Anti-her3 dual payload antibody-drug conjugate and preparation method therefor and use thereof

Abstract

The present invention relates to an anti-HER3 dual payload antibody-drug conjugate (HER3-dpADC) having a structure of formula (I), and a preparation method therefor and the use thereof, wherein the dual loads comprise a topoisomerase inhibitor and a tyrosine kinase inhibitor, respectively.

Description

Anti-HER3 dual-loaded antibody conjugate and its preparation method and use Technical Field

[0001] The present invention relates to an anti-HER3 dual-loaded antibody-drug conjugate (HER3-dpADC) and a preparation method and use thereof. Background Art

[0002] Lung cancer is one of the most common malignancies in the world, with over 2 million cases and nearly 1.8 million deaths worldwide each year. Approximately 85% of lung cancer patients are diagnosed with non-small cell lung cancer (NSCLC). Over the past decade, significant progress has been made in the treatment of NSCLC. The discovery of driver mutations in lung cancer has made personalized targeted therapy possible. Detecting oncogenic driver genes in lung cancer patients and receiving appropriate targeted therapy is crucial (Kris MG, et al. JAMA. 2014 311(19):1998-2006.).

[0003] The epidermal growth factor (EGFR) gene encodes a receptor tyrosine kinase. Activated EGFR can phosphorylate various substrates, thereby activating multiple downstream signaling pathways related to DNA synthesis, cell survival, and cell proliferation, such as the PI3K-AKT-mTOR signaling pathway and the RAS-RAF-MEK-ERK signaling pathway. Studies have shown that targeted therapies targeting specific EGFR mutations can inhibit tumor progression and provide good clinical prognosis, leading to the development of EGFR tyrosine kinase inhibitors (TKIs). In 2019, osimertinib was approved in the United States for the first-line treatment of patients with metastatic non-small cell lung cancer harboring EGFR mutations (exon 19 deletion or exon 21 L858R mutation). Patients who took osimertinib for 18.9 months developed resistance again. Resistance mechanisms include the EGFR C797S mutation and activation of other bypass signaling pathways, such as HER2 amplification, MET amplification, PI3K mutation, and BRAF mutation. In addition, patients may also transform from non-small cell lung cancer to small cell lung cancer and develop drug resistance (Wang M, et al. Nat Med. 2021 27(8): 1345-1356.).

[0004] Antibody-drug conjugates (ADCs) combine the tumor-targeting specificity of monoclonal antibodies with the potent cell-killing activity of cytotoxic drugs (payloads). By coupling the two together through a chemical linker, they can achieve precise targeting while also possessing strong anti-tumor activity. To overcome the drug resistance of a single toxic small molecule and effectively improve the therapeutic window of ADC drugs, single-payload ADCs can be upgraded to dual-payload ADCs (dpADCs). The design concept of dual-payload ADCs is to produce a synergistic effect or overcome the drug resistance of a single payload and improve the therapeutic window by coupling two small molecules with different molecular mechanisms (Nilchan N, Li X, et al. Antib Ther. 2019 2(4):71-78.; Yamazaki CM, et al. Nat Commun. 2021 12(1):3528.).

[0005] Patients with non-small cell lung cancer who have been treated with EGFR TKIs are often accompanied by increased expression levels of human epidermal growth factor receptor 3 (HER3) (Yonesaka K, Tanizaki J, et al. Clin Cancer Res. 2022 28(2): 390-403.). Developed by Daiichi Sankyo, the ADC drug U3-1402 (HER3-DXd), which targets HER3, showed an overall response rate of approximately 30% in patients with NSCLC who had undergone EGFR TKI treatment (Yu HA, Yang JC, et al. Future Oncol. 202319(19): 1319-1329.). This means that 70% of patients who have been treated with EGFR TKIs will still experience disease progression, and there is still a huge unmet clinical need. Recent studies have shown that short-term pretreatment with osimertinib can promote the upregulation of HER3 expression levels on the cell membrane. Preclinical in vitro and in vivo studies have shown that the combination of osimertinib and HER3-DXd has stronger anti-tumor activity than HER3-DXd or osimertinib alone (Haikala HM, Lopez T, et al. Cancer Res. 2022 82(1):130-141.). Summary of the Invention

[0006] In a first aspect, the present invention provides a dual-loaded antibody-drug conjugate having the following structure:

[0007] in,

[0008] Ab is an anti-HER3 antibody or an antigen-binding fragment thereof;

[0009] P1 and P2 are payloads, each independently selected from a topoisomerase inhibitor and a tyrosine kinase inhibitor, provided that the two are different;

[0010] L1 is a linker that is connected to the Ab in a site-specific manner based on the N-glycosylation site in the Fc region of the antibody;

[0011] L2 is a linker that is connected to Ab in a site-directed coupling manner based on ligase catalysis;

[0012] a and b are each independently selected from an integer from 1 to 10;

[0013] q and t are each independently selected from an integer of 1-10.

[0014] In a second aspect, the present invention provides a pharmaceutical composition comprising the dual-loaded antibody-drug conjugate according to the first aspect of the present invention and at least one pharmaceutically acceptable carrier.

[0015] In a third aspect, the present invention relates to use of the dual-loaded antibody-drug conjugate of the first aspect of the present invention or the pharmaceutical composition of the second aspect of the present invention in the preparation of a drug for treating a HER3-positive disease; preferably, the disease is sensitive or resistant to tyrosine kinase inhibitors.

[0016] In a fourth aspect, the present invention also relates to a method for treating HER3-positive diseases, which comprises administering a therapeutically effective amount of the dual-loaded antibody-drug conjugate of the first aspect of the present invention or the pharmaceutical composition of the second aspect of the present invention to an individual in need thereof; preferably, the individual / disease is sensitive or resistant to tyrosine kinase inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 shows the HIC-HPLC analysis results of ADC-1.

[0018] FIG2 shows the SEC-HPLC analysis results of ADC-1.

[0019] FIG3 shows the HIC-HPLC analysis results of ADC-2.

[0020] FIG4 shows the SEC-HPLC analysis results of ADC-2.

[0021] Figure 5 shows the RP-HPLC analysis results of dpADC-1; wherein, Ab1-LC represents the light chain fragment of antibody Ab1, Ab1-HC represents the heavy chain fragment of antibody Ab1, Ab1-LC-LP1 represents the fragment of antibody Ab1 light chain bound to LP1, and Ab1-HC-LP2 represents the fragment of antibody Ab1 heavy chain bound to LP2.

[0022] FIG6 shows the SEC-HPLC analysis results of dpADC-1.

[0023] Figure 7 shows the bystander killing effects of ADC-1, ADC-2, and dpADC-1. Figure 7(a) shows the bystander killing effects of ADC-1, ADC-2, and dpADC-1 on HER3-positive HCC827-hERBB3 cells and HER3-negative PC-9 cells, respectively; Figure 7(b) shows the killing effects of ADC-1, ADC-2, and dpADC-1 on HCC827-hERBB3 cells; Figure 7(c) shows the killing effects of ADC-1, ADC-2, and dpADC-1 on PC-9 cells.

[0024] Figure 8 shows the in vivo efficacy results of osimertinib, ADC-2, and dpADC-1 in an osimertinib-resistant NSCLC PDX model expressing HER3. Figure 8(a) shows the effects of osimertinib, ADC-2, and dpADC-1 on tumor volume in an osimertinib-resistant NSCLC PDX model expressing HER3; Figure 8(b) shows the effects of osimertinib, ADC-2, and dpADC-1 on mouse body weight in an osimertinib-resistant NSCLC PDX model expressing HER3.

[0025] Figure 9 shows the in vivo efficacy results of osimertinib, ADC-2, and dpADC-1 in the osimertinib-sensitive NSCLC PC-9CDX model with low HER3 expression. Figure 9(a) shows the effects of osimertinib, ADC-2, and dpADC-1 on tumor volume in the osimertinib-sensitive NSCLC PC-9CDX model with low HER3 expression; Figure 9(b) shows the effects of osimertinib, ADC-2, and dpADC-1 on mouse body weight in the osimertinib-sensitive NSCLC PC-9CDX model with low HER3 expression. DETAILED DESCRIPTION

[0026] General Definitions and Terminology

[0027] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms related to chemical synthesis, molecular biology, and laboratory procedures used herein are those widely used in the relevant fields and are common procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0028] As used herein, "at least one" or "one or more" may mean 1, 2, 3, 4, 5, 6, 7, 8 or more.

[0029] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0030] As used herein, the term "and / or" connecting multiple elements should be understood to include both individual and combined options. In other words, "and / or" includes "and" and "or." For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. More elements qualified with "and / or" are understood in a similar manner and include any one thereof and any combination thereof.

[0031] Unless otherwise stated, any numerical value or numerical range, such as concentration or concentration range, is in any case understood to be modified by the term "about". Therefore, numerical value generally includes ± 10% of the value. As used herein, the use of numerical ranges explicitly includes all possible subranges, all single numerical values ​​within the range, including integers and fractions within the range, unless the context clearly indicates otherwise. Unless otherwise stated, the numerical ranges listed herein are intended to include all integers and fractions (decimals) within the endpoints and ranges of the range. For example, the expression "a is an integer of 1-20" means that a is any integer of 1-20, and for example a can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Other similar expressions such as i, i1, i2, j, j1, j2, m and n etc. should also be understood in a similar manner.

[0032] The term "optionally" means that the subsequently described event may or may not occur, and that the description includes instances where said event or circumstance occurs or does not occur.

