Targeted polypeptide-drug conjugate and its use

Abstract

Provided is a targeted polypeptide-drug conjugate having a structure shown in Formula I. A method for preparing the conjugate and a composition comprising the conjugate are provided. The conjugate and the composition can be used to treat a variety of diseases and conditions, such as tumors. Also provided is the use of the targeted polypeptide-drug conjugate, or together with other tumor therapies or drugs, in the preparation of a drug for treating tumors.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a conjugate of a targeting polypeptide and a small molecule therapeutic drug, and its use in preparing a drug. Background Art

[0002] Camptothecin (CPT) is an alkaloid extracted from the bark and fruit of the Chinese plant Camptotheca acuminata by American chemists in 1966. It has excellent anti-tumor activity and its chemical structure is

[0003] It can inhibit topoisomerase I and exert anti-tumor effects. Camptothecin derivatives can be modified rationally to improve their anti-tumor activity and can be used as a potential candidate drug molecule. However, small molecule drugs usually have problems such as low targeting, being cleared by the body's circulatory system before reaching the tumor site, killing normal cells, or having serious toxic side effects.

[0004] With the development of drugs that more effectively target and kill tumor cells, tumor treatment has made significant progress. Antibody-drug conjugates (ADCs), composed of antibodies and drug chemicals through a linker, not only have the anti-tumor efficacy of highly cytotoxic small molecule drugs, but also combine the high selectivity, stability and favorable pharmacokinetic characteristics of antibodies. However, due to the limited number of antigens on the surface of tumor cells (about 5,000-10 per cell), the number of ADCs is relatively small, and the number of ADCs is relatively small. 6 The average drug-to-antibody ratio (DAR) of ADC is limited to 3.5-4, that is, the amount of drug transferred into tumor cells by ADC is very low, and ADC combined with cytotoxic drugs may cause clinical failure. Therefore, it is urgent to develop more effective antibody-drug conjugates. Summary of the invention

[0005] The present application provides a targeted polypeptide-drug conjugate, and a method for preparing the same and a composition. The conjugates, methods and compositions disclosed in the present application can be used to treat a variety of diseases and conditions, including diseases and conditions in which there are selectable targeting moieties, such as tumors. The present application also provides the use of the conjugates together with other tumor therapies or drugs in the preparation of drugs for treating tumors. The conjugates of the present application have at least one of the following properties: (1) the ability to bind to tumor antigens with high affinity, (2) excellent in vitro target cell killing activity, inhibiting tumor cell growth, (3) excellent in vivo activity in inhibiting tumor cell growth and (4) good homogeneity.

[0006] In one aspect, the present application provides a conjugate having a structure shown in Formula I:

[0007]

[0008] Among them, R 1 is a methyl group substituted by halogen; R 2 Selected from hydrogen, C 1 -C 6 Alkyl and substituted C 1 -C 6 Alkyl; R 3 Selected from hydrogen, amino, C 1 -C 6 Alkyl and substituted C 1 -C 6 Alkyl; Mab is a targeting polypeptide; L is a linker.

[0009] In certain embodiments, the conjugate has a structure shown in Formula II:

[0010]

[0011] Among them, R 1 is a methyl group substituted by halogen; R 2 Selected from hydrogen, C 1 -C 6 Alkyl and substituted C 1 -C 6 Alkyl; R 3 Selected from hydrogen, amino, C 1 -C 6 Alkyl and substituted C 1 -C 6 alkyl.

[0012] In certain embodiments, R 1 For-CF 2 H.

[0013] In certain embodiments, the conjugate has a structure shown in Formula III:

[0014]

[0015] In certain embodiments, the Mab comprises an antibody or an antigen-binding fragment thereof.

[0016] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0017] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab) 2 , Fv and ScFv fragments.

[0018] In certain embodiments, the Mab specifically binds to a tumor-specific antigen.

[0019] In certain embodiments, the tumor-specific antigen is selected from the group consisting of CLDN18.2, Nectin 4, Her2 and Trop2.

[0020] In certain embodiments, the Mab comprises an antibody heavy chain HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 19, 25, 31 and 39.

[0021] In certain embodiments, the Mab comprises an antibody heavy chain HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 18, 24, 30 and 38.

[0022] In certain embodiments, the Mab comprises an antibody heavy chain HCDR1, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 17, 23, 29 and 37.

[0023] In certain embodiments, the Mab comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 5, 32 and 40.

[0024] In certain embodiments, the Mab comprises an antibody heavy chain, and the antibody heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 9, 11, 13 and 15.

[0025] In certain embodiments, the Mab comprises an antibody light chain LCDR3, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 22, 28, 35 and 43.

[0026] In certain embodiments, the Mab comprises an antibody light chain LCDR2, and the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 21, 27, 34 and 42.

[0027] In certain embodiments, the Mab comprises an antibody light chain LCDR1, and the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 20, 26, 33 and 41.

[0028] In certain embodiments, the Mab comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 36 and 44.

[0029] In certain embodiments, the Mab comprises an antibody light chain, and the antibody light chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 10, 12, 14 and 16.

[0030] On the other hand, the present application provides a method for producing the conjugate, comprising:

[0031] Obtain the compound shown in formula IV:

[0032]

[0033] Among them, R 1 is a methyl group substituted by halogen; R 2 Selected from hydrogen, C 1 -C 6 Alkyl and substituted C 1 -C 6 Alkyl; R 3 Selected from hydrogen, amino, C 1 -C 6 Alkyl and substituted C 1 -C 6 Alkyl; Mab is a targeting polypeptide; L is a linker; the compound represented by formula IV is conjugated with the Mab to obtain the conjugate.

[0034] In another aspect, the present application provides a pharmaceutical composition comprising the conjugate described herein.

[0035] On the other hand, the present application provides use of the conjugate or the pharmaceutical composition described in the present application in the preparation of a drug for treating tumors.

[0036] On the other hand, the present application provides the use of the conjugate together with other tumor therapies or drugs in the preparation of drugs for treating tumors.

[0037] In certain embodiments, the other tumor therapy or drug is selected from the group consisting of chemotherapy, radiotherapy, miRNA and oligonucleotides.

[0038] In certain embodiments, the tumor comprises a solid tumor and / or a non-solid tumor. For example, the tumor is a solid tumor.

[0039] In certain embodiments, the tumor is selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, prostate cancer, and gastric cancer.

[0040] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

[0042] Figure 1 Shown is the binding activity of the conjugate described in the present application to Her2 on Ba / F2 cells;

[0043] Figure 2 Shown is the binding activity of the conjugate described in the present application to Trop2 on 293T cells;

[0044] Figure 3 What is shown is the activity of the conjugate described in the present application in inhibiting SK-BR-3 cells in vitro;

[0045] Figure 4 What is shown is the activity of the conjugate described in the present application in inhibiting NCI-N87 cells in vitro;

[0046] Figure 5 What is shown is the activity of the conjugate described in the present application in inhibiting MDA-MB-468 cells in vitro;

[0047] Figure 6 What is shown is the activity of the conjugate described in the present application in inhibiting BXPC-3 cells in vitro;

[0048] Figure 7 What is shown is the activity of the conjugate described in the present application in inhibiting COLO-205 cells in vitro;

[0049] Figure 8 The results show that the conjugate described in the present application inhibits the activity of HCC1954 cells in vitro.

[0050] Fig. 9 What is shown is that HB010 inhibits breast cancer growth;

[0051] Fig.10 Shown is the change in body weight of breast cancer mice after administration of the conjugate of the present application;

[0052] Fig.11 It is shown that the conjugate HB060 described in the present application inhibits breast cancer growth;

[0053] Fig.12 Shown is the change in body weight of breast cancer mice after administration of the conjugate of the present application;

[0054] Fig.13 It is shown that the conjugate HB030 described in the present application inhibits breast cancer growth;

[0055] Fig.14Shown is the change in body weight of breast cancer mice after administration of the conjugate of the present application;

[0056] Fig.15 It is shown that the conjugate HB030 described in the present application inhibits breast cancer growth;

[0057] Fig.16 Shown is the change in body weight of breast cancer mice after administration of the conjugate of the present application;

[0058] Fig.17 It is shown that the conjugate HB010 described in the present application inhibits the growth of gastric cancer;

[0059] Fig.18 Shown is the weight change of gastric cancer mice after administration of the conjugate of the present application;

[0060] Fig.19 It is shown that the conjugate HB050 described in the present application inhibits the growth of gastric cancer;

[0061] Fig. 20 Shown is the weight change of gastric cancer mice after administration of the conjugate of the present application;

[0062] Fig.21 It is shown that the conjugate HB050 described in the present application inhibits the growth of gastric cancer;

[0063] Fig. 22 Shown is the weight change of gastric cancer mice after administration of the conjugate of the present application;

[0064] Fig.23 It is shown that the conjugate HB030 described in the present application inhibits the growth of pancreatic cancer;

[0065] Fig.24 Shown is the change in body weight of pancreatic cancer mice after administration of the conjugate of the present application;

[0066] Fig.25 It is shown that the conjugate HB050 described in the present application inhibits the growth of pancreatic cancer;

[0067] Fig.26 Shown is the change in body weight of pancreatic cancer mice after administration of the conjugate of the present application;

[0068] Fig. 27 It is shown that the conjugate HB060 described in the present application inhibits prostate cancer growth;

[0069] Fig.28 Shown is the weight change of prostate cancer mice after administration of the conjugate of the present application;

[0070] Fig.29 It is shown that the conjugate HB060 described in the present application inhibits prostate cancer growth;

[0071] Fig.30 Shown is the change in body weight of prostate cancer mice after administration of the conjugate of the present application. DETAILED DESCRIPTION

[0072] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0073] Definition of terms

[0074] In this application, the term "targeting polypeptide" generally refers to a polypeptide that specifically binds or selectively binds to a target molecule, cell, particle, tissue or aggregate. For example, the targeting polypeptide can be an antibody, antibody fragment, bispecific antibody, antigen binding fragment or other antibody-based molecule or compound. Other examples of targeting moieties can also be known in the art, such as aptamers, avimers, receptor binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins.

[0075] In the present application, the term "humanized antibody" generally refers to a recombinant protein in which the CDRs of an antibody from a species (e.g., a mouse antibody) are transferred from the heavy and light chains of the species to the variable regions of human heavy and light chains. The constant region of the antibody molecule is derived from the constant region of a human antibody. In certain embodiments, specific residues of the variable region of the humanized antibody may be modified, especially residues that contact or approach the CDR sequence, for example, by replacing corresponding residues from original murines, rodents, non-primates, or other antibodies.

