WO2021228141A1 - 抗体药物缀合物及其制备方法和用途 - Google Patents

抗体药物缀合物及其制备方法和用途 Download PDF

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WO2021228141A1
WO2021228141A1 PCT/CN2021/093348 CN2021093348W WO2021228141A1 WO 2021228141 A1 WO2021228141 A1 WO 2021228141A1 CN 2021093348 W CN2021093348 W CN 2021093348W WO 2021228141 A1 WO2021228141 A1 WO 2021228141A1
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antibody
cancer
seq
sequence
tumor
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PCT/CN2021/093348
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English (en)
French (fr)
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肖亮
薛彤彤
王晶翼
王成
刘登念
田强
宋帅
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四川科伦博泰生物医药股份有限公司
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Priority to MX2022014332A priority Critical patent/MX2022014332A/es
Priority to EP21804105.1A priority patent/EP4151235A1/en
Priority to CA3178406A priority patent/CA3178406A1/en
Priority to CN202180032620.1A priority patent/CN115715202A/zh
Application filed by 四川科伦博泰生物医药股份有限公司 filed Critical 四川科伦博泰生物医药股份有限公司
Priority to PE2022002658A priority patent/PE20230373A1/es
Priority to AU2021270940A priority patent/AU2021270940A1/en
Priority to CR20220580A priority patent/CR20220580A/es
Priority to JP2022568760A priority patent/JP7407973B2/ja
Priority to US17/998,772 priority patent/US20230338565A1/en
Priority to KR1020227043736A priority patent/KR20230012000A/ko
Priority to IL298184A priority patent/IL298184A/en
Publication of WO2021228141A1 publication Critical patent/WO2021228141A1/zh
Priority to CONC2022/0016208A priority patent/CO2022016208A2/es
Priority to DO2022000251A priority patent/DOP2022000251A/es
Priority to JP2023213677A priority patent/JP2024037960A/ja

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    • A—HUMAN NECESSITIES
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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
    • A61K47/6863—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 the tumour determinant being from stomach or intestines cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the technical field of biomedicine. Specifically, the present invention relates to an anti-Claudin 18.2 antibody-drug conjugate (ADC) and a composition containing the ADC, and their uses.
  • ADC anti-Claudin 18.2 antibody-drug conjugate
  • the treatment of gastric cancer is mainly based on surgical resection.
  • chemotherapy is the main treatment method, but under existing conditions, chemotherapy can only improve symptoms and prolong survival.
  • biomacromolecule drugs except for Trastuzumab, Ramucirumab, Pembrolizumab, etc., which have been approved, most of the other targeted therapy drugs for gastric cancer have unsatisfactory results or It is still in the early stages of clinical research.
  • the core defect of existing drugs is the unmet clinical needs of non-surgical treatment.
  • the treatment options for advanced or recurrent gastric cancer are very limited, the prognosis is extremely poor, and the mortality rate is high.
  • gastric cancer drugs are currently in great demand.
  • Claudin 18.2 (CLDN 18.2) is a member of the Claudin protein family.
  • Claudin family proteins are a class of proteins that mediate tight junctions between cells.
  • Claudin proteins of different subtypes are expressed in different tissues and are related to different types of cancer.
  • Claudin 18.2 is only expressed in gastric mucosal cells in normal tissues and not expressed in other normal tissues.
  • Claudin 18.2 is highly expressed in 70% of primary gastric adenocarcinoma and its metastases. It is also expressed in other cancers such as pancreatic cancer, esophageal cancer and non-small cell lung cancer. The highly specific expression of Claudin 18.2 in tumor tissues makes Claudin 18.2 a very good target for tumor immunotherapy.
  • ADC Claudin 18.2 antibody-drug conjugate
  • the inventors connected an antibody with high affinity and specific binding to Claudin 18.2 to a biologically active molecule through a linker to obtain a class of antibody-drug conjugates targeting Claudin 18.2 and a composition containing the conjugate.
  • the conjugate or composition has high killing activity against some tumor cells expressed by Claudin 18.2.
  • the conjugate can effectively inhibit the growth of Claudin 18.2 positive tumors, especially gastric cancer or gastric adenocarcinoma, and has high safety.
  • the present invention provides an antibody-drug conjugate that specifically binds to human CLDN 18.2, the structure of which is shown in formula (I),
  • D is a biologically active molecule fragment
  • L is a linker
  • ⁇ is selected from an integer between 1-10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); preferably, ⁇ is selected from an integer between 1-8 (such as 1, 2, 3, 4, 5, 6, 7 or 8);
  • A is an antibody or an antigen-binding fragment thereof that specifically binds to human CLDN 18.2.
  • the antibody or antigen-binding fragment thereof that specifically binds to human CLDN18.2 comprises:
  • CDR-L1, CDR-L2 and CDR-L3 contained in the VL (light chain variable region) shown in SEQ ID NO: 15 or 24;
  • the following three light chain CDRs (1) the CDR-L1 or its variants containing amino acid mutations, (1) the CDR-L2 or its variants containing amino acid mutations, CDR-L3 described in (1) or a variant containing amino acid mutations compared with it;
  • At least one of the three heavy chain CDRs and/or three light chain CDRs described in (2) contains amino acid mutations compared with the corresponding CDRs in (1), and the amino acid mutations are one or several amino acids
  • the substitution, deletion or addition for example, substitution, deletion or addition of 1, 2, or 3 amino acids
  • the antibody or antigen-binding fragment containing the mutation can still specifically bind to CLDN 18.2; preferably, specifically Combines human CLDN 18.2; preferably, the substitution is a conservative substitution.
  • the CDRs are defined according to the IMGT or AbM numbering system.
  • the antibody or antigen-binding fragment thereof further includes framework regions (FRs) from human immunoglobulins.
  • FRs framework regions
  • the antibody or antigen-binding fragment thereof comprises:
  • the three light chain CDRs defined by the IMGT numbering system are as follows: CDR-L1 with the sequence of SEQ ID No: 4, CDR-L2 with the sequence of SEQ ID No: 5, and CDR-L3 with the sequence of SEQ ID No: 6;
  • CDR-L1 described in (1-1) or its variants containing amino acid mutations
  • CDR-L2 described in (1-1) or its variants containing amino acid mutations
  • At least one of the three heavy chain CDRs and/or three light chain CDRs contains an amino acid mutation compared with the corresponding CDR in (1-1), and the amino acid mutation is a substitution of one or several amino acids , Deletion or addition (for example, substitution, deletion or addition of 1, 2, or 3 amino acids); the antibody or antigen-binding fragment containing the mutation can still specifically bind to human CLDN 18.2; preferably, the The substitution is a conservative substitution;
  • the three light chain CDRs defined by the AbM numbering system are as follows: CDR-L1 with the sequence of SEQ ID No: 10, CDR-L2 with the sequence of SEQ ID No: 11, and CDR-L3 with the sequence of SEQ ID No: 12;
  • CDR-L1 described in (2-1) or its variants containing amino acid mutations and CDR-L2 described in (2-1) or its variants containing amino acid mutations
  • CDR-L3 described in (2-1) or a variant containing amino acid mutations compared with it A variant of CDR-L3 described in (2-1) or a variant containing amino acid mutations compared with it;
  • At least one of the three heavy chain CDRs and/or three light chain CDRs contains an amino acid mutation compared with the corresponding CDR in (2-1), and the amino acid mutation is a substitution of one or several amino acids , Deletion or addition (for example, substitution, deletion or addition of 1, 2 or 3 amino acids); the antibody or antigen-binding fragment containing the mutation can still specifically bind to human CLDN 18.2; preferably, the Substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof further includes framework regions (FRs) from human immunoglobulins.
  • FRs framework regions
  • the antibody or antigen-binding fragment thereof comprises:
  • VH and/or VL where CDRs are defined according to the IMGT numbering system:
  • (1-1) VH containing the following 3 CDRs: CDR-H1 with the sequence of SEQ ID No: 1, CDR-H2 with the sequence of SEQ ID Nos: 2 or 21, and CDR of the sequence SEQ ID No: 3 -H3; and/or,
  • VL containing the following 3 CDRs: CDR-L1 with the sequence of SEQ ID No: 4, CDR-L2 with the sequence of SEQ ID No: 5, and CDR-L3 with the sequence of SEQ ID No: 6;
  • At least one CDR contains a mutation, which is a substitution, deletion or addition of one or several amino acids or any combination thereof (for example, 1 One, two or three amino acid substitutions, deletions or additions or any combination thereof); preferably, the substitution is a conservative substitution; the antibody or antigen-binding fragment containing the mutation can still specifically bind to CLDN 18.2 ; Preferably, it specifically binds to human CLDN 18.2;
  • VH and/or VL where CDRs are defined according to the AbM numbering system:
  • VL containing the following 3 CDRs: CDR-L1 with the sequence of SEQ ID No: 10, CDR-L2 with the sequence of SEQ ID No: 11, and CDR-L3 with the sequence of SEQ ID No: 12;
  • At least one CDR contains a mutation, which is a substitution, deletion or addition of one or several amino acids or any combination thereof (for example, 1 One, two or three amino acid substitutions, deletions or additions or any combination thereof); preferably, the substitution is a conservative substitution; the antibody or antigen-binding fragment containing the mutation can still specifically bind to CLDN 18.2 ; Preferably, it specifically binds to human CLDN 18.2.
  • a mutation which is a substitution, deletion or addition of one or several amino acids or any combination thereof (for example, 1 One, two or three amino acid substitutions, deletions or additions or any combination thereof); preferably, the substitution is a conservative substitution; the antibody or antigen-binding fragment containing the mutation can still specifically bind to CLDN 18.2 ; Preferably, it specifically binds to human CLDN 18.2.
  • the VH and/or VL of the antibody or antigen-binding fragment thereof includes framework regions (FRs) from human or murine immunoglobulins.
  • the antibody or antigen-binding fragment thereof comprises:
  • the VH contained in the antibody or antigen-binding fragment thereof has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%. %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; and/or, the antibody or antigen-binding fragment thereof Compared with the VL described in (1), the contained VL has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%.
  • the antibody or antigen-binding fragment thereof containing the same sequence can still specifically bind to CLDN 18.2; preferably, Specifically bind to human CLDN 18.2;
  • the VH contained in the antibody or antigen-binding fragment has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, one, two , 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); and/or, the VL contained in the antibody or antigen-binding fragment thereof is compared with the VL described in (1), Have one or several amino acid substitutions, deletions or additions or any combination thereof (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); preferably, the The substitutions are conservative substitutions.
  • the mutation-containing antibody or antigen-binding fragment thereof can still specifically bind to CLDN 18.2; preferably, it specifically binds to human CLDN 18.2.
  • the antibody or antigen-binding fragment thereof may further comprise a constant region derived from a mammalian (e.g., murine or human) immunoglobulin or a variant thereof.
  • the antibody may comprise:
  • the CH (heavy chain constant region) of a human immunoglobulin or a variant thereof which has one or more amino acid substitutions, deletions or additions (for example, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions); and/or ,
  • CL light chain constant region of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions (for example, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions).
  • the constant region is changed, for example, mutated to modify the properties of the anti-CLDN18.2 antibody molecule (e.g., to change one or more of the following properties: Fc receptor binding, antibody glycosylation, cysteine Number of amino acid residues, effector cell function or complement function).
  • the function can be changed by replacing at least one amino acid residue in the constant region of the antibody with a different residue, for example, changing the affinity of the antibody to the effector ligand (such as FcR or complement C1q), thereby changing the effector function (such as enhancing ).
  • the constant region is altered to alter (eg enhance) antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular phagocytosis (ADCP).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • the CH is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.
  • the antibody or antigen-binding fragment thereof comprises the heavy chain constant region of human IgG1.
  • the antibody or antigen-binding fragment thereof comprises CH as shown in SEQ ID NO: 16 or a variant thereof, which has conservative substitutions of up to 20 amino acids compared with SEQ ID NO: 16 (For example, conservative substitutions of up to 15, up to 10, or up to 5 amino acids; for example, conservative substitutions of 1, 2, 3, 4, or 5 amino acids).
  • the CH containing mutations retains substantially the same function as SEQ ID NO: 16.
  • the CL is selected from the light chain constant region of kappa or lambda.
  • the CL is a kappa light chain constant region (e.g., human kappa light chain).
  • the antibody or antigen-binding fragment thereof comprises CL as shown in SEQ ID NO: 17, or a variant thereof, which has conservative substitutions of up to 20 amino acids compared to SEQ ID NO: 17 (For example, conservative substitutions of up to 15, up to 10, or up to 5 amino acids; for example, conservative substitutions of 1, 2, 3, 4, or 5 amino acids).
  • the CL containing the mutation retains substantially the same function as SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof comprises CH as shown in SEQ ID NO: 16 and/or CL as shown in SEQ ID NO: 17.
  • the antibody includes a heavy chain composed of the VH of the sequence shown in SEQ ID NO: 13 and the CH of SEQ ID NO: 16, and,
  • the antibody includes a heavy chain consisting of the VH of the sequence shown in SEQ ID NO: 14 and the CH of SEQ ID NO: 16, and,
  • the antibody or antigen-binding fragment thereof comprises:
  • the heavy chain which comprises an amino acid sequence selected from the following:
  • sequence shown in SEQ ID NO: 18 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity;
  • sequence shown in SEQ ID NO: 20 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (1-2) and (2-2) above are conservative substitutions.
  • the CH containing mutations retains substantially the same function as SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof comprises:
  • the heavy chain which comprises an amino acid sequence selected from the following:
  • sequence shown in SEQ ID NO: 19 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity;
  • sequence shown in SEQ ID NO: 20 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (1-2) and (2-2) above are conservative substitutions.
  • the aforementioned antibodies or antigen-binding fragments containing mutations or identical sequences can still specifically bind to human CLDN 18.2.
  • the antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', (Fab') 2 , Fv fragments, disulfide-linked Fv (dsFv), diabody, bispecific Sex antibodies and multispecific antibodies.
  • the (DL) ⁇ - A structure is as shown in formula (II):
  • each of R 1 and R 2 is independently hydrogen (such as protium or deuterium), halogen, carboxylic acid, sulfonic acid, cyano, C 1-6 alkyl, halogenated C 1-6 alkyl, cyano substituted C 1-6 alkyl (for example -CH 2 CN), C 1-6 alkoxy, C 2-10 alkenyl or C 2-10 alkynyl;
  • Z 1 is an amino acid or a peptide composed of 2-10 amino acids ;
  • X 1 and x 2 are each independently 0,1,2,3,4,5 or 6; and L 1 is connected at the position D 1, L 1 is at position 2 is connected to L 2;
  • L 2 is Wherein, y 1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and L 2 is connected to L 1 at position 1, and L 2 is connected to L 3 at position 2;
  • L 3 is selected from 5-12 membered heteroaromatic ring
  • L 4 is Wherein Z 2 is selected from C 1-6 alkylene, C 2-10 alkenylene, C 2-10 alkynylene and C 3-8 cycloalkylene; R 3 is selected from hydrogen (such as protium or deuterium) and C 1-6 alkyl; Z 3 does not exist or is selected from C 1-6 alkylene; or, R 3 and Z 3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; ⁇ is 0, 1 , 5, or 6, and at the second position E and L 4 is connected, is connected at a position L 4 and L 3;
  • each R 4 is independently hydrogen (such as protium or deuterium), ⁇ is 0, 1, 2, 3, 4, 5, or 6, and the 2 position of E is connected to A, and the 1 position of E is connected to L 4 Connected
  • n 1 , m 2 and m 3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • A, D and ⁇ are as described above.
  • the (DL) ⁇ - A structure is as shown in formula (III):
  • R 5 and R 6 are each independently hydrogen (such as protium or deuterium) or C 1-6 alkyl; x 3 is 1, 2, 3, 4, 5 or 6; and, if L 1 'is present, it Connect with D at position 1 and connect with L 1 at position 2.
  • each of R 1 and R 2 is independently hydrogen (such as protium or deuterium), halogen, carboxylic acid, sulfonic acid, cyano, C 1-6 alkyl, halogenated C 1-6 alkyl, cyano substituted C 1-6 alkyl (for example -CH 2 CN), C 1-6 alkoxy, C 2-10 alkenyl or C 2-10 alkynyl;
  • Z 1 is an amino acid or a peptide composed of 2-10 amino acids ;
  • X 1 and x 2 are each independently 0, 1 , 2, 3, 4, 5 or 6; and, at position 1 of L 1 is connected to L 1 '(when L 1 ' exists), or, L 1 Connected to D at position 1 of L (when L 1 'does not exist); Connected to L 5 at position 2 of L 1;
  • L 5 is Wherein, R 7 is hydrogen or C 1-6 alkyl, or R 7 is connected to the N atom on ⁇ -C to form a 5-6 membered heterocyclic group; x 4 is 1, 2, 3, 4, 5 or 6; y 1 is 0, 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10; and L 5 is connected to L 1 at position 1, and L 5 is connected to L 3 at position 2;
  • L 3 is selected from 5-12 membered heteroaromatic ring
  • L 4 is Wherein Z 2 is selected from C 1-6 alkylene, C 2-10 alkenylene, C 2-10 alkynylene and C 3-8 cycloalkylene; R 3 is selected from hydrogen (such as protium or deuterium) and C 1-6 alkyl; Z 3 does not exist or is selected from C 1-6 alkylene; or, R 3 and Z 3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; ⁇ is 0, 1 , 5, or 6, and at the second position E and L 4 is connected, is connected at a position L 4 and L 3;
  • each R 4 is independently hydrogen (such as protium or deuterium), ⁇ is 0, 1, 2, 3, 4, 5, or 6, and the 2 position of E is connected to A, and the 1 position of E is connected to L 4 Connected
  • n 1 , m 2 , m 3 and m 4 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • A, D and ⁇ are as described above.
  • L 1 in the antibody-drug conjugate represented by formula (II) or formula (III) is Wherein, Z 1 is an amino acid or a peptide composed of 2-5 amino acids, wherein the amino acid is selected from Lys, Cit, Val, D-Val, Phe, Leu, Gly, Ala and Asn; preferably, Z 1 is selected from Cit , Lys, Cit-Val and Ala-Val.
  • L 1 is N
  • L 1 is N
  • L 2 in the antibody-drug conjugate represented by formula (II) is And m 1 is 1.
  • L 3 in the antibody-drug conjugate represented by formula (II) or formula (III) is a 5-6 membered heteroaromatic ring, and m 2 is 1.
  • L 3 is triazole, and m 2 is 1.
  • L 4 in the antibody-drug conjugate represented by formula (II) or formula (III) is Wherein Z 2 is a C 1-6 alkylene group, Z 3 is a C 1-6 alkylene group; and m 3 is 1.
  • L 4 is And m 3 is 1.
  • L 1 ′ in the antibody-drug conjugate represented by formula (III) is
  • L 5 in the antibody-drug conjugate represented by formula (III) is Wherein, x 4 is 1, 2, 3 , 4, 5, or 6; y 1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 5 in the antibody-drug conjugate represented by formula (III) is
  • D in the antibody-drug conjugate represented by formula (III) is
  • D-[L 1 -(L 2 ) m1 -(L 3 ) m2 -(L 4 ) m3 -E]- in the antibody-drug conjugate represented by formula (II) is
  • the structure of the antibody-drug conjugate represented by formula (II) is:
  • ⁇ is selected from an integer between 1-10, for example, ⁇ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and A is the aforementioned antibody that specifically binds to human CLDN 18.2 or its Antigen-binding fragments.
  • the antibody-drug conjugate represented by formula (II) is represented by the following formula:
  • ⁇ is an integer from 1 to 10, for example, ⁇ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and A is 2C6.9-hz21.
  • the structure of the antibody-drug conjugate represented by formula (III) is:
  • ⁇ is selected from an integer between 1-10, for example, ⁇ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and A is the aforementioned antibody that specifically binds to human CLDN 18.2 or its Antigen-binding fragments.
  • the antibody-drug conjugate represented by formula (III) is represented by the following formula:
  • ⁇ is an integer from 1 to 10, for example, ⁇ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and A is 2C6.9-hz21.
  • the structure of the antibody-drug conjugate represented by formula (III) is:
  • ⁇ is selected from an integer between 1-10, for example, ⁇ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and A is the aforementioned antibody that specifically binds to human CLDN 18.2 or its Antigen-binding fragments.
  • the antibody-drug conjugate represented by formula (III) is represented by the following formula:
  • ⁇ is an integer from 1 to 10, for example, ⁇ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and A is 2C6.9-hz21.
  • the present invention also provides a composition containing one or more of the above-mentioned antibody-drug conjugates, in which the biologically active molecule fragments (ie D in formula (I)) specifically bind to
  • the molar ratio (DAR value) of the antibody of CLDN18.2 or its antigen-binding fragment (i.e., A in formula (I)) is a decimal or integer between 1-10 (for example, a decimal or integer between 1-8).
  • the composition further contains the aforementioned antibody or antigen-binding fragment thereof.
  • the composition contains 2C6.9-TL001. In some embodiments, the composition is 2C6.9-TL001.
  • the composition contains 2C6.9-TL002. In some embodiments, the composition is 2C6.9-TL002.
  • the composition contains 2C6.9-TL003. In some embodiments, the composition is 2C6.9-TL003.
  • the antibody or antigen-binding fragment thereof of the present invention may be derivatized, for example, linked to another molecule (for example, another polypeptide or protein).
  • another molecule for example, another polypeptide or protein.
  • the derivatization (eg, labeling) of the antibody or antigen-binding fragment thereof will not adversely affect its binding to CLDN 18.2 (especially human CLDN 18.2). Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms.
  • bispecific antibody is produced by cross-linking two or more antibodies (of the same type or of different types).
  • Methods for obtaining bispecific antibodies are well known in the art, and examples thereof include, but are not limited to, chemical cross-linking methods, cell engineering methods (hybrid hybridoma methods), or genetic engineering methods.
  • Another type of derivatized antibody is a labeled antibody.
  • the antibody or antigen-binding fragment thereof of the present invention can be linked to a detectable label.
  • the detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means.
  • Such labels are well known in the art, examples of which include, but are not limited to, enzymes (for example, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radioactive nuclear (E.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin ( PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g.
  • enzymes for example, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.
  • radioactive nuclear E.g., 3H, 125I, 35S, 14C or 32P
  • the detectable label as described above can be detected by methods known in the art.
  • a radioactive label can be detected using photographic film or a scintillation calculator, and a fluorescent label can be detected using a light detector to detect the emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric markers are detected by simply visualizing colored markers.
  • such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
  • the detectable label as described above can be connected to the antibody or antigen-binding fragment thereof of the present invention through linkers of different lengths to reduce potential steric hindrance.
  • the antibody or antigen-binding fragment thereof contained in the antibody-drug conjugate of the present invention is one or more of the aforementioned derivatized antibodies or antigen-binding fragments thereof.
  • the present invention also provides a pharmaceutical composition.
  • the pharmaceutical composition contains one or more of the aforementioned antibody-drug conjugates or compositions.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition further contains other ingredients with anti-tumor activity.
  • the antibody-drug conjugate or composition and other ingredients with anti-tumor activity are in the same formulation unit, or in different formulation units. Therefore, the antibody-drug conjugate or composition of the present invention and other ingredients with anti-tumor activity can be administered simultaneously, separately or sequentially.
  • the other ingredients with anti-tumor activity are biologically active polypeptides or active tablets or chemotherapeutic drugs.
  • the biologically active polypeptide is selected from immune checkpoint inhibitors (e.g., PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody), or cytokines (e.g., interferon , IL-2, IL-15, GM-CSF, IL-7, IL-12, IL-18, IL-21).
  • immune checkpoint inhibitors e.g., PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody
  • cytokines e.g., interferon , IL-2, IL-15, GM-CSF, IL-7, IL-12, IL-18, IL-21.
  • the chemotherapeutic drug is selected from one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, and albumin-bound paclitaxel.
  • the other ingredients with anti-tumor activity are a combination of epirubicin, oxaliplatin and 5-fluorouracil, or a combination of oxaliplatin, leucovorin and 5-fluorouracil.
  • the antibody-drug conjugate or composition in the pharmaceutical composition of the invention is sufficient in a subject:
  • tumor cells especially gastric cancer cells, such as gastric adenocarcinoma cells
  • tumor cells especially gastric cancer cells, such as gastric adenocarcinoma cells
  • the present invention provides the use of the antibody-drug conjugate, the composition, and the pharmaceutical composition in the preparation of drugs for the prevention and/or treatment and/or adjuvant treatment of tumors.
  • the tumor is selected from solid tumors, hematological tumors, and metastatic, refractory, or recurrent lesions of cancer.
  • the tumor or cancer is selected from the group consisting of esophageal cancer, gastrointestinal cancer, gastric adenocarcinoma, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g. non-small cell lung cancer), liver cancer, gastric cancer, stomach cancer Esophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, cholangiocarcinoma, gallbladder Cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g. multiple myeloma), sarcoma, glioblastoma, leukemia.
  • lung cancer e.g. non-small cell lung cancer
  • liver cancer gastric cancer
  • the tumor is selected from gastric cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer).
  • gastric cancer gastric adenocarcinoma
  • GEJ gastroesophageal junction
  • esophageal cancer e.g., esophageal cancer
  • gastrointestinal cancer e.g., pancreatic cancer
  • lung cancer e.g., non-small cell lung cancer
  • the tumor is gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma, such as locally advanced unresectable or metastatic gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma.
  • GEJ gastroesophageal junction
  • the tumor is CLDN18.2 positive, and further, the tumor is HER2 negative.
  • the tumor is HER2 negative.
  • the present invention provides a method of preventing and/or treating tumors in a subject. In another aspect, the invention provides a method of delaying tumor progression in a subject. In another aspect, the present invention provides a method of reducing or inhibiting tumor recurrence in a subject. The method described above includes administering an effective amount of the antibody-drug conjugate, composition or pharmaceutical composition of the present invention to a subject in need thereof.
  • the method described above further comprises administering a second therapy to the subject, the second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy , Viral therapy, adjuvant therapy and any combination thereof.
  • the second therapy can be applied separately or in combination with the methods described above; or, the second therapy can be applied separately or in combination with the methods described above simultaneously or sequentially.
  • the second therapy is chemotherapy.
  • the chemotherapy drug is selected from one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, and albumin-bound paclitaxel.
  • the chemotherapy drug is a combination of epirubicin, oxaliplatin and 5-fluorouracil, or a combination of oxaliplatin, leucovorin and 5-fluorouracil.
  • the combined dosing regimen of oxaliplatin, leucovorin, and 5-fluorouracil is selected from FOLFOX4, FOLFOX6, or m FOLFOX6.
  • the second therapy is immunotherapy.
  • the immunotherapy drugs are selected from immune checkpoint inhibitors (e.g., PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody), or cytokines (e.g., interference Vegetarian, IL-2, IL-15, GM-CSF, IL-7, IL-12, IL-18, IL-21).
  • immune checkpoint inhibitors e.g., PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody
  • cytokines e.g., interference Vegetarian, IL-2, IL-15, GM-CSF, IL-7, IL-12, IL-18, IL-21.
  • the tumor is selected from solid tumors, hematological tumors, and metastatic, refractory, or recurring lesions of cancer.
  • the tumor or cancer is selected from the group consisting of esophageal cancer, gastrointestinal cancer, gastric adenocarcinoma, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g. non-small cell lung cancer), liver cancer, gastric cancer, stomach cancer Esophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, cholangiocarcinoma, gallbladder Cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g. multiple myeloma), sarcoma, glioblastoma, leukemia.
  • lung cancer e.g. non-small cell lung cancer
  • liver cancer gastric cancer
  • the tumor is selected from gastric cancer, gastric adenocarcinoma, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, and lung cancer (e.g., non-small cell lung cancer).
  • gastric cancer gastric adenocarcinoma
  • GEJ gastroesophageal junction
  • esophageal cancer e.g., gastrointestinal cancer
  • pancreatic cancer e.g., non-small cell lung cancer
  • the tumor is gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma, such as locally advanced unresectable or metastatic gastric cancer, gastric adenocarcinoma, or gastroesophageal junction (GEJ) adenocarcinoma.
  • GEJ gastroesophageal junction
  • the tumor is CLDN18.2 positive, and further, the tumor is HER2 negative.
  • the tumor is HER2 negative.
  • the antibody-drug conjugate, composition or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders , Granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections and concentrated solutions for injections), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical composition of the present invention should be sterile and stable under the conditions of production and storage.
  • a preferred dosage form is injection. Such injection may be a sterile injection solution.
  • a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the antibody-drug conjugate or composition in a suitable solvent, and optionally, simultaneously incorporating other desired ingredients (including but not Limited to pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization.
  • the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for storage and use.
  • sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.
  • the antibody-drug conjugate of the present invention may be present in a pharmaceutical composition in a unit dosage form for ease of administration.
  • the antibody-drug conjugate, composition or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal , Intraleaf sheath, intracytoplasmic reticulum, groin, bladder, topical (eg powder, ointment or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (eg, intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
  • the route and/or manner of administration will vary according to the intended purpose.
  • the antibody-drug conjugate, composition or pharmaceutical composition of the present invention is administered by intravenous infusion or injection.
  • the pharmaceutical composition of the present invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of the antibody-drug conjugate or composition of the present invention.
  • “Prophylactically effective amount” refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease.
  • “Therapeutically effective amount” refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who has already suffered from the disease.
  • the therapeutically effective amount of the antibody-drug conjugate or composition of the present invention may vary according to the following factors: the severity of the disease to be treated, the overall state of the patient’s own immune system, and the patient’s general conditions such as age, weight, and gender , The way the drug is administered, and other treatments that are administered at the same time, and so on.
  • the dosage regimen can be adjusted to obtain the best objective response (for example, therapeutic or preventive response).
