation results of different payloads for potential drug resistance in the Elacridar-resistant HCT15 cell line.

[0081] Figure 4 shows the bystander effect results of HER3-ADC conjugated with different linkers and payloads.

[0082] FIG5 shows a schematic structural diagram of the HER3-MB3 molecule of Example 1.

[0083] FIG6 shows the relative expression levels of HER3 in tumor cell lines.

[0084] FIG. 7 shows the results of HER3-MB3 killing of NUGC4 in vitro.

[0085] FIG8 shows the results of HER3-MB3 killing of HCC1569 cells in vitro.

[0086] FIG9 shows the results of HER3-MB3 killing of NCI-H508 cells in vitro.

[0087] FIG10 shows the results of HER3-MB3 killing of SW620 cells in vitro.

[0088] FIG11 shows the results of HER3-MB3 killing in vitro in HCC827WT and HCC827+HER3OX.

[0089] FIG. 12 shows the bystander effect of HER3-MB3.

[0090] FIG13 shows the efficacy results of HER3-MB3 in the SW620 mouse tumor model.

[0091] FIG14 shows the results of body weight changes of HER3-MB3 in the SW620 mouse tumor model.

[0092] FIG15 shows the efficacy results of HER3-MB3 in the NUGC4 mouse tumor model.

[0093] FIG16 shows the results of body weight changes of HER3-MB3 in the NUGC4 mouse tumor model.

[0094] FIG17 shows the relationship between the blood concentration of HER3-ADC in mice and time.

[0095] FIG18 shows the toxicokinetic study of HER3-MB3 in cynomolgus monkeys. DETAILED DESCRIPTION

[0096] Certain embodiments will now be described in detail, examples of which are illustrated in the accompanying specific embodiments. Although the embodiments of the present invention will be described, it should be understood that they are not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to encompass all alternatives, modifications, and equivalents, which may be included within the scope of the present invention defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. If one or more incorporated documents and similar materials differ from or contradict the present disclosure, including but not limited to defined terms, term usage, described technology, etc., the present disclosure shall prevail.

[0097] It will be appreciated that certain features of the present invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0098] I. Definition

[0099] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0100] The term "or" refers to a single element of the listed alternative elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed alternative elements.

[0101] As used herein, the term "about" refers to a value within a range of ±10% of a given numerical value.

[0102] As used herein, the terms "include", "comprise", "have" and similar expressions do not exclude unrecited elements. These terms also include the situation that the composition consists of only the listed elements.

[0103] As used herein, the term "HER3" (human epidermal growth factor receptor 3, also known as ErbB3) is a receptor protein tyrosine kinase and belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes HER1 (also known as EGFR), HER2, and HER4. HER3 is a transmembrane receptor and consists of an extracellular ligand binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, and an intracellular protein tyrosine kinase domain (TKD) and a C-terminal phosphorylation domain. HER3 has been found to be overexpressed in several types of cancer (e.g., breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer, and bladder cancer). The expression of HER2 / HER3 has been shown to correlate with progression from non-invasive to invasive stages.

[0104] As used herein, the term "antibody" is used in its broadest sense and includes immunoglobulins or other types of molecules comprising one or more antigen-binding domains that specifically bind to an antigen, and is a protein or polypeptide that exhibits binding specificity to a specific antigen. Specific examples of antibodies may include complete antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies such as bispecific antibodies, and the like. Classical antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. Based on the conservative differences in the amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region is used to recognize and bind to the antigen, and the constant region (e.g., Fc fragment) is used to initiate downstream effects, such as antibody-dependent cellular cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, there are three local regions, each with a higher degree of variation in amino acid composition and arrangement order, which are key positions for antibody-antigen binding and are therefore also referred to as complementary determining regions (CDRs). The amino acid sequences of the CDRs can be readily determined using art-recognized numbering schemes, such as Kabat, Chothia, IMGT, AbM, or Contact. In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme. Herein, the three heavy chain complementary determining regions are referred to as HCDR1, HCDR2, and HCDR3, respectively, and the three light chain complementary determining regions are referred to as LCDR1, LCDR2, and LCDR3, respectively.

[0105] Based on the amino acid sequence of the constant region of its heavy chains, antibodies can be divided into five major classes: IgA, IgD, IgE, IgG, and IgM. These antibody classes are further divided into subclasses based on the size of the hinge region, the location of interchain disulfide bonds, and molecular weight, such as IgG1, IgG2a, IgG2b, and IgG3. Based on the amino acid composition and arrangement of the constant region of its light chains, light chains can be divided into two types: kappa and lambda. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known in the art.

[0106] As used herein, an "antigen-binding fragment" of an antibody refers to an amino acid fragment of an antibody molecule that participates in antigen-specific binding, such as Fab, Fab', and F(ab')2. Those skilled in the art know how to obtain these antigen-binding fragments. For example, a classical antibody molecule can be digested with papain to obtain a Fab fragment, and with pepsin to obtain a F(ab')2. A Fab' fragment can be formed by treating the F(ab')2 with a reducing agent to break the disulfide bonds within the hinge region.

[0107] As used herein, the term "single-chain antibody (scFv)" refers to a single-chain antibody consisting of the variable regions of the heavy and light chains of an antibody linked by a short peptide. Properly folded, the variable regions from the heavy and light chains interact through non-covalent bonds to form the Fv fragment, allowing the scFv to retain its affinity for the antigen.

[0108] As used herein, the term "single domain antibody (sdAb)", or also referred to as "VHH antibody", refers to an antibody molecule with antigen binding ability, including a heavy chain variable region but no light chain. Structurally, a single domain antibody can also be considered a fragment of a classic four-chain antibody molecule. Single domain antibodies were first discovered in camelids, and subsequently, researchers discovered more single domain antibodies with antigen binding ability by screening antibody libraries (such as phage display libraries). Single domain antibodies have some advantages over ordinary antibody molecules (e.g., classic antibody molecules), such as but not limited to: a smaller molecular weight, which makes it easy to reach tissues or parts that are difficult for ordinary antibody molecules to reach when used in the human body, or the ability to access antigenic epitopes in proteins or polypeptides that are difficult for ordinary antibody molecules to access; more stable, and able to withstand, for example, changes in temperature and pH, as well as the effects of denaturants and proteases.

[0109] As used herein, the term "Fc fragment" refers to the handle region of the Y-shaped classical antibody molecule, i.e., a crystallizable fragment (Fc), including the second and third constant domains (CH2 and CH3 domains) of the heavy chain. The antibody Fc region can be obtained by hydrolyzing the antibody molecule with a proteolytic enzyme (such as papain). In some instances, the Fc region may include a hinge, CH2, and CH3. When the Fc region includes a hinge, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment may be from IgG, IgM, IgD, IgE, or IgA. In some instances, the Fc region is from IgG1, IgG2, IgG3, or IgG4. "Fc fragment" also includes variant Fc fragments from natural Fc fragments that have been modified but still retain their effector functions. "Variant Fc fragment" comprises an amino acid sequence having at least one amino acid change in the amino acid sequence of a natural Fc fragment. In some instances, the variant Fc fragment has at least one amino acid substitution compared to the parent Fc fragment (native Fc fragment), for example, about 1 to about 10 amino acids are substituted in the parent Fc fragment, and preferably about 1 to about 5 amino acid substitutions. In some instances, the variant Fc fragment Fc region has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the parent Fc fragment. The effector functions of the "Fc fragment" may include binding to Fc receptors, Clq binding and complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediating phagocytosis, and the like.

[0110] As used herein, the term "mouse antibody" refers to an antibody whose variable region and constant region (if any) are derived from mouse or rat immunoglobulin sequences. Mouse antibodies can be conveniently obtained by immunizing mice or rats with the corresponding antigen and isolating the target antibody therefrom. Alternatively, after immunizing mice or rats with the corresponding antigen, cells expressing the target antibody (such as B cells) are isolated and cultured to obtain the target antibody. Alternatively, after immunizing mice or rats with the corresponding antigen, cells expressing the target antibody are isolated and cultured, and the cells are fused with immortalized cells such as myeloma cells to obtain hybridoma cells. Cultivating hybridoma cells can obtain the target antibody (such as monoclonal antibody) for a long time and in large quantities. In some embodiments, the "mouse antibody" is a mouse antibody. "Humanized antibody" refers to a chimeric antibody obtained by artificially modifying a non-human antibody, i.e., an antibody whose variable region and constant region (if any) are not derived from human immunoglobulins, so that it contains the amino acid sequence of a human antibody. A humanized antibody may contain the constant region and / or framework region of a human antibody. Humanized antibodies can be obtained through genetic engineering, for example, by replacing the constant regions of a mouse antibody with those of a human antibody and / or replacing the framework regions of a mouse antibody with those of a human antibody. Such humanization generally does not affect the binding specificity of the original antibody to the corresponding antigen, and therefore such antigens are also included within the scope of the present invention.

[0111] As used herein, the term "monoclonal antibody" refers to an antibody that is homogeneous and directed only against a specific antigenic epitope. Compared to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous character of the antibody and is not to be construed as requiring the antibody to be produced by any particular method. The monoclonal antibodies of the present invention can be produced by hybridoma methods or recombinant DNA methods well known in the art, or obtained by screening methods described elsewhere herein.

[0112] As used herein, the term "immunoconjugate" refers to an antibody or antigen-binding fragment thereof that is linked to other chemical groups or peptides. For the purposes of the present invention, the chemical groups or peptides can facilitate the detection of the antibody or antigen-binding fragment thereof, or facilitate the detection of the immune complex (i.e., antigen-antibody complex) formed by the antibody or antigen-binding fragment thereof and the corresponding antigen (e.g., type IV collagen α1 chain), and therefore can include a "detectable label." A "detectable label" can be used to indicate the presence or content of the corresponding antigen in a sample, or to track the location of the corresponding antigen in a cell or in the body. Examples of detectable labels include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP); fluorescent groups (e.g., FAM, FITC) or fluorescent proteins (e.g., GFP); and radioactive isotopes (e.g., 3H, 14C, 35S). When the detectable label is an enzyme, the presence or content of the antibody or antigen-binding fragment thereof linked to the enzyme can be determined by the enzymatic activity of the enzyme.

[0113] As used herein, the term "purification tag" refers to an amino acid sequence that facilitates the separation of a polypeptide or protein of interest from a cell culture or supernatant expressing the polypeptide or protein of interest. Examples include, but are not limited to, His6 tags, Flag tags, MBP tags, GST tags, SUMO tags, and the like.

[0114] For antibodies or antigen-binding fragments thereof, "binding," "directed against," or "specifically binds" means that one molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher binding affinity for another molecule (e.g., an antigen) relative to other molecules present in the environment. A molecule can bind to, be directed against, or specifically bind to more than one molecule. For example, a bispecific antibody can have a higher binding affinity for two different antigens relative to other molecules. The binding affinity of an antibody for an antigen can be measured by a number of parameters, such as the EC50 value or KD value of the antibody binding to the antigen.

[0115] EC 50 Concentration for 50% of maximal effect refers to the concentration that produces 50% of the maximal effect. In enzyme-linked immunosorbent assays (ELISAs), when used to indicate the binding ability of an antibody molecule to its corresponding antigen, it refers to the antibody concentration that produces half the maximum detection signal (e.g., colorimetric or fluorescence intensity). The lower the EC50 value, the greater the binding affinity for the antigen.