[0076] In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies in the cluster are identical, except for a small amount of natural mutations that may exist. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which generally have different antibodies for different determinants), each monoclonal antibody is for a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins.

[0077] In this application, the term "single-chain antibody" or "scFv" generally refers to an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L Such scFv molecules may have the general structure: NH 2 -V L -Connector-V H -COOH or NH 2 -VH-Linker-VL-COOH.

[0078] The term "antibody" as used herein generally includes whole antibodies and any antigen-binding fragments or single chains thereof. It may typically include an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. The VH and VL regions may be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conservative regions called framework regions (FRs). Each VH and VL may be composed of three CDRs and four FR regions, which may be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The term "antibody" may also encompass antibodies, digested fragments thereof, designated portions, and variants thereof, including antibody mimetics or antibody portions comprising structures and / or functions that mimic antibodies or designated fragments or portions thereof, including single-chain antibodies and single-domain antibodies and fragments thereof. Functional fragments include antigen-binding fragments directed against preselected targets.

[0079] The term "antigen binding fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antigen binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv and single-chain Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antigen binding fragments.

[0080] In this application, the term "Fab" generally refers to a fragment containing a heavy chain variable domain and a light chain variable domain, and also contains a constant domain of a light chain and the first constant domain (CH1) of a heavy chain; the term "Fab'" generally refers to a fragment different from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) at the carboxyl terminus of the heavy chain CH1 domain; the term "F(ab') 2 " generally refers to a dimer of Fab', an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region. The term "Fv" generally refers to the smallest antibody fragment containing a complete antigen recognition and binding site. In some cases, the fragment can be composed of a dimer of a heavy chain variable region and a light chain variable region in tight non-covalent association; the term "dsFv" generally refers to a disulfide-stabilized Fv fragment, the bond between a single light chain variable region and a single heavy chain variable region is a disulfide bond. The term "dAb fragment" generally refers to an antibody fragment consisting of a VH domain. In the present application, the term "scFv" generally refers to a monovalent molecule formed by covalently linking and pairing a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have a general structure: NH 2 -VL-Linker-VH-COOH or NH 2 -VH-Linker-VL-COOH.

[0081] In the present application, the term "linker" generally refers to a bond, molecule or group of molecules that bind two separated entities (such as the targeting polypeptide and camptothecin small molecule in the present application) to each other. The linker can provide an optimal interval for the two entities, or can also provide an unstable connection that separates the two entities from each other. Unstable connections include photocleavable groups, acid-labile parts, base-labile parts and enzyme-cleavable groups. In certain embodiments, the linker can refer to any reagent or molecule that connects the targeting polypeptide and the pharmaceutically active part (such as, camptothecin small molecule). The linker can form a covalent bond or a non-covalent bond with the two entity parts. An ideal linker can be any reagent or molecule that remains stable, does not affect the functionality of the targeting polypeptide or the pharmaceutically active part, or does not produce additional unwanted functions. The linker can be degradable (cleavable) or non-cleavable (non-cleavable). Common linkers may include, but are not limited to, valine-citrulline linker (vc), valine-alanine linker (va), maleimidomethyl cyclohexane-1-carboxylate (MCC), maleimidocaproyl (mc), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), or 4-(4-acetylphenoxy)butanoic acid ( ... acid-N, AcBut-N).

[0082] In this application, the term "chimeric antibody" generally refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. Fragments of the antibodies are also included, which exhibit the desired biological activity (i.e., the ability to specifically bind to a tumor antigen).

[0083] In this application, the term "fully human antibody" generally refers to antibodies expressed by animals by transferring genes encoding human antibodies into genetically engineered antibody gene-deficient animals. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

[0084] In this application, the term "specific binding" generally refers to binding to a specific target without cross-reactivity to other targets. For example, when an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" an antigen when it binds to an epitope more easily through its antigen binding domain than it would bind to a random, unrelated epitope. Selective reactivity generally refers to preferential binding to a specific target.

[0085] In this application, the term "tumor-specific antigen" generally refers to a protein that is primarily or exclusively expressed on tumor cells. Mainly expressed refers to relatively higher expression on tumor cells than on normal somatic cells, while exclusively expressed refers to expression on tumor cells of a protein that is not detected on normal somatic cells by standard means known in the art.

[0086] In the present application, the term "conjugate" refers to a polypeptide (e.g., peptide, nucleic acid, protein, enzyme, sugar, polysaccharide, lipid, glycoprotein and lipoprotein) and a chemical moiety that are covalently or non-covalently (e.g., via a linker) linked into a larger molecule. The chemical moiety that can be included in the conjugate can be a therapeutic agent or a cytotoxic agent, non-limiting examples of which can be: mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, anti-hormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors and radiosensitizers.

[0087] In the present application, the term "Her2" generally refers to a type I transmembrane protein belonging to the epidermal growth factor receptor family, also known as c-erbB2, ErbB2 or Neu (Slamon, et al., Science 235 (1987) 177-182; Swiss-Prot P04626). In the present application, the term "Her2" may also encompass homologues, variants and isoforms of Her2, including spliced ​​isoforms. The term "Her2" also includes proteins having one or more sequences in Her2 homologues, variants and isoforms, and fragments of the sequence, as long as the variant protein (including isoforms), homologous protein and / or fragment can be recognized by one or more Her-specific antibodies (such as Pertuzumab or Trastuzumab). Her2 is associated with tumor transformation in human breast cancer cells, such as overexpression of Her2 protein has been detected in patients with breast cancer, gastric cancer, pancreatic cancer, ovarian cancer, peritoneal cancer or colon cancer. An exemplary heavy chain variable region of Pertuzumab may include the amino acid sequence shown in SEQ ID NO.3, and an exemplary light chain variable region of Pertuzumab may include the amino acid sequence shown in SEQ ID NO.4; an exemplary heavy chain variable region of Trastuzumab may include the amino acid sequence shown in SEQ ID NO.1, and an exemplary light chain variable region of Trastuzumab may include the amino acid sequence shown in SEQ ID NO.2.

[0088] In the present application, the terms "Trop2", "TROP2" generally refer to a single-pass transmembrane type I cell membrane protein, also known as tumor-associated calcium signal transducer 2 (TACSTD2), GA733-1, EGP-1 or MIS1. In the present application, the term "Trop2" may also cover homologs, variants and isoforms of Trop 2, including spliced ​​isoforms. The term "Trop" also includes proteins having one or more sequences of Trop 2 homologs, variants and isoforms, as well as fragments of the sequence, as long as the variant protein (including isoforms), homologous protein and / or fragment can be recognized by one or more Trop-specific antibodies (such as hRS7 or hTINA1). Trop2 may be human Trop2, and the DNA sequence and amino acid sequence of human Trop2 can be obtained on public databases, for example, see NCBI accession numbers NM_002353 and NP_002344. An exemplary heavy chain variable region of hRS7 may comprise the amino acid sequence shown in SEQ ID NO.5, and an exemplary light chain variable region of hRS7 may comprise the amino acid sequence shown in SEQ ID NO.6; an exemplary heavy chain variable region of hTINA1 may comprise the amino acid sequence shown in SEQ ID NO.7, and an exemplary light chain variable region of hTINA1 may comprise the amino acid sequence shown in SEQ ID NO.8. DETAILED DESCRIPTION OF THE INVENTION

[0090] In one aspect, the present application provides a conjugate having a structure of Formula I:

[0091]

[0092] Targeting peptides

[0093] The conjugate of the present application has the structure of Formula I, wherein Mab is a targeting polypeptide.

[0094] In some cases, Mab can be an antibody (including fully human antibodies, humanized antibodies, non-human antibodies or chimeric antibodies), or an antigen-binding fragment thereof (including enzymatically or recombinantly produced fragments), or a binding protein incorporating a sequence from an antibody or its antigen-binding fragment. The antibody or its antigen-binding protein can be multivalent and multispecific or multivalent and monospecific.

[0095] In some cases, the Mab can be a monoclonal antibody. In more specific cases, the targeting polypeptide can be a multivalent and / or multispecific monoclonal antibody. The targeting polypeptide can be a mouse, chimeric, humanized or fully human monoclonal antibody, and the antibody can be a complete, fragment (Fab', Fab, F(ab) 2 、F(ab') 2 The conjugates, methods, compositions and uses described herein can utilize any of a variety of antibodies known in the art. The antibodies used can be commercially available from a variety of known sources.

[0096] In some cases, the Mab can be a chimeric antibody. A chimeric antibody is a recombinant protein in which the variable region of a human antibody is replaced by, for example, the variable region of a mouse antibody, and includes the complementary determining region (CDR) of the mouse antibody. Chimeric antibodies exhibit reduced immunogenicity and increased stability when administered to a subject. Methods for constructing chimeric antibodies are well known in the art.

[0097] Chimeric antibodies can be humanized by transferring the mouse CDRs of the mouse immunoglobulin heavy and light chain variable regions to the corresponding domains of human antibodies. The mouse framework region (FR) of the chimeric antibody is also replaced with a human FR sequence. In some cases, the Mab can be a humanized antibody. In order to retain the stability and antigenic specificity of the humanized antibody, one or more human FR residues can be replaced with the equivalent residues of the mouse. Humanized antibodies can be used for therapeutic treatment of the patient. The technology for producing humanized antibodies is well known in the art.

[0098] In some cases, the Mab can be a fully human antibody. Methods for producing fully human antibodies using a combination of methods or using transgenic animals transformed with human immunoglobulin loci are known in the art. The fully human antibody is expected to show less side effects than chimeric antibodies or humanized antibodies and act as a substantially endogenous human antibody in vivo.