  • the best objective response for example, therapeutic or preventive response
  • it can be administered in a single dose, it can be administered multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.
  • the typical non-limiting range of the therapeutically or prophylactically effective amount of the antibody-drug conjugate or composition of the present invention is 0.02-100 mg/kg, for example 0.1-100 mg/kg, 0.1-50 mg/kg, or 1-50 mg/kg. It should be noted that the dosage may vary depending on the type and severity of the symptoms to be treated. In addition, those skilled in the art understand that for any particular patient, the specific dosing regimen should be adjusted over time according to the needs of the patient and the professional evaluation of the doctor; the dosage range given here is for illustrative purposes only, and is not limited The use or scope of the pharmaceutical composition of the present invention.
  • the subject may be a mammal, such as a human.
  • FR Antibody framework region amino acid residues other than CDR residues in the variable region of the antibody
  • Kabat The immunoglobulin comparison and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991 ).
  • Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classic rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. People (1989) Nature 342:878-883).
  • IMGT is based on the international immunogenetics information system (The international ImMunoGeneTics information) initiated by Lefranc et al. (IMGT)) for the numbering system, please refer to Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
  • conjugate or “conjugate” are interchangeable and refer to a substance obtained by connecting a biologically active molecule to an antibody through a linker, and the English name is ADC (antibody-drug conjugate).
  • the linker can be connected to the antibody through various chemical bonds.
  • the linker is connected by forming a thioether bond with the sulfhydryl group of the antibody.
  • ADC molecules such as 2C6.9-TL001, 2C6.9-TL002, or 2C6.9-TL003 ADC molecules
  • -S- only represents the thioether bond formed by the linker and the sulfhydryl group of the antibody, It does not mean that -S- is part of the linker.
  • ADC of the present application can be represented by (DL) ⁇ - A, where D is a biologically active molecular fragment; L is a linker; A is an antibody or antigen-binding fragment that specifically binds to human CLDN 18.2; ⁇ is selected from 1. An integer between -10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. ⁇ refers to the number of (DL) attached to each antibody molecule, which is an integer. However, in the ADC preparation process, each antibody molecule may be connected to a different number of (DL). Therefore, generally speaking, the product of the ADC is a composition coupled with a different number of (DL) antibodies. In practice, DAR is usually used to represent the average value of the number of antibodies (DL) attached.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a light chain (LC) and a heavy chain (HC)).
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the constant region of the light chain consists of a domain CL.
  • the constant domain does not directly participate in the binding of antibodies and antigens, but exhibits a variety of effector functions, such as mediating immunoglobulins and host tissues or factors, including various cells of the immune system (for example, effector cells) and classical complement The combination of the first component (C1q) of the system.
  • VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form an antigen binding site.
  • the allocation of amino acids in each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.
  • CDR complementarity determining region
  • the precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J.Mol. Biol. 196:901-917; Chothia et al.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDR contained in the antibody or antigen-binding fragment thereof of the present invention is preferably determined by the IMGT or AbM numbering system.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/ Or compete with the full-length antibody for specific binding to the antigen, which is also referred to as the "antigen-binding portion".
  • an antibody such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/ Or compete with the full-length antibody for specific binding to the antigen, which is also referred to as the "antigen-binding portion”.
  • antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining regions (CDR) Fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), and such polypeptides, It contains at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • full-length antibody means an antibody composed of two “full-length heavy chains” or “heavy chains” and two “full-length light chains” or “light chains”.
  • full-length heavy chain or “heavy chain” refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, and a hinge region ( HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is of an IgE isotype, it optionally also includes a heavy chain constant region CH4 domain.
  • the "full-length heavy chain” is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction.
  • a “full-length light chain” or “light chain” is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
  • the two pairs of full-length antibody chains are connected by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full-length heavy chains.
  • the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
  • the full-length antibody of the present invention includes two antigen binding sites formed by a pair of VH and VL respectively, and the two antigen binding sites specifically recognize/bind the same antigen.
  • the term “Fd fragment” means an antibody fragment composed of VH and CH1 domains;
  • the term “dAb fragment” means an antibody fragment composed of VH domains (Ward et al., Nature 341:544 546 (1989));
  • the term “Fab fragment” means an antibody fragment composed of VL, VH, CL and CH1 domains;
  • the term “F(ab')2 fragment” means a fragment comprising a disulfide bridge connected by a hinge region The antibody fragment of two Fab fragments;
  • the term “Fab' fragment” means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain The Fd fragment (consisting of VH and CH1 domains) is composed.
  • Fv fragment means an antibody fragment composed of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a variable region (such as a Fd fragment, which contains only three CDRs specific to an antigen) can recognize and bind to an antigen, although its affinity may be lower than the complete binding site.
  • Fc fragment means that the second and third constant regions of the first heavy chain of an antibody are formed by disulfide bonding of the second and third constant regions of the second heavy chain. Antibody fragments. The Fc fragment of an antibody has many different functions, but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (see, for example, Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • GGGGS linker having an amino acid sequence
  • Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol.
  • di-scFv refers to an antibody fragment formed by linking two scFvs.
  • the term "diabody” means that its VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow it to be between the two domains of the same chain. Pairing, thereby forcing the domain to pair with the complementary domain of the other chain and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) ), and Poljak RJ et al., Structure 2:1121-1123 (1994)).
  • Each of the aforementioned antibody fragments maintains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • bispecific antibody refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or antibody analog through a coupling arm.
  • the coupling method includes but It is not limited to chemical reaction, gene fusion, protein fusion, polypeptide fusion and enzymatic.
  • Multispecific antibodies include, for example, trispecific antibodies and tetraspecific antibodies. The former is an antibody with three different antigen binding specificities, and the latter is an antibody with four different antigen binding specificities.
  • antibody mimetics refers to the same specific binding to an antigen as an antibody, but without the structure of an antibody. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa.
  • DARPin and fynomer are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa.
  • the designed ankyrin repeat protein (DARPin) is linked to IgG antibody, scFv-Fc antibody fragment or a combination thereof, such as CN104341529A.
  • the anti-IL-17a fynomer binds to the anti-IL-6R antibody, such as WO2015141862A1.
  • the technique for obtaining antibodies can use conventional techniques known to those skilled in the art (for example, recombinant DNA technology or enzymatic or chemical fragmentation methods) to obtain the antigen of the antibody from a given antibody (for example, the antibody provided by the present invention). Binding fragments (for example, the above-mentioned antibody fragments), and specifically screening for antigen-binding fragments of antibodies in the same manner as used for intact antibodies.
  • the terms “monoclonal antibody”, “monoclonal antibody”, and “mAb” have the same meaning and are used interchangeably, which refers to a group of highly homologous antibody molecules.
  • An antibody or a fragment of an antibody that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
  • the monoclonal antibody has high specificity for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which usually include at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen.
  • the modifier "monoclonal” only indicates that the antibody is characterized as being obtained from a group of highly homologous antibodies, and cannot be understood as requiring any specific method to prepare the antibody.
  • the monoclonal antibody of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256:495,1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage Antibody library technology (see, for example, Clackson et al. Nature352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).
  • monoclonal antibodies can be prepared as follows. First immunize mice or other suitable host animals with immunogen (add adjuvant when necessary).
  • the injection method of immunogen or adjuvant is usually subcutaneous multi-point injection or intraperitoneal injection.
  • the immunogen can be pre-conjugated to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host.
  • the adjuvant may be Freund's adjuvant or MPL-TDM or the like. After the animal is immunized, the body will produce lymphocytes that secrete antibodies that specifically bind to the immunogen. In addition, lymphocytes can also be obtained by in vitro immunization.
  • hybridoma cells prepared above can be inoculated into a suitable culture medium for growth, and the culture medium preferably contains one or more substances capable of inhibiting the growth of unfused, parental myeloma cells.
  • HGPRT hypoxanthine guanine phosphotransferase
  • HAT medium hypoxanthine, aminopterin, and thymine
  • the preferred myeloma cells should have the characteristics of high fusion rate, stable antibody secretion ability, and sensitivity to HAT medium.
  • murine myeloma such as MOP-21 or MC-11 mouse tumor-derived strains (THE Salk Institute Cell Distribution Center, San Diego, Calif.
  • Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the Scatchard assay described by Munson et al., Anal. Biochem. 107:220 (1980) can be used to determine the affinity of monoclonal antibodies.
  • the target cell line can pass the standards described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Dilution method for subcloning.
  • Suitable culture medium can be DMEM or RPMI-1640.
  • hybridoma cells can also grow in animals in the form of ascites tumors.
  • immunoglobulin purification methods such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, etc.
  • the monoclonal antibodies secreted by subclonal cells can be removed from the cell culture medium, Separated from ascites or serum.
  • Monoclonal antibodies can also be obtained through genetic engineering recombination technology. Using nucleic acid primers that specifically bind to the heavy and light chain genes of the monoclonal antibody for PCR amplification, DNA molecules encoding the heavy and light chain genes of the monoclonal antibody can be isolated from hybridoma cells. Insert the resulting DNA molecule into an expression vector, then transfect host cells (such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin), and culture them under appropriate conditions. Recombinantly expressed target antibodies can be obtained.
  • host cells such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin
  • Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Subsequently or as an alternative, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column, and the immunospecific antibody can be purified by immunoaffinity chromatography.
  • the purification of immunoglobulin can refer to, for example, D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).
  • murine antibody refers to the fusion of B cells derived from immunized mice with myeloma cells, followed by screening of mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies. Then proceed to screening, antibody preparation and antibody purification. Or, after the antigen invades the mouse body, B cells differentiate and proliferate to form plasma cells, which can produce and secrete antibodies. Stimulated by specific antigens, the production of antibodies is due to the interaction of various immune cells caused by antigens invading the human body, so that the B cells in the lymphocytes differentiate and proliferate to form plasma cells, which can produce and secrete antibodies.
  • chimeric antibody refers to an antibody whose light chain or/and part of its heavy chain is derived from an antibody (which can be derived from a specific species or belong to a certain species).
  • a specific antibody class or subclass), and another part of the light chain or/and heavy chain is derived from another antibody (which can be derived from the same or different species or belong to the same or different antibody class or subclass), but no matter However, it still retains the binding activity to the target antigen (USP 4,816,567 to Capability et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from the first antibody (e.g., murine antibody), and the heavy chain and The light chain variable region is derived from a second antibody (e.g., a human antibody).
  • first antibody e.g., murine antibody
  • second antibody e.g., a human antibody
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology with the sequence of a human antibody.
  • CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from human source.
  • Immunoglobulin (receptor antibody) Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to improve immune cell activity, and ability to enhance immune response.
  • the donor antibody can be a mouse, rat, rabbit, or non-human primate with expected properties (eg, antigen specificity, affinity, reactivity, ability to improve immune cell activity, and/or ability to enhance immune response) Animal-like (e.g., cynomolgus monkey) antibodies.
  • Humanized antibodies can not only retain the expected properties of non-human donor antibodies (such as murine antibodies), but also effectively reduce the immunogenicity of non-human donor antibodies (such as murine antibodies) in human subjects. Therefore, it is particularly advantageous.
  • the expected properties of the humanized antibody eg, antigen specificity, affinity, reactivity, ability to improve immune cell activity, and/or The ability to enhance immune response
  • a high degree of humanization for example, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% humanization degree
  • the framework region (FR) of the humanized antibody of the present invention may include both the amino acid residues of the human acceptor antibody and the corresponding non-human donor antibody.
  • the humanized antibody of the present invention can be prepared according to the sequence of the murine monoclonal antibody prepared above.
  • DNA encoding the heavy and light chains can be obtained from target murine hybridomas and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.
  • the murine immunoglobulin variable region can be linked to the human immunoglobulin constant region using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.).
  • the DNA encoding VH is operably linked to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene.
  • the sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ), DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region.
  • the DNA encoding VL is operably linked to another DNA molecule encoding the light chain constant region CL to obtain the full-length light chain gene (and the Fab light chain gene).
  • the sequence of the human light chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ), DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.
  • transgenic animals can also be used, which can produce no endogenous immunoglobulin after immunization and can produce a complete human antibody library.
  • JH antibody heavy chain joining region
  • Non-limiting examples of the above-mentioned transgenic animals include HuMAb mice (Medarex, Inc.), which contain human immunoglobulin genes encoding unrearranged human heavy chain ( ⁇ and ⁇ ) and ⁇ light chain immunoglobulin sequences. Locus (miniloci), plus targeted mutations that inactivate endogenous mu and ⁇ chain loci (see, for example, Lonberg et al. (1994) Nature 368(6474):856-859); or carrying human heavy chain transgenes and humans The light chain transchromosome "KM MouseTM” (see patent application WO02/43478). Other methods for humanization of antibodies also include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227: 381; Marks et al., J. Mol. Biol. 1991, 222: 581-597; Vaughan et al., 1996 , Nature Biotech 14: 309).
  • degree of humanization is an index used to evaluate the number of non-human amino acid residues in a humanized antibody.
  • the degree of humanization of a humanized antibody can be predicted by the IMGT website DomainGapAlign to predict the homology of the variable region sequence with the human V domain.
  • homologous antibody refers to a variant of an antibody, which contains the same amino acid sequence as the amino acid sequence of the antibody or antigen-binding fragment thereof provided herein. Source, and wherein the variant retains the desired functional properties of the anti-CLDN18.2 antibody of the present invention.
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) or the half maximum effect concentration (EC50) of the interaction.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • Both the "binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio of kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). Any effective method can be used to measure KD, kon and kdis values.
  • bioluminescence interferometry e.g., ForteBio's Octet method
  • surface plasmon resonance technology such as Biacore
  • Kinexa can also be used to measure the dissociation constant.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • the molecules are the same at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453 which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput.
  • conservative substitution means an amino acid substitution that does not adversely affect or change the expected properties of a protein/polypeptide comprising an amino acid sequence.
  • a variant of an antibody obtained by conservatively substituting amino acids retains its source sequence.
  • Biological activity such as specifically binding to CLDN 18.2.
  • conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties, including The ability to form covalent bonds or hydrogen bonds, etc.) is replaced by residues.
  • Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamate), uncharged polar side chains (e.g., glycine , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • alanine, valine, leucine, isoleucine Acid, proline, phenylalanine, methionine), beta branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999) ; And Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which is incorporated herein by reference).
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancement Agents, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugar, NaCl and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol) and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, paraben, chlorobutanol, phenol, sorbic acid and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredients in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , Lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate).
  • prevention refers to a method performed in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., tumor, infection, or autoimmune disease) in a subject.
  • a disease or disorder or symptom e.g., tumor, infection, or autoimmune disease
  • treatment refers to a method performed in order to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing (ie, no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the disease Status, and relief of symptoms (whether partial or full), whether detectable or undetectable.
  • treatment can also refer to prolonging survival compared to expected survival (if not receiving treatment).
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject e.g., human
  • the subject has tumors, infections, or autoimmune diseases, or is at risk of suffering from the aforementioned diseases.
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., tumor, infection, or autoimmune disease
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who have already suffered from the disease. It is completely within the abilities of those skilled in the art to determine such an effective amount.
  • an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general conditions such as age, weight and sex, the way the drug is administered, and other treatments administered simultaneously and many more.
  • immune cell includes cells that have hematopoietic origin and play a role in an immune response, such as lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes , Macrophages, eosinophils, mast cells, basophils and granulocytes.
  • the term "immune response” refers to immune cells (such as lymphocytes, antigen-presenting cells, phagocytes or granulocytes) and soluble macromolecules (including antibodies, cytokines) produced by immune cells or the liver. , And complement), which results in the selective damage, destruction or destruction of invasive pathogens, cells or tissues infected by pathogens, cancer cells, or normal human cells or tissues under autoimmune or pathological inflammation Cleared from the human body.
  • the term "antigen-specific T cell response” refers to an immune response produced by a T cell, and the response is generated when the T cell specific antigen stimulates the T cell.
  • Non-limiting examples of the response produced by T cells upon antigen-specific stimulation include the proliferation of T cells and the production of cytokines such as IL-2.
  • effector function refers to those biological activities attributable to the Fc region (natural sequence Fc region or amino acid sequence variant Fc region) of an antibody, and which vary with the antibody The same type varies.
  • antibody effector functions include, but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP) , Down-regulation of cell surface receptors (such as B cell receptors), B cell activation, cytokine secretion, half-life/clearance of antibodies and antigen-antibody complexes, etc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • Down-regulation of cell surface receptors such as B cell receptors
  • B cell activation such as B cell activation
  • cytokine secretion half-life/clearance of antibodies and antigen-antibody complexes, etc.
  • cancer and “tumor” are used interchangeably and refer to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues, and may metastasize to distant parts of the body through the lymphatic system or bloodstream. Cancers include benign and malignant cancers as well as dormant tumors or micrometastasis. Cancer also includes hematological tumors, especially hematological malignancies.
  • lymphoma includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as spleen cancer and lymph node tumors.
  • exemplary lymphomas include B-cell lymphoma and T-cell lymphoma.
  • B-cell lymphoma including, for example, Hodgkin's lymphoma.
  • T-cell lymphoma including, for example, cutaneous T-cell lymphoma.
  • Hematological malignancies also include leukemias, such as secondary leukemia or acute lymphocytic leukemia.
  • myeloma eg, multiple myeloma
  • other hematological and/or B cell or T cell related cancers include myeloma (eg, multiple myeloma) and other hematological and/or B cell or T cell related cancers.
  • the term "pharmaceutically acceptable” means that when the molecular body, molecular fragment or composition is properly administered to animals or humans, they will not produce adverse, allergic or other adverse reactions.
  • Specific examples of substances that can be used as pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, and the like.
  • the combination therapy includes combining the antibody-drug conjugate, composition or pharmaceutical composition of the present invention with one or more therapeutic agents (e.g., chemotherapeutics) or other prevention or treatment modes (e.g., , Radiotherapy) combined use.
  • therapeutic agents e.g., chemotherapeutics
  • other prevention or treatment modes e.g., Radiotherapy
  • Exemplary therapeutic agents for the second therapy may include chemotherapeutic agents (e.g., mitotic inhibitors), alkylating agents (e.g., Nitrogen Mustard), antimetabolites (e.g., folate analogs), natural products (e.g., Vinca alkaloid Vinca Alkaloid), various reagents (such as platinum coordination complexes), hormones and antagonists (such as adrenal corticosteroids), immunomodulators (such as Bropirimine, Upjohn), etc.
  • chemotherapeutic agents e.g., mitotic inhibitors
  • alkylating agents e.g., Nitrogen Mustard
  • antimetabolites e.g., folate analogs
  • natural products e.g., Vinca alkaloid Vinca Alkaloid
  • various reagents such as platinum coordination complexes
  • hormones and antagonists such as adrenal corticosteroids
  • immunomodulators such as Bropirimine, Upjohn
  • Combination therapies include therapeutic agents that affect the immune response (e.g., enhance or activate the response) and therapeutic agents that affect (e.g., inhibit or kill) tumor/cancer cells.
  • Combination therapy can reduce the possibility of drug-resistant cancer cells.
  • Combination therapy may allow the dose of one or more of the agents to be reduced to reduce or eliminate the adverse effects associated with one or more of the agents.
  • Such combination therapies can have a synergistic therapeutic or preventive effect on underlying diseases, disorders or conditions.
  • “combination” includes therapies that can be administered separately, for example, separately formulated for separate administration (for example, can be provided in a kit), and therapies that can be administered together as a single formulation (ie, "co-formulation") .
  • the antibody-drug conjugate, composition or pharmaceutical composition of the present invention can be administered sequentially.
  • the antibody-drug conjugate, composition or pharmaceutical composition may be administered simultaneously.
  • the antibody-drug conjugate of the present invention can be used in any combination with at least one other (active) agent.
  • HER2 negative refers to the absence of a large amount of HER2 protein on the cell surface, including IHC 1+, or IHC 2+/FISH negative, as well as IHC 0 to 1+ and IHC 1+ to 2+.
  • halogen includes fluorine, chlorine, bromine, and iodine.
  • C 1-6 alkyl means a straight-chain or branched alkyl group containing 1-6 carbon atoms, including, for example, "C 1-4 alkyl” and "C 1-3 alkyl” Etc. Specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methyl Butyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl Base, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl Group, 2-
  • C 1-6 alkylene refers to a divalent group obtained by losing two hydrogen atoms of a straight or branched chain alkane containing 1-6 carbon atoms, including, for example, “C 1-4 alkylene “Group”, “C 1-3 alkylene” and the like, specific examples include but are not limited to: methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene Base or 1,6-hexylene, etc.
  • C 2-10 alkenyl refers to a straight-chain or branched alkenyl group having at least one double bond and a carbon number of 2-10, including, for example, “C 2-6 alkenyl”, “ C 2-4 alkenyl” and so on.
  • Examples include but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl Alkenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexenyl Dienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, etc.
  • C 2-10 alkenylene refers to a divalent group obtained by losing two hydrogen atoms in an olefin containing 2-10 carbon atoms. Including, for example, “C 2-8 alkenylene”, “C 4-6 alkenylene” and the like. Examples thereof include, but are not limited to: 1,5-pentenylene, 1,5-penten-2-enyl, 1,6-hexenylene, and the like.
  • C 2-10 alkynyl refers to a straight-chain or branched alkynyl group having at least one triple bond and a carbon number of 2-10, including, for example, “C 2-6 alkynyl", “ C 2-4 alkynyl” and so on. Examples include but are not limited to: ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3 -Hexynyl, 5-methyl-2-hexynyl, etc.
  • C 2-10 alkynylene refers to a divalent group obtained by losing two hydrogen atoms of an alkyne containing 2-10 carbon atoms. Including, for example, “C 2-8 alkynylene", “C 4-6 alkynylene” and the like. Examples thereof include, but are not limited to: 1,5-pentynylene, 1,5-pentynylene-2-ynyl, 1,6-hexynylene, and the like.
  • C 1-6 alkoxy refers to a group having a structure of C 1-6 alkyl-O-, wherein C 1-6 alkyl is as defined above. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy Group, hexyloxy group, etc.
  • the term "5-12 membered heteroaryl” refers to an aromatic cyclic group containing 5-12 ring members, and at least one of the ring members is a heteroatom selected from N, O, and S. Specific examples include, but are not limited to, 5-10 membered heteroaryl, 5-10 membered nitrogen-containing heteroaryl, 5-6 membered oxygen-containing heteroaryl, etc., such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazole Group, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1, 2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrimidinyl, pyrida
  • the present invention is not limited to the specific methodologies, protocols, cell lines, vectors, or reagents described herein, as they can vary.
  • the terms used herein are only used for the purpose of describing specific embodiments, not for limiting the scope of the present invention.
  • Figure 1A shows the flow cytometric detection of HEK293T-Claudin 18.2 monoclonal stable cell line.
  • Figure 1B shows the flow cytometric assay of L929-Claudin 18.2 monoclonal stable cell line.
  • Figure 1C shows the flow cytometric detection of KATOIII-Claudin 18.2 monoclonal stable cell line.
  • Figure 1D shows the flow cytometric detection of NCI-N87-Claudin 18.2 monoclonal stable cell line.
  • Figure 1E shows the Western blot detection of HEK293T-Claudin 18.1 monoclonal stable cell line.
  • Figure 2 shows 2C6.9-hz21, IMAB362 antibody affinity detection (flow cytometry).
  • Figure 3 shows the 2C6.9-hz21, IMAB362 antibody affinity detection (L929-Claudin 18.2 cells).
  • Figure 4 shows the specificity of 2C6.9-hz21 antibody detected by flow cytometry.
  • Figure 5 shows the CDC killing activity of 2C6.9-hz21 and IMAB362 on HEK293T-Claudin 18.2 cells.
  • Figure 6 shows the detection of ADCC activity of 2C6.9-hz21 and IMAB362 antibodies (HEK293T-Claudin 18.2 cells).
  • Figure 7 shows the HPLC-SEC detection profile of 2C6.9-TL001 (DAR: 3.79).
  • Figure 8 shows the HPLC-SEC detection profile of 2C6.9-TL001 (DAR: 7.12).
  • Figure 9 shows the test results of the affinity between 2C6.9-TL001 (DAR: 7.12) and Claudin 18.2 on the cell membrane surface.
  • Figure 10A shows the detection result of 2C6.9-TL001 (DAR: 7.12) on the killing activity of HEK293T-Claudin 18.2 cells.
  • Figure 10B shows the detection result of 2C6.9-TL001 (DAR: 3.79) on the killing activity of HEK293T-Claudin 18.2 cells.
  • Figure 10C shows the detection result of 2C6.9-TL001 (DAR: 7.12) on the killing activity of HEK293T-Claudin 18.1 cells.
  • Figure 10D shows the detection results of 2C6.9-TL002 and 2C6.9-TL003 on HEK293T-Claudin 18.2 cell killing activity.
  • Figure 10E shows the detection results of 2C6.9-TL002 and 2C6.9-TL003 on HEK293T-Claudin 18.1 cell killing activity.
  • Figure 11A shows the results of the detection of 2C6.9-TL001 (DAR: 7.12) on the killing activity of NUGC-4 cells.
  • Figure 11B shows the results of the detection of the killing activity of 2C6.9-TL001 (DAR: 3.79) on NUGC-4 cells.
  • Figure 11C shows the detection results of 2C6.9-TL002 and 2C6.9-TL003 on the killing activity of NUGC-4 cells.
  • Figure 12A shows the change of tumor volume in each group of mice in the subcutaneous NCI-N87-Claudin18.2 cell transplantation tumor model of Balb/c Nude mice (*: P ⁇ 0.05; ****: P ⁇ 0.0001).
  • Figure 12B shows the changes in body weight of mice in each group in the Balb/c Nude mouse subcutaneous NCI-N87-Claudin18.2 cell transplantation tumor model.
  • Figure 12C shows the results of comparing the efficacy of 2C6.9-TL001 and 2C6.9 mAb + chemotherapy in the CDX model for 11 days in vivo (****: P ⁇ 0.0001).
  • Figure 12D shows the comparison of the efficacy of 2C6.9-TL001 and 2C6.9 mAb+chemotherapy in the 21-day CDX model (****: P ⁇ 0.0001).
  • Figure 12E shows the change of tumor volume in each group of mice in the CDX (NUGC-4) model with different DAR values of 2C6.9-TL001 on 21 days (****: P ⁇ 0.001).
  • Figure 12F shows the changes in the body weight of mice in each group for 21 days in the CDX (NUGC-4) model with different DAR values of 2C6.9-TL001.
  • Figure 12G shows the Balb/c Nude mouse subcutaneously Changes in tumor volume of mice in each group in the gastric cancer GA0006 PDX model at 17 days (****: P ⁇ 0.0001).
  • Figure 12H shows the Balb/c Nude mouse subcutaneously Changes in body weight of mice in each group in 17 days of gastric cancer GA0006 PDX model.
  • Figure 12I shows the Balb/c Nude mouse subcutaneously Changes in tumor volume of mice in each group in 24 days in the GA0006 PDX model of gastric cancer (****: P ⁇ 0.0001).
  • Figure 12J shows the Balb/c Nude mouse subcutaneously Changes in body weight of mice in each group in 24 days in the GA0006 PDX model of gastric cancer.
  • Figure 12K shows the change of tumor volume in each group of mice in the subcutaneous NCI-N87-Claudin18.2 cell transplantation tumor model of Balb/c Nude mice (***: P ⁇ 0.001; ****: P ⁇ 0.0001) .
  • Figure 12L shows the changes in body weight of mice in each group in the Balb/c Nude mouse subcutaneous NCI-N87-Claudin18.2 cell transplantation tumor model.
  • Figure 12M shows the change of tumor volume in each group of mice in the subcutaneous HEK293T-Claudin18.2 cell transplantation tumor model of Balb/c Nude mice (**: P ⁇ 0.01; ****: P ⁇ 0.0001).
  • Figure 12N shows the changes in body weight of mice in each group in the subcutaneous HEK293T-Claudin18.2 cell transplantation tumor model of Balb/c Nude mice.
  • Figure 120 shows the changes in tumor volume of each group of mice in the Balb/c Nude mouse subcutaneous NUGC-4 cell transplantation tumor model (****: P ⁇ 0.0001).
  • Figure 12P shows the changes in body weight of mice in each group in the Balb/c Nude mouse subcutaneous NUGC-4 cell transplantation tumor model.
  • Figure 13 shows the HPLC-SEC detection profile of 2C6.9-TL001 (DAR: 7.40).
  • Methanesulfonyl chloride (462mg, 12.77mmol, purity about 70%) was added dropwise to a solution of belotecan hydrochloride (3g, 6.38mmol) and triethylamine (2.58g, 25.54mmol) in dichloromethane (40mL) at room temperature Under the conditions, the reaction is 2h.
  • compound 3-2 (200mg, 0.8mmol) was dissolved in a mixed solution of tetrahydrofuran and water (4mL/4mL), lithium hydroxide monohydrate (235mg, 5.6mmol) was added, the reaction was stirred at room temperature for 4h, diluted with water, Extract with ethyl acetate (20mL ⁇ 2), adjust the pH to 3 with 1N hydrochloric acid for the aqueous phase, extract with ethyl acetate (20mL ⁇ 3), combine the organic phases, wash with saturated brine (20mL ⁇ 2), and dry with anhydrous sodium sulfate. The desiccant was filtered off, and the solvent was evaporated under reduced pressure to obtain 120 mg of the title compound.
