WO2022228563A1 - 靶向Nectin-4的抗体药物偶联物及其制备方法和用途 - Google Patents

靶向Nectin-4的抗体药物偶联物及其制备方法和用途 Download PDF

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WO2022228563A1
WO2022228563A1 PCT/CN2022/090450 CN2022090450W WO2022228563A1 WO 2022228563 A1 WO2022228563 A1 WO 2022228563A1 CN 2022090450 W CN2022090450 W CN 2022090450W WO 2022228563 A1 WO2022228563 A1 WO 2022228563A1
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antibody
seq
nectin
cdr
fragment
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PCT/CN2022/090450
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English (en)
French (fr)
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周伟
谭小钉
刘大涛
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江苏迈威康新药研发有限公司
迈威(上海)生物科技股份有限公司
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Application filed by 江苏迈威康新药研发有限公司, 迈威(上海)生物科技股份有限公司 filed Critical 江苏迈威康新药研发有限公司
Priority to KR1020237041036A priority Critical patent/KR20240004708A/ko
Priority to CA3217112A priority patent/CA3217112A1/en
Priority to BR112023022549A priority patent/BR112023022549A2/pt
Priority to JP2023566911A priority patent/JP2024517776A/ja
Priority to AU2022267613A priority patent/AU2022267613A1/en
Priority to EP22795032.6A priority patent/EP4331615A1/en
Publication of WO2022228563A1 publication Critical patent/WO2022228563A1/zh

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Definitions

  • the present invention relates to antibody-drug conjugates, in particular, the present invention relates to antibody-drug conjugates targeting poliovirus receptor-like molecule 4 (Nectin-4) and preparation methods and uses thereof.
  • poliovirus receptor-like molecule 4 Nectin-4
  • Nectins are a novel class of cell adhesion proteins that together or alone regulate cell junctions with cadherins, family members include Nectin-1, -2, -3, -4, all of which It consists of a ring structure with 3 Ig, a transmembrane region and a cytoplasmic tail region.
  • Nectin-4 is specifically expressed in embryos, placenta and tumor cells, and it has been found that it is closely related to the occurrence and development of various tumor cells. For example, pathological section analysis of 2394 tumor patients found that Nectin-4 is widely expressed in bladder cancer, breast cancer, and pancreatic cancer patient populations. Therefore, Nectin-4 has become an important target for the diagnosis and treatment of many tumors or cancers.
  • Antibody-drug conjugate is a kind of anti-tumor drugs (such as cytotoxic agents, cytostatic agents, small molecule chemotherapeutics) that use the specific recognition ability of antibodies to specific antigens on the surface of tumor cells. etc.) are delivered to tumor target cells, causing intracellular accumulation and release, and then the technology of precise tumor killing.
  • Antibody-drug conjugates generally consist of three parts: antibodies or antibody-like ligands, small molecule drugs, and linkers (linkers) that couple the antibodies or antibody-like ligands with the drugs.
  • Antibody-drug conjugates have been considered to be the most potential anti-tumor drugs due to their suitable molecular weight, high stability, strong targeting, and low toxicity and side effects.
  • the antibody needs to specifically recognize the lesion site, has low immunosensitization, and can undergo efficient and rapid endocytosis; the antibody-drug linker has high stability in blood and needs to be specific in the targeted cells. It is activated and efficiently releases small molecule drugs, otherwise it will cause unacceptable levels of toxicity to normal cells; the coupled small molecule drugs need to have strong cell killing ability, etc.
  • highly active cytotoxic small-molecule drugs are usually linked to lysine residues on the antibody surface or cysteine residues in the hinge region through linkers.
  • the drug/ligand ratio (DAR) was 2-4.
  • T-DM1 average DAR of 3.5
  • DM1 microtubule inhibitor maytansine
  • reducing agents DTT, TCEP, etc.
  • DTT interchain disulfide bonds
  • TCEP TCEP
  • the resulting conjugate is not a homogeneous product, but is composed of various components.
  • the DAR of the main component is 0, 2, 4, 6, 8, and corresponding to each specific DAR component there are isomers formed due to different attachment sites.
  • the heterogeneity of antibody drug conjugate products can lead to the heterogeneity of pharmacokinetic properties, potency and toxicity among the individual components. For example, components with higher DARs are cleared faster in the body and lead to higher toxicity.
  • Enfortumab Vedotin is often associated with fever, pruritus, peripheral neuropathy, dry eye, and neutropenia. These adverse reactions are directly related to the excessive linking of small molecules to antibodies and the unstable linking mode.
  • the technical problem to be solved by the present invention is to obtain a high-affinity antibody that specifically binds to human Nectin-4 through hybridoma screening and humanization technology, wherein the antibody is designed to minimize the number of murine amino acids through humanization design , in order to have better in vivo safety and application prospects; on this basis, further screen antibodies with stronger endocytosis effect, and prepare them with small molecule chemical drugs into antibody-drug conjugates (antibody-drug conjugates) , using the targeting of the antibody to Nectin-4 expressing cells and the strong endocytosis effect through the target, combined with the action of the small molecule chemical drug, to achieve excellent tumor killing effect.
  • an object of the present invention is to provide antibodies or fragments thereof that specifically bind to Nectin-4; wherein, the fragments of the antibody encompass various functional fragments of the antibody, such as antigen-binding portions thereof, such as Fab, F(ab' )2 or scFv fragments.
  • Another object of the present invention is to provide an antibody-drug conjugate or a salt thereof targeting Nectin-4 prepared by using the antibody or its fragment.
  • the present invention provides an antibody-drug conjugate targeting Nectin-4 or a salt thereof, comprising an anti-Nectin-4 antibody or a fragment thereof covalently linked to the drug.
  • the anti-Nectin-4 antibody or fragment thereof comprises a heavy chain and a light chain
  • the heavy chain and the light chain respectively comprise the following heavy chain complementarity determination Regions 1 to 3 (CDR-H1, CDR-H2 and CDR-H3) and Light Chain Complementarity Determining Regions 1 to 3 (CDR-L1, CDR-L2 and CDR-L3):
  • the antibody drug conjugate or salt thereof provided by the present invention has the molecular formula Ab-[L-CTD]m, wherein Ab represents the anti-Nectin-4 antibody or fragment thereof, L represents the linker, CTD represents the drug, and m Indicates the average number of drug linkages relative to each molecule of Ab.
  • the CTD is a cytotoxic drug; preferably, the CTD is one or more selected from the group consisting of microtubule inhibitors MMAE, DM1, DM4, Tublysin, amantine, calicheamicin, eribulin and derivatives of said drugs; topoisomerase inhibitors SN38, ixitecan and derivatives of said drugs; and DNA binding agent PBD , Doxorubicin and derivatives of said drugs.
  • m is 1.0-5.0, preferably 3.0-4.2, more preferably 3.5-4.5, still more preferably 3.8-4.2, still more preferably 3.9-4.1, particularly preferably 4.0.
  • the heavy chain and light chain of the anti-Nectin-4 antibody or its fragment respectively comprise a heavy chain variable region (VH) and a light chain variable region region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or a variant thereof,
  • the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8 or a variant thereof.
  • the heavy chain variable region (VH) and light chain variable region (VL) of the anti-Nectin-4 antibody or its fragment respectively comprise :
  • a "variant of an amino acid sequence” means at least 75% sequence identity to said amino acid sequence (eg at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity, etc. any percent identity > 75%).
  • the anti-Nectin-4 antibody or fragment thereof of the present invention comprises at least a heavy chain variable region and a light chain variable region, both of which include the above-mentioned CDRs and spaced framework regions (FRs), and the The arrangement is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the "at least 75% sequence identity" results in at most 25% difference in amino acid sequence with respect to the heavy chain variable region and the light chain variable region contained in the anti-Nectin-4 antibody or fragment thereof of the present invention Can be present in any framework region in either the heavy chain variable region or the light chain variable region.
  • the up to 25% difference may exist in any antibody or fragment of the invention other than the heavy chain variable region and the light chain variable region domain or sequence.
  • the differences can result from amino acid deletions, additions or substitutions at any position, where the substitutions can be conservative or non-conservative.
  • the anti-Nectin-4 antibody or its fragment can be a monoclonal antibody, single-chain antibody, diabody, single-domain antibody, nano-body against Nectin-4 Any form of antibody, fully or partially humanized antibody or chimeric antibody, or the antibody or fragment thereof is a half-antibody against Nectin-4 or an antigen-binding fragment of a half-antibody, such as a single-chain variable fragment (single-chain variable fragment).
  • the fragment is any fragment of the antibody that can specifically bind to Nectin-4.
  • the Nectin-4 is mammalian Nectin-4, preferably primate Nectin-4, more preferably human Nectin-4.
  • the anti-Nectin-4 antibody or fragment thereof may further comprise a constant region.
  • the anti-Nectin-4 antibody or fragment thereof further comprises human or murine heavy chain constant region (CH) and/or light chain constant region (CL), more preferably selected from the group consisting of IgG, IgA, IgM, Heavy chain constant region of IgD or IgE and/or light chain constant region of kappa or lambda type.
  • the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain The constant region is of the kappa type.
  • the antibody is an immunoglobulin, in particular IgA, IgD, IgE, IgG or IgM, such as a human subtype of IgA, IgD, IgE, IgG or IgM, more preferably a human IgG1, IgG2, IgG3 or IgG4 subtype type.
  • the anti-Nectin-4 antibody or fragment thereof comprises a heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof.
  • the anti-Nectin-4 antibody or fragment thereof comprises a light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 10 or a variant thereof.
  • a "variant of an amino acid sequence" refers to an amino acid sequence having at least 75% sequence identity to said amino acid sequence.
  • antibody drug conjugate or its salt provided by the present invention has the structure shown in the following formula Ia and/or Ib:
  • Ab is the anti-Nectin-4 antibody or fragment thereof
  • Ar' is any one selected from the group consisting of a substituted or unsubstituted C6-C10 arylene group and a substituted or unsubstituted 5-12 membered heteroarylene group, wherein the substitution means that the hydrogen atom on the group is replaced by one or substituted by a plurality of substituents selected from any one of the following: halogen (F, Cl, Br or I), halogenated alkyl (such as halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, such as trifluoromethyl) and alkoxy (such as C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy);
  • halogen F, Cl, Br or I
  • alkyl such as halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, such as trifluoromethyl
  • alkoxy such as C1-C6 alkoxy, preferably
  • L 1 is -O(CH2CH2O)n- connected to the Ar' group, wherein n is selected from any integer from 1-24, preferably any integer from 1-10, more preferably any integer from 3-5;
  • L 2 is an enzyme cleavage fragment, such as a dipeptide or a tripeptide or a tetrapeptide or its combination with a self-releasing structural fragment (that is, a polypeptide fragment consisting of 2-4 amino acids or a combination with a self-releasing structural fragment), such as Val- Ala, Val-Ala-PAB, Val-Cit, Val-Cit-PAB, Phe-Lys-PAB, Ala-Ala-Ala, Gly-Gly-Phe-Gly(GGFG), MAC glucuronide phenol.
  • a self-releasing structural fragment that is, a polypeptide fragment consisting of 2-4 amino acids or a combination with a self-releasing structural fragment
  • L2 - CTD is VcMMAE, GGFG-Dxd or VC-seco-DUBA.
  • the heteroatom is N.
  • Ar' is a substituted or unsubstituted C6 arylene group or a substituted or unsubstituted 6-membered heteroarylene group.
  • the antibody drug conjugate or salt thereof provided by the present invention has the following structure:
  • the present invention provides a kind of preparation method of the antibody-drug conjugate of targeting Nectin-4 or its salt, and described antibody-drug conjugate or its salt has the structure shown in formula Ia and Ib:
  • the method includes the following steps:
  • step (2) Cross-linking the drug linker (linker-drug conjugate) with the reduced antibody or its fragment obtained in step (1) in a mixture of buffer and organic solvent to obtain targeting Nectin-4 of antibody-drug conjugates.
  • the Nectin-4 antibody or its fragment is as defined above;
  • the drug linker has the structure shown in the following formula Ic:
  • R is X or R'S, wherein X is halogen (F, Cl, Br or I), preferably Br or I; R' is substituted or unsubstituted C6-C10 aryl or substituted or unsubstituted 5-12-membered heterocyclic Aryl, wherein the substitution means that the hydrogen atoms on the group are replaced by one or more substituents selected from any of the following: alkyl (eg C1-C6 alkyl, preferably C1-C4 alkyl group), alkoxy (such as C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy), halogen (F, Cl, Br or I), ester, amide and cyano;
  • alkyl eg C1-C6 alkyl, preferably C1-C4 alkyl group
  • alkoxy such as C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy
  • halogen F, Cl,
  • R is R'S, wherein R' is phenyl or substituted phenyl, and in substituted phenyl, the substituent is selected from alkyl (eg C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (eg C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), halogen (F, Cl, Br or I), ester, amide and cyano; preferably, R' is Phenyl, 4-methylformyl substituted phenyl or 4-formylmorpholine substituted phenyl
  • Ar', L 1 , L 2 and CTD are as defined above.