[0099] The Mab described herein may be an antibody fragment. Antibody fragments may be obtained by conventional methods, such as digesting a full-length antibody with pepsin or papain. For example, antibody fragments may be produced by enzymatic cleavage of an antibody with pepsin to provide a fragment denoted as F(ab'). 2 5S fragment. The fragment can be further cleaved using a thiol reducing agent, and optionally using a blocking group of the thiol group produced by the disulfide bond cleavage to produce a 3.5S Fab' monovalent fragment. For example, two monovalent Fab fragments and an Fc fragment are produced using pepsin cleavage. Other methods of cleaving antibodies can also be used, such as separating the heavy chain to form a monovalent light chain-heavy chain fragment, further cleaving the fragment, or other enzymatic techniques, chemical techniques or genetic techniques, as long as the fragment binds to the antigen recognized by the intact antibody. In some cases, the Mab can be a single-chain antibody. These single-chain antibodies can be prepared by constructing a structural gene comprising a DNA sequence encoding a VH domain and a VL domain connected by an oligonucleotide linker sequence. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as Escherichia coli. The recombinant host cell synthesizes a single polypeptide chain in which a linker peptide bridges two variable domains. Methods for producing scFv are well known in the art.

[0100] In some cases, the Mab can be modified, for example, by deleting, increasing or replacing parts of the antibody. The modification can change a certain property of the antibody, such as making the number of small molecule parts connected to the targeting part reach the desired number, reducing ADCC after enhancement, enhancing or reducing CDC. In certain embodiments, the sequence of the antibody, such as the Fc part of the antibody, can be changed to optimize the physiological characteristics of the conjugate, such as serum half-life. The method of replacing the amino acid sequence in the protein is widely known in the art, for example, by site-directed mutagenesis. In certain embodiments, the change may include adding or removing one or more glycosylation sites in the Fc sequence. In other embodiments, specific amino acid substitutions can be made in the Fc sequence.

[0101] In certain cases, the Mab is capable of recognizing or binding to a marker or tumor-associated antigen, and is primarily or only capable of recognizing expression on cells that are diseased relative to normal tissues, and is capable of being internalized by the cells.

[0102] In some cases, the target or antigen that the Mab can recognize or bind to can include: carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp, Her2, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, lF5MAb), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6, alpha-fetoprotein (AFP), VEGF, ED-B fibronectin (e.g., L19), EGP-1, EGP-2 (e.g., 17-1A), EGF receptor (ErbB1), ErbB2, ErbB3, factor H, FHL-1, Flt-3, folate receptor, Ga733, GROB-1, hypoxia-inducible factor (HIF), HM1.24, insulin IL-1R, IFN-γ, IFN-α, IFN-β, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, ganglioside, HCG, L243-bound HLA-DR antigen, CD66 antigen (CD66a-d or a combination thereof), MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration inhibitory factor (MIF), MUC1, MU C2, MUC3, MUC4, MUC5, placental growth factor (P1GF), PSA (prostate-specific antigen), PSMA, PAM4 antigen, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenreich antigen, tumor necrosis antigen, tumor angiogenesis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, RANTES, T101, as well as cancer stem cell antigens, complement factors C3, C3a, C3b, C5a, C5, and oncogene products.

[0103] In some cases, the Mab can specifically bind to a tumor-specific antigen. In some cases, the targeting polypeptide can be an antibody or an antigen-binding fragment thereof that reacts with an antigen or antigenic epitope expressed on a tumor cell. Available tumor-specific antigens include the tumor-specific antigen can be selected from CLDN18.2, Nectin 4, CD22, CD30, CD33, Her2, Mesothelin, PSMA and Trop2. For example, the tumor-specific antigen can be selected from the following group: CLDN18.2, Nectin 4, Her2 and Trop2.

[0104] In certain instances, the Mab can be internalized and then re-expressed, processed and presented on the cell surface, allowing for continued cellular uptake of the conjugate and increasing conjugate circulation. Targeting polypeptides that can be used in the present application include, but are not limited to, the following polypeptides or antibodies: LL1 antibody (anti-CD74), LL2 and RFB4 antibodies (anti-CD22), RS7 antibody (anti-epithelial glycoprotein-1 (EGP-1)), PAM-4 and KC4 antibodies (both anti-mucin), MN-14 antibody (anti-carcinoembryonic antigen (CEA, also known as CD66e), Mu-9 antibody (anti-colon-specific antigen-p), Immu31 antibody (anti-alpha-fetoprotein), TAG-72 antibody (such as CC49), Tn antibody, J591 or HuJ591 (anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IXMab) and hL243 (anti-HLA-DR).

[0105] For example, the Mab may comprise an antibody HCDR3, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 19, 25, 31 and 39.

[0106] For example, the Mab may comprise an antibody HCDR2, and the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 18, 24, 30 and 38.

[0107] For example, the Mab may comprise antibody HCDR1, and the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 17, 23, 29 and 37.

[0108] For example, the Mab in the conjugate of the present application may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 respectively comprise any one group of amino acid sequences selected from the following groups:

[0109] (1) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18 and HCDR3: SEQ ID NO: 19;

[0110] (2) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24 and HCDR3: SEQ ID NO: 25;

[0111] (3) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30 and HCDR3: SEQ ID NO: 31; and

[0112] (4) HCDR1:SEQ ID NO:37, HCDR2:SEQ ID NO:38 and HCDR3:SEQ ID NO:39.

[0113] The Mab in the conjugate of the present application may comprise an antibody heavy chain variable region VH, and the VH may comprise an amino acid sequence shown in any one of SEQ ID NOs: 1, 5, 32 and 40.

[0114] For example, the Mab may comprise antibody LCDR3, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 22, 28, 35, and 43.

[0115] For example, the Mab may comprise antibody LCDR2, and the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 21, 27, 34 and 42.

[0116] For example, the Mab may comprise antibody LCDR1, and the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 20, 26, 33 and 41.

[0117] For example, the Mab in the conjugate of the present application may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 respectively comprise any one group of amino acid sequences selected from the following groups:

[0118] (1) LCDR1:SEQ ID NO:20, LCDR2:SEQ ID NO:21 and LCDR3:SEQ ID NO:22;

[0119] (2) LCDR1: SEQ ID NO: 26, LCDR2: SEQ ID NO: 27 and LCDR3: SEQ ID NO: 28;

[0120] (3) LCDR1: SEQ ID NO: 33, LCDR2: SEQ ID NO: 34 and LCDR3: SEQ ID NO: 35; and

[0121] (4) LCDR1:SEQ ID NO:41, LCDR2:SEQ ID NO:42 and LCDR3:SEQ ID NO:43.

[0122] The Mab in the conjugate of the present application may comprise an antibody light chain variable region VL, and the VL may comprise an amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 36 and 44.

[0123] For example, the Mab in the conjugate of the present application may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise any one group of amino acid sequences selected from the following groups:

[0124] (1) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, HCDR3: SEQ ID NO: 19, LCDR1: SEQ ID NO: 20, LCDR2: SEQ ID NO: 21 and LCDR3: SEQ ID NO: 22;

[0125] (2) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, HCDR3: SEQ ID NO: 25, LCDR1: SEQ ID NO: 26, LCDR2: SEQ ID NO: 27 and LCDR3: SEQ ID NO: 28;

[0126] (3) HCDR1:SEQ ID NO:29, HCDR2:SEQ ID NO:30, HCDR3:SEQ ID NO:31, LCDR1:SEQ ID NO:33, LCDR2:SEQ ID NO:34 and LCDR3:SEQ ID NO:35; and

[0127] (4) HCDR1:SEQ ID NO:37, HCDR2:SEQ ID NO:38, HCDR3:SEQ ID NO:39, LCDR1:SEQ ID NO:41, LCDR2:SEQ ID NO:42 and LCDR3:SEQ ID NO:43.

[0128] For example, the Mab in the conjugate described in the present application may include VH and VL, and the VH may include the amino acid sequence shown in SEQ ID NO: 1, and the VL may include the amino acid sequence shown in SEQ ID NO: 2. For example, the Mab in the conjugate described in the present application may include VH and VL, and the VH may include the amino acid sequence shown in SEQ ID NO: 5, and the VL may include the amino acid sequence shown in SEQ ID NO: 6. For example, the Mab in the conjugate described in the present application may include VH and VL, and the VH may include the amino acid sequence shown in SEQ ID NO: 32, and the VL may include the amino acid sequence shown in SEQ ID NO: 36. For example, the Mab in the conjugate described in the present application may include VH and VL, and the VH may include the amino acid sequence shown in SEQ ID NO: 40, and the VL may include the amino acid sequence shown in SEQ ID NO: 44.

[0129] The Mab in the conjugate described in the present application may include an antibody heavy chain and an antibody light chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO:9, and the antibody light chain may include the amino acid sequence shown in SEQ ID NO:10. For example, the Mab in the conjugate described in the present application may include an antibody heavy chain and an antibody light chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO:11, and the antibody light chain may include the amino acid sequence shown in SEQ ID NO:12. For example, the Mab in the conjugate described in the present application may include an antibody heavy chain and an antibody light chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO:13, and the antibody light chain may include the amino acid sequence shown in SEQ ID NO:14. For example, the Mab in the conjugate described in the present application may include an antibody heavy chain and an antibody light chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO:15, and the antibody light chain may include the amino acid sequence shown in SEQ ID NO:16.

[0130] Camptothecin derivatives

[0131] The conjugate of the present application has a structure of formula I, wherein R 1 It may be a methyl group substituted by halogen (eg, a methyl group substituted by chlorine, a methyl group substituted by fluorine, a methyl group substituted by bromine, a methyl group substituted by iodine); R 2 Can be selected from hydrogen, C 1 -C 6 Alkyl and substituted C 1 -C 6 Alkyl; R 3 Can be selected from hydrogen, amino, C 1 -C 6 Alkyl and substituted C1 -C 6 Alkyl. The amino group described in the present application may include a -NH 2 , may also include -NH 2 A group in which one or two hydrogen atoms are replaced by an alkyl group, for example, -N(CH 3 ) 2 NHCH 3 、-N(CH 2 CH 3 ) 2 、-N(CH 2 CH 2 CH 3 ) 2 、-NHCH 2 CH 3 or -NHCH 2 CH 2 CH 3 Etc. 1 -C 6 The alkyl group may be a straight chain alkyl group or a branched chain alkyl group, including but not limited to methyl, ethyl, n-propyl, isopropyl, tert-butyl and the like.

[0132] In this application, the term "substituted C 1 -C 6 "Alkyl" may refer to C 1- C 6 A group in which at least one hydrogen atom in an alkyl group is substituted by a halogen, an amino group, a hydroxyl group, an alkoxy group or an aryl group.