  • Step 1 Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propargyl)hex-5-ynamide (Compound 3-5)
  • Step 2 (4-((S)-35-azido-2-(4-(((4-methoxyphenyl) benzhydryl)amino)butyl)-4,8-diox ((S)-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazatripentacoylamino)benzyl)((S)-4-ethyl -11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4 ': Synthesis of 6,7] indazino[1,2-b]quinolin-4-yl)carbonate (Compound 33-1)
  • Step 3 ((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetra Hydrogen-1H-pyrano[3',4':6,7]indazino[1,2-b]quinolin-4-yl)(4-((S)-2-(4-(( (4-Methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yne Amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonane Synthesis of oxy-3,9-diaza pentapentadecyl amido)benzyl)carbonate (compound 33-2
  • Step 4 (4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yne Amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-Nine Oxa-3,9-diaza pentapentadecylamido)benzyl)((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl) -3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inazino[1,2-b]quinoline-4 -Base) carbonate (compound TL001) synthesis
  • Step 1 (S)-(4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4': Synthesis of 6,7] indazino[1,2-b]quinolin-9-yl)(4-nitrophenyl) carbonate
  • Step 2 (S)-tert-butyl (4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran [3', Synthesis of 4':6,7] indazino[1,2-b]quinolin-9-yl)ethane-1,2-diyl bis(methyl carbamate)
  • Step 3 (S)-N-methyl-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran [3 Synthesis of',4':6,7]indazino[1,2-b]quinolin-9-yl)(2-(methylamino)ethyl)carbamate trifluoroacetate
  • Step 4 (4-((S)-2-((S)-2-(4-(1-(26-azide-3,6,9,12,15,18,21,24-octaoxy Heterohexadecyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)((S)-4,11-diethyl-4-hydroxyl -3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]inazino[1,2-b]quinoline-9- Synthesis of ethane-1,2-diyl bis(methyl carbamate)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Step 5 ((S)-4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4': 6,7]Indazino[1,2-b]quinolin-9-yl)(4-((S)-2-((S)-3-methyl-2-(4-(1-( 26-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl )-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidin-4-yl)butyramido)butyramido)propionamido)benzyl)ethyl Synthesis of Alkyl-1,2-Diyl Bis(Methyl Carbamate) Trifluoroacetate
  • the reaction solution was purified by preparative high performance liquid chromatography.
  • the preparation solution was lyophilized and dissolved in dichloromethane (2mL), added with trifluoroacetic acid (0.2mL), and stirred at 25°C for 0.5h. Purified by preparative high performance liquid chromatography, the preparation solution was freeze-dried to obtain 16 mg of the title compound.
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% trifluoroacetic acid)
  • Mobile phase A acetonitrile
  • mobile phase B water
  • Step 2 4-(4-((S)-1-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino) Synthesis of tert-butyl -3-methyl-1-oxobutyl-2-yl)amino)-4-oxobutyl)piperidine-1-carboxylate
  • Step 3 (S)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methyl-2- Synthesis of (4-(piperidin-4-yl)butyramido)butyramide trifluoroacetate
  • reaction solution was concentrated under reduced pressure, the residue was dissolved in acetonitrile (30 mL), potassium carbonate (1.22 g, 8.85 mmol) was added, and the reaction was carried out at 25° C. for 2 h. Suction filtration, the filter cake was washed with acetonitrile, the filtrate was collected, and concentrated under reduced pressure to obtain 900 mg of the title compound.
  • Step 4 (S)-2-(4-(1-(26-azido-3,6,9,12,15,18,21,24-octaoxahexadecyl)piperidine- 4-yl)butyramido)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutan Synthesis of amide
  • Step 5 (4-((S)-2-((S)-2-(4-(1-(26-azide-3,6,9,12,15,18,21,24-octaoxy Synthesis of Heterohexadecyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)(4-nitrophenyl)carbonate
  • Step 6 (4-((S)-2-((S)-2-(4-(1-(26-azide-3,6,9,12,15,18,21,24-octaoxy Heterohexadecyl)piperidin-4-yl)butyramido)-3-methylbutyramido)propionamido)benzyl)2-((S)-4-ethyl-4-hydroxy- 3,14-Dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]inazino[1,2-b]quinolin-11-yl) Synthesis of ethyl isopropyl carbamate
  • Step 7 (4-((S)-2-((S)-3-methyl-2-(4-(1-(26-(4-((6-(2-(methylsulfonyl) (Pyrimidine-5-yl)hex-5-ynyl amido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24 -Octaoxahexadecyl)piperidin-4-yl)butyramido)butyramido)propionamido)benzyl)2-((S)-4-ethyl-4-hydroxy-3, 14-Dioxy-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]inazino[1,2-b]quinolin-11-yl)ethyl Synthesis of Isopropyl Carbamate Trifluoroacetate
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% trifluoroacetic acid)
  • the human Claudin 18.2 monoclonal antibody in the present invention is a humanized monoclonal antibody, including 2C6.9-hz11 and 2C6.9-hz21.
  • the CDR, variable region sequence and constant region sequence information are shown in Table 1:
  • Table 1 Summary table of sequence information
  • the human Claudin 18.2 complete coding sequence (gene number: NM_001002026.2, synthesized by Nanjing GenScript Biotechnology Co., Ltd.) and the human Claudin 18.1 complete coding sequence (gene number: NM_016369) .3, synthesized by Nanjing GenScript Biotechnology Co., Ltd.) cloned into the lentiviral vector pLVX-IRES-puro, and passed the literature (Mohammadi Z etl., Mol Biotechnol.
  • Lentiviral packaging system prepares the virus, and after the virus is obtained, the HEK293T, L929, KATOIII and NCI-N87 cells are respectively infected, and the monoclonal HEK293T-Claudin 18.1, HEK293T-Claudin 18.2, L929 are obtained through puromycin screening and monoclonal selection.
  • the human Claudin 18.2 and human Claudin 18.1 plasmids were transfected into BaF/3 cells (DSMZ, Cat#ACC300) using the 4D-Nucleofector X Transfection Kit (Lonza, Cat#V4XC-3012), and 1.25 mg/ mL hygromycin (Thermo Fisher Sci.Cat# 10687010) was screened. After 12 days of screening, single clones were sorted to obtain monoclonal BaF/3-Claud 18.1 and BaF/3-Claud 18.2 cell lines.
  • HEK293T-Claudin 18.1 uses Western Blot identification (detection antibody: Proteintech, 66167-1-Ig), other cell lines use flow cytometry (flow cytometer: Beckman, CytoFlex; detection antibody IMAB362, sequence from patent: CN 101312989B ) For identification. As shown in Figures 1A-1D, the flow cytometry results show that HEK293T-Claudin 18.2, L929-Claudin 18.2, KATOIII-Claudin 18.2, and NCI-N87-Claudin 18.2 all have a high positive rate (close to 100%) and good uniformity. Monoclonal cell line can be used for subsequent experiments.
  • the invention uses DNA immunization and cellular immunization to immunize wild-type mice to obtain anti-human Claudin 18.2 murine monoclonal antibodies.
  • Each Balb/c mouse was injected with 100 ⁇ g plasmid containing the complete coding sequence of human Claudin 18.2 by way of tail vein injection.
  • the serum titer was detected by flow cytometry.
  • the mice with higher titer were selected for booster immunization with BaF/3-Claudin18.2 overexpression cell line 3-5 days before fusion.
  • the standard fusion process was used to fuse mouse spleen cells with Sp2/0 (ATCC, Cat#CRL-1581) mouse myeloma cell line using PEG fusion, and then HAT pressure screening was performed, and flow was performed 10-14 days later. ⁇ filtering.
  • the human gastric cancer cell line NUGC4 (purchased from the Japanese JCRB cell bank, catalog number: JCRB0834) endogenously expresses Claudin 18.2 protein, and is widely used to detect the binding of antibodies to endogenous Claudin 18.2 and develop functional detection methods. NUGC4 cells were used to detect candidate clones, and finally 7 subclones were selected as candidate clones. After further affinity detection, 2C6.9M was selected for variable region amplification and humanization.
  • the Pierce Rapid Isotyping kit (Thermo Fisher Sci.Cat#26179) was used to identify 2C6.9M. The identification results showed that the heavy chain was of IgG1 subtype, and the light chain was of Kappa subtype.
  • the number of hybridoma cells was cultured to about 8000, the cells were lysed and the first strand cDNA was synthesized using a cDNA reverse transcription kit (Thermo Fisher Sci. Cat# 18080-200).
  • the primers were used to amplify VH and VK (VL Kappa) genes from cDNA.
  • the PCR products were purified by DNA purification kit (Qiagen, Cat#28104) and ligated to TOPO vector (Thermo Fisher Sci.Cat#K457540). Approximately 12 clones were picked for each ligation reaction and sequenced. The sequence was analyzed by Vector NTI 11.5 (Thermo Fisher Sci.) and Sequencer 5.4.6 (Genecodes). The obtained murine antibody 2C6.9M variable region sequence and CDR sequence are shown in Table 2.
  • the mouse antibody 2C6.9M was humanized by the CDR grafted antibody humanization method.
  • the humanization transformation involves the following steps: compare the amino acid sequence of the mouse monoclonal antibody with the amino acid sequence of the human germline antibody to find a sequence with high homology and superior physical and chemical properties, and use it as a human embryonic line. Framework sequence; analyze and investigate HLA-DR affinity, select the human embryonic framework sequence with low affinity; then transplant the six CDRs of the mouse antibody to the selected heavy chain and light chain framework sequences.
  • the heavy chain and light chain CDR regions of the murine antibody 2C6.9M were respectively grafted onto the FR framework of the corresponding humanized template.
  • the humanized heavy chain and light chain templates of 2C6.9M are human germline gene sequences IGHV4-59*01 (see IMGT accession number AB019438) and IGKV4-1*01 (see IMGT accession number Z00023), respectively.
  • variable region sequence of the murine antibody 2C6.9M is shown in SEQ ID NO: 23 and SEQ ID NO: 24, and its CDR sequence is shown in SEQ ID NO: 1 to SEQ ID NO: 12.
  • the present invention modified the 2C6.9 CDR-H2 amino acid sequence, and the modified sequence is shown in SEQ ID NO: 21 and SEQ ID NO: 22.
  • two humanized antibodies were constructed, named 2C6.9-hz11 and 2C6.9-hz21.
  • the heavy chain constant region of each antibody is a human wild-type IgG1 heavy chain constant region (SEQ ID NO: 16), and the light chain constant region of the antibody is a human wild-type IgG1 kappa light chain constant region (SEQ ID NO: 17).
  • variable region, constant region, and heavy and light chain amino acid sequences of each antibody are shown in Table 1.
  • the heavy chain and light chain amino acid sequences of the 2C6.9 humanized antibody are linked into the plasmid pcDNA3.4 through codon optimization synthesis cDNA (entrusted to Nanjing GenScript Biotechnology Co., Ltd.).
  • the pcDNA3.4 corresponding to the heavy chain and light chain were simultaneously transfected into Expi293F cells (purchased from Thermo), and the supernatant after the cell culture was purified by protein A (MabSelect SuRe, GE) affinity chromatography column to obtain 2C6 .9 humanized monoclonal antibody.
  • HEK293T-Claudin 18.2 cells were used to detect the affinity of 2C6.9-hz21 with human Claudin 18.2 on the cell membrane.
  • the specific steps are as follows: digest HEK293T-Claudin18.2 cells, centrifuge and resuspend, wash twice with PBS; resuspend the cells with PBS (containing 1% BSA), pour 300,000 cells/well into 96-well tip bottom plate, 50 ⁇ l per well Pave 20 wells; add 50 ⁇ L 2C6.9-hz21 and IMAB362 antibodies to each well.
  • the final concentration starts at 1000nM and is diluted by 3 times.
  • Antibody name EC50(nM) Max MFI 2C6.9-hz21 5.767 165866.3 IMAB362 8.217 156715.7
  • Human Claudin 18.2 is a four-time transmembrane membrane protein with a complex structure.
  • Cell ELISA is used for detection to ensure the complete configuration of the Claudin 18.2 antigen molecule.
  • the L929-Claudin 18.2 stable cell line constructed in 6.1 was selected for detection.
  • Antibody name EC50(nM) Maximum signal value (OD450) 2C6.9-hz21 0.12 0.92 IMAB362 0.15 0.53
  • the present invention also detects the specificity of the candidate antibody through flow cytometry.
  • the specific steps are as follows: digest HEK293T, HEK293T-human Claudin 18.1, HEK293T-human Claudin 18.2 cells, centrifuge and resuspend, and wash twice with PBS; resuspend the cells with PBS (containing 1% BSA), each of which takes 300,000 cells Add candidate antibody with a final concentration of 1000nM, mix well, and react for 1 hour at 4°C in the dark; wash 3 times with PBS, add FITC-labeled anti-human Fc secondary antibody (brand: BioLegend, catalog number: 409322), and incubate at 4°C in the dark for 0.5 Hour; PBS wash 3 times, flow cytometry (flow cytometer brand: Beckman, model: Cytoflex) detection.
  • 2C6.9 is an IgG1 subtype, which can effectively activate the classical pathway of complement and exert the complement dependent cytotoxicity (CDC) function.
  • CDC complement dependent cytotoxicity
  • the present invention uses guinea pig serum with rich complement content (purchased from Zhengzhou Beiji, product number: S0001) as the material for experiment.
  • the specific experimental steps are as follows: Take HEK293T-Claudin 18.2 cells, adjust the cell density after centrifugation, and plate 5 ⁇ 10 4 /well overnight; prepare DMEM+20% guinea pig serum medium on the second day, and use this medium to dilute 2C6.9-hz21 And IMAB362 antibody, starting at 20 ⁇ g/mL, 2 times dilution, 10 concentration points; remove the original culture medium of HEK293T-Claudin 18.2 cells, add the antibody diluted in the previous step to the corresponding well, 100 ⁇ L/well; set Completely kill the positive control group, that is, add 10 ⁇ l/well lysis buffer; after standing for 3 hours in a 37°C, 5% CO 2 incubator, add CellTiter-Glo Luminescent (CTG, purchased from Promega, catalog number: G7573) staining solution Stain, 50 ⁇ l/well, mix well for 30 seconds, place at room temperature for 1 minute, measure the fluorescence signal value with
  • Antibody name EC50 value (ng/mL) 2C6.9-hz21 1363 IMAB362 3317
  • ADCC antibody-dependent cellular cytotoxicity
  • 2C6.9 is an IgG1 subtype with strong antibody-dependent cell-mediated cytotoxicity (ADCC) activity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the specific experimental procedures are as follows: Take HEK293T-Claudin 18.2 cells, adjust the cell density after centrifugation, and plate 1 ⁇ 10 4 cells/well overnight; remove the medium from the plate the next day, take NK92MI-CD16a cells (Huabo Bio) and centrifuge with Resuspend in empty MEMA medium, adjust the cell density to 1 ⁇ 10 6 /mL, add 50 ⁇ l/well to the corresponding well; dilute 2C6.9-hz21 and IMAB362 antibody with empty MEMA medium, HEK293T-Claudin18.2 cells 40 ⁇ g/ ml start, 5-fold dilution, 10 concentration points; NUGC-4 cells start at 2mg/mL, 5-fold dilution, 11 concentration points; add 50 ⁇ L/well of diluted antibody to the corresponding well, put it at 37°C, After standing for 5.5 hours in a 5% CO 2 cell incubator, add lysis buffer to the positive control well, and after standing for another 0.5 hours, add lactate dehydr
  • Antibody name EC50 value (ng/mL) Maximum kill rate (%) 2C6.9-hz21 67.67 30 IMAB362 57.66 20
  • the TL001 prepared in Example 3 is coupled with the 2C6.9 humanized monoclonal antibody to prepare 2C6.9-TL001.
  • the preparation method is as follows:
  • Buffer replacement Use 30KDa 50ml ultrafiltration tube (Millipore) to replace 2C6.9-TL001 with 10mM histidine-histidine hydrochloride + 8% sucrose (pH6.0) buffer , The replacement factor is 15 times; collect samples, add 10% Tween-20 to make the final concentration of Tween-20 in the sample 0.02% (M/V).
  • Liquid chromatography column ThermoMAbPac RP 3.0*100mm;
  • Mobile phase B 0.1%FA/2%H2O/98%ACN
  • Switching valve 0-3min to waste, 3-22min to MS, 22-30min to waste
  • Mass spectrometer model AB Sciex Triple TOF 5600+;
  • the antibody light chain (LC) in 2C6.9-TL001 is coupled with 0 to 1 toxin (the ratio of LC and DAR1 are 57.8% and 42.2% respectively)
  • the heavy chain (HC) is coupled with 0 to 4 toxins (the proportions of HC, DAR1, DAR2, DAR3, and DAR4 are 22.5%, 30.3%, 25.0%, 21.9%, 0.3%, respectively), to calculate the coupling between the antibody and the toxin
  • the feeding ratio of TL001 and 2C6.9-hz21 is 9:1
  • the light chain of the antibody in 2C6.9-TL001 is coupled with 0 to 1 toxin (the ratio of LC and DAR1 is 7.5% and 92.5% respectively)
  • heavy chain Coupling 0 to 4 toxins the proportions of HC, DAR1, DAR2, DAR3, and DAR4 are 2.5%, 10.2%, 9.8%, 76.4%, 1.1%, respectively
  • the coupling ratio of antibody to toxin (DAR) is calculated as 7.12.
  • the coupling feed ratio is 9:1
  • the antibody light chain in 2C6.9-TL001 is coupled with 0 to 1 toxin (the ratio of LC, DAR1, respectively) 1.5%, 24.0%)
  • heavy chain coupled with 0 to 4 toxins the proportions of HC, DAR1, DAR2, DAR3, and DAR4 are 0.7%, 2.1%, 14.3%, 54.5%, 2.8%, respectively
  • the antibody to toxin coupling ratio (DAR) was 7.40.
  • the ADC molecular structure in 2C6.9-TL001 is as follows:
  • ⁇ is an integer from 1 to 10
  • A is 2C6.9-hz21.
  • the conjugate was detected by SEC-HPLC.
  • Liquid chromatography column TSKgel G3000SWxl, 300*7.8mm, 5 ⁇ m;
  • Injection volume 40 ⁇ l; isocratic operation: 30min.
  • the SEC chromatograms of 2C6.9-TL001 (DAR: 3.79) and 2C6.9-TL001 (DAR: 7.12) are shown in Figure 7-8, and the SEC chromatograms of 2C6.9-TL001 (DAR: 7.40) are shown in Figure 13
  • the molecular weight of the main coupling product is about 150kD, that is, the 2C6.9-TL001 obtained by coupling TL001 and 2C6.9-hz21 still maintains the intact structure of the antibody.
  • the TL002 and TL003 prepared in Example 4 and Example 5 were coupled with the humanized monoclonal antibody 2C6.9 using the same preparation method to prepare 2C6.9-TL002 and 2C6.9-TL003. Preparation and detection methods As mentioned above, the final DAR values of 2C6.9-TL002 and 2C6.9-TL003 are 6.95 and 7.03, respectively.
  • the ADC molecular structure in 2C6.9-TL002 is as follows:
  • ⁇ is an integer from 1 to 10
  • A is 2C6.9-hz21.
  • the ADC molecular structure in 2C6.9-TL003 is as follows:
  • ⁇ is an integer from 1 to 10
  • A is the 2C6.9 antibody.
  • the present invention uses a cell ELISA method to detect the affinity of 2C6.9-TL001 (DAR: 7.12) with Claudin 18.2 on the cell membrane surface.
  • the specific experimental steps are as follows: The adherent growth of the L929-Claudin 18.2 cell line was digested with 2mM EDTA, resuspended to 2*10 ⁇ 5 cells/ml, 100 ⁇ L per well was plated in a 96-well plate, and incubated overnight at 37°C; Remove the medium, wash once with PBS, add 100 ⁇ L 4% formaldehyde to each well for 30 minutes at room temperature, remove formaldehyde, wash twice with PBS, add 100 ⁇ L PBS (containing 2% BSA) to block for 2 hours; remove the blocking solution, use PBS (containing 2%BSA) diluted to be tested 2C6.9-TL001, 9.375 ⁇ g/mL starting, 4-fold dilution, 9 concentration points, 100 ⁇ L/well added to the well, 37°C for 2
  • HEK293T-Claudin 18.2 and HEK293T-Claudin 18.1 cells are selected to detect the killing activity of 2C6.9-TL001 on Claudin 18.2 high expression cell lines.
  • the specific experimental steps are as follows: the day before the experiment, the cells were diluted with DMEM+10% FBS, 100 ⁇ L/well, and 1*10 ⁇ 4 cells per well were plated overnight; the second day, the ADC molecules were diluted with DMEM basal medium, 150 ⁇ g/mL (DAR : 7.12) or 262.5 ⁇ g/mL (DAR: 3.79) starting, 4-fold dilution, 11 concentration points, add to the corresponding wells, 100 ⁇ L per well, the final serum concentration is 5%; 37°C, 5% CO 2 incubator Incubate in medium for 48 hours; add CCK8 (Rhinogen), 20 ⁇ l/well, 37°C, 5% CO 2 incubator for 0.5-2.5 hours, read with a microplate reader (MD) 450nm every half
  • the experimental results are shown in Figure 10A, Figure 10B and Figure 10C.
  • the 2C6.9-TL001 molecule can effectively kill HEK293T-Claudin18.2 cells.
  • DAR value is 7.12
  • its EC 50 value is 473ng/mL
  • DAR value is 7.12
  • its EC 50 value was 794.1ng/mL.
  • the EC50 value of 2C6.9-TL001 (DAR: 7.12) molecule against HEK293T-Claudin 18.1 was 3900 ng/mL
  • the 2C6.9-TL001 (DAR: 7.12) molecule against Claudin 18.2 cells was significantly higher than Claudin 18.1. Cells (about an 8-fold difference), indicating that this killing effect is specific to Claudin 18.2.
  • Both 2C6.9-TL002 and 2C6.9-TL003 can effectively kill HEK293T-Claudin 18.2 cells, and their EC 50 values are 628.9ng/mL and 540.2ng/mL respectively; 2C6 The EC50 values of 9-TL002 and 2C6.9-TL003 on HEK293T-Claudin18.1 killing activity were 30590ng/mL and 8258ng/mL, respectively. The killing activity of the two on HEK293T-Claudin 18.2 cells was significantly higher than that of Claudin 18.1 cells, indicating that the killing effect of the two is Claudin 18.2 specific.
  • the present invention selects gastric cancer cell line NUGC-4 to detect the killing activity of 2C6.9-TL001 on the endogenously expressing Claudin 18.2 cells.
  • the specific experimental procedures are as follows: the day before the experiment, the cells were diluted with RPMI 1640+10% FBS, 100 ⁇ L/well, and 1*10 ⁇ 4 cells per well were plated overnight; the next day, the 2C6.9-TL001 molecules were gradually diluted with RPMI 1640 medium, Start at 1000 ⁇ g/mL (DAR 7.12) or 1750 ⁇ g/mL (DAR: 3.79), 3 times dilution, 11 concentration points, add to the corresponding wells, 100 ⁇ L per well, the final serum concentration is 5%; 37°C, 5% Incubate in a CO 2 incubator for 72 hours; add CCK8 (Rhinogen), 20 ⁇ l/well, 37°C, 5% CO 2 incubator for 0.5-2.5 hours, read on a microplate reader (MD) at 450nm every half hour and import
  • the killing activity of 2C6.9-TL002 and 2C6.9-TL003 on NUGC-4 cells was detected by the similar experimental method described above.
  • the initial ADC concentration was 500 ⁇ g/mL, diluted 4 times, and 11 concentration points.
  • the experimental results are shown in Figure 11C. Both 2C6.9-TL002 and 2C6.9-TL003 can effectively kill NUGC-4 cells, and their EC 50 values are 54.92 ⁇ g/mL and 123.94 ⁇ g/mL, respectively.
  • the present invention selects NUGC-4 cells to detect the internalization activity of the 2C6.9 antibody.
  • the specific experimental steps are as follows: take NUGC-4 cells, trypsinize and count, resuspend the cells in PBS (containing 1% BSA), adjust the cell density to 3 ⁇ 10 6 /mL; take 100 ⁇ L of the resuspended cells and add the final concentration to 100 ⁇ g/mL 2C6.9-hz21 antibody, with human IgG of the same type as a negative control, incubate on ice for 1 hour; after incubation, wash with pre-cooled PBS 3 times and use NUGC-4 cell culture medium (1640+10% FBS) Resuspend the cells and divide the cells into two parts.
  • endocytosis group [1-(MFI 37°C antibody group -MFI 37°C control group )/(MFI antibody group on ice- MFI ice control group)] ⁇ 100% Internalization rate.
  • the present invention uses Cancer cell line-derived xenograft (CDX) model and patient-derived xenograft (PDX) model to evaluate the anti-tumor effect of ADC molecules.
  • CDX Cancer cell line-derived xenograft
  • PDX patient-derived xenograft
  • NCI-N87-Claudin18.2+Balb/c Nude mouse CDX model Use RPMI1640 medium containing 10% fetal bovine serum (FBS) to culture NCI-N87-Claudin18 under the conditions of 37°C and 5% CO 2. 2 cells. Cells in exponential growth phase were collected, PBS resuspended cells according to the amount of 5 ⁇ 10 6 / is only (suspended in 0.1ml PBS), were inoculated subcutaneously in female Balb / c Nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) Establish a subcutaneous transplanted tumor model, and when the average tumor volume reaches 70-100mm 3 , randomly group according to the tumor volume, 7 in each group.
  • FBS fetal bovine serum
  • the day of grouping is marked as day 0 (Day 0), and the groups are Human IgG1 isotype control antibody (negative control) group (abbreviated as IgG1), 2C6.9-TL001 (DAR: 7.12) 1mg/kg group and 3mg/kg group, 2C6.9-TL002 (DAR: 6.95) 1mg/kg group and 3mg/kg group. All samples were injected via tail vein, twice a week, for a total of 6 administrations.
  • TGI tumor growth inhibition rate
  • TGI(%) [1-(V T end- V T beginning )/(V C end- V C beginning )]*100%
  • V T end mean tumor volume at the end of the experiment in the treatment group
  • V T start mean tumor volume at the beginning of the treatment group
  • V C end the mean tumor volume at the end of the experiment in the negative control group
  • V C start mean tumor volume at the start of administration of the negative control group
  • T/C(%)(tumor volume) (T t /T 0 )/(C t /C 0 ) ⁇ 100%
  • T 0 the average tumor volume of the treatment group at the initial (ie P0)
  • T t The average tumor volume of the treatment group at each measurement
  • C t The average tumor volume of the negative control group at each measurement.
  • 2C6.9-TL001 (DAR: 7.12) has a significant inhibitory effect on the tumor growth of the NCI-N87-Claudin 18.2 gastric cancer xenograft model, and is dose-dependent.
  • TGI tumor inhibition rate
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • PR partial tumor regression
  • CR full tumor regression.
  • P value is compared with IgG1 group; N/A: not applicable; ns: P>0.05, no statistical difference.
  • the NCI-N87-Claudin18.2 subcutaneous transplanted tumor model was established and grouped according to the method in experimental example 12.1.
  • the day of grouping is marked as Day 0, and the groups are Human IgG1 isotype control antibody (negative control) group (IgG1 for short), paclitaxel group (albumin-binding type), 2C6.9 monoclonal antibody and paclitaxel combined group and 2C6.9-TL001 (DAR: 7.12) group. All samples were injected through the tail vein twice a week for 3 weeks. The dosages are shown in Table 9.
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • PR partial tumor regression
  • CR full tumor regression.
  • P value is compared with the IgG1 group; N/A: not applicable; ns: P>0.05, no statistical difference.
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • PR partial tumor regression
  • CR full tumor regression.
  • the P value is compared with the IgG1 group
  • N/A Not applicable.
  • NUGC-4 cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO 2. Cells in exponential growth phase were collected, PBS resuspended cells according to the amount of 5 ⁇ 10 6 / is only (suspended in 0.1ml PBS), were inoculated subcutaneously in female Balb / c Nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) Establish a subcutaneous transplanted tumor model, and when the average tumor volume reaches about 70-100 mm 3 , randomly group them according to the tumor volume, with 7 in each group.
  • FBS fetal bovine serum
  • the day of grouping is marked as day 0 (Day 0), and the group is Human IgG1 isotype control antibody (negative control) group (abbreviated as IgG1), 2C6.9-TL001 (DAR: 3.79) 5.25 mg/kg group, 2C6.9- TL001 (DAR: 3.79) 17.5 mg/kg group, 2C6.9-TL001 (DAR: 7.12) 3 mg/kg group, 2C6.9-TL001 (DAR: 7.12) 10 mg/kg group (ADC drugs with different DAR values follow the same toxin Carrying out the designed dose for the load). All samples were injected through the tail vein, twice a week, for a total of 3 weeks. The dosages are shown in Table 11.
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • PR partial tumor regression
  • CR full tumor regression.
  • P value is compared with IgG1 group
  • N/A Not applicable.
  • mice There are 3 groups: Human IgG1 isotype control antibody (negative control) group (IgG1 10mg/kg), 2C6.9-TL001-DAR7.12 3mg/kg group and 10mg/kg group. All samples were injected via tail vein, twice a week, for a total of 5 administrations. After the administration, observe and regularly measure the tumor volume and body weight of the mice. The measurement method is as described in Experimental Example 12.1.
  • 2C6.9-TL001-DAR7.12 has a significant inhibitory effect on the tumor growth of the GA0006 gastric cancer PDX model, and is dose-dependent.
  • the tumor inhibition rate (TGI) of the 3 mg/kg group was as high as 94.72%, and 4 of them were partially resolved; the TGI of the 10 mg/kg group reached 124.49%, All 7 tumors regressed, indicating that 2C6.9-TL001 can effectively inhibit tumor growth. All treatment groups had no significant weight loss during the observation period, and the animals were well tolerated.
  • Table 12 Gastric cancer GA0006+Balb/c Nude mouse PDX model
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • PR partial tumor regression
  • CR full tumor regression.
