WO2021115426A1 - 抗密蛋白抗体药物偶联物及其医药用途 - Google Patents

抗密蛋白抗体药物偶联物及其医药用途 Download PDF

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WO2021115426A1
WO2021115426A1 PCT/CN2020/135680 CN2020135680W WO2021115426A1 WO 2021115426 A1 WO2021115426 A1 WO 2021115426A1 CN 2020135680 W CN2020135680 W CN 2020135680W WO 2021115426 A1 WO2021115426 A1 WO 2021115426A1
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seq
variable region
antibody
cancer
heavy chain
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PCT/CN2020/135680
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English (en)
French (fr)
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杨阳
许建烟
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CA3162754A priority Critical patent/CA3162754A1/en
Priority to MX2022006893A priority patent/MX2022006893A/es
Priority to JP2022535591A priority patent/JP2023505708A/ja
Priority to BR112022011032A priority patent/BR112022011032A2/pt
Priority to KR1020227022162A priority patent/KR20220113728A/ko
Priority to US17/782,980 priority patent/US20230054458A1/en
Priority to CN202080077964.XA priority patent/CN114650845B/zh
Priority to AU2020402006A priority patent/AU2020402006A1/en
Priority to EP20898395.7A priority patent/EP4074345A4/en
Publication of WO2021115426A1 publication Critical patent/WO2021115426A1/zh

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    • A—HUMAN NECESSITIES
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    • 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
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    • 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
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    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
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    • 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
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    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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    • 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
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    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • 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
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to anti-claudin antibody drug conjugates, especially anti-Claudin 18.2 antibody-exiticam analog conjugates, preparation methods thereof, pharmaceutical compositions containing antibody drug conjugates, and their use in preparation Use in drugs for treating diseases or disorders mediated by Claudin 18.2; especially in the preparation of anticancer drugs.
  • Claudin-18 (CLDN18) is a protein encoded by the Claudin18 gene in humans. It belongs to the family of cell tight junction proteins and can control the molecular flow between layers of cells. Claudin protein structure includes four transmembrane regions, two extracellular loops, and its N-terminal and C-terminal are in the cytoplasm.
  • Claudin-18 has two splice variants, Claudin 18.1 and Claudin 18.2, and there are only eight amino acid differences between the two sequences in the first extracellular loop.
  • the expression distribution of Claudin 18.1 and Claudin 18.2 is different.
  • Claudin 18.1 is selectively expressed in normal lung cells, and Claudin 18.2 is highly restricted in normal cells, but in a variety of tumors (gastric cancer, lung cancer, pancreatic cancer, etc.) Frequent ectopic activation and overexpression.
  • Claudin 18.2 is considered as a potential therapeutic target for gastric cancer and other cancer types. The discovery of this target also provides a new option for the treatment of gastric cancer.
  • Antibody-drug conjugate connects monoclonal antibodies or antibody fragments with biologically active cytotoxins through stable chemical linkers, making full use of the specificity of antibody binding to normal cells and tumor cell surface antigens.
  • ADC Antibody-drug conjugate
  • the present disclosure relates to the ADC of an anti-Claudin 18.2 antibody and its use, wherein an ADC drug coupled with an anti-Claudin 18.2 antibody or an antigen-binding fragment and a cytotoxic substance exenotecan analog is provided.
  • the purpose of this disclosure is to provide a ligand-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof:
  • Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2- , -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group group, cycloalkyl group and heterocyclic group; form a cycloalkyl or heterocyclic group, or, R a and R b are carbon atoms connected thereto;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic group, aryl and heteroaryl;
  • R 2 is selected from hydrogen atom, halogen, Haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; alternatively, R 1 and R 2 form together with the carbon atom to which they are attached Cycloalkyl or heterocyclic group;
  • R a and R 2 together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;
  • n is an integer from 0 to 4.
  • n is from 1 to 10, n is a decimal or integer;
  • Pc is an anti-Claudin 18.2 antibody or an antigen-binding fragment thereof.
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 3, and the light chain variable region includes the same sequence as SEQ ID NO:
  • the light chain variable region shown in the sequence 4 has LCDR1, LCDR2 and LCDR3 with the same sequence; or
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region includes the same sequence as SEQ ID NO:
  • the light chain variable region shown in the 6 sequence has LCDR1, LCDR2 and LCDR3 of the same sequence.
  • the heavy chain variable region includes the sequences of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and the light chain variable region includes sequences as LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; or
  • the heavy chain variable region includes the sequences of HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and the light chain variable region includes sequences as The LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 3 or is at least 90% identical to it, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 4 or is identical to it. At least 90% identity;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 24 or is at least 90% identical to it, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 21 or is identical to it. At least 90% identity;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 5 or is at least 90% identical to it, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 6 or is identical to it. At least 90% identical; or
  • variable region of the heavy chain is shown in SEQ ID NO: 31 or is at least 90% identical to it
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 28 or is identical to it. At least 90% identity.
  • a humanized antibody comprising a framework region derived from a human antibody or a framework region variant thereof, the framework region variant being on the light chain framework region and/or the heavy chain framework region of the human antibody, respectively Have at most 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid back mutations;
  • the framework region variant comprises a mutation selected from the following (a) or (b):
  • the light chain variable region contains one or more amino acid back mutations optionally selected from 22S, 85I and 87H, and/or the heavy chain variable region contains optionally selected from 48I, 82T and One or more amino acids in 69M are backmutated; or
  • the light chain variable region contains one or more amino acid back mutations optionally from 4L or 22S, and/or the heavy chain variable region contains optionally from 38K, 40R, 48I, 66K, One or more amino acids in 67A, 69L, 71L and 73K are backmutated;
  • the framework region variant comprises a mutation selected from the following:
  • the light chain variable region contains an amino acid back mutation selected from 4L.
  • the heavy chain variable region sequence is as shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27 and the light chain variable region sequence is as shown in SEQ ID NO : 21, SEQ ID NO: 22 or SEQ ID NO: 23;
  • the heavy chain variable region sequence is as shown in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 and the light chain variable region sequence is as SEQ ID NO : 28, SEQ ID NO: 29 or SEQ ID NO: 30;
  • the anti-Claudin 18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as shown below:
  • Sequences such as SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45.
  • the heavy chain and sequence are shown as SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID
  • h1901-11 comprises the heavy chain whose amino acid sequence is shown in SEQ ID NO: 44, and the light chain whose sequence is shown in SEQ ID NO: 41; or
  • h1902-5 Contains the heavy chain whose amino acid sequence is shown in SEQ ID NO: 49, and the light chain whose sequence is shown in SEQ ID NO: 47.
  • the antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), and disulfide bond stabilized The modified V area (dsFv).
  • n can be between 1-10 Integer or decimal between, n can be the mean value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • n is a decimal or integer from 2 to 8, preferably a decimal or integer from 3 to 8, more preferably a decimal or integer from 5 to 9, or preferably a decimal or integer from 2 to 7.
  • n is a decimal or integer from 3.5 to 4.5.
  • Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, or an alkyl group;
  • R 1 is haloalkyl or C 3-6 cycloalkyl
  • R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 cycloalkyl group;
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • m 0 or 1.
  • the O end of Y is connected to the joint unit L.
  • a ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -.
  • L 1 is selected from -(succinimidyl-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- or -C (O)-WC(O)-, where W is selected from C 1-8 alkyl, C 1-8 alkyl-C 3-6 cycloalkyl or linear heteroalkyl of 1 to 8 chain atoms, so
  • the heteroalkyl group contains 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 alkyl group, C 1-8 alkyl group-C 3-6 cycloalkyl group or 1 to 8
  • the linear heteroalkyl groups of chain atoms are each independently optionally further selected from one or more of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl. Substituents are substituted.
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O) -, -S(CH 2 )p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20.
  • L 3 is a peptide residue composed of 2 to 7 amino acids, wherein the amino acid is selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, and glutathione.
  • L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH 2 ) t -or a chemical bond, where t is An integer from 1 to 6.
  • R 3 , R 4, and R 5 are the same or different, and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.
  • L 1 is selected from -(succinimidyl-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- or -C(O)-WC( O)-, wherein W is selected from a C 1-8 alkyl group, a C 1-8 alkyl-cycloalkyl group or a linear heteroalkyl group of 1 to 8 atoms, and the heteroalkyl group contains 1 to 3 selected from N, O or S heteroatoms, wherein the C 1-8 alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxy, cyano, amino, alkyl, Substituted by one or more substituents of chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20;
  • L 3 is a peptide residue composed of 2 to 7 amino acids, wherein the amino acid is selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid
  • the amino acid residue formed by the amino acid in the acid is optionally further selected from one of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl. Replaced by multiple substituents;
  • L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH 2 ) t -or a chemical bond, wherein t is an integer from 1 to 6;
  • R 3 , R 4 and R 5 are the same or different, and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group.
  • L 1 is s 1 is an integer from 2 to 8;
  • L 2 is a chemical bond
  • L 3 is a tetrapeptide residue; preferably, L 3 is a tetrapeptide residue of GGFG (SEQ ID No. 55);
  • L 4 is -NR 5 (CR 6 R 7 )t-, R 5 , R 6 or R 7 are the same or different, and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2;
  • the L 1 terminal is connected to Pc, and the L 4 terminal is connected to Y.
  • the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any one of the preceding items has the general formula (Pc-L a- YD) Ligand-drug conjugate or pharmaceutically acceptable salt thereof:
  • W, L 2 , L 3 , R 5 , R 6 , and R 7 are as defined in the aforementioned joint unit -L-;
  • Pc, n, R 1 , R 2 , and m are as defined in the general formula (Pc-LYD).
  • the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any one of the preceding items has the general formula (Pc-L b -YD) Ligand-drug conjugate or pharmaceutically acceptable salt thereof:
  • s 1 is an integer from 2 to 8;
  • Pc, R 1 , R 2 , R 5 to R 7 , m and n are as defined in the general formula (Pc-L a -YD).
  • Pc and n are as defined in the general formula (Pc-L-Y-D).
  • a ligand-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof is provided, and the ligand-drug conjugate is selected from:
  • n is as defined in the general formula (Pc-L-Y-D); antibody h1902-5 and h1901-11 are as defined above.
  • the present disclosure further provides a method for preparing a ligand-drug conjugate represented by the general formula (Pc-L a -YD) or a pharmaceutically acceptable salt thereof, which includes the following steps:
  • Pc is the anti-Claudin 18.2 antibody or antigen-binding fragment thereof as described above; Pc' is obtained after Pc is reduced;
  • W, L 2 , L 3 , R 1 , R 2 , R 5 to R 7 , m and n are as defined in the general formula (Pc-L a -YD).
  • the present disclosure further provides a method for preparing the antibody drug conjugate represented by the general formula (Pc-L'-D), which includes the following steps:
  • Pc is the aforementioned anti-Claudin 18.2 antibody or antigen-binding fragment thereof;
  • n is as defined in the general formula (Pc-L-Y-D).
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding items, and one or more pharmaceutically acceptable excipients Agent, diluent or carrier.
  • the present disclosure provides the use of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same as any one of the foregoing as a medicine.
  • the drug is used to treat Claudin 18.2 mediated diseases or conditions; wherein the Claudin 18.2 mediated diseases or conditions are preferably Claudin 18.2 high expression cancers.
  • the drug is used to treat cancer.
  • the cancer is preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer , Neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, Colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma , Myeloma, multiple myeloma, myelodysplastic syndrome, Keukenb's tumor, myeloproliferative tumor, s
  • the present disclosure provides that the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same as described in any one of the preceding items is prepared for the treatment of Claudin 18.2 mediated diseases or disorders.
  • the disease or disorder mediated by Claudin 18.2 is Claudin 18.2 high expression cancer.
  • the disease is preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer , Neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, Colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma , Myeloma, multiple myeloma, myelodysplastic syndrome, Keukenb's tumor, myeloproliferative tumor, s
  • the present disclosure provides the use of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same as described in any one of the preceding items in the preparation of a medicament for the treatment or prevention of tumors;
  • the tumor and cancer are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor , Throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, Kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma,
  • the present disclosure further relates to a method for treating and/or preventing tumors, the method comprising administering to a subject in need thereof a therapeutically effective dose or a prophylactically effective dose of the ligand as described in any one of the preceding-
  • the drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the drug conjugate preferably, the tumor is a cancer related to the high expression of Claudin 18.2.
  • the present disclosure further relates to a method for treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective dose or a preventive effective dose of the ligand-drug couple as described in any one of the preceding items.
  • the active compound for example, the ligand-drug conjugate according to the present disclosure, or a pharmaceutically acceptable salt thereof
  • the active compound can be prepared into a form suitable for administration by any appropriate route, and the active compound is preferably in a unit dose Or it is a way that subjects can self-administer in a single dose.
  • the unit dosage form of the present disclosure can be tablet, capsule, cachet, bottled syrup, powder, granule, lozenge, suppository, regenerated powder or liquid preparation.
  • the dosage of the active compound or composition used in the treatment method of the present disclosure will generally vary with the severity of the disease, the weight of the subject, and the efficacy of the active compound.
  • a suitable unit dose may be 0.1 mg to 1000 mg.
  • the pharmaceutical composition of the present disclosure may contain one or more excipients selected from the following ingredients: fillers, diluents, binders, wetting agents, disintegrants or excipients, etc. .
  • the composition may contain 0.1 to 99% by weight of the active compound.
  • the Claudin 18.2 antibody and antibody-drug conjugate provided in this disclosure have good affinity with cell surface antigens, good endocytosis efficiency and strong tumor suppression efficiency, and have a wider window of drug application, which is suitable for clinical use. Drug application.
  • Figure 1 FACS test results of humanized antibody binding to human Claudin 18.2 at the cellular level.
  • FIG. 1 NUGC4 endocytosis experiment of humanized antibody.
  • Figure 3A to Figure 3C ADCC effect detection of antibodies in NUGC4 cells with different expression levels of Claudin 18.2.
  • Figure 3A shows the ADCC effect detection of antibody in wild-type NUGC4 cells (Claudin18.2 low expression);
  • Figure 3B shows the ADCC effect detection of antibody in Claudin18.2 medium expression NUGC4 cells;
  • Figure 3C shows the high expression of antibody in Claudin 18.2 Detection of ADCC effect in NUGC4 cells.
  • Figure 4 The results of the tumor suppression experiment of ADC-1 of the present disclosure.
  • Figure 5 The results of the tumor suppression experiment of ADC-2 of the present disclosure.
  • trade name When a trade name is used in this disclosure, it is intended to include the formulation of the trade name product, the generic drug and the active drug portion of the trade name product.
  • drug refers to a chemical substance that can change or find out the physiological function and pathological state of the body, and can be used to prevent, diagnose and treat diseases.
  • Drugs include cytotoxic drugs. There is no strict boundary between drugs and poisons. Toxicants refer to chemical substances that have a toxic effect on the body and harm human health in a small dose. Any excessive dose of drugs can produce toxic reactions. Cytotoxic drugs are substances that inhibit or prevent the function of cells and/or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at a sufficient concentration, but due to lack of specificity, while killing tumor cells, it will also cause normal cell apoptosis, leading to serious side effects.
  • Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants or animals, radioisotopes (such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu radioisotopes), toxin drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes.
  • toxins such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants or animals, radioisotopes (such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu radioisotopes)
  • toxin drugs such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi
  • linker unit refers to a chemical structural fragment or bond that is connected to a ligand (such as an antibody or an antigen-binding fragment thereof) at one end and a drug at the other end. It can also be connected to other linkers before connecting to Drugs are connected.
  • ligand such as an antibody or an antigen-binding fragment thereof
  • the joint may include one or more joint members.
  • exemplary linker building blocks include 6-maleimidohexanoyl ("MC"), maleimidopropionyl ("MP”), valine-citrulline (“val-cit” or “vc "), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl 4-(2-pyridylthio)pentanoate ( “SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”, also referred to herein as "MCC”), and N- Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”).
  • MC 6-maleimidohexanoyl
  • MP maleimidopropionyl
  • val-cit valine-citrulline
  • the linker can include extensions, spacers and amino acid units, and can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers such as hydrazone
  • protease-sensitive such as peptidase-sensitive
  • light-labile linkers dimethyl linkers
  • disulfide-containing linkers Chargei et al., Cancer Research 52:127-131
  • Joint components include but are not limited to:
  • MC 6-maleimidohexanoyl
  • Val-Cit or "vc” valine-citrulline (an exemplary dipeptide in a protease cleavable linker),
  • Citrulline 2-amino-5-ureidovaleric acid
  • PAB p-aminobenzyloxycarbonyl (an example of a "self-sacrificing" linker assembly)
  • Me-Val-Cit N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to prevent it from being cleaved by cathepsin B),
  • MC(PEG)6-OH maleimidohexanoyl-polyethylene glycol (can be attached to antibody cysteine),
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SMCC Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • ligand-drug conjugate refers to a ligand that is connected to a drug with biological activity through a linking unit.
  • the "ligand-drug conjugate” is preferably an antibody-drug conjugate (ADC), which refers to connecting a monoclonal antibody or antibody fragment to a toxic drug with biological activity through a connecting unit.