[0133] In some cases, R 3 Can be selected from hydrogen, amino, C 1 -C 4 Alkyl or substituted C 1 -C 4 alkyl; in other cases, R 3 Can be selected from hydrogen, amino or Among them, R 4 and R 5 R may be selected from hydrogen, C1-C6 alkyl, substituted C1-C6 alkyl or aryl. 3 The binding site of R1 can be any one of the three unsubstituted sites on the benzene ring, including the ortho or meta position of R1. 3 The binding site can be a small molecule C 9 Position. For example, R 3 It may be hydrogen.

[0134] In some cases, R 2 Can be selected from hydrogen, C 1 -C 6Alkyl and substituted C 1 -C 6 Similarly, C 1 -C 6 The alkyl group may be a straight chain alkyl group or a branched chain alkyl group, including but not limited to methyl, ethyl, n-propyl, isopropyl, tert-butyl, etc. 1 -C 6 The alkyl group may refer to a group in which at least one hydrogen atom in the C1-C6 alkyl group is substituted by a halogen, an amino group, a hydroxyl group, an alkoxy group or an aryl group. 2 Can be selected from hydrogen, C 1 -C 4 Alkyl or substituted C 1 -C 4 Alkyl; for example, R 2 Can be selected from Et, Among them, R 4 and R 5 can be selected from hydrogen, C 1 -C 6 Alkyl, substituted C 1 -C 6 Alkyl or aryl. For example, R 2 Can be -CH 2 CH 3 .

[0135] In some cases, R 1 It may be a methyl group substituted by a halogen. 1 It can be methyl substituted by fluorine, chlorine, bromine or iodine. In some cases, R1 can be methyl substituted by 1, 2 or 3 halogens. For example, R 1 Can be selected from -CF 2 H, -CCl 2 H, -CBr 2 H, -CFH 2 , -CBrH 2 , -CClH 3 , -CF 3 , -CCl 3 or -CBr 3 In certain embodiments, R 1 Can be -CF 2 H.

[0136] For example, the small molecule in the conjugate described in the present application can be the following structure:

[0137] Linker

[0138] The targeting polypeptide and the small molecule portion may be connected via a linker L. In certain embodiments, the linker L may comprise a defined polyethylene glycol (PEG) portion, an L-amino acid, and an additional spacer.

[0139] In some cases, the determined PEG portion is a PEG comprising a determined number of monomeric units, wherein the determined PEG can be a low molecular weight PEG, in some cases, can comprise 1-30 monomeric units, for example, 1-12 monomeric units. The determined PEG can have a reactive group (e.g., carboxylic acid or hydroxyl) at one end.

[0140] In some cases, the L-amino acid can be selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine ​​and proline. In some embodiments, the number of the L-amino acid can be 0, 1, 2, 3, 4 or more. For example, the number of the L-amino acid can be 1, and the 1 L-amino acid can be selected from any one of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine ​​and proline.

[0141] In some cases, the additional spacer may be selected from ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol or substituted or unsubstituted ethylenediamine. If it includes a hydroxyl group, it is attached to the hydroxyl or amino group of the drug in the form of a carbonate or carbamate, respectively. In some cases, the additional spacer is a substituted ethylenediamine derived from an L-amino acid in which the carboxylic acid group is replaced by a hydroxymethyl moiety. The additional spacer may be derived from any of the following L-amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine ​​and proline. In other cases, the additional spacer may be a foldable portion injected into 4-aminobenzyl alcohol or in the benzylic position with C1-C 10 Alkyl substituted 4-aminobenzyl alcohol, substituted 4-aminobenzyl alcohol is linked to L-amino acid or a polypeptide comprising a plurality of L-amino acid moieties via its amino group; wherein the N-terminus is linked to a cross-linker terminating in a targeting polypeptide moiety. For example, in the linker L, the additional spacer can be derived from substituted 4-aminobenzyl alcohol, the substituted 4-aminobenzyl alcohol being replaced by hydrogen or a selected from C 1 -C 10Alkyl substitution of alkyl.

[0142] In certain embodiments, the linker L may have the following structure:

[0143]

[0144] method

[0145] On the other hand, the present application provides a method for producing the conjugate, which may include: obtaining a compound represented by formula IV:

[0146]

[0147] Among them, R 1 It may be a methyl group substituted by halogen; R 2 Can be selected from hydrogen, C 1 -C 6 Alkyl and substituted C 1 -C 6 Alkyl; R 3 Can be selected from hydrogen, amino, C 1 -C 6 Alkyl and substituted C 1 -C 6 Alkyl; Mab can be a targeting polypeptide; L can be a linker; the compound represented by formula IV is conjugated with the Mab to obtain the conjugate.

[0148] The present application provides a method for preparing compound IV, which may include: 1) preparing a small molecule shown in formula I, 2) synthesizing a linker, and 3) obtaining a compound shown in formula IV.

[0149] The preparation of the small molecule of formula I may include reacting a 10-hydroxycamptothecin compound with a sulfonylating agent to obtain a sulfonate intermediate. In some cases, the structural formula of the 10-hydroxycamptothecin compound may be

[0150]

[0151] The structural formula of the sulfonate intermediate can be Among them, R 1 Can be selected from hydrogen, amino, C 1 -C 6 Alkyl or substituted C 1 -C 6 Alkyl, R 1 The binding site of R can be any of the three unsubstituted sites on the benzene ring; 2 Can be selected from hydrogen, C 1 -C 6 Alkyl or substituted C 1-C 6 Alkyl; R 3 Selected from hydrogen, acyl, C 1 -C 6 Alkyl, substituted C 1 -C 6 Alkyl; R 6 It may be selected from methanesulfonyl, p-toluenesulfonyl or trifluoromethanesulfonyl.

[0152] The sulfonylating agent can sulfonylate the C10 hydroxyl group of the 10-hydroxycamptothecin compound to obtain a leaving group -OTf, -OTs or -OMs, which is convenient for substitution with a halogen-substituted methyl group in the subsequent step. The sulfonylating agent can include a sulfonyl chloride reagent, a sulfonic acid reagent, a sulfonic anhydride reagent and a sulfonamide reagent, and according to the specific sulfonyl group, it can include at least one of methanesulfonyl chloride, p-toluenesulfonyl chloride, trifluoromethanesulfonyl chloride, methanesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, methanesulfonic anhydride, p-toluenesulfonic anhydride, trifluoromethanesulfonic anhydride and N-phenylbis(trifluoromethanesulfonimide).

[0153] The temperature for the reaction of 10-hydroxycamptothecin compounds with the sulfonylating agent can be 60-100° C., and the reaction time can be 2-8 hours. Under the above reaction conditions, the sulfonylation reaction is more complete, and the sulfonate intermediate can be obtained in high yield. For example, the molar ratio of the 10-hydroxycamptothecin compounds to the sulfonylating agent can be 1:1-1.5. Excess sulfonylating agent can ensure the full reaction of the 10-hydroxycamptothecin compounds and improve the utilization rate of raw materials.

[0154] The preparation of the small molecule represented by formula I may also include reacting the sulfonate intermediate with a halogen-substituted methyl reagent to obtain a 10-halomethylcamptothecin compound.

[0155] The preparation method described in the present application may also include synthesizing a linker L. In the present application, a defined polyethylene glycol (PEG) portion (i.e., a PEG containing a defined number of monomer units) is placed between the small molecule and the targeting polypeptide, wherein the defined PEG is a low molecular weight PEG, and in some cases, contains 1-30 monomer units, for example, 1-12 monomer units. The linker can enhance the solubility of the small molecule.

[0156] In some embodiments, one end of the determined polyethylene glycol portion of the L may carry different reactive groups, for example, a carboxyl or hydroxyl group. The polyethylene glycol portion may be connected to the amino group of an amino alcohol, and the hydroxyl group of the amino alcohol is connected to the hydroxyl group of a carbonate on a small molecule. In some cases, one end of the different reactive groups of the polyethylene glycol portion may be connected to the N-terminal of an L-amino acid or a polypeptide, and the C-terminal is connected to the amino group of the amino alcohol, and the hydroxyl group of the amino alcohol is connected to the carbonate or carbamate connected to the small molecule, respectively. In some cases, the final conjugate that can be used for conjugation with a targeting polypeptide can be provided via an acetylene-azide cycloaddition reaction with a small molecule. In some embodiments, the targeting polypeptide-coupling group is designed as a thiol or thiol reactive group.

[0157] The preparation method described in the present application may also include obtaining a compound shown in Formula IV, and conjugating the targeting portion and the small molecule in Formula I through the linker L.

[0158] Pharmaceutical compositions and uses

[0159] On the other hand, the present application provides a pharmaceutical composition comprising the conjugate. The conjugate or drug can be formulated according to known methods for preparing pharmaceutically usable compositions, whereby the immunoconjugate is mixed with a pharmaceutically suitable excipient in a mixture. Sterile phosphate buffered saline is an example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those skilled in the art.

[0160] On the other hand, the present application provides the use of the conjugate or pharmaceutical composition described herein in the preparation of a drug for preventing or treating a tumor. The present application also provides a method for preventing or treating a tumor, the method comprising administering the conjugate or pharmaceutical composition described herein to a subject in need thereof. Tumors that can be treated with drugs prepared using the conjugate or pharmaceutical composition described herein may include solid tumors and / or non-solid tumors, for example, the solid tumor may be selected from the following groups: prostate cancer, breast cancer, pancreatic cancer, colon cancer and gastric cancer. The conjugate described herein can be used to inhibit the growth, progression and / or metastasis of tumors, especially those cancers listed above.

[0161] On the other hand, the present application provides the use of the conjugate and other tumor therapies or drugs in the preparation of drugs for preventing or treating tumors. The present application also provides a method for preventing or treating tumors, the method comprising administering the conjugate or pharmaceutical composition described in the present application to a subject in need, as well as other tumor therapies or drugs. The tumor may include solid tumors and / or non-solid tumors, for example, the solid tumor may be selected from the following groups: prostate cancer, breast cancer, pancreatic cancer, colon cancer and gastric cancer. Depending on the disease state and the tolerance of the conjugate, the conjugate may be administered once or repeatedly, and may also be used in combination with other tumor therapies or drugs, which may include, for example, surgery, external radiation, radioimmunotherapy, chemotherapy, antisense therapy, interfering RNA therapy, gene therapy, etc. Each combination will be suitable for the tumor type, stage, patient condition and previous therapy, as well as other factors considered by the attending physician. In certain embodiments, the other tumor therapies or drugs are selected from the following groups: chemotherapy, radiotherapy, miRNA and oligonucleotides.