  • drug linker is any one selected from the following:
  • the preparation method comprises the following steps:
  • Antibody reduction adding a reducing agent with a molar equivalent of ⁇ 5.5 times the antibody to the phosphate buffer containing the antibody at a concentration of 5-30 mg/mL, and reacting for 1.5-2 h, wherein the reducing agent is selected from TCEP, DTT, 2 - one or more of MEA and DTBA;
  • Antibody coupling replace the reduced antibody in step a into phosphate buffer at pH 6.5-7.8, and dilute the antibody to a concentration of 3.5-15 mg/mL, dissolve 4.5-6.5 in an organic co-solvent
  • the drug-containing linker of times the antibody molar equivalent is added to the antibody diluent, and the reaction is stirred at 15-35 ° C for ⁇ 0.5 h, wherein the organic co-solvent is one selected from DMA, DMSO, DMF and ACN or variety;
  • Hydrophobic chromatography The antibody conjugated product is purified by hydrophobic chromatography using a hydrophobic filler.
  • the preparation method further comprises the following steps after step b or after step c:
  • the displacements in steps a, b and d are carried out using molecular sieves, ultrafiltration centrifuge tubes or ultrafiltration membrane packs.
  • the hydrophobic filler that can be used in step c is Butyl Sepharose HP, Capto Phenyl Impres or Butyl Sepharose FF.
  • the present invention provides an anti-Nectin-4 antibody or a fragment thereof.
  • the above descriptions and definitions for the anti-Nectin-4 antibody or fragment thereof contained in the antibody-drug conjugate targeting Nectin-4 or its salt are applicable to the anti-Nectin-4 antibody or fragment thereof in this aspect .
  • the anti-Nectin-4 antibodies or fragments thereof provided by the present invention comprise heavy chains and light chains, which respectively comprise heavy chain complementarity determining regions 1 to 3 (CDR-H1, CDR-H2 and CDR-H3) and light chain complementarity determining regions 1 to 3 (CDR-L1, CDR-L2 and CDR-L3):
  • amino acid sequences are respectively CDR-H1, CDR-H2 and CDR-H3 shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 13; and, the amino acid sequences are respectively as shown in SEQ ID NO: 14, SEQ ID NO: 13 CDR-L1, CDR-L2 and CDR-L3 shown in NO: 15 and SEQ ID NO: 16.
  • the heavy chain and light chain of the anti-Nectin-4 antibody or fragment thereof comprise a heavy chain variable region (VH) and a light chain variable region (VL), respectively, wherein the heavy chain variable region (VH ) comprising the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7 or a variant thereof, the light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 8 or its variants.
  • VH heavy chain variable region
  • VL light chain variable region
  • the heavy chain variable region (VH) and light chain variable region (VL) of the anti-Nectin-4 antibody or fragment thereof respectively comprise:
  • the anti-Nectin-4 antibody or fragment thereof also has each of the features defined above in the present application with respect to the antibody component in the antibody drug conjugate.
  • the present invention provides a nucleic acid molecule comprising the light chain variable region, the heavy chain variable region, the heavy chain variable region, the heavy chain variable region, and the light chain variable region contained in the anti-Nectin-4 antibody or its fragment encoding the present invention or the nucleotide sequence of the light chain.
  • the nucleic acid molecules of the present invention can be cloned into vectors, which in turn transform or transfect host cells. Accordingly, in yet another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention.
  • the vectors can be eukaryotic expression vectors, prokaryotic expression vectors, artificial chromosomes, phage vectors, and the like.
  • the vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells or enter into host cells in any way, for purposes such as preservation or expression of antibodies. Accordingly, in yet another aspect, the present invention provides a host cell comprising, or transformed or transfected with, the nucleic acid molecule and/or vector of the present invention.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
  • the anti-Nectin-4 antibody or fragment thereof provided by the present invention can be obtained by any method known in the art.
  • the heavy chain variable region and/or light chain variable region of the antibody can be obtained first from the nucleic acid molecule provided by the present invention, or the heavy chain and/or light chain of the antibody molecule can be obtained, and then combined with the antibody molecule
  • the optional other domains of the antibody are assembled into antibodies; or, in the host cell provided by the invention, the variable region of the heavy chain and/or the variable region of the light chain of the antibody molecule or the variable region of the heavy chain and/or the heavy chain of the antibody molecule are expressed
  • the host cell is cultured with the light chain to assemble into the antibody.
  • the method further comprises the step of recovering the antibody molecule produced.
  • the antibody drug conjugate targeting Nectin-4 or its salt, the anti-Nectin-4 antibody or its fragment, nucleic acid molecule, vector or host cell provided by the present invention can be included in the composition, more particularly in the In pharmaceutical preparations, it can be used for various purposes according to actual needs. Therefore, in another aspect, the present invention also provides a composition comprising the antibody drug conjugate targeting Nectin-4 of the present invention or a salt thereof, an antibody against Nectin-4 or Fragments, nucleic acid molecules, vectors and/or host cells thereof.
  • the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.
  • the present invention also provides the antibody drug conjugates targeting Nectin-4 or their salts, anti-Nectin-4 antibodies or fragments thereof, nucleic acid molecules, vectors, host cells and/or compositions for preparation Use in medicines for the treatment of tumors.
  • the present invention also provides a method for treating tumors, the method comprising administering the Nectin-4-targeting antibody-drug conjugate or a salt thereof, an anti-nectin-4 antibody provided by the present invention to a subject in need thereof Antibodies to Nectin-4 or fragments thereof, nucleic acid molecules, vectors, host cells and/or compositions.
  • the subject is a mammal, preferably a primate, more preferably a human.
  • the tumor is a tumor or cancer associated with high expression of Nectin-4; preferably, the tumor or cancer is any one selected from the group consisting of bladder cancer, breast cancer, ovarian cancer, pancreatic cancer, hepatocyte Cancer, Gastric Cancer, Non-Hodgkin Lymphoma, Hodgkin Lymphoma, Acute Lymphocytic Leukemia, Anaplastic Large Cell Lymphoma, Multiple Myeloma, Prostate Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, Malignant Melanoma , squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, prostate cancer, colorectal cancer, glioma, mesothelioma.
  • bladder cancer breast cancer, ovarian cancer, pancreatic cancer, hepatocyte Cancer, Gastric Cancer, Non-Hodgkin Lymphoma, Hodgkin Lymphoma, Acute Lymphocytic Leukemia, Anaplastic Large Cell Lymphoma,
  • Figure 1 shows the characterization results of ADC 3d, wherein 1A: HIC pattern; 1B: SEC pattern; 1C: NR-CE-SDS pattern; 1D: LCMS pattern.
  • Figure 2 shows the in vivo efficacy results of different ADCs.
  • Figure 3 shows the in vivo efficacy results of different ADCs.
  • Figure 4 shows the in vivo efficacy results of different ADCs.
  • Figure 5 shows the in vivo efficacy results of different ADCs.
  • Figure 6 shows the in vivo efficacy results of different ADCs.
  • the recombinant protein human Nectin-4 (purchased from Inshore Protein, Cat: CJ19) was used to immunize mice, and then the B cells of the immunized mice were fused with SP20 myeloma cells prepared in advance, and the ability to bind human Nectin-4 antigen was screened. positive hybridoma cells, and finally obtain a positive hybridoma cell line that secretes only one antibody.
  • the heavy chain variable region sequence of the murine anti-human Nectin-4 monoclonal antibody and the heavy chain constant region sequence (SEQ ID NO: 9) of the published human monoclonal antibody IgG1 subclass were spliced together, and constructed into mammalian In the cell expression vector; the light chain variable region sequence of the murine anti-human Nectin-4 monoclonal antibody and the light chain constant region sequence of the published human monoclonal antibody ⁇ subclass (SEQ ID NO: 10) are spliced together , constructed into mammalian cell expression vectors.
  • the heavy chain vector and light chain vector of the constructed anti-human Nectin-4 chimeric antibody were paired and mixed, and PEI was used to transfect HEK293 cells. After about 7 days, the cell supernatant was collected and purified using Mabselect to obtain the anti-human Nectin-4 chimeric antibody.
  • the amino acid sequence regions of the six antigenic complementarity determinants (CDRs) of the heavy and light chains of the murine antibody and the framework regions supporting the conservative three-dimensional conformation of the antibody were determined. Then search for the known human antibody sequence through analysis, select the human antibody heavy chain variable region sequence that is most similar to the mouse antibody, such as IGHV1
  • the chain CDRs are combined with the human antibody framework regions to ultimately generate humanized antibody heavy chain variable region sequences.
  • a humanized antibody light chain variable region sequence was generated.
  • the individual amino acids of the framework region are changed from human source to murine source by back mutation, and/or the existing post-translational modification sites and other individual amino acids are modified according to needs, and finally an alternative humanized version is obtained.
  • Heavy chain variable region and light chain variable region are changed from human source to murine source by back mutation, and/or the existing post-translational modification sites and other individual amino acids are modified according to needs, and finally an alternative humanized version is obtained.
  • Heavy chain variable region and light chain variable region are changed from human source to murine source by back mutation, and/or the existing post-translational modification sites and other individual amino acids are modified according to needs, and finally an alternative humanized version is obtained.
  • humanized heavy chain variable regions and light chain variable regions are combined in pairs, referring to the preparation of chimeric antibodies, to obtain humanized antibodies.
  • target cancer cell endocytosis experiments target cancer cell proliferation inhibition experiments for human Nectin-4, and ADCs made with known drug-containing linkers to screen for antigen binding, endocytosis activity or pharmacodynamics, the following results were screened.
  • Humanized antibody, CDRs are underlined:
  • VH (SEQ ID NO: 1; CDRs are: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, according to the CHOTHIA definition method)
  • VL (SEQ ID NO: 2; CDRs are: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, according to the CHOTHIA definition method)
  • VH (SEQ ID NO: 3; CDRs are: SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 13, according to the CHOTHIA definition method)
  • VL (SEQ ID NO: 4; CDRs are: SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 16, according to the CHOTHIA definition method)
  • VH (SEQ ID NO:5; CDRs are: SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:13, according to the KABAT definition method)
  • VL (SEQ ID NO:6; CDRs are: SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, according to the KABAT definition method)
  • VH (SEQ ID NO:7; CDRs are: SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:13, according to the KABAT definition method)
  • VL (SEQ ID NO: 8; CDRs are: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, according to the KABAT definition method)
  • the drug-containing linker MC-VC-MMAE was purchased from MCE.
  • the antibody sample was replaced with a Sephadex G-25-supported NAP-25 desalting column to pH 7.4 containing 50 mM sodium chloride, 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, and the antibody was mixed. The concentration was diluted to 10 mg/ml. 10 ml of an antibody sample of 100 mg in total was taken, and an aqueous solution of 10 mg/ml of TCEP (Sigma-Aldrich) was added at an antibody-reducing agent molar ratio of 1:10 equivalents.
  • TCEP Sigma-Aldrich
  • reaction solution was replaced with a Sephadex G25 desalting column into a buffer solution containing 50 mM sodium chloride, 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate at pH 7.0.
  • the above-mentioned reduced antibody was diluted to 5 mg/mL, and 1.33 ml of N,N-dimethylacetamide (DMA) with a total reaction volume of 7.4% was added in turn as a pre-solvent, and the molar ratio of antibody-small molecule drug was 1 to 5.5 times.
  • An equivalent solution of DMA-drug linker mixture containing 10 mg/mL drug linker was added. Stir at room temperature for 60 minutes.
  • the NAP-25 desalting column of Sephadex G-25 carrier replaces the reaction solution with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH 8.0, removes the excess drug linker, and heats it in a water bath at 37°C for 3 hours.
  • Sample purification The above samples were concentrated using AMICOM ultrafiltration centrifuge tubes, and the samples were concentrated to about 15 mg/mL. Add 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 3M ammonium phosphate buffer solution to a conductivity of 74 ms/cm. Loaded on a Butyl-Sepharose 4 FF packing (purchased from GE Healthcare) hydrophobic column, using phase A is 50mM disodium hydrogen phosphate-sodium dihydrogen phosphate+0.45M ammonium sulfate; phase B is 50mM disodium hydrogen phosphate-dihydrogen phosphate sodium buffer solution. Phase B 0-80% linear gradient 12 times the column volume, B phase 100% isocratic gradient elution, the main peak was collected.