[0116] The KD value can also be used to measure the binding affinity between an antibody and its antigen. The KD value is the equilibrium dissociation constant between the antibody and its antigen, i.e., the ratio of koff / kon. Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody.

[0117] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers composed of amino acid residues. Full-length proteins and fragments thereof are encompassed within this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and similar modifications. In addition, for the purposes of the present invention, "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions, and substitutions (which are generally conservative in practice), as long as the protein retains the desired activity. These modifications may be purposeful, such as through site-directed mutagenesis, or may be accidental, such as through mutations in the host producing the protein or errors due to PCR amplification.

[0118] As used herein, the term "functional variant" refers to a variant molecule obtained by introducing one or more amino acid insertions, deletions or substitutions into a parent protein molecule (e.g., a natural protein molecule), which still retains at least part of the function of the parent protein molecule (especially the function of interest, such as the ability to bind to the corresponding antigen). For example, a functional variant of an antibody molecule may retain at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the antigen binding ability of its parent molecule, or even have a higher binding ability than the parent molecule. In some embodiments, a functional variant of an antibody molecule may retain at least 80%, 85%, 90%, 95% or even 100% or more of the antigen binding affinity of its parent molecule. For an antibody molecule or its antigen-binding fragment, a functional variant typically includes amino acid changes in the variable region framework sequence and / or constant region, but does not exclude the possibility of making one or a few amino acid changes to the CDR region sequence.

[0119] As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0120] As used herein, the term "vector" refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (e.g., a nucleic acid, a plasmid, or a virus, etc.). A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of the polynucleotide of interest (such as a promoter and / or enhancer), and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric analysis, such as β-galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0121] As used herein, the term "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, CHO cells, HEK-293 cells, BHK cells, or PER-C6 cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical to the original parent cell (in terms of morphology or genomic DNA complement) due to natural, accidental, or deliberate mutations. Host cells can be isolated cells or cell lines, and also include cells transfected in vivo with the nucleic acid molecules or expression vectors provided herein.

[0122] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also called "sequence identity") refers to the amount of consistency between two amino acid or nucleotide sequences (such as a query sequence and a reference sequence), generally expressed as a percentage. Usually, before calculating the percentage of consistency between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If the amino acid residues or bases in the two sequences are the same at a certain comparison position, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the comparison window to obtain sequence consistency. In other algorithms, the number of gaps and / or gap lengths are also taken into account. Commonly used sequence comparison algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purpose of the present invention, the publicly available alignment software BLAST (available from https: / / www.ncbi.nlm.nih.gov / ) can be used to obtain optimal sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using default settings.

[0123] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a cell population is characterized by unregulated cell growth. As used herein, the terms "cancer cell" and "tumor cell" refer to the total number of cells from a tumor, including tumorigenic stem cells (cancer stem cells) and non-tumorigenic cells that make up the majority of the tumor cell population. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, lung cancer, such as small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, melanoma, head and neck squamous cell carcinoma, and various types of head and neck cancer.

[0124] As used herein, the term "tumor" refers to any mass of tissue resulting from excessive growth or proliferation of cells, whether benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

[0125] The terms "cancer" and "tumor" are used interchangeably herein and include both solid and hematological tumors.

[0126] As used herein, the term "metastasis" refers to the process by which cancer spreads or metastasizes from its site of origin to other areas of the body as similar cancerous lesions develop in new locations. "Metastatic" cells are cells that have lost their adhesive contacts with neighboring cells and migrated from the primary site of the disease via the blood or lymph to invade neighboring body structures.

[0127] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein to refer to a human subject.

[0128] As used herein, the terms "treat" and "prevent" refer to: 1) curing, slowing, alleviating symptoms and / or stopping the progression of a diagnosed pathological condition or disease; 2) pre-implemented methods to prevent, delay or slow the development of a target pathological condition or disease. Therefore, people in need of treatment include those who already have the disease; those who are susceptible to the disease; and those in need of prevention. A subject is successfully "treated" according to the methods of the present disclosure if the patient exhibits one or more of the following: a reduction or complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs (these include the spread of cancer cells to soft tissue and bone); inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with a specific cancer; reduction in morbidity and mortality; and improvement in quality of life. In the context of cancer, the term "prevention" refers to the administration of medical treatments such as drugs to a subject, particularly a subject at risk of cancer, before the onset of symptoms or pathological conditions associated with the cancer.

[0129] As used herein, the term "antibody-drug conjugate" refers to a targeted biologic drug obtained by connecting a bioactive compound fragment (drug molecule) to an antibody or its antigen-binding fragment through a linker. The linker, also known as a linker, can break in a specific environment (such as a low pH environment in the cell) or under a specific action (such as the action of a lysosomal protease), thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment. ADC can utilize the high affinity and specificity of the target antibody to recognize tumor cell surface antigens, and deliver bioactive compounds, such as small molecule cytotoxic drugs, to tumor cells through strong intracellular internalization, thereby achieving precise and efficient killing of tumor cells.

[0130] As used herein, the term "linker" refers to a fragment that connects a biologically active compound fragment (drug molecule) to an antibody or antigen-binding fragment thereof. It should be understood that the linker has a functional group that can form a bond with a functional group of the antibody or antigen-binding fragment thereof before being attached to the antibody or antigen-binding fragment thereof.

[0131] The term "linker-payload" refers to a compound formed by linking a payload, such as a drug (eg, a small molecule drug), to a linker.

[0132] As used herein, the term "biologically active compound fragment" refers to a bioactive compound fragment in an antibody-drug conjugate (or antibody-drug conjugate, ADC) known in the art, which can form a biologically active drug, such as a small molecule cytotoxic drug, after cleavage / degradation / enzymatic cleavage of the linker between tumor tissues or within tumor cells.

[0133] As used herein, the term "DAR (drug to antibody ratio)" is also referred to as "drug to antibody ratio" and is also referred to herein as "linkage number", which refers to the number of drug moieties coupled to the antibody in an antibody-drug conjugate molecule. DAR can vary and will be limited by the number of available sites on the antibody. It should be understood that DAR or "linkage number" should be a natural number greater than 0, i.e., a positive integer. In some embodiments, the antibody-drug conjugates described herein have a DAR of 1-15, such as 1-14, 2-13, 3-12, 4-11, 5-10, 6-9 or 7-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or a numerical range with any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 as endpoints. During the conjugation process, a heterogeneous mixture of different antibody-drug conjugate (ADC) molecules (i.e., ADC molecules with different DARs) is typically produced. Therefore, the term "antibody-drug conjugate (ADC)" also refers to such a mixture of ADC molecules with different DARs. The term "average DAR" refers to the average value of the DARs of a population of ADC molecules in such a mixture, also referred to in this disclosure as the "average number of attachments." As is well known in the art, DAR and drug loading distribution can be determined, for example, by using hydrophobic interaction chromatography (HIC) or reversed phase high-performance liquid chromatography (RP-HPLC), with HIC being particularly suitable for determining the average DAR. In some embodiments, the average DAR (average number of attachments) of the antibody-drug conjugates described herein is a value between 1-15, such as a value in the range of 1-14, 2-13, 3-12, 4-11, 5-10, 6-9, or 7-8, such as a value within a numerical range with any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 as endpoints; it should be understood that the "value" includes integers and decimals. A value between 1-15 refers to any value between 1-15, including integers and decimals, and includes endpoints.In some embodiments, the average DAR (average number of connections) is, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, or a value selected from a range having two of these values ​​as endpoints.

[0134] The term "drug combination" refers to a non-fixed combination product or a fixed combination product. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antibody drug conjugate of the present invention, its stereoisomer, or its pharmaceutically acceptable salt or solvate and (ii) other therapeutic agents) are administered to the patient simultaneously, without specific time restrictions or at the same or different time intervals, in sequence as separate entities, wherein such administration provides two or more active agents at a preventive or therapeutically effective level in the patient's body. In some embodiments, the antibody drug conjugate of the present invention, its stereoisomer, or its pharmaceutically acceptable salt or solvate and other therapeutic agents used in the drug combination are administered at a level that does not exceed that when they are used alone. The term "fixed combination" means that two or more active agents are administered to the patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0135] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or regulates (e.g., activates) an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0136] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0137] The term "small molecule drug" refers to low molecular weight organic compounds that can modulate biological processes, particularly alter or prevent pathological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD, more preferably less than 500 kD. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.

[0138] The term "alkyl" as used herein refers to a fully saturated branched or unbranched hydrocarbon group. The alkyl group preferably contains 1 to 16 carbon atoms, such as 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0139] The term "alkylene" refers to an alkyl group as defined above, but it is divalent, i.e., has two single bonds connected to two other groups. Non-limiting examples of alkylene include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(-CH2CH3)-, or -CH2CH(-CH3)-.

[0140] The term "alkenyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and including at least one double bond and no triple bonds. Alkenyl groups preferably contain 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0141] The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and at least one triple bond. Alkynyl groups preferably contain 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

[0142] The term "halogen" or "halo" refers to fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I).

[0143] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogen groups as defined herein. Halogenated alkyl groups may preferably be monohalogenated alkyl, dihalogenated alkyl, or polyhalogenated alkyl (including perhalogenated alkyl). Monohalogenated alkyl groups may contain one iodine, bromine, chlorine, or fluorine in the alkyl group. Dihalogenated alkyl and polyhalogenated alkyl groups may contain two or more identical halogen atoms or a combination of different halo groups in the alkyl group. Preferably, polyhalogenated alkyl groups contain up to 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhalogenated alkyl groups refer to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0144] The term "haloalkenyl" refers to an alkenyl group, as defined herein, substituted with one or more halo groups, as defined herein. The term "haloalkynyl" refers to an alkynyl group, as defined herein, substituted with one or more halo groups, as defined herein. The meaning of "halo" as defined for "haloalkyl" applies to both "haloalkenyl" and "haloalkynyl."

[0145] The term "polyol group" refers to an alkyl group as defined above containing a plurality (e.g. 2-10, e.g. 3, 4, 5, 6, 7 or 8) of hydroxyl groups, which optionally contains 1 or more (e.g. 2, 3 or 4) other groups (e.g. amino, carbonyl). Non-limiting examples of "polyol groups" include, for example Among them, the chiral center without the indicated stereo configuration can be R or S configuration, preferably

[0146] The term "amino acid" refers to naturally occurring and synthetic amino acids. Amino acids can be L or D isomers. Conventional amino acids are referred to herein in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Also, in this disclosure, amino acids are generally referred to by single-letter and three-letter abbreviations known in the art. For example, the amino acid can be selected from phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro) and glutamine (Gln).

[0147] "Anti-tumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, especially small molecule cytotoxic agents or chemotherapeutic agents, such as camptothecin compounds such as those disclosed in WO2021 / 173773, WO2022180581, CN 102574866, etc., Exitecan (a topoisomerase I inhibitor Exatecan), Dxd (a new topoisomerase I inhibitor Exatecan derivative); Auristatin compounds such as monomethyl auristatin E (MMAE); or Maytansine compounds such as small molecule microtubule inhibitor DM1. It should be understood that anti-tumor compounds can be substituted by isotopes including but not limited to deuterium, tritium, etc. For example, after substitution with deuterium, the carbon-deuterium bond replaces the carbon-hydrogen bond. Since the former is more stable than the latter, the substitution can directly affect the absorption, distribution, metabolism and excretion properties of certain drugs, thereby improving the efficacy, safety and tolerability of the drug. Therefore, the "anti-tumor compound" of the present application may include compounds substituted with deuterium.

