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WO2025162493A1 - 抗Nectin-4抗体、配体药物偶联物及其应用 - Google Patents

抗Nectin-4抗体、配体药物偶联物及其应用

Info

Publication number
WO2025162493A1
WO2025162493A1 PCT/CN2025/075858 CN2025075858W WO2025162493A1 WO 2025162493 A1 WO2025162493 A1 WO 2025162493A1 CN 2025075858 W CN2025075858 W CN 2025075858W WO 2025162493 A1 WO2025162493 A1 WO 2025162493A1
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WIPO (PCT)
Prior art keywords
seq
amino acid
heavy chain
light chain
antibody
Prior art date
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Pending
Application number
PCT/CN2025/075858
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English (en)
French (fr)
Inventor
焦娇
孙英杰
车黎明
崔梦函
王佳星
路腾飞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gan and Lee Pharmaceuticals Co Ltd
Original Assignee
Gan and Lee Pharmaceuticals Co Ltd
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Publication of WO2025162493A1 publication Critical patent/WO2025162493A1/zh
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to the field of medical technology, and in particular to an antibody targeting nectin-4, a ligand-drug conjugate and applications thereof.
  • Nectin-4 also known as poliovirus receptor-related protein 4 (PVRL4), is a type I transmembrane protein belonging to the Nextins family of immunoglobulin-like molecules. Studies have found that nectin-4 has limited expression in healthy adult tissues but is overexpressed in various malignancies, including bladder cancer, breast cancer, and lung cancer. As a tumor-associated inducer, it is associated with various aspects of tumor progression, including proliferation, metastasis, and epithelial-mesenchymal transition, making it an ideal anti-tumor drug target.
  • PVRL4 poliovirus receptor-related protein 4
  • ADCs Antibody-drug conjugates
  • mAb humanized monoclonal antibody
  • cytotoxic payload a cytotoxic payload
  • linker connecting unit or connecting structure
  • the antibody is coupled to the cytotoxic drug through the linker, which together gives the ADC cell targeting and cell-killing potency, thereby achieving precise and efficient elimination of cancer cells. It has become a hot topic in anti-cancer drug research and development.
  • An ideal ADC drug should remain stable in the bloodstream, accurately reach the therapeutic target, and ultimately release the cytotoxic payload near the target (e.g., cancer cells).
  • target antigen, antibody, cytotoxic payload, linker, and conjugation method will affect the ultimate efficacy and safety of the ADC.
  • linkers and DAR values are one of the key factors in improving the drug activity and pharmacokinetic properties of ADCs.
  • DAR drug-to-antibody ratio
  • ADCs with high DAR values are generally thermally unstable, prone to aggregation, and have accelerated clearance rates in the body, resulting in reduced efficacy and increased toxicity.
  • EV Enfortumab vedotin
  • nectin-4 targeted drugs under development worldwide, of which more than 20 are ADC drugs, and 9 have entered clinical trials.
  • Enfortumab vedotin (EV) is currently the only marketed drug targeting nectin-4. It is composed of an anti-nectin-4 antibody and a microtubule inhibitor MMAE linked by a dipeptide linker. It is approved by the FDA for the treatment of locally advanced or metastatic urothelial carcinoma [2] , and its combination therapy with Keytruda is approved for first-line treatment.
  • EV has shown excellent clinical potential, there has been no breakthrough in other solid tumors such as breast cancer (ORR of 15.6% in the HR-positive/HER2-negative breast cancer cohort and 19% in the TNBC cohort).
  • EV has been observed to cause severe and fatal skin adverse reactions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) [3] , and can also lead to adverse reactions such as hyperglycemia, lung inflammation, and peripheral neuropathy. Its safety needs to be improved.
  • SJS Stevens-Johnson syndrome
  • TEN toxic epidermal necrolysis
  • MMAE MMAE or camptothecin derivatives as cytotoxic payloads [4] , which poses a risk of drug resistance. Therefore, it is urgent to develop a Nectin-4-targeted ADC drug with a differentiated payload to achieve better clinical benefits, overcome tumor resistance, and better meet patient needs.
  • the first aspect of the present invention provides an antibody or an antigen-binding fragment thereof, comprising: an antibody or an antigen-binding fragment thereof that specifically binds to nectin-4, comprising:
  • HCDRs heavy chain complementarity determining regions
  • LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:4, 6, 8, 101, 103, 105, 29, 30, 31, 32, 37, 38, 39, 44, 45, 46, or 47.
  • an antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprising:
  • HCDR3 comprising or consisting of any one of the group consisting of the following amino acid sequences: SEQ ID NO: 13, 19, 25, 84, 90, and 96;
  • LCDR3 comprises any amino acid sequence in the group consisting of the following amino acid sequences or consists of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 16, 22, 28, 87, 93, and 99.
  • HCDR1 comprising or consisting of any one of the group consisting of the following amino acid sequences: SEQ ID NO: 11, 17, 23, 82, 88, and 94;
  • LCDR1 comprising or consisting of any one of the group consisting of the following amino acid sequences: SEQ ID NOs: 14, 20, 26, 85, 91, and 97;
  • the antibody or antigen-binding fragment thereof further comprises:
  • HCDR2 comprising or consisting of any one of the group consisting of the following amino acid sequences: SEQ ID NO: 12, 18, 24, 83, 89, and 95; and
  • LCDR2 comprises any amino acid sequence in the group consisting of the following amino acid sequences or consists of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 15, 21, 27, 86, 92, and 98.
  • the antibody or antigen-binding fragment thereof comprises:
  • the antibody or antigen-binding fragment thereof comprises:
  • HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 11,
  • HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 12,
  • HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 13,
  • LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 14,
  • LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 15, and
  • LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 16; or
  • HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 17,
  • HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 18,
  • HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 19,
  • LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 20,
  • LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 21, and
  • LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 22; or
  • HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 23,
  • HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 24,
  • HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 25,
  • LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 26,
  • LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 27, and
  • LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 28; or
  • HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 82,
  • HCDR2 comprising or consisting of the sequence shown in SEQ ID NO:83,
  • HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 84,
  • LCDR1 comprising or consisting of the sequence shown in SEQ ID NO:85,
  • LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 86, and
  • LCDR3 comprising or consisting of the sequence shown in SEQ ID NO:87; or
  • HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 88,
  • HCDR2 comprising or consisting of the sequence shown in SEQ ID NO:89,
  • HCDR3 comprising or consisting of the sequence shown in SEQ ID NO:90,
  • LCDR1 comprising or consisting of the sequence shown in SEQ ID NO:91,
  • LCDR2 comprising or consisting of the sequence shown in SEQ ID NO:92, and
  • LCDR3 comprising or consisting of the sequence shown in SEQ ID NO:93; or
  • HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 94,
  • HCDR2 comprising or consisting of the sequence shown in SEQ ID NO:95,
  • HCDR3 comprising or consisting of the sequence shown in SEQ ID NO:96,
  • LCDR1 comprising or consisting of the sequence shown in SEQ ID NO:97,
  • LCDR2 comprising or consisting of the sequence shown in SEQ ID NO:98, and
  • LCDR3 comprising or consisting of the sequence shown in SEQ ID NO:99.
  • Another embodiment of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to nectin-4, comprising:
  • a heavy chain variable region wherein the amino acid sequence of the heavy chain variable region has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 3, 5, 7, 100, 102, 104, 33, 34, 35, 36, 40, 41, 42, 43, 48, 49, 50, and 51; preferably, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 3, 5, 7, 100, 102, 104, 33, 34, 35, 36, 40, 41, 42, 43, 48, 49, 50, or 51.
  • the amino acid sequence of the framework region of the heavy chain variable region has the same HCDR amino acid sequence as that of SEQ ID NO: 3, 5, 7, 100, 102, 104, 33, 34, 35, 36, 40, 41, 42, 43, 48, 49, 50, or 51, and the amino acid sequence of the framework region of the heavy chain variable region has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the framework region of the amino acid sequence of SEQ ID NO: 3, 5, 7, 100, 102, 104, 33, 34, 35, 36, 40, 41, 42, 43, 48, 49, 50, or 51; preferably, the heavy chain variable region has the sequence as shown in SEQ ID NO: 3, 5, 7, 100, 102, 104, 33, 34, 35, 36, 40, 41, 42, 43, 48, 49, 50, or 51; and/or
  • a light chain variable region wherein the amino acid sequence of the light chain variable region has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 4, 6, 8, 101, 103, 105, 29, 30, 31, 32, 37, 38, 39, 44, 45, 46, and 47; preferably, the amino acid sequence of the light chain variable region has a sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, 101, 103, 105, 29, 30, 31, 32, 37, 38, 39,
  • the light chain variable region has the same LCDR as 44, 45, 46, or 47
  • the framework region of the light chain variable region has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the framework region of sequence SEQ ID NO:4, 6, 8, 101, 103, 105, 29, 30, 31, 32, 37, 38, 39, 44, 45, 46, or 47; preferably, the light chain variable region has a sequence as
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the amino acid sequence pair of the heavy chain variable region and the light chain variable region is selected from any one of the groups consisting of the following amino acid sequence pairs:
  • the antibody or antigen-binding fragment thereof comprises a heavy chain constant region or a variant thereof and/or a light chain constant region or a variant thereof, preferably comprising a murine or humanized heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region is an IgG heavy chain constant region, and/or the light chain constant region is a ⁇ or ⁇ light chain constant region; preferably, the heavy chain constant region is an IgG1 heavy chain constant region, and/or the light chain constant region is a ⁇ light chain constant region; preferably, the amino acid sequence of the humanized heavy chain constant region is as shown in SEQ ID NO:9, and/or the amino acid sequence of the humanized light chain constant region is as shown in SEQ ID NO:10.
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', Fab'-SH, Fv , scFv , and F(ab') 2 .
  • the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a bispecific antibody.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain and/or a light chain, wherein:
  • the amino acid sequence of the heavy chain has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO:78, and SEQ ID NO:79; preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:106, SEQ ID NO:108, SEQ ID
  • the amino acid sequence of the light chain has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 73, S SEQ ID NO:74, and SEQ ID NO:75; preferably, the amino acid sequence of the light chain is as shown in SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:107SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:73
  • it comprises a heavy chain and a light chain
  • the amino acid sequence pairs of the heavy chain and the light chain are selected from any one of the groups consisting of the following amino acid sequence pairs: SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 106 and SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111.
  • HCDRs and LCDRs are defined according to the Kabat definition scheme, the Chothia definition scheme, the Abm definition scheme, the IMGT definition scheme and/or the Contact definition scheme; preferably, the HCDRs and LCDRs are defined according to the Kabat definition scheme or the Chothia definition scheme.
  • An expression vector comprising the nucleic acid according to claim 14.
  • a host cell comprising the expression vector according to claim 15.
  • a method for producing an antibody or an antigen-binding fragment thereof comprising culturing the host cell according to claim 16, and recovering the antibody or antigen-binding fragment thereof expressed thereby from the culture.
  • the host cell is a prokaryotic cell or a eukaryotic cell, preferably, the host cell is an Escherichia coli cell, a yeast cell, an insect cell, a plant cell or a mammalian cell; preferably, the host cell is a Chinese hamster ovary cell (CHO), a CHO cell variant, a 293 cell or an NSO cell.
  • CHO Chinese hamster ovary cell
  • the present invention also provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, and an effector molecule portion, wherein the effector molecule portion is coupled to the antibody or antigen-binding fragment thereof; preferably, the effector molecule portion is selected from a cytotoxic drug portion, a drug portion for treating autoimmune diseases, and an anti-inflammatory drug portion.
  • the present invention also provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate is as shown in Formula 1:
  • Ab is an antibody or antigen-binding fragment that targets nectin-4.
  • Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or an antibody comprising a heavy chain as shown in SEQ ID NO: 80 and a light chain as shown in SEQ ID NO: 81, or an antibody comprising a heavy chain as shown in SEQ ID NO: 112 and a light chain as shown in SEQ ID NO: 113.
  • Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.
  • n is an integer or decimal selected from 1-20; preferably m is an integer or decimal selected from 1-12;
  • L is a chemical linking structure
  • D is an effector molecule portion; preferably, D is selected from a cytotoxic drug portion, a drug portion for treating autoimmune diseases, and an anti-inflammatory drug portion; preferably, D is a cytotoxic drug portion; preferably, D is selected from a topoisomerase I (TOP1) inhibitor portion, a microtubule polymerization inhibitor portion, a topoisomerase II (TOP2) inhibitor portion, a dihydrofolate reductase inhibitor portion, a DNA alkylating agent portion, a thymidine synthetase inhibitor portion, a purine nucleoside synthetase inhibitor portion, a ribonucleotide reductase inhibitor portion, a DNA polymerase inhibitor portion, an RNA polymerase II inhibitor portion, and other compound portions capable of inhibiting cell proliferation; preferably, D is selected from (Eribulin portion) and The wavy line indicates the connection point between D and L.
  • TOP1 topoisomerase I
  • L is Among them , the represents the connection point with the antibody or antigen-binding fragment thereof, L 5c Indicates the connection point with D;
  • T a is selected from Wherein the left side of the T a structure Indicates the connection point with the antibody or antigen-binding fragment thereof, the right side of the T a structure Indicates the connection point with Mc ;
  • Mc is selected from a single bond, a 6-10 membered arylene, a 5-13 membered heteroarylene, a 3-15 membered cycloalkylene, and a 3-15 membered heterocycloalkylene, said arylene, heteroarylene, cycloalkylene, and heterocycloalkylene being unsubstituted or optionally substituted with one or more Ra ;
  • L 1c is selected from a combination of 1, 2, 3 or more of a single bond, alkynylene, alkenylene, -NR a -, -O-, -C(O)-, -NR a -C(O)-, alkylene and heteroalkylene, wherein the alkynylene, alkenylene, alkylene, and heteroalkylene are unsubstituted or optionally substituted with one or more Ra ;
  • L 2c is selected from single bonds, -(CH 2 ) n7 -(OCH 2 CH 2 ) n8 -O-(CH 2 ) n9 -Cy 3 -(CH 2 ) n10 -C(O)-, -(CH 2 ) n7 -(CH 2 CH 2 O) n8 -(CH 2 ) n9 -Cy 3 -(CH 2 ) n10 -C(O)-, -(CH 2 ) n7 -Cy 3 -(CH 2 CH 2 O) n8 -(CH 2 ) n9 -C(O)-, -(CH 2 ) n11 -(OCH 2 CH 2 ) n12 -O-(CH 2 ) n13 -Cy 3 -(CH 2 ) n14 -(OCH 2 CH 2 ) n15 -C(O)-, -(CH 2 ) n7 -(OCH 2
  • n7, n9, n10, n11, n13 and n14 are each independently selected from 0, 1, 2, 3, 4, 5 and 6 at each occurrence;
  • n12 and n15 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, and n12 and n15 are not 0 at the same time;
  • j is independently selected at each occurrence from 0, 1, 2, 3, 4, 5, and 6;
  • Cy 3 is selected from 6-10 membered arylene, 5-13 membered heteroarylene, 3-15 membered cycloalkylene and 3-15 membered heterocycloalkylene, said arylene, heteroarylene, cycloalkylene and heterocycloalkylene being unsubstituted or optionally substituted with 1, 2, 3, 4, 5 or 6 R a ;
  • Mc , L1c , and L2c are not single bonds at the same time;
  • L 4c is a peptide residue consisting of amino acids or -NR a -alkylene-NR a -, wherein the alkylene, and amino acids are unsubstituted or optionally further substituted with one or more Ra ;
  • L 5c is selected from a single bond, -N(R b )-(CH 2 ) g -O-(CH 2 ) g -OC(O)-, -N(R b )-(CH 2 ) g -O-(CH 2 -CH 2 -O) g -C(O)-,
  • n is independently selected from any integer between 1 and 30; preferably, each occurrence of n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28;
  • g is independently selected at each occurrence from 1, 2, 3, 4, 5, and 6;
  • R a and R b are each independently selected from H, a deuterium atom, F, Cl, Br, I, C 1 -C 6 alkyl, C 1 -C 6 deuterated alkyl, C 2 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 alkoxy, hydroxy, nitro, cyano, amino, carboxyl, C 3 -C 6 cycloalkyl, 3-6 membered heterocyclyl, C 1 -C 6 alkylamino, C 1 -C 6 alkyl-C (O) -, C 1 -C 6 alkyl-OC (O) -, C 1 -C 6 alkyl-NHC (O) -, -C 1 -6 alkylene-C (O) -NH 2 , C 1 -6 alkylOC (O) NH-, -C 1 -6 alkylene-C (O) -NH 2
  • Mc is selected from a single bond, phenylene, 5-7 membered heteroarylene, 3-7 membered cycloalkylene and 3-7 membered heterocycloalkylene, wherein the arylene, heteroarylene, cycloalkylene and heterocycloalkylene are unsubstituted or optionally substituted with 1, 2, 3, 4, 5 or 6 Ra ; preferably, Mc is selected from a single bond, and/or
  • L 1c is selected from a single bond, C 2-6 alkynylene, C 2-6 alkenylene, -NR a -, -O-, -C(O)-, -NR a -C(O)-, C 1-6 alkylene and C 1-6 heteroalkylene, wherein the alkynylene, alkenylene, alkylene and heteroalkylene are unsubstituted or optionally substituted with 1, 2, 3, 4, 5 or 6 R a ; preferably, L 1c is selected from a single bond, C 2-3 alkynylene, C 2-3 alkenylene, -NR a -, -C(O)-, -NR a -C(O)-, C 1-3 alkylene and C 1-3 heteroalkylene, wherein the alkynylene, alkenylene, alkylene and heteroalkylene are unsubstituted or optionally substituted with 1, 2, 3, 4, 5 or 6 R a a replaced; and/or
  • Cy 3 is selected from Preferably, Cy 3 is selected from and/or
  • L 4c is a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids or -NRa-C 1-6 alkylene-NRa-, wherein the amino acid is selected from D-alanine, L-alanine, phenylalanine, glycine, valine, lysine, leucine, citrulline, serine, glutamic acid, aspartic acid, arginine and asparagine, and the alkylene and amino acid are unsubstituted or optionally substituted with 1, 2, 3, 4, 5 or 6 Ra ; preferably, L 4c is selected from -NH-CH 2 CH 2 -NH-, and/or
  • L 5c is selected from: a single bond, and/or
  • Ra and Rb are each independently selected at each occurrence from H, cyano, a deuterium atom, F, Cl, Br, I, carboxyl, hydroxyl, amino, C1 - C3 alkylamino, C1 - C3 alkyl and C1 - C3 alkoxy.
  • Mc is selected from a single bond, and/or
  • L 1c is selected from a single bond, C 2-3 alkynylene, C 2-3 alkenylene, -NR a -, -C(O)-, -NR a -C(O)-, C 1-3 alkylene and C 1-3 heteroalkylene, wherein the alkynylene, alkenylene, alkylene and heteroalkylene are unsubstituted or optionally substituted with 1, 2, 3, 4, 5 or 6 Ra ; and/or
  • L 2c is selected from a single bond, wherein n17 is independently selected from 6, 8, 12 and 24 at each occurrence;
  • L 4c is selected from -NH-CH 2 CH 2 -NH-, and/or
  • L 5c is selected from: a single bond, and/or
  • Ra and Rb are each independently selected at each occurrence from H, cyano, a deuterium atom, F, Cl, Br, I, carboxyl, hydroxyl, amino, C1 - C3 alkylamino, C1 - C3 alkyl and C1 - C3 alkoxy.
  • L is selected from the following structures:
  • n8 and n5 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 at each occurrence.
  • n is any integer or decimal from 1 to 10; preferably, m is any integer or decimal from 2 to 8; preferably, m is any integer or decimal from 2 to 6; and/or
  • D is selected from the group consisting of a topoisomerase I (TOP1) inhibitor portion, a tubulin polymerization inhibitor portion, a topoisomerase II (TOP2) inhibitor portion, a dihydrofolate reductase inhibitor portion, a DNA alkylating agent portion, a thymidine synthetase inhibitor portion, a purine nucleoside synthetase inhibitor portion, a ribonucleotide reductase inhibitor portion, a DNA polymerase inhibitor portion, an RNA polymerase II inhibitor portion, and other compound portions capable of inhibiting cell proliferation; preferably, D is selected from the group consisting of (Eribulin portion) and Preferably, D is (Eribulin portion), where the wavy line indicates the point where D and L are connected.
  • TOP1 topoisomerase I
  • TOP2 topoisomerase II
  • D is selected from the group consisting of (Eribulin portion) and Preferably, D is (E
  • the ligand-drug conjugate is selected from the following structures:
  • Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1-13, or an antibody comprising the heavy chain shown in SEQ ID NO: 80 and the light chain shown in SEQ ID NO: 81, or an antibody comprising the heavy chain shown in SEQ ID NO: 112 and the light chain shown in SEQ ID NO: 113;
  • m and m1 are each independently selected from integers or decimals from 1 to 20 at each occurrence; preferably, m and m1 are each independently selected from integers or decimals from 1 to 12 at each occurrence; preferably, m and m1 are each independently selected from integers or decimals from 1 to 10 at each occurrence; preferably, m and m1 are each independently selected from integers or decimals from 2 to 8 at each occurrence; preferably, m and m1 are each independently selected from integers or decimals from 2 to 6 at each occurrence; preferably, m and m1 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 at each occurrence; and
  • n1, n8 and n5 are each independently selected from 1, 2, 3, 4, 5, 6, 7, and 8 at each occurrence.
  • Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13; preferably, Ab is an antibody comprising a heavy chain and a light chain, and the amino acid sequence pairs of the heavy chain and the light chain in the antibody are selected from any one of the groups consisting of the following amino acid sequence pairs: SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 106 and SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 110.
  • m and m1 are each independently selected from integers or decimals from 1 to 20 at each occurrence; preferably, m and m1 are each independently selected from integers or decimals from 1 to 12 at each occurrence; preferably, m and m1 are each independently selected from integers or decimals from 1 to 10 at each occurrence; preferably, m and m1 are each independently selected from integers or decimals from 2 to 8 at each occurrence; preferably, m and m1 are each independently selected from integers or decimals from 2 to 6 at each occurrence; preferably, m and m1 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 at each occurrence.
  • the present invention also provides an antibody or an antigen-binding fragment thereof, comprising:
  • the present invention also provides an antibody or an antigen-binding fragment thereof, comprising:
  • HCDR3 comprising or consisting of any one of the group consisting of the following amino acid sequences: SEQ ID NO: 13, 19, and 25;
  • LCDR3 comprising or consisting of any one of the group consisting of the following amino acid sequences: SEQ ID NOs: 16, 22, and 28;
  • the antibody or antigen-binding fragment thereof comprises:
  • HCDR1 wherein the HCDR1 comprises or consists of any one of the following amino acid sequences: SEQ ID NO: 11, 17, 23; and
  • LCDR1 wherein the LCDR1 comprises or consists of any one of the following amino acid sequences: SEQ ID NO: 14, 20, 26;
  • the antibody or antigen-binding fragment thereof further comprises:
  • HCDR2 comprises or consists of any one of the following amino acid sequences: SEQ ID NO: 12, 18, 24; and
  • LCDR2 comprises any amino acid sequence in the group consisting of the following amino acid sequences or consists of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 15, 21, 27.
  • it comprises:
  • the amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:53; or
  • the amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:55; or
  • the amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:57;
  • the antibody or antigen-binding fragment thereof comprises:
  • HCDR1 comprising or consisting of SEQ ID NO: 11,
  • HCDR2 comprising or consisting of SEQ ID NO: 12,
  • HCDR3 comprising or consisting of SEQ ID NO: 13,
  • LCDR1 comprising or consisting of SEQ ID NO: 14,
  • LCDR2 comprising or consisting of SEQ ID NO: 15, and
  • LCDR3 comprising or consisting of SEQ ID NO: 16; or
  • HCDR1 comprising or consisting of SEQ ID NO: 17,
  • HCDR2 comprising or consisting of SEQ ID NO: 18,
  • HCDR3 comprising or consisting of SEQ ID NO: 19,
  • LCDR1 comprising or consisting of SEQ ID NO: 20,
  • LCDR2 comprising or consisting of SEQ ID NO: 21, and
  • LCDR3 comprising or consisting of SEQ ID NO: 22; or
  • HCDR1 represented by or consisting of SEQ ID NO: 23
  • HCDR2 comprising or consisting of SEQ ID NO: 24,
  • HCDR3 comprising or consisting of SEQ ID NO: 25,
  • LCDR1 comprising or consisting of SEQ ID NO: 26,
  • LCDR2 comprising or consisting of SEQ ID NO: 27, and
  • LCDR3 comprising or consisting of SEQ ID NO:28.
  • the present invention also provides an antibody or an antigen-binding fragment thereof, comprising:
  • a heavy chain variable region wherein the amino acid sequence of the heavy chain variable region is at least 80% identical to any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 33, 34, 35, 36, 40, 41, 42, 43, 48, 49, 50, 51; preferably, the heavy chain variable region has a CDRs region amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 40, 41, 42, 43, 48, 49, 50, 51.
  • the amino acid sequence of the framework region of the heavy chain variable region has at least 80% identity with the framework region of the amino acid sequence SEQ ID NO: 33, 34, 35, 36, 40, 41, 42, 43, 48, 49, 50, 51; preferably, the heavy chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 33, 34, 35, 36, 40, 41, 42, 43, 48, 49, 50, 51; and/or
  • a light chain variable region wherein the amino acid sequence of the light chain variable region is at least 80% identical to any one of the amino acid sequences selected from the group consisting of the following amino acid sequences: SEQ ID NO: 29, 30, 31, 32, 37, 38, 39, 44, 45, 46, 47; preferably, the amino acid sequence of the light chain variable region is at least 80% identical to the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 37, 38, 39, 44, 45, 46, 47.
  • the framework region of the light chain variable region is at least 80% identical to the framework region of SEQ ID NO: 29, 30, 31, 32, 37, 38, 39, 44, 45, 46, 47; preferably, the light chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 29, 30, 31, 32, 37, 38, 39, 44, 45, 46, 47;
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the amino acid sequence pair of the heavy chain variable region/light chain variable region is selected from any one of the group consisting of the following amino acid sequence pairs:
  • it comprises a heavy chain constant region and/or a light chain constant region, preferably it comprises a murine or humanized heavy chain constant region and/or a light chain constant region; preferably, the amino acid sequence of the humanized heavy chain constant region is as shown in SEQ ID NO: 9, and the amino acid sequence of the humanized light chain constant region is as shown in SEQ ID NO: 10; preferably, the antibody or antigen-binding fragment thereof, its heavy chain/light chain amino acid sequence pair is selected from any one of the groups consisting of the following amino acid sequence pairs: SEQ ID NO: 60 and SEQ ID NO: 5 8.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof or any one of the above-mentioned ligand-drug conjugates or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable excipient.
  • the present invention also provides use of any of the antibodies or antigen-binding fragments thereof described above, any of the ligand-drug conjugates or pharmaceutically acceptable salts thereof described above, or the pharmaceutical composition described above in the preparation of a medicament for treating and/or preventing a disease or condition mediated by Nectin-4; preferably, the disease or condition mediated by Nectin-4 is breast cancer, bladder cancer, or lung cancer; preferably, the breast cancer is triple-negative breast cancer.
  • the present invention also provides the antibody or antigen-binding fragment thereof described in any of the foregoing, the ligand-drug conjugate or pharmaceutically acceptable salt thereof described in any of the foregoing, or the pharmaceutical composition described in any of the foregoing, for use in treating and/or preventing a disease or condition mediated by Nectin-4; preferably, the disease or condition mediated by Nectin-4 is breast cancer, bladder cancer or lung cancer; preferably, the breast cancer is triple-negative breast cancer.
  • the present invention also provides a method for treating and/or preventing a disease or condition mediated by Nectin-4, comprising administering to a subject in need thereof a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof described above, any of the ligand-drug conjugates or pharmaceutically acceptable salts thereof described above, or the pharmaceutical composition described above; preferably, the disease or condition mediated by Nectin-4 is breast cancer, bladder cancer, or lung cancer; preferably, the breast cancer is triple-negative breast cancer.
  • Figure 1 is a diagram of specific detection of AGSM6 protein.
  • Figure 2 is a diagram of specific detection of h10592-2 protein.
  • Figure 3 is a species cross diagram of h10592-2.
  • Figure 4 is a species cross diagram of AGSM6.
  • Figure 5 shows the HIC analysis results of ADC drug (Enfortumab vedotin).
  • FIG. 6 shows the analysis results of NECTIN 4-ADC-11HIC.
  • FIG. 7 shows the analysis results of NECTIN 4-ADC-18HIC.
  • Figure 8 shows the analysis results of NECTIN 4-ADC-26HIC.
  • FIG9 is a graph showing the changes in tumor volume in the MDA-MB-468 model.
  • FIG10 is a graph showing the changes in tumor volume in the MDA-MB-453 model.
  • FIG11 shows the changes in tumor volume of the HT1376 model at a dose of 4 mg/kg.
  • FIG12 shows the changes in tumor volume of the HT1376 model at a dose of 8 mg/kg.
  • FIG13 is a graph showing the changes in HT1376 tumor volume at different doses.
  • FIG14 is a graph showing the changes in tumor volume in the RT4/Nectin4 model.
  • FIG15 is the acute toxicity survival curve of BALB/C mice.
  • Figure 16 is the body weight change curve of BALB/C mice in acute toxicity.
  • FIG17 is a long-term toxicity survival curve of BALB/C mice.
  • FIG18 is a curve showing the body weight changes of BALB/C mice after long-term toxicity.
  • FIG19 shows the tumor volume inhibition rate of the MDA-MB-453 model.
  • FIG20 shows the MDA-MB-468 tumor volume inhibition rate.
  • Figure 21 is the survival curve of the humanized mouse toxicology MTD experiment.
  • Figure 22 shows the body weight change rate in the humanized mouse toxicology MTD experiment.
  • Figure 23 shows the survival curve of repeated-dose toxicology experiments in humanized mice.
  • Figure 24 shows the body weight change rate in repeated-dose toxicology experiments in humanized mice.
  • FIG25 shows the pharmacokinetic results of MMAE and Eribulin free payload in BALB/c mice.
  • FIG26 shows the pharmacokinetic results of plasma ADC and total antibody in BALB/c mice.
  • Figure 27 shows the pharmacokinetic test results of HT1376 tumor-bearing mice.
  • the antibody-drug conjugate of the present invention achieves an average DAR value of 3.5-4.5 by adopting a novel bridged maleimide linker and a cleavable linker, significantly improving the homogeneity of the antibody-drug conjugate and specifically releasing the cytotoxic payload under the action of tumor cell lysosomes, thereby improving the safety of the drug while ensuring efficacy;
  • the Nectin4-targeted antibody-drug conjugate of the present invention with Eribulin as the payload has a significant inhibitory effect on triple-negative breast cancer, breast cancer, and bladder cancer tumors.
  • the antibody-drug conjugate of the present invention has better efficacy than the positive control drug Enfortumab vedotin and the positive control drug 9MW2821.
  • the antibody-drug conjugate of the present invention has a TGI of 100% and 99.73% in the triple-negative breast cancer MDA-MB-468 CDX model and the breast cancer MDA-MB-453 CDX model, respectively, showing significant efficacy.
  • the antibody-drug conjugate of the present invention has better efficacy than Enfortumab vedotin.
  • the tumor inhibition rate of the antibody-drug conjugate of the present invention is comparable to that of Enfortumab vedotin at a dose of 5 mg/kg.
  • the antibody-drug conjugate of the present invention has better safety.
  • obvious adverse reactions and mouse deaths were observed in the Enfortumab vedotin control group, while no mouse deaths were observed in the antibody-drug conjugate group obtained by the present invention, and the survival rate was high, that is, it has better safety and can reduce the serious side effects of Nectin4 target skin toxicity to a certain extent.
  • any polypeptide chain is described herein as having an amino acid sequence starting at the N-terminus and ending at the C-terminus.
  • antibody and antigen-binding portion thereof herein is used in the broadest sense to include various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.
  • antibody refers to an immunoglobulin molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains: CH1, CH2, and CH3.
  • Each light chain comprises a light chain variable region (LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CL1).
  • the VH and VL regions are further divided into highly variable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL domain is composed of three CDRs and four FRs, arranged from amino-terminal to carboxyl-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDRs of VH are HCDR1, HCDR2, and HCDR3, and the three CDRs of VL are LCDR1, LCDR2, and LCDR3.
  • the amino acid assignments of each domain are generally consistent with the following definitions: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol., 196: 901-917 (1987); Chothia et al., Nature, 342: 878-883 (1989).
  • IgA, IgD, IgE, IgG, and IgM are five main types of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the amino acid sequence at the C-terminus of the antibody molecule is relatively stable, and this region is called the constant region.
  • the constant region is identical for the same antibody.
  • the constant region of the antibody light chain consists of one Ig domain; the constant region of the heavy chain consists of 3-4 Ig domains in series and a hinge region for added flexibility.
  • IgA, IgE, and IgG have three domains (CH1, CH2, CH3), while IgD and IgM have four domains (CH1, CH2, CH3, CH4).
  • the heavy chain constant domains or heavy chain constant regions corresponding to different types of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • IgG molecules can be degraded into two Fab segments and one Fc segment by the action of papain.
  • the Fab segment is composed of the variable region of the antibody light chain, the constant region of the light chain, the variable region of the heavy chain, and the constant region of the heavy chain.
  • the variable region is the site of antigen binding, and therefore the Fab segment is also called the antigen-binding segment.
  • the Fc segment contains protein sequences common to all antibody molecules as well as determinants unique to each class.
  • the Fc segment has various biological activities, including complement binding, Fc receptor binding, and placental translocation.
  • an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (such as Nectin 4 protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by certain fragments of a full-length antibody.
  • binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains; (ii) a F(ab') 2 fragment, i.e., a bivalent fragment consisting of two Fab fragments linked by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the single-arm VL and VH domains of an antibody; (v) a dAb fragment consisting of the VH domain; and (vi) CDRs.
  • a Fab fragment i.e., a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains
  • a F(ab') 2 fragment i.e., a bivalent fragment consisting of two Fab fragments linked by a disulfide bond
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected together by a synthetic linker through recombinant methods to form a single connected chain, wherein the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv)).
  • single-chain Fv single-chain Fv
  • Such single-chain antibodies are also included in the term "antigen-binding portion" of an antibody.
  • Other forms of single-chain antibodies, such as bispecific antibodies, are also included.
  • Different analyses can be used to determine or roughly estimate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.
  • the Kabat definition is a standard for numbering residues in antibodies and is commonly used to determine CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000).
  • the Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the position of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989).
  • the AbM definition uses an integrated suite of computer programs produced by the Oxford Molecular Group that simulates antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.
  • AbM defines modeling of antibody tertiary structure from primary sequence using a known database and an ab initio method, such as that described in Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999).
  • Contact definitions are based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).
  • the antibodies of the present invention are not limited in their origin and may be antibodies derived from any animal, such as human antibodies, mouse antibodies, and rat antibodies. They may also be recombinant antibodies, such as chimeric antibodies and humanized antibodies. Humanized antibodies are preferred.
  • chimeric antibody refers to an antibody comprising the variable regions of the heavy and light chains of mammals other than humans, such as mouse antibodies, and the constant regions of the heavy and light chains of human antibodies.
  • Chimeric antibodies can be prepared using known methods. For example, chimeric antibodies can be prepared by inserting the genes of hybridoma clones into appropriate vectors and then introducing them into a host. Specifically, reverse transcriptase is used to synthesize cDNA of the antibody variable region (V) from the mRNA of the hybridoma. When DNA encoding the target antibody V region is obtained, it is connected to DNA encoding the desired human antibody constant region (C region) and then inserted into an expression vector.
  • V antibody variable region
  • the DNA encoding the antibody V region can be inserted into a DNA expression vector comprising the human antibody C region. Insert it into an expression vector so that it is expressed under the mediation of the expression regulatory region.
  • the host cell is transformed with the expression vector to express the chimeric antibody.
  • humanized antibody refers to an antibody containing at least one, and usually two, nearly complete variable regions, in which all or nearly all of the corresponding CDR regions are derived from non-human antibodies, and all or nearly all of the FR regions are derived from human antibodies. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components.
  • affinity refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to the intrinsic binding affinity of a 1:1 interaction between members of a reactive binding pair (e.g., an antibody and an antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by an equilibrium dissociation constant ( KD ), a dissociation constant (Kd), or an association constant (Ka). Affinity can be measured by general methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
  • surface plasmon resonance refers to an optical phenomenon that allows real-time interactive analysis by detecting changes in protein concentration in a biosensor matrix, for example using a BiacoreTM system.
  • neutralizing or blocking antibody refers to an antibody whose binding to a NECTIN4 protein results in inhibition of the biological activity of the NECTIN4 protein. This inhibition of the biological activity of the NECTIN4 protein can be assessed by measuring one or more indicators of NECTIN4 protein biological activity well known in the art, such as NECTIN4 protein-induced cell activation and antibody binding to the NECTIN4 protein (see Examples below).
  • Sequence similarity of polypeptides is typically measured using sequence analysis software.
  • Protein analysis software uses similarity measurements of various substitutions, deletions, and other modifications, including conservative amino acid substitutions, to match similar sequences.
  • GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different biological species. See, for example, GCG version 6.1.
  • the FASTA program in GCG version 6.1 can also be used to compare polypeptide sequences using default or recommended parameters.
  • FASTA (such as FASTA2 and FASTA3) provides alignments and percentages of sequence identity between query and searched sequences based on the best overlapping regions (Pearson (2000) supra).
  • another preferred computer program is the BLAST computer program, particularly BLASTP or TBLASTN, using default parameters for the comparison.
  • nucleic acids or fragments thereof mean that the nucleotide sequence is identical in at least about 80%, more preferably at least about 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide substitutions, insertions or deletions, as calculated using any of the following sequence identity calculation programs, such as FASTA, BLAST or Gap.
  • the terms “substantially similar” or “substantially similar” mean that two peptide sequences share at least 80% sequence identity, more preferably at least 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, when optimally aligned using programs such as Gap or BESTFIT using default gap weights.
  • Non-identical residue positions can differ by amino acid substitutions, deletions, or insertions, and more preferably, non-identical residue positions differ by conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
  • conservative amino acid substitutions do not substantially alter the functional properties of the protein.
  • the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art.
  • amino acid groups containing side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl groups: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine.
  • Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.
  • a conservative substitution can be any change that has a positive value in the PAM250 log-likelihood matrix disclosed by Gonnet et al. (1992) Science 256:1443-1445.
  • a "moderately conservative" substitution is any change with a non-negative value in the PAM250 log-likelihood matrix.
  • vector is intended to refer to any molecule or entity (eg, a nucleic acid, plasmid, phage, or virus) used to transfer protein coding information into a host cell.
  • molecule or entity eg, a nucleic acid, plasmid, phage, or virus
  • expression vector or "expression construct” refers to a vector suitable for transforming a host cell and containing nucleic acid sequences that direct and/or regulate the expression of one or more operably linked heterologous coding regions.
  • Expression vectors may include, but are not limited to, sequences that affect or regulate transcription and translation; and, if introns are present, sequences that affect RNA splicing of the operably linked coding region.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom (regardless of the number of passages).
  • the nucleic acid content of the progeny may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as that screened or selected for in the original cell are included herein.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell.
  • a cell is "transfected" when the exogenous DNA is introduced into the cell membrane.
  • transfection techniques are well known in the art. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.
  • treatment includes therapeutic treatment, prophylactic treatment, and use in reducing the risk of a subject developing a disease or other risk factors. Treatment does not require a complete cure of the disease, but includes embodiments in which symptoms are alleviated or underlying risk factors are mitigated.
  • prevent does not require 100% elimination of the likelihood of an event. Rather, it means that the likelihood of an event occurring is reduced in the presence of the compound or method.
  • secondary antibody refers to a second antibody that binds to an antibody (primary antibody). Its primary function is to detect the presence of the antibody and amplify the primary antibody signal. Secondary antibodies utilize the antigenic nature of antibodies, which are large proteins, to immunize a xenobiotic animal. These secondary antibodies are reactive against all antibodies (e.g., IgG, IgM, or IgA) from a specific species (e.g., mouse).
  • drug refers to a chemical substance that can alter or identify physiological functions and pathological states of the body and can be used to prevent, diagnose, and treat disease.
  • Drugs include cytotoxic drugs. There is no strict distinction between drugs and poisons. Poisons are chemical substances that can have toxic effects on the body at relatively low doses, potentially damaging human health. Excessive doses of any drug can produce toxic reactions.
  • Cytotoxic drugs are substances that inhibit or prevent cellular function and/or cause cell death or destruction. Cytotoxic drugs can, in principle, kill tumor cells at sufficiently high concentrations. However, due to their lack of specificity, they can also induce apoptosis of normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, radioactive isotopes, chemotherapeutic agents, antibiotics, and nucleolytic enzymes.
  • linker unit or “linking fragment” or “linking unit” refers to a fragment or bond in a chemical structure that is connected to a ligand at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug.
  • ligand-drug conjugate refers to a ligand linked to a biologically active drug via a stable linker.
  • the "ligand-drug conjugate” is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linker.
  • ADC antibody-drug conjugate
  • Drug loading also known as the drug-to-antibody ratio (DAR) is the average number of drugs conjugated to each antibody in the ADC. It can be, for example, in the range of about 1 to about 12 drugs conjugated to each antibody, and in certain embodiments, in the range of about 1 to about 8 drugs conjugated to each antibody, preferably in the range of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. Exemplarily, the drug loading can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments of the present invention, the drug loading can be expressed as m or m1, which is a decimal or integer. Drug loading can be determined by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay, and HPLC.
  • hydrophilic unit refers to -CH2 - CH2 -O-, hydrophilic amino acids, glucosamine, glycosyl, phosphate, sulfonate or a combination thereof; preferably, the hydrophilic unit is selected from -CH2 - CH2- O-, sarcosine, alanine, serine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamate, glucosamine, glycosyl, phosphate, sulfonate or a combination thereof.
  • a vector refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto.
  • a vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected.
  • a vector is a viral vector, in which another DNA segment can be connected to a viral genome.
  • Vectors disclosed herein can autonomously replicate in the host cell into which they have been introduced (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after introducing the host cell, thereby replicating (e.g., non-additional mammalian vectors) with the host genome.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms.
  • C 1-6 alkyl refers to a saturated straight or branched chain hydrocarbon group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6 carbon atoms).
  • C 1-6 alkyl can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl, etc.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms are selected from nitrogen, oxygen, or sulfur, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms.
  • monocyclic heterocyclyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclyls include spirocyclic, fused, and bridged heterocyclyls.
  • aryl refers to a 6- to 15-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated ⁇ electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, with phenyl being preferred.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 15 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen.
  • the heteroaryl group is preferably 5- to 10-membered, more preferably 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like.
  • the heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring.
  • haloalkyl refers to an alkyl group substituted with one or more halogens.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • substituted and “substituted” mean that one or more (e.g., one, two, three, or four) hydrogen atoms on the designated atom are replaced with a group selected from the indicated group, provided that the designated atom's normal valency in the present context is not exceeded and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
  • substituent may be (1) unsubstituted, or (2) substituted. If an atom or group is described as optionally substituted with one or more of the substituents listed, one or more hydrogen atoms on the atom or group may be replaced with independently selected, optional substituents. If a substituent is described as "independently selected from” or “each independently is,” each substituent is selected independently of the others. Thus, each substituent may be the same as or different from another substituent.
  • R groups such as, but not limited to, R2, R3, Rh, Ri, Rx, and/or Ry
  • each R is selected independently of the others, i.e., may be the same or different.
  • the same is true for the selection of numerical values such as d, g, m, and n.
  • the point of attachment of a substituent may be from any suitable position of the substituent.
  • a “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active ingredient is administered and which is suitable, within the scope of sound medical judgment, for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • active ingredient refers to a chemical entity that is effective in treating one or more symptoms of a target disorder or condition.
  • the term "effective amount” refers to an amount of active ingredient that, after administration, will achieve the desired effect to some extent, such as alleviating one or more symptoms of the condition being treated or preventing the appearance of the condition or its symptoms.
  • PBS refers to phosphate buffer solution
  • PBST refers to PBS solution with Tween-20 added.
  • BSA stands for bovine serum albumin.
  • BSA blocking solution is made from bovine serum albumin.
  • TMB refers to 3,3’,5,5’-Tetramethylbenzidine solution.
  • MEM medium refers to Minimum Essential Medium, which is a commonly used medium in animal cell culture.
  • FBS refers to fetal bovine serum
  • ND (Not Detected) means that the antibody did not exhibit in vitro cell neutralization activity under the experimental conditions.
  • the pCDNA3.1(+) expression vector was purchased from Invitrogen with the catalog number V79020.
  • Mouse myeloma cells SP2/0 were purchased from the American Type Culture Collection with the catalog number CRL-1581.
  • HEp-2 cells were purchased from ATCC, catalog number CCL-23.
  • 293F cells were purchased from ATCC, catalog number CRL-1573.
  • Nectin4 protein Based on the gene sequence of Nectin4 protein in the NCBI database, the expression gene with a His tag was chemically synthesized and combined with the pCDNA3.1(+) expression vector to construct an expression plasmid for Nectin4 protein. After transfection into 293F cells, the cell expression supernatant was cultured and collected, concentrated, and purified using a nickel column (Cytiva) to obtain Nectin4 protein, the amino acid sequence of which is shown in SEQ ID NO:1, and the gene sequence of which is shown in SEQ ID NO:2.
  • Nectin4 protein was used for the first immunization. Before immunization, Sigma Adjuvant System adjuvant (Sigma) was resuspended in 1ml PBS, and then 1ml Nectin4 protein (1mg/ml) was added as an antigen. The mixture was emulsified in equal volumes and injected intraperitoneally and subcutaneously. Each mouse received 100 ⁇ g each time. Immunization was performed once every two weeks. After the third immunization, blood was collected to detect the titer, and a total of four immunizations were performed. The spleen of the mouse was taken for fusion. 72h before fusion, the immunization was boosted again, and Nectin4 antigen protein was injected intraperitoneally once, 100 ⁇ L/mouse.
  • Spleen cells from immunized mice were fused with SP2/0 mouse myeloma cells.
  • the spleen was ground to obtain a splenocyte suspension, which was then mixed with SP2/0 mouse myeloma cells in logarithmic growth at a 1:1 ratio.
  • the two cells were fused by electrofusion to generate hybridoma cells.
  • the fused cell suspension was then diluted to 5,000-10,000 cells/ml and evenly plated in a 96-well plate.
  • the fusion medium was DMEM complete selection medium supplemented with HAT and 20% FBS. After incubation at 37°C, 5% CO2 for 7 days, positive hybridoma cell lines were screened using indirect ELISA. The cell culture medium was changed the day before screening.
  • cell supernatants were assayed using an indirect ELISA method.
  • Cells with strong binding to the nectin 4 antigen protein were selected, promptly expanded, and cryopreserved.
  • Monoclonal cell lines were obtained through subcloning using the limiting dilution method.
  • Supernatants from the monoclonal cell lines were collected and analyzed by Biacore analysis to determine their binding activity to the nectin 4 antigen.
  • Monoclonal hybridoma cell lines mAb10591, mAb10592, and mAb10593 with high in vitro activity were selected; the monoclonal antibody sequences were determined, and then humanized, recombinantly expressed, and activity evaluated.
  • the process of hybridoma sequencing is as follows. Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) (according to the kit instructions) and reverse transcribed (PrimeScript "Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sent to a sequencing company for sequencing. The amino acid sequence corresponding to the obtained DNA sequence is shown in SEQ ID NO: 3-8:
  • mAb10591 murine heavy chain variable region (SEQ ID NO: 3):
  • mAb10591 murine light chain variable region (SEQ ID NO: 4):
  • mAb10592 murine heavy chain variable region (SEQ ID NO: 5):
  • mAb10592 murine light chain variable region (SEQ ID NO: 6):
  • mAb10593 murine heavy chain variable region (SEQ ID NO: 7):
  • a chimeric antibody was constructed using a fusion PCR method: the variable region of the mouse antibody in Example 1 was connected with the coding genes of the heavy chain constant region and the human ⁇ light chain constant region of the human IgG1 antibody (heavy chain constant region amino acid sequence: SEQ ID NO: 9, light chain constant region amino acid sequence: SEQ ID NO: 10) to construct a fusion gene, the fusion gene was amplified and cloned into the pCDNA3.1(+) expression vector to construct an expression plasmid for the chimeric antibody, and after transfection into 293F cells, the supernatant of the cell expression was cultured and collected, concentrated, and purified by Protein A (Cytiva) to obtain the corresponding chimeric antibody.
  • Protein A Protein A
  • Heavy chain constant region of human IgG1 antibody (SEQ ID NO: 9):
  • the mouse CDR regions of mAb10591, mAb10592, and mAb10593 were retained, and their FR regions were humanized.
  • the steps for humanizing the FR regions were as follows:
  • variable regions of humanized antibodies were compared using NCBI Ig Blast, and the human germline sequence with the highest homology was selected for CDR transplantation. In the first round of humanization, all the mouse antibody FR sequences were replaced with humanized ones.
  • the humanized light chain variable region sequence was VL1 (SEQ ID NO: 29), or VL3 (SEQ ID NO: 31) and the heavy chain variable region sequence was VH1 (SEQ ID NO: 33), or VH3 (SEQ ID NO: 35); and then recombined with the IgG constant region to form a complete antibody.
  • VL1 was mutated to obtain VL2 (mutation pattern: M4L, Y48V)
  • VH1 was mutated to obtain VH2 (mutation pattern: F24V, S30T, A49A, T73N)
  • VL3 was mutated to obtain VL4 (mutation pattern: I48V, F70Y)
  • VH1 was mutated to obtain VH2 (mutation pattern: S30T, I37V, A49G, T73N).
  • Exemplary backmutation patterns and combinations are as follows:
  • Table 2 Humanized mAb10591 and reverse mutations* *All amino acid position numbers in the table are based on the Kabat numbering convention. In the F24V of the heavy chain variable region, 24 is the 24th position in the Kabat numbering convention. Bit.
  • the corresponding heavy chain variable regions in the table above can be linked to the human IgG1 heavy chain constant region set forth in SEQ ID NO:9 to form the heavy chain of a full-length antibody, and the corresponding light chain variable regions can be linked to the human kappa light chain constant region set forth in SEQ ID NO:10 to form the light chain of a full-length antibody.
  • the heavy chain variable regions and light chain variable regions can also be linked to other heavy chain constant regions and light chain constant regions, respectively, to form a full-length antibody.
  • the humanized light chain variable region sequence was VL5 (SEQ ID NO: 37) or VL7 (SEQ ID NO: 39) and the heavy chain variable region sequence was VH5 (SEQ ID NO: 40) or VH7 (SEQ ID NO: 42); then they were recombined with the IgG constant region to form a complete antibody.
  • VL5 was mutated to obtain VL6 (mutation mode: Y53K)
  • VH5 was mutated to obtain VH6
  • VH7 was mutated to obtain VH8 (mutation mode: M48I, V68A, I70L, R72V, T74K).
  • backmutation modes and combinations are as follows:
  • Table 4 Humanized mAb10592 and reverse mutations* *All amino acid position numbers in the table are based on the Kabat numbering convention.
  • the corresponding heavy chain variable regions in the table above can be linked to the human IgG1 heavy chain constant region set forth in SEQ ID NO:9 to form the heavy chain of a full-length antibody, and the corresponding light chain variable regions can be linked to the human kappa light chain constant region set forth in SEQ ID NO:10 to form the light chain of a full-length antibody.
  • the heavy chain variable regions and light chain variable regions can also be linked to other heavy chain constant regions and light chain constant regions, respectively, to form a full-length antibody.
  • the humanized light chain variable region sequence was VL9 (SEQ ID NO: 44) or VL11 (SEQ ID NO: 46) and the heavy chain variable region sequence was VH9 (SEQ ID NO: 48) or VH11 (SEQ ID NO: 50); and then recombined with the IgG constant region to form a complete antibody.
  • VL9 was mutated to obtain VL10 (mutation mode: F70Y)
  • VH9 was mutated to obtain VH10
  • VL11 was mutated to obtain VL12 (mutation mode: L47I)
  • VH11 was mutated to obtain VH12 (mutation mode: V37I, M48I, V68A, S70L, R72V, T74I, Y95F).
  • Exemplary backmutation modes and combinations are as follows:
  • the corresponding heavy chain variable regions in the table above can be linked to the human IgG1 heavy chain constant region set forth in SEQ ID NO:9 to form the heavy chain of a full-length antibody, and the corresponding light chain variable regions can be linked to the human kappa light chain constant region set forth in SEQ ID NO:10 to form the light chain of a full-length antibody.
  • the heavy chain variable regions and light chain variable regions can also be linked to other heavy chain constant regions and light chain constant regions, respectively, to form a full-length antibody.
  • the full-length antibody sequence is as follows:
  • the above antibodies were cloned, expressed and purified respectively using conventional gene cloning and recombinant expression methods.
  • Part 2 ADC part
  • Examples 2-12-2-19 were synthesized according to the synthetic route of Example 2-11.
  • the structures and LC-MS results are shown in the table below.
  • Example 2-21 was synthesized by referring to the synthetic route of Example 2-20.
  • the structural formula and LC-MS results are shown in the table below.
  • Example 2-24 was synthesized by referring to Example 2-23 and the synthetic route.
  • the structural formula and LC-MS are shown in the table below.
  • Example 2-27 was synthesized by referring to the synthetic route of Example 2-26. Its structure and LC-MS information are shown in the table below.
  • Examples 2-28 were synthesized by referring to the synthetic route of Example 7. Their structures and LC-MS results are shown in the table below.
  • Examples 2-31-2-36 were synthesized by referring to the synthetic route of Example 2-11, and their structural formulas are shown in the table below.
  • the affinity of chimeric and humanized antibodies for Nectin 4 was detected using capture antibodies.
  • the antibodies were affinity captured using a Sensor Chip Protein A biosensor chip (Cytiva, 29650263), and then the antigen Nectin4-his was passed over the chip surface.
  • the reaction signal was detected in real time using a Biacore K8 instrument (Cytiva) to obtain the binding and dissociation curves. After each experimental cycle of dissociation was completed, the chip was washed and regenerated with regeneration buffer. After the experiment, the data was fitted with the (1:1) Langmuir model using GE Biacore K8 Evaluation version 3.0 software to obtain the affinity value.
  • the affinity of chimeric and humanized antibodies to proteins is shown in Tables 20 and 21 below:
  • the exemplary antibodies provided by the present invention have comparable or even better affinity for Nectin 4 than the positive control AGSM6.
  • the binding activity of the antibodies was detected by ELISA.
  • the process is briefly described as follows: the purified Nectin4-His antigen protein from Example 1 was coated at a coating concentration of 1 ⁇ g/mL. After blocking with 2% BSA overnight, serially diluted antibodies to be tested were added for incubation (the starting concentration of the antibodies in Table 15 was 10 ⁇ g/mL, 2-fold serial dilution). After incubation at 37°C for 1 hour, mouse anti-human IgG (immunoglobulin G) Fab antibody (Mouse Anti-Human IgG Fab Antibody) (12H3C4A6) [HRP] (Gensher, A01855-200) was added as a secondary antibody and incubated for 1 hour.
  • TMB Solerbo, RP1200-1000 mL
  • stop solution Boyuntian, P0215
  • the absorbance at OD450 was read on a microplate reader, and the binding EC50 value of each antibody was calculated using GraphPad Prism9 to compare the binding activities of the antibodies. The results are shown in Table 22.
  • the exemplary antibodies provided by the present invention have comparable or even better Nectin4 binding activity than the positive control AGSM6.
  • the cell line used in this experiment was T47D cells purchased from Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60397, a nectin4 high-expressing cell line.
  • the difference in endocytosis was obtained by comparing the mean fluorescence intensity (MFI) of the samples incubated at 37°C with that of the samples incubated at 4°C.
  • MFI mean fluorescence intensity
  • the exemplary antibodies provided by the present invention have comparable or even better endocytosis efficiency than the positive control AGSM6.
  • the exemplary antibody h10591-2 provided by the present invention has good specificity and does not bind to the same family proteins hNectin-1/2/3, but only binds to hNectin-4-His.
  • Nectin-4 has high homology in different species.
  • hNectin-4-His monkey cNectin-4 (ACRO,NE4-C52H4), and mouse mNectin-4 (ACRONE4-M52H3) proteins were coated, followed by serial dilutions of AGSM6 and h10591-2 antibodies.
  • the positive antibodies provided by the present invention have cross-reactivity with humans, monkeys, and rats, and weak cross-reactivity with mice, which is consistent with literature reports; the exemplary antibody h10591-2 cross-reactivity with humans and monkeys, but not with mice and rats.
  • Antibody 1 of this patent is AGSM6 prepared with reference to Example 2 of WO2012047724, and its heavy chain and light chain amino acid sequences are shown above.
  • Antibody 2 of this patent is hH2L1, which was prepared with reference to the sequence of Example 1 of CN117942409A.
  • the amino acid sequences of its heavy chain and light chain are shown in SEQ ID NO: 112 and SEQ ID NO: 113, respectively.
  • the nectin-4 antibody selected for example in this patent is h10591-2 prepared in Example 3.
  • the antibody-drug conjugates shown in Table 28 were prepared, wherein the NECTIN 4-ADC-1 to NECTIN 4-ADC-4, NECTIN 4-ADC-7-1, NECTIN 4-ADC-8, NECTIN 4-ADC-9-1, and NECTIN 4-ADC-10 conjugated antibodies were antibody 1, and the NECTIN 4-ADC-7-2, NECTIN 4-ADC-9-2, and NECTIN 4-ADC-22 to NECTIN 4-ADC-24 conjugated antibodies were h10591-2.
  • m or m1 is the drug-to-antibody ratio (DAR).
  • the antibody was diluted to 5 mg/mL in 1 mM EDTA-PBS buffer.
  • 5 mM TCEP was added at an 8-fold molar equivalent ratio for cleavage, and the mixture was shaken at 37°C for 2 h. After cleavage, 5 mg/mL of the linker-payload was added at a 6-fold molar equivalent ratio.
  • DMSO was also added to the system to a 10% DMSO content. After mixing at room temperature for 2 h, the sample was centrifuged to remove the precipitate and desalted. The final sample was subjected to quantitative analysis, SEC, and HIC analysis.
  • the antibody-drug conjugates shown in Table 29 were prepared, wherein the antibody conjugated to 9MW2821 and NECTIN 4-ADC-29 was antibody 2hH2L1, the antibody conjugated to NECTIN 4-ADC-5-1, NECTIN 4-ADC-6, and NECTIN 4-ADC-28 was antibody 1AGSM6, and the antibody conjugated to NECTIN 4-ADC-5-2, NECTIN 4-ADC-11 to NECTIN 4-ADC-21, NECTIN 4-ADC-25, NECTIN 4-ADC-26, and NECTIN 4-ADC-27 was h10591-2.
  • the maleimide ring of the double conjugated bridge part ADC has two forms: open ring and closed ring.
  • the maleimide of the linker part in ADC is hydrolyzed to form (Closed loop) becomes (Open loop).
  • the ADCs listed in Table 29-1 of the present invention exist in both open-ring and closed-ring forms.
  • the maleimide hydrolysis rate in nectin 4-ADC-14, nectin 4-ADC-15, nectin 4-ADC-14, and nectin 4-ADC-20 ADCs exceeded 80%, with nectin 4-ADC-11 completely hydrolyzed. This indicates that the introduction of aromatic groups can effectively control the hydrolysis of the maleimide moiety and improve ADC stability.
  • HPLC-HIC analysis was used to determine whether the coupling was successful and to detect the DAR value; SEC-HPLC was used to detect the purity of ADC.
  • MDA-MB-468 (breast cancer cells, Kebai, CBP60387) was used. This cell line has a high expression of Nectin 4.
  • Cell suspensions were prepared in fresh cell culture medium containing 10% FBS at a density of 5 ⁇ 104 cells/mL. 100 ⁇ L was added to each well of a 96-well cell culture plate (Fisherries Scientific, 310109027) and incubated at 37°C in 5% CO2 for 24 hours. ADC samples were prepared in fresh culture medium to a 4 ⁇ M concentration. Using this as the starting concentration, the samples were serially diluted tenfold in PBS for a total of nine concentrations. 50 ⁇ L of culture medium was aspirated from each well and 50 ⁇ L of the ADC solution was added, resulting in a 2 ⁇ M ADC concentration in the first well. The final volume was 100 ⁇ L per well.
  • the cells were incubated at 37°C in 5% CO2 for 5 days. The supernatant was discarded, and 100 ⁇ L of CCK8 (Biyuntian, C0042) was added to each well. The cells were incubated at 37°C for 4 hours. Chemiluminescence was measured on a microplate reader (TECAN, Spark) and data were analyzed using Graphpad Prism 5 software. The results are shown in Table 23 below.
  • MDA-MB-453 (breast cancer cells, Kebai, CBP60386) was used. This cell line has a high expression of Nectin 4.
  • Cell suspensions were prepared in fresh cell culture medium containing 10% FBS at a density of 5 ⁇ 104 cells/mL. 100 ⁇ L was added to each well of a 96-well cell culture plate (Fisherries Scientific, 310109027) and incubated at 37°C in 5% CO2 for 24 hours. ADC samples were prepared in fresh culture medium to a 0.8 ⁇ M concentration. Using this as the starting concentration, the samples were serially diluted fivefold with PBS for a total of nine concentrations. 50 ⁇ L of culture medium was aspirated from each well and 50 ⁇ L of the ADC solution was added, resulting in a starting ADC concentration of 0.4 ⁇ M in the first well. The final volume was 100 ⁇ L per well.
  • the cells were incubated at 37°C in 5% CO2 for 5 days. The supernatant was discarded, and 100 ⁇ L of CCK8 (Biyuntian, C0042) was added to each well. The cells were incubated at 37°C for 4 hours. Chemiluminescence was measured on a microplate reader (TECAN, Spark) and data were analyzed using Graphpad Prism 5 software. The results are shown in Table 23 below.
  • HT1376 (bladder cancer cells, Kebai, CBP60310) was used, which is a cell line with medium to high expression of Nectin 4.
  • ADC samples were prepared in fresh culture medium at 0.4 ⁇ M, 2 ⁇ M, and 7 ⁇ M concentrations. These concentrations were used as the initial concentrations, and a five-fold serial dilution was performed using PBS for a total of nine concentrations. 50 ⁇ L of culture medium was aspirated from each well, and 50 ⁇ L of the ADC solution was added, resulting in a 5 ⁇ M ADC concentration in the initial well.
  • the final volume was 100 ⁇ L per well. Incubate at 37°C in 5% CO2 for 3 days. All supernatants were discarded, and 100 ⁇ L of CCK8 (Biyuntian, C0042) was added to each well. The cells were incubated at 37°C for 4 hours. Chemiluminescence was read on a microplate reader (TECAN, Spark), and data were analyzed using Graphpad Prism 5 software. The experimental results are shown in Table 23 below.
  • T47D ductal breast carcinoma cell line, Kebai, CBP60397
  • Kebai ductal breast carcinoma cell line
  • CBP60397 ductal breast carcinoma cell line
  • Test Example 8 In vivo efficacy experiment of the ADC compound of the present invention
  • V 1/2L long diameter ⁇ (L short diameter) 2
  • RTV Relative tumor volume
  • Relative tumor proliferation rate T/C (%) TRTV/CRTV ⁇ 100%
  • Tumor inhibition rate (%) (CRTV-TRTV)/CRTV (%)
  • Tumor weight inhibition rate (%) (1-average tumor weight of the treatment group/average tumor weight of the control group) * 100%
  • V0 and VT are the tumor volumes at the beginning and end of the experiment, respectively.
  • CRTV and TRTV are the relative tumor volumes of the blank control group (PBS) and the experimental group, respectively, at the end of the experiment.
  • mice BALB/c-Nude nude mice were subcutaneously inoculated with human triple-negative breast cancer MDA-MB-468 cells (Kebai) (1 x 10 ⁇ 7/200 ⁇ L/mouse, in a 50% growth factor-reduced artificial basement membrane) in the right flank. After 7 days of inoculation, tumors grew to approximately 135 mm3 . The animals were then randomly divided into groups (D0), with 6 mice per group.
  • the exemplary compound NECTIN 4-ADC-11 of the present disclosure has a significant tumor growth inhibitory effect at a dose of 0.5-2 mg/kg. Its tumor growth inhibitory effect is significantly better than that of enfortumab vedotin. Even the tumor inhibitory effect of 0.5 mg/kg NECTIN 4-ADC-11 is better than that of 2 mg/kg enfortumab vedotin.
  • the TGI of 2 mg/kg NECTIN 4-ADC-11 is as high as 100%.
  • mice BALB/c-Nude nude mice were subcutaneously inoculated with human breast cancer MDA-MB-453 cells (Kebai) (1 x 10 ⁇ 7/200 ⁇ L/mouse, in a 50% growth factor-reduced artificial basement membrane) in the right flank. After inoculation, tumors were allowed to grow for 7 days. Once the tumor volume reached approximately 120 mm3 , the animals were randomly divided into groups (D0), with 5 mice per group.
  • the drug was administered via tail vein injection, with a single dose of 1 mg/kg administered in parallel, and observation was continued for 20 days. Tumor volume and body weight were measured twice a week and the data were recorded.
  • the exemplary antibody-drug conjugates NECTIN 4-ADC-11 and NECTIN4-ADC-22 disclosed herein have significant tumor growth inhibitory effects and have better tumor inhibitory effects than Enfortumab vedotin.
  • mice BALB/c-Nude nude mice were subcutaneously inoculated with human breast cancer HT1376 cells (Kebai) (1 x 10 ⁇ 7/200 ⁇ L/mouse, in a 50% growth factor-reduced artificial basement membrane) in the right flank. After inoculation, tumors were allowed to grow for 8 days. Once the tumor volume reached approximately 120 mm3 , the animals were randomly divided into groups (D0), with 5 mice per group.
  • the drug was administered by tail vein injection, with multiple doses of 8 mg/kg and 4 mg/kg administered in parallel, and single injection administered, for 21 days. Tumor volume and body weight were measured twice weekly and the data were recorded.
  • the exemplary antibody-drug conjugates NECTIN 4-ADC-7-2, NECTIN 4-ADC-9-2, and NECTIN 4-ADC-11 disclosed herein have significant tumor growth inhibitory effects, and their tumor inhibitory effects are better than or equivalent to those of Enfortumab vedotin.
  • mice BALB/c-Nude nude mice were subcutaneously inoculated with human breast cancer HT1376 cells (Kebai) (1 x 10 ⁇ 7/200 ⁇ L/mouse, in a 50% growth factor-reduced artificial basement membrane) in the right flank. After inoculation, tumors were allowed to grow for 8 days. Once the tumor volume reached approximately 120 mm3 , the animals were randomly divided into groups (D0), with 6 mice per group.
  • the drug was administered by tail vein injection, with multiple doses of 5 mg/kg, 2.5 mg/kg, and 1 mg/kg administered in parallel, and observed for 21 days. Tumor volume and body weight were measured twice weekly, and the data were recorded to calculate the tumor volume inhibition rate.
  • mice were subcutaneously inoculated with human bladder transitional cell papilloma cells (RT4/Nectin 4) (9 x 10 ⁇ 6/200 ⁇ L/mouse, supplemented with a 50% growth factor-reduced artificial basement membrane) in the right flank. After 7 days of inoculation, tumors grew to approximately 110-120 mm3 in volume. The animals were then randomly divided into groups (D0), with 5 mice per group.
  • RT4/Nectin 4 human bladder transitional cell papilloma cells
  • the animals were injected via tail vein at a single dose of 5 mg/kg and observed for 21 days. Tumor volume and body weight were measured twice weekly and the data were recorded.
  • the exemplary antibody-drug conjugate NECTIN 4-ADC-11 disclosed in the present invention has a significant tumor growth inhibitory effect in the RT4/Nectin 4 CDX model, and its tumor inhibitory effect is significantly better than that of Enfortumab vedotin.
  • mice were subcutaneously inoculated with human breast cancer MDA-MB-453 cells (Kebai) (1 ⁇ 10 7 /200 ⁇ L/mouse, with 50% reduced growth factor artificial basement membrane) in the right flank. After inoculation, tumors grew for 6 days. When the tumor volume reached approximately 135 mm 3 , the animals were randomly divided into groups (D0), with 6 mice per group.
  • the exemplary antibody-drug conjugate NECTIN 4-ADC-11 disclosed in the present invention can effectively inhibit the growth of MDA-MB-453 transplanted tumors in tumor-bearing nude mice at doses of 0.5-2 mg/kg in a dose-dependent manner, and its efficacy is significantly better than that of positive drugs.
  • mice were subcutaneously inoculated with human triple-negative breast cancer cells (MDA-MB-468) (1 ⁇ 10 7 /200 ⁇ L/mouse, with 50% reduced growth factor artificial basement membrane) in the right flank. After cell inoculation, tumors grew for 10-11 days. When the tumor volume reached approximately 115-135 mm 3 , the animals were randomly divided into groups (D0), with 5 mice per group.
  • MDA-MB-468 human triple-negative breast cancer cells
  • the exemplary antibody-drug conjugate NECTIN 4-ADC-11 disclosed herein can effectively inhibit the growth of MDA-MB-468 transplanted tumors in tumor-bearing nude mice at doses of 1 mg/kg and 2 mg/kg in a dose-dependent manner, and its efficacy is significantly better than that of the positive drug 9MW2821.
  • Acute toxicity studies were conducted in 6-8 week-old BALB/c mice treated with Vital River. Enfortumab vedotin, NECTIN 4-ADC-9-2, NECTIN 4-ADC-11, and NECTIN 4-ADC-22 were administered once via tail vein injection at a dose of 100 mg/kg in a volume of 200 ⁇ L per group. Four experimental groups (6 mice each, 3 males and 3 females) were administered, respectively.
  • Body weight recorded once before administration and twice a week after administration; the recording time is Day 0, Day 4, Day 7, Day 12 and Day 14.
  • Acute toxicity studies were conducted in 6-8 week-old BALB/c mice.
  • Enfortumab vedotin, NECTIN 4-ADC-9-2, and NECTIN 4-ADC-11 were administered via tail vein injection at a dose of 60 mg/kg in a volume of 200 ⁇ L per group. Dosing was repeated twice (day 0 and day 7) for 14 days.
  • the solvent control group (PBS group) consisted of four mice (two males and two females), while the experimental groups consisted of six mice (three males and three females).
  • Enfortumab vedotin, NECTIN 4-ADC-9-2, and NECTIN 4-ADC-11 were administered, respectively.
  • Body weight once before administration, once a week after administration, and once before autopsy (twice a week in this experiment)
  • NECTIN 4-ADC-9-2, NECTIN 4-ADC-11, and NECTIN 4-ADC-22 disclosed herein had survival rates comparable to those of the vehicle group during the observation period, had good safety, and were significantly superior to Enfortumab vedotin in terms of mouse survival rate.
  • Administration route and frequency Tail vein injection, once a week, with the day of administration being Day 0. The results are shown in Figures 21 and 22.
  • the exemplary antibody-drug conjugate NECTIN 4-ADC-11 disclosed in this patent (with an MTD of up to 80 mg/kg) has a better safety profile than the positive control enfortumab vedotin (MTD ⁇ 70 mg/kg).
  • Administration route Tail vein injection, once a week, the day of administration is Day 1, and the drug is administered on Days 1, 8, 15, and 22, for a total of 4 times.
  • Frequency of administration Multiple doses, once a week;
  • the exemplary antibody-drug conjugate NECTIN 4-ADC-11 of this application is well tolerated and has a higher tolerance dose than the positive drug Enfortumab vedotin.
  • the degree of weight loss at the same dose is lower than that of the positive drug, and it can reduce the serious side effects of Nectin 4 target skin toxicity to a certain extent.
  • mice After a single injection of NECTIN 4-ADC-11 and control via the tail vein of mice, their basic pharmacokinetic characteristics in mice were observed.
  • Dosage frequency and method Single administration via tail vein.
  • the first blood draw from each animal was done via the orbital venous plexus; the blood volume was 0.15 mL/time point.
  • the second blood draw was done by enucleation of the eyeball; the blood volume was 0.5 mL/time point.
  • three mice were used as a cohort, with each cohort consisting of 24 mice.
  • mice were subcutaneously inoculated with human breast cancer HT1376 cells (Kebai) (1 ⁇ 10 7 /200 ⁇ L/mouse, with a 50% growth factor-reduced artificial basement membrane). After 17 days of inoculation, tumors grew to approximately 250-300 mm 3 in volume. The animals were then randomly divided into 24 groups (D0), with 3 animals per group. (PBS, Enfortumab vedotin, and NECTIN 4-ADC-11 were administered.)
  • the drug was administered by tail vein injection, with a single dose of 6 mg/kg.
  • the antibody-drug conjugate NECTIN 4-ADC-11 of the present invention specifically accumulates in tumors, has lower payload exposure in serum than the positive control drug Enfortumab vedotin, and has higher safety in vivo.

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Abstract

提供了抗Nectin-4抗体、配体-药物偶联物及其应用。抗Nectin-4抗体、配体-药物偶联物可用于Nectin-4介导的疾病或病症的预防和/或治疗。

Description

抗Nectin-4抗体、配体药物偶联物及其应用
本申请要求如下优先权：
2024年1月29日提交的中国专利CN202410116532.5、
2024年5月8日提交的中国专利CN 202410564053.X、
2024年10月8日提交的中国专利CN202411388187.7、
2024年10月14日提交的中国专利CN 202411429924.4、
2024年12月6日提交的中国专利CN 202411783791.X、
2024年10月14日提交的中国专利CN202411429096.3、
2024年12月9日提交的中国专利CN202411795873.6、
2024年12月27日提交的中国专利CN202411947010.6。
技术领域
本发明涉及医药技术领域，具体涉及靶点为Nectin-4的抗体、配体药物偶联物及其应用。
背景技术
Nectin-4，又称脊髓灰质炎病毒受体相关蛋白4(PVRL4)，是一种属于免疫球样蛋白样分子Nextins家族的I型跨膜蛋白。研究发现，Nectin-4在成人健康组织中表达有限，但在膀胱癌、乳腺癌、肺癌等多种恶性肿瘤中过表达，并且作为肿瘤相关诱导剂与增殖、转移、上皮-间质细胞转化等肿瘤进展的各个方面相关，是一种理想的抗肿瘤药物靶点。
目前FDA批准以Nectin-4作为癌症治疗的靶标的药物仅有维汀-恩弗妥单抗(Enfortumabvedotin)用于晚期泌尿道上皮癌的二线治疗。因此，发现该靶点的一些新的抗体序列，用于下一步的成药性分析，是本领域亟待解决的技术问题。
抗体药物偶联物(ADC)由人源化单克隆抗体(mAb)、细胞毒性有效载荷(Cytotoxic payload)以及连接子(连接单元或连接结构)(Linker)三部分组成，抗体通过连接子与细胞毒性药物偶联，共同赋予ADC细胞靶向性和杀死细胞的效力，从而实现精准高效消灭癌细胞，现已成为抗癌药物研发的热点之一。理想的ADC药物在血液循环中应保持稳定，准确地达到治疗靶点，并最终在靶点(例如癌细胞)附近释放细胞毒性有效载荷(Payload)。目标抗原、抗体、细胞毒性有效载荷、连接子及缀合方式中的每个要素都会影响ADC的最终疗效和安全性。
其中，连接子及DAR值的选择是改善ADC药物活性和药代动力学性质的关键因素之一。增加药物抗体比率(drug to antibody ratio，DAR)尽管从理论上讲可以提高ADC的活性，但高DAR值的ADC通常热稳定性差、容易聚集、体内清除速率加快，导致药效降低和毒性增加。研究表明，药物-连接子系统的强疏水性可能是导致这一现象的主要原因。为了使ADC同时具有理想活性和良好的药代动力学特性，一种亲水性的药物-连接子系统来使高DAR值ADC在体内具有良好的抗肿瘤活性、安全性及药代动力学特性是急需的。
目前，全球在研的Nectin-4靶向药物共28款，其中有20余款为ADC药物，并有9款进入临床。Enfortumab vedotin(EV)是目前唯一一个靶向Nectin-4的上市药物，它由抗Nectin-4抗体与微管蛋白抑制剂MMAE通过二肽连接子连接而成，被FDA批准用于治疗局部晚期或转移性尿路上皮癌[2]，其与Keytruda的联合疗法被批准用于一线治疗。虽然EV显示出优异的临床潜力，但在乳腺癌等其他实体瘤中尚无突破进展(HR阳性/HER2阴性乳腺癌队列ORR为15.6％，TNBC队列ORR为19％)。在临床应用中，EV观察到严重和致命的皮肤不良反应如Stevens-Johnson综合征(SJS)和中毒性表皮坏死松解症(TEN)[3]，并且还会导致高血糖、肺部炎症以及周围神经病变等不良反应的发生，安全性有待提高。除此以外，目前在研的其他靶向Nectin-4的ADC药物如9MW2821、SYS6002等虽然取得了优于EV的临床疗效，但全部选用MMAE或喜树碱类衍生物作为细胞毒性荷载[4]，存在耐药风险。因此，亟需开发一种差异化payload的Nectin-4靶向ADC药物，以取得更好的临床获益，并克服肿瘤耐药性，更好地满足患者需求。
发明内容
本发明第一方面提供了一种抗体或其抗原结合片段，其包含：特异性结合Nectin-4的抗体或其抗原结合片段，其包含：
SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、或51所示的重链可变区内所含的三个重链互补决定区(HCDR)HCDR1、HCDR2和HCDR3；和/或
SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、或47所示的轻链可变区内所含的三个轻链互补决定区(LCDR)LCDR1、LCDR2和LCDR3。
在另外一个实施例中提供了特异性结合Nectin-4的抗体或其抗原结合片段，其包含：
1)HCDR3，所述HCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:13、19、25、84、90、和96；和
2)LCDR3，所述LCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:16、22、28、87、93、和99。
3、如权利要求2述的抗体或其抗原结合片段，其进一步包含：
1)HCDR1，所述HCDR1包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:11、17、23、82、88、和94；和
2)LCDR1，所述LCDR1包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:14、20、26、85、91、和97；
优选地，所述抗体或其抗原结合片段还包含：
3)HCDR2，所述HCDR2包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:12、18、24、83、89、和95；和
4)LCDR2，所述LCDR2包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:15、21、27、86、92、和98。
在一些实施方案中，所述的抗体或其抗原结合片段，其包含：
1)SEQ ID NO:3所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:4所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
2)SEQ ID NO:5所示的重链可变区所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:6所示的轻链可变区所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
3)SEQ ID NO:7所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:8所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
4)SEQ ID NO:100所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:101所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
5)SEQ ID NO:102所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:103所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
6)SEQ ID NO:104所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:105所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
7)SEQ ID NO:33所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:29所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
8)SEQ ID NO:34所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:30所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
9)SEQ ID NO:35所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:31所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
10)SEQ ID NO:36所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:32所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
11)SEQ ID NO:40所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:37所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
12)SEQ ID NO:41所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:38所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
13)SEQ ID NO:42所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:39所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
14)SEQ ID NO:43所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:37所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
15)SEQ ID NO:43所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:38所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
16)SEQ ID NO:43所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:39所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
17)SEQ ID NO:48所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:44所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
18)SEQ ID NO:49所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:45所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
19)SEQ ID NO:50所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:46所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
20)SEQ ID NO:51所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和SEQ ID NO:47所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3。
在一些实施方案中，所述的抗体或其抗原结合片段，其包含：
1)包含SEQ ID NO:11所示序列或由其组成的HCDR1，
包含SEQ ID NO:12所示序列或由其组成的HCDR2，
包含SEQ ID NO:13所示序列或由其组成的HCDR3，
包含SEQ ID NO:14所示序列或由其组成的LCDR1，
包含SEQ ID NO:15所示序列或由其组成的LCDR2，和
包含SEQ ID NO:16所示序列或由其组成的LCDR3；或
2)包含SEQ ID NO:17所示序列或由其组成的HCDR1，
包含SEQ ID NO:18所示序列或由其组成的HCDR2，
包含SEQ ID NO:19所示序列或由其组成的HCDR3，
包含SEQ ID NO:20所示序列或由其组成的LCDR1，
包含SEQ ID NO:21所示序列或由其组成的LCDR2，和
包含SEQ ID NO:22所示序列或由其组成的LCDR3；或
3)包含SEQ ID NO:23所示序列或由其组成的HCDR1，
包含SEQ ID NO:24所示序列或由其组成的HCDR2，
包含SEQ ID NO:25所示序列或由其组成的HCDR3，
包含SEQ ID NO:26所示序列或由其组成的LCDR1，
包含SEQ ID NO:27所示序列或由其组成的LCDR2，和
包含SEQ ID NO:28所示序列或由其组成的LCDR3；或
4)包含SEQ ID NO:82所示序列或由其组成的HCDR1，
包含SEQ ID NO:83所示序列或由其组成的HCDR2，
包含SEQ ID NO:84所示序列或由其组成的HCDR3，
包含SEQ ID NO:85所示序列或由其组成的LCDR1，
包含SEQ ID NO:86所示序列或由其组成的LCDR2，和
包含SEQ ID NO:87所示序列或由其组成的LCDR3；或
5)包含SEQ ID NO:88所示序列或由其组成的HCDR1，
包含SEQ ID NO:89所示序列或由其组成的HCDR2，
包含SEQ ID NO:90所示序列或由其组成的HCDR3，
包含SEQ ID NO:91所示序列或由其组成的LCDR1，
包含SEQ ID NO:92所示序列或由其组成的LCDR2，和
包含SEQ ID NO:93所示序列或由其组成的LCDR3；或
6)包含SEQ ID NO:94所示序列或由其组成的HCDR1，
包含SEQ ID NO:95所示序列或由其组成的HCDR2，
包含SEQ ID NO:96所示序列或由其组成的HCDR3，
包含SEQ ID NO:97所示序列或由其组成的LCDR1，
包含SEQ ID NO:98所示序列或由其组成的LCDR2，和
包含SEQ ID NO:99所示序列或由其组成的LCDR3。
本发明的另外一个实施方案提供特异性结合Nectin-4的抗体或其抗原结合片段，其包含：
重链可变区，所述重链可变区的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％、至少85％、至少90％、或至少95％的序列同一性：SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、和51；优选地，所述重链可变区具有与SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、或51所示氨基酸序列相同的HCDR氨基酸序列、且所述重链可变区的框架区的氨基酸序列与氨基酸序列SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、或51的框架区有至少80％、至少85％、至少90％、或至少95％的序列同一性；优选地，所述重链可变区具有如SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、或51所示的序列；和/或
轻链可变区，所述轻链可变区的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％、至少85％、至少90％、或至少95％的序列同一性：SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、和47；优选地，所述轻链可变区的氨基酸序列具有与氨基酸序列SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、或47相同的LCDR、且所述轻链可变区的框架区与序列SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、或47的框架区有至少80％、至少85％、至少90％、或至少95％的序列同一性；优选地，所述轻链可变区具有如SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、或47所示的序列。
在一些实施方案中，所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区和轻链可变区的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：
SEQ ID NO:3和SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8、SEQ ID NO:100和SEQ ID NO:101、SEQ ID NO:102和SEQ ID NO:103、SEQ ID NO:104和SEQ ID NO:105、SEQ ID NO:33和SEQ ID NO:29、SEQ ID NO:34和SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:32、SEQ ID NO:40和SEQ ID NO:37、SEQ ID NO:41和SEQ ID NO:38、SEQ ID NO:42和SEQ ID NO:39、SEQ ID NO:43和SEQ ID NO:37、SEQ ID NO:43和SEQ ID NO:38、SEQ ID NO:43和SEQ ID NO:39、SEQ ID NO:48和SEQ ID NO:44、SEQ ID NO:49和SEQ ID NO:45、SEQ ID NO:50和SEQ ID NO:46、和SEQ ID NO:51和SEQ ID NO:47。
在一些实施方案中，所述的抗体或其抗原结合片段，其包含重链恒定区或其变体和/或轻链恒定区或其变体，优选其包含鼠源的或人源化的重链恒定区和/或轻链恒定区；优选地，所述重链恒定区为IgG重链恒定区，和/或所述轻链恒定区为κ或λ轻链恒定区；优选地，所述重链恒定区为IgG1重链恒定区，和/或所述轻链恒定区为κ轻链恒定区；优选地，所述人源化的重链恒定区的氨基酸序列如SEQ ID NO:9所示，和/或所述人源化的轻链恒定区的氨基酸序列如SEQ ID NO:10所示。
在一些实施方案中，所述的抗体或其抗原结合片段，所述抗原结合片段选自Fab、Fab'、Fab'-SH、Fv、scFv和F(ab')2。
在一些实施方案中，抗体或其抗原结合片段，所述抗体为单克隆抗体、嵌合抗体、人源化抗体、全人源抗体或双特异性抗体。
在一些实施方案中，所述的抗体或其抗原结合片段，其包含重链和/或轻链，其中，
所述重链的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％、至少85％、至少90％、或至少95％的序列同一性：SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、和SEQ ID NO:79；优选，所述重链的氨基酸序列如SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、或SEQ ID NO:79所示；和/或
所述轻链的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％、至少85％、至少90％、或至少95％的序列同一性：SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:107SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、和SEQ ID NO:75；优选，所述轻链的氨基酸序列如SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:107SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、或SEQ ID NO:75所示。
在一些实施方案中，其包含重链和轻链，所述重链和轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:52和SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57、SEQ ID NO:106和SEQ ID NO:107、SEQ ID NO:108和SEQ ID NO:109、SEQ ID NO:110和SEQ ID NO:111、SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:60和SEQ ID NO:59、SEQ ID NO:61和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:58、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:65和SEQ ID NO:64、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:64、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:64、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:64、SEQ ID NO:69和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:63、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:63、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:76和SEQ ID NO:75、SEQ ID NO:77和SEQ ID NO:72、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:75、SEQ ID NO:78和SEQ ID NO:72、SEQ ID NO:78和SEQ ID NO:73、SEQ ID NO:78和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、SEQ ID NO:79和SEQ ID NO:72、SEQ ID NO:79和SEQ ID NO:73、SEQ ID NO:79和SEQ ID NO:74、和SEQ ID NO:79和SEQ ID NO:75，优选地，其重链和轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、和SEQ ID NO:79和SEQ ID NO:72。
13、如权利要求1-12任一项所述的抗体或其抗原结合片段，其中所述HCDR和LCDR根据Kabat定义方案、Chothia定义方案、Abm定义方案、IMGT定义方案和/或Contact(接触)定义方案定义；优选，所述HCDR和LCDR根据Kabat定义方案或Chothia定义方案定义。
14、核酸，其编码如权利要求1-13中任一项所述的抗体或其抗原结合片段。
15、表达载体，其包含如权利要求14所述的核酸。
16、宿主细胞，其包含如权利要求15所述的表达载体。
17、生产抗体或其抗原结合片段的方法，其包含培养如权利要求16所述的宿主细胞，并从培养物中回收由此表达的抗体或其抗原结合片段。
在一些实施方案中，其中所述宿主细胞为原核细胞或真核细胞，优选地，所述宿主细胞为大肠杆菌细胞、酵母细胞、昆虫细胞、植物细胞或哺乳动物细胞；优选，所述宿主细胞为中国仓鼠卵巢细胞(CHO)、CHO细胞变体、293细胞或NSO细胞。
本发明还提供配体药物偶联物或其药学上可接受的盐，其中，所述配体药物偶联物包含权利要求1-13中任一项所述的抗体或其抗原结合片段、以及效应分子部分，其中，所述效应分子部分偶联至所述抗体或其抗原结合片段；优选所述效应分子部分选自细胞毒性药物部分、治疗自身免疫疾病的药物部分和抗炎的药物部分。
本发明还提供配体药物偶联物或其药学上可接受的盐，所述配体药物偶联物如式1所示，
其中，
Ab为靶向Nectin-4相结合的抗体或抗原结合片段，优选地，Ab为权利要求1-13中任一项所述的抗体或其抗原结合片段、或为包含如SEQ ID NO:80所示的重链和如SEQ ID NO:81所示的轻链的抗体、或为包含如SEQ ID NO:112所示的重链和如SEQ ID NO:113所示的轻链的抗体；优选地，Ab为权利要求1-13中任一项所述的抗体或其抗原结合片段；
m选自1-20的整数或小数；优选m选自1-12的整数或小数；
L为化学连接结构；
D为效应分子部分部分；优选D选自细胞毒性药物部分、治疗自身免疫疾病的药物部分和抗炎的药物部分；优选地，D为细胞毒性药物部分；优选地，D选自拓扑异构酶I(TOP1)抑制剂部分、微管蛋白聚合抑制剂部分、拓扑异构酶Ⅱ(TOP2)抑制剂部分、二氢叶酸还原酶抑制剂部分、DNA烷化剂部分、胸腺核苷合成酶抑制剂部分、嘌呤核苷合成酶抑制剂部分、核苷酸还原酶抑制剂部分、DNA多聚酶抑制剂部分、RNA聚合酶Ⅱ抑制剂部分和其他能够抑制细胞增殖的化合物部分；优选地，D选自(艾日布林部分)和其中波浪线表示D与L的连接点。
在一些实施方案中，其中，
L为其中Ta处的表示与所述抗体或其抗原结合片段的连接点，L5c处的表示与D的连接点；
Ta选自 其中所述Ta结构中左侧的表示与所述抗体或其抗原结合片段的连接点，所述Ta结构中右侧的表示与Mc的连接点；
Mc选自单键、6-10元亚芳基、5-13元亚杂芳基、3-15元亚环烷基和3-15元亚杂环烷基，所述亚芳基、亚杂芳基、亚环烷基和亚杂环烷基，未被取代或任选地被一个或多个Ra取代；
L1c选自单键、亚炔基、亚烯基、-NRa-、-O-、-C(O)-、-NRa-C(O)-、亚烷基和亚杂烷基中的1个、2个、3个或更多个的组合，其中所述亚炔基、亚烯基、亚烷基、和亚杂烷基未被取代或任选地被一个或多个Ra取代；
L2c选自单键、-(CH2)n7-(OCH2CH2)n8-O-(CH2)n9-Cy3-(CH2)n10-C(O)-、-(CH2)n7-(CH2CH2O)n8-(CH2)n9-Cy3-(CH2)n10-C(O)-、-(CH2)n7-Cy3-(CH2CH2O)n8-(CH2)n9-C(O)-、-(CH2)n11-(OCH2CH2)n12-O-(CH2)n13-Cy3-(CH2)n14-(OCH2CH2)n15-C(O)-、-(CH2)n7-(OCH2CH2)n8-(CH2)n10-C(O)-、 中的1个、2个、3个或更多个组合；
n7、n9、n10、n11、n13和n14每次出现时各自独立地选自0、1、2、3、4、5和6；
n8和n17每次出现时各自独立地选自1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27和28；
n12和n15每次出现时各自独立地选自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14和15，且n12和n15不同时为0；
j每次出现时各自独立地选自0、1、2、3、4、5和6；
Cy3选自6-10元亚芳基、5-13元亚杂芳基、3-15元亚环烷基和3-15元亚杂环烷基，所述亚芳基、亚杂芳基、亚环烷基和亚杂环烷基未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；
Mc、L1c、和L2c不同时为单键；
L4c为由氨基酸构成的肽残基或-NRa-亚烷基-NRa-，其中所述亚烷基、和氨基酸未被取代或任选地进一步被一个或多个Ra取代；
L5c选自单键、-N(Rb)-(CH2)g-O-(CH2)g-O-C(O)-、-N(Rb)-(CH2)g-O-(CH2-CH2-O)g-C(O)-、
n每次出现时各自独立地选自1-30间的任一整数；优选地，n每次出现时各自独立地选自1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27和28；
X1选自
g每次出现时各自独立地选自1、2、3、4、5和6；和
Ra和Rb每次出现时各自独立选自H、氘原子、F、Cl、Br、I、C1-C6烷基、C1-C6氘代烷基、C2-C6杂烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、羟基、硝基、氰基、氨基、羧基、C3-C6环烷基、3-6元杂环基、C1-C6烷基氨基、C1-C6烷基-C(O)-、C1-C6烷基-O-C(O)-、C1-C6烷基-NHC(O)-、-C1-6亚烷基-C(O)-NH2、C1-6烷基O-C(O)NH-、-C1-6亚烷基-NHC(O)-NH2、C1-C6烷基-NHC(O)-NH-、C6-C15芳基和5-13元杂芳基，其中所述的C1-C6烷基、C2-C6杂烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、C3-C6环烷基、3-6元杂环基、C1-C6烷基氨基、C6-C15芳基和5-13元杂芳基各自独立地任选被选自F、Cl、Br、I、C1-C6烷基、C2-C6杂烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、羟基、C1-C6卤代烷基、C1-C6羟烷基、氰基、氨基、硝基、C3-C6环烷基、3-6元杂环基、C1-C6烷基氨基、C6-C15芳基和5-13元杂芳基中的1个、2个、3个或多个取代基所取代。
在一些实施方案中，，其中，
Mc选自单键、亚苯基、5-7元亚杂芳基、3-7元亚环烷基和3-7元亚杂环烷基，所述亚芳基、亚杂芳基、亚环烷基和亚杂环烷基，未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；优选地，Mc选自单键、和/或
L1c选自单键、C2-6亚炔基、C2-6亚烯基、-NRa-、-O-、-C(O)-、-NRa-C(O)-、C1-6亚烷基和C1-6亚杂烷基中1个、2个、3个或更多个的组合，其中所述亚炔基、亚烯基、亚烷基和亚杂烷基未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；优选地，L1c选自单键、C2-3亚炔基、C2-3亚烯基、-NRa-、-C(O)-、-NRa-C(O)-、C1-3亚烷基和C1-3亚杂烷基中1个、2个、3个或多个的组合，其中所述亚炔基、亚烯基、亚烷基和亚杂烷基未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；和/或
Cy3选自 优选地，Cy3选自和/或
L4c为由2、3、4、5、6或7个氨基酸构成的肽残基或-NRa-C1-6亚烷基-NRa-，其中所述氨基酸选自D-丙氨酸、L-丙氨酸、苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、亮氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸、精氨酸和天冬酰胺，所述亚烷基和氨基酸未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；优选地，L4c选自-NH-CH2CH2-NH-、 和/或
L5c选自：单键、 和/或
Ra和Rb每次出现时各自独立地选自H、氰基、氘原子、F、Cl、Br、I、羧基、羟基、氨基、C1-C3烷基氨基、C1-C3烷基和C1-C3烷氧基。
在一些实施方案中，其中，
Mc选自单键、和/或
L1c选自单键、C2-3亚炔基、C2-3亚烯基、-NRa-、-C(O)-、-NRa-C(O)-、C1-3亚烷基和C1-3亚杂烷基中1个、2个、3个或多个的组合，其中所述亚炔基、亚烯基、亚烷基和亚杂烷基未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；和/或
L2c选自单键、 其中，n17每次出现时各自独立地选自6、8、12和24；和/或
L4c选自-NH-CH2CH2-NH-、 和/或
L5c选自：单键、 和/或
Ra和Rb每次出现时各自独立地选自H、氰基、氘原子、F、Cl、Br、I、羧基、羟基、氨基、C1-C3烷基氨基、C1-C3烷基和C1-C3烷氧基。
在一些实施方案中，其中，L选自下述结构：





其中，n8和n5每次出现时各自独立地选自1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27和28。
在一些实施方案中，其中，
m为1至10任意整数或小数；优选地，m为2至8任意整数或小数；优选地，m为2至6任意整数或小数；和/或
D选自拓扑异构酶I(TOP1)抑制剂部分、微管蛋白聚合抑制剂部分、拓扑异构酶Ⅱ(TOP2)抑制剂部分、二氢叶酸还原酶抑制剂部分、DNA烷化剂部分、胸腺核苷合成酶抑制剂部分、嘌呤核苷合成酶抑制剂部分、核苷酸还原酶抑制剂部分、DNA多聚酶抑制剂部分、RNA聚合酶Ⅱ抑制剂部分和其他能够抑制细胞增殖的化合物部分；优选地，D选自(艾日布林部分)和优选地，D为(艾日布林部分)，其中波浪线表示D与L的连接点。
在一些实施方案中，其中，所述配体-药物偶联物选自下述结构：










其中，Ab为权利要求1-13中任一项所述的抗体或其抗原结合片段、或包含如SEQ ID NO:80所示的重链和如SEQ ID NO:81所示的轻链的抗体、或包含如SEQ ID NO:112所示的重链和如SEQ ID NO:113所示的轻链的抗体；
m和m1每次出现时各自独立地选自1-20的整数或小数；优选地，m和m1每次出现时各自独立地选自1-12的整数或小数；优选地，m和m1每次出现时各自独立地选自1至10的整数或小数；优选地，m和m1每次出现时各自独立地选自2至8整数或小数；优选地，m和m1每次出现时各自独立地选自2-6的整数或小数；优选地m和m1每次出现时各自独立地选自1、2、3、4、5、6、7、8、9和10；和
n1、n8和n5每次出现时各自独立地选自1、2、3、4、5、6、7、和8。
在一些实施方案中，其中，
Ab为权利要求1-13中任一项所述的抗体或其抗原结合片段；优选地，Ab为包含重链和轻链的抗体，所述抗体中的重链和轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:52和SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57、SEQ ID NO:106和SEQ ID NO:107、SEQ ID NO:108和SEQ ID NO:109、SEQ ID NO:110和SEQ ID NO:111、SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:60和SEQ ID NO:59、SEQ ID NO:61和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:58、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:65和SEQ ID NO:64、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:64、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:64、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:64、SEQ ID NO:69和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:63、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:63、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:76和SEQ ID NO:75、SEQ ID NO:77和SEQ ID NO:72、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:75、SEQ ID NO:78和SEQ ID NO:72、SEQ ID NO:78和SEQ ID NO:73、SEQ ID NO:78和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、SEQ ID NO:79和SEQ ID NO:72、SEQ ID NO:79和SEQ ID NO:73、SEQ ID NO:79和SEQ ID NO:74、和SEQ ID NO:79和SEQ ID NO:75；优选，所述抗体中的重链和轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、和SEQ ID NO:79和SEQ ID NO:72；和/或
m和m1每次出现时各自独立地选自1-20的整数或小数；优选地，m和m1每次出现时各自独立地选自1-12的整数或小数；优选地，m和m1每次出现时各自独立地选自1至10的整数或小数；优选地，m和m1每次出现时各自独立地选自2至8整数或小数；优选地，m和m1每次出现时各自独立地选自2-6的整数或小数；优选地m和m1每次出现时各自独立地选自1、2、3、4、5、6、7、8、9和10。
本发明还提供抗体或其抗原结合片段，其包含：
氨基酸序列为SEQ ID NO:52、54、或56的肽段内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3；和/或
氨基酸序列为SEQ ID NO:53、55、或57的肽段内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3。
本发明还提供抗体或其抗原结合片段，其包含：
1)HCDR3，所述HCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:13、19、和25；和
2)LCDR3，所述LCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:16、22、和28；
优选地，所述的抗体或其抗原结合片段，其包含：
1)HCDR1，所述HCDR1包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:11、17、23；和
2)LCDR1，所述LCDR1包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:14、20、26；
优选地，所述抗体或其抗原结合片段还包含：
3)HCDR2，所述HCDR2包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:12、18、24；和
4)LCDR2，所述LCDR2包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:15、21、27。
在一些实施方案中，其包含：
1)氨基酸序列为SEQ ID NO:52所示肽段内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
氨基酸序列为SEQ ID NO:53所示肽段内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
2)氨基酸序列为SEQ ID NO:54所示肽段内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
氨基酸序列为SEQ ID NO:55所示肽段内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
3)氨基酸序列为SEQ ID NO:56所示肽段内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
氨基酸序列为SEQ ID NO:57所示肽段内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；
优选地，所述的抗体或其抗原结合片段，其包含：
1)包含SEQ ID NO:11或由其组成的HCDR1，
包含SEQ ID NO:12或由其组成的HCDR2，
包含SEQ ID NO:13或由其组成的HCDR3，
包含SEQ ID NO:14或由其组成的LCDR1，
包含SEQ ID NO:15或由其组成的LCDR2，和
包含SEQ ID NO:16或由其组成的LCDR3；或
2)包含SEQ ID NO:17或由其组成的HCDR1，
包含SEQ ID NO:18或由其组成的HCDR2，
包含SEQ ID NO:19或由其组成的HCDR3，
包含SEQ ID NO:20或由其组成的LCDR1，
包含SEQ ID NO:21或由其组成的LCDR2，和
包含SEQ ID NO:22或由其组成的LCDR3；或
3)包含SEQ ID NO:23所示或由其组成的HCDR1，
包含SEQ ID NO:24或由其组成的HCDR2，
包含SEQ ID NO:25或由其组成的HCDR3，
包含SEQ ID NO:26或由其组成的LCDR1，
包含SEQ ID NO:27或由其组成的LCDR2，和
包含SEQ ID NO:28或由其组成的LCDR3。
本发明还提供抗体或其抗原结合片段，其包含：
重链可变区，所述重链可变区的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％同一性：SEQ ID NO:33、34、35、36、40、41、42、43、48、49、50、51；优选地，所述重链可变区具有与SEQ ID NO:33、34、35、36、40、41、42、43、48、49、50、51氨基酸序列相同的CDRs区氨基酸序列、且所述重链可变区的框架区的氨基酸序列与氨基酸序列SEQ ID NO:33、34、35、36、40、41、42、43、48、49、50、51的框架区有至少80％同一性的序列；优选地，所述重链可变区具有选自由下述氨基酸序列所构成的组中的任一氨基酸序列：SEQ ID NO:33、34、35、36、40、41、42、43、48、49、50、51；和/或
轻链可变区，所述轻链可变区的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％同一性：SEQ ID NO:29、30、31、32、37、38、39、44、45、46、47；优选地，所述轻链可变区的氨基酸序列具有与氨基酸序列SEQ ID NO:29、30、31、32、37、38、39、44、45、46、47相同的CDRs区、且所述轻链可变区的框架区与序列SEQ ID NO:29、30、31、32、37、38、39、44、45、46、47的框架区有至少80％同一性；优选地，所述轻链可变区具有选自由下述氨基酸序列所构成的组中的任一氨基酸序列：SEQ ID NO:29、30、31、32、37、38、39、44、45、46、47；
优选地，所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区/轻链可变区的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：
SEQ ID NO:33和SEQ ID NO:29、SEQ ID NO:34和SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:32、SEQ ID NO:40和SEQ ID NO:37、SEQ ID NO:41和SEQ ID NO:38、SEQ ID NO:42和SEQ ID NO:39、SEQ ID NO:43和SEQ ID NO:37、SEQ ID NO:43和SEQ ID NO:38、SEQ ID NO:43和SEQ ID NO:39、SEQ ID NO:48和SEQ ID NO:44、SEQ ID NO:49和SEQ ID NO:45、SEQ ID NO:50和SEQ ID NO:46、SEQ ID NO:51和SEQ ID NO:47。
在一些实施方案中，其包含重链恒定区和/或轻链恒定区，优选其包含鼠源的或人源化的重链恒定区和/或轻链恒定区；优选地，所述人源化的重链恒定区的氨基酸序列如SEQ ID NO:9所示，所述人源化的轻链恒定区的氨基酸序列如SEQ ID NO:10所示；优选地所述的抗体或其抗原结合片段，其重链/轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:60和SEQ ID NO:59、SEQ ID NO:61和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:58、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:65和SEQ ID NO:64、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:64、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:64、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:64、SEQ ID NO:69和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:63、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:63、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:76和SEQ ID NO:75、SEQ ID NO:77和SEQ ID NO:72、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:75、SEQ ID NO:78和SEQ ID NO:72、SEQ ID NO:78和SEQ ID NO:73、SEQ ID NO:78和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、SEQ ID NO:79和SEQ ID NO:72、SEQ ID NO:79和SEQ ID NO:73、SEQ ID NO:79和SEQ ID NO:74、SEQ ID NO:79和SEQ ID NO:75，优选地，其重链/轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、SEQ ID NO:79和SEQ ID NO:72。
本发明还提供药物组合物，其包含前文所述的抗体或其抗原结合片段或前文任意一项所述的配体-药物偶联物或其药学上可接受的盐、和可药用赋形剂。
本发明还提供前文中任一项所述的抗体或其抗原结合片段、前文中任意一项所述的配体-药物偶联物或其药学上可接受的盐、或前文所述的药物组合物在制备用于治疗和/或预防Nectin-4介导的疾病或病症的药物中的用途；优选，所述Nectin-4介导的疾病或病症为乳腺癌、膀胱癌或肺癌；优选地，所述乳腺癌为三阴性乳腺癌。
本发明还提供前文任一项所述的抗体或其抗原结合片段、前文任意一项所述的配体-药物偶联物或其药学上可接受的盐、或前文所述的药物组合物，其用于治疗和/或预防Nectin-4介导的疾病或病症；优选，所述Nectin-4介导的疾病或病症为乳腺癌、膀胱癌或肺癌；优选地，所述乳腺癌为三阴性乳腺癌。
本发明还提供治疗和/或预防Nectin-4介导的疾病或病症的方法，包括对有需要的受试者施用治疗有效量的前文任一项所述的抗体或其抗原结合片段、前文中任意一项所述的配体-药物偶联物或其药学上可接受的盐、或前文所述的药物组合物；优选，所述Nectin-4介导的疾病或病症为乳腺癌、膀胱癌或肺癌；优选地，所述乳腺癌为三阴性乳腺癌。
附图说明
图1为AGSM6蛋白特异性检测图。
图2为h10592-2蛋白特异性检测图。
图3为h10592-2种属交叉图。
图4为AGSM6种属交叉图。
图5为ADC药物(Enfortumab vedotin)HIC分析结果图。
图6为NECTIN 4-ADC-11HIC分析结果图。
图7为NECTIN 4-ADC-18HIC分析结果图。
图8为NECTIN 4-ADC-26HIC分析结果图。
图9为MDA-MB-468模型肿瘤体积变化图。
图10为MDA-MB-453模型肿瘤体积变化图。
图11为给药剂量为4mg/kg时HT1376模型瘤体积变化。
图12为给药剂量为8mg/kg时HT1376模型瘤体积变化。
图13为不同剂量下HT1376肿瘤体积变化图。
图14为RT4/Nectin4模型肿瘤体积变化图。
图15为BALB/C小鼠急性毒性存活曲线。
图16为BALB/C小鼠急性毒性体重变化曲线。
图17为BALB/C小鼠长期毒性存活曲线。
图18为BALB/C小鼠长期毒性体重变化曲线。
图19为MDA-MB-453模型肿瘤体积抑制率。
图20为MDA-MB-468肿瘤体积抑制率。
图21为人源化小鼠毒理MTD实验生存曲线。
图22为人源化小鼠毒理MTD实验体重变化率。
图23为人源化小鼠重复剂量毒理实验生存曲线。
图24为人源化小鼠重复剂量毒理实验体重变化率。
图25为MMAE和艾日布林在BALB/c小鼠体内游离有效载荷(Payload)药代动力学结果。
图26为BALB/c小鼠体内血浆ADC及总抗体药代动力学结果。
图27为HT1376荷瘤鼠药代动力学检测结果。
本发明有益效果：
①本发明的抗体药物偶联物通过采用一种新型桥连马来酰亚胺连接头及可裂解连接子，实现了平均DAR值3.5-4.5，显著提高了抗体药物偶联物的均一性，并在肿瘤细胞溶酶体的作用下特异性释放细胞毒性有效载荷，在保证疗效的同时提高了药物的安全性；
②本发明的以Eribulin为Payload的靶向Nectin4抗体药物偶联物对三阴乳腺癌、乳腺癌和膀胱癌肿瘤均有明显的抑制作用，在三阴乳腺癌MDA-MB-468 CDX模型中，本发明的抗体药物偶联物与阳性对照药Enfortumab vedotin、阳性对照药9MW2821相比，均具有更好的药效。其中，2mg/kg剂量下本发明的抗体药物偶联物在三阴乳腺癌MDA-MB-468 CDX模型及乳腺癌MDA-MB-453 CDX模型中，TGI分别为100％和99.73％，药效显著。在膀胱癌HT1376 CDX模型中，与Enfortumab vedotin相比本发明的抗体药物偶联物具有更好的药效，2.5mg/kg剂量下本发明的抗体药物偶联物与5mg/kg剂量的Enfortumab vedotin肿瘤抑制率相当。
③与Enfortumab vedotin相比，本发明的抗体药物偶联物具有更好的安全性，在BALB/C小鼠及人源化小鼠毒理实验中，Enfortumab vedotin对照组观察到明显的不良反应并出现了小鼠死亡，而本发明获得的抗体药物偶联物组无小鼠死亡，存活率高，即具有更优异的安全性，并且可以在一定程度上降低Nectin4靶点皮肤毒性严重的副作用。
定义
除非另外指明，在此将任何多肽链描述为具有起始于N-末端并终止于C-末端的氨基酸序列。
以最宽泛的意义使用本文中的术语“抗体和其抗原结合部分”，其包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、和抗体片段，只要它们显示所希望的抗原-结合活性即可。
本文所用的术语“抗体”指由四条多肽链即由二硫键互连的两条重链(H)和两条轻链(L)组成的免疫球蛋白分子。每条重链包含重链可变区(HCVR或VH)和重链恒定区。重链恒定区包含CH1、CH2和CH3三个结构域。每条轻链包含轻链可变区(LCVR或VL)和轻链恒定区。轻链恒定区包含一个结构域(CL1)。VH和VL区可进一步分成被称为互补决定区(Complementarity determining region(s)，CDR(s))的高变区，其中散布着较保守的被称为框架区(framework region，FR)的区域。每个VH和VL由三个CDR和四个FR组成，从氨基酸末端到羧基末端按下列次序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4，其中VH的三个CDRs为HCDR1，HCDR2和HCDR3，VL的三个CDRs为LCDR1，LCDR2和LCDR3。每一结构域的氨基酸分配通常与如下定义一致：Kabat Sequences of Proteins of Immunological Interest(具有免疫学意义的Kabat蛋白序列)(National Institutes of Health，Bethesda，Md.(1987和1991))或Chothia&Lesk，J.Mol.Biol.，196：901-917(1987)；Chothia等，Nature，342：878-883(1989)。
存在五种主要的抗体类型：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可以进一步分为亚类(同种型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。抗体分子C端氨基酸序列相对稳定，该区域称为恒定区。同一种抗体的恒定区是相同的。抗体轻链的恒定区由一个Ig结构域构成；重链的恒定区由3-4个串联的Ig结构域及一个用于增加灵活性的铰链区构成。IgA、IgE、IgG有三个结构域(CH1、CH2、CH3)，IgD、IgM有四个结构域(CH1、CH2、CH3、CH4)。
对应于免疫球蛋白的不同类型的重链恒定结构域或重链恒定区分别被称为α、δ、ε、γ和μ。IgG分子在木瓜蛋白酶的作用下可以被降解为两个Fab段及一个Fc段。Fab段由抗体轻链的可变区、轻链的恒定区、重链的可变区及重链恒定区构成。可变区是与抗原结合的部位，因此Fab段又称为抗原结合段。Fc段包含了所有抗体分子共有的蛋白质序列以及各个类别独有的决定簇。Fc段有多种生物学活性，具有结合补体、结合Fc受体、通过胎盘等作用。
本文所用的术语抗体的“抗原结合部分”(或简称“抗体部分”或“抗体片段”)指抗体中保留了与抗原(如Nectin4蛋白)特异性结合能力的一个或多个片段。业已证明，抗体的抗原结合功能可由全长抗体的某些片段来实现。抗体的“抗原结合部分”这一术语所涵盖的结合片段包括(i)Fab片段，即由VL、VH、CL1和CH1结构域组成的单价片段；(ii)F(ab’)2片段，即由铰链区的二硫键连接的两个Fab片段组成的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂VL和VH结构域组成的Fv片段；(v)由VH结构域组成的dAb片段；以及(vi)CDR。此外，尽管，Fv片段的两个结构域VL和VH是由不同的基因编码的，但它们可通过重组方法，由一种合成的接头连接在一起而成为单独的相连的链，其中VL和VH区配对而形成单价分子(称为单链Fv(scFv))。这样的单链抗体也涵盖在抗体的“抗原结合部分”的术语范围内。其他形式的单链抗体，如双特异抗体也涵盖在内。
可采用不同的分析来确定或粗略估计CDR区。所述方法实例包括但不限于Kabat定义、Chothia定义、AbM定义和接触定义(contact definition)。Kabat定义为用于在抗体中为残基编号的标准，通常用于确定CDR区。参见例如Johnson&Wu，Nucleic Acids Res.，28：214-8(2000)。Chothia定义与Kabat定义类似，但Chothia定义考虑某些结构环区的位置。参见例如Chothia等，J.Mol.Biol.，196：901-17(1986)；Chothia等，Nature，342：877-83(1989)。AbM定义使用由Oxford Molecular Group制作的模仿抗体结构的整合的计算机程序组。参见例如Martin等，Proc Natl Acad Sci(USA)，86：9268-9272(1989)；“AbMTM，A Computer Program for Modeling Variable Regions of Antibodies(AbMTM，用于为抗体可变区建模的计算机程序)”Oxford，UK；Oxford Molecular，Ltd。AbM定义用已知数据库和从头开始法(ab initio method)由一级序列建立抗体三级结构的模型，所述方法例如为在以下文献中所述的方法：Samudrala等，“Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach(用联合的分级方法从头开始预测蛋白结构)”PROTEINS，Structure，Function andGenetics Suppl.，3：194-198(1999)。接触定义基于对有效的复合体晶体结构的分析。参见例如MacCallum等，J.Mol.Biol.，5：732-45(1996)。
本发明的抗体，对其来源没有限定，可以是人抗体、小鼠抗体、大鼠抗体等来自任何动物的抗体。还可以是嵌合抗体或人源化抗体等重组抗体。优选的是人源化抗体。
术语“嵌合抗体”是指包含人以外的哺乳动物、例如小鼠抗体的重链、轻链的可变区和人抗体的重链、轻链的恒定区的抗体。嵌合抗体可以利用已知的方法来制备。例如，由杂交瘤克隆体的基因，将其插入到适当的载体中，再将其导入宿主中，即可制备嵌合抗体。具体而言，使用逆转录酶由杂交瘤的mRNA合成抗体可变区(V)区的cDNA。得到编码目标抗体V区的DNA时，连接其与编码所期望的人抗体恒定区(C区)的DNA，再将其插入到表达载体中。或者，可以将编码抗体V区的DNA插入到包含人抗体C区的DNA表达载体中。将其插入到表达载体中，使之在表达调节区的调解下表达。接下来，利用该表达载体转化宿主细胞，可以使嵌合抗体表达。
术语“人源化抗体”是指含有至少一个、通常是两个几乎完整的可变区，其中对应的所有或几乎所有的CDR区是来自非人源抗体，其中的全部或几乎全部的FR区是来自人源抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。
术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。
术语“亲和力”是指分子(例如，抗体)的单个结合位点及其结合伙伴(例如，抗原)之间的非共价相互作用的总和的强度。除非另有说明，如本文使用的，“结合亲和力”是指反应结合对(例如，抗体和抗原)的成员之间的1:1相互作用的固有结合亲和力。分子X对其伙伴Y的亲和力通常可以由平衡解离常数(KD)、解离常数(Kd)或结合常数(Ka)表示。亲和力可以通过本领域中已知的一般方法测量，包括本文中描述的那些。用于测量结合亲和力的具体说明性和示例性实施方案在下文中描述。
术语“表面等离子共振”是指一种光学现象，它使得可通过检测一种生物传感器基质中蛋白浓度的改变来进行实时互动分析，例如使用BiacoreTM系统进行分析。
术语“中和”或“阻断”抗体意指其与NECTIN4蛋白的结合造成对NECTIN4蛋白生物活性抑制的抗体。这种NECTIN4蛋白生物活性的抑制可通过测量本领域众所周知的一个或多个NECTIN4蛋白生物活性指标，如NECTIN4蛋白诱导的细胞活化和抗体与NECTIN4蛋白结合等指标来评估(参阅下面的实施例)。
多肽的序列相似性也称为序列同一/源性，通常是由序列分析软件测量的。蛋白质分析软件利用各种取代、删除和其他修饰，包括保守的氨基酸取代的相似性测量值来对相似的序列进行匹配，例如，GCG软件包含诸如Gap和Bestfit等程序，可用默认参数来确定密切相关的多肽例如来自不同生物种的同源多肽的序列同源性或序列同一性，参阅如GCG6.1版。还可以用GCG6.1版中的FASTA程序，用默认或建议的参数来比较多肽序列。FASTA(如FASTA2和FASTA3)提供了查询的和搜索到的序列之间最佳迭合区域的比对和序列同一性百分数(Pearson(2000)如上)。当对本发明的序列和包含大量源自不同生物的序列的数据库进行比较时，另一种优选的计算程序是计算机程序BLAST，尤其是BLASTP或TBLASTN，在比较时采用默认参数。
术语“基本同一性”或“基本上相同”当其指核酸或其片段时，表示当以适当的核苷酸替换、插入或删除与另一个核酸(或其互补链)进行最佳比对时，用下述任何的序列同一性计算程序如FASTA、BLAST或Gap计算，在至少约80％，更优选在至少约80％，85％，91％，92％，93％，94％，95％，96％，97％，98％或99％的核苷酸碱基中具有核苷酸序列同一性。
当应用于多肽时，术语“基本相似”或“基本上相似”意为当以Gap或BESTFIT等程序用默认空位权重进行最佳比对时，两个肽序列至少具有80％的序列同一性，更优选的是至少具有80％，85％，91％，92％，93％，94％，95％，96％，97％，98％或99％的序列同一性。不相同的残基位置的区别可以为氨基酸的取代、删除或插入，更优选的是，不相同的残基位置的区别在于保守的氨基酸取代。“保守的氨基酸取代”是这样的取代，其中氨基酸残基被另一个含有类似化学性质(如电荷或疏水性)的侧链(R基团)的氨基酸残基所取代。一般来说，保守的氨基酸取代不会从实质上改变蛋白质的功能性质。在两个或两个以上氨基酸序列由于保守的取代而不同的情况下，可向上调节序列同一性百分数或类似程度，以修正取代的保守性。进行这种调节的方法为本领域的专业人员所熟知。含有具有类似化学性质侧链的氨基酸群的实例包括(1)脂肪族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；(2)脂肪族-羟基族：丝氨酸和苏氨酸；(3)含酰胺侧链：天冬酰胺和谷氨酰胺；(4)芳香族侧链：苯丙氨酸、酪氨酸和色氨酸；(5)碱性侧链：赖氨酸、精氨酸和组氨酸；(6)酸性侧链：天冬氨酸和谷氨酸，以及(7)含硫侧链：半胱氨酸和甲硫氨酸。优选的保守的氨基酸取代群为：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸-缬氨酸、谷氨酸-天冬氨酸，以及天冬酰胺-谷氨酰胺。或者，保守的取代可以是Gonnet等人(1992)Science256:1443-1445所披露的PAM250对数-似然矩阵中具有正值的任何变化。“中度保守的”取代是PAM250对数-似然矩阵中具有非负值的任何变化。
术语“载体”意指用于将蛋白编码信息转移到宿主细胞的任何分子或实体(例如核酸、质粒、噬菌体或病毒)。
术语“表达载体”或“表达构建体”是指适于转化宿主细胞并含有指导和/或调控一个或多个可操作地与其连接的异源编码区的表达的核酸序列的载体。表达载体可包括但不限于：影响或调控转录、翻译的序列；和若存在内含子，则影响可操作地与其连接的编码区的RNA剪接的序列。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换使用，是指向其中引入外源核酸的细胞，包括所述细胞的子代。宿主细胞包括“转化子”和“转化细胞”，其包括原代转化的细胞和从其衍生的子代(不考虑传代数)。子代的核酸含量可以不完全与母细胞相同，而是可以含有突变。具有与对于在最初细胞中筛选或选择的相同功能或生物活性的突变体子代包括在本文中。
术语“转染”意指细胞吸收外来或外源DNA，当将所述外源DNA引入到细胞膜内时细胞就被“转染”了。多种转染技术在本领域是众所周知。参见例如Graham等，1973，Virology 52：456；Sambrook等，2001，Molecular Cloning：A Laboratory Manual(分子克隆：实验室手册)；Davis等，1986，Basic Methods in Molecular Biology(分子生物学基本方法)，Elsevier；Chu等，1981，Gene 13：197。所述技术可用于将一种或多种外源DNA部分引入到合适的宿主细胞中。
术语“治疗”包括治疗性治疗、预防性治疗及在降低受治疗者发展疾病的风险或其他风险因素中的应用。治疗不需要完全治愈疾病，而是包括在其中减轻症状或减轻潜在风险因素的实施方案。
术语“预防”不需要100％消除事件的可能性。更准确地说，它表示在所述化合物或方法存在下事件发生的可能性降低了。
术语“二抗”是指第二抗体，第二抗体用于和抗体(一抗)结合，即抗体的抗体，其主要作用是检测抗体的存在，放大一抗的信号。二抗是利用抗体是大分子的蛋白质具有抗原性的性质，去免疫异种动物，由异种动物的免疫系统产生的针对于此抗体的免疫球蛋白。二抗针对某一特定物种(如小鼠)的所有抗体(如IgG、IgM或IgA等)均具有反应性。
术语“药物”是指可以改变或查明机体的生理功能及病理状态，可用以预防，诊断和治疗疾病的化学物质。药物包括细胞毒性药物。药物和毒物之间并无严格界限，毒物是指在较小剂量即对机体产生毒害作用，损害人体健康的化学物质，任何药物剂量过大都可产生毒性反应。
细胞毒性药物，即抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。细胞毒性药物包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素，化疗药物，抗生素和核溶酶。
术语“接头单元”或“连接片段”或“连接单元”是指一端与配体连接而另一端与药物相连的化学结构中的片段或键，也可以连接其他接头后再与药物相连。
术语“配体-药物偶联物”，指配体通过稳定的连接单元与具有生物活性的药物相连。在本公开中“配体-药物偶联物”优选为抗体-药物偶联物(antibody drug conjugate，ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的毒性药物相连。
载药量，也称药物抗体比例(Drug-to-Antibody Ratio，DAR)，即ADC中每个抗体所偶联的药物的平均数量。其可在例如每个抗体偶联约1至约12个药物的范围内，并且在某些实施方案中，在每个抗体偶联约1至约8个药物的范围内，优选自2-8，2-7，2-6，2-5，2-4，3-4，3-5，5-6，5-7，5-8和6-8的范围。示例性地，载药量可以为1，2，3，4，5，6，7，8，9，或10的均值。在本发明的一些实施方式中，载药量可表示为m或m1，是小数或整数。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC测定载药量。
术语“亲水性单元”，指-CH2-CH2-O-、亲水性氨基酸、葡糖胺、糖基、磷酸根、磺酸根或其组合；优选地，所述亲水性单元选自-CH2-CH2-O-、肌氨酸、丙氨酸、丝氨酸、天冬酰胺、谷氨酰胺、酪氨酸、赖氨酸、精氨酸、组氨酸、天冬氨酸、谷氨酸葡糖胺、糖基、磷酸根、磺酸根或其组合。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。术语“C1-6烷基”指具有1-6个碳原子(例如1、2、3、4、5或6个碳原子)的饱和直链或支链烃基。例如“C1-6烷基”可以是甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、新戊基或正己基等。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个(例如1、2或3个)环原子为选自氮、氧或硫，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选环烷基环包含3至10个环原子。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“芳基”指具有共轭的π电子体系的6至15元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基，优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环。
术语“杂芳基”指包含1至4个杂原子、5至15个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环。
术语“卤代烷基”指烷基被一个或多个卤素取代。
术语“氘代烷基”指烷基被一个或多个氘原子取代。
术语“卤素”指氟、氯、溴或碘。
术语“取代”和“取代的”指所指定的原子上的一个或多个(例如一个、两个、三个或四个)氢被从所指出的基团的选择代替，条件是未超过所指定的原子在当前情况下的正常原子价并且所述取代形成稳定的化合物。取代基和/或变量的组合仅仅当这种组合形成稳定的化合物时才是允许的。
如果取代基被描述为“任选地…被取代”，则取代基可(1)未被取代，或(2)被取代。如果某个原子或基团被描述为任选地被取代基列表中的一个或多个取代，则该原子或基团上的一个或多个氢可被独立地选择的、任选的取代基替代。如果取代基被描述为“独立地选自”或“各自独立地为”，则各取代基互相独立地加以选择。因此，各取代基可与另一(其他)取代基相同或不同。例如，某个取代基或取代位置或者不同的取代基或取代位置具有可能相同或不同符号指代的R基团(例如但不限于R2、R3、Rh、Ri、Rx和/或Ry)的选择时，各个R之间独立地加以选择，即可以相同也可以不同。关于数值如d、g、m、n的选择也是如此。
除非指明，否则如本文中所使用，取代基的连接点可来自取代基的任意适宜位置。
当取代基的键显示为穿过环中连接两个原子的键时，则这样的取代基可键连至该可取代的环中的任一成环原子。
术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的或开放式的，且不排除其它未列举的元素或方法步骤。本领域技术人员应当理解，上述术语如“包括”涵盖“由…组成”的含义。
本发明中“药学上可接受的载体”是指与活性成分一同给药的稀释剂、辅剂、赋形剂或媒介物，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激、过敏反应或与合理的益处/风险比相应的其它问题或并发症。术语“活性成分”、“治疗剂”，“活性物质”或“活性剂”是指一种化学实体，它可以有效地治疗目标病症或病况的一种或多种症状。
如本文中所使用的术语“有效量”(例如“治疗有效量”或“预防有效量”)指给药后会在一定程度上实现预期效果的活性成分的量，例如缓解所治疗病症的一种或多种症状或预防病症或其症状的出现。
除非另外说明，否则如本文中所使用，术语“治疗”意指逆转、减轻、抑制该术语所应用的病症或病况或者所述病症或病况的一种或多种症状的进展，或预防所述病症或病况或者所述病症或病况的一种或多种症状。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
缩略语
PBS是指磷酸盐缓冲溶液(Phosphate Buffer Solution)。
PBST是指PBS溶液中加入Tween-20。
BSA是指牛血清蛋白(Bovine SerumAlbumin)。BSA封闭液是由牛血清白蛋白配制而成。
TMB是指3,3’,5,5’-四甲基联苯胺(3,3’,5,5’-Tetramethylbenzidine)溶液。
MEM培养基是指低限基本培养基(Minimum Essential Medium)，是动物细胞培养中的常用培养基。
FBS是指胎牛血清(Fetal Bovine Serum)。
ND(NotDetected)是指在实验条件下，抗体未展现出体外细胞中和活性。
试剂与仪器
生物材料来源
pCDNA3.1(+)表达载体购自Invitrogen，货号为V79020。
小鼠骨髓瘤细胞SP2/0购自美国菌种保藏中心，货号为CRL-1581。
HEp-2细胞购自ATCC，货号CCL-23。
293F细胞购自ATCC，货号CRL-1573。
第一部分：Nectin4抗体部分
实施例1.杂交瘤细胞株的筛选
1、Nectin4重组蛋白的表达纯化
根据NCBI数据库内的Nectin4蛋白的基因序列，化学合成加入His标签后的表达基因，与pCDNA3.1(+)表达载体构建Nectin4蛋白的表达质粒，转染293F细胞后，培养并收集细胞表达的上清液，浓缩后经镍柱(Cytiva)纯化，得到Nectin4蛋白，其氨基酸序列如SEQ ID NO:1所示，其基因序列如SEQ ID NO:2所示。

2、杂交瘤细胞的构建
取6-8周龄的BALB/C雌性小鼠，饲养一周后进行免疫，首次免疫使用Nectin4蛋白，免疫前用1ml PBS重悬Sigma Adjuvant System佐剂(Sigma)，再加入1ml Nectin4蛋白(1mg/ml)作为抗原等体积混合乳化，经腹腔和皮下两点注射，每只每次注射100μg。每两周免疫一次。第三次免疫后采血检测效价，共免疫四次。取小鼠脾脏做融合。融合前72h再次加强免疫，经腹腔注射Nectin4抗原蛋白1次，100μL/只。
取免疫后的小鼠脾脏细胞与小鼠骨髓瘤细胞SP2/0相融合。先把脾脏研磨得到脾细胞悬液，与处于对数生长期的SP2/0小鼠骨髓瘤细胞1：1混合，经电融合将两种细胞融合一起得杂交瘤细胞，然后把融合细胞液稀释为5000-10000个细胞/ml均匀铺到96孔板中。融合培养基为含HAT和20％FBS的DMEM完全筛选培养基。37℃,5％CO2,孵育7天后,采用间接ELISA方法进行阳性杂交瘤细胞株筛选，筛选前一天进行细胞换液。
3、单克隆细胞株产生
根据杂交瘤细胞生长密度，采用间接ELISA方法检测细胞上清。选择与Nectin4抗原蛋白结合能力强的细胞,及时进行扩增冻存。采用有限稀释法，经过亚克隆获得单克隆细胞株。收集单克隆细胞株上清，进行Biacore实验，检测单克隆细胞株与Nectin4抗原的结合活性。
4、鼠源抗体
挑选出体外活性高的单克隆杂交瘤细胞株mAb10591，mAb10592，mAb10593；测定的单克隆抗体序列,再进行人源化、重组表达和活性评价。杂交瘤测序的过程如下。收集对数生长期杂交瘤细胞,用Trizol(Invitrogen,15596-018)提取RNA(按照试剂盒说明书步骤)并进行反转录(PrimeScript"Reverse Transcriptase,Takara,cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326 Rev.B 0503)进行PCR扩增,送测序公司测序。得到的DNA序列对应的氨基酸序列SEQ ID NO:3-8所示:
mAb10591鼠源重链可变区(SEQ ID NO:3)：
mAb10591鼠源轻链可变区(SEQ ID NO:4)：
mAb10592鼠源重链可变区(SEQ ID NO:5)：
mAb10592鼠源轻链可变区(SEQ ID NO:6)：
mAb10593鼠源重链可变区(SEQ ID NO:7)：
mAb10593鼠源轻链可变区(SEQ ID NO:8)：
mAb10594鼠源重链可变区(SEQ ID NO:100)：
mAb10594鼠源轻链可变区(SEQ ID NO:101)：
mAb10595鼠源重链可变区(SEQ ID NO:102)：
mAb10595鼠源轻链可变区(SEQ ID NO:103)：
mAb10596鼠源重链可变区(SEQ ID NO:104)：
mAb10596鼠源轻链可变区(SEQ ID NO:105)：
表1鼠源抗体的CDR序列

实施例2.嵌合抗体的制备
1、嵌合抗体的构建和表达
利用融合PCR方法构建嵌合抗体：将上述实施例1的鼠源抗体可变区与人IgG1抗体的重链恒定区和人源κ轻链恒定区连接(重链恒定区氨基酸序列：SEQ ID NO:9，轻链恒定区氨基酸序列：SEQ ID NO:10)的编码基因连接构建融合基因，将融合基因进行扩增，并克隆入pCDNA3.1(+)表达载体，构建嵌合抗体的表达质粒，转染293F细胞后，培养并收集细胞表达的上清液，浓缩后经Protein A(Cytiva)纯化，得到相应的嵌合抗体。
人IgG1抗体的重链恒定区(SEQ ID NO:9):
人源κ轻链恒定区(SEQ ID NO:10):
实施例3.抗体人源化改造
1、人源化抗体的制备
保留抗体mAb10591、mAb10592、mAb10593中鼠的CDR区，对其的FR区进行人源化移植改造，FR区人源化移植改造的步骤如下：
(1)通过Bioluminate软件中的Prediction功能对鼠源抗体可变区重轻链序列进行同源建模，并通过Reliable report功能对同源建模的可信度进行评价；在此评价基础上，进一步对模型进行优化，得到优化后的抗体可变区模型。
(2)通过NCBI Ig Blast对待人源化抗体可变区进行比对，选定同源性最高的Human germline序列进行CDR移植，并在首轮人源化过程中将鼠源抗体FR序列全部进行人源化替换。
(3)通过CDR grafting中advanced option选项，对框架区FR中Canonical Structure Region、Vernier Zone等关键位点进行预测。
mAb10591的人源化改造和回复突变设计
选择适当的人抗体种系,对mAb10591鼠源抗体进行人源化改造,将鼠源抗体mAb10591的CDR区移植到选择的人源化模板上,得到人源化可变区,其人源化轻链可变区序列为VL1(SEQ ID NO:29),或VL3(SEQ ID NO:31)和重链可变区序列为VH1(SEQ ID NO:33),或VH3(SEQ ID NO:35)；再与IgG恒定区重组,形成完整抗体。同时,对人源化抗体的V区中FR区进行回复突变,对VL1突变得到VL2(突变方式为M4L,Y48V)，对VH1突变得到VH2(突变方式为F24V,S30T,A49A,T73N)，对VL3突变得到VL4(突变方式为I48V,F70Y)，对VH1突变得到VH2(突变方式为S30T,I37V,A49G,T73N)，示例性回复突变方式及组合如下:
表2 mAb10591人源化抗体及回复突变*

*表格中所有氨基酸位置编号为Kabat编号规则的编号,重链可变区的F24V中,24为Kabat规则的第24
位。
表3 mAb10591人源化抗体轻链可变区和重链可变区序列

上表中对应重链可变区可与SEQ ID NO:9所示的人IgG1重链恒定区连接形成全长抗体的重链,轻链可变区与SEQ ID NO:10所示的人κ轻链恒定区连接形成全长抗体的轻链。在其他实施方案中,重链可变区和轻链可变区也可与其他重链恒定区和轻链恒定区分别连接形成全长抗体。
2.2 mAb10592的人源化改造和回复突变设计
选择适当的人抗体种系,对mAb10592鼠源抗体进行人源化改造,将鼠源抗体mAb10592的CDR区移植到选择的人源化模板上,得到人源化可变区,其人源化轻链可变区序列为VL5(SEQ ID NO:37),或VL7(SEQ ID NO:39)和重链可变区序列为VH5(SEQ ID NO:40)，或VH7(SEQ ID NO:42)；再与IgG恒定区重组,形成完整抗体。同时,对人源化抗体的V区中FR区进行回复突变,对VL5突变得到VL6(突变方式为Y53K)，对VH5突变得到VH6(突变方式为M48I，V68A,M70L,R72V,T74K)，对VH7突变得到VH8(突变方式为M48I，V68A,I70L,R72V,T74K)，示例性回复突变方式及组合如下:
表4 mAb10592人源化抗体及回复突变*

*表格中所有氨基酸位置编号为Kabat编号规则的编号。
表5 mAb10592人源化抗体轻链可变区和重链可变区序列

上表中对应重链可变区可与SEQ ID NO:9所示的人IgG1重链恒定区连接形成全长抗体的重链,轻链可变区与SEQ ID NO:10所示的人κ轻链恒定区连接形成全长抗体的轻链。在其他实施方案中,重链可变区和轻链可变区也可与其他重链恒定区和轻链恒定区分别连接形成全长抗体。
2.3 mAb10593的人源化改造和回复突变设计
选择适当的人抗体种系,对mAb10593鼠源抗体进行人源化改造,将鼠源抗体mAb10593的CDR区移植到选择的人源化模板上,得到人源化可变区,其人源化轻链可变区序列为VL9(SEQ ID NO:44),或VL11(SEQ ID NO:46)和重链可变区序列为VH9(SEQ ID NO:48),或VH11(SEQ ID NO:50)；再与IgG恒定区重组,形成完整抗体。同时,对人源化抗体的V区中FR区进行回复突变,对VL9突变得到VL10(突变方式为F70Y)，对VH9突变得到VH10(突变方式为V37I,M48I,V68A,M70L,T72V,T74I,Y95F)，对VL11突变得到VL12(突变方式为L47I)，对VH11突变得到VH12(突变方式为V37I,M48I,V68A,S70L,R72V,T74I,Y95F)，示例性回复突变方式及组合如下:
表6 mAb10593人源化抗体及回复突变*

*表格中所有氨基酸位置编号为Kabat编号规则的编号。
表7 mAb10593人源化抗体轻链可变区和重链可变区序列

上表中对应重链可变区可与SEQ ID NO:9所示的人IgG1重链恒定区连接形成全长抗体的重链,轻链可变区与SEQ ID NO:10所示的人κ轻链恒定区连接形成全长抗体的轻链。在其他实施方案中,重链可变区和轻链可变区也可与其他重链恒定区和轻链恒定区分别连接形成全长抗体。
示例性的，抗体全长序列如下：
嵌合抗体ch1059-1
ch1059-1重链(SEQ ID NO:52)
ch1059-1轻链(SEQ ID NO:53)
嵌合抗体ch1059-2
ch10592重链(SEQ ID NO:54)

ch1059-2轻链(SEQ ID NO:55)
嵌合抗体ch1059-3
ch1059-3重链(SEQ ID NO:56)
ch1059-3轻链(SEQ ID NO:57)
嵌合抗体ch1059-4
ch1059-4重链(SEQ ID NO:106)
ch1059-4轻链(SEQ ID NO:107)
嵌合抗体ch1059-5
ch1059-5重链(SEQ ID NO:108)
ch1059-5轻链(SEQ ID NO:109)
嵌合抗体ch1059-6
ch1059-6重链(SEQ ID NO:110)
ch1059-6轻链(SEQ ID NO:111)
mAb10591人源化抗体
表8 mAb10591人源化抗体
全长抗体轻重链序列如下所示:
表9 mAb10591人源化抗体轻链和重链序列

表10 mAb10592人源化抗体
全长抗体轻重链序列如下所示:
表11 mAb10592人源化抗体轻链和重链序列


表12 mAb10593人源化抗体
全长抗体轻重链序列如下所示:
表13 mAb10593人源化抗体轻链和重链序列


本专利公开抗体1AGSM6(参照WO2012047724实施例2序列制备)
AGSM6重链(SEQ ID NO:80)
AGSM6轻链(SEQ ID NO:81)
用常规的基因克隆、重组表达的方法分别克隆、表达、纯化上述抗体。
第二部分：ADC部分
实施例2-1：
化合物1
合成路线：
中间体1-3的合成：
将1-1(200mg，0.46mmol)用DMF(2mL)溶解，依次加入1-2(甲磺酸艾日布林，351mg，0.46mmol)、HATU(209mg，0.55mmol)和DIEA(227μL，1.38mmol)，室温反应过夜。反向柱色谱纯化，减压蒸馏除去溶剂得到白色固体1-3(190mg，36％)。LC-MS(ESI):m/z found[M+H]+＝1148.9。
中间体1-4的合成：
将1-3(79mg，0.058mmol)用DCM(5mL)溶解，加入HCl/EA(4M，72.5μL)，室温反应4h。减压蒸馏除去溶剂，未经进一步纯化直接用于下一步。LC-MS(ESI):m/z found[M+H]+＝1048.75。
化合物1的合成：
将中间体1-4(25mg，0.024mmol)用DMF(2mL)溶解，依次加入马来酰亚胺-二聚乙二醇-羧酸(6.13mg，0.024mmol)、HATU(10.88mg，0.029mmol)和DIEA(19.7μL，0.119mmol)，室温反应3h。Pre-HPLC制备，冻干，得到白色固体化合物1(6.7mg，21.8％)。LC-MS(ESI):m/z found[M+H]+＝1288.00。
实施例2-2：
化合物2
合成路线：
中间体2-3的合成：
将化合物2-1(500mg，1.22mmol)用DMF(10mL)溶解，依次加入化合物2-2(150mg，1.22mmol)、HATU(464mg，1.22mmol)和DIEA(604μL，3.66mmol)，室温反应5h。反向柱色谱纯化(H2O：MeCN＝1:2)，冻干得到淡黄色固体2-3(580mg，92.3％)。LC-MS(ESI):m/z found[M+2H]+＝517.2。
中间体2-4的合成：
将化合物2-3(580mg，1.12mmol)用DMF(8mL)溶解，依次加入二(对硝基苯)碳酸酯(410mg，1.35mmol)和DIEA(743μL，4.50mmol)，室温反应4h。反向柱色谱纯化(H2O：MeCN＝30:70)，冻干得黄色固体2-4(502mg，66.0％)。LC-MS(ESI):m/z found[M+H]+＝681.2。
中间体2-5的合成：
将化合物2-4(50mg，0.074mmol)用DMF(3mL)溶解，依次加入甲磺酸艾日布林(54mg，0.074mmol)、HOBt(10mg，0.074mmol)和DIEA(24μL，0.147mmol)，室温反应2h。反相柱色谱纯化(H2O：MeCN＝1:2)，冻干得到白色固体2-5(60mg，64.5％)。LC-MS(ESI):m/z found[M+H]+＝1272.85。
中间体2-6的合成：
将化合物2-5(60mg，0.047mmol)用DMF(2mL)溶解，冰浴并氮气保护下加入二乙胺(50μL)，室温反应2h。减压蒸馏除去二乙胺后，反相柱色谱纯化(H2O：MeCN＝2:1)，冻干得到无色油状物2-6(25mg，51％)。LC-MS(ESI):m/z found[M+H]+＝1049.85。
化合物2的合成：
将中间体2-6(25mg，0.024mmol)用DMF(2mL)溶解，依次加入马来酰亚胺-二聚乙二醇-羧酸(6mg，0.024mmol)、HATU(9mg，0.024mmol)和DIEA(12μL，0.072mmol)，室温反应3h。Pre-HPLC制备，冻干，得到白色固体化合物2(3.5mg，11.3％)。LC-MS(ESI):m/z found[M+H]+＝1289.90。
实施例2-3：
化合物3
合成路线：
中间体3-2的合成：
将化合物3-1(500mg，1.47mmol)用MeCN(7.5mL)溶解，冰浴下依次加入HOSU(178mg，1.55mmol)和DCC(319mg，1.55mmol)后转室温反应4h。抽滤除去白色不溶固体，减压蒸馏浓缩滤液，得到白色固体3-2未经进一步纯化直接用于下一步。LC-MS(ESI):m/z found[M+H2O]+＝454.2。
中间体3-3的合成：
将中间体3-2(700mg，1.148mmol)用DMF(5mL)溶解，依次加入Nε-(叔丁氧基羰基)-L-赖氨酸(283mg，1.148mmol)和DIEA(190μL)，室温反应5h，反相柱色谱纯化(H2O：MeCN＝2:3)，冻干得白色固体3-3(570mg，82.4％)。LC-MS(ESI):m/z found[M+H]+＝567.2。
中间体3-4的合成：
将中间体3-3(200mg，0.35mmol)用DMF溶解，依次加入4-氨基苄醇(43.1mg，0.35mmol)、HATU(51.7mg，0.399mmol)和DIEA(65.9μL,0.399mmol)，室温反应至原料消失。反向柱色谱纯化(63％MeCN：H2O)，冻干得白色固体3-4(210.6mg，89.4％)。LC-MS(ESI):m/z found[M+H]+＝673.55。
中间体3-5的合成：
将中间体3-4(210mg，0.312mmol)用DMF(3mL)溶解，冰浴并氮气保护下依次加入二(对硝基苯)碳酸酯(105mg，0.344mmol)和DIEA(81mg，0.625mmol)，室温反应过夜。反相柱色谱纯化(H2O：MeCN＝1:3)，冻干得白色固体3-5(117mg，44.1％)。LC-MS(ESI):m/z found[M+H]+＝837.6。
中间体3-6的合成：
将中间体3-5(110mg，0.131mmol)用DMF(3mL)溶解，依次加入甲磺酸艾日布林(96mg，0.131mmol)、HOBt(18mg，0.131mmol)和DIEA(34mg，0.262mmol)，室温反应3h。反相柱色谱纯化(H2O：MeCN＝2:3)，冻干得白色固体3-6(70mg，37.4％)。LC-MS(ESI):m/z found[M-56+H]+＝1385.95。
中间体3-7的合成：
将3-6(60mg，0.042mmol)用DMF(3mL)溶解，冰浴氮气保护下加入二乙胺(75μL)室温反应2h。反相柱色谱纯化(H2O：MeCN＝1:1)，冻干得无色固体3-7(25mg，45.5％)。LC-MS(ESI):m/z found[M+H]+＝1207.00。
中间体3-8的合成：
将中间体3-7(25mg，0.022mmol)用DMF溶解，依次加入马来酰亚胺-PEG2-琥珀酰亚胺酯(8mg，0.022mmol)和DIEA(7mg，0.044mmol)，室温反应3h。反相柱色谱纯化(H2O：MeCN＝2:3)，冻干得无色油状物3-8(20mg，62.5％)。LC-MS(ESI):m/z found[M-100+H]+＝1347.15。
化合物3的合成：
将中间体3-8(10mg，0.007mmol)用DCM(1mL)溶解，加入TFA(0.4mL)，室温反应2h。减压蒸馏除去溶剂，少量DMF溶解，Pre-HPLC制备，冻干得白色固体化合物3。LC-MS(ESI):m/z found[M+H]+＝1346.00。
实施例2-4：
化合物4
合成路线：
中间体4-2的合成：
冰浴下，将4-1(3g，7.93mmol)缓慢加入HBr-AcOH(33％，20mL)中，室温下反应过夜。用DCM稀释，加水萃取，饱和碳酸氢钠溶液和饱和氯化钠溶液洗涤有机相，减压蒸馏浓缩得棕色油状物4-2(3.1g，98.7％)。LC-MS(ESI):m/z found[M+H]+＝397.0。
中间体4-4的合成：
将5-甲酰水杨酸(4-3，400mg，2.4mmol)用DMF(5mL)溶解，冰浴并氮气保护下加入单Boc-乙二胺(460mg，2.9mmol)、EDCI(560mg，2.9mmol)和HOBt(400mg，2.9mmol)，室温反应5h。反相柱色谱纯化(H2O：MeCN＝3:2)，冻干得到淡黄色固体4-4(530mg，71.7％)。LC-MS(ESI):m/z found[M+H-56]+＝253.0。
中间体4-5的合成：
将4-4(300mg，0.974mmol)用MeCN(10mL)溶解，冰浴并氮气保护下依次加入中间体4-2(425mg，1.071mmol)和Ag2O(451mg，1.948mmol)，室温反应过夜。硅藻土过滤，减压蒸馏浓缩滤液，正相柱纯化(DCM:EA＝1:1)，减压蒸馏得到白色油状物4-5(498mg，82.0％)。LC-MS(ESI):m/z found[M+H-100]+＝525.2。
中间体4-6的合成：
将中间体4-5(250mg，0.477mmol)用EA溶解，加入Pd/C，氢气气氛下室温反应过夜。硅藻土过滤，正相柱纯化(DCM:MeOH＝19:1)，减压蒸馏得到无色固体4-6(150mg，50.3％)。LC-MS(ESI):m/z found[M+H-100]+＝527.0。
中间体4-7的合成：
将中间体4-6(150mg，0.285mmol)用DMF(3mL)溶解，冰浴并氮气保护下加入二(对硝基苯)碳酸酯(96mg,0.313mmol)和DIEA(94μL)，后转室温反应过夜。反向柱色谱纯化，冻干，得白色固体4-7(210mg，93.0％)。LC-MS(ESI):m/z found[M+H-100]+＝692.2。
中间体4-8的合成：
将4-7(210mg，0.265mmol)用DMF溶解，依次加入Eribulin(194mg，0.265mmol)、HOBt(36mg，0.265mmol)和DIEA(88μL)，室温反应2h。反向柱色谱纯化(H2O:MeCN＝1:3)，冻干得无色固体4-8(228mg，62.3％)。LC-MS(ESI):m/z found[M-100/2+H]+＝641.8。
中间体4-9的合成：
将4-8(180mg，0.13mmol)用MeOH(2mL)溶解，加入LiOH·H2O(54mg，1.3mmol)和H2O(2mL)，室温反应1.5h。加入0.5M盐酸将pH调至5，旋去甲醇，残留物用乙酸乙酯萃取，减压浓缩，反向柱色谱纯化(H2O:MeCN＝2:3)，冻干得到白色固体4-9(157mg，97.5％)。LC-MS(ESI):m/z found[M+H]+＝1242.85。
中间体4-10的合成：
将4-9(157mg，0.126mmol)用DCM(10mL)溶解，加入TFA(1mL)，室温反应1.5h。反向柱色谱纯化(H2O:MeCN＝2:3)，冻干得黄色固体4-10(60mg，41.7％)。LC-MS(ESI):m/z found[M+H]+＝1142.80。
化合物4的合成：
将4-10(30mg，0.026mmol)用DMF(2mL)溶解，依次加入马来酰亚胺-PEG2-琥珀酰亚胺酯(9mg，0.026mmol)和DIEA(6.79mg，0.053mmol)，室温反应3h。Pre-HPLC纯化，冻干得白色固体化合物4(2.7mg，7.5％)。LC-MS(ESI):m/z found[M+H]+＝1381.95。
实施例2-5：
化合物5
合成路线：
中间体5-3的合成：
将化合物5-1(200mg，1.3mmol)用MeOH(20mL)溶解，加入化合物5-2(384mg，1.58mmol)，室温反应1.5h。减压蒸馏除去溶剂，DCM打浆，抽滤得到棕色固体5-3(436mg，93.7％)。LC-MS(ESI):m/zfound[M+H]+＝359.2。
中间体5-4的合成：
将中间体5-3(100mg，0.278mmol)用DCM溶解，加入DCC(28.9mg，0.138mmol)，室温反应30min后加入2-[2-(炔丙基氧)乙氧基]乙胺(22.1μL，0.153mmol)继续室温反应2h。减压浓缩，少量MeOH溶解，反相柱色谱纯化(37％MeCN:H2O)，冻干得棕红色固体5-4(75mg，46.1％)。LC-MS(ESI):m/z found[M+H]+＝486.15。
中间体5-5的合成：
将叠氮-乙二醇-乙酸(9.7mg，0.048mmol)用DMF溶解，加入HATU(21.7mg，0.057mmol)室温搅拌5min后，依次加入DIEA(18.5mg，0.143mmol)和中间体2-6(50mg，0.048mmol)，继续室温反应2h。Pre-HPLC制备，冻干得无色油状物5-5(40mg，68％)。LC-MS(ESI):m/z found[M+H]+＝1234.85。
化合物5的合成：
将中间体5-5(40mg，0.324mmol)和5-4(31mg，0.648mmol)用THF(0.5mL)和EtOH(1.5mL)溶解，依次加入CuSO4·5H2O(8mg，0.324mmol)和Ascorbic acid(6mg，0.324mmol)，氮气保护下室温反应2h。Pre-HPLC制备，冻干得白色固体化合物5(8mg，14.4％)。LC-MS(ESI):m/z found[M+H]+＝1719.95。
实施例2-6：
化合物6
合成路线：
中间体6-1的合成：
除将原料替换为2-叠氮乙酸外，其余实验步骤与实施例5中的中间体5-5的合成相同，得到浅紫色固体6-1(16mg，49％)。LC-MS(ESI):m/z found[M+H]+＝1132.80。
化合物6的合成：
除将中间体5-5替换成中间体6-1外，其余步骤与化合物5的合成相同，得到白色固体化合物6(1.94mg，27.3％)。
实施例2-7：
化合物7
合成路线：
中间体7-2的合成：
室温下，将2-氨基-1-(4-硝基-苯)-乙酮盐酸盐(7-1，2g,9.23mmol)用DCM(20mL)溶解，将K2CO3(3.8g,27.7mmol)用H2O(8mL)溶解,冷却后加入K2CO3溶液和(Boc)2O(2.012g,9.23mmol)，转室温反应2h，。TLC监测原料反应完全，反应液用DCM萃取，饱和NaCl洗，无水Na2SO4干燥，抽滤，有机相浓缩后中压液相色谱纯化(石油醚：乙酸乙酯＝83：17)，浓缩得产物7-2(980mg，产率37.9％)。LC-MS(ESI):m/z found[(M-100)+H]+＝181.2。
中间体7-3的合成：
室温下，将化合物7-2(980mg,3.5mmol)用EtOH(20mL)溶解，加入NaBH4(200mg,5.28mmol)后，在N2保护下室温反应2h。LC-MS监测原料反应完全，反应液用EA/H2O萃取，有机相用饱和食盐水洗涤，无水Na2SO4干燥浓缩，中压液相色谱纯化(石油醚：乙酸乙酯＝3：1)，浓缩得产物7-3(590mg，产率60％)。LC-MS(ESI):m/z found[(M-56)+H]+＝227.20。
中间体7-4的合成：
室温下，将化合物7-3(590mg,2.09mmol)用MeOH(10mL)溶解，加入Pd/C(22.2mg,0.209mmol)后在H2条件下室温反应4h，LC-MS监测原料反应完全，将反应液过滤后，滤液浓缩，中压液相色谱纯化(石油醚：乙酸乙酯＝3：7)纯化，浓缩得产物7-4(450mg，产率85％)。LC-MS(ESI):m/z found[(M-56)+H]+＝179.20。
中间体7-5的合成：
室温下，将化合物7-4(450mg,1.78mmol)用DMF(4mL)溶解，搅拌5min后依次加入DIEA(692mg,5.34mmol)和N-[芴甲氧羰基]-L-缬氨酰-L-丙氨酸(732mg,1.78mmol)，室温反应2h。反向柱色谱纯化(48％MeCN in H2O)，浓缩得产物7-5(1.05g，产率91％)。LC-MS(ESI):m/z found[(M-100)+H]+＝546.5。
中间体7-6的合成：
室温下，将化合物7-5(800mg,1.24mmol)用MeOH(5mL)溶解，加入4M的HCl/EA(3mL,12.4mmol)后室温反应4h，反向柱色谱纯化(50％MeCN in H2O)，冻干得产物7-6(380mg，产率56％)。LC-MS(ESI):m/z found[M+H]+＝545.2。
中间体7-7的合成：
室温下，将化合物7-6(380mg,0.698mmol)用DMF(5mL)溶解，加入DIEA(270.5mg,2.09mmol)和甲基-PEG8-NHS(355.5mg,0.698mmol)后室温反应2h，LC-MS监测原料基本反应完全，反应液通过反向柱色谱纯化(42％MeCN in H2O)，冻干得产物7-7(350mg，产率53.4％)。LC-MS(ESI):m/z found[M+H]+＝939.7。
中间体7-8的合成：
室温下，将化合物7-7(350mg,0.373mmol)用DMF(4mL)溶解，加入DIEA(48mg,0.373mmol)和二(对硝基苯)碳酸酯(226.7mg,0.746mmol)后室温反应2h，通过反向柱色谱纯化(50％MeCN in H2O)，冻干得产物7-8(120mg，产率29％)。LC-MS(ESI):m/z found[M+H]+＝1104.70。
中间体7-9的合成：
室温下，将化合物7-8(60mg,0.054mmol)用DMF(2mL)溶解，加入HOBT(8.1mg,0.059mmol),DIEA(21mg,0.163mmol)和甲磺酸艾日布林(40mg,0.054mmol)后室温反应2h，LC-MS监测原料反应完全，反应液通过反向C-18柱(45％MeCN in H2O)纯化，冻干得产物7-9(47mg，产率51％)。LC-MS(ESI):m/z found[M/2+H]+＝848.45。
中间体7-10的合成：
室温下，将化合物7-9(47mg,0.0277mmol)用DMF(1mL)溶解，加入二乙胺(125μl)后室温反应4h，将反应液过滤后，通过Pre-HPLC纯化，冻干得产物7-10(25mg，产率62％)。LC-MS(ESI):m/zfound[M/2+H]+＝737.15。
化合物7的合成：
室温下，将化合物7-10(10mg,6.79μmol)用DMF(1mL)溶解，加入DIEA(1.75mg,13.58μmol)和马来酰亚胺-PEG2-琥珀酰亚胺酯(7.2mg,20.37μmol)后室温反应4h，LC-MS监测原料不在减少后，将反应液过滤，通过Pre-HPLC纯化，冻干得产物7(3.3mg，产率28.4％)。LC-MS(ESI):m/z found[M/2+H]+＝857.10。1H NMR(400MHz,DMSO-d6)δ9.93(s,1H),8.38(d,J＝8.0Hz,1H),8.19(d,J＝8.0Hz,1H),8.01–7.90(m,1H),7.87(d,J＝8.0Hz,1H),7.61(d,J＝8.0Hz,1H),7.55(d,J＝8.0Hz,1H),7.23(d,J＝8.0Hz,1H),7.02(d,J＝4.0Hz,4H),6.55(s,1H),5.52(s,2H),5.32(t,J＝4.0Hz,3H),5.02(d,J＝20.0Hz,2H),4.83(s,2H),4.75(s,2H),4.63(s,2H),4.58–4.50(m,4H),4.25(d,J＝12.0Hz,2H),4.13–4.07(m,4H),3.84–3.62(m,6H),3.57–3.52(m,8H),3.49(d,J＝4.0Hz,32H),3.47–3.38(m,4H),3.24(s,4H),3.23(s,6H),2.93(s,4H),2.83(d,J＝8.0Hz,2H),2.67(t,J＝4.0Hz,2H),2.35–2.30(m,2H),2.00(dd,J＝12.0,4.0Hz,4H),1.91(d,J＝4.0Hz,2H),1.67(d,J＝12.0Hz,2H),1.32–1.28(m,2H),1.23(s,4H),1.03(d,J＝8.0Hz,6H),0.88–0.83(m,3H).
实施例2-8：
化合物8
合成路线：
化合物8的合成：
除将马来酰亚胺-PEG2-琥珀酰亚胺酯替换成6-(马来酰亚胺基)己酸琥珀酰亚胺酯外，其余实验步骤与实施例7的合成相同，得到白色固体化合物8(2.2mg，19.5％)。LC-MS(ESI):m/z found[M/2+H]+＝834.20。
实施例2-9：
化合物9
合成路线：
中间体9-3的合成：
将化合物9-1(250mg，0.736mmol)用DMF溶解，冰浴并氮气保护下加入化合物9-2(300mg，0.590mmol)和DIEA(124mg，0.874mmol)，冰浴反应1h后转室温反应4h。反相柱色谱纯化(H2O:MeCN＝3:2)，冻干得淡黄色油状物9-3(401mg，74.2％)。LC-MS(ESI):m/z found[M+H]+＝735.3。
中间体9-5的合成：
将化合物9-4(300mg，0.499mmol)用DMF溶解，依次加入二(对硝基苯)碳酸酯(604mg,0.998mmol)和DIEA(130μL，0.749mmol)，室温反应2h。EA/H2O萃取，饱和NaCl水溶液洗涤，无水Na2SO4干燥，抽滤，减压蒸馏浓缩，正相柱色谱纯化(DCM:MeOH＝93:7)，减压蒸馏除去溶剂得白色固体9-5(249mg，65％)。LC-MS(ESI):m/z found[M+H]+＝767.2。
中间体9-6的合成：
将化合物9-5(27.9mg，0.007mmol)和甲磺酸艾日布林(30mg，0.007mmol)用DMF(3mL)溶解，依次计入HOBt(5.4mg，0.008mmol)和DIEA(18.9μL，0.022mmol)，室温反应2h。DCM/H2O萃取(30mL×3)，饱和NaCl水溶液洗涤(20mL×3)，无水Na2SO4干燥，抽滤，减压浓缩，正相柱色谱纯化(DCM:MeOH＝91:9)，减压蒸馏除去溶剂得白色固体9-6(40mg，81％)。LC-MS(ESI):m/z found[M+H]+＝1357.80。
中间体9-7的合成：
将化合物9-6(10mg，0.007mmol)用DMF(2mL)溶解，加入DBU(1.5μL，0.110mmol)，室温反应1.5h。反应完全后未经纯化直接用于下一步。LC-MS(ESI):m/z found[M+H]+＝1135.86。
中间体9-8的合成：
将9-7(8.35mg，0.007mmol)用DMF溶解，依次加入中间体9-3(5.4mg，0.007mmol)、HATU(3.9mg，0.010mmol)和DIEA(1.2μL，0.007mmol)，室温反应2.5h。Pre-HPLC制备，冻干得白色固体9-8(4mg，30.9％)。LC-MS(ESI):m/z found[M/2+H]+＝927.2。
中间体9-9的合成：
将中间体9-8(24mg，0.013mmol)用DMF溶解，加入DBU(2.8μL)后室温反应2.5h。Pre-HPLC制备，冻干得白色固体9-9(10mg，48％)。LC-MS(ESI):m/z found[M/2+H]+＝816.05。
化合物9的合成：
将中间体9-9(5mg，0.003mmol)和马来酰亚胺基乙酸N-琥珀酰亚胺酯(0.93mg，0.004mmol)用DMF(2mL)溶解，室温反应2.5h。Pre-HPLC制备，冻干得白色固体化合物9(3mg，56％)。LC-MS(ESI):m/zfound[M/2+H]+＝884.6。1H NMR(400MHz,DMSO-d6)δ10.01(s,1H),8.46–8.40(m,1H),8.20(d,J＝8.0Hz,1H),7.95(d,J＝8.0Hz,1H),7.85(t,J＝12.0Hz,1H),7.57(d,J＝8.0Hz,2H),7.27(d,J＝8.0Hz,2H),7.11(s,1H),5.99(dd,J＝8.0,4.0Hz,1H),5.05(s,2H),5.00(s,2H),4.93(s,2H),4.83(s,2H),4.75(s,2H),4.64(s,2H),4.55(d,J＝4.0Hz,2H),4.35(d,J＝8.0Hz,4H),4.26(d,J＝12.0Hz,2H),4.18(d,J＝4.0Hz,2H),4.10(s,3H),4.07(s,2H),4.02(d,J＝4.0Hz,1H),3.84–3.66(m,4H),3.58(t,J＝16.0Hz,3H),3.49(s,30H),3.47(s,2H),3.44–3.41(m,2H),3.24(s,2H),3.08–2.87(m,6H),2.84(d,J＝8.0Hz,1H),2.76–2.67(m,2H),2.33(d,J＝8.0Hz,2H),2.30(s,2H),2.23(s,2H),2.20–2.10(m,2H),1.98(dd,J＝20.0,8.0Hz,4H),1.92(s,4H),1.75(dd,J＝12.0,4.0Hz,2H),1.71–1.65(m,4H),1.65–1.55(m,4H),1.48(d,J＝8.0Hz,2H),1.25(d,J＝12.0Hz,3H),1.03(d,J＝4.0Hz,3H),0.84(dd,J＝12.0,8.0Hz,6H).
实施例2-10：
化合物10
合成路线：
化合物10的合成：
将化合物10-1(3.3mg，0.012mmol)用DMF溶解，依次加入HOSU(1.99mg，0.017mmol)和EDCI(3.2mg，0.017mmol)，室温反应2h。后加入中间体9-9(10mg，0.006mmol)和DIEA(1.01μL，0.017mmol)，室温反应过夜。Pre-HPLC制备，冻干得白色固体化合物10(2mg，17.7％)。LC-MS(ESI):m/z found[M/2+H]+＝941.35。
实施例2-11：
化合物11
合成路线：
中间体11-3的合成：
将11-1(100mg，0.326mmol)用THF(3mL)溶解，加入催化量的DMF，0℃且氮气保护下滴加草酰氯(138μL，0.038mmol)，后转室温反应1h，溶液由浑浊变为澄清，减压浓缩。将酰氯中间体用DCM(2mL)溶解，0℃下加入TEA(453μL，3.26mmol)和吗啉(11-2，143μL，1.632mmol)，后转室温反应2h。DCM/H2O萃取，饱和NaCl洗，无水Na2SO4干燥，抽滤，减压浓缩，正相柱纯化(MeOH：DCM)，减压浓缩得浅棕色油状物11-3(141mg，97.3％)。LC-MS(ESI):m/z found[M+H]+＝445.0。
中间体11-4的合成：
将11-3(2.89g，6.5mmol)用EtOH(25mL)溶解，分两次加入NaBH4(1.967g，52mmol)，每次间隔0.5h，氮气保护下室温反应过夜。饱和NH4Cl水溶液淬灭，EA萃取，无水Na2SO4干燥，抽滤，减压浓缩，正相柱纯化(10％MeOH：90％DCM)。减压浓缩，得淡黄色油状物11-4(1.25g，41.1％)。LC-MS(ESI):m/z found[M+H]+＝224.2。
中间体11-7的合成：
将11-5(200mg，1.142mmol)和11-6(340.8mg，1.142mmol)用DMF(5mL)溶解，加入K2CO3(316mg，2.284mmol)后100℃回流过夜。1N稀盐酸/DCM萃取，无水Na2SO4干燥，抽滤，减压浓缩，正相柱纯化(15％EA：85％PE)，减压浓缩，得淡黄色油状物11-7(234.7mg，68.2％)。LC-MS(ESI):m/z found[M+H]+＝302.2。
中间体11-8的合成：
将11-7(235mg，0.780mmol)用EtOH(8mL)溶解，依次加入H2O(2mL)、Fe(174.3mg，3.12mmol)和NH4Cl(66.8mg，1.248mml)后升温至80℃回流5h。硅藻土抽滤，滤液减压浓缩，正相柱纯化(EA：PE)，减压浓缩得淡黄色油状物11-8(127.2mg，60.1％)。LC-MS(ESI):m/z found[M+H]+＝272.2。
中间体11-9的合成：
将11-8(23mg，0.085mmol)用1mLAcOH溶解，加入2,3-二溴马来酸酐(21.7mg，0.085mmol)后升温至110℃回流3h。减压蒸馏，正相柱纯化(EA：PE)，减压浓缩得白色固体11-9(32.9mg，76.0％)。LC-MS(ESI):m/z found[M+Na]+＝526.8。
中间体11-10的合成：
将11-9(32.9mg，0.065mmol)用2mL DCM溶解，0℃下缓慢加入11-4(28.9mg，0.129mmol)和DIEA(21.3μL，0.129mmol)，0℃下搅拌5min后转室温反应2h。减压蒸馏，正相柱纯化，减压浓缩得橘黄色固体11-10(16.3mg，31.6％)。LC-MS(ESI):m/z found[M+H]+＝794.0。
中间体11-13的合成：
将11-11(1.87g，4.93mmol)用DMF(10mL)溶解，依次加入11-12(0.5g，4.93mmol)和EEDQ(1.46g，5.9mmol)，氮气保护下室温反应过夜。反相柱(20％ACN：H2O)纯化，减压浓缩得白色固体产物11-13(1.8g，78.9％)。LC-MS(ESI):m/z found[M+H]+＝463.5。
中间体11-14的合成：
将化合物11-13(525mg，1.14mmol)溶于DMF(4mL)中，加入(PNP)2CO(380mg，1.25mmol)，DIEA(376μL，2.27mmol)后室温反应3小时。LC-MS监测反应完全后，反相柱层析纯化(H2O:ACN＝1:1)，得到白色固体产物(590mg，82.6％)。LC-MS(ESI):m/z found[M+H]+＝628.45。中间体11-15的合成：
将化合物11-14(300mg，0.48mmol)溶于DMF(3mL)中，，加入HOBt(64mg，0.48mmol)，DIEA(80μL，0.96mmol)和甲磺酸艾日布林(400mg，0.48mmol)，冰浴反应3h。LC-MS监测到反应完全后，反相柱色谱纯化，冻干得到白色固体产物(220mg，37.7％)。LC-MS(ESI):m/z found[M+H]+＝1219.60。
化合物11的合成：
将化合物11-15(200mg，0.16mmol)溶于DMSO(3mL)中，加入11-10(120mg，0.16mmol)，CuSO4·5H2O(48mg，0.20mmol)，抗坏血酸(72mg，0.40mmol)。氮气保护下，室温反应2小时。LC-MS监测到反应完全后，HPLC制备纯化，冻干，得到黄色固体产物(54.1mg，16.8％)。LC-MS(ESI):m/z found[M/2+H]+＝1007.30。1H NMR(400MHz,DMSO-d6)δ10.01(s,1H),8.44(d,J＝8.7Hz,1H),8.32(d,J＝7.4Hz,1H),8.02(s,1H),7.57(d,J＝8.6Hz,2H),7.31(s,8H),7.28(s,1H),7.25(d,J＝6.1Hz,2H),7.22(d,J＝2.5Hz,1H),7.09(s,1H),6.00(s,1H),5.20(d,J＝1.7Hz,2H),5.05(d,J＝2.4Hz,1H),5.00(s,1H),4.94(d,J＝2.4Hz,2H),4.84(s,1H),4.75(d,J＝1.8Hz,1H),4.64(td,J＝4.2,3.7,1.8Hz,1H),4.56(t,J＝4.2Hz,1H),4.52(s,2H),4.39(q,J＝7.8Hz,1H),4.29(tt,J＝10.8,6.2Hz,4H),4.17(td,J＝10.2,4.2Hz,1H),4.13–4.07(m,3H),4.05–3.97(m,1H),3.84–3.75(m,3H),3.74–3.69(m,4H),3.63–3.55(m,21H),3.25(d,J＝4.0Hz,6H),2.98(h,J＝10.4,8.1Hz,4H),2.84(dd,J＝9.7,2.0Hz,1H),2.74(td,J＝18.0,16.9,8.0Hz,2H),2.62–2.54(m,1H),2.36–2.12(m,6H),2.03–1.88(m,7H),1.79–1.59(m,7H),1.59–1.40(m,5H),1.39–1.26(m,5H),1.04(d,J＝6.4Hz,3H),0.87(dd,J＝12.4,6.7Hz,6H).
实施例2-12-2-19参照实施例2-11合成路线合成，结构及LC-MS结果见下表。
表14实施例2-12-2-19结构式及LC-MS


实施例2-20：
化合物20
合成路线：
中间体20-3的合成：
室温下，将20-2(500mg)溶在THF/DMF(5mL/5mL)中，加入DIEA(750mg，2eq)和HATU(1.32g，1.2eq)，室温搅拌20分钟后加入20-1(482mg，1.2eq),室温反应过夜。将反应液倒入水(40V)中，EA(40V)萃取两次，有机相合并后，用无水硫酸钠干燥。正相柱层析纯化，得到粗品，粗品经反相柱层析纯化得到黄色固体(400mg，47.2％)。LC-MS(ESI):m/z found[M+H]+＝293。
中间体20-4的合成：
室温下，将20-3(350mg，1eq)溶在EtOH/H2O(14mL/3.5mL)中，加入NH4Cl(128.2mg，2eq)和Fe(268.5mg，4eq)，78℃搅拌6小时。LCMS监测，反应完成。反应液过滤后，用水(60V)和EA(40V)萃取，有机相干燥后浓缩，得到红色油状物(280mg，88.8％)。LC-MS(ESI):m/z found[M+H]+＝263。
中间体20-5的合成：
室温下，将20-4(250mg，1eq)溶在AcOH(10V)中,加入二溴马来酸酐(243mg，1eq),120℃反应1小时。LCMS监测反应完成，将反应液倒入水(30V)中，EA(20V)萃取两次，有机相合并后干燥，硅胶拌样，正向柱层析纯化，浓缩后得到黄色固体(320mg，67.3％)。LC-MS(ESI):m/z found[M+H]+＝501。
中间体20-6的合成：
室温下，将20-5(20mg，1eq)溶在DCM(1.5ml)中，氮气置换，冰浴下加入11-4(17.84mg，2eq)和DIEA(10.32mg，2eq)，保持冰浴20min，后转室温搅拌2小时。LCMS监测反应完成，信号正确。将反应液通过反相柱层析纯化，冻干后得到黄色固体(20mg，63.77％)。LC-MS(ESI):m/z found[M+H]+＝785。
化合物20的合成：
室温下，将20-6(5.5mg，1eq)溶于DMSO(0.5ml)中，加入11-15(10mg，1.2eq)，CuSO4·5H2O(1.76mg，1eq)和抗坏血酸(1.24mg，1eq)，室温下搅拌反应1.5小时。LCMS和TLC监测反应，反应完成。HPLC制备纯化，得到黄色固体(6mg，42.8％)。LC-MS(ESI):m/z found[1/2M+H]+＝1002。
参照实施例2-20合成路线合成实施例2-21，结构式及LC-MS结果见下表。
表15实施例2-21结构式及LC-MS
实施例2-22：
化合物22
合成路线：
中间体22-3的合成:
室温下，将22-1(3g,1eq)置于单口瓶中，加入AcOH(10V)，超声至固体全部溶解后，加入22-2(2.2g,1eq)，氮气置换三次，油浴加热120℃搅拌1.5h。TLC显示反应完全。反应液冷却至室温后，加入水(20V)和EA(20V)，饱和食盐水(10V)，萃取分液，有机相暂存，水相再用EA(20V)萃取分液，有机相合并，用无水硫酸钠干燥后，3倍质量硅胶拌样，通过柱层析纯化(EA/PE＝21％)，得到黄色油状物(3.5g，62.6％)。LC-MS(ESI):m/z found[M+H]+＝426。
中间体22-4的合成：
室温下，将22-3(3g,1eq)溶于THF中，加入叠氮基乙酸叔丁酯(1.3g,1.2eq),CuSO4·5H2O(1.76g,1eq)和抗坏血酸(1.24g,1eq)，氮气置换，室温下搅拌反应1.5小时。LCMS和TLC监测反应，反应完成，MS信号正确。将反应液倒入水(80V)中，加入EA(80V)和饱和食盐水(40V)，萃取分液两次，有机相合并后浓缩，拌硅胶(200-300目)，再经过正相柱层析纯化,得到黄色油状物(2.58g，62.6％)。LC-MS(ESI):m/z found[M+H]+＝583。
中间体22-5的合成：
室温下，将22-4(2.5g,1eq)溶在Acetone(40V)中，加入NaN3(1.11g,6eq)，氮气保护，室温反应16小时。取样LCMS监测，反应完成。将反应液倒入水(80V)中，加入EA(60V)萃取分液三次，有机相合并后用无水硫酸钠干燥，正向柱色谱纯化，减压浓缩后得到黄绿色油状物(1.85g，84.8％)。LC-MS(ESI):m/zfound[M+H]+＝507。
中间体22-6的合成：
室温下，将22-7(1.85g)溶在EtOH(30V)中，加入Pd-C(1.85g)，氢气置换三次，室温搅拌2小时。LCMS监测反应完成，信号正确。将反应液过滤后浓缩,得到红色油状物(1.6g，82.9％)。LC-MS(ESI):m/zfound[M+H]+＝455。
中间体22-7的合成：
室温下，将22-6(400mg，1eq)溶在ACN(35V)中，加入1,4-二溴-2,3-丁二酮(215mg，1.2eq)室温下反应2小时。LCMS监测反应完成，信号正确。将反应液倒入水(70V)中，EA(50V)萃取，有机相合并后干燥，2g硅胶拌样，正向柱层析纯化(75％EA保持)，浓缩后得到黄绿色油状物(200mg，34.3％)。LC-MS(ESI):m/z found[M+H]+＝663。
中间体22-8的合成：
室温下，将22-7(125mg)溶于DCM(8mL)中,加入TFA(5mL),室温下搅拌反应16小时。LCMS监测，反应完成。室温下用乙腈置换旋干，HPLC制备纯化，直接冻干，得到白色固体(25mg，21.9％)。LC-MS(ESI):m/z found[M+H]+＝607。
化合物22的合成：
将化合物22-8(14mg，1eq)用DCM溶解，依次加入EEDQ(3mg，1.5eq)和VC-PABL-Eribulin(10mg，0.8eq)，室温反应2h，LC-MS监测反应完成，pre-HPLC制备，冻干得白色固体(1.1mg，4.9％)。LC-MS(ESI):m/z found[M/2+H]+＝862.60。
实施例2-23：
化合物23
合成路线：
中
间体23-2的合成：
室温下，将23-1(500mg，2.448mmol)溶于乙酸(5mL)中，加入2,3-二溴马来酸酐(22-2，626.4mg，2.448mmol)，升温到40℃反应过夜。反应完全后，减压浓缩，少量DMF溶解，干法正相柱层析纯化(PE:EA＝67:33)得白色固体产物(744.1mg,68.75％)。LC-MS(ESI):m/z found[M-56]+＝387.0。
中间体23-3的合成：
冰浴下，将23-2(20mg，0.0452mmol)溶在DCM(3mL)中，保持冰浴缓慢加入DIEA(15.76μL，0.0905mmol)和11-4(20.21mg，0.0905mmol)，换气三次，搅拌5min后室温反应2小时。反应完全后，将反应液减压浓缩后用DMF溶解，pre-HPLC纯化(ACN:H2O＝54:46)得到淡黄色固体(13.2mg,80.29％)。LC-MS(ESI):m/z found[M+H]+＝727.4。
中间体23-4的合成：
冰浴下，将23-3(13.2mg，0.018mmol)溶于DMF(2.5mL)中,加入HATU(8.21mg，0.0216mmol),搅拌30min后，依次加入2-[2-(炔丙基氧)乙氧基]乙胺(2.6mg，0.018mmol)和DIEA(3.135μL，0.018mmol)，室温反应2h。反应完全后，将反应液减压干燥，用DMF溶解，pre-HPLC纯化(ACN:H2O＝46:54)得到黄色固体(10.1mg，65.27％)。LC-MS(ESI):m/z found[M+H]+＝852.2
中间体23-5的合成：
室温下，将23-4(10.8mg，0.0126mmol)溶于DCM(2.5mL)中,加入HCl/EA溶液(0.1mL),室温反应1h。反应完全后，将反应液减压干燥，少量DMF溶解，pre-HPLC纯化(ACN:H2O＝23:77)得到黄色固体(6.9mg，72.4％)。LC-MS(ESI):m/z found[M+H]+＝752.45。
化合物23的合成：
室温下，将23-5(6.5mg，0.0086mmol)溶于EtOH(6mL)和THF(2mL)混合溶液中，加入11-15(12.6mg，0.0104mmol)，五水硫酸铜(2.157mg，0.0086mmol)和抗坏血酸(1.5mg，0.0086mmol),氮气置换三次，室温反应3h。反应完全后，将反应液减压干燥，少量DMF溶解，pre-HPLC纯化(ACN:H2O＝26:74)，冻干得到黄色固体(4.18mg，24.54％)。LC-MS(ESI):m/z found[M+H/2]+＝986.3。
除替换相应反应原料外，参照实施例2-23和合成路线合成实施例2-24，结构式及LC-MS见下表。
表16实施例2-24结构式及LC-MS
实施例2-25：
化合物25
合成路线：
中间体25-2的合成：
将25-1(1g，0.824mmol)溶于DMF(4mL)中，加入9-1(280mg，0.824mmol)，DIEA(150μL，0.906mmol)，室温反应4小时。反应完全后，反相柱色谱纯化，减压浓缩得到无色油状产物(1.15g，97.0％)。LC-MS(ESI):m/z found[M+1]+＝1440.2。
中间体25-3的合成：
将化合物25-2(150mg，0.104mmol)溶于DMF(1.5mL)中，加入HOSU(16mg,0.141mmol)，EDCI(27mg,0.141mmol)，室温反应2小时后，加入DIEA(18μL,0.104mmol)，11-11(53mg，0.141mmol)，室温反应2小时。反应完全后，反相柱层析纯化(H2O:ACN＝1:1)，得到白色固体产物(143mg，76.5％)。LC-MS(ESI):m/z found[M/2+1]+＝901.65。
中间体25-4的合成：
将化合物25-3(143mg，0.079mmol)溶于DMF(2mL)中，加入二乙胺(50μL)，室温反应3小时。LCMS监测到反应完全后，反相柱层析纯化(H2O:ACN＝2:1)，得到无色油状产物(111mg，89.0％)。LC-MS(ESI):m/z found[M/2+1]+＝790.60。
中间体25-5的合成：
将叠氮乙酸(2.5mg，0.025mmol)溶于乙腈(2mL)中，加入HATU(11.4mg，0.030mmol)，DIEA(8.4μL，0.051mmol)，室温反应0.5小时后，加入化合物25-4(40mg，0.025mmol)，室温反应3.5小时。LCMS监测到反应完全后，反相柱层析纯化(H2O:ACN＝3:2)，得到无色油状产物(28.7mg，69.1％)。LC-MS(ESI):m/z found[M+18/2+1]+＝840.55。
中间体25-6的合成：
将25-5(45mg，0.024mmol)溶于DMF(1.5mL)中，加入(PNP)2CO(36mg，0.096mmol)，DIEA(10μL，0.048mmol)，50℃反应过夜。LCMS监测到反应完全后，反相柱层析纯化(H2O:ACN＝1:1)，得到无色油状产物(26mg，59.4％)。LC-MS(ESI):m/z found[M/2+1]+＝923.10。
中间体25-7的合成：
将25-6(26mg，0.014mmol)溶于DMF(1mL)中，加入甲磺酸艾日布林(12mg，0.014mmol)，HOBT(3mg，0.021mmol)，DIEA(3.5μL，0.021mmol)，室温反应过夜。LCMS监测到反应完全后，反相柱层析纯化(H2O:ACN＝1:1)，得到白色固体产物(30.6mg，90.5％)。LC-MS(ESI):m/z found[M/2+1]+＝1218.45。
化合物25的合成：
将化合物25-7(15mg，0.006mmol)溶于DMSO(1.5mL)中，加入11-10(5mg，0.006mmol)，五水硫酸铜(2mg，0.0072mmol)，抗坏血酸(2.5mg，0.0144mmol)。氮气保护下，室温反应2小时。LCMS监测到反应完全后，HPLC制备纯化，冻干，得到黄色固体产物(2.05mg，10.6％)。LC-MS(ESI):m/z found[M/3+1]+＝1071.75。
实施例2-26：
化合物26
合成路线：
中间体26-2的合成：
将化合物26-1(2g，3.571mmol)用THF(20mL)溶解，加入NaH(171mg，7.125mmol)后冰浴下搅拌1h，后加入溴乙腈(843mg，7.14mmol)升温至室温反应3h。LC-MS监测反应完全后，在冰浴下滴加水淬灭，用EA萃取，无水Na2SO4干燥，抽滤，浓缩得黄色油状物(2.1g，99％)。LC-MS(ESI):m/z found[M+1]+＝599.60。
中间体26-3的合成：
将26-2(1.2g，2.006mmol)和(2S)-N-FMoc-6-叠氮基己酸(800mg)溶解在THF(5mL)和EtOH(15mL)中，依次加入五水硫酸铜(505mg，2.028mmol)和抗坏血酸(357mg，2.027mmol)后，在氮气保护下室温反应1.5h。LC-MS监测反应完全后，过滤除去不溶固体，滤液减压浓缩后加入DMF溶解，反相柱色谱纯化，浓缩得绿色油状液体(1.86g，92.3％)。LC-MS(ESI):m/z found[M+1]+＝993.43。
中间体26-4的合成：
将26-3(1.8g，1.814mmol)用DMF(12mL)溶解，依次加入HATU(1.0g，2.631mmol)、11-11(711mg，1.873mmol)和DIEA(604mg，4.682mmol)，室温反应5h。LC-MS监测反应完全，反相柱纯化，减压浓缩得白色固体(2.3g，93.6％)。LC-MS(ESI):m/z found[M/2+1]+＝678.45。
中间体26-5的合成：
将化合物26-4(2.3g，1.698mmol)溶DMF(15mL)溶解，加入二乙胺(0.4mL)，室温反应1.5h，LC-MS监测反应完全，反相柱色谱纯化，减压浓缩得黄色固体(1.4g，73％)。LC-MS(ESI):m/z found[M/2+1]+＝567.10。
中间体26-6的合成：
将化合物26-5(2.0g，1.768mmol)溶解在DMF(12mL)中，依次加入马来酰亚胺-PEG2-琥珀酰亚胺酯(843mg，3.345mmol)和DIEA(171mg，2.495mmol)，室温反应3h。LC-MS监测反应完全，反相柱色谱纯化，减压浓缩得白色固体(1.1g，65％)。LC-MS(ESI):m/z found[M/2+1]+＝635.85。
中间体26-7的合成：
将26-6(555mg，2.006mmol)用DMF(5mL)溶解，依次加入(PNP)2CO(492mg，2.028mmol)和DIEA(505mg，2.028mmol)，升温至50℃反应2h后转室温反应过夜。LC-MS监测反应完全，反相柱色谱纯化，减压浓缩得白色固体(430mg，92.3％)。LC-MS(ESI):m/z found[M/2+1]+＝769.43。
化合物26的合成：
将化合物26-7(50mg，0.032mmol)用DMF溶解，依次加入甲磺酸艾日布林(26mg，0.032mmol)、HOBt(4mg，0.032mmol)和DIEA(8mg，0.064mmol)，室温反应2h。LC-MS监测反应完全，pre-HPLC制备，冻干得黄色固体。LC-MS(ESI):m/z found[M/2+1]+＝1064.85。1H NMR(400MHz,DMSO-d6)δ10.03(s,1H),8.05(q,J＝20.0,8.0Hz,3H),7.78(d,J＝8.0Hz,1H),7.59–7.53(m,2H),7.30–7.20(m,2H),7.10(t,J＝8.0Hz,1H),7.02(s,1H),6.55(s,1H),5.97(t,J＝8.0Hz,1H),5.42(s,2H),5.08–4.98(m,2H),4.92(s,2H),4.83(s,1H),4.75(s,1H),4.63(td,J＝4.0,4.0Hz,1H),4.55(t,J＝4.0Hz,1H),4.50(s,2H),4.38(d,J＝8.0Hz,2H),4.30(t,J＝8.0Hz,3H),4.21–4.15(m,2H),4.10(dd,J＝8.0,4.0Hz,2H),4.02(s,1H),3.86–3.73(m,3H),3.71–3.62(m,2H),3.56–3.47(m,56H),3.44–3.39(m,4H),3.24(d,J＝4.0Hz,6H),3.02(s,1H),2.95(d,J＝4.0Hz,4H),2.84(d,J＝8.0Hz,1H),2.74(s,2H),2.67(m,1H),2.37–2.29(m,4H),2.25(d,J＝16.0Hz,2H),2.20–2.12(m,2H),2.02–1.89(m,6H),1.79(t,J＝8.0Hz,2H),1.68(dd,J＝24.0,12.0Hz,6H),1.48(dd,J＝24.0,8.0Hz,3H),1.32(d,J＝12.0Hz,4H),1.28–1.11(m,9H),1.03(d,J＝4.0Hz,3H),0.83(dd,J＝12.0,8.0Hz,6H).
除替换相应反应原料外，参照实施例2-26合成路线合成实施例2-27，其结构及LC-MS信息见下表。
表17实施例2-27结构式及LC-MS
参照实施例7合成路线合成实施例2-28，其结构及LC-MS结果见下表。
表18实施例2-28结构式及LC-MS
实施例2-29：
化合物29
合成路线：
中间体29-3的合成：
室温下，将29-1(300mg，1.292mmol)、29-2(334μL，3.876mmol)和四丁基溴化铵(10mg，0.031mmol)溶于甲苯(3mL)和5％氢氧化钠溶液(3mL)中，保持室温反应过夜。TLC监测反应完全。反应液中加入20mL水，再加入适量乙酸乙酯洗涤萃取分液，收集有机相减压浓缩，正相柱层析纯化得白色固体产物(164mg,47％)。
中间体29-4的合成：
室温下，将29-3(50mg，0.185mmol)、碘甲烷(12μL，0.185mmol)和碳酸铯(121mg，0.370mmol)溶于DMF(5mL)中，保持室温反应过夜。TLC监测反应完全。反应液中加入少量乙酸乙酯稀释，再加入适量水洗涤萃取分液，收集有机相减压浓缩得到粗产品(62mg,100％)。
中间体29-5的合成：
室温下，将29-4(62mg，0.218mmol)溶解在DCM(3mL)和TFA(3mL)中，室温反应一小时。减压浓缩将溶剂除去(加入甲苯带走大部分TFA)。再加入AcOH(5mL)将混合物溶解，加入二溴马来酸酐(53mg，0.207mmol)随后升温至110℃反应2小时。TLC监测反应完全。反应液减压浓缩除去大量乙酸，加入少量DCM溶解通过正相柱层析纯化(PE:EA＝60:40)得到产品(24mg,46％)。LC-MS(ESI):m/z found[M+H]+＝322.8。
中间体29-6的合成：
将叠氮-乙二醇-乙酸(214mg，1.054mmol)用DMF(4mL)溶解，依次加入EEDQ(226mg，1.264mmol)和11-11(400mg，1.054mmol)，氮气保护下室温反应16h。LC-MS检测反应完全，反相柱色谱纯化，减压浓缩得白色固体产物(160mg，26.8％)。LC-MS(ESI):m/z found[M+H]+＝563.45。
中间体29-7的合成：
将29-6(25mg，0.044mmol)用DMF溶解，加入(PNP)2CO(40mg，0.133mmol)和DIEA(12mg，0.089mmol)，室温反应4h。LC-MS检测反应完全后，反相柱色谱纯化，减压浓缩得白色固体产物(17mg，52％)。
中间体29-8的合成：
将29-7(50mg，1eq)用DMF溶解，加入HOBt(9.2mg，1.1eq)室温搅拌15min后依次加入甲磺酸艾日布林(56mg，1eq)和DIEA(17.7mg，2eq)，室温反应2h。LC-MS检测反应完全后，反相柱色谱纯化，冻干得白色固体(50mg，55％)。LC-MS(ESI):m/z found[M+H]+＝1320.6。
化合物29的合成：
室温下，将29-5(2.5mg，0.0078mmol)、29-8(10.3mg，0.0078mmol)、五水硫酸铜(1.9mg，0.0078mmol)和维生素C(1.4mg，0.0078mmol)溶于干燥四氢呋喃(2mL)、乙醇(6mL)中，氮气置换三次后保持室温反应2小时。LCMS监测反应完全，减压浓缩，少量DMF溶解，pre-HPLC纯化得黄色固体产物(7.02mg,55％)。LC-MS(ESI):m/z found[M/2+H]+＝822.05。
实施例2-30：
化合物30
合成路线：
中间体30-1的合成：
冰浴下，将29-5(24mg，0.075mmol)用DCM(5mL)溶解，加入11-4(34mg，0.149mmol)和DIPEA(26μL，0.149mmol)，冰浴下反应半小时后升温至室温反应2小时。LC-MS监测反应，反应完全后反应液减压浓缩除去溶剂，少量甲醇溶解，反相柱色谱纯化(ACN:H2O＝30:70)，干燥得金黄色固体(45mg,98％)。LC-MS(ESI):m/z found[M+H]+＝607.0。
化合物30的合成：
室温下，将30-1(5mg，0.0082mmol)、29-8(11mg，0.0082mmol)、五水硫酸铜(2.1mg，0.0082mmol)和维生素C(1.5mg，0.0082mmol)溶于干燥四氢呋喃(2mL)、乙醇(6mL)中，氮气置换三次后保持室温反应2小时。LCMS监测反应完全减压浓缩，少量DMF溶解，pre-HPLC纯化冻干得黄色固体产物(7.56mg,63％)。LC-MS(ESI):m/z found[M/2+H]+＝964.70。
实施例31-35：
除替换相应反应原料外，参照实施例2-11合成路线合成实施例2-31-2-36，其结构式见下表。
表19实施例2-31-2-35结构式及LC-MS

实施例2-37：
化合物37
合成路线：
中间体37-2的合成：
将37-1(5g，1.0eq)溶于50mLDCM中，依次加入乙二醇(8.4g，10eq)和PPTS(6.82g，2.1eq)后室温反应过夜。TLC监测反应完全，减压浓缩后DMF溶解，反相柱色谱纯化，冻干得白色固体产物(4.68g，93％)。
中间体37-3的合成：
将37-2(650mg)用DCM(10mL)溶解，加入DEA(1mL)后室温反应1.5h。TLC监测反应完全，减压浓缩得粗品淡黄色油状物662mg，直接用于下一步。LC-MS(ESI):m/z found[M+H]+＝148.2。
中间体37-6的合成：
室温下，将37-4(1g,1.7mmol)溶解在DMF(4mL)中，加入HOSU(215.2mg,1.87mmol)和EDCI(358mg,1.87mmol)，室温反应6小时，后加入化合物37-5(578mg,1.7mmol)和DIEA(444.3μL，2.55mmol)，室温反应2小时。LCMS监测原料反应完全。反相柱色谱纯化，减压浓缩得到白色油状物(1.142g,73.77％)。LC-MS(ESI):m/z found[M+H]+＝911.8。
中间体37-7的合成：
室温下，将37-6(1.142g,1.25mmol)溶解在ACN(10mL)中，加入HOSU(216.4mg,1.88mmol)和DCC(388mg,1.88mmol)，室温反应2小时，之后将反应液过滤，收集滤液减压干燥，粗品用DMF(5mL)溶解，加入DIEA(327.5μL，1.88mmol)和甘氨酰甘氨酰-L-苯丙氨酸(420mg,1.5mmol)，室温反应2小时。LCMS检测原料反应完全。反相柱色谱纯化，减压浓缩得到白色油状物(1.076g,73.22％)。MS(ESI):m/z found[M+H/2]+＝587.2。
中间体37-8的合成：
室温下，将37-7(400mg，0.34mmol)溶于DMF(2mL)中,加入哌啶(337μL，3.41mmol)，室温反应2小时。LC-MS检测原料反应完全，反应液通过pre-HPLC制备纯化，冻干得到白色油状物(159.3mg,49.14％)。LC-MS(ESI):m/z found[M+H]+＝950.7。
中间体37-9的合成：
室温下，将37-8(45mg，0.0474mmol)溶于DMF(2.5mL)中,依次加入叠氮乙酸NHS酯(18.8mg，0.0947mmol)和DIEA(16.5μL，0.0947mmol)，室温反应1.5小时。反应完全后，反应液通过pre-HPLC制备，冻干得到白色油状物(19.4mg,39.65％)。LC-MS(ESI):m/z found[M+H]+＝1033.75。
中间体37-10的合成：
室温下，将37-9(50mg，0.048mmol)用THF(2mL)溶解，加入DCC(12mg，0.058mmol)和HOSU(7mg，0.058mmol)室温反应2h。反应完全后，过滤反应液，浓缩后用入DMF溶解，加入DIEA(10μL，0.058mmol)和37-3(11mg，0.073mmol)，反应2h，LCMS监测反应完成。Pre-HPLC制备，冻干得白色油状产物(15mg，26.8％)。LC-MS(ESI):m/z found[M+H]+＝1163.3。
中间体37-11的合成：
室温下，将37-10(286mg,0.246mmol)溶于DMF(4mL),加入二(对硝基苯)碳酸酯(523mg,1.72mmol)后滴加DIEA(128μL,0.74mmol)，升温至50℃，氮气保护下反应过夜。LCMS监测反应完成，将反应液过滤，取滤液反向柱色谱纯化，冻干得到无色固体(148mg,44.7％)。LC-MS(ESI):m/z found[M+H]+＝1345.6。
中间体37-12的合成：
室温下，将37-11(170mg,0.214mmol)用DMSO(5mL)溶解，加入11-10(283mg,0.26mmol)、五水硫酸铜(54mg,0.214mmol)和Vc(39mg,0.214mmol)，氮气保护下室温反应1h，TLC监测原料反应完成。反相柱色谱纯化，冻干得黄色固体(278mg，62.3％)。LC-MS(ESI):m/z found[M+H]+＝2122.2。
化合物37的合成：
将37-2(1eq)用DMA溶解，依次加入甲磺酸艾日布林(1eq)和DIEA(2eq)，室温反应2h。LC-MS监测基本反应完全，Pre-HPLC纯化，冻干后得黄色固体。试验例1：嵌合抗体与人源化抗体亲和力检测
嵌合抗体与人源化抗体对Nectin 4亲和力的检测，采用捕获抗体的形式。用Sensor Chip Protein A生物传感芯片(Cytiva，29650263)亲和捕获抗体，然后于芯片表面流经抗原Nectin4-his，用Biacore K8仪器(Cytiva)实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用再生缓冲液将芯片洗净再生。实验结束后用GE Biacore K8 Evaluation version 3.0软件以(1:1)Langmuir模型拟合数据，得出亲和力数值。嵌合抗体和人源化抗体与蛋白的亲和力见下表20和21：
表20嵌合抗体亲和力检测结果
表21人源化抗体亲和力检测结果

通过表20和21可以看出，本发明所提供的示例性抗体相对于阳性对照AGSM6具有相当甚至更好的对Nectin4的亲和力。
试验例2：结合活性检测
通过ELISA方法检测抗体的结合活性，过程简述如下，包被实施例1纯化Nectin4-His抗原蛋白，包被浓度为1ug/mL，2％BSA过夜封闭后，分别加入梯度稀释的待检测抗体孵育(表15抗体的起始浓度为10ug/mI,2倍梯度稀释)，37℃孵育1小时后，加入小鼠抗人IgG(免疫球蛋白G)Fab段抗体(Mouse Anti-Human IgG Fab Antibody)(12H3C4A6)[HRP](金斯瑞，A01855-200)二抗孵育1h,随后加入TMB(索莱宝，RP1200-1000mL)显色，用终止液(碧云天，P0215)终止显色，酶标仪读取OD450的吸光度后，通过GraphPad Prism9计算各抗体的结合EC50数值比较各抗体的结合活性，结果见表22。
表22嵌合抗体结合活性结果
表23人源化抗体结合活性结果
通过表23可以看出，本发明所提供的示例性抗体相对于阳性对照AGSM6具有相当甚至更好的Nectin4结合活性。
试验例3：内吞效率测定
该实验中使用的细胞系为购于南京科佰生物科技有限公的T47D细胞，货号：CBP60397，Nectin4高表达细胞系。
用FACS(2％FBS+PBS)染色缓冲液制取细胞悬液，密度为2×106cells/mL，将细胞悬液以50μL/孔铺于96孔U形底细胞板(UWP043096)中，每个样品设置一组平行实验。使用FACS染色缓冲液稀释一抗至浓度为首孔10ug/ml，5倍梯度稀释。取50μL一抗稀释液分别加入到96孔U形底细胞板中，轻柔混匀后冰浴60min。加入200μLFACS染色缓冲液洗涤细胞，4℃、300g离心5min，弃上清，重复洗涤2次。每孔中加入30μLFACS重悬细胞，37℃孵育6h，使结合于细胞表面的抗体内化。使用另一个在4℃孵育相同时间的样品作为无内化的阴性对照。6h后，将对应的37℃和4℃孵育的样品移至冰上，并加入170μL冰FACS，终止内化。4℃、300g离心5min沉淀细胞。按1：200的比例加入二抗，100μL/孔。4℃避光孵育30min。加入200μLFACS染色缓冲液洗涤细胞，移除未结合的抗体。4℃、300g离心5min沉淀细胞，弃上清。重复洗涤2次。将染色后细胞重悬于70μL冰PBS中。细胞洗涤完成后，尽快使用流式细胞仪分析。
通过37℃孵育样品相对于4℃孵育样品的平均荧光强度(MFI)降低水平得出内吞差值。如表24所示：
表24嵌合抗体内吞结果
表25人源化抗体内吞结果
通过表24和25可以看出，本发明所提供的示例性抗体相对于阳性对照AGSM6具有相当甚至更好的内吞效率。
试验例4：特异性检测
本试验通过ELISA方法检测不同抗体与人Nectin-4同家族蛋白hNectin-1(ACRO，PV1-H5223)、hNectin-2(ACRO,PV2-H52E2)、hNectin-3(ACRO，PV3-H52E4)、hNectin-4-His的结合情况，确认抗体的特异性。方法和过程参考试验例3，包被目的抗原(1ug/mL)，封闭后加入梯度稀释抗体(起始浓度为10ug/mL，10倍梯度稀释)，结果如表26和图1、图2所示：
表26人源化抗体特异性检测结果
通过表26可以看出，本发明所提供的示例性抗体h10591-2抗体具有良好的特异性，不与同家族蛋白hNectin-1/2/3结合，只与hNectin-4-His结合。
试验例5：种属特异性检测
Nectin-4在不同种属具有较高的同源性。
通过ELISA方法检测不同抗体与不同种属Nectin-4蛋白的结合情况。分别包被hNectin-4-His，猴cNectin-4蛋白(ACRO,NE4-C52H4)和小鼠mNectin-4蛋白(ACRONE4-M52H3)，梯度稀释抗体AGSM6和h10591-2，并加入小鼠抗人IgG(免疫球蛋白G)Fab段抗体(MouseAnti-Human IgG Fc Antibody)[HRP]作为二抗显色，结果如表27和图3、图4所示。
表27人源化抗体种属特异性检测结果
通过表27可以看出，本发明所提供的阳性抗体与人、猴、大鼠有交叉，与小鼠弱交叉，与文献报道一致；示例性抗体h10591-2与人、猴交叉，与大小鼠不交叉。
试验例6 NECTIN4 ADC偶联
本专利抗体1为AGSM6参照WO2012047724实施例2制备得到，其重链和轻链氨基酸序列分别如前文所示。
本专利抗体2为hH2L1，参照CN117942409A实施例1序列制备得到，其重链和轻链氨基酸序列分别如SEQ ID NO:112和SEQ ID NO:113所示。
hH2L1重链(SEQ ID NO:112)
hH2L1轻链(SEQ ID NO:113)
本专利示例性所选用Nectin-4抗体为实施例3所制得的h10591-2。
1.1基于部分还原的半胱氨酸偶联
使用1mM EDTA-PBS缓冲液将抗体稀释至5mg/mL，按照2倍摩尔当量比加入5mM TCEP进行断键，37℃震荡2h。断键完成后，按照4倍摩尔当量比加入5mg/mL的Linker-payload，同时在体系中补加DMSO，使体系中DMSO的含量为10％。室温混匀2h后，样品离心，去除沉淀后，进行脱盐处理。最终收获的样品进行定量、SEC、HIC检测。
制备得到表28所示的抗体-药物偶联物，其中，NECTIN 4-ADC-1～NECTIN 4-ADC-4、NECTIN 4-ADC-7-1、NECTIN 4-ADC-8、NECTIN 4-ADC-9-1、NECTIN 4-ADC-10偶联抗体为抗体1，NECTIN 4-ADC-7-2、NECTIN 4-ADC-9-2、NECTIN 4-ADC-22～NECTIN 4-ADC-24偶联抗体为h10591-2。
表28基于部分还原的半胱氨酸偶联ADC编号及结构式



注：m或m1为药物-抗体比率(Drug-to-Antibody Ratio，DAR)。
1.2基于双共轭桥连的偶联
使用1mM EDTA-PBS缓冲液将抗体稀释至5mg/mL，按照8倍摩尔当量比加入5mM TCEP进行断键，37℃震荡2h。断键完成后，按照6倍摩尔当量比加入5mg/mL的Linker-payload，同时在体系中补加DMSO，使体系中DMSO的含量为10％。室温混匀2h后，样品离心，去除沉淀后，进行脱盐处理。最终收获的样品进行定量、SEC、HIC检测。
制备得到表29所示的抗体-药物偶联物，其中，9MW2821、NECTIN 4-ADC-29偶联抗体为抗体2hH2L1，NECTIN4-ADC-5-1、NECTIN4-ADC-6、NECTIN 4-ADC-28偶联抗体为抗体1AGSM6，NECTIN 4-ADC-5-2、NECTIN 4-ADC-11～NECTIN 4-ADC-21、NECTIN 4-ADC-25、NECTIN 4-ADC-26、NECTIN 4-ADC-27偶联抗体为h10591-2。
表29基于双共轭桥连的ADC编号及结构式







注：m为药物-抗体比率；
双共轭桥联部分ADC的马来酰亚胺环有开环和闭环两种形式，在制备过程中，ADC中的接头部分马来酰亚胺发生水解，由(闭环)变成(开环)。
使用LC-MS检测本发明的ADC中闭环结构和开环结构比例，如下表29-1：
表29-1

结论：本发明表29-1中的ADC，存在开环和闭环两种形式。Nectin 4-ADC-14、Nectin 4-ADC-15、Nectin4-ADC-14和Nectin 4-ADC-20ADC中马来酰亚胺水解比例大于80％，其中Nectin 4-ADC-11完全水解。表明，通过引入芳香基团可有效控制马来酰亚胺部分的水解反应，提高ADC稳定性。
1.3 ADC纯度及DAR值测试
通过HPLC-HIC分析，确定是否偶联成功并检测DAR值；通过SEC-HPLC法检测ADC纯度。
表30-1本公开ADC HIC及SEC结果

对照ADC(Enfortumab vedotin)及本公开ADC偶联物HIC分析结果如图5-8所示。
结论：从图5-8的结果来看，本公开的抗体偶联药物(如NECTIN 4-ADC-11、NECTIN 4-ADC-18、NECTIN4-ADC-26等)与Enfortumab vedotin相比，具有更优异的DAR值均一性。
试验例7：本发明ADC对肿瘤细胞体外增殖抑制测试
1.MDA-MB-468细胞活性抑制实验
使用MDA-MB-468(乳腺癌细胞，科佰，CBP60387)，此细胞为Nectin 4中高表达细胞系。
用含10％FBS的新鲜细胞培养基制取细胞悬液，密度分别为5×104cells/mL，以每孔100μL加入到96孔细胞培养板(fisheries scientific,310109027)中，5％二氧化碳37℃培养24h。ADC样品用新鲜培养基分别配制成4μΜ。以此为首浓度，用PBS十倍梯度稀释，共9个浓度。每孔吸出50μL培养液，再补加50μL的上述ADC溶液，故首孔ADC浓度为2μΜ，终体积为每孔100μL。5％二氧化碳37℃培养5天。弃去所有上清，每孔加入100μL CCK8(碧云天,C0042)，37℃静置孵育4小时，酶标仪(TECAN,Spark)上读取化学发光，利用Graphpad Prism 5软件进行数据分析，实验结果见下表23。
2.MDA-MB-453细胞活性抑制实验
使用MDA-MB-453(乳腺癌细胞，科佰，CBP60386)，此细胞为Nectin 4中高表达细胞系。
用含10％FBS的新鲜细胞培养基制取细胞悬液，密度分别为5×104cells/mL，以每孔100μL加入到96孔细胞培养板(fisheries scientific,310109027)中，5％二氧化碳37℃培养24h。ADC样品用新鲜培养基分别配制成0.8μΜ。以此为首浓度，用PBS五倍梯度稀释，共9个浓度。每孔吸出50μL培养液，再补加50μL的上述ADC溶液，故首孔ADC浓度为0.4μΜ，终体积为每孔100μL。5％二氧化碳37℃培养5天。弃去所有上清，每孔加入100μL CCK8(碧云天,C0042)，37℃静置孵育4小时，酶标仪(TECAN,Spark)上读取化学发光，利用Graphpad Prism 5软件进行数据分析，实验结果见下表23。
3.HT1376细胞活性抑制实验
使用HT1376(膀胱癌细胞，科佰，CBP60310)，此细胞为Nectin 4中高表达细胞系。
用含10％FBS的新鲜细胞培养基制取细胞悬液，密度分别为3×104cells/mL，以每孔100μL加入到96孔细胞培养板(fisheries scientific,310109027)中，5％二氧化碳37℃培养24h。ADC样品用新鲜培养基分别配制成0.4μΜ，2μΜ，7μΜ。以此为首浓度，用PBS五倍梯度稀释，共9个浓度。每孔吸出50μL培养液，再补加50μL的上述ADC溶液，故首孔ADC浓度为5μΜ，终体积为每孔100μL。5％二氧化碳37℃培养3天。弃去所有上清，每孔加入100μL CCK8(碧云天,C0042)，37℃静置孵育4小时，酶标仪(TECAN,Spark)上读取化学发光，利用Graphpad Prism 5软件进行数据分析，实验结果见下表23。
4.T47D细胞活性抑制实验
使用使用T47D(乳腺管癌细胞，科佰，CBP60397)，此细胞为Nectin 4中高表达细胞系。
用含10％FBS的新鲜细胞培养基制取细胞悬液，密度分别为1×105cells/mL，以每孔100μL加入到96孔细胞培养板(fisheries scientific,310109027)中，5％二氧化碳37℃培养24h。ADC样品用新鲜培养基分别配制成0.4μΜ，2μΜ，7μΜ。以此为首浓度，用PBS五倍梯度稀释，共9个浓度。每孔吸出50μL培养液，再补加50μL的上述ADC溶液，故首孔ADC浓度为5μΜ，终体积为每孔100μL。5％二氧化碳37℃培养3天。弃去所有上清，每孔加入100μL CCK8(碧云天,C0042)，37℃静置孵育4小时，酶标仪(TECAN,Spark)上读取化学发光，利用Graphpad Prism 5软件进行数据分析，实验结果见下表23。
表23：本公开部分抗体药物偶联物在不同细胞系中的IC50值


注：“-”表示未检测。
结论：以上数据表明，本公开制备得到的抗体药物偶联物在Nectin 4表达的多种细胞(例如乳腺癌、膀胱癌细胞)中具有优异的抑制活性，且其抑制活性显著优于对照药物Enfortumab vedotin。并且，相同抗体下，以Eribulin为有效载荷的ADC(Nectin 4-ADC-11)比以MMAE为paylaod的ADC(Nectin 4-ADC-27)具有更好的肿瘤抑制活性。
试验例8：本发明ADC化合物体内药效实验
本试验数据统计使用Excel 2016统计软件：平均值以average计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算。其中各指标计算公式如下：
肿瘤体积(V)：V＝1/2L长径×(L短径)2
相对肿瘤体积(RTV)：RTV＝VT/V0
相对肿瘤增殖率T/C(％)＝TRTV/CRTV×100％
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)
瘤重抑瘤率(％)＝(1-给药组平均瘤重/对照组平均瘤重)*100％
其中V0、VT分别为试验开始时及试验结束时的肿瘤体积。CRTV、TRTV分别为试验结束时的空白对照组(PBS)及实验组的相对肿瘤体积。
1.MDA-MB-468细胞CDX模型小鼠体内药效评价
在BALB/c-Nude裸小鼠右肋部皮下接种人三阴乳腺癌细胞MDA-MB-468(科佰)(1*10^7/200μL/只，具有50％的低生长因子人工基底膜)。接种细胞后，肿瘤生长7天，瘤体积长至135mm3左右后将动物随机分组(D0)，每组6只。
采用尾静脉注射给药，多剂量平行给药2mg/kg、1mg/kg、0.5mg/kg，单针给药，持续观测21天。每周测量2次肿瘤体积和体重，记录数据，计算肿瘤体积抑制率。数据统计使用Excel 2016统计软件：平均值以average计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算。
结果如图9和表24所示。
表24 MDA-MB-468肿瘤体积抑制率
结论：在三阴乳腺癌MDA-MB-468CDX模型中，本公开示例性化合物NECTIN 4-ADC-11在0.5-2mg/kg剂量下具有明显的肿瘤生长抑制作用，其对肿瘤的生长抑制作用显著优于Enfortumab vedotin，甚至0.5mg/kg NECTIN 4-ADC-11对肿瘤的抑制作用相较2mg/kg Enfortumab vedotin对肿瘤的抑制作用更优，其2mg/kg的NECTIN 4-ADC-11 TGI高达100％。
2.MDA-MB-453细胞CDX模型小鼠体内药效评价
在BALB/c-Nude裸小鼠右肋部皮下接种人乳腺癌细胞MDA-MB-453(科佰)(1*10^7/200μL/只，具有50％的低生长因子人工基底膜)。接种细胞后，肿瘤生长7天，瘤体积长至120mm3左右后将动物随机分组(D0)，每组5只。
采用尾静脉注射给药，单剂量平行给药1mg/kg，单针给药，持续观测20天。每周测量2次肿瘤体积和体重，记录数据。
结果如图10所示：
结论：在乳腺癌MDA-MB-453CDX模型中，本公开示例性的抗体药物偶联物NECTIN 4-ADC-11、NECTIN4-ADC-22具有明显的肿瘤生长抑制作用，相较于Enfortumab vedotin具有更好的肿瘤抑制作用。
3.HT1376细胞CDX模型小鼠体内药效评价
在BALB/c-Nude裸小鼠右肋部皮下接种人乳腺癌细胞HT1376(科佰)(1*10^7/200μL/只，具有50％的低生长因子人工基底膜)。接种细胞后，肿瘤生长8天，瘤体积长至120mm3左右后将动物随机分组(D0)，每组5只。
采用尾静脉注射给药，多剂量平行给药8mg/kg、4mg/kg，单针给药，持续观测21天。每周测量2次肿瘤体积和体重，记录数据。
结果如图11-12所示。
结论：在HT1376 CDX模型中，本公开示例性的抗体药物偶联物NECTIN 4-ADC-7-2、NECTIN 4-ADC-9-2、NECTIN 4-ADC-11具有明显的肿瘤生长抑制作用，其对肿瘤的抑制作用优于Enfortumab vedotin或相当。
4.HT1376-CDX模型小鼠体内药效评价
在BALB/c-Nude裸小鼠右肋部皮下接种人乳腺癌细胞HT1376(科佰)(1*10^7/200μL/只，具有50％的低生长因子人工基底膜)。接种细胞后，肿瘤生长8天，瘤体积长至120mm3左右后将动物随机分组(D0)，每组6只。
采用尾静脉注射给药，多剂量平行给药5mg/kg、2.5mg/kg、1mg/kg，单针给药，持续观测21天。每周测量2次肿瘤体积和体重，记录数据，计算肿瘤体积抑制率。
结果如图13所示。
表25 HT1376肿瘤体积抑制率
结论：实验结果表明，在HT1376 CDX模型中，不同给药剂量下本公开示例性的抗体药物偶联物NECTIN 4-ADC-11对肿瘤生长有优异的抑制作用，且在2.5mg/kg给药剂量下的药效比5mg/kg的Enfortumab vedotin组更优。
5.RT4/Nectin 4细胞CDX小鼠模型体内药效评价
在BALB/c-Nude裸小鼠右肋部皮下接种人膀胱移行细胞乳头瘤细胞RT4/Nectin 4(科佰)(9*10^6/200μL/只，具有50％的低生长因子人工基底膜)。接种细胞后，肿瘤生长7天，瘤体积长至110-120mm3左右后将动物随机分组(D0)，每组5只。
采用尾静脉注射给药，单剂量给药5mg/kg，单针给药，持续观测21天。每周测量2次肿瘤体积和体重，记录数据。
结果如图14所示。
结论：本公开示例性的抗体药物偶联物NECTIN 4-ADC-11在RT4/Nectin 4 CDX模型具有明显的肿瘤生长抑制作用，其对肿瘤抑制作用明显优于Enfortumab vedotin。
6、MDA-MB-453细胞CDX模型小鼠体内药效评价
a.在BALB/c-Nude裸鼠右肋部皮下接种人乳腺癌细胞MDA-MB-453(科佰)(1×107/200μL/只，具有50％的低生长因子人工基底膜)。接种细胞后，肿瘤生长6天，瘤体积长至135mm3左右后将动物随机分组(D0)，每组6只。
b.采用尾静脉注射给药，给药剂量分别为2mg/kg、1mg/kg、0.5mg/kg，单针给药。每周测量2次肿瘤体积和体重，记录数据，计算肿瘤体积抑制率。
结果如图19和表26所示。
表26 MDA-MB-453肿瘤体积抑制率

结论：本公开示例性的抗体药物偶联物NECTIN 4-ADC-11在0.5-2mg/kg剂量下均能有效抑制荷瘤裸鼠中MDA-MB-453移植瘤的生长，且呈剂量依赖性，药效显著优于阳性药。
7、MDA-MB-468荷瘤裸鼠动物药效
a.在BALB/c-Nude裸鼠右肋部皮下接种人三阴性乳腺癌细胞MDA-MB-468(科佰)(1×107/200μL/只，具有50％的低生长因子人工基底膜)。接种细胞后，肿瘤生长10-11天，瘤体积长至115-135mm3左右后将动物随机分组(D0)，每组5只。
b.采用尾静脉注射给药，给药剂量分别为2mg/kg、1mg/kg，单针给药。每周测量2次肿瘤体积和体重，记录数据，计算肿瘤体积抑制率。
结果如图20和表27所示。
表27 MDA-MB-468肿瘤体积抑制率
结论：本公开示例性的抗体药物偶联物NECTIN 4-ADC-11在1mg/kg和2mg/kg剂量下均能有效抑制荷瘤裸鼠中MDA-MB-468移植瘤的生长，且呈剂量依赖性，其药效显著优于阳性药9MW2821。
试验例9 ADC药物毒理学实验
1.BALB/c小鼠急性毒性实验
选用维通利华6-8周的BALB/c小白鼠进行急毒实验，采用尾静脉注射给药，给药剂量为100mg/kg，给药体积为200μL/组，给药1次，观察14天。溶剂对照组(PBS组)2只小鼠(雌雄各1只)；实验组每组6只小鼠(雌雄各3只)，共有4组，分别给药Enfortumab vedotin、NECTIN 4-ADC-9-2、NECTIN 4-ADC-11、NECTIN4-ADC-22。
检测指标：
笼旁观察每天两次(包括观察死亡和濒死)，根据存活情况绘制生存曲线图；
详细临床观察：2次/周；
体重：给药前记录1次，给药后每周记录2次；记录时间为Day 0、Day 4、Day 7、Day 12和Day 14。
2.BALB/c小鼠长毒实验
选用维通利华6-8周的BALB/c小白鼠进行急毒实验，采用尾静脉注射给药，给药剂量为60mg/kg，给药体积为200μL/组，给药2次(day0,day7)，观察14天。溶剂对照组(PBS组)4只小鼠(雌雄各2只)；实验组每组6只小鼠(雌雄各3只)，共有3组，分别给药Enfortumab vedotin、NECTIN 4-ADC-9-2、NECTIN 4-ADC-11。
检测指标：
笼旁观察每天两次(包括观察死亡和濒死)，根据存活情况绘制生存曲线图；
详细临床观察：2次/周；
体重：给药前1次。给药后每周1次，及解剖前1次(本次实验2次/周)
急性毒性试验和长期毒性试验的结果如图15-18所示。
结论：本公开示例性的抗体药物偶联物NECTIN 4-ADC-9-2、NECTIN 4-ADC-11和NECTIN 4-ADC-22在观察期间存活率与载体组相当，具有良好的安全性，小鼠生存率等方面显著优于Enfortumab vedotin。
3.人源化小鼠毒理最大耐受量(MTD)实验
(一)实验分组
表28
(二)实验方法
给药途径及频率：尾静脉注射，每周1次，给药当天为Day 0。结果如图21、22所示。
结论：本专利公开示例性的抗体偶联药物NECTIN 4-ADC-11(的耐受性良好，其MTD＝可达80mg/kg)。与阳性对照Enfortumab vedotin(MTD＜70mg/kg)相比，具有更好的安全性。
4.Nectin4人源化小鼠毒理重复给药实验
实验分组
表29
(二)实验方法
给药途径：尾静脉注射，每周1次，给药当天为Day 1，分别于Day 1、8、15、22给药，共给药4次。
给药频率：多次给药，每周一次；
实验时长：4周。
每日毒性观察，包小鼠生存情况、体重变化、皮肤毒性等。
检测指标：
结果见图23、图24。
表30皮肤毒性发病率
结论：本申请示例性抗体偶联药物NECTIN 4-ADC-11的耐受性良好，且相较于阳性药Enfortumab vedotin具有更高的耐受剂量，同等剂量下体重降低程度低于阳性药，且可以在一定程度上降低Nectin4靶点皮肤毒性严重的副作用。
试验例10：药代动力学测试
1、BALB/c小鼠药代动力学
仪器和试剂：
表31
实验步骤：
通过小白鼠尾静脉分别单次注射给予NECTIN 4-ADC-11和对照后，观察其在小鼠体内的药代动力学基本特征。
a.分组、剂量以及给药体积如下表所示：
表32
b.给药次数及方式：单次尾静脉给药。
c.取血点：5min、1h、4h、8h、24h、48h、96h(Day4)、168h(Day7)、240h(Day10)、336h(Day14)、504h(Day21)、672h(Day28)
d.采血方式：每只动物首次取血采用眼眶静脉丛采血；采血体积：0.15mL/时间点。第二次采血使用摘眼球取血；采血体积：0.5mL/时间点。8种受试物，每种3只作为一个队列，一个队列24只老鼠。
e.血样处理和检测：血清采集完成后，静置，于1h小时血清明显分层后，4000rpm离心10min取上清。获得的血清样品分两份冻存于-80℃冰箱，试验结束后进行游离小分子的检测及ADC和总抗体的检测。
结果如图25、图26所示。
表33 BALB/c小鼠体内血浆ADC药代动力学结果

结论：实验结果显示，给药剂量为1mg/kg、2.5mg/kg、5mg/kg或10mg/kg时，在小鼠药代动力学中，两种药物、ADC及总抗体在体内的释放均具有剂量依赖性，且本专利本发明抗体偶联药物抗体药物偶联物NECTIN4-ADC-11具有更低的游离有效载荷(pPayload)脱落、较低的清除率和更长的半衰期。由此可见，本专利本发明抗体偶联药物抗体药物偶联物NECTIN 4-ADC-11具有更高的稳定性，并且有望降低体内循环中游离Ppayload的提前释放，降低脱靶毒性。。
2、HT1376荷瘤鼠药代动力学
(一)仪器和试剂
表34
(二)实验步骤
a.在BALB/c-Nude裸小鼠右肋部皮下接种人乳腺癌细胞HT1376(科佰)(1×107/200μL/只，具有50％的低生长因子人工基底膜)。接种细胞后，肿瘤生长17天，瘤体积长至250-300mm3左右后将动物随机分组(D0)，每组3只，共24组。(PBS,Enfortumab vedotin,NECTIN 4-ADC-11共3种药)
b.采用尾静脉注射给药，单剂量给药6mg/kg。
c.采血点设计：1h、6h、24h、48h、72h、168h、240h、336h。
d.摘眼球取血800μL，解剖取瘤，研磨，离心，取上清，Elisa检测TAB和ADC含量，LC-MS检测游离小分子含量。
结果见图27所示。
结论：本发明中的抗体偶联药物抗体药物偶联物NECTIN 4-ADC-11在肿瘤中有特异性蓄积，在血清中有效载荷暴露量低于阳性对照药Enfortumab vedotin，在体内具有更高的安全性。
本发明已经通过上述实施例进行了说明，但应当理解的是，上述实施例只是用于举例和说明的目的，而非意在将本发明限制于所描述的实施例范围内。此外本领域技术人员可以理解的是，本发明并不局限于上述实施例，根据本发明的教导还可以做出更多的变型和修改，这些变型和修改均落在本发明所要求保护的范围以内。本发明的保护范围由附属的权利要求书及其等效范围所界定。

Claims (36)

  1. 特异性结合Nectin-4的抗体或其抗原结合片段，其包含：
    SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、或51所示的重链可变区内所含的三个重链互补决定区(HCDR)HCDR1、HCDR2和HCDR3；和/或
    SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、或47所示的轻链可变区内所含的三个轻链互补决定区(LCDR)LCDR1、LCDR2和LCDR3。
  2. 特异性结合Nectin-4的抗体或其抗原结合片段，其包含：
    1)HCDR3，所述HCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:13、19、25、84、90、和96；和
    2)LCDR3，所述LCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:16、22、28、87、93、和99。
  3. 如权利要求2述的抗体或其抗原结合片段，其进一步包含：
    1)HCDR1，所述HCDR1包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:11、17、23、82、88、和94；和
    2)LCDR1，所述LCDR1包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:14、20、26、85、91、和97；
    优选地，所述抗体或其抗原结合片段还包含：
    3)HCDR2，所述HCDR2包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:12、18、24、83、89、和95；和
    4)LCDR2，所述LCDR2包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:15、21、27、86、92、和98。
  4. 如权利要求1-3任一项所述的抗体或其抗原结合片段，其包含：
    1)SEQ ID NO:3所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:4所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    2)SEQ ID NO:5所示的重链可变区所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:6所示的轻链可变区所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    3)SEQ ID NO:7所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:8所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    4)SEQ ID NO:100所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:101所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    5)SEQ ID NO:102所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:103所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    6)SEQ ID NO:104所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:105所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    7)SEQ ID NO:33所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:29所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    8)SEQ ID NO:34所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:30所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    9)SEQ ID NO:35所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:31所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    10)SEQ ID NO:36所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:32所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    11)SEQ ID NO:40所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:37所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    12)SEQ ID NO:41所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:38所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    13)SEQ ID NO:42所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:39所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    14)SEQ ID NO:43所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:37所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    15)SEQ ID NO:43所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:38所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    16)SEQ ID NO:43所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:39所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    17)SEQ ID NO:48所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:44所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    18)SEQ ID NO:49所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:45所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    19)SEQ ID NO:50所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:46所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    20)SEQ ID NO:51所示的重链可变区内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    SEQ ID NO:47所示的轻链可变区内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3。
  5. 如权利要求1-4中任一项所述的抗体或其抗原结合片段，其包含：
    1)包含SEQ ID NO:11所示序列或由其组成的HCDR1，
    包含SEQ ID NO:12所示序列或由其组成的HCDR2，
    包含SEQ ID NO:13所示序列或由其组成的HCDR3，
    包含SEQ ID NO:14所示序列或由其组成的LCDR1，
    包含SEQ ID NO:15所示序列或由其组成的LCDR2，和
    包含SEQ ID NO:16所示序列或由其组成的LCDR3；或
    2)包含SEQ ID NO:17所示序列或由其组成的HCDR1，
    包含SEQ ID NO:18所示序列或由其组成的HCDR2，
    包含SEQ ID NO:19所示序列或由其组成的HCDR3，
    包含SEQ ID NO:20所示序列或由其组成的LCDR1，
    包含SEQ ID NO:21所示序列或由其组成的LCDR2，和
    包含SEQ ID NO:22所示序列或由其组成的LCDR3；或
    3)包含SEQ ID NO:23所示序列或由其组成的HCDR1，
    包含SEQ ID NO:24所示序列或由其组成的HCDR2，
    包含SEQ ID NO:25所示序列或由其组成的HCDR3，
    包含SEQ ID NO:26所示序列或由其组成的LCDR1，
    包含SEQ ID NO:27所示序列或由其组成的LCDR2，和
    包含SEQ ID NO:28所示序列或由其组成的LCDR3；或
    4)包含SEQ ID NO:82所示序列或由其组成的HCDR1，
    包含SEQ ID NO:83所示序列或由其组成的HCDR2，
    包含SEQ ID NO:84所示序列或由其组成的HCDR3，
    包含SEQ ID NO:85所示序列或由其组成的LCDR1，
    包含SEQ ID NO:86所示序列或由其组成的LCDR2，和
    包含SEQ ID NO:87所示序列或由其组成的LCDR3；或
    5)包含SEQ ID NO:88所示序列或由其组成的HCDR1，
    包含SEQ ID NO:89所示序列或由其组成的HCDR2，
    包含SEQ ID NO:90所示序列或由其组成的HCDR3，
    包含SEQ ID NO:91所示序列或由其组成的LCDR1，
    包含SEQ ID NO:92所示序列或由其组成的LCDR2，和
    包含SEQ ID NO:93所示序列或由其组成的LCDR3；或
    6)包含SEQ ID NO:94所示序列或由其组成的HCDR1，
    包含SEQ ID NO:95所示序列或由其组成的HCDR2，
    包含SEQ ID NO:96所示序列或由其组成的HCDR3，
    包含SEQ ID NO:97所示序列或由其组成的LCDR1，
    包含SEQ ID NO:98所示序列或由其组成的LCDR2，和
    包含SEQ ID NO:99所示序列或由其组成的LCDR3。
  6. 特异性结合Nectin-4的抗体或其抗原结合片段，其包含：
    重链可变区，所述重链可变区的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％、至少85％、至少90％、或至少95％的序列同一性：SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、和51；优选地，所述重链可变区具有与SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、或51所示氨基酸序列相同的HCDR氨基酸序列、且所述重链可变区的框架区的氨基酸序列与氨基酸序列SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、或51的框架区有至少80％、至少85％、至少90％、或至少95％的序列同一性；优选地，所述重链可变区具有如SEQ ID NO:3、5、7、100、102、104、33、34、35、36、40、41、42、43、48、49、50、或51所示的序列；和/或
    轻链可变区，所述轻链可变区的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％、至少85％、至少90％、或至少95％的序列同一性：SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、和47；优选地，所述轻链可变区的氨基酸序列具有与氨基酸序列SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、或47相同的LCDR、且所述轻链可变区的框架区与序列SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、或47的框架区有至少80％、至少85％、至少90％、或至少95％的序列同一性；优选地，所述轻链可变区具有如SEQ ID NO:4、6、8、101、103、105、29、30、31、32、37、38、39、44、45、46、或47所示的序列。
  7. 如权利要求6所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区和轻链可变区的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：
    SEQ ID NO:3和SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8、SEQ ID NO:100和SEQ ID NO:101、SEQ ID NO:102和SEQ ID NO:103、SEQ ID NO:104和SEQ ID NO:105、SEQ ID NO:33和SEQ ID NO:29、SEQ ID NO:34和SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:32、SEQ ID NO:40和SEQ ID NO:37、SEQ ID NO:41和SEQ ID NO:38、SEQ ID NO:42和SEQ ID NO:39、SEQ ID NO:43和SEQ ID NO:37、SEQ ID NO:43和SEQ ID NO:38、SEQ ID NO:43和SEQ ID NO:39、SEQ ID NO:48和SEQ ID NO:44、SEQ ID NO:49和SEQ ID NO:45、SEQ ID NO:50和SEQ ID NO:46、和SEQ ID NO:51和SEQ ID NO:47。
  8. 如权利要求1-7任一项所述的抗体或其抗原结合片段，其包含重链恒定区或其变体和/或轻链恒定区或其变体，优选其包含鼠源的或人源化的重链恒定区和/或轻链恒定区；优选地，所述重链恒定区为IgG重链恒定区，和/或所述轻链恒定区为κ或λ轻链恒定区；优选地，所述重链恒定区为IgG1重链恒定区，和/或所述轻链恒定区为κ轻链恒定区；优选地，所述人源化的重链恒定区的氨基酸序列如SEQ ID NO:9所示，和/或所述人源化的轻链恒定区的氨基酸序列如SEQ ID NO:10所示。
  9. 如权利要求1-8任一项所述的抗体或其抗原结合片段，所述抗原结合片段选自Fab、Fab'、Fab'-SH、Fv、scFv和F(ab')2。
  10. 如权利要求1-9任一项所述的抗体或其抗原结合片段，所述抗体为单克隆抗体、嵌合抗体、人源化抗体、全人源抗体或双特异性抗体。
  11. 如权利要求1-10任一项所述的抗体或其抗原结合片段，其包含重链和/或轻链，其中，
    所述重链的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％、至少85％、至少90％、或至少95％的序列同一性：SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、和SEQ ID NO:79；优选，所述重链的氨基酸序列如SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、或SEQ ID NO:79所示；和/或
    所述轻链的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％、至少85％、至少90％、或至少95％的序列同一性：SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:107SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、和SEQ ID NO:75；优选，所述轻链的氨基酸序列如SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:107SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、或SEQ ID NO:75所示。
  12. 如权利要求1-11任一项所述的抗体或其抗原结合片段，其包含重链和轻链，所述重链和轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:52和SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57、SEQ ID NO:106和SEQ ID NO:107、SEQ ID NO:108和SEQ ID NO:109、SEQ ID NO:110和SEQ ID NO:111、SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:60和SEQ ID NO:59、SEQ ID NO:61和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:58、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:65和SEQ ID NO:64、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:64、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:64、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:64、SEQ ID NO:69和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:63、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:63、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:76和SEQ ID NO:75、SEQ ID NO:77和SEQ ID NO:72、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:75、SEQ ID NO:78和SEQ ID NO:72、SEQ ID NO:78和SEQ ID NO:73、SEQ ID NO:78和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、SEQ ID NO:79和SEQ ID NO:72、SEQ ID NO:79和SEQ ID NO:73、SEQ ID NO:79和SEQ ID NO:74、和SEQ ID NO:79和SEQ ID NO:75，优选地，其重链和轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、和SEQ ID NO:79和SEQ ID NO:72。
  13. 如权利要求1-12任一项所述的抗体或其抗原结合片段，其中所述HCDR和LCDR根据Kabat定义方案、Chothia定义方案、Abm定义方案、IMGT定义方案和/或Contact(接触)定义方案定义；优选，所述HCDR和LCDR根据Kabat定义方案或Chothia定义方案定义。
  14. 核酸，其编码如权利要求1-13中任一项所述的抗体或其抗原结合片段。
  15. 表达载体，其包含如权利要求14所述的核酸。
  16. 宿主细胞，其包含如权利要求15所述的表达载体。
  17. 生产抗体或其抗原结合片段的方法，其包含培养如权利要求16所述的宿主细胞，并从培养物中回收由此表达的抗体或其抗原结合片段。
  18. 如权利要求17所述的方法，其中所述宿主细胞为原核细胞或真核细胞，优选地，所述宿主细胞为大肠杆菌细胞、酵母细胞、昆虫细胞、植物细胞或哺乳动物细胞；优选，所述宿主细胞为中国仓鼠卵巢细胞(CHO)、CHO细胞变体、293细胞或NSO细胞。
  19. 配体药物偶联物或其药学上可接受的盐，其中，所述配体药物偶联物包含权利要求1-13中任一项所述的抗体或其抗原结合片段、以及效应分子部分，其中，所述效应分子部分偶联至所述抗体或其抗原结合片段；优选所述效应分子部分选自细胞毒性药物部分、治疗自身免疫疾病的药物部分和抗炎的药物部分。
  20. 配体药物偶联物或其药学上可接受的盐，所述配体药物偶联物如式1所示，
    其中，
    Ab为靶向Nectin-4相结合的抗体或抗原结合片段，优选地，Ab为权利要求1-13中任一项所述的抗体或其抗原结合片段、或为包含如SEQ ID NO:80所示的重链和如SEQ ID NO:81所示的轻链的抗体、或为包含如SEQ ID NO:112所示的重链和如SEQ ID NO:113所示的轻链的抗体；优选地，Ab为权利要求1-13中任一项所述的抗体或其抗原结合片段；
    m选自1-20的整数或小数；优选m选自1-12的整数或小数；
    L为化学连接结构；
    D为效应分子部分部分；优选D选自细胞毒性药物部分、治疗自身免疫疾病的药物部分和抗炎的药物部分；优选地，D为细胞毒性药物部分；优选地，D选自拓扑异构酶I(TOP1)抑制剂部分、微管蛋白聚合抑制剂部分、拓扑异构酶Ⅱ(TOP2)抑制剂部分、二氢叶酸还原酶抑制剂部分、DNA烷化剂部分、胸腺核苷合成酶抑制剂部分、嘌呤核苷合成酶抑制剂部分、核苷酸还原酶抑制剂部分、DNA多聚酶抑制剂部分、RNA聚合酶Ⅱ抑制剂部分和其他能够抑制细胞增殖的化合物部分；优选地，D选自 其中波浪线表示D与L的连接点。
  21. 如权利要求20所述的配体药物偶联物或其药学上可接受的盐，其中，
    L为其中Ta处的表示与所述抗体或其抗原结合片段的连接点，L5c处的表示与D的连接点；
    Ta选自 其中所述Ta结构中左侧的表示与所述抗体或其抗原结合片段的连接点，所述Ta结构中右侧的表示与Mc的连接点；
    Mc选自单键、6-10元亚芳基、5-13元亚杂芳基、3-15元亚环烷基和3-15元亚杂环烷基，所述亚芳基、亚杂芳基、亚环烷基和亚杂环烷基，未被取代或任选地被一个或多个Ra取代；
    L1c选自单键、亚炔基、亚烯基、-NRa-、-O-、-C(O)-、-NRa-C(O)-、亚烷基和亚杂烷基中的1个、2个、3个或更多个的组合，其中所述亚炔基、亚烯基、亚烷基、和亚杂烷基未被取代或任选地被一个或多个Ra取代；
    L2c选自单键、-(CH2)n7-(OCH2CH2)n8-O-(CH2)n9-Cy3-(CH2)n10-C(O)-、-(CH2)n7-(CH2CH2O)n8-(CH2)n9-Cy3-(CH2)n10-C(O)-、-(CH2)n7-Cy3-(CH2CH2O)n8-(CH2)n9-C(O)-、-(CH2)n11-(OCH2CH2)n12-O-(CH2)n13-Cy3-(CH2)n14-(OCH2CH2)n15-C(O)-、-(CH2)n7-(OCH2CH2)n8-(CH2)n10-C(O)-、 中的1个、2个、3个或更多个组合；
    n7、n9、n10、n11、n13和n14每次出现时各自独立地选自0、1、2、3、4、5和6；
    n8和n17每次出现时各自独立地选自1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27和28；
    n12和n15每次出现时各自独立地选自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14和15，且n12和n15不同时为0；
    j每次出现时各自独立地选自0、1、2、3、4、5和6；
    Cy3选自6-10元亚芳基、5-13元亚杂芳基、3-15元亚环烷基和3-15元亚杂环烷基，所述亚芳基、亚杂芳基、亚环烷基和亚杂环烷基未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；
    Mc、L1c、和L2c不同时为单键；
    L4c为由氨基酸构成的肽残基或-NRa-亚烷基-NRa-，其中所述亚烷基、和氨基酸未被取代或任选地进一步被一个或多个Ra取代；
    L5c选自单键、-N(Rb)-(CH2)g-O-(CH2)g-O-C(O)-、-N(Rb)-(CH2)g-O-(CH2-CH2-O)g-C(O)-、
    n每次出现时各自独立地选自1-30间的任一整数；优选地，n每次出现时各自独立地选自1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27和28；
    X1选自
    g每次出现时各自独立地选自1、2、3、4、5和6；和
    Ra和Rb每次出现时各自独立选自H、氘原子、F、Cl、Br、I、C1-C6烷基、C1-C6氘代烷基、C2-C6杂烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、羟基、硝基、氰基、氨基、羧基、C3-C6环烷基、3-6元杂环基、C1-C6烷基氨基、C1-C6烷基-C(O)-、C1-C6烷基-O-C(O)-、C1-C6烷基-NHC(O)-、-C1-6亚烷基-C(O)-NH2、C1-6烷基O-C(O)NH-、-C1-6亚烷基-NHC(O)-NH2、C1-C6烷基-NHC(O)-NH-、C6-C15芳基和5-13元杂芳基，其中所述的C1-C6烷基、C2-C6杂烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、C3-C6环烷基、3-6元杂环基、C1-C6烷基氨基、C6-C15芳基和5-13元杂芳基各自独立地任选被选自F、Cl、Br、I、C1-C6烷基、C2-C6杂烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、羟基、C1-C6卤代烷基、C1-C6羟烷基、氰基、氨基、硝基、C3-C6环烷基、3-6元杂环基、C1-C6烷基氨基、C6-C15芳基和5-13元杂芳基中的1个、2个、3个或多个取代基所取代。
  22. 根据权利要求21所述的配体-药物偶联物或其药学上可接受的盐，其中，
    Mc选自单键、亚苯基、5-7元亚杂芳基、3-7元亚环烷基和3-7元亚杂环烷基，所述亚芳基、亚杂芳基、亚环烷基和亚杂环烷基，未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；优选地，Mc选自单键、和/或
    L1c选自单键、C2-6亚炔基、C2-6亚烯基、-NRa-、-O-、-C(O)-、-NRa-C(O)-、C1-6亚烷基和C1-6亚杂烷基中1个、2个、3个或更多个的组合，其中所述亚炔基、亚烯基、亚烷基和亚杂烷基未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；优选地，L1c选自单键、C2-3亚炔基、C2-3亚烯基、-NRa-、-C(O)-、-NRa-C(O)-、C1-3亚烷基和C1-3亚杂烷基中1个、2个、3个或多个的组合，其中所述亚炔基、亚烯基、亚烷基和亚杂烷基未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；和/或
    Cy3选自 优选地，Cy3选自和/或
    L4c为由2、3、4、5、6或7个氨基酸构成的肽残基或-NRa-C1-6亚烷基-NRa-，其中所述氨基酸选自D-丙氨酸、L-丙氨酸、苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、亮氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸、精氨酸和天冬酰胺，所述亚烷基和氨基酸未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；优选地，L4c选自-NH-CH2CH2-NH-、 和/或
    L5c选自：单键、 和/或
    Ra和Rb每次出现时各自独立地选自H、氰基、氘原子、F、Cl、Br、I、羧基、羟基、氨基、C1-C3烷基氨基、C1-C3烷基和C1-C3烷氧基。
  23. 根据权利要求21或22所述的配体-药物偶联物或其药学上可接受的盐，其中，
    Mc选自单键、和/或
    L1c选自单键、C2-3亚炔基、C2-3亚烯基、-NRa-、-C(O)-、-NRa-C(O)-、C1-3亚烷基和C1-3亚杂烷基中1个、2个、3个或多个的组合，其中所述亚炔基、亚烯基、亚烷基和亚杂烷基未被取代或任选地被1个、2个、3个、4个、5个或6个Ra取代；和/或
    L2c选自单键、
    和其中，n17每次出现时各自独立地选自6、8、12和24；和/或
    L4c选自-NH-CH2CH2-NH-、 和/或
    L5c选自：单键、 和/或
    Ra和Rb每次出现时各自独立地选自H、氰基、氘原子、F、Cl、Br、I、羧基、羟基、氨基、C1-C3烷基氨基、C1-C3烷基和C1-C3烷氧基。
  24. 根据权利要求20-23任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，L选自下述结构：




    其中，n、n1、n8和n5每次出现时各自独立地选自1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27和28。
  25. 根据权利要求20-24中任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，
    m为1至10任意整数或小数；优选地，m为2至8任意整数或小数；优选地，m为2至6任意整数或小数；和/或
    D选自拓扑异构酶I(TOP1)抑制剂部分、微管蛋白聚合抑制剂部分、拓扑异构酶Ⅱ(TOP2)抑制剂部分、二氢叶酸还原酶抑制剂部分、DNA烷化剂部分、胸腺核苷合成酶抑制剂部分、嘌呤核苷合成酶抑制剂部分、核苷酸还原酶抑制剂部分、DNA多聚酶抑制剂部分、RNA聚合酶Ⅱ抑制剂部分和其他能够抑制细胞增殖的化合物部分；优选地，D选自 优选地，D为其中波浪线表示D与L的连接点。
  26. 如权利要求20-25任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，所述配体-药物偶联物选自下述结构：













    其中，Ab为权利要求1-13中任一项所述的抗体或其抗原结合片段、或包含如SEQ ID NO:80所示的重链和如SEQ ID NO:81所示的轻链的抗体、或包含如SEQ ID NO:112所示的重链和如SEQ ID NO:113所示的轻链的抗体；
    m和m1每次出现时各自独立地选自1-20的整数或小数；优选地，m和m1每次出现时各自独立地选自1-12的整数或小数；优选地，m和m1每次出现时各自独立地选自1至10的整数或小数；优选地，m和m1每次出现时各自独立地选自2至8整数或小数；优选地，m和m1每次出现时各自独立地选自2-6的整数或小数；优选地m和m1每次出现时各自独立地选自1、2、3、4、5、6、7、8、9和10；和n1、n8和n5每次出现时各自独立地选自1、2、3、4、5、6、7、和8。
  27. 根据权利要求20-26任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，
    Ab为权利要求1-13中任一项所述的抗体或其抗原结合片段；优选地，Ab为包含重链和轻链的抗体，所述抗体中的重链和轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:52和SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57、SEQ ID NO:106和SEQ ID NO:107、SEQ ID NO:108和SEQ ID NO:109、SEQ ID NO:110和SEQ ID NO:111、SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:60和SEQ ID NO:59、SEQ ID NO:61和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:58、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:65和SEQ ID NO:64、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:64、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:64、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:64、SEQ ID NO:69和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:63、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:63、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:76和SEQ ID NO:75、SEQ ID NO:77和SEQ ID NO:72、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:75、SEQ ID NO:78和SEQ ID NO:72、SEQ ID NO:78和SEQ ID NO:73、SEQ ID NO:78和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、SEQ ID NO:79和SEQ ID NO:72、SEQ ID NO:79和SEQ ID NO:73、SEQ ID NO:79和SEQ ID NO:74、和SEQ ID NO:79和SEQ ID NO:75；优选，所述抗体中的重链和轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、和SEQ ID NO:79和SEQ ID NO:72；和/或
    m和m1每次出现时各自独立地选自1-20的整数或小数；优选地，m和m1每次出现时各自独立地选自1-12的整数或小数；优选地，m和m1每次出现时各自独立地选自1至10的整数或小数；优选地，m和m1每次出现时各自独立地选自2至8整数或小数；优选地，m和m1每次出现时各自独立地选自2-6的整数或小数；优选地m和m1每次出现时各自独立地选自1、2、3、4、5、6、7、8、9和10。
  28. 抗体或其抗原结合片段，其包含：
    氨基酸序列为SEQ ID NO:52、54、或56的肽段内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3；和/或
    氨基酸序列为SEQ ID NO:53、55、或57的肽段内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3。
  29. 抗体或其抗原结合片段，其包含：
    1)HCDR3，所述HCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:13、19、和25；和
    2)LCDR3，所述LCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:16、22、和28；
    优选地，所述的抗体或其抗原结合片段，其包含：
    1)HCDR1，所述HCDR1包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:11、17、23；和
    2)LCDR1，所述LCDR1包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:14、20、26；
    优选地，所述抗体或其抗原结合片段还包含：
    3)HCDR2，所述HCDR2包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:12、18、24；和
    4)LCDR2，所述LCDR2包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:15、21、27。
  30. 如权利要求28-29任一项所述的抗体或其抗原结合片段，其包含：
    1)氨基酸序列为SEQ ID NO:52所示肽段内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    氨基酸序列为SEQ ID NO:53所示肽段内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    2)氨基酸序列为SEQ ID NO:54所示肽段内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    氨基酸序列为SEQ ID NO:55所示肽段内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；或
    3)氨基酸序列为SEQ ID NO:56所示肽段内所含的三个重链互补决定区HCDR1、HCDR2与HCDR3，和
    氨基酸序列为SEQ ID NO:57所示肽段内所含的三个轻链互补决定区LCDR1、LCDR2与LCDR3；
    优选地，所述的抗体或其抗原结合片段，其包含：
    1)包含SEQ ID NO:11或由其组成的HCDR1，
    包含SEQ ID NO:12或由其组成的HCDR2，
    包含SEQ ID NO:13或由其组成的HCDR3，
    包含SEQ ID NO:14或由其组成的LCDR1，
    包含SEQ ID NO:15或由其组成的LCDR2，和
    包含SEQ ID NO:16或由其组成的LCDR3；或
    2)包含SEQ ID NO:17或由其组成的HCDR1，
    包含SEQ ID NO:18或由其组成的HCDR2，
    包含SEQ ID NO:19或由其组成的HCDR3，
    包含SEQ ID NO:20或由其组成的LCDR1，
    包含SEQ ID NO:21或由其组成的LCDR2，和
    包含SEQ ID NO:22或由其组成的LCDR3；或
    3)包含SEQ ID NO:23所示或由其组成的HCDR1，
    包含SEQ ID NO:24或由其组成的HCDR2，
    包含SEQ ID NO:25或由其组成的HCDR3，
    包含SEQ ID NO:26或由其组成的LCDR1，
    包含SEQ ID NO:27或由其组成的LCDR2，和
    包含SEQ ID NO:28或由其组成的LCDR3。
  31. 抗体或其抗原结合片段，其包含：
    重链可变区，所述重链可变区的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％同一性：SEQ ID NO:33、34、35、36、40、41、42、43、48、49、50、51；优选地，所述重链可变区具有与SEQ ID NO:33、34、35、36、40、41、42、43、48、49、50、51氨基酸序列相同的CDRs区氨基酸序列、且所述重链可变区的框架区的氨基酸序列与氨基酸序列SEQ ID NO:33、34、35、36、40、41、42、43、48、49、50、51的框架区有至少80％同一性的序列；优选地，所述重链可变区具有选自由下述氨基酸序列所构成的组中的任一氨基酸序列：SEQ ID NO:33、34、35、36、40、41、42、43、48、49、50、51；和/或
    轻链可变区，所述轻链可变区的氨基酸序列与选自由下述氨基酸序列所构成的组中的任一氨基酸序列有至少80％同一性：SEQ ID NO:29、30、31、32、37、38、39、44、45、46、47；优选地，所述轻链可变区的氨基酸序列具有与氨基酸序列SEQ ID NO:29、30、31、32、37、38、39、44、45、46、47相同的CDRs区、且所述轻链可变区的框架区与序列SEQ ID NO:29、30、31、32、37、38、39、44、45、46、47的框架区有至少80％同一性；优选地，所述轻链可变区具有选自由下述氨基酸序列所构成的组中的任一氨基酸序列：SEQ ID NO:29、30、31、32、37、38、39、44、45、46、47；
    优选地，所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区/轻链可变区的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：
    SEQ ID NO:33和SEQ ID NO:29、SEQ ID NO:34和SEQ ID NO:30、SEQ ID NO:35和SEQ ID NO:31、SEQ ID NO:36和SEQ ID NO:32、SEQ ID NO:40和SEQ ID NO:37、SEQ ID NO:41和SEQ ID NO:38、SEQ ID NO:42和SEQ ID NO:39、SEQ ID NO:43和SEQ ID NO:37、SEQ ID NO:43和SEQ ID NO:38、SEQ ID NO:43和SEQ ID NO:39、SEQ ID NO:48和SEQ ID NO:44、SEQ ID NO:49和SEQ ID NO:45、SEQ ID NO:50和SEQ ID NO:46、SEQ ID NO:51和SEQ ID NO:47。
  32. 如权利要求28-31任一项所述的抗体或其抗原结合片段，其包含重链恒定区和/或轻链恒定区，优选其包含鼠源的或人源化的重链恒定区和/或轻链恒定区；优选地，所述人源化的重链恒定区的氨基酸序列如SEQ ID NO:9所示，所述人源化的轻链恒定区的氨基酸序列如SEQ ID NO:10所示；优选地所述的抗体或其抗原结合片段，其重链/轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:60和SEQ ID NO:59、SEQ ID NO:61和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:58、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:65和SEQ ID NO:64、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:64、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:64、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:64、SEQ ID NO:69和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:63、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:63、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:76和SEQ ID NO:75、SEQ ID NO:77和SEQ ID NO:72、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:75、SEQ ID NO:78和SEQ ID NO:72、SEQ ID NO:78和SEQ ID NO:73、SEQ ID NO:78和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、SEQ ID NO:79和SEQ ID NO:72、SEQ ID NO:79和SEQ ID NO:73、SEQ ID NO:79和SEQ ID NO:74、SEQ ID NO:79和SEQ ID NO:75，优选地，其重链/轻链的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:60和SEQ ID NO:58、SEQ ID NO:61和SEQ ID NO:59、SEQ ID NO:62和SEQ ID NO:59、SEQ ID NO:65和SEQ ID NO:63、SEQ ID NO:66和SEQ ID NO:63、SEQ ID NO:67和SEQ ID NO:63、SEQ ID NO:68和SEQ ID NO:63、SEQ ID NO:69和SEQ ID NO:64、SEQ ID NO:70和SEQ ID NO:64、SEQ ID NO:71和SEQ ID NO:64、SEQ ID NO:76和SEQ ID NO:72、SEQ ID NO:76和SEQ ID NO:73、SEQ ID NO:76和SEQ ID NO:74、SEQ ID NO:77和SEQ ID NO:73、SEQ ID NO:77和SEQ ID NO:74、SEQ ID NO:78和SEQ ID NO:75、SEQ ID NO:79和SEQ ID NO:72。
  33. 药物组合物，其包含权利要求1-13或28-32中任一项所述的抗体或其抗原结合片段或权利要求19-27中任意一项所述的配体-药物偶联物或其药学上可接受的盐、和可药用赋形剂。
  34. 权利要求1-13或28-32中任一项所述的抗体或其抗原结合片段、权利要求19-27中任意一项所述的配体-药物偶联物或其药学上可接受的盐、或权利要求28所述的药物组合物在制备用于治疗和/或预防Nectin-4介导的疾病或病症的药物中的用途；优选，所述Nectin-4介导的疾病或病症为乳腺癌、膀胱癌或肺癌；优选地，所述乳腺癌为三阴性乳腺癌。
  35. 权利要求1-13或28-32中任一项所述的抗体或其抗原结合片段、权利要求19-27中任意一项所述的配体-药物偶联物或其药学上可接受的盐、或权利要求28所述的药物组合物，其用于治疗和/或预防Nectin-4介导的疾病或病症；优选，所述Nectin-4介导的疾病或病症为乳腺癌、膀胱癌或肺癌；优选地，所述乳腺癌为三阴性乳腺癌。
  36. 治疗和/或预防Nectin-4介导的疾病或病症的方法，包括对有需要的受试者施用治疗有效量的权利要求1-13或28-32中任一项所述的抗体或其抗原结合片段、权利要求19-27中任意一项所述的配体-药物偶联物或其药学上可接受的盐、或权利要求28所述的药物组合物；优选，所述Nectin-4介导的疾病或病症为乳腺癌、膀胱癌或肺癌；优选地，所述乳腺癌为三阴性乳腺癌。
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