  • the above-mentioned Sephadex G-25 carrier desalting step and the AMICOM ultrafiltration centrifuge tube are replaced with HYDRO-30kD (Sartorius) ultrafiltration membrane packs under the condition that the reaction equivalence ratio remains unchanged. Other main parameters remain unchanged.
  • the concentration of the antibody drug conjugate can be obtained by measuring the UV absorbance at 280 nm and the characteristic absorption wavelength of small molecules, and then performing the following calculation.
  • DAR is known in, is the molar absorption coefficient of the antibody at 280 nm
  • a 280 is the UV absorption value of the antibody-drug conjugate at 280 nm
  • a 248 is the UV absorption value of the antibody-drug conjugate at the characteristic absorption wavelength of the drug-containing joint at 248 nm
  • DAR is known in, is the molar absorption coefficient of the antibody at 280 nm
  • a 280 is the UV absorption value of the antibody-drug conjugate at 280 nm
  • a 251 is the UV absorption value of the antibody-drug conjugate at the characteristic absorption wavelength of the drug-containing joint at 251 nm
  • BL20E is the product of compound C-3 above after removing the leaving group R and opening the maleimide ring.
  • the concentration of the antibody-drug conjugate has the following relationship:
  • the concentration of antibody drug conjugate (g/L) is obtained Substitute the DAR value into the formula to obtain the protein concentration.
  • MW ADC is the molecular weight of the antibody-drug conjugate
  • MW ab is the molecular weight of the antibody
  • MW D is the molecular weight of the drug-containing linker
  • Sample preparation The sample was diluted to 2.0 mg/ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for HPLC analysis;
  • Sample preparation The sample was diluted to 2.0 mg/ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for HPLC analysis;
  • Sample processing Take an appropriate amount of sample into an ultrafiltration tube, replace with 50mM NH4HCO3 replacement buffer (pH7.1), add buffer, ultrafiltration centrifuge (13000g*5min). Add 8 ⁇ l of PNGase F enzyme to the replaced samples, incubate at 37°C for 5 h for deglycosysis treatment, after incubation, centrifuge at 12000 rpm for 5 min, take the supernatant into the injection vial as the test sample for use.
  • Mobile phase 50mM ammonium acetate, pH 7.0;
  • Injection volume 2 ⁇ L
  • Sheath gas temperature 325°C
  • Sample processing After the sample was diluted to about 1.0 mg/ml with mobile phase, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken for injection and analysis.
  • anti-Nectin-4 antibody Ref prepared according to the sequence of Enfortumab in WHO Drug Information
  • 42D20-hz63, 42D20-hz10, hH2L1, hH1L2mut1, IgG1 Bio-Rad Antibodies, product No. MCA928, 100 mg each, respectively, coupled with drug-containing linkers C-1 and C-3 to obtain site-directed conjugated ADC drugs 1a, 1b, 1c, 1d, 1e, 1f and 2a, 2b, 2c, 2d, 2e.
  • sample name Measured molecular weight system content 3d-before hydrolysis 155015 The system is a mixture of Ia and Ib 3d-After hydrolysis 155034
  • the system is Ib single substance
  • the molecular weight measured by mass spectrometry shows that the sample is a mixture of structures represented by formula Ia and Ib before hydrolysis, and the maleimide is completely ring-opened after hydrolysis to obtain a single substance represented by formula Ib.
  • Example group 3 In vitro killing effect and in vivo efficacy of antibody drug conjugates
  • BT474 cells Human breast ductal carcinoma cells BT474 cells (purchased from ATCC) were used in this experiment. The density of BT474 cells was adjusted to 15 ⁇ 10 4 with complete medium (RPMI 1640 medium 45ml, 5ml FBS, used after mixing), 100 ⁇ l/well was added to the cell culture plate, and cultured overnight. The next day, the ADC samples were diluted with the above-mentioned complete medium to 50ug/ml, respectively, followed by 4-fold gradient dilution, a total of 9 gradients were added with zero point, and 3 replicate wells were set for all samples.
  • complete medium RPMI 1640 medium 45ml, 5ml FBS, used after mixing
  • mice (BALB/cJGpt-Foxn1nu/Gpt, 4-5 weeks) were inoculated with human breast ductal carcinoma cells BT474 cells (purchased from ATCC) and human breast cancer cells MDA-MB-468 (purchased from ATCC), respectively. mold. After the tumor grows to 100-200 mm 3 , the mice were administered intravenously (IV) with a dose of 10 mL/kg; the solvent group was given the same volume of solvent (physiological saline); the specific dosage and dosing schedule are as follows . The tumor volume was measured twice a week, the mice were weighed, and the data were recorded.
  • IV intravenously
  • the experimental index is to investigate the effect of drugs on tumor growth, and the specific index is T/C% or tumor inhibition rate TGI (%).
  • the tumor diameter was measured twice a week with a vernier caliper, and the tumor volume (V) was calculated as:
  • V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width, respectively.
  • T/C(%) (TT 0 )/(CC 0 ) ⁇ 100, where T and C are the tumor volumes at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • TGI Tumor inhibition rate
  • TGI tumor inhibition rate
  • Partial tumor regression was defined if the tumor was smaller than the initial volume, that is, T ⁇ T 0 or C ⁇ C 0 ; complete tumor regression (CR) was defined if the tumor disappeared completely.
  • BT474 was used for modeling, and grouping comparison was conducted on the conjugated ADC products of different drug-containing linkers and different anti-Nectin-4 antibodies.
  • MDA-MB-468 was used for modeling, and grouping comparison was conducted on the conjugated ADC products of different drug-containing linkers and different anti-Nectin-4 antibodies.
  • Example group 4 Evaluation of the efficacy of antibody drug conjugates 3d on candidate drugs in different tumor models
  • Nude mice (BALB/cJGpt) were treated with human breast cancer cells MDA-MB-468 (purchased from ATCC), lung cancer cells NCI-H322 (purchased from ATCC), and bladder cancer cells T24/nectin-4 (Cell Bank of Chinese Academy of Sciences). - Foxn1nu/Gpt, 4-5 weeks) for inoculation modeling.
  • a group comparison study was performed using the antibody drug conjugate 3d and the control drug PADCVE (Enfortumab Vedotin; purchased from Seattle Gentics) (as shown in Table 9). Mice were administered intravenously (IV) respectively, and the administration volume was 10 mL/kg; the solvent group was administered the same volume of solvent (physiological saline); the specific administration dosage and administration schedule are shown in Table 9. The tumor volume was measured twice a week, the mice were weighed, and the data were recorded.
  • PADCVE Enfortumab Vedotin
  • Example 1 In vivo efficacy study of Nectin-4-targeting antibody drug conjugates in an animal model of breast cancer
  • MDA-MB-468 was used for modeling according to the methods of groups 4.1 and 4.2 of this example, and group comparison studies were performed on 3d and PADCVE. The results are shown in Figure 4 and Table 10.
  • Example 2 In vivo efficacy study of targeting Nectin-4 antibody-drug conjugates in an animal model of lung cancer
  • Modeling was performed using NCI-H322 according to the methods of Groups 4.1 and 4.2 of this example, and a group comparison study was performed on 3d and PADCVE. The results are shown in Figure 5 and Table 11.
  • Example 1 Affinity analysis of the Fab functional domain of the antibody drug conjugate targeting Nectin-4
  • Human Nectin4 protein was diluted to 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, OnM with corresponding running reagents.
  • the diluted human Nectin4 protein was injected into the experimental channel and the reference channel at a flow rate of 30 ⁇ L/min, binding for 120 s and dissociation for 300 s.
  • KD values for each antibody were calculated using Biocore 8K analysis software.
  • the reference channel is used for background subtraction.
  • FcRn human FcyR I (CD64), human FcyR IIa (human CD32a (H167)), human FcyR IIb (human CD32b) and human FcyR were determined using BIAcore IIIa (human CD16a(V176)).
  • the reference channel (FC1) does not require ligand capture.
  • the sample to be tested was diluted with the corresponding running reagent, and the diluted sample was sequentially injected into the experimental channel and the reference channel at a flow rate of 30 ⁇ L/min, and the binding and dissociation were carried out for corresponding time. KD values for each antibody were calculated using Biocore 8K analysis software.
  • the reference channel (FC1) is used for background subtraction.
  • Table 14 shows the affinity test results of hH2L1, 3d and Padcev with Fc receptors. The results showed that there was no significant difference in the affinity of hH2L1 and four batches of 3d to Fc receptors; the overall affinity of 3d and one batch of Padcev to Fc ⁇ R IIIa was slightly worse than that of hH2L1, and there was no significant difference in the affinities of other receptors among the three.
  • the stably transfected cell line PC-3-Nectin4(2-4) (self-made) cells with high expression of Nectin4 were used as target cells to detect the drug endocytosis of HH2L1, 3D and PADCEV.
  • Experimental method take the target cells grown to log phase, collect the cells by centrifugation, adjust the density to 5 ⁇ 10 5 cells/ml with complete medium (F-12K medium containing 10% FBS and 250 ⁇ g/ml G418), respectively. Pipette 1 ml of the cell suspension into an EP tube, ie 5 x 10 5 cells, then centrifuge, discard the supernatant, and wash twice with PBS. Take 200 ⁇ l of each of hH2L1, 3d and PADCEV samples and add them to the cells, and then put them at 4 °C (as the control group) and 37 °C (as the experimental group), respectively.