[0148] "Deuterium-substituted" means that a hydrogen in the molecule is replaced by deuterium, eg, 1 or more hydrogens, eg, 1-10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) are replaced by deuterium.

[0149] II. Antibody-Drug Conjugates

[0150] The present disclosure provides an antibody-drug conjugate having formula (I), a stereoisomer or a pharmaceutically acceptable salt or solvate thereof:

[0151] Ab-(LD) n (Ⅰ)

[0152] in,

[0153] Ab is an antibody or antigen-binding fragment thereof that binds to HER3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 are respectively identified by the Kabat numbering scheme, wherein

[0154] HCDR1 comprises or consists of the amino acid sequence of GYYWS (SEQ ID NO: 1);

[0155] HCDR2 comprises or consists of the amino acid sequence of EINHSGSTNYNPSLKS (SEQ ID NO: 2);

[0156] HCDR3 comprises or consists of the amino acid sequence of DKWTWYFDL (SEQ ID NO: 3);

[0157] LCDR1 comprises or consists of the amino acid sequence of RSSQSVLYSSSNRNYLA (SEQ ID NO: 4);

[0158] LCDR2 comprises or consists of the amino acid sequence of WASTRES (SEQ ID NO: 5);

[0159] LCDR3 comprises or consists of the amino acid sequence of QQYYSTPRT (SEQ ID NO: 6);

[0160] L is a linker having the structure shown below: -ZE-NH-CH2-,

[0161] Wherein Z is connected to Ab, -CH2- is connected to D;

[0162] Z is selected from

[0163] where m a1 and m a2 independently selected from integers from 0 to 20, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0164] m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8,

[0165] Preferred wherein m is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5; the carbonyl group at the right end is covalently linked to E;

[0166] E is a peptide residue having 2-10 amino acids, wherein the peptide residue is optionally substituted with one or more (e.g., 2, 3, or 4) groups selected from C 1-6 an alkyl group and a polyol group, wherein the N-terminus of the peptide residue is covalently linked to Z,

[0167] D is a cytotoxic compound having the formula -QL 2 -L 1 -D 1 structure,

[0168] wherein Q is -O- or -S-;

[0169] L 1 Does not exist or -(C1-C 10 alkylene)-;

[0170] L 2 Does not exist, *-(C1-C 10 Alkylene)-C(O)N(R 5 )-or*-(C1-C 10 Alkylene)-N(R 5 )C(O)-; wherein * indicates that the terminus is covalently linked to Q; and R 5 is H or C1-C6 alkyl,

[0171] Among them D 1 It has the structure shown in formula (D-1):

[0172] where R 1 Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;

[0173] R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and-SR 4 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 ; or R 2 and R 3 Together they form -O(CH2) n1 O- or -O(CF2) n1 O-, where n1 is 1 or 2;

[0174] R 4 is selected from H or C1-C4 alkyl; and

[0175] n represents DAR, and n is a natural number selected from 1-15, for example, a natural number in the range of 1-14, 2-13, 3-12, 4-11, 5-10, 6-9, 7-8, for example, a natural number in a numerical range with any two values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 as endpoints, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0176] It should be understood that a peptide residue having 2 to 10 amino acids means a peptide residue consisting of 2 to 10 amino acid residues.

[0177] In some embodiments, in the antibody-drug conjugate of formula (I) of the present disclosure, the Ab comprises a heavy chain variable region (V H ), the heavy chain variable region

[0178] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7; or

[0179] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7; or

[0180] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 7, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) does not exceed 10, for example, does not exceed 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids,

[0181] The amino acid sequence shown in SEQ ID NO: 7 is as follows:

[0182] The nucleic acid sequence encoding this amino acid sequence is as follows:

[0183] In some embodiments, in the antibody-drug conjugate of formula (I) of the present disclosure, the Ab comprises a light chain variable region (V L ), the light chain variable region

[0184] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; or

[0185] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 9; or

[0186] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 9, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) does not exceed 10, for example, does not exceed 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids,

[0187] The amino acid sequence shown in SEQ ID NO: 9 is as follows:

[0188] The nucleic acid sequence encoding this amino acid sequence is as follows:

[0189] In some embodiments, in the antibody-drug conjugate of formula (I) of the present disclosure, the Ab further comprises a heavy chain constant region (C H ), the heavy chain constant region

[0190] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11; or

[0191] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11; or

[0192] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 11, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) is no more than 10, for example, no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids,

[0193] The amino acid sequence shown in SEQ ID NO: 11 is as follows:

[0194] In some embodiments, in the antibody-drug conjugate of formula (I) of the present disclosure, the Ab further comprises a light chain constant region (C L ), the light chain constant region

[0195] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12; or

[0196] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12; or

[0197] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 12, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) does not exceed 10, for example, not more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids,

[0198] The amino acid sequence shown in SEQ ID NO: 12 is as follows

[0199] In some embodiments, in the antibody-drug conjugate of formula (I) disclosed herein, the Ab comprises a heavy chain (HC), which comprises or consists of a heavy chain variable region and a heavy chain constant region described herein.

[0200] In some embodiments, in the antibody-drug conjugate of formula (I) of the present disclosure, the Ab comprises a heavy chain (HC)

[0201] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13; or

[0202] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 13; or

[0203] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 13, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) does not exceed 10, for example, not more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids,

[0204] The amino acid sequence shown in SEQ ID NO: 13 is as follows:

[0205] In some embodiments, in the antibody-drug conjugate of formula (I) disclosed herein, the Ab comprises a light chain (LC), which comprises a light chain variable region and a light chain constant region as described herein, or consists of the light chain variable region and the light chain constant region.

[0206] In some embodiments, in the antibody-drug conjugate of formula (I) of the present disclosure, the Ab comprises a light chain (LC)

[0207] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14; or

[0208] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 14; or

[0209] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 14, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) is no more than 10, for example, no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids,

[0210] The amino acid sequence shown in SEQ ID NO: 14 is as follows:

[0211] In some embodiments, in the antibody-drug conjugate of formula (I) disclosed herein, the Ab specifically binds to HER3 and comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, in the antibody-drug conjugate of formula (I) disclosed herein, the Ab specifically binds to HER3 and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 9.

[0212] In some embodiments, the Ab further comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 11, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the Ab further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, and the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12.

[0213] In some embodiments, in the antibody-drug conjugate of formula (I) disclosed herein, the Ab specifically binds to HER3 and comprises a heavy chain and / or a light chain, wherein the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 13, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 14.

[0214] In some embodiments, in the antibody-drug conjugate of formula (I) of the present disclosure, the Ab specifically binds to HER3 and comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 14.

[0215] In some embodiments, in the antibody-drug conjugate of formula (I) disclosed herein, the Ab is a chimeric antibody, a humanized antibody, or a human antibody.

[0216] In some embodiments, the Ab is a monoclonal antibody or an antigen-binding fragment thereof.

[0217] In some embodiments, R 1 H, R 2 is a C1-C6 alkyl group or a C1-C6 alkoxy group, R 3 is halogen, preferably -F.

[0218] In some embodiments, D 1 It has the structure shown in formula (D-2):

[0219] where R 1 、R 2 and R 3 As defined above.

[0220] In some embodiments, D 1 It has the structure shown in formula (D-3):

[0221] In some embodiments, D 1 It has the structure shown in formula (D-4):

[0222] In some embodiments, -L 2 -L 1 -is -(C1-C6 alkylene)-, *-(C1-C6 alkylene)-C(O)N(R 5 )-(C1-C6 alkylene)-or *-(C1-C6 alkylene)-N(R 5 )C(O)-(C1-C6 alkylene)-, wherein * indicates that the end is covalently linked to Q;

[0223] R 5 It is H or C1-C6 alkyl.

[0224] In some embodiments, -L 2 -L 1 - is -(C1-C6 alkylene)-.

[0225] In some embodiments, -QL 2 -L 1 - is -OCH2-CH2-CH2-CH2-. It should be understood that the left side of this group is connected to L.

[0226] In some embodiments, wherein Z is linked to Ab, said -CH2- is linked to Q;

[0227] Z is selected from

[0228] where m a1 and m a2 independently selected from integers from 0 to 20, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0229] m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8,

[0230] Preferred wherein m is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5; the carbonyl group at the right end is covalently linked to E;

[0231] In some embodiments, E is a peptide residue having 2, 3, or 4 amino acids.

[0232] In some embodiments, the amino acid is selected from, for example, glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine, and proline, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group and optionally with 1 C 1-6 In some embodiments, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group and optionally substituted with 1 C 1-6 Alkyl substitution.

[0233] In some embodiments, the polyol group is selected from Preferably

[0234] In some embodiments, the substituted glutamine or glutamic acid has the structure shown below:

[0235] where R 6 is H or C1-C6 alkyl;

[0236] Preferably

[0237] where R 6 It is H or C1-C6 alkyl.

[0238] In some embodiments, E is: -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Cit-, -Ala-Ala-, -Ala- Cit-, -Ala-Lys-, -Ala-Val-, -Asn-Cit-, -Asp-Cit-, -Asn-Lys-, -Asp-Val-, -Cit-Ala-, -Cit-Asn-, -Cit-Asp-, -Cit-Cit-, -Cit-Lys-, -Cit-Ser-, -Cit-Val-, -Glu-Val-, -Glu-Gly-, -Ile-Cit-, -Ile-Pro-, -Ile-Val-, - Leu-Cit-, -Lys-Cit-, -Phe-Arg-, -Phe-Cit-, -Phe-Lys-, -Pro-Lys-, -Ser-Cit-, -Trp-Cit-, -Ala-Val-, -Val -Asp-, -Cit-Val-, -Val-Glu-, -Val-Lys-, -Gly-Gly-Gly-, -Gly-Gly-Arg-, -Phe-Lys-Gly-, -Leu-Lys-Gly-, - Leu-Leu-Gly-, -Glu-Val-Cit-, -Cit-Ala-Glu-, -Val-Lys-Gly-, -Val-Lys-Ala-, -Val-Gly-Gly-, -Val-Cit-G ly-, -Val-Gln-Gly-, -Val-Glu-Gly-, -Val-Lys-Gly-, -Val-Lys-Leu-, -Ala-Ala-Ala-, -Asn-Ala-Ala-, -Gly- Gly-Gly-Gly-, -Gly-Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, -Gly-Val-Lys-Gly-, -A1a-Leu-A1a-Leu-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Phe-Gly-Gly-, and -Val-Lys-Gly-Gly, wherein Gln and / or Glu are optionally substituted with 1 polyol group and optionally substituted with 1 C 1-6 Alkyl substitution,

[0239] Preferably, the substituted Gln or Glu has a structure as shown in Formula (G-1a), Formula (G-1b) or Formula (G-1c) above.

[0240] It will be understood that the E group is shown covalently linked to Z via the left amino acid (N-terminus).

[0241] In some embodiments, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, or where R 6 is H or C1-C6 alkyl, wherein these E groups are covalently linked to Z via the left N terminus.

[0242] In some embodiments, -ZE-NH-CH2- has the structure

[0243] It will be understood that the left end of the structure is connected to the Ab moiety.