  • ADC antibody-drug conjugate
  • the antibody can be coupled to the drug directly or via a linker.
  • the average number of drug modules per antibody (average drug load or drug loading, which can be represented by the value of n), and its range can be, for example, from about 0 to about 20 drug modules per antibody. In some embodiments, it is per antibody.
  • Each antibody is coupled to 1 to about 10 drug modules, and in certain embodiments, each antibody is coupled to 1 to about 8 drug modules.
  • average drug load refers to the average amount of cytotoxic drug loaded on each ligand in the ligand-drug conjugate molecule, and can also be expressed as the ratio of drug amount to antibody amount.
  • the amount can range from 0-12, preferably 1-10 cytotoxic drugs per ligand (Pc).
  • the drug loading is expressed as n, which can also be referred to as a DAR (Drug-antibody Ratio) value.
  • n can be a non-zero integer from 0 to 12 or a decimal number, preferably an integer between 1-10 Or a decimal; more preferably 2 to 8, which can be an integer or a decimal; most preferably 3 to 8, which can be an integer or a decimal.
  • Exemplary are the mean values of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • Conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC characteristics can be used to identify the average number of drug products per ADC molecule after the coupling reaction.
  • Claudin 18 (CLD18) molecule (Genbank accession number: splice variant 1 (CLD18A1): NP_057453, NM016369, and splice variant 2 (CLD18A2 or Claudin18.2): NM_001002026, NP_001002026) are intrinsic transmembrane proteins located in the epithelium and The tight junctions of the endothelium. In tight junctions, occludin and claudin are the most important transmembrane protein components. Due to the strong intercellular adhesion properties of claudins, they create a primary barrier that prevents and controls the paracellular transport of solutes and restricts the lateral diffusion of membrane lipids and proteins to maintain cell polarity. The proteins that form tight junctions participate in the tissue structure of the epithelium. According to reports, these proteins are almost inaccessible to antibodies in well-structured epithelium, but become exposed in tumor cells.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding The heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain.
  • the same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into ⁇ chain or ⁇ chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the full-length antibody heavy chain and light chain varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are a constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • fully human antibody means “fully human antibody”, “fully human antibody” or “fully human antibody”, also known as “fully human monoclonal antibody”, the variable region and constant region of the antibody are of human origin.
  • monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the related technologies of fully human antibody preparation mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies.
  • the binding fragment contained in the "antigen-binding fragment” is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), disulfide bond stabilized V region (dsFv) and antigen-binding fragments of peptides containing CDRs, examples include (i) Fab fragments, monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, including through hinge A bivalent fragment of two Fab fragments connected by a disulfide bridge in the region; (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VH and VL domains of one arm of an antibody
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, recombination methods can be used to connect them through a synthetic linker so that it can be produced as a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chain referred to as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • single chain antibodies are also intended to be included in the term "antigen-binding fragments" of antibodies.
  • the antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (for example, cleaving the amino acid residue at position 224 of the H chain), wherein the H chain The part on the N-terminal side and the L chain are joined together by a disulfide bond.
  • protease papain for example, cleaving the amino acid residue at position 224 of the H chain
  • F(ab')2 is obtained by digesting the lower part of the disulfide bond in the hinge region of IgG with the enzyme pepsin. It has a molecular weight of about 100,000 and has antigen-binding activity, and contains two connected at the hinge position. Antibody fragments of the Fab region.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of F(ab')2 described above.
  • the Fab' can be produced by inserting the DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express Fab'.
  • single chain antibody means a molecule comprising an antibody heavy chain variable domain (or VH) and an antibody light chain variable domain (or VL) connected by a linker.
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of a repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448) .
  • linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions in the variable domain of an antibody that mainly contribute to antigen binding.
  • CDR CDR
  • HCDR1, HCDR2, HCDR3 CDR1, LCDR2, LCDR3
  • LCDR1, LCDR2, LCDR3 three CDRs
  • Any of a variety of well-known schemes can be used to determine the amino acid sequence boundaries of the CDRs.
  • One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • CDR Kabat definition of CDR only applies to CDR1, CDR2, and CDR3 of the light chain variable domain, and CDR2 and CDR3 of the heavy chain variable domain. It also includes "Chothia” numbering rules, "ABM” numbering rules, "contact” numbering rules (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001) and ImmunoGenTics (IMGT) numbering rules (Lefranc MP) , Dev. Comp. Immunol., 27, 55-77 (2003) and so on.
  • antibody framework refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitope or "antigenic determinant” refers to a site on an antigen that is bound by an immunoglobulin or antibody. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular B iology, Volume 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of about less than 10 -7 M, for example, about less than 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • KD refers to the dissociation equilibrium constant of the antibody-antigen interaction.
  • the antibodies (or antigen-binding fragments) of the present disclosure bind to Claudin 18.2 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M or 10 -9 M, (or its epitope )
  • KD dissociation equilibrium constant
  • the affinity of the antibody to the cell surface antigen is determined by the FACS method to determine the KD value.
  • nucleic acid molecule refers to a DNA molecule or an RNA molecule.
  • the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • Amino acid sequence identity refers to the process of aligning amino acid sequences, introducing gaps when necessary to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of sequence identity.
  • the alignment can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 Megalign
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after being introduced into the host cell, so as to follow The host genome replicates together (e.g., a non-episomal mammalian vector).
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • the human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, Lefranc, Obtained on G., The Immunoglobulin FactsBook, Academic Press, 2001ISBN012441351.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into host cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the N-terminal site of the Fc region.
  • Positive clones are expanded in the culture medium of the bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF column for purification. Wash away non-specifically bound components.
  • the bound antibody was eluted by the pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • peptide refers to a fragment of a compound between amino acids and a protein. It is composed of two or more amino acid molecules connected to each other through peptide bonds, and is a structural and functional fragment of a protein.
  • sucrose refers to a biological macromolecule composed of three elements of C, H, and O, which can be divided into monosaccharides, disaccharides and polysaccharides.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably containing 1 to 10 carbons The most preferred is an alkyl group containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5, or 6 carbon atoms).
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-Dimethylpropyl, 2,2-Dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhe
  • lower alkyl groups containing 1 to 6 carbon atoms More preferred are lower alkyl groups containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl.
  • Alkyl groups may be substituted or unsubstituted.
  • substituents When substituted, substituents may be substituted at any available attachment point.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkanes Group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein the alkyl group is as defined above.
  • alkylene refers to a saturated linear or branched aliphatic hydrocarbon group, which has two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which is A straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, more preferably containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 Or 6 carbon atoms) alkylene.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 -) CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -) Wait.
  • the alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably independently optionally selected from alkyl, alkenyl, alkynyl , Alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy , Cycloalkylthio, heterocycloalkylthio and oxo groups are substituted by one or more substituents.
  • alkoxy refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where the definition of alkyl or cycloalkyl is as described above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio And heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent.
  • the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 Carbon atoms, most preferably 3 to 8 carbon atoms (including 3, 4, 5, 6, 7, or 8 carbon atoms).
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups and cyclooctyl groups, etc.; polycyclic cycloalkyls include spiro, fused, and bridged cycloalkyls.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer of 0, 1 or 2) heteroatoms, but does not include the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms (1, 2, 3 or 4 heteroatoms); more preferably the cycloalkyl ring contains 3 to 10 ring atoms (including 3 , 4, 5, 6, 7, 8, 9, or 10 ring atoms).
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • spiroheterocyclic group refers to a polycyclic heterocyclic group that shares one atom (called a spiro atom) between 5- to 20-membered monocyclic rings, in which one or more ring atoms are selected from nitrogen, oxygen or S(O ) Heteroatoms of m (where m is an integer of 0 to 2), and the remaining ring atoms are carbon. It can contain one or more double bonds, but none of the rings have a fully conjugated ⁇ -electron system. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • the spiro heterocyclic group is classified into a single spiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a single spiro heterocyclic group and a dispiro heterocyclic group. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospiro heterocyclic group.
  • Non-limiting examples of spiroheterocyclic groups include:
  • fused heterocyclic group refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system.
  • One or more rings may contain one or more Double bonds, but none of the rings have a fully conjugated ⁇ -electron system, where one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer of 0, 1, or 2), The remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members (7-membered, 8-membered, 9-membered, or 10-membered ring).
  • bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group.
  • fused heterocyclic groups include:
  • bridged heterocyclic group refers to a 5- to 14-membered polycyclic heterocyclic group with any two rings sharing two atoms that are not directly connected. It may contain one or more double bonds, but none of the rings has a complete common A conjugated ⁇ -electron system in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is an integer of 0, 1 or 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members (7-membered, 8-membered, 9-membered, or 10-membered ring).
  • bridged heterocyclic groups include:
  • the heterocyclic ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, non-limiting examples thereof include:
  • the heterocyclic group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio and oxo.
  • aryl refers to a 6 to 14-membered all-carbon monocyclic or fused polycyclic (that is, rings that share adjacent pairs of carbon atoms) groups with a conjugated ⁇ -electron system, preferably 6 to 10-membered (6-membered) , 7-membered, 8-membered, 9-membered or 10-membered), such as phenyl and naphthyl, preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples thereof include:
  • the aryl group may be substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycle Alkylthio.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms (1, 2, 3 or 4 heteroatoms) and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur And nitrogen.
  • Heteroaryl groups are preferably 5- to 10-membered (5-membered, 6-membered, 7-membered, 8-membered, 9-membered or 10-membered heteroaryl), more preferably 5-membered or 6-membered, such as furyl, thienyl, pyridyl , Pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl and tetrazolyl, etc.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • Heteroaryl groups may be optionally substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio And heterocycloalkylthio.
  • amino protecting group is to keep the amino group unchanged when other parts of the molecule react, and to protect the amino group with a group that is easy to remove.
  • Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1-3 substituents (1, 2, or 3 substituents) selected from halogen, alkoxy, or nitro.
  • the amino protecting group is preferably 9-fluorenylmethyloxycarbonyl.
  • haloalkyl refers to the replacement of hydrogen on an alkyl group with one or more halogens, where the alkyl group is as defined above.
  • deuterated alkyl refers to the replacement of hydrogen on an alkyl group with one or more deuterium atoms, where the alkyl group is as defined above.
  • hydroxyalkyl means that the hydrogen on an alkyl group is replaced by one or more hydroxy groups, where the alkyl group is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH 2 .
  • nitro refers to -NO 2 .
  • cyano refers to -CN.
  • acylamino refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may but does not have to be present, and the description includes the case where the heterocyclic group is substituted by an alkyl group and the case where the heterocyclic group is not substituted by an alkyl group.
  • Substituted refers to one or more hydrogen atoms in the group, preferably at most 5, more preferably 1, 2, or 3 hydrogen atoms are independently substituted with substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without too much effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • pharmaceutically acceptable salt refers to the salt of the ligand-drug conjugate of the present disclosure, or the salt of the active compound described in the present disclosure, and such salts are used in a subject It is safe and effective, and has due biological activity.
  • the ligand-drug conjugate of the present disclosure contains at least one amino group, so it can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloric acid Salt, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, phosphoric acid Dihydrogen salt, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate , P-toluenesulfonate.
  • the cytotoxic drug is coupled to the sulfhydryl group of the antibody through a linking unit.
  • ligand cytotoxic drug conjugates including:
  • the term "pharmaceutically acceptable carrier” used for the drugs of the present disclosure refers to a system that can change the way the drug enters the subject and the distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ.
  • the drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to contain drug molecules and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • excipient is an addendum other than the active compound in the pharmaceutical composition, and can also be referred to as an excipient.
  • excipients Such as binders, fillers, disintegrants, lubricants in tablets; base parts in semi-solid preparations ointments and creams; preservatives, antioxidants, correctives, fragrances, etc. in liquid preparations
  • Cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
  • diluent is also called a filler, and its main purpose is to increase the weight and volume of the tablet.
  • the addition of diluent not only guarantees a certain volume, but also reduces the deviation of the dosage of the main components, and improves the compression molding of the drug.
  • an absorbent should be added to absorb the oily substance to keep it in a "dry” state to facilitate the manufacture of tablets.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • a sterile injectable aqueous solution Among the acceptable solvents and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile oil-in-water injection microemulsion in which the active ingredient is dissolved in an oil phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is added to a mixture of water and glycerin to form a microemulsion.
  • the injection or microemulsion can be injected into the bloodstream of the subject by local large-volume injection.
  • a continuous intravenous delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM. 5400 intravenous pump.
  • the pharmaceutical composition may be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration.
  • the suspension can be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents mentioned above.
  • the sterile injection preparation may also be a sterile injection solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, for example, a solution prepared in 1,3-butanediol.
  • sterile fixed oil can be conveniently used as a solvent or suspending medium. For this purpose, any blended fixed oil including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be used to prepare injections.
  • the preparation method of the compound represented by the general formula (Pc-L a -YD) includes the following steps:
  • the reducing agent is preferably TCEP, and in particular, the disulfide bond on the antibody is preferably reduced;
  • Pc, W, L 2 , L 3 , R 1 , R 2 , R 5 to R 7 , m and n are as defined in the general formula (Pc-L a -YD).
  • Example 1-1 Construction of claudin18.2 highly expressing cell line
  • Cells with the same expression level of Claudin 18.2 with a tumor IHC score of 3 are high-expressing cells; cells with the same level of expression of Claudin 18.2 with a tumor IHC score of 2 are medium-expressing cells.
  • the FACS detection of Claudin18.2 expression on the surface of NUGC4 cells infected with lentivirus the NUGC4/hClaudin18.2 monoclonal cell line with high Claudin18.2 expression was selected.
  • the expression of Claudin 18.2 on the surface of wild-type NUGC4 cells was detected by FACS, and the NUGC4 clone cell line with medium expression of Claudin 18.2 was selected, and wild-type NUGC4 was a cell with low expression of Claudin 18.2.
  • the selected monoclonal cell lines will be expanded and cultured, and frozen and stored for subsequent experiments.
  • Example 1-2 Production of anti-human claudin18.2 monoclonal antibody
  • the anti-human Claudin 18.2 monoclonal antibody was produced by immunizing mice.
  • Experimental SJL white mice female, 6-8 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Feeding environment SPF level. After the mice are purchased, they are kept in a laboratory environment for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 °C; humidity 40-60%. Mice that have adapted to the environment were immunized according to the following protocol.
  • the immune antigen is huClaudin18.2-HEK293 cells (HEK-293 stably transfected cell line transfected with human Claudin18.2 plasmid).
  • Immunization program before immunizing cells for the first time, use Gold Adjuvant (Sigma Cat No. T2684) 0.1mL/mouse was injected intraperitoneally (IP), half an hour later, each mouse was injected intraperitoneally (IP) with 0.1mL normal saline diluted to 1 ⁇ 10 8 /mL cells liquid. After the cells are blown evenly, they are inoculated, and the time is 0, 14, 28, 42, and 56 days. Blood was collected on the 21st, 35th, 49th, and 63rd day, and the antibody titer in the mouse serum was determined by ELISA.
  • Gold Adjuvant Sigma Cat No. T2684
  • mice with high antibody titer in the serum and the titer tending to the plateau were selected for spleen cell fusion.
  • the immunization was boosted, and 1 ⁇ 10 7 cells were injected intraperitoneally (IP).
  • the optimized PEG-mediated fusion procedure was used to combine splenic lymphocytes with myeloma cells Sp2/0 cells ( CRL-8287 TM ) was fused to obtain hybridoma cells.
  • the hybridoma cells obtained by the fusion were resuspended in complete medium (IMDM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 0.5-1 ⁇ 10 6 /mL, and 100 ⁇ L/well was seeded in a 96-well plate After incubating at 37°C and 5% CO 2 for 3-4 days, supplement the HAT complete medium with 100 ⁇ L/well, and continue to culture for 3-4 days until a needle-like clone is formed. Remove the supernatant, add 200 ⁇ L/well of HT complete medium (IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), culture at 37°C, 5% CO 2 for 3 days, and perform ELISA detection.
  • complete medium IMDM medium containing 20%
  • the hybridoma culture supernatant was detected by the combined ELISA method.
  • Select the cells that have strong binding ability to huClaudin18.2-HEK293 cells and at the same time do not bind to HEK293 cells for expansion and cryopreservation in time, and perform subcloning two to three times until a single cell clone is obtained.
  • the hybridoma clones were screened through the above experiments, and the antibodies were further prepared by the serum-free cell culture method, and the antibodies were purified according to the purification examples for use in the test examples.
  • Example 1-3 Humanization of murine antibody
  • the monoclonal hybridoma cell lines mAb1901 and mAb1902 with high in vitro activity were selected, the monoclonal antibody sequence was cloned, and then humanization, recombinant expression and activity evaluation were carried out.
  • the procedure for cloning sequences from hybridomas is as follows.
  • the logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) (according to the instructions of the kit), and reverse transcription (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sent to a sequencing company for sequencing.