[0033] The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. An alkyl group can have 1 to 60 carbon atoms, for example, a group having 1 to 20 carbon atoms is referred to as "C1-C 20 Alkyl (C 1-20Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or isomers thereof.

[0034] A divalent free radical is a radical obtained by removing a hydrogen atom from a carbon atom with free valence electrons from a corresponding monovalent free radical. A divalent free radical has two attachment sites to the rest of the molecule. For example, "alkylene" or "alkylene group" refers to a saturated straight or branched divalent hydrocarbon radical. Examples of "alkylene" include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H8-), and thiazolinyl (-CH2-). 10 -), hexamethylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene or ethylpropylene, etc.

[0035] The term "cycloalkyl" refers to a cyclic saturated aliphatic group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. A cycloalkyl group can have 3 to 10 carbon atoms, i.e., "C3-C 10 "Cycloalkyl" refers to a divalent cycloalkyl radical, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. "Cycloalkylene" refers to a divalent cycloalkyl radical.

[0036] The term "heterocyclyl" refers to a cycloalkyl group in which one or more carbon atoms are replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, such as azepine, oxa-, or thiirane, azepine, oxa-, or thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, or tetrahydrothiopyranyl. "Heterocyclylene" refers to a divalent heterocycloalkyl group.

[0037] As used herein, when a group is combined with another group, the connection between the groups can be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination can be connected to other parts of the molecule through any suitable atom in the structure, preferably through a specified chemical bond. For example, when two or more groups selected from -CR 1’ R 2’ -、C 1-10 Alkylene, C 4-10 Cycloalkylene, 4-10 membered heterocyclylene and divalent groups of -(CO)- are combined together to form a combination, and two or more divalent groups can form a linear connection with each other, such as -CR 1’ R 2’ -C 1-10 Alkylene-(CO)-, -CR 1’ R 2’ -C 4-10 Cycloalkylene-(CO)-, -CR 1’ R 2’ -C 4-10 Cycloalkylene-C 1-10 Alkylene-(CO)-, etc. The resulting divalent structure can be further linked to other parts of the molecule.

[0038] When multiple identical letters representing chemical groups appear in the same chemical formula, they are independently selected and not necessarily the same. For example, multiple M in this application are independently selected from LKa-L 2 ―L 1 ―B―P; and multiple L 2 They are also independent of each other and not necessarily the same.

[0039] As used herein, the term "amino acid" includes "natural amino acids" and "unnatural amino acids."

[0040] The term "natural amino acids" refers to amino acids, which are protein-building amino acids, including the common twenty amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as the less common selenocysteine ​​and pyrrolysine.

[0041] As used herein, the term "unnatural amino acid" refers to an amino acid that is not a protein-forming amino acid. Specifically, the term refers to an amino acid that is not a natural amino acid as defined above.

[0042] HER3 refers to human epidermal growth factor receptor-3, which belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family.

[0043] As used herein, "antibody" refers to an immunoglobulin or its fragment, which specifically binds to an antigenic epitope through at least one antigen binding site. Antibody encompasses antibody fragments. As used herein, the term "antibody" includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, nanobodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0044] An intact antibody or full-length antibody essentially comprises an antigen-binding variable region and a light chain constant region (C L ) and heavy chain constant region (C H ), which may include C H 1, C H 2. C H 3 and C H 4, depending on the subtype of the antibody. The antigen-binding variable region (also called fragment variable region, Fv fragment) usually contains the light chain variable region (V L ) and heavy chain variable region (V H The constant region can be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. The variable region recognizes and interacts with the target antigen. The constant region can be recognized and interacted with by the immune system.

[0045] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites) and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments may comprise a portion of an intact antibody, preferably its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, V H and C H 1 domain, Fd fragment, Fv fragment, single domain antibody (dAb) fragment and isolated complementary determining region (CDR). Fab fragment is an antibody fragment obtained by papain digestion of full-length immunoglobulin, or a fragment with the same structure produced by, for example, recombinant expression. Fab fragment contains light chain (including V L and C L ) and another chain, wherein the other chain comprises the variable region of the heavy chain (V H ) and a constant region of the heavy chain (C H1). F(ab')2 fragments are antibody fragments obtained by digesting immunoglobulins with pepsin at pH 4.0-4.5, or fragments with the same structure produced by, for example, recombinant expression. F(ab')2 fragments essentially comprise two Fab fragments, wherein each heavy chain portion comprises several additional amino acids, including cysteine ​​that forms a disulfide bond connecting the two fragments. Fab' fragments are fragments comprising half of a F(ab')2 fragment (a heavy chain and a light chain). The antibody fragment may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, bispecific antibodies, Fd and Fd' fragments, and other fragments, including modified fragments. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (for example, by replacing the corresponding region), obtains an antibody that immunospecifically binds to an antigen.

[0046] Immunoglobulin G (IgG) molecules are composed of two light chains and two heavy chains connected by disulfide bonds and non-covalent bonds. Studies have shown that IgG is a glycoprotein. H There is a conserved glycosylation site at the asparagine 297 (Asn297 / N297) position of 2, and the site-specific connection of different molecules to the antibody can be achieved through sugar chain remodeling at this site.

[0047] In particular, the dual-carrier antibody-drug conjugates of the present application can be site-specifically conjugated to any naturally N-glycosylated site in the antibody Fc region. Molecules containing sugar chains in the antibody Fc region (including but not limited to antibodies, bispecific antibodies, Fc fusion proteins, single-chain antibodies, nanobodies, etc.) can be linked to the cargo using the methods of the present invention. Therefore, the antibodies of the present application are not particularly limited, other than being anti-HER3 antibodies; they can be naturally occurring antibodies as long as their Fc regions contain sugar chains.

[0048] In addition, the antibodies of the present invention can also be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant technology / genetic engineering technology, phage display technology, synthetic technology, or other techniques known in the art. For example, genetically engineered recombinant antibodies can be expressed in a suitable culture system (e.g., E. coli or mammalian cells). The genetic engineering can refer to, for example, the introduction of a ligase-specific recognition sequence at its terminus.

[0049] Small molecule compounds refer to molecules of a size comparable to organic molecules commonly used in pharmaceuticals. The term does not encompass biological macromolecules (e.g., proteins, nucleic acids, etc.), but does encompass low molecular weight peptides or derivatives thereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Typically, small molecule compounds have a molecular weight of, for example, about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, or about 200 to about 500 Da.

[0050] Cytotoxins are substances that inhibit or prevent the expression activity, cellular function, and / or cause cell destruction. Cytotoxins commonly used in ADCs are more toxic than chemotherapy drugs. In some embodiments, the payload of the present invention is a topoisomerase inhibitor.

[0051] In the present invention, the amino acid sequences of CDRs are shown according to the Kabat definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on antibody sequence variability (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), AbM (Martin, ACR and J. Allen (2007) "Bioinformatics tools for antibody engineering," in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp.95–118), Contact (MacCallum, RM et al., (1996) J. Mol. Biol. 262:732-745), IMGT (Lefranc, M.-P., 2011(6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. In this article, multiple CDR numbering systems may be used for the same variable region, such as Chothia, Abm, Kabat, Contact, and IMGT. It should be understood by those skilled in the art that although the CDRs defined by different numbering systems may be different, the CDRs corresponding to the same numbering system represent effective antigen binding sites that can bind to antigen epitopes.Unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined by any of the above-mentioned known schemes described herein. Although the scope of protection claimed in the claims of the present invention is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0052] Thus, when referring to antibodies defined by specific CDR sequences defined herein, the scope of said antibodies also encompasses antibodies whose variable region sequences comprise said specific CDR sequences, but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0053] As used herein, the terms "framework region" and "framework region" are used interchangeably. As used herein, the terms "framework region," "framework region," or "FR" residues refer to those amino acid residues in the antibody variable region excluding the CDR sequences as defined above.

[0054] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a sulfhydryl group that can form a disulfide bond or bridge a second sulfhydryl group.

[0055] As used herein, "percent (%) sequence identity" or "sequence identity" of amino acid sequences has an art-recognized definition and refers to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). This can be determined using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0056] The non-essential regions in a polypeptide can be modified, for example, by substitution, addition and / or deletion of one or more amino acids without altering the function of the polypeptide. Suitable conservative amino acid substitutions are known to those skilled in the art in peptides or proteins, and generally can be made without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in the non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0057] "Spacer" refers to a structure located between different structural modules that can spatially separate the structural modules. The definition of a spacer does not limit whether it has a certain function or whether it can be cut or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, wherein the non-amino acid structure can be, but is not limited to, an amino acid derivative or analog. "Spacer sequence" refers to an amino acid sequence that serves as a spacer, examples of which include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids, such as GA, or for example, GGGGS (SEQ ID No: 15), GGGGSGGGGS (SEQ ID No: 16), GGGGSGGGGSGGGGS (SEQ ID No: 17), etc. A self-immolative spacer (e.g., self-immolative spacer Sp1) is a covalent component that causes two chemical bonds to be cleaved sequentially after activation of the protective portion in the precursor: the protective portion (e.g., the cleavable sequence) is removed after activation, triggering a cascade of decomposition reactions, resulting in the release of smaller molecules in a sequential order. Examples of self-immolative spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0058] If a binding molecule, e.g., an antibody, or fragment, variant, or derivative thereof, preferentially binds to an epitope to the extent that it blocks binding of the reference antibody or antigen-binding fragment to the epitope, then the binding molecule, e.g., an antibody, or fragment, variant, or derivative thereof, is said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope. Competitive inhibition can be determined by any method known in the art, e.g., a competition ELISA assay. The binding molecule can be said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0059] Terms such as "treating" or "treating" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, alleviate, reduce the symptoms of an existing, diagnosed pathological condition or disorder, and / or arrest or slow the progression of an existing, diagnosed pathological condition or disorder. Terms such as "preventing," "preventing," "avoiding," "containment," and the like refer to preventative or prophylactic measures that prevent the progression of an undiagnosed target pathological condition or disorder. Thus, a "subject in need thereof" can include a subject already suffering from a disease; a subject susceptible to a disease; and a subject in need of prevention of a disease.