[0244] In some embodiments, in the antibody-drug conjugate of formula (I) disclosed herein, L is a linker that can connect the antibody portion (Ab) to the biologically active compound portion. In some embodiments, the linker L has the following structure:

[0245] In some embodiments, the linker L is connected to the cysteine ​​side chain of the antibody (Ab) via a maleimide linker, for example, via an -S- bond. In this case, the antibody-drug conjugate of formula (I) can also be represented by the following formula:

[0246] Ab'-(SLD) n (I')

[0247] wherein Ab' is as defined above for Ab, and L, D and n are as defined above.

[0248] In some embodiments, in the antibody-drug conjugates of formula (I) disclosed herein, D is a biologically active compound, for example, an active compound that has a biological effect on tumors (also known as an anti-tumor compound). The anti-tumor compound can be a cytotoxic compound. In some embodiments, the cytotoxic compound D has the following structure:

[0249] In some embodiments, the antibody-drug conjugates of the present disclosure have the following structure:

[0250] wherein Ab and n are as defined in the present disclosure.

[0251] In some embodiments, n is a natural number selected from 1-10, for example, a natural number in a numerical range consisting of any two values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 as endpoints, for example, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, n is 7, or n is 8, or n is 9.

[0252] In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 2-8, 3-8, 4-9, or 7.5-8.5.

[0253] The antibody-drug conjugates provided herein are described with reference to general formulae and specific compounds. In addition, the antibody-drug conjugates of the present disclosure may exist in a variety of different forms or derivatives, all of which are within the scope of the present disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, positional isomers, prodrugs, solvated forms, different crystalline forms or polymorphs, and active metabolites.

[0254] As used herein, unless otherwise indicated, the term "pharmaceutically acceptable salt" includes salts that maintain the biological effectiveness of the free acid / base form of the specific compound and are not undesirable in biological or other aspects. Pharmaceutically acceptable salts can include salts formed with inorganic bases or acids and organic bases or acids. In the case where the antibody-drug conjugates of the present disclosure contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically acceptable salts. Therefore, the antibody-drug conjugates of the present disclosure containing acidic groups (e.g., carboxyl groups) can exist in salt form and can be used according to the present invention, for example, alkali metal salts, alkaline earth metal salts, aluminum salts or ammonium salts. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts or salts with ammonia or organic amines (e.g., ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids). For example, these salts are readily obtained by reacting a compound having an acidic group with a suitable base (e.g., lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide). Other alkaline salts of the antibody-drug conjugates of the present disclosure include, but are not limited to, copper (I), copper (II), iron (II), iron (III), manganese (II), and zinc salts. The antibody-drug conjugates of the present disclosure contain one or more basic groups, such as groups that can be protonated, and can exist in the form of salts and can be used in accordance with the present disclosure in the form of addition salts thereof with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, malonic acid, maleic acid, malic acid, pamoic acid, mandelic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, as well as others known to those skilled in the art. The salts formed are especially hydrochloride, chloride, hydrobromide, bromide, iodide, sulfate, phosphate, methanesulfonate (mesylate), toluenesulfonate, carbonate, bicarbonate, formate, acetate, sulfoacetate, triflate, oxalate, malonate, maleate, succinate, tartrate, malate, pamoate, mandelate, fumarate, lactate, citrate, glutarate, stearate, aspartate and glutamate. In addition, the stoichiometry of the salts formed by the antibody-drug conjugates of the present disclosure can be an integer multiple or a non-integer multiple of 1.

[0255] Antibody-drug conjugates of the present disclosure containing basic nitrogen-containing groups can be synthesized using reagents such as C 1-4Quaternization of alkyl halides, for example, methyl, ethyl, isopropyl and tert-butyl chloride, bromine and iodine; di-C 1-4 Alkyl sulfates, such as dimethyl sulfate, diethyl sulfate, and dipentyl sulfate; C 10-18 Alkyl halides, such as decyl, dodecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and aryl C 1-4 Alkyl halides, such as benzyl chloride and phenethyl bromide.

[0256] If the antibody-drug conjugates of the present disclosure contain both acidic and basic groups in the molecule, then in addition to the above-mentioned salt forms, the present disclosure also includes internal salts or betaines (zwitterions). The corresponding salts can be obtained by conventional methods known to those skilled in the art, for example, by contacting them with organic or inorganic acids or bases in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present disclosure also includes all salts of the antibody-drug conjugates of the present disclosure, which are not directly suitable for pharmaceutical use due to low physiological compatibility, but can be used, for example, as intermediates in chemical reactions or for preparing pharmaceutically acceptable salts. For a review of more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, 2002).

[0257] The antibody-drug conjugate of formula (I) and its pharmaceutically acceptable salts can exist in unsolvated and solvated forms. As used herein, the term "solvate" refers to a molecular complex comprising an antibody-drug conjugate of formula (I), its stereoisomers or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable solvent molecules. For example, the term "hydrate" is used when the solvent is water.

[0258] The antibody-drug conjugate of formula (I) can have one or more chiral (asymmetric) centers. The present disclosure encompasses all stereoisomeric forms of the antibody-drug conjugate of formula (I). The asymmetric centers present in the antibody-drug conjugate of formula (I) can have (R) or (S) configurations independently of each other. When the bond of the chiral carbon is described as a straight line in the structural formula of the present disclosure, or when the compound name is described in the absence of the (R) or (S) chiral name of the chiral carbon, it should be understood that the (R) and (S) configurations of each such chiral carbon and therefore each enantiomer or diastereomer and mixtures thereof are included in the formula or name. The production of a specific stereoisomer or mixture thereof can be identified in the example of obtaining such stereoisomers or mixtures, but this in no way limits all stereoisomers and mixtures thereof to be included within the scope of the present disclosure. When the bond of a chiral carbon is depicted as a triangular solid line or a dashed line in the structural formula of the present disclosure, or when the compound name is depicted with the (R) or (S) chiral designation of the chiral carbon, it should be understood that the compound represented by the structural formula or name has a definite stereo configuration at the chiral carbon position and is to be distinguished from other stereoisomers, enantiomers, diastereomers, or mixtures thereof.

[0259] The present disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, such as mixtures of enantiomers and / or diastereomers in all ratios. Therefore, enantiomers are enantiomerically pure forms (as left-handed and right-handed enantiomers), racemic forms, and mixtures of two enantiomers in all ratios of the disclosed subject matter. In the case of cis / trans isomers, the present disclosure includes mixtures of all ratios of cis-form and trans-form and these forms. If necessary, single stereoisomers can be prepared by conventional methods (such as by chromatography or crystallization, by using stereochemically uniform synthetic starting materials or by stereoselective synthesis). Optionally, derivatization can be carried out before stereoisomer separation. The separation of stereoisomer mixtures can be carried out in an intermediate step during the synthesis of the antibody-drug conjugate of formula (I), or can be carried out on the final racemic product. Absolute stereochemistry can be determined by X-ray crystallography of crystalline products or crystalline intermediates which have been derivatized, if necessary, with reagents containing stereocenters of known configuration. Alternatively, absolute stereochemistry can be determined by vibrational circular dichroism (VCD) spectroscopy.

[0260] Unless otherwise indicated, structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, compounds in which one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Such compounds are termed "isotopic variants." The present disclosure is intended to include all pharmaceutically acceptable isotopic variants of the antibody-drug conjugates of Formula (I). Examples of isotopes suitable for inclusion in the antibody-drug conjugates of the present disclosure include, but are not limited to, isotopes of hydrogen, such as 2 H (i.e., D, deuterium) and 3 H (i.e. tritium); carbon, e.g. 11 C. 13 C and 14 C; chlorine, e.g. 36 Cl; fluorine, e.g. 18 F; iodine, e.g. 123 I and 125 I; nitrogen, e.g. 13 N and 15 N; oxygen, e.g. 15 O. 17 O and 18 O; phosphorus, e.g. 32 P; and sulfur, e.g. 35 Certain isotopic variants of the antibody-drug conjugates of formula (I), such as those incorporating radioactive isotopes, are useful in drug and / or substrate tissue distribution studies. In particular, those having isotopic variations that are observed only upon substitution with heavier isotopes, such as deuterium ( 2 H or D) replaces hydrogen) in the depicted structure of different compounds can provide certain therapeutic advantages, for example, due to greater metabolic stability, increased in vivo half-life or reduced dosage requirements, and thus can be used in some specific situations. Isotopic variants of the antibody-drug conjugates of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples and by using appropriate isotopically labeled reagents instead of the non-labeled reagents previously employed for synthesis.

[0261] When describing the structures of compounds herein, the undesignated hydrogen atoms in the shown formula are D (i.e., 2 H), it is generally understood that the hydrogen at this position is a hydrogen isotope " 1 hydrogen( 1 H)" or in a form with natural isotopic abundance in its natural state. The structures shown indicate that the hydrogen atoms are D (i.e., 2 H, deuterium), it should be understood that the hydrogen at this position is a hydrogen isotope " 2 hydrogen( 2H, D, deuterium)" or in a form in which deuterium is present at a greater isotopic abundance than natural deuterium (e.g., a deuterium abundance of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or 100%).

[0262] Pharmaceutically acceptable solvates according to the present disclosure may include those wherein the solvent of crystallization may be isotopically substituted, for example D2O, d6-acetone, d6-DMSO.

[0263] III. Use and Administration

[0264] The antibody-drug conjugates disclosed herein (antibody-drug conjugates of Formula (I) or stereoisomers thereof) or pharmaceutically acceptable salts or solvates thereof, including mixtures thereof in all ratios, can be used as pharmaceuticals. They have been found to exhibit pharmacological activity that targets HER3 and thereby kills cancer cells that overexpress HER3. Through this activity, the antibody-drug conjugates disclosed herein can be used to treat conditions or diseases associated with HER3 activity, such as HER3-positive tumors.

[0265] Therefore, the antibody-drug conjugates of the present disclosure that can target HER3 are particularly suitable for treating diseases and conditions associated with HER3 activity, such as cancers and tumors, including but not limited to the following: gastric cancer, breast cancer, colorectal cancer, and lung cancer.

[0266] The antibody-drug conjugates of the present disclosure can be administered in an amount effective to treat the diseases or conditions described herein. The antibody-drug conjugates of the present disclosure can be administered as the antibody-drug conjugate itself, or alternatively, as a pharmaceutically acceptable salt. For the purpose of administration and dosing, the antibody-drug conjugates of the present disclosure (antibody-drug conjugates of formula (I), stereoisomers thereof) or pharmaceutically acceptable salts, solvates thereof will be referred to simply as the antibody-drug conjugates of the present disclosure.

[0267] The antibody-drug conjugates of the present disclosure are administered by any suitable route in the form of a pharmaceutical composition suitable for such route and at a dose effective for the intended treatment. The antibody-drug conjugates of the present disclosure can be administered orally, rectally, vaginally, parenterally or topically.

[0268] As used herein, the term "administer" refers to absorbing, ingesting, injecting, inhaling, implanting or otherwise introducing an antibody-drug conjugate of the present disclosure or its pharmaceutical composition. The term "treating" refers to reversing, alleviating, delaying the onset of "pathological conditions" (e.g., diseases, disorders or conditions, or one or more signs or symptoms thereof) described herein or inhibiting their progression. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease or condition have developed or have been observed. In other embodiments, treatment may be performed in the absence of signs or symptoms of the disease or condition. For example, susceptible individuals may be treated before the onset of symptoms (e.g., according to a history of symptoms and / or according to genetic or other susceptibility factors). Treatment may also be continued after the symptoms subside, for example, to delay or prevent recurrence. As used herein, the terms "disease," "disorder," "condition," and "pathological condition" are used interchangeably.