  • the amino acid sequence corresponding to the obtained hybridoma DNA sequence is shown in SEQ ID NO: 3-6:
  • mAb1901 murine light chain variable region (SEQ ID NO: 4)
  • mAb1902 murine light chain variable region (SEQ ID NO: 6)
  • the above murine heavy chain variable region and light chain variable region are respectively connected with the heavy chain constant region and human kappa light chain constant region of the following human IgG1 antibody to form chimeric antibodies ch1901 and ch1902.
  • the constant region is selected from the following sequences:
  • the heavy chain constant region of human IgG1 antibody (SEQ ID NO: 7)
  • Human ⁇ light chain constant region (SEQ ID NO: 8)
  • humanization of murine monoclonal antibodies is carried out according to the methods published in many documents in this field.
  • human constant domains are used instead of parental (murine antibody) constant domains
  • human germline antibody sequences are selected based on the homology of the murine antibody and the human antibody, and CDR grafting is performed.
  • candidate molecules with good activity are selected for humanization, and the results are as follows.
  • amino acid residues of the VH/VL CDR in Table 1 are determined and annotated by the Kabat numbering system.
  • the CDR sequence of the murine antibody is as described in Table 1:
  • Antibody mAb1901 HCDR1 DYGIH(SEQ ID NO: 9) HCDR2 YISRGSSTIYYADTVKG (SEQ ID NO: 10) HCDR3 GGYDTRNAMDY(SEQ ID NO:11) LCDR1 KSSQSLLNSGNQKNYLA (SEQ ID NO: 12) LCDR2 GASTRAS(SEQ ID NO: 13)
  • the heavy and light chain variable region sequences were compared with the antibody Germline database to obtain a human germline template with high homology.
  • the human germline light chain framework region is derived from the human kappa light chain gene.
  • the corresponding heavy chain variable region is connected to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody
  • the light chain variable region is connected to the human ⁇ light chain shown in SEQ ID NO: 8
  • the chain constant regions are joined to form the light chain of the full-length antibody.
  • the variable region of the heavy chain and the variable region of the light chain can also be linked to other heavy chain constant regions and light chain constant regions, respectively, to form a full-length antibody.
  • the corresponding heavy chain variable region is connected with the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody
  • the light chain variable region is connected with the human ⁇ light chain shown in SEQ ID NO: 8
  • the chain constant regions are joined to form the light chain of the full-length antibody.
  • ch1901 heavy chain (SEQ ID NO: 35)
  • ch1902 heavy chain (SEQ ID NO: 37)
  • Table 6 shows the humanized antibodies of mAb1901:
  • the heavy chain of the humanized antibody h1901-1 in the table is H1, the light chain is L1, and so on.
  • the full-length antibody light and heavy chain sequences of the humanized antibody of mAb1901 are shown in Table 7 below:
  • Table 8 shows the humanized antibodies of mAb1902:
  • the heavy chain of the humanized antibody h1902-1 in the table is H11, the light chain is L11, and so on.
  • the positive control antibody of this disclosure is IMAB-362 (from WO2016166122)
  • IMAB-362 heavy chain (SEQ ID NO: 53):
  • IMAB-362 light chain (SEQ ID NO: 54):
  • the above-mentioned antibodies were cloned, expressed and purified by conventional gene cloning and recombinant expression methods.
  • the experimental methods that do not indicate specific conditions in the embodiments of this disclosure usually follow conventional conditions or conditions suggested by raw material or commodity manufacturers.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR is measured with Bruker AVANCE-400 nuclear magnetic instrument, the solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), the internal standard is tetramethyl Based on silane (TMS), the chemical shift is given in units of 10 -6 (ppm).
  • DMSO-d6 deuterated dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS tetramethyl Based on silane
  • HPLC determination uses Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150 ⁇ 4.6mm chromatographic column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 ⁇ 4.6mm chromatographic column).
  • the UV-HPLC measurement uses a Thermonanodrop2000 ultraviolet spectrophotometer.
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used in thin layer chromatography (TLC) is 0.15mm to 0.2mm, and the size of the thin layer chromatography separation and purification product is 0.4mm. To 0.5mm silica gel plate.
  • the known starting materials of the present disclosure can be synthesized using or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Darui Chemicals and other companies.
  • the reactions are all carried out under an argon atmosphere or a nitrogen atmosphere.
  • the argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1L.
  • the hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1L.
  • the pressure hydrogenation reaction uses Parr 3916EKX hydrogenator and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenator.
  • the hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.
  • the microwave reaction uses the CEM Discover-S 908860 microwave reactor.
  • the solution in the reaction refers to an aqueous solution.
  • reaction temperature is room temperature.
  • Room temperature is the most suitable reaction temperature, and the temperature range is 20°C to 30°C.
  • the preparation of the PBS buffer with pH 6.5 in the examples: take KH 2 PO 4 8.5g, K 2 HPO 4 .3H 2 O 8.56g, NaCl 5.85g, and EDTA 1.5g in a bottle, dilute the volume to 2L, and ultrasonic Dissolve all of it, shake well and get it.
  • the eluent system of column chromatography and the developing solvent system of thin-layer chromatography used to purify compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, C: petroleum ether and In the ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or alkaline reagents can also be added for adjustment.
  • Q-TOF LC/MS uses Agilent 6530 accurate mass quadrupole-time-of-flight mass spectrometer and Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 ⁇ m, 2.1 ⁇ 75mm column).
  • the reaction solution was concentrated under reduced pressure, and the obtained crude compound 2 was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc), B-acetonitrile , Gradient elution, flow rate: 18 mL/min), collect the corresponding components, and concentrate under reduced pressure to obtain the title product (2-A: 1.5 mg, 2-B: 1.5 mg).
  • reaction was quenched by adding 5 mL of water to the reaction solution, the reaction solution was extracted with ethyl acetate (10 mL ⁇ 3), the organic phases were combined, washed with saturated sodium chloride solution (5 mL ⁇ 2), and the organic phase was dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with the developing solvent system B to obtain the title product 4 (2.5 mg, yield: 80.9%).
  • the benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104mg, 0.54mmol; prepared by the method disclosed in the patent application "US2005/20645”) and 2-((((9H-fluoren-9-yl) Methoxy) carbonyl) amino) acetamido) methyl acetate 8b (100 mg, 0.27 mmol; prepared by the method disclosed in the patent application "CN105829346A”) was added to the reaction flask, 5 mL of tetrahydrofuran was added, argon replaced three times, ice The temperature of the water bath was lowered to 0-5°C, potassium tert-butoxide (61mg, 0.54mmol) was added, the ice bath was removed, and the temperature was raised to room temperature and stirred for 10 minutes.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/min ), the corresponding components were collected, and concentrated under reduced pressure to obtain the title product 8 (2 mg, yield: 39.0%).
  • the obtained residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, extracted with ethyl acetate (10 mL ⁇ 3), the organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with the developing solvent system C to obtain the title product 9b (48 mg, yield: 19%).
  • Dissolve 9d (19 mg, 22.6 ⁇ mol) in 2 mL of dichloromethane, add 1 mL of diethylamine, and stir at room temperature for 2 hours.
  • the reaction solution was concentrated under reduced pressure, 1 mL of toluene was added and concentrated under reduced pressure, repeated twice.
  • the solid residue was concentrated under reduced pressure and pulled dry by an oil pump to obtain the crude title product 9e (17 mg), which was directly used in the next reaction without purification.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min), the corresponding components were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4 mg, 9-B: 1.7 mg).
  • UV-HPLC is used to calculate the DAR value n in some ADC embodiments of this disclosure, and the details are as follows:
  • UV spectrophotometry using instrument: Thermonanodrop2000 UV spectrophotometer
  • the principle is that the total absorbance of the ADC stock solution at a certain wavelength is equal to the drug and monoclonal antibody at this wavelength.
  • the sum of the absorbance value namely:
  • a 280nm ⁇ mab-280 bC mab + ⁇ Drug-280 bC Drug
  • ⁇ Drug-280 The average molar extinction coefficient of the drug at 280nm is 5100;
  • ⁇ mab-280 The average molar extinction coefficient of single antigen solution at 280nm is 214600;
  • C mab concentration of single antigen solution
  • the optical path length is 1 cm.
  • a 370nm ⁇ mab-370 bC mab + ⁇ Drug-370 bC Drug
  • ⁇ Drug-370 The average molar extinction coefficient of the drug at 370nm is 19000;
  • ⁇ mab-370 The extinction coefficient of single antigen solution at 370nm is 0;
  • C mab concentration of single antigen solution
  • the optical path length is 1 cm.
  • the drug load can be calculated.
  • Drug load C Drug /C mab .
  • the DAR value calculation method of some ADC embodiments of this disclosure adopts RP-HPLC (reversed-phase high performance liquid chromatography), which is specifically as follows:
  • Naked antibody (unconjugated antibody) and ADC sample for test were reduced by adding 4 ⁇ L DDT (sigma), and then bathed in 37°C water for 1 hour. After completion, they were taken out into the inner cannula.
  • DDT standard-dielectric chromatograph
  • Agilent 1200 for detection select Agilent PLRP-S 1000A 8 ⁇ m 4.6*250mm column temperature: 80°C; DAD detector wavelength 280nm; flow rate: 1mL/min; sample volume: 40 ⁇ L; afterwards By comparing the spectra of the sample and the naked antibody, the positions of the light and heavy chains are distinguished, and then the spectra of the test samples are integrated to calculate the DAR value n.
  • Preparation example Take 5.78mg of DTT, add 150 ⁇ L of purified water to fully dissolve it, prepare 0.25M DTT solution, and store at -20°C.
  • Preparation example Measure 1000mL purified water in a graduated cylinder, add 1mL TFA (sigma), mix thoroughly before use, and store at 2-8°C for 14 days.
  • Preparation example Measure 1000mL acetonitrile in a graduated cylinder, add 1mL TFA, mix thoroughly and use, store at 2-8°C for 14 days.
  • the positions of the light chain and the heavy chain are distinguished, and then the spectra of the test samples are integrated to calculate the DAR value (n).
  • HC DAR ⁇ (number of connected drugs*peak area percentage)/sum of HC peak area
  • Embodiment 3-1, 3-2 ADC-1, ADC-2
  • Embodiment 3-5 ADC-5
  • histidine-acetic acid-Tris/EDTA buffer containing antibody h1902-5 (pH 7.2 10mM histidine-acetic acid-Tris and 2.5mM EDTA buffer; 20.6g/L, 6.49 L, 0.91mmol) was added to the prepared TCEP histidine buffer (10mM histidine buffer; 1.717mM, 1.16L1.99mmol), placed in a constant temperature water bath, stirred at 12 °C for 2 hours, stop Reaction to obtain Intermediate I solution.
  • the cation eluate was subjected to 7-fold equal volume ultrafiltration (a 30KD polycellulose membrane package was used for the ultrafiltration membrane package) to obtain the exemplary product ADC-9 of h1902-5-9-A.
  • the average value calculated by RP-HPLC: n 4.1.
  • the drug loading obtained in this example is a non-limiting example. Those skilled in the art can obtain different DAR values (1-10, preferably 1-8, more preferably 2-8, 2- 7) The conjugate.
  • Test Example 1 Cell-level ELISA binding experiment
  • sample diluent pH7.4PBS containing 1% r milk
  • HRP-labeled goat anti-human secondary antibody Jackson Immuno Research, 109-035-003
  • Test Example 2 Antibody cell-level binding experiment
  • the NUGC4 cells stably expressing Claudin 18.2 were prepared with FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH 7.4 PBS (Sigma, P4417-100TAB)) into a cell suspension of 1 ⁇ 10 6 /mL, 100 ⁇ L /Well was added to a 96-well round bottom plate (Corning, 3795). After centrifugation to remove the supernatant, add 50 ⁇ L/well of the Claudin 18.2 antibody to be tested in different concentrations diluted with FACS buffer, and incubate for 1 hour in a refrigerator at 4°C in the dark.
  • FACS buffer 2% fetal bovine serum (Gibco, 10099141) pH 7.4 PBS (Sigma, P4417-100TAB)
  • Test Example 4 Antibody affinity determination based on flow cytometry
  • HEK293/hClaudin18.2 cells were collected in a U-bottom 96-well plate, with 1 ⁇ 2 ⁇ 10 5 cells per well.
  • Add the human Claudin 18.2 antibody with an initial concentration of 5 ⁇ g/mL, 2 ⁇ gradient dilution (12 concentration points), and incubate for 1 hour at 4°C.
  • the positive control is IMAB362, and a negative control without antibody is set.
  • the antibody was removed by centrifugation, and then 100 ⁇ L/well FITC anti-human IgG Fc antibody (200 ⁇ ) was added, incubated at 4°C for 30 minutes in the dark, washed twice with PBS+2% FBS, and then ready for flow cytometry detection.
  • the antibody was diluted in the above-mentioned phenol red-free medium to prepare a 3 ⁇ antibody dilution solution, and 25 ⁇ L/well of antibody was added to the cell plate. Incubate in a 37°C, 5% CO 2 incubator for 0.5 hours.
  • Collect effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells), centrifuge at 1000 rpm, and resuspend for counting. Resuspend the cells at a density of 3 ⁇ 10 6 cells/mL in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum). Add 25 ⁇ L of cells (7.5 ⁇ 10 4 Cells/well). Incubate for 6 hours in a 37°C, 5% CO 2 incubator.
  • FBS New Zealand ultra-low IgG fetal bovine serum
  • Test Example 6 Inhibition test of compound on tumor cell proliferation in vitro
  • the purpose of this experiment is to detect the drug compound of the present disclosure, against U87MG cells (glioma cells, Cell Bank of Chinese Academy of Sciences, Catalog#TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, catalog number HTB-30) Inhibitory activity of in vitro proliferation.
  • the cells were treated in vitro with different concentrations of compounds, and after 6 days of culture, CTG ( Luminescent Cell Viability Assay, Promega, article number: G7573)
  • the reagent detects the proliferation of cells, and evaluates the in vitro activity of the compound based on the IC50 value.
  • the following takes the in vitro proliferation inhibition test method for U87MG cells as an example to illustrate the method for the in vitro proliferation inhibition activity test of the compound of the present disclosure on tumor cells. This method is also applicable to, but not limited to, the in vitro proliferation inhibitory activity test on other tumor cells.
  • the initial concentration of the small molecule compound is 500 nM, and the dispensing method is as follows.
  • the initial concentration of ADC is 10 nM or 500 nM, and the dispensing method is as follows.
  • the first column of the 96-well plate add 100 ⁇ L of different samples to be tested, with a sample concentration of 100 nM or 5 ⁇ M; add 100 ⁇ L of PBS to each well of the second column to the 11th column. Take 50 ⁇ L of the sample from the first column to 100 ⁇ L of PBS in the second column, mix well, take 50 ⁇ L to the third column, and so on, dilute it 3 times to the 10th column.
  • Table 15 The IC 50 value of the small molecule fragments in the present disclosure on the inhibition of SK-BR-3 cell and U87 cell proliferation
  • the small molecule fragments in the present disclosure have obvious proliferation inhibitory activity on SK-BR-3 cells and U87 cells, and the chiral center has a certain effect on the inhibitory activity of the compound.
  • the CellTiter-Glo Luminescence Cell Viability Assay was used to detect the killing effect of ADC molecules on human gastric cancer cell lines in vitro.
  • collect NUGC4-claudin18.2 low expression, NUGC4-claudin18.2 expression, NUGC4-claudin18.2 high expression cells adjust the density to 2.5 ⁇ 10 4 /mL, and add 90 ⁇ L/mL to the 96-well white transparent bottom plate.
  • Well about 2500 cells per well. Incubate overnight at 37°C in a 5% CO 2 incubator.
  • Test Example 8 Evaluation of the efficacy of ADC molecules in vivo
  • Balb/c was inoculated naked and subcutaneously in the right rib with human gastric cancer cell NUGC4 (Claudin 18.2 medium expression) cells (5 ⁇ 10 6 containing 50% matrigel matrigel/mouse), grouped on day 0, 8 rats/group, 5 groups in total .
  • the average tumor volume is about 84.41mm 3 .
  • ADC was injected intraperitoneally for a total of 3 times, each of which was injected with 10g/0.1mL according to body weight, and was administered on day 0, day 4, and day 11, respectively.
  • ADC was injected intraperitoneally, 4 times in total, 5 days apart, each injected 10g/0.1mL according to body weight.
  • the tumor volume and body weight were measured twice a week, and the data was recorded.
  • V tumor volume
  • Relative volume (RTV) VT/V0
  • Tumor inhibition rate (%) (CRTV-TRTV)/CRTV (%)
  • V0 and VT are the tumor volume at the beginning of the experiment (the day of the first dosing is the 0th day) and the tumor volume at the time of measurement and counting, respectively.
  • CRTV and TRTV are the relative tumor volumes of the blank control group and the experimental group at the end of the experiment, respectively. The results are shown in Table 17 and Figure 4 and Figure 5.

