WO2023232080A1 - 抗cldn18.2抗体及其抗体药物偶联物和用途 - Google Patents

抗cldn18.2抗体及其抗体药物偶联物和用途 Download PDF

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WO2023232080A1
WO2023232080A1 PCT/CN2023/097470 CN2023097470W WO2023232080A1 WO 2023232080 A1 WO2023232080 A1 WO 2023232080A1 CN 2023097470 W CN2023097470 W CN 2023097470W WO 2023232080 A1 WO2023232080 A1 WO 2023232080A1
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seq
antibody
amino acid
acid sequence
antigen
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French (fr)
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萧翠珍
秦超
麦思琪
俞金泉
李胜峰
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百奥泰生物制药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • 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
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to anti-CLDN18.2 antibodies, antibody-drug conjugates and their uses.
  • Antibody drug conjugates consist of three parts: the antibody, the cytotoxic molecule and the linker connecting the two (Thomas, A., et al. (2016), Lancet Oncol 17(6):e254-e262). Each of the three has a unique function: the antibody needs to specifically bind to tumor cells, the cytotoxic molecule needs to be sufficiently active and broad-spectrum against tumor cells, and the connector needs to have unique functionality, be stable in the blood circulation, and reach the tumor. Cells can effectively release cytotoxic molecules (Chari, R.V. (2008), Acc Chem Res 41(1):98-107). Only by rational construction of the three can good clinical effects be achieved (Singh, S.K., et al. (2015), Pharm Res 32(11):3541-3571; Hamilton, G.S. (2015), Biologicals 43(5):318-332).
  • Cell junction claudin 18.2 (claudin 18.2 or CLDN18.2) is one of the current targets for tumor treatment development (Sahin U., et al. Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development, Clin Cancer Res.,2008,14(23):7624-34).
  • an antibody or antigen-binding unit of the invention is capable of recognizing and binding human CLDN18.2.
  • the invention provides anti-CLDN18.2 antibodies or antigen-binding units comprising one or more amino acid sequences of (a)-(f):
  • VH CDR1 which contains the amino acid sequence shown in any one of SEQ ID NO: 1-6;
  • VH CDR2 which includes the amino acid sequence shown in any one of SEQ ID NO: 7-13;
  • VH CDR3 which includes the amino acid sequence shown in any one of SEQ ID NO: 14-21;
  • VL CDR1 which includes the amino acid sequence shown in any one of SEQ ID NO: 22-29;
  • VL CDR2 which includes the amino acid sequence shown in any one of SEQ ID NO: 30-37;
  • VL CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 38-45.
  • the antibody or antigen-binding unit comprises a VH CDR1 as set forth in any one of SEQ ID NO: 1-6, a VH CDR2 as set forth in any one of SEQ ID NO: 7-13 and VH CDR3 as shown in any of SEQ ID NOs: 14-21.
  • the antibody or antigen binding unit comprises a VL CDR1 as set forth in any one of SEQ ID NO:22-29, a VL CDR2 as set forth in any one of SEQ ID NO:30-37 and VL CDR3 as shown in any of SEQ ID NO:38-45.
  • the antibody or antigen-binding unit comprises a VH CDR1 as set forth in any one of SEQ ID NO: 1-6, a VH CDR2 as set forth in any one of SEQ ID NO: 7-13 , VH CDR3 as shown in any one of SEQ ID NO: 14-21, VL CDR1 as shown in any one of SEQ ID NO: 22-29, VL as shown in any one of SEQ ID NO: 30-37 CDR2 and VL CDR3 as shown in any one of SEQ ID NO:38-45.
  • the antibody or antigen binding unit comprises a VH CDR1 as set forth in SEQ ID NO:2, a VH CDR2 as set forth in SEQ ID NO:8, a VH CDR2 as set forth in SEQ ID NO:15 VH CDR3, VL CDR1 as shown in SEQ ID NO:26, VL CDR2 as shown in SEQ ID NO:34 and VL CDR3 as shown in SEQ ID NO:42.
  • the antibody or antigen binding unit comprises a VH CDR1 as set forth in SEQ ID NO:3, a VH CDR2 as set forth in SEQ ID NO:9, a VH CDR2 as set forth in SEQ ID NO:16 VH CDR3, VL CDR1 as shown in SEQ ID NO:25, VL CDR2 as shown in SEQ ID NO:33 and VL CDR3 as shown in SEQ ID NO:41.
  • the antibody or antigen binding unit comprises a VH CDR1 as shown in SEQ ID NO:6, a VH CDR2 as shown in SEQ ID NO:13, a VH CDR2 as shown in SEQ ID NO:20 VH CDR3, VL CDR1 as shown in SEQ ID NO:28, VL CDR2 as shown in SEQ ID NO:36 and VL CDR3 as shown in SEQ ID NO:44.
  • the antibody or antigen binding unit comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR2 as set forth in SEQ ID NO:12, a VH CDR2 as set forth in SEQ ID NO:19 VH CDR3, VL CDR1 as shown in SEQ ID NO:29, VL CDR2 as shown in SEQ ID NO:37 and VL CDR3 as shown in SEQ ID NO:45.
  • the antibody or antigen binding unit comprises a VH CDR1 as set forth in SEQ ID NO:4, a VH CDR2 as set forth in SEQ ID NO:11, a VH CDR2 as set forth in SEQ ID NO:18 VH CDR3, VL CDR1 as shown in SEQ ID NO:27, VL CDR2 as shown in SEQ ID NO:35 and VL CDR3 as shown in SEQ ID NO:43.
  • the antibody or antigen binding unit comprises a VH CDR1 as set forth in SEQ ID NO:3, a VH CDR2 as set forth in SEQ ID NO:9, a VH CDR2 as set forth in SEQ ID NO:16 VH CDR3, VL CDR1 as shown in SEQ ID NO:26, VL CDR2 as shown in SEQ ID NO:34 and VL CDR3 as shown in SEQ ID NO:42.
  • the antibody or antigen binding unit comprises a VH CDR1 as set forth in SEQ ID NO:3, a VH CDR2 as set forth in SEQ ID NO:9, a VH CDR2 as set forth in SEQ ID NO:16 VH CDR3, VL CDR1 as shown in SEQ ID NO:24, VL CDR2 as shown in SEQ ID NO:32 and VL CDR3 as shown in SEQ ID NO:40.
  • the antibody or antigen binding unit comprises a VH CDR1 as shown in SEQ ID NO: 1, a VH CDR2 as shown in SEQ ID NO: 7, a VH CDR2 as shown in SEQ ID NO: 14 VH CDR3, VL CDR1 as shown in SEQ ID NO:22, VL CDR2 as shown in SEQ ID NO:30 and VL CDR3 as shown in SEQ ID NO:38.
  • the antibody or antigen binding unit comprises a VH CDR1 as shown in SEQ ID NO: 1, a VH CDR2 as shown in SEQ ID NO: 7, a VH CDR2 as shown in SEQ ID NO: 14 VH CDR3, VL CDR1 as shown in SEQ ID NO:23, VL CDR2 as shown in SEQ ID NO:31 and VL CDR3 as shown in SEQ ID NO:39.
  • the antibody or antigen binding unit comprises a VH CDR1 as set forth in SEQ ID NO:2, a VH CDR2 as set forth in SEQ ID NO:10, a VH CDR2 as set forth in SEQ ID NO:17 VH CDR3, VL CDR1 as shown in SEQ ID NO:27, VL CDR2 as shown in SEQ ID NO:35 and VL CDR3 as shown in SEQ ID NO:43.
  • the antibody or antigen binding unit comprises a VH CDR1 as set forth in SEQ ID NO:2, a VH CDR2 as set forth in SEQ ID NO:8, a VH CDR2 as set forth in SEQ ID NO:21 VH CDR3, VL CDR1 as shown in SEQ ID NO:26, VL CDR2 as shown in SEQ ID NO:34 and SEQ ID NO:42 VL CDR3.
  • the antibody or antigen-binding unit is a murine antibody, a chimeric antibody, or a humanized antibody.
  • the antibody or antigen-binding unit comprises a heavy chain variable region and/or a light chain variable region.
  • the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NO:46-55, or is identical to an amino acid sequence set forth in any one of SEQ ID NO:46-55.
  • the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NO:56-64, or is identical to an amino acid sequence set forth in any one of SEQ ID NO:56-64 An amino acid sequence that has at least 80% or 90% identity when compared to an amino acid sequence, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 56-64.
  • the heavy chain variable region includes an amino acid sequence as shown in any one of SEQ ID NO:46-55, and the light chain variable region includes an amino acid sequence as shown in any one of SEQ ID NO:56-64 The amino acid sequence shown in any one.
  • the antibody or antigen-binding unit comprises a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:47, and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:60.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:48, and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:59.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:52, and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:62.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:51 and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:63.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:50 and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:61.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:48 and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:60.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:48, and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:58.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:46, and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:56.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:46, and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:57.
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:61.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:53, and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:64.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:54, and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:64.
  • the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO:55 and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO:64.
  • the antibody or antigen-binding unit further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.
  • the light chain is of the kappa or lambda type.
  • the light chain constant region is a kappa or lambda chain constant region.
  • the antibody or antigen binding unit is IgG, One of the isotypes IgM, IgA, IgE or IgD.
  • the isotype is IgGl, IgG2, IgG3 or IgG4.
  • the antibody is an IgG1 antibody.
  • the Fc is a variant Fc region.
  • a variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions, relative to the parent Fc region.
  • amino acid modifications to the Fc region alter the effector function activity relative to the activity of the parent Fc region.
  • a variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization or cell binding.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-mediated cytotoxicity
  • phagocytosis opsonization or cell binding.
  • Fc region amino acid modifications can alter the affinity of a variant Fc region for an Fc ⁇ R (Fc ⁇ receptor) relative to the parent Fc region.
  • the Fc region is derived from IgGl or IgG4.
  • the antibody or antigen binding unit is an isolated antibody or antigen binding unit. In one or more embodiments, the antibody or antigen binding unit is scFV, Fab, F(ab) 2 or IgGl. In one or more embodiments, the antibody or antigen-binding unit is a monoclonal antibody.
  • the heavy chain constant region contains one or more of the following amino acid mutations: N297A, L234A, L235A, P329G (EU numbering). In one or more embodiments, the heavy chain constant region comprises the following amino acid mutation: N297A (EU numbering). In one or more embodiments, the heavy chain constant region comprises the following amino acid mutations: L234A, L235A and P329G (EU numbering).
  • the heavy chain constant region comprises an amino acid sequence as set forth in any one of SEQ ID NO:65-67, or is identical to an amino acid sequence set forth in any one of SEQ ID NO:65-67 Compared to an amino acid sequence that has at least 80% or 90% identity, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 65-67.
  • the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 68, or has at least 80% or at least 90% compared to the amino acid sequence set forth in SEQ ID NO: 68 An identical amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 68.
  • the heavy chain constant region comprises an amino acid sequence as set forth in any one of SEQ ID NO:65-67, and the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO:68 sequence.
  • the heavy chain constant region includes the amino acid sequence set forth in SEQ ID NO:65 and the light chain constant region includes the amino acid sequence set forth in SEQ ID NO:68.
  • the heavy chain constant region includes the amino acid sequence set forth in SEQ ID NO:66, and the light chain constant region includes the amino acid sequence set forth in SEQ ID NO:68.
  • the heavy chain constant region includes the amino acid sequence set forth in SEQ ID NO:67 and the light chain constant region includes the amino acid sequence set forth in SEQ ID NO:68.
  • the heavy chain of the antibody comprises an amino acid sequence as set forth in any one of SEQ ID NO: 69-71, or an amino acid sequence identical to any one of SEQ ID NO: 69-71 Compared to an amino acid sequence that has at least 80% or 90% identity, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 69-71.
  • the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:72, or is at least 80% or 90% identical to the amino acid sequence set forth in SEQ ID NO:72. A unique amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:72.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in any one of SEQ ID NO:69-71, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:72 sequence. In one or more embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:69 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:72. In one or more embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:70, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:72 acid sequence. In one or more embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:71 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:72.
  • the antibodies described herein contain two sequence-identical heavy chains and two sequence-identical light chains.
  • One or more embodiments also provide a polynucleotide encoding an antibody or antigen binding unit described herein, or a portion thereof.
  • the polynucleotide is an isolated polynucleotide.
  • One or more embodiments also provide an expression vector comprising a polynucleotide encoding an antibody or antigen-binding unit described herein.
  • One or more embodiments also provide a cell comprising a polynucleotide encoding an antibody or antigen-binding unit as described herein.
  • the vector, cell or microorganism is an isolated vector, cell or microorganism.
  • the cells are CHO cells, HEK cells (such as HEK293F cells), BHK cells, Cos1 cells, Cos7 cells, CV1 cells or murine L cells.
  • the cells are CHO cells.
  • the invention also provides a method for producing the antibody, which comprises culturing a host cell comprising a nucleic acid encoding the antibody in a culture medium.
  • the method further includes purifying the antibody. Purification can be performed using conventional methods, such as centrifuging the cell suspension and collecting the supernatant, and then centrifuging again to further remove impurities. Methods such as Protein A affinity columns and ion exchange columns can be used to purify antibody proteins.
  • One or more embodiments also provide diagnostic methods and uses.
  • a method for detecting CLDN18.2 expression in a sample is provided, contacting the sample with the antibody or antigen-binding unit, such that the antibody or antigen-binding unit binds to CLDN18.2, and detecting it Combined, that is, the content of CLDN18.2 in the sample.
  • use of the antibody or antigen-binding unit in the preparation of a kit for diagnosis or prognosis of cancer or tumors is provided.
  • a diagnostic or prognostic kit comprising the antibody or antigen binding unit is provided.
  • the present invention also provides an antibody drug conjugate (ADC), which contains the antibody or antigen-binding unit of the present invention coupled to a drug through a linker, or a pharmaceutically acceptable salts or solvates.
  • ADC antibody drug conjugate
  • the linker is a cleavable linker.
  • the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutritional factor, a steroid drug, a drug for treating autoimmune diseases, an anti-inflammatory drug, or a drug for treating infectious diseases.
  • the drug is an anti-cancer drug.
  • the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.
  • the tubulin inhibitor is selected from the group consisting of dolastatins, auristatins, and maytansines.
  • the drug is an auristatin, selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) or auristatin (AF ).
  • auristatin selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) or auristatin (AF ).
  • the drug is a DNA damaging agent, such as calicheamicins, duocarmycins, and antromycin derivatives PBD (pyrrolobenzodiazepine, pyrrolocene and diazepine).
  • a DNA damaging agent such as calicheamicins, duocarmycins, and antromycin derivatives PBD (pyrrolobenzodiazepine, pyrrolocene and diazepine).
  • the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, irinotecan derivatives, camptothecin, 9-amino Camptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyecliptaline, topotetherine Can, letotecan, bellotecan, ixotecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosyl Amino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-propene Amide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-
  • the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, bellotecan, irinotecan, 22-hydroxyecliptaline Or Ixotecan.
  • the drug has an amino group or an amino group substituted by an alkyl group, which is connected to the linker through an amide bond.
  • the drug is or its stereoisomers, where
  • X 1 and X 2 are each independently:
  • Amino an amino group substituted by an amino protecting group, a C1-C6 aminoalkyl group optionally substituted in the amino portion by an amino protecting group or a C1-C6 alkyl group,
  • a C1-C6 aminoalkylamino group optionally substituted in the amino portion by an amino protecting group or a C1-C6 alkyl group,
  • C1-C6 alkyl attached to a heterocycle optionally substituted by one or more C1-C6 alkyl, C1-C6 alkoxy, amino, halogen, nitro or cyano,
  • C1-C6 alkylamino group connected to a heterocyclic ring is optionally substituted by C1-C6 alkyl, C1-C6 alkoxy group, the amino group is optionally protected by an amino protecting group, halogen, nitro, cyano group or protective group substitution,
  • Amino-substituted heterocyclyl the nitrogen atom or amino portion of the heterocyclic part is optionally substituted by a protecting group or one or more C1-C6 alkyl groups,
  • Heterocyclic amino group, the nitrogen atom or amino part of the heterocyclic part is optionally substituted by a protecting group or a C1-C6 alkyl group,
  • Carbamoyl optionally substituted by a carbamoyl protecting group or C1-C6 alkyl,
  • X 3 is C1-C6 alkyl
  • X 4 is H, -(CH 2 ) q -CH 3 , -(CHR n ) q -CH 3 , C3-C8 carbocyclic group, -O-(CH 2 ) q -CH 3 , arylene group -CH 3 , -(CH 2 ) q -arylene-CH 3 , -arylene-(CH 2 ) q -CH 3 , -(CH 2 ) q -(C3-C8 carbocyclyl)-CH 3 , -( C3-C8 carbocyclyl)-(CH 2 ) q -CH 3 , C3-C8 heterocyclyl, -(CH 2 ) q -(C3-C8 heterocyclyl)-CH 3 , -(C3-C8 heterocycle base)-(CH 2 ) q -CH 3 , -(CH 2 ) q C(O)NR n (CH 2 ) q
  • y 0, 1 or 2;
  • Y is O, S or CR 1 R 2 , where R 1 and R 2 are each independently H or C1-C6 alkyl;
  • s and t are each independently 0, 1, or 2, but not 0 at the same time.
  • X 4 is H or C1-C6 alkyl.
  • the heterocycle is azetidine, niverazine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.
  • the amino protecting group is formyl, acetyl, trityl, tert-butoxycarbonyl, benzyl or p-methoxybenzyloxycarbonyl.
  • the drug is Or its stereoisomer, wherein X 1 and X 2 are each independently C1-C6 alkyl, halogen or -OH; ** is connected to the linker.
  • the drug is Or its stereoisomer, wherein X 1 and X 2 are each independently C1-C6 alkyl, halogen or -OH; ** is connected to the linker.
  • X 1 and X 2 are each -CH 3 .
  • X 1 and X 2 are each independently F, Cl, Br, or I.
  • X 1 and X 2 are each F.
  • each of X 1 and X 2 is independently -CH 3 , F, or -OH.
  • X 1 and X 2 are each independently F or -CH 3 .
  • X 1 is -CH 3 and X 2 is F.
  • the invention provides an antibody drug conjugate, which has the structure shown in Formula I, or its stereoisomer, or its pharmaceutically acceptable salt or solvate:
  • Abu is an antibody or antigen-binding unit; the antibody or antigen-binding unit specifically binds CLDN18.2; D is a drug;
  • M is Among them, * is connected to Abu, ** is connected to B, and R is selected from: -(CH 2 ) r -, -(CHR m ) r -, C3-C8 carbocyclic group, -O-(CH 2 ) r -, arylene group , -(CH 2 ) r -arylene-, -arylene-(CH 2 )r-, -(CH 2 ) r -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl) )-(CH 2 ) r -, C3-C8 heterocyclyl, -(CH 2 ) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH 2 ) r -, -(CH 2 ) r C(O)NR m (CH 2 ) r -, -(CH 2 CH 2 O)
  • B is For example Among them, * connects to M, ** connects to L, and *** connects to G;
  • L is -(AA) i -(FF) f -, where AA is an amino acid or polypeptide, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; each FF is independently Wherein each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO 2 or halogen; z is 0, 1, 2, 3 or 4; f is 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10; where * connects AA and ** connects D;
  • G is where n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;
  • p is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutritional factor, a steroid drug, a drug for treating autoimmune diseases, an anti-inflammatory drug, or a drug for treating infectious diseases.
  • the drug is an anti-cancer drug.
  • the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.
  • the tubulin inhibitor is selected from the group consisting of dolastatins, auristatins, and maytansines.
  • the drug is an auristatin, such as MMAE, MMAF or AF.
  • the drug is a DNA damaging agent, such as calicheamicin, duocarmycin, and antromycin derivative PBD (pyrrolobenzodiazepine, pyrrolobenzodiazepine). aza).
  • a DNA damaging agent such as calicheamicin, duocarmycin, and antromycin derivative PBD (pyrrolobenzodiazepine, pyrrolobenzodiazepine). aza).
  • the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin Tothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyecliptaline, topotecan, letotecan, bello Tecan, ixotecan, ixotecan derivatives, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino) Phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(
  • the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, bellotecan, irinotecan, 22-hydroxyecliptaline Or Ixotecan.
  • the drug is Tubulysin, taxane drug derivatives, leptomycine derivatives, CC-1065 and its analogs, Amatoxin, spliceosome inhibitors, benzodiazepines (PBD) ) dimers, doxorubicin, methotrexate, vincristine, vinblastine, daunorubicin, mitomycin C, melphalan or chlorambucil derivatives.
  • PBD benzodiazepines
  • the drug has an amino group or an amino group substituted by an alkyl group, which is linked to the FF through an amide bond.
  • the drug is in one or more embodiments.
  • X 1 and X 2 are each independently:
  • Amino an amino group substituted by an amino protecting group, a C1-C6 aminoalkyl group optionally substituted in the amino portion by an amino protecting group or a C1-C6 alkyl group,
  • a C1-C6 aminoalkylamino group optionally substituted in the amino portion by an amino protecting group or a C1-C6 alkyl group,
  • C1-C6 alkyl attached to a heterocycle optionally substituted by one or more C1-C6 alkyl, C1-C6 alkoxy, amino, halogen, nitro or cyano,
  • C1-C6 alkylamino group connected to a heterocyclic ring is optionally substituted by C1-C6 alkyl, C1-C6 alkoxy group, the amino group is optionally protected by an amino protecting group, halogen, nitro, cyano group or protective group substitution,
  • Amino-substituted heterocyclyl the nitrogen atom or amino portion of the heterocyclic part is optionally substituted by a protecting group or one or more C1-C6 alkyl groups,
  • Heterocyclic amino group, the nitrogen atom or amino part of the heterocyclic part is optionally substituted by a protecting group or a C1-C6 alkyl group,
  • Carbamoyl optionally substituted by a carbamoyl protecting group or C1-C6 alkyl,
  • X 3 is C1-C6 alkyl
  • X 4 is H, -(CH 2 ) q -CH 3 , -(CHR n ) q -CH 3 , C3-C8 carbocyclic group, -O-(CH 2 ) q -CH 3 , arylene group -CH 3 , -(CH 2 ) q -arylene-CH 3 , -arylene-(CH 2 ) q -CH 3 , -(CH 2 ) q -(C3-C8 carbocyclyl)-CH 3 , -( C3-C8 carbocyclyl)-(CH 2 ) q -CH 3 , C3-C8 heterocyclyl, -(CH 2 ) q -(C3-C8 heterocyclyl)-CH 3 , -(C3-C8 heterocycle base)-(CH 2 ) q -CH 3 , -(CH 2 ) q C(O)NR n (CH 2 ) q
  • y 0, 1 or 2;
  • Y is O, S or CR 1 R 2 , where R 1 and R 2 are each independently H or C1-C6 alkyl;
  • s and t are each independently 0, 1, or 2, but not 0 at the same time.
  • X 4 is H or C1-C6 alkyl.
  • the heterocycle is azetidine, niverazine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.
  • the amino protecting group is formyl, acetyl, trityl, tert-butoxycarbonyl, benzyl or p-methoxybenzyloxycarbonyl.
  • the drug is Where X 1 and X 2 are each independently C1-C6 alkyl, halogen or -OH; ** is connected to L.
  • the drug is Where X 1 and X 2 are each independently C1-C6 alkyl, halogen or -OH; ** is connected to L.
  • X 1 and X 2 are each -CH 3 .
  • X 1 and X 2 are each independently F, Cl, Br, or I.
  • X 1 and X 2 are each F.
  • each of X 1 and X 2 is independently -CH 3 , F, or -OH.
  • X 1 and X 2 are each independently F or -CH 3 .
  • X 1 is -CH 3 and X 2 is F.
  • R is -( CH2 ) r- .
  • R is -(CH 2 ) r -, r is 1 or 5.
  • each AA is independently selected from the following amino acid or peptide sequences: Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile -Cit, Trp, Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala -Leu-Ala-Leu, ⁇ -Ala-Leu-Ala-Leu and Gly-Phe-Leu-Gly.
  • i 1
  • AA is Val-Cit and i is 1.
  • each FF is independently
  • Each R F is independently C1-C6 alkyl, C1-C6 alkoxy, -NO 2 or halogen; where * is connected to AA and ** is connected to D.
  • halogen is F and z is 0, 1, 2, 3, or 4.
  • R F is -CH 3 , F, -NO 2 or -OCH 3 .
  • z is zero.
  • z is 1 or 2.
  • each FF is independently Among them, * connects AA and ** connects D.
  • f is 1.
  • FF is f is 1; where * connects AA and ** connects D.
  • L is Among them, * connects to B and ** connects to D.
  • L is Among them, * connects to B and ** connects to D.
  • G is n is 4-12.
  • n is 4-8.
  • n 4.
  • n 8.
  • p is 2-8.
  • p is 4-8.
  • p is 6-8.
  • p is 7-8.
  • the Abu is an antibody or antigen-binding unit provided by the invention.
  • the antibody drug conjugate has the structure shown in Formula I-1 or Formula I-2 or its stereoisomer or its pharmaceutically acceptable salt or solvate:
  • Abu is the antibody or antigen-binding unit provided by the present invention.
  • R is selected from: -(CH 2 ) r -, -(CHR m )r-, C3-C8 carbocyclyl, -O-(CH 2 ) r -, arylene, -(CH 2 ) r -arylene Base-, -arylene-(CH 2 )r-, -(CH 2 ) r -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH 2 ) r -, C3 -C8 heterocyclyl, -(CH 2 ) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH 2 ) r -, -(CH 2 ) r C(O) NR m (CH 2 ) r -, -(CH 2 CH 2 O) r -, -(CH 2 CH 2 O) r -CH 2 -
  • n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24;
  • p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • R is -( CH2 ) r- .
  • R is -(CH 2 ) r -, r is 1 or 5.
  • n is 4-12.
  • n is 4-8.
  • n 4.
  • n 8.
  • p is 2-8.
  • p is 4-8.
  • p is 6-8.
  • p is 7-8.
  • the antibody drug conjugate has the structure shown in Formula I-3 or Formula I-4 or its stereoisomer or its pharmaceutically acceptable salt or solvate:
  • Abu is the antibody or antigen-binding unit provided by the present invention.
  • n is an integer from 1 to 24, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24;
  • p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • n is 4-12.
  • n is 4-8.
  • n 4.
  • n 8.
  • p is 2-8.
  • p is 4-8.
  • p is 6-8.
  • p is 7-8.
  • the antibody drug conjugate has a structure represented by Formula I-5, I-6, I-7, I-8, I-9, I-10, or I-11 or its stereoisomers or its pharmaceutically acceptable salts or solvates:
  • Abu is the antibody or antigen-binding unit provided by the present invention.
  • p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell nutritional factor, a steroid drug, a drug for treating autoimmune diseases, an anti-inflammatory drug or a drug for treating infectious diseases.
  • the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.
  • tubulin inhibitor is selected from the group consisting of dolastatin, auristatin, and maytansine.
  • the drug is an auristatin, such as MMAE, MMAF or AF.
  • the drug is a DNA damaging agent, such as calicheamicins, duocarmycins, and antromycin derivatives PBD (pyrrolobenzodiazepine, pyrrolobenzodiazepine).
  • a DNA damaging agent such as calicheamicins, duocarmycins, and antromycin derivatives PBD (pyrrolobenzodiazepine, pyrrolobenzodiazepine).
  • the drug is a DNA topoisomerase inhibitor or a salt thereof, such as irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10 -Hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyecliptaline, topotecan, letotecan, bellotecan, et Xitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazole Linone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(pheny
  • the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, bellotecan, irinotecan, 22-hydroxyecliptaline or ixotecan Kang.
  • the drug is Tubulysin, taxane drug derivatives, leptomycine derivatives, CC-1065 and its analogs, Amatoxin, spliceosome inhibitors, benzodiazepine (PBD) dimer doxorubicin, methotrexate, vincristine, vinblastine, daunorubicin, mitomycin C, melphalan, or chlorambucil derivatives.
  • X 1 and X 2 are each independently:
  • Amino an amino group substituted by an amino protecting group, a C1-C6 aminoalkyl group optionally substituted in the amino portion by an amino protecting group or a C1-C6 alkyl group,
  • a C1-C6 aminoalkylamino group optionally substituted in the amino portion by an amino protecting group or a C1-C6 alkyl group,
  • C1-C6 alkyl attached to a heterocycle optionally substituted by one or more C1-C6 alkyl, C1-C6 alkoxy, amino, halogen, nitro or cyano,
  • C1-C6 alkylamino group connected to a heterocyclic ring is optionally substituted by C1-C6 alkyl, C1-C6 alkoxy group, the amino group is optionally protected by an amino protecting group, halogen, nitro, cyano group or protective group substitution,
  • Amino-substituted heterocyclyl the nitrogen atom or amino portion of the heterocyclic part is optionally substituted by a protecting group or one or more C1-C6 alkyl groups,
  • Heterocyclic amino group, the nitrogen atom or amino part of the heterocyclic part is optionally substituted by a protecting group or a C1-C6 alkyl group,
  • Carbamoyl optionally substituted by a carbamoyl protecting group or C1-C6 alkyl,
  • X 3 is C1-C6 alkyl
  • X 4 is H, -(CH 2 ) q -CH 3 , -(CHR n ) q -CH 3 , C3-C8 carbocyclic group, -O-(CH 2 ) q -CH 3 , arylene group -CH 3 , -(CH 2 ) q -arylene-CH 3 , -arylene-(CH 2 ) q -CH 3 , -(CH 2 ) q -(C3-C8 carbocyclyl)-CH 3 , -( C3-C8 carbocyclyl)-(CH 2 ) q -CH 3 , C3-C8 heterocyclyl, -(CH 2 ) q -(C3-C8 heterocyclyl)-CH 3 , -(C3-C8 heterocycle base)-(CH 2 ) q -CH 3 , -(CH 2 ) q C(O)NR n (CH 2 ) q
  • y 0, 1 or 2;
  • Y is O, S or CR 1 R 2 , where R 1 and R 2 are each independently H or C1-C6 alkyl;
  • s and t are each independently 0, 1, or 2, but not 0 at the same time.
  • X 4 is H or C1-C6 alkyl.
  • the heterocycle is azetidine, glycolazine, morpholine, pyrrolidine, piperidine, imidazole, thiazole, oxazole, or pyridine.
  • the amino protecting group is formyl, acetyl, trityl, tert-butoxycarbonyl, benzyl or p-methoxybenzyloxycarbonyl.
  • the drug is Wherein X 1 and X 2 are each independently C1-C6 alkyl, halogen or -OH; ** is connected to other parts of the antibody drug conjugate.
  • the drug is Wherein X 1 and X 2 are each independently C1-C6 alkyl, halogen or -OH; ** is connected to other parts of the antibody drug conjugate.
  • the C1-C6 alkyl group is -CH3 .
  • the halogen is F.
  • each of X 1 and X 2 is independently -CH 3 , F, or -OH.
  • X 1 and X 2 are each -CH 3 .
  • X 1 and X 2 are each independently F, Cl, Br, or I.
  • X 1 and X 2 are each F.
  • X 1 and X 2 are each independently F or -CH 3 .
  • X 1 is -CH 3 and X 2 is F.
  • the antibody drug conjugate has the formula I-12, I-13, I-14, I-15, I-16, I-17, I-18, I-19, The structure represented by I-20, I-21, I-22, I-23, I-24, or I-25 or its stereoisomer or its pharmaceutically acceptable salt or solvate:
  • Abu is the antibody or antigen-binding unit provided by the present invention.
  • p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • p is 2-8.
  • p is 4-8.
  • p is 6-8.
  • p is 7-8.
  • One or more embodiments provide antibodies or antigen-binding units or antibody-drug conjugates for use as medicaments.
  • the antibodies or antigen-binding units or antibody drug conjugates of the invention are used in combination with one or more other therapies.
  • Suitable additional therapies include existing pharmaceutical and/or surgical therapies for specific applications such as cancer.
  • the antibody or antigen binding unit or antibody drug conjugate is used in combination with one or more other chemotherapeutic or anti-neoplastic agents.
  • the other chemotherapeutic agent is radiation therapy.
  • the chemotherapeutic agent is a cell death inducing agent.
  • the antibody or antigen binding unit or antibody drug conjugate and other agents are prepared as a single therapeutic composition and the antibody or antigen binding unit or antibody drug conjugate and other agents are administered simultaneously.
  • Other reagents are prepared as independent therapeutic compositions, for example, they are prepared as independent therapeutic compositions, and the antibody or antigen-binding unit or antibody-drug conjugate and other reagents are administered simultaneously.
  • agent, or the antibody or antigen-binding unit or antibody-drug conjugate and other agent are administered at different times during the treatment regimen.
  • the antibody or antigen-binding unit or antibody-drug conjugate is administered before the other agent is administered, the antibody or antigen-binding unit or antibody-drug conjugate is administered after the other agent is administered, or in an alternating schedule.
  • the antibody or antigen-binding unit or antibody-drug conjugate and other agent are administered in a single dose or in multiple doses.
  • the present invention also provides a pharmaceutical composition, which includes the antibody or antigen-binding unit or antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier, excipient and/or or excipients.
  • the pharmaceutical composition further comprises optional other anti-cancer drugs.
  • the pharmaceutical composition provided by the present invention can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelium or skin mucosa (such as oral mucosa, rectum and intestinal mucosa, etc.), and can be co-administered with other biologically active agents. .
  • the pharmaceutical composition may be administered intravenously, orally, rectally, parenterally, intracerebrally, intravaginally, intraperitoneally, externally (e.g., via powder, ointment, drops) or transdermal patch), orally, or by oral or nasal spray.
  • the disease is a disease associated with CLDN18.2 expression. In one or more embodiments, the disease is a disease associated with overexpression of CLDN18.2. In one or more embodiments, the disease is a cancer or tumor expressing CLDN18.2. In one or more embodiments, the disease is a cancer or tumor that overexpresses CLDN18.2.
  • the disease is a disease associated with CLDN18.2 expression.
  • the disease is cancer or tumor.
  • the disease is a disease associated with overexpression of CLDN18.2.
  • the disease is a cancer or tumor expressing CLDN18.2.
  • the disease is a cancer or tumor that overexpresses CLDN18.2.
  • One or more embodiments provide a method of treating and/or preventing disease, the method comprising administering to a patient in need thereof an effective amount of an antibody or antigen-binding unit, biological material (selected from polynucleotides, Expression vectors and cells), antibody drug conjugates and/or pharmaceutical compositions containing the same.
  • the disease is cancer or tumor.
  • the disease is a disease associated with CLDN18.2 expression.
  • the disease is a disease associated with overexpression of CLDN18.2.
  • the disease is a cancer or tumor expressing CLDN18.2.
  • the disease is a cancer or tumor that overexpresses CLDN18.2.
  • the effective amount refers to the amount of active compound or agent that results in the biological or pharmaceutical effects of the tissue, system, animal, individual, and human being sought by the researcher, veterinarian, physician, or other clinician. With response, this involves treating a disease. In one or more embodiments, the effective amount is about 0.1 to 100 mg/kg.
  • cancers include, but are not limited to, solid tumors, hematological cancers, and metastatic lesions.
  • the cancer or tumor includes, but is not limited to, bladder cancer, ovarian cancer (such as ovarian adenocarcinoma and ovarian teratocarcinoma), lung cancer (such as small cell lung cancer and non-small cell lung cancer), adenocarcinoma, gastric cancer, Breast cancer, liver cancer, pancreatic cancer, skin cancer (such as basal cell carcinoma and squamous cell carcinoma), malignant melanoma, head and neck cancer, sarcoma (such as synovial sarcoma and carcinosarcoma), cholangiocarcinoma, kidney cancer (clear cell renal cell carcinoma) carcinoma and papillary renal cell carcinoma), colon cancer, small bowel cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer and gallbla
  • the antibody or antigen binding unit or antibody drug conjugate can be formulated into a pharmaceutical composition and administered to the patient in a form suitable for the chosen route of administration, such as parenteral. , intravenously (iv), intramuscularly, topically or subcutaneously.
  • the patient is treated for one cycle of treatment. In one or more embodiments, the patient receives multiple (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) treatment cycles treat. In one or more embodiments, the patient receives treatment until the condition resolves and treatment is no longer required.
  • the antibody or antigen-binding unit or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by injection. In one or more embodiments, the antibody or antigen-binding unit or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by subcutaneous (s.c.) injection, intraperitoneal (i.p.) injection, parenteral injection, intra-arterial injection Administration by injection or intravenous (i.v.) injection. In one or more embodiments, the antibody or antigen-binding unit or antibody-drug conjugate or pharmaceutical composition containing the same is administered by infusion. In one or more embodiments, the antibody or antigen-binding unit or antibody-drug conjugate or pharmaceutical composition containing the same is administered by bolus injection.
  • the antibody or antigen-binding unit or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by intravenous injection. In one or more embodiments, the antibody or antigen-binding unit or antibody-drug conjugate or pharmaceutical composition comprising the same is administered by intravenous infusion.
  • the amount of antibody or antigen-binding unit or antibody-drug conjugate administered will depend on the nature of the drug, the extent to which the cell surface triggers internalization, transport and release of the drug, as well as the disease being treated and the condition of the patient (e.g. age, sex , weight, etc.).
  • the antibody or antigen-binding unit or antibody-drug conjugate or pharmaceutical composition containing the same is administered by intravenous (i.v.) infusion (i.e., intravenous infusion).
  • the intravenous infusion duration is about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 55 minutes, about 60 minutes , about 65 minutes, about 70 minutes, about 75 minutes, about 81 minutes, about 87 minutes, about 90 minutes, about 95 minutes, or a range between any two of these values (inclusive of the endpoints) or any value therein.
  • the intravenous infusion time is ⁇ 30 minutes.
  • the intravenous infusion time is ⁇ 60 minutes.
  • the intravenous infusion time is ⁇ 90 minutes.
  • the invention provides a pharmaceutical composition suitable for injection, such as a push-type pharmaceutical composition or an infusion (drip) type, comprising the antibody or antigen-binding unit or antibody-drug conjugate.
  • a pharmaceutical composition suitable for injection such as a push-type pharmaceutical composition or an infusion (drip) type, comprising the antibody or antigen-binding unit or antibody-drug conjugate.
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (here water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include solvents or dispersion media such as physiological saline, bacteriostatic water or phosphate buffered saline (PBS), ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixture.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be implemented by containing antibacterial and/or antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • the pharmaceutical composition contains at least 0.1% of antibody or antigen binding unit or antibody drug conjugate. The percentage of antibody can vary and is between about 2% and 90% by weight of a given dosage form. The amount of antibody or antigen-binding unit or antibody-drug conjugate in such a pharmaceutical composition may be an effective amount for administration.
  • the invention provides a method for preparing a pharmaceutical composition: respectively, the antibody or antigen-binding unit or antibody-drug conjugate described herein and a pharmaceutically acceptable carrier (such as water for injection, physiological salt water, etc.) and mix.
  • a pharmaceutically acceptable carrier such as water for injection, physiological salt water, etc.
  • the invention provides a kit comprising an antibody or antigen-binding unit as described herein, an antibody-drug conjugate, or a pharmaceutical composition comprising the same, and instructions for instructing administration to a patient.
  • One or more embodiments also provide an article of manufacture comprising an antibody or antigen-binding unit, an antibody-drug conjugate, or a pharmaceutical composition comprising the same according to the invention;
  • Figure 1 shows the dose curve of anti-CLDN18.2 chimeric antibody binding to KATOIII cells, and the ordinate represents the average fluorescence intensity.
  • Figure 2 shows the cell binding specificity results of anti-CLDN18.2 humanized antibodies, in which H239H-2a+K-6a is represented as antibody H239H-2a-K-6a, and H239H-2b+K-6a is represented as antibody H239H-2b. -K-6a.
  • Figure 3 shows the dose binding curve of anti-CLDN18.2 antibody to KATOIII cells; wherein CLDN18.2Ab represents antibody H239H-2b-K-6a-1.
  • Figure 4 shows the dose curve of ADC inhibiting the proliferation of CHO-CLDN18.2 cells.
  • Figure 5 shows the viable cell number statistics of the bystander effect of ADC.
  • Figure 6 shows the growth curve (mean ⁇ standard error) of tumor volume in each group of mice in the GA0006 xenograft model.
  • Figure 7 shows the tumor weight (mean ⁇ standard error) of mice in each group in the GA0006 xenograft model.
  • an entity refers to one or more such entities, e.g. "an antibody” should be understood to mean one or more antibodies, therefore, the term “a” (or “an” ), “one or more” and “at least one” may be used interchangeably herein.
  • the term “comprises” or “includes” means that the antibody, composition, method, etc. includes the listed elements, such as components or steps, but does not exclude others.
  • Consisting essentially of means that the antibody, composition, or method excludes other elements that have a fundamental impact on the characteristics of the combination, but does not exclude elements that do not substantially affect the antibody, composition, or method.
  • Consisting of means the exclusion of elements not specifically enumerated.
  • antibody refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.
  • Antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single chain antibodies, Fab, Fab- and F(ab') 2 fragments, Fv and Fab expression libraries.
  • antibodies includes a wide variety of biochemically distinguishable polypeptides.
  • the antibodies, antigen-binding units or derivatives disclosed in the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope binding Fragments (eg Fab-like, Fab'-like and F(ab')-like 2 ), single-chain Fvs-like (scFv).
  • mAb refers to a population of antibody molecules that contain only one molecular species of the antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementation of monoclonal antibodies The determining region (CDR) is the same in all molecules of the population. MAbs contain antigen-binding sites capable of immunoreacting with specific epitopes of the antigen.
  • single chain antibody refers to an antibody composed of an antibody heavy chain variable region (VH) and a light chain variable region (VL) connected through a 15-20 amino acid linker.
  • Linkers can be rich in glycine to increase flexibility, and rich in serine or threonine to increase solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa.
  • the protein has had the constant region removed and linkers introduced, it retains the specificity of the original immunoglobulin.
  • ScFv molecules are generally known in the art and are described, for example, in US Patent 5,892,019.
  • classes of heavy chains include gamma, mu, alpha, delta or epsilon (gamma, mu, alpha, delta, epsilon), of which there are also subclasses (eg ⁇ 1- ⁇ 4).
  • the nature of this chain determines the "class" of the antibody: IgG, IgM, IgA, IgG, or IgE.
  • Immunoglobulin subclasses such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and the functional specificities conferred are known. All immunoglobulin species are within the scope of the invention.
  • the immunoglobulin molecule is of IgG type.
  • Two heavy chains and two light chains are connected by disulfide bonds in a "Y" configuration, where the light chain begins at the "Y" mouth and continues through the variable region surrounding the heavy chain.
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can be combined with a kappa or lambda light chain.
  • immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are joined by covalent bonds, and the "tail" portions of the two heavy chains are bonded by covalent disulfide bonds or Non-covalent bonding.
  • the amino acid sequence extends from the N-terminus at the fork end of the Y configuration to the C-terminus at the bottom of each chain.
  • the variable region of the immunoglobulin kappa light chain is V ⁇ ; the variable region of the immunoglobulin lambda light chain is V ⁇ .
  • the antibody light chain variable region (VL) and heavy chain variable region (VH) determine antigen recognition and specificity.
  • the light chain constant region (CL) and heavy chain constant region (CH) confer important biological properties, such as secretion, transplacental movement, Fc receptor binding, complement binding, etc. By convention, the numbering of constant regions increases as they become farther away from the antibody's antigen-binding site, or amino terminus.
  • the N-terminal part is the variable region and the C-terminal part is the constant region; the CH3 and CL domains actually contain the carboxyl termini of the heavy and light chains respectively.
  • each antigen-binding domain is short, short, “complementarity-determining regions” or “CDRs” that form the antigen-binding domain, assuming the antibody assumes its three-dimensional configuration in an aqueous environment.
  • the remaining other amino acids in the antigen-binding domain known as the "framework" ("FR") region, show less inter-molecular variability.
  • FR framework
  • Most of the framework region adopts a ⁇ -sheet conformation, and the CDRs form a ring structure connected to it, or in some cases form part of the ⁇ -sheet structure.
  • the framework region forms a scaffold that positions the CDR in the correct orientation through non-covalent interactions between chains.
  • the antigen-binding domain with CDRs in specific positions forms a surface complementary to the epitope on the antigen that promotes non-covalent binding of the antibody to its epitope.
  • each heavy chain and light chain has three CDRs, which are called VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3.
  • the heavy chain variable region In order of position, usually the heavy chain variable region includes VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3 and VH FR4, and the light chain variable region includes VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3 and VL FR4.
  • VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3 and VH FR4 For a given heavy chain or light chain variable region, one of ordinary skill in the art can identify the amino acids containing the CDR and framework regions by known methods (see Kabat, E., et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
  • the framework and CDR regions of humanized antibodies do not necessarily correspond to the parent sequence exactly.
  • the donor antibody CDR or consensus framework can be mutated through the substitution, insertion and/or deletion of at least one amino acid residue, so that the CDR at this site or the framework residues do not correspond to the donor antibody or consensus framework.
  • at least 80%, at least 85%, more at least 90%, or at least 95% of the humanized antibody residues will correspond to those residues of the parent FR and CDR sequences.
  • the term "consensus framework" refers to the framework regions in consensus immunoglobulin sequences.
  • the term "consensus immunoglobulin sequence” refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see e.g. For example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). Within the immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either one may be included in the consensus sequence.
  • CDR complementarity determining region
  • Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody.
  • One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence without relying on experimental data other than the sequence itself.
  • “Kabat number” refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983).
  • Antibodies can also use the EU or Chothia numbering system.
  • the antibodies disclosed in the present invention can be derived from any animal, including but not limited to fish, birds and mammals.
  • the antibody is a human, mouse, donkey, rabbit, goat, camel, llama, horse or chicken antibody.
  • the variable regions may be of condricthoid origin (eg, from sharks).
  • Heavy chain constant region includes at least one of a CH1 domain, a hinge (eg, upper, middle and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment.
  • the heavy chain constant region of an antibody can be derived from different immunoglobulin molecules.
  • the heavy chain constant region of an antibody may include a CH1 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule.
  • the heavy chain constant region may include a hinge region derived partially from an IgG1 molecule and partially from an IgG3 molecule.
  • a portion of the heavy chain may comprise a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG4 molecule.
  • Light chain constant region includes a portion of the amino acid sequence derived from an antibody light chain.
  • the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.
  • a "light chain-heavy chain pair” refers to a collection of light and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.
  • Disulfide bond refers to a covalent bond formed between two sulfur atoms.
  • the thiol group of a cysteine can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are linked by disulfide bonds.
  • a “chimeric antibody” refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial, or modified) are derived from a second species.
  • the variable regions are from a non-human source (eg, mouse or primate) and the constant regions are from a human source.
  • epitope includes any protein-determining region capable of specifically binding to an immunoglobulin or fragment thereof or to a T cell receptor.
  • Epitope-determining regions usually consist of chemically active surface groups of molecules (such as amino acids or sugar side chains) and usually have specific three-dimensional structural properties as well as specific charge properties.
  • the term “specifically binds” or “immunoreacts against” or “against” refers to the non-covalent interaction that occurs between an immunoglobulin molecule and one or more epitopes of its target antigen.
  • the strength or affinity of an immunological binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, where a smaller KD represents greater affinity.
  • KD equilibrium dissociation constant
  • the immunobinding properties of selected polypeptides can be quantified using methods well known in the art. One such method requires measuring the rates of antigen binding site/antigen complex formation and dissociation, where those rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions.
  • both the "association rate constant” ( kon ) and the “dissociation rate constant” ( koff ) can be determined by calculating the concentration and the actual association and dissociation rates (see Malmqvist, M., Nature 361:186-87 (1993)).
  • the k off /k on ratio eliminates all affinity-independent parameters and is equal to the equilibrium dissociation constant KD (see generally Davies et al. (1990) Annual Rev Biochem 59:439-473).
  • Specific binding can be measured by radioligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assays, or similar assays known to those skilled in the art.
  • isolated used in the present invention with respect to cells, nucleic acids, polypeptides, etc., such as “isolated” DNA, RNA, and polypeptides, refers to one or more of the other components in the natural environment of the cell, such as DNA or RNA. Various separated molecules.
  • isolated as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or cell culture media when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is intended to include nucleic acid fragments that do not exist in their native state and do not exist in their native state.
  • isolated is also used herein to refer to cells or polypeptides separated from other cellular proteins or tissues.
  • Isolated polypeptide is intended to include purified and recombinant polypeptides.
  • Isolated polypeptides and the like are generally prepared by at least one purification step.
  • the purity of the isolated nucleic acid, polypeptide, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or these values The range between any two values (inclusive) or any value therein.
  • encoding when applied to a polynucleotide, refers to a polynucleotide that is said to "encode” a polypeptide that, in its native state or when manipulated by methods well known to those skilled in the art, is transcribed and/or Or translation can produce the polypeptide and/or fragments thereof.
  • recombinant refers to a polypeptide or polynucleotide and means a form of the polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may be combined to produce polynucleotides that do not normally exist or Peptides.
  • Amino acid refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which may be encoded by a nucleic acid directly or in the form of a precursor.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called the "degeneracy of the genetic code.”
  • Amino acids include natural amino acids and unnatural amino acids.
  • the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd edition, edited by E.S. Golub and D.R.Gren, Sinauer Associates, Sunderland, Mass. (1991)). Twenty stereoisomers of conventional amino acids (e.g., D-amino acids), unnatural amino acids (such as ⁇ -, ⁇ -disubstituted amino acids), N-alkylamino acids, and other unconventional amino acids may also be suitable for use in this disclosure. Components of polypeptides.
  • Examples of unconventional amino acids include: citrulline (Cit), 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine amino acid, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, ⁇ -N-methylarginine and other similar Amino acids and imino acids (e.g. 4-hydroxyproline).
  • the left-hand direction is the amino-terminal direction
  • the right-hand direction is the carboxyl-terminal direction, consistent with standard usage and convention.
  • Conventional (or natural) amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D ), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
  • polypeptide is intended to encompass the singular “polypeptide” as well as the plural “polypeptide” and refers to a molecule formed from amino acid monomers linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any single chain or chains of two or more amino acids and does not refer to a specific length of the product.
  • the definition of “polypeptide” includes peptide, dipeptide, tripeptide, oligopeptide, "protein,””amino acid chain” or any other term used to refer to two or more amino acid chains, and the term “polypeptide” may Used instead of or interchangeably with any of the above terms.
  • polypeptide is also intended to refer to the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur.
  • a polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it does not have to be translated from a specified nucleic acid sequence and may be produced by any means including chemical synthesis.
  • amino acid or polypeptide when an amino acid or polypeptide is a component of a molecule (such as an antibody or ADC), the amino acid or polypeptide refers to the amino acid residue or polypeptide residue (whether stated or not), that is, it is in relation to other parts of the molecule.
  • some of its groups such as a hydrogen atom of its amino group and/or the hydroxyl group of its carboxyl group
  • covalent bonds such as amide bonds
  • substantially identical means that two peptide sequences share at least 80% sequence identity, preferably at least 90% sequence identity when optimally aligned, such as by the GAP or BESTFIT programs using default gap weights. identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity.
  • a polynucleotide is composed of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when the polynucleotide is RNA Thymine is replaced with uracil (U).
  • a "polynucleotide sequence” may be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used in bioinformatics applications, such as for functional genomics and homology searches.
  • polynucleotide refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or the like.
  • Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.
  • genes or gene fragments e.g.
  • polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modifications are present, structural modifications to the nucleotide may be made before or after assembly of the polynucleotide.
  • sequence of nucleotides can be interrupted by non-nucleotide components.
  • the polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component.
  • This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include double-stranded forms and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • a polynucleotide or polynucleotide sequence has a certain percentage (eg, 90%, 95%, 98% or 99%) of "identity or sequence identity” to another sequence It refers to the percentage of bases (or amino acids) in the two sequences being compared that are identical when the sequences are compared.
  • the alignment and percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described in Ausubel et al. eds. (2007), Current Protocols in Molecular Biology. It is preferred to use the default parameters for comparison.
  • Biologically equivalent polynucleotides are polynucleotides that share the percentage identity specified above and encode a polypeptide with the same or similar biological activity.
  • amino acid sequence of the antibody or immunoglobulin molecule are encompassed by the present disclosure, provided that the identity of the amino acid sequence remains at least 90%, such as at least 92%, 95%, 98% or 99%.
  • the changes are conservative amino acid substitutions.
  • Conservative amino acid substitutions are substitutions that occur within a family of related amino acids in their side chains.
  • amino acids encoded by genes are roughly divided into the following categories: (1) Acidic amino acids are aspartate and glutamate; (2) Basic amino acids are lysine, arginine, and histidine; (3) Non-polar amino acids Sexual amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar amino acids are Glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.
  • amino acids include (i) the aliphatic-hydroxyl family of serine and threonine; (ii) the amide-containing family of asparagine and glutamine; (iii) the aliphatic family of alanine, valine, Leucine and isoleucine; and (iv) phenylalanine, tryptophan and tyrosine of the aromatic family.
  • the conservative amino acid substitution group is: valine-leucine-isoleucine, benzene Alanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartate, and asparagine-glutamine.