[0269] Those skilled in the art can determine the dosage level to be administered by routine experiments. The dosage regimen of the antibody-drug conjugates of the present disclosure and / or the compositions comprising the antibody-drug conjugates is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the specific antibody-drug conjugate used. Therefore, the dosage regimen can be very different. For example, the dosage level of the antibody-drug conjugates of the present disclosure can be from about 0.001 to about 100 mg / kg (i.e., mg / kg body weight) per day. In some embodiments, the total daily dose of the antibody-drug conjugates of the present disclosure administered in a single or divided dose can be from about 0.001 to about 10 mg / kg. It is not uncommon that the administration of the antibody-drug conjugates of the present disclosure can be repeated multiple times in one day.

[0270] In some embodiments, the antibody-drug conjugates of the present disclosure may be used in combination with one or more other therapeutic agents. In some embodiments, non-limiting examples of such other therapeutic agents include chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, and immunomodulators. These therapeutic agents may be administered before, after, or simultaneously with the administration of the antibody-drug conjugates of the present disclosure.

[0271] IV pharmaceutical compositions, drug combinations, and kits

[0272] In some aspects, the present disclosure relates to pharmaceutical compositions comprising an antibody-drug conjugate of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, as provided herein, and at least one pharmaceutically acceptable carrier or excipient, also referred to as a "pharmaceutical composition of the present invention."

[0273] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier or excipient that can be used to prepare a pharmaceutical composition, which is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes carriers or excipients that are acceptable for veterinary use as well as human pharmaceutical use. As used herein, a pharmaceutically acceptable carrier or excipient includes one or more such carriers or excipients. The specific carrier or excipient used will depend on the manner and purpose of the application of the antibody-drug conjugate of the present disclosure. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, for example, Ansel, Howard C et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R. et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. One or more of buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavorings, flavorings, diluents and other known additives may also be included to improve the therapeutic performance of the drug (i.e., the antibody-drug conjugate or pharmaceutical composition provided herein) or to aid in the production of a pharmaceutical product (i.e., a drug).

[0274] The compositions of the present disclosure can be formulated into a variety of forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, suppositories, etc. The form depends on the intended mode of administration and therapeutic application.

[0275] The pharmaceutical compositions of the present disclosure can be prepared by any well-known pharmaceutical techniques (e.g., effective formulations and administration procedures). The above considerations on effective formulations and administration procedures are well known in the art and are described in standard textbooks. For example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients, 3rd ed., American Pharmaceutical Association, Washington, 1999, the formulation of drug products is discussed.

[0276] In one aspect, the present invention also provides a drug combination or a drug combination product comprising an antibody-drug conjugate of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, and one or more other therapeutic agents. Other therapeutic agents that can be combined with the molecules of the present invention or administered in combination encompass various therapeutic agents for treating tumors, such as chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0277] In yet another aspect, the present disclosure relates to a kit or reagent kit for treating diseases and conditions associated with HER3 activity, such as cancer, comprising an antibody-drug conjugate of Formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof as provided herein, or a pharmaceutical composition comprising an antibody-drug conjugate of Formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof as provided herein, optionally a container, and optionally a package insert or label indicating treatment.

[0278] V Treatment Methods

[0279] In another aspect, the present disclosure relates to a method of treating diseases and conditions associated with HER3 activity, such as cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate of Formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present invention as provided herein.

[0280] As used herein, the term "subject in need thereof" refers to a subject who has a disease or condition associated with HER3 activity, such as cancer, or a subject who has an increased risk of developing a disease or condition associated with HER3 activity relative to the population as a whole. In some embodiments, the subject is a warm-blooded animal. In some embodiments, the warm-blooded animal is a mammal. In some embodiments, the warm-blooded animal is a human.

[0281] As used herein, the term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody drug conjugate or pharmaceutical composition or drug combination are outweighed by the therapeutically beneficial effects. Relative to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100%.

[0282] As used herein, the term "diseases and conditions associated with HER3 activity" refers to any pathophysiological condition associated with the activity or expression of HER3, for example, any pathophysiological condition in which inhibition of HER3 would be beneficial. In some embodiments, the disease and condition associated with HER3 activity is cancer. In some embodiments, the disease and condition associated with HER3 activity is a HER3-positive tumor, for example, a HER3-overexpressing tumor. In some embodiments, the subject (particularly an adult subject) has HER3 expression, particularly HER3 overexpression. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) HER3 (e.g., compared to healthy subjects). In some embodiments, the subject has (e.g., elevated levels of HER3 in a biological sample (e.g., tumor cells or tumor tissue) (e.g., elevated levels, e.g., by greater than 10%, greater than 20%, greater than 50%, greater than 80%, greater than 90%, greater than 1-fold, greater than 2-fold, greater than 3-fold, greater than 5-fold, greater than 10-fold, or a range between these values ​​(e.g., 10%-2-fold), e.g., nucleic acid or protein levels or activity) HER3 (e.g., compared to HER3 in a biological sample (e.g., a corresponding tissue or cell in a healthy subject), or compared to adjacent healthy tissue or cells of the subject). In some embodiments, the HER3-positive tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer, and bladder cancer. In some embodiments, the HER3-positive tumor is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, colon cancer, and epidermal cell carcinoma.

[0283] In another aspect, the present disclosure relates to an antibody-drug conjugate of formula (I) as provided herein, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present invention, for use in treating diseases and conditions associated with HER3 activity, such as cancer or tumors, wherein the diseases and conditions associated with HER3 activity are as defined herein, e.g., HER3-positive tumors, e.g., selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer, and bladder cancer.

[0284] In another aspect, the present disclosure relates to the use of an antibody-drug conjugate of formula (I) as provided herein, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present invention in the preparation of a medicament for treating diseases and conditions associated with HER3 activity, such as cancer or tumors, wherein the diseases and conditions associated with HER3 activity are as defined herein; for example, HER3-positive tumors, for example, selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer and bladder cancer.

[0285] VI. Preparation of ADC molecules of the present invention

[0286] Another aspect of the present invention provides a method for preparing the antibody-drug conjugate of formula (I).

[0287] In some embodiments, the method comprises the steps of:

[0288] (a) adding the antibody Ab to a buffer solution, adding a reducing agent, and then incubating;

[0289] (b) adding a linker-payload to the reaction solution in step (a) for coupling to obtain a crude product; and

[0290] (c) optionally purifying the crude product to obtain the antibody drug conjugate of the present invention;

[0291] wherein Ab is as defined above.

[0292] It will be understood that the linker-payload reacts with Ab to provide the -LD moiety in the compound of Formula I, and where -LD is clearly defined, the structure of the linker-payload can be determined.

[0293] In some embodiments, the buffer solution in step a) is a PBS buffer, preferably, having a pH of 5.0-9.0, such as 6.0-8.0.

[0294] In some embodiments, the reducing agent of step a) is TCEP.

[0295] In some embodiments, the linker-payload has the following structure: Z'-E-NH-CH2-QL 2 -L 1 -D 1 , where E, Q, L 2 、L 1 、D 1 As defined above, Z' is as defined above for Z, except that the maleimide group in Z is replaced by Replace with

[0296] In some embodiments, the steps are performed under the specific reaction conditions disclosed in the Examples.

[0297] It should be noted that embodiments in which the ranges or specific values ​​of the specific reaction conditions disclosed in the examples are varied by 100%, 80%, 60%, 40%, 20% or 10% are also contemplated by the present invention.

[0298] These and other aspects and embodiments of the present invention are described in the accompanying drawings and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art.

[0299] Example

[0300] The technical solutions of the present invention will be further described in detail below by way of examples and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased on the market. Those skilled in the art will appreciate that the methods and materials described below are merely exemplary and should not be construed as limiting the scope of the present invention.

[0301] The embodiments of the present invention are not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

[0302] Example 1. Preparation and detection of antibody molecules and ADCs

[0303] Example 1.1 Preparation of HER3 monoclonal antibody

[0304] Nucleic acid fragments encoding the HER3 heavy and light chains were inserted into the vector pcDNA3.1 (Invitrogen, V790-20) to generate anti-HER3 heavy and light chain plasmids. The heavy and light chain plasmids were transiently transfected into ExpiCHO cells (Gibco, A29133) using FectoPRO Transfection Reagent (Polyplus, 101000007). After 7 days, the cell fermentation broth was obtained, clarified by filtration, and captured using a Hitrap Mabselect Sure chromatography column (GE Healthcare, 11-0034-95) to obtain the antibody.

[0305] Table 1. HER3 protein purity and yield

[0306] Example 1.2 HER3-MB3 conjugate preparation

[0307] The reducing agent TCEP (Sigma, C4706) was added to the HER3 antibody prepared as above at a ratio of 20:1 (M / M), and the reduction reaction was carried out in a water bath at 25°C / 2h. MB3 (for its preparation, see Example 4 of WO2021173773A1, where the payload in MB3 is MB1) was added to the system at a ratio of 1:14 (M / M) for HER3 antibody:Drug-linker (MB3). The system was adjusted to have a DMSO concentration of 10% (v / v) and the reaction was carried out in a water bath at 25°C / 1h. According to the relative molar ratio of MB3, 20 times N-acetylcysteine ​​(Sigma, A8199) was added and the reaction was carried out at room temperature for 15min to quench the unreacted MB3 in the reaction system. The reaction was then quenched with Zeba TM A desalting centrifugal column (Thermo, 40K MWCO) was used to remove impurities and replace 20 mM pH 5.5 histidine buffer. Finally, the prepared and purified HER3-MB3 was sterilized by filtration using 0.2 uM PVDF (Millipore, SLGVR13SL) and stored at -40°C.

[0308] The structure of the HER3-MB3 molecule is shown below:

[0309] Wherein Ab is Patritumab (the antibody prepared in Example 1.1), and n is mainly 8.

[0310] FIG5 shows a schematic diagram of the HER3-MB3 molecule of Example 1.

[0311] Table 2. HER3-MB3 quality detection

[0312] Example 1.3 Preparation of other ADCs

[0313] ADCs with different linkers and payloads (e.g., different HER3-ADCs) were prepared using different liners and payloads as shown in Table 3, and antibodies (e.g., HER3 antibody Patritumab or BL antibody Izalontamab) based on methods known in the prior art. The liners and payloads in the table are well known to those skilled in the art of ADCs and are commercially available and / or synthesized according to prior art.

[0314] 1. Preparation of Patritumab-DM4 (HER3-DM4) conjugate

[0315] Patritumab antibody was replaced in 20mM phosphate buffer, pH 7.0. SPDB-DM4 was added to the system at a ratio of 1:8 (M / M) to drug-linker (SPDB-DM4). The DMA concentration was adjusted to 15% (v / v). The coupling reaction was carried out in a water bath at 30°C for 16 hours. Impurities were removed using a Zeba™ desalting spin column (Thermo, 40K MWCO) and the buffer was replaced with 20mM histidine buffer, pH 5.5. Finally, the purified Patritumab-DM4 was sterilized by filtration using 0.2μM PPVDF (Millipore, SLGVR13SL) and stored at -40°C.