  • endocytosis percentage at tx time point (fluorescence value of sample at 1-37°C/fluorescence value of sample at 4°C ) ⁇ 100% (tx represents the x time that the primary antibody was incubated with the cells)

Abstract

一种靶向脊髓灰质炎病毒受体样分子4(Nectin-4)的抗体药物偶联物，该抗体药物偶联物可用于制备治疗与Nectin-4有关的疾病的药物。所述抗体药物偶联物具有对Nectin-4的强靶向性和经由该靶点的强内吞效应，具有优异的肿瘤杀伤效果。

Description

靶向Nectin-4的抗体药物偶联物及其制备方法和用途
相关申请的交叉引用
本专利申请要求于2021年4月30日提交的申请号为CN202110481199.4的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明涉及抗体药物偶联物，具体而言，本发明涉及靶向脊髓灰质炎病毒受体样分子4(Nectin-4)的抗体药物偶联物及其制备方法和用途。
背景技术
Nectins(脊髓灰质炎病毒受体样分子)是一类新颖的细胞粘附蛋白，与钙粘蛋白共同或单独调节细胞连接，家族成员包括Nectin-1、-2、-3、-4，它们都由具有3个Ig的环状结构、1个跨膜区、胞质尾区组成。在这些成员中，Nectin-4特异性表达于胚胎、胎盘以及肿瘤细胞中，已有研究发现其与多种肿瘤细胞的发生、发展有着密切联系。例如，对2394例肿瘤患者进行病理切片分析，发现Nectin-4广泛表达于膀胱癌、乳腺癌以及胰腺癌患者人群中。因此，Nectin-4已成为许多肿瘤或癌症的诊断和治疗的一个重要靶点。
抗体药物偶联物(Antibody-drug conjugate；ADC)是一种利用抗体对肿瘤细胞表面特定抗原的特异性识别能力，精准地将抗肿瘤药物(如细胞毒性剂、细胞抑制剂、小分子化疗物等)递送到肿瘤靶细胞，使之发生胞内积蓄并释放，进而精准杀伤肿瘤的技术。抗体药物偶联物一般由三部分组成：抗体或抗体类配体，小分子药物，将抗体或抗体类配体与药物偶联起来的连接子(接头)。由于分子量大小合适，稳定性高，靶向性强，毒副作用小，抗体药物偶联物已被认为是最具潜力的抗肿瘤药物。
但是，成功开发ADC也存在诸多必须考虑且解决的问题。例如，抗体需特异性地识别病变部位，免疫致敏性低，能够高效迅速地发生细胞内吞作用；抗体-药物接头，在血液中稳定性要高并需在所靶向的细胞中特异地被激活并高效释放小分子药物，否则会对正常细胞产生不可接受水平的毒性；所偶联的小分子药物需具有强细胞杀伤能力等。在目前进入临床试验的抗体药物偶联物中，高活性的细胞毒性小分子药物通常是通过连接子连接在抗体表面的赖氨酸残基或者铰链区域的半胱氨酸残基处，最佳药物/配体比值(DAR)为2-4。但是，抗体表面大量的赖氨酸残基(超过80个)以及偶联反应的非选择性，导致偶联数目和位点不确定，进而导致生成的抗体药物偶联物不均一。例如，由 抗HER2靶向药物曲妥珠单抗与微管抑制剂美坦新(DM1)制成的抗体药物偶联物T-DM1(平均DAR为3.5)，其DAR分布为0-8。同样，虽然抗体铰链区的链间二硫键只有四对，但为了达到最佳平均DAR(2-4)的要求，需要对链间二硫键进行部分还原。但是，现有还原剂(DTT、TCEP等)无法选择性地还原链间二硫键，因此生成的偶联物也不是均一的产物，而是由多种组分组成，主要组分的DAR为0、2、4、6、8，而且对应每一种特定DAR的组分都存在由于连接位点不同而形成的异构体。抗体药物偶联物产品的不均一性可导致各成员组分之间药物动力学性质、效价以及毒性的不均一性。例如，具有较高DAR的组分在体内被清除得更快，并导致更高的毒性。
目前，西雅图基因公司与安斯泰来合作，利用该公司特有的连接子mc-vc-MMAE与anti-Nectin-4抗体enfortumab进行随机偶联，得到一种anti-Nectin-4抗体药物偶联物Enfortumab Vedotin(Padcev)。临床试验结果显示，在接受化疗以及PD-1/PD-L1抑制剂治疗的患者中，接受Enfortumab Vedotin的患者的中位总生存期为12.9个月，比化疗对照组延长了3.9个月，显示了良好的肿瘤治疗效果。然而，临床研究同时发现，使用Enfortumab Vedotin往往伴随发热、皮肤瘙痒、周围神经病、干眼症、中性粒细胞下降。这些不良反应与小分子与抗体连接过量、连接方式不稳定直接相关。
因此，本领域迫切需要提供高效、简单、实用的化学偶联方法用于靶向Nectin-4的抗体-药物偶联物研究与开发。
发明内容
本发明要解决的技术问题是，通过杂交瘤筛选和人源化技术，获得特异性结合人Nectin-4的高亲和力抗体，其中通过人源化设计，使该抗体最大程度减少鼠源氨基酸的数目，以拥有更好的体内安全性和应用前景；在此基础上，进一步筛选具有更强细胞内吞效应的抗体，将其与小分子化学药物制备成抗体药物偶联物(antibody-drug conjugate)，利用该抗体对Nectin-4表达细胞的靶向性和经由靶点的强的内吞效应，结合该小分子化学药物的作用，以达到优异的肿瘤杀伤效果。
因此，本发明的一个目的是提供特异性结合Nectin-4的抗体或其片段；其中，所述抗体的片段涵盖抗体的各种功能性片段，例如其抗原结合部分，如Fab、F(ab’)2或scFv片段。本发明的另一个目的是提供采用该抗体或其片段制备的靶向Nectin-4的抗体药物偶联物或其盐。
本发明的技术方案如下。
一方面，本发明提供一种靶向Nectin-4的抗体药物偶联物或其盐，其包含与药物共 价连接的抗Nectin-4的抗体或其片段。
在本发明提供的抗体药物偶联物或其盐中，所述抗Nectin-4的抗体或其片段包含重链和轻链，所述重链和轻链分别包含如下所示的重链互补决定区1至3(CDR-H1、CDR-H2和CDR-H3)和轻链互补决定区1至3(CDR-L1、CDR-L2和CDR-L3)：
(i)氨基酸序列分别如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的CDR-L1、CDR-L2和CDR-L3；
(ii)氨基酸序列分别如SEQ ID NO:11、SEQ ID NO:17和SEQ ID NO:13所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:15和SEQ ID NO:16所示的CDR-L1、CDR-L2和CDR-L3；
(iii)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:13所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的CDR-L1、CDR-L2和CDR-L3。
优选地，本发明提供的抗体药物偶联物或其盐具有分子式Ab-[L-CTD]m，其中Ab表示所述抗Nectin-4的抗体或其片段，L表示接头，CTD表示药物，m表示相对于每一分子Ab的药物平均连接数。
优选地，在本发明提供的抗体药物偶联物或其盐中，CTD为细胞毒药物；优选地，CTD为选自以下的一种或多种：微管抑制剂类MMAE、DM1、DM4、Tublysin、鹅膏蕈碱、卡奇霉素、艾日布林及所述药物的衍生物；拓扑异构酶抑制剂类SN38、依喜替康及所述药物的衍生物；以及DNA结合剂PBD、多柔比星及所述药物的衍生物。
m为1.0-5.0，优选为3.0-4.2，更优选为3.5-4.5，进一步优选为3.8-4.2，又优选为3.9-4.1，特别优选为4.0。
优选地，在本发明提供的抗体药物偶联物或其盐中，所述抗Nectin-4的抗体或其片段的重链和轻链分别包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:7所示的氨基酸序列或其变体，所述轻链可变区(VL)包含SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6或SEQ ID NO:8所示的氨基酸序列或其变体。
更优选地，在本发明提供的抗体药物偶联物或其盐中，所述抗Nectin-4的抗体或其片段的重链可变区(VH)和轻链可变区(VL)分别包含：
(i)如SEQ ID NO:1所示的氨基酸序列或其变体；和，如SEQ ID NO:2所示的氨基酸序列或其变体；
(ii)如SEQ ID NO:3所示的氨基酸序列或其变体；和，如SEQ ID NO:4所示的氨基酸序列或其变体；
(iii)如SEQ ID NO:5所示的氨基酸序列或其变体；和，如SEQ ID NO:6所示的氨基酸序列或其变体；或
(iv)如SEQ ID NO:7所示的氨基酸序列或其变体；和，如SEQ ID NO:8所示的氨基酸序列或其变体。
本发明的上下文中，“氨基酸序列的变体”是指与所述氨基酸序列具有至少75％序列同一性(例如至少80％、优选至少85％、更优选至少90％、进一步优选至少91％、92％、93％、94％、95％、96％、97％、98％或甚至99％同一性等≥75％的任何百分比的同一性)的氨基酸序列。
特别地，本发明抗Nectin-4的抗体或其片段至少包含重链可变区和轻链可变区，二者均包括上述CDR以及间隔的框架区(framework region，FR)，各个结构域的排列方式为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。因此，就本发明抗Nectin-4的抗体或其片段所包含的重链可变区和轻链可变区而言，所述“至少75％序列同一性”导致的氨基酸序列的至多25％差异可存在于重链可变区或轻链可变区中的任意框架区中。或者，就本发明抗Nectin-4的抗体或其片段的整体而言，所述至多25％差异可存在于本发明的抗体或其片段中重链可变区和轻链可变区以外的任意结构域或序列中。所述差异可以由任何位置的氨基酸缺失、添加或置换产生，其中置换可以是保守置换或非保守置换。
在本发明提供的抗体药物偶联物或其盐中，所述抗Nectin-4的抗体或其片段可以为针对Nectin-4的单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式，或者，所述抗体或其片段为针对Nectin-4的半抗体或半抗体的抗原结合片段，例如单链可变片段(single-chain variable fragment,scFv)、二价单链可变片段(bivalent single-chain variable fragment,BsFv)、二硫键稳定的可变片段(disulfide-stabilized variable fragment,dsFv)、(二硫键稳定的可变片段) 2((dsFv) 2)、抗原结合片段(antigen-binding fragment,Fab)、Fab'片段(Fab')、(Fab'片段) 2(F(ab') 2)或可变片段(variable fragment,Fv)。关于本发明提供的抗体的片段，优选地，所述片段为抗体的能够特异性结合Nectin-4的任何片段。并且，所述Nectin-4为哺乳动物Nectin-4，优选灵长类动物Nectin-4，更优选人Nectin-4。
优选地，在本发明提供的抗体药物偶联物或其盐中，所述抗Nectin-4的抗体或其片段还可以包含恒定区。优选地，所述抗Nectin-4的抗体或其片段还包含人或鼠的重链恒定区(CH)和/或轻链恒定区(CL)，更优选地包含选自IgG、IgA、IgM、IgD或IgE的 重链恒定区和/或κ或λ型轻链恒定区。
优选地，所述抗体为单克隆抗体，优选为鼠源、嵌合或人源化的单克隆抗体；更优选地，所述单克隆抗体的重链恒定区为IgG1或IgG4亚型，轻链恒定区为κ型。或者，例如，所述抗体为免疫球蛋白，具体为IgA、IgD、IgE、IgG或IgM，例如IgA、IgD、IgE、IgG或IgM的人亚型，更优选为人IgG1、IgG2、IgG3或IgG4亚型。
根据本发明的具体实施方式，所述抗Nectin-4的抗体或其片段包含重链恒定区，所述重链恒定区包含如SEQ ID NO:9所示的氨基酸序列或其变体。或者，所述抗Nectin-4的抗体或其片段包含轻链恒定区，所述轻链恒定区包含如SEQ ID NO:10所示的氨基酸序列或其变体。如上文所限定，“氨基酸序列的变体”是指与所述氨基酸序列具有至少75％序列同一性的氨基酸序列。
进一步地，本发明提供的抗体药物偶联物或其盐具有如下式Ia和/或Ib所示的结构：
Figure PCTCN2022090450-appb-000001
和/或
Figure PCTCN2022090450-appb-000002
其中：
Ab是所述抗Nectin-4的抗体或其片段；
Ar’为选自如下的任一个：取代或未取代的C6-C10亚芳基和取代或未取代的5-12元亚杂芳基，其中所述取代指基团上的氢原子被一个或多个取代基所取代，所述取代基选自如下的任一个：卤素(F、Cl、Br或I)、卤代烷基(例如卤代C1-C6烷基、优选卤代C1-C4烷基，例如三氟甲基)和烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，例如甲氧基)；
L 1为连接于Ar’基团上的-O(CH2CH2O)n-，其中n选自1-24中任一整数，优选为1-10、 更优选3-5中的任意整数；
L 2为酶切片段，如二肽或三肽或四肽或其与自释放结构片段的组合(即2-4个氨基酸组成的多肽片段或其与自释放结构片段的组合)，如Val-Ala、Val-Ala-PAB、Val-Cit、Val-Cit-PAB、Phe-Lys-PAB、Ala-Ala-Ala、Gly-Gly-Phe-Gly(GGFG)、MAC glucuronide phenol。