  • the P value is compared with the IgG1 group
  • N/A Not applicable.
  • the experimental results are shown in Table 13 and Figures 12I and 12J.
  • 2C6.9-TL001-DAR7.12 has a significant inhibitory effect on the tumor growth of the GA0006 gastric cancer PDX model, and is dose-dependent.
  • the tumor inhibition rate (TGI) of the 3 mg/kg group was as high as 103.76%, and 6 tumors were partially resolved; the 10 mg/kg group TGI reached 113.70%, and all 7 tumors resolved, indicating 2C6.9- TL001 can effectively inhibit tumor growth. All treatment groups had no significant weight loss during the observation period, and the animals were well tolerated.
  • Table 13 Gastric cancer GA0006+Balb/c Nude mouse PDX model
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • PR partial tumor regression
  • CR full tumor regression.
  • the P value is compared with the IgG1 group
  • N/A Not applicable.
  • the NCI-N87-Claudin18.2 subcutaneous transplanted tumor model was established according to the method in Experimental Example 12.1.
  • the day of grouping is marked as Day 0, and the group is Human IgG1 isotype control antibody (negative control) group (IgG1 for short), 2C6.9-TL001 (DAR: 7.40) 0.3 mg/kg group, 1 mg/kg group And 3mg/kg group. All samples were injected via tail vein, twice a week, for a total of 6 administrations. The dosages are shown in Table 14.
  • the experimental results are shown in Table 14 and Figures 12K and 12L. Stopping the drug after 6 doses extends the observation to the 31st day (31st day after the first dose). Compared with the negative control group, 2C6.9-TL001 0.3 mg
  • the tumor inhibition rate (TGI) in the 1 mg/kg group was 34.04%, while the TGI in the 1 mg/kg group and 3 mg/kg group were as high as 122.57% (partial regression of tumors in all mice) and 184.22% (all tumors in 4 mice subsided, 4 hours). The mouse tumor partially subsided).
  • the mice in all treatment groups had no significant weight loss during the observation period, and the animals had good tolerance.
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • PR partial tumor regression
  • CR full tumor regression.
  • the P value is compared with the IgG1 group
  • N/A Not applicable.
  • HEK293T-Claudin 18.2 cells (human embryonic kidney cells) were cultured in DMEM medium containing 3 ⁇ g/mL puromycin and 10% fetal bovine serum (FBS) at 37° C. and 5% CO 2. Collect exponential growth phase cells, resuspend in PBS, and inoculate subcutaneously in female Balb/c Nude mice (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) according to the amount of 1 ⁇ 10 7 cells per mouse (suspended in 0.1ml PBS) Establish a subcutaneous transplanted tumor model. When the average tumor volume reaches about 120-140mm 3 , randomly group them according to the tumor volume, with 8 in each group.
  • FBS fetal bovine serum
  • the day of grouping is marked as Day 0, and the group is Human IgG1 isotype control antibody (negative control) group (IgG1 for short), 2C6.9-TL001 (DAR: 7.40) 0.3 mg/kg group, 1 mg/kg group And 3mg/kg group. All samples were injected through the tail vein, twice a week, for a total of 6 administrations. The dosages are shown in Table 15.
  • the experimental results are shown in Table 15 and Figures 12M and 12N.
  • 2C6.9-TL001 DAR: 7.40
  • HEK293T-Claudin18 The tumor growth of human embryonic kidney cell transplantation tumor model has a significant inhibitory effect, and it is dose-dependent.
  • 2C6.9-TL001 The tumor inhibition rate (TGI) of the 0.3 mg/kg group was 70.99% (all tumors in 1 mouse disappeared); while the TGI of the 1 mg/kg group and the 3 mg/kg group were 94.98% (2 mice Partial tumor regression) and 182.81% (2 mice with partial tumor regression, 6 mice with all tumor regression).
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • PR partial tumor regression
  • CR full tumor regression.
  • the P value is compared with the IgG1 group
  • N/A Not applicable.
  • NUGC-4 cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO 2. Collect exponential growth phase cells, resuspend in PBS, and inoculate subcutaneously in female Balb/c Nude mice (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) according to the cell volume of 5 ⁇ 10 6 per mouse (suspended in 0.1ml PBS) , Establish a subcutaneous transplanted tumor model. When the average tumor volume reaches about 80mm 3 , randomly group them according to the tumor volume, with 8 in each group.
  • FBS fetal bovine serum
  • the day of grouping was recorded as Day 0, and the groups were Human IgG1 isotype control antibody (negative control) group (abbreviated as IgG1), 2C6.9-TL001 (DAR: 7.40) 3 mg/kg group and 10 mg/kg group. All samples were injected through the tail vein, twice a week, for a total of 6 administrations. The dosages are shown in Table 16.
  • 2C6.9-TL001 (DAR: 7.40) is effective against NUGC-4 gastric cancer.
  • the tumor growth of the transplanted tumor model has a significant inhibitory effect, and it is dose-dependent.
  • 2C6.9-TL001 The tumor inhibition rate (TGI) of the 3mg/kg group was 56.62%; while the TGI of the 10mg/kg group was 90.28% (2 mice with partial tumor regression).
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • PR partial tumor regression
  • CR full tumor regression.
  • the P value is compared with the IgG1 group
  • N/A Not applicable.
  • 2C6.9-TL001 in the present invention can effectively inhibit the growth of Claudin 18.2 positive expression tumors in a dose-dependent manner, and has good safety.

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Abstract

一种抗CLDN18.2抗体-药物缀合物及其制备方法和用途，该抗体-药物缀合物及含有该缀合物的组合物能够有效结合CLDN18.2并抑制Claudin18.2阳性肿瘤生长。进一步涉及包含该抗体-药物缀合物或组合物的药物组合物，以及该药物组合物在制备预防和/或治疗肿瘤药物中的用途。

Description

抗体药物缀合物及其制备方法和用途
本申请是以CN申请号为202010410633.5，申请日为2020年5月15日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本发明属于生物医药技术领域。具体而言，本发明涉及抗Claudin 18.2抗体-药物缀合物(ADC)及含有所述ADC的组合物，以及它们的用途。
背景技术
胃癌的治疗主要以手术切除为主，对于不可切除或复发胃癌，化疗是主要的治疗手段，但现有条件下化疗仅能改善症状，延长生存期。目前国际上无一致公认的进展期胃癌标准化疗方案。生物大分子药物方面除曲妥珠单抗(Trastuzumab)、雷莫芦单抗(Ramucirumab)、帕博丽珠单抗(Pembrolizumab)等获批外，其它胃癌靶向治疗药物大多结果不甚理想或尚处于临床研究的早期阶段。现有药物的核心缺陷是非手术治疗未满足的临床需求，晚期或复发性胃癌的治疗选择非常有限，预后极差，死亡率高。作为高发癌症，目前胃癌药物需求极大。
Claudin 18.2(CLDN 18.2)是Claudin蛋白家族成员。Claudin家族蛋白是一类介导细胞与细胞之间紧密连接的蛋白，不同亚型的Claudin蛋白表达于不同的组织，与不同类型的癌症有关。Claudin 18.2在正常组织中的表达仅限于胃粘膜细胞，不表达于其它正常组织中。Claudin 18.2高表达于70％的原发性胃腺癌及其转移瘤中，在其它一些癌症如胰腺癌，食管癌和非小细胞肺癌中也有表达。Claudin 18.2在肿瘤组织中的高特异性表达使Claudin 18.2成为肿瘤免疫治疗的非常好的靶点。
发明内容
本申请的发明人在PCT/CN2019/126495中记载了与Claudin 18.2具有高特异性和高亲和力的抗体，该申请全部内容以参考方式并入本文并成为本申请内容的一部分。本申请的发明人经过大量创造性的劳动，进一步发明了Claudin 18.2抗体-药物缀合物(ADC)，为肿瘤的治疗提供了安全有效的药物选择。
具体地，发明人将高亲和力和特异性结合Claudin 18.2的抗体与生物活性分子通过连接子连接，得到一类靶向Claudin 18.2的抗体-药物缀合物以及含有所述缀合物的组合物。所述缀合物或组合物对一些Claudin 18.2表达的肿瘤细胞具有较高的杀伤活性。在动物体内试验中，所述缀合物能够有效抑制Claudin 18.2阳性肿瘤生长，尤其是胃癌或胃腺癌，并且具有较高的安全性。
抗体-药物缀合物
在一个方面，本发明提供特异性结合人CLDN18.2的抗体-药物缀合物，其结构如式(I)所示，
(D-L) γ-A
式(I)
其中，D为生物活性分子片段；L为连接子；
γ选自1-10之间的整数(例如1、2、3、4、5、6、7、8、9或10)；优选地，γ选自1-8之间的整数(例如1、2、3、4、5、6、7或8)；
A为特异性结合人CLDN18.2的抗体或其抗原结合片段。
在一些实施方案中，特异性结合人CLDN18.2的抗体或其抗原结合片段包含：
(1)如下的三个重链CDRs：如SEQ ID NOs:13，14或23所示的VH(重链可变区)所含的CDR-H1、CDR-H2以及CDR-H3；和/或，
如下的三个轻链CDRs：如SEQ ID NO:15或24所示的VL(轻链可变区)所含的CDR-L1、CDR-L2以及CDR-L3；
或，
(2)如下的三个重链CDRs：(1)中所述的CDR-H1或与其相比含有氨基酸突变的变体，(1)中所述的CDR-H2或与其相比含有氨基酸突变的变体，和(1)中所述的CDR-H3或与其相比含有氨基酸突变的变体；和/或，
如下的三个轻链CDRs：(1)中所述的CDR-L1或与其相比含有氨基酸突变的变体，(1)中所述的CDR-L2或与其相比含有氨基酸突变的变体，和(1)中所述的CDR-L3或与其相比含有氨基酸突变的变体；
其中，(2)中所述的三个重链CDRs和/或三个轻链CDRs中至少一个CDR与(1)中相应的CDR相比含有氨基酸突变，所述氨基酸突变为一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；所述含有突变的抗体或其抗原结合片段仍然能特异地结合CLDN18.2；优选地，特异地结合人CLDN18.2；优选地，所述的置换是保守置换。
在一些实施方案中，所述CDRs根据IMGT或AbM编号系统定义。
在一些实施方案中，所述抗体或其抗原结合片段还包括来自人的免疫球蛋白的构架区(FRs)。
在一些实施方案中，所述抗体或其抗原结合片段包含：
(1-1)按IMGT编号系统定义的如下三个重链CDRs：序列为SEQ ID No：1的CDR-H1，序列为SEQ ID Nos：2或21的CDR-H2，和序列为SEQ ID No：3的CDR-H3；和/或，
按IMGT编号系统定义的如下三个轻链CDRs：序列为SEQ ID No：4的CDR-L1，序列为SEQ ID No：5的CDR-L2，和序列为SEQ ID No：6的CDR-L3；
或，
(1-2)如下的三个重链CDRs：(1-1)中所述的CDR-H1或与其相比含有氨基酸突变的变体，(1-1)中所述的CDR-H2或与其相比含有氨基酸突变的变体，和(1-1)中所述的CDR-H3或与其相比含有氨基酸突变的变体；和/或，
如下的三个轻链CDRs：(1-1)中所述的CDR-L1或与其相比含有氨基酸突变的变体，(1-1)中所述的CDR-L2或与其相比含有氨基酸突变的变体，和(1-1)中所述的CDR-L3或与其相比含有氨基酸突变的变体；
其中，所述的三个重链CDRs和/或三个轻链CDRs中至少一个CDR与(1-1)中相应的CDR相比含有氨基酸突变，所述氨基酸突变为一个或几个氨基酸的置换、缺失或添加(例如1个，2 个或3个氨基酸的置换、缺失或添加)；所述含有突变的抗体或其抗原结合片段仍然能特异地结合人CLDN18.2；优选地，所述的置换是保守置换；
或，
(2-1)按AbM编号系统定义的如下三个重链CDRs：序列为SEQ ID No：7的CDR-H1，序列为SEQ ID Nos：8或22的CDR-H2，和序列为SEQ ID No：9的CDR-H3；和/或，
按AbM编号系统定义的如下三个轻链CDRs：序列为SEQ ID No：10的CDR-L1，序列为SEQ ID No：11的CDR-L2，和序列为SEQ ID No：12的CDR-L3；
或，
(2-2)如下的三个重链CDRs：(2-1)中所述的CDR-H1或与其相比含有氨基酸突变的变体，(2-1)中所述的CDR-H2或与其相比含有氨基酸突变的变体，和(2-1)中所述的CDR-H3或与其相比含有氨基酸突变的变体；和/或，
如下的三个轻链CDRs：(2-1)中所述的CDR-L1或与其相比含有氨基酸突变的变体，(2-1)中所述的CDR-L2或与其相比含有氨基酸突变的变体，和(2-1)中所述的CDR-L3或与其相比含有氨基酸突变的变体；
其中，所述的三个重链CDRs和/或三个轻链CDRs中至少一个CDR与(2-1)中相应的CDR相比含有氨基酸突变，所述氨基酸突变为一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；所述含有突变的抗体或其抗原结合片段仍然能特异地结合人CLDN18.2；优选地，所述的置换是保守置换。
在一些实施方案中，所述抗体或其抗原结合片段还包括来自人的免疫球蛋白的构架区(FRs)。
在一些实施方案中，所述抗体或其抗原结合片段包含：
(1)下述的VH和/或VL，其中CDRs按IMGT编号系统定义：
(1-1)：包含如下3个CDRs的VH：序列为SEQ ID No：1的CDR-H1，序列为SEQ ID Nos：2或21的CDR-H2，和序列为SEQ ID No：3的CDR-H3；和/或，
包含如下3个CDRs的VL：序列为SEQ ID No：4的CDR-L1，序列为SEQ ID No：5的CDR-L2，和序列为SEQ ID No：6的CDR-L3；
或，
(1-2)：与(1-1)中所述的VH或VL相比，至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个或3个氨基酸的置换、缺失或添加或其任意组合)；优选地，所述的置换为保守置换；所述含有突变的抗体或其抗原结合片段仍然能特异地结合CLDN18.2；优选地，特异性地结合人CLDN18.2；
或，
(2)下述的VH和/或VL，其中CDRs按AbM编号系统定义：
(2-1)：包含如下3个CDRs的VH：序列为SEQ ID No：7的CDR-H1，序列为SEQ ID Nos：8或22的CDR-H2,和序列为SEQ ID No：9的CDR-H3；和/或，
包含如下3个CDRs的VL：序列为SEQ ID No：10的CDR-L1，序列为SEQ ID No：11的 CDR-L2,和序列为SEQ ID No：12的CDR-L3；
或，
(2-2)：与(2-1)中所述的VH或VL相比，至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个或3个氨基酸的置换、缺失或添加或其任意组合)；优选地，所述的置换为保守置换；所述含有突变的抗体或其抗原结合片段仍然能特异地结合CLDN18.2；优选地，特异性地结合人CLDN18.2。
在一些实施方案中，所述抗体或其抗原结合片段的VH和/或VL中包括来自人或鼠的免疫球蛋白的构架区(FRs)。
在一些实施方案中，所述抗体或其抗原结合片段包含：
(1)SEQ ID NO：13或14所示的VH；和/或，SEQ ID NO：15所示的VL；
或者，
(2)所述抗体或其抗原结合片段所包含的VH与(1)中所述的VH相比，具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的同一性；和/或，所述抗体或其抗原结合片段所包含的VL与(1)中所述的VL相比，具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的同一性；所述含有同一性序列的抗体或其抗原结合片段仍然能特异地结合CLDN18.2；优选地，特异性地结合人CLDN18.2；
再或者，
(3)所述抗体或其抗原结合片段所包含的VH与(1)中所述的VH相比，具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；和/或，所述抗体或其抗原结合片段所包含的VL与(1)中所述的VL相比，具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；优选地，所述的置换是保守置换。所述含有突变的抗体或其抗原结合片段仍然能特异地结合CLDN18.2；优选地，特异性地结合人CLDN18.2。
在以上任一方面中，所述抗体或其抗原结合片段可以进一步包含来源于哺乳动物(例如，鼠或人)免疫球蛋白的恒定区或其变体。
在一些实施方案中，所述抗体或包含：
(1)人免疫球蛋白的CH(重链恒定区)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和/或，
(2)人免疫球蛋白的CL(轻链恒定区)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至 多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)。
在一些实施方案中，恒定区被改变，例如被突变，以修饰抗CLDN18.2抗体分子的性质(例如改变下列中的一个或更多个特性：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能)。可以通过将抗体恒定区中的至少一个氨基酸残基替换为不同残基，产生功能改变，例如，改变抗体对效应子配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如增强)。在一些实施方案中，恒定区被改变从而改变(例如增强)抗体依赖性细胞介导的细胞毒性(ADCC)、补体依赖的细胞毒性(CDC)和/或抗体依赖性细胞吞噬作用(ADCP)。
在一些实施方案中，所述CH是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。
在一些实施方案中，所述抗体或其抗原结合片段包含人IgG1的重链恒定区。
在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：16所示的CH或其变体，所述变体与SEQ ID NO：16相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。所述含有突变的CH保留大体与SEQ ID NO：16相同的功能。
在一些实施方案中，所述CL选自κ或λ的轻链恒定区。
在一些实施方案中，所述CL是κ轻链恒定区(例如人κ轻链)。
在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：17所示的CL或其变体，所述变体与SEQ ID NO：17相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。所述含有突变的CL保留大体与SEQ ID NO：17相同的功能。
在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：16所示的CH和/或如SEQ ID NO：17所示的CL。
在一些实施方案中，所述抗体包括由SEQ ID NO：13所示序列的VH和SEQ ID NO：16所示的CH组成的重链，和，
由SEQ ID NO：15所示序列的VL和SEQ ID NO：17所示的CL组成的轻链。
在一些实施方案中，所述抗体包括由SEQ ID NO：14所示序列的VH和SEQ ID NO：16所示的CH组成的重链，和，
由SEQ ID NO：15所示序列的VL和SEQ ID NO：17所示的CL的轻链。
在一些实施方案中，所述抗体或其抗原结合片段包含：
(1)重链，其包含选自下列的氨基酸序列：
(1-1)SEQ ID NO：18所示的序列；
(1-2)与SEQ ID NO：18所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例 如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(1-3)与SEQ ID NO：18所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和
(2)轻链，其包含选自下列的氨基酸序列：
(2-1)SEQ ID NO：20所示的序列；
(2-2)与SEQ ID NO：20所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(2-3)与SEQ ID NO：20所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在一些实施方案中，上述(1-2)和(2-2)中所述的置换是保守置换。所述含有突变的CH保留大体与SEQ ID NO：16相同的功能。
在一些实施方案中，所述抗体或其抗原结合片段包含：
(1)重链，其包含选自下列的氨基酸序列：
(1-1)SEQ ID NO：19所示的序列；
(1-2)与SEQ ID NO：19所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(1-3)与SEQ ID NO：19所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和
(2)轻链，其包含选自下列的氨基酸序列：
(2-1)SEQ ID NO：20所示的序列；
(2-2)与SEQ ID NO：20所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(2-3)与SEQ ID NO：20所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在一些实施方案中，上述(1-2)和(2-2)中所述的置换是保守置换。优先地，上述含有突变或同一性序列的抗体或其抗原结合片段仍然能特异地结合人CLDN18.2。
在一些实施方案中，所述抗体或其抗原结合片段选自ScFv、Fab、Fab’、(Fab’) 2、Fv片段、二硫键连接的Fv(dsFv)、双抗体(diabody)、双特异性抗体和多特异性抗体。
在一些实施方案中，所述(D-L) γ-A结构如式(II)所示：
{D-[L 1-(L 2) m1-(L 3) m2-(L 4) m3-E]} γ-A
式(II)
其中，
L 1为
Figure PCTCN2021093348-appb-000001
其中，各R 1和R 2各自独立地为氢(例如氕或氘)、卤素、羧酸、磺酸、氰基、C 1-6烷基、卤代C 1-6烷基、氰基取代的C 1-6烷基(例如-CH 2CN)、C 1-6烷氧基、C 2-10烯基或C 2-10炔基；Z 1为氨基酸或2-10个氨基酸组成的肽；x 1和x 2各自独立地为0、1、2、3、4、5或6；并且L 1的1位置处与D相连、L 1的2位置处与L 2相连；
L 2为
Figure PCTCN2021093348-appb-000002
其中，y 1为0、1、2、3、4、5、6、7、8、9或10；并且L 2的1位置处与L 1相连、L 2的2位置处与L 3相连；
L 3选自5-12元杂芳环；
L 4为
Figure PCTCN2021093348-appb-000003
其中Z 2选自C 1-6亚烷基、C 2-10亚烯基、C 2-10亚炔基和C 3-8亚环烷基；R 3选自氢(例如氕或氘)和C 1-6烷基；Z 3不存在或者选自C 1-6亚烷基；或者，R 3与Z 3连同其所连接的氮原子形成4-8元杂环基；α为0、1、2、3、4、5或6，并且L 4的2位置处与E相连、L 4的1位置处与L 3相连；
E为
Figure PCTCN2021093348-appb-000004
其中，各R 4独立地为氢(例如氕或氘)，β为0、1、2、3、4、5或6，并且E的2位置处与A相连、E的1位置处与L 4相连；
m 1、m 2和m 3各自独立地为0、1、2、3、4、5、6、7、8、9或10；
A，D和γ如前文所述。
在一些实施方案中，所述(D-L) γ-A结构如式(III)所示：
{D-[(L 1’) m4-L 1-(L 5) m5-(L 3) m2-(L 4) m3-E]} γ-A
式(III)
其中，
L 1’为
Figure PCTCN2021093348-appb-000005
其中，R 5和R 6各自独立地为氢(例如氕或氘)或C 1-6烷基；x 3为1、2、3、4、5或6；并且，如果L 1’存在，其1位置处与D相连、2位置处与L 1相连；
L 1为
Figure PCTCN2021093348-appb-000006
其中，各R 1和R 2各自独立地为氢(例如氕或氘)、卤素、羧酸、磺酸、氰基、C 1-6烷基、卤代C 1-6烷基、氰基取代的C 1-6烷基(例如-CH 2CN)、C 1-6烷氧基、C 2-10烯基或C 2-10炔基；Z 1为氨基酸或2-10个氨基酸组成的肽；x 1和x 2各自独立地为0、1、2、3、4、5或6；并且，L 1的1位置处与L 1’相连(当L 1’存在时)，或者，L 1的1位置处与D相连(当L 1’不存在时)；L 1的2位置处与L 5相连；
L 5为
Figure PCTCN2021093348-appb-000007
其中，R 7为氢或C 1-6烷基，或者R 7与其γ-C上的N原子连接形成5-6元杂环基；x 4为1、2、3、4、5或6；y 1为0、1、2、3、4、5、6、7、8、9或10；并且L 5的1位置处与L 1相连、L 5的2位置处与L 3相连；
L 3选自5-12元杂芳环；
L 4为
Figure PCTCN2021093348-appb-000008
其中Z 2选自C 1-6亚烷基、C 2-10亚烯基、C 2-10亚炔基和C 3-8亚环烷基；R 3选自氢(例如氕或氘)和C 1-6烷基；Z 3不存在或者选自C 1-6亚烷基；或者，R 3与Z 3连同其所连接的氮原子形成4-8元杂环基；α为0、1、2、3、4、5或6，并且L 4的2位置处与E相连、L 4的1位置处与L 3相连；
E为
Figure PCTCN2021093348-appb-000009
其中，各R 4独立地为氢(例如氕或氘)，β为0、1、2、3、4、5或6，并且E的2位置处与A相连、E的1位置处与L 4相连；
m 1、m 2、m 3和m 4各自独立地为0、1、2、3、4、5、6、7、8、9或10；
A，D和γ如前文所述。
在一些实施方案中，式(II)或式(III)所示的抗体-药物缀合物中的L 1为
Figure PCTCN2021093348-appb-000010
其中，Z 1为氨基酸或2-5个氨基酸组成的肽，其中所述氨基酸选自Lys、Cit、Val、D-Val、Phe、Leu、Gly、Ala和Asn；优选地，Z 1选自Cit、Lys、Cit-Val和Ala-Val。
在一些实施方案中，L 1为
Figure PCTCN2021093348-appb-000011
在一些实施方案中，L 1为
Figure PCTCN2021093348-appb-000012