[0060] As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

[0061] As used herein, the term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule or other drug that is effective for "treating" a disease or condition in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; block or stop cancer cell division, reduce or block the increase in tumor size; inhibit, for example, suppress, block, prevent, stop, delay or reverse cancer cell infiltration into peripheral organs, including, for example, the spread of cancer to soft tissue and bone; inhibit, for example, suppress, block, prevent, shrink, stop, delay or reverse tumor metastasis; inhibit, for example, suppress, block, prevent, stop, delay or reverse tumor growth; alleviate one or more symptoms associated with cancer to some extent, reduce morbidity and mortality; improve quality of life; or a combination of these effects. To the extent that a drug prevents growth and / or kills existing cancer cells, it can refer to cytostatic and / or cytotoxic.

[0062] As used herein, the terms "subject," "patient," or "individual" generally include humans and non-human animals, and preferably include mammals (e.g., non-human primates, including marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, and baboons, macaques, chimpanzees, orangutans, gorillas; cows; horses; sheep; pigs; chickens; cats; dogs; mice; rats; rabbits; guinea pigs, etc.), including chimeric and transgenic animals and disease models. The term "subject" preferably refers to a non-human primate or a human, most preferably a human.

[0063] The antibody numbers used herein (such as Ab1 and Ab2, etc.) are only used to distinguish or identify antibodies or products, and are not intended to indicate that such identification is a feature of the antibodies or products of the present invention. It will be understood by those skilled in the art that, for example, other antibodies or products may also use such identification for the purpose of distinction or identification, but do not refer to the same or equivalent antibodies or products. Similarly, the similar numbering or identification used in the embodiments is only for illustrative convenience, and the antibodies or products of the present invention are defined by the features described in the appended claims.

[0064] Double-loaded antibody-drug conjugate of the present invention

[0065] The present invention provides a dual-load antibody-drug conjugate having the following structure:

[0066] in,

[0067] Ab is an anti-HER3 antibody or an antigen-binding fragment thereof;

[0068] P1 and P2 are payloads, each independently selected from a topoisomerase inhibitor and a tyrosine kinase inhibitor, provided that the two are different;

[0069] L1 is a linker that is connected to the Ab in a site-specific manner based on the N-glycosylation site in the Fc region of the antibody;

[0070] L2 is a linker that is connected to Ab in a site-directed coupling manner based on ligase catalysis;

[0071] a and b are each independently selected from an integer from 1 to 10;

[0072] q and t are each independently selected from an integer of 1-10.

[0073] In some embodiments, (P1) a -L1- has the following structure

[0074] in,

[0075] * represents the end connected to the antibody; preferably, the -NHC(O)CH2- at the * end is part of the amino acid sequence of the antibody Fc region;

[0076] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 Each independently selected from H, C 1-6 Alkoxy or OH;

[0077] R 7 is hydrogen or α-L-fucosyl;

[0078] L 1-a is a connector, and L 1-a It is connected to the terminal sugar chain of the antibody via the terminal -NH-.

[0079] In some embodiments, the -NHC(O)CH2- at the *terminal end is part of an asparagine at position 297 in the Fc region of an antibody. In a specific embodiment, the -NHC(O)CH2- at the *terminal end is part of an asparagine at position 297 in the Fc region of an antibody.

[0080] In some embodiments, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 Each is independently selected from H, methoxy or OH.

[0081] In some embodiments, R 1 、R 2 、R 3 、R 4 、R 5 、R6 Each is independently selected from H or OH, and satisfies the following conditions:

[0082] R 1 and R 2 Different, R 3 and R 4 Different and R 5 and R 6 In some embodiments, R 7 It is α-L-fucosyl.

[0083] In some embodiments, (P1) a -L1- is selected from the following structures:

[0084] in,

[0085] * represents one end connected to the antibody; preferably, the -NHC(O)CH2- at the * end is derived from an amino acid in the Fc region of the antibody; more preferably, the -NHC(O)CH2- at the * end is derived from an asparagine in the Fc region of the antibody;

[0086] R 7 It is hydrogen or α-L-fucosyl.

[0087] In some embodiments, the -NHC(O)CH2- at the * end is derived from asparagine at position 297 of the antibody Fc region. 7 It is α-L-fucosyl.

[0088] In some embodiments, -L 1-a -(P1) a L in 1-a As a connector, there is no special requirement for its structure, as long as it can play a connecting role.

[0089] In some embodiments, -L 1-a -(P1) a L in 1-a It is a linker that forms an amide bond with the sugar chain carboxyl group through the terminal N and contains an enzyme-cleavable group, for example, selected from Gly-Gly-Phe-Gly, Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu and Ala-Ala-Ala and combinations thereof; the preferred cleavable group is Gly-Gly-Phe-Gly.

[0090] In some embodiments, -L 1-a -(P1) a Has the following structure:

[0091] in,

[0092] Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i1 -(CO)-; or each independently unsubstituted or -CO-(PEG) on the side chain j1 -R 11 Substituted natural amino acids or oligomeric natural amino acids having a degree of polymerization of 2-10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0093] -(PEG) i1 -and-(PEG) j1 - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 alkylene;

[0094] M is hydrogen or LKa-L 2 ―L 1 ―B―P1;

[0095] Q is NH2 or L 2 ―L 1 ―B―P1;

[0096] Provided that the following is not included: M is hydrogen and Q is NH2;

[0097] Each LKa is absent or independently selected (For example, ); wherein h is an integer selected from 1-3, such as 1, 2 or 3;

[0098] opSu is or a mixture thereof; wherein * represents and L 2 The connecting part;

[0099] B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1’ R 2’ -、C 1-10 Alkylene, C 4-10 Cycloalkylene, 4-10 membered heterocyclylene and -(CO)-;

[0100] L 1independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;

[0101] L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination:

[0102] 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3’ R 4’ -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5’ -、 C 4-10 Cycloalkylene, 4-10 membered heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent;

[0103] 2) Amino acid residue sequence, i.e. -*(AA) n **—, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid. m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3;

[0104] Preferably, -L 2 -L 1 -B- represents -NH-(PEG)2-C2H4-(CO)-Gly-Gly-Phe-Gly-;

[0105] R 1’ 、R 2’ 、R 3’ 、R 4’ 、R 5’ 、R 6’ 、R 7’ 、R 8 、R 9are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1’ and R 2’ and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3’ and R 4’ and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group;

[0106] R 11 It is C 1-10 an alkyl group, preferably a methyl group;

[0107] d is 0, 1, 2, 3, 4, 5, or 6;

[0108] Each i1 is independently an integer selected from 1-20, more preferably 4;

[0109] Each j1 is independently an integer selected from 1-20, more preferably 12.

[0110] In some embodiments, B, L 1 and L 2 At least one of them is not "not present".

[0111] In some embodiments, L 1 A cleavable sequence comprising an amino acid sequence cleavable by an enzyme, wherein the amino acid sequence cleavable by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In some embodiments, the amino acid sequence cleavable by the enzyme is selected from -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof; a preferred amino acid sequence cleavable by the enzyme is -Gly-Gly-Phe-Gly-. In some embodiments, L 1 is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably, -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof. In some embodiments, L 1 means -Gly-Gly-Phe-Gly-.

[0112] In some embodiments, L 2is the amino acid residue sequence, i.e. -*(AA) n **—, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid. m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the oligosaccharide structure. In a preferred embodiment, L 2 It is Gly, and there is -(C2H4-O) between its amino group and α-carbon m -(CH2) p -, where m is 2 and p is 1.

[0113] In some embodiments, AA is independently any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His, or any combination thereof at each occurrence. In some embodiments, n is an integer from 1 to 50, preferably an integer from 1 to 30, preferably an integer from 1 to 20, preferably an integer from 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In a preferred embodiment, L 2 is Gly, and n is 3.

[0114] In some embodiments, Sp1 is selected from PABC (p-aminobenzyloxycarbonyl), acetal, heteroacetal and combinations thereof; preferably, Sp1 is acetal, heteroacetal or PABC; further preferably, the heteroacetal is selected from N,O-heteroacetal; more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is linked to an amino acid sequence that can be cleaved by the enzyme, U does not exist, or is CH2, O, S or NH, preferably O or S.

[0115] In some embodiments, B is absent, or is -NH-CH2-U-, or is -NH-CH2-U-(CH2) g -(CO)-, wherein g is 1, 2, 3, 4, 5 or 6, U is absent or is CH2, O, S or NH, preferably O or S. In some embodiments, B is absent. In some embodiments, B is 1), 2), or a combination of 1) and 2): 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1’ R 2’ -、C1-10 In some embodiments, B is -NH-CH2-U- or -NH-CH2-U-(CH2) g -(CO)-, U is absent or is CH2, O, S or NH, preferably O or S. In some embodiments, B is connected to the payload via an amide bond, an ester bond, or an ether bond. In some embodiments, B is selected from (-PABC-), -NH-CH2-U- or -NH-CH2-U-(CH2) g -(CO)-; wherein g is 1, 2, 3, 4, 5 or 6; U is absent or is CH2, O, S or NH, preferably O or S. In some preferred embodiments, B is -NH-CH2-O-.