优选地，L 2-CTD为VcMMAE、GGFG-Dxd或VC-seco-DUBA。
优选地，在Ar’取代或未取代的5-12元亚杂芳基时，杂原子为N。
优选地，Ar’为取代或未取代的C6亚芳基或取代或未取代的6元亚杂芳基。
根据本发明的具体实施方式，本发明提供的抗体药物偶联物或其盐具有如下结构：
偶联物ADC-1：
Figure PCTCN2022090450-appb-000003
偶联物ADC-2：
Figure PCTCN2022090450-appb-000004
偶联物ADC-3：
Figure PCTCN2022090450-appb-000005
偶联物ADC-4：
Figure PCTCN2022090450-appb-000006
偶联物ADC-5：
Figure PCTCN2022090450-appb-000007
偶联物ADC-6：
Figure PCTCN2022090450-appb-000008
偶联物ADC-7：
Figure PCTCN2022090450-appb-000009
另一方面，本发明提供一种靶向Nectin-4的抗体药物偶联物或其盐的制备方法，所 述抗体药物偶联物或其盐具有式Ia和Ib所示的结构：
Figure PCTCN2022090450-appb-000010
和/或
Figure PCTCN2022090450-appb-000011
所述方法包括以下步骤：
(1)使抗Nectin-4的抗体或其片段与还原试剂在缓冲液中反应，得到经还原后的抗体或其片段；
(2)使药物接头(连接子-药物缀合物)与步骤(1)得到的经还原后的抗体或其片段在缓冲液与有机溶剂的混合液中进行交联，得到靶向Nectin-4的抗体药物偶联物。
在本发明提供的制备方法中，所述Nectin-4的抗体或其片段为如上文所限定；所述药物接头具有如下式Ic所示的结构：
Figure PCTCN2022090450-appb-000012
其中：
R为X或R’S，其中X为卤素(F、Cl、Br或I)，优选为Br或I；R’为取代或未取代的C6-C10芳基或者取代或未取代的5-12元杂芳基，其中所述取代指基团上的氢原子被一个或多个取代基所取代，所述取代基选自如下的任一个：烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，例如甲氧基)、卤素(F、Cl、Br或I)、酯基、酰胺基和氰基；
优选地，R为R’S，其中R’为苯基或取代的苯基，在取代的苯基中，取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧 基，优选甲氧基)、卤素(F、Cl、Br或I)、酯基、酰胺基和氰基；优选地，R’为苯基、4-甲基甲酰基取代苯基
Figure PCTCN2022090450-appb-000013
或4-甲酰基吗啉取代苯基
Figure PCTCN2022090450-appb-000014
Ar’、L 1、L 2和CTD如上文所限定。
在本发明的上下文中，“药物接头”、“[L-D]”、“含药接头”和“连接子-药物缀合物”等可互换使用。根据本发明的具体实施方式，所述药物接头为选自如下的任一个：
Figure PCTCN2022090450-appb-000015
Figure PCTCN2022090450-appb-000016
Figure PCTCN2022090450-appb-000017
Figure PCTCN2022090450-appb-000018
Figure PCTCN2022090450-appb-000019
优选地，所述制备方法包括以下步骤：
a.抗体还原：将≥5.5倍抗体摩尔当量的还原剂加入到含5-30mg/mL浓度抗体的磷酸盐缓冲液中，反应1.5-2h，其中所述还原剂为选自TCEP、DTT、2-MEA和DTBA中的一种或多种；
b.抗体偶联：将步骤a中经还原的抗体置换到pH 6.5-7.8的磷酸盐缓冲液中，并将抗体稀释到3.5-15mg/mL浓度，将溶解于有机共溶剂中的4.5-6.5倍抗体摩尔当量的含药接头加入到所述抗体稀释液中，在15-35℃下搅拌反应≥0.5h，其中所述有机共溶剂为选自DMA、DMSO、DMF和ACN中的一种或多种；
c.疏水层析：使用疏水填料对抗体偶联产物进行疏水层析纯化。
进一步优选地，所述制备方法在步骤b之后或步骤c之后还包括以下步骤：
d.水解：将抗体偶联产物置换到pH 7.4-9.0的磷酸盐缓冲液中，在35±10℃下加热2-24h，得到水解产物。
优选地，步骤a、b和d中的置换采用分子筛、超滤离心管或超滤膜包进行。优选地，步骤c中可采用的疏水填料为Butyl Sepharose HP、Capto Phenyl Impres或Butyl Sepharose FF。
又一方面，本发明提供一种抗Nectin-4的抗体或其片段。上文就靶向Nectin-4的抗体药物偶联物或其盐中包含的抗Nectin-4的抗体或其片段进行的描述与定义均适用于这一方面的抗Nectin-4的抗体或其片段。关于具体序列，本发明提供的抗Nectin-4的抗体或其片段包含重链和轻链，所述重链和轻链分别包含如下所示的重链互补决定区1至3(CDR-H1、CDR-H2和CDR-H3)和轻链互补决定区1至3(CDR-L1、CDR-L2和CDR-L3)：
氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:13所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的CDR-L1、CDR-L2和CDR-L3。
优选地，所述抗Nectin-4的抗体或其片段的重链和轻链分别包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)包含SEQ ID NO:5或SEQ ID NO:7所示的氨基酸序列或其变体，所述轻链可变区(VL)包含SEQ ID NO:6或SEQ ID NO:8所示的氨基酸序列或其变体。
更优选地，所述抗Nectin-4的抗体或其片段的重链可变区(VH)和轻链可变区(VL)分别包含：
(A)如SEQ ID NO:5所示的氨基酸序列或其变体；和，如SEQ ID NO:6所示的氨基酸序列或其变体；或
(B)如SEQ ID NO:7所示的氨基酸序列或其变体；和，如SEQ ID NO:8所示的氨基酸序列或其变体。
进一步地，这一方面，所述抗Nectin-4的抗体或其片段还具有本申请上文中关于抗体药物偶联物中的抗体成分所限定的各个特征。
根据本发明提供的上述抗体，本发明提供一种核酸分子，其包含编码本发明所述的抗Nectin-4的抗体或其片段中包含的轻链可变区、重链可变区、重链或轻链的核苷酸序列。
本发明的核酸分子可以被克隆到载体中，进而转化或转染宿主细胞。因此，还一方面，本发明提供一种载体，其包含本发明的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。
本发明的载体或核酸分子可以用于转化或转染宿主细胞或者以任何方式进入宿主细胞内，用于保存或表达抗体等目的。因此，又一方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明的核酸分子和/或载体，或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
本发明提供的抗Nectin-4的抗体或其片段可以利用本领域已知的任何方法获得。例如，可以先由本发明提供的核酸分子获得所述抗体的重链可变区和/或轻链可变区，或者获得所述抗体分子的重链和/或轻链，然后与所述抗体分子的任选其他结构域组装成抗体；或者，在允许本发明提供的宿主细胞表达所述抗体分子的重链可变区和/或轻链可变区或者所述抗体分子的重链和/或轻链以组装成所述抗体的情况下，培养所述宿主细胞。任选地，所述方法还包括回收产生的抗体分子的步骤。
本发明提供的靶向Nectin-4的抗体药物偶联物或其盐、抗Nectin-4的抗体或其片段、核酸分子、载体或宿主细胞可以被包含在组合物中，更特别地被包含在药物制剂中，从而根据实际需要用于各种目的。因此，在又一方面，本发明还提供一种组合物，所述组合物包含本发明提供的本发明的靶向Nectin-4的抗体药物偶联物或其盐、抗Nectin-4的抗体或其片段、核酸分子、载体和/或宿主细胞。优选地，所述组合物为药物组合物，其可选地还包含药学上可接受的载体、辅料或赋形剂。
再一方面，本发明还提供所述靶向Nectin-4的抗体药物偶联物或其盐、抗Nectin-4的抗体或其片段、核酸分子、载体、宿主细胞和/或组合物在制备用于治疗肿瘤的药物中的用途。或者，本发明还提供一种用于治疗肿瘤的方法，所述方法包括向有此需要的受试者施用本发明提供的所述靶向Nectin-4的抗体药物偶联物或其盐、抗Nectin-4的抗体或其片段、核酸分子、载体、宿主细胞和/或组合物。其中，所述受试者为哺乳类动物，优选地为灵长类动物，更优选为人。
优选地，所述肿瘤为与Nectin-4高表达相关的肿瘤或癌症；优选地，所述肿瘤或癌症为选自如下的任一种：膀胱癌，乳腺癌，卵巢癌，胰腺癌，肝细胞癌，胃癌，非霍奇金淋巴瘤，霍奇金淋巴瘤，急性淋巴细胞性白血病，间变性大细胞淋巴瘤，多发性骨髓瘤，前列腺癌，非小细胞肺癌，小细胞肺癌，恶性黑色素瘤，鳞状细胞癌，胶质母细胞瘤，肾细胞癌，胃肠道肿瘤，前列腺癌，直结肠癌，神经胶质瘤，间皮瘤。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了ADC 3d的表征结果，其中1A：HIC图谱；1B：SEC图谱；1C：NR-CE-SDS图谱；1D：LCMS图谱。
图2显示了不同ADC的体内药效结果。
图3显示了不同ADC的体内药效结果。
图4显示了不同ADC的体内药效结果。
图5显示了不同ADC的体内药效结果。
图6显示了不同ADC的体内药效结果。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例组1 anti-Nectin-4抗体的筛选与制备
采用重组蛋白人Nectin-4(购自近岸蛋白，Cat：CJ19)免疫小鼠，然后获取被免疫小鼠的B细胞与提前准备好的SP20骨髓瘤细胞融合，筛选能够结合人Nectin-4抗原的阳性杂交瘤细胞，最后获得只分泌一个抗体的阳性杂交瘤细胞株。
将分泌抗人Nectin-4抗体的杂交瘤细胞扩大培养后，提取细胞总RNA，反转录成cDNA；PCR扩增抗体轻链可变区IgVL(κ)和重链可变区VH序列，纯化扩增产物后连接至T载体并转化大肠杆菌细胞，菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体的重轻链可变区序列。
将鼠源抗人Nectin-4单克隆抗体的重链可变区序列和公开发表的人单克隆抗体IgG1亚类的重链恒定区序列(SEQ ID NO:9)拼接在一起，构建到哺乳动物细胞表达载体中；将鼠源抗人Nectin-4单克隆抗体的轻链可变区序列和公开发表的人单克隆抗体κ亚类的轻链恒定区序列(SEQ ID NO:10)拼接在一起，构建到哺乳动物细胞表达载体中。构建好的抗人Nectin-4嵌合抗体的重链载体和轻链载体配对混合，使用PEI转染HEK293细胞，约7天后收集细胞上清，使用Mabselect纯化得到抗人Nectin-4嵌合抗体。
综合抗体编码方案，确定鼠源抗体的重轻链6个抗原互补决定簇(CDR)的氨基酸序列区域与支撑抗体保守三维构象的框架区。随后通过分析搜索已知人源抗体序列，选择与鼠源抗体最为相似接近的人源抗体重链可变区序列，如IGHV1|IGHJ4*01，选择其抗体框架区序列作为模板，将鼠源抗体重链CDR与人源抗体框架区结合，最终生成人源化抗体重链可变区序列。同样过程，生成人源化抗体轻链可变区序列。根据结合活性变化，根据需要将框架区个别氨基酸进行从人源改回鼠源的回复突变，和/或对存在的翻译后修饰位点等个别氨基酸进行修改，最终获得备选的人源化的重链可变区和轻链可变区。
将人源化的重链可变区和轻链可变区两两组合，参考嵌合抗体的制备，获得人源化抗体。通过针对人Nectin-4的体外细胞结合实验、靶标癌细胞内吞实验、靶标癌细胞增殖抑制实验、与已知含药接头制成ADC筛选抗原结合、内吞活性或药效等，筛选出如下人源化抗体，下划线显示CDR：
42D20-hz63
VH(SEQ ID NO:1；CDR依次为：SEQ ID NO:11，SEQ ID NO:12，SEQ ID NO:13，根据CHOTHIA定义方式)
Figure PCTCN2022090450-appb-000020
VL(SEQ ID NO:2；CDR依次为：SEQ ID NO:14，SEQ ID NO:15，SEQ ID NO:16，根据CHOTHIA定义方式)
Figure PCTCN2022090450-appb-000021
42D20-hz10：
VH(SEQ ID NO:3；CDR依次为：SEQ ID NO:11，SEQ ID NO:17，SEQ ID NO:13，根据CHOTHIA定义方式)
Figure PCTCN2022090450-appb-000022
VL(SEQ ID NO:4；CDR依次为：SEQ ID NO:18，SEQ ID NO:15，SEQ ID NO:16，根据CHOTHIA定义方式)
Figure PCTCN2022090450-appb-000023
hH2L1：
VH(SEQ ID NO:5；CDR依次为：SEQ ID NO:19，SEQ ID NO:20，SEQ ID NO:13，根据KABAT定义方式)
Figure PCTCN2022090450-appb-000024
VL(SEQ ID NO:6；CDR依次为：SEQ ID NO:14，SEQ ID NO:15，SEQ ID NO:16，根据KABAT定义方式)
Figure PCTCN2022090450-appb-000025