在一些实施方案中，式(II)所示的抗体-药物缀合物中的L 2为
Figure PCTCN2021093348-appb-000013
且m 1为1。
在一些实施方案中，式(II)或式(III)所示的抗体-药物缀合物中的L 3为5-6元杂芳环，且m 2为1。
在一些实施方案中，L 3为三氮唑，且m 2为1。
在一些实施方案中，式(II)或式(III)所示的抗体-药物缀合物中的L 4为
Figure PCTCN2021093348-appb-000014
其中Z 2为C 1-6亚烷基，Z 3为C 1-6亚烷基；且m 3为1。
在一些实施方案中，L 4为
Figure PCTCN2021093348-appb-000015
且m 3为1。
在一些实施方案中，式(III)所示的抗体-药物缀合物中的L 1’为
Figure PCTCN2021093348-appb-000016
在一些实施方案中，式(III)所示的抗体-药物缀合物中的L 5为
Figure PCTCN2021093348-appb-000017
其中，x 4为1、2、3、4、5或6；y 1为1、2、3、4、5、6、7、8、9或10。
在一些实施方案中，式(III)所示的抗体-药物缀合物中的L 5为
Figure PCTCN2021093348-appb-000018
在一些实施方案中，式(I)所示的抗体-药物缀合物中的D为
Figure PCTCN2021093348-appb-000019
在一些实施方案中，式(II)所示的抗体-药物缀合物中的D为
Figure PCTCN2021093348-appb-000020
在一些实施方案中，式(III)所示的抗体-药物缀合物中的D为
Figure PCTCN2021093348-appb-000021
Figure PCTCN2021093348-appb-000022
在一些实施方案中，式(II)所示的抗体-药物缀合物中D-[L 1-(L 2) m1-(L 3) m2-(L 4) m3-E]-为
Figure PCTCN2021093348-appb-000023
在一些实施方案中，式(III)所示的抗体-药物缀合物中D-[(L 1’) m4-L 1-(L 5) m5-(L 3) m2-(L 4) m3-E]-为
Figure PCTCN2021093348-appb-000024
在一些实施方案中，式(II)所示的抗体-药物缀合物的结构为：
Figure PCTCN2021093348-appb-000025
其中，γ选自1-10之间的整数，例如γ为1、2、3、4、5、6、7、8、9或10，A为前述特异性结合人CLDN18.2的抗体或其抗原结合片段。
在一些实施方案中，式(II)所示的抗体-药物缀合物如下式所示：
Figure PCTCN2021093348-appb-000026
其中γ为1～10的整数，例如γ为1、2、3、4、5、6、7、8、9或10，A为2C6.9-hz21。
在一些实施方案中，式(III)所示的抗体-药物缀合物的结构为：
Figure PCTCN2021093348-appb-000027
其中，γ选自1-10之间的整数，例如γ为1、2、3、4、5、6、7、8、9或10，A为前述特异性结合人CLDN18.2的抗体或其抗原结合片段。
在一些实施方案中，式(III)所示的抗体-药物缀合物如下式所示：
Figure PCTCN2021093348-appb-000028
其中γ为1～10的整数，例如γ为1、2、3、4、5、6、7、8、9或10，A为2C6.9-hz21。
在一些实施方案中，式(III)所示的抗体-药物缀合物的结构为：
Figure PCTCN2021093348-appb-000029
其中，γ选自1-10之间的整数，例如γ为1、2、3、4、5、6、7、8、9或10，A为前述特异性结合人CLDN18.2的抗体或其抗原结合片段。
在一些实施方案中，式(III)所示的抗体-药物缀合物如下式所示：
Figure PCTCN2021093348-appb-000030
其中γ为1～10的整数，例如γ为1、2、3、4、5、6、7、8、9或10，A为2C6.9-hz21。
本发明还提供一种组合物，其含有一个或多个上述的抗体-药物缀合物，所述组合物中的生物活性分子片段(即式(I)中所述的D)与特异性结合CLDN18.2的抗体或其抗原结合片段(即式(I)中所述的A)的摩尔比(DAR值)为1-10之间的小数或整数(例如1-8之间的小数或整数，例如1.0,1.5,2.0,2.5，3.0，3.1，3.2，3.3，3.4，3.5，3.6，3.7，3.79，3.8，3.9，4.0，4.1，4.2，4.3，4.4，4.5，4.6，4.7，4.8，4.9，5.0，5.1，5.2，5.3，5.4，5.5，5.6，5.7，5.8，5.9，6.0，6.1，6.2，6.3，6.4，6.5，6.6，6.7，6.8，6.9，6.95，7.0，7.03，7.1，7.12，7.2，7.3，7.40，7.5，7.6，7.7，7.8，7.9或8.0)。
DAR值又称为抗体药物偶联比，其是指平均每个抗体所连接的(D-L)的数量，其可以通过本领域已知方法进行测定和计算。例如通过LC-MS、HIC等测定抗体和(D-L)偶联后的分子量，分别计算偶联不同数量(D-L)的轻链和重链的比例，并通过公式DAR＝(轻链DAR1*1+…+轻链 DARn 1*n 1)*2+(重链DAR1*1+…+DARn 2*n 2)*2计算得出DAR值；其中DARn 1表示偶联n 1个(D-L)的轻链占轻链部分的比例，DARn 2表示偶联n 2个(D-L)的重链占重链部分的比例。
在一些实施方案中，所述组合物中进一步含有前文所述的抗体或其抗原结合片段。
在一些实施方案中，所述组合物中含有2C6.9-TL001。在一些实施方案中，所述组合物为2C6.9-TL001。
在一些实施方案中，所述组合物中含有2C6.9-TL002。在一些实施方案中，所述组合物为2C6.9-TL002。
在一些实施方案中，所述组合物中含有2C6.9-TL003。在一些实施方案中，所述组合物为2C6.9-TL003。
本发明的抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会不利影响其对CLDN18.2(特别是人CLDN18.2)的结合。因此，本发明的抗体或其抗原结合片段还意欲包括此类衍生化的形式。
一种类型的衍生化抗体(例如，双特异性抗体)是通过交叉连接2个或多个抗体(属于同一类型或不同类型)而产生的。获得双特异性抗体的方法是本领域公知的，其实例包括但不限于，化学交联法、细胞工程法(杂交杂交瘤法)或基因工程法。
另一种类型的衍生化抗体是标记的抗体。例如，可以将本发明的抗体或其抗原结合片段连接至可检测的标记。本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯类化合物、磁珠(例如，
Figure PCTCN2021093348-appb-000031
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。如上所述的可检测的标记可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中，可通过不同长度的接头(linker)将如上所述的可检测的标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
在某些实施方案中，本发明的抗体-药物缀合物中包含的抗体或其抗原结合片段为一种或一种以上前述衍生化抗体或其抗原结合片段。
治疗方法和药物组合物
在另一个方面，本发明还提供了药物组合物。
在一些实施方案中，所述药物组合物含有一个或多个上述的抗体-药物缀合物或组合物。
在一些优选地实施方案中，所述药物组合物进一步包含药学上可接受的载体和/赋形剂。
在一些优选地实施方案中，所述药物组合物还包含其它具有抗肿瘤活性的成分。在一些实施方案中，所述抗体-药物缀合物或组合物与其它具有抗肿瘤活性的成分在同一制剂单元中，或者在不同的制剂单元中。因此，本发明的抗体-药物缀合物或组合物与其它具有抗肿瘤活性的成分可以同时、分开或相继施用。
在一些实施方案中，所述其它具有抗肿瘤活性的成分是生物活性多肽或其活性片或化疗药物。在一些实施方案中，所述生物活性多肽选自免疫检查点抑制剂(例如，PD-1抗体、PD-L1抗体、CTLA-4抗体、LAG-3抗体)，或细胞因子(例如，干扰素、IL-2、IL-15、GM-CSF、IL-7、IL-12、IL-18、IL-21)。在一些实施方案中，所述化疗药物选自表柔比星、奥沙利铂、卡培他滨、5-氟尿嘧啶、亚叶酸、紫杉醇和白蛋白结合型紫杉醇中的一种或多种。在一些实施方案中，所述其它具有抗肿瘤活性的成分为表柔比星、奥沙利铂和5-氟尿嘧啶的组合，或奥沙利铂、亚叶酸和5-氟尿嘧啶的组合。
在另一个方面，本发明的药物组合物中的抗体-药物缀合物或组合物足以在受试者中：
(a)诱导肿瘤细胞(尤其是胃癌细胞，例如胃腺癌细胞)凋亡；
(b)抑制肿瘤细胞(尤其是胃癌细胞，例如胃腺癌细胞)增殖；
(c)诱发和/或增加补体依赖性细胞毒活性；
(d)诱发和/或增加抗体依赖性细胞毒活性；
(e)抑制CLDN18.2的表达和活化；
(f)抑制CLDN18.2介导的信号传导；或
(g)(a)-(f)的任意组合。
在另一个方面，本发明提供所述的抗体-药物缀合物、所述组合物、药物组合物在制备预防和/或治疗和/或辅助治疗肿瘤的药物中的用途。
在一些实施方案中，所述肿瘤选自实体肿瘤、血液肿瘤和癌症的转移性、难治性或复发性病灶。
在一些实施方案中，所述肿瘤或癌症选自食管癌、胃肠癌、胃腺癌、胰腺癌、甲状腺癌、结直肠癌、肾癌、肺癌(例如非小细胞肺癌)、肝癌、胃癌、胃食管交界处(GEJ)腺癌、头颈癌、膀胱癌、乳腺癌、子宫癌、宫颈癌、卵巢癌、前列腺癌、睾丸癌、生殖细胞癌、骨癌、皮肤癌、胸腺癌、胆管癌、胆囊癌、黑素瘤、间皮瘤、淋巴瘤、骨髓瘤(例如多发性骨髓瘤)、肉瘤、神经胶质母细胞瘤、白血病。
在一些实施方案中，所述肿瘤选自胃癌、胃腺癌、胃食管交界处(GEJ)腺癌、食管癌、胃肠癌、胰腺癌、肺癌(例如非小细胞肺癌)。
在一些实施方案中，所述肿瘤为胃癌、胃腺癌或胃食管交界处(GEJ)腺癌，例如局部晚期不可切除或转移的胃癌、胃腺癌或胃食管交界处(GEJ)腺癌。
在一些实施方案中，所述肿瘤为CLDN18.2阳性，进一步，所述肿瘤为HER2阴性。
在一些实施方案中，所述肿瘤为HER2阴性。
在另一个方面，本发明提供了一种在受试者中预防和/或治疗肿瘤的方法。在另一个方面，本发明提供了一种在受试者中延迟肿瘤进展的方法。在另一个方面，本发明提供了一种在受试者中降低或抑制肿瘤复发的方法。以上所述方法包括向有此需要的受试者施用有效量的本发明所述的抗体-药物缀合物、组合物或药物组合物。
在一些实施方案中，以上所述方法还包括向所述受试者施用第二疗法，所述第二疗法选自手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其任意组合。
在一些实施方案中，所述第二疗法可以与以上所述的方法分开或联合应用；或，所述第二疗法可以与以上所述的方法分开或联合应用同时或相继应用。
在一些实施方案中，所述第二疗法为化疗。在一些实施方案中，所述化疗的药物选自表柔比星、奥沙利铂、卡培他滨、5-氟尿嘧啶、亚叶酸、紫杉醇和白蛋白结合型紫杉醇中的一种或多种。在一些实施方案中，所述化疗的药物为表柔比星、奥沙利铂和5-氟尿嘧啶的组合，或奥沙利铂、亚叶酸和5-氟尿嘧啶的组合。在一些实施方案中，奥沙利铂、亚叶酸和5-氟尿嘧啶的组合给药方案选自FOLFOX4、FOLFOX6或m FOLFOX6。
在一些实施方案中，所述第二疗法为免疫疗法。在一些实施方案中，所述免疫疗法的药物选自免疫检查点抑制剂(例如，PD-1抗体、PD-L1抗体、CTLA-4抗体、LAG-3抗体)，或细胞因子(例如，干扰素、IL-2、IL-15、GM-CSF、IL-7、IL-12、IL-18、IL-21)。
在一些实施方案中，所述肿瘤选自实体肿瘤、血液肿瘤，和癌症的转移性、难治性或复发性病灶。
在一些实施方案中，所述肿瘤或癌症选自食管癌、胃肠癌、胃腺癌、胰腺癌、甲状腺癌、结直肠癌、肾癌、肺癌(例如非小细胞肺癌)、肝癌、胃癌、胃食管交界处(GEJ)腺癌、头颈癌、膀胱癌、乳腺癌、子宫癌、宫颈癌、卵巢癌、前列腺癌、睾丸癌、生殖细胞癌、骨癌、皮肤癌、胸腺癌、胆管癌、胆囊癌、黑素瘤、间皮瘤、淋巴瘤、骨髓瘤(例如多发性骨髓瘤)、肉瘤、神经胶质母细胞瘤、白血病。
在一些实施方案中，所述肿瘤选自胃癌、胃腺癌、胃食管交界处(GEJ)腺癌、食管癌、胃肠癌、胰腺癌和肺癌(例如非小细胞肺癌)。
在一些实施方案中，所述肿瘤为胃癌、胃腺癌或胃食管交界处(GEJ)腺癌，例如局部晚期不可切除或转移的胃癌、胃腺癌或胃食管交界处(GEJ)腺癌。
在一些实施方案中，所述肿瘤为CLDN18.2阳性，进一步，所述肿瘤为HER2阴性。
在一些实施方案中，所述肿瘤为HER2阴性。
本发明的抗体-药物缀合物、组合物或药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的抗体-药物缀合物或组合物，以及任选地，同时掺入其它期望的成分(包括但不限于，pH调 节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如无菌无热原水。
此外，本发明的抗体-药物缀合物可以以单位剂量形式存在于药物组合物中，以便于施用。
本发明的抗体-药物缀合物、组合物或药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在一个优选的实施方案中，本发明的抗体-药物缀合物、组合物或药物组合物通过静脉输注或注射给予。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明的抗体-药物缀合物或组合物。“预防有效量”是指，足以预防，阻止，或延迟疾病的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明的抗体-药物缀合物或组合物的治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其它治疗等等。
在本发明中，可调整给药方案以获得最佳目的反应(例如治疗或预防反应)。例如，可以单次给药，可以在一段时间内多次给药，或者可以随治疗情况的紧急程度按比例减少或增加剂量。
本发明的抗体-药物缀合物或组合物的治疗或预防有效量的典型非极限范围是0.02～100mg/kg，例如0.1～100mg/kg，0.1～50mg/kg，或1～50mg/kg。应注意的是，剂量可随需要治疗的症状的类型和严重性不同而发生变化。此外，本领域技术人员理解，对于任一特定患者，特定的给药方案应根据患者需要和医生的专业评价而随时间调整；此处给出的剂量范围只用于举例说明目的，而不限定本发明药物组合物的使用或范围。
在本发明中，所述受试者可以为哺乳动物，例如人。
缩略词
CDR         免疫球蛋白可变区中的互补决定区
FR          抗体构架区：抗体可变区中除CDR残基以外的氨基酸残基
VH          抗体重链可变区
VL          抗体轻链可变区
IgG         免疫球蛋白G
AbM         AbM CDR定义方式来源于Martin的相关研究(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268–9272)，此定义方法整合了Kabat及Chothia两者的部分定义。
Kabat       由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service, National Institutes of Health,Bethesda,Md.,1991)。
Chothia     由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
IMGT        基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information
Figure PCTCN2021093348-appb-000032
(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
mAb         单克隆抗体
EC50        产生50％功效或结合的浓度
HRP         辣根过氧化物酶
CDR-H1      免疫球蛋白重链可变区中的互补决定区1
CDR-H2      免疫球蛋白重链可变区中的互补决定区2
CDR-H3      免疫球蛋白重链可变区中的互补决定区3
CDR-L1      免疫球蛋白轻链可变区中的互补决定区1
CDR-L2      免疫球蛋白轻链可变区中的互补决定区2
CDR-L3      免疫球蛋白轻链可变区中的互补决定区3
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、生物化学、核酸化学、免疫学等实验操作除非特殊说明为相应领域内常规使用的操作。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“偶联物”或“缀合物”可以互换，指的是生物活性分子通过连接子与抗体连接得到的物质，英文名称为ADC(antibody-drug conjugate)。
所述连接子可以通过各种化学键与抗体连接。例如，在一些实施方式中，所述连接子通过与抗体的巯基形成硫醚键相连接。在一些具体ADC分子(如2C6.9-TL001、2C6.9-TL002、或2C6.9-TL003中的ADC分子)结构式中的-S-仅表示连接子与抗体的巯基形成的硫醚键，并不代表-S-是连接子中的一部分。
本申请ADC的结构式可以以(D-L) γ-A表示，其中，D为生物活性分子片段；L为连接子；A为特异性结合人CLDN18.2的抗体或其抗原结合片段；γ选自1-10之间的整数，例如1、2、3、4、5、6、7、8、9或10。γ是指每一个抗体分子所连接的(D-L)的个数，其为整数。然而，在ADC制备过程中，各抗体分子可能连接不同数量的(D-L)，因此，一般而言，ADC的产物是偶联了不同数量的(D-L)的抗体的组合物。在实践中，通常用DAR表示抗体所连接(D-L)数量的平均值。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻 链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia & Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883，或AbM，Martin的相关研究(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268–9272)的定义。在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。这些氨基酸残基的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia & Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)，IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)，或，Martin的相关研究(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268–9272)，此定义方法整合了Kabat及Chothia两者的部分定义，最早应用在Oxford Molecular抗体建模软件中(Martin A C R.Protein sequence and structure analysis of antibody variable domains[M]//Antibody engineering.Springer,Berlin,Heidelberg,2010:33-51.)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过IMGT或AbM编号系统确定。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语抗体的“抗原结合片段”是指抗体的片段的多肽,例如全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’) 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)、线性抗体(linear antibody)、纳米抗体(技术来自Domantis)、结构域抗体(技术来自Ablynx)、和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述 于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”或“重链”和两条“全长轻链”或“轻链”组成的抗体。其中，“全长重链”或“重链”是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”或“轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种，例如人；也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc片段”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-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，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其它接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.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。在一些情况下，scFv的VH与VL之间还可以存在二硫键。如本文中所使用的，术语“di-scFv”是指，由两个scFv连接形成的抗体片段。
如本文中所使用的，术语“双抗体(diabody)”意指，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA  90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
如本文中所使用的，“双特异性抗体”，指由第一抗体(片段)和第二抗体(片段)或抗体类似物通过偶联臂所形成的缀合物，偶联的方式包括但不限于化学反应、基因融合，蛋白融合，多肽融合和酶促。“多特异性抗体”包括例如：三特异性抗体和四特异性抗体，前者是具有三种不同抗原结合特异性的抗体，而后者是具有四种不同抗原结合特异性的抗体。
如本文中所使用的，“抗体类似物(antibody mimetics)”，指与抗体一样特异性结合抗原，但却没有抗体结构。它们通常是人工肽或蛋白质，摩尔质量约为3至20kDa。例如，锚蛋白重复蛋白(DARPin)和fynomer。经设计的锚蛋白重复蛋白(DARPin)，与IgG抗体，scFv-Fc抗体片段相连或其组合，如CN104341529A。抗IL-17a的fynomer与抗IL-6R抗体结合，如WO2015141862A1。
在本文中，获得抗体的技术可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
如本文中所使用的，术语“单克隆抗体”、“单抗”、“mAb”具有相同的含义且可互换使用可互换，其是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。此外，修饰语“单克隆”仅表明该抗体的特征为从高度同源的抗体群中获得，不能理解为需要通过任何特定方法来制备所述抗体。
本发明的单克隆抗体可以通过多种技术进行制备，例如杂交瘤技术(参见，例如Kohler等人.Nature,256:495,1975)，重组DNA技术(参见，例如美国专利申请4,816,567)，或噬菌体抗体库技术(参见，例如Clackson等.Nature352：624-628,1991，或Marks等.J.Mol.Biol.222：581-597,1991)。
例如，可以如下来制备单克隆抗体。首先用免疫原(必要时候添加佐剂)免疫注射小鼠或其它合适的宿主动物。免疫原或佐剂的注射方式通常为皮下多点注射或腹腔注射。可将免疫原预先偶联到某些已知蛋白，如血清白蛋白或大豆胰酶抑制剂上，以增强抗原在宿主内的免疫原性。佐剂可以是弗氏佐剂或MPL-TDM等。动物在接受免疫后，体内将产生分泌特异性结合免疫原的抗体的淋巴细胞。另外，淋巴细胞也可以利用体外免疫获得。收集目的淋巴细胞，并用合适的融合剂，如PEG，使其与骨髓瘤细胞融合以获得杂交瘤细胞(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)。上述制备的杂交瘤细胞可以接种到合适的培养液中生长，培养液中优选含有一种或多种能够抑制未融合的、母体骨髓瘤细胞生长的物质。例如，对于缺乏次黄嘌呤鸟嘌呤磷酸转移酶(HGPRT或HPRT)的母体骨髓瘤细胞，在培养液中添加次黄嘌呤、氨基喋呤和胸腺嘧啶(HAT培养基)等物质将可以抑制HGPRT-缺陷细胞的生长。优选的骨髓瘤细胞应该具有融合率高，抗体分泌能力稳定，对HAT培养液敏感等特征。其中，骨髓瘤细胞首选鼠源骨髓瘤，如MOP-21或MC-11小鼠肿瘤衍生株(THE Salk Institute Cell Distribution Center,San Diego,Calif.USA)，和SP-2/0或X63-Ag8-653细胞株(American Type Culture Collection,Rockville,Md.USA)。另外也有研究报道，利用人骨髓瘤和 人鼠异源骨髓瘤细胞株制备人单抗(Kozbor,J.Immunol.,133:3001(1984)；Brodeur et al.,Monoclonal Antibody Production Techniques and Applications,pp.51-63,Marcel Dekker,Inc.,New York,1987)。生长杂交瘤细胞的培养液用于检测针对特异抗原的单抗的产生。测定杂交瘤细胞产生的单抗的结合特异性的方法包括例如，免疫沉淀或体外结合试验，如放射免疫试验(RIA)、酶联免疫吸附试验(ELISA)。例如，可利用Munson等在Anal.Biochem.107:220(1980)描述的Scatchard分析法来测定单抗的亲和力。当确定了杂交瘤产生的抗体的特异性、亲和力和反应性之后，目的细胞株可以通过(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)所描述的标准的有限稀释法进行亚克隆化。合适的培养液可以是DMEM或RPMI-1640等。另外，杂交瘤细胞还可以腹水瘤的形式在动物体内生长。利用传统的免疫球蛋白纯化方法，如蛋白A琼脂糖凝胶、羟基磷灰石层析、凝胶电泳、透析或亲和层析等，可以将亚克隆细胞分泌的单抗从细胞培养液、腹水或血清中分离出来。
还可以通过基因工程重组技术获得单克隆抗体。利用特异性结合单抗重链和轻链基因的核酸引物进行PCR扩增，可以从杂交瘤细胞中分离得到编码单抗重链和轻链基因的DNA分子。将所得的DNA分子插入表达载体内，然后转染宿主细胞(如E.coli细胞、COS细胞、CHO细胞、或其它不产生免疫球蛋白的骨髓瘤细胞)，并在合适的条件下进行培养，可以获得重组表达的目标抗体。
抗体可通过公知的技术，例如使用蛋白A或蛋白G的亲和层析进行纯化。随后或作为替代，可将特异性抗原(该抗体识别的靶分子)或其抗原表位固定在柱上，并通过免疫亲合层析法来纯化免疫特异性抗体。免疫球蛋白的纯化可参考例如D.Wilkinson(The Scientist,published by The Scientist,Inc.,Philadelphia Pa.,Vol.14,No.8(Apr.17,2000),pp.25-28)。
如本文中所使用的，术语“鼠源抗体”是指，来源于免疫接种过的小鼠的B细胞与骨髓瘤细胞融合，继而筛选出既能无限增殖又能分泌抗体的鼠杂交融合细胞，进而进行筛选、抗体制备和抗体纯化。或者由于抗原侵入小鼠体后B细胞分化增殖而形成浆细胞，浆细胞可产生分泌抗体。由特异性抗原刺激产生，抗体的产生是由于抗原侵入人体后引起各种免疫细胞相互作用，使淋巴细胞中的B细胞分化增殖而形成浆细胞，浆细胞可产生分泌抗体。
如本文中所使用的，术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链可变区来自第二抗体(例如人抗体)。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)的小鼠、大鼠、兔或非人灵长 类动物(例如，食蟹猴)抗体。
人源化抗体既能够保留非人源供体抗体(例如鼠源抗体)的预期性质，又能够有效降低非人源供体抗体(例如鼠源抗体)在人受试者中的免疫原性，因此，是特别有利的。然而，由于供体抗体的CDR与受体抗体的FR之间的匹配问题，人源化抗体的预期性质(例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)通常低于非人源供体抗体(例如鼠源抗体)。
因此，尽管本领域的研究人员已对抗体的人源化展开了深入的研究，并取得了一些进展(参见例如，Jones et al.,Nature,321:522 525(1986)；Reichmann et al.,Nature,332:323 329(1988)；Presta,Curr.Op.Struct.Biol.,2:593 596(1992)；and Clark,Immunol.Today 21:397 402(2000))，但是，如何对某一供体抗体进行充分的人源化，以使得所产生的人源化抗体既具有尽可能高的人源化程度，又能够尽可能地保留供体抗体的预期性质，现有技术并没有提供详尽的指导。技术人员需要针对具体供体抗体进行摸索、探究和改造，付出大量的创造性劳动才有可能获得，既具有高人源化程度(例如至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的人源化程度)、又保留具体供体抗体的预期性质的人源化抗体。
在本发明中，为了使人源化抗体尽可能保留供体抗体的性质(包括例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)，本发明的人源化抗体中构架区(FR)可以既包含人源受体抗体的氨基酸残基，也包含相应的非人源供体抗体的氨基酸残基。
本发明的人源化抗体可以根据上述制备的鼠单克隆抗体的序列进行制备。编码重链和轻链的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
为制备嵌合抗体，可使用本领域已知的方法将鼠免疫球蛋白可变区连接至人免疫球蛋白恒定区(参见例如Cabilly等人的美国专利No.4,816,567)。例如，将编码VH的DNA可操作的连接至编码重链恒定区的另一DNA分子以获得全长重链基因。人重链恒定区基因的序列是本领域已知的(参见例如Kabat,E.A.等人(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)，包含这些区的DNA片段可以通过标准PCR扩增获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区，但是通常优选为IgG1或IgG4恒定区。例如，将编码VL的DNA可操作的连接至编码轻链恒定区CL的另一DNA分子以获得全长轻链基因(以及Fab轻链基因)。人轻链恒定区基因的序列是本领域已知的(参见例如Kabat，E.A.等人(1991)Sequences of Proteins of Immunological Interest，Fifth Edition，U.S.Department of Health and Human Services，NIH Publication No.91-3242)，包含这些区的DNA片段可以通过标准PCR扩增获得。轻链恒定区可以是κ或λ恒定区，但通常优选为κ恒定区。
为制备人源化抗体，可以使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539；Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370；以及Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)。或者，还可以利用转基因动物，其能够在免疫后不产生内源性免疫球蛋白、并且能够产生完整人抗体库。例如，已有报道在嵌合和种系突变小鼠中抗体重链连接区(JH)基因的纯合缺失 可以完全抑制了内源性抗体产生，然后将人种系免疫球蛋白基因阵列转移到所述种系突变小鼠中将导致该小鼠在遇到抗原刺激时产生人抗体(参见例如，Jakobovits等，1993，Proc.Natl.Acad.Sci.USA 90：2551；Jakobovits等，1993，Nature362：255-258；Bruggermann等，1993，Year in Immunology 7：33；和Duchosal等，1992，Nature 355：258)。上述转基因动物的非限制性实例包括，HuMAb小鼠(Medarex,Inc.)，其含有编码未重排的人重链(μ和γ)和κ轻链免疫球蛋白序列的人免疫球蛋白基因微型基因座(miniloci)，加之使内源μ和κ链基因座失活的靶向突变(参见例如Lonberg等人(1994)Nature 368(6474):856-859)；或携带人重链转基因和人轻链转染色体的“KM小鼠TM”(参见专利申请WO02/43478)。其它抗体人源化改造的方法还包括噬菌体展示技术(Hoogenboom等，1991，J.Mol.Biol.227：381；Marks等，J.Mol.Biol.1991，222：581-597；Vaughan等，1996，Nature Biotech 14：309)。