  • amino acid substitutions have the following effects: (1) reduce sensitivity to proteolysis, (2) reduce sensitivity to oxidation, (3) alter binding affinity for protein complex formation, ( 4) alter binding affinity, and (5) impart or improve other physicochemical or functional properties of such analogs.
  • Analogues may include various muteins whose sequences differ from naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in naturally occurring sequences, preferably in portions of the polypeptide outside of domains forming intermolecular contacts.
  • Conservative amino acid substitutions should not significantly alter the structural properties of the parent sequence (e.g., the replaced amino acids should not tend to disrupt the helical structure present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence).
  • Examples of artificially identified secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (edited by Creighton, W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (edited by C. Branden and J. Tooze) , Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).
  • the number of amino acids substituted by conservative amino acids of VL and VH can be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11 , about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values (inclusive) or any value therein.
  • the number of amino acids substituted by conservative amino acids in the heavy chain constant region, light chain constant region, heavy chain or light chain can be about 1, about 2, about 3, about 4, about 5, about 6, about 8 About 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, About 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values, inclusive, or any value therein.
  • agent means a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material.
  • labeled refers to the incorporation of a detectable label, e.g., by incorporation of a radioactively labeled amino acid, or attachment to an avidin that may be labeled (e.g., containing a fluorescent label or that may be optically labeled). Methods or calorimetry to detect the enzymatic activity of streptavidin) to detect the biotinyl moiety of the polypeptide. In some cases, markers or labels may also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I ), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase), chemiluminescence Tag, biotinyl group, predetermined polypeptide epitope recognized by the secondary reporter gene (e.g., leucine zipper pair sequence, secondary antibody binding site, metal binding domain, epitope tag).
  • labels are attached via spacer arms of various lengths to reduce possible steric hindrance.
  • the term "agent” or “drug” refers to a compound or composition capable of inducing a desired therapeutic effect when appropriately
  • EC 50 is the concentration for 50% of maximal effect (EC 50 ), which refers to the concentration that can cause 50% of the maximum effect.
  • IC 50 represents the 50% inhibitory concentration, that is, the concentration of a drug or inhibitor required to inhibit half of a specified biological process.
  • Treatment means therapeutic treatment and prophylactic or preventative measures designed to prevent, slow down, ameliorate or halt adverse physiological changes or disorders, such as the progression of a disease, including but not limited to the following whether detectable or undetectable
  • the results include alleviation of symptoms, reduction in disease severity, stabilization of disease status (i.e. no worsening), delay or slowdown of disease progression, improvement, alleviation, reduction or disappearance of disease status (whether partial or complete), prolongation and Expected survival without treatment, etc.
  • Patients in need of treatment include patients who already have a condition or disorder, are susceptible to a condition or disorder, or are in need of prevention of a condition or disorder for which they can or are expected to benefit from administration of an antibody or pharmaceutical composition disclosed herein for detection. , patients who benefit from the diagnostic process and/or treatment.
  • cancer means or is intended to describe a physiological state in mammals that is typically characterized by uncontrolled cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, or leukemia. More specific examples of such cancers include, but are not limited to, colorectal cancer, lung cancer, ovarian cancer, uterine cancer, endometrial cancer, salivary gland cancer, peritoneal cancer, fallopian tube cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer Cancer, laryngeal cancer, lung adenocarcinoma, lung squamous cell carcinoma, liver cancer, hepatocellular carcinoma, gastrointestinal cancer, glioblastoma, breast cancer, brain cancer, kidney cancer, renal cell carcinoma, colon cancer, rectal cancer, prostate cancer carcinoma, vulvar cancer, testicular cancer, squamous cell carcinoma, small cell lung cancer, cervical cancer, bladder cancer, retin
  • overexpressed or “overexpressed” interchangeably refer to a gene that is typically transcribed or translated at a detectably higher level in certain cells, such as cancer cells, compared to normal cells.
  • Overexpression can be overexpression of protein or RNA (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability and altered protein degradation), as well as localized overexpression due to altered protein trafficking patterns (nuclear localization increased) and enhanced functional activity, e.g., increased enzymatic hydrolysis of substrates.
  • Overexpression can be 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90% or more compared to normal cells or control cells.
  • the antibodies or antigen-binding units or antibody drug conjugates of the invention are used to treat solid tumors that may express CLDN18.2.
  • tumor overexpressing CLDN18.2 refers to tumors (including benign tumors and cancers) that overexpress CLDN18.2.
  • expression of CLDN18.2 in a tumor sample that is above background levels in immune tissue indicates that the tumor is a tumor that overexpresses CLDN18.2.
  • Methods for detecting CLDN18.2 expression in tumors are known in the art, such as immunohistochemical assays.
  • CLDN18.2 negative cells are cells in the cell sample that lack CLDN18.2 above background (eg, as determined by immunohistochemical techniques).
  • administering means delivering a substance (eg, an anti-CLDN18.2 antibody or ADC) for a therapeutic purpose (eg, treating a CLDN18.2-related disease).
  • Modes of administration may be parenteral, enteral and topical.
  • Parenteral administration is usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intrasaccular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, intravesicular Subarachnoid, intraspinal, and intrasternal injections and infusions.
  • the term "effective amount” or “therapeutically effective amount” refers to an amount of a drug, such as an antibody or ADC, that is sufficient to reduce or ameliorate the severity and/or severity of a condition (e.g., cancer) or one or more symptoms thereof. or duration; to prevent the progression of a condition; to cause resolution of a condition; to prevent the recurrence, development, onset or progression of one or more symptoms associated with a condition; to detect a condition; or to enhance or improve another therapy (e.g., a prophylactic or therapeutic agent) Amount of preventive or therapeutic effect.
  • a condition e.g., cancer
  • ADC an antibody or ADC
  • an effective amount of the antibody can inhibit tumor growth (e.g., inhibit an increase in tumor volume); reduce tumor growth (e.g., reduce tumor volume); reduce the number of cancer cells; and/or alleviate cancer-related symptoms to a certain extent. one or more symptoms.
  • an effective amount may improve disease-free survival (DFS), improve overall survival (OS), or reduce the likelihood of relapse.
  • DFS disease-free survival
  • OS overall survival
  • patient and “subject” are used interchangeably and refer to any mammal in need of diagnosis, prognosis or treatment, including but not limited to humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, Niu et al.
  • the term "in need” means that a patient has been identified as needing a particular method or treatment. In some embodiments, identification can be by any diagnostic means. Patients may require any of the methods and treatments described in this article.
  • the term "administration” refers to the administration of a substance for a therapeutic purpose (eg, to treat a tumor).
  • tumor-treating drug refers to an agent that has the functional property of inhibiting the development or progression of tumors in the human body, especially malignant (cancerous) lesions such as carcinoma, sarcoma, lymphoma or leukemia. Inhibition of metastasis is in many cases a property of antineoplastic agents.
  • pharmaceutically acceptable carrier generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary agent, etc.
  • carrier refers to a diluent, adjuvant, excipient or vehicle with which the active ingredient can be administered to a patient.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • water is the preferred carrier.
  • Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, skimmed milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. .
  • the compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite.
  • These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, and the like.
  • compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides.
  • Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, which is hereby incorporated by reference.
  • Such compositions will contain a clinically effective dose of the antibody or antigen-binding unit or ADC, preferably in purified form, together with an appropriate amount of carrier to provide a dosage form suitable for the patient.
  • the formulation should be suitable for the mode of administration.
  • the parent preparation may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.
  • antibody drug conjugate refers to a binding protein (such as an antibody or antigen-binding unit) linked to one or more chemical agents, which may optionally be therapeutic or cytotoxic agents.
  • the ADC includes an antibody, a drug (eg, a cytotoxic drug), and a linker that enables the drug to be attached or coupled to the antibody.
  • Non-limiting examples of drugs that may be included in the ADC are mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemical Protective agents, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors (e.g., TEC-family kinase inhibitors and serine/threonine amino acid kinase inhibitors) and radiosensitizers.
  • mitotic inhibitors e.g., anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemical Protective agents, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents
  • antibody drug conjugate and “ADC” are used interchangeably.
  • anti-CLDN18.2 antibody drug conjugate and “anti-CLDN18.2 ADC” are used interchangeably and refer to an ADC containing an antibody that specifically binds CLDN18.2, wherein the antibody is conjugated to one or more drugs.
  • the anti-CLDN18.2 ADC comprises an antibody conjugated to ixotecan.
  • an anti-CLDN18.2 antibody or ADC binds to CLDN18.2 (eg, human CLDN18.2).
  • DAR drug-antibody conjugation ratio
  • the term “drug-antibody conjugation ratio” or “DAR” refers to the amount of drug (eg, ixotecan) of the ADC that is attached to the antibody.
  • the DAR of an ADC can range from 1 to 10, but depending on the number of attachment sites on the antibody, higher loadings (e.g., 20) are also possible.
  • the term DAR may be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs. In some embodiments, the value is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the average number of small molecule drug bindings that is, the average drug binding number of the antibody, or the average drug-antibody coupling ratio
  • its value is selected from about 0 to about 10, or about 2 to about 8.
  • the drug-to-antibody conjugation ratio is from about 3 to about 6.
  • the drug-to-antibody conjugation ratio is from about 6 to about 8, or from about 7 to about 8.
  • the DAR value can be expressed as p in this article.
  • the DAR value of the ADC is available using UV-visible Absorption spectrometry (UV-Vis), high-performance liquid chromatography-hydrophobic chromatography (HPLC-HIC), high-performance liquid chromatography-reversed-phase chromatography (RP-HPLC), liquid chromatography-mass spectrometry (LC-MS) and other determinations. These techniques are documented in Ouyang, J. Methods Mol Biol, 2013, 1045: p. 275-83.
  • substituents are defined below.
  • substituents e.g., alkyl, alkenyl, alkynyl, alkoxy, aminoalkoxy, aminoalkyl, aminoalkylamino, alkylamino, heterocyclyl, heterocyclicamino, and The number of carbon atoms in an aryl group
  • Cx-Cy or “Cx-y”
  • x is the minimum number of carbon atoms
  • y is the maximum number of carbon atoms.
  • C1-C6 alkyl refers to an alkyl group containing from 1 to 6 carbon atoms.
  • a substituent is described as "substituted," a hydrogen atom on the carbon or nitrogen is replaced by a non-hydrogen group.
  • a substituted alkyl substituent is an alkyl substituent in which at least one hydrogen atom on the alkyl group is replaced with a non-hydrogen group.
  • a monofluoroalkyl group is an alkyl group substituted with one fluoro group
  • a difluoroalkyl group is an alkyl group substituted with two fluoro groups. It should be appreciated that if more than one substitution is present on a substituent, each substitution may be the same or different (unless otherwise stated).
  • substituents include, but are not limited to, hydroxyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 aminoalkoxy, halogen, nitro, cyano , thiol, alkylthio, amino, C1-C6 aminoalkyl, C1-C6 aminoalkylamino, C1-C6 alkyl connected to heterocycle, C1-C6 alkylamino connected to heterocycle, heterocyclyl , amino-substituted heterocyclyl, heterocyclic amino, carbamoyl, morpholin-1-yl, piperidin-1-yl, -(CH 2 ) q -CH 3 , -(CHR n ) q -CH 3 , C3-C8 carbocycly
  • Alkyl refers to a saturated aliphatic hydrocarbon group, and the term includes straight and branched chain hydrocarbon groups.
  • C1-C20 alkyl such as C1-C6 alkyl.
  • C1-C20 alkyl refers to an alkyl group with 1 to 20 carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 Alkyl groups of 18 carbon atoms, 19 carbon atoms or 20 carbon atoms.
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, and the like.
  • the alkyl group may be unsubstituted or substituted by one or more substituents, including but not limited to alkyl, alkoxy, cyano, hydroxyl, carbonyl, carboxyl, aryl, heteroaryl, Amino, halogen, sulfonyl, sulfinyl, phosphono, etc.
  • alkenyl by itself or as part of another substituent means an unsaturated branched, straight-chain or cyclic alkyl group having at least one carbon-carbon double bond removed from a single carbon atom of the parent alkene obtained from a hydrogen atom.
  • Typical alkenyl groups include but are not limited to vinyl; propenyl (such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-ene -2-yl, cycloprop-1-en-1-yl); cycloprop-2-en-1-yl; butenyl (such as but-1-en-1-yl, but-1-en-2 -yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl , but-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobut-1,3-dien-1-yl, etc. )wait.
  • propenyl such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-ene -2-y
  • alkynyl by itself or as part of another substituent refers to an unsaturated branched, straight-chain, or cyclic alkyl group having at least one carbon-carbon triple bond formed through a single carbon atom of the parent alkyne. Obtained by removing one hydrogen atom.
  • Typical alkynyl groups include but are not limited to ethynyl; propynyl (such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.); butynyl (such as but-1-yn-1-yl) -yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.), etc.
  • Carbocyclyl refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical composed only of carbon and hydrogen atoms, which May include fused or bridged ring systems having 3 to 15 carbon atoms, for example having 3 to 10 (eg 3, 4, 5, 6, 7, 8, 9 or 10) carbon atoms, and which are saturated or unsaturated and connected to the remainder of the molecule by a single bond.
  • Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • Polycyclic free radicals include, for example, adamantyl, norbornyl, decalinyl, and the like.
  • the carbocyclic group may be optionally substituted with one or more substituents independently selected from the following: alkyl, halogen, haloalkyl, cyano, nitro, oxo, aryl , aralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl.
  • Aryl refers to an all-carbon monocyclic or all-carbon fused ring with a fully conjugated ⁇ electron system, usually having 5 to 14 carbon atoms, for example, 6, 10, 12, 14 carbon atoms.
  • Aryl groups may be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxyl, carboxyl, aryl, aralkyl, amine, Halogen, sulfonyl, sulfinyl, phosphonoyl.
  • unsubstituted aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl.
  • Heterocyclyl refers to a stable 3- to 18-membered aromatic or nonaromatic ring substituent consisting of 2 to 8 (e.g., 2, 3, 4, 5, 6, 7 or 8) carbon atoms and It consists of 1 to 6 (1, 2, 3, 4, 5 or 6) heteroatoms selected from nitrogen, oxygen and sulfur.
  • the heterocyclyl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl group may be any is optionally oxidized; the nitrogen atoms are optionally quaternized; and the heterocyclyl group may be partially or fully saturated.
  • heterocyclyl groups include, but are not limited to, dioxolanyl, dioxinyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, iso Thiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl , oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, piperrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, 1,2,4-thiadiazole -5(4H)-yl, tetrahydrofuryl, trioxanyl, trithialkyl,
  • heterocyclyl may be optionally substituted with one or more substituents selected from the group consisting of: alkyl, alkenyl, halogen, haloalkyl, cyano, oxo, thioxo , nitro, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted hetero Arylalkyl.
  • substituents selected from the group consisting of: alkyl, alkenyl, halogen, haloalkyl, cyano, oxo, thioxo , nitro, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted hetero Arylalkyl.
  • Alkoxy refers to the formula -O-(alkyl), where alkyl is alkyl as defined herein.
  • alkoxy groups are methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy , tert-butoxy. Alkoxy groups may be substituted or unsubstituted.
  • Halogen refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).
  • Amino refers to -NH2 .
  • Cyano refers to -CN.
  • Niro refers to -NO 2 .
  • Carboxyl refers to -COOH.
  • Stereoisomers refer to isomeric compounds that have the same order of atoms connected, but different arrangements of atoms in space. Stereoisomers can have one or more stereocenters and each center can exist as R or S. Stereoisomers can also be cis-trans isomers. Stereoisomers of the compounds provided herein include all diastereomeric, enantiomeric, and cis-trans isomeric forms thereof, any one or mixture thereof.
  • Pharmaceutically acceptable salts include pharmaceutically acceptable salts of the compounds with a wide variety of organic and inorganic counterions well known in the art.
  • Merely exemplary salts include, when the molecule contains acidic functional groups, organic or inorganic salts. Such as lithium salt, sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, isopropylamine, trimethylamine, diethylamine, triethylamine, triethylamine Propylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine , ethylenediamine, glucosamine, methylglucamine, theobromine, purinepiperazine, piperidine, N-ethyl, piperidine, polyamine resin and tetraalkylammonium salt, etc.; and when the molecule contains basic functional groups When used, organic or
  • acids include sulfuric acid, nitric acid, phosphoric acid, propionic acid, glycolic acid, pyruvic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, Ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.
  • These salts can generally be prepared by conventional methods by reacting, for example, a suitable acid or base with the compound.
  • Solvates include hydrates.
  • the antibodies or antigen-binding units of the invention are capable of specifically binding to CLDN18.2.
  • the antibody or antigen-binding unit is a murine antibody, a chimeric antibody, or a humanized antibody.
  • the antibody or antigen-binding unit of the invention has one or more of the following properties: a) specifically binds to CLDN18.2; b) high affinity; c) strong ADCC activity; d) Strong CDC activity.
  • an antibody or antigen-binding unit comprises an amino acid sequence having one or more modifying groups.
  • the antibodies or antigen-binding units disclosed herein may contain flexible linker sequences, or may be modified to add functional groups (eg, PEG, drugs, toxins, or tags).
  • the antibodies and antigen-binding units disclosed in the present invention include modified derivatives, that is, modified by covalent linkage of any type of molecule to the antibody or antigen-binding unit, where the covalent linkage does not prevent the antibody or antigen-binding unit from binding to the epitope.
  • antibodies or antigen binding units can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to Cell ligands or other proteins, etc. Any of numerous chemical modifications can be performed by existing technologies, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
  • the antibody or antigen-binding unit may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.
  • Antibodies or antigen-binding units can be detectably labeled by coupling them to chemiluminescent compounds. The presence of the chemiluminescently labeled antibody or antigen-binding unit is then determined by detecting the luminescence that occurs during the chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts and oxalate esters.
  • antibodies or antigen-binding units are prepared that do not elicit a deleterious immune response in the animal to be treated (eg, a human).
  • the antibodies, antigen-binding units, or derivatives disclosed herein are modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, that retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans.
  • CDRs complementarity-determining regions
  • the framework residues in the human framework region will be derived from the CDR donor antibody Replacement of corresponding residues of the body, such as residues that improve antigen binding.
  • framework substitutions can be identified by methods well known in the art, such as by modeling the interaction of CDRs and framework residues to identify framework residues that are important for antigen binding and by sequence comparison to identify aberrant framework residues at specific positions ( See U.S. Patent 5,585,089; the entire contents of which are incorporated herein by reference).
  • Antibodies can be humanized using a variety of techniques well known in the art, such as CDR grafting (WO1991009967; US Patents 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP592,106; EP519,596), and chain rearrangement ( U.S. Patent 5,565,332), the entire contents of which are incorporated herein by reference.
  • Deimmunization can also be used to reduce the immunogenicity of antibodies.
  • the term "deimmunization” includes altering antibodies to modify T cell epitopes (see eg WO2000034317A2).
  • the heavy chain variable region sequence and the light chain variable region sequence from the starting antibody are analyzed and a "map" of human T cell epitopes from each variable region is generated, showing the epitopes relative to complementarity determining regions (CDRs) and the location of other key residues within the sequence.
  • CDRs complementarity determining regions
  • a series of alternative heavy chain variable domain sequences and light chain variable domain sequences containing combinations of amino acid substitutions are designed and subsequently incorporated into a series of binding polypeptides.
  • Genes containing the complete heavy and light chains with modified variable regions and human constant regions are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce intact antibodies.
  • the antibodies are then compared in appropriate biochemical and biological experiments to identify the optimal antibody.
  • scFv The preparation of scFv can be seen in the technology for producing single-chain units (U.S. Patent 4,946,778).
  • Single-chain units are formed by bridging the heavy and light chain segments of the Fv region with amino acids, resulting in a single-chain fusion peptide.
  • Techniques for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 240:1038-1041 (1988)).
  • scFv single chain Fv
  • antibodies examples include those described in U.S. Patent Nos. 4,946,778 and 5,258,498.
  • chimeric, humanized, or fully human antibodies may be used.
  • Chimeric antibodies are a class of molecules in which different parts of the antibody are derived from different animal species, such as an antibody that has the variable region of a murine monoclonal antibody and the constant region of a human immunoglobulin.
  • Methods of producing chimeric antibodies are known in the art, see U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference.
  • hybridoma technology is used to prepare the antibodies of the invention.
  • Monoclonal antibodies are prepared using, for example, hybridoma methods, such as described by Kohler and Milstein, Nature, 256:495 (1975).
  • mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to induce the production of lymphocytes or antibodies that specifically bind the immunizing agent.
  • Immunizing agents will typically include protein antigens, fragments thereof, or fusion proteins thereof.
  • peripheral blood lymphocytes are used; if non-human mammalian sources are desired, spleen lymphocytes or lymph node cells are used.
  • splenic lymphocytes are used; the lymphocytes are then fused to an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin.
  • Hybridoma cells can be cultured in a suitable culture medium, which in some embodiments contains one or more substances that inhibit the growth or survival of unfused immortalized cells.
  • a suitable culture medium typically includes hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT)
  • HGPRT or HPRT hypoxanthine-guanine phosphoribosyltransferase
  • the hybridoma's culture medium typically includes hypoxanthine, aminopterin, and thymine (“HAT medium"), so The substances prevent the growth of HGPRT-deficient cells.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Podbard, Anal. Biochem., 107:220 (1980).
  • the clones can be subcloned using a limiting dilution procedure and grown using standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103) .
  • Media suitable for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium.
  • monoclonal antibodies secreted by subclones can be isolated or purified by conventional techniques, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity Chromatography.
  • Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies described herein can be isolated and sequenced using conventional methods (eg, by using oligonucleotide probes capable of binding specifically to the genes of the heavy and light chains of the antibody).
  • DNA encoding the antibodies described herein can also be designed and synthesized based on the antibody sequence by conventional methods.
  • the isolated or synthesized DNA is inserted into an expression vector, which is then transfected into host cells such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, simian COS cells, PER, which do not otherwise produce immunoglobulins.
  • NS0 cells, SP2/0, YB2/0 or myeloma cells to obtain synthetic monoclonal antibodies in recombinant host cells.
  • hybridoma technology is used to prepare the antibody or antigen-binding unit of the invention, and a protein preparation containing CLDN18.2 is used to immunize mice. After immunization, the spleen lymphocytes and myeloma of the mice are removed. The cells were fused, and the hybridomas were screened by CLDN18.2 protein, and the positive hybridomas were limitedly diluted and further subcloned to re-identify the binding ability of the hybridoma strains to CLDN18.2 protein to prepare anti-CLDN18.2 Antibody.
  • the mouse is a female Balb/c mouse 6-8 weeks old.
  • the binding specificity of the antibodies or antigen-binding units disclosed in the present invention can be detected by in vitro experiments, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • in vitro experiments such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • Chimeric antibodies are a class of molecules in which different parts of the antibody are derived from different animal species, such as an antibody that has the variable region of a murine monoclonal antibody and the constant region of a human immunoglobulin.
  • Methods of producing chimeric antibodies are known in the art, see U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816397, the entire contents of which are incorporated herein by reference.
  • Human antibodies can also be produced by transgenic mice that do not express functional endogenous immunoglobulins but express human immunoglobulin genes.
  • human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells either randomly or by homologous recombination.
  • human variable, constant and diversity regions can be introduced into mouse embryonic stem cells.
  • Mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional by introduction of human immunoglobulin loci via homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies.
  • the modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice.
  • Transgenic mice are immunized in a conventional manner with a selected antigen, eg, all or part of the desired polypeptide target.
  • Antigen-targeting monoclonal antibodies can be obtained from immunized transgenic mice using conventional hybridoma technology.
  • Transgenic mice carry human immunoglobulin transgenes that rearrange during B cell differentiation, with subsequent class switching and somatic mutations. Therefore, using this technology it is possible to generate IgG, IgA, IgM and IgE antibodies that can be used therapeutically.
  • DNA encoding the desired monoclonal antibody can be readily isolated using conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding murine antibody heavy and light chains) and It is sequenced. Once isolated, the DNA can be placed into expression vectors and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins. in cells. Isolated DNA (which may be synthetic as described herein) can also be used to prepare sequences for the constant and variable regions of antibodies, as described in U.S.
  • prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins. in cells.
  • Isolated DNA (which may be synthetic as described here
  • Patent 5,658,570 the entire contents of which are incorporated herein by reference. This method selects RNA is extracted from cells and converted into cDNA, and then amplified by PCR using Ig-specific primers. Suitable probes for this purpose are also described in US Patent 5,658,570.
  • one or more CDRs of the antibody or antigen-binding unit of the invention can be inserted into the framework region, for example, into the human framework region to construct a humanized non-fully human antibody.
  • the framework regions may be naturally occurring or shared framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a series of human framework regions).
  • Some polynucleotides may encode a combination of framework regions and CDRs to produce an antibody that specifically binds to at least one epitope of the target antigen.
  • one or more amino acid substitutions within the framework regions one can select amino acid substitutions that improve the binding of the antibody to its antigen.
  • this method can be used to substitute or delete cysteine residues in one or more variable regions involved in the formation of interchain disulfide bonds, thereby generating antibody molecules lacking one or more interchain disulfide bonds.
  • Other modifications to polynucleotides within the skill of the art are also encompassed by the present invention.
  • Antibodies can be produced using conventional recombinant DNA techniques.
  • Vectors and cell lines for producing antibodies can be selected, constructed and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, the full text of which is included Supplementary content is incorporated by reference into the full text.
  • the DNA encoding the antibody can be designed and synthesized according to the amino acid sequence of the antibody described herein according to conventional methods, placed into an expression vector, and then transfected into host cells, and the transfected cells are cultured in the culture medium. Host cells produce monoclonal antibodies.
  • an expression antibody vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence.
  • Efficient transcription can be obtained through the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus. Promoters from other cells such as muscle can also be used. Kinesin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, pLNCX, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL , PMSG, pRSVcat, pSV2dhfr, pBC12MI or pCS2, etc. Commonly used mammalian host cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells.
  • the inserted gene fragment needs to contain a selection marker.
  • selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes, so that Screening and isolation of successfully transfected cells.
  • the constructed plasmid is transfected into host cells without the above genes, and then cultured in a selective medium. The successfully transfected cells grow in large quantities and produce the desired protein.
  • mutations can be introduced into the nucleotide sequences encoding the antibodies of the invention using standard techniques known to those skilled in the art, including, but not limited to, site-directed mutagenesis resulting in amino acid substitutions and PCR-mediated mutations.
  • Variants including derivatives encoding substitutions of less than 50 amino acids, substitutions of less than 40 amino acids, substitutions of less than 30 amino acids, substitutions of less than 30 amino acids, relative to the original heavy chain variable region and light chain variable region Substitutions of 25 amino acids, substitutions of less than 20 amino acids, substitutions of less than 15 amino acids, substitutions of less than 10 amino acids, substitutions of less than 5 amino acids, substitutions of less than 4 amino acids, substitutions of less than 3 Substitution of amino acids or substitution of less than 2 amino acids.
  • mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.
  • the antibody or antigen-binding unit of the present invention can be coupled with a drug to form an anti-CLDN18.2 antibody drug conjugate (anti-CLDN18.2 ADC).
  • anti-CLDN18.2 ADC anti-CLDN18.2 antibody drug conjugate
  • ADCs can selectively deliver one or more drugs to target tissues (e.g., cancers or tumors expressing CLDN18.2)
  • target tissues e.g., cancers or tumors expressing CLDN18.2
  • ADCs antibody-drug conjugates
  • the antibody drug conjugate (ADC) of the present invention includes an anti-CLDN18.2 antibody or antigen-binding unit described herein and at least one drug (eg, ixotecan).
  • Antigen-binding fragments or antigen-binding units of the anti-CLDN18.2 antibodies of the invention can be conjugated to drugs described herein. Accordingly, in one or more embodiments, an antigen-binding fragment or antigen-binding unit of an anti-CLDN18.2 antibody described herein is coupled to a drug via a linker to form an anti-CLDN18.2 ADC.
  • Anti-CLDN18.2 ADCs of the invention comprise an antibody or antigen-binding unit that specifically binds CLDN18.2 (eg, human CLDN18.2) linked to one or more drugs.
  • the specificity of the ADC can be determined by the specificity of the antibody (eg, anti-CLDN18.2 antibody) or antigen-binding fragment.
  • an anti-CLDN18.2 antibody is linked to one or more drugs (e.g., DNA topoisomerase inhibitors) that are delivered to the expression CLDN18.2 cells, especially cancer cells expressing CLDN18.2.
  • an anti-CLDN18.2 antibody drug conjugate comprises an anti-CLDN18.2 antibody coupled to a drug (eg, ixotecan) via a linker.
  • a drug eg, ixotecan
  • the anti-CLDN18.2 antibodies described herein provide the ADC with the ability to bind CLDN18.2 so that drugs attached to the antibody can be delivered to CLDN18.2-expressing cells, particularly CLDN18.2-expressing cancer cells.
  • the ADC is a structure as shown in Formula I or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof.
  • Abu is antibody H239H-2b-K-6a-1, H239H-2b-K-6a-2, or H239H-2b-K-6a-3.
  • the ADC is ADC1, ADC2, ADC3, ADC4, ADC5 or ADC6 or a pharmaceutically acceptable salt or solvate thereof.
  • the antibody-drug conjugate of the present invention has a significant bystander effect.
  • the invention also provides methods for preparing intermediates and antibody-drug conjugates.
  • the intermediates and antibody-drug conjugates of the present invention can be prepared by known preparations and methods.
  • methods for preparing intermediate compounds 1-7 are as follows.
  • the first step the compound of general formula 1-1 and the compound of general formula 1-1’ are reacted under alkaline conditions to obtain the compound of general formula 1-2;
  • the second step the compound of general formula 1-2 and the general formula (AA) i -(FF 1 ) f react in the presence of a condensing agent and under alkaline conditions to obtain the compound of general formula 1-3;
  • the third step remove the amino protecting group W 1 of the compound of general formula 1-3 to obtain the compound of general formula 1-4;
  • Step 4 react the compound of general formula 1-4 and the compound of general formula 1-5 under alkaline conditions to obtain the compound of general formula 1-6;
  • Step 5 The compound of general formula 1-6 and bis(p-nitrobenzene) carbonate are reacted under alkaline conditions to obtain the compound of general formula 1-7.
  • W 1 is an amino protecting group, such as 9-fluorenylmethoxycarbonyl
  • W 2 is a carboxylic acid active ester, such as succinimide ester.
  • R F is F.
  • z is zero.
  • z is 1 or 2.
  • the intermediate is:
  • R and n are as described in this article.
  • the intermediate is:
  • R and n are as described in this article.
  • the intermediate is:
  • n is as described in this article.
  • the intermediate is:
  • n is as described in this article.
  • the above-mentioned alkaline conditions can be provided by reagents, which include organic bases and inorganic bases.
  • the organic bases include but are not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide.
  • the inorganic bases include but are not limited to sodium hydride and potassium phosphate. , sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide.
  • the above-mentioned condensation agent can be selected from N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea, 4-(4,6-dimethyl Oxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N ',N'-Tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N,N ',N'-Tetramethylurea hexafluorophosphate, 2-(7-
  • methods for preparing intermediate compounds 1-8 are as follows.
  • n, AA, R, i, f, FF 2 , FF and D are as described herein.
  • the intermediate shown is:
  • R, n and D are as described in this article.
  • the intermediate is:
  • R, n and D are as described in this article.
  • the intermediate is:
  • n and D are as described in this article.
  • the intermediate is:
  • n and D are as described in this article.
  • the above-mentioned alkaline conditions can be provided by reagents, which include organic bases and inorganic bases.
  • the organic bases include but are not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide.
  • the inorganic bases include but are not limited to sodium hydride and potassium phosphate. , sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide.
  • the above-mentioned condensation agent can be selected from N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea, 4-(4,6-dimethyl Oxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N ',N'-Tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N,N ',N'-Tetramethylurea hexafluorophosphate, 2-(7-
  • ADC1-9 is prepared as follows.
  • n, AA, R, i, f, FF, D and Abu are as described in this article.
  • Antibody drug conjugates can be purified using conventional methods, such as preparative high performance liquid chromatography (prep-HPLC) and other methods.
  • pre-HPLC preparative high performance liquid chromatography
  • the above-mentioned weakly acidic conditions can be provided by reagents, which include organic acids and inorganic acids.
  • the organic acids include but are not limited to acetic acid, benzoic acid, tartaric acid, oxalic acid, malic acid, citric acid, ascorbic acid, citric acid, citrus acid, etc.
  • the inorganic acids include but are not limited to carbonic acid, nitrous acid, acetic acid , hypochlorous acid, hydrofluoric acid, sulfurous acid, hydrosulfuric acid, silicic acid, metasilicic acid, phosphoric acid, metaphosphoric acid, sodium bicarbonate, sodium bisulfite.
  • the invention also provides methods and uses of treatment.
  • methods are provided for treating or ameliorating various types of cancer or tumor-related diseases, comprising administering to a patient in need thereof an effective dose of an anti-CLDN18.2 antibody or antigen binding unit or ADC.
  • the use of anti-CLDN18.2 antibodies or antigen-binding units or ADCs in treating or ameliorating related diseases such as cancer or tumors is provided.
  • the application of the anti-CLDN18.2 antibody or antigen-binding unit or ADC in the preparation of a drug for treating or improving related diseases such as cancer or tumors is provided.
  • the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, antigen-binding fragment or derivative used, the patient's age and weight, general health, sex and diet, as well as the timing of administration, Frequency of excretion, combination of medications, and severity of the specific condition being treated. Judgment of these factors is left to a medical practitioner, including those with ordinary skill in the art.
  • the dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease and the desired effect.
  • the dosage used can be determined by pharmacological and pharmacokinetic principles well known in the art.
  • the antibody or antigen-binding unit or ADC of the invention is administered to a patient at a dose of 0.01 mg/kg to 100 mg/kg of patient body weight per administration. In some embodiments, administration is every 1 week, 2 weeks, 3 weeks, or monthly.
  • Methods of administration of antibodies or antigen-binding units or ADCs include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural injection, and oral administration.
  • the antibody, antigen-binding unit, ADC or composition may be administered by any convenient route, such as by infusion or bolus injection, absorbed through the epithelium or mucosal membranes of the skin (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be combined with other biologically active agents are administered together.
  • the pharmaceutical composition containing the antibody, antigen-binding unit, and ADC of the present invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, externally (such as by powder, ointment, drops or transdermal patch), given by mouth or by oral or nasal spray.
  • parenteral refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the mode of administration may be systemic or local.
  • the antibodies, antigen-binding units, ADCs or pharmaceutical compositions of the present invention can be applied topically to the area requiring treatment; by, but not limited to, the following methods: local infusion during surgery, for example, local application in combination with post-operative wound dressings, by injection, This is done through a catheter, by means of suppositories or by means of implants, which are porous, non-porous or gel-like materials, including membranes (eg silicone rubber membranes) or fibers.
  • care must be taken to use materials that do not absorb the protein.
  • compositions of the invention comprise a nucleic acid or polynucleotide encoding an antibody or antigen-binding unit, which can be administered in vivo by constructing it as part of a suitable nucleic acid expression vector to facilitate Expression of the protein it encodes is then made intracellular by administration of part of the vector, for example by using a retroviral vector (see U.S. Patent 4,980,286), or by direct injection, or by using microparticle bombardment (e.g.
  • nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.
  • Methods for treating disease are typically tested in vitro and include administering an antibody, antigen-binding unit, ADC or derivative of the invention, followed by testing for the desired therapeutic or preventive activity in vivo in an acceptable animal model, and finally administering in the human body.
  • Suitable animal models, including transgenic animals are known to those of ordinary skill in the art.
  • in vitro assays used to demonstrate the therapeutic use of the antibodies or antigen-binding units or ADCs of the invention include the effects of the antibodies or antigen-binding units or ADCs on cell lines or patient tissue samples.
  • the effects of antibodies or antigen-binding units or ADCs on cell lines and/or tissue samples can be detected using techniques known to those skilled in the art, such as those disclosed elsewhere in this invention.
  • in vitro assay experiments that may be used to determine whether a specific antibody or antigen-binding unit or ADC is administered include in vitro cell culture experiments in which patient tissue samples are grown in culture and exposed to or otherwise administered with a compound, and observe the effects of this compound on tissue samples.
  • Various known delivery systems may be used to administer the antibodies or antigen-binding units or ADCs of the invention or polynucleotides encoding the antibodies or antigen-binding units of the invention, e.g., encapsulated in liposomes, microparticles, microcapsules, capable of expressing the compounds Recombinant cells, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviruses or other vectors, etc.
  • an anti-CLDN18.2 antibody or antigen-binding unit or ADC of the invention may be combined with other therapeutic or prophylactic regimens, including administering one or more antibodies or antigen-binding units or ADCs of the invention and one or Various other therapeutic agents or methods are used together or in combination.
  • other treatment options include, but are not limited to, radiation therapy, chemotherapy, hormonal therapy, and the like.
  • the antibody or antigen-binding unit or ADC can be administered with the other therapeutic agent either simultaneously or separately.
  • the antibody or antigen-binding unit or ADC of the invention may be administered before or after administration of another other therapeutic agent.
  • an antibody or antigen-binding unit or ADC of the invention may be administered in combination with a chemotherapeutic agent.
  • the antibodies or antigen-binding units or ADCs of the invention can be administered in combination with cytokines, chemokines, or costimulatory molecules.
  • an antibody or antigen-binding unit or ADC of the invention may be administered in combination with an immunotherapeutic agent.
  • CLDN18.2 Expression of CLDN18.2 is observed in certain cancer tumor samples, and patients who positively express CLDN18.2 respond to treatment with anti-CLDN18.2 antibodies or antigen-binding units or ADCs of the invention. Therefore, the antibodies or antigen-binding units of the invention can also be used for diagnosis and prognosis.
  • a sample containing cells may be obtained from a patient, who may be a cancer patient or a patient undergoing diagnosis.
  • the cells are cells of tumor tissue or tumor mass, blood sample, urine sample, or any sample from a patient.
  • the sample can be incubated with the antibody or antigen-binding unit of the invention under conditions that allow the antibody or antigen-binding unit to interact with CLDN18.2 protein that may be present in the sample.
  • Methods such as ELISA can be used to detect the presence and expression of CLDN18.2 protein in a sample using anti-CLDN18.2 antibodies or antigen-binding units.
  • the presence of CLDN18.2 protein in a sample can be used to diagnose cancer, as an indication that the patient is suitable for antibody treatment, or as an indication that the patient has responded (or has not) responded to cancer treatment.
  • the test can be done once, twice, or more often at specific stages after starting cancer treatment to indicate the progress of treatment.
  • compositions comprise an effective dose of an anti-CLDN18.2 antibody (or antigen binding unit) or ADC and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains 0.1%-90% anti-CLDN18.2 antibody or antigen-binding unit or ADC.
  • the pharmaceutical composition further includes an anti-cancer agent (eg, immune checkpoint inhibitor).
  • the term "pharmaceutically acceptable” refers to materials listed in the Pharmacopoeia for use in animals, particularly in humans.
  • a “pharmaceutically acceptable carrier” will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid.
  • the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous injection into the human body.
  • Compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include solubilizers and local anesthetics such as lidocaine to relieve pain at the injection site.
  • the active ingredients are supplied singly or mixed together in unit dosage form, such as as a dry lyophilized powder or anhydrous concentrate in sealed containers (such as ampoules or sachets) indicating the quantity of active agent.
  • the composition may be dispensed from an infusion bottle containing sterile pharmaceutical grade water or saline.
  • ampoules of sterile water or saline for injection may be used, allowing the active ingredients to be mixed prior to administration.
  • the compounds of the invention may be formulated in neutral or salt form.
  • Pharmaceutically acceptable salts include salts with anions derived from, for example, hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts with anions derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2 -Salts formed from cations such as ethylaminoethanol, histidine, and procaine.
  • variable and constant regions of the anti-CLDN18.2 antibody is shown in Table 1.
  • VH and CH make up the heavy chain of the antibody
  • VL and CL make up the light chain of the antibody.
  • CDR summary of the anti-CLDN18.2 antibody is shown in Table 2.
  • the composition of the CDR region See Table 3, and the variable region and constant region sequences are shown in Table 4.
  • the light chains of antibodies H239H-2b-K-6a-1, H239H-2b-K-6a-2 and H239H-2b-K-6a-3 have the same sequence, and their amino acid sequences are as shown in SEQ ID NO: 72 is shown, the corresponding gene sequence is shown in SEQ ID NO:73; the amino acid sequence of the heavy chain of antibody H239H-2b-K-6a-1 is shown in SEQ ID NO:69, and the corresponding gene sequence is shown in SEQ ID NO:74 Shown; the amino acid sequence of the heavy chain of antibody H239H-2b-K-6a-2 is as SEQ ID NO:70 shown, the corresponding gene sequence is shown in SEQ ID NO:75; the amino acid sequence of the heavy chain of antibody H239H-2b-K-6a-3 is shown in SEQ ID NO:71, and the corresponding gene sequence is shown in SEQ ID NO:76 As shown (see Table 5 and Table 6). Those starting with "CH" represent chimeric antibodies (mouse variable region + human constant
  • ribosome binding site (kozak sequence: GCCGCCACC, SEQ ID NO:77) and signal peptide gene sequence to the 5' end of the antibody gene sequence.
  • Light chain signal peptide MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO:78), the corresponding nucleotide sequence is: atggacatgagggtgctggcccagctgctgggactgctgctgctgtgcttcccaggcgccagatgc (SEQ ID NO:79);
  • Heavy chain signal peptide MGWSLILLFLVAVATRVLS (SEQ ID NO:80), heavy chain gene sequence addition of antibodies H239H-2b-K-6a-1, H239H-2b-K-6a-2 and H239H-2b-K-6a-3
  • the signal peptide gene sequence is: atgggttggtctcttatccttctattcctggttgcagtggcaacaagagtgctttct (SEQ ID NO:81).
  • the signal peptide gene sequence added to the heavy chain gene sequence of other antibodies is: atgggttggtctctgattctcctgtttctggtggcagtggctacaagagtcctgtca, SEQ ID NO:82).
  • the antibody heavy chain and light chain gene sequences are connected into the expression vector to construct full-length expression plasmids for the light and heavy chains.
  • the plasmid expressing the antibody light and heavy chains was transiently transfected into HEK293F cells at a ratio of 1:1, and the antibody was cultured and purified.
  • CB14 refers to the synthesis of CB07 in Example 3, replacing 4,7,10,13,16,19,22,25-octaoxahexadecanoic acid-N-succinimide ester with 4,7 ,10,13-Tetraxatetradecanoic acid-N-succinimide ester. Finally, a white solid CB14 was obtained.
  • reaction bottle R2 The solution in reaction bottle R2 was added dropwise to reaction bottle R1, and then reaction bottle R2 was washed with N,N-dimethylformamide (1 mL), and the washing liquid was added to reaction bottle R1. Then weigh 1-hydroxybenzotriazole (60 mg, 0.44 mmol) and pyridine (0.5 mL) and add them to reaction bottle R1. Stir at 0-5°C for 10 min, then rise to room temperature and stir for 5.5 h. After the reaction is complete, concentrate under reduced pressure at 35°C. Remove solvent. It was purified by preparative high performance liquid chromatography (prep-HPLC) and freeze-dried to obtain a white powder.
  • prep-HPLC preparative high performance liquid chromatography
  • Flow cytometry was used to detect the binding ability of anti-CLDN18.2 antibodies and ADC to cells expressing CLDN18.2, and further detect the specificity of not binding to CLDN18.1.
  • An overview of the experimental steps is as follows: Collect CLDN18.2 positive cells (KATOIII or CHO-CLDN18.2) and negative cells (CHO-CLDN18.1), wash them once with PBS, and resuspend the cells in PBS, 100,000 cells/50 ⁇ l/well , spread in a 96-well V-bottom plate (Cat.
  • IMAB362 Compared with the positive antibody IMAB362 (whose sequence is the same as the sequence of the antibody expressed by hybridoma cell 175D10 in patent US20090169547), the chimeric antibody CH239H-2-K-6 has a better binding ability to CLDN18.2 on KATOIII cells. Good: IMAB362 and CH239H-2-K-6 have EC50 values of 8.082nM and 6.290nM respectively.
  • the humanized antibodies H239H-2a-K-6a and H239H-2b-K-6a were effective against CLDN18.2-positive cells KATOIII and CHO-CLDN18.2
  • the binding ability is basically the same as that of chimeric antibody CH239H-2-K-6, and the affinity has not decreased; antibody CH239H-2-K-6, antibody H239H-2a-K-6a, antibody H239H-2b-K-6a and KATOIII
  • the EC 50 values of cell binding are 6.108nM, 6.203nM and 6.920nM respectively;
  • the EC 50 values of antibody CH239H-2-K-6, antibody H239H-2a-K-6a, antibody H239H-2b-K-6a, and positive antibody IMAB362 binding to CHO-CLDN18.2 cells are 19.92nM, 22.83nM, respectively. 23.31nM, 30.02nM.
  • humanized antibodies H239H-2a-K-6a and H239H-2b-K-6a only bind to CLDN18.2 and do not bind to CLDN18.1.
  • the EC 50 value of humanized antibody H239H-2b-K-6a-1 binding to KATOIII cells is 3.887nM; the EC 50 values of ADC1, ADC2, ADC3, and ADC4 binding to KATOIII cells are respectively : 4.531nM, 7.763nM, 7.322nM, 6.194nM.
  • the nucleic acid sequence corresponding to the above human CLDN18.2 was synthesized, and the restriction site HindIII and EcoRI sequences were added to both ends of the sequence, and then constructed into the pcDNA3.1 expression vector (Invitrogen, Cat. No. V79020), and then transferred by electroporation. Dye into CHO cells (Life technologies, Cat. No.: A13696-01).
  • the electroporation conditions are: voltage 300V, time 17 milliseconds, 4mm electroporation cup. After 48 hours, add 50 ⁇ M MSX (methionine iminosulfone) screening pressure. After 2 weeks , screen positive cells. Use FACS detection to screen high-expressing cell lines.
  • this antibody can recognize the same intracellular segment of CLDN18.1 and CLDN18.2, so CHO-CLDN18.2 cells can be used as a positive control) at room temperature. Incubate for 2 hours, wash 3 times with PBST, then soak the membrane in goat anti-rabbit HRP antibody (Cat. No.: ab6721, abcam), incubate at room temperature for 2 hours, wash 3 times with PBST and then use DAB (Cat. No.: AR1000, Boster Biotechnology) for visualization. color. The finally obtained cells were named CHO-CLDN18.1 cells.
  • Example 15 ADCC activity of anti-CLDN18.2 antibody (antibody dependent cell-mediated cytotoxicity)
  • ADCC Reporter Bioassay was selected to evaluate the ADCC activity of CLDN18.2 antibody.
  • This is a bioluminescent reporter gene detection system that replaces NK cells with artificially constructed effector cells and cooperates with highly sensitive detection reagents to quantify the biological activity of therapeutic antibody drugs based on the ADCC mechanism of action in their activation pathways. It is a A mechanism of action detection method.
  • Cells expressing CLDN18.2 (CHO-CLDN18.2) were used as target cells, and Jurkat-NFAT-luc-Fc ⁇ RIIIa was used as effector cells (Fc ⁇ RIIIa (sequence from NCBI, NP_001121065.1) was transferred to Jurkat through lentivirus infection.
  • CHO-CLDN18.1 cells were used as cell negative control, IMAB362 was used as antibody positive control, and IgG isotype (Cat. No.: BE0297, Bio cell) was used as antibody negative control.
  • Collect CHO-CLDN18.2 cells wash them once with PBS, use RPMI1640 medium to adjust the cell density to 6.6 ⁇ 10 5 /ml, 45 ⁇ l/well, and spread them on a 96-well flat-bottom plate. Pre-dilute the antibody to 50 ⁇ g/ml with RPMI1640 culture medium, and then dilute it 4 times downward for a total of 9 gradients, and use the well without antibody as a control well.
  • Human serum complement (Cat. No.: Quidel, A113) with a final concentration of 5% was used as the source of complement.
  • Anti-CLDN18.2 ADC was incubated with CLDN18.2-expressing cells, and the number of viable cells was detected using CCK-8 (Cat. No.: CK04, DojinDo) reagent, and the inhibition rate was counted.
  • CCK-8 Cat. No.: CK04, DojinDo
  • the experimental steps are summarized as follows: Collect CHO-CLDN18.2 cells in the logarithmic growth phase, centrifuge at 800 rpm for 5 minutes, remove the supernatant, wash once with CD-CHO-AGT (Cat. No.: 12490; life technologies) culture medium, centrifuge at 800 rpm for 5 minutes. , go to the supernatant. Resuspend the cells in 0.5% FBS (Cat.
  • Inhibition rate % (1-absorbance value of sample well/absorbance value of control well) ⁇ 100.
  • the final concentrations of ADC are 10 nM, 1 nM, and 0.1 nM.
  • the wells without ADC are used as negative controls. Place in a 37°C, 5% CO2 incubator for 5 days, digest and collect the cells with trypsin (Cat. No.: 25200-072; gibco), and count the total number of viable cells.
  • trypsin Cat. No.: 25200-072; gibco
  • cells were treated with FITC-labeled anti-Trop2 antibody (the heavy and light chain sequences of the anti-Trop2 antibody are SEQ ID NO:83 and SEQ ID NO:84, respectively, see Table 7) Stain (KATOIII is Trop2-positive cells) and incubate on ice for 30 min.
  • BLI Bio-Layer Interferometry, biofilm layer surface interference
  • the instrument uses Octet Platform, Fortebio, Octet QK, S/N33-8094-2244, Equipment ID TBIO001.
  • the sensor is SA Biosensor, Fortebio, Cat#18-5019.
  • Fc receptors include: FcRn, ACRO Biosystems, Cat#FCM-H8286; Fc ⁇ RIa, ACRO Biosystems, Cat#FCA-H82E8; Fc ⁇ RIIa H131, ACRO Biosystems, Cat#CDA-H82E6; Fc ⁇ RIIa R131, ACRO Biosystems, Cat#CDA-H82E7 ; Fc ⁇ RIIb, ACRO Biosystems, Cat#CDB-H82E0; Fc ⁇ RIIIa 158V, ACRO Biosystems, Cat#CDA-H82E9; Fc ⁇ RIIIa 158F, ACRO Biosystems, Cat#CDA-H82E8; Fc ⁇ RIIIb(NA2), ACRO Biosystems, Cat#CDB-H82E a.
  • Example 20 ADC in Anti-tumor effect of gastric cancer GA0006 xenograft in female BALB/c nude mouse animal model
  • TGI TV % [1-(Ti-T0)/(Ci-C0)] ⁇ 100 where T0 and C0 are the average tumor volumes on the day (day 0) of the administration group and vehicle control group respectively, Ti and Ci respectively It is the average tumor volume on the 14th day in the drug treatment group and the vehicle control group.
  • the average tumor volume of the control group (Group 1) of this model reached 628.62mm 3 after 14 days of administration.
  • each treatment group also demonstrated different degrees of anti-tumor efficacy after 14 days of administration, as shown in Figure 7.