[0162] Chemotherapy that can be used in combination with the conjugate includes chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotic agents, antiangiogenic agents and pro-apoptotic agents, such as doxorubicin, methotrexate, paclitaxel, other camptothecins, and other forms of these and other classes of anticancer agents, etc. Other chemotherapeutic drugs may include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, etc. The chemotherapy can be part of the conjugate described in the present application, or alternatively can be used in combination before, at the same time, or after the conjugate.

[0163] Radiotherapy that can be used in combination with the conjugates described herein can include any mechanism for inducing local DNA damage in tumor cells, such as gamma-radiation, X-rays, UV-radiation, microwaves, electron emission, etc. Radiotherapy can also include combination therapy using targeted delivery of radioisotopes to tumor cells, and can be used in combination with or as part of the conjugates. Optionally, the radiotherapy can be administered as a single dose or as multiple consecutive doses.

[0164] Oligonucleotides that can be used in conjunction with the conjugates described herein can include any suitable short chain of nucleotides (including nucleotides within deoxyribonucleic acid DNA or ribonucleic acid RNA), for example, antisense oligonucleotides, small interfering RNA, ribozymes, deoxyribozymes, antigenes, CpG oligonucleotides, transcription factor decoys and nucleic acid aptamers.

[0165] Suitable routes of administration of the conjugate include, but are not limited to, oral, parenteral, rectal, transmucosal, enteral administration, intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal or intraocular injection. For example, the route of administration is parenteral. One may administer the compound in a local rather than a systemic manner, for example, via direct injection of the compound into a solid tumor.

[0166] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0167] Example

[0168] Example 1 Preparation of conjugate

[0169] 1.1 Linker Synthesis

[0170] 1.5 g of commercially available Fmoc-Lys-(MMT)-OH (CAS#167393-62-6) and 0.3 g of 4-aminobenzyl alcohol (CAS#623-04-1) were dissolved in 15 ml of dichloromethane at room temperature, and EEDQ (CAS#16357-59-8) was added to the mixture and stirred for 4 h. After extraction, 1.67 g of the intermediate product (Compound I) was obtained by flash chromatography as a white foam. Compound I was characterized as follows: ESI-MS m / e 745.8 (M+H).

[0171]

[0172] 1.5 g of the intermediate product was dissolved in diethylamine (10 mL, CAS#109-89-7) and stirred for 2 h. After removing the solvent, the residue was washed with hexane to obtain 0.97 g of the intermediate product (Compound II) as a colorless precipitate. Compound II was characterized as follows: ESI-MS m / e 549.2 (M+Na).

[0173]

[0174] 0.57 g of compound II was coupled with 0.63 g of commercially available PEG-N3 (O-(2-azidoethyl)-O'-(N-diglycolyl-2-aminoethyl) heptane glycol) in dichloromethane using EEDQ (CAS#16357-59-8). The solvent was removed and flash chromatography gave 0.99 g of linker (compound III) as a light yellow oil with a yield of 85%. Compound III was characterized as follows: ESI-MS: m / e 1082.7 (M+Na), m / e 1061.3 (M+H).

[0175]

[0176] 1.2Linker-Small Molecule Preparation

[0177] Compound V was prepared from compound IV (camptothecin derivative, for the preparation method, please refer to CN108690036A) using triphosgene (CAS#1122-58-3) and DMAP (CAS#32315-10-9).

[0178]

[0179] Under argon protection, 0.5 g of compound V and 0.95 g of compound III were reacted in 10 mL of dichloromethane for 10 min. The mixture was purified by flash chromatography to obtain 0.98 g of a light yellow oil (compound VI, yield 68%). Compound VI was characterized as follows: ESI-MS: m / e 1513.7 (M+H).

[0180]

[0181] In the presence of CuBr (0.01 g, CAS#7787-70-4), DIEA (0.01 mL, CAS#7087-68-5) and triphenylphosphine (0.02 g, CAS#603-35-0), compound VI (0.88 g) was reacted with 4-(N-maleimidomethyl)-N-(propargyl)-cyclohexane-1-amide (0.25 g) in dichloromethane for 18 h. It was then washed with EDTA (CAS#139-33-3). Flash chromatography gave compound VII (0.95 g, 85% yield) as a yellow foam. Compound VII was characterized as follows: ESI-MS m / e 1788.1 (M+H).

[0182]

[0183] Dichloroacetic acid (0.6 mL, CAS#79-43-6) and anisole (0.06 mL, CAS#100-66-3) were dissolved in dichloromethane (6 mL), 0.5 g of compound VII was deprotected, and precipitated with ether to obtain 0.4 g of compound VIII as a pale yellow powder with a yield of 94%. Compound VIII was characterized as follows: ESI-MS m / e 1517.2 (M+2H), m / e 758.8 ([M+2H] / 2).

[0184]

[0185] 1.3 Antibody reduction

[0186] Method 1 or method 2 can be used to obtain mildly reduced antibodies anti-Her2 antibodies trastuzumab, pertuzumab, anti-Trop2 antibodies hRS7, and hTINA1. Among them, the amino acid sequence of the heavy chain variable region of trastuzumab is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of pertuzumab is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of hRS7 antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 6; the amino acid sequence of the heavy chain variable region of hTINA1 antibody is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 8.

[0187] Method 1: In 40 mM PBS (Na 2 HPO 4 、NaH 2 PO 4 Each antibody was reduced with 50-70 times molar excess of dithiothreitol (DTT, CAS#3483-12-3) for 45 min in 5% paraformaldehyde (pH=7.4) solution. The reduced product was purified by size exclusion chromatography and diafiltration. The buffer was then replaced with an appropriate buffer at pH 6.5. Finally, the thiol content was determined by Ellman's test (DTNB, CAS69-78-3) and the absorbance at 412 nm was measured.

[0188] Method 2: The antibody was reduced with Tris (2-carboxyethyl) phosphine (TCEP, CAS# 51805-45-9) in phosphate buffer in the pH range of 5-7.

[0189] MAb→Reduced MAb

[0190] 1.4 Conjugation

[0191] Using 7-15% v / v DMSO as a co-solvent, the reduced antibody was reacted with a 10-15-fold molar excess of compound VIII and incubated at room temperature for 20 minutes. The conjugate was purified by size exclusion chromatography, hydrophobic separation, and finally by ultrafiltration-dialysis. The camptothecin absorption of the product was determined by the absorbance at 366nm using a UV spectrophotometer, which was correlated with the standard value, and the protein concentration was determined from the absorbance at 280nm, removing the camptothecin absorption. The substitution ratio of small molecule drug / antibody was thus determined.

[0192] The purified conjugate was stored as a lyophilized formulation in glass vials, capped under vacuum and stored in a -20 °C freezer.

[0193]

[0194] The obtained conjugates were named HB010 (the antibody is trastuzumab), HB020 (the antibody is pertuzumab), HB030 (the antibody is hRS7), HB040 (the antibody is hTINA1), HB050 (the antibody is anti-CLDN18.2 antibody) and HB060 (the antibody is anti-nectin 4 antibody).

[0195] At the same time, compound IV was replaced with SN-38 (CAS#86639-52-3), and the control conjugates HB011 (the antibody is trastuzumab), HB021 (the antibody is pertuzumab), HB031 (the antibody is hRS7), HB041 (the antibody is hTINA1), HB051 (the antibody is anti-CLDN18.2 antibody) and HB061 (the antibody is anti-nectin 4 antibody) were prepared in the same manner.

[0196] According to the above method, the difluoromethyl compound IV is replaced by a monofluoromethyl compound, a chlorinated compound, or a brominated compound to prepare a conjugate.

[0197] The prepared conjugate and drug-antibody ratios are as follows:

[0198] Table 1 Exemplary conjugates and their DAR values

[0199]

[0200]

[0201] Example 2 Cell Binding Activity

[0202] The binding activity of the conjugate obtained in Example 1 with the antigen was detected. The cells in the logarithmic growth phase were harvested, and the cell viability was detected by trypan blue exclusion method to ensure that the cell viability was above 90%. After centrifugation at 1000 r / min for 5 min, the supernatant was discarded; the cells were washed once with PBS; the cells were resuspended with FACS Buffer and counted; the density of 5×10 6 cells / mL of cell suspension; 80 μL of cell suspension was added to each 96-well plate; a blank control group (without antibody or secondary antibody), a secondary antibody control group (without antibody but with secondary antibody) and an experimental group (with antibody and secondary antibody) were set up.

[0203] 20 μL of the test substance with different gradient concentrations was added to the experimental group, and the final concentration was 20 μg / ml, 3.16-fold dilution, and 8 concentrations. An equal volume of FACS buffer was added to the other control groups; after mixing, the cells were incubated at 4°C in the dark for 40 min; the cells were washed 3 times with FACS buffer, 400 μL each time, centrifuged at 1000 r / min for 5 min, and finally resuspended with 100 μL FACS buffer; then 5 μL of PE-labeled secondary antibody (PE anti-human IgG Fc antibody) was added to the experimental group and the secondary antibody control group, and an equal volume of FACS buffer was added to the blank control group; after mixing, the cells were incubated at 4°C in the dark for 40 min; then, the cells were washed 3 times with FACS buffer, 400 μL each time, centrifuged at 1000 r / min for 5 min, and finally resuspended with 250 μL FACS buffer; YEL-HLog (Excitation Laser: 488nm Blue Laser) was detected by flow cytometry. Finally, FACS data were analyzed using FlowJo software.

[0204] The binding activity of the sample to be tested with the antigen is Figure 1-2 As shown in Table 2 below. The results show that the binding EC of the conjugate HB010 described in the present application to Her2 50 The values ​​were not significantly different from those of the control molecule HB011. The EC values ​​of HB030 and TROP2 binding were 50 Slightly higher than the control molecule HB031.

[0205] The results showed that the conjugates connected with different Her2 antibodies or Trop2 antibodies can also effectively bind to the antigen. After the difluoromethyl substitution is replaced with other halogen substitutions (such as chlorine, bromine), the resulting conjugates also have antigen binding activity.

[0206] Table 2 Binding activity of conjugates to antigens

[0207]

[0208] Example 3 In vitro antitumor activity

[0209] The in vitro antitumor activity of the conjugate obtained in Example 1 was detected. Cells in the logarithmic growth phase were collected by cold light detection and counted by a platelet counter. Cell viability was detected by trypan blue exclusion to ensure that the cell viability was above 90%. The commercially available cell lines, culture media and cell inoculation numbers were shown in Table 3 below.

[0210] Table 3 Culture conditions of cancer cell lines

[0211] Cell lines Cell Type Number of cells / well Culture medium MDA-MB-468 Breast cancer 3000 RPMI1640+10%FBS HCC1954 Breast cancer 3000 RPMI1640+10%FBS SK-BR-3 Breast cancer 5000 RPMI1640+10%FBS BXPC-3 Pancreatic cancer 5000 DMEM + 10% FBS COLO205 Colon cancer 3000 RPMI1640+10%FBS NCI-N87 Gastric cancer 5000 RPMI1640+10%FBS

[0212] Add 90 μl of cell suspension to each 96-well plate and place the cells in the 96-well plate at 37°C and 5% CO. 2 , 95% humidity overnight. Prepare a concentration gradient dilution of the drug to be tested solution, add 10 μl of the drug to be tested to each well of the 96-well plate inoculated with cells, and set up three replicate wells for each concentration. Place the cells in the 96-well plate with the drug added at 37°C and 5% CO 2 , 95% humidity conditions for 72 hours, then add CTG reagent (Promega, Cat#G7572) for measuring luminescent cell viability for analysis. Melt the CTG reagent and equilibrate the cell plate to room temperature for 30 minutes, add an equal volume of CTG solution to each well, shake on an orbital shaker for 5 minutes to lyse the cells, place the cells at room temperature for 20 minutes, and read the cold light signal using a SpectraMax (MD, 2104-0010A) multi-label microplate reader. GraphPad Prism5.0 software was used to analyze the data, and nonlinear S-curve regression was used to fit the data to obtain a dose-effect curve, and the IC50 value was calculated from this. The calculation results are shown in the following table. Figure 3-8 and as shown in Table 4 below.

[0213] The results showed that HB010 and HB030 had significant inhibitory effects on a variety of tumor cells. The anti-tumor effect of HB010 on breast cancer and gastric cancer cell lines was not significantly different from that of the control molecule HB011, while the anti-tumor effect of HB030 on breast cancer, pancreatic cancer, colon cancer and gastric cancer cell lines was better than that of the control molecule HB031.

[0214] The results showed that conjugates connected to different Her2 antibodies or Trop2 antibodies can also effectively inhibit tumor cell growth. After replacing the difluoromethyl substitution with other halogen substitutions (such as chlorine and bromine), the resulting conjugates also have an inhibitory effect on cancer cell lines.

[0215] Table 4 In vitro activity of the conjugates in inhibiting cancer cell growth

[0216]

[0217] Example 4 Pharmacokinetic Experiment

[0218] The conjugate obtained in Example 1 was intravenously injected into mice, and blood samples were collected before administration and within a certain period of time after the injection. The serum was separated and the concentration of the drug in the serum was determined by Elisa method, and the pharmacokinetic parameters were calculated by software.

[0219] The results show the half-life of the conjugate in mice.

[0220] Example 5 In vivo antitumor activity

[0221] Mice with breast cancer, pancreatic cancer, colon cancer or gastric cancer xenografts were treated with the conjugate prepared in Example 1. The therapeutic effect of the conjugate was observed. The results showed that the conjugate described in the present application can effectively treat tumors in vitro.

[0222] Example 6 The conjugate of the present application inhibits tumor growth in breast cancer mice

[0223] 6.1SK-BR-3 Breast Cancer

[0224] (1) Fifty 5- to 6-week-old female BALB / c nude mice were acclimated to SPF conditions for 5 days and inoculated under the armpit with 4 × 10 6 SK-BR-3 breast cancer cells. Feed sterilized feed and 10ppm sodium hypochlorite sterilized tap water. When the tumor volume is about 130mm 3 At the same time, the mice were randomly divided into 5 groups, with 10 mice in each group: normal saline group, irinotecan (5 mg / kg) group, HB011 (0.5 mg / kg) group, HB010 (0.5 mg / kg) group, and HB010 (0.05 mg / kg) group. According to the corresponding dose, each mouse was injected into the tail vein twice a week for 4 consecutive weeks, and then observed for 10 days after drug withdrawal. The long and short diameters of the tumor were measured once a week with an electronic digital caliper, and the weight of the nude mice was weighed once with an electronic scale.

[0225] Tumor volume (mm 3 )=1 / 2×long diameter(mm)×[short diameter(mm)] 2

[0226] Percentage change of tumor volume (%) = (weekly tumor volume - first administration tumor volume) / first administration tumor volume × 100%

[0227] The results are as follows Figure 9-10 As shown: the weight of nude mice in each group increased positively over time, and there was no significant difference in weight between the groups at each time point, and no serious toxicity was observed in each group. The tumor volume of the HB010 (0.5 mg / kg) group became smaller than that of the other groups, and its average percentage change was statistically significantly different from that of the other groups (P<0.05 or 0.001); the tumor volume of the HB010 (0.05 mg / kg) group was smaller than that of the saline group, but larger than that of the irinotecan (5 mg / kg) group or the HB011 (0.5 mg / kg) group. And at the same dose, HB010 was slightly better than HB011 in inhibiting the growth of breast cancer. Fig. 9 The mean percentage of tumor volume in the HB010 (0.5 mg / kg) group was compared with that in the other groups *P < 0.05, #P < 0.001; Fig.10 There was no statistically significant difference in the average body weight of the HB010 (0.5 mg / kg) group compared with the other groups.

[0228] The results showed that HB010 had a better inhibitory effect on SK-BR-3 breast cancer than irinotecan and HB011, with a certain dose-effect relationship, and no toxic reactions were observed.

[0229] (2) Alternatively, 6×10 6 SK-BR-3 breast cancer cells were inoculated into female nude mice and the mice were observed for 10 days after drug withdrawal to detect the inhibitory effects of HB060 and HB061 on tumors.

[0230] The results are as follows Figure 11-12 As shown, the weight of nude mice in each group increased positively over time, and there was no significant difference in weight between the groups at each time point, and no serious toxicity was observed in each group. The tumor volume of the HB060 (0.5 mg / kg) group became smaller than that of the other groups, and its average percentage change was statistically significantly different from that of the other groups (P<0.05 or 0.001); the tumor volume of the HB060 (0.05 mg / kg) group was smaller than that of the saline group, but larger than that of the irinotecan (5 mg / kg) group or the HB061 (0.5 mg / kg) group. And at the same dose, HB060 has a better effect on inhibiting the growth of breast cancer than HB061. Fig.11 The mean percentage of tumor volume in the HB060 (0.5 mg / kg) group was compared with that in the other groups *P < 0.05, #P < 0.001; Fig.12 There was no statistically significant difference in the average body weight of the HB060 (0.5 mg / kg) group compared with the other groups.

[0231] 6.2 MDA-MB-468 Breast Cancer

[0232] (1) Inoculate 2×10 6 MDA-MB-468 breast cancer cells were inoculated into female nude mice and observed for 17 days after drug withdrawal. The mice were given drugs according to the method in 6.1 and the changes in tumor volume and average body weight of the mice were detected.

[0233] The results are as follows Figure 13-14 As shown: the weight of nude mice in each group increased positively over time, and there was no significant difference in weight between the groups at each time point, and no serious toxic reactions were observed in the groups. The tumor volume of the HB030 (0.5 mg / kg) group became smaller than that of the other groups, and its average percentage change was statistically significantly different from that of the other groups (P<0.05 or 0.001); the tumor volume of the HB030 (0.05 mg / kg) group was smaller than that of the saline group, similar to that of the irinotecan (5 mg / kg) group, and larger than that of the HB031 (0.5 mg / kg) group. And at the same dose, the effect of HB030 in inhibiting breast cancer growth is similar to or even slightly better than that of HB031. Fig.13The average percentage of tumor volume in the HB030 (0.5 mg / kg) group was compared with that in the other groups *P < 0.05, #P < 0.001; Fig.14 There was no statistically significant difference in the average body weight of the HB030 (0.5 mg / kg) group compared with the other groups.

[0234] (2) Inoculate 6×10 6 MDA-MB-468 breast cancer cells were injected into female nude mice once every 5 days through the tail vein and observed for 10 days after drug withdrawal. Figure 13-14 As shown, the tumor inhibition effect of HB030 (0.2 mg / kg) was significantly better than that of other groups, and its anti-tumor activity was at least 2.5 times that of HB031 (0.5 mg / kg). No toxicity was observed in any group. Fig.15 The mean percentage of tumor volume in the HB030 (0.2 mg / kg) group was compared with that in the other groups *P < 0.001; Fig.16 There was no statistically significant difference in the average body weight of the HB030 (0.2 mg / kg) group compared with the other groups.

[0235] Figure 13-16 The results showed that HB030 had a better inhibitory effect on MDA-MB-468 breast cancer than irinotecan, anti-Trop2 antibody and HB031, with a certain dose-effect relationship, and no toxic reactions were observed.

[0236] Example 7 The conjugate of the present application inhibits tumor growth in gastric cancer mice

[0237] 7.1 NCI-N87 Gastric Cancer

[0238] (1) Inoculate 4×10 6 The NCI-N87 gastric cancer cells were transferred to female nude mice and observed for 10 days after drug withdrawal to detect the inhibitory effects of HB010 and HB011 on tumors.

[0239] The results are as follows Figure 17-18 As shown, the weight of nude mice in each group increased positively over time, and there was no significant difference in weight between the groups at each time point, and no serious toxicity was observed in each group. The tumor volume of the HB010 (0.5 mg / kg) group became smaller than that of the other groups, and its average percentage change was statistically significantly different from that of the other groups (P<0.05 or 0.001); the tumor volume of the HB010 (0.05 mg / kg) group was smaller than that of the saline group, but larger than that of the irinotecan (5 mg / kg) group or the HB011 (0.5 mg / kg) group. And at the same dose, HB010 was slightly better than HB011 in inhibiting the growth of gastric cancer. Fig.17The mean percentage of tumor volume in the HB010 (0.5 mg / kg) group was compared with that in the other groups *P < 0.05, #P < 0.001; Fig.18 There was no statistically significant difference in the average body weight of the HB010 (0.5 mg / kg) group compared with the other groups.

[0240] The results showed that HB010 had a better inhibitory effect on NCI-N87 gastric cancer than irinotecan and HB011, with a certain dose-effect relationship, and no toxic reactions were observed.

[0241] (2) Inoculate 6×10 6 The NCI-N87 gastric cancer cells were transferred to female nude mice and observed for 17 days after drug withdrawal to detect the inhibitory effects of HB050 and HB051 on tumors.

[0242] The results are as follows Figure 19-20 As shown, the weight of nude mice in each group increased positively over time, and there was no significant difference in weight between the groups at each time point, and no serious toxicity was observed in each group. The tumor volume of the HB050 (0.5 mg / kg) group became smaller than that of the other groups, and its average percentage change was statistically significantly different from that of the other groups (P<0.05 or 0.001); the tumor volume of the HB050 (0.05 mg / kg) group was smaller than that of the saline group, but larger than that of the irinotecan (5 mg / kg) group or the HB051 (0.5 mg / kg) group. And at the same dose, HB050 has a better effect on inhibiting the growth of gastric cancer than HB051. Fig.19 The mean percentage of tumor volume in the HB050 (0.5 mg / kg) group was compared with that in the other groups *P < 0.05, #P < 0.001; Fig. 20 There was no statistically significant difference in the average body weight of the HB050 (0.5 mg / kg) group compared with the other groups.

[0243] The results showed that HB050 had a better inhibitory effect on NCI-N87 gastric cancer than irinotecan and HB051, with a certain dose-effect relationship, and no toxic reactions were observed.

[0244] (3) Inoculate 6×10 6 NCI-N87 gastric cancer cells were injected into male nude mice once every 5 days through the tail vein. The mice were observed for 10 days after drug withdrawal. Figure 21-22 As shown, the tumor inhibition effect of HB050 (0.2 mg / kg) was significantly better than that of the other groups, and its anti-tumor activity was at least 2.5 times that of HB051 (0.5 mg / kg). No toxicity was observed in any group. Fig.21The mean percentage of tumor volume in the HB050 (0.2 mg / kg) group was compared with that in the other groups *P < 0.001; Fig. 22 There was no statistically significant difference in the average body weight of the HB050 (0.2 mg / kg) group compared with the other groups.

[0245] Example 8 The conjugate of the present application inhibits tumor growth in pancreatic cancer mice

[0246] 8.1BXPC-3 Pancreatic Cancer

[0247] (1) Inoculate 4×10 6 BXPC-3 pancreatic cancer cells were transplanted into female nude mice and observed for 17 days after drug withdrawal to detect the inhibitory effects of HB030 and HB031 on tumors.

[0248] The results are as follows Figure 23-24 As shown, the weight of nude mice in each group increased positively over time, and there was no significant difference in weight between the groups at each time point, and no serious toxicity was observed in each group. The tumor volume of the HB030 (0.5 mg / kg) group became smaller than that of the other groups, and its average percentage change was statistically significantly different from that of the other groups (P<0.05 or 0.001); the tumor volume of the HB030 (0.05 mg / kg) group was smaller than that of the saline group, but larger than that of the irinotecan (5 mg / kg) group or the HB031 (0.5 mg / kg) group. And at the same dose, HB030 has a better effect on inhibiting the growth of pancreatic cancer than HB031. Fig.23 The average percentage of tumor volume in the HB030 (0.5 mg / kg) group was compared with that in the other groups *P < 0.05, #P < 0.001; Fig.24 There was no statistically significant difference in the average body weight of the HB030 (0.5 mg / kg) group compared with the other groups.

[0249] The results showed that HB030 had a better inhibitory effect on BXPC-3 pancreatic cancer than irinotecan and HB031, with a certain dose-effect relationship, and no toxic reactions were observed.

[0250] (1) Inoculate 6×10 6 BXPC-3 pancreatic cancer cells were transplanted into female nude mice and observed for 17 days after drug withdrawal to detect the inhibitory effects of HB050 and HB051 on tumors.

[0251] The results are as follows Figure 25-26As shown, the weight of nude mice in each group increased positively over time, and there was no significant difference in weight between the groups at each time point, and no serious toxicity was observed in each group. The tumor volume of the HB050 (0.5 mg / kg) group became smaller than that of the other groups, and its average percentage change was statistically significantly different from that of the other groups (P<0.05 or 0.001); the tumor volume of the HB050 (0.05 mg / kg) group was smaller than that of the saline group, but larger than that of the irinotecan (5 mg / kg) group or the HB051 (0.5 mg / kg) group. And at the same dose, HB050 has a better effect on inhibiting the growth of pancreatic cancer than HB051. Fig.25 The mean percentage of tumor volume in the HB050 (0.5 mg / kg) group was compared with that in the other groups *P < 0.05, #P < 0.001; Fig.26 There was no statistically significant difference in the average body weight of the HB050 (0.5 mg / kg) group compared with the other groups.

[0252] The results showed that HB050 had a better inhibitory effect on BXPC-3 pancreatic cancer than irinotecan and HB051, with a certain dose-effect relationship, and no toxic reactions were observed.

[0253] Example 9 The conjugate of the present application inhibits tumor growth in prostate cancer mice

[0254] 9.1PC3 Prostate Cancer

[0255] (1) Inoculate 6×10 6 PC3 prostate cancer cells were transplanted into male nude mice and the mice were observed for 10 days after drug withdrawal to detect the inhibitory effects of HB050 and HB051 on tumors.

[0256] The results are as follows Figure 27-28 As shown, the weight of nude mice in each group increased positively over time, and there was no significant difference in weight between the groups at each time point, and no serious toxicity was observed in each group. The tumor volume of the HB050 (0.5 mg / kg) group became smaller than that of the other groups, and its average percentage change was statistically significantly different from that of the other groups (P<0.05 or 0.001); the tumor volume of the HB050 (0.05 mg / kg) group was smaller than that of the saline group, but larger than that of the irinotecan (5 mg / kg) group or the HB051 (0.5 mg / kg) group. And at the same dose, HB050 has a better effect on inhibiting the growth of prostate cancer than HB051. Fig. 27 The mean percentage of tumor volume in the HB050 (0.5 mg / kg) group was compared with that in the other groups *P < 0.05, #P < 0.001; Fig.28 There was no statistically significant difference in the average body weight of the HB050 (0.5 mg / kg) group compared with the other groups.

[0257] The results showed that HB050 had a better inhibitory effect on PC3 prostate cancer than irinotecan and HB051, with a certain dose-effect relationship, and no toxic reactions were observed.

[0258] (2) Inoculate 6×10 6 PC3 prostate cancer cells were implanted into male nude mice and injected into the tail vein once every 5 days. The mice were observed for 10 days after drug withdrawal. Figure 29-30 The results showed that the tumor inhibition effect of HB060 (0.2 mg / kg) was significantly better than that of the other groups, and its anti-tumor activity was at least 2.5 times that of HB061 (0.5 mg / kg). No toxicity was observed in any group. Fig.29 The mean percentage of tumor volume in the HB060 (0.2 mg / kg) group was compared with that in the other groups *P < 0.001; Fig.30 There was no statistically significant difference in the average body weight of the HB060 (0.2 mg / kg) group compared with the other groups.