如本文中所使用的，术语“人源化程度”是用于评价人源化抗体中非人源氨基酸残基的数量的指标。人源化抗体的人源化程度例如可通过IMGT网站DomainGapAlign来预测可变区序列与人V结构域的同源性。
如本文中所使用的，“同源抗体”指抗体的变体，其包含的重链和轻链可变区所包含的氨基酸序列与本文中所提供的抗体或其抗原结合片段的氨基酸序列同源，且其中所述变体保留了本发明抗CLDN18.2抗体的期望的功能特性。
用于比较的序列比对方法在本领域是熟知的。多种程序和比对算法描述于：Smith TF和Waterman MS,Adv.Appl.Math.,2:482,1981；Higgins DG和Sharp PM,CABIOS5:151,1989。Altschul SF等,Nature Genet.,6:119,1994提供了序列比对方法和同源性计算的详细思路。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)或半最大效应浓度(EC50)表示。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用生物发光干涉测量法(例如ForteBio Octet法)来测量解离常数。除此以外还可用表面等离子共振技术(例如Biacore)或Kinexa来测量解离常数。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可 通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换，通过保守置换氨基酸得到的抗体的变体保留其来源序列的生物学活性，如特异性地与CLDN18.2结合。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明 胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤、感染或自身免疫性疾病)在受试者体内的发生而实施的方法。
如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有肿瘤、感染或自身免疫性疾病，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤、感染或自身免疫性疾病)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤、感染或自身免疫性疾病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其它治疗等等。
如本文中所使用的，术语“免疫细胞”包括具有造血的起源并在免疫应答中起作用的细胞，例如淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
如本文中所使用的，术语“免疫应答”是指，免疫细胞(例如淋巴细胞、抗原呈递细胞、吞噬细胞或粒细胞)以及由免疫细胞或肝脏所产生的可溶性大分子(包括抗体、细胞因子、以及补体)的作用，该作用导致对侵入性病原体、被病原体感染的细胞或组织、癌细胞、或者在自身免疫或病理炎症情况下的正常人类细胞或组织的选择性损害、破坏或将它们从人体中清除。在本发明中，术语“抗原特异性T细胞应答”指由T细胞产生的免疫应答，该应答产生于当该T细胞特异的抗原对该T细胞的刺激之时。由T细胞在抗原特异性刺激时产生的反应的非限制性实例包括T细胞的增殖以及细胞因子(例如IL-2)的产生。
如本文中所使用的，术语“效应子功能(effector function)”是指，那些可归因于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)的生物学活性，且其随抗体同种型而变化。抗体效应子功能的例子包括但不限于：Fc受体结合亲和性、抗体依赖性细胞介导的细胞毒性(ADCC)、补体依赖的细胞毒性(CDC)、抗体依赖性细胞吞噬作用(ADCP)、细胞表面受体(例如B细胞受体)的下调、B细胞活化、细胞因子分泌、抗体和抗原-抗体复合物的半衰期/清除率等。改变抗体的效应子功能的方法是本领域已知的，例如通过在Fc区引入突变来完成。
术语“癌症”“肿瘤”可互换使用，其是指以体内异常细胞的不受控生长为特征的一大类疾病。不受管制的细胞分裂可能导致恶性肿瘤或侵入邻近组织的细胞的形成，并可能通过淋巴系统或血流转移到身体的远端部位。癌症包括良性和恶性癌症以及休眠肿瘤或微转移。癌症也包括血液肿瘤，尤其是血 液学恶性肿瘤。
术语“血液学恶性肿瘤”包括淋巴瘤，白血病，骨髓瘤或淋巴恶性肿瘤，以及脾癌和淋巴结肿瘤。示例性淋巴瘤包括B细胞淋巴瘤和T细胞淋巴瘤。B细胞淋巴瘤，包括例如霍奇金淋巴瘤。T细胞淋巴瘤，包括例如皮肤T细胞淋巴瘤。血液学恶性肿瘤还包括白血病，例如继发性白血病或急性淋巴细胞性白血病。血液恶性肿瘤还包括骨髓瘤(例如多发性骨髓瘤)及其它血液学和/或B细胞或T细胞相关的癌症。
如本文中所使用的，术语“药学上可接受的”指当分子本体、分子片段或组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。可作为药学上可接受的载体或其组分的一些物质的具体示例包括糖类(如乳糖)、淀粉、纤维素及其衍生物、植物油、明胶、多元醇(如丙二醇)、海藻酸等。
在本文中，组合疗法包括将本发明涵盖抗体-药物缀合物、组合物或药物组合物与一种或多种第二疗法的治疗剂(例如化学治疗剂)或其它预防或治疗模式(例如，放疗)组合使用。
第二疗法示例性的治疗剂可包括化学治疗剂(例如有丝分裂抑制剂)、烷化剂(例如氮芥Nitrogen Mustard)、抗代谢物(例如叶酸类似物)、天然产物(例如长春花生物碱Vinca Alkaloid)、多种试剂(例如铂配位络合物)、激素和拮抗剂(例如肾上腺皮质类固醇)、免疫调节剂(例如溴匹立明Bropirimine,Upjohn)等。其它抗癌治疗包括特异性靶向癌细胞的其它抗体。
在此类组合疗法中，各种治疗剂经常具有不同的互补作用机制，组合疗法可能导致协同效应。组合疗法包含影响免疫反应(例如增强或活化反应)之治疗剂及影响(例如抑制或杀死)肿瘤/癌细胞之治疗剂。组合疗法可降低抗药性癌细胞发生的可能性。组合疗法可允许试剂中的一种或多种试剂剂量减少，以减少或消除与试剂中之一或多种相关的不良作用。此类组合疗法可对潜在疾病、病症或病状具有协同的治疗或预防作用。
在本文中，“组合”包括可以分开施用的疗法，例如针对单独投药分开调配(例如，可以在套组中提供)，及可以按单一调配物(亦即“共调配物”)一起施用的疗法。在一些实施方案中，本发明的抗体-药物缀合物、组合物或药物组合物可依次序施用。在其它实施方案中，抗体-药物缀合物、组合物或药物组合物可同时施用。本发明的抗体-药物缀合物可以与至少一种其它(活性)药剂以任何方式组合使用。
HER2阴性指的是细胞表面没有大量的HER2蛋白，包括IHC 1+，或IHC 2+/FISH阴性，还包括IHC 0至1+及IHC 1+至2+的范围。
在本文中，术语“卤素”包括氟、氯、溴、碘。
在本文中，术语“C 1-6烷基”表示直链或支链的含有1-6个碳原子的烷基，包括例如“C 1-4烷基”、“C 1-3烷基”等，具体实例包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、2-甲基丁基、新戊基、1-乙基丙基、正己基、异己基、3-甲基戊基、2-甲基戊基、1-甲基戊基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、1,2-二甲基丙基等。
在本文中，术语“C 1-6亚烷基”表示含1-6个碳原子的直链或支链烷烃失去两个氢原子得到的二价基团，包括例如“C 1-4亚烷基”、“C 1-3亚烷基”等，具体实例包括但不限于：亚甲基、亚乙基、1,3-亚丙 基、1,4-亚丁基、1,5-亚戊基或1,6-亚己基等。
在本文中，术语“C 2-10烯基”是指含有至少一个双键且碳原子数为2-10的直链或支链的烯基，包括例如“C 2-6烯基”、“C 2-4烯基”等。其实例包括但不限于：乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、2-丁烯基、1,3-丁二烯基、1-戊烯基、2-戊烯基、3-戊烯基、1,3-戊二烯基、1,4-戊二烯基、1-己烯基、2-己烯基、3-己烯基、1,4-己二烯基、环戊烯基、1,3-环戊二烯基、环己烯基、1,4-环己二烯基等。
在本文中，术语“C 2-10亚烯基”是指含有2-10个碳原子的烯烃失去两个氢原子得到的二价基团。包括例如“C 2-8亚烯基”、“C 4-6亚烯基”等。其实例包括但不限于：1,5-亚戊烯基、1,5-亚戊-2-烯基、1,6-亚己烯基等。
在本文中，术语“C 2-10炔基”是指含有至少一个三键且碳原子数为2-10的直链或支链的炔基，包括例如“C 2-6炔基”、“C 2-4炔基”等。其实例包括但不限于：乙炔基、丙炔基、2-丁炔基、2-戊炔基、3-戊炔基、4-甲基-2-戊炔基、2-己炔基、3-己炔基、5-甲基-2-己炔基等。
在本文中，术语“C 2-10亚炔基”是指含有2-10个碳原子的炔烃失去两个氢原子得到的二价基团。包括例如“C 2-8亚炔基”、“C 4-6亚炔基”等。其实例包括但不限于：1,5-亚戊炔基、1,5-亚戊-2-炔基、1,6-亚己炔基等。
在本文中，术语“C 1-6烷氧基”是指具有C 1-6烷基-O-结构的基团，其中C 1-6烷基如前文中所定义。具体实例包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基、己氧基等。
在本文中，术语“5-12元杂芳基”是指含有5-12个环成员的芳香环状基团，并且其中至少一个环成员为选自N、O和S的杂原子。具体实例包括但不限于5-10元杂芳基、5-10元含氮杂芳基、5-6元含氧杂芳基等，例如呋喃基、噻吩基、吡咯基、噻唑基、异噻唑基、噻二唑基、噁唑基、异噁唑基、噁二唑基、咪唑基、吡唑基、1,2,3-三唑基、1,2,4-三唑基、1,2,3-噁二唑基、1,2,4-噁二唑基、1,2,5-噁二唑基、1,3,4-噁二唑基、吡啶基、嘧啶基、哒嗪基、吡嗪基、1,2,3-三嗪基、1,3,5-三嗪基、1,2,4,5-四嗪基等。
本发明并不限于本文所描述的特定方法学、方案、细胞系、载体、或试剂，因为它们可以有所变化。另外，本文所用的术语只用于描述特定实施方式的目的，而不是用于限定本发明的范围。
本文中(说明书及权利要求)，单数形式“一个”、“一种”和“所述”，除非上下文中有清楚的不同的说明，包括其复数形式，例如"一个宿主细胞"包括多个这样的宿主细胞。另外中文中没有对应的英文单复数语法规则，一个名词的单复数需根据上下文或实际情况判断，因此，英译本中，一个名词前面加上了“one or more than one”很可能是正确的。
附图说明
图1A显示了HEK293T-Claudin 18.2单克隆稳定细胞系流式检测。
图1B显示了L929-Claudin 18.2单克隆稳定细胞系流式检测。
图1C显示了KATOIII-Claudin 18.2单克隆稳定细胞系流式检测。
图1D显示了NCI-N87-Claudin 18.2单克隆稳定细胞系流式检测。
图1E显示了HEK293T-Claudin 18.1单克隆稳定细胞系Western blot检测。
图2显示了2C6.9-hz21、IMAB362抗体亲和力检测(流式细胞技术)。
图3显示了2C6.9-hz21、IMAB362抗体亲和力检测(L929-Claudin 18.2细胞)。
图4显示了通过流式细胞技术检测2C6.9-hz21抗体的特异性。
图5显示了2C6.9-hz21、IMAB362对HEK293T-Claudin 18.2细胞CDC杀伤活性测定。
图6显示了2C6.9-hz21、IMAB362抗体ADCC活性检测(HEK293T-Claudin 18.2细胞)。
图7显示了2C6.9-TL001(DAR：3.79)的HPLC-SEC检测图谱。
图8显示了2C6.9-TL001(DAR：7.12)的HPLC-SEC检测图谱。
图9显示了2C6.9-TL001(DAR：7.12)与细胞膜表面Claudin 18.2的亲和力检测结果。
图10A显示了2C6.9-TL001(DAR：7.12)对HEK293T-Claudin 18.2细胞杀伤活性检测结果。
图10B显示了2C6.9-TL001(DAR：3.79)对HEK293T-Claudin 18.2细胞杀伤活性检测结果。
图10C显示了2C6.9-TL001(DAR：7.12)对HEK293T-Claudin 18.1细胞杀伤活性检测结果。
图10D显示了2C6.9-TL002、2C6.9-TL003对HEK293T-Claudin 18.2细胞杀伤活性检测结果。
图10E显示了2C6.9-TL002、2C6.9-TL003对HEK293T-Claudin 18.1细胞杀伤活性检测结果。
图11A显示了2C6.9-TL001(DAR：7.12)对NUGC-4细胞杀伤活性检测结果。
图11B显示了2C6.9-TL001(DAR：3.79)对NUGC-4细胞杀伤活性检测结果。
图11C显示了2C6.9-TL002、2C6.9-TL003对NUGC-4细胞杀伤活性检测结果。
图12A显示了Balb/c Nude小鼠皮下NCI-N87-Claudin18.2细胞移植瘤模型中各组小鼠肿瘤体积的变化情况(*：P<0.05；****：P<0.0001)。
图12B显示了Balb/c Nude小鼠皮下NCI-N87-Claudin18.2细胞移植瘤模型中各组小鼠体重的变化情况。
图12C显示了2C6.9-TL001与2C6.9单抗+化疗在CDX模型11天体内药效比较结果(****：P<0.0001)。
图12D显示了2C6.9-TL001与2C6.9单抗+化疗在CDX模型21天体内药效比较结果(****：P<0.0001)。
图12E显示了不同DAR值2C6.9-TL001在CDX(NUGC-4)模型中21天各组小鼠肿瘤体积的变化情况(****：P<0.001)。
图12F显示不同DAR值2C6.9-TL001在CDX(NUGC-4)模型中21天各组小鼠体重的变化情况。
图12G显示了Balb/c Nude小鼠皮下
Figure PCTCN2021093348-appb-000033
胃癌GA0006 PDX模型中17天各组小鼠肿瘤体积的变化情况(****：P<0.0001)。
图12H显示了Balb/c Nude小鼠皮下
Figure PCTCN2021093348-appb-000034
胃癌GA0006 PDX模型中17天各组小鼠体重的变化情况。
图12I显示了Balb/c Nude小鼠皮下
Figure PCTCN2021093348-appb-000035
胃癌GA0006 PDX模型中24天各组小鼠肿瘤体积的变化情况(****：P<0.0001)。
图12J显示了Balb/c Nude小鼠皮下
Figure PCTCN2021093348-appb-000036
胃癌GA0006 PDX模型中24天各组小鼠体重的变化情况。
图12K显示了Balb/c Nude小鼠皮下NCI-N87-Claudin18.2细胞移植瘤模型中各组小鼠肿瘤体积的变化情况(***：P<0.001；****：P<0.0001)。
图12L显示了Balb/c Nude小鼠皮下NCI-N87-Claudin18.2细胞移植瘤模型中各组小鼠体重的变化情况。
图12M显示了Balb/c Nude小鼠皮下HEK293T-Claudin18.2细胞移植瘤模型中各组小鼠肿瘤体积的变化情况(**：P<0.01；****：P<0.0001)。
图12N显示了Balb/c Nude小鼠皮下HEK293T-Claudin18.2细胞移植瘤模型中各组小鼠体重的变化情况。
图12O显示了Balb/c Nude小鼠皮下NUGC-4细胞移植瘤模型中各组小鼠肿瘤体积的变化情况(****：P<0.0001)。
图12P显示了Balb/c Nude小鼠皮下NUGC-4细胞移植瘤模型中各组小鼠体重的变化情况。
图13显示了2C6.9-TL001(DAR：7.40)的HPLC-SEC检测图谱。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例一 生物活性分子(TOXIN-1)的合成
Figure PCTCN2021093348-appb-000037
将甲磺酰氯(462mg,12.77mmol，纯度70％左右)滴加入盐酸贝洛替康(3g,6.38mmol)和三乙胺(2.58g,25.54mmol)的二氯甲烷(40mL)溶液中，室温条件下反应2h。抽滤，滤饼用二氯甲烷(3mL)洗三次，得到2.2g的(S)-N-(2-(4-乙基-4-羟基-3,14-二酮-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲嗪并[1,2-b]喹啉-11-基)乙基)-N-异丙基甲磺酰胺(TOXIN-1)。
结构表征数据如下：
1H NMR(400MHz,DMSO-d 6)δ8.32(d,J＝8.4Hz,1H),8.20(dd,J＝8.4,1.2Hz,1H),7.93-7.84(m,1H),7.79(t,J＝7.6Hz,1H),7.35(s,1H),6.56(s,1H),5.44(d,J＝9.2Hz,4H),3.98(p,J＝6.7Hz,1H),3.50(t,J＝8.0Hz,2H),3.42-3.35(m,2H),3.00(s,3H),1.93-1.82(m,2H),1.15(d,J＝6.7Hz,6H),0.88(t, J＝7.3Hz,3H).
ESI-MS(m/z):512.2[M+H] +.
[α] D 20为+28.19°(c＝0.101g/100mL,CH 3CN).
实施例二 6-(2-(甲磺酰基)嘧啶-5-基)-5-己炔酸(化合物3-4)的合成
Figure PCTCN2021093348-appb-000038
步骤一：6-(2-(甲硫基)嘧啶-5-基)-5-己炔酸甲酯(化合物3-2)的合成
室温下，将5-己炔酸甲酯(500mg，3.97mmol)，5-溴-2-甲硫基嘧啶溶于N,N-二甲基甲酰胺(3mL)中，依次加入三乙胺(3mL)，碘化亚铜(75mg，0.4mmol)和二三苯基膦二氯化钯(279mg，0.4mmol)，氮气保护下升温至95℃搅拌反应6h，加水淬灭，乙酸乙酯萃取(20mL×3)，合并有机相，饱和食盐水洗涤(20mL×2)，无水硫酸钠干燥，滤除干燥剂，减压蒸除溶剂，制备液相色谱纯化，得300mg标题化合物。ESI-MS(m/z):251.3[M+H] +。
步骤二：6-(2-(甲硫基)嘧啶-5-基)-5-己炔酸(化合物3-3)的合成
室温下，将化合物3-2(200mg，0.8mmol)溶于四氢呋喃和水混合溶液中(4mL/4mL)，加入氢氧化锂一水合物(235mg，5.6mmol)，室温搅拌反应4h，加水稀释，乙酸乙酯萃取(20mL×2)，水相用1N盐酸调pH＝3，乙酸乙酯(20mL×3)萃取，合并有机相，饱和食盐水洗涤(20mL×2)，无水硫酸钠干燥，滤除干燥剂，减压蒸除溶剂，得120mg标题化合物。
步骤三：6-(2-(甲磺酰基)嘧啶-5-基)-5-己炔酸(化合物3-4)的合成
室温下，将化合物3-3(20mg，0.085mmol)溶于二氯甲烷(4mL)中，加入间氯过氧苯甲酸(22mg，0.127mmol)，加毕，室温搅拌反应过夜，制备液相色谱纯化，得20mg标题化合物。ESI-MS(m/z):269.1[M+H] +。
实施例三 (4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五烷酰胺基)苄基)((S)-4-乙基-11-(2-(N-异丙基甲基磺酰胺基)乙基)-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲嗪并[1,2-b]喹啉-4-基)碳酸酯(化合物TL001)的合成
Figure PCTCN2021093348-appb-000039
步骤一：6-(2-(甲基磺酰基)嘧啶-5-基)-N-(炔丙基)己-5-炔酰胺(化合物3-5)的合成
25℃下将炔丙胺(189mg,3.4mmol)和化合物3-4(800mg,2.83mmol)溶于二氯甲烷(10mL)中，依次加入N,N-二异丙基乙胺(738mg,5.67mmol)，O-(7-氮杂苯并三唑-1-基)-N,N,N′,N′-四甲基脲六氟磷酸酯(1.63g,4.25mmol)，搅拌反应2h。反应液减压浓缩，残余物经快速硅胶柱纯化(乙酸乙酯/石油醚＝3/1)得700mg标题化合物。ESI-MS(m/z):306.1[M+H] +。
步骤二：(4-((S)-35-叠氮基-2-(4-(((4-甲氧基苯基)二苯甲基)氨基)丁基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五烷酰氨基)苄基)((S)-4-乙基-11-(2-(N-异丙基甲磺酰胺基)乙基)-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4'：6,7]吲嗪并[1,2-b]喹啉-4-基)碳酸酯(化合物33-1)的合成
25℃氮气保护下，将TOXIN-1(250mg,0.49mmol)溶于二氯甲烷(10mL)中，降温至0℃，加入4-二甲氨基吡啶(478mg,3.91mmol)的二氯甲烷(3mL)溶液，然后缓慢滴加三光气(72mg,0.24mmol)的二氯甲烷(10mL)溶液，加毕，0℃搅拌反应20min，反应液用氮气吹20min。加入(S)-2-(32-叠氮基-5-氧代-3,9,12,15,18,21,24,27,30-九氧杂-6-氮杂三十二烷酰胺基)-N-(4-(羟甲基)苯基)-6-(((4-甲氧基苯基)二苯甲基)氨基)己酰胺(518mg,0.49mmol)的二氯甲烷(7mL)溶液，加毕，0℃搅拌反应1h。反应液减压浓缩，残余物经制备高效液相色谱纯化得500mg标题化合物。ESI-MS(m/z):1597.5[M+H] +。
步骤三：((S)-4-乙基-11-(2-(N-异丙基甲磺酰胺基)乙基)-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并 [3',4':6,7]吲嗪并[1,2-b]喹啉-4-基)(4-((S)-2-(4-(((4-甲氧基苯基)二苯基甲基)氨基)丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-壬氧基-3,9-二氮杂三十五烷酰胺基)苄基)碳酸酯(化合物33-2)的合成
室温下，将化合物33-1(80mg，0.05mmol)和3-5片段(23mg,0.075mmol)溶于二甲基亚砜和水(2.0mL：0.5mL)中，加入溴化亚铜(11mg，0.08mmol)，搅拌反应1h。经制备高效液相色谱纯化，得30mg标题化合物。ESI-MS(m/z):815.9[(M-273)/2+H] +。
步骤四：(4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五烷酰胺基)苄基)((S)-4-乙基-11-(2-(N-异丙基甲基磺酰胺基)乙基)-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲嗪并[1,2-b]喹啉-4-基)碳酸酯(化合物TL001)的合成
将化合物33-2(30mg，0.02mmol)溶于二氯甲烷(1.0mL)反应液中，加入三氟乙酸(0.2mL)，室温反应30min。经制备高效液相色谱纯化，得20.0mg标题化合物的三氟乙酸盐。其结构表征如下：
1H NMR(400MHz,DMSO-d 6)δ10.18(s,1H),9.10(s,2H),8.38(t,J＝5.56Hz,1H),8.32(d,J＝8.40Hz,1H),8.22–8.20(m,2H),8.09(t,J＝5.68Hz,1H),7.91–7.87(m,2H),7.82–7.78(m,1H)，7.69(brs,3H),7.61(d,J＝8.56Hz,2H),7.32(d,J＝8.56Hz,2H),7.06(s,1H),5.56(d,J＝16.96Hz,1H),5.51(d,J＝16.96Hz,1H),5.47(d,J＝19.28Hz,1H),5.42(d,J＝19.28Hz,1H),5.14(d,J＝12.20Hz,1H),5.07(d,J＝12.16Hz,1H),4.48(t,J＝5.24Hz,2H),4.46–4.43(m,1H),4.29(d,J＝5.60Hz,2H),4.08–3.95(m,5H),3.79(t,J＝5.28Hz,2H),3.51–3.43(m,32H),3.40(s,3H),3.39–3.35(m,2H),3.30–3.26(m,2H),3.00(s,3H),2.82–2.74(m,2H),2.56(t,J＝7.08Hz,2H),2.29(t,J＝7.36Hz,2H),2.23–2.13(m,2H),1.82(p,J＝7.24Hz,2H),1.78–1.63(m,2H),1.61–1.49(m,2H),1.42–1.27(m,2H),1.15(d,J＝6.80Hz,3H),1.13(d,J＝6.76Hz,3H),0.90(t,J＝7.32Hz,3H).
ESI-MS(m/z):816.0[M/2+H] +.
[α] D 20为-19.55°(c＝1.000g/100mL,CH 3CN).
实施例四 ((S)-4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)(4-((S)-2-((S)-3-甲基-2-(4-(1-(26-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)丁酰胺基)丙酰胺基)苄基)乙烷-1,2-二基双(甲基氨基甲酸酯)三氟乙酸盐的合成(TL002)
Figure PCTCN2021093348-appb-000040
步骤一：(S)-(4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)(4-硝基苯基)碳酸酯的合成
(S)-4,11-二乙基-4,9-二羟基-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-3,14(4H,12H)-二酮(150mg,0.37mmol)溶于二氯甲烷(15mL)中，加入二异丙基乙胺(96.81mg,0.74mmol)，再加入双(4-硝基苯基)碳酸酯(127.92mg,0.41mmol)的二氯甲烷(15mL)溶液，25℃反应3h。反应液浓缩，得标题化合物207mg。直接用于下一步反应。
ESI-MS(m/z):558.1[M+H] +。
步骤二：(S)-叔丁基(4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)乙烷-1,2-二基双(甲基氨基甲酸酯)的合成
将(S)-(4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)(4-硝基苯基)碳酸酯(207mg,0.33mmol)溶于二氯甲烷(10mL)中，加二异丙基乙胺(130.87mg,1.00mmol)和N-甲基-N-[2-(甲胺基)乙基]氨基甲酸叔丁酯(71.34mg,0.37mmol)，25℃搅拌12h。反应液浓缩，残余物经硅胶柱层析色谱法纯化(洗脱剂：二氯甲烷/甲醇＝9/1)，得标题化合物207mg。
ESI-MS(m/z):607.3[M+H] +。
步骤三：(S)-N-甲基-(4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)(2-(甲氨基)乙基)氨基甲酸酯三氟乙酸盐的合成
(S)-叔丁基(4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)乙烷-1,2-二基双(甲基氨基甲酸酯)(207mg,0.19mmol)溶于二氯甲烷(8mL)，加入三氟乙酸(2mL)，25℃反应2h。反应液浓缩，得标题化合物200mg。直接用于下一步反应。
ESI-MS(m/z):507.2[M+H] +。
步骤四：(4-((S)-2-((S)-2-(4-(1-(26-叠氮-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)-3-甲基丁酰胺基)丙酰胺基)苄基)((S)-4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)乙烷-1,2-二基双(甲基氨基甲酸酯)的合成
(4-((S)-2-((S)-2-(4-(1-(26-叠氮-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)-3-甲基丁酰胺基)丙酰胺基)苄基)(4-硝基苯基)碳酸酯(150mg,0.12mmol)溶于N,N-二甲基甲酰胺(3mL)中，加入1-羟基苯并三唑(34.03mg,0.25mmol)和二异丙基乙胺(48.82mg,0.37mmol)，再加入(S)-N-甲基-(4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)(2-(甲氨基)乙基)氨基甲酸酯三氟乙酸盐(102.11mg,0.12mmol)，25℃反应16h。反应液用制备高效液相色谱仪纯化，制备液经冷冻干燥，得标题化合物44mg。
ESI-MS(m/z):1400.7[M+H] +。
色谱柱：Waters XBridge Prep C18 OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 10 90 28
3.00 10 90 28
19.00 90 10 28
步骤五：((S)-4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)(4-((S)-2-((S)-3-甲基-2-(4-(1-(26-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)丁酰胺基)丙酰胺基)苄基)乙烷-1,2-二基双(甲基氨基甲酸酯)三氟乙酸盐的合成
(4-((S)-2-((S)-2-(4-(1-(26-叠氮-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)-3-甲基丁酰胺基)丙酰胺基)苄基)((S)-4,11-二乙基-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-9-基)乙烷-1,2-二基双(甲基氨基甲酸酯)(44mg,0.30mmol)和6-(2-(甲基磺酰基)嘧啶-5-基)-N-炔丙基)己酰胺(14.09mg,0.045mmol)溶于二甲亚砜(3mL)和水(0.75mL)中，加入溴化亚铜(8.65mg,0.06mmol),25℃反应1h。反应液用制备高效液相色谱仪纯化，制备液冻干后溶于二氯甲烷(2mL)中，加三氟乙酸(0.2mL)，25℃下搅拌0.5h。制备高效液相色谱仪纯化，制备液经冷冻干燥，得标题化合物16mg。
ESI-MS(m/z):853.6[M/2+H] +。
色谱柱：Waters SunFire Prep C18 ODS 5μm 19x50mm
流动相A：乙腈；流动相B：水(0.05％三氟乙酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 10 90 28
4.00 10 90 28
20.00 90 10 28
实施例五 (4-((S)-2-((S)-3-甲基-2-(4-(1-(26-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔基酰胺基)甲基)-1H-1,2,3-三唑-1-基)-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)丁酰胺基)丙酰胺基)苄基)2-((S)-4-乙基-4-羟基-3,14-二氧-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-11-基)乙基异丙基氨基甲酸酯三氟乙酸盐(TL003)
Figure PCTCN2021093348-appb-000041
步骤一：(S)-2-氨基-N-((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙基-2-基)-3-甲基丁酰胺的合成
室温下，将((9H-芴-9-基)甲基)((S)-1-(((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙基-2-基)氨基)-3-甲基-1-氧代丁基-2-基)氨基甲酸酯(2.00g，3.88mmol)溶于N,N-二甲基甲酰胺(12mL)中，加哌啶(3mL)，25℃反应2h。将反应液倒入水中，析出固体，抽滤，经制备高效液相色谱仪纯化，制备液经冷冻干燥，得标题化合物810mg。
ESI-MS(m/z):294.0[M+H] +。
色谱柱：Waters SunFire Prep C18 ODS 8μm 45x450mm
流动相A：乙腈；流动相B：水
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 7 93 60
7.00 7 93 60
50.00 50 50 60
步骤二：4-(4-((S)-1-((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙基-2-基)氨基)-3-甲基-1-氧代丁基-2-基)氨基)-4-氧代丁基)哌啶-1-甲酸叔丁酯的合成
室温下，将(S)-2-氨基-N-((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙基-2-基)-3-甲基丁酰胺(810mg，2.76mmol)、4-(1-(叔丁氧羰基)哌啶-4-基)丁酸和2-乙氧基-1-乙氧基碳基-1,2-二氢喹啉溶于二氯甲烷(8mL)和甲醇(8mL)中，45℃反应2h。反应液减压浓缩，残余物经硅胶层析色谱柱(洗脱剂：二氯甲烷/甲醇＝15/1)纯化，得标题化合物1.31g。
ESI-MS(m/z):546.8[M+H] +。
步骤三：(S)-N-((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙基-2-基)-3-甲基-2-(4-(哌啶-4-基)丁酰胺基)丁酰胺三氟乙酸盐的合成