[0116] In some embodiments, Ld2 and each Ld1 are independently selected from a bond, or

[0117] Each of i1, j1 and k1 is independently selected from an integer of 1-20.

[0118] In some embodiments, each i1, j1, and k1 is independently selected from an integer from 1 to 20. In some embodiments, each i1, j1, and k1 is independently selected from an integer from 1 to 12.

[0119] In some embodiments, each i1 is independently selected from an integer from 2 to 8; particularly 4.

[0120] In some embodiments, each j1 is independently selected from an integer between 8 and 12; particularly 8 or 12.

[0121] In some embodiments, each k1 is independently selected from an integer of 1-7; particularly 1 or 3 or 5.

[0122] In some embodiments, Ld2 and each Ld1 are independently selected from a bond; or a C with an amino group and a carbonyl group at both ends. 1-20 Alkylene, or a PEG fragment of a certain length with an amino group and a carbonyl group at both ends (represented as -(PEG) i -), or one or more natural amino acids, each of which is independently unsubstituted or substituted with a PEG fragment of a certain length (represented as -CO-(PEG) j -)replace.

[0123] In some embodiments, -(PEG) i1 -Contains -(O-C2H4) i1 -or-(C2H4-O) i1 -, and optionally appended with C at one end 1-10 Alkylene; -(PEG)j -Contains -(O-C2H4) j1 -or-(C2H4-O) j1 -, and optionally appended with C at one end 1-10 In a specific embodiment, -(PEG) i - contains -C2H4-(O-C2H4) i1 -or-(C2H4-O) i1 -C2H4-.

[0124] In a specific embodiment, Ld1 is In a specific embodiment, Ld2 is

[0125] In some embodiments, LKa is wherein h is an integer selected from 1-3, for example, 1, 2 or 3.

[0126] In a specific embodiment, LKa is Where * represents and L 2 The connecting part.

[0127] In a specific embodiment, Q is NH2. In a specific embodiment, d is 1.

[0128] In some embodiments, -L 1-a -(P1) a Has the following structure:

[0129] in,

[0130] P1 is the load;

[0131] Each i1 is independently selected from an integer of 1-20; preferably, i1 is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; more preferably, i1 is 4;

[0132] Each i 1’ are independently selected from integers of 1-20; preferably, i 1’ is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; more preferably, i 1’ is 2;

[0133] j1 is an integer selected from 1-20; preferably, j1 is an integer selected from 2-16, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; more preferably, j1 is 12;

[0134] m1 is an integer selected from 1-8, for example 1, 2, 3, 4, 5, 6, 7 or 8; preferably, m1 is an integer selected from 1-5; more preferably, m1 is 3;

[0135] h is an integer selected from 1-3, for example 1, 2 or 3; preferably, h is 1 or 2.

[0136] In some preferred embodiments, -L 1-a -(P1) a Has the following structure:

[0137] Among them, P1, i1, i 1’ , j1, m1 are as defined herein.

[0138] In some embodiments, P1 is selected from

[0139] Preferred

[0140] In a specific embodiment, -L 1-a -(P1) a With the following structure

[0141] In some embodiments, -L2-(P2) b for

[0142] in,

[0143] M is LKa-L 2 ―L 1 ―B―P2;

[0144] b is an integer selected from 1-20;

[0145] The L 2 and Ld2 are as defined in the present invention.

[0146] In some embodiments, L 1 A cleavable sequence comprising an amino acid sequence cleavable by an enzyme, wherein the amino acid sequence cleavable by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. 1 It is -Val-Ala-.

[0147] In some embodiments, L 2 is the amino acid residue sequence, i.e. -*(AA) n**-, n is an integer from 1 to 100, and AA is independently any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His, or any combination thereof. In some embodiments, n is an integer from 1 to 50, preferably an integer from 1 to 30, preferably an integer from 1 to 20, preferably an integer from 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In a preferred embodiment, L 2 is Gly, and n is 3.

[0148] In some embodiments, L 2 Selected from -NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3’ R 4’ -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5’ -、 C 4-10 Cycloalkylene, 4-10 membered heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 In a specific embodiment, L 2 for wherein i2 is an integer selected from 1-20; preferably, i2 is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; more preferably, i2 is 4.

[0149] In some embodiments, in a specific embodiment, Ld2 is wherein j2 is selected from an integer of 1-20; preferably, j2 is an integer selected from 2-16, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; more preferably, j2 is 12.

[0150] In a specific embodiment, Lka is absent.

[0151] In one embodiment, B is a self-immolative spacer Sp1, wherein Sp1 is selected from PABC (p-aminobenzyloxycarbonyl), acetal, heteroacetal, and combinations thereof; preferably, Sp1 is acetal, heteroacetal, or PABC; further preferably, the heteroacetal is selected from N,O-heteroacetal; more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is linked to an enzymatically cleavable amino acid sequence, and U is absent or is CH2, O, S, or NH, preferably O or S. In a specific embodiment, B is -PABC-.

[0152] In some embodiments, -L 2 -L 1 -B- represents -NH-(PEG)4-(C2H4)2-(CO)-Val-Ala-PABC-, and the structure of -PABC- is

[0153] In some embodiments, -L2-(P2) b for

[0154] in,

[0155] P2 is the load;

[0156] i2 is an integer selected from 1-20; preferably, i2 is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; more preferably, i2 is 4;

[0157] j2 is an integer selected from 1-20; preferably, j2 is an integer selected from 2-16, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; more preferably, j2 is 12;

[0158] m2 is an integer selected from 1-8, for example 1, 2, 3, 4, 5, 6, 7 or 8; preferably, m2 is an integer selected from 1-5; more preferably, m2 is 3.

[0159] In some embodiments, P2 is selected from lapatinib, neratinib, pyrotinib, afatinib, gefitinib, erlotinib and osimertinib, or derivatives thereof; preferably osimertinib or a derivative thereof.

[0160] In some embodiments, -L2-(P2) b for

[0161] In some embodiments, (P1) a-L1- is linked to the anti-HER3 antibody or antigen-binding fragment thereof by an enzyme or a mutant thereof, wherein the enzyme or mutant thereof is N-acetylglucosamine endohydrolase, or a mutant thereof. In a preferred embodiment, the N-acetylglucosamine endohydrolase is selected from Endo H, Endo D, Endo F2, Endo M, Endo Om, Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC), Endo CC1, Endo CC2, or mutants thereof. In a more preferred embodiment, the N-acetylglucosamine endohydrolase is selected from Endo F3, Endo S and Endo S2, or mutants thereof.

[0162] In some embodiments, (P2) b -G in L2- m2 The P2 portion is a recognition sequence for the ligase acceptor or donor substrate, which promotes the ligase-catalyzed b -L2- Enzyme-catalyzed coupling with a targeting molecule. The targeting molecule is optionally modified and comprises a corresponding recognition sequence for the ligase acceptor or donor substrate. In some embodiments, (P2) b -L2- is linked to the anti-HER3 antibody or antigen-binding fragment thereof by an enzyme or a mutant thereof, wherein the enzyme or mutant thereof is a transpeptidase or a mutant thereof. In a preferred embodiment, the enzyme or mutant thereof is Sortase A, Sortase B, Sortase C, Sortase D, and Sortase L. plantarum, or a mutant thereof. In a more preferred embodiment, the enzyme or mutant thereof is Sortase A. The type of ligase corresponds to the ligase recognition sequence, thereby achieving specific conjugation between different molecules or structural fragments.

[0163] In some embodiments, the Ab comprises an antibody with a C-terminal modification: the modification comprises the antibody, a spacer (SP), and a ligase donor substrate recognition sequence linked in sequence, or the antibody and a ligase donor substrate recognition sequence linked in sequence.

[0164] In some embodiments, the spacer is selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS; preferably, the spacer is GA.

[0165] In some embodiments, the ligase donor substrate recognition sequence is LPX1TGX2 (SEQ ID NO: 18), wherein X1 is any natural or unnatural amino acid, and X2 is absent or an amino acid fragment containing 1-10 amino acids. m2 During conjugation, the peptide bond upstream of the glycine in the LPX1TGX2 sequence is cleaved by the ligase, and the resulting intermediate is ligated to G m2 A new peptide bond is generated at the free N-terminus, and the resulting amino acid sequence is LPXTG m2 .

[0166] In some embodiments, the ligase is Sortase A, which catalyzes a reaction represented by the following scheme:

[0167] The triangle represents a portion of the antibody; and the pentagon represents (P2) b -L2- part. m2, X1 and X2 are as defined above. m2 During conjugation, the peptide bond upstream of glycine in the LPX1TGX2 sequence is cleaved by Sortase A, and the resulting intermediate is linked to G m2 A new peptide bond is generated at the free N-terminus, and the resulting amino acid sequence is LPXTG m2 (SEQ ID NO:19). Sequence G m2 and LPX1TGX2 are as defined above.

[0168] In particular, the ligase donor substrate recognition sequence is LPETG (SEQ ID NO: 20) or LPETGG (SEQ ID NO: 21).