[0316] 2. Preparation of Patritumab-DM1 (HER3-DM1) Conjugate

[0317] Patritumab antibody was replaced in 20mM phosphate buffer, pH 7.0. SPDB-DM1 was added to the system at a ratio of 1:10 (M / M) to drug-linker (SMCC-DM1). The DMA concentration was adjusted to 15% (v / v). The coupling reaction was carried out in a water bath at 30°C for 16 hours. Impurities were removed using a Zeba™ desalting spin column (Thermo, 40K MWCO) and the buffer was replaced with 20mM histidine buffer, pH 5.5. Finally, the purified Patritumab-DM1 was sterilized by filtration using 0.2μM PVDF (Millipore, SLGVR13SL) and stored at -40°C.

[0318] 3. Preparation of Patritumab-MMAE (HER3-MMAE) Conjugate

[0319] The reducing agent TCEP (Sigma, C4706) was added to the Patritumab antibody at a ratio of 2:1 (M / M) and the reaction was carried out in a water bath at 25°C for 1 hour. MC-VC-PAB-MMAE was then added to the system at a ratio of Patritumab antibody to drug-linker (MC-VC-PAB-MMAE) of 1:6 (M / M), and the DMSO concentration was adjusted to 10% (v / v). The reaction was carried out in a water bath at 25°C for 1 hour. Impurities were removed using a Zeba™ desalting spin column (Thermo, 40K MWCO) and replaced with a 20mM pH 5.5 histidine buffer. Finally, the purified Patritumab-MMAE was sterilized by filtration using 0.2μM PVDF (Millipore, SLGVR13SL) and stored at -40°C.

[0320] 4. Preparation of Patritumab-DXd (HER3-DXd) Conjugate

[0321] The reducing agent TCEP (Sigma, C4706) was added to the Patritumab antibody at an 8:1 (M / M) ratio and reduced in a water bath at 25°C for 1 hour. MC-GGFG-Dxd was then added to the system at a Patritumab antibody:drug-linker (MC-GGFG-Dxd) ratio of 1:10 (M / M), adjusting the DMSO concentration to 10% (v / v) and incubating in a water bath at 25°C for 1 hour. Impurities were removed using a Zeba™ desalting spin column (Thermo, 40K MWCO) and replaced with 20 mM histidine buffer, pH 5.5. Finally, the purified Patritumab-Dxd was sterilized by filtration using 0.2-μM PVDF (Millipore, SLGVR13SL) and stored at -40°C.

[0322] Replace Patritumab-MMAF with a dedicated linker-drug, and its preparation method is the same as Patritumab-MMAE; replace Izalontamab, and the BL-DXd coupling preparation method is the same as Patritumab-DXd method.

[0323] Table 3. ADC different linker and payload information

[0324] Example 1.4 HER3-MB3 affinity detection

[0325] Thin-layer interferometry (BLI) was used to measure the affinity (KD) of HER3-MB3 for human (Sino Biological, 10201-H08H), monkey (Sino Biological, 90043-K08H), mouse (Sino Biological, 51003-M08H), and rat (Sino Biological, 80111-R08H) HER3. Before the experiment, an appropriate number of AHC sensors (18-5060, Sartorius) were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). HER3-MB3 and the corresponding antigen were diluted to 100 nM. SD buffer, HER3-MB3, and the corresponding antigen were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a Fortebio Octet Red96e, and KD analysis was performed using a ForteBio Octet.

[0326] Table 4. HER3-MB3 affinity for HER3 in different species

[0327] Example 2. Screening of Antibody-Drug Conjugates

[0328] In Example 1, various antibody-drug conjugates were designed and synthesized, such as HER3 antibodies conjugated to cytotoxic drug molecules such as DXd, MMAE, MMAF, DM1, and DM4. In this example, HER3-MB3 (Patritumab-MB3) was ultimately selected through in vitro pharmacodynamic sequence screening, experimental comparisons of various aspects, including the presence of potential drug resistance in the payload and bystander effects of different linker-payloads.

[0329] 1. Antibody sequence screening

[0330] In vitro killing assay method

[0331] (1) 2000 cells / 100 μl were plated on a 96-well low-adhesion plate (Corning, CLS7007-24EA) and 3D cell culture was performed for two days to allow the suspended cells to form cell clusters.

[0332] (2) Add the prepared drug molecules (maximum concentration 100 nM, 4-fold dilution) diluted in advance to the corresponding cell wells, mix well, and culture at 37°C with 5% CO2 for 7 days.

[0333] (3) After 7 days of culture, add the Cell-Titer reagent (Promega, G7572) prepared in advance to the corresponding cell well plate and keep it at room temperature in the dark for 15-25 minutes.

[0334] (4) Transfer the Cell-Titer and cell mixture into a 96-well white-bottom plate (NUNC, 136101) and detect using a microplate reader (Molecular Devices, Spectra MAXi3).

[0335] 2. Does the payload have potential drug resistance?

[0336] Elacridar is an orally active Pgp glycoprotein that can be used to study the effects of efflux transporters on drugs; the acclimated Elacridar-resistant cell line HCT15 was screened and used to evaluate whether the payload has potential drug resistance.

[0337] Experimental methods

[0338] (1) Take a 96-well white-bottom plate (NUNC, 136101), plate 1000 cells / well, and culture at 37°C 5% CO2 overnight.

[0339] (2) Add the prepared payload drug molecules (maximum concentration 100 nM, 3-fold dilution) diluted in advance to the corresponding cell well plates containing 0.5 uM Elacridar or not containing 0.5 uM Elacridar, mix well, and culture at 37°C 5% CO2 for four days.

[0340] (3) After four days of culture and killing, the Cell-Titer reagent (Promega, G7572) prepared in advance was added to the corresponding cell well plate, kept in the dark at room temperature for 15-25 minutes, and detected by microplate reader (Molecular Devices, Spectra MAXi3).

[0341] 3. Linker-payload bystander effect

[0342] In the SW480 / SW480HER3-OX system, the HER3-MB3 bystander effect was performed.

[0343] Construction of SW480HER3-OX cell line: Lentvirus+HER3 lentivirus was constructed and packaged, SW480 was infected with Lentvirus+HER3 virus, and the SW480HER3-OX stable cell line was obtained through pressure screening.

[0344] Experimental methods

[0345] (1) 80,000 cells / SW480 and 100,000 cells / SW480+HER3 cells were plated in six-well plates and cultured overnight.

[0346] (2) Add the prepared drugs diluted in advance to the corresponding cell wells, with the final drug concentration of 5 nM, mix well, and culture at 37°C with 5% CO2 for 5 days.

[0347] (3) On the 5th day, the cells were digested and the total cell number was counted; according to the HER3 expression level, the percentage relationship between SW480HER3-OX and SW480 was marked by FACS (BD, Celesta).

[0348] Absolute cell number calculation method:

[0349] SW480HER3-OX=N*FACS(HER3 + -SW480HER3OX)*100

[0350] SW480=N*FACS(HER3 - -SW480)*100

[0351] Experimental results:

[0352] The results from Figures 1 and 2 show that HER3-ADC (Patritumab-MB3 and Paritumab-DXd) have better in vitro efficacy than BL-DXd (Izalontamab-DXd) in SW620 and MCF-7, and HER3-MB3 (Patritumab-MB3) has better in vitro efficacy than HER3-DXd (Patritumab-DXd). The results from Figure 3 show that MB1 has lower potential resistance than MMAF, MMAE, DM1, DM4, and DXd. The results from Figure 4 show that HER3-MB3 (Patritumab-MB3) has a stronger bystander effect. Based on the results from Figures 1 to 4, the antibody-drug conjugate HER3-MB3 was ultimately selected.

[0353] Example 3. Detection of HER3 expression abundance in major tumor cell lines

[0354] In addition to being related to drug-linker technology, the anti-tumor effect of HER3-MB3 is also related to the intensity and abundance of HER3 expression in tumor cells.

[0355] HER3 relative expression analysis was performed in various tumor cell lines. Among the analyzed cell lines, the order of HER3 overall expression was gastric cancer, breast cancer, colorectal cancer, lung cancer, and pancreatic cancer. The experimental results are shown in Figure 6.

[0356] Experimental methods:

[0357] 1. Resuspend the cells in FACS buffer and plate 100,000-200,000 cells in a 96-well plate (Corning, CLS3799-50EA). Add 10 μg / ml HER3 antibody (Patritumab) and 10 μg / ml IgG to the plate and incubate at 4°C for 1 hour.

[0358] 2. Wash twice with PBS, add the prepared APC-anti human Fc antibody (Biolegend, 410712) to the corresponding cell wells, and incubate at 4°C for 30-40 minutes.

[0359] 3. Wash twice with PBS and detect by FACS (BD, Celesta). The results are shown in Figure 6.

[0360] Cell Source:

[0361] Gastric cancer cell lines: (1). NUGC4: CoBier, CBP60012; (2). KATO-III: Cell Bank of Chinese Academy of Sciences, SCSP-573; (3). NCI-N87: CoBier, CBP60491; (4). MKN45: CoBier, CBP60488; (5). MGC-803: CoBier, CBP60485.

[0362] Breast cancer cell lines: (6). MDA-MB-453: CoBier, CBP60386; (7). MCF-7: CoBier, CBP60380; (8). HCC1569: CoBier, CBP60372; (9). JIMT-1: CoBioer, CBP60378; (10). MDA-MB-231: Chinese Academy of Sciences Cell Bank, SCSP-5043.

[0363] Colorectal cancer cell lines: (11).SW620: CoBioer, CBP60036; (12).HT55: HT55, CBP60012; (13).LOVO: Cell Bank of the Chinese Academy of Sciences, TCHu 82; (14).HCT-15: Shanghai Cell Bank of the Chinese Academy of Sciences, TCHu133; (15).GP2D: CoBioer, CBP60010; (16).H508: CoBioer, CBP60795; (17).LS174T: CoBioer, CBO60033; (18).SW480: Cell Bank of the Chinese Academy of Sciences, SCSP-5033; (19).Colo320: CoBioer, CBP61130.

[0364] Lung cancer cell lines: (20). Calu-3: CoBioer, CBP60086; (21). H358: CoBioer, CBP60136; (22). DMS53: CoBioer, CBP60172; (23). HCC827: ATCC, CRL-2868; (24). NCI-H1975: ATCC, CRL-5908; (25). A431: ATCC, CRL-1555; (26). A549: CoBioer, CBP60084; (27). NCI-H441: CoBioer, CBP60137; (28).NCI-H2030:ATCC,CRL-5914; (29).NCI-H292:Cell Bank of Chinese Academy of Sciences,SCSP-582; (30).NCI-H4006:ATCC,CRL-2871; (31).NCI-H1703:ATCC,CRL-5889; (32).SK-MES-1:CoBioer,CBP60152; (33).Calu-1:ATCC,HTB-54; (34).PC-9:Shanghai Yubo Biotechnology,YB-H3210; (35).HCC2279:CoBioer,CBP60098.

[0365] Pancreatic cancer cell lines: (36).Capan-1:CoBioer,CBP60543; (37).T3M4:CoBioer,CBP60692; (38).Capan-2:CoBioer,CBP60834; (39).CFPAC-1:CoBioer,CBP60665; (40).Miapaca2:CoBioer,CBP60544; (41).AsPc-1:CoBioer,CBP60546; (42).HuP-T4:CoBioer,CBP60540; (43).PSN-1:CoBioer,CBP61215; (44).BxPc-3:CoBioer,CBP60542; (45).PANC-1:CoBioer,CBP60545.