hL2H1mut1：
VH(SEQ ID NO:7；CDR依次为：SEQ ID NO:19，SEQ ID NO:20，SEQ ID NO:13，根据KABAT定义方式)
Figure PCTCN2022090450-appb-000026
Figure PCTCN2022090450-appb-000027
VL(SEQ ID NO:8；CDR依次为：SEQ ID NO:14，SEQ ID NO:15，SEQ ID NO:16，根据KABAT定义方式)
Figure PCTCN2022090450-appb-000028
实施例组2抗体偶联药物的制备与理化表征
示例性地，使用以下方法进行实验。
2.1含药接头C-1、C-2、C-3、C-4的制备
Figure PCTCN2022090450-appb-000029
Figure PCTCN2022090450-appb-000030
化合物按照WO2018/095422A1公开的方法合成，产物均为黄色固体，LC-MS(ESI):M+1分别为1927(C-1)、1987(C-2)、1963(C-3)和1995(C-4)。
含药接头MC-VC-MMAE购自MCE。
Figure PCTCN2022090450-appb-000031
2.2定点偶联的抗体药物偶联物的制备
Figure PCTCN2022090450-appb-000032
根据专利申请CN202011046911X所述的抗体药物偶联物制备方法：将抗体经还原打开二硫键，再与含药接头进行偶联形成抗体药物偶联物，再经水解打开马来酰亚胺环，最后经纯化得到DAR＝4的抗体药物偶联物(发明专利申请CN202011046911X的全部内容以引用方式并入本文)。
具体示例性方法如下：
样品还原与偶联：将抗体样品使用Sephadex G-25载体的NAP-25脱盐柱置换至pH值7.4含有50mM的氯化钠、50mM磷酸二氢钠-磷酸氢二钠缓冲溶液中，并将抗体浓度稀释至10mg/ml。取10ml共计100mg的抗体样品，以抗体-还原剂摩尔比1比10当量添加10mg/ml的TCEP(Sigma-Aldrich)的水溶液。孵育2小时后，使用Sephadex G25脱盐柱将反应溶液进行置换，置换至pH 7.0含有50mM氯化钠，50mM磷酸二氢钠-磷酸氢二钠的缓冲溶液中。
将上述还原后的抗体稀释至5mg/mL，依次加入反应总体积7.4％的1.33ml N,N-二甲基乙酰胺(DMA)作为预溶剂、以抗体-小分子药物摩尔比1比5.5倍当量添加含10mg/mL药 物接头的DMA-药物接头混合溶液。室温搅拌60分钟。Sephadex G-25载体的NAP-25脱盐柱将反应液置换至pH8.0的磷酸氢二钠-磷酸二氢钠缓冲溶液，除去过量的药物接头，37℃水浴加热3小时。在水浴加热前后，分别进行质谱分析偶联产物结构。不同偶联产物的分析结果均表明，在水浴之前偶联产物为式Ia结构，水浴之后，得到完全开环的式Ib结构，即100％水解。
样品纯化：将上述样品使用AMICOM超滤离心管进行浓缩，将样品浓缩至15mg/mL左右。添加50mM磷酸氢二钠-磷酸二氢钠+3M磷酸铵缓冲溶液至电导率为74ms/cm。上样至Butyl-Sepharose 4 FF填料(购自GE Healthcare)疏水柱，使用A相为50mM磷酸氢二钠-磷酸二氢钠+0.45M硫酸铵；B相为50mM磷酸氢二钠-磷酸二氢钠的缓冲溶液。B相0-80％线性梯度12倍柱体积、B相100％等度梯度洗脱，对主峰进行收集。
使用AMICOM超滤离心管将最终样品置换至pH值7.4的50mM的磷酸氢二钠-磷酸二氢钠缓冲液中，并使用0.22um的滤膜进行过滤(Sartorius stedim Ministart)。
如实际反应规模≥1g，在反应当量比不变的情况下，将上述Sephadex G-25载体脱盐步骤以及AMICOM超滤离心管替换为HYDRO-30kD(Sartorius)超滤膜包。其他主要参数不变。
2.3定点偶联的抗体药物偶联物的理化表征
a.紫外分光光度法测定药物抗体偶联比(UV-DAR法)与浓度
抗体药物偶联物浓度可通过测定在280nm及小分子特征吸收波长下的UV吸光度，然后进行下述计算而得到。
a1.随机偶联药物的药物抗体偶联比(DAR)测定
根据文献[Clin Cancer Res.2004 Oct 15；10(20):7063-70]可知DAR
Figure PCTCN2022090450-appb-000033
Figure PCTCN2022090450-appb-000034
其中，
Figure PCTCN2022090450-appb-000035
为抗体在280nm下摩尔吸收系数，A 280为抗体药物偶联物在280nm下紫外吸收值，A 248为抗体药物偶联物在含药接头特征吸收波长248nm下紫外吸收值，
Figure PCTCN2022090450-appb-000036
为抗体在含药接头特征吸收波长248nm下摩尔吸收系数，
Figure PCTCN2022090450-appb-000037
为含药接头在其特征吸收波长248nm下摩尔吸收系数，
Figure PCTCN2022090450-appb-000038
为含药接头在280nm下摩尔吸收系数。
其中：
Figure PCTCN2022090450-appb-000039
a2.定点偶联药物的药物抗体偶联比(DAR)测定
根据文献[Clin Cancer Res.2004 Oct 15；10(20):7063-70]可知DAR
Figure PCTCN2022090450-appb-000040
Figure PCTCN2022090450-appb-000041
其中，
Figure PCTCN2022090450-appb-000042
为抗体在280nm下摩尔吸收系数，A 280为抗体药物偶联物在280nm下紫外吸收值，A 251为抗体药物偶联物在含药接头特征吸收波长251nm下紫外吸收值，
Figure PCTCN2022090450-appb-000043
为抗体在含药接头特征吸收波长251nm下摩尔吸收系数，
Figure PCTCN2022090450-appb-000044
为含药接头在其特征吸收波长251nm下摩尔吸收系数，
Figure PCTCN2022090450-appb-000045
为含药接头在280nm下摩尔吸收系数。
其中：
Figure PCTCN2022090450-appb-000046
注：BL20E为上文中C-3化合物去掉离去基团R并且马来酰亚胺环开环后的产物。
a3.药物浓度测定
由于某一波长下的总吸光度等于存在于体系内的所有吸收化学物质种类的吸光度的和(吸光度的加成性)，所以假设在抗体与含药接头偶联前后，抗体及含药接头的摩尔吸光系数不发生变化时，抗体药物偶联物浓度有如下关系：
Figure PCTCN2022090450-appb-000047
因此，抗体药物偶联物摩尔浓度(mol/L)
Figure PCTCN2022090450-appb-000048
由此得到抗体药物偶联物浓度(g/L)
Figure PCTCN2022090450-appb-000049
Figure PCTCN2022090450-appb-000050
将DAR值代入式中即得蛋白浓度。
其中，MW ADC为抗体药物偶联物分子量，MW ab为抗体分子量，MW D为含药接头分子量。
b-1.定点偶联药物疏水色谱HIC-HPLC测定DAR值
样品制备：样品用流动相B稀释至2.0mg/ml后，12000rpm离心10min，取上清用于HPLC分析；
色谱柱：Sepax Proteomix HIC Butyl-NP5，5μm，4.6mm*35mm；
流动相A：0.025M磷酸盐+1.2M硫酸铵(pH 7.0)；
流动相B：0.025M磷酸盐(pH 7.0)；
流动相C：100％IPA；
流速：0.8mL/min；
检测波长：280nm；
柱温：30℃；
上样量：20μL。
HIC色谱梯度
Figure PCTCN2022090450-appb-000051
DAR计算公式：
DAR＝∑(加权峰面积)/100，即DAR＝(D0峰面积比*0+D1峰面积比*1+D2峰面积比*2+D3峰面积比*3+D4峰面积比*4+D5峰面积比*5+D6峰面积比*6+D7峰面积比*7+D8峰面积比*8)/100。
b-2.随机偶联药物疏水色谱HIC-HPLC测定DAR值
样品制备：样品用流动相B稀释至2.0mg/ml后，12000rpm离心10min，取上清用于HPLC分析；
色谱柱：TOSOH Butyl NPR，2.5μm，4.6mm*100mm；
流动相A：125mM磷酸盐+2.5M硫酸铵(pH 6.8)；
流动相B：125mM磷酸盐(pH 6.8)；
流动相C：100％IPA；
流动相D：H 2O；
流速：0.7mL/min；
检测波长：280nm；
柱温：30℃；
上样量：10μL。
HIC色谱梯度
Figure PCTCN2022090450-appb-000052
Figure PCTCN2022090450-appb-000053
DAR计算公式：
DAR＝∑(加权峰面积)/100，即DAR＝(D0峰面积比*0+D1峰面积比*1+D2峰面积比*2+D3峰面积比*3+D4峰面积比*4+D5峰面积比*5+D6峰面积比*6+D7峰面积比*7+D8峰面积比*8)/100。
c.质谱法LC-MS测定DAR值
样品处理：取适量样品于超滤管中，采用50mM NH4HCO3置换缓冲液(pH7.1)进行置换，补加缓冲液，超滤离心(13000g*5min)。加入8μl的PNGase F酶至置换后的样品中，37℃孵育5h进行脱糖处理，孵育结束后，12000rpm离心5min，取上清液于进样小瓶中作为供试样品待用。
色谱柱：
Figure PCTCN2022090450-appb-000054
PolyHYDROXYETHYLA Column,
Figure PCTCN2022090450-appb-000055
5um,2.1mm×200mm；
流动相：50mM乙酸铵，pH 7.0；
运行时间：10min；
流速：0.1mL/min；
进样体积：2μL；
柱温：25℃；
检测波长：280nm；
离子化方式：ESI positive
干燥气体温度：325℃
干燥气体流速：8L/min
雾化器压力：20psig
鞘气温度：325℃；
鞘气流量：12L/min；
扫描设置：900-8000m/z。
d.分子排阻色谱SEC-HPLC测定分子大小异质性
样品处理：样品用流动相稀释至约1.0mg/ml后，12000rpm离心10min，取上清进样分析。
色谱柱：TOSOH，TSKgel G3000SW XL，5μm，7.8mm*300mm；
流动相：100mM PB+200mM盐酸精氨酸，5％异丙醇(pH 6.8)；
流速：0.6mL/min；
检测波长：280nm；
柱温：30℃；
上样量：20uL；
洗脱时间：25min；
洗脱梯度：等度洗脱。
e.非还原毛细管电泳NR-CE-SDS测定纯度
根据《中国药典四部》通则3127单抗分子大小变异体测定法测定。
实施例1靶向Nectin-4的抗体药物偶联物的制备和理化分析
根据本实施例组2.2所述，将anti-Nectin-4抗体Ref(根据WHO Drug Information中Enfortumab的序列进行制备)、42D20-hz63、42D20-hz10、hH2L1、hH1L2mut1、IgG1(Bio-Rad Antibodies，产品编号MCA928)各100mg，分别与含药接头C-1和C-3进行偶联，得到定点偶联ADC药物1a、1b、1c、1d、1e、1f和2a、2b、2c、2d、2e。
根据本实施例组2.2，采用专利申请CN202011046911X所述的抗体药物偶联物制备方法，将anti-Nectin-4抗体hH2L1与C-3进行放大制备，获得样品3d。
根据专利US 2009/0010945 A1方法，将anti-Nectin-4抗体42D20-hz63、42D20-hz10、IgG1各100mg，Ref、hH2L1、hH1L2mut1各500mg分别与含药接头MC-VC-MMAE进行偶联，得到随机偶联ADC药物4a、4b、4c、4d、4e、4f。
分别使用本实施例组2中2.3a、2.3b-1、2.3b-2、2.3d以及2.3c、2.3e对得到的定点偶联ADC药物和随机偶联ADC药物进行各项理化性质测定，结果分别见表1至表3以及图1。
表1.靶向Nectin-4的抗体药物偶联物的质量表征结果
ADC 抗体 含药接头 UV DAR HIC DAR SEC
1a Ref(Enfortumab) C-1 3.7 4.0 99.1
1b 42D20-hz10 C-1 4.2 4.0 98.7
1c 42D20-hz63 C-1 4.1 4.0 97.9
1d hH2L1 C-1 4.3 4.0 99.7
1e hH1L2mut1 C-1 4.4 4.0 99.3
1f IgG1 C-1 4.3 4.0 98.7
2a Ref(Enfortumab) C-3 3.9 4.0 99.3
2b 42D20-hz10 C-3 3.9 4.0 99.1
2c 42D20-hz63 C-3 4.0 4.0 98.5
2d hH2L1 C-3 4.0 4.0 99.4
2e hH1L2mut1 C-3 3.9 4.0 98.8
2f IgG1 C-3 3.7 4.0 97.9
3d hH2L1 C-3 4.0 4.0 98.7
表2.靶向Nectin-4的对照抗体药物偶联物的质量表征结果
ADC 抗体 含药接头 UV DAR HIC DAR SEC
4a Ref(Enfortumab) MC-VC-MMAE 4.2 4.0 98.2
4b 42D20-hz10 MC-VC-MMAE 4.0 3.7 96.9