[0169] In a specific embodiment, the anti-HER3 antibody (Ab) has a terminal modification of GALPETG at the light chain terminus for achieving Sortase A-catalyzed site-directed conjugation.

[0170] In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and light chain variable region (V L ),in,

[0171] The heavy chain variable region comprises:

[0172] i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1;

[0173] ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 2;

[0174] iii) a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3;

[0175] and / or

[0176] The light chain variable region comprises:

[0177] i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 4;

[0178] ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 5;

[0179] iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0180] In some embodiments, the heavy chain variable region of the anti-HER3 antibody or antigen-binding fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7.

[0181] In some embodiments, the light chain variable region of the anti-HER3 antibody or antigen-binding fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8.

[0182] In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof comprises a heavy chain constant region (C H ), which comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 9. In a preferred embodiment, the anti-HER3 antibody or antigen-binding fragment thereof comprises a heavy chain constant region (C H ), which comprises the amino acid sequence of SEQ ID NO:9.

[0183] In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof comprises a light chain constant region (C L), which comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 10. In a preferred embodiment, the anti-HER3 antibody or antigen-binding fragment thereof comprises a light chain constant region (C L ), which comprises the amino acid sequence of SEQ ID NO:10.

[0184] In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11 or SEQ ID NO: 13. In a preferred embodiment, the anti-HER3 antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 13. In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 14. In a preferred embodiment, the anti-HER3 antibody or antigen-binding fragment thereof comprises a light chain comprising SEQ ID NO: 12 or SEQ ID NO: 14.

[0185] In some embodiments, the dual-loaded antibody-drug conjugates of the present invention have the following structure:

[0186] in,

[0187] Ab is as defined in the present invention;

[0188] R 7 is hydrogen or α-L-fucosyl; preferably, R 7 It is α-L-fucosyl.

[0189] q and t are each independently selected from an integer of 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0190] Preferably,

[0191] q is 1 or 2; more preferably, q is 2;

[0192] t is 1 or 2; more preferably, t is 2.

[0193] Pharmaceutical composition

[0194] Another object of the present invention is to provide a pharmaceutical composition comprising a preventive or therapeutically effective amount of the dual-loaded antibody-drug conjugate of the present invention, and optionally at least one pharmaceutically acceptable carrier.

[0195] The pharmaceutical composition of the present invention can be administered in any manner as long as it achieves the effect of preventing, alleviating, preventing or treating the symptoms of humans or animals. For example, various suitable dosage forms can be prepared according to the route of administration, particularly injections such as lyophilized powder injections, injections or sterile injection powders.

[0196] The term "pharmaceutically acceptable" means that it does not produce undue toxicity, irritation or allergic reaction when in contact with patient tissues within the scope of normal medical judgment, has a reasonable ratio of advantages to disadvantages, and is effective for the intended use.

[0197] The term "pharmaceutically acceptable carrier" refers to any carrier material that is pharmaceutically acceptable and does not interfere with the biological activity and performance of the dual-loaded antibody-drug conjugates of the present invention. Examples of aqueous carriers include, but are not limited to, buffered saline. Pharmaceutically acceptable carriers also include substances that allow the composition to approach physiological conditions, such as pH adjusters and buffers, toxicity modifiers, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0198] In some embodiments, the drug / antibody ratio (DAR) of the pharmaceutical composition of the present invention is an integer or non-integer of 0-20, such as about 0-about 10, about 0-about 8, about 0-about 6, about 0-about 4, about 0-about 3, about 0-about 2, or about 0-about 1. In some embodiments, the DAR of the dual-loaded antibody-drug conjugate of the present invention is about 1.78, about 1.93, about 3.83, about 3.89, or about 5.76. In a particular embodiment, the DAR of the dual-loaded antibody-drug conjugate of the present invention is about 5.76.

[0199] Treatment methods and uses

[0200] The dual-loaded antibody-drug conjugates of the present invention can be used for the medical treatment and / or diagnosis of HER3-positive diseases. HER3-positive diseases susceptible to treatment with the dual-loaded antibody-drug conjugates of the present invention include tumors characterized by specific tumor-associated antigens or cell surface receptors. These tumor cells can be recognized by the targeting moiety of the dual-loaded antibody-drug conjugates of the present invention and, in turn, killed by the payload / cytotoxin in the dual-loaded antibody-drug conjugates.

[0201] Therefore, in another aspect, the present invention also provides the use of the dual-loaded antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease, disorder or condition, wherein the disease, disorder or condition is selected from HER3-positive disease.

[0202] In another aspect, the present invention provides a dual-loaded antibody-drug conjugate of the present invention or a pharmaceutical composition of the present invention for treating HER3-positive diseases.

[0203] In a further aspect, the present invention provides a method for treating HER3-positive diseases, comprising administering to an individual in need thereof an effective amount of the dual-loaded antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention.

[0204] In some embodiments, the subject is sensitive or resistant to a tyrosine kinase inhibitor.

[0205] In some embodiments, the HER3 -positive disease is further sensitive or resistant to a tyrosine kinase inhibitor.

[0206] In some embodiments, the HER3-positive disease of the present invention includes breast cancer, ovarian cancer, colon cancer, gastric cancer, lung cancer, skin cancer or pancreatic cancer; preferably lung cancer.

[0207] The dosage of the dual-loaded antibody-drug conjugate administered to a subject can be adjusted to a considerable extent. The dosage can vary depending on the specific route of administration and the needs of the subject and can be subject to the judgment of a healthcare professional.

[0208] Dosage and kit

[0209] The dual-loaded antibody conjugates and pharmaceutical compositions according to the present invention will be administered alone or in combination with additional therapeutic agents in an effective amount by any common and acceptable means known in the art. The effective amount may vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors known to those skilled in the art.

[0210] As a general example, a daily dosage of about 0.001 to about 100 mg / kg body weight can be used, or more particularly about 0.03 to 2.5 mg / kg body weight. In larger mammals, such as humans, the daily dosage can be in the range of about 0.5 mg to about 2000 mg.

[0211] The dual-loaded antibody conjugates and pharmaceutical compositions of the present invention are typically administered in the form of a pharmaceutical composition comprising a pharmaceutically active ingredient and various other pharmaceutically acceptable components, for example, see Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred or desired form depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also include a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a carrier commonly used to formulate a pharmaceutical composition for administration to animals or humans. The choice of diluent does not affect the biological activity of the combination. Examples of diluents include, but are not limited to, distilled water, physiological phosphate-buffered saline, Ringer's solution, glucose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0212] The dual-loaded antibody-drug conjugates and pharmaceutical compositions of the present invention can be administered in the form of pharmaceutical compositions by any conventional route; for example, enterally, such as orally, for example, in the form of tablets or capsules; parenterally, for example, in the form of injectable solutions or suspensions; or topically, for example, through the eyes or nasal cavity, for example, in the form of emulsions, gels, ointments, creams or suppositories.

[0213] In some embodiments, the pharmaceutical composition is a solution of the active ingredient, including a suspension or dispersion, such as an isotonic aqueous solution. For a lyophilized composition comprising only the active ingredient or comprising the active ingredient and a carrier (such as mannitol), a dispersion or suspension can be prepared before use.

[0214] The limiting examples of carriers include fillers, such as sugars, such as lactose, sucrose, mannitol or sorbitol, cellulose preparations and / or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate, and binders, such as starches, such as corn, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinyl pyrrolidone, and / or if necessary, disintegrants, such as the above-mentioned starches, carboxymethyl starch, cross-linked polyvinyl pyrrolidones, alginic acid or its salts, such as sodium alginate. Other carriers include, but are not limited to, rheology modifiers and lubricants, such as silicic acid, talc, stearic acid or its salts, such as magnesium or calcium stearate, and / or polyethylene glycol or its derivatives.

[0215] The present invention also provides a pharmaceutical combination, such as a kit, comprising a) a first agent, which is a dual-loaded antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt thereof, and b) at least one additional agent. The kit may further comprise instructions for administration thereof. Beneficial effects

[0216] The present invention provides a novel HER3 dual-loaded ADC drug (HER3-dpADC) containing a DNA topoisomerase I inhibitor and an EGFR TKI, prepared using two enzymatic site-directed coupling technologies. The HER3 dual-loaded ADC has stable properties, good homogeneity, a simple preparation process, and can be linearly scaled up at low cost. In vitro experiments have shown that the HER3-dpADC is more effective than the HER3 single-loaded ADC in killing HER3-negative cells containing EGFR mutations. In vivo experimental results show that the HER3-dpADC has a higher therapeutic window than the HER3 single-loaded ADC in mouse tumor models that are sensitive or resistant to TKIs (such as osimertinib). This indicates that HER3-dpADC is a potential and promising new treatment option that can be used to treat patients who are sensitive or resistant to TKIs (such as osimertinib).

[0217] Example

[0218] The solution of the present invention is further described in detail below with reference to specific embodiments.

[0219] It should be noted that the following embodiments are merely examples for the purpose of clearly illustrating the technical solutions of the present invention and are not intended to limit the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the description of the present invention. It is not necessary and impossible to enumerate all embodiments here, and any obvious variations or modifications derived therefrom remain within the scope of protection of the present invention.

[0220] Unless otherwise specified, the instruments, equipment, and reagents used herein are commercially available and the reagents used were used directly without further purification.