[0366] HCC827+HER3 (HCC827 HER3OX) overexpressing cell line: Construct and package lentivirus+HER3 lentivirus, infect HCC827 with lentivirus+HER3 virus, and obtain stably transfected HCC827+HER3 cells through pressure screening and sorting.

[0367] Example 4. In vitro efficacy study of HER3-MB3 in tumor cell lines

[0368] Experimental methods:

[0369] 1. In vitro killing assay method

[0370] (1) 2000 cells / 100 μl were plated on a 96-well low-adhesion plate (Corning, CLS7007-24EA) and 3D cell culture was performed for two days to allow the suspended cells to form cell clusters.

[0371] (2) Add the prepared drug molecules (maximum concentration 25 nM, 4-fold dilution) diluted in advance to the corresponding cell wells, mix well, and culture at 37°C with 5% CO2 for 7 days.

[0372] (3) After 7 days of culture, add the Cell-Titer reagent (Promega, G7572) prepared in advance to the corresponding cell well plate and keep it at room temperature in the dark for 15-25 minutes.

[0373] (4) Transfer the Cell-Titer and cell mixture into a 96-well white-bottom plate (NUNC, 136101) and detect using a microplate reader (Molecular Devices, Spectra MAXi3).

[0374] Experimental results:

[0375] From the results in Figures 7-10, it was found that the in vitro pharmacodynamic activity of HER3-MB3 was positively correlated with the HER3 expression intensity. The higher the HER3 expression, the stronger the in vitro pharmacodynamic activity of HER3-MB3, indicating that HER3-MB3 can be used to treat patients with HER3-positive tumors.

[0376] The results in Figure 7 show that HER3-MB3 has anti-tumor activity in gastric cancer. In the NUGC4 tumor cell line, the in vitro activity of HER3-DXd (U3-1402) is far superior. At the highest concentration of 25nM, the maximum tumor killing rate of HER3-MB3 is 73.34±3.21%, and the IC 50 is 0.55nM; the maximum tumor killing rate of HER3-DXd is 45.52±5.82%, IC 50 It is 2.02nM.

[0377] The results in Figure 8 show that HER3-MB3 has anti-tumor activity in breast cancer. In the HCC1569 tumor cell line, its in vitro activity is far superior to that of HER3-DXd (U3-1402). At the highest concentration of 25nM, the maximum tumor killing rate of HER3-MB3 is 77.16±0.62%, and the IC 50 is 0.07nM; the maximum tumor killing rate of HER3-DXd is 72.90±1.62%, IC50 It is 0.20nM.

[0378] The results in Figures 9 and 10 show that HER3-MB3 exhibits antitumor activity in colorectal cancer, with in vitro efficacy superior to that of HER3-DXd (U3-1402). In the NCI-H508 tumor cell line, at a 25 nM dose, the maximum in vitro tumor killing rates of HER3-MB3 and HER3-DXd were 68.07 ± 1.45% and 43.27 ± 2.93%, respectively. In the SW620 tumor cell line, at a 25 nM dose, the maximum in vitro tumor killing rates of HER3-MB3 and HER3-DXd were 30.21 ± 2.34% and 25.00 ± 2.68%, respectively.

[0379] The results in FIG11 show that HER3-MB3 has significant anti-tumor activity in the HER3-overexpressing HCC827 cell line.

[0380] From the in vitro killing results of other breast cancer and colorectal cancer cells in Table 5, it was found that HER3-MB3 had a stronger in vitro efficacy than HER3-DXd (U3-1402).

[0381] Table 5. HER3-MB3 killing results in tumor cell lines

[0382] Example 5. HER3-MB3 bystander effect experiment

[0383] The bystander effect refers to the fact that while ADC kills and inhibits positive target tumor cells, some toxin molecules are released from the positive target cells and enter nearby negative target tumor cells, further exerting a stronger anti-tumor effect.

[0384] As shown in Figure 12, by comparing different drugs, it was found that MB3 showed better anti-tumor activity and bystander effect.

[0385] Experimental methods:

[0386] HER3-MB3 bystander effect assay in the SW480 / SW480HER3-OX system:

[0387] (1) 80,000 cells / SW480 and 100,000 cells / SW480+HER3 cells were plated in six-well plates and cultured overnight.

[0388] (2) Add the prepared drugs diluted in advance to the corresponding cell wells, with the final drug concentration of 5 nM, mix well, and culture at 37°C with 5% CO2 for 5 days.

[0389] (3) On the 5th day, the cells were digested and the total cell number was counted; according to the HER3 expression level, the percentage relationship between SW480HER-OX and SW480 was marked by FACS (BD, Celesta).

[0390] Absolute cell number calculation method:

[0391] SW480HER3-OX=N*FACS(HER3 + -SW480ER3OX)*100

[0392] SW480=N*FACS(HER3 - -SW480)*100

[0393] Example 6. HER3-MB3 efficacy experiment in mice

[0394] SW620 in vivo efficacy experiment

[0395] To verify the in vivo efficacy of HER3-MB3 in a colorectal cancer tumor model, a SW620 CDX mouse tumor model was established using BALB / C-Nude (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., certificate: NO.110011221105802464) mice.

[0396] Experimental methods:

[0397] SW620 tumor cells were subcultured and resuspended in equal proportions of PBS and Matrigel Matix. 6E6 cells / 200ul were inoculated into the right abdomen of mice. The mice were grouped and intraperitoneally administered on the 9th day. The administration cycle was once a week for a total of three times. Tumor size and body weight were measured twice a week.

[0398] The tumor inhibition rate (TGI%) was calculated as follows: TGI% = 100%*(tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - initial tumor volume of the control group).

[0399] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V = L*W2 / 2

[0400] Experimental results

[0401] On day 21 after SW620 cell modeling, the mouse tumor inhibition rate was calculated to evaluate the in vivo efficacy of HER3-MB3. As shown in Figure 13, at a dose of 10 mg / kg, the maximum tumor inhibition rates of HER3-MB3 and HER3-DXd (U3-1402) were 111.80% and 108.76%, respectively. HER3-MB3 was slightly more effective than HER3-DXd (U3-1403). At a dose of 10 mg / kg, IgG-MB3 showed non-specific tumor killing. At a dose of 3 mg / kg, the maximum tumor inhibition rates of HER3-MB3 and HER3-DXd (U3-1402) were 71.43% and 72.43%, respectively. The results in Figure 14 show that under high and low dose conditions, there was no significant change in the body weight of HER3-MB3 and HER3-DXd mice, indicating that HER3-ADC is safe.

[0402] In vivo efficacy experiments of NUGC4

[0403] To verify the in vivo efficacy of HER3-MB3 in a gastric cancer tumor model, a NUGC4 CDX mouse tumor model was established using NOG mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., NO.110011221107474276).

[0404] Experimental methods:

[0405] NUGC4 tumor cells were subcultured and resuspended in equal proportions of PBS and Matrigel Matix. 4E6 cells / 200ul / mouse were inoculated into the right abdomen of mice. The mice were grouped and intraperitoneally administered on the 18th day. The administration cycle was once a week for a total of three times. The tumor size and body weight were measured twice a week.

[0406] The tumor inhibition rate (TGI%) was calculated as follows: TGI% = 100%*(tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - initial tumor volume of the control group).

[0407] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V = L*W2 / 2

[0408] Experimental results

[0409] On day 21 after NUGC4 cell modeling, mouse tumor inhibition rates were calculated to evaluate the in vivo efficacy of HER3-MB3. As shown in Figure 15, at a dose of 10 mg / kg, the maximum tumor inhibition rates of HER3-MB3 and HER3-DXd (U3-1402) were 99.27% ​​and 99.93%, respectively. At a dose of 3 mg / kg, HER3-MB3 was significantly more effective than HER3-DXd, with maximum tumor inhibition rates of 81.31% for HER3-MB3 and 46.61% for HER3-DXd. At a high dose of 10 mg / kg, both IgG-MB3 and IgG-DXd exhibited non-specific tumor killing. The results in Figure 16 show that at both high and low doses, there was no significant weight change in mice treated with HER3-MB3 or HER3-DXd, indicating the safety of the HER3-ADC.

[0410] Example 7. PK of HER3-MB3 in mice

[0411] To verify the pharmacokinetics (PK) of HER3-MB3 and HER3-DXd in mice, mice were administered with a dose of 10 mg / kg HER3-MB3 and 10 mg / kg HER3-DXd through the tail vein. Orbital blood was collected and serum was prepared at 5 min, 30 min, 2 h, 6 h, 24 h, 48 h, 96 h, 168 h, 336 h and 504 h, and the blood drug concentration was detected using the HER3 mAb and HER3-ADC detection methods, respectively.

[0412] Mouse serum HER3 mAb PK assay

[0413] 1.1 μg / ml HER3 protein (Acrobiosystem, ER3-H82E6) was plated on a 96-well ELISA plate (Thermo, 442404) and incubated overnight at 4°C.

[0414] 2. Wash with PBST (PBS solution containing 0.05% Tween-20), wash three times.

[0415] 3. Block with blocking solution (PBST containing 5% skim milk powder) at room temperature for 2 hours.

[0416] 4. Wash three times with washing solution, add standard (HER3-MB3, HER3-DXd) and serum sample to be tested, and incubate at room temperature for 2 hours.

[0417] 5. Wash 6 times with detergent and incubate with Goat anti-human IgG-Fc-HRP (Bethyl, A80-104P) at room temperature for 1 hour.

[0418] 6. Wash 6 times with washing solution, add TMB (Solarbau, PR1200), and color development for 5-10 minutes in the dark.

[0419] 7. Add ELISA stop solution (Solarbio, C1058) and read OD450 / OD620 nm on a microplate reader (Thermo, Multiskan FC).

[0420] Mouse serum HER3-ADC PK detection method

[0421] 1. 1ug / ml Payload antibody (Innovent, 18E7C9) was spread on a 96-well ELISA plate (Thermo, 442404) and incubated overnight at 4°C.

[0422] 2. Wash with PBST (PBS solution containing 0.05% Tween-20), wash three times.

[0423] 3. Block with blocking solution (PBST containing 5% skim milk powder) at room temperature for 2 hours.

[0424] 4. Wash three times with washing solution, add standard (HER3-MB3, HER3-DXd) and serum sample to be tested, and incubate at room temperature for 2 hours.

[0425] 5. Wash 6 times with washing buffer, add 1 μg / ml HER3 protein (Acrobiosystem, ER3-H82E6), and incubate at room temperature for 2 hours.

[0426] 6. Wash 6 times with washing buffer and incubate with SA HRP (BD, 554066) at room temperature for 1 hour.

[0427] 7. Wash 6 times with washing solution, add TMB (Solarbau, PR1200), and color development for 5-10 minutes in the dark.

[0428] 8. Add ELISA stop solution (Solarbio, C1058) and detect OD450 / OD620 nm using a microplate reader (Thermo, Multiskan FC).

[0429] Experimental results:

[0430] As shown in Figure 17 and the data results in Table 6, the total HER3 antibody and HER3-ADC concentrations in mouse blood were detected using the above different detection methods. HER3-MB3 outperformed HER3-DXd in mice, further indicating that HER3-MB3 has better in vivo efficacy than HER3-DXd.