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Abstract

本发明公开了抗CLDN18.2抗体及其抗体药物偶联物和用途，属于生物医药领域。在一些实施方案中，所述抗体药物偶联物为式I的化合物或其立体异构体或其药学上可接受的盐或溶剂合物。所述抗CLDN18.2抗体、抗体药物偶联物可用于治疗CLDN18.2表达相关的疾病。

Description

抗CLDN18.2抗体及其抗体药物偶联物和用途 技术领域
本发明涉及抗CLDN18.2抗体、抗体药物偶联物及其用途。
背景技术
靶向治疗癌症、免疫缺陷和感染性疾病等是目前精准医疗关注的核心。多年来许多文献报道了利用细胞表面受体结合分子作为药物输送工具与细胞毒素分子形成偶联物(共轭体)，进行靶向输送细胞毒素分子进攻各类致病细胞(Allen,T.M.and Cullis,P.R.,(2004)Science,303(5665),1818-22；Hu,Q.Y.,et al.(2016)，Chem Soc Rev 45(6):1691-1719.)。
抗体药物偶联物由三部分组成：抗体，细胞毒素分子和连接二者之间的连接体(Thomas,A.,et al.(2016)，Lancet Oncol 17(6):e254-e262)。三者之间各具有独特的功能：抗体需要对肿瘤细胞特异性结合，细胞毒素分子需要对肿瘤细胞足够的活性和广谱性，连接体需要独特的功能性，在血液循环中稳定，到达肿瘤细胞后有效释放细胞毒素分子(Chari,R.V.(2008)，Acc Chem Res 41(1):98-107)，三者合理构建才能取得良好的临床效果(Singh,S.K.,et al.(2015)，Pharm Res 32(11):3541-3571；Hamilton,G.S.(2015)，Biologicals 43(5):318-332)。
细胞连接密蛋白18.2(密蛋白18.2或CLDN18.2)是目前治疗肿瘤研发的靶点之一(Sahin U.,et al.Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development,Clin Cancer Res.,2008,14(23):7624-34)。
发明内容
本发明提供了特异性结合CLDN18.2的抗体或抗原结合单元。在一个或多个实施方式中，本发明的抗体或抗原结合单元能够识别并结合人CLDN18.2。
在一个或多个实施方式中，本发明提供抗CLDN18.2抗体或抗原结合单元，所述抗体或抗原结合单元包含(a)-(f)中的一个或多个氨基酸序列：
(a)VH CDR1，其包含如SEQ ID NO:1-6任一项所示的氨基酸序列；
(b)VH CDR2，其包含如SEQ ID NO:7-13任一项所示的氨基酸序列；
(c)VH CDR3，其包含如SEQ ID NO:14-21任一项所示的氨基酸序列；
(d)VL CDR1，其包含如SEQ ID NO:22-29任一项所示的氨基酸序列；
(e)VL CDR2，其包含如SEQ ID NO:30-37任一项所示的氨基酸序列；
(f)VL CDR3，其包含如SEQ ID NO:38-45任一项所示的氨基酸序列。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:1-6任一项所示的VH CDR1、如SEQ ID NO:7-13任一项所示的VH CDR2和如SEQ ID NO:14-21任一项所示的VH CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:22-29任一项所示的VL CDR1、如SEQ ID NO:30-37任一项所示的VL CDR2和如SEQ ID NO:38-45任一项所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:1-6任一项所示的VH CDR1、如SEQ ID NO:7-13任一项所示的VH CDR2、如SEQ ID NO:14-21任一项所示的VH CDR3、如SEQ ID NO:22-29任一项所示的VL CDR1、如SEQ ID NO:30-37任一项所示的VL CDR2和如SEQ ID NO:38-45任一项所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:2所示的VH CDR1、如SEQ ID NO:8所示的VH CDR2、如SEQ ID NO:15所示的VH CDR3、如SEQ ID NO:26所示的VL CDR1、如SEQ ID NO:34所示的VL CDR2和如SEQ ID NO:42所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:9所示的VH CDR2、如SEQ ID NO:16所示的VH CDR3、如SEQ ID NO:25所示的VL CDR1、如SEQ ID NO:33所示的VL CDR2和如SEQ ID NO:41所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:6所示的VH CDR1、如SEQ ID NO:13所示的VH CDR2、如SEQ ID NO:20所示的VH CDR3、如SEQ ID NO:28所示的VL CDR1、如SEQ ID NO:36所示的VL CDR2和如SEQ ID NO:44所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:5所示的VH CDR1、如SEQ ID NO:12所示的VH CDR2、如SEQ ID NO:19所示的VH CDR3、如SEQ ID NO:29所示的VL CDR1、如SEQ ID NO:37所示的VL CDR2和如SEQ ID NO:45所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:4所示的VH CDR1、如SEQ ID NO:11所示的VH CDR2、如SEQ ID NO:18所示的VH CDR3、如SEQ ID NO:27所示的VL CDR1、如SEQ ID NO:35所示的VL CDR2和如SEQ ID NO:43所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:9所示的VH CDR2、如SEQ ID NO:16所示的VH CDR3、如SEQ ID NO:26所示的VL CDR1、如SEQ ID NO:34所示的VL CDR2和如SEQ ID NO:42所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:9所示的VH CDR2、如SEQ ID NO:16所示的VH CDR3、如SEQ ID NO:24所示的VL CDR1、如SEQ ID NO:32所示的VL CDR2和如SEQ ID NO:40所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:7所示的VH CDR2、如SEQ ID NO:14所示的VH CDR3、如SEQ ID NO:22所示的VL CDR1、如SEQ ID NO:30所示的VL CDR2和如SEQ ID NO:38所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:7所示的VH CDR2、如SEQ ID NO:14所示的VH CDR3、如SEQ ID NO:23所示的VL CDR1、如SEQ ID NO:31所示的VL CDR2和如SEQ ID NO:39所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:2所示的VH CDR1、如SEQ ID NO:10所示的VH CDR2、如SEQ ID NO:17所示的VH CDR3、如SEQ ID NO:27所示的VL CDR1、如SEQ ID NO:35所示的VL CDR2和如SEQ ID NO:43所示的VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元包含如SEQ ID NO:2所示的VH CDR1、如SEQ ID NO:8所示的VH CDR2、如SEQ ID NO:21所示的VH CDR3、如SEQ ID NO:26所示的VL CDR1、如SEQ ID NO:34所示的VL CDR2和如SEQ ID NO:42所示的 VL CDR3。
在一个或多个实施方式中，所述抗体或抗原结合单元是鼠源抗体、嵌合抗体或人源化抗体。
在一个或多个实施方式中，所述抗体或抗原结合单元包含重链可变区和/或轻链可变区。在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:46-55任一项所示的氨基酸序列，或与SEQ ID NO:46-55任一项所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:46-55任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。在一个或多个实施方式中，所述轻链可变区包含如SEQ ID NO:56-64任一项所示的氨基酸序列，或与SEQ ID NO:56-64任一项所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:56-64任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:46-55任一项所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:56-64任一项所示的氨基酸序列。
在一个或多个实施方式中，所述抗体或抗原结合单元包含重链可变区和轻链可变区。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:47所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:60所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:48所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:59所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:52所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:62所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:51所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:63所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:50所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:61所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:48所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:60所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:48所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:58所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:46所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:46所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:57所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:49所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:61所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:53所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:64所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:54所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:64所示的氨基酸序列。
在一个或多个实施方式中，所述重链可变区包含如SEQ ID NO:55所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:64所示的氨基酸序列。
在一个或多个实施方式中，所述抗体或抗原结合单元还包含重链恒定区、轻链恒定区、Fc区或其组合。在一些实施方案中，轻链是κ或λ型。在一个或多个实施方式中，轻链恒定区是κ或λ链恒定区。在一个或多个实施方式中，所述抗体或抗原结合单元是IgG、 IgM、IgA、IgE或IgD其中一种同种型。在一个或多个实施方式中，同种型是IgG1、IgG2、IgG3或IgG4。在一个或多个实施方式中，所述抗体为IgG1抗体。
在一个或多个实施方式中，Fc是变体Fc区。在一个或多个实施方式中，相对于亲本Fc区，变体Fc区具有一个或多个氨基酸修饰，如取代、缺失或插入。在一个或多个实施方式中，相对于亲本Fc区活性，Fc区的氨基酸修饰改变了效应功能活性。在一个或多个实施方式中，变体Fc区可以具有改变的(即，增加的或降低的)抗体依赖性细胞毒性(ADCC)、补体介导的细胞毒性(CDC)、吞噬作用、调理作用或细胞结合。在一个或多个实施方式中，相对于亲本Fc区，Fc区氨基酸修饰可以改变变体Fc区对FcγR(Fcγ受体)的亲和力。在一个或多个实施方式中，所述Fc区来源于IgG1或IgG4。
在一个或多个实施方式中，所述抗体或抗原结合单元为分离的抗体或抗原结合单元。在一个或多个实施方式中，所述抗体或抗原结合单元为scFV、Fab、F(ab)2或IgG1。在一个或多个实施方式中，所述抗体或抗原结合单元为单克隆抗体。
在一个或多个实施方式中，所述重链恒定区包含如下一种或多种氨基酸突变：N297A、L234A、L235A、P329G(EU编号)。在一个或多个实施方式中，所述重链恒定区包含如下氨基酸突变：N297A(EU编号)。在一个或多个实施方式中，所述重链恒定区包含如下氨基酸突变：L234A、L235A和P329G(EU编号)。
在一个或多个实施方式中，所述重链恒定区包含如SEQ ID NO:65-67任一项所示的氨基酸序列，或与SEQ ID NO:65-67任一项所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:65-67任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一个或多个实施方式中，所述轻链恒定区包含如SEQ ID NO:68所示的氨基酸序列，或与SEQ ID NO:68所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:68所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一个或多个实施方式中，所述重链恒定区包含如SEQ ID NO:65-67任一项所示的氨基酸序列，所述轻链恒定区包含如SEQ ID NO:68所示的氨基酸序列。在一个或多个实施方式中，所述重链恒定区包含如SEQ ID NO:65所示的氨基酸序列，所述轻链恒定区包含如SEQ ID NO:68所示的氨基酸序列。在一个或多个实施方式中，所述重链恒定区包含如SEQ ID NO:66所示的氨基酸序列，所述轻链恒定区包含如SEQ ID NO:68所示的氨基酸序列。在一个或多个实施方式中，所述重链恒定区包含如SEQ ID NO:67所示的氨基酸序列，所述轻链恒定区包含如SEQ ID NO:68所示的氨基酸序列。
在一个或多个实施方式中，所述抗体的重链包含如SEQ ID NO:69-71任一项所示的氨基酸序列，或与SEQ ID NO:69-71任一项所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:69-71任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。在一个或多个实施方式中，所述抗体的轻链包含如SEQ ID NO:72所示的氨基酸序列，或与SEQ ID NO:72所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:72所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一个或多个实施方式中，所述抗体的重链包含如SEQ ID NO:69-71任一项所示的氨基酸序列，所述抗体的轻链包含如SEQ ID NO:72所示的氨基酸序列。在一个或多个实施方式中，所述抗体的重链包含如SEQ ID NO:69所示的氨基酸序列，所述抗体的轻链包含如SEQ ID NO:72所示的氨基酸序列。在一个或多个实施方式中，所述抗体的重链包含如SEQ ID NO:70所示的氨基酸序列，所述抗体的轻链包含如SEQ ID NO:72所示的氨基 酸序列。在一个或多个实施方式中，所述抗体的重链包含如SEQ ID NO:71所示的氨基酸序列，所述抗体的轻链包含如SEQ ID NO:72所示的氨基酸序列。
在一个或多个实施方式中，本文所述抗体含有两条序列相同的重链和两条序列相同的轻链。
一个或多个实施方式还提供一种多聚核苷酸，所述多聚核苷酸编码本文所述的抗体或抗原结合单元或其一部分。在一个或多个实施方式中，所述多聚核苷酸为分离的多聚核苷酸。
一个或多个实施方式还提供一种表达载体，所述表达载体包含编码本文所述的抗体或抗原结合单元的多聚核苷酸。
一个或多个实施方式还提供一种细胞，所述细胞包含编码如本文所述的抗体或抗原结合单元的多聚核苷酸。
在一个或多个实施方式中，载体、细胞或微生物为分离的载体、细胞或微生物。在一个或多个实施方式中，所述细胞为CHO细胞、HEK细胞(如HEK293F细胞)、BHK细胞、Cos1细胞、Cos7细胞、CV1细胞或鼠L细胞。在一个或多个实施方式中，所述细胞为CHO细胞。
本发明还提供了制备所述抗体的方法，其包括在培养基里培养包含编码所述抗体的核酸的宿主细胞。在一些实施方案中，所述方法还包括纯化所述抗体。纯化可以采用常规方法进行，例如先离心细胞悬液并收集上清液，再次离心进一步去除杂质。Protein A亲和柱和离子交换柱等方法可以用于纯化抗体蛋白。
一个或多个实施方式还提供了诊断方法和用途。在一个或多个实施方式中，提供了检测样品中CLDN18.2表达的方法，使样品与所述抗体或抗原结合单元进行接触，使得所述抗体或抗原结合单元结合CLDN18.2，并检测其结合，即样品中CLDN18.2的含量。在一个或多个实施方式中，提供了所述抗体或抗原结合单元在制备用于诊断或预后癌症或肿瘤试剂盒中的应用。在一些实施方式中，提供了一种包含所述抗体或抗原结合单元的诊断或预后试剂盒。
在一个或多个实施方式中，本发明还提供一种抗体药物偶联物(ADC)，其包含通过接头与药物偶联的本发明所述的抗体或抗原结合单元，或其药学上可接受的盐或溶剂合物。
在一个或多个实施方式中，所述接头为可裂解的接头。
在一个或多个实施方式中，所述药物为抗癌药物、细胞毒性药物、细胞分化因子、干细胞营养因子、类固醇类药物、治疗自身免疫疾病的药物、抗炎症药物或治疗传染性疾病的药物。
在一个或多个实施方式中，所述药物为抗癌药物。
在一个或多个实施方式中，所述药物为微管蛋白抑制剂、DNA损伤剂或DNA拓扑异构酶抑制剂。
在一个或多个实施方式中，所述微管蛋白抑制剂选自海兔毒素(dolastatin)、奥瑞他汀类(auristatin)、及美登素类(maytansine)。
在一个或多个实施方式中，所述药物为奥瑞他汀类(auristatin)，选自一甲基澳瑞他汀E(MMAE)、一甲基澳瑞他汀F(MMAF)或澳瑞他汀(AF)。
在一个或多个实施方式中，所述药物为DNA损伤剂，例如卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD(pyrrolobenzodiazepine，吡咯并苯并二氮杂)。
在一个或多个实施方式中，所述药物为DNA拓扑异构酶抑制剂或其盐，例如伊立替康、伊立替康盐酸盐、依喜替康衍生物，喜树碱、9-氨基喜树碱、9-硝基喜树碱、10-羟基喜树碱、9-氯-10-羟基喜树碱、喜树碱类衍生物SN-38、22-羟基旱莲木碱、拓扑替康、勒托替康、贝洛替康、依喜替康、硅基高喜树碱(homosilatecan)、6,8-二溴-2-甲基-3-[2-(D-吡喃木糖基氨基)苯基]-4(3H)-喹唑啉酮、2-氰基-3-(3,4-二羟基苯基)-N-(苯基甲基)-(2E)-2-丙烯酰胺、2-氰基-3-(3,4-二羟基苯基)-N-(3-羟基苯基丙基)-(E)-2-丙烯酰胺、12-β-D-吡喃萄萄糖基-12,13-二氢-2,10-二羟基-6-[[2-羟基-1-(羟基甲基)乙基]氨基]-5H-吲哚并[2,3-a]吡咯并[3,4-c]咔唑-5,7(6H)-二酮、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺二盐酸盐、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺。
在一个或多个实施方式中，所述DNA拓扑异构酶抑制剂为喜树碱、10-羟基喜树碱、拓扑替康、贝洛替康、伊立替康、22-羟基旱莲木碱或依喜替康。
在一个或多个实施方式中，所述药物具有氨基或被一个烷基取代氨基，其与接头通过酰胺键连接。
在一个或多个实施方式中，所述药物为或其立体异构体，其中
X1和X2各自独立地为：
H，
羟基，
C1-C6烷基，
被一个或多个羟基、卤素、硝基或氰基取代的C1-C6烷基，
C2-C6烯基，
C2-C6炔基，
C1-C6烷氧基，
C1-C6氨基烷氧基，
卤素，
硝基，
氰基，
巯基，
烷硫基，
氨基，被氨基保护基取代的氨基，在氨基部分任选被氨基保护基或C1-C6烷基取代的C1-C6氨基烷基，
在氨基部分任选被氨基保护基或C1-C6烷基取代的C1-C6氨基烷基氨基，
连接至杂环的C1-C6烷基，所述杂环任选被一个或多个C1-C6烷基、C1-C6烷氧基、氨基、卤素、硝基或氰基取代，
连接至杂环的C1-C6烷基氨基，所述杂环任选被C1-C6烷基、C1-C6烷氧基取代，所述氨基任选被氨基保护基、卤素、硝基、氰基或保护基取代，
氨基取代的杂环基，其在杂环部分的氮原子或氨基部分任选被保护基或一个或多个C1-C6烷基取代，
杂环氨基，其在杂环部分的氮原子或氨基部分任选被保护基或C1-C6烷基取代，
任选被氨基甲酰基保护基或C1-C6烷基取代的氨基甲酰基，
吗啉-1-基，或
哌啶-1-基；
X3为C1-C6烷基；
X4为H、-(CH2)q-CH3、-(CHRn)q-CH3、C3-C8碳环基、-O-(CH2)q-CH3、亚芳基-CH3、-(CH2)q-亚芳基-CH3、-亚芳基-(CH2)q-CH3、-(CH2)q-(C3-C8碳环基)-CH3、-(C3-C8碳环基)-(CH2)q-CH3、C3-C8杂环基、-(CH2)q-(C3-C8杂环基)-CH3、-(C3-C8杂环基)-(CH2)q-CH3、-(CH2)qC(O)NRn(CH2)q-CH3、-(CH2CH2O)q-CH3、-(CH2CH2O)q-CH2-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH2-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH2-CH3、或-(CH2CH2O)qC(O)NRn(CH2)q-CH3；其中各Rn独立为H、C1-C6烷基、C3-C8碳环基、苯基或苄基；并且各q独立为1、2、3、4、5、6、7、8、9或10；
**与所述接头连接；
y为0、1或2；
Y为O、S或CR1R2，其中R1和R2各自独立地为H或C1-C6烷基；
s和t各自独立为0、1或2，但不同时为0。
在一个或多个实施方式中，X4为H或C1-C6烷基。
在一个或多个实施方式中，所述杂环为氮杂环丁烷、乙醛嗪(niverazine)、吗啉、吡咯烷、哌啶、咪唑、噻唑、噁唑或吡啶。
在一个或多个实施方式中，所述氨基保护基为甲酰基、乙酰基、三苯甲基、叔丁氧基羰基、苄基或对甲氧基苄氧基羰基。
在一个或多个实施方式中，所述药物为 或其立体异构体，其中X1和X2各自独立地为C1-C6烷基、卤素或-OH；**与所述接头连接。
在一个或多个实施方式中，所述药物为或其立体异构体，其中X1和X2各自独立地为C1-C6烷基、卤素或-OH；**与所述接头连接。
在一个或多个实施方式中，X1和X2各自为-CH3。
在一个或多个实施方式中，X1和X2各自独立地为F、Cl、Br或I。
在一个或多个实施方式中，X1和X2各自为F。
在一个或多个实施方式中，X1和X2各自独立地为-CH3、F或-OH。
在一个或多个实施方式中，X1和X2各自独立地为F或-CH3。
在一个或多个实施方式中，X1为-CH3及X2为F。
在一个或多个实施方式中，本发明提供的抗体药物偶联物，其具有式I所示的结构、或其立体异构体、或其药学上可接受的盐或溶剂合物：

其中
Abu为抗体或抗原结合单元；所述抗体或抗原结合单元特异性结合CLDN18.2；D为药物；
M为其中*连接Abu，**连接B，R选自：-(CH2)r-、-(CHRm)r-、C3-C8碳环基、-O-(CH2)r-、亚芳基、-(CH2)r-亚芳基-、-亚芳基-(CH2)r-、-(CH2)r-(C3-C8碳环基)-、-(C3-C8碳环基)-(CH2)r-、C3-C8杂环基、-(CH2)r-(C3-C8杂环基)-、-(C3-C8杂环基)-(CH2)r-、-(CH2)rC(O)NRm(CH2)r-、-(CH2CH2O)r-、-(CH2CH2O)r-CH2-、-(CH2)rC(O)NRm(CH2CH2O)r-、-(CH2)rC(O)NRm(CH2CH2O)r-CH2-、-(CH2CH2O)rC(O)NRm(CH2CH2O)r-、-(CH2CH2O)rC(O)NRm(CH2CH2O)r-CH2-和-(CH2CH2O)rC(O)NRm(CH2)r-；其中各Rm独立为H、C1-C6烷基、C3-C8碳环基、苯基或苄基；并且各r独立为1、2、3、4、5、6、7、8、9或10；
B为例如为其中*连接M，**连接L，***连接G；
L为-(AA)i-(FF)f-，其中，AA为氨基酸或多肽，i是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20；各FF独立为 其中各RF独立为C1-C6烷基、C1-C6烷氧基、-NO2或卤素；z为0、1、2、3或4；f为1、2、3、4、5、6、7、8、9或10；其中*连接AA，**连接D；
G为其中n为1-24的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24；
p为1-10的整数，例如1、2、3、4、5、6、7、8、9或10。
在一个或多个实施方式中，药物为抗癌药物、细胞毒性药物、细胞分化因子、干细胞营养因子、类固醇类药物、治疗自身免疫疾病的药物、抗炎症药物或治疗传染性疾病的药物。
在一个或多个实施方式中，药物为抗癌药物。
在一个或多个实施方式中，药物为微管蛋白抑制剂、DNA损伤剂或DNA拓扑异构酶抑制剂。
在一个或多个实施方式中，所述微管蛋白抑制剂选自海兔毒素(dolastatin)、奥瑞他汀(auristatin)类、美登素(maytansine)类。
在一个或多个实施方式中，药物为奥瑞他汀(auristatin)类，例如MMAE、MMAF或AF。
在一个或多个实施方式中，药物为DNA损伤剂，例如卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD(pyrrolobenzodiazepine，吡咯并苯并二氮杂)。
在一个或多个实施方式中，药物为DNA拓扑异构酶抑制剂或其盐，例如伊立替康、伊立替康盐酸盐、喜树碱、9-氨基喜树碱、9-硝基喜树碱、10-羟基喜树碱、9-氯-10-羟基喜树碱、喜树碱类衍生物SN-38、22-羟基旱莲木碱、拓扑替康、勒托替康、贝洛替康、依喜替康、依喜替康衍生物，硅基高喜树碱(homosilatecan)、6,8-二溴-2-甲基-3-[2-(D-吡喃木糖基氨基)苯基]-4(3H)-喹唑啉酮、2-氰基-3-(3,4-二羟基苯基)-N-(苯基甲基)-(2E)-2-丙烯酰胺、2-氰基-3-(3,4-二羟基苯基)-N-(3-羟基苯基丙基)-(E)-2-丙烯酰胺、12-β-D-吡喃萄萄糖基-12,13-二氢-2,10-二羟基-6-[[2-羟基-1-(羟基甲基)乙基]氨基]-5H-吲哚并[2,3-a]吡咯并[3,4-c]咔唑-5,7(6H)-二酮、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺二盐酸盐、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺。
在一个或多个实施方式中，所述DNA拓扑异构酶抑制剂为喜树碱、10-羟基喜树碱、拓扑替康、贝洛替康、伊立替康、22-羟基旱莲木碱或依喜替康。
在一个或多个实施方式中，药物为Tubulysin类、紫杉类药物衍生物、leptomycine衍生物、CC-1065及其类似物、Amatoxin类、剪接体抑制剂、苯(并)二卓氮(PBD)二聚体类、阿霉素、甲氨蝶呤，长春新碱，长春碱，柔红霉素，丝裂霉素C，马法兰或苯丁酸氮芥衍生物。
在一个或多个实施方式中，药物具有氨基或被一个烷基取代氨基，其与FF通过酰胺键连接。
在一个或多个实施方式中，药物为其中
X1和X2各自独立地为：
H，
羟基，
C1-C6烷基，
被一个或多个羟基、卤素、硝基或氰基取代的C1-C6烷基，
C2-C6烯基，
C2-C6炔基，
C1-C6烷氧基，
C1-C6氨基烷氧基，
卤素，
硝基，
氰基，
巯基，
烷硫基，
氨基，被氨基保护基取代的氨基，在氨基部分任选被氨基保护基或C1-C6烷基取代的C1-C6氨基烷基，
在氨基部分任选被氨基保护基或C1-C6烷基取代的C1-C6氨基烷基氨基，
连接至杂环的C1-C6烷基，所述杂环任选被一个或多个C1-C6烷基、C1-C6烷氧基、氨基、卤素、硝基或氰基取代，
连接至杂环的C1-C6烷基氨基，所述杂环任选被C1-C6烷基、C1-C6烷氧基取代，所述氨基任选被氨基保护基、卤素、硝基、氰基或保护基取代，
氨基取代的杂环基，其在杂环部分的氮原子或氨基部分任选被保护基或一个或多个C1-C6烷基取代，
杂环氨基，其在杂环部分的氮原子或氨基部分任选被保护基或C1-C6烷基取代，
任选被氨基甲酰基保护基或C1-C6烷基取代的氨基甲酰基，
吗啉-1-基，或
哌啶-1-基；
X3为C1-C6烷基；
X4为H、-(CH2)q-CH3、-(CHRn)q-CH3、C3-C8碳环基、-O-(CH2)q-CH3、亚芳基-CH3、-(CH2)q-亚芳基-CH3、-亚芳基-(CH2)q-CH3、-(CH2)q-(C3-C8碳环基)-CH3、-(C3-C8碳环基)-(CH2)q-CH3、C3-C8杂环基、-(CH2)q-(C3-C8杂环基)-CH3、-(C3-C8杂环基)-(CH2)q-CH3、-(CH2)qC(O)NRn(CH2)q-CH3、-(CH2CH2O)q-CH3、-(CH2CH2O)q-CH2-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH2-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH2-CH3或-(CH2CH2O)qC(O)NRn(CH2)q-CH3；其中各Rn独立为H、C1-C6烷基、C3-C8碳环基、苯基或苄基；并且各q独立为1、2、3、4、5、6、7、8、9或10；
**与L连接；
y为0、1或2；
Y为O、S或CR1R2，其中R1和R2各自独立地为H或C1-C6烷基；
s和t各自独立为0、1或2，但不同时为0。
在一个或多个实施方式中，X4为H或C1-C6烷基。
在一个或多个实施方式中，所述杂环为氮杂环丁烷、乙醛嗪(niverazine)、吗啉、吡咯烷、哌啶、咪唑、噻唑、噁唑或吡啶。
在一个或多个实施方式中，所述氨基保护基为甲酰基、乙酰基、三苯甲基、叔丁氧基羰基、苄基或对甲氧基苄氧基羰基。
在一个或多个实施方式中，药物为其中X1和X2各自独立地为C1-C6烷基、卤素或-OH；**与L连接。
在一个或多个实施方式中，药物为其中X1和X2各自独立地为C1-C6烷基、卤素或-OH；**与L连接。
在一个或多个实施方式中，X1和X2各自为-CH3。
在一个或多个实施方式中，X1和X2各自独立地为F、Cl、Br或I。
在一个或多个实施方式中，X1和X2各自为F。
在一个或多个实施方式中，X1和X2各自独立地为-CH3、F或-OH。
在一个或多个实施方式中，X1和X2各自独立地为F或-CH3。
在一个或多个实施方式中，X1为-CH3及X2为F。
在一个或多个实施方式中，R为-(CH2)r-。
在一个或多个实施方式中，R为-(CH2)r-，r为1或5。
在一个或多个实施方式中，各AA独立选自以下的氨基酸或肽序列：Val-Cit、Val-Lys、Phe-Lys、Lys-Lys、Ala-Lys、Phe-Cit、Leu-Cit、Ile-Cit、Trp、Cit、Phe-Ala、Phe-Phe-Lys、D-Phe-Phe-Lys、Gly-Phe-Lys、Leu-Ala-Leu、Ile-Ala-Leu、Val-Ala-Val、Ala-Leu-Ala-Leu、β-Ala-Leu-Ala-Leu和Gly-Phe-Leu-Gly。
在一个或多个实施方式中，i为1。
在一个或多个实施方式中，AA为Val-Cit，i为1。
在一个或多个实施方式中，各FF独立为 其中各RF独立为C1-C6烷基、C1-C6烷氧基、-NO2或卤素；其中*连接AA，**连接D。
在一个或多个实施方式中，卤素为F，z为0、1、2、3或4。
在一个或多个实施方式中，RF为-CH3、F、-NO2或-OCH3。
在一个或多个实施方式中，z为0。
在一个或多个实施方式中，z为1或2。
在一个或多个实施方式中，各FF独立为 其中*连接AA，**连接D。
在一个或多个实施方式中，f为1。
在一个或多个实施方式中，FF为f为1；其中*连接AA，**连接D。
在一个或多个实施方式中，L为其中*连接B，**连接D。
在一个或多个实施方式中，L为其中*连接B，**连接D。
在一个或多个实施方式中，G为n为4-12。
在一个或多个实施方式中，n为4-8。
在一个或多个实施方式中，n为4。
在一个或多个实施方式中，n为8。
在一个或多个实施方式中，p为2-8。
在一个或多个实施方式中，p为4-8。
在一个或多个实施方式中，p为6-8。
在一个或多个实施方式中，p为7-8。
在一个或多个实施方式中，所述Abu为本发明提供的抗体或抗原结合单元。
在一个或多个实施方式中，所述抗体药物偶联物具有式I-1或式I-2所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物：
其中
Abu为本发明提供的抗体或抗原结合单元；
R选自：-(CH2)r-、-(CHRm)r-、C3-C8碳环基、-O-(CH2)r-、亚芳基、-(CH2)r-亚芳基-、-亚芳基-(CH2)r-、-(CH2)r-(C3-C8碳环基)-、-(C3-C8碳环基)-(CH2)r-、C3-C8杂环基、-(CH2)r-(C3-C8杂环基)-、-(C3-C8杂环基)-(CH2)r-、-(CH2)rC(O)NRm(CH2)r-、-(CH2CH2O)r-、-(CH2CH2O)r-CH2-、-(CH2)rC(O)NRm(CH2CH2O)r-、-(CH2)rC(O)NRm(CH2CH2O)r-CH2-、-(CH2CH2O)rC(O)NRm(CH2CH2O)r-、-(CH2CH2O)rC(O)NRm(CH2CH2O)r-CH2-和-(CH2CH2O)rC(O)NRm(CH2)r-；其中各Rm独立为H、C1-C6烷基、C3-C8碳环基、苯基或苄基；并且各r独立为1、2、3、4、5、6、7、8、9或10；
D为药物；
n为1-24的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24；
p为1-10，例如1、2、3、4、5、6、7、8、9或10。
在一个或多个实施方式中，R为-(CH2)r-。
在一个或多个实施方式中，R为-(CH2)r-，r为1或5。
在一个或多个实施方式中，n为4-12。
在一个或多个实施方式中，n为4-8。
在一个或多个实施方式中，n为4。
在一个或多个实施方式中，n为8。
在一个或多个实施方式中，p为2-8。
在一个或多个实施方式中，p为4-8。
在一个或多个实施方式中，p为6-8。
在一个或多个实施方式中，p为7-8。
在一个或多个实施方式中，所述抗体药物偶联物具有式I-3或式I-4所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物：
其中
Abu为本发明提供的抗体或抗原结合单元；
D为药物；
n为1-24的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24；
p为1-10，例如1、2、3、4、5、6、7、8、9或10。
在一个或多个实施方式中，n为4-12。
在一个或多个实施方式中，n为4-8。
在一个或多个实施方式中，n为4。
在一个或多个实施方式中，n为8。
在一个或多个实施方式中，p为2-8。
在一个或多个实施方式中，p为4-8。
在一个或多个实施方式中，p为6-8。
在一个或多个实施方式中，p为7-8。
在一个或多个实施方式中，所述抗体药物偶联物具有式I-5、I-6、I-7、I-8、I-9、I-10、或I-11所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物：


其中
Abu为本发明提供的抗体或抗原结合单元；
D为药物；
p为1-10，例如1、2、3、4、5、6、7、8、9或10。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，p为2-8。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，p为4-8。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，p为6-8。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，p为7-8。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，药物为抗癌药物、细胞毒性药物、细胞分化因子、干细胞营养因子、类固醇类药物、治疗自身免疫疾病的药物、抗炎症药物或治疗传染性疾病的药物。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，药物为微管蛋白抑制剂、DNA损伤剂或DNA拓扑异构酶抑制剂。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，所述微管蛋白抑制剂选自海兔毒素(dolastatin)、奥瑞他汀(auristatin)类、及美登素(maytansine)类。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，药物为奥瑞他汀(auristatin)类，例如MMAE、MMAF或AF。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，药物为DNA损伤剂，例如卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD(pyrrolobenzodiazepine，吡咯并苯并二氮杂)。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，药物为DNA拓扑异构酶抑制剂或其盐，例如伊立替康、伊立替康盐酸盐、喜树碱、9-氨基喜树碱、9-硝基喜树碱、10-羟基喜树碱、9-氯-10-羟基喜树碱、喜树碱类衍生物SN-38、22-羟基旱莲木碱、拓扑替康、勒托替康、贝洛替康、依喜替康、硅基高喜树碱(homosilatecan)、6,8-二溴-2-甲基-3-[2-(D-吡喃木糖基氨基)苯基]-4(3H)-喹唑啉酮、2-氰基-3-(3,4-二羟基苯基)-N-(苯基甲基)-(2E)-2-丙烯酰胺、2-氰基-3-(3,4-二羟基苯基)-N-(3-羟基苯基丙基)-(E)-2-丙烯酰胺、12-β-D-吡喃萄萄糖基-12,13-二氢-2,10-二羟基-6-[[2-羟基-1-(羟基甲基)乙基]氨基]-5H-吲哚并[2,3-a]吡咯并[3,4-c]咔唑-5,7(6H)-二酮、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺二盐酸盐、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，所述DNA拓扑异构酶抑制剂为喜树碱、10-羟基喜树碱、拓扑替康、贝洛替康、伊立替康、22-羟基旱莲木碱或依喜替康。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，药物为Tubulysin类、紫杉类药物衍生物、leptomycine衍生物、CC-1065及其类似物、Amatoxin类、剪接体抑制剂、苯(并)二卓氮(PBD)二聚体类、阿霉素、甲氨蝶呤，长春新碱，长春碱，柔红霉素，丝裂霉素C，马法兰，或苯丁酸氮芥衍生物。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，药物为其中
X1和X2各自独立地为：
H，
羟基，
C1-C6烷基，
被一个或多个羟基、卤素、硝基或氰基取代的C1-C6烷基，
C2-C6烯基，
C2-C6炔基，
C1-C6烷氧基，
C1-C6氨基烷氧基，
卤素，
硝基，
氰基，
巯基，
烷硫基，
氨基，被氨基保护基取代的氨基，在氨基部分任选被氨基保护基或C1-C6烷基取代的C1-C6氨基烷基，
在氨基部分任选被氨基保护基或C1-C6烷基取代的C1-C6氨基烷基氨基，
连接至杂环的C1-C6烷基，所述杂环任选被一个或多个C1-C6烷基、C1-C6烷氧基、氨基、卤素、硝基或氰基取代，
连接至杂环的C1-C6烷基氨基，所述杂环任选被C1-C6烷基、C1-C6烷氧基取代，所述氨基任选被氨基保护基、卤素、硝基、氰基或保护基取代，
氨基取代的杂环基，其在杂环部分的氮原子或氨基部分任选被保护基或一个或多个C1-C6烷基取代，
杂环氨基，其在杂环部分的氮原子或氨基部分任选被保护基或C1-C6烷基取代，
任选被氨基甲酰基保护基或C1-C6烷基取代的氨基甲酰基，
吗啉-1-基，或
哌啶-1-基；
X3为C1-C6烷基；
X4为H、-(CH2)q-CH3、-(CHRn)q-CH3、C3-C8碳环基、-O-(CH2)q-CH3、亚芳基-CH3、-(CH2)q-亚芳基-CH3、-亚芳基-(CH2)q-CH3、-(CH2)q-(C3-C8碳环基)-CH3、-(C3-C8碳环基)-(CH2)q-CH3、C3-C8杂环基、-(CH2)q-(C3-C8杂环基)-CH3、-(C3-C8杂环基)-(CH2)q-CH3、-(CH2)qC(O)NRn(CH2)q-CH3、-(CH2CH2O)q-CH3、-(CH2CH2O)q-CH2-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH2-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH2-CH3、或-(CH2CH2O)qC(O)NRn(CH2)q-CH3；其中各Rn独立为H、C1-C6烷基、C3-C8碳环基、苯基或苄基；并且各q独立为1、2、3、4、5、6、7、8、9或10；
**与所述抗体药物偶联物其他部分连接；
y为0、1或2；
Y为O、S或CR1R2，其中R1和R2各自独立地为H或C1-C6烷基；
s和t各自独立为0、1或2，但不同时为0。
在一个或多个实施方式中，X4为H或C1-C6烷基。
在一个或多个实施方式中，所述杂环为氮杂环丁烷、乙醛嗪、吗啉、吡咯烷、哌啶、咪唑、噻唑、噁唑或吡啶。
在一个或多个实施方式中，所述氨基保护基为甲酰基、乙酰基、三苯甲基、叔丁氧基羰基、苄基或对甲氧基苄氧基羰基。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，药物为其中X1和X2各自独立地为C1-C6烷基、卤素或-OH；**与所述抗体药物偶联物其他部分连接。
在一个或多个式I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、或I-11的实施方式中，药物为其中X1和X2各自独立地为C1-C6烷基、卤素或-OH；**与所述抗体药物偶联物其他部分连接。
在一个或多个实施方式中，所述C1-C6烷基为-CH3。
在一个或多个实施方式中，所述卤素为F。
在一个或多个实施方式中，X1和X2各自独立地为-CH3、F或-OH。
在一个或多个实施方式中，X1和X2各自为-CH3。
在一个或多个实施方式中，X1和X2各自独立地为F、Cl、Br或I。
在一个或多个实施方式中，X1和X2各自为F。
在一个或多个实施方式中，X1和X2各自独立地为F或-CH3。
在一个或多个实施方式中，X1为-CH3及X2为F。
在一个或多个实施方式中，所述抗体药物偶联物具有式I-12、I-13、I-14、I-15、I-16、I-17、I-18、I-19、I-20、I-21、I-22、I-23、I-24、或I-25所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物：




其中
Abu为本发明提供的抗体或抗原结合单元；
p为1-10，例如1、2、3、4、5、6、7、8、9或10。
在一个或多个实施方式中，p为2-8。
在一个或多个实施方式中，p为4-8。
在一个或多个实施方式中，p为6-8。
在一个或多个实施方式中，p为7-8。
一个或多个实施方式提供了抗体或抗原结合单元或抗体药物偶联物，其用作药物。
在一个或多个实施方式中，本发明抗体或抗原结合单元或抗体药物偶联物与一种或多种其它疗法联用。合适的其它疗法包括现有的用于特定应用(如癌症)的药物和/或手术疗法。例如，所述抗体或抗原结合单元或抗体药物偶联物与一种或多种其它化疗或抗肿瘤试剂联用。或者，其它化疗剂是放射疗法。在一个或多个实施方式中，所述化疗剂是细胞死亡诱导剂。
在一个或多个实施方式中，所述抗体或抗原结合单元或抗体药物偶联物和其它试剂被制备为单个治疗组合物，并同时给予所述抗体或抗原结合单元或抗体药物偶联物和其它试剂。或者，所述抗体或抗原结合单元或抗体药物偶联物和其它试剂彼此独立，例如分别制备为独立的治疗组合物，并同时给予所述抗体或抗原结合单元或抗体药物偶联物和其它试 剂，或在治疗方案期间在不同时间给予所述抗体或抗原结合单元或抗体药物偶联物和其它试剂。例如，在给予其它试剂前给予所述抗体或抗原结合单元或抗体药物偶联物，在给予其它试剂后给予所述抗体或抗原结合单元或抗体药物偶联物，或在以交替的方案给予所述抗体或抗原结合单元或抗体药物偶联物和其它试剂。本文中，以单个剂量或多个剂量给予所述抗体或抗原结合单元或抗体药物偶联物和其它试剂。
在一个或多个实施方式中，本发明还提供了一种药物组合物，其包含本发明所述抗体或抗原结合单元、或抗体药物偶联物以及药物可接受的载体、赋形剂和/或辅料。在一个或多个实施方式中，所述药物组合物还包含任选的其他抗癌药物。本发明提供的药物组合物可以通过任何方便的途径施用，例如通过输注或推注，通过上皮或皮肤粘膜(例如口腔粘膜、直肠和肠粘膜等)吸收，并且可以与其他生物活性剂共同施用。因此，所述药物组合物可以静脉给药、口服给药、直肠给药、肠胃外给药、脑内给药、阴道内给药、腹腔内给药、外敷(如通过粉末，软膏，滴剂或透皮贴剂)、口腔给药或通过口服或鼻腔喷雾给药。
一个或多个实施方式提供了本文所述抗体或抗原结合单元、生物材料(选自多聚核苷酸、表达载体和细胞)、抗体药物偶联物、药物组合物中的至少一种在制备治疗和/或预防疾病的药物中的应用。在一个或多个实施方式中，所述疾病为CLDN18.2表达相关的疾病。在一个或多个实施方式中，所述疾病为CLDN18.2过表达相关的疾病。在一个或多个实施方式中，所述疾病为表达CLDN18.2的癌症或肿瘤。在一个或多个实施方式中，所述疾病为过表达CLDN18.2的癌症或肿瘤。
一个或多个实施方式提供了本文所述抗体或抗原结合单元、生物材料(选自多聚核苷酸、表达载体和细胞)、抗体药物偶联物和/或包含其的药物组合物在治疗和/或预防疾病中的应用。在一个或多个实施方式中，所述疾病为CLDN18.2表达相关的疾病。在一个或多个实施方式中，所述疾病为癌症或肿瘤。在一个或多个实施方式中，所述疾病为CLDN18.2过表达相关的疾病。在一个或多个实施方式中，所述疾病为表达CLDN18.2的癌症或肿瘤。在一个或多个实施方式中，所述疾病为过表达CLDN18.2的癌症或肿瘤。
一个或多个实施方式提供了治疗和/或预防疾病的方法，该方法包括向有需要的患者给药有效量的本文所述抗体或抗原结合单元、生物材料(选自多聚核苷酸、表达载体和细胞)、抗体药物偶联物和/或包含其的药物组合物。在一个或多个实施方式中，所述疾病为癌症或肿瘤。在一个或多个实施方式中，所述疾病为CLDN18.2表达相关的疾病。在一个或多个实施方式中，所述疾病为CLDN18.2过表达相关的疾病。在一个或多个实施方式中，所述疾病为表达CLDN18.2的癌症或肿瘤。在一个或多个实施方式中，所述疾病为过表达CLDN18.2的癌症或肿瘤。在一个或多个实施方式中，所述有效量是指活性化合物或药剂的量，其导致研究人员、兽医、医生或其他临床医生正寻求的组织、系统、动物、个体以及人的生物或药用响应，这包含治疗一种疾病。在一个或多个实施方式中，所述有效量是约0.1～100mg/kg。
癌症的实例包括但不限于实体瘤、血液学癌及转移性病灶。在一些实施方式中，所述癌症或肿瘤包括但不限于膀胱癌、卵巢癌(如卵巢腺癌和卵巢畸胎癌)、肺癌(如小细胞肺癌和非小细胞肺癌)、腺癌、胃癌、乳腺癌、肝癌、胰腺癌、皮肤癌(如基底细胞癌和鳞状细胞癌)、恶性黑色素瘤、头颈癌、肉瘤(如滑膜肉瘤和癌肉瘤)、胆管癌、肾癌(透明细胞肾细胞癌和乳头状肾细胞癌)、结肠癌、小肠癌、睾丸胚胎性癌、胎盘绒毛膜癌、宫颈癌、睾丸癌、子宫癌、食道癌和胆囊癌细胞等。
在一个或多个实施方式中，可以将抗体或抗原结合单元或抗体药物偶联物配制成药物组合物，并以适合于所选给药途径的形式向患者给药，给药途径例如肠胃外、静脉内(iv)、肌肉内、局部或皮下。
在一个或多个实施方式中，患者接受一个治疗周期治疗。在一个或多个实施方式中，患者接受多个(例如2个、3个、4个、5个、6个、7个、8个、9个、10个、11个或12个)治疗周期治疗。在一个或多个实施方式中，患者接受治疗直至病症得到缓解而不再需要治疗。
在一个或多个实施方式中，所述抗体或抗原结合单元或抗体药物偶联物或包含其的药物组合物是通过注射给药。在一个或多个实施方式中，所述抗体或抗原结合单元或抗体药物偶联物或包含其的药物组合物是通过皮下(s.c.)注射、腹膜内(i.p.)注射、肠胃外注射、动脉内注射或静脉内(i.v.)注射等方式给药。在一个或多个实施方式中，所述抗体或抗原结合单元或抗体药物偶联物或包含其的药物组合物是输液方式进行给药。在一个或多个实施方式中，所述抗体或抗原结合单元或抗体药物偶联物或包含其的药物组合物是推注方式进行给药。在一个或多个实施方式中，所述抗体或抗原结合单元或抗体药物偶联物或包含其的药物组合物是通过静脉注射给药。在一个或多个实施方式中，所述抗体或抗原结合单元或抗体药物偶联物或包含其的药物组合物是通过静脉输注给药。抗体或抗原结合单元或抗体药物偶联物的给药量将取决于药物的性质、细胞表面触发药物的内在化、运输和释放的程度，以及所治疗的疾病和患者的状况(如年龄、性别、体重等)。
在一个或多个实施方式中，所述抗体或抗原结合单元或抗体药物偶联物或包含其的药物组合物是通过静脉内(i.v.)输液方式(即静脉输注)进行给药。在一个或多个实施方式中，静脉内输液持续时间为约10分钟、约15分钟、约20分钟、约25分钟、约30分钟、约40分钟、约50分钟、约55分钟、约60分钟、约65分钟、约70分钟、约75分钟、约81分钟、约87分钟、约90分钟、约95分钟，或这些数值中任何两个值之间的范围(包括端点)或其中任何值。在一个或多个实施方式中，静脉输注时间≤30分钟。在一个或多个实施方式中，静脉输注时间≥60分钟。在一个或多个实施方式中，静脉输注时间≥90分钟。
在一个或多个实施方式中，本发明提供了包含所述抗体或抗原结合单元或抗体药物偶联物的适合注射用的药物组合物，如推注型药物组合物或输液(滴注)型药物组合物。适于注射用途的药物组合物包括无菌水性溶液(在此是水溶性的)或分散体以及用于即时制备无菌注射液或分散体的无菌粉末。对于静脉内施用，合适的载体包括生理盐水、抑菌水或磷酸盐缓冲盐水(PBS)、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)的溶剂或分散介质，及其适宜的混合物。在一个或多个实施方式中，药物组合物还包括药学可接受的载体。在一个或多个实施方式中，药学上可接受的载体可以包含抗细菌剂和/或抗真菌剂，如对羟基苯甲酸酯、氯代丁醇、苯酚、抗坏血酸、硫柳汞等来实现。在一个或多个实施方式中，药物组合物至少包含0.1％的抗体或抗原结合单元或抗体药物偶联物。抗体的百分比可以变化，并且为给定剂型重量的约2％至90％之间。这种药物组合物中的抗体或抗原结合单元或抗体药物偶联物的量可以为给药的有效量。
在一个或多个实施方式中，本发明提供了药物组合物的制备方法：分别将本文所述的抗体或抗原结合单元或抗体药物偶联物与药学上可接受的载体(例如注射用水，生理盐水等)混合。上述抗体或抗原结合单元或抗体药物偶联物与药学上可接受的载体的混合方法是本领域通常已知的。
在一个或多个实施方式中，本发明提供了一种试剂盒，包含本文所述抗体或抗原结合单元、抗体药物偶联物或包含其的药物组合物以及用于指导患者给药的说明书。
一个或多个实施方式还提供了制品，其包含本发明所述抗体或抗原结合单元、抗体药物偶联物或者包含其的药物组合物；
容器；和
包装插页、说明书或标签，其指示本发明所述抗体或抗原结合片段、抗体药物偶联物或者包含其的药物组合物用于治疗和/预防疾病。
附图说明
图1表示抗CLDN18.2嵌合抗体对KATOIII细胞结合的剂量曲线，纵坐标表示平均荧光强度。
图2为抗CLDN18.2人源化抗体细胞结合特异性结果，其中，H239H-2a+K-6a表示为抗体H239H-2a-K-6a，H239H-2b+K-6a表示为抗体H239H-2b-K-6a。
图3表示抗CLDN18.2抗体对KATOIII细胞的剂量结合曲线；其中CLDN18.2Ab表示抗体H239H-2b-K-6a-1。
图4表示ADC对CHO-CLDN18.2细胞增殖抑制剂量曲线。
图5表示ADC旁观者效应的活细胞数统计。
图6表示在GA0006异种移植模型中各组小鼠肿瘤体积的生长曲线(平均值±标准误)。
图7表示在GA0006异种移植模型中各组小鼠肿瘤重量(平均值±标准误)。
具体实施方式
除非另有定义，本发明中使用的科学和技术术语的含义是本领域技术人员所通常理解的含义。通常，本文所述的细胞培养、分子生物学以及蛋白质纯化使用的命名和技术是本领域公知且普遍使用的。对于重组DNA、寡核苷酸合成和细胞培养与转化(如电穿孔、脂质转染)，使用了标准技术。酶促反应和纯化技术根据生产商的说明书或本领域普遍使用或本文所述的方法进行。前述技术和方法通常根据本领域公知且本说明书中引用和讨论的多部综合和较具体的文献中描述的那样使用。参见例如Sambrook等，Molecular Cloning:A Laboratory Manual(《分子克隆：实验室手册》)(第2版，冷泉港实验室出版社(Cold Spring Harbor Laboratory Press)，纽约冷泉港(1989))。
定义
应当注意的是，术语“一种”实体是指一种或多种该实体，例如“一种抗体”应当被理解为一种或多种抗体，因此，术语“一种”(或“一个”)、“一种或多种”和“至少一种”可以在本文中互换使用。
本文所用的术语“包含”或“包括”意味着抗体、组合物或方法等包括所列举的元素，例如组份或步骤，但不排除其它。“基本上由……组成”意味着抗体、组合物或方法等排除对组合的特征有根本影响的其它元素，但不排除对抗体、组合物或方法等无本质上影响的元素。“由……组成”意味着排除未特别列举的元素。
本文所用术语“抗体”指免疫球蛋白(Ig)分子和免疫球蛋白分子的免疫活性部分，即包含特异性结合抗原(与其免疫反应)的抗原结合位点的分子。抗体包括但不限于单克隆抗体、嵌合抗体、dAb(结构域抗体)、单链抗体、Fab、Fab-和F(ab’)2片段、Fv和Fab表达库。
术语“抗体”包括可以在生物化学上区分的各种广泛种类的多肽。
本发明公开的抗体、抗原结合单元或衍生物包括但不限于多克隆、单克隆、多特异性、全人源、人源化、灵长类化、嵌合抗体、单链抗体、表位结合片段(例如类Fab、类Fab'和类F(ab')2)、类单链Fvs(scFv)。
术语“单克隆抗体”(mAb)是指一群这样的抗体分子：其只含有由独特的轻链基因产物和独特的重链基因产物组成的抗体分子中的一种分子种类。具体地，单克隆抗体的互补 决定区(CDR)在该群体的所有分子中是相同的。MAb含有能够与抗原的特定表位发生免疫反应的抗原结合位点。
术语“单链抗体”(scFv)是指由抗体重链可变区(VH)和轻链可变区(VL)通过15～20个氨基酸的接头(linker)连接而成的抗体。接头可以富含甘氨酸以增加柔韧性，以及富含丝氨酸或苏氨酸以增加溶解性，并且可以连接VH的N端和VL的C端，反之亦然。尽管该蛋白质被除去了恒定区和引入了接头，但其保留了原始免疫球蛋白的特异性。ScFv分子通常是本领域中已知的，例如在美国专利5,892,019中有相关描述。
本领域技术人员将会理解，重链的类别包括gamma、mu、alpha、delta或epsilon(γ、μ、α、δ、ε)，其中还有一些亚类(例如γ1-γ4)。该链的性质决定了抗体的“种类”分别为IgG、IgM、IgA、IgG或IgE。免疫球蛋白亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgG5等已被充分表征并且赋予的功能特异性也已知。所有的免疫球蛋白种类都在本发明公开的保护范围内。在一个或多个实施方式中，免疫球蛋白分子的种类为IgG。两条重链和两条轻链通过二硫键以“Y”构型连接，其中轻链从“Y”口开始并延续通过可变区包围重链。轻链可以分为kappa(κ)或lambda(λ)。每个重链可以与κ或λ轻链结合。一般来说，当由杂交瘤、B细胞或基因工程宿主细胞生产免疫球蛋白时，其轻链和重链通过共价键结合，两条重链的“尾巴”部分通过共价二硫键或非共价键结合。在重链中，氨基酸序列从Y构型的叉状末端的N末端延伸至每条链底部的C末端。免疫球蛋白κ轻链可变区为Vκ；免疫球蛋白λ轻链可变区为Vλ。
抗体轻链可变区(VL)和重链可变区(VH)决定了抗原识别和特异性。轻链恒定区(CL)和重链恒定区(CH)赋予重要的生物学性质，如分泌、经胎盘移动、Fc受体结合、补体结合等。按照惯例，恒定区的编号随着它们变得更远离抗体的抗原结合位点或氨基末端而增加。N端部分是可变区，C端部分是恒定区；CH3和CL结构域实际上分别包含重链和轻链的羧基端。
在天然存在的抗体中，假设抗体在含水环境中呈现其三维构型时，存在于每个抗原结合域中的六个“互补决定区”或“CDR”是形成抗原结合结构域的短的、非连续的与抗原特异性结合的氨基酸序列。抗原结合结构域中被称为“框架”(“FR”)区域的剩余其它氨基酸显示出较小的分子间可变性。框架区大部分采用β-折叠构象，CDR形成与之连接的环状结构，或在某些情况下形成β折叠结构的一部分。因此，框架区通过形成支架从而通过链间非共价相互作用使CDR定位在正确的方位上。具有特定位置的CDR的抗原结合域形成了与抗原上的表位互补的表面，该互补表面促进抗体和其抗原表位的非共价结合。通常在抗体分子中，每条重链和轻链有三个CDR，分别被称为VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2以及VL CDR3。按照位置顺序，通常重链可变区包含VH FR1、VH CDR1、VH FR2、VH CDR2、VH FR3、VH CDR3以及VH FR4，轻链可变区包含VL FR1、VL CDR1、VL FR2、VL CDR2、VL FR3、VL CDR3以及VL FR4。对于给定的重链或轻链可变区，本领域普通技术人员都可以通过已知方法鉴定出包含CDR和框架区的氨基酸(参见Kabat,E.,et al.,U.S.Department of Health and Human Services,Sequences of Proteins of Immunological Interest,(1983)和Chothia and Lesk,J.Mol.Biol.,196:901-917(1987))。
人源化抗体的框架区及CDR区不必准确对应于亲本序列，例如供体抗体CDR或共有框架可通过至少一个氨基酸残基的取代、插入和/或缺失而突变诱发，使得该位点的CDR或框架残基不对应于供体抗体或共有框架。通常，至少80％、至少85％、更至少90％或至少95％的人源化抗体残基将对应于亲本FR及CDR序列的那些残基。如本文使用的，术语“共有框架”是指共有免疫球蛋白序列中的框架区。如本文使用的，术语“共有免疫球蛋白序列”是指由相关免疫球蛋白序列家族中最频繁出现的氨基酸(或核苷酸)形成的序列(参见例 如，Winnaker,From Genes to Clones[从基因到克隆](Verlagsgesellschaft,Weinheim,德国1987))。在免疫球蛋白家族中，共有序列中的各位置由该家族中最频繁出现于该位置的氨基酸占据。若两个氨基酸同等频繁地出现，则共有序列中可包括任一个。
在本领域中使用和/或接受的术语有两个或多个定义的情况下，除非明确地对立指出，否则本文使用的术语的定义包括所有这些含义。一个具体的例子是使用“互补决定区”(“CDR”)一词来描述在重链和轻链多肽的可变区内发现的非连续的抗原结合位点。这一特定区域在Kabat et al.,U.S.Dept.of Health and Human Services,Sequences of Proteins of Immunological Interest(1983)和Chothia等在J.Mol.Biol.196:901-917(1987)有相关描述，其通过引用全部并入本文。
Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以不依赖于序列本身以外的其他实验数据将该“Kabat编号”系统应用到任何可变区序列。“Kabat编号”是指由Kabat et al.,U.S.Dept.of Health and Human Services在“Sequence of Proteins of Immunological Interest”(1983)提出的编号系统。抗体还可以用EU或Chothia编号系统。
本发明公开的抗体可以来源于任何动物，包括但不限于鱼类、鸟类和哺乳动物。较佳地，抗体是人源、鼠源、驴源、兔源、山羊源、骆驼源、美洲驼源、马源或鸡源抗体。在另一实施方案中，可变区可以是软骨鱼纲(condricthoid)来源(例如来自鲨鱼)。
“重链恒定区”包括CH1结构域、铰链(例如上、中和/或下铰链区)结构域、CH2结构域、CH3结构域，或变体或片段中的至少一种。抗体的重链恒定区可以来源于不同的免疫球蛋白分子。例如，抗体的重链恒定区可以包括源自IgG1分子的CH1结构域和源自IgG3分子的铰链区。在另一实施方案中，重链恒定区可以包括部分源自IgG1分子和部分源自IgG3分子的铰链区。在另一实施方案中，部分重链可以包括部分源自IgG1分子和部分源自IgG4分子的嵌合铰链区。
“轻链恒定区”包括来自抗体轻链的一部分氨基酸序列。较佳地，轻链恒定区包含恒定κ结构域或恒定λ结构域中的至少一个。“轻链-重链对”是指可通过轻链的CL结构域和重链的CH1结构域之间的二硫键形成二聚体的轻链和重链的集合。
“二硫键”指两个硫原子之间形成的共价键。半胱氨酸的硫醇基团可以与第二个硫醇基团形成二硫键或桥接。在大多数天然存在的IgG分子中，CH1和CL区通过二硫键连接。
“嵌合抗体”指其可变区从第一个物种中获得或衍生，而其恒定区(可以是完整的、部分的或修饰过的)来源于第二个物种的任何抗体。某些实施方案中，可变区来自非人源(例如小鼠或灵长类动物)，而恒定区来自人源。
本文所用术语“表位”包括任意能够特异性结合免疫球蛋白或其片段或T细胞受体的蛋白决定区。表位决定区通常由分子的化学活性表面基团(如氨基酸或糖侧链)组成且通常有特定的三维结构性质以及特定的电荷性质。
如本文所用，术语“特异性结合”或“发生免疫反应”或“针对”是指在免疫球蛋白分子与其目标抗原的一个或多个抗原决定簇之间发生的非共价相互作用。免疫学结合相互作用的强度或亲和力可以以相互作用的平衡解离常数(KD)表示，其中较小的KD代表较大的亲和力。所选多肽的免疫结合特性可使用本领域熟知方法进行定量。一种此类方法需要测量抗原结合位点/抗原复合物形成和解离的速率，其中那些速率取决于复合物配偶体的浓度、相互作用的亲和力和在两个方向同等影响该速率的几何参数。因此，“结合速率常数”(kon)和“解离速率常数”(koff)两者可通过计算浓度和实际的缔合和解离速率测定(参见Malmqvist,M.,Nature 361:186-87(1993))。koff/kon比率能够消除所有与亲和力无关的参数，并且等于平衡解离常数KD(通常参见Davies等人(1990)Annual Rev Biochem 59:439-473)。 特异性结合可由放射性配体结合测定、表面等离子体共振(SPR)、流式细胞术结合测定、或本领域技术人员已知的类似测定所测。
本发明中关于细胞、核酸、多肽等所使用的术语“分离的”，例如“分离的”DNA、RNA、多肽是指分别于细胞天然环境中的其它组分如DNA或RNA中的一种或多种所分离的分子。本发明使用的术语“分离的”还指当通过重组DNA技术产生时基本上不含细胞材料、病毒材料或细胞培养基的核酸或肽，或化学合成时的化学前体或其他化学品。此外，“分离的核酸”意在包括不以天然状态存在的核酸片段，并且不会以天然状态存在。术语“分离的”在本发明中也用于指从其他细胞蛋白质或组织分离的细胞或多肽。分离的多肽意在包括纯化的和重组的多肽。分离的多肽等通常通过至少一个纯化步骤制备。在一个或多个实施方式中，分离的核酸、多肽等的纯度至少为约50％、约60％、约70％、约80％、约90％、约95％、约99％，或这些数值中的任何两个值之间的范围(包括端值)或其中任何值。
术语“编码”应用于多聚核苷酸时，是指被称为“编码”多肽的多聚核苷酸，在其天然状态或当通过本领域技术人员公知的方法操作时，经转录和/或翻译可以产生该多肽和/或其片段。
术语“重组”涉及多肽或多聚核苷酸，意指天然不存在的多肽或多聚核苷酸的形式，不受限制的实施例可以通过组合产生通常并不存在的多聚核苷酸或多肽。
“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。
如本文所用，二十种常规氨基酸及其缩写遵循常规用法。参见Immunology-A Synthesis(第2版，E.S.Golub和D.R.Gren编辑，Sinauer Associates,Sunderland,Mass.(1991))。二十种常规氨基酸的立体异构体(例如，D-氨基酸)、非天然氨基酸(诸如α-、α-二取代氨基酸)、N-烷基氨基酸及其它非常规氨基酸也可为适用于本公开多肽的组分。非常规氨基酸的示例包括：瓜氨酸(Cit)、4-羟脯氨酸、γ-羧基谷氨酸盐、ε-N,N,N-三甲基赖氨酸、ε-N-乙酰赖氨酸、O-磷酸丝氨酸、N-乙酰丝氨酸、N-甲酰甲硫氨酸、3-甲基组氨酸、5-羟赖氨酰、σ-N-甲基精氨酸及其它类似的氨基酸和亚氨基酸(例如4-羟脯氨酸)。在本文所用的多肽表示方法中，左手方向为氨基末端方向，并且右手方向为羧基末端方向，与标准用法和惯例一致。常规(或天然)氨基酸包括丙氨酸(三字母代码：Ala，一字母代码：A)、精氨酸(Arg，R)、天冬酰胺(Asn，N)、天冬氨酸(Asp，D)、半胱氨酸(Cys，C)、谷氨酰胺(Gln，Q)、谷氨酸(Glu，E)、甘氨酸(Gly，G)、组氨酸(His，H)、异亮氨酸(Ile，I)、亮氨酸(Leu，L)、赖氨酸(Lys，K)、甲硫氨酸(Met，M)、苯丙氨酸(Phe，F)、脯氨酸(Pro，P)、丝氨酸(Ser，S)、苏氨酸(Thr，T)、色氨酸(Trp，W)、酪氨酸(Tyr，Y)和缬氨酸(Val，V)。
术语“多肽”旨在涵盖单数的“多肽”以及复数的“多肽”，并且是指由通过酰胺键(也称为肽键)线性连接的氨基酸单体形成的分子。术语“多肽”是指两个或更多个氨基酸的任何单条链或多条链，并且不涉及产物的特定长度。因此，“多肽”的定义中包括肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指两个或多个氨基酸链的任何其他术语，并且术语“多肽”可以用来代替上述任何一个术语，或者与上述任何一个术语交替使用。术语“多肽”也意在指多肽表达后修饰的产物，包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰。多肽可以源自天然生物来源或通过重组技术产生，但其不必从指定的核酸序列翻译所得，它可能以包括化学合成的任何方式产生。
本领域技术人员理解当氨基酸或多肽作为一个分子(如抗体或ADC)的组成部分时，氨基酸或多肽指氨基酸残基或多肽残基(不论是否写明)，即其在与该分子的其他部分结合时，其部分基团(例如其氨基的一个氢原子和/或羧基的羟基)因其与分子的其他部分形成共价键(如酰胺键)而失去后的剩余部分。
当应用于多肽时，术语“基本上相同”是指两个肽序列在诸如通过GAP或BESTFIT程序使用默认空位权重进行最佳比对时，共享至少80％序列同一性，优选至少90％序列同一性，更优选至少95％序列同一性，并最优选至少99％序列同一性。
多聚核苷酸是由四个碱基的特定序列组成：腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)，或当多聚核苷酸是RNA时胸腺嘧啶换为尿嘧啶(U)。“多聚核苷酸序列”可以以多聚核苷酸分子的字母表示。该字母表示可以被输入到具有中央处理单元的计算机中的数据库中，并用于生物信息学应用，例如用于功能基因组学和同源性搜索。
术语“多核苷酸”、“多聚核苷酸”和“寡核苷酸”可互换使用，是指任何长度的核苷酸的聚合形式，无论是脱氧核糖核苷酸还是核糖核苷酸或其类似物。多聚核苷酸可以具有任何三维结构并且可以执行已知或未知的任何功能。以下是不受限制的多聚核苷酸的实施例：基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA、dsRNA、siRNA、miRNA、重组多聚核苷酸、分支的多聚核苷酸、质粒、载体、DNA、RNA、核酸探针和引物。多聚核苷酸可以包含修饰的核苷酸，例如甲基化的核苷酸和核苷酸类似物。如果存在该修饰，则对核苷酸的结构修饰可以在组装多聚核苷酸之前或之后进行。核苷酸的序列可以被非核苷酸组分中断。聚合后可以进一步修饰多聚核苷酸，例如通过与标记组分缀合。这个术语也指双链和单链分子。除另有说明或要求外，本公开的任何多聚核苷酸的实施例包括双链形式和已知或预测构成双链形式的两种可互补单链形式中的每一种。
多聚核苷酸或多聚核苷酸序列(或多肽或抗体序列)与另一序列有具有一定百分比(例如90％、95％、98％或者99％)的“同一性或序列同一性”是指当序列比对时，所比较的两个序列中该百分比的碱基(或氨基酸)相同。可以使用目测或本领域已知的软件程序来确定该比对和同一性百分比或序列同一性，比如Ausubel et al.eds.(2007)在Current Prot ocols in Molecular Biology中所述的软件程序。优选使用默认参数进行比对。其中一种比对程序是使用默认参数的BLAST，例如BLASTN和BLASTP，两者使用下列默认参数：Geneticcode＝standard；filter＝none；strand＝both；cutoff＝60；expect＝10；Matrix＝BLOSUM62；Descriptions＝50sequences；sortby＝HIGHSCORE；Databases＝non-redundant；GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+Swi ssProtein+SPupdate+PIR。生物学上等同的多聚核苷酸是具有上述指定百分比的同一性并编码具有相同或相似生物学活性的多肽的多聚核苷酸。
抗体或免疫球蛋白分子的氨基酸序列的微小变化都涵盖在本公开之内，条件是氨基酸序列的同一性保持至少90％，如至少92％、95％、98％或99％。在一些实施方案中，变化为保守氨基酸取代。保守氨基酸取代是在其侧链中相关的氨基酸家族内发生的取代。基因编码的氨基酸大致分以下类：(1)酸性氨基酸为天冬氨酸盐、谷氨酸盐；(2)碱性氨基酸为赖氨酸、精氨酸、组氨酸；(3)非极性氨基酸为丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)；以及(4)无电荷的极性氨基酸为甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸。其它家族的氨基酸包括(i)脂肪族-羟基家族的丝氨酸和苏氨酸；(ii)含酰胺家族的天冬酰胺和谷氨酰胺；(iii)脂肪族家族的丙氨酸、缬氨酸、亮氨酸和异亮氨酸；以及(iv)芳族家族的苯丙氨酸、色氨酸和酪氨酸。在一些实施方案中，保守氨基酸取代组为：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。例如，可以合理地预测用异亮氨酸或缬氨酸单独的置换亮氨酸，用谷氨酸盐置换天冬氨酸盐，用丝氨酸置换苏氨酸，或用一个结构相关的氨基酸类似的置换一个氨基酸，且对所得分子的结合或特性不会有重要影响，特别是该置换不涉及结合位点内的氨基酸。氨基酸改变是否产生功能性肽可以容易地通过测定多肽衍生物的比活性来确定。所述测定在本文进行了详细描述。抗体或免疫球蛋白分子的片段或类似物可由本领域普通技术人员容易地制备。
在一些实施方案中，氨基酸取代具有如下效果：(1)降低对蛋白水解作用的敏感性，(2)降低对氧化作用的敏感性，(3)改变用于形成蛋白复合物的结合亲和力，(4)改变结合亲和力，和(5)赋予或改进此类类似物的其它物理化学或功能特性。类似物可包括序列不同于天然存在的肽序列的各种突变蛋白。例如，可在天然存在的序列(优选在形成分子间接触的结构域之外的多肽部分中)中进行单个或多个氨基酸取代(优选保守氨基酸取代)。保守氨基酸取代不应当显著改变亲本序列的结构特性(例如，置换的氨基酸不应当趋于破坏亲本序列中存在的螺旋结构，或破坏表征亲本序列的其它类型二级结构)。人工识别的多肽的二级和三级结构的示例描述于Proteins,Structures and Molecular Principles(Creighton编辑，W.H.Freeman and Company,New York(1984))；Introduction to Protein Structure(C.Branden和J.Tooze编辑，Garland Publishing,New York,N.Y.(1991))；和Thornton等人Nature 354:105(1991)。
VL、VH的保守氨基酸取代的氨基酸数目可以为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端值)或其中任何值。重链恒定区、轻链恒定区、重链或轻链的保守氨基酸取代的氨基酸数目可以为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约18个、约19个、约22个、约24个、约25个、约29个、约31个、约35个、约38个、约41个、约45个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端值)或其中任何值。
本文所用术语“试剂”表示化学化合物、化学化合物混合物、生物大分子或由生物材料制得的提取物。
如本文所用，术语“标记”或“经标记的”是指掺入可检测标记，例如，通过掺入放射性标记的氨基酸，或者附着于可由标记的亲和素(例如，含有荧光标记或可由光学方法或量热法检测的酶活性的链霉亲和素)检测的生物素基部分的多肽。在某些情况下，标记物或标记也可为治疗性的。标记多肽和糖蛋白的各种方法是本领域已知的并且可以使用。用于多肽的标记物的示例包括但不限于以下项：放射性同位素或放射性核素(例如，3H、14C、15N、35S、90Y、99Tc、111In、125I、131I)，荧光标记物(例如，FITC、罗丹明、镧系磷光体)，酶标记物(例如，辣根过氧化酶、β-半乳糖苷酶、荧光素酶、碱性磷酸酶)，化学发光标记，生物素酰基，被二级报告基因识别的预定多肽表位(例如，亮氨酸拉链对序列、二级抗体结合位点、金属结合结构域、表位标签)。在一些实施方案中，标记通过各种长度的间隔臂连接以减小可能的空间位阻。术语“药剂”或“药物”是指适当施用于患者时能够诱导期望的治疗效果的化合物或组合物。
“约”指相关技术领域技术人员容易知道的相应数值的常规误差范围。在一些实施方式中，本文中提到“约”指所描述的数值以及其±10％、±5％或±1％的范围。
“EC50”即半最大效应浓度(concentration for 50％of maximal effect,EC50)是指能引起50％最大效应的浓度。
“IC50”表示50％抑制浓度，即对指定的生物过程抑制一半时所需的药物或者抑制剂的浓度。
“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防、减缓、改善或停止不良的生理改变或紊乱，例如疾病的进程，包括但不限于以下无论是可检测还是不可检测的结果，症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善、缓和、减轻或消失(无论是部分还是全部)、延长与不接受治疗时预期的生存期限等。需要治疗的患者包括已经患有病症或紊乱的患者，容易患有病症或紊乱的患者，或者需要预防该病症或紊乱的患者，可以或预期从施用本发明公开的抗体或药物组合物用于检测、诊断过程和/或治疗中受益的患者。
术语“癌症”意指或意在描述哺乳动物的生理状态，其典型特征是细胞生长失控。癌症的实例包括但不限于癌、淋巴瘤、母细胞瘤、肉瘤或白血病。此类癌症的更具体实例包括但不限于大肠癌、肺癌、卵巢癌、子宫癌、子宫内膜癌、唾液腺癌、腹膜癌、输卵管癌、胰腺癌、甲状腺癌、头颈鳞形细胞癌、鼻咽癌、喉癌、肺腺癌、肺鳞癌、肝癌、肝细胞癌、胃肠道癌、成胶质细胞瘤、乳腺癌、脑癌、肾癌、肾细胞癌、结肠癌、直肠癌、前列腺癌、外阴癌、睾丸癌、鳞状细胞癌、小细胞肺癌、子宫颈癌、膀胱癌、视网膜母细胞瘤、神经胶母细胞瘤、间皮瘤、口腔上皮样癌、绒毛膜癌和头颈癌。
术语“过表达”或“过表达的”可互换地指一种基因，与正常细胞相比，其通常在某些细胞，如癌细胞中以可检测到的更高水平的转录或翻译。过表达可以是蛋白质或RNA的过表达(由于增加的转录，转录后加工，翻译，翻译后加工，改变的稳定性和改变的蛋白质降解)，以及蛋白质运输模式改变导致的局部过表达(核定位增加)和增强的功能活性，例如，如增加底物的酶水解。与正常细胞或对照细胞相比，过表达可以是5％、10％、20％、30％、50％、60％、70％、80％、90％或更多。在某些实施例中，本发明的抗体或抗原结合单元或抗体药物偶联物用于治疗可能表达CLDN18.2的实体瘤。
如本文使用，术语“过表达CLDN18.2的肿瘤”是指过表达CLDN18.2的肿瘤(包括良性肿瘤和癌症)。在一些实施例中，肿瘤样品中的CLDN18.2表达高于免疫组织的背景水平(例如用免疫组织化学染色测定)表明肿瘤是过表达CLDN18.2的肿瘤。检测肿瘤中CLDN18.2表达的方法是本领域已知的，例如免疫组织化学测定法。在一些实施例中，“CLDN18.2阴性细胞”为细胞样品中缺乏高于背景的CLDN18.2的细胞(例如通过免疫组织化学技术测定)。
如本文所用，术语“给予”意指递送物质(例如，抗CLDN18.2抗体或ADC)以实现治疗目的(例如，治疗CLDN18.2相关疾病)。给予方式可以是肠胃外、肠内和局部。肠胃外给予通常是通过注射，包括但不限于静脉、肌肉内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、以及胸骨内注射和输注。
如本文所用，术语“有效量”或“治疗有效量”是指药物，例如抗体或ADC的量，该量足以降低或改善病症(例如癌症)或其一种或多种症状的严重程度和/或持续时间；预防病症进展；引起病症消退；预防与病症相关的一种或多种症状复发、发展、发作或进展；检测病症；或增强或改善另一疗法(例如预防剂或治疗剂)的预防或治疗效果的量。例如，抗体的有效量可以抑制肿瘤生长(例如，抑制肿瘤体积的增加)；减少肿瘤生长(例如，减小肿瘤体积)；减少癌细胞的数量；和/或在一定程度上缓解与癌症相关的一种或多种症状。例如，有效量可以改善无病生存(DFS)、改善总体存活(OS)或降低复发的可能性。
术语“患者”和“受试者”可互换使用，是指需要诊断、预后或治疗的任何哺乳动物，包括但不限于人类、狗、猫、豚鼠、兔子、大鼠、小鼠、马、牛等。
如本文所用，术语“有需要”是指已将患者鉴定为需要特定方法或治疗。在一些实施例中，可以通过任何诊断方式进行识别。在本文描述的任何方法和治疗中，患者可能需要。
本文所用术语“给药”是指施用一种物质以实现治疗目的(例如，治疗肿瘤)。
本文所用术语“治疗肿瘤的药物”是指具有抑制肿瘤在人体中发展或累进的功能特性的试剂，尤其是恶性(癌性)病变，诸如癌、肉瘤、淋巴瘤或白血病。抑制转移在很多情况下是抗肿瘤药的特性。
术语“药物可接受的载体”通常指是任何类型的无毒固体、半固体或液体填充剂、稀释剂、包封材料或制剂助剂等。
术语“载体”是指可以与活性成分一起施用于患者的稀释剂、佐剂、赋形剂或载体。这此类药物载体可以是无菌液体，如水和油，包括石油、动植物或合成来源的油，如花生油、大豆油、矿物油、芝麻油等。当药物组合物静脉内给药时，水是优选的载体。盐水溶液和葡萄糖水溶液和甘油溶液也可用作液体载体，特别是用于注射溶液。合适的药物赋形剂包括淀粉、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。如有需要，组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。抗菌剂如苯甲醇或对羟基苯甲酸甲酯、抗氧化剂如抗坏血酸或亚硫酸氢钠。这些组合物可以采取溶液、悬液、乳剂、片剂、丸剂、胶囊、散剂、缓释制剂等形式。该组合物可以用传统的粘合剂和载体如甘油三酯配制成栓剂。口服制剂可以包括标准载体，例如药物等级的甘露糖醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。合适的药物载体的实例在E.W.Martin的Remington's Pharmaceutical Sciences中有描述，在此通过引用并入本发明。此类组合物将含有临床有效剂量的抗体或抗原结合单元或ADC，优选以纯化后的形式，连同合适数量的载体，以提供适合于患者的给药形式。该制剂应该适用于给药模式。亲本制剂可以封装在安瓿瓶、一次性注射器或由玻璃或塑料制成的多剂量小瓶中。
术语“抗体药物偶联物”或“ADC”是指与一个或多个化学药物连接的结合蛋白(如抗体或抗原结合单元)，其可以任选地为治疗剂或细胞毒性剂。在优选的实施例中，ADC包括抗体，药物(例如细胞毒性药物)和能够使药物与抗体附接或偶联的接头。可包含在ADC中的药物的非限制性实例是有丝分裂抑制剂、抗肿瘤抗生素、免疫调节剂、用于基因治疗的载体、烷化剂、抗血管生成剂、抗代谢物、含硼剂、化学保护剂、激素、抗激素剂、皮质类固醇、光活性治疗剂、寡核苷酸、放射性核素剂、拓扑异构酶抑制剂、激酶抑制剂(例如，TEC-家族激酶抑制剂和丝氨酸/苏氨酸激酶抑制剂)和放射增敏剂。
术语“抗体药物偶联物”和“ADC”可互换使用。术语“抗CLDN18.2抗体药物偶联物”、“抗CLDN18.2ADC”可互换使用，是指包含特异性结合CLDN18.2的抗体的ADC，其中抗体与一种或多种药物偶联。在一个或多个实施方式中，抗CLDN18.2 ADC包含与依喜替康偶联的抗体。在一个或多个实施方式中，抗CLDN18.2抗体或ADC与CLDN18.2(例如人CLDN18.2)结合。
术语“药物抗体偶联比”或“DAR”是指ADC的与抗体附接的药物(例如，依喜替康)的数量。ADC的DAR可以在1到10的范围内，但是取决于抗体上的连接位点的数量，更高的负载(例如20)也是可能的。提及负载到单个抗体上的药物的数量时，或可替代地，提及一组ADC的平均或均值DAR时，可以使用术语DAR。在一些实施方式中，其值选自1、2、3、4、5、6、7、8、9或10。当考虑小分子药物的平均结合个数时，即抗体的药物平均结合数，或称为平均药物抗体偶联比，其值选自约0至约10，或约2至约8。在一些实施方式中，药物抗体偶联比为约3至约6。在另一些实施方式中，药物抗体偶联比为约6至约8，或约7至约8。DAR值在本文可用p表示。ADC的DAR值可利用紫外可见 吸收光谱法(UV-Vis)、高效液相色谱-疏水色谱(HPLC-HIC)、高效液相色谱-反相色谱(RP-HPLC)、液相色谱质谱联用(LC-MS)等测定。这些技术在Ouyang,J.Methods Mol Biol,2013,1045:p.275-83中有记载。
各种取代基如下定义。在一些情况下，取代基(例如，烷基、烯基、炔基、烷氧基、氨基烷氧基、氨基烷基、氨基烷基氨基、烷基氨基、杂环基、杂环氨基、和芳基)中碳原子的数量由前缀“Cx-Cy”或“Cx-y”指示，其中x是碳原子的最小值并且y是碳原子的最大值。因此，例如，“C1-C6烷基”是指含有从1至6个碳原子的烷基。如果取代基被描述为“被……取代的”，则碳或氮上的氢原子被非氢基团取代。例如，被取代的烷基取代基是烷基取代基，其中烷基上的至少一个氢原子被非氢基团取代。用于说明，单氟烷基是被一个氟基取代的烷基，并且二氟烷基是被两个氟基取代的烷基。应该认识到，如果在取代基上存在一个以上的取代，则每个取代可以是相同的或不同的(除非另有说明)。如果取代基被描述为“任选被……取代”，则取代基可以是(1)未被取代的或(2)被取代的。可能的取代基包括但不限于羟基、C1-C6烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、C1-C6氨基烷氧基、卤素、硝基、氰基、巯基、烷硫基、氨基、C1-C6氨基烷基、C1-C6氨基烷基氨基、连接至杂环的C1-C6烷基、连接至杂环的C1-C6烷基氨基、杂环基、氨基取代的杂环基、杂环氨基、氨基甲酰基、吗啉-1-基、哌啶-1-基、-(CH2)q-CH3、-(CHRn)q-CH3、C3-C8碳环基、-O-(CH2)q-CH3、亚芳基-CH3、-(CH2)q-亚芳基-CH3、-亚芳基-(CH2)q-CH3、-(CH2)q-(C3-C8碳环基)-CH3、-(C3-C8碳环基)-(CH2)q-CH3、C3-C8杂环基、-(CH2)q-(C3-C8杂环基)-CH3、-(C3-C8杂环基)-(CH2)q-CH3、-(CH2)qC(O)NRn(CH2)q-CH3、-(CH2CH2O)q-CH3、-(CH2CH2O)q-CH2-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH2-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH2-CH3、或-(CH2CH2O)qC(O)NRn(CH2)q-CH3；其中各Rn独立为H、C1-C6烷基、C3-C8碳环基、苯基或苄基；并且各q独立为1、2、3、4、5、6、7、8、9或10。
“烷基”指饱和脂肪族烃基基团，该术语包括直链和支链烃基。例如，C1-C20烷基，如C1-C6烷基。C1-C20烷基指具有1至20个碳原子的烷基，例如具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子、6个碳原子、7个碳原子、8个碳原子、9个碳原子、10个碳原子、11个碳原子、12个碳原子、13个碳原子、14个碳原子、15个碳原子、16个碳原子、17个碳原子、18个碳原子、19个碳原子或20个碳原子的烷基。烷基的非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、新戊基、正己基等。所述烷基可以是非取代的或被一个或多个取代基所取代，所述取代基包括但不限于烷基、烷氧基、氰基、羟基、羰基、羧基、芳基、杂芳基、胺基、卤素、磺酰基、亚磺酰基、膦酰基等。
术语“烯基”本身或作为另一取代基的一部分是指具有至少一个碳-碳双键的不饱和支链、直链或环状烷基，双键通过从母体烯烃的单个碳原子上除去一个氢原子而得到。典型的烯基包括但不限于乙烯基；丙烯基(如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基、丙-2-烯-2-基、环丙-1-烯-1-基)；环丙-2-烯-1-基；丁烯基(如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基-丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1,3-二烯-1-基、丁-1,3-二烯-2-基、环丁-1-烯-1-基、环丁-1-烯-3-基、环丁-1,3-二烯-1-基等)等。
术语“炔基”本身或作为另一取代基的一部分是指具有至少一个碳-碳三键的不饱和支链、直链或环状烷基，三键通过从母体炔烃的单个碳原子上除去一个氢原子而得到。典型的炔基包括但不限于乙炔基；丙炔基(如丙-1-炔-1-基、丙-2-炔-1-基等)；丁炔基(如丁-1-炔-1-基、丁-1-炔-3-基、丁-3-炔-1-基等)等。
“碳环基”是指仅仅由碳和氢原子组成的稳定的非芳香族的单环或多环的烃自由基，其 可包括稠合或桥接环系统，具有3至15个碳原子，例如具有3至10个(例如3、4、5、6、7、8、9或10个)碳原子，并且其是饱和的或不饱和的并且通过单键与分子的剩余部分连接。单环自由基包括，例如环丙基、环丁基、环戊基、环己基、环庚基和环辛基。多环自由基包括，例如金刚烷基、降冰片基、十氢萘基等。当在说明书中具体说明时，碳环基可被独立地选自下列中的一种或多种取代基任选取代：烷基、卤素、卤代烷基、氰基、硝基、氧代、芳基、芳烷基、碳环基、碳环基烷基、杂环基、杂环基烷基、杂芳基、杂芳基烷基。
“芳基”指具有完全共轭的π电子体系的全碳单环或全碳稠合环，通常具有5-14个碳原子，例如，6个、10个、12个、14个碳原子。芳基可以是非取代的，或被一个或多个取代基所取代，所述取代基包括但不限于烷基、烷氧基、氰基、羟基、羧基、芳基、芳烷基、胺基、卤素、磺酰基、亚磺酰基、膦酰基。非取代的芳基实例包括但不限于苯基、萘基和蒽基。
“杂环基”是指稳定的3元至18元芳香性或非芳香性环取代基，其由2至8个(例如2、3、4、5、6、7或8个)碳原子和选自氮、氧和硫中的1至6个(1、2、3、4、5或6个)杂原子组成。除非在说明书中另外具体说明，杂环基可以是单环、双环、三环或四环环系统，其可以包括稠合或桥接环系统；且杂环基中的氮、碳或硫原子可以任选地被氧化；氮原子任选地被季铵化；且杂环基可以部分或完全饱和。此类杂环基的实例包括但不限于，二氧戊环基、二噁英基、噻吩基[1,3]二噻烷基、十氢异喹啉基、咪唑啉基、咪唑烷基、异噻唑烷基、异噁唑烷基、吗啉基、八氢吲哚基、八氢异吲哚基、2-氧代哌嗪基、2-氧代哌啶基、2-氧代吡咯烷基、噁唑烷基、哌啶基、哌嗪基、4-哌啶酮基、哌咯烷基、吡唑烷基、奎宁环基、噻唑烷基、1,2,4-亚噻二唑-5(4H)-基、四氢呋喃基、三氧杂环己基、三噻烷基、三嗪烷基(triazinanyl)、四氢吡喃基、硫代吗啉基、硫代吗啉基(thiamorpholinyl)、1-氧代-硫代吗啉基和1,1-二氧代-硫代吗啉基。当在说明书中具体说明时，杂环基可以被选自下列中的一个或多个取代基任选取代：烷基、烯基、卤素、卤代烷基、氰基、氧代、硫代(thioxo)、硝基、芳基、芳烷基、环烃基、环烃基烷基、任选取代的杂环基、任选取代的杂环基烷基、任选取代的杂芳基、任选取代的杂芳基烷基。
“烷氧基”是指式-O-(烷基)，其中烷基为本文定义的烷基。烷氧基的非限制性列举为甲氧基、乙氧基、正丙氧基、1-甲基乙氧基(异丙氧基)、正丁氧基、异丁氧基、仲丁氧基、叔丁氧基。烷氧基可为取代的或未取代的。
“卤素”是指氟(F)、氯(Cl)、溴(Br)、或碘(I)。
“氨基”指-NH2。
“氰基”，是指-CN。
“硝基”指-NO2。
“羟基”，是指-OH。
“羧基”，是指-COOH。
“巯基”是指-SH。
“羰基”是指C＝O。
“立体异构体”指具有相同原子连接顺序，但原子在空间排列不相同的异构体化合物。立体异构体可具有一个或多个立体中心并且每个中心可以以R或S存在，立体异构体也可以是顺反异构体。本文提供的化合物立体异构体包括其所有非对映异构、对映异构和顺反异构形式的任何一个或它们的混合物。
药学上可接受的盐包括化合物与该技术领域众所周知的各种各样的有机和无机抗衡离子产生的药学上可接受的盐，仅仅示例性盐包括，当分子含有酸性官能团时，有机或无机盐如锂盐、钠盐﹑钾盐﹑钙盐﹑镁盐﹑铵盐﹑异丙胺﹑三甲胺﹑二乙胺基﹑三乙胺﹑三 丙胺、乙醇胺、2-二甲胺基乙醇、2-二乙胺基乙醇、二环己基胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、胆碱、甜菜碱、乙二胺、葡糖胺、甲葡糖胺、可可碱、嘌呤哌嗪、哌啶、N-乙基、哌啶、多胺树脂及四烷基铵盐等；以及当分子含有碱性官能团时，有机或无机酸盐如盐酸盐、氢溴酸盐、酒石酸盐、甲磺酸盐、醋酸盐、马来酸盐和草酸盐。酸的其它非限制例子包括硫酸、硝酸、磷酸、丙酸、乙醇酸、丙酮酸、丙二酸、琥珀酸、富马酸、酒石酸、柠檬酸、苯甲酸肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸水杨酸等。这些盐通常可以通过常规方法通过使例如适当的酸或碱与该化合物反应来制备。溶剂合物包括水合物。
本文中的其它化学术语根据本领域的常规用法使用，如The McGraw-Hill Dictionary of Chemical Terms(《麦格劳-希尔化学术语词典》)(Parker,S.编，格劳-希尔公司(McGraw-Hill)，旧金山(1985))。
本文中引用的所有出版物，和专利全部内容通过参考并入本文用于所有目的。
抗CLDN18.2抗体
在一个或多个实施方式中，本发明的抗体或抗原结合单元能够与CLDN18.2特异性结合。在一个或多个实施方式中，所述抗体或抗原结合单元是鼠源抗体、嵌合抗体或人源化抗体。在一个或多个实施方式中，本发明的抗体或抗原结合单元具有以下一种或多种特性：a)与CLDN18.2特异性结合；b)高亲和力；c)强的ADCC活性；d)强的CDC活性。
在一个或多个实施方式中，抗体或抗原结合单元包含的氨基酸序列具有一个或多个修饰基团。例如，本发明公开的抗体或抗原结合单元可以包含有韧性的接头序列，或者可以被修饰以添加功能性基团(例如PEG、药物、毒素或标签)。
本发明公开的抗体、抗原结合单元包括被修饰的衍生物，即通过任何类型的分子与抗体或抗原结合单元的共价连接进行修饰，其中共价连接不会阻止抗体或抗原结合单元与表位结合。包括但不限制以下实例，抗体或抗原结合单元可以被糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割、连接至细胞配体或其他蛋白质等。众多化学修饰中的任一种修饰可以通过现有技术进行，包括但不限于特异性化学裂解、乙酰化、甲酰化、衣霉素的代谢合成等。
在一个或多个实施方式中，抗体或抗原结合单元可以与治疗剂、药物前体、肽、蛋白质、酶、病毒、脂类、生物反应调节剂、药剂或PEG缀合。
抗体或抗原结合单元可通过将其偶联至化学发光化合物来被可检测地标记。然后通过检测在化学反应过程中出现的发光从而确定化学发光标记的抗体或抗原结合单元的存在。化学发光标记化合物的实例包括鲁米诺、异鲁米诺、芳香吖啶酯、咪唑、吖啶盐和草酸酯。
抗体的制备
在某些实施方案中，制备的抗体或抗原结合单元不会在待治疗的动物(例如人类)中引起有害的免疫应答。在一个或多个实施方式中，本发明公开的抗体、抗原结合单元、或衍生物使用本领域公认的技术修饰以降低其免疫原性。例如，抗体可以被人源化、灵长类化、去免疫化或者可以制备嵌合抗体。这些类型的抗体来源于非人抗体，通常是鼠类或灵长类抗体，其保留或基本保留亲本抗体的抗原结合特性但在人体中免疫原性较低。其可以通过多种方法来实现，包括(a)将整个非人源的可变区移植到人源的恒定区以产生嵌合抗体；(b)将一个或多个非人类互补决定区(CDR)的至少一部分移植到人源的框架和恒定区中，保留或不保留关键的框架残基；或(c)移植整个非人源的可变区，但通过用类人源的部分置换表面残基从而“隐藏”它们。通常人框架区中的框架残基将被来自CDR供体抗 体的相应残基取代，比如能够改善抗原结合的残基。这些框架替换可以通过本领域公知的方法鉴定，例如通过模拟CDR和框架残基的相互作用以鉴定对抗原结合起重要作用的框架残基和通过序列对比以鉴定特定位置上异常的框架残基(参考美国专利5,585,089；其全部内容通过引用并入本文)。可以使用本领域公知的多种技术使抗体人源化，例如CDR移植(WO1991009967；美国专利5,225,539,5,530,101和5,585,089)，修复或者表面重排(EP592,106；EP519,596，以及链的重排(美国专利5,565,332)，其全部内容通过引用并入本文。
去免疫化也可用于降低抗体的免疫原性。在本发明中，术语“去免疫化”包括改变抗体以修饰T细胞表位(参见例如WO2000034317A2)。例如，分析来自起始抗体的重链可变区序列和轻链可变区序列，并产生来自每个可变区的人T细胞表位“图谱”，显示表位相对于互补决定区(CDRs)和序列内其它关键残基的位置。分析来自T细胞表位图的单个T细胞表位，以鉴定具有较低改变抗体活性风险的可选择的氨基酸取代。设计包含氨基酸取代组合的一系列可选的重链可变区序列和轻链可变区序列，随后将这些序列掺入到一系列结合多肽中。然后将包含修饰过的可变区和人类恒定区的完整重链和轻链的基因克隆到表达载体中，随后将质粒转入细胞系以产生完整的抗体。然后利用合适的生物化学和生物学实验中比较抗体，鉴定出最佳的抗体。
scFv的制备可参见生产单链单元的技术(美国专利4,946,778)。通过氨基酸桥接Fv区的重链和轻链片段形成单链单元，产生单链融合肽。也可以使用在大肠杆菌中组装功能性Fv片段的技术(Skerra et al.,Science 240:1038-1041(1988))。
可用于生产单链Fv(scFv)和抗体的技术的实例包括如美国专利4,946,778和5,258,498中所述。对于包括在人体内使用抗体和体外检测实验的某些用途，可以使用嵌合抗体、人源化抗体或全人源抗体。嵌合抗体是抗体的不同部分源自不同动物物种的一类分子，例如具有鼠源单克隆抗体的可变区和人源免疫球蛋白恒定区的抗体。生产嵌合抗体的方法是本领域已知的，参见美国专利5,807,715、4,816,567和4,816,397，其全部内容通过引用并入本文。
在一个或多个实施方式中，采用杂交瘤技术来制备以产生本发明的抗体。使用例如杂交瘤方法来制备单克隆抗体，诸如Kohler和Milstein,Nature,256:495(1975)所述。在杂交瘤方法中，通常用免疫剂免疫小鼠、仓鼠或其它合适的宿主动物，以引起淋巴细胞产生或能产生特异性结合免疫剂的抗体。
免疫剂将通常包括蛋白抗原、其片段、或其融合蛋白。通常，如果期望人源细胞，则使用外周血淋巴细胞；如果期望非人哺乳动物源，则使用脾淋巴细胞或淋巴结细胞。在一些实施方案中，采用脾淋巴细胞；然后用适合的融合剂，例如聚乙二醇，将淋巴细胞与永生化细胞系融合，以形成杂交瘤细胞(Goding，Monoclonal Antibodies:Principles and Practice,Academic Press,(1986)第59-103页)。永生化细胞系通常是转化的哺乳动物细胞，特别是啮齿动物、牛和人来源的骨髓瘤细胞。通常，采用大鼠或小鼠骨髓瘤细胞系。在一些实施方案中，采用脾淋巴细胞和小鼠的骨髓瘤细胞进行融合。杂交瘤细胞可在适当的培养基中培养，一些实施方式中培养基含有一种或多种抑制未融合的永生化细胞生长或存活的物质。例如，如果亲本细胞缺乏次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(HGPRT或HPRT)，则杂交瘤的培养基通常包括次黄嘌呤、氨基喋呤和胸腺嘧啶(“HAT培养基”)，所述物质防止HGPRT-缺陷的细胞生长。在一些实施方案中，杂交瘤细胞所产生的单克隆抗体的结合特异性通过免疫沉淀或通过体外结合测定，如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA)来测定。此类技术和测定是本领域已知的。单克隆抗体的结合亲和力可例如通过Munson和Podbard,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。此外，在单克隆抗体的 治疗应用中，鉴定对靶抗原具有高度特异性和高结合亲和力的抗体是重要的。
在鉴定出期望的杂交瘤细胞之后，可用有限稀释步骤将所述克隆进行亚克隆并用标准方法使其生长(参见Goding,Monoclonal Antibodies:Principles and Practice,Academic Press,(1986)第59-103页)。适用于该目的的培养基包括例如Dulbecco改良的Eagle培养基和RPMI-1640培养基等。
在一个或多个实施方式中，可通过常规技术手段分离或纯化由亚克隆分泌的单克隆抗体，如蛋白A-琼脂糖凝胶、羟基磷灰石层析、凝胶电泳、透析或亲和层析。
单克隆抗体还可通过重组DNA方法制备，例如美国专利No.4,816,567中所述。编码本文所述单克隆抗体的DNA可使用常规方法来分离和测序(例如，通过使用能够与抗体的重链和轻链的基因特异性结合的寡核苷酸探针)。编码本文所述抗体的DNA还可以按常规方法根据抗体序列设计合成。将分离或合成的DNA插入表达载体中，然后将其转染到宿主细胞例如不另外产生免疫球蛋白的中国仓鼠卵巢(CHO)细胞、人胚胎肾(HEK)293细胞、猿COS细胞、PER.NS0细胞、SP2/0、YB2/0或骨髓瘤细胞中，从而在重组宿主细胞中获得合成的单克隆抗体。
在一个或多个实施方式中，采用杂交瘤技术来制备以产生本发明的抗体或抗原结合单元，使用含有CLDN18.2的蛋白制剂免疫小鼠，免疫后去小鼠的脾淋巴细胞与骨髓瘤细胞进行融合，通过CLDN18.2蛋白对杂交瘤进行筛选，并对阳性的杂交瘤进行有限稀释，进一步亚克隆，再次鉴定杂交瘤株与CLDN18.2蛋白的结合能力，以制备产生抗CLDN18.2抗体。在一个或多个实施方式中，小鼠为6-8周龄的雌性Balb/c小鼠。
本发明公开的抗体或抗原结合单元的结合特异性可以通过体外实验，例如免疫共沉淀、放射免疫实验(RIA)或酶联免疫吸附实验(ELISA)来检测。
对于包括在人体内使用抗体和体外检测实验的某些用途，一些实施方式中可以使用嵌合抗体、人源化抗体或全人源抗体。嵌合抗体是抗体的不同部分源自不同动物物种的一类分子，例如具有鼠源单克隆抗体的可变区和人源免疫球蛋白恒定区的抗体。生产嵌合抗体的方法是本领域已知的，参见美国专利5,807,715、4,816,567和4,816397，其全部内容通过引用并入本文。
还可以转基因小鼠来生产人源抗体，所述小鼠不能表达功能性内源性免疫球蛋白但能表达人类免疫球蛋白基因。例如，人重链和轻链免疫球蛋白基因复合物可以随机引入或通过同源重组引入到小鼠胚胎干细胞。或者，除了人重链和轻链基因之外，还可以将人的可变区、恒定区和多样性区域引入小鼠胚胎干细胞中。小鼠重链和轻链的免疫球蛋白基因可以通过同源重组引入人免疫球蛋白基因座而丧失功能。特别地，JH区域的纯合缺失可以防止内源抗体的产生。将修饰过的胚胎干细胞扩增并显微注射进囊胚中以产生嵌合小鼠。然后培育嵌合小鼠以产生表达人源抗体的纯合后代。用选择出的抗原例如全部或部分期望的多肽靶点以常规方式免疫转基因小鼠。可以使用常规杂交瘤技术从免疫的转基因小鼠获得靶向抗原的单克隆抗体。转基因小鼠携带的人免疫球蛋白转基因在B细胞分化过程中重排，随后发生类别转换和体细胞突变。因此，使用这种技术可以产生可用于治疗的IgG、IgA、IgM和IgE抗体。
在另一些实施方案中，使用常规方法(例如使用能够特异性结合编码鼠抗体重链和轻链的基因的寡核苷酸探针)，可以容易地分离编码所需单克隆抗体的DNA并对其进行测序。一旦分离出来，DNA可以被置于表达载体中，然后被转染到原核或真核宿主细胞如大肠杆菌细胞、猿猴COS细胞、中国仓鼠卵巢(CHO)细胞或不另外产生免疫球蛋白的骨髓瘤细胞中。分离的DNA(如本文所述可以是合成的)也可用于制备抗体的恒定区和可变区的序列，如美国专利5,658,570中所述，其全部内容通过引用并入本文。该方法从所选 细胞中提取RNA并转化成cDNA，然后使用Ig特异性引物通过PCR技术进行扩增。适于此目的的合适的探针在美国专利5,658,570中也有所提及。
此外，使用常规重组DNA技术，可将本发明的抗体或抗原结合单元的一个或多个CDR插入框架区，例如插入到人类框架区以构建人源化非全人源抗体。框架区可以是天然存在的或共有的框架区，优选人类框架区(参见Chothia et al.,J.Mol.Biol.278:457-479(1998)，其列出一系列人类框架区)。一些多核苷酸可以编码框架区和CDR组合产生的与目标抗原的至少一个表位特异性结合的抗体。在框架区内进行一个或多个氨基酸取代，可以选择能够改善抗体与其抗原结合的氨基酸取代。另外，可用此法进行参与链间二硫键形成的一个或多个可变区中半胱氨酸残基的取代或缺失，从而产生缺少一个或多个链间二硫键的抗体分子。本领域技术范围内的对多核苷酸进行的其他改变也涵盖于本发明中。
此外，在Newman,Biotechnology 10:1455-1460(1992)中公开了另一种生产重组抗体的高效方法，特别地，该技术能产生含有猴可变区和人恒定区序列的灵长类抗体，该参考文献的全部内容通过引用并入本文。此外，该技术也在共同转让的美国专利5,658,570、5,693,780和5,756,096中有所提及，每个专利的全部内容通过引用并入本文。
抗体可以通过使用常规重组DNA技术制备。使用本领域技术人员公知的技术可以选择、构建和培养生产抗体的载体及细胞系等。这些技术在各种实验室手册和主要出版物中均有描述，例如Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells，D.L.Hacker,F.M.Wurm,in Reference Module in Life Sciences,2017，其全部内容包括补充内容通过引用并入全文。
在一个或多个实施方式中，可以按常规方法根据本文所述抗体氨基酸序列设计合成编码抗体的DNA，将其置入表达载体中，然后转染宿主细胞，在培养基中培养被转染的宿主细胞产生单克隆抗体。在一些实施方案中，表达抗体载体包括至少一个启动子元件，抗体编码序列，转录终止信号和polyA尾。其他元件包括增强子，Kozak序列及插入序列两侧RNA剪接的供体和受体位点。可以通过SV40的前期和后期启动子，来自逆转录病毒的长末端重复序列如RSV、HTLV1、HIVI及巨细胞病毒的早期启动子来获得高效的转录，也可应用其它一些细胞的启动子如肌动蛋白启动子。合适的表达载体可包括pIRES1neo，pRetro-Off，pRetro-On，PLXSN，pLNCX，pcDNA3.1(+/-)，pcDNA/Zeo(+/-)，pcDNA3.1/Hygro(+/-)，PSVL，PMSG，pRSVcat，pSV2dhfr，pBC12MI或pCS2等。常使用的哺乳动物宿主细胞包括HEK293细胞、Cos1细胞、Cos7细胞、CV1细胞、鼠L细胞和CHO细胞等。
在一个或多个实施方式中，插入基因片段需含有筛选标记，常见的筛选标记包括二氢叶酸还原酶、谷氨酰胺合成酶、新霉素抗性、潮霉素抗性等筛选基因，以便于转染成功的细胞的筛选分离。将构建好的质粒转染到无上述基因的宿主细胞，经过选择性培养基培养，转染成功的细胞大量生长，产生想要获得的目的蛋白。
此外，可以使用本领域技术人员已知的标准技术在编码本发明所述抗体的核苷酸序列中引入突变，包括但不限于导致氨基酸取代的定点突变和PCR介导的突变。变体(包括衍生物)编码相对于原重链可变区和轻链可变区来说少于50个氨基酸的取代、少于40个氨基酸的取代、少于30个氨基酸的取代、少于25个氨基酸的取代、少于20个氨基酸的取代、少于15个氨基酸的取代、少于10个氨基酸的取代、少于5个氨基酸的取代、少于4个氨基酸的取代、少于3个氨基酸的取代或少于2个氨基酸的取代。或者可以沿着全部或部分编码序列时随机引入突变，例如通过饱和突变，以及可以筛选所得突变体的生物活性以鉴定保留活性的突变体。
抗体药物偶联物
本发明的抗体或抗原结合单元可以与药物偶联形成抗CLDN18.2抗体药物偶联物(抗CLDN18.2ADC)。由于抗体药物偶联物(ADC)能够选择性地将一种或多种药物递送至靶组织(例如，表达CLDN18.2的癌症或肿瘤)，抗体药物偶联物(ADC)可以增加抗体在治疗疾病(例如癌症)中的治疗功效。在一个或多个实施方式中，本发明的抗体药物偶联物(ADC)，其包含本文所述的抗CLDN18.2抗体或抗原结合单元和至少一种药物(例如依喜替康)。
本发明的抗CLDN18.2抗体的抗原结合片段或抗原结合单元可以与本文所述的药物偶联。因此，在一个或多个实施方式中，本文所述的抗CLDN18.2抗体的抗原结合片段或抗原结合单元通过接头与药物偶联形成抗CLDN18.2ADC。
本发明的抗CLDN18.2ADC包含与一个或多个药物连接的特异性结合CLDN18.2(例如人CLDN18.2)的抗体或抗原结合单元。ADC的特异性可以由抗体(例如抗CLDN18.2抗体)或抗原结合片段的特异性决定。在一个或多个实施方式中，抗CLDN18.2抗体与一种或多种药物(例如DNA拓扑异构酶抑制剂)连接，所述药物(例如DNA拓扑异构酶抑制剂)被递送至表达CLDN18.2的细胞，特别是表达CLDN18.2的癌细胞。在一个或多个实施方式中，抗CLDN18.2抗体药物偶联物包含通过接头与药物(例如依喜替康)偶联的抗CLDN18.2抗体。本文所述的抗CLDN18.2抗体为ADC提供了结合CLDN18.2的能力，使得附着于抗体的药物可以被递送至表达CLDN18.2的细胞，特别是表达CLDN18.2的癌细胞。
在一个或多个实施方式中，ADC为如式I所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物。
其中Abu、M、B、G、L、D、p如本文所定义。
在一个或多个实施方式中，Abu为抗体H239H-2b-K-6a-1、H239H-2b-K-6a-2或H239H-2b-K-6a-3。
在一个或多个实施方式中，ADC为ADC1、ADC2、ADC3、ADC4、ADC5或ADC6或其药学上可接受的盐或溶剂合物。
在一个或多个实施方式中，本发明所述抗体偶联药物具有明显的旁观者效应。
合成方法
本发明还提供了中间体及抗体药物偶联物的制备方法。本发明的中间体及抗体药物偶联物可以通过已知制剂和方法制备。在一个或多个实施方式中，制备中间体化合物1-7的方法如下所述。
第一步：通式1-1化合物和通式1-1’化合物在碱性条件下反应得到通式1-2化合物；
第二步：通式1-2化合物和通式(AA)i-(FF1)f在缩合剂存在下、在碱性条件下反应得到通式1-3化合物；
第三步：去除通式1-3化合物氨基保护基W1得到通式1-4化合物；
第四步：通式1-4化合物和通式1-5化合物在碱性条件下反应得到通式1-6化合物；
第五步：通式1-6化合物和二(对硝基苯)碳酸酯在碱性条件下反应得到通式1-7化合物。
其中
W1为氨基保护基，例如为9-芴甲氧羰基；W2为羧酸活性酯，例如为琥珀酰亚胺酯。
其中，n、AA、R、i、f如本文所述，各FF1独立为 其中各RF独立为C1-C6烷基、C1-C6烷氧基、-NO2或卤素；z为0、1、 2、3或4；其中*连接AA；各FF2独立为 其中各RF独立为C1-C6烷基、C1-C6烷氧基、-NO2或卤素；z为0、1、2、3或4；其中*连接AA；
在一个或多个实施方式中，RF为F。
在一个或多个实施方式中，z为0。
在一个或多个实施方式中，z为1或2。
在一个或多个实施方式中，所述中间体为：
其中，R、n如本文所述。
在一个或多个实施方式中，所述中间体为：
其中，R、n如本文中所述。
在一个或多个实施方式中，所述中间体为：