[0221] 1. Preparation of Linker-Payload (LP)

[0222] Example 1: Preparation of Linker Payload 1 (LP1, Compound 10)

[0223] The structure of LP1 is as follows:

[0224] Step A: Synthesis of compound 10c

[0225] Compound 10a (4.1 g, 10 mmol, CAS No. 150114-97-9) and 4-aminobenzyl alcohol (compound 10b, 2.46 g, 20 mmol) were dissolved in a mixed solution of DCM / MeOH (200 mL, v / v), and then EEDQ (4.94 g, 20 mmol, CAS No. 16357-59-8) was added, and the reaction system was stirred at room temperature. The reaction was monitored by high performance liquid chromatography (HPLC) until the reaction was complete. The reaction mixture was filtered, and the filtered solid was washed three times with Et2O to obtain a white solid compound 10c (4 g, yield 78%). MS (ESI) C 30 H 34 O5N3[(M+H)] + Calculated value: 516.25, measured value: 516.38.

[0226] Step B: Synthesis of compound 10d

[0227] Compound 10c (2 g, 3.89 mmol) was dissolved in DMF (10 mL) with stirring. DBU (296 mg, 1.9 mmol, CAS No. 6674-22-2) was added, and the reaction system was stirred at room temperature for 2 h. After completion of the reaction, the reaction mixture was used directly in the next step without further purification.

[0228] Step C: Synthesis of compound 10e

[0229] PPTS (961 mg, 1.9 mmol, CAS No. 24057-28-1) was added to the mixture of the previous reaction, and the reaction was stirred at room temperature for 5 minutes to neutralize the DBU in the previous reaction. Fmoc-PEG4-COOH (1.5 g, 3.1 mmol), EDCI (891 mg, 4.66 mmol, CAS No. 25952-53-8) and HOBt (638 mg, 4.66 mmol, CAS No. 2592-95-2) were then added to the reaction system, and the reaction was stirred at room temperature for 12 hours. The reaction was monitored by HPLC until complete. The reaction system was diluted with DCM, washed with saturated NaCl solution, and the organic phase was collected and dried over anhydrous Na2SO4. The resulting organic phase was concentrated and purified by silica gel chromatography to give a light yellow solid compound 10e (1.8 g, two-step yield 62%). MS (ESI) C 41 H 54 N4NaO 10 [(M+Na)] + Calculated value: 785.37, measured value: 785.46.

[0230] Step D: Synthesis of compound 10g

[0231] Compound 10e (900 mg, 1.2 mmol) and bis(4-nitrophenyl) carbonate (compound 10f, 715 mg, 2.36 mmol, CAS No. 5070-13-3) were dissolved in DCM (15 mL), and DIEA (304 mg, 2.36 mmol) was added. The reaction system was stirred at room temperature under nitrogen for 12 h. The reaction was monitored by HPLC until completion. The reaction system was diluted with DCM, washed with saturated NaCl solution, and the organic phase was collected and dried over anhydrous Na2SO4. The resulting organic phase was concentrated and purified by silica gel chromatography to give a yellow solid compound 10g (800 mg, 73% yield). MS (ESI) C 49 H 58 N4NaO 14 [(M+Na)] + Calculated value: 950.01, measured value: 950.36.

[0232] Step E: Synthesis of compound 10i

[0233] Compound 10g (525 mg, 0.56 mmol) and compound 10h (304 mg, 0.63 mmol, AZD7550, CAS No. 1421373-99-0, commercially available) were dissolved in DMF (8 mL), DIEA (158 mg, 1.2 mmol) was added, and the reaction system was stirred at room temperature under nitrogen for 12 h. The reaction was monitored by HPLC until completion. The reaction system was diluted with DCM, washed with saturated NaCl solution, and the organic phase was collected and dried over anhydrous Na2SO4. The resulting organic phase was concentrated and purified by silica gel chromatography to give a yellow solid compound 10i (467 mg, 66% yield). MS (ESI) C 69 H 84 N 11 O 13 [(M+H)] + Calculated value: 1274.63, measured value: 1274.61.

[0234] Step F: Synthesis of compound 10j

[0235] Compound 10i (300 mg, 0.23 mmol) was dissolved in DMF (3 mL), and DBU (18 mg, 0.11 mmol) was added. The reaction system was stirred at room temperature for 2 h. The reaction was monitored by HPLC until completion. The reaction system was concentrated and purified by silica gel chromatography to obtain a yellow solid compound 10j (230 mg, 87% yield). MS (ESI) C 54 H 74 N11 O 11 [(M+H)] + Calculated value: 1052.56, measured value: 1052.49.

[0236] Step G: Synthesis of compound 101

[0237] Compound 10j (120 mg, 0.12 mmol) and compound 10k (152 mg, 0.13 mmol, readily prepared by peptide solid-phase synthesis (SPPS)) were dissolved in DMF (2 mL). DIEA (77 mg, 0.6 mmol) and HATU (68 mg, 0.18 mmol, CAS No. 148893-10-1) were added, and the reaction system was stirred at room temperature for 2 h. The reaction was monitored by HPLC until completion. The reaction mixture was used directly in the next step without further purification.

[0238] Step H: Synthesis of compound 10

[0239] Et2NH (200 μL) was added to the mixture from the previous step and stirred at room temperature for 1 h. The reaction was monitored by HPLC until completion. The reaction system was directly purified by preparative high performance liquid chromatography (prep-HPLC) to obtain yellow solid compound 10 (120 mg, 51% yield for the two-step reaction). MS (ESI) C 94 H 148 N 16 O 29 [(M+2H) / 2] + Calculated value: 983.53, measured value: 983.93.

[0240] Example 2: Preparation of Linker Payload 2 (LP2)

[0241] The LP2 structure is as follows:

[0242] The preparation method thereof can refer to the preparation of LP-6 in Example 9 of WO2023232144A1, and the entire text of WO2023232144A is incorporated herein by reference.

[0243] 2. Antibody Selection and Preparation

[0244] The HER3 antibody Ab2 sequence is derived from Partritumab, synthesized by Bio-Innovative Biotechnology. Ab1 is an engineered modification of the light chain terminus of Ab2. For detailed preparation, purification, and identification methods, please refer to Example 1 of patent CN 106856656 B, which is incorporated herein by reference in its entirety.

[0245] Table 1: Antibody sequence list

[0246] 3. Preparation and Characterization of ADCs

[0247] Table 2: ADC list

[0248] Example 3: Preparation and Characterization of ADC-1 (Ab1-LP1)

[0249] 3.1 Preparation of ADC-1

[0250] ADC-1 is prepared by catalyzing the coupling reaction between antibody Ab1 and LP1 using Sortase or its variants (preferably a modified transpeptidase). Specifically, the antibody and LP1 are thoroughly mixed in a 1× endonuclease buffer at an appropriate molar ratio (1:1 to 1:100), then added to an immobilized enzyme medium and mixed thoroughly. The immobilized enzyme medium contains an immobilized endonuclease, which catalyzes the coupling reaction between antibody Ab1 and LP1. The coupling reaction is carried out at 4-40°C for 0.5-20 hours. After the reaction is completed, the solid-phase coupling reaction mixture is centrifuged, removed, and purified, ultrafiltered, or dialyzed to remove unreacted drug intermediates. The purified ADC-1 is stored in 1× PBS (pH 7.4) buffer at 4°C or -80°C.

[0251] 3.2 Detection and Characterization of ADC-1

[0252] 3.2.1 HIC-HPLC analysis of ADC-1

[0253] A Proteomix HIC Butyl-NP5 4.6*100mm 5μm Non-Porous column (manufacturer: Saifen, PN: 431NP5-4610) was used; 1.5M ammonium sulfate + 20mM phosphate buffer, pH 7.0 was used as mobile phase A; 20mM phosphate buffer, pH 7.0: isopropanol = 7:3 (v / v) was used as mobile phase B; the flow rate was 0.8mL / min; a gradient method was used: phase B increased from 10% to 100% within 8 minutes; and the detection wavelength was selected at 280nm to detect the DAR distribution of the ADC drug ADC-1.

[0254] The test results are shown in Figure 1. The unconjugated cytotoxic antibody Ab1 is less than 7%; the conjugated product is mainly DAR2, and the overall DAR value of the ADC drug is about 1.78.

[0255] 3.2.2 SEC-HPLC analysis of ADC-1

[0256] A TSKgel G3000SWXL 7.8mm ID*30cm, 5μm column (Manufacturer: Tosoh, PN: 0008541) was used as the mobile phase; 2*PBS:methanol = 9:1 (v / v); room temperature; isocratic; flow rate 1.0mL / min; run time 15min; 280nm was selected as the detection wavelength to analyze the high molecular weight aggregation of ADC drugs.

[0257] The test results are shown in FIG2 . The high molecular weight aggregates in the ADC drug are less than 5%, and the peak at 8.05 min of the ADC sample mainly exists in the form of monomers.

[0258] Example 4: Preparation and Characterization of ADC-2 (Ab2-LP2)

[0259] 4.1 Preparation of ADC-2

[0260] ADC drug ADC-2 is prepared based on the coupling reaction of antibody Ab2 and LP2 catalyzed by glycosidase (preferably a modified endonuclease). Specifically, in a 1× endonuclease buffer, the antibody and LP2 are thoroughly mixed in an appropriate molar ratio (1:1 to 1:100), and the solid-phase enzyme medium is added and mixed. The matrix of the solid-phase enzyme medium has an immobilized endonuclease, which catalyzes the coupling reaction of antibody Ab2 and LP2. The coupling reaction in the mixed state is carried out at 4-40°C for 0.5-20 hours. After the reaction is completed, centrifuge, remove the solid-phase coupling reaction mixture, and purify, ultrafilter or dialyze to remove unreacted drug intermediates. The purified ADC-2 is stored in a buffer solution at 4°C or -80°C.