[0431] Table 6. PK parameters of 10 mg / kg HER3-ADC in mice

[0432] Example 8. Toxicokinetics of HER3-MB3 in cynomolgus monkeys and rats (TK)

[0433] Compared with HER3-DXd in vitro efficacy data, in vivo efficacy data, bystander effect and in vivo pharmacokinetic data in mice, HER3-MB3 has better efficacy data than HER3-DXd. To further verify the safety of HER3-MB3, according to the experimental plan designed by Innovent, Zhaoyan (Suzhou) New Drug Research Center Co., Ltd. was commissioned to conduct a pre-toxicological safety study of HER3-MB3 in cynomolgus monkeys, as shown in Table 7.

[0434] Table 7. HER3-MB3 pre-toxicology study plan

[0435] Experimental results:

[0436] According to clinical observations, no deaths or serious toxic side effects occurred in crab-eating macaques at 30mg / kg HER3-MB3 and 80mg / kg HER3-MB3, indicating that HER3-MB3 has a good safety profile.

[0437] According to Table 8 and Figure 18, the accumulation of HER3-MB3 in cynomolgus monkeys increased with increasing dose. In cynomolgus monkeys, the exposure of HER3-MB was basically the same as that of total HER3 mAb: st -30 mg / kg (ADC / TAB=90%), 1 st -80mg / kg (ADC / TAB=109%), 2 nd -30 mg / kg (ADC / TAB=95%), 2 nd -80 mg / kg (ADC / TAB=96%).

[0438] Table 8. Main parameters of HER3-MB3 pre-toxicity TK

[0439] The foregoing description is intended to be merely illustrative of the principles of the invention. Furthermore, since many modifications and variations will be apparent to those skilled in the art, it is not intended to limit the invention to the exact configuration and process described above. Therefore, all suitable modifications and equivalents are intended to fall within the scope of the invention as defined by the appended claims.

[0440] All publications, patents, and patent applications cited herein are incorporated by reference into this disclosure in their entirety.The amino acid sequences of the antibody molecules mentioned herein and the nucleic acid sequences encoding the antibody molecules are as follows.

[0441] HER3 antibody HCDR1 amino acid sequence SEQ ID NO: 1

[0442] HER3 antibody HCDR2 amino acid sequence SEQ ID NO: 2

[0443] HER3 antibody HCDR3 amino acid sequence SEQ ID NO: 3

[0444] HER3 antibody LCDR1 amino acid sequence SEQ ID NO: 4

[0445] HER3 antibody LCDR2 amino acid sequence SEQ ID NO: 5

[0446] HER3 antibody LCDR3 amino acid sequence SEQ ID NO: 6

[0447] HER3 antibody heavy chain variable region amino acid sequence SEQ ID NO: 7

[0448] Nucleotide coding sequence of the HER3 antibody heavy chain variable region SEQ ID NO: 8

[0449] HER3 antibody light chain variable region amino acid sequence SEQ ID NO: 9

[0450] Nucleotide coding sequence of the HER3 antibody light chain variable region SEQ ID NO: 10

[0451] HER3 antibody heavy chain constant region amino acid sequence SEQ ID NO: 11

[0452] HER3 antibody light chain constant region amino acid sequence SEQ ID NO: 12

[0453] HER3 antibody heavy chain amino acid sequence SEQ ID NO: 13

[0454] HER3 antibody light chain amino acid sequence SEQ ID NO: 14

Claims

1. An antibody-drug conjugate having formula (I), a stereoisomer or a pharmaceutically acceptable salt or solvate thereof: Ab-(LD) n (Ⅰ) in, Ab is an antibody or an antigen-binding fragment thereof that binds to HER3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the light chain variable regions LCDR1, LCDR2 and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, L is a linker having the following structure: -ZE-NH-CH2-, Wherein Z is connected to Ab, -CH2- is connected to D; Z is selected from Where m a1 and m a2 independently selected from integers from 0 to 20; m is an integer selected from 1 to 10, The carbonyl group on the right end of Z is covalently linked to E; E is a peptide residue comprising 2-10 amino acids, wherein the peptide residue is optionally substituted by one or more groups selected from C 1-6 an alkyl group and a polyol group, wherein the N-terminus of the peptide residue is covalently linked to Z, D is a cytotoxic compound having the formula -QL 2 -L 1 -D 1 The structure, Wherein Q is -O- or -S-; L 1 is not present or -(C1-C 10 Alkylene)-; L 2 Does not exist, *-(C1-C 10 Alkylene)-C(O)N(R 5 )-or*-(C1-C 10 Alkylene)-N(R 5 )C(O)-; wherein * indicates that the terminus is covalently linked to Q; and R 5 is H or C1-C6 alkyl, Where D 1 It has the structure shown in formula (D-1): Where R 1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl; R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 4 and-SR 4 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 4 ; or R 2 and R 3 Together they form -O(CH2) n1 O- or -O(CF2) n1 O-, where n1 is 1 or 2; R 4 is selected from H or C1-C4 alkyl; and n represents DAR, and n is a natural number selected from 1-15.

2. The antibody-drug conjugate, its stereoisomer, or pharmaceutically acceptable salt or solvate according to claim 1, wherein the heavy chain variable region of the Ab (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7; or (iii) comprises or consists of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 7, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions.

3. The antibody-drug conjugate according to claim 1 or 2, its stereoisomer or pharmaceutically acceptable salt or solvate, wherein the light chain variable region of the Ab (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 9; or (iii) comprises or consists of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 9, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions.

4. The antibody-drug conjugate, stereoisomer, pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the heavy chain variable region of the Ab comprises or consists of the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region of the Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:

9.

5. The antibody-drug conjugate, stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 4, wherein the Ab further comprises a heavy chain constant region, the heavy chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 11; or (iii) comprising an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 11, or consisting of said amino acid sequence.

6. The antibody-drug conjugate, stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein the Ab further comprises a light chain constant region, the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12; or (iii) comprises an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 12, or consists of said amino acid sequence.

7. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, wherein the Ab comprises a heavy chain, wherein the heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 13; or (iii) comprises an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 13, or consists of said amino acid sequence.

8. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 7, wherein the Ab comprises a light chain, wherein the light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 14; or (iii) comprises an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 14, or consists of said amino acid sequence.

9. The antibody-drug conjugate, stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 8, wherein the Ab comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:

14.

10. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 9, wherein the Ab is selected from an antigen-binding fragment Fv, Fab, Fab', or F(ab')2; a single-chain antibody scFv; or a single-domain antibody VHH.

11. The antibody-drug conjugate, stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 10, wherein the linker is linked to Ab via a cysteine ​​residue side chain.

12. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 11, wherein R 1 For H, R 2 is C1-C6 alkyl or C1-C6 alkoxy, R 3 is halogen, preferably -F.

13. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 12, wherein D 1 It has the structure shown in formula (D-2): Where R 1 , R 2 and R 3 As defined in any one of claims 1 to 12.

14. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 13, wherein D 1 It has the structure shown in formula (D-4):

15. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 14, wherein -L 2 -L 1 - is -(C1-C6 alkylene)-, *-(C1-C6 alkylene)-C(O)N(R 5 )-(C1-C6 alkylene)-or *-(C1-C6 alkylene)-N(R 5 )C(O)-(C1-C6 alkylene)-, wherein * indicates that the end is covalently linked to Q; R 5 It is H or C1-C6 alkyl.

16. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 15, wherein -L 2 -L 1 - is -(C1-C6 alkylene)-.

17. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 16, wherein -QL 2 -L 1 - is -OCH2-CH2-CH2-CH2-.

18. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 17, wherein Z is Wherein m is an integer from 1 to 10.

19. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 18, wherein E is a peptide residue consisting of 2, 3 or 4 amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine and proline, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group and optionally substituted with 1 C 1-6 Alkyl substitution; Preferably, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group and optionally with 1 C 1- 6 Alkyl substitution.

20. The antibody-drug conjugate, stereoisomer, pharmaceutically acceptable salt or solvate thereof according to claim 19, wherein the substituted glutamine or glutamic acid has the structure shown below: Where R 6 is H or C1-C6 alkyl; Preferably Where R 6 It is H or C1-C6 alkyl.

21. The antibody-drug conjugate, stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 20, wherein E is: -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Val-Cit-, -Ala-Ala-, -Ala-Cit-, -Ala-Lys-, -Ala-Val-, -Asn-Cit-, -Asp-Cit-, -Asn-Lys-, -Asp-Val -, -Cit-Ala-, -Cit-Asn-, -Cit-Asp-, -Cit-Cit-, -Cit-Lys-, -Cit-Ser-, -Cit-Val-, -Glu-Val-, -Glu-Gly-, -Ile-Cit-, -Ile-Pro-, - Ile-Val-, -Leu-Cit-, -Lys-Cit-, -Phe-Arg-, -Phe-Cit-, -Phe-Lys-, -Pro-Lys-, -Ser-Cit-, -Trp-Cit-, -Ala-Val-, -Val-Asp-, -Cit- Val-, -Val-Glu-, -Val-Lys-, -Gly-Gly-Gly-, -Gly-Gly-Arg-, -Phe-Lys-Gly-, -Leu-Lys-Gly-, -Leu-Leu-Gly-, -Glu-Val-Cit-, -Cit -Ala-Glu-, -Val-Lys-Gly-, -Val-Lys-Ala-, -Val-Gly-Gly-, -Val-Cit-Gly-, -Val-Gln-Gly-, -Val-Glu-Gly-, -Val-Lys-Gly-, -Val-L -ys-Leu-, -Ala-Ala-Ala-, -Asn-Ala-Ala-, -Gly-Gly-Gly-Gly-, -Gly-Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, -Gly-Val-Lys-Gly-, -A1a-Leu-A1a-Leu-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Phe-Gly-Gly-, and -Val-Lys-Gly-Gly, wherein Gln and / or Glu are optionally substituted with 1 polyol group and optionally with 1 C 1-6 Alkyl substitution.

22. The antibody-drug conjugate according to claim 21, its stereoisomer or pharmaceutically acceptable salt or solvate, in, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-,,-Gly-Gly-Phe-Gly-, -Val-Ala- or Where R 6 is H or C1-C6 alkyl, wherein these E groups are covalently linked to Z via the left N terminus.

23. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 22, Among them, -ZE-NH-CH2- has the following structure Its left end is connected to the Ab part.

24. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 23, wherein the antibody-drug conjugate has the following structure: wherein Ab is as defined in any one of claims 1-23, n is DAR and n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

25. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 24, wherein the antibody-drug conjugate has an average DAR of 2-10, 4-9, or 7.5-8.

5.

26. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 25, its stereoisomer or pharmaceutically acceptable salt or solvate, and a pharmaceutically acceptable carrier or excipient.

27. A method for treating or preventing a disease or condition associated with HER3 activity, the method comprising administering to a subject an effective amount of an antibody-drug conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 25, or a pharmaceutical composition according to claim 26.

28. The method of claim 27, wherein the disease or condition associated with HER3 activity is a HER3-positive tumor.

29. The method of claim 28, wherein the HER3-positive tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer, and bladder cancer.

30. The method according to any one of claims 27-29, wherein the method further comprises co-administering one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs and immunomodulators.

31. The method of claim 30, wherein the antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof and one or more therapeutic agents are administered simultaneously or sequentially in any order.

32. The method of claims 27-31, wherein the method further comprises administering to the patient one or more treatment modalities selected from radiation therapy or surgery.

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