室温下，将4-(4-((S)-1-((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙基-2-基)氨基)-3-甲基-1-氧代丁基-2-基)氨基)-4-氧代丁基)哌啶-1-甲酸叔丁酯(1.3g，2.14mmol)溶于二氯甲烷(20mL)中，加三氟乙酸(5mL)，25℃反应2h。将反应液减压浓缩，残余物溶于乙腈(30mL)中，加碳酸钾(1.22g，8.85mmol)，25℃反应2h。抽滤，滤饼用乙腈洗涤，收集滤液，减压浓缩，得标题化合物900mg。
ESI-MS(m/z):446.9[M+H] +。
步骤四：(S)-2-(4-(1-(26-叠氮基-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)-N-((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙基-2-基)-3-甲基丁酰胺的合成
室温下，将(S)-N-((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙基-2-基)-3-甲基-2-(4-(哌啶-4-基)丁酰胺基)丁酰胺三氟乙酸盐(487mg，0.78mmol)和26-叠氮基-3,6,9,12,15,18,21,24-八氧杂二十六烷基对甲苯磺酸酯(619mg，0.94mmol)溶于乙腈(20mL)中，加碳酸钾(655mg，4.69mmol)，16℃反应6h。反应液减压浓缩，残余物经硅胶层析色谱柱(洗脱剂：二氯甲烷/甲醇＝8/1)纯化，得标题化合物586mg。
ESI-MS(m/z):868.5[M+H] +。
步骤五：(4-((S)-2-((S)-2-(4-(1-(26-叠氮-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)-3-甲基丁酰胺基)丙酰胺基)苄基)(4-硝基苯基)碳酸酯的合成
室温下，将(S)-2-(4-(1-(26-叠氮-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)-N-((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙基-2-基)-3-甲基丁酰胺(585mg，0.64mmol)溶于二氯甲烷(30mL)中，加N,N-二异丙基乙胺(334.31mg，2.56mmol)，然后滴加二(对硝基苯)碳酸酯(602.35mg，1.92mmol)的二氯甲烷(30mL)溶液，25℃反应6h。反应液减压浓缩，残余物加入甲基叔丁基醚，抽滤，得标题化合物760mg。
ESI-MS(m/z):1033.4[M+H] +。
步骤六：(4-((S)-2-((S)-2-(4-(1-(26-叠氮-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)-3-甲基丁酰胺基)丙酰胺基)苄基)2-((S)-4-乙基-4-羟基-3,14-二氧-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-11-基)乙基异丙基氨基甲酸酯的合成
室温下，将(4-((S)-2-((S)-2-(4-(1-(26-叠氮-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)-3-甲基丁酰胺基)丙酰胺基)苄基)(4-硝基苯基)碳酸酯(150mg，0.12mmol)溶于N,N-二甲基甲酰胺(3mL)中，加1-羟基苯并三唑(34.03mg，0.25mmol)和N,N-二异丙基乙胺(48.82mg，0.37mmol)，然后加(S)-4-乙基-4-羟基-11-(2-(异丙基氨基)乙基)-1,12-二氢-14H-吡喃[3',4'：6,7]吲嗪并[1,2-b]喹啉-3,14(4H)-二酮盐酸盐(59.79mg，0.12mmol)，反应液室温搅拌过夜。反应液经制备高效液相色谱仪纯化，制备液冷冻干燥，得标题化合物64mg。
ESI-MS(m/z):1327.6[M+H] +。
步骤七：(4-((S)-2-((S)-3-甲基-2-(4-(1-(26-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔基酰胺基)甲基)-1H-1,2,3-三唑-1-基)-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)丁酰胺基)丙酰胺基)苄基)2-((S)-4-乙基-4-羟基-3,14-二氧-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-11-基)乙基异丙基氨基甲酸酯三氟乙酸盐的合成
室温下，将(4-((S)-2-((S)-2-(4-(1-(26-叠氮-3,6,9,12,15,18,21,24-八氧杂二十六烷基)哌啶-4-基)丁酰胺基)-3-甲基丁酰胺基)丙酰胺基)苄基)2-((S)-4-乙基-4-羟基-3,14-二氧-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲嗪并[1,2-b]喹啉-11-基)乙基异丙基氨基甲酸酯(64mg，0.046mmol)和6-(2-(甲基磺酰基)嘧啶-5-基)-N-(丙-2-炔-1-基)己酰胺(21.63mg，0.069mmol)溶于二甲亚砜(4mL)和水(1mL)中，加溴化亚铜(13.27mg，0.092mmol)，25℃反应1h。反应液经制备高效液相色谱仪纯化，制备液经冷冻干燥，得标题化合物35mg。ESI-MS(m/z):1632.8[M+H] +。
色谱柱：Waters SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％三氟乙酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 10 90 28
2.00 10 90 28
18.00 90 10 28
实施例六 靶向人Claudin 18.2单克隆抗体的制备
本发明中靶向人Claudin 18.2单克隆抗体为人源化单克隆抗体，包括2C6.9-hz11和2C6.9-hz21，其CDR、可变区序列及恒定区序列信息如表1所示：
表1：序列信息简表
Figure PCTCN2021093348-appb-000042
Figure PCTCN2021093348-appb-000043
6.1人Claudin 18.2和人Claudin 18.1过表达细胞系的构建及鉴定
6.1.1人Claudin18.2和人Claudin18.1过表达细胞系构建
为验证人Claudin 18.2抗体的特异性及功能，将人Claudin 18.2完整编码序列(基因编号：NM_001002026.2，由南京金斯瑞生物科技有限公司合成)和人Claudin 18.1完整编码序列(基因编号：NM_016369.3，由南京金斯瑞生物科技有限公司合成)克隆到慢病毒载体pLVX-IRES-puro上，并通过文献所述(Mohammadi Z etl.,Mol Biotechnol.2015Sep；57(9):793-800.)慢病毒包装系统制备病毒，获得病毒后分别感染HEK293T、L929、KATOIII及NCI-N87细胞，通过嘌呤霉素筛选及单克隆挑选，获得单克隆的HEK293T-Claudin 18.1、HEK293T-Claudin 18.2、L929-Claudin 18.2、KATOIII-Claudin 18.2、NCI-N87-Claudin 18.2稳定细胞系。将人Claudin 18.2、人Claudin 18.1质粒通过4D-Nucleofector X转染试剂盒(Lonza,Cat#V4XC-3012)转染BaF/3细胞(DSMZ,Cat#ACC300)，转染48小时后加1.25mg/mL hygromycin(Thermo Fisher Sci.Cat# 10687010)进行筛选，筛选12天后分选得到单克隆，获得单克隆的BaF/3-Claud18.1和BaF/3-Claud18.2细胞系。
6.1.2人Claudin18.2和人Claudin18.1过表达细胞系检测
HEK293T-Claudin 18.1采用Western Blot鉴定(检测抗体：Proteintech，66167-1-Ig)，其它细胞系采用流式细胞仪(流式细胞仪：Beckman，CytoFlex；检测抗体IMAB362，序列来自于专 利：CN 101312989B)进行鉴定。如图1A-1D所示，流式结果依次显示HEK293T-Claudin 18.2、L929-Claudin 18.2、KATOIII-Claudin 18.2、NCI-N87-Claudin 18.2均获得阳性率高(接近100％)、均一性较好的单克隆细胞系，可用于后续实验。Western Blot结果(图1E)显示三株HEK293T-Claudin 18.1稳定细胞系均过表达人Claudin 18.1，其中HEK293T-Claudin 18.1-1C2单克隆表达量较高，可用于后续实验。
6.2抗人Claudin18.2鼠源单克隆抗体的制备
本发明采用DNA免疫和细胞免疫的方式免疫野生型小鼠获得抗人Claudin18.2鼠源单克隆抗体。每只Balb/c小鼠通过尾静脉注射的方式注射100μg含人Claudin18.2完整编码序列的质粒，第四次免疫和第六次免疫后，通过流式分析检测血清滴度。选取滴度较高的小鼠用BaF/3-Claudin18.2过表达细胞系在融合前3-5天进行加强免疫。采用标准的融合流程用PEG融合的方式将小鼠脾脏细胞和Sp2/0(ATCC,Cat#CRL-1581)小鼠骨髓瘤细胞系进行融合，之后采取HAT加压筛选，10-14天后进行流式筛选。
通过流式细胞仪(购自Sartorius，型号：iQue Screener Plus)筛选了6000个杂交瘤克隆上清，获得43个能够识别HEK293T-Claudin18.2细胞系的阳性杂交瘤克隆进行亚克隆。选择HEK293T-Claudin18.2和HEK293T-Claudin18.1细胞系进一步通过流式细胞仪筛选获得只结合人Claudin18.2而不结合人Claudin18.1的阳性克隆14个。最后通过有限稀释法进行亚克隆分选以获得单克隆。
人胃癌细胞系NUGC4(购自日本JCRB细胞库，货号：JCRB0834)内源性表达Claudin18.2蛋白，被广泛用于检测抗体与内源性Claudin18.2的结合以及功能检测方法开发。采用NUGC4细胞对候选克隆进行了检测，最终选取了7个亚克隆作为候选克隆。经进一步亲和力检测后，选取2C6.9M进行可变区扩增及人源化。
为了检测候选杂交瘤克隆的抗体亚型，采用Pierce Rapid Isotyping试剂盒(Thermo Fisher Sci.Cat#26179)对2C6.9M进行了鉴定。鉴定结果表明其重链为IgG1亚型，轻链为Kappa亚型。
杂交瘤细胞培养到8000个左右，细胞裂解并采用cDNA反转录试剂盒(Thermo Fisher Sci.Cat# 18080-200)合成第一链cDNA。采用引物从cDNA中PCR方式扩增VH和VK(VL Kappa)基因，PCR产物通过DNA纯化试剂盒(Qiagen,Cat#28104)纯化，并连接到TOPO载体上(Thermo Fisher Sci.Cat#K457540)。每一个连接反应大概挑取12个克隆进行测序。序列通过Vector NTI 11.5(Thermo Fisher Sci.)和Sequencer 5.4.6(Genecodes)进行分析。获得鼠源抗体2C6.9M可变区序列以及CDR序列如表2所示。
表2：2C6.9可变区及CDR氨基酸序列(SEQ ID NO：)
Figure PCTCN2021093348-appb-000044
6.3鼠源抗体2C6.9M的人源化
采用CDR移植抗体人源化改造方法对鼠源抗体2C6.9M进行人源化改造。简言之，人源化改造涉 及以下步骤：把鼠源单克隆抗体的氨基酸序列与人胚系抗体氨基酸序列进行比对，找出同源性高，理化性质较优的序列，作为人胚胎系框架序列；分析考察HLA-DR亲和性，选出亲和力低的人胚胎系框架序列；再将鼠源抗体的六个CDR分别移植到选定的重链及轻链框架序列上。
具体而言，将鼠源抗体2C6.9M的重链和轻链CDR区分别移植到对应的人源化模板的FR框架上。2C6.9M的重链、轻链人源化模板分别为人胚系基因序列IGHV4-59*01(参见IMGT登录号AB019438)及IGKV4-1*01(参见IMGT登录号Z00023)。
进一步利用计算机模拟技术，应用分子对接分析可变区及其周边的框架氨基酸序列，考察其空间立体结合方式。通过计算静电力，范德华力，亲疏水性和熵值，分析该鼠源抗体氨基酸序列中可与Claudin18.2作用以及维护空间构架的关键氨基酸，并在移植后的抗体中，保留这些鼠源的氨基酸。也即，对上述人源化模板的FR区氨基酸残基进行了一系列的回复突变，以使人源化抗体尽可能保留鼠源抗体的抗原结合能力。
鼠源抗体2C6.9M的可变区序列如SEQ ID NO：23、SEQ ID NO：24所示，其CDR序列如SEQ ID NO：1至SEQ ID NO：12所示。为在不影响亲和力的情况下避免异构体的发生，本发明将2C6.9CDR-H2氨基酸序列进行改造，改造后序列如SEQ ID NO：21、SEQ ID NO：22所示。最终构建了2株人源化抗体，分别命名为2C6.9-hz11、2C6.9-hz21。各抗体的重链恒定区均为人野生型IgG1重链恒定区(SEQ ID NO：16)，抗体的轻链恒定区为人野生型IgG1κ轻链恒定区(SEQ ID NO：17)。
2C6.9各抗体的可变区，恒定区以及重轻链氨基酸序列如表1所示。
本发明将2C6.9的人源化抗体的重链和轻链氨基酸序列通过密码子优化合成cDNA连接入质粒pcDNA3.4(委托南京金斯瑞生物科技有限公司)。重链和轻链对应的pcDNA3.4同时转染到Expi293F细胞中(购买自Thermo公司)，将该细胞培养后的上清通过protein A(MabSelect SuRe,GE)亲和层析柱纯化后获得2C6.9的人源化单克隆抗体。
6.4 2C6.9的人源化单克隆抗体亲和力检测
采用HEK293T-Claudin 18.2细胞检测2C6.9-hz21与细胞膜上人Claudin 18.2亲和力。具体步骤如下：消化HEK293T-Claudin18.2细胞，离心重悬，PBS洗两次；用PBS(含1％BSA)重悬细胞，按300000细胞/孔铺细胞至96孔尖底板中，每孔50μl体积，铺20个孔；在每个孔中分别加入50μL 2C6.9-hz21及IMAB362抗体，终浓度按1000nM起始，3倍梯度稀释，共11个浓度点；以Human IgG作为阴性对照；混匀，4℃避光反应1小时；PBS洗3次，加入FITC标记的抗人Fc二抗(品牌：BioLegend，货号：409322)，4℃避光孵育0.5小时；PBS洗3次，流式上机(流式细胞仪品牌：Beckman，型号：Cytoflex)检测。
实验结果如图2及表3所示，通过流式细胞技术检测2C6.9-hz21与HEK293T-Claudin 18.2结合亲和力EC50值小于IMAB362，最大荧光信号值大于IMAB362，表明2C6.9-hz21与细胞膜上Claudin18.2亲和力优于IMAB362。
表3：人源化抗体2C6.9-hz21结合HEK293T-Claudin 18.2流式亲和力检测
抗体名称 EC50(nM) Max MFI
2C6.9-hz21 5.767 165866.3
IMAB362 8.217 156715.7
6.5 2C6.9的人源化单克隆抗体亲和力及特异性测定
人Claudin 18.2为4次跨膜的膜蛋白，结构较为复杂，采用细胞ELISA的方法进行检测，以保证Claudin 18.2抗原分子的完整构型。选用6.1中构建的L929-Claudin 18.2稳定细胞系进行检测。具体实验步骤如下：将贴壁生长的L929-Claudin 18.2细胞系用2mM EDTA消化，重悬至2×10 5细胞/毫升，每孔100μL铺于96孔板中，37℃培养过夜；第二天去掉培养基，PBS清洗一次，每孔加入100μL 4％甲醛室温固定30分钟，去甲醛，PBS清洗两次，加入100μL PBS(含2％BSA)于37℃封闭2小时；去封闭液，用PBS(含2％BSA)稀释待测抗体，1μM起始，4倍稀释，11个浓度点，100μL/每孔，37℃孵育2小时；250μL PBST清洗5次，每次静置2分钟；PBS(含2％BSA)稀释辣根过氧化物酶(HRP)标记的抗人IgG二抗(HRP-anti-Human IgG，Jackson ImmunoResearch，109-035-003)，1:10000稀释，100μL/孔，37℃孵育1小时；250μL PBST清洗6次，每次静置2分钟；加入100μL TMB显色液(Thermo，34029)到对应的孔中，37℃显色20分钟；加入50μL 2mol/L H 2SO 4终止，酶标仪(MD，SpectraMax M2)450nm读数，结果导入Graphpad Prism进行曲线拟合。
实验结果如图3及表4所示，表明人源化抗体2C6.9-hz21与人Claudin18.2亲和力显著优于IMAB362。同样的实验测得人源化抗体2C6.9-hz11与抗体2C6.9-hz21亲和力接近(结果略)。
表4：人源化抗体2C6.9-hz21结合L929-Claudin 18.2细胞ELISA亲和力检测
抗体名称 EC50(nM) 最大信号值(OD450)
2C6.9-hz21 0.12 0.92
IMAB362 0.15 0.53
本发明还通过流式细胞技术检测了候选抗体的特异性。具体步骤如下：消化HEK293T、HEK293T-人Claudin 18.1、HEK293T-人Claudin18.2细胞，离心重悬，PBS洗两次；用PBS(含1％BSA)重悬细胞，每种细胞分别取300000个细胞加入终浓度为1000nM的候选抗体，混匀，4℃避光反应1小时；PBS洗3次，加入FITC标记的抗人Fc二抗(品牌：BioLegend，货号：409322)，4℃避光孵育0.5小时；PBS洗3次，流式细胞仪检测(流式细胞仪品牌：Beckman，型号：Cytoflex)检测。
实验结果如图4所示，2C6.9-hz21能够特异性地结合人Claudin 18.2，而不结合人Claudin 18.1。
6.6 2C6.9的人源化抗体的补体依赖性细胞毒性(CDC)检测
2C6.9为IgG1亚型，能够有效激活补体经典途径，发挥补体依赖的细胞毒性(complement dependent cytotoxicity，CDC)功能。为测定抗体2C6.9的CDC效应，本发明以补体含量丰富的豚鼠血清(购自郑州百基，货号：S0001)为材料进行实验。具体实验步骤如下：取HEK293T-Claudin 18.2细胞，离心后调整细胞密度，5×10 4/孔铺板过夜；第二天配制DMEM+20％豚鼠血清培养基，用此培养基稀释2C6.9-hz21及IMAB362抗体，20μg/mL起始，2倍稀释，10个浓度点；去除原来培养HEK293T-Claudin 18.2细胞的培养基，将上一步中稀释好的抗体加入至对应孔中，100μL/孔；设置完全杀伤的阳性对照组，即加入10μl/孔裂解缓冲液；37℃，5％CO 2培养箱静置培养3小时后，加入CellTiter-Glo Luminescent(CTG，购自Promega，货号：G7573)染液染色，50μl/ 孔，混匀30秒，于室温放置1分钟后用酶标仪(MD，SpectraMax M2)测定荧光信号值，结果导入Graphpad Prism进行曲线拟合。
实验结果如图5和表6所示，2C6.9-hz21的CDC活性优于对照抗体IMAB362。
表6：抗Claudin18.2人源化抗体2C6.9-hz21CDC活性检测
抗体名称 EC50值(ng/mL)
2C6.9-hz21 1363
IMAB362 3317
6.7 2C6.9的人源化抗体的抗体依赖性细胞毒性(ADCC)活性测定
2C6.9为IgG1亚型，具有较强的抗体依赖性细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)活性。为测定2C6.9-hz21的ADCC活性，本发明采用NK细胞杀伤的方法进行检测。具体实验步骤如下：取HEK293T-Claudin 18.2细胞，离心后调整细胞密度，1×10 4细胞/孔铺板过夜；第二天去除板中培养基，取NK92MI-CD16a细胞(华博生物)离心，用空MEMA培养基重悬，调整细胞密度至1×10 6/mL，50μl/孔加入至对应孔中；用空MEMA培养基稀释2C6.9-hz21及IMAB362抗体，HEK293T-Claudin18.2细胞40μg/ml起始，5倍稀释，10个浓度点；NUGC-4细胞2mg/mL起始，5倍稀释，11个浓度点；将稀释好的抗体50μL/孔加入对应孔中，放入37℃、5％CO 2细胞培养箱中静置培养5.5小时后，在阳性对照孔中加入lysis buffer，再静置0.5小时后，加入乳酸脱氢酶(LDH)检测试剂(东仁化学，CK12)，50μl/孔，每10分钟酶标仪(MD，SpectraMax M2)490nm读数,结果导入Graphpad Prism进行曲线拟合。
实验结果如图6及表7所示，抗体2C6.9-hz21在HEK293T-Claudin 18.2上的ADCC细胞杀伤活性优于IMAB362。
表7：抗Claudin18.2人源化抗体2C6.9-hz21ADCC活性测定
抗体名称 EC50值(ng/mL) 最大杀伤率(％)
2C6.9-hz21 67.67 30
IMAB362 57.66 20
实施例七 靶向人Claudin 18.2ADC(2C6.9-ADC)的制备
本发明采用实施例三中制备获得的TL001与2C6.9的人源化单克隆抗体进行偶联，制备2C6.9-TL001。制备方法如下：
(1)偶联：取2C6.9-hz21抗体30mg，添加20mM PB+105mM NaCl+100mM依地酸二钠溶液，pH 7.7，用2M Tris溶液调节pH至7.7，然后，用20mM PB+105mM NaCl pH7.7溶液稀释(依地酸二钠终浓度为5mM，抗体终浓度为15mg/mL)，混匀；然后，加入10mM TCEP溶液混匀，室温放置一定时间(30或90分钟)；向上述溶液体系加入溶解于二甲基亚砜中的TL001(与抗体摩尔比：5:1或9:1)，混匀，室温静置2小时，得到偶联后样品，命名为2C6.9-TL001。
(2)缓冲液置换：用30KDa 50ml超滤管(Millipore)对2C6.9-TL001进行缓冲液置换，置换液为10mM组氨酸-盐酸组氨酸+8％蔗糖(pH6.0)缓冲液，置换倍数15倍；收集样品，加入10％吐温-20使样品中吐温-20最终浓度0.02％(M/V)。
(3)检测：
对置换后的2C6.9-TL001进行LC-MS分子量分析，条件如下：
色谱测定条件：
液相色谱柱：Thermo MAbPac RP 3.0*100mm；
流动相A：0.1％FA/98％H2O/2％ACN；
流动相B：0.1％FA/2％H2O/98％ACN；
流速：0.25ml/min；样品室温度：8℃；柱温：60℃；进样量：1μl；
时间(min) 2 20 22 25 26 30
流动相A(％) 75 60 5 5 75 75
流动相B(％) 25 40 95 95 25 25
切换阀：0-3min to waste，3-22min to MS，22-30min to waste
质谱测定条件：
质谱型号：AB Sciex Triple TOF 5600+；
参数：GS1 35；GS2 35；CUR 30；TEM 350；ISVF 5500；DP 200；CE 10；m/z 600-4000；Time bins to sum 40。
TL001与2C6.9-hz21偶联后得到的2C6.9-TL001的轻链、重链理论分子量及实测分子量(重链以主要糖型G0F计算)见下表：
Figure PCTCN2021093348-appb-000045
当TL001与2C6.9-hz21偶联投料比为5:1时，2C6.9-TL001中抗体轻链(LC)偶联0～1个毒素(LC、DAR1比例分别为57.8％，42.2％)、重链(HC)偶联0～4个毒素(HC、DAR1、DAR2、DAR3、DAR4比例分别为22.5％、30.3％、25.0％、21.9％、0.3％)，由此计算抗体与毒素的偶联比(DAR)为3.79，计算公式：DAR＝轻链DAR1*2+重链(DAR1*1+DAR2*2+DAR3*3+DAR4*4)*2。
当TL001与2C6.9-hz21偶联投料比为9:1时，2C6.9-TL001中抗体轻链偶联0～1个毒素(LC、DAR1比例分别为7.5％，92.5％)、重链偶联0～4个毒素(HC、DAR1、DAR2、DAR3、DAR4比例分别为2.5％、10.2％、9.8％、76.4％、1.1％)，由此计算抗体与毒素的偶联比(DAR)为7.12。
当TL001与2C6.9-hz21，偶联投料比为9:1时，经偶联和阳离子层析后、2C6.9-TL001中抗体轻链偶联0～1个毒素(LC、DAR1比例分别为1.5％，24.0％)、重链偶联0～4个毒素(HC、DAR1、DAR2、DAR3、DAR4比例分别为0.7％、2.1％、14.3％、54.5％、2.8％)，由此计算的抗体与毒素的偶联比(DAR)为7.40。
2C6.9-TL001中的ADC分子结构如下:
Figure PCTCN2021093348-appb-000046
其中γ为1～10的整数，A为2C6.9-hz21。
通过SEC-HPLC对缀合物进行SEC检测。
色谱条件：
液相色谱柱：TSKgel G3000SWxl，300*7.8mm，5μm；
流动相：90mmol/L Na2HPO4，30mmol/L NaH2PO4，200mM NaCl，5％乙腈；
流速：0.8ml/min；检测波长：280nm；柱温：室温；样品室温度：8℃；
进样量：40μl；等度运行：30min。
2C6.9-TL001(DAR：3.79)及2C6.9-TL001(DAR：7.12)的SEC色谱图如图7-8所示，2C6.9-TL001(DAR：7.40)的SEC色谱图如图13所示，根据SEC保留时间及峰面积比例，确认主要偶联产物分子量约为150kD，即TL001与2C6.9-hz21偶联得到的2C6.9-TL001，仍然保持抗体的完整结构。
采用相同制备方法将实施例四及实施例五中制备获得的TL002、TL003与人源化单克隆抗体2C6.9进行偶联，制备2C6.9-TL002、2C6.9-TL003，制备及检测方法如上所述，最终获得2C6.9-TL002、2C6.9-TL003的DAR值分别为6.95、7.03。
2C6.9-TL002中的ADC分子结构如下:
Figure PCTCN2021093348-appb-000047
其中γ为1～10的整数，A为2C6.9-hz21。
2C6.9-TL003中的ADC分子结构如下:
Figure PCTCN2021093348-appb-000048
其中γ为1～10的整数，A为2C6.9抗体。
实施例八 2C6.9-TL001的亲和力检测
本发明采用细胞ELISA法检测2C6.9-TL001(DAR：7.12)与细胞膜表面Claudin 18.2亲和力。具体实验步骤如下：将贴壁生长的L929-Claudin 18.2细胞系用2mM EDTA消化，重悬至2*10^5细胞/毫升，每孔100μL铺于96孔板中，37℃培养过夜；第二天去掉培养基，PBS清洗一次，每孔加入100μL 4％甲醛室温固定30分钟，去甲醛，PBS清洗两次，加入100μL PBS(含2％BSA)封闭2小时；去封闭液，用PBS(含2％BSA)稀释待测2C6.9-TL001，9.375μg/mL起始，4倍稀释，9个浓 度点，100μL/孔加入孔中，37℃孵育2小时；250μL PBST清洗5次，每次静置2分钟；PBS(2％BSA)稀释辣根过氧化物酶(HRP)标记的抗人IgG二抗(HRP-anti-Human IgG，Jackson ImmunoResearch)，1:10000稀释，100μL/孔，37℃孵育1小时；250μL PBST清洗6次，每次静置2分钟；加入100μL TMB显色液(Thermo)到对应的孔中，37℃显色20分钟；加入50μL 2mol/L H 2SO 4终止，酶标仪(MD)450nm读数并导入Graphpad Prism进行曲线拟合。实验结果如图9所示，2C6.9-TL001(DAR：7.12)细胞膜表面Claudin 18.2亲和力EC 50值为39.94ng/mL。偶联TL001后抗体2C6.9-hz21仍然具有优良的Claudin18.2亲和力。
实施例九 2C6.9-ADC对Claudin18.2高表达肿瘤细胞系的杀伤活性检测
本发明选取HEK293T-Claudin 18.2、HEK293T-Claudin 18.1细胞检测2C6.9-TL001对Claudin18.2高表达细胞系的杀伤活性。具体实验步骤如下：实验前一天用DMEM+10％FBS稀释细胞，100μL/孔，每孔1*10^4细胞铺板过夜；第二天用DMEM基础培养基梯度稀释ADC分子，150μg/mL(DAR：7.12)或262.5μg/mL(DAR：3.79)起始，4倍稀释，11个浓度点，加入对应孔中，每孔100μL，最终血清浓度为5％；37℃、5％CO 2培养箱中培养48小时；加入CCK8(Rhinogen)，20μl/孔，37℃、5％CO 2培养箱中孵育0.5-2.5小时，每半小时用酶标仪(MD)450nm读数并导入Graphpad Prism进行曲线拟合。
实验结果如图10A、图10B及图10C所示，2C6.9-TL001分子能够有效杀伤HEK293T-Claudin18.2细胞，当DAR值为7.12时，其EC 50值为473ng/mL；当DAR值为3.79时，其EC 50值为794.1ng/mL。同时，2C6.9-TL001(DAR：7.12)分子对HEK293T-Claudin18.1杀伤活性EC50值为3900ng/mL；2C6.9-TL001(DAR：7.12)分子对Claudin 18.2细胞杀伤活性明显高于Claudin 18.1细胞(相差约8倍)，表明这种杀伤作用是Claudin 18.2特异性的。
用上文所述相同的实验方法检测2C6.9-TL002、2C6.9-TL003对HEK293T-Claudin 18.2、HEK293T-Claudin 18.1细胞的杀伤活性。实验结果如图10D、图10E所示，2C6.9-TL002、2C6.9-TL003均能够有效杀伤HEK293T-Claudin 18.2细胞，其EC 50值为分别为628.9ng/mL、540.2ng/mL；2C6.9-TL002、2C6.9-TL003对HEK293T-Claudin18.1杀伤活性EC50值分别为30590ng/mL、8258ng/mL。两者对HEK293T-Claudin 18.2细胞杀伤活性明显高于Claudin 18.1细胞，表明两者的杀伤作用是Claudin 18.2特异性的。
实施例十 2C6.9-抗体-药物缀合物对内源表达Claudin 18.2细胞系的杀伤活性检测
本发明选取胃癌细胞系NUGC-4检测2C6.9-TL001对内源性表达Claudin 18.2细胞的杀伤活性。具体实验步骤如下：实验前一天用RPMI 1640+10％FBS稀释细胞，100μL/孔，每孔1*10^4细胞铺板过夜；第二天用RPMI 1640培养液梯度稀释2C6.9-TL001分子，1000μg/mL(DAR为7.12)或1750μg/mL(DAR：3.79)起始，3倍稀释，11个浓度点，加入对应孔中，每孔100μL，最终血清浓度为5％；37℃、5％CO 2培养箱中培养72小时；加入CCK8(Rhinogen)，20μl/孔，37℃、5％CO 2培养箱中孵育0.5-2.5小时，每半小时酶标仪(MD)450nm读数并导入Graphpad Prism进行曲线拟合。实验结果如图11A及11B所示，2C6.9-TL001分子能够有效杀伤NUGC-4细胞，当DAR值为7.12时，其EC 50值为2.383μg/mL；当DAR值为3.79时，其EC 50值为10.01μg/mL。
用上文所述相似的实验方法检测2C6.9-TL002、2C6.9-TL003对NUGC-4细胞的杀伤活性，ADC起始浓度为500μg/mL，4倍稀释，11个浓度点。实验结果如图11C所示，2C6.9-TL002、2C6.9-TL003均能够有效杀伤NUGC-4细胞，其EC 50值为分别为54.92μg/mL、123.94μg/mL。
实施例十一 2C6.9-hz21抗体内化检测
本发明选取NUGC-4细胞检测2C6.9抗体的内化活性。具体实验步骤如下：取NUGC-4细胞，胰酶消化计数，用PBS(含1％BSA)重悬细胞，调整细胞密度至3×10 6/mL；取100μL重悬后细胞，加入终浓度为100μg/mL的2C6.9-hz21抗体，以同型Human IgG作为阴性对照，冰上孵育1小时；孵育完成后用预冷PBS洗3次，用NUGC-4细胞培养基(1640+10％FBS)重悬细胞并将细胞分为两部分，一部分放置在37℃细胞培养箱中孵育4小时(内吞组)，另一部分继续在冰上孵育4小时(亲和力组)；孵育结束后，预冷PBS洗3次，用50uL PBS(含1％BSA)重悬细胞，加入抗人荧光二抗(BioLegend)，4℃孵育半小时；孵育结束后，预冷PBS洗3次，流式上机检测(Beckman)，按公式：内吞(％)＝[1-(MFI 37℃抗体组-MFI 37℃对照组)/(MFI 冰上抗体组–MFI 冰上对照组)]×100％计算出抗体在内化率。实验结果表明2C6.9-hz21在NUGC-4细胞上4小时的内化比例为37.79％。说明2C6.9-hz21偶联药物后的缀合物具有将药物内化进入细胞，并杀伤肿瘤细胞的潜力。
实施例十二 2C6.9-抗体-药物缀合物体内药效检测
本发明使用Cancer cell line-derived xenograft(CDX)模型和patient-derived xenograft(PDX)模型评价ADC分子的抗肿瘤作用。