4c 42D20-hz63 MC-VC-MMAE 4.2 3.8 94.1
4d hH2L1 MC-VC-MMAE 4.1 4.2 98.5
4e hH1L2mut1 MC-VC-MMAE 4.1 4.3 99.3
4f IgG1 MC-VC-MMAE 4.2 4.2 99.5
表3. 3d水解前和水解后质谱表征结果
样品名称 实测分子量 体系内容物
3d-水解前 155015 体系内为Ia和Ib混合物
3d-水解后 155034 体系内为Ib单一物质
通过质谱实测分子量可知，样品水解前为式Ia和Ib所示结构的混合物，水解后马来酰亚胺完全开环，得到如式Ib所示单一物质。
实施例组3抗体药物偶联物的体外杀伤作用与体内药效研究
示例性地，使用以下方法进行实验。
3.1体外杀伤作用研究方法
本实验利用人乳腺导管癌细胞BT474细胞(购自ATCC)。用完全培养基(RPMI 1640培养基45ml，5ml FBS，混匀后使用)将BT474细胞密度调到15×10 4之后，100μl/孔加入细胞培养板中，过夜培养。第二天，用上述完全培养基稀释ADC样品，分别稀释到50ug/ml，之后再4倍梯度稀释，共9个梯度加零点，所有样品均设3个复孔。设阴性对照(细胞+培养基)和空白对照(无细胞，纯培养基)；将上述稀释后的ADC样品依次加入到过夜培养的细胞培养板中，100μl/孔。然后置于细胞培养箱中孵育96h。取出细胞培养板，40μl/孔加入MTS后，37℃培养箱反应2-4h；取出细胞板，在490nm处读取OD值。
3.2体内药效研究方法
分别使用人乳腺导管癌细胞BT474细胞(购自ATCC)、人乳腺癌细胞MDA-MB-468(购自ATCC)对裸小鼠(BALB/cJGpt-Foxn1nu/Gpt，4-5周)进行接种造模。待肿瘤生长至100-200mm 3后，对小鼠静脉注射(IV)给药，给药体积10mL/kg；溶剂组给予相同体积的溶剂(生理盐水)；具体给药剂量和给药方案见下文。每周测2次肿瘤体积，称小鼠体重，记录数据。
实验结束、达到实验终点或肿瘤体积达到1500mm 3，CO 2麻醉处死动物，随后解剖取瘤并拍照。
3.3体内药效统计方法
实验指标为考察药物对肿瘤生长的影响，具体指标为T/C％或抑瘤率TGI(％)。
每周二次用游标卡尺测量肿瘤直径，肿瘤体积(V)计算公式为：
V＝1/2×a×b 2，其中a、b分别表示长、宽。
T/C(％)＝(T-T 0)/(C-C 0)×100，其中T、C为实验结束时的肿瘤体积；T 0、C 0为实验开始时的肿瘤体积。
抑瘤率(TGI)(％)＝100-T/C(％)。
当肿瘤出现消退时，抑瘤率(TGI)(％)＝100-(T-T 0)/T 0×100
如果肿瘤比起始体积缩小，即T<T 0或C<C 0时，即定义为肿瘤部分消退(PR)；如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。
实施例1对靶向Nectin-4抗体药物偶联物的体外杀伤作用研究
根据本实施例组3中3.1的方法对不同含药接头与不同anti-Nectin-4抗体的偶联ADC产物进行分组比较研究。发现：
使用不同抗体与MC-VC-MMAE形成ADC时，使用抗体hH2L1与对照抗体Ref(Enfortumab)的体外杀伤效果处于相当的水平，略优于使用抗体42D20-hz10、42D20-hz63以及hH1L2mut1。进一步地，通过对2a、1a、2e的比较可知，C-3、C-1体内活性相当。结果见表4。
表4.不同抗体与含药接头的体外杀伤研究
Figure PCTCN2022090450-appb-000056
Figure PCTCN2022090450-appb-000057
实施例2对靶向Nectin-4抗体药物偶联物的体内药效研究(1)
根据本实施例组3中3.2和3.3的方法使用BT474进行造模，对不同含药接头与不同anti-Nectin-4抗体的偶联ADC产物进行分组比较研究。
表5.不同ADC药物分组与给药方案
Figure PCTCN2022090450-appb-000058
结果见图2和表6。
表6.不同ADC的体内药效结果
Figure PCTCN2022090450-appb-000059
注：表中P值为与溶剂组相比。
实施例3对靶向Nectin-4抗体药物偶联物的体内药效研究(2)
根据实施例组3中3.2和3.3的方法使用MDA-MB-468进行造模，对不同含药接头与不同anti-Nectin-4抗体的偶联ADC产物进行分组比较研究。
表7.不同ADC的药物分组与给药方案
Figure PCTCN2022090450-appb-000060
结果见图3和表8。
表8.不同ADC的体内药效结果
Figure PCTCN2022090450-appb-000061
注：表中P值为与溶剂组相比；TV：肿瘤体积，mm 3。
实施例组4抗体药物偶联物3d在不同肿瘤模型对候选药物的药效评估
示例性地，使用以下方法进行实验。
4.1药物分组与实验设计
分别使用人乳腺癌细胞MDA-MB-468(购自ATCC)、肺癌细胞NCI-H322(购自ATCC)、膀胱癌细胞T24/nectin-4(中国科学院细胞库)对裸小鼠(BALB/cJGpt-Foxn1nu/Gpt，4-5周)进行接种造模。
使用抗体药物偶联物3d与对照药物PADCVE(Enfortumab Vedotin；购自Seattle Gentics)进行分组比较研究(如表9)。分别对小鼠静脉注射(IV)给药，给药体积10mL/kg；溶剂组给予相同体积的溶剂(生理盐水)；具体给药剂量和给药方案见表9。每周测2次肿瘤体积，称小鼠体重，记录数据。
实验结束、达到实验终点或肿瘤体积达到1500mm 3，CO 2麻醉处死动物，随后解剖取瘤并拍照。
表9.不同ADC的药物分组与给药方案
Figure PCTCN2022090450-appb-000062
4.2体内药效统计方法
同实施例组3中的3.3。
实施例1乳腺癌动物模型中对靶向Nectin-4抗体药物偶联物的体内药效研究
根据本实施例组4.1和4.2的方法使用MDA-MB-468进行造模，对3d和PADCVE进行分组比较研究。结果见图4和表10。
表10.不同ADC的体内药效结果
Figure PCTCN2022090450-appb-000063
注：表中P值为与溶剂组相比；TV：肿瘤体积，mm 3；PR：部分消退。
实施例2肺癌动物模型中对靶向Nectin-4抗体药物偶联物的体内药效研究
根据本实施例组4.1和4.2的方法使用NCI-H322进行造模，对3d和PADCVE进行分组比较研究。结果见图5和表11。
表11.不同ADC的体内药效结果
Figure PCTCN2022090450-appb-000064
Figure PCTCN2022090450-appb-000065
注：表中P值为与溶剂组相比；TV：肿瘤体积，mm 3；PR：部分消退。
实施例3膀胱癌动物模型中对靶向Nectin-4抗体药物偶联物的体内药效研究
根据上述实施例组4.1和4.2的方法使用T24/nectin-4进行造模，对3d和PADCVE进行分组比较研究。结果见图6和表12。
表12.不同ADC的体内药效结果
Figure PCTCN2022090450-appb-000066
注：表中P值为与溶剂组相比。
实施例组5抗体偶联药物的功能评估
实施例1靶向Nectin-4抗体药物偶联物的Fab功能区亲和力分析
使用BIAcore测定3d和Padcev与抗原重组人Nectin 4胞外区的亲和力。
通过脱盐柱及运行试剂1(10Mm N-(2-羟乙基)哌嗪-N-2磺酸，150mM氯化钠，3mM乙二胺四乙酸，0.005％吐温-20，pH调至7.4)将人Nectin4蛋白(购自novoprotein)进行缓冲液置换。将待测样品用运行试剂1稀释至5μg/mL，并以10μL/min的流速依次注入到His捕获芯片实验通道约400RU。参比通道不需要进行配体的捕获。将人Nectin4蛋白用相应运行试剂稀释至50、25、12.5、6.25、3.125、1.563、0.781、0.391、0nM。将稀释后的人Nectin4蛋白以30μL/min的流速注入到实验通道和参比通道，结合120s和解离300s。使用Biocore 8K分析软件计算每个抗体的KD值。参比通道用于背景的扣减。
hH2L1、3d和Padcev与人Nectin 4亲和力检测结果见表13。结果显示，hH2L1、3d以及Padcev与人Nectin 4的亲和力(KD)均为nM级别，批间一致性较好，且hH2L1 与3d亲和力基本一致，说明ADC偶联工艺没有显著影响抗体与Nectin 4的结合；Padcev的结合速率ka高于3d，但解离速率kd高于3d，总体亲和力3d略优于Padcev，是Padcev的2倍左右。
表13.与抗原的亲和力检测结果
分组 ka(1/Ms) kd(1/s) KD(nM)
hH2L1 5.62E+05 1.45E-03 2.58
3d 5.89E+05 1.42E-03 2.41
PADCEV 1.05E+06 5.12E-03 4.87
实施例2靶向Nectin-4抗体药物偶联物的Fc功能区分析
使用BIAcore测定hH2L1、3d和Padcev与Fc受体的亲和力，Fc受体包括：FcRn、人FcγR I(CD64)、人FcγR IIa(人CD32a(H167))、人FcγR IIb(人CD32b)和人FcγR IIIa(人CD16a(V176))。将人FcγR I(CD64)、FcγR IIa(CD32a(H167))、FcγR IIb(CD32b)和FcγR IIIa(CD16a(V176))蛋白用运行试剂1(10mM N-(2-羟乙基)哌嗪-N-2磺酸，150mM氯化钠，3mM乙二胺四乙酸，0.005％吐温-20，pH调至7.4)稀释至0.25μg/mL，FcRn蛋白用运行试剂2(2mM磷酸二氢钠，10mM磷酸氢二钠，137mM氯化钠，2.7mM氯化钾，0.05％吐温-20，pH调至6.0)稀释至0.25μg/mL并以10μL/min的流速依次注入到His捕获芯片实验通道(FC2)约40RU。参比通道(FC1)不需要进行配体的捕获。将待测样品用相应运行试剂稀释，稀释后的样品依次以30μL/min的流速注入到实验通道和参比通道，结合和解离相应时间。使用Biocore 8K分析软件计算每个抗体的KD值。参比通道(FC1)用于背景的扣减。
hH2L1、3d和Padcev与Fc受体亲和力检测结果见表14。结果显示，hH2L1及4批3d与Fc受体的亲和力批间无显著差异；3d及1批Padcev与FcγR IIIa的亲和力整体略差于hH2L1，其余受体的亲和力三者之间无明显差异。
表14.与Fc受体的亲和力检测结果
Figure PCTCN2022090450-appb-000067
实施例3靶向Nectin-4抗体偶联药物内吞活性研究
以高表达Nectin4的稳转细胞株PC-3-Nectin4(2-4)(自制)细胞作为靶细胞检测 HH2L1、3D及PADCEV的药物内吞作用。
实验方法：取生长至对数期的靶细胞，离心收集细胞，以完全培养基(含有10％FBS和250μg/ml G418的F-12K培养基)调整密度至5×10 5个/ml，分别吸取1ml细胞悬液至EP管中，即5×10 5个细胞，然后离心，弃上清，并用PBS清洗两遍。取样品hH2L1、3d及PADCEV各200μl加入细胞中，然后分别置于4℃(作为对照组)和37℃(作为实验组)，反应2h后加入荧光标记二抗Goat Anti human IgG Fc-FITC(购自Sigma)4℃孵育1h，最后利用流式细胞仪检测荧光强度，按公式计算样品内吞率：tx时间点的内吞百分比＝(1-37℃样品的荧光值/4℃样品的荧光值)×100％(tx表示一抗与细胞孵育的x时间)
结果表明，通过与无关抗体比较，PC-3-Nectin4(2-4)细胞表面有较高水平的靶蛋白Nectin4的表达，且与目的ADC、裸抗及PADCEV均有较强的结合能力。从目前的结果来看，3d及三批ADC原液与PADCEV的药物内吞效应之间无明显差异。
实验结果见表15。
表15.hH2L1、3d和PADCEV的内吞作用检测结果
Figure PCTCN2022090450-appb-000068
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (21)

  1. 一种靶向Nectin-4的抗体药物偶联物或其盐，其包含与药物共价连接的抗Nectin-4的抗体或其片段；
    其中，所述抗Nectin-4的抗体或其片段包含重链和轻链，所述重链和轻链分别包含如下所示的重链互补决定区1至3(CDR-H1、CDR-H2和CDR-H3)和轻链互补决定区1至3(CDR-L1、CDR-L2和CDR-L3)：
    (i)氨基酸序列分别如SEQ ID NO 11:、SEQ ID NO:12和SEQ ID NO:13所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的CDR-L1、CDR-L2和CDR-L3；
    (ii)氨基酸序列分别如SEQ ID NO:11、SEQ ID NO:17和SEQ ID NO:13所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:15和SEQ ID NO:16所示的CDR-L1、CDR-L2和CDR-L3；
    (iii)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:13所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的CDR-L1、CDR-L2和CDR-L3。