Abstract

抗密蛋白抗体药物偶联物及其医药用途。具体而言，涉及通式(Pc-L-Y-D)所示的配体-药物偶联物，其中Pc为抗Claudin18.2抗体或其抗原结合片段，L、Y和n如说明书中所定义。

Description

抗密蛋白抗体药物偶联物及其医药用途
本申请要求2019年12月12日提交的中国专利申请(申请号CN201911273041.7)和2020年09月30日提交的中国专利申请(申请号CN202011060513.3)的优先权。
技术领域
本披露涉及抗密蛋白抗体药物偶联物，尤其是抗Claudin18.2抗体-依喜替康类似物偶联物、其制备方法、包含抗体药物偶联物的药物组合物、及其用于制备治疗Claudin18.2介导的疾病或病症的药物中的用途；尤其在用于制备抗癌药物中的用途。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然地构成现有技术。
密蛋白18(Claudin-18，CLDN18)是一种在人类中由Claudin18基因编码的蛋白质，属于细胞紧密连接蛋白家族，可以控制层细胞之间的分子流动。Claudin蛋白结构中包括四个跨膜区域、两个细胞外环，其N末端和C末端在胞浆内。
Claudin-18具有两个剪接变体，分别为Claudin 18.1和Claudin 18.2，两者序列之间仅在第一个细胞外环有八个氨基酸的差异。Claudin 18.1和Claudin 18.2的表达分布有所不同，Claudin 18.1在正常肺的细胞中选择性表达，Claudin 18.2在正常细胞中表达高度受限，但在多种肿瘤(胃癌、肺癌和胰腺癌等)中频繁异位激活和过表达。Claudin18.2被认为是胃癌和其他癌症类型的潜在治疗靶点，此靶点的发现也为胃癌的治疗提供了一种新的选择。
抗体-药物偶联物(antibody drug conjugate,ADC)将单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒性物质的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体-药物偶联物能更精准地结合肿瘤细胞并降低将对正常细胞的影响。
目前已有靶向Claudin18.2的抗体及ADC药物的专利报道，如WO2020200196A1、WO2016166122和WO2016165762。但是，目前仍需要开发更有效且更具安全性的抗Claudin18.2抗体-药物偶联物，以更好地用于Claudin18.2相关肿瘤的治疗。
发明内容
本披露涉及抗Claudin18.2抗体的ADC及其用途，其中提供与抗Claudin18.2 抗体或抗原结合片段与细胞毒性物质依喜替康类似物偶联的ADC药物。
因此，本披露的目的为提供一种通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2020135680-appb-000001
其中：
Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-和-S-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基和杂环基；或者，R a和R b与其相连接的碳原子一起形成环烷基或杂环基；
R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
或者，R a和R 2与其相连的碳原子一起形成环烷基或杂环基；
m为0至4的整数；
n为1至10，n是小数或整数；
L为接头单元；
Pc为抗Claudin18.2抗体或其抗原结合片段。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
i)所述重链可变区包含与如SEQ ID NO：3序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：4序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3；或
ii)所述重链可变区包含与如SEQ ID NO：5序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：6序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3。
在本公开的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
iii)所述重链可变区包含序列分别如SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含序列分别如SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14所示的LCDR1、LCDR2和LCDR3；或
iv)所述重链可变区包含序列分别如SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含序列分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体是鼠源抗体、嵌合抗体或人源化抗体。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
(1)所述重链可变区氨基酸序列如SEQ ID NO:3所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:4所示或与其有至少90％同一性；
(2)所述重链可变区氨基酸序列如SEQ ID NO:24所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:21所示或与其有至少90％同一性；
(3)所述重链可变区氨基酸序列如SEQ ID NO:5所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:6所示或与其有至少90％同一性；或
(4)所述重链可变区氨基酸序列如SEQ ID NO:31所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:28所示或与其有至少90％同一性。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体为人源化抗体，所述人源化抗体包含来源自人抗体的框架区或其框架区变体，所述框架区变体为在人抗体的轻链框架区和/或重链框架区上分别具有至多10个(例如1、2、3、4、5、6、7、8、9、10)氨基酸的回复突变；
优选地，所述框架区变体包含选自以下(a)或(b)所述的突变：
(a)所述轻链可变区中包含任选自22S、85I和87H中的一个或更多个氨基酸回复突变，和/或所述重链可变区中包含任选自48I、82T和69M中的一个或更多个氨基酸回复突变；或
(b)所述轻链可变区中包含任选自4L或22S一个或更多个氨基酸回复突变， 和/或所述重链可变区中包含任选自38K、40R、48I、66K、67A、69L、71L和73K中的一个或更多个氨基酸回复突变；
优选地，所述框架区变体包含选自以下所述的突变：
(a-1)所述轻链可变区中包含22S、85I和87H的氨基酸回复突变，和所述重链可变区中包含48I和82T的氨基酸回复突变；或
(b-1)所述轻链可变区中包含选自4L的氨基酸回复突变。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段包含如下所示的重链可变区和轻链可变区：
(vii)所述重链可变区序列如SEQ ID NO:3所示和所述轻链可变区序列如SEQ ID NO：4所示；
(viii)所述重链可变区序列如SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:27所示和所述轻链可变区序列如SEQ ID NO：21、SEQ ID NO:22或SEQ ID NO:23所示；
(ix)所述重链可变区序列如SEQ ID NO:5所示和所述轻链可变区序列如SEQ ID NO：6所示；或
(x)所述重链可变区序列如SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33或SEQ ID NO:34所示和所述轻链可变区序列如SEQ ID NO：28、SEQ ID NO:29或SEQ ID NO:30所示；
优选地，所述抗Claudin18.2抗体或其抗原结合片段包含如下所示的重链可变区和轻链可变区：
(xi)所述重链可变区序列如SEQ ID NO:31所示和所述轻链可变区序列如SEQ ID NO:29所示；或
(xii)所述重链可变区序列如SEQ ID NO:26所示和所述轻链可变区序列如SEQ ID NO:23所示。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段，包含抗体重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；更优选地，所述抗体包含序列如SEQ ID NO:7所示的重链恒定区和序列如SEQ ID NO:8所示的轻链恒定区；最优选所述抗体包含：与具有SEQ ID NO:35或SEQ ID NO:42所示氨基酸序列的重链具有至少90％同一性的重链，和与具有SEQ ID NO:36或SEQ ID NO:39所示氨基酸序列的轻链有至少90％同一性的轻链；或
与具有SEQ ID NO:37或SEQ ID NO:49所示氨基酸序列的重链具有至少90％同一性的重链，和与具有SEQ ID NO:38或SEQ ID NO:46所示氨基酸序列的轻链 有至少90％同一性的轻链。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段，包含：
(c)序列如SEQ ID NO:35所示的重链和序列如SEQ ID NO：36所示的轻链；
(d)序列如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44或SEQ ID NO:45所示的重链和序列如SEQ ID NO：39、SEQ ID NO:40或SEQ ID NO:41所示的轻链；
(e)序列如SEQ ID NO:37所示的重链和序列如SEQ ID NO：38所示的轻链；或
(f)序列如SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51或SEQ ID NO:52所示的重链和序列如SEQ ID NO：46、SEQ ID NO:47或SEQ ID NO:48所示的轻链。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体选自：
h1901-11：包含氨基酸序列如SEQ ID NO:44所示的重链，和序列如SEQ ID NO:41所示的轻链；或
h1902-5：包含氨基酸序列如SEQ ID NO:49所示的重链，和序列如SEQ ID NO:47所示的轻链。
在本披露的一些实施方案中，所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)和二硫键稳定化的V区(dsFv)。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中n可以为1-10之间的整数或小数，n可以为1、2、3、4、5、6、7、8、9或10的均值。n为2至8的小数或整数，优选为3至8的小数或整数，更优选为5至9的小数或整数，或优选为2至7的小数或整数。在一些实施方案中，n为3.5至4.5的小数或整数。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，
其中：
Y为-O-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素或烷基；
R 1为卤代烷基或C 3-6环烷基；
R 2选自氢原子、卤代烷基或C 3-6环烷基；
或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
m为0或1。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配 体-药物偶联物或其药学上可接受的盐，其中Y选自：
Figure PCTCN2020135680-appb-000002
其中Y的O端与接头单元L相连。
在本披露的一些实施方案中，提供的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中接头单元-L-为-L 1-L 2-L 3-L 4-。
在一些实施方案中，L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 3-W-C(O)-或-C(O)-W-C(O)-，其中W选自C 1-8烷基、C 1-8烷基-C 3-6环烷基或1至8个链原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述的C 1-8烷基、C 1-8烷基-C 3-6环烷基或1至8个链原子的直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代。
在一些实施方案中，L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-或化学键，其中p 1为1至20的整数。
在一些实施方案中，L 3为由2至7个氨基酸构成的肽残基，其中所述的氨基酸选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代。
在一些实施方案中，L 4选自-NR 5(CR 6R 7) t-、-C(O)NR 5-、-C(O)NR 5(CH 2) t-或化学键，其中t为1至6的整数。
在一些实施方案中，R 3、R 4和R 5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基。
在一些实施方案中，R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 3-W-C(O)-或-C(O)-W-C(O)-，其中W选自C 1-8烷基、C 1-8烷基-环烷基或1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-或化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸构成的肽残基，其中所述的氨基酸选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
L 4选自-NR 5(CR 6R 7) t-、-C(O)NR 5、-C(O)NR 5(CH 2) t-或化学键，其中t为1至6的整数；
R 3、R 4和R 5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1为
Figure PCTCN2020135680-appb-000003
s 1为2至8的整数；
L 2为化学键；
L 3为四肽残基；优选的，L 3为GGFG的四肽残基(SEQ ID No.55)；
L 4为-NR 5(CR 6R 7)t-，R 5、R 6或R 7相同或不同，且各自独立地为氢原子或烷基，t为1或2；
其中所述的L 1端与Pc相连，L 4端与Y相连。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中-L-为：
Figure PCTCN2020135680-appb-000004
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中-L-Y-任选自：
Figure PCTCN2020135680-appb-000005
Figure PCTCN2020135680-appb-000006
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L a-Y-D)所示的配体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2020135680-appb-000007
其中：
W、L 2、L 3、R 5、R 6、R 7如前所述接头单元-L-中所定义；
Pc、n、R 1、R 2、m如通式(Pc-L-Y-D)中所定义。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L b-Y-D)所示的配体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2020135680-appb-000008
其中：
s 1为2至8的整数；
Pc、R 1、R 2、R 5～R 7、m和n如通式(Pc-L a-Y-D)所中所定义。
在本披露的一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，所述配体-药物偶联物选自：
Figure PCTCN2020135680-appb-000009
其中Pc和n如通式(Pc-L-Y-D)中所定义。
在本披露的一些实施方案中，提供的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，所述配体-药物偶联物选自：
Figure PCTCN2020135680-appb-000010
Figure PCTCN2020135680-appb-000011
其中，n如通式(Pc-L-Y-D)中所定义；抗体h1902-5，h1901-11如前所定义。
本披露本披露进一步提供一种制备如通式(Pc-L a-Y-D)所示的配体-药物偶联物或其药学上可接受的盐的方法，其包括以下步骤：
Figure PCTCN2020135680-appb-000012
Pc’与通式(L a-Y-D)所示的化合物进行偶联反应，得到通式(Pc-L a-Y-D)所示的化合物；
其中：
Pc为如上所述的抗Claudin18.2抗体或其抗原结合片段；Pc’为Pc经还原后所得；
W、L 2、L 3、R 1、R 2、R 5～R 7、m和n如通式(Pc-L a-Y-D)中所定义。
本披露进一步提供一种制备如通式(Pc-L‘-D)所示的抗体药物偶联物的方法，其包括以下步骤：
Figure PCTCN2020135680-appb-000013
Pc还原后，与通式(L’-D)偶联反应，得到化合物；其中：
Pc为如前所述的抗Claudin18.2抗体或其抗原结合片段；
n如通式(Pc-L-Y-D)中所定义。
另一方面，本披露提供一种药物组合物，其包含如前任一项所述的配体-药物偶联物或其药学上可接受的盐，以及一种或多种可药用的赋形剂、稀释剂或载体。
另一方面，本披露提供如前任一项所述的配体-药物偶联物或其药学上可接受的盐或包含其的药物组合物作为药物的用途。在一些实施方案中，其中所述药物用于治疗Claudin18.2介导的疾病或病症；其中所述Claudin18.2介导的疾病或病症优选Claudin18.2高表达癌症。在一些实施方案中，其中所述药物用于治疗癌症。在一些实施方案中，其中所述癌症优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
另一方面，本披露提供如前任一项所述的配体-药物偶联物或其药学上可接受的盐或包含其的药物组合物在制备用于治疗Claudin18.2介导的疾病或病症的药物中的用途。其中所述Claudin18.2介导的疾病或病症为Claudin18.2高表达癌症。在一些实施方案中，其中所述疾病优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
另一方面，本披露提供如前任一项所述的配体-药物偶联物或其药学上可接受的盐或包含其的药物组合物在制备用于治疗或预防肿瘤的药物中的用途；其中所述肿瘤和癌症优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
另一方面，本披露进一步涉及一种用于治疗和/或预防肿瘤的方法，该方法包括向需要其的受试者施用治疗有效剂量或预防有效剂量的如前任一项所述的配体- 药物偶联物或其药学上可接受的盐或包含其的药物组合物；优选其中所述的肿瘤为与Claudin18.2高表达相关的癌症。
另一方面，本披露进一步涉及一种用于治疗或预防癌症的方法，该方法包括向需要其的受试者施用治疗有效剂量或预防有效剂量的如前任一项所述的配体-药物偶联物或其药学上可接受的盐或包含其的药物组合物；其中所述肿瘤和癌症优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
可将活性化合物(例如根据本披露所述的配体-药物偶联物、或其药学上可接受的盐)制成适合于通过任何适当途径给药的形式，活性化合物优选是以单位剂量的方式，或者是以受试者能够以单剂进行自我给药的方式。本披露的单位剂量的方式可以是片剂、胶囊、扁囊剂、瓶装药水、药粉、颗粒剂、锭剂、栓剂、再生药粉或液体制剂。
本披露治疗方法中所用活性化合物或组合物的施用剂量通常将随疾病的严重性、受试者的体重和活性化合物的功效而改变。不过，作为一般性指导，合适的单位剂量可以是0.1mg至1000mg。
本披露的药物组合物除活性化合物外，可含有一种或多种辅料，所述辅料选自以下成分：填充剂、稀释剂、粘合剂、润湿剂、崩解剂或赋形剂等。根据给药方法的不同，组合物可含有0.1至99重量％的活性化合物。
本披露提供的Claudin18.2抗体及抗体药物偶联物具有与细胞表面抗原良好的亲和力，良好的细胞内吞效率和很强的肿瘤抑制效率，并且具有更宽的药物应用窗口，适于临床的药物应用。
附图说明
图1：人源化抗体在细胞水平与人Claudin18.2结合的FACS检测结果。
图2：人源化抗体的NUGC4细胞内吞实验。
图3A至图3C：抗体在不同Claudin18.2表达程度的NUGC4细胞中的ADCC效应检测。图3A为抗体在野生型NUGC4细胞(Claudin18.2低表达)中的ADCC效应检测；图3B为抗体在Claudin18.2中等表达NUGC4细胞中的ADCC效应检测；图3C为抗体在Claudin18.2高表达NUGC4细胞中的ADCC效应检测。
图4：本披露ADC-1的抑瘤实验结果。
图5：本披露ADC-2的抑瘤实验结果。
具体实施方式
一、术语
除非另有限定，本文所用的所有技术和科学术语均与本披露所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本披露，但本文描述了优选的方法和材料。描述和要求保护本披露时，依据以下定义使用下列术语。
当本披露中使用商品名时，旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“药物”是指可以改变或查明机体的生理功能及病理状态，可用以预防，诊断和治疗疾病的化学物质。药物包括细胞毒性药物。药物和毒物之间并无严格界限，毒物是指在较小剂量即对机体产生毒害作用，损害人体健康的化学物质，任何药物剂量过大都可产生毒性反应。细胞毒性药物，即抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质。细胞毒性药物原则上在足够的浓度下可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。细胞毒性药物包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)，毒素药物，化疗药物，抗生素和核溶酶。
术语“接头单元”、“接头”或连接片段”是指一端与配体(如抗体或其抗原结合片段)连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。
接头可以包含一种或多种接头构件。示例性的接头构件包括6-马来酰亚氨基己酰基(“MC”)、马来酰亚氨基丙酰基(“MP”)、缬氨酸-瓜氨酸(“val-cit”或“vc”)、丙氨酸-苯丙氨酸(“ala-phe”)、对氨基苄氧羰基(“PAB”)、N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(“SPP”)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(“SMCC”，在本文中也称作“MCC”)和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(“SIAB”)。接头可以包括延伸物、间隔物和氨基酸单元，可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细 胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
缩写
接头组件包括但不限于：
MC＝6-马来酰亚氨基己酰基，结构如下：
Figure PCTCN2020135680-appb-000014
Val-Cit或“vc”＝缬氨酸-瓜氨酸(蛋白酶可切割接头中的示例二肽)，
瓜氨酸＝2-氨基-5-脲基戊酸，
PAB＝对氨基苄氧羰基(“自我牺牲”接头组件的示例)，
Me-Val-Cit＝N-甲基-缬氨酸-瓜氨酸(其中接头肽键已经修饰以防止其受到组织蛋白酶B的切割)，
MC(PEG)6-OH＝马来酰亚氨基己酰基-聚乙二醇(可附着于抗体半胱氨酸)，