12.1 NCI-N87-Claudin18.2+Balb/c Nude小鼠CDX模型用含10％胎牛血清(FBS)的RPMI1640培养液，在37℃，5％CO 2的条件下培养NCI-N87-Claudin18.2细胞。收集指数生长期细胞，PBS重悬，按照5×10 6/只的细胞量(悬浮于0.1ml PBS)，皮下接种于雌性Balb/c Nude小鼠(北京维通利华实验动物技术有限公司)，建立皮下移植瘤模型，待肿瘤平均体积达到70～100mm 3时，根据瘤体积随机分组，每组7只。分组当天记为第0天(Day 0)，组别为Human IgG1同型对照抗体(阴性对照)组(简称IgG1)、2C6.9-TL001(DAR：7.12)1mg/kg组和3mg/kg组、2C6.9-TL002(DAR：6.95)1mg/kg组和3mg/kg组。所有样品均为尾静脉注射，每周2次，共给药6次。
给药后每周2次用游标卡尺测量肿瘤直径，并按如下计算公式计算肿瘤体积：V＝0.5a×b 2，其中a和b分别表示肿瘤的长径和短径。每天观察记录动物死亡情况。
采用以下公式计算肿瘤生长抑制率TGI(％)，用于评价抑瘤疗效：
TGI(％)＝[1-(V T末-V T始)/(V C末-V C始)]*100％
其中 V T末：处理组实验结束时肿瘤体积均值
V T始：处理组给药开始时肿瘤体积均值
V C末：阴性对照组实验结束时肿瘤体积均值
V C始：阴性对照组给药开始时肿瘤体积均值
采用以下公式计算相对肿瘤增殖率T/C(％)，用于评价抑瘤疗效：
T/C(％)(瘤体积)＝(T t/T 0)/(C t/C 0)×100％
其中，T 0：初始(即P0)时处理组的平均瘤体积
T t：每次测量时处理组的平均瘤体积
C 0：初始(即P0)时阴性对照组的平均瘤体积
C t：每次测量时阴性对照组的平均瘤体积。
实验结果如表8及图12A、12B所示，2C6.9-TL001(DAR：7.12)对NCI-N87-Claudin18.2胃癌移植瘤模型的肿瘤生长有显著的抑制作用，且呈剂量依赖性。与阴性对照组相比，6次给药后(第21天)，2C6.9-TL001 1mg/kg组的肿瘤抑制率(TGI)已高达96.03％，且有4只小鼠肿瘤部分消退；而3mg/kg组TGI达到133.50％，3只小鼠肿瘤部分消退，4只全部消退。2C6.9-TL002 3mg/kg组TGI为40.11％，1mg/kg组无明显抑瘤作用。所有治疗组小鼠在观察期内均无明显的体重下降，动物耐受性良好。
表8：NCI-N87-Claudin18.2+Balb/c Nude小鼠CDX模型
Figure PCTCN2021093348-appb-000049
注：TGI：肿瘤生长抑制率；T/C：相对肿瘤增殖率；PR：肿瘤部分消退；CR：肿瘤全部消退。P值为与IgG1组比较结果；N/A：不适用；ns：P>0.05,无统计学差异。
12.2 2C6.9-TL001与2C6.9单抗+化疗CDX模型体内药效比较
按实验例12.1中方法建立NCI-N87-Claudin18.2皮下移植瘤模型并进行分组。分组当天记为第0天(Day 0)，组别为Human IgG1同型对照抗体(阴性对照)组(简称IgG1)、紫杉醇组(白蛋白结合型)、2C6.9单抗与紫杉醇联用组及2C6.9-TL001(DAR：7.12)组。所有样品均为尾静脉注射，每周2次，共给药3周，剂量如表9所示。
实验结果如表9及图12C所示，给药11天后，与阴性对照组相比，3个给药组均能够显著抑制肿瘤生长，2C6.9-TL001(DAR：7.12)组最为显著。2C6.9-TL001(DAR：7.12)组的肿瘤抑制率(TGI)已高达121.68％，且7只小鼠中6只肿瘤部分消退。
表9：2C6.9-TL001与2C6.9单抗+化疗CDX模型体内药效比较
Figure PCTCN2021093348-appb-000050
注：TGI：肿瘤生长抑制率；T/C：相对肿瘤增殖率；PR：肿瘤部分消退；CR：肿瘤全部消退。P 值为与IgG1组比较结果；N/A：不适用；ns：P>0.05,无统计学差异。
给药21天后，与阴性对照组相比，3个给药组均能够显著抑制肿瘤生长，2C6.9-TL001(DAR：7.12)组最为显著。2C6.9-TL001(DAR：7.12)组的肿瘤抑制率(TGI)已高达125.73％，且7只小鼠中5只肿瘤全部消退，2只肿瘤部分消退。实验结果如表10及图12D所示。
表10：2C6.9-TL001与2C6.9单抗+化疗CDX模型体内药效比较
Figure PCTCN2021093348-appb-000051
注：TGI：肿瘤生长抑制率；T/C：相对肿瘤增殖率；PR：肿瘤部分消退；CR：肿瘤全部消退。P值为与IgG1组比较结果；N/A：不适用。
12.3不同DAR值2C6.9-TL001CDX模型体内药效比较
用含10％胎牛血清(FBS)的RPMI1640培养液，在37℃，5％CO 2的条件下培养NUGC-4细胞。收集指数生长期细胞，PBS重悬，按照5×10 6/只的细胞量(悬浮于0.1ml PBS)，皮下接种于雌性Balb/c Nude小鼠(北京维通利华实验动物技术有限公司)，建立皮下移植瘤模型，待肿瘤平均体积达到70-100mm 3左右时，根据瘤体积随机分组，每组7只。分组当天记为第0天(Day 0)，组别为Human IgG1同型对照抗体(阴性对照)组(简称IgG1)、2C6.9-TL001(DAR：3.79)5.25mg/kg组、2C6.9-TL001(DAR：3.79)17.5mg/kg组、2C6.9-TL001(DAR：7.12)3mg/kg组、2C6.9-TL001(DAR：7.12)10mg/kg组(不同DAR值ADC药物按照同等毒素载量进行设计剂量给药)。所有样品均为尾静脉注射，每周2次，共给药3周，剂量如表11所示。
实验结果如表11及图12E、12F所示，同等毒素载量的情况下，给药21天后，2C6.9-TL001高DAR值(7.12)药效与低DAR值(3.79)药效基本相当。所有治疗组小鼠在观察期内均无明显的体重下降，动物耐受性良好。
表11：不同DAR值2C6.9-TL001CDX模型体内药效比较
Figure PCTCN2021093348-appb-000052
注：TGI：肿瘤生长抑制率；T/C：相对肿瘤增殖率；PR：肿瘤部分消退；CR：肿瘤全部消退。P 值为与IgG1组比较结果；N/A：不适用。
12.4
Figure PCTCN2021093348-appb-000053
胃癌GA0006+Balb/c Nude小鼠PDX模型
从
Figure PCTCN2021093348-appb-000054
胃癌异种移植模型GA0006(中美冠科生物技术(太仓)有限公司，高表达Claudin18.2的胃肿瘤来源于一名57岁女性患者)荷瘤小鼠收取肿瘤组织，切成直径为3×3×3mm的瘤块接种于Balb/c裸小鼠右前肩胛处皮下。当荷瘤鼠平均肿瘤体积到达约150-250mm 3时，根据肿瘤大小随机分组，每组7只，分组当天定义为第0天(Day 0)。组别为3组：Human IgG1同型对照抗体(阴性对照)组(IgG1 10mg/kg)、2C6.9-TL001-DAR7.12 3mg/kg组和10mg/kg组。所有样品均为尾静脉注射，每周2次，共给药5次。给药后观察并定期测量小鼠肿瘤体积及体重，测量方法如实验例12.1所述。
实验结果如表12及图12G、12H所示，2C6.9-TL001-DAR7.12对GA0006胃癌PDX模型的肿瘤生长有显著的抑制作用，且呈剂量依赖性。与阴性对照组相比，5次给药后(第17天)，3mg/kg组的肿瘤抑制率(TGI)已高达94.72％，且有4只部分消退；10mg/kg组TGI达到124.49％，7只肿瘤全部消退，表明2C6.9-TL001可高效抑制肿瘤生长。所有治疗组在观察期内均无明显的体重下降，动物耐受性良好。
表12：
Figure PCTCN2021093348-appb-000055
胃癌GA0006+Balb/c Nude小鼠PDX模型
Figure PCTCN2021093348-appb-000056
注：TGI：肿瘤生长抑制率；T/C：相对肿瘤增殖率；PR：肿瘤部分消退；CR：肿瘤全部消退。P值为与IgG1组比较结果；N/A：不适用。
实验结果如表13及图12I、12J所示，给药后第24天，2C6.9-TL001-DAR7.12对GA0006胃癌PDX模型的肿瘤生长有显著的抑制作用，且呈剂量依赖性。与阴性对照组相比，3mg/kg组的肿瘤抑制率(TGI)已高达103.76％，且有6只部分消退；10mg/kg组TGI达到113.70％，7只肿瘤全部消退，表明2C6.9-TL001可高效抑制肿瘤生长。所有治疗组在观察期内均无明显的体重下降，动物耐受性良好。
表13：
Figure PCTCN2021093348-appb-000057
胃癌GA0006+Balb/c Nude小鼠PDX模型
Figure PCTCN2021093348-appb-000058
注：TGI：肿瘤生长抑制率；T/C：相对肿瘤增殖率；PR：肿瘤部分消退；CR：肿瘤全部消退。P值为与IgG1组比较结果；N/A：不适用。
12.5 NCI-N87-Claudin18.2+Balb/c Nude小鼠CDX模型体内药效评价
按实验例12.1中方法建立NCI-N87-Claudin18.2皮下移植瘤模型，待肿瘤平均体积达到140mm 3左右时，根据瘤体积随机分组，每组8只。分组当天记为第0天(Day 0)，组别为Human IgG1同型对照抗体(阴性对照)组(简称IgG1)，2C6.9-TL001(DAR：7.40)0.3mg/kg组、1mg/kg组和3mg/kg组。所有样品均为尾静脉注射，每周2次，共给药6次，剂量如表14所示。
实验结果如表14及图12K和12L所示，6次给药后停药延长观察至第31天(首次给药后第31天)，与阴性对照组相比，2C6.9-TL001 0.3mg/kg组的肿瘤抑制率(TGI)为34.04％；而1mg/kg组和3mg/kg组TGI分别高达122.57％(全部小鼠肿瘤部分消退)和184.22％(4小鼠肿瘤全部消退，4小鼠肿瘤部分消退)。所有治疗组小鼠在观察期内均无明显的体重下降，动物耐受性良好。
表14：NCI-N87-Claudin18.2+Balb/c Nude小鼠CDX模型体内药效评价
Figure PCTCN2021093348-appb-000059
注：TGI：肿瘤生长抑制率；T/C：相对肿瘤增殖率；PR：肿瘤部分消退；CR：肿瘤全部消退。P值为与IgG1组比较结果；N/A：不适用。
12.6 HEK293T-Claudin18.2+Balb/c Nude小鼠CDX模型体内药效评价
用含3μg/mL puromycin和10％胎牛血清(FBS)的DMEM培养液，在37℃，5％CO 2的条件下培养HEK293T-Claudin18.2细胞(人胚肾细胞)。收集指数生长期细胞，PBS重悬，按照1×10 7/只的细胞量(悬浮于0.1ml PBS)，皮下接种于雌性Balb/c Nude小鼠(浙江维通利华实验动物技术有限公司)，建立皮下移植瘤模型，待肿瘤平均体积达到120-140mm 3左右时，根据瘤体积随机分组，每组8只。分组当天记为第0天(Day 0)，组别为Human IgG1同型对照抗体(阴性对照)组(简称IgG1)，2C6.9-TL001(DAR：7.40)0.3mg/kg组、1mg/kg组和3mg/kg组。所有样品均为尾静脉注射，每周2次，共给药6次，剂量如表15所示。
实验结果如表15及图12M和12N所示，与阴性对照组相比，6次给药后(首次给药后第21天)，2C6.9-TL001(DAR：7.40)对HEK293T-Claudin18.2人胚肾细胞移植瘤模型的肿瘤生长有显著的抑制作用，且呈剂量依赖性。2C6.9-TL001 0.3mg/kg组的肿瘤抑制率(TGI)为70.99％(1只小鼠肿瘤全部消退)；而1mg/kg组和3mg/kg组TGI分别为94.98％(2只小鼠肿瘤部分消退)和182.81％(2只小鼠肿瘤部分消退，6只小鼠肿瘤全部消退)。
表15：HEK293T-Claudin18.2+Balb/c Nude小鼠CDX模型体内药效评价
Figure PCTCN2021093348-appb-000060
Figure PCTCN2021093348-appb-000061
注：TGI：肿瘤生长抑制率；T/C：相对肿瘤增殖率；PR：肿瘤部分消退；CR：肿瘤全部消退。P值为与IgG1组比较结果；N/A：不适用。
12.7 NUGC-4+Balb/c Nude小鼠CDX模型体内药效评价
用含10％胎牛血清(FBS)的RPMI1640培养液，在37℃，5％CO 2的条件下培养NUGC-4细胞。收集指数生长期细胞，PBS重悬，按照5×10 6/只的细胞量(悬浮于0.1ml PBS)，皮下接种于雌性Balb/c Nude小鼠(浙江维通利华实验动物技术有限公司)，建立皮下移植瘤模型，待肿瘤平均体积达到80mm 3左右时，根据瘤体积随机分组，每组8只。分组当天记为第0天(Day 0)，组别为Human IgG1同型对照抗体(阴性对照)组(简称IgG1)，2C6.9-TL001(DAR：7.40)3mg/kg组和10mg/kg组。所有样品均为尾静脉注射，每周2次，共给药6次，剂量如表16所示。
实验结果如表16及图12O和12P所示，与阴性对照组相比，6次给药后(首次给药后第21天)，2C6.9-TL001(DAR：7.40)对NUGC-4胃癌移植瘤模型的肿瘤生长有显著的抑制作用，且呈剂量依赖性。2C6.9-TL001 3mg/kg组的肿瘤抑制率(TGI)为56.62％；而10mg/kg组TGI为90.28％(2只小鼠肿瘤部分消退)。
表16：NUGC-4+Balb/c Nude小鼠CDX模型体内药效评价
Figure PCTCN2021093348-appb-000062
注：TGI：肿瘤生长抑制率；T/C：相对肿瘤增殖率；PR：肿瘤部分消退；CR：肿瘤全部消退。P值为与IgG1组比较结果；N/A：不适用。
综上所述，无论是CDX模型还是PDX模型，本发明中2C6.9-TL001均能剂量依赖性地有效抑制Claudin18.2阳性表达肿瘤生长，且具有良好的安全性。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的保护范围由所附权利要求及其任何等同物给出。

Claims (24)

  1. 抗体-药物缀合物，其结构如式(I)所示，
    (D-L) γ-A
    式(I)
    其中，D为生物活性分子片段；L为连接子；
    γ选自1-10之间的整数；优选地，γ选自1-8之间的整数(例如1、2、3、4、5、6、7或8)；
    A为特异性结合人CLDN18.2的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (1)下述的VH和/或VL，其中CDR按IMGT编号系统定义：
    (1-1)：包含如下3个CDRs的VH：序列为SEQ ID No：1的CDR-H1，序列为SEQ ID Nos：2或21的CDR-H2，和序列为SEQ ID No：3的CDR-H3；和/或，
    包含如下3个CDRs的VL：序列为SEQ ID No：4的CDR-L1，序列为SEQ ID No：5的CDR-L2，和序列为SEQ ID No：6的CDR-L3；
    或，
    (1-2)：与(1-1)中所述的VH或VL相比，至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个或3个氨基酸的置换、缺失或添加或其任意组合)；所述含有突变的抗体或其抗原结合片段仍然能特异地结合人CLDN18.2；
    或，
    (2)下述的VH和/或VL，其中CDR按AbM编号系统定义：
    (2-1)：包含如下3个CDRs的VH：序列为SEQ ID No：7的CDR-H1，序列为SEQ ID Nos：8或22的CDR-H2，和序列为SEQ ID No：9的CDR-H3；和/或，
    包含如下3个CDRs的VL：序列为SEQ ID No：10的CDR-L1，序列为SEQ ID No：11的CDR-L2，和序列为SEQ ID No：12的CDR-L3；
    或，
    (2-2)：与(2-1)中所述的VH或VL相比，至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个或3个氨基酸的置换、缺失或添加或其任意组合)；所述含有突变的抗体或其抗原结合片段仍然能特异地结合人CLDN18.2；
    优选地，所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH和/或VL中包括来自人或鼠的免疫球蛋白的构架区(FRs)。
  2. 权利要求1的抗体-药物缀合物，其中：
    (1)所述抗体或其抗原结合片段包含SEQ ID NOs：13或14所示的VH；和/或，SEQ ID NO：15所示的VL；
    (2)所述抗体或其抗原结合片段所包含的VH与(1)中所述的VH相比，具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、 至少97％、至少98％、至少99％、或100％的同一性；和/或，所述抗体或其抗原结合片段所包含的VL与(1)中所述的VL相比，具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的同一性；
    或者，
    (3)所述抗体或其抗原结合片段所包含的VH与(1)中所述的VH相比，具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；和/或，所述抗体或其抗原结合片段所包含的VL与(1)中所述的VL相比，具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；优选地，所述的置换是保守置换。
  3. 权利要求1-2任一项的抗体-药物缀合物，其中，所述抗体包含：
    (1)人免疫球蛋白的CH(重链恒定区)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和/或，
    (2)人免疫球蛋白的CL(轻链恒定区)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；
    优选地，所述CH是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；
    优选地，所述抗体包含人IgG1的重链恒定区；
    优选地，所述抗体包含如SEQ ID NO：16所示的CH或其变体，所述变体与SEQ ID NO：16相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)；
    优选地，所述CL是κ轻链恒定区；
    优选地，所述抗体包含如SEQ ID NO：17所示的CL或其变体，所述变体与SEQ ID NO：17相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)；
    更优选的，所述抗体包含如SEQ ID NO：16所示的CH和/或如SEQ ID NO：17所示的CL。
  4. 权利要求1-3任一项所述的抗体-药物缀合物，其中，所述抗体为：
    (1)包括SEQ ID NO：13所示序列的VH和SEQ ID NO：16所示序列的CH的重链，和，包括SEQ ID NO：15所示序列的VL和SEQ ID NO：17所示序列的CL的轻链；
    或者，
    (2)包括SEQ ID NO：14所示序列的VH和SEQ ID NO：16所示序列的CH的重链，和，包括SEQ ID NO：15所示序列的VL和SEQ ID NO：17所示序列的CL的轻链。
  5. 权利要求1-4任一项所述的抗体-药物缀合物，其中，所述抗体或其抗原结合片段包含：
    (1)重链，其包含选自下列的氨基酸序列：
    (1-1)SEQ ID NO：18或SEQ ID NO：19所示的序列；
    (1-2)与SEQ ID NO：18或SEQ ID NO：19所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (1-3)与SEQ ID NO：18或SEQ ID NO：19所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和
    (2)轻链，其包含选自下列的氨基酸序列：
    (2-1)SEQ ID NO：20所示的序列；
    (2-2)与SEQ ID NO：20所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (2-3)与SEQ ID NO：20所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(1-2)和(2-2)中所述的置换是保守置换。
  6. 权利要求1-5任一项所述的抗体-药物缀合物，其中，所述抗体或其抗原结合片段选自ScFv、Fab、Fab’、(Fab’) 2、Fv片段、二硫键连接的Fv(dsFv)、双抗体(diabody)、双特异性抗体和多特异性抗体。
  7. 权利要求1-6任一项的抗体-药物缀合物，其结构如式(II)所示，
    {D-[L 1-(L 2) m1-(L 3) m2-(L 4) m3-E]} γ-A
    式(II)
    其中，
    L 1为
    Figure PCTCN2021093348-appb-100001
    其中，各R 1和R 2各自独立地为氢(例如氕或氘)、卤素、羧酸、磺酸、氰基、C 1-6烷基、卤代C 1-6烷基、氰基取代的C 1-6烷基(例如-CH 2CN)、C 1-6烷氧基、C 2-10烯基或C 2-10炔基；Z 1为氨基酸或2-10个氨基酸组成的肽；x 1和x 2各自独立地为0、1、2、3、4、5或6；并且L 1的1位置处与D相连、L 1的2位置处与L 2相连；
    L 2为
    Figure PCTCN2021093348-appb-100002
    其中，y 1为0、1、2、3、4、5、6、7、8、9或10；并且L 2的1位置处与L 1相连、L 2的2位置处与L 3相连；
    L 3选自5-12元杂芳环；
    L 4为
    Figure PCTCN2021093348-appb-100003
    其中，其中Z 2选自C 1-6亚烷基、C 2-10亚烯基、C 2-10亚炔基和C 3-8亚环烷基；R 3选自氢(例如氕或氘)和C 1-6烷基；Z 3不存在或者选自C 1-6亚烷基；或者，R 3与Z 3连同其所连接的氮原子形成4-8元杂环基；α为0、1、2、3、4、5或6，并且L 4的2位置处与E相连、L 4的1位置处与L 3相连；
    E为
    Figure PCTCN2021093348-appb-100004
    其中，各R 4独立地为氢(例如氕或氘)，β为0、1、2、3、4、5或6，并且E的2位置处与A相连、E的1位置处与L 4相连；
    m 1、m 2和m 3各自独立地为0、1、2、3、4、5、6、7、8、9或10；
    A如权利要求1-6任一项中所定义；
    D和γ如权利要求1中所定义。
  8. 权利要求1-6任一项的抗体-药物缀合物，其结构如式(III)所示：
    {D-[(L 1’) m4-L 1-(L 5) m5-(L 3) m2-(L 4) m3-E]} γ-A
    式(III)
    其中，
    L 1’为
    Figure PCTCN2021093348-appb-100005
    其中，R 5和R 6各自独立地为氢(例如氕或氘)或C 1-6烷基；x 3为1、2、3、4、5或6；并且，如果L 1’存在，其1位置处与D相连、2位置处与L 1相连；
    L 1为
    Figure PCTCN2021093348-appb-100006
    其中，各R 1和R 2各自独立地为氢(例如氕或氘)、卤素、羧酸、磺酸、氰基、C 1-6烷基、卤代C 1-6烷基、氰基取代的C 1-6烷基(例如-CH 2CN)、C 1-6烷氧基、C 2-10烯基或C 2-10炔基；Z 1为氨基酸或2-10个氨基酸组成的肽；x 1和x 2各自独立地为0、1、2、3、4、5或6；并且，L 1的1位置处与L 1’相连(当L 1’存在时)，或者，L 1的1位置处与D相连(当L 1’不存在时)；L 1的2位置处与L 5相连；
    L 5为
    Figure PCTCN2021093348-appb-100007
    其中，R 7为氢或C 1-6烷基，或者R 7与其 γ-C上的N原子连接形成5-6元杂环基；x 4为1、2、3、4、5或6；y 1为0、1、2、3、4、5、6、7、8、9或10；并且L 5的1位置处与L 1相连、L 5的2位置处与L 3相连；
    L 3选自5-12元杂芳环；
    L 4为
    Figure PCTCN2021093348-appb-100008
    其中Z 2选自C 1-6亚烷基、C 2-10亚烯基、C 2-10亚炔基和C 3-8亚环烷基；R 3选自氢(例如氕或氘)和C 1-6烷基；Z 3不存在或者选自C 1-6亚烷基；或者，R 3与Z 3连同其所连接的氮原子形成4-8元杂环基；α为0、1、2、3、4、5或6，并且L 4的2位置处与E相连、L 4的1位置处与L 3相连；
    E为
    Figure PCTCN2021093348-appb-100009
    其中，各R 4独立地为氢(例如氕或氘)，β为0、1、2、3、4、5或6，并且E的2位置处与A相连、E的1位置处与L 4相连；
    m 1、m 2、m 3和m 4各自独立地为0、1、2、3、4、5、6、7、8、9或10；
    A如权利要求1-6任一项中所定义；
    D和γ如权利要求1中所定义。
  9. 权利要求7-8任一项所述的抗体-药物缀合物，其中，L 1为
    Figure PCTCN2021093348-appb-100010
    其中，Z 1为氨基酸或2-5个氨基酸组成的肽，其中所述氨基酸选自Lys、Cit、Val、D-Val、Phe、Leu、Gly、Ala和Asn；优选地，Z 1选自Cit、Lys、Cit-Val和Ala-Val；
    优选地，L 1为
    Figure PCTCN2021093348-appb-100011
    优选地，L 1为
    Figure PCTCN2021093348-appb-100012
  10. 权利要求7-9任一项所述的抗体-药物缀合物，其中，L 2为
    Figure PCTCN2021093348-appb-100013
    且m 1为1。
  11. 权利要求7-10任一项所述的抗体-药物缀合物，其中，L 3为5-6元杂芳环，且m 2为1；
    优选地，L 3为三氮唑，且m 2为1。
  12. 权利要求7-11任一项所述的抗体-药物缀合物，其中，L 4为
    Figure PCTCN2021093348-appb-100014
    Z 2为C 1-6亚烷基，Z 3为C 1-6亚烷基，且m 3为1；
    优选地，L 4为
    Figure PCTCN2021093348-appb-100015
    且m 3为1。
  13. 权利要求8-12任一项所述的抗体-药物缀合物，其中，L 1’为
    Figure PCTCN2021093348-appb-100016
  14. 权利要求8-13任一项所述的抗体-药物缀合物，其中，L 5为
    Figure PCTCN2021093348-appb-100017
    其中，x 4为1、2、3、4、5或6；y 1为1、2、3、4、5、6、7、8、9或10；
    优选地，L 5为
    Figure PCTCN2021093348-appb-100018
  15. 权利要求1-14任一项所述的抗体-药物缀合物，其中，D为
    Figure PCTCN2021093348-appb-100019
    优选地，D为
    Figure PCTCN2021093348-appb-100020
  16. 权利要求7的抗体-药物缀合物，其中，式(II)中D-[L 1-(L 2) m1-(L 3) m2-(L 4) m3-E]-为
    Figure PCTCN2021093348-appb-100021
  17. 权利要求8的抗体-药物缀合物，其中，式(III)中D-[(L 1’) m4-L 1-(L 5) m5-(L 3) m2-(L 4) m3-E]-为
    Figure PCTCN2021093348-appb-100022
  18. 权利要求1-17任一项的抗体-药物缀合物，其中所述抗体-药物缀合物选自：
    Figure PCTCN2021093348-appb-100023
    Figure PCTCN2021093348-appb-100024
    其中，γ选自1-10之间的整数，例如γ为1、2、3、4、5、6、7、8、9或10。
  19. 权利要求1-18任一项所述的抗体-药物缀合物，其中，所述抗体或其抗原结合片段带有标记；优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  20. 组合物，其含有权利要求1-19任一项所述的抗体-药物缀合物，所述组合物中的生物活性分子片段与特异性结合CLDN18.2的抗体或其抗原结合片段的摩尔比(DAR值)为1-10之间的小数或整数(例如3-8之间的小数或整数，例如1.0，1.5，2.0，2.5，3.0，3.1，3.2，3.3，3.4，3.5，3.6，3.7，3.79，3.8，3.9，4.0，4.1，4.2，4.3，4.4，4.5，4.6，4.7，4.8，4.9，5.0，5.1，5.2，5.3，5.4，5.5，5.6，5.7，5.8，5.9，6.0，6.1，6.2，6.3，6.4，6.5，6.6，6.7，6.8，6.9，6.95，7.0，7.03，7.1，7.12，7.2，7.3，7.40，7.5，7.6，7.7，7.8，7.9或8.0)。
  21. 药物组合物，其含有权利要求1-19任一项所述的抗体-药物缀合物、或权利要求20所述的组合物，以及药学上可接受的载体和/或赋形剂。
  22. 权利要求1-19任一项所述的抗体-药物缀合物、权利要求20所述的组合物、或权利要求21所述的药物组合物在制备预防和/或治疗和/或辅助治疗肿瘤的药物中的用途；
    可选地，所述药物还包括另外的抗肿瘤活性成分；优选地，所述抗体-药物缀合物、组合物或药物组合物与另外的抗肿瘤活性成分分开、同时或相继施用；优选地，所述另外的抗肿瘤活性成分是生物活性多肽或其活性片段，或化疗药物；更优选地，所述生物活性多肽选自免疫检查点抑制剂(例如，PD-1抗体、PD-L1抗体、CTLA-4抗体、LAG-3抗体)，或细胞因子(例如，干扰素、IL-2、IL-15、GM-CSF、IL-7、IL-12、IL-18、IL-21)；优选地，所述化疗药物选自表柔比星、奥沙利铂、卡培他滨、5-氟尿嘧啶、亚叶酸、紫杉醇和白蛋白结合型紫杉醇中的一种或多种。
  23. 根据权利要求22的用途，其中所述肿瘤选自实体肿瘤、血液肿瘤和癌症的转移性、难治性或复发性病灶；
    优选的，所述肿瘤或癌症选自食管癌、胃肠癌、胰腺癌、甲状腺癌、结直肠癌、肾癌、肺癌(例如非小细胞肺癌)、肝癌、胃癌、胃腺癌、胃食管交界处(GEJ)腺癌、头颈癌、膀胱癌、乳腺癌、子宫癌、宫颈癌、卵巢癌、前列腺癌、睾丸癌、生殖细胞癌、骨癌、皮肤癌、胸腺癌、胆管癌、胆囊癌、黑素瘤、间皮瘤、淋巴瘤、骨髓瘤(例如多发性骨髓瘤)、肉瘤、神经胶质母细胞瘤和白血病；
    优选地，所述肿瘤选自胃癌、胃腺癌、胃食管交界处(GEJ)腺癌、食管癌、胃肠癌、胰腺癌，肺癌 (例如非小细胞肺癌)；
    优选地，所述肿瘤为胃癌、胃腺癌或胃食管交界处(GEJ)腺癌，例如局部晚期不可切除或转移的胃癌、胃腺癌或胃食管交界处(GEJ)腺癌；
    优选地，所述肿瘤为CLDN18.2阳性，更优选地，所述肿瘤为HER2阴性。
  24. 一种用于在受试者中预防和/或治疗肿瘤、和/或延迟肿瘤进展、和/或降低或抑制肿瘤复发的方法，所述方法包括向有此需要的受试者施用有效量的权利要求1-19任一项所述的抗体-药物缀合物、权利要求20所述的组合物、或21所述的药物组合物；
    可选地，所述方法还包括向所述受试者施用第二疗法，所述第二疗法选自手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其任意组合；所述方法与所述第二疗法分开、联合、同时或相继应用；
    优选地，所述免疫疗法包括应用生物活性多肽，更优选地，所述生物活性多肽选自免疫检查点抑制剂(例如，PD-1抗体、PD-L1抗体、CTLA-4抗体、LAG-3抗体)，或细胞因子(例如，干扰素、IL-2、IL-15、GM-CSF、IL-7、IL-12、IL-18、IL-21)；
    优选地，所述化疗选自应用表柔比星、奥沙利铂、卡培他滨、5-氟尿嘧啶、亚叶酸、紫杉醇和白蛋白结合型紫杉醇中的一种或多种；
    优选地，所述肿瘤选自实体肿瘤、血液肿瘤和癌症的转移性、难治性或复发性病灶；
    优选的，所述肿瘤或癌症选自食管癌、胃肠癌、胰腺癌、甲状腺癌、结直肠癌、肾癌、肺癌(例如非小细胞肺癌)、肝癌、胃癌、胃腺癌、胃食管交界处(GEJ)腺癌、头颈癌、膀胱癌、乳腺癌、子宫癌、宫颈癌、卵巢癌、前列腺癌、睾丸癌、生殖细胞癌、骨癌、皮肤癌、胸腺癌、胆管癌、胆囊癌、黑素瘤、间皮瘤、淋巴瘤、骨髓瘤(例如多发性骨髓瘤)、肉瘤、神经胶质母细胞瘤、白血病；
    优选地，所述肿瘤选自胃癌、胃腺癌、胃食管交界处(GEJ)腺癌、食管癌、胃肠癌、胰腺癌，肺癌(例如非小细胞肺癌)；
    优选地，所述肿瘤为胃癌、胃腺癌或胃食管交界处(GEJ)腺癌，例如局部晚期不可切除或转移的胃癌、胃腺癌或胃食管交界处(GEJ)腺癌；
    优选地，所述肿瘤为CLDN18.2阳性，更优选地，所述肿瘤为HER2阴性。
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WO2023143208A1 (zh) * 2022-01-26 2023-08-03 苏州宜联生物医药有限公司 一种药物连接体偶联物的制备方法
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WO2024027708A1 (zh) * 2022-08-02 2024-02-08 诺纳生物（苏州）有限公司 Msln抗体药物偶联物
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