[0261] 4.2 ADC-2 Assay Characterization

[0262] 4.2.1 HIC-HPLC analysis of ADC-2

[0263] A Sepax Proteomix HIC Butyl-NP5, 4.6×100mm 5μm column (manufacturer: Sepax, PN: 431NP5-4610) was used; mobile phase A was 1.5M ammonium sulfate + 20mM phosphate buffer, pH 7.0; mobile phase B was 20mM phosphate buffer, pH 7.0: isopropanol = 7:3 (v / v); the flow rate was 0.8mL / min; a gradient method was used: phase B increased from 10% to 70% within 8 minutes; and the detection wavelength was selected at 280nm to detect the DAR distribution of the ADC drug ADC-2.

[0264] The test results are shown in FIG3 . The unconjugated cytotoxic antibody Ab2 is less than 4%; the conjugated product is mainly DAR4, and the overall DAR value of the ADC drug is about 3.89.

[0265] 4.2.2 SEC-HPLC Analysis of ADC-2

[0266] A TSKgel G3000SWXL 7.8mm ID*30cm, 5μm column (Manufacturer: Tosoh, PN: 0008541) was used as the mobile phase; 2*PBS:acetonitrile = 9:1 (v / v); room temperature; isocratic; flow rate 1.0mL / min; run time 15min; 280nm was selected as the detection wavelength to analyze the high molecular weight aggregation of ADC drugs.

[0267] The test results are shown in FIG4 . The high molecular weight aggregates in the ADC drug ADC-2 are less than 5%, and the peak at 7.90 min of the ADC sample mainly exists in the form of monomers.

[0268] Example 5: Preparation and Characterization of dpADC-1 (Ab1-LP1+LP2)

[0269] 5.1 Preparation of dpADC-1

[0270] Sortase (preferably a modified transpeptidase) and endoglycosidase (preferably a modified endoglycosidase) are used to catalyze the coupling reaction of antibody Ab1 with LP1 and LP2 to prepare the ADC drug dpADC-1. Antibody Ab1 is thoroughly mixed with LP1 and LP2 in a 1× buffer at an appropriate molar ratio (1:1 to 1:100, respectively). The immobilized transpeptidase and endoglycosidase medium are then added and mixed thoroughly. The immobilized transpeptidase and endoglycosidase on the matrix of the immobilized enzyme medium catalyze the coupling reaction of antibody Ab1 with LP1 and LP2. The coupling reaction is carried out at 4-40°C for 0.5-20 hours. After completion of the reaction, the solid-phase coupling reaction mixture is centrifuged, removed, and purified, ultrafiltered, or dialyzed to remove unreacted drug intermediates. The purified ADC drug dpADC-1 is stored in 1× PBS (pH 7.4) at 4°C or -80°C.

[0271] 5.2 Detection and Characterization of dpADC-1

[0272] 5.2.1 RP-HPLC analysis of dpADC-1

[0273] The DAR distribution of the ADC drug dpADC-1 was determined using a MAbPac™ RP 4μm, 2.1×100mm reversed-phase chromatography column (Thermo, PN: 088647). Mobile phase A consisted of 0.1% HCOOH and 0.025% TFA in water, and mobile phase B consisted of 0.1% HCOOH and 0.025% TFA in acetonitrile. The flow rate was 0.5 mL / min, the column temperature was 80°C, and a gradient method was used: phase B increased from 20% to 50% over 30 minutes. The detection wavelength was 280 nm. Prior to injection, dpADC-1 was reduced with dithiothreitol (DTT).

[0274] The test results are shown in Table 3 and Figure 5 below. The sum of the relative peak areas of the heavy and light chains of the unconjugated cytotoxic antibody Ab1 is less than 8%; the conjugated product is mainly DAR (2+4), and the overall DAR value of the dpADC drug is about 5.76 (LC+HC=1.93+3.83).

[0275] Table 3 RP-HPLC results of dpADC-1

[0276] 5.2.2 SEC-HPLC Analysis of dpADC-1

[0277] A TSKgel G3000SWXL 7.8 mm ID*30 cm, 5 μm column (Manufacturer: Tosoh, PN: 0008541) was used; 2×PBS + 10% ACN was used as the mobile phase; room temperature; isocratic; flow rate 1.0 mL / min; run time 15 min; and detection wavelength 280 nm were selected to analyze the high molecular weight aggregation of ADC drugs.

[0278] The test results are shown in FIG6 . The high molecular weight aggregates in the ADC drug are less than 1%, and the peak at 7.81 min of the ADC sample mainly exists in the form of monomers.

[0279] IV. ADC Activity Testing

[0280] Example 6: In vivo bystander effect experiment of ADCs

[0281] HCC827 cells (human non-small cell lung cancer cells, HCC827-hERBB3) overexpressing human HER3 protein were used as HER3-positive cells, and PC-9 cells (human lung cancer cells) were used as HER3-negative cells. HER3-positive cells and HER3-negative cells in the logarithmic growth phase were seeded in a 96-well plate at a ratio of 1:2, and the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours. After the incubation period, 1 nM and 10 nM of ADC-1, ADC-2, and dpADC-1 were added to the 96-well plate, respectively. The 96-well plate after administration was placed in a 37°C, 5% CO2 incubator for 144 hours, and then detected by flow cytometry.

[0282] The test results are shown in Figure 7. The experimental results show that dpADC-1 and ADC-2 achieved 100% cytotoxicity against HER3-positive cells, while ADC-1 had a 17% cytotoxicity against HER3-positive cells. At 1 nM, dpADC-1 and ADC-2 had comparable cytotoxicity against HER3-negative cells, with ADC-1 showing a higher cytotoxicity. At 10 nM, dpADC-1 exhibited a higher cytotoxicity against HER3-negative cells than both ADC-1 and ADC-2. These results suggest that HER3 dual-loaded ADCs exhibit superior in vitro cytotoxicity compared to single-loaded ADCs.

[0283] Example 7 In vivo efficacy of ADCs in mice in an osimertinib-resistant NSCLC PDX model expressing HER3

[0284] 7.1 In vivo Passaging and Group Dosing of Human Lung Cancer Transplants

[0285] Human lung cancer transplants were cut into approximately 3mm x 3mm x 3mm (approximately 45-60mg) pieces of tumor tissue and inoculated subcutaneously into NU / NU mice. The mice were observed and tumor growth was monitored. On day 27 of inoculation, the average tumor volume of the tumor-bearing mice was 163mm. 3 The grouping and dosing schedule are as follows:

[0286] Table 4: In vivo experimental protocol

[0287] Note: Dosage volume: adjusted according to the weight of tumor-bearing mice (0.2 mL / 20 g); IV: tail vein injection; PO: oral administration; Q3W: once every 3 weeks; QD: once a day; in each dosing group, the solvent used for IV was PBS, and the solvent used for PO was 20% PEG400 + 80% PBS.

[0288] 7.2 Tumor volume and mouse body weight measurement

[0289] Tumor volume and tumor-bearing mouse weight measurement: Use vernier calipers to measure twice a week. The tumor volume is calculated as V = 0.5a × b 2 , a and b represent the long diameter and wide diameter of the tumor, respectively.

[0290] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : RTV of vehicle group). The relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV=V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., d0), V t is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0291] Tumor growth inhibition rate TGI (%) = [1-(T i -T0) / (V i -V0)]×100,T i is the average tumor volume of the compound group after the start of administration, T0 is the average tumor volume of the compound group at the first administration, V0 is the average tumor volume of the vehicle group at the first administration, and V i is the average tumor volume of the vehicle group after the start of drug administration.

[0292] The body weight of all tumor-bearing mice was measured every day. At the same time, the relative change ratio of the body weight of mice after administration was calculated: RCBW (%) = (BW i –BW0) / BW0×100, BW i BW0 is the body weight after the start of drug administration, and BW1 is the body weight at the first drug administration.

[0293] 7.3 Conclusion

[0294] The experimental results are shown in Figure 8. On day 28 after administration, dpADC-1 demonstrated superior tumor suppression compared to ADC-2 and osimertinib alone. This demonstrates that HER3-dpADCs offer significant advantages over single-dose HER3-loaded ADCs for the treatment of osimertinib-resistant NSCLC. There was no significant difference in mouse body weight between all drug-treated and control groups, demonstrating the favorable safety profile of HER3-dpADCs.

[0295] Example 8 In vivo efficacy study of ADCs in osimertinib-sensitive NSCLC PC-9CDX model with low HER3 expression

[0296] 8.1 Cell culture

[0297] PC-9 tumor cells (human lung cancer cells, Riken, Manassas, VA, cat# RCB4455) were cultured in vitro as monolayers at 37°C in 5% CO₂ in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0298] 8.2 Tumor inoculation and animal grouping

[0299] PC-9 tumor cells (10×10 6 Animals were randomly divided into groups and treatment started on day 6 after tumor inoculation, when the average tumor volume reached approximately 172 mm. 3 The animals were randomized into groups using Excel-based randomization software, with stratified randomization based on tumor volume. Each group consisted of 5 tumor-bearing mice. The mice were dosed according to the pre-determined schedule shown in Table 5.

[0300] Table 5 In vivo experimental plan

[0301] Note:

[0302] aN: number of animals in each group;

[0303] b. Dose volume: Adjust the dose volume based on the standard of 10 mL / kg body weight;

[0304] c. The duration of the experiment is 28 days.

[0305] IV: tail vein injection; PO: oral administration; in each administration group, the solvent used for IV was PBS, and the solvent used for PO was 0.5% HPMC+0.1% Tween80.

[0306] 8.3 Tumor Measurements and Endpoints

[0307] The primary endpoint was to see if tumor growth could be slowed or if the mice could be cured. Tumor volume was measured twice weekly in two dimensions using calipers and expressed in mm using the following formula: 3 The unit represents the tumor volume: V = 0.5axb 2 , where a and b are the long and wide diameters of the tumor, respectively.

[0308] The relative tumor proliferation rate (T / C) of each group was calculated using the following formula: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : RTV of the control group; RTV: relative tumor volume). RTV=V t / V0, V0 is the average tumor volume on the first day of treatment, V t is the average tumor volume on a given day, T RTV with C RTV The data are from the same day.

[0309] The tumor growth inhibition rate (TGI) of each group was calculated using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100;T i is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on the first day of treatment, V i Is with T i The mean tumor volume of the control group on the same day, V0 is the mean tumor volume of the control group on the first day of treatment.

[0310] Tumor weights were measured at the end of the study. 重量 The value (percentage) is calculated using the following formula: T / C 重量 % = T 重量 / C 重量 x 100%, where T 重量 and C 重量 are the average tumor weights of the treatment and control groups, respectively.

[0311] 8.4 Conclusion

[0312] The experimental results are shown in Figure 9. On day 28 after administration, dpADC-1 demonstrated superior tumor suppression in mice compared to the HER3-loaded ADC-2 alone and osimertinib alone. This demonstrates that HER3-dpADC offers significant advantages over HER3-loaded ADCs for the treatment of osimertinib-sensitive NSCLC. There was no significant difference in mouse body weight between all drug-treated and control groups, demonstrating the favorable safety profile of HER3-dpADC.

Claims

1. A dual-loaded antibody-drug conjugate having the following structure: in, Ab is an anti-HER3 antibody or an antigen-binding fragment thereof; P1 and P2 are payloads, each independently selected from a topoisomerase inhibitor and a tyrosine kinase inhibitor, provided that the two are different; L1 is a linker that is connected to the Ab in a site-specific manner based on the N-glycosylation site in the Fc region of the antibody; L2 is a linker that is connected to Ab in a site-directed coupling manner based on ligase catalysis; a and b are each independently selected from an integer from 1 to 10; q and t are each independently selected from an integer of 1-10.

2. The dual-loaded antibody-drug conjugate according to claim 1, wherein (P1) a -L1- has the following structure in, * represents the end connected to the antibody; preferably, the -NHC(O)CH2- at the * end is part of the amino acid in the Fc region of the antibody; preferably, the -NHC(O)CH2- at the * end is part of the asparagine in the Fc region of the antibody; more preferably, the -NHC(O)CH2- at the * end is part of the asparagine at position 297 in the Fc region of the antibody; R 1 、R 2 、R 3 、R 4 、R 5 、R 6 Each independently selected from H, C 1-6 Alkoxy or OH; preferably, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 are each independently selected from H, methoxy or OH; more preferably, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 Each is independently selected from H or OH, and satisfies the following conditions: R 1 and R 2 Different, R 3 and R 4 Different and R 5 and R 6 different; R 7 is hydrogen or α-L-fucosyl; preferably, R 7 is α-L-fucosyl; L 1-a is a connector, and L 1-a It is connected to the terminal sugar chain of the antibody via the terminal -NH-.

3. The dual-loaded antibody-drug conjugate according to claim 2, wherein: (P1) a -L1- is selected from the following structures in, * represents the end connected to the antibody; preferably, the -NHC(O)CH2- at the * end is derived from an amino acid in the Fc region of the antibody; more preferably, the -NHC(O)CH2- at the * end is derived from asparagine in the Fc region of the antibody; further preferably, the -NHC(O)CH2- at the * end is derived from asparagine at position 297 in the Fc region of the antibody; R 7 is hydrogen or α-L-fucosyl; preferably, R 7 It is α-L-fucosyl.

4. The dual-loaded antibody-drug conjugate according to claim 2 or 3, wherein: -L 1-a -(P1) a With the following structure in, Each i1 is independently selected from an integer of 1-20; preferably, i1 is an integer selected from 1-10; more preferably, i1 is 4; Each i 1’ are independently selected from integers of 1-20; preferably, i 1’ is an integer selected from 1-10; more preferably, i 1’ is 2; j1 is an integer selected from 1-20; preferably, j1 is an integer selected from 2-16; more preferably, j1 is 12; m1 is an integer selected from 1-8; preferably, m1 is an integer selected from 1-5; more preferably, m1 is 3; h is an integer selected from 1-3; preferably, h is 1 or 2; Preferably, -L 1-a -(P1) a Has the following structure:

5. The dual-loaded antibody-drug conjugate according to claim 1 or 4, wherein: P1 is selected from 6. The dual-loaded antibody-drug conjugate according to claim 4, wherein -L 1-a -(P1) a With the following structure 7. The dual-loaded antibody-drug conjugate according to claim 1, wherein: -L2-(P2) b for in, P2 is the load; i2 is an integer selected from 1-20; preferably, i2 is an integer selected from 1-10; more preferably, i2 is 4; j2 is an integer selected from 1-20; preferably, j2 is an integer selected from 2-16; more preferably, j2 is 12; m2 is an integer selected from 1-8; preferably, m2 is an integer selected from 1-5; more preferably, m2 is 3.

8. The dual-loaded antibody-drug conjugate according to claim 1 or 7, wherein: P2 is selected from lapatinib, neratinib, pyrotinib, afatinib, gefitinib, erlotinib and osimertinib, or derivatives thereof; preferably osimertinib or a derivative thereof.

9. The dual-loaded antibody-drug conjugate according to claim 1, wherein -L2-(P2) b for 10. The dual-loaded antibody-drug conjugate according to claim 1, wherein: (P1) a -L1- is linked to the anti-HER3 antibody or antigen-binding fragment thereof by enzyme or mutant catalysis; Wherein, the enzyme or its mutant is N-acetylglucosamine endohydrolase, or its mutant; Preferably, the N-acetylglucosamine endohydrolase is selected from Endo H, Endo D, Endo F2, Endo M, Endo Om, Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd) and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC), Endo CC1, Endo CC2, or mutants thereof; or Further preferably, the N-acetylglucosamine endohydrolase is selected from Endo F3, Endo S and Endo S2, or mutants thereof; and / or, (P2) b -L2- is linked to the anti-HER3 antibody or antigen-binding fragment thereof by an enzyme or a mutant thereof, wherein the enzyme or the mutant thereof is a transpeptidase or a mutant thereof; Preferably, the enzyme or its mutant is Sortase A, Sortase B, Sortase C, Sortase D and Sortase L. plantarum or a mutant thereof; Further preferably, the enzyme or its mutant is Sortase A, and the anti-HER3 antibody has a terminal modification of GALPETG at the end of the light chain to achieve site-directed coupling catalyzed by Sortase A.

11. The dual-loaded antibody-drug conjugate according to claim 1, wherein: The anti-HER3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and light chain variable region (V L ),in, The heavy chain variable region comprises: i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; iii) a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and / or The light chain variable region comprises: i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; iii) LCDR3 comprising the amino acid sequence of SEQ ID NO:

6.

12. The dual-loaded antibody-drug conjugate according to claim 11, wherein The heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or The light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

8.

13. The dual-loaded antibody-drug conjugate according to claim 11 or 12, wherein: The anti-HER3 antibody or antigen-binding fragment thereof comprises a heavy chain constant region (C H ), which comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; and / or The anti-HER3 antibody or antigen-binding fragment thereof comprises a light chain constant region (C L ), which comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

10.

14. The dual-loaded antibody-drug conjugate according to any one of claims 11 to 13, wherein The anti-HER3 antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 13; and / or The anti-HER3 antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12 or SEQ ID NO: 14; Preferably, The anti-HER3 antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 13; and / or The anti-HER3 antibody or antigen-binding fragment thereof comprises a light chain comprising SEQ ID NO: 12 or SEQ ID NO:

14.

15. The dual-loaded antibody-drug conjugate according to any one of claims 1 to 14, having the following structure: in, Ab as defined in claim 1; R 7 is hydrogen or α-L-fucosyl; preferably, R 7 is α-L-fucosyl; q and t are independently selected from integers of 1-10; Preferably, q is 1 or 2, more preferably, q is 2; t is 1 or 2, more preferably, t is 2. 16 . A pharmaceutical composition comprising the dual-loaded antibody-drug conjugate according to any one of claims 1 to 15 and at least one pharmaceutically acceptable carrier.

17. Use of the dual-loaded antibody-drug conjugate according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16 in the preparation of a drug for treating HER3-positive diseases; Preferably, the disease is sensitive or resistant to tyrosine kinase inhibitors.

18. A method for treating a HER3-positive disease, comprising administering to an individual in need thereof a therapeutically effective amount of the dual-loaded antibody-drug conjugate according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16; Preferably, the individual / disease is sensitive or resistant to tyrosine kinase inhibitors.

19. The use according to claim 17 or the method according to claim 18, wherein The disease includes breast cancer, ovarian cancer, colon cancer, stomach cancer, lung cancer, skin cancer or pancreatic cancer; preferably lung cancer.

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