SPP＝N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯，
SPDP＝N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯，
SMCC＝琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯，
IT＝亚氨基硫烷。
术语“配体-药物偶联物”指配体通过连接单元与具有生物活性的药物相连。在本披露中“配体-药物偶联物”优选为抗体-药物偶联物(antibody drug conjugate，ADC)，指将单克隆抗体或者抗体片段通过连接单元与具有生物活性的毒性药物相连。抗体可直接地或经接头地偶联至药物。每个抗体的平均药物模块数(平均药物载荷或载药量，可用n值表示)，其范围可以是例如每个抗体偶联约0到约20个药物模块，在某些实施方案中是每个抗体偶联1个到约10个药物模块，在某些实施方案中是每个抗体偶联1个到约8个药物模块。
术语“平均药物载荷”或“载药量”是指配体-药物偶联物分子中每个配体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个配体(Pc)连接0-12个，优选1-10个细胞毒性药物。在本公开的实施方案中，载药量表示为n，也可称为DAR(Drug-antibody Ratio)值，n可以为0至12的非零整数或小数，优选为1-10之间的整数或小数；更优选为2至8，可以为整数，也可以为小数；最优选为3至8，可以为整数，也可以为小数。示例性的为1、2、3、4、5、6、7、8、9、10的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物品均数量。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中 所述。
密蛋白18(CLD18)分子(Genbank登记号：剪接变体1(CLD18A1)：NP_057453、NM016369，以及剪接变体2(CLD18A2或Claudin18.2)：NM_001002026、NP_001002026)是内在跨膜蛋白，位于上皮和内皮的紧密连接中。在紧密连接中，闭合蛋白(occludin)和密蛋白是最主要的跨膜蛋白组分。由于密蛋白强胞间粘附特性，它们产生了防止和控制溶质的细胞旁转运并限制膜脂和蛋白质侧向扩散以维持细胞极性的一级屏障。形成紧密连接的蛋白质参与了上皮的组织结构。据报道，这些蛋白质在构造良好的上皮中几乎无法接近抗体，但在肿瘤细胞中则变得暴露出来。
术语“抗体”指免疫球蛋白，是由两条重链和两条轻链通过链间二硫键连接而成的四肽链结构。根据免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
全长抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
术语“完全人源抗体”、“完全人抗体”或“全人抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。全人源抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
术语“抗原结合片段”是指抗体的保持结合抗原的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段”中包含的结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段，示例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv 片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
通常，Fab是通过用蛋白酶木瓜蛋白酶(例如，切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的部分和L链通过二硫键结合在一起。
通常，F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中二硫键的下方部分而获得的，其分子量约为100,000，并具有抗原结合活性，并包含在铰链位置相连的两个Fab区的抗体片段。
通常，Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。
此外，可以通过将编码Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或VH)和抗体轻链可变结构域(或VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本披露的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological  interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3，以及重链可变结构域的CDR2和CDR3。还包括“Chothia”编号规则、“ABM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Dev.Comp.Immunol.，27，55-77(2003)等。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上被免疫球蛋白或抗体结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合。
术语“KD”是指抗体-抗原相互作用的解离平衡常数。通常，本披露的抗体(或抗原结合片段)以小于大约10 -7M，例如小于大约10 -8M或10 -9M的解离平衡常数(KD)结合Claudin18.2，(或其表位)例如，在本披露中抗体与细胞表面抗原的亲和力采用FACS法测定KD值。
术语“核酸分子”是指DNA分子或RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
氨基酸序列“同一性”指，比对氨基酸序列过程中，在必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基相同的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引 入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，Lefranc,G.,The Immunoglobulin FactsBook,Academic Press，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本披露工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染宿主细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的N端位点。阳性的克隆在生物反应器的培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
术语“肽”是指介于氨基酸和蛋白质之间的化合物片段，由2个或2个以上氨基酸分子通过肽键相互连接而成，是蛋白质的结构与功能片段。
术语“糖”是指由C、H、O三种元素组成的生物大分子，可分为单糖、二糖和多糖等。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子(包含1个、2个、3个、4个、5个或6个碳原子)的烷基。非限制性示例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基 丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子(包含1个、2个、3个、4个、5个或6个碳原子)的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性示例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、 杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基和杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子(包含3个、4个、5个、6个、7个或8个碳原子)。单环环烷基的非限制性示例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基和环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0、1或2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1至4个是杂原子(1个、2个、3个或4个杂原子)；更优选环烷基环包含3至10个环原子(包含3个、4个、5个、6个、7个、8个、9个或10个环原子)。单环杂环基的非限制性示例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“螺杂环基”指5至20元的单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。螺杂环基的非限制性示例包括：
Figure PCTCN2020135680-appb-000015
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0、1或2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(7元、8元、9元或10元环)。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。稠杂环基的非限制性示例包括：
Figure PCTCN2020135680-appb-000016
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0、1或2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(7元、8元、9元或10元环)。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更优选为双环或三环。桥杂环基的非限制性示例包括：
Figure PCTCN2020135680-appb-000017
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性示例包括：
Figure PCTCN2020135680-appb-000018
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元(6元、7元、8元、9元或10元)，例如苯基和萘基，优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性示例包括：
Figure PCTCN2020135680-appb-000019
Figure PCTCN2020135680-appb-000020
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基和杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子(1个、2个、3个或4个杂原子)、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元(5元、6元、7元、8元、9元或10元杂芳基)，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基和四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性示例包括：
Figure PCTCN2020135680-appb-000021
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基和杂环烷硫基。
术语“氨基保护基”是为了使分子其它部位进行反应时氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性实施例包含9-芴甲氧羰基、叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基(1个、2个或3个取代基)所取代。所述氨基保护基优选为9-芴甲氧羰基。
术语“卤代烷基”指烷基上的氢被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基上的氢被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟烷基”指烷基上的氢被一个或多个羟基取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。
术语“硝基”指-NO 2。
术语“氰基”指-CN。
术语“酰氨基”指-C(O)N(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1个、2个或3个氢原子彼此独立地被取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的盐”或“可药用盐”是指本披露配体-药物偶联物的盐，或本披露中所述的活性化合物的盐，这类盐用于受试者时具有安全性和有效性，且具有应有的生物活性，本披露配体-药物偶联物至少含有一个氨基，因此可以与酸形成盐，可药用盐的非限制性示例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
本披露的一个实施方式中，细胞毒性药物通过连接单元偶联在抗体的巯基上。
可以用以下非限制性方法控制配体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
常规的药物组合物的制备见中国药典。
术语“药学上可接受的载体”用于本披露的药物，是指能改变药物进入受试者的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的示例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物组合物中除活性化合物以外的附加物，也可称为辅 料。如片剂中的粘合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐、微晶纤维素等。
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入受试者的血流中。或者，最好按可保持本披露化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的示例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1,3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。为此目的，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
二、合成方法
为了完成合成目的，采用如下的合成技术方案：
通式(Pc-L a-Y-D)所示的化合物的制备方法，其包括如下步骤：
Figure PCTCN2020135680-appb-000022
Pc还原后，与通式(L a-Y-D)偶联反应，得到通式(Pc-L a-Y-D)所示的化合物； 还原剂优选TCEP，特别地，优选还原抗体上的二硫键；
其中：Pc、W、L 2、L 3、R 1、R 2、R 5～R 7、m和n如通式(Pc-L a-Y-D)中所定义。
在以上说明书中提出了本披露一种或多种实施方案的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本披露，但是以下描述优选的方法和材料。通过说明书和权利要求书，本披露的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本披露所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本披露的优选实施方案。这些实施例不应以任何方式理解为限制本披露的范围，本披露的范围由权利要求书限定。
具体实施方式
一、抗体的制备
实施例1-1：claudin18.2高表达细胞株构建
将pCDH-hClaudin18.2慢病毒表达载体质粒与pVSV-G,pCMV-dR8.91慢病毒系统包装载体用Lipofectamine 3000转染试剂转染至病毒包装细胞293T中；收集含有病毒的培养基上清，过滤并进行超高速离心；使用浓缩后的病毒感染人胃印戒细胞癌细胞株NUGC4，经puromycin筛选两至三周，再进行FACS单细胞分选。根据肿瘤IHC评分来区分Claudin18.2表达程度。与肿瘤IHC评分为3分的肿瘤Claudin18.2表达水平相当的细胞为高表达细胞；与肿瘤IHC评分为2分的肿瘤Claudin18.2表平水平相当的细胞为中等表达细胞。根据通过FACS检测慢病毒感染的NUGC4细胞表面的Claudin18.2表达，挑选出Claudin18.2表达量高的NUGC4/hClaudin18.2单克隆细胞株。同时通过FACS检测野生型NUGC4细胞表面的Claudin18.2表达，挑选出Claudin18.2表达量中等的NUGC4克隆细胞株，野生型NUGC4为Claudin18.2低表达量细胞。
将挑选出的单克隆细胞株扩大培养，冻存备库以便后续实验。
Claudin18.2序列Genbank:NP_001002026：(SEQ ID NO:1)
Figure PCTCN2020135680-appb-000023
Claudin18.2DNA序列：(SEQ ID NO:2)
Figure PCTCN2020135680-appb-000024
Figure PCTCN2020135680-appb-000025
Figure PCTCN2020135680-appb-000026
Figure PCTCN2020135680-appb-000027
实施例1-2：抗人claudin18.2单克隆抗体产生
1免疫
抗人Claudin18.2单克隆抗体通过免疫小鼠产生。实验用SJL白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。免疫抗原为huClaudin18.2-HEK293细胞(转染人Claudin18.2质粒的HEK-293稳转细胞株)。
免疫方案：首次免疫细胞前，用
Figure PCTCN2020135680-appb-000028
Gold Adjuvant(Sigma Cat No. T2684)0.1mL/只注射小鼠腹膜内(IP)，半小时后每只小鼠腹膜内(IP)注射0.1mL生理盐水稀释至1×10 8/mL浓度的细胞液。细胞吹散均匀后进行接种，时间为第0、14、28、42、56天。于第21，35，49，63天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第4-5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射1×10 7细胞。
2脾细胞融合
采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2020135680-appb-000029
CRL-8287 TM)进行融合得到杂交瘤细胞。融合所得的杂交瘤细胞以0.5-1×10 6/mL的密度用完全培养基(含20％FBS、1×HAT、1×OPI的IMDM培养基)重悬，100μL/孔种于96孔板中，37℃，5％CO 2孵育3-4天后，补充HAT完全培养基100μL/孔，继续培养3-4天至形成针尖般克隆。去除上清，加入200μL/孔的HT完全培养基(含20％FBS、1×HT和1×OPI的IMDM培养基)，37℃，5％CO 2培养3天后进行ELISA检测。
3杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法检测杂交瘤培养上清。选择与huClaudin18.2-HEK293细胞结合能力强，同时与HEK293细胞没有结合的细胞及时进行扩增冻存，进行二到三次亚克隆直至获得单细胞克隆。
每次亚克隆细胞也均需进行细胞结合实验检测。通过以上实验筛选得到杂交瘤克隆，用无血清细胞培养法进一步制备抗体，按纯化示例纯化抗体，供在检测例中使用。
实施例1-3：鼠源抗体的人源化
挑选出体外活性高的单克隆杂交瘤细胞株mAb1901，mAb1902，克隆其中的单克隆抗体序列，再进行人源化，重组表达和活性评价。
从杂交瘤中克隆序列过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,15596-018)提取RNA(按照试剂盒说明书步骤)，反转录(PrimeScript TM Reverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增后送测序公司测序。得到的杂交瘤细胞DNA序列对应的氨基酸序列SEQ ID NO:3-6所示：
mAb1901鼠源重链可变区(SEQ ID NO:3)
Figure PCTCN2020135680-appb-000030
mAb1901鼠源轻链可变区(SEQ ID NO:4)
Figure PCTCN2020135680-appb-000031
Figure PCTCN2020135680-appb-000032
mAb1902鼠源重链可变区(SEQ ID NO:5)
Figure PCTCN2020135680-appb-000033
mAb1902鼠源轻链可变区(SEQ ID NO:6)
Figure PCTCN2020135680-appb-000034
上述鼠源重链可变区和轻链可变区分别与下述人IgG1抗体的重链恒定区和人源κ轻链恒定区连接形成嵌合抗体ch1901和ch1902。
恒定区选自以下序列：
人IgG1抗体的重链恒定区：(SEQ ID NO:7)
Figure PCTCN2020135680-appb-000035
人源κ轻链恒定区：(SEQ ID NO:8)
Figure PCTCN2020135680-appb-000036
鼠源单克隆抗体人源化如本领域许多文献公示的方法进行。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种系抗体序列，进行CDR移植。本发明选择活性好的候选分子进行人源化，结果如下。
1、鼠源抗体的CDR区
表1中VH/VL CDR的氨基酸残基由Kabat编号系统确定并注释。
鼠源抗体的CDR序列如表1所述：
表1鼠源抗体的CDR序列
抗体 mAb1901
HCDR1 DYGIH(SEQ ID NO:9)
HCDR2 YISRGSSTIYYADTVKG(SEQ ID NO:10)
HCDR3 GGYDTRNAMDY(SEQ ID NO:11)
LCDR1 KSSQSLLNSGNQKNYLA(SEQ ID NO:12)
LCDR2 GASTRAS(SEQ ID NO:13)
LCDR3 QNDLYYPLT(SEQ ID NO:14)
抗体 mAb1902
HCDR1 SYWMH(SEQ ID NO:15)
HCDR2 MIHPNSGSTNYNEKFKGR(SEQ ID NO:16)
HCDR3 LKTGNSFDY(SEQ ID NO:17)
LCDR1 KSSQSLLNSGNQKNYLT(SEQ ID NO:18)
LCDR2 WASTRES(SEQ ID NO:19)
LCDR3 QNAYTYPFT(SEQ ID NO:20)
2、选择人种系FR区序列
在所获得的鼠源抗体VH/VLCDR典型结构的基础上，将重、轻链可变区序列与抗体Germline数据库比较，获得同源性高的人种系模板。其中人类种系轻链框架区来自人κ轻链基因。
2.1mAb1901的人源化改造和回复突变设计
选择适当的人抗体种系，对mAb1901鼠源抗体进行人源化改造，将鼠源抗体mAb1901的CDR区移植到选择的人源化模板上，替换人源化可变区，再与IgG恒定区重组，形成完整抗体。同时，对人源化抗体的V区中FR区进行回复突变，示例性回复突变方式及组合如下：
表2 mAb1901人源化抗体及回复突变 *
Figure PCTCN2020135680-appb-000037
*表格中所有氨基酸位置编号为Kabat编号规则的编号，重链可变区的N82T中，82为Kabat规则的第82A位。
表3 mAb1901人源化抗体轻链可变区和重链可变区序列
Figure PCTCN2020135680-appb-000038
Figure PCTCN2020135680-appb-000039
上表中对应重链可变区与SEQ ID NO：7所示的人IgG1重链恒定区连接形成全长抗体的重链，轻链可变区与SEQ ID NO：8所示的人κ轻链恒定区连接形成全长抗体的轻链。在其他实施方案中，重链可变区和轻链可变区也可与其他重链恒定区和轻链恒定区分别连接形成全长抗体。
2.2mAb1902的人源化改造和回复突变设计