其中，n如本文所述。
在一个或多个实施方式中，所述中间体为：
其中，n如本文所述。
上述碱性条件可以用试剂来提供，该试剂包括有机碱和无机碱类，所述有机碱类包括但不限于三乙胺、二乙胺、N-甲基吗啉、吡啶、六氢吡啶、N,N-二异丙基乙胺、正丁基锂、二异丙基氨基锂、醋酸钾、叔丁醇钠或叔丁醇钾，所述无机碱类包括但不限于氢化钠、磷酸钾、碳酸钠、碳酸钾、碳酸铯、氢氧化钠和氢氧化锂。
上述缩合剂可以选自N,N,N',N'-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲、4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐、1-羟基苯并三唑和1-(3-二甲氨基丙基)-3-乙 基碳二亚胺盐酸盐、N,N'-二环己基碳化二亚胺、N,N'-二异丙基碳二酰亚胺、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯、1-羟基苯并三唑、1-羟基-7-偶氮苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲脲六氟磷酸酯、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、苯并三氮唑-1-基氧基三(二甲基氨基)磷鎓六氟磷酸盐或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷。
在一个或多个实施方式中，制备中间体化合物1-8方法如下所述。
其中，n、AA、R、i、f、FF2、FF、D如本文所述。
通式1-7化合物和D在缩合剂存在下、在碱性条件下反应得到通式1-8化合物。
在一个或多个实施方式中，所示中间体为：
其中，R、n、D如本文所述。
在一个或多个实施方式中，所述中间体为：
其中，R、n、D如本文所述。
在一个或多个实施方式中，所述中间体为：
其中，n、D如本文所述。
在一个或多个实施方式中，所述中间体为：
其中，n、D如本文所述。
上述碱性条件可以用试剂来提供，该试剂包括有机碱和无机碱类，所述有机碱类包括但不限于三乙胺、二乙胺、N-甲基吗啉、吡啶、六氢吡啶、N,N-二异丙基乙胺、正丁基锂、二异丙基氨基锂、醋酸钾、叔丁醇钠或叔丁醇钾，所述无机碱类包括但不限于氢化钠、磷酸钾、碳酸钠、碳酸钾、碳酸铯、氢氧化钠和氢氧化锂。
上述缩合剂可以选自N,N,N',N'-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲、4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐、1-羟基苯并三唑和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N,N'-二环己基碳化二亚胺、N,N'-二异丙基碳二酰亚胺、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯、1-羟基苯并三唑、1-羟基-7-偶氮苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲脲六氟磷酸酯、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、苯并三氮唑-1-基氧基三(二甲基氨基)磷鎓六氟磷酸盐或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷。
在一个或多个实施方式中，制备ADC1-9方法如下所述。
其中，n、AA、R、i、f、FF、D、Abu如本文所述。
通式1-8化合物和Abu在弱酸性条件下偶联得到通式1-9化合物。本发明提供的抗体上的链间二硫键被硫醇类还原剂还原打开，产生游离的巯基，然后与通式1-8化合物发生偶联反应，得到通式1-9所示抗体药物偶联物。
抗体药物偶联物可以采用常规方法进行纯化，例如用制备型高效液相色谱(prep-HPLC)等方法。
上述弱酸性条件可以用试剂来提供，该试剂包括有机酸和无机酸类，所述有机酸类包括但不限于乙酸、苯甲酸、酒石酸、草酸、苹果酸、柠檬酸、抗坏血酸、柠檬酸、枸椽酸、水杨酸、咖啡酸、山梨酸、奎宁酸、齐墩果酸、丁二酸、绿原酸、甲酸、丙酸，所述无机酸类包括但不限于碳酸、亚硝酸、乙酸、次氯酸、氢氟酸，亚硫酸、氢硫酸、硅酸、偏硅酸、磷酸、偏磷酸、碳酸氢钠、亚硫酸氢钠。
治疗方法
本发明还提供了治疗方法和用途。在一个或多个实施方式中，提供了用于治疗或改善各种类型的癌症或肿瘤等相关疾病的方法，所述方法包括向有需要的患者施用有效剂量的抗CLDN18.2抗体或抗原结合单元或ADC。在一个或多个实施方式中，提供了抗CLDN18.2抗体或抗原结合单元或ADC在用于治疗或改善癌症或肿瘤等相关疾病中的应用。在一个或多个实施方式中，提供了所述抗CLDN18.2抗体或抗原结合单元或ADC在制备用于治疗或改善癌症或肿瘤等相关疾病的药物中的应用。
对于任何特定患者的具体剂量和治疗方案将取决于各种因素，包括所使用的特定抗体、抗原结合片段或衍生物、患者的年龄和体重、一般健康状况、性别和饮食，以及给药时间、排泄频率、药物组合，以及所治疗的特定疾病的严重程度。由包括在本领域普通技术人员范围内的医疗护理人员对这些因素进行判断。所述剂量还将取决于待治疗的个体患者、给药途径、制剂类型、所用化合物的特性、疾病的严重程度以及所需的效果。所用剂量可以通过本领域熟知的药理学和药代动力学原理确定。在一些实施方案中，本发明抗体或抗原结合单元或ADC施用于患者的剂量为每次0.01mg/kg至100mg/kg患者体重。在一些实施方案中，每1星期、2星期、3星期、或每月给药一次。
抗体或抗原结合单元或ADC的施用方法包括但不限于真皮内、肌肉、腹腔、静脉、皮下、鼻腔、硬脊膜外注射和口服。抗体、抗原结合单元、ADC或组合物可以通过任何方便的途径施用，例如通过输注或推注，通过上皮或皮肤粘膜(例如口腔粘膜、直肠和肠粘膜等)吸收，并且可以与其他生物活性剂共同施用。因此，含有本发明的抗体、抗原结合单元、ADC的药物组合物可以口服给药、直肠给药、肠胃外给药、脑池内给药、阴道内给药、腹腔内给药、外敷(如通过粉末，软膏，滴剂或透皮贴剂)、口腔给药或通过口服或鼻腔喷雾给药。
本发明使用的术语“肠胃外”是指包括静脉内、肌肉内、腹腔内、胸骨内、皮下和关节内注射和输注的施用方式。
施用方式可以是全身施用或局部施用。
本发明抗体、抗原结合单元、ADC或药物组合物可以局部施用于需要治疗的区域；可以通过但不限于以下方式：手术期间局部输注，例如与手术后伤口敷料联合的局部应用，通过注射，通过导管，借助栓剂或借助植入物来实现，所述植入物是多孔的、无孔的或凝胶状的材料，包括膜(例如硅橡胶膜)或纤维。一些实施方式中，当施用本发明的蛋白质(包括抗体)时，必须注意使用不吸收蛋白质的材料。
在一个或多个实施方式中，本发明组合物包含编码抗体或抗原结合单元的核酸或多聚核苷酸，可以通过将其构建为合适的核酸表达载体的一部分来体内施用所述核酸以促进其编码的蛋白质的表达，然后通过下述方式施用上述部分载体使其变为胞内部分，例如通过使用逆转录病毒载体(参见美国专利4,980,286)，或通过直接注射，或通过使用微粒轰击(例如基因枪；Biolistic，Dupont)，或用脂质或细胞表面受体或转染试剂包被，或者通过与已知进入细胞核的同源异型盒类肽连接施用(参见例如Joliot et al.,1991,Proc.Natl.Acad.Sci.USA 88:1864-1868)等等。可选地，核酸可以通过同源重组在引入细胞内并整合至宿主细胞DNA中用于表达。
通常在进行体外测试用于治疗疾病的方法，包括施用本发明所述抗体、抗原结合单元、ADC或衍生物，然后在可接受的动物模型中体内测试期望的治疗性或预防性活性，最后施用于人体。合适的动物模型(包括转基因动物)是本领域普通技术人员所公知的。例如，用于证明本发明所述抗体或抗原结合单元或ADC的治疗用途的体外测定包括抗体或抗原结合单元或ADC对细胞系或患者组织样品的影响。抗体或抗原结合单元或ADC对细胞系和/或组织样品的作用可以利用本领域技术人员已知的技术进行检测，例如本发明其他部分公开的技术。根据本发明的内容，可用于确定是否施用特异性抗体或抗原结合单元或ADC的体外测定实验包括体外细胞培养实验，其中患者组织样品在培养物中培养，并暴露于或以其他方式施用化合物，并观察这种化合物对组织样品的影响。
各种已知输送系统可用于施用本发明抗体或抗原结合单元或ADC或编码本发明抗体或抗原结合单元的多核苷酸，例如包封于脂质体、微粒、微胶囊、能够表达所述化合物的重组细胞、受体介导的内吞作用(参见例如Wu and Wu,1987,J.Biol.Chem.262:4429-4432)、作为逆转录病毒或其它载体的一部分的核酸的构建等。
联合疗法
在一个或多个实施方式中，本发明抗CLDN18.2抗体或抗原结合单元或ADC可以结合其它治疗或预防方案，包括施用一种或多种本发明抗体或抗原结合单元或ADC以及一种或多种其它治疗剂或方法一起使用或组合使用。在一个或多个实施方式中，其他治疗方案包括但不限于放射疗法、化学疗法、激素疗法等。对于组合治疗，抗体或抗原结合单元或ADC可以与其它治疗剂可同时或分开施用。当分开施用时，可以在施用另一种其它治疗剂之前或之后施用本发明抗体或抗原结合单元或ADC。
在一个或多个实施方式中，本发明抗体或抗原结合单元或ADC可以与化疗剂组合施用。在一个或多个实施方式中，本发明抗体或抗原结合单元或ADC可以与细胞因子、趋化因子或共刺激分子组合施用。在一个或多个实施方式中，本发明抗体或抗原结合单元或ADC可以与免疫治疗剂组合施用。
诊断方法
在某些癌症肿瘤样品中观察到CLDN18.2的表达，并且阳性表达CLDN18.2的患者对使用本发明的抗CLDN18.2抗体或抗原结合单元或ADC的治疗有响应。因此，本发明的抗体或抗原结合单元也可以用于诊断和预后。
包含细胞的样品可以从患者体内获得，该患者可以是癌症患者或待诊断的患者。细胞是肿瘤组织或肿瘤块、血液样本、尿液样本或来自患者的任何样本的细胞。在选择性地对样品进行预处理之后，可以在允许抗体或抗原结合单元与可能存在于样品中的CLDN18.2蛋白相互作用的条件下，将样品与本发明的抗体或抗原结合单元一起孵育。可以使用诸如ELISA的方法，利用抗CLDN18.2抗体或抗原结合单元来检测样品中CLDN18.2蛋白的存在和表达量。
样品中CLDN18.2蛋白的存在(任意的含量或浓度)可以用于诊断癌症，其可以作为患者适用抗体治疗的指示，或作为患者已经(或没有)对癌症治疗作出反应的指示。对于预后方法，可以在开始癌症治疗后的特定阶段进行一次、两次或更多次地检测，以指示治疗的进展。
组合物
本发明还提供了药物组合物。这样的组合物包含有效剂量的抗CLDN18.2抗体(或抗原结合单元)或ADC和药学上可接受的载体。在一些实施方案中，药物组合物包含0.1％-90％的抗CLDN18.2抗体或抗原结合单元或ADC。在一些实施方案中，药物组合物还包含抗癌剂(例如免疫检查点抑制剂)。
在一个或多个实施方式中，术语“药学上可接受的”是指药典中列出的用于动物、特别是用于人类的物料。此外，“药学上可接受的载体”通常将是任何类型的无毒固体、半固体或液体填充剂、稀释剂、包封材料或制剂助剂。
在一个或多个实施方式中，根据常规步骤将组合物配制成适合静脉内注射于人体的药物组合物。用于静脉内给药的组合物是在无菌等渗水性缓冲液中的溶液。组合物还可包含增溶剂和局部麻醉剂如利多卡因，从而缓解注射部位的疼痛。一般而言，有效成分以单位剂量形式单独供给或混在一起供给，如以干燥的冻干粉末或无水浓缩物的形式装在可指示活性剂份量的密封容器(如安瓿瓶或小袋)中。在通过输注施用组合物的情况下，可以用含有无菌药用级水或盐水的输液瓶来分装组合物。在通过注射施用组合物的情况下，可以使用注射用的无菌水或盐水的安瓿瓶，使得可以在施用之前混合有效成分。
本发明的化合物可以配制成中性的或盐的形式。药学上可接受的盐包括与衍生自如盐酸、磷酸、乙酸、草酸、酒石酸等的阴离子形成的盐，以及与衍生自如钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、2-乙氨基乙醇、组氨酸、普鲁卡因等的阳离子形成的盐。
实施例
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径或已知方法得到。
实施例1.抗体的制备
抗CLDN18.2抗体的可变区和恒定区组成见表1，VH和CH组成抗体的重链，VL和CL组成抗体的轻链；抗CLDN18.2抗体的CDR汇总见表2，CDR区组成见表3，可变区、恒定区序列见表4。其中，抗体H239H-2b-K-6a-1、H239H-2b-K-6a-2和H239H-2b-K-6a-3的轻链具有相同的序列，其氨基酸序列如SEQ ID NO:72所示，对应的基因序列如SEQ ID NO:73所示；抗体H239H-2b-K-6a-1重链的氨基酸序列如SEQ ID NO:69所示，对应的基因序列如SEQ ID NO:74所示；抗体H239H-2b-K-6a-2重链的氨基酸序列如SEQ ID NO:70 所示，对应的基因序列如SEQ ID NO:75所示；抗体H239H-2b-K-6a-3重链的氨基酸序列如SEQ ID NO:71所示，对应的基因序列如SEQ ID NO:76所示(见表5和表6)。以“CH”开头的表示嵌合抗体(鼠源的可变区+人源的恒定区)，以“H”开头的表示人源化抗体(人源化的可变区+人源的恒定区)。
表1抗CLDN18.2抗体的可变区和恒定区组成
表2VH CDR1-3和VL CDR1-3

表3抗体CDR区的组成
表4可变区和恒定区的氨基酸序列


表5示例的抗CLDN18.2抗体的重链和轻链序列

表6示例的抗CLDN18.2抗体的基因序列


在抗体基因序列5’端添加核糖体结合位点(kozak序列：GCCGCCACC，SEQ ID NO:77)及信号肽的基因序列。
轻链信号肽，MDMRVLAQLLGLLLLCFPGARC(SEQ ID NO:78)，对应核苷酸序列为：atggacatgagggtgctggcccagctgctgggactgctgctgctgtgcttcccaggcgccagatgc(SEQ ID NO:79)；
重链信号肽，MGWSLILLFLVAVATRVLS(SEQ ID NO:80)，抗体H239H-2b-K-6a-1、H239H-2b-K-6a-2和H239H-2b-K-6a-3的重链基因序列添加的信号肽基因序列为：atgggttggtctcttatccttctattcctggttgcagtggcaacaagagtgctttct(SEQ ID NO:81)，其它抗体的重链基因序列添加的信号肽基因序列为：atgggttggtctctgattctcctgtttctggtggcagtggctacaagagtcctgtca，SEQ ID NO:82)。
在抗体基因序列3’端设计终止密码子TGA。
将抗体重链和轻链基因序列连接至表达载体中，构建成轻、重链全长表达质粒。将表达抗体轻重链的质粒以1:1的比例瞬时转染HEK293F细胞，培养并纯化得到抗体。
实施例2.化合物(1S,9S)-1-氨基-9-乙基-4,5-二氟-9-羟基-1,2,3,9,12,15-六氢-10H,13H苯并[de]哌喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-10,13-二酮盐酸盐(D-1)的合成步骤
1.N,N'-(3,4-二氟-8-氧代-5,6,7,8-四氢萘-1,7-二基)二乙酰胺的合成
氮气氛围中，将叔丁醇钾四氢呋喃溶液(42ml，1M)加入干燥反应瓶中搅拌，降温至0-5℃。将溶于四氢呋喃(25ml)的N-(3,4-二氟-8-氧代-5,6,7,8-四氢萘-1-基)乙酰胺(CAS号：143655-49-6，5g，21mmol)，缓慢滴加到反应瓶中，随后滴加亚硝酸叔丁酯(4.32g，2eq)(控温0-5℃)，升温至15-20℃搅拌2小时(h)。反应完全后，降温至0-5℃，滴加醋酸(25ml)、醋酸酐(25ml)(控温不超过10℃)，滴完搅拌20min。保持5-10℃，根据N-(3,4-二氟-8-氧代-5,6,7,8-四氢萘-1-基)乙酰胺的量加锌粉(8eq)，20-25℃搅拌1h。过滤，乙酸乙酯(50ml)漂洗固体，降温至0-5℃，滴加15％NaCO3水溶液(50ml)洗三次，乙酸乙酯(25ml)萃取，合并有机相，饱和NaCl水溶液洗涤。加入乙酸乙酯(10ml)，40℃搅拌30min，缓慢降至0-5℃搅拌2h。过滤，固体用乙酸乙酯/石油醚(1/2，10ml)洗涤。真空干燥得灰色粉末(2.1g，33.7％)。LC-MS:[M+H]+＝297。
2.N-(8-氨基-5,6-二氟-1-氧代-1,2,3,4-四氢萘-2-基)乙酰胺的合成
将N,N'-(3,4-二氟-8-氧代-5,6,7,8-四氢萘-1,7-二基)二乙酰胺(500mg，1.68mmol)加入2M盐酸乙醇溶液(5ml)，50℃搅拌4h，检测反应完全后，加水(7.5ml)，降温至0-5℃，滴加三乙胺(1.03g)搅拌3h。过滤，分别用40％冷乙醇水溶液(3ml)，水(3ml)洗涤。真空干燥得灰色粉末(320mg，74.6％)。LC-MS:[M+H]+＝255。
3.N-((9S)-9-乙基-4,5-二氟-9-羟基-10,13-二氧-2,3,9,10,13,15-六氢-1H,12H苯并[de]哌喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺的合成
氮气氛围下将N-(8-氨基-5,6-二氟-1-氧代-1,2,3,4-四氢萘-2-基)乙酰胺(1.1g，1eq)、(S)-4-乙基-4-羟基-7,8-二氢-1H-哌喃并[3,4-f]吲哚嗪-3,6,10(4H)-三酮(1.15g，1eq)及甲苯(50ml)加入到反应瓶，升温至回流，搅拌1h后加入4-甲基苯磺酸吡啶(100mg)，继续搅拌回流20h。降至室温搅拌1h。过滤，固体分别用丙酮(10ml)，冷乙醇(5ml)洗涤。真空干燥得灰褐色粉末(1.1g，53％)。LC-MS:[M+H]+＝482。
4. (1S,9S)-1-氨基-9-乙基-4,5-二氟-9-羟基-1,2,3,9,12,15-六氢-10H,13H苯并[de]哌喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-10,13-二酮盐酸盐的合成
将N-((9S)-9-乙基-4,5-二氟-9-羟基-10,13-二氧-2,3,9,10,13,15-六氢-1H,12H苯并[de]哌喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺(1.1g，2.28mmol)、6M盐酸水溶液(44ml)加入到反应瓶，氮气氛围下回流搅拌4h。浓缩除去溶剂，HPLC纯化得到白色粉末(200mg，18％)。LC-MS:[M+H]+＝440。
实施例3. 4-((30S,33S,36S)-30-(2-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-33-异丙基-26,31,34-三氧-36-(3-脲丙基)-2,5,8,11,14,17,20,23-八氧-27,32,35-三氮杂三十七烷-37-胺基)苯甲基(4-硝基苯基)碳酸酯(CB07)的合成步骤
1)(S)-30-((((9H-芴-9-基)甲氧基)羰基)氨基)-26-氧-2,5,8,11,14,17,20,23-八氧杂-27-氮杂-三十一烷-31-酸(CB01)的合成。
0-5℃氮气保护下，将8.14g N2-芴甲氧羰基-L-2,4-二氨基丁酸(CB00-2)用40mL二甲基甲酰胺(DMF)溶解，加入10g 4,7,10,13,16,19,22,25-八氧杂二十六烷酸-N-琥珀酰亚胺酯(CB00-3)和10mL DMF，保持0-5℃滴加6.5mL DIPEA，加完1h后室温下搅拌反应4h，反应完成后减压除去DMF，硅胶柱层析(二氯甲烷和甲醇体积比20:1作为洗脱溶剂)得到淡黄色油状液体14.2g CB01。
2)[(S)-1-[[(S)-1-[[4-(羟甲基)苯基]氨基]-1-氧代-5-脲基戊烷-2-基]氨基]-3-甲基-1-氧代丁-2-基]氨基甲酸(9H-芴-9-基)甲酯(CB02)的合成。
室温氮气保护下，11g(S)-2-((S)-2-((((9H-芴-9-基)甲氧基)羰基)氨基)-3-甲基丁酰氨基)-5-脲基戊酸(CB00-4)和5.5g对氨基苯甲醇(CB00-5)用400mL二氯甲烷和200mL甲醇溶解，机械搅拌下，分批加入17g 2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(EEDQ)，避光下反应15h。反应完成后减压除去溶剂得到糊状固体，硅胶柱层析(二氯甲烷和甲醇体积比20:1作为洗脱溶剂)得到灰白色固体11.9g。
3)(S)-2-((S)-2-氨基-3-甲基丁酰胺基)-N-(4-(羟基甲基)苯基)-5-脲基戊酰胺(CB03)的合成。
室温氮气保护下，11.9g[(S)-1-[[(S)-1-[[4-(羟甲基)苯基]氨基]-1-氧代-5-脲基戊烷-2-基]氨基]-3-甲基-1-氧代丁-2-基]氨基甲酸(9H-芴-9-基)甲酯(CB02)中加入300mL乙腈，搅拌下滴加18mL哌啶，滴加完毕后室温下反应2h。反应完成后减压蒸馏除去溶剂和哌啶，硅胶柱层析(二氯甲烷和甲醇体积比20:1作为洗脱溶剂)得到白色固体7.5g CB03。
4)(9H-芴-9-基)甲基((30S,33S,36S)-41-氨基-36-((4-(羟甲基)苯基)氨基甲酰基)-33-异丙基-26,31,34,41-四氧-2,5,8,11,14,17,20,23-八氧杂-27,32,35,40-四氮杂-30-基)氨基甲酸酯(CB04)的合成。
0℃氮气氛围下，将14.2g(S)-30-((((9H-芴-9-基)甲氧基)羰基)氨基)-26-氧-2,5,8,11,14,17,20,23-八氧杂-27-氮杂-三十一烷-31-酸(CB01)溶解于100mL DMF中，分批加入11g N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲(HATU)，搅拌反应30min后，加入7.5g的(S)-2-((S)-2-氨基-3-甲基丁酰胺基)-N-(4-(羟基甲基)苯基)-5-脲基戊酰胺(CB03)，保持0℃下反应2.5h。反应完成后减压蒸馏除去溶剂，硅胶柱层析(二氯甲烷和甲醇体积比10:1作为洗脱溶剂)得到固体9.66g CB04。
5)N-((S)-3-氨基-4(((S)-1-(((S)-1-((4-(羟甲基)苯基)胺基)-1-氧代-5-脲基戊烷-2-基)胺基)-3-甲基-1-氧丁烷-2-基)胺基)-4-氧基)-2,5,8,11,14,17,20,23-八氧杂二十六烷-26-酰胺(CB05)的合成。
室温氮气保护下，9.66g的(9H-芴-9-基)甲基((30S,33S,36S)-41-氨基-36-((4-(羟甲基)苯基)氨基甲酰基)-33-异丙基-26,31,34,41-四氧-2,5,8,11,14,17,20,23-八氧杂-27,32,35,40-四氮杂-30-基)氨基甲酸酯(CB04)溶解于50mL DMF，加入12mL二乙胺，搅拌反应1.5h。反应完成后减压蒸馏除去溶剂，硅胶柱层析(二氯甲烷和甲醇体积比7.5:1作为洗脱溶剂)得到淡黄色固体7.7g CB05。
6)N-((S)-3-(2-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-4-(((S)-1-(((S)-1-((4-(羟甲基)苯基)胺基)-1-氧-5-脲基戊烷-2-基)胺基)-3-甲基-1-丁酮-2-基)胺基)-4-氧代)-2,5,8,11,14,17,20,23-八氧杂二十六烷-26-酰胺(CB06)的合成。
7.7g N-((S)-3-氨基-4(((S)-1-(((S)-1-((4-(羟甲基)苯基)胺基)-1-氧代-5-脲基戊烷-2-基)胺基)-3-甲基-1-氧丁烷-2-基)胺基)-4-氧基)-2,5,8,11,14,17,20,23-八氧杂二十六烷-26-酰胺(CB05)溶于40mL DMF中，0-5℃氮气氛围下，分批加入马来酰亚胺基乙酸琥珀酰亚胺酯(CB00-1),保持0-5℃下反应4h。反应完成后减压蒸馏除去溶剂，硅胶柱层析(二氯甲烷和甲醇体积比10:1作为洗脱溶剂)得到淡黄色固体9.5g CB06。
7)4-((30S,33S,36S)-30-(2-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-33-异丙基-26,31,34-三氧-36-(3-脲丙基)-2,5,8,11,14,17,20,23-八氧-27,32,35-三氮杂三十七烷-37-胺基)苯甲基(4-硝基苯基)碳酸酯(CB07)的合成。
9.5g N-((S)-3-(2-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-4-(((S)-1-(((S)-1-((4-(羟甲基)苯基)胺基)-1-氧-5-脲基戊烷-2-基)胺基)-3-甲基-1-丁酮-2-基)胺基)-4-氧代)-2,5,8,11,14,17,20,23-八氧杂二十六烷-26-酰胺(CB06)溶于50mL DMF中，0℃氮气氛围下，加入14.0g二(对硝基苯)碳酸酯((PNP)2CO)，溶解后再加入8.2mL N,N-二异丙基乙胺(DIPEA)，保持0℃反应4h。反应完成后减压蒸馏除去溶剂，硅胶柱层析(二氯甲烷和甲醇体积比8:1作为洗脱溶剂)得到白色固体2.6g CB07。
实施例4. 4-((18S,21S,24S)-18-(2-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-21-异丙基-14,19,22-三氧-24-(3-脲丙基)-2,5,8,11-四氧-15,20,23-三氮杂二十五烷-25-胺基)苯甲基(4-硝基苯基)碳酸酯(CB14)的合成
CB14的制备参考实施例3中CB07的合成，将4,7,10,13,16,19,22,25-八氧杂二十六烷酸-N-琥珀酰亚胺酯替换为4,7,10,13-四氧杂十四烷酸-N-琥珀酰亚胺酯。最终得到白色固体CB14。
实施例5.中间体(CB07-Exatecan)的合成
4-(30S,33S,36S)-30-(2-(2,5-二氧杂-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-33-异丙基-26,31,34-三氧杂-36-(3-脲丙基)-2,5,8,11,14,17,20,23-八氧杂-27,32,35-三氮杂庚三氨基乌头-37-基)苯甲酰基(1s,9s)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃[3’,4’:6,7]吲哚嗪并[1,2-b]喹啉-1-氨基甲酸酯的合成。
将4-((30S,33S,36S)-30-(2-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-33-异丙基-26,31,34-三氧-36-(3-脲丙基)-2,5,8,11,14,17,20,23-八氧-27,32,35-三氮杂三十七烷-37-胺基)苯甲基(4-硝基苯基)碳酸酯(CB07，2.6g，2.21mmol)与N,N-二甲基甲酰胺(23ml)加入反应瓶R1，氮气保护下搅拌降温至0-5℃。同时取(1S,9S)-1-氨基-9-乙基-5-氟-9-羟基-4-甲基-1,2,3,9,12,15-六氢-10H,13H-苯并[3’,4’:6,7]吲哚嗪并[1,2-b]喹啉-10,13-二酮甲磺酸盐(依喜替康甲磺酸盐，0.98g，1.84mmol，Advanced ChemBlocks公司)与N,N-二甲基甲酰胺(5ml)加入到另一个反应瓶R2，0-5℃滴加三乙胺(230mg，2.27mmol)，搅拌至全溶后。将反应瓶R2中溶液滴加到反应瓶R1中，然后用N,N-二甲基甲酰胺(2ml)洗涤反应瓶R2后，洗涤液加入到反应瓶R1中。再称取1-羟基苯并三唑(497mg，3.68mmol)、吡啶(1.45g，18.4mmol)加入到反应瓶R1。0-5℃搅拌10min，升至室温搅拌5.5h反应完全后，35℃减压浓缩除去溶剂。用制备型高效液相色谱(prep-HPLC)纯化，冻干，得到白色粉末(1.6g，59％)。LC-MS:[1/2M+H]+＝737。
实施例6.中间体(CB07-D-1)的合成
4-(30S,33S,36S)-30-(2-(2,5-二氧杂-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-33-异丙基-26,31,34-三氧杂-36-(3-脲丙基)-2,5,8,11,14,17,20,23-八氧杂-27,32,35-三氮杂庚三氨基乌头-37-基)苯甲酰基(1S,9S)-9-乙基-4,5-二氟-9-羟基-10,13-二氧-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃[3’,4’:6,7]吲哚嗪并[1,2-b]喹啉-1-氨基甲酸酯的合成
将4-((30S,33S,36S)-30-(2-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-33-异丙基-26,31,34-三氧-36-(3-脲丙基)-2,5,8,11,14,17,20,23-八氧-27,32,35-三氮杂三十七烷-37-胺基)苯甲基(4-硝基苯基)碳酸酯(CB07，220mg，0.189mmol)与N,N-二甲基甲酰胺(5ml)加入反应瓶R1，氮气保护下搅拌降温至0-5℃。同时取(1S,9S)-1-氨基-9-乙基-4,5-二氟-9-羟基-1,2,3,9,12,15-六氢-10H,13H苯并[de]哌喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-10,13-二酮盐酸盐(D-1)(90mg，0.189mmol)与N,N-二甲基甲酰胺(5ml)加入到另一个反应瓶R2，0-5℃滴加三乙胺3滴，搅拌至全溶后。将反应瓶R2中溶液滴加到反应瓶R1中，再称取1-羟基苯并三唑(60mg，0.44mmol)、吡啶(0.5ml)加入到反应瓶R1。0-5℃搅拌10min，升至室温搅拌3h反应完全后，减压浓缩除去溶剂。用制备型高效液相色谱(prep-HPLC)纯化，冻干，得到白色粉末(55mg，20％)。LC-MS:[1/2M+H]+＝739。
实施例7.中间体(CB14-Exatecan)的合成
4-(18S,21S,24S)-18-(2-(2,5-二氧六环-2,5-二氢-1h-吡咯-1-基)乙酰氨基)-21-异丙基-14,19,22-三氧代-24-(3-脲基丙基)-2,5,8,11-四氧-15,20,23-三氮杂二十五烷-25-胺基)-(1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃[3’,4’:6,7]吲哚嗪并[1,2-b]喹啉-1-氨基甲酸酯的合成。
类似地，将4-((18S,21S,24S)-18-(2-(2,5-二氧-2,5-二氢-1H-吡咯-1-基)乙酰氨基)-21-异丙基-14,19,22-三氧-24-(3-脲丙基)-2,5,8,11-四氧-15,20,23-三氮杂二十五烷-25-胺基)苯甲基(4-硝基苯基)碳酸酯(190mg，0.19mmol)与N,N-二甲基甲酰胺(23mL)加入反应瓶R1，氮气保护下搅拌降温至0-5℃。同时取(1S,9S)-1-氨基-9-乙基-5-氟-9-羟基-4-甲基-1,2,3,9,12,15-六氢-10H,13H-苯并[3’,4’:6,7]吲哚嗪并[1,2-b]喹啉-10,13-二酮甲磺酸盐(依喜替康甲磺酸盐；101mg，0.19mmol；Advanced ChemBlocks公司)与N,N-二甲基甲酰胺(5mL)加入到另一个反应瓶R2，0-5℃滴加三乙胺(3滴)，搅拌至全溶后。将反应瓶R2中溶液滴加到反应瓶R1中，然后用N,N-二甲基甲酰胺(1mL)洗涤反应瓶R2后，洗涤液加入到反应瓶R1中。再称取1-羟基苯并三唑(60mg，0.44mmol)、吡啶(0.5mL)加入到反应瓶R1。0-5℃搅拌10min，升至室温搅拌5.5h反应完全后，35℃减压浓缩除去溶剂。用制备型高效液相色谱(prep-HPLC)纯化，冻干，得到白色粉末。
实施例8.ADC1(Abu为抗体H239H-2b-K-6a-1)的合成
取5ml的11.5mg/ml的抗体H239H-2b-K-6a-1，加入100μl的0.1M EDTA水溶液，根据抗体的物质的量，加入2.7摩尔当量的10mM TCEP水溶液，25℃恒温箱反应2h，超滤换液，去除体系中的还原剂TCEP。随后，根据抗体的物质的量加入6摩尔当量的10mM CB07-Exatecan的二甲基乙酰胺(DMA)溶液进行偶联，加入有机溶剂DMA至其体积占偶联体系的20％，25℃恒温箱反应2h15min，加入0.1M NAC(N-乙酰半胱氨酸)水溶液至其终浓度为1.5mM，反应15分钟后终止反应。
纯化后ADC1的DAR即p通过RP-UPLC检测约为4.05。
实施例9.ADC2(Abu为抗体H239H-2b-K-6a-1)的合成
取15.2ml的11.5mg/ml的抗体H239H-2b-K-6a-1，加入326μl的0.1M EDTA水溶液，根据抗体的物质的量，加入7摩尔当量的10mM TCEP水溶液，25℃恒温箱反应2h，超滤换液，去除体系中的还原剂TCEP。随后，根据抗体的物质的量加入12摩尔当量的10mM CB07-Exatecan的二甲基乙酰胺(DMA)溶液进行偶联，加入有机溶剂DMA至其体积占偶联体系的20％，25℃恒温箱反应2.5h，加入0.1M NAC水溶液至其终浓度为2mM，反应15分钟后终止反应。
纯化后ADC2的DAR即p通过RP-UPLC检测约为7.49。
实施例10.ADC3(Abu为抗体H239H-2b-K-6a-1)的合成
取2.5ml的11.5mg/ml的抗体H239H-2b-K-6a-1，加入50μl的0.1M EDTA水溶液，根据抗体的物质的量，加入6摩尔当量的15mM TCEP水溶液，25℃恒温箱反应2h，超滤换液，去除体系中的还原剂TCEP。随后，根据抗体的物质的量加入12摩尔当量的10mM CB07-D-1的二甲基乙酰胺(DMA)溶液进行偶联，加入有机溶剂DMA至其体积占偶联体系的20％，25℃恒温箱反应2.5h，加入0.1M NAC水溶液至其终浓度为2mM，反应15分钟后终止反应。
纯化后ADC3的DAR即p通过RP-UPLC检测约为7.65。
实施例11.ADC4(Abu为抗体H239H-2b-K-6a-1)的合成
取5ml的11.5mg/ml的抗体H239H-2b-K-6a-1，加入100μl的0.1M EDTA水溶液，根据抗体的物质的量，加入2.7摩尔当量的10mM TCEP水溶液，25℃恒温箱反应2h，超滤换液，去除体系中的还原剂TCEP。随后，根据抗体的物质的量加入6摩尔当量的10mM MC-GGFG-DXD(Deruxtecan,MCE,Cat.#HY-13631E/CS-0045125)的二甲基乙酰胺(DMA)溶液进行偶联，加入有机溶剂DMA至其体积占偶联体系的20％，25℃恒温箱反应2h，加入0.1M NAC水溶液至其终浓度为1.5mM，反应15分钟后终止反应。
纯化后ADC4的DAR即p通过RP-UPLC检测约为3.43。
实施例12.ADC5(Abu为抗体H239H-2b-K-6a-1)的合成
取5ml的11.5mg/ml的抗体H239H-2b-K-6a-1，加入100μl的0.1M EDTA水溶液，根据抗体的物质的量，加入7摩尔当量的10mM TCEP水溶液，25℃恒温箱反应2h，超滤换液，去除体系中的还原剂TCEP。随后，根据抗体的物质的量加入12摩尔当量的10mM MC-GGFG-DXD(Deruxtecan,MCE,Cat.#HY-13631E/CS-0045125)的二甲基乙酰胺(DMA)溶液进行偶联，加入有机溶剂DMA至其体积占偶联体系的20％，25℃恒温箱反应2h，加入0.1M NAC水溶液至其终浓度为2mM，反应15分钟后终止反应。
纯化后ADC5的DAR即p通过RP-UPLC检测约为7.67。
实施例13.ADC6(Abu为抗体H239H-2b-K-6a-2)的合成
取3ml的7.89mg/ml的抗体H239H-2b-K-6a-2，加入60μl的0.1M EDTA水溶液，根据抗体的物质的量，加入7摩尔当量的10mM TCEP水溶液，25℃恒温箱反应2h，超滤换液，去除体系中的还原剂TCEP。随后，根据抗体的物质的量加入15摩尔当量的100mM CB07-Exatecan的二甲基乙酰胺(DMA)溶液进行偶联，25℃恒温箱反应2h，加入0.1M NAC水溶液至其终浓度为2mM，反应15分钟后终止反应。
纯化后ADC6的DAR即p通过RP-UPLC检测约为7.19。
实施例14.抗CLDN18.2抗体与细胞表面表达的CLDN18.2结合
利用流式细胞法，检测抗CLDN18.2抗体及ADC对表达CLDN18.2细胞的结合能力，并进一步检测其不结合CLDN18.1的特异性。实验步骤概况如下：收集CLDN18.2阳性细胞(KATOIII或CHO-CLDN18.2)及阴性细胞(CHO-CLDN18.1)，用PBS洗一遍后，用PBS重悬细胞，100000个细胞/50μl/孔，铺于96孔V底板中(货号：3897，Costar)，用PBS分别预稀释抗体及ADC至333.2nM，然后2倍往下稀释9个梯度，50μl/孔加入到上述96孔V底板的细胞中，混匀(抗体或ADC终浓度为：166.6、83.3、41.6、20.8、10.4、5.2、2.6、1.3、0.65、0.325nM)，于4℃孵育1h后，用PBS洗2次，接着加入100μl 1:500稀释的羊抗人IgG-Fc PE荧光二抗(货号：12-4998-82，eBioscience)，于4℃孵育1h后，用PBS洗2次，之后使用CytoFLEX流式细胞仪(型号：AOO-1-1102，贝克曼)进行分析。
1)嵌合抗体与表达CLDN18.2细胞的检测见图1，嵌合抗体CH239H-2-K-6、CH239H-9-K-4、CH394H2-K-1、CH372H6-K-1、CH239H-9-K-6、CH239H-9-K-1、CH101H1-K-1和CH372H1-K-1均能结合KATOIII细胞上的CLDN18.2，其中嵌合抗体CH239H-2-K-6的结合活性更好。
2)与阳性抗体IMAB362(其序列与专利US20090169547中杂交瘤细胞175D10表达的抗体的序列是相同的)相比，嵌合抗体CH239H-2-K-6与KATOIII细胞上的CLDN18.2结合能力更好：IMAB362和CH239H-2-K-6的EC50值分别为8.082nM和6.290nM。
3)嵌合抗体CH239H-2-K-6进行人源化之后，人源化抗体H239H-2a-K-6a和H239H-2b-K-6a对CLDN18.2阳性细胞KATOIII和CHO-CLDN18.2的结合能力与嵌合抗体CH239H-2-K-6基本一致，亲和力并没有下降；抗体CH239H-2-K-6、抗体H239H-2a-K-6a、抗体H239H-2b-K-6a与KATOIII细胞结合的EC50值分别为6.108nM、6.203nM和6.920nM； 抗体CH239H-2-K-6、抗体H239H-2a-K-6a、抗体H239H-2b-K-6a、阳性抗体IMAB362与CHO-CLDN18.2细胞结合的EC50值分别为19.92nM、22.83nM、23.31nM、30.02nM。
4)如图2所示，人源化抗体H239H-2a-K-6a和H239H-2b-K-6a只结合CLDN18.2且不结合CLDN18.1。
5)如图3所示，人源化抗体H239H-2b-K-6a-1对KATOIII细胞结合的EC50值为3.887nM；ADC1、ADC2、ADC3、ADC4对KATOIII细胞结合的EC50值分别为：4.531nM、7.763nM、7.322nM、6.194nM。
CHO-CLDN18.2稳定细胞系构建：
人CLDN18.2全长氨基酸序列，(来自NCBI，NM_001002026.3)：
对应的核酸序列如下：
合成上述人CLDN18.2对应的核酸序列，并在序列两端添加酶切位点HindIII和EcoRI的序列，然后构建到pcDNA3.1表达载体(Invitrogen，货号为V79020)上，接着通过电转的方法转染到CHO细胞(Life technologies，货号：A13696-01)中，电转条件为：电压300V，时间17毫秒，4mm电转杯，48h后加入50μM的MSX(蛋氨酸亚氨基代砜)筛选压，2周后，筛选阳性细胞。利用FACS检测，筛选高表达的细胞株。收集细胞，用PBS(磷酸缓冲液，1L PBS含NaCl 8.0g，Na2HPO40.9g，KH2PO40.156g，KCl 0.125g，pH 7.2-7.4)洗一遍后，加入3μg/ml抗体IMAB362，4℃孵育1h后，用PBS洗2遍，加入100μl 1:500稀释的羊抗人IgG-Fc PE荧光二抗(货号：12-4998-82，eBioscience)，4℃孵育1h后，用PBS洗2遍，C6流式细胞仪(BD，型号：C6Flow cytometer)分析。将最终得到的细胞命名为CHO-CLDN18.2细胞。
CHO-CLDN18.1稳定细胞系构建：
人CLDN18.1全长氨基酸序列，(来自NCBI，NM_016369.4)：
对应的核酸序列如下：
合成上述人CLDN18.1对应的核酸序列，并在序列两端添加酶切位点HindIII和EcoRI的序列，然后构建到pcDNA3.1表达载体上，通过电转的方法转染到CHO细胞中，电转条件为：电压300V，时间17毫秒，4mm电转杯，48h后加入50μM的MSX筛选压，2周后，筛选阳性细胞。利用DotBlot点杂交法检测，收集细胞，提取细胞裂解上清，同时以空白CHO细胞裂解上清作为阴性对照，CHO-CLDN18.2细胞裂解上清作为阳性对照，取20μl细胞裂解液点在已活化的PVDF膜(millipore)上，静置3～5min，使膜将蛋白充分吸附，将膜浸泡在5％脱脂奶粉中，置于37℃封闭2h，用PBST(PBS+0.05％吐温)洗3次，接着将膜浸泡在兔抗人Claudin18抗体(货号：ab230224，abcam，该抗体能识别CLDN18.1和CLDN18.2相同的胞内段，故CHO-CLDN18.2细胞可作为阳性对照)中室温孵育2h，用PBST洗3次，之后将膜浸泡在羊抗兔HRP抗体(货号：ab6721，abcam)中，室温孵育2h，PBST洗3次后用DAB(货号：AR1000，博士德生物)进行显色。将最终得到的细胞命名为CHO-CLDN18.1细胞。
实施例15.抗CLDN18.2抗体的ADCC活性(antibody dependent cell-mediated cytotoxicity)
本实验选择ADCC Reporter Bioassay评价CLDN18.2抗体的ADCC活性。这是一种生物发光报告基因检测系统，以人工构建的效应细胞替代NK细胞，配合高灵敏度的检测试剂，可定量基于ADCC作用机制的治疗性抗体药在其活化途径中的生物活性，是一种作用机制检测法。利用表达CLDN18.2的细胞(CHO-CLDN18.2)为靶细胞，Jurkat-NFAT-luc-FcγRIIIa为效应细胞(通过慢病毒侵染的方法将FcγRIIIa(序列来自NCBI，NP_001121065.1)转到Jurkat(中国科学院上海细胞库，Clone E6-1)细胞上，并用0.3μg/ml的嘌呤霉素(货号：A11138-03，Gibco)，筛选阳性克隆，命名为Jurkat-FcγRIIIa；接着通过电转染的方法将含有荧光素报告基因的质粒pGL4.30[luc2P/NFAT-RE/Hygro](货号：E848A，Promega)转染到Jurkat-FcγRIIIa细胞上，并用潮霉素B(货号：A600230-0001，生工生物)筛选阳性克隆，最后得到的细胞命名为Jurkat-NFAT-luc-FcγRIIIa)，通过ONE-GloTM Luciferase Assay System(货号：E6120-100ml，Promega)进行检测。具体步骤如下：收集CHO-CLDN18.2细胞，用RPMI1640培养基洗1遍，然后用RPMI1640培养基调整细胞密度为1.2×106/ml，25μl/孔接种到96孔白板中(货号：3917，Costar)，即3万/孔，轻拍混匀。用1％FBS的RPMI1640培养基作为抗体稀释液，将抗体预稀释至10μg/ml，然后5倍往下稀释8个梯度，50μl/孔加入至上述细胞板中，轻拍混匀。接着收集对数生长期的Jurkat-NFAT-luc-FcγRIIIa细胞，用RPMI1640培养基洗1遍，然后用RPMI1640培养基调整细胞密度2.4×106/ml，25μl/孔加入上述细胞培养板中，即6万/孔，混匀。将培养板置于37℃、5％CO2培养箱培养6h后，取出放置于室温条件下10分钟，让细胞培养板恢复到室温温度。加入50μl ONE-GLO底物(货号：E6120-100ml，promega)，置于酶标仪(型号：SpectraMax M3，Molecular Devices)读取Luminesecence相对荧光单位RLU。
CHO-CLDN18.1细胞作为细胞阴性对照，IMAB362作为抗体阳性对照，IgG同种型(货号：BE0297，Bio cell)作为抗体阴性对照。
结果显示，抗体H239H-2b-k-6a具有很强的ADCC作用，且抗体H239H-2b-k-6a比阳性抗体IMAB362的ADCC效应强：抗体IMAB362、抗体H239H-2b-k-6a对应的EC50值分别为0.7953nM和0.06253nM。
实施例16.抗CLDN18.2抗体的CDC活性(complement dependent cytoxicity)
收集CHO-CLDN18.2细胞，用PBS洗一遍，用RPMI1640培养基调整细胞密度为6.6×105/ml，45μl/孔，铺于96孔平底板。用RPMI1640培养基预稀释抗体至50μg/ml，然后4倍往下稀释，共9个梯度，并以不加抗体孔作为对照孔。以终浓度为5％的人血清补体(货号：Quidel，A113)作为补体来源，37℃孵育5h后，加入CCK-8试剂(货号：CK04，DojinDo)，检测活细胞数，并计算细胞杀伤率[％细胞杀伤率＝(1-抗体孔读数/对照孔读数)×100]。
结果显示，抗体H239H-2b-k-6a在补体存在的情况下存在显著的CDC效应，抗体H239H-2b-k-6a比阳性抗体IMAB362的CDC效应强：抗体IMAB362、抗体H239H-2b-k-6a对应的IC50值分别为5.867nM和0.2909nM。
实施例17.抗CLDN18.2ADC增殖抑制实验
抗CLDN18.2ADC与表达CLDN18.2的细胞共孵育，通过CCK-8(货号：CK04，DojinDo)试剂检测活细胞数，计数抑制率。实验步骤概况如下：收集对数生长期的CHO-CLDN18.2细胞，800rpm，离心5min，去上清，用CD-CHO-AGT(货号：12490；life technologies)培养基洗一遍，800rpm，离心5min，去上清。用0.5％FBS(货号：FSP500；Excell Bio)CD-CHO-AGT培养基重悬细胞，调整细胞密度为5×104个/ml，100μl/孔铺于96孔细胞培养板中(货号：3599；costar)。加入用0.5％FBS CD-CHO-AGT梯度稀释好的ADC，100μl/孔，ADC终浓度为：500、250、125、62.5、31.25、15.625、7.813、3.9、1.95nM。置于37℃，5％CO2培养箱中，培养5天后，每孔加入20μl CCK-8，37℃放置1h，于酶标仪(型号：SpectraMax M3，Molecular Devices)读取OD450nm吸光值，计算细胞抑制率。抑制率％＝(1-样品孔吸光值/对照孔吸光值)×100。
结果显示，不同ADC对CHO-CLDN18.2细胞有明显的增殖抑制作用，见图4。
实施例18.ADC旁观者效应
在体外共培养CLDN18.2阳性细胞KATOIII和阴性细胞HGC-27的条件下，观察不同ADC对这两个细胞的杀伤情况。实验步骤概况如下：收集对数生长期的KATOIII(CLDN18.2阳性细胞)和HGC-27(CLDN18.2阴性细胞)，用2％FBS RPMI 1640重悬细胞，铺于6孔板(货号：3516；costar)中，KATOIII每孔12万个细胞，HCG-27每孔8万个细胞。置于37℃、5％CO2培养箱培养过夜，第二天加入3ml ADC，ADC终浓度为10nM、1nM、0.1nM，并以不加ADC孔作为阴性对照。置于37℃，5％CO2培养箱，培养5天，用胰蛋白酶(货号：25200-072；gibco)消化收集细胞，计算活细胞总数。为了确定总存活细胞中KATOIII和HGC-27的比例，将细胞用FITC标记的抗Trop2抗体(抗Trop2抗体的重链和轻链序列分别为SEQ ID NO:83和SEQ ID NO:84，见表7)染色(KATOIII是Trop2阳性细胞)并在冰上孵育30min，洗涤后，使用CytoFLEX流式细胞仪(型号：AOO-1-1102，贝克曼)测量染色细胞的荧光信号。根据每个处理孔中KATOIII阳性和HGC-27阴性细胞的数量和比例，计算KATOIII和HGC-27细胞的数量，结果见图5。
结果显示：不同ADC对阳性细胞和阴性细胞都有杀伤作用，其中ADC2对阴性细胞的旁观者效应最强。
表7抗Trop2抗体的氨基酸序列
实施例19.抗CLDN18.2抗体及其Fc突变抗体对Fc受体的亲和力测定
采用BLI(Bio-Layer Interferometry，生物膜层表面干涉)技术，检测抗CLDN18.2抗体及其Fc突变抗体对Fc受体的亲和力。仪器使用Octet Platform，Fortebio，Octet QK，S/N33-8094-2244，Equipment ID TBIO001。传感器为SA Biosensor，Fortebio，Cat#18-5019。Fc受体包括：FcRn，ACRO Biosystems，Cat#FCM-H8286；FcγRIa，ACRO Biosystems，Cat#FCA-H82E8；FcγRIIa H131，ACRO Biosystems，Cat#CDA-H82E6；FcγRIIa R131，ACRO Biosystems，Cat#CDA-H82E7；FcγRIIb，ACRO Biosystems，Cat#CDB-H82E0；FcγRIIIa 158V，ACRO Biosystems，Cat#CDA-H82E9；FcγRIIIa 158F，ACRO Biosystems，Cat#CDA-H82E8；FcγRIIIb(NA2)，ACRO Biosystems，Cat#CDB-H82Ea。
将SA传感器放置在PBS中预湿10min后捕获Fc受体，接着对抗体进行结合及解离信号采集，采集数据后，用仪器的数据分析软件Acquisition 8.2对数据进行分析，得出亲和常数KD。检测结果见表8。
结果显示：抗体H239H-2b-K-6a-1的Fc突变不影响抗体对FcRn的结合。N297A突变后，抗体H239H-2b-K-6a-2对FcγRIa的亲和力下降约100倍，对FcγRII及FcγRIII都不结合了。LALA P329G突变后，抗体H239H-2b-K-6a-3均不结合FcγRI、FcγRII及FcγRIII。
表8抗CLDN18.2抗体及其Fc突变抗体对Fc受体的亲和常数KD(M)