[0259] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments currently listed in this application are obvious to those of ordinary skill in the art and are retained within the scope of the appended claims and their equivalents. Sequence Listing <110> Beijing Haibu Pharmaceutical Technology Co., Ltd. <120> Targeted polypeptide-drug conjugate and its use <130> 0152-PA-002CN <160> 44 <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab heavy chain variable region <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab light chain variable region <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Pertuzumab heavy chain variable region <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Pertuzumab light chain variable region <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 5 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> hRS7 heavy chain variable region <400> 5 Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Ser Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Variable region of hRS7 light chain <400> 6 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 7 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of hTINA1 <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30 Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60 Lys Gly Arg Val Thr Leu Ser Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> hTINA1 light chain variable region <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 9 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab heavy chain <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 10 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab light chain <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 11 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> hRS7 heavy chain <400> 11 Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 12 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Light chain of hRS7 antibody <400> 12 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 13 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> anti-nectin-4 antibody heavy chain <400> 13 Gln Val Gly Leu Ala Gln Ser Gly Ala Glu Leu Gly Arg Pro Gly Gly 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Ile Gln Leu Ser Thr Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Tyr Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala 115 120 125 Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu 130 135 140 Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly 145 150 155 160 Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp 165 170 175 Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro 180 185 190 Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys 195 200 205 Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro 210 215 220 Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro 245 250 255 Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val 260 265 270 Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala 290 295 300 Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys 305 310 315 320 Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser 325 330 335 Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro 340 345 350 Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val 355 360 365 Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly 370 375 380 Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp 405 410 415 Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His 420 425 430 Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 435 440 445 <210> 14 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Anti-nectin-4 antibody light chain <400> 14 Asp Ile Val Met Thr Gln Ala Val Phe Ser Asn Pro Gln Thr Leu Gly 1 5 10 15 Thr Ser Gly Ser Ile Ser Cys Arg Ser Ser Lys Ser Val Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Ala Tyr Trp Tyr Leu Gln Lys Pro Gly Gly Ser 35 40 45 Pro Gln Leu Gly Ile Tyr His Met Ser Asn Leu Ala Ser Ala Val Pro 50 55 60 Asp Arg Phe Thr Ser Ser Val Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Ala Glu Val Glu Asp Val Gln Ala Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Phe Thr Phe Gly Leu Gly Thr Lys Ala Glu Thr Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gln Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Val Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Leu Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 Pro Ile Val Lys Thr Phe Asn Arg Asn Glu Cys 210 215 <210> 15 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 antibody heavy chain <400> 15 Met Asp Trp Gln Trp Asn Leu Gln Phe Leu Met Val Gly Ala Leu Ser 1 5 10 15 Ile Val Gly Ala Ile Gln Gln Val Leu Ser Ala Pro Glu Leu Lys Lys 20 25 30 Pro Ala Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Val Tyr Thr Phe 35 40 45 Thr Asn Tyr Gly Met Asn Trp Ala Lys Leu Gly Pro Leu Lys Gly Leu 50 55 60 Lys Trp Met Gln Trp Ile Asn Thr Asn Thr Ala Glu Pro Thr Tyr Val 65 70 75 80 Glu Glu Phe Lys Ala Arg Phe Gln Phe Ser Leu Glu Thr Ser Leu Ser 85 90 95 Thr Gly Tyr Leu Ala Ile Asn Asn Leu Lys Asn Glu Asp Thr Val Thr 100 105 110 Tyr Phe Cys Gln Arg Leu Gly Phe Gly Asn Leu Met Asp Tyr Trp Leu 115 120 125 Val Ala Thr Ser Val Thr Val Ser Ser Leu Ser Thr Lys Ala Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gln Gly Thr Ala 145 150 155 160 Ala Leu Leu Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> 16 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 antibody light chain <400> 16 Met Glu Ser Gly Thr Gly Leu Leu Met Ser Gln Ala Phe Trp Gly Ser 1 5 10 15 Ala Thr Cys Val Asp Ile Gln Met Thr Ala Ser Pro Ser Ser Leu Thr 20 25 30 Gly Thr Ala Val Glu Lys Leu Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Ala Leu Asn Ser Gly Asn Gln Lys Asn Tyr Gln Thr Trp Tyr Gln Ala 50 55 60 Lys Pro Gln Ala Pro Pro Lys Leu Leu Ile Tyr Trp Val Ser Thr Arg 65 70 75 80 Glu Ser Leu Gln Pro Asp Arg Phe Thr Gly Ser Val Ser Ala Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Ala Leu Gly Glu Asp Gln Val Ala Tyr 100 105 110 Tyr Cys Ala Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gln Gly Val Thr 115 120 125 Lys Leu Glu Leu Lys Arg Thr Val Gln Gly Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Ala Leu Lys Ser Val Thr Gln Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Ala Trp Lys Val 165 170 175 Asp Asn Gln Leu Gly Ser Gly Asn Ser Ala Glu Ser Gln Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab HCDR1 <400> 17 Gly Phe Asn Ile Lys Asp Thr Tyr Ile His 1 5 10 <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab HCDR2 <400> 18 Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab HCDR3 <400> 19 Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab LCDR1 <400> 20 Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala 1 5 10 <210> twenty one <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab LCDR2 <400> twenty one Serve me, I'll be there soon 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Trastuzumab LCDR3 <400> twenty two Gln Gln His Tyr Thr Thr Pro Pro Thr 1 5 <210> twenty three <211> 11 <212> PRT <213> Artificial Sequence <220> <223> hRS7 HCDR1 <400> twenty three Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 1 5 10 <210> twenty four <211> 18 <212> PRT <213> Artificial Sequence <220> <223> hRS7 HCDR2 <400> twenty four Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe Lys 1 5 10 15 Gly Arg <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> hRS7 HCDR3 <400> 25 Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> hRS7 LCDR1 <400> 26 Lys Ala Ser Gln Asp Val Ser Ile Ala Val Ala 1 5 10 <210> 27 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> hRS7 LCDR2 <400> 27 Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hRS7 LCDR3 <400> 28 Gln Gln His Tyr Ile Thr Pro Leu Thr 1 5 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Anti-nectin-4 antibody HCDR1 <400> 29 Ser Gly Tyr Thr Phe Thr Thr Tyr Trp Met Gln Trp 1 5 10 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Anti-nectin-4 antibody HCDR2 <400> 30 Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys 1 5 10 <210> 31 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> anti-nectin-4 antibody HCDR3 <400> 31 Tyr Cys Ala Arg Glu Tyr Tyr Gly Leu Asp Tyr Trp 1 5 10 <210> 32 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> anti-nectin-4 antibody VH <400> 32 Gln Val Gly Leu Ala Gln Ser Gly Ala Glu Leu Gly Arg Pro Gly Gly 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Ile Gln Leu Ser Thr Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Tyr Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 33 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Anti-nectin-4 antibody LCDR1 <400> 33 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr 1 5 10 15 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Anti-nectin-4 antibody LCDR2 <400> 34 Tyr His Met Ser Asn Leu Ala Ser Gly 1 5 <210> 35 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Anti-nectin-4 antibody LCDR3 <400> 35 Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Phe 1 5 10 <210> 36 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Anti-nectin-4 antibody VL <400> 36 Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr His Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Thr Lys 100 105 110 <210> 37 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 antibody HCDR1 <400> 37 Gln Ser Ala Pro Glu Leu Lys Lys Pro Ala Glu Thr Val 1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 Antibody HCDR2 <400> 38 Asn Trp Val Lys Gln Gly Pro Ala Lys Gly 1 5 10 <210> 39 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 Antibody HCDR3 <400> 39 Phe Cys Gly Arg Leu Gly Phe Ala Asn Ala Met Asp Tyr 1 5 10 <210> 40 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 antibody VH <400> 40 Met Asp Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Gly Ala Gln Ser 1 5 10 15 Ile Gln Gly Gln Ile Gln Leu Val Gln Ser Ala Pro Glu Leu Lys Lys 20 25 30 Pro Ala Glu Thr Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Ala Met Asn Trp Val Lys Gln Gly Pro Ala Lys Gly Leu 50 55 60 Lys Trp Met Ala Trp Ile Asn Thr Asn Thr Ala Glu Pro Thr Tyr Gly 65 70 75 80 Glu Glu Phe Lys Gly Arg Phe Gly Phe Ser Leu Glu Thr Ser Ala Ser 85 90 95 Thr Gly Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Val Thr 100 105 110 Tyr Phe Cys Gly Arg Leu Gly Phe Ala Asn Ala Met Asp Tyr Trp Ala 115 120 125 Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 antibody LCDR1 <400> 41 Met Thr Gln Ser Pro Ser Ser Leu Thr Gly 1 5 10 <210> 42 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 antibody LCDR2 <400> 42 Gln Lys Pro Ala Gln Pro Pro Lys Leu Leu Ile Tyr Trp 1 5 10 <210> 43 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 antibody LCDR3 <400> 43 Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly 1 5 10 <210> 44 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 Antibody VL <400> 44 Met Glu Ser Gln Thr Gln Ala Leu Met Ser Leu Leu Phe Trp Gly Ser 1 5 10 15 Ala Thr Cys Val Asp Ile Ala Met Thr Gln Ser Pro Ser Ser Leu Thr 20 25 30 Gly Thr Ala Val Glu Lys Ala Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln 50 55 60 Lys Pro Ala Gln Pro Pro Lys Leu Leu Ile Tyr Trp Gly Ser Thr Arg 65 70 75 80 Glu Ser Ala Val Pro Asp Arg Phe Thr Ala Ser Val Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Gly Gln Gly Glu Asp Leu Val Ala Tyr 100 105 110 Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Val Ala Thr 115 120 125 Lys Leu Glu Leu Lys 130

Claims

1. A conjugate having a structure shown in formula III: Formula III; The MAb comprises an antibody or an antigen-binding fragment thereof, wherein the MAb specifically binds to a tumor-specific antigen, wherein the tumor-specific antigen is selected from the group consisting of Her2, Trop2, CLDN18.2 and Nectin 4.

2. The conjugate according to claim 1, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody and a humanized antibody. The conjugate according to claim 1 , wherein the antibody is a fully human antibody.

4. The conjugate of claim 1, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv and ScFv fragments.

5. The conjugate according to claim 1 or 2, wherein the MAb comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are any one group of amino acid sequences selected from the following groups (1)-(4): (1) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, HCDR3: SEQ ID NO: 19, LCDR1: SEQ ID NO: 20, LCDR2: SEQ ID NO: 21 and LCDR3: SEQ ID NO: 22; (2) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 24, HCDR3: SEQ ID NO: 25, LCDR1: SEQ ID NO: 26, LCDR2: SEQ ID NO: 27 and LCDR3: SEQ ID NO: 28; (3) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, HCDR3: SEQ ID NO: 31, LCDR1: SEQ ID NO: 33, LCDR2: SEQ ID NO: 34 and LCDR3: SEQ ID NO: 35; and (4) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 38, HCDR3: SEQ ID NO: 39, LCDR1: SEQ ID NO: 41, LCDR2: SEQ ID NO: 42 and LCDR3: SEQ ID NO:

43.

6. The conjugate according to claim 1 or 2, wherein the MAb comprises an antibody heavy chain variable region VH and a light chain variable region VL, and the VH and VL are any one of the following amino acid sequences (1) to (4): (1) the VH is the amino acid sequence shown in SEQ ID NO: 1, and the VL is the amino acid sequence shown in SEQ ID NO: 2; (2) the VH is the amino acid sequence shown in SEQ ID NO: 5, and the VL is the amino acid sequence shown in SEQ ID NO: 6; (3) the VH is the amino acid sequence shown in SEQ ID NO: 32, and the VL is the amino acid sequence shown in SEQ ID NO: 36; (4) The VH is the amino acid sequence shown in SEQ ID NO: 40, and the VL is the amino acid sequence shown in SEQ ID NO:

44.

7. The conjugate according to claim 1 or 2, wherein the MAb comprises an antibody heavy chain and an antibody light chain, and the antibody heavy chain and the antibody light chain are any one of the following amino acid sequences (1) to (4): (1) The antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 9, and the antibody light chain has the amino acid sequence shown in SEQ ID NO: 10; (2) The antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 11, and the antibody light chain has the amino acid sequence shown in SEQ ID NO: 12; (3) The heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO: 13, and the light chain of the antibody has the amino acid sequence shown in SEQ ID NO: 14; (4) The antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 15, and the antibody light chain has the amino acid sequence shown in SEQ ID NO:

16.

8. A method for producing the conjugate of claim 1, comprising: The compound shown in formula VIII is obtained: Formula VIII, The compound represented by formula VIII is conjugated with the MAb to obtain the conjugate of claim 1.

9. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 7.

10. Use of the conjugate according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing or treating a tumor, wherein the tumor is selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, gastric cancer and prostate cancer.

11. Use of the conjugate of any one of claims 1-7 together with other tumor therapies or other drugs in the preparation of a drug for preventing or treating tumors, wherein the tumor is selected from the following group: breast cancer, pancreatic cancer, colon cancer, gastric cancer and prostate cancer, wherein the other tumor therapy is selected from the following group: chemotherapy and radiotherapy, and wherein the other drug is an oligonucleotide.

12. Use of the conjugate of any one of claims 1-7 together with other tumor therapies or other drugs in the preparation of a drug for preventing or treating tumors, wherein the tumor is selected from the following group: breast cancer, pancreatic cancer, colon cancer, gastric cancer and prostate cancer, wherein the other tumor therapies are selected from the following group: chemotherapy and radiotherapy, and wherein the other drug is miRNA.

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