选择适当的人抗体种系，对mAb1902鼠源抗体进行人源化改造，将鼠源抗体mAb1902的CDR区移植到选择的人源化模板上，替换人源化可变区，再与IgG恒定区重组，形成完整抗体。同时，对人源化抗体的V区中FR区进行回复突变，示例性回复突变方式及组合如下：
表4 mAb1902人源化抗体及其回复突变设计 *
Figure PCTCN2020135680-appb-000040
*表格中所有氨基酸位置编号为Kabat编号规则的编号。
表5 mAb1902人源化抗体轻链可变区和重链可变区序列
Figure PCTCN2020135680-appb-000041
Figure PCTCN2020135680-appb-000042
上表中对应重链可变区与SEQ ID NO：7所示的人IgG1重链恒定区连接形成全长抗体的重链，轻链可变区与SEQ ID NO：8所示的人κ轻链恒定区连接形成全长抗体的轻链。
嵌合抗体ch1901
ch1901重链：(SEQ ID NO:35)
Figure PCTCN2020135680-appb-000043
ch1901轻链(SEQ ID NO:36)
Figure PCTCN2020135680-appb-000044
Figure PCTCN2020135680-appb-000045
嵌合抗体ch1902
ch1902重链(SEQ ID NO:37)
Figure PCTCN2020135680-appb-000046
ch1902轻链(SEQ ID NO:38)
Figure PCTCN2020135680-appb-000047
表6展示了mAb1901的人源化抗体：
表6 mAb1901人源化抗体
轻重链 H1 H2 H3 H4
L1 h1901-1 h1901-2 h1901-3 h1901-4
L2 h1901-5 h1901-6 h1901-7 h1901-8
L3 h1901-9 h1901-10 h1901-11 h1901-12
备注：表中人源化抗体h1901-1的重链为H1，轻链为L1，其它以此类推。mAb1901的人源化抗体的全长抗体轻重链序列如下表7所示：
表7 mAb1901人源化抗体轻链和重链序列
Figure PCTCN2020135680-appb-000048
Figure PCTCN2020135680-appb-000049
Figure PCTCN2020135680-appb-000050
表8展示了mAb1902的人源化抗体：
表8 mAb1902人源化抗体
轻重链 H11 H12 H13 H14
L11 h1902-1 h1902-2 h1902-3 h1902-4
L12 h1902-5 h1902-6 h1902-7 h1902-8
L13 h1902-9 h1902-10 h1902-11 h1902-12
备注：表中人源化抗体h1902-1的重链为H11，轻链为L11，其它以此类推。
mAb1902的人源化抗体轻重链序列如下表9所示：
表9 mAb1901人源化抗体轻链和重链序列
Figure PCTCN2020135680-appb-000051
Figure PCTCN2020135680-appb-000052
Figure PCTCN2020135680-appb-000053
本披露阳性对照抗体为IMAB-362(来自WO2016166122)
IMAB-362重链(SEQ ID NO:53)：
Figure PCTCN2020135680-appb-000054
IMAB-362轻链(SEQ ID NO:54)：
Figure PCTCN2020135680-appb-000055
Figure PCTCN2020135680-appb-000056
用常规基因克隆、重组表达的方法分别克隆、表达、纯化上述抗体。
二、化合物的制备
本披露实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
化合物的结构是通过核磁共振(NMR)或质谱(MS)来确定的。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)，化学位移是以10 -6(ppm)作为单位给出。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo,型号：Finnigan LCQ advantage MAX)。
UPLC的测定用Waters Acquity UPLC SQD液质联用仪。
HPLC的测定使用安捷伦1200DAD高压液相色谱仪(Sunfire C18 150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18 150×4.6mm色谱柱)。
UV-HPLC的测定使用Thermo nanodrop2000紫外分光光度计。
增殖抑制率及IC 50值的测定用PHERA starFS酶标仪(德国BMG公司)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm至0.2mm，薄层层析分离纯化产品采用的规格是0.4mm至0.5mm硅胶板。
柱层析一般使用烟台黄海200～300目硅胶为载体。
本披露的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH&Co.KG，Acros Organnics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中如无特殊说明，反应均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中如无特殊说明，反应中的溶液是指水溶液。
实施例中如无特殊说明，反应的温度为室温。
室温为最适宜的反应温度，温度范围是20℃至30℃。
实施例中pH＝6.5的PBS缓冲液的配制：取KH 2PO 4 8.5g，K 2HPO 4.3H 2O 8.56g，NaCl 5.85g，EDTA 1.5g置于瓶中，定容至2L，超声波使其全部溶解，摇匀即得。
纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂的体系包括：A：二氯甲烷和异丙醇体系，B：二氯甲烷和甲醇体系，C：石油醚和乙酸乙酯体系，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和酸性或碱性试剂等进行调节。
本披露部分化合物是通过Q-TOF LC/MS来表征的。Q-TOF LC/MS使用安捷伦6530精确质量数四级杆-飞行时间质谱仪和安捷伦1290-Infinity超高效液相色谱仪(安捷伦Poroshell 300SB-C8 5μm,2.1×75mm色谱柱)。
本披露抗体药物偶联物的Y-D药物部分参见PCT/CN2019/107873，相关的化合物合成及测试引用至本专利。其中的非限制性实施例合成引用如下：
实施例1
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环丙烷-1-甲酰胺1
Figure PCTCN2020135680-appb-000057
向依喜替康甲磺酸盐1b(2.0mg，3.76μmol，采用专利申请“EP0737686A1”公开的方法制备而得)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-羟基环丙基甲酸1a(1.4mg，3.7μmol，采用公知的方法“Tetrahedron Letters,25(12),1269-72；1984”制备 而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(3.8mg，13.7μmol)，加毕，在0-5℃搅拌反应2小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(8mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物1(1.6mg，产率：82.1％)。
MS m/z(ESI):520.2[M+1]
1H NMR(400MHz,CDCl 3):δ7.90-7.84(m,1H),7.80-7.68(m,1H),5.80-5.70(m,1H),5.62-5.54(m,2H),5.44-5.32(m,2H),5.28-5.10(m,2H),3.40-3.15(m,3H),2.44(s,3H),2.23(t,1H),2.06-1.75(m,2H),1.68-1.56(m,1H),1.22-1.18(m,2H),1.04-0.98(m,2H),0.89(t,3H)。
实施例2
(S)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺2-A
(R)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺2-B
Figure PCTCN2020135680-appb-000058
向1b(4mg，7.53μmol)中加入2mL乙醇和0.4mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啉，搅拌至反应液变澄清。向反应液中依次加入2-环丙基-2-羟基乙酸2a(2.3mg，19.8μmol，采用专利申请“WO2013106717”公开的方法制备而得)、1-羟基苯并三唑(3mg，22.4μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(4.3mg，22.4μmol)，加毕，在0-5℃搅拌反应1小时。撤去冰水浴，加热至30℃搅拌2小时。反应液减压浓缩，所得到的粗品化合物2用高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18OBD 5μm 19*250mm；流动相：A-水(10mmol NH 4OAc)，B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(2-A：1.5mg，2-B：1.5mg)。
MS m/z(ESI):534.0[M+1]。
单一构型化合物2-B(较短保留时间)
UPLC分析：保留时间1.06分钟，纯度：88％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.37(d,1H),7.76(d,1H),7.30(s,1H),6.51(s,1H),5.58-5.56(m,1H),5.48(d,1H),5.41(s,2H),5.32-5.29(m,2H),3.60(t,1H),3.19-3.13(m,1H),2.38(s,3H),2.20-2.14(m,1H),1.98(q,2H),1.87-1.83(m,1H),1.50-1.40(m,1H),1.34-1.28(m,1H),0.86(t,3H),0.50-0.39(m,4H)。
单一构型化合物2-A(较长保留时间)
UPLC分析：保留时间1.10分钟，纯度：86％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.35(d,1H),7.78(d,1H),7.31(s,1H),6.52(s,1H),5.58-5.53(m,1H),5.42(s,2H),5.37(d,1H),5.32(t,1H),3.62(t,1H),3.20-3.15(m,2H),2.40(s,3H),2.25-2.16(m,1H),1.98(q,2H),1.87-1.82(m,1H),1.50-1.40(m,1H),1.21-1.14(m,1H),0.87(t,3H),0.47-0.35(m,4H)。
实施例3
(S)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟-2-羟基丙酰胺3-A
(R)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟-2-羟基丙酰胺3-B
Figure PCTCN2020135680-appb-000059
向1b(5.0mg，9.41μmol)中添加2mL乙醇和0.4mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啡啉，搅拌至反应液变澄清。向反应液中依次加入3,3,3-三氟-2-羟基丙酸3a(4.1mg，28.4μmol，供应商Alfa)、1-羟基苯并三唑(3.8mg，28.1μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(5.4mg，28.2μmol)，加毕，在0-5℃搅拌反应10分钟。撤去冰水浴，加热至30℃搅拌8小时。反应液减压浓缩，所得到的粗品化合物3用高效液相色谱法纯化(分离条件：色谱 柱：XBridge Prep C18OBD 5μm 19*250mm；流动相：A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(3-A:1.5mg，3-B:1.5mg)。
MS m/z(ESI):561.9[M+1]。
单一构型化合物(较短保留时间)
UPLC分析：保留时间1.11分钟，纯度：88％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.94(d,1H),7.80(d,1H),7.32(s,1H),7.20(d,1H),6.53(s,1H),5.61-5.55(m,1H),5.45-5.23(m,3H),5.15-5.06(m,1H),4.66-4.57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.26-2.20(m,1H),2.16-2.08(m,1H),2.02-1.94(m,1H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H)。
单一构型化合物(较长保留时间)
UPLC分析：保留时间1.19分钟，纯度：90％(色谱柱：ACQUITY UPLC BEHC18 1.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.97(d,1H),7.80(d,1H),7.31(s,1H),7.16(d,1H),6.53(s,1H),5.63-5.55(m,1H),5.45-5.20(m,3H),5.16-5.07(m,1H),4.66-4.57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.22-2.14(m,1H),2.04-1.95(m,2H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H)。
实施例4
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环戊烷-1-甲酰胺4
Figure PCTCN2020135680-appb-000060
向1b(3.0mg，5.64μmol)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃， 滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-羟基-环戊烷甲酸4a(2.2mg，16.9μmol，采用专利申请“WO2013106717”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(4.7mg，16.9μmol)，加毕，在0-5℃搅拌反应1小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(10mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物4(2.5mg，产率：80.9％)。
MS m/z(ESI):548.0[M+1]。
1H NMR(400MHz,CDCl 3):δ7.73-7.62(m,2H),5.75-5.62(m,1H),5.46-5.32(m,2H),5.26-5.10(m,1H),3.30-3.10(m,1H),2.43(s,3H),2.28-2.20(m,2H),2.08-1.84(m,8H),1.69-1.58(m,2H),1.04-1.00(m,2H),0.89(t,3H)。
实施例5
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-(羟甲基)环丙烷-1-甲酰胺5
Figure PCTCN2020135680-appb-000061
向1b(2.0mg，3.76μmol)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-(羟甲基)-环戊烷甲酸5a(0.87mg，7.5μmol，采用专利申请“WO201396771”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(2mg，7.24μmol)，加毕，在0-5℃搅拌反应2小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(8mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物5(1.0mg，产率：50％)。
MS m/z(ESI):533.9[M+1]。
1H NMR(400MHz,CDCl 3):δ8.07(s,1H),7.23-7.18(m,2H),6.71-6.64(m,1H),6.55-6.51(m,1H),5.36-5.27(m,2H),4.67-4.61(m,2H),3.53-3.48(m,1H),3.30-3.22(m,2H),3.18-3.13(m,1H),2.71-2.61(m,2H),2.35-2.28(m,1H),2.04-1.91(m,4H),1.53-1.40(m,3H),0.91-0.75(m,4H)。
实施例6
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-(羟基甲基)环丁烷-1-甲酰胺6
Figure PCTCN2020135680-appb-000062
向1b(3.0mg，5.64μmol)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-(羟基甲基)环丁烷-1-甲酸6a(2.2mg，16.9μmol；采用文献“Journal of the American Chemical Society,2014,vol.136,#22,p.8138-8142”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(4.7mg，16.9μmol)，加毕，在0-5℃搅拌反应1小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(10mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物6(2.1mg，产率：67.9％)。
MS m/z(ESI):548.0[M+1]。
1H NMR(400MHz,DMSO-d 6):δ7.85-7.62(m,1H),6.88(br，1H),5.87-5.48(m，2H),5.47-5.33(m，1H),5.31-5.06(m，1H),4.25-3.91(m,2H),3.25(br,1H),2.60-2.32(m,3H),2.23(t,1H),2.15-1.95(m,3H),1.70-1.56(m,2H),1.41-1.17(m,9H),1.03(s,1H),0.95-0.80(m,2H)。
实施例7
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环丁烷-1-甲酰胺7
Figure PCTCN2020135680-appb-000063
向1b(3.0mg，5.64μmol)中添加2mL乙醇和0.4mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啡啉，搅拌至反应液变澄清。向反应液中依次加入1-羟基环丁烷甲酸7a(2.0mg，17.22μmol，供应商药石)，1-羟基苯并三唑(2.3mg，17.0μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(3.2mg，16.7μmol)，加毕，在0-5℃搅拌反应10分钟。撤去冰水浴，常温搅拌2小时。反应液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物7(2.5mg，产率：83.1％)。
MS m/z(ESI):534.0[M+1]。
1H NMR(400MHz,DMSO-d 6):δ8.28(d,1H),7.75(d,1H),7.29(s,1H),6.51(s,1H),6.12(s,1H),5.59-5.51(m,1H),5.41(s,2H),5.20-5.01(m,2H),3.27-3.17(m,1H),3.15-3.05(m,1H),2.71-2.63(m,1H),2.37(s,3H),2.12-2.05(m,1H),2.03-1.94(m,2H),1.92-1.78(m,4H),1.50-1.42(m,1H),0.90-0.83(m,4H)。
实施例8
1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺8
Figure PCTCN2020135680-appb-000064
第一步
1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丙烷-1-羧酸苄酯8c
将1-羟基环丙烷-1-羧酸苄酯8a(104mg，0.54mmol；采用专利申请“US2005/20645”公开的方法制备而得)和2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲基乙酸酯8b(100mg，0.27mmol；采用专利申请“CN105829346A”公开的方法制备而得)加入反应瓶，加入5mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(61mg，0.54mmol)，撤去冰浴，升至室温搅拌10分钟，加入20mL冰水，用乙酸乙酯(5mL×2)和氯仿(5mL×5)萃取，合并有机相并浓缩。所得残余物溶于3mL 1,4-二氧六环中，加入0.6mL水，加入碳酸氢钠(27mg，0.32mmol)和氯甲酸-9-芴甲酯(70mg，0.27mmol)，室温搅拌1小时。加入20mL水，用乙酸乙酯(8mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系B纯化所得残余物，得到标题产物8c(100mg，产率：73.6％)。
MS m/z(ESI):501.0[M+1]。
第二步
1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丙烷-1-羧酸8d
将8c(50mg，0.10mmol)溶于3mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(25mg，含量10％)，氢气置换三次，室温搅拌反应1小时。反应液用硅 藻土过滤，滤饼用四氢呋喃淋洗，滤液浓缩，得到标题产物8d(41mg，产率：100％)。
MS m/z(ESI):411.0[M+1]。
第三步
(9H-芴-9-基)甲基(2-(((1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯8e
将1b(7mg，0.013mmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入8d(7mg，0.017mmol)的0.5mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(7mg，0.026mmol)，冰浴搅拌反应35分钟。加入10mL水，用乙酸乙酯(5mL×3)萃取，有机相用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物8e(8.5mg，产率78.0％)。
MS m/z(ESI):828.0[M+1]。
第四步
1-((2-氨基乙酰氨基)甲氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺8f
将8e(4mg，4.84μmol)溶于0.2mL二氯甲烷中，加入0.1mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次，加入3mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩得到粗品标题产物8f(2.9mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):606.0[M+1]。
第五步
1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺8