实施例20.ADC在胃癌GA0006异种移植雌性BALB/c裸小鼠动物模型中的抗肿瘤作用
测试不同ADC在胃癌异种移植模型GA0006荷瘤小鼠体内的抗肿瘤效果。将直径为2～3mm的瘤块接种于BALB/c裸小鼠右前肩胛处皮下。当荷瘤鼠平均肿瘤体积到达约139.15mm3时，将小鼠随机分组。分组当天设定为第0天，给药开始于第0天，单次给药。监测小鼠的体重和肿瘤的生长情况。GA0006异种移植模型各治疗组和对照组肿瘤生长情况见图6。肿瘤体积计算公式：肿瘤体积(mm3)＝1/2×(a×b2)(其中a表示长径，b表示短径)。TGITV％＝[1-(Ti-T0)/(Ci-C0)]×100其中T0及C0分别是给药组及溶媒对照组分组当天(第0天)的平均肿瘤体积，Ti及Ci分别是给药组及溶媒对照组第14天时的平均肿瘤体积。nsP>0.05，*P<0.05，**P<0.01及***P<0.001与溶媒对照组肿瘤体积相比。
该模型的对照组(第1组)在给药14天后平均肿瘤体积达到628.62mm3。
10mg/kg和5mg/kg ADC2(即第2组和第3组)给药14天后，平均肿瘤体积分别为78.17mm3和91.47mm3，相对肿瘤抑制率分别TGI为112.45％(P＝3.43e-09***)和109.75％(P＝6.53e-08***)。
10mg/kg和5mg/kg ADC6(即第4组和第5组)给药14天后，平均肿瘤体积分别为25.77mm3和78.07mm3，相对肿瘤抑制率分别TGI为123.17％(P＝7.58e-12***)和112.48％(P＝1.23e-08***)。
10mg/kg和5mg/kg ADC3(即第6组和第7组)给药14天后，平均肿瘤体积分别为185.69mm3和284.02mm3，相对肿瘤抑制率分别TGI为90.50％(P＝5.92e-04***)和70.40％(P＝3.51e-01ns)。
5mg/kg ADC5(即第8组)给药14天后，平均肿瘤体积为296.86mm3，相对肿瘤抑制率TGI为67.79％(P＝3.51e-0ns)。
与肿瘤体积数据趋势一致，以肿瘤重量变化为指标，在给药14天后各治疗组同样展示了不同程度的抗肿瘤药效，见图7。

Claims (36)

  1. 一种抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元特异性结合CLDN18.2，并且包含(a)-(f)中的一个或多个氨基酸序列：
    (a)VH CDR1，其包含如SEQ ID NO:1-6任一项所示的氨基酸序列；
    (b)VH CDR2，其包含如SEQ ID NO:7-13任一项所示的氨基酸序列；
    (c)VH CDR3，其包含如SEQ ID NO:14-21任一项所示的氨基酸序列；
    (d)VL CDR1，其包含如SEQ ID NO:22-29任一项所示的氨基酸序列；
    (e)VL CDR2，其包含如SEQ ID NO:30-37任一项所示的氨基酸序列；
    (f)VL CDR3，其包含如SEQ ID NO:38-45任一项所示的氨基酸序列。
  2. 如权利要求1所述的抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元包含如SEQ ID NO:1-6任一项所示的VH CDR1、如SEQ ID NO:7-13任一项所示的VH CDR2和如SEQ ID NO:14-21任一项所示的VH CDR3。
  3. 如权利要求1或2所述的抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元包含如SEQ ID NO:22-29任一项所示的VL CDR1、如SEQ ID NO:30-37任一项所示的VL CDR2和如SEQ ID NO:38-45任一项所示的VL CDR3。
  4. 一种抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元特异性结合CLDN18.2，其中所述抗体或抗原结合单元包含如SEQ ID NO:2所示的VH CDR1、如SEQ ID NO:8所示的VH CDR2、如SEQ ID NO:15所示的VH CDR3、如SEQ ID NO:26所示的VL CDR1、如SEQ ID NO:34所示的VL CDR2和如SEQ ID NO:42所示的VL CDR3；或所述抗体或抗原结合单元包含如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:9所示的VH CDR2、如SEQ ID NO:16所示的VH CDR3、如SEQ ID NO:25所示的VL CDR1、如SEQ ID NO:33所示的VL CDR2和如SEQ ID NO:41所示的VL CDR3；或
    所述抗体或抗原结合单元包含如SEQ ID NO:6所示的VH CDR1、如SEQ ID NO:13所示的VH CDR2、如SEQ ID NO:20所示的VH CDR3、如SEQ ID NO:28所示的VL CDR1、如SEQ ID NO:36所示的VL CDR2和如SEQ ID NO:44所示的VL CDR3；或
    所述抗体或抗原结合单元包含如SEQ ID NO:5所示的VH CDR1、如SEQ ID NO:12所示的VH CDR2、如SEQ ID NO:19所示的VH CDR3、如SEQ ID NO:29所示的VL CDR1、如SEQ ID NO:37所示的VL CDR2和如SEQ ID NO:45所示的VL CDR3；或
    所述抗体或抗原结合单元包含如SEQ ID NO:4所示的VH CDR1、如SEQ ID NO:11所示的VH CDR2、如SEQ ID NO:18所示的VH CDR3、如SEQ ID NO:27所示的VL CDR1、如SEQ ID NO:35所示的VL CDR2和如SEQ ID NO:43所示的VL CDR3；或
    所述抗体或抗原结合单元包含如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:9所示的VH CDR2、如SEQ ID NO:16所示的VH CDR3、如SEQ ID NO:26所示的VL CDR1、如SEQ ID NO:34所示的VL CDR2和如SEQ ID NO:42所示的VL CDR3；或
    所述抗体或抗原结合单元包含如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:9所示的VH CDR2、如SEQ ID NO:16所示的VH CDR3、如SEQ ID NO:24所示的VL CDR1、如SEQ ID NO:32所示的VL CDR2和如SEQ ID NO:40所示的VL CDR3；或
    所述抗体或抗原结合单元包含如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:7所示的VH CDR2、如SEQ ID NO:14所示的VH CDR3、如SEQ ID NO:22所示的VL CDR1、如SEQ ID NO:30所示的VL CDR2和如SEQ ID NO:38所示的VL CDR3；或
    所述抗体或抗原结合单元包含如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:7所示的VH CDR2、如SEQ ID NO:14所示的VH CDR3、如SEQ ID NO:23所示的VL CDR1、 如SEQ ID NO:31所示的VL CDR2和如SEQ ID NO:39所示的VL CDR3；或
    所述抗体或抗原结合单元包含如SEQ ID NO:2所示的VH CDR1、如SEQ ID NO:10所示的VH CDR2、如SEQ ID NO:17所示的VH CDR3、如SEQ ID NO:27所示的VL CDR1、如SEQ ID NO:35所示的VL CDR2和如SEQ ID NO:43所示的VL CDR3；或
    所述抗体或抗原结合单元包含如SEQ ID NO:2所示的VH CDR1、如SEQ ID NO:8所示的VH CDR2、如SEQ ID NO:21所示的VH CDR3、如SEQ ID NO:26所示的VL CDR1、如SEQ ID NO:34所示的VL CDR2和如SEQ ID NO:42所示的VL CDR3。
  5. 如权利要求1-4任一项所述的抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元包含重链可变区和/或轻链可变区；其中，
    所述重链可变区包含如SEQ ID NO:46-55任一项所示的氨基酸序列，或与SEQ ID NO:46-55任一项所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:46-55任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述轻链可变区包含如SEQ ID NO:56-64任一项所示的氨基酸序列，或与SEQ ID NO:56-64任一项所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:56-64任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  6. 一种抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元特异性结合CLDN18.2，并且包含重链可变区和轻链可变区；其中，
    所述重链可变区包含如SEQ ID NO:47所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:60所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:48所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:59所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:52所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:62所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:51所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:63所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:50所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:61所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:48所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:60所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:48所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:58所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:46所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:46所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:57所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:49所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:61所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:53所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:64所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:54所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:64所示的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO:55所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:64所示的氨基酸序列。
  7. 如权利要求1-6任一项所述的抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元为人源化抗体或抗原结合单元。
  8. 如权利要求1-7任一项所述的抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元为IgG同种型；或者，所述抗体或抗原结合单元为IgG1或IgG4同种型。
  9. 如权利要求1-8任一项所述的抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元包含重链恒定区和/或轻链恒定区。
  10. 如权利要求9所述的抗体或抗原结合单元，其特征在于，所述重链恒定区包含如下一种或多种氨基酸突变：N297A、L234A、L235A、P329G；或者，所述重链恒定区包含如下氨基酸突变：L234A、L235A和P329G。
  11. 如权利要求1-9任一项所述的抗体或抗原结合单元，其特征在于，所述抗体或抗原结合单元包含重链恒定区和轻链恒定区；其中，
    所述重链恒定区包含如SEQ ID NO:65-67任一项所示的氨基酸序列，或与SEQ ID NO:65-67任一项所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:65-67任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述轻链恒定区包含如SEQ ID NO:68所示的氨基酸序列，或与SEQ ID NO:68所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:68所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  12. 如权利要求1-3任一项所述的抗体或抗原结合单元，其特征在于，所述抗体的重链包含如SEQ ID NO:69-71任一项所示的氨基酸序列，或与SEQ ID NO:69-71任一项所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:69-71任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗体的轻链包含如SEQ ID NO:72所示的氨基酸序列，或与SEQ ID NO:72所示的氨基酸序列相比具有至少80％或90％同一性的氨基酸序列，或与SEQ ID NO:72所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  13. 一种抗体或抗原结合单元，其特征在于，所述抗体的重链包含如SEQ ID NO:69-71任一项所示的氨基酸序列；和/或所述抗体的轻链包含如SEQ ID NO:72所示的氨基酸序列。
  14. 一种生物材料，为：
    一种多聚核苷酸，其特征在于，所述多聚核苷酸编码如权利要求1-13任一项所述的抗体或抗原结合单元；或，
    一种表达载体，其特征在于，所述表达载体包含编码如权利要求1-13任一项所述的抗体或抗原结合单元的多聚核苷酸；或，
    一种细胞，其特征在于，所述细胞包含编码如权利要求1-13任一项所述的抗体或抗原结合单元的多聚核苷酸；或者，所述细胞为CHO细胞或HEK293细胞。
  15. 一种抗体药物偶联物，其特征在于，包含通过接头与药物偶联的权利要求1-13任一项所述的抗体或抗原结合单元，或其药学上可接受的盐或溶剂合物；或者，所述接头为可裂解的接头。
  16. 一种抗体药物偶联物，其特征在于，具有如式I所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物：
    其中，
    Abu为结合CLDN18.2的抗体或抗原结合单元；
    D为药物；
    M为其中*连接Abu，**连接B，R选自：-(CH2)r-、-(CHRm)r-、C3-C8碳环基、-O-(CH2)r-、亚芳基、-(CH2)r-亚芳基-、-亚芳基-(CH2)r-、-(CH2)r-(C3-C8碳环基)-、-(C3-C8碳环基)-(CH2)r-、C3-C8杂环基、-(CH2)r-(C3-C8杂环基)-、-(C3-C8杂环基)-(CH2)r-、-(CH2)rC(O)NRm(CH2)r-、-(CH2CH2O)r-、-(CH2CH2O)r-CH2-、-(CH2)rC(O)NRm(CH2CH2O)r-、-(CH2)rC(O)NRm(CH2CH2O)r-CH2-、-(CH2CH2O)rC(O)NRm(CH2CH2O)r-、-(CH2CH2O)rC(O)NRm(CH2CH2O)r-CH2-和-(CH2CH2O)rC(O)NRm(CH2)r-；其中各Rm独立为H、C1-C6烷基、C3-C8碳环基、苯基或苄基；并且各r独立为1、2、3、4、5、6、7、8、9或10；
    B为其中*连接M，**连接L，***连接G；
    L为-(AA)i-(FF)f-，其中，AA为氨基酸或多肽，i是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20；各FF独立为 其中各RF独立为C1-C6烷基、C1-C6烷氧基、-NO2或卤素；z为0、1、2、3或4，其中*连接AA，**连接D；f为1、2、3、4、5、6、7、8、9或10；
    G为其中n为1-24；
    p为1-10。
  17. 如权利要求16所述的抗体药物偶联物，其特征在于，所述Abu为权利要求1-13任一项所述的抗体或抗原结合单元。
  18. 如权利要求16或17所述的抗体药物偶联物，其特征在于，所述B为其中*连接M，**连接L，***连接G。
  19. 如权利要求16-18任一项所述的抗体药物偶联物，其特征在于，各AA独立选自以下氨基酸或肽序列：Val-Cit、Val-Lys、Phe-Lys、Lys-Lys、Ala-Lys、Phe-Cit、Leu-Cit、Ile-Cit、Trp、Cit、Phe-Ala、Phe-Phe-Lys、D-Phe-Phe-Lys、Gly-Phe-Lys、Leu-Ala-Leu、Ile-Ala-Leu、Val-Ala-Val、Ala-Leu-Ala-Leu、β-Ala-Leu-Ala-Leu和Gly-Phe-Leu-Gly。
  20. 如权利要求19所述的抗体药物偶联物，其特征在于，AA为Val-Cit。
  21. 如权利要求16-20任一项所述的抗体药物偶联物，其特征在于，i为1。
  22. 如权利要求16-21任一项所述的抗体药物偶联物，其特征在于，FF独立为 其中*连接AA，**连接D。
  23. 如权利要求22所述的抗体药物偶联物，其特征在于，FF为其中*连接AA，**连接D。
  24. 如权利要求16-23任一项所述的抗体药物偶联物，其特征在于，f为1。
  25. 如权利要求24所述的抗体药物偶联物，其特征在于，L为其中*连接B，**连接D。
  26. 一种抗体药物偶联物，其特征在于具有式I-1或I-2所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物，其中
    所述式I-1为：
    所述式I-2为：
    其中
    Abu为权利要求1-13任一项所述的抗体或抗原结合单元；
    R选自：-(CH2)r-、-(CHRm)r-、C3-C8碳环基、-O-(CH2)r-、亚芳基、-(CH2)r-亚芳基-、-亚芳基-(CH2)r-、-(CH2)r-(C3-C8碳环基)-、-(C3-C8碳环基)-(CH2)r-、C3-C8杂环基、-(CH2)r-(C3-C8杂环基)-、-(C3-C8杂环基)-(CH2)r-、-(CH2)rC(O)NRm(CH2)r-、-(CH2CH2O)r-、-(CH2CH2O)r-CH2-、-(CH2)rC(O)NRm(CH2CH2O)r-、-(CH2)rC(O)NRm(CH2CH2O)r-CH2-、-(CH2CH2O)rC(O)NRm(CH2CH2O)r-、-(CH2CH2O)rC(O)NRm(CH2CH2O)r-CH2-和-(CH2CH2O)rC(O)NRm(CH2)r-；其中各Rm独立为H、C1-C6烷基、C3-C8碳环基、苯基或苄基；并且各r独立为1、2、3、4、5、6、7、8、9或10；
    D为药物；
    n为1-24的整数；
    p为1-10。
  27. 如权利要求16-26任一项所述的抗体药物偶联物，其特征在于，所述R为-(CH2)r-，所述r为1或5。
  28. 一种抗体药物偶联物，其特征在于具有式I-3或I-4所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物，其中
    所述式I-3为：
    所述式I-4为：
    其中
    Abu为权利要求1-13任一项所述的抗体或抗原结合单元；
    D为药物；
    n为1-24的整数；
    p为1-10。
  29. 一种抗体药物偶联物，其特征在于具有式I-5、I-6、I-7、I-8、I-9、I-10或I-11所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物，其中
    所述式I-5为：
    所述式I-6为：
    所述式I-7为：
    所述式I-8为：
    所述式I-9为：
    所述式I-10为：
    或所述式I-11为：
    其中
    Abu为权利要求1-13任一项所述的抗体或抗原结合单元；
    D为药物；
    p为1-10。
  30. 如权利要求15-29任一项所述的抗体药物偶联物，其特征在于，所述药物为抗癌药物、细胞毒性药物、细胞分化因子、干细胞营养因子、类固醇类药物、治疗自身免疫疾病的药物、抗炎症药物或治疗传染性疾病的药物；或所述药物为抗癌药物；或所述药物为微管蛋白抑制剂、DNA损伤剂或DNA拓扑异构酶抑制剂；或所述微管蛋白抑制剂选自海兔毒素、奥瑞他汀类、及美登素类；或所述药物为奥瑞他汀类，选自MMAE、MMAF或AF；或所述药物为DNA损伤剂，选自卡奇霉素类、倍癌霉素类、安曲霉素类衍生物PBD；或所述药物为DNA拓扑异构酶抑制剂或其盐，选自伊立替康、伊立替康盐酸盐、依喜替康衍生物，喜树碱、9-氨基喜树碱、9-硝基喜树碱、10-羟基喜树碱、9-氯-10-羟基喜树碱、喜树碱类衍生物SN-38、22-羟基旱莲木碱、拓扑替康、勒托替康、贝洛替康、依喜替康、硅基高喜树碱、6,8-二溴-2-甲基-3-[2-(D-吡喃木糖基氨基)苯基]-4(3H)-喹唑啉酮、2-氰基-3-(3,4-二羟基苯基)-N-(苯基甲基)-(2E)-2-丙烯酰胺、2-氰基-3-(3,4-二羟基苯基)-N-(3-羟基苯基丙基)-(E)-2-丙烯酰胺、12-β-D-吡喃萄萄糖基-12,13-二氢-2,10-二羟基-6-[[2-羟基-1-(羟基甲基)乙基]氨基]-5H-吲哚并[2,3-a]吡咯并[3,4-c]咔唑-5,7(6H)-二酮、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺二盐酸盐、N-[2-(二甲基氨基)乙基]-4-吖啶甲酰胺；或所述DNA拓扑异构酶抑制剂为喜树碱、10-羟基喜树碱、拓扑替康、贝洛替康、伊立替康、22-羟基旱莲木碱或依喜替康；或其药学上可接受的盐或溶剂合物。
  31. 如权利要求15-29任一项所述的抗体药物偶联物，其特征在于，所述药物为其中
    X1和X2各自独立地为：
    H，
    羟基，
    C1-C6烷基，
    被一个或多个羟基、卤素、硝基或氰基取代的C1-C6烷基，
    C2-C6烯基，
    C2-C6炔基，
    C1-C6烷氧基，
    C1-C6氨基烷氧基，
    卤素，
    硝基，
    氰基，
    巯基，
    烷硫基，
    氨基，被氨基保护基取代的氨基，在氨基部分任选被氨基保护基或C1-C6烷基取代的C1-C6氨基烷基，
    在氨基部分任选被氨基保护基或C1-C6烷基取代的C1-C6氨基烷基氨基，
    连接至杂环的C1-C6烷基，所述杂环任选被一个或多个C1-C6烷基、C1-C6烷氧基、氨基、卤素、硝基或氰基取代，
    连接至杂环的C1-C6烷基氨基，所述杂环任选被C1-C6烷基、C1-C6烷氧基取代，所述氨基任选被氨基保护基、卤素、硝基、氰基或保护基取代，
    氨基取代的杂环基，其在杂环部分的氮原子或氨基部分任选被保护基或一个或多个C1-C6烷基取代，
    杂环氨基，其在杂环部分的氮原子或氨基部分任选被保护基或C1-C6烷基取代，
    任选被氨基甲酰基保护基或C1-C6烷基取代的氨基甲酰基，
    吗啉-1-基，或
    哌啶-1-基；
    X3为C1-C6烷基；
    X4为H、-(CH2)q-CH3、-(CHRn)q-CH3、C3-C8碳环基、-O-(CH2)q-CH3、亚芳基-CH3、-(CH2)q-亚芳基-CH3、-亚芳基-(CH2)q-CH3、-(CH2)q-(C3-C8碳环基)-CH3、-(C3-C8碳环基)-(CH2)q-CH3、C3-C8杂环基、-(CH2)q-(C3-C8杂环基)-CH3、-(C3-C8杂环基)-(CH2)q-CH3、-(CH2)qC(O)NRn(CH2)q-CH3、-(CH2CH2O)q-CH3、-(CH2CH2O)q-CH2-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH3、-(CH2)qC(O)NRn(CH2CH2O)q-CH2-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH3、-(CH2CH2O)qC(O)NRn(CH2CH2O)q-CH2-CH3、或-(CH2CH2O)qC(O)NRn(CH2)q-CH3；其中各Rn独立为H、C1-C6烷基、C3-C8碳环基、苯基或苄基；并且各q独立为1、2、3、4、5、6、7、8、9或10；或者，X4为H或C1-C6烷基；
    **与所述抗体药物偶联物其他部分连接；
    y为0、1或2；
    Y为O、S或CR1R2，其中R1和R2各自独立地为H或C1-C6烷基；
    s和t各自独立为0、1或2，但不同时为0；
    或所述药物为其中X1和X2各自独立地为C1-C6烷基、卤素或-OH；或所述C1-C6烷基为-CH3；或所述卤素为F；**与所述 抗体药物偶联物其他部分连接；
    或所述药物为其中X1和X2各自独立地为C1-C6烷基、卤素或-OH；或所述C1-C6烷基为-CH3；或所述卤素为F；**与所述抗体药物偶联物其他部分连接。
  32. 如权利要求16-28任一项所述的抗体药物偶联物，其特征在于，n为4-12；或n为4-8；或n为4或8。
  33. 一种抗体药物偶联物，其特征在于，具有如式I-12、I-13、I-14、I-15、I-16、I-17、I-18、I-19、I-20、I-21、I-22、I-23、I-24或I-25的所示的结构或其立体异构体或其药学上可接受的盐或溶剂合物，其中所述式I-12、I-13、I-14、I-15、I-16、I-17、I-18、I-19、I-20、I-21、I-22、I-23、I-24或I-25为：





    其中
    Abu为权利要求1-13任一项所述的抗体或抗原结合单元；
    p为1-10。
  34. 如权利要求16-33任一项所述的抗体药物偶联物，其特征在于，p为2-8；或p为4-8；或p为6-8；或p为7-8。
  35. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1-13任一项所述的抗体或抗原结合单元或权利要求15-34任一项所述的抗体药物偶联物，以及药学上可接受的载体、赋形剂和/或辅料。
  36. 权利要求1-13任一项所述的抗体或抗原结合单元、权利要求15-34任一项所述的抗体药物偶联物或权利要求35所述的药物组合物在治疗和/或预防疾病或在制备治疗和/或预防疾病的药物中的应用；或者，所述疾病为CLDN18.2表达相关的疾病；或者，所述疾病为CLDN18.2过表达相关的疾病；或者，所述疾病为癌症或肿瘤；或者，所述癌症或肿瘤为表达CLDN18.2的癌症或肿瘤；或者，所述癌症或肿瘤选自膀胱癌、卵巢癌、肺癌、腺癌、胃癌、乳腺癌、肝癌、胰腺癌、皮肤癌、恶性黑色素瘤、头颈癌、肉瘤、胆管癌、肾癌、结肠癌、小肠癌、睾丸胚胎性癌、胎盘绒毛膜癌、宫颈癌、睾丸癌、子宫癌、食道癌和胆囊癌细胞。
PCT/CN2023/097470 2022-06-01 2023-05-31 抗cldn18.2抗体及其抗体药物偶联物和用途 WO2023232080A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
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CN107667118A (zh) * 2015-04-15 2018-02-06 加尼梅德药物有限公司 包含针对密封蛋白18.2之抗体的药物缀合物
CN111110862A (zh) * 2018-11-01 2020-05-08 上海健信生物医药科技有限公司 抗cldn18．2抗体的药物偶联体及其制备方法和用途
CN114269389A (zh) * 2019-11-05 2022-04-01 礼新医药科技(上海)有限公司 靶向紧密连接蛋白18.2的抗体药物偶联物
CN115429893A (zh) * 2021-06-02 2022-12-06 百奥泰生物制药股份有限公司 药物偶联物及其用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107667118A (zh) * 2015-04-15 2018-02-06 加尼梅德药物有限公司 包含针对密封蛋白18.2之抗体的药物缀合物
CN111110862A (zh) * 2018-11-01 2020-05-08 上海健信生物医药科技有限公司 抗cldn18．2抗体的药物偶联体及其制备方法和用途
CN114269389A (zh) * 2019-11-05 2022-04-01 礼新医药科技(上海)有限公司 靶向紧密连接蛋白18.2的抗体药物偶联物
CN115429893A (zh) * 2021-06-02 2022-12-06 百奥泰生物制药股份有限公司 药物偶联物及其用途

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