  2. 根据权利要求1所述的抗体药物偶联物或其盐，其特征在于，所述抗体药物偶联物或其盐具有分子式Ab-[L-CTD]m，其中Ab表示所述抗Nectin-4的抗体或其片段，L表示接头，CTD表示药物，m表示相对于每一分子Ab的药物平均连接数；
    优选地，CTD为细胞毒药物，优选地，CTD为选自以下的一种或多种：微管抑制剂类MMAE、DM1、DM4、Tublysin、鹅膏蕈碱、卡奇霉素、艾日布林及所述药物的衍生物；拓扑异构酶抑制剂类SN38、依喜替康及所述药物的衍生物；DNA结合剂PBD、多柔比星及所述药物的衍生物；
    优选地，m为1.0-5.0，优选为3.0-4.2，更优选为3.5-4.5，进一步优选为3.8-4.2，又优选为3.9-4.1，特别优选为4.0。
  3. 根据权利要求1或2所述的抗体药物偶联物或其盐，其特征在于，所述抗Nectin-4的抗体或其片段的重链和轻链分别包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:7所示的氨基酸序列或其变体，所述轻链可变区(VL)包含SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6或SEQ ID NO:8所示的氨基酸序列或其变体；
    优选地，所述抗Nectin-4的抗体或其片段的重链可变区(VH)和轻链可变区(VL)分别包含：
    (i)如SEQ ID NO:1所示的氨基酸序列或其变体；和，如SEQ ID NO:2所示的氨基 酸序列或其变体；
    (ii)如SEQ ID NO:3所示的氨基酸序列或其变体；和，如SEQ ID NO:4所示的氨基酸序列或其变体；
    (iii)如SEQ ID NO:5所示的氨基酸序列或其变体；和，如SEQ ID NO:6所示的氨基酸序列或其变体；或
    (iv)如SEQ ID NO:7所示的氨基酸序列或其变体；和，如SEQ ID NO:8所示的氨基酸序列或其变体。
  4. 根据权利要求1至3中任一项所述的抗体药物偶联物或其盐，其特征在于，所述抗Nectin-4的抗体或其片段为针对Nectin-4的单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式；或者，所述抗体或其片段为针对Nectin-4的半抗体或半抗体的抗原结合片段，例如scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv；
    优选地，所述Nectin-4为哺乳动物Nectin-4，优选灵长类动物Nectin-4，更优选人Nectin-4。
  5. 根据权利要求1至4中任一项所述的抗体药物偶联物或其盐，其特征在于，所述抗体为单克隆抗体，优选为鼠源、嵌合或人源化的单克隆抗体；更优选地，所述单克隆抗体的重链恒定区为IgG1或IgG4亚型，轻链恒定区为κ型；
    或者，所述抗体为免疫球蛋白，具体为IgA、IgD、IgE、IgG或IgM，例如IgA、IgD、IgE、IgG或IgM的人亚型，更优选为人IgG1、IgG2、IgG3或IgG4亚型；
    优选地，所述抗Nectin-4的抗体或其片段包含重链恒定区，所述重链恒定区包含如SEQ ID NO:9所示的氨基酸序列或其变体；或者，所述抗Nectin-4的抗体或其片段包含轻链恒定区，所述轻链恒定区包含如SEQ ID NO:10所示的氨基酸序列或其变体。
  6. 根据权利要求1至5中任一项所述的抗体药物偶联物或其盐，其特征在于，所述抗体药物偶联物或其盐具有如下式Ia和/或Ib所示的结构：
    Figure PCTCN2022090450-appb-100001
    和/或
    Figure PCTCN2022090450-appb-100002
    其中：
    Ab是所述抗Nectin-4的抗体或其片段；
    Ar’为选自如下的任一个：取代或未取代的C6-C10亚芳基和取代或未取代的5-12元亚杂芳基，其中所述取代指基团上的氢原子被一个或多个取代基所取代，所述取代基选自如下的任一个：卤素(F、Cl、Br或I)、卤代烷基(例如卤代C1-C6烷基、优选卤代C1-C4烷基，例如三氟甲基)和烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，例如甲氧基)；
    L 1为连接于Ar’基团上的-O(CH2CH2O)n-，其中n选自1-24中任一整数，优选为1-10、更优选3-5中的任意整数；
    L 2为酶切片段，如二肽或三肽或四肽或其与自释放结构片段的组合(即2-4个氨基酸组成的多肽片段或其与自释放结构片段的组合)，如Val-Ala、Val-Ala-PAB、Val-Cit、Val-Cit-PAB、Phe-Lys-PAB、Ala-Ala-Ala、Gly-Gly-Phe-Gly(GGFG)、MAC glucuronide phenol。
  7. 根据权利要求1至6中任一项所述的抗体药物偶联物或其盐，其特征在于，L 2-CTD为VcMMAE、GGFG-Dxd或VC-seco-DUBA；
    优选地，在Ar’取代或未取代的5-12元亚杂芳基时，杂原子为N；
    优选地，Ar’为取代或未取代的C6亚芳基或取代或未取代的6元亚杂芳基；
    更优选地，所述抗体药物偶联物或其盐具有如下结构：
    偶联物ADC-1：
    Figure PCTCN2022090450-appb-100003
    偶联物ADC-2：
    Figure PCTCN2022090450-appb-100004
    偶联物ADC-3：
    Figure PCTCN2022090450-appb-100005
    偶联物ADC-4：
    Figure PCTCN2022090450-appb-100006
    偶联物ADC-5：
    Figure PCTCN2022090450-appb-100007
    偶联物ADC-6：
    Figure PCTCN2022090450-appb-100008
    偶联物ADC-7：
    Figure PCTCN2022090450-appb-100009
  8. 一种靶向Nectin-4的抗体药物偶联物或其盐的制备方法，所述抗体药物偶联物或其盐具有式Ia和Ib所示的结构：
    Figure PCTCN2022090450-appb-100010
    和/或
    Figure PCTCN2022090450-appb-100011
    所述方法包括以下步骤：
    (1)使抗Nectin-4的抗体或其片段与还原试剂在缓冲液中反应，得到经还原后的抗体或其片段；
    (2)使药物接头(连接子-药物缀合物)与步骤(1)得到的经还原后的抗体或其片段 在缓冲液与有机溶剂的混合液中进行交联，得到靶向Nectin-4的抗体药物偶联物。
  9. 根据权利要求8所述的制备方法，其特征在于，所述药物接头具有如下式Ic所示的结构：
    Figure PCTCN2022090450-appb-100012
    其中：
    R为X或R’S，其中X为卤素(F、Cl、Br或I)，优选为Br或I；R’为取代或未取代的C6-C10芳基或者取代或未取代的5-12元杂芳基，其中所述取代指基团上的氢原子被一个或多个取代基所取代，所述取代基选自如下的任一个：烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，例如甲氧基)、卤素(F、Cl、Br或I)、酯基、酰胺基和氰基；
    优选地，R为R’S，其中R’为苯基或取代的苯基，在取代的苯基中，取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、卤素(F、Cl、Br或I)、酯基、酰胺基和氰基；优选地，R’为苯基、4-甲基甲酰基取代苯基
    Figure PCTCN2022090450-appb-100013
    或4-甲酰基吗啉取代苯基
    Figure PCTCN2022090450-appb-100014
    Ar’、L 1、L 2和CTD如权利要求6至8中任一项所限定。
  10. 根据权利要求8或9所述的制备方法，其特征在于，所述药物接头为选自如下的任一个：
    Figure PCTCN2022090450-appb-100015
    Figure PCTCN2022090450-appb-100016
    Figure PCTCN2022090450-appb-100017
    Figure PCTCN2022090450-appb-100018
    Figure PCTCN2022090450-appb-100019
  11. 根据权利要求8至10中任一项所述的制备方法，其特征在于，所述制备方法包括以下步骤：
    a.抗体还原：将≥5.5倍抗体摩尔当量的还原剂加入到含5-30mg/mL浓度抗体的磷酸盐缓冲液中，反应1.5-2h，其中所述还原剂为选自TCEP、DTT、2-MEA和DTBA中的一种或多种；
    b.抗体偶联：将步骤a中经还原的抗体置换到pH 6.5-7.8的磷酸盐缓冲液中，并将抗体稀释到3.5-15mg/mL浓度，将溶解于有机共溶剂中的4.5-6.5倍抗体摩尔当量的含药接头加入到所述抗体稀释液中，在15-35℃下搅拌反应≥0.5h，其中所述有机共溶剂为选自DMA、DMSO、DMF和ACN中的一种或多种；
    c.疏水层析：使用疏水填料对抗体偶联产物进行疏水层析纯化；
    优选地，所述制备方法在步骤b之后或步骤c之后还包括以下步骤：
    d.水解：将抗体偶联产物置换到pH 7.4-9.0的磷酸盐缓冲液中，在35±10℃下加热2-24h，得到水解产物。
  12. 一种抗Nectin-4的抗体或其片段，其包含重链和轻链，所述重链和轻链分别包含如下所示的重链互补决定区1至3(CDR-H1、CDR-H2和CDR-H3)和轻链互补决定区1至3(CDR-L1、CDR-L2和CDR-L3)：
    氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:13所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的CDR-L1、CDR-L2和CDR-L3。
  13. 根据权利要求12所述的抗Nectin-4的抗体或其片段，其特征在于，所述抗Nectin-4的抗体或其片段的重链和轻链分别包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)包含SEQ ID NO:5或SEQ ID NO:7所示的氨基酸序列或其变体，所述轻链可变区(VL)包含SEQ ID NO:6或SEQ ID NO:8所示的氨基酸序列或其变体；
    优选地，所述抗Nectin-4的抗体或其片段的重链可变区(VH)和轻链可变区(VL)分别包含：
    (A)如SEQ ID NO:5所示的氨基酸序列或其变体；和，如SEQ ID NO:6所示的氨基酸序列或其变体；或
    (B)如SEQ ID NO:7所示的氨基酸序列或其变体；和，如SEQ ID NO:8所示的氨基酸序列或其变体；
    进一步优选地，所述抗Nectin-4的抗体或其片段如权利要求4或5中任一项所限定。
  14. 一种核酸分子，其包含编码权利要求12或13所述的抗Nectin-4的抗体或其片段中包含的轻链可变区、重链可变区、重链或轻链的核苷酸序列。
  15. 一种载体，其包含权利要求14所述的核酸分子。
  16. 一种宿主细胞，其包含权利要求14所述的核酸分子和/或权利要求15所述的载体。
  17. 一种组合物，所述组合物包含权利要求1至7中任一项所述的靶向Nectin-4的抗体药物偶联物或其盐、权利要求12或13所述的抗Nectin-4的抗体或其片段、权利要求14所述的核酸分子、权利要求15所述的载体和/或权利要求16所述的宿主细胞。
  18. 权利要求1至8中任一项所述的靶向Nectin-4的抗体药物偶联物或其盐、权利要求12或13所述的抗Nectin-4的抗体或其片段、权利要求14所述的核酸分子、权利要求15所述的载体、权利要求16所述的宿主细胞和/或权利要求17所述的组合物在制备用于治疗肿瘤的药物中的用途。
  19. 根据权利要求18所述的用途，其特征在于，所述肿瘤为与Nectin-4高表达相关的肿瘤或癌症；
    优选地，所述肿瘤或癌症为选自如下的任一种：膀胱癌，乳腺癌，卵巢癌，胰腺癌，肝细胞癌，胃癌，非霍奇金淋巴瘤，霍奇金淋巴瘤，急性淋巴细胞性白血病，间变性大细胞淋巴瘤，多发性骨髓瘤，前列腺癌，非小细胞肺癌，小细胞肺癌，恶性黑色素瘤，鳞状细胞癌，胶质母细胞瘤，肾细胞癌，胃肠道肿瘤，前列腺癌，直结肠癌，神经胶质瘤，间皮瘤。
  20. 一种用于治疗肿瘤的方法，所述方法包括向有此需要的受试者施用权利要求1至8中任一项所述的靶向Nectin-4的抗体药物偶联物或其盐、权利要求12或13所述的抗Nectin-4的抗体或其片段、权利要求14所述的核酸分子、权利要求15所述的载体、权利要求16所述的宿主细胞和/或权利要求17所述的组合物。
  21. 根据权利要求20所述的方法，其特征在于，所述受试者为哺乳类动物，优选地为灵长类动物，更优选为人；
    优选地，所述肿瘤为与Nectin-4高表达相关的肿瘤或癌症；
    优选地，所述肿瘤或癌症为选自如下的任一种：膀胱癌，乳腺癌，卵巢癌，胰腺癌，肝细胞癌，胃癌，非霍奇金淋巴瘤，霍奇金淋巴瘤，急性淋巴细胞性白血病，间变性大细胞淋巴瘤，多发性骨髓瘤，前列腺癌，非小细胞肺癌，小细胞肺癌，恶性黑色素瘤，鳞状细胞癌，胶质母细胞瘤，肾细胞癌，胃肠道肿瘤，前列腺癌，直结肠癌，神经胶质瘤，间皮瘤。
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