将粗品8f(2.9mg，4.84μmol)溶于0.5mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入(S)-2(-2-(-2-(6-(2,5-二氧代-1H-吡咯-1-基)已酰氨基)乙酰氨基)乙酰氨基)-3-苯基丙酸8g(2.7mg，5.80μmol，采用专利申请“EP2907824”公开的方法制备而得)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(2.7mg，9.67μmol)，冰浴搅拌反应30分钟，撤去冰浴，升至室温搅拌15分钟。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩得到标题产物8(2mg， 产率：39.0％)。
MS m/z(ESI):1060.0[M+1]。
1H NMR(400MHz,DMSO-d 6):δ9.01(d,1H),8.77(t,1H),8.21(t,1H),8.08-7.92(m,2H),7.73(d,1H),7.28(s,1H),7.24-7.07(m,4H),6.98(s,1H),6.50(s,1H),5.61(q,1H),5.40(s,2H),5.32(t,1H),5.12(q,2H),4.62(t,1H),4.52(t,1H),4.40-4.32(m,1H),3.73-3.47(m,8H),3.16-3.04(m,2H),2.89(dd,1H),2.69-2.55(m,2H),2.37-2.23(m,4H),2.12-1.93(m,4H),1.90-1.74(m,2H),1.52-1.38(m,4H),1.33-1.11(m,5H),0.91-0.81(m,4H)。
实施例9
N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-A
N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-B
Figure PCTCN2020135680-appb-000065
第一步
2-环丙基-2-羟基乙酸苄酯9a
将2a(1.3g，11.2mmol；采用专利申请“WO2013/106717”公开的方法制备而得)溶于50mL乙腈中，依次加入碳酸钾(6.18g，44.8mmol)，溴化苄(1.33mL，11.2mmol)和四丁基碘化铵(413mg，1.1mmol)。将反应液室温搅拌48小时，通过硅藻土过滤，滤饼用乙酸乙酯(10mL)淋洗，合并滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物9a(2g，产率：86.9％)。
第二步
10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯9b
将9a(120.9mg,0.586mmol)和8b(180mg，0.489mmol)加入反应瓶，加入4mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(109mg，0.98mmol)，撤去冰浴，升至室温搅拌40分钟，加入10mL冰水，用乙酸乙酯(20mL×2)和氯仿(10mL×5)萃取，合并有机相并浓缩。所得残余物溶于4mL二氧六环中，加入2mL水，加入碳酸氢钠(49.2mg，0.586mmol)和氯甲酸-9-芴甲酯(126mg，0.49mmol)，室温搅拌2小时。加入20mL水，用乙酸乙酯(10mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物9b(48mg，产率：19％)。
MS m/z(ESI):515.0[M+1]。
第三步
10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸9c
将9b(20mg，0.038mmol)溶于4.5mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(12mg，含量10％，干型)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液浓缩，得到粗品标题产物9c(13mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):424.9[M+1]。
第四步
(9H-芴-9-基)甲基(2-(((1-环丙基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-2-氧代乙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯9d
将1b(10mg，18.8μmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入粗品9c(13mg，30.6μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(16.9mg，61.2μmol)，冰浴搅拌反应40分钟。加入10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物9d(19mg，产率：73.6％)。
MS m/z(ESI):842.1[M+1]。
第五步
2-((2-氨基乙酰氨基)甲氧基)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺9e
将9d(19mg，22.6μmol)溶于2mL二氯甲烷中，加入1mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入1mL甲苯并减压浓缩，重复两次。往残余物中加入3mL正己烷打浆，静置后倾倒出上层清液，保留固体。将固体残余物减压浓缩，油泵拉干得到粗品标题产物9e(17mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):638.0[M+18]。
第六步
N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-A
N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-B
将粗品9e(13.9mg，22.4μmol)溶于0.6mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入8g(21.2mg，44.8μmol)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(18.5mg，67.3μmol)，冰浴搅拌反应10分钟，撤去冰浴，升至室温搅拌1小时，反应生成化合物9。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(9-A：2.4mg，9-B：1.7mg)。
MS m/z(ESI):1074.4[M+1]。
单一构型化合物9-A(较短保留时间)：
UPLC分析:保留时间1.14分钟，纯度：85％(色谱柱：ACQUITY UPLC BEHC181.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,1H),4.54-4.40(m,2H),3.76-3.64(m，4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,1H),2.80-2.62(m,1H),2.45-2.30(m,3H),2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H)。
单一构型化合物9-B(较长保留时间)：
UPLC分析:保留时间1.16分钟，纯度：89％(色谱柱：ACQUITY UPLC BEHC181.7μm 2.1*50mm，流动相：A-水(5mmol NH 4OAc),B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.13(m,3H),6.99(s,1H),6.55-6.48(m,1H),5.60-5.50(m,1H),5.41(s,2H),5.35-5.15(m,2H),4.78-4.68(m,1H),4.60-4.40(m,2H),3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H),3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13(m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,4H),0.91-0.79(m,3H),0.53-0.34(m,4H)。
三、抗Claudin18.2抗体ADC偶联物的制备
ADC原液药物载药量分析
一、UV-HPLC方法
本披露部分ADC实施例的DAR值n计算方法采用了UV-HPLC，具体如下：
1、测定方法：
将装有琥珀酸钠缓冲液的比色皿分别置于参比吸收池和样品测定吸收池中后，扣除溶剂空白后，再将装有供试品溶液的比色皿置于样品测定吸收池中，测定280nm和370nm处吸光度。
2、结果计算：采用紫外分光光度法(使用仪器：Thermo nanodrop2000紫外分光光度计)测定ADC原液载量，其原理是在某波长下ADC原液的总吸光值等于药物与单克隆抗体在该波长下吸光值的加和，即：
(1)A 280nm＝ε mab-280bC mab+ε Drug-280bC Drug
ε Drug-280：药物在280nm平均摩尔消光系数5100；
C Drug：药物的浓度；
ε mab-280：单抗原液在280nm平均摩尔消光系数214600；
C mab：单抗原液的浓度；
b：光程长度为1cm。
同理可以得到样品在370nm下的总吸光值方程：
(2)A 370nm＝ε mab-370bC mab+ε Drug-370bC Drug
ε Drug-370：药物在370nm平均摩尔消光系数19000；
C Drug：药物的浓度；
ε mab-370：单抗原液在370nm消光系数为0；
C mab：单抗原液的浓度；
b：光程长度为1cm。
由⑴和⑵两种方程结合单克隆抗体和药物在两个检测波长下的消光系数和浓度数据可以计算出药物的载量。
药物载量＝C Drug/C mab。
二、RP-HPLC方法
本披露部分ADC实施例的DAR值计算方法采用了RP-HPLC(反相高效液相色谱)，具体如下：
1、测定方法：
裸抗(未缀合的抗体)和供测试ADC样品(浓度为1mg/mL)，加入4μL DDT(sigma)还原，37℃水浴1小时，结束后取出到内插管中。使用高效液相色谱仪Agilent 1200进行检测，色谱柱选用Agilent PLRP-S 1000A 8μm 4.6*250mm，柱温：80℃；DAD检测器波长280nm；流速：1mL/min；进样量为：40μL；之后通过样品与裸抗的谱图比对，区分出轻重链的位置，然后对检测样品的谱图进行积分，计算出DAR值n。
2、溶液配制
1)0.25M DTT溶液：
配制示例：取DTT 5.78mg，加入150μL纯化水充分溶解后，配得0.25M DTT溶液，-20℃保存。
2)流动相A(0.1％TFA水溶液)：
配制示例：量筒量取1000mL纯化水，加入1mL TFA(sigma)，充分混匀后使用，2-8℃保存14天。
3)流动相B(0.1％TFA乙腈溶液)：
配制示例：量筒量取1000mL乙腈，加入1mL TFA，充分混匀后使用，2-8℃保存14天。
3、数据分析
通过样品与裸抗的谱图比对，区分出轻链与重链的位置，然后对检测样品的谱图进行积分，计算出DAR值(n)。
计算公式如下：
Figure PCTCN2020135680-appb-000066
LC峰面积总和＝LC峰面积+LC+1峰面积
HC峰面积总和＝HC峰面积+HC+1峰面积+HC+2峰面积+HC+3峰面积
LC DAR＝Σ(连接药物数*峰面积百分比)/LC峰面积总和
HC DAR＝Σ(连接药物数*峰面积百分比)/HC峰面积总和
DAR＝LC DAR+HC DAR。
Claudin18.2抗体-药物偶联物制备实施例
实施例3-1、3-2：ADC-1、ADC-2
Figure PCTCN2020135680-appb-000067
在37℃条件下，向含抗体h1902-5的PBS缓冲液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，320.0mL，21.62μmol)中加入三(2-羧乙基)膦(TCEP)水溶液(10mM，11.03mL，110.3μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(350mg，303mol)溶解于13.2mL乙腈和6.6mL DMSO中，加入到降温至25℃的上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。
将得到的反应液先后用5L的PBS缓冲液(50mM，pH＝6.5，4％乙腈，2％DMSO)和5L的琥珀酸缓冲液(10mM，pH＝5.3)进行超滤膜纯化，除去小分子，加入蔗糖至60mg/mL、吐温-20至0.2mg/mL，最终制备得到通式抗体药物偶联物h1902-5-9-A的示例性产物ADC-1(10mM、pH＝5.3琥珀酸；10mg/mL，2.626g)。收率：81.81％。
UV-HPLC计算平均值：n＝6.8。
使用上述方法，可以用抗体h1901-11代替h1902-5，和化合物9-A制备得到通式抗体药物偶联物h1901-11-9-A的示例性产物ADC-2，DAR值n＝7.1。
实施例3-3 ADC-3
在37℃条件下，向抗体h1901-11的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1mL，67.5nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，10.1μL，101nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(0.58mg，540nmol)溶解于34μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到抗体药物偶联物h1901-11-9-A的示例性产物ADC-3的PBS缓冲液(0.72mg/mL，11.2mL)，于4℃储存。RP-HPLC计算平均值：n＝2.51。
实施例3-4 ADC-4
在37℃条件下，向抗体h1901-11的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1mL，67.5nmol)加入配置好的的三(2-羧乙基)膦(TCEP)的水溶液(10mM，16.9μL，169nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(0.73mg，680nmol)溶解于43μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到抗体药物偶联物h1901-11-9-A的示例性产物ADC-4的PBS缓冲液(0.62mg/mL，12.5mL)，于4℃储存。RP-HPLC计算平均值：n＝4.06。
实施例3-5 ADC-5
在37℃条件下，向抗体h1901-11的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1mL，67.5nmol)加入配置好的的三(2-羧乙基)膦(TCEP)的水溶液(10mM，35.8μL，358nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(1.09mg，1015nmol)溶解于64μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到抗体药物偶联物h1901-11-9-A的示例性产物ADC-5的PBS缓冲液(0.54mg/mL，12.5mL)，于4℃储存。RP-HPLC计算平均值：n＝6.8。
实施例3-6 ADC-6
在37℃条件下，向抗体h1902-5的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.08mL，72.9nmol)加入配置好的的三(2-羧乙基)膦(TCEP)的水溶液(10mM，10.9μL，109nmol)，置于水浴振荡器，于37℃下振荡 反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(0.63mg，587nmol)溶解于40μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到h1902-5-9-A的示例性产物ADC-6的PBS缓冲液(0.7mg/mL，13.0mL)，于4℃储存。RP-HPLC计算平均值：n＝2.69。
实施例3-7 ADC-7
在37℃条件下，向抗体h1902-5的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.08mL，72.9nmol)加入配置好的的三(2-羧乙基)膦(TCEP)的水溶液(10mM，18.3μL，183nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(0.79mg，736nmol)溶解于50μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到h1902-5-9-A的示例性产物ADC-7的PBS缓冲液(0.6mg/mL，14.0mL)，于4℃储存。RP-HPLC计算平均值：n＝4.25。
实施例3-8 ADC-8
在37℃条件下，向抗体h1902-5的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.08mL，72.9nmol)加入配置好的的三(2-羧乙基)膦(TCEP)的水溶液(10mM，38.7μL，387nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(1.18mg，1099nmol)溶解于70μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到h1902-5-9-A的示例性产物ADC-8的PBS缓冲液(0.56mg/mL，14.2mL)，于4℃储存。RP-HPLC计算平均值：n＝7.01。
实施例3-9 ADC-9
在12℃条件下，向含抗体h1902-5的组氨酸-醋酸-Tris/EDTA缓冲液(pH 7.2的10mM组氨酸-醋酸-Tris和2.5mM EDTA的缓冲液；20.6g/L，6.49L，0.91mmol)中加入配制好的TCEP组氨酸缓冲液(10mM组氨酸缓冲液；1.717mM，1.16L1.99mmol)，置于恒温水浴锅中，于12℃下搅拌反应2小时，停止反应，得到中间体I溶液。
将化合物9-A(4.72g，4.39mmol)溶解于0.38L DMSO中，生成化合物9-A 的DMSO溶液。向上述中间体I溶液中预加0.38L DMSO，再加入上述化合物9-A的DMSO溶液，置于恒温水浴锅中，于12℃下搅拌反应1小时，停止反应。
将上述反应液经Capto S Impact阳离子层析柱纯化，分别用9个柱体积的含有10％(v/v)DMSO的0.05M醋酸缓冲液(pH＝5.0)和6个柱体积0.05M醋酸缓冲液(pH＝5.0)洗涤，再用0.05M醋酸、0.30M氯化钠缓冲液(pH＝5.5)进行洗脱，去除反应液中游离毒素和残留溶剂。在22℃下，将阳离子洗脱液进行7倍体积等体积超滤(超滤膜包采用30KD的聚纤维素膜包)得到h1902-5-9-A的示例性产物ADC-9。RP-HPLC计算平均值：n＝4.1。
本实施例获得的载药量为非限制性实施例，本领域的技术人员根据反应条件和试剂的调整，获得不同DAR值(1-10，优选1-8，更优选2-8，2-7)的偶联物。
生物学评价
测试例1：细胞Cell水平ELISA结合实验
Cell based ELISA实验被用来检测Claudin18.2抗体的结合特性。将稳转表达Claudin18.2的NUGC4细胞培养于96孔细胞板中，待生长至90％密度时加入4％多聚甲醛固定细胞1小时，用PBST缓冲液(pH 7.4PBS含0.05％Tween-20)洗板3次后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μL/孔，37℃孵育箱孵育2.5小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板3次后，加入50μL/孔用样品稀释液(pH7.4PBS含1％r牛奶)稀释的不同浓度待测抗体，放于37℃孵育箱孵育2小时。孵育结束后用PBST洗板5次，加入100μL/孔用样品稀释液稀释的HRP标记的羊抗人二抗(Jackson Immuno Research,109-035-003)，37℃孵育1小时。用PBST洗板6次后，加入50μL/孔TMB显色底物(KPL,52-00-03)，于室温孵育10-15分钟，加入50μL/孔1M H 2SO 4终止反应，用MD Versa Max TM酶标仪在450nm处读取吸收值，计算Claudin18.2抗体对Claudin18.2的结合EC50值。
表10杂交瘤抗体结合活性
抗体 IMAB362 ch1901 ch1902
Emax 1.175 1.399 1.272
EC50(nM) 0.108 0.098 0.074
表11 mAb1901人源化抗体结合活性
抗体 IMAB362 h1901-2 h1901-3 h1901-4 h1901-6
Emax 1.115 1.039 1.1055 0.986 0.937
EC50(nM) 0.086 0.076 0.22 0.201 0.091
抗体 h1901-7 h1901-8 h1901-11 h1901-12  
Emax 0.921 1.047 1.44 1.22  
EC50(nM) 0.166 0.091 0.076 0.116  
表12 mAb1902人源化抗体结合活性
抗体 IMAB362 h1902-1 h1902-2 h1902-3 h1902-4 h1902-5
Emax 0.88 0.87 0.88 0.84 0.82 0.90
EC50(nM) 0.187 0.113 0.107 0.175 0.087 0.098
抗体 h1902-6 h1902-7 h1902-8 h1902-9 h1902-10  
Emax 0.78 0.75 0.89 0.75 0.89  
EC50(nM) 0.141 0.121 0.132 0.137 0.133  
测试例2：抗体细胞水平结合实验
将稳转表达Claudin18.2的NUGC4细胞用FACS缓冲液(2％胎牛血清(Gibco，10099141)pH7.4PBS(Sigma,P4417-100TAB))制备成1×10 6/mL的细胞悬液，100μL/孔加入96孔圆底板(Corning，3795)中。离心去除上清后加入50μL/孔用FACS缓冲液稀释的不同浓度待测Claudin18.2抗体，放于4℃冰箱中避光孵育1小时。以FACS缓冲液300g离心洗涤3次后，加入工作浓度的Alexa Fluor 488羊抗人IgG(H+L)(invitrogen,A-11013)，放于4℃冰箱中避光孵育40分钟。以FACS缓冲液300g离心洗涤3次后，在BD FACS CantoII流式细胞仪上检测几何平均数荧光强度，计算Claudin18.2抗体对稳转表达Claudin18.2的NUGC4细胞的结合EC50值，结果见图1。
测试例3：抗体内吞实验
将预标记了DyLight 488NHS Ester(thermofisher，46403)的待测Claudin18.2抗体，以5μg/mL终浓度加入1×10 6/mL稳转表达Claudin18.2的NUGC4细胞中，放于冰上避光孵育1小时，以预冷的FACS缓冲液(pH7.4PBS，2％胎牛血清)离心洗涤3次，去上清后加入预热的完全培养基，放入37℃5％CO 2细胞培养箱。分别在0、0.5、1、2、4小时后取出细胞，放置于冰上避光保存。待样品全部收集后，300g低温离心去除上清，加入洗脱缓冲液(pH1.7 0.05M甘氨酸，0.1M氯化钠)后，室温孵育7分钟，以FACS缓冲液300g离心洗涤1次，在BD FACS CantoII流式细胞仪上检测几何平均数荧光强度，计算Claudin18.2抗体对稳转表达Claudin18.2的NUGC4细胞的内吞效率。结果显示(见图2)，人源化抗体具有良好的细胞内吞效率。
测试例4：基于流式细胞技术的抗体亲和力测定
实验当天收集HEK293/hClaudin18.2细胞于U底96孔板中，每孔1～2×10 5个细胞。加入起始浓度5μg/mL，2×梯度稀释(12个浓度点)的人Claudin18.2抗体，4℃孵育1小时，阳性对照为IMAB362，同时设置不加抗体的阴性对照。离心去除抗体，再加入100μL/孔FITC抗人IgG Fc抗体(200×)，4℃避光孵育30分钟， 用PBS+2％FBS清洗两遍后准备进行流式细胞仪检测。启动BD FACS CantoII，预热完成后打开BD FACSDiva软件，建立一个新的实验，检测HEK293/hClaudin18.2阴性对照样品，调节FSC及SSC电压至适当的数值并保存。根据Quantum TM FITC-5MESF Kit说明书，分别检测空白样品B及标准曲线1，调节FITC电压至适当的数值并保存。在保存的电压下检测U底96孔板中的样品，记录数据。使用Flowjo软件分析实验数据得到Geo平均值，根据Quantum TM FITC-5MESF Kit说明书拟合MESF-Geo Mean标准曲线，根据FITC抗人IgG Fc抗体的浓度荧光值计算出与HEK293/hClaudin18.2细胞结合的人Claudin18.2抗体的摩尔浓度及游离抗体浓度，利用Scatchard作图法计算抗体的Bmax和解离常数KD。结果见表13。
表13人源化抗体细胞水平亲和力
抗体 IMAB362 h1901-11 h1902-5
KD(nM) 10.2 6.8 1.64
测试例5：抗体的ADCC效应评价
消化各种NUGC4细胞(高中低表达Claudin18.2)，1000rpm离心后，重悬计数。将细胞以3×10 5细胞/mL的密度重悬在添加10％FBS(新西兰超低IgG胎牛血清，Gibco,1921005PJ)的无酚红RPMI 1640中(Gibco,11835-030)。在96孔板(Corning,3903)中，每孔加入25μL细胞(7500个/孔)。将抗体稀释在上述无酚红培养基中，配制成3×的抗体稀释液，向细胞板中加入25μL/孔的抗体。在37℃、5％CO 2培养箱中孵育0.5小时。
收集效应细胞(FcrR3A-V158-NFAT-RE-Jurkat细胞)，1000rpm离心后，重悬计数。将细胞以3×10 6细胞/mL的密度重悬在添加10％FBS(新西兰超低IgG胎牛血清)的无酚红RPMI 1640中，在实验板中每孔加入25μL细胞(7.5×10 4个细胞/孔)。在37℃、5％CO 2培养箱中孵育6小时。
向实验板的每个孔中加入75μL/孔的Bright-Glo(Promega,E2610)，用酶标仪(PerkinElmer,VITOR3)检测化学发光(luminescence)。
结果显示(见表14和图3A-图3C)，在低(图3A)-中(图3B)-高(图3C)不同程度Claudin18.2表达的NUGC4细胞中，抗体h1901-11和h1902-5均显示出很强的ADCC活性。
表14抗体在Claudin18.2不同表达程度的NUGC4细胞中的ADCC效应
单位IC50(ng/mL)
Claudin18.2表达程度 h1901-11 h1902-5 IMAB362
低表达 22.42 35.46 183.4
中等表达 15.35 30.00 210.4
高表达 26.17 32.16 132.6
测试例6：化合物对肿瘤细胞体外增殖抑制测试
一、测试目的
本实验的目的是为了检测本披露药物化合物，对U87MG细胞(胶质瘤细胞，中科院细胞库，Catalog#TCHu138)和SK-BR-3肿瘤细胞(人乳腺癌细胞，ATCC，货号HTB-30)体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经6天培养后，采用CTG(
Figure PCTCN2020135680-appb-000068
Luminescent Cell Viability Assay，Promega，货号：G7573)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
二、实验方法
下面以对U87MG细胞体外增殖抑制测试方法为例，用于举例说明本披露化合物对肿瘤细胞进行体外增殖抑制活性测试的方法。本方法同样适用于，但不限于对其它肿瘤细胞进行体外增殖抑制活性测试。
1、细胞培养：U87MG和SK-BR-3细胞分别用10％FBS的EMEM培养基(GE，货号SH30024.01)和含10％FBS的McCoy's 5A培养基(Gibco，货号16600-108)培养。
2、细胞准备。取对数生长期的U87MG和SK-BR-3细胞，用PBS(磷酸盐缓冲液，上海源培生物科技股份有限公司)洗涤1次之后，加入2-3mL胰蛋白酶(0.25％Trypsin-EDTA(1x)，Gibico,Life Technologies公司)消化2-3分钟，待细胞消化完全后，加入10-15mL细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5分钟，弃上清，接着加入10-20mL细胞培养液将细胞重悬，制成单细胞悬液。
3、细胞铺板。将U87MG和SK-BR-3单细胞悬液混匀，用细胞培养液分别调整活细胞密度至2.75×10 3cells/mL和8.25×10 3细胞/mL，将密度调整过后的细胞悬液混匀，以180μL/孔加入96孔细胞培养板。96孔板外周孔只加入200μL培养基。将培养板在培养箱培养24小时(37℃，5％CO 2)。
4、化合物准备。用DMSO(二甲基亚砜，上海泰坦科技股份有限公司)溶解化合物，配制成初始浓度为10mM的存储液。
小分子化合物的起始浓度为500nM，配药方法如下。
在96孔U型底配药板第一列中分别加入30μL不同待测样品，样品浓度为100μM；第2列至第11列每孔中加入20μL DMSO。取第一列样品10μL至第二列20μL DMSO中，混匀，取10μL至第三列中，以此类推至第10列。将配药板中的药每孔取5μL至95μL EMEM培养基中，混匀，待用。
ADC的起始浓度为10nM或500nM，配药方法如下。
在96孔板第一列中分别加入100μL不同待测样品，样品浓度为100nM或5μM；第2列至第11列每孔中加入100μL PBS。取第一列样品50μL至第二列100μL PBS中，混匀，取50μL至第三列中，以此类推3倍稀释至第10列。
5、加样操作。向培养板中加入20μL配置的不同浓度的待测样品，每个样品两复孔。将培养板在培养箱孵育6天(37℃，5％CO 2)。
6、显色操作。取出96孔细胞培养板，向每孔加入90μL CTG溶液，室温孵育10分钟。
7、读板操作。取出96孔细胞培养板，置于酶标仪(BMG labtech，PHERAstar FS)中，用酶标仪测定化学发光。
三、数据分析
用Microsoft Excel，Graphpad Prism 5对数据进行处理分析。实施例结果参见下表。
表15本披露中的小分子片段对SK-BR-3细胞和U87细胞体外增殖抑制的IC 50值
Figure PCTCN2020135680-appb-000069
结论：本披露中的小分子片段对SK-BR-3细胞和U87细胞具有明显的增殖抑制活性，手性中心对化合物的抑制活性有一定影响。
测试例7：ADC分子细胞活性实验
本实验通过利用CellTiter-Glo Luminescence Cell Viability Assay检测ADC分子在体外对人胃癌细胞株的杀伤作用。第一天，收集NUGC4-claudin18.2低表达，NUGC4-claudin18.2中表达，NUGC4-claudin18.2高表达细胞，调整密度为2.5×10 4/mL，在96孔白色透明底板中加入90μL/孔，约为2500个细胞每孔。37℃，5％CO 2培养箱过夜培养。第二天，在U底96孔板中稀释样品，起始浓度为5μM，4×梯度稀释，9个浓度点，向细胞板中加入10μL/孔稀释好的样品。37℃，5％CO 2培养6天。第八天，取出细胞培养板，加入50μL/孔Cell Titer-Glo Reagent，室温放置2～3分钟，在PHERAstar FS读板器上读取荧光数值。利用GraphPad Prism软件进行数据分析。见表16。
表16本披露的ADC分子体外细胞杀伤作用
Figure PCTCN2020135680-appb-000070
体内活性生物学评价
测试例8：ADC分子体内药效评价
Balb/c裸在右肋部皮下接种人胃癌细胞NUGC4(Claudin18.2中等表达)细胞(5×10 6含50％matrigel基质胶/只)，第0日分组，8只/组，共5组。平均瘤体积约84.41mm 3。
ADC腹腔注射，共给药3次，每只按体重注射10g/0.1mL，分别于第0日，4日，11日给药。
分组当天ADC腹腔注射，共给药4次，间隔5天给药，每只按体重注射10g/0.1mL。
每周测量2次瘤体积和体重，记录数据。
使用Excel 2003统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对体积(RTV)＝VT/V0
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)
其中V0、VT分别为实验开始时(首次给药当天为第0天)及测量计数时的肿瘤体积。CRTV、TRTV分别为实验结束时的空白对照组及实验组的相对肿瘤体积。结果见表17和图4、图5。
表17 ADC抑瘤实验结果
Figure PCTCN2020135680-appb-000071
Figure PCTCN2020135680-appb-000072
vs空白：**p<0.01。

Claims (26)

  1. 一种通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐：
    Figure PCTCN2020135680-appb-100001
    其中：
    Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-和-S-(CR aR b) m-CR 1R 2-C(O)-；
    R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基和杂环基；或者，R a和R b与其相连接的碳原子一起形成环烷基或杂环基；
    R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
    或者，R a和R 2与其相连的碳原子一起形成环烷基或杂环基；
    m为0至4的整数；
    n为1至10，n是小数或整数；
    L为接头单元；
    Pc为抗Claudin18.2抗体或其抗原结合片段。
  2. 根据权利要求1所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    i)所述重链可变区包含与如SEQ ID NO：3序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：4序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3；或
    ii)所述重链可变区包含与如SEQ ID NO：5序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：6序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3。
  3. 根据权利要求1或2所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其 药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    iii)所述重链可变区包含序列分别如SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含序列分别如SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14所示的LCDR1、LCDR2和LCDR3；或
    iv)所述重链可变区包含序列分别如SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含序列分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3。
  4. 根据权利要求1至3中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体是鼠源抗体、嵌合抗体或人源化抗体。
  5. 根据权利要求1至4中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区氨基酸序列如SEQ ID NO:3所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:4所示或与其有至少90％同一性；
    (2)所述重链可变区氨基酸序列如SEQ ID NO:24所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:21所示或与其有至少90％同一性；
    (3)所述重链可变区氨基酸序列如SEQ ID NO:5所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:6所示或与其有至少90％同一性；或
    (4)所述重链可变区氨基酸序列如SEQ ID NO:31所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:28所示或与其有至少90％同一性。
  6. 根据权利要求1至5中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体为人源化抗体，所述人源化抗体包含来源自人抗体的框架区或其框架区变体，所述框架区变体为在人抗体的轻链框架区和/或重链框架区上分别具有至多10个氨基酸的回复突变；
    优选地，所述框架区变体包含选自以下(a)或(b)所述的突变：
    (a)所述轻链可变区中包含任选自22S、85I和87H中的一个或更多个氨基酸回复突变，和/或所述重链可变区中包含任选自48I、82T和69M中的一个或更多个氨基酸回复突变；或
    (b)所述轻链可变区中包含任选自4L和22S中的一个或更多个氨基酸回复突变，和/或所述重链可变区中包含任选自38K、40R、48I、66K、67A、69L、71L和73K中的一个或更多个氨基酸回复突变；
    优选地，所述框架区变体包含选自以下所述的突变：
    (a-1)所述轻链可变区中包含22S、85I和87H的氨基酸回复突变，和所述重链可变区中包含48I和82T的氨基酸回复突变；或
    (b-1)所述轻链可变区中包含4L的氨基酸回复突变。
  7. 根据权利要求1至6中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段包含如下所示的重链可变区和轻链可变区：
    (vii)所述重链可变区序列如SEQ ID NO:3所示和所述轻链可变区序列如SEQ ID NO：4所示；
    (viii)所述重链可变区序列如SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:27所示和所述轻链可变区序列如SEQ ID NO：21、SEQ ID NO:22或SEQ ID NO:23所示；
    (ix)所述重链可变区序列如SEQ ID NO:5所示和所述轻链可变区序列如SEQ ID NO：6所示；或
    (x)所述重链可变区序列如SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33或SEQ ID NO:34所示和所述轻链可变区序列如SEQ ID NO：28、SEQ ID NO:29或SEQ ID NO:30所示；
    优选地，所述抗Claudin18.2抗体或其抗原结合片段包含如下所示的重链可变区和轻链可变区：
    (xi)所述重链可变区序列如SEQ ID NO:31所示和所述轻链可变区序列如SEQ ID NO:29所示；或
    (xii)所述重链可变区序列如SEQ ID NO:26所示和所述轻链可变区序列如SEQ ID NO:23所示。
  8. 根据权利要求1至7中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段包含抗体重链恒定区和轻链恒定区；
    优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；
    更优选地，所述抗Claudin18.2抗体或其抗原结合片段包含序列如SEQ ID NO:7所示的重链恒定区和序列如SEQ ID NO:8所示的轻链恒定区；
    最优选所述抗Claudin18.2抗体或其抗原结合片段包含：与如SEQ ID NO:35或SEQ ID NO:42所示的重链具有至少90％序列同一性的重链，和与如SEQ ID NO:36或SEQ ID NO:39所示的轻链具有至少90％序列同一性的轻链；或
    与如SEQ ID NO:37或SEQ ID NO:49所示的重链具有至少90％序列同一性的重链，和与如SEQ ID NO:38或SEQ ID NO:46所示的轻链有至少90％序列同一性的轻链。
  9. 根据权利要求1至8中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体或其抗原结合片段，包含：
    (c)序列如SEQ ID NO:35所示的重链和序列如SEQ ID NO：36所示的轻链；
    (d)序列如SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44或SEQ ID NO:45所示的重链和序列如SEQ ID NO：39、SEQ ID NO:40或SEQ ID NO:41所示的轻链；
    (e)序列如SEQ ID NO:37所示的重链和序列如SEQ ID NO：38所示的轻链；或
    (f)序列如SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51或SEQ ID NO:52所示的重链和序列如SEQ ID NO：46、SEQ ID NO:47或SEQ ID NO:48所示的轻链。
  10. 根据权利要求1至9中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中所述抗Claudin18.2抗体选自:
    h1901-11：包含氨基酸序列如SEQ ID NO:44所示的重链，和如SEQ ID NO:41所示的轻链；或
    h1902-5：包含氨基酸序列如SEQ ID NO:49所示的重链，和如SEQ ID NO:47所示的轻链。
  11. 根据权利要求1至10中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中n为2至8的小数或整数，优选为3.5至4.5的小数或整数。
  12. 根据权利要求1至11中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，
    其中：
    Y为-O-(CR aR b) m-CR 1R 2-C(O)-；
    R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素或烷基；
    R 1为卤代烷基或C 3-6环烷基；
    R 2选自氢原子、卤代烷基或C 3-6环烷基；
    或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
    m为0或1。
  13. 根据权利要求1至12中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中Y选自：
    Figure PCTCN2020135680-appb-100002
    其中Y的O端与接头单元L相连。
  14. 根据权利要求1至13中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
    L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 3-W-C(O)-或-C(O)-W-C(O)-，其中W选自C 1-8烷基、C 1-8烷基-C 3-6环烷基和1至8个链原子的直链杂烷基，所述杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、C 1-8烷基-C 3-6环烷基或1至8个链原子的直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-或化学键，其中p 1为1至20的整数；
    L 3为由2至7个氨基酸构成的肽残基，其中所述的氨基酸选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
    L 4选自-NR 5(CR 6R 7) t-、-C(O)NR 5-、-C(O)NR 5(CH 2) t-和化学键，其中t为1至6的整数；
    R 3、R 4和R 5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
    R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基。
  15. 根据权利要求1至14中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
    L 1为
    Figure PCTCN2020135680-appb-100003
    s 1为2至8的整数；
    L 2为化学键；
    L 3为四肽残基；优选为GGFG的四肽残基；
    L 4为-NR 5(CR 6R 7)t-，R 5、R 6或R 7相同或不同，且各自独立地为氢原子或烷基，t为1或2；
    其中L 1端与Pc相连，L 4端与Y相连。
  16. 根据权利要求1至15中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中-L-为：
    Figure PCTCN2020135680-appb-100004
  17. 根据权利要求1至16中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其中-L-Y-任选自：
    Figure PCTCN2020135680-appb-100005
  18. 根据权利要求1至17中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L a-Y-D)所示的配体-药物偶联物或其药学上可接受的盐：
    Figure PCTCN2020135680-appb-100006
    其中：
    W、L 2、L 3、R 5、R 6、R 7如权利要求14所定义；
    Pc、n、R 1、R 2、m如权利要求1中所定义。
  19. 根据权利要求1至18中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L b-Y-D)所示的配体-药物偶联物或其药学上可接受的盐：
    Figure PCTCN2020135680-appb-100007
    其中：
    s 1为2至8的整数；
    Pc、R 1、R 2、R 5～R 7、m和n如权利要求18中所定义。
  20. 根据权利要求1至19中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，所述配体-药物偶联物选自：
    Figure PCTCN2020135680-appb-100008
    Figure PCTCN2020135680-appb-100009
    其中Pc和n如权利要求1中所定义。
  21. 根据权利要求1至20中任一项所述的通式(Pc-L-Y-D)所示的配体-药物偶联物或其药学上可接受的盐，所述配体-药物偶联物选自：
    Figure PCTCN2020135680-appb-100010
    其中，n如权利要求1所定义；抗体h1902-5，h1901-11如权利要求10中定义。
  22. 一种制备如通式(Pc-L a-Y-D)所示的配体-药物偶联物或其药学上可接受的盐的方法，其包括以下步骤：
    Figure PCTCN2020135680-appb-100011
    Pc’与通式(L a-Y-D)所示的化合物进行偶联反应，得到通式(Pc-L a-Y-D)所示的化合物；
    其中：
    Pc为抗Claudin18.2抗体或其抗原结合片段；Pc’为Pc经还原后所得；
    W、L 2、L 3、R 1、R 2、R 5～R 7、m和n如权利要求18中所定义。
  23. 一种药物组合物，其包含根据权利要求1至21中任一项所述的配体-药物偶联物或其药学上可接受的盐，以及一种或多种药学上可接受的赋形剂、稀释剂或载体。
  24. 根据权利要求1至21中任一项所述的配体-药物偶联物或其药学上可接受的盐或根据权利要求23所述的药物组合物，在制备用于治疗Claudin18.2介导的疾病或病症的药物中的用途。
  25. 根据权利要求24所述的用途，其中所述Claudin18.2介导的疾病或病症为Claudin18.2高表达癌症。
  26. 根据权利要求1至21中任一项所述的配体-药物偶联物或其药学上可接受的盐或根据权利要求23所述的药物组合物在制备治疗和/或预防肿瘤和癌症的药物中的用途，其中所述肿瘤和癌症优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌 肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
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