WO2020063988A1 - Cldn18的抗体和化疗药物的联合治疗 - Google Patents

Cldn18的抗体和化疗药物的联合治疗 Download PDF

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WO2020063988A1
WO2020063988A1 PCT/CN2019/109214 CN2019109214W WO2020063988A1 WO 2020063988 A1 WO2020063988 A1 WO 2020063988A1 CN 2019109214 W CN2019109214 W CN 2019109214W WO 2020063988 A1 WO2020063988 A1 WO 2020063988A1
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seq
amino acid
acid sequence
variable region
chain variable
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PCT/CN2019/109214
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French (fr)
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李宗海
王华茂
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科济生物医药（上海）有限公司
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Priority to KR1020217013109A priority Critical patent/KR20210072027A/ko
Priority to US17/281,372 priority patent/US20220033491A1/en
Priority to CN201980065066.XA priority patent/CN112888458A/zh
Priority to EP19866728.9A priority patent/EP3858384A4/en
Priority to JP2021517792A priority patent/JP2022511394A/ja
Publication of WO2020063988A1 publication Critical patent/WO2020063988A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to a combination therapy of CLDN18 antibody and chemotherapeutic drug.
  • Gastric cancer is one of the cancers with a high incidence worldwide. According to statistics from the World Health Organization's Cancer Control Project, as many as 7 million patients die from cancer each year worldwide, of which 700,000 die from gastric cancer.
  • German biotechnology company Ganymed reported the combination of chemotherapy drugs with EOX (epirubicin, oxaliplatin, and capecitabine) and antibodies targeting Claudin 18.2 for gastric cancer
  • EOX epirubicin, oxaliplatin, and capecitabine
  • EP2852408B also reported the use of EOF (epirubicin
  • chemotherapeutic drugs Staurexin
  • CLDN cell junction clin
  • chemotherapeutic drugs usually have large cytotoxicity, and the patients are poorly tolerated, the use of multiple chemotherapeutic drugs brings a great risk of clinical application.
  • the object of the present invention is to provide a combination therapy of antibodies and chemotherapeutic drugs for CLDN18, which is used to effectively treat diseases related to the expression of CLD18A.
  • a method for treating a CLDN18-positive tumor administering to an individual in need thereof an antibody that specifically recognizes CLDN18, 5-fluorouracil or a prodrug thereof or an active metabolite thereof, and oxaliplatin Or a prodrug or an active metabolite thereof; or an antibody in need of specific recognition of CLDN18, 5-fluorouracil or a prodrug thereof or an active metabolite thereof, oxaliplatin or a prodrug thereof or an active metabolite thereof, And a taxane drugs.
  • an individual in need thereof is given an antibody that specifically recognizes CLDN18, 5-fluorouracil or a prodrug thereof, and oxaliplatin or a prodrug thereof is given to an individual in need, an antibody that specifically recognizes CLDN18, 5 -Fluorouracil or a prodrug thereof, oxaliplatin or a prodrug thereof, and a taxane drug.
  • the prodrug of 5-fluorouracil is capecitabine.
  • the CLDN18 is CLDN18.2.
  • the antibody specifically recognizes CLDN18.2 and does not recognize CLDN18.1.
  • the antibodies of CLDN18, 5-fluorouracil or a prodrug thereof or an active metabolite thereof, oxaliplatin or a prodrug thereof or an active metabolite thereof are administered in any order.
  • antibodies to 5-fluorouracil or its prodrug or its active metabolite, oxaliplatin, CLDN18 are administered on the same day.
  • the CLDN18 antibody is administered at a dose of 500-1200 mg / m 2 / time. In a preferred example, it is 500-1000 mg / m 2 / time. In a preferred example, it is 500-900 mg / m 2 / time. In a preferred example, it is 600-800 mg / m 2 / time.
  • the CLDN18 antibody is administered at a dose of 20-300 mg / kg / time. In a preferred example, it is 20 to 100 mg / kg / time. In a preferred example, it is 20-50 mg / kg / time.
  • the 5-fluorouracil or its prodrug or its active metabolite is administered at a dose of 300-900 mg / m 2 / time.
  • 5-fluorouracil is administered in an amount of 300-800 mg / m 2 / time.
  • capecitabine is administered at 600-700 mg / m 2 / time. In a preferred example, it is 625 mg / m 2 / time.
  • the 5-fluorouracil or its prodrug or its active metabolite is administered at a dose of 50 to 70 mg / kg / time.
  • 5-fluorouracil is administered in an amount of 50 to 70 mg / kg / time.
  • the application amount of 5-fluorouracil is 50 to 60 mg / kg / time.
  • the oxaliplatin or its prodrug or its active metabolite is administered at a dose of 50-300 mg / m 2 / time. In a preferred example, oxaliplatin is administered at 100 to 200 mg / m 2 / time. In a more preferred example, oxaliplatin is administered at 130 mg / m 2 / times.
  • the oxaliplatin or its prodrug or its active metabolite is administered at a dose of 1 to 10 mg / kg / time. In a preferred example, oxaliplatin is administered at 1 to 5 mg / kg / time.
  • the antibody that specifically recognizes CLDN18 is a humanized antibody or a chimeric antibody.
  • the antibody that specifically recognizes CLDN18 comprises:
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 16 HCDR2 shown in the amino acid sequence of NO: 17, 40, or 41
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 34 HCDR2 shown in the amino acid sequence of SEQ ID NO: 35 or 42
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 36 and amino acid sequence of SEQ ID NO: 37
  • LCDR2 shown in the amino acid sequence of SEQ ID NO: 39
  • amino acid sequence of SEQ ID NO: 39 or
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 25 LCDR1, LCDR2 shown in the amino acid sequence of SEQ ID NO: 26, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 27; or,
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 28 HCDR2 shown in the amino acid sequence of SEQ ID NO: 29, HCDR3 shown in the amino acid sequence of SEQ ID NO: 30, and HCDR3 shown in the amino acid sequence of SEQ ID NO: 31 LCDR1, LCDR2 shown in the amino acid sequence of SEQ ID NO: 32, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 33; or,
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 43 HCDR2 shown in the amino acid sequence of SEQ ID NO: 44
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 46 LCDR1, LCDR2 shown in the amino acid sequence of SEQ ID NO: 47
  • LCDR3 shown in the amino acid sequence of SEQ ID NO: 48 or,
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 49 HCDR2 shown in the amino acid sequence of SEQ ID NO: 50
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 51 and amino acid sequence of SEQ ID NO: 52
  • LCDR3 shown in the amino acid sequence of SEQ ID NO: 54 LCDR1, LCDR3 shown in the amino acid sequence of SEQ ID NO: 53
  • LCDR3 shown in the amino acid sequence of SEQ ID NO: 54 LCDR1, LCDR3 shown in the amino acid sequence of SEQ ID NO: 53
  • the antibody that specifically recognizes CLDN18 comprises:
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 34 HCDR2 shown in the amino acid sequence of SEQ ID NO: 35 or 42
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 36 and amino acid sequence of SEQ ID NO: 37
  • LCDR2 shown in the amino acid sequence of SEQ ID NO: 39
  • amino acid sequence of SEQ ID NO: 39 or
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 43
  • HCDR2 shown in the amino acid sequence of SEQ ID NO: 44
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 45
  • amino acid sequence of SEQ ID NO: 46 The LCDR1 shown in the amino acid sequence of SEQ ID NO: 47 and the LCDR3 shown in the amino acid sequence of SEQ ID NO: 48.
  • the antibody that specifically recognizes CLDN18 contains:
  • the antibody that specifically recognizes CLDN18 contains:
  • the antibody that specifically recognizes CLDN18 contains:
  • the antibody that specifically recognizes CLDN18 is selected from:
  • the CLDN18-positive tumors include: gastric cancer, pancreatic cancer, esophageal cancer, and lung cancer.
  • the taxane drug is selected from paclitaxel, albumin-bound paclitaxel, and docetaxel; preferably, it is albumin-bound paclitaxel.
  • kits combination for treating CLDN18-positive tumors.
  • the kit combination includes a kit A and a kit B.
  • the kit A includes an antibody that specifically recognizes CLDN18.
  • the kit B includes a chemotherapeutic drug, the chemotherapeutic drug is: 5-fluorouracil or a prodrug thereof or an active metabolite thereof, oxaliplatin or a prodrug thereof or an active metabolite thereof; or, 5-fluorouracil or a prodrug thereof Or its active metabolite, oxaliplatin or its prodrug or its active metabolite, and a taxane drug.
  • the kit A and the kit B are administered to an individual in need, wherein the kit B includes 5-fluorouracil or a prodrug thereof, and oxaliplatin or a prodrug thereof; or to an individual in need thereof Kit A and Kit B, wherein Kit B includes 5-fluorouracil or a prodrug thereof, oxaliplatin or a prodrug thereof, and a taxane drug.
  • the prodrug of 5-fluorouracil is capecitabine.
  • the CLDN18 is CLDN18.2.
  • the antibody specifically recognizes CLDN18.2 and does not recognize CLDN18.1.
  • kit A and kit B are administered in no particular order.
  • 5-fluorouracil or its prodrug or its active metabolite, oxaliplatin-containing kit B, and CLDN18-containing antibody kit A are administered on the same day.
  • the CLDN18 antibody is administered at a dose of 500 to 1200 mg / m 2 / time. In a preferred example, it is 500-1000 mg / m 2 / time. In a preferred example, it is 500-900 mg / m 2 / time. In a preferred example, it is 600-800 mg / m 2 / time.
  • the CLDN18 antibody is administered at a dose of 20 to 300 mg / kg / time. In a preferred example, it is 20 to 100 mg / kg / time. In a preferred example, it is 20-50 mg / kg / time.
  • the 5-fluorouracil or prodrug or active metabolite thereof is administered at a dose of 300 to 900 mg / m 2 / time.
  • 5-fluorouracil is administered in an amount of 300-800 mg / m 2 / time.
  • capecitabine is administered at 600-700 mg / m 2 / time. In a preferred example, it is 625 mg / m 2 / time.
  • the 5-fluorouracil or prodrug or active metabolite thereof is administered at a dose of 50 to 70 mg / kg / time.
  • 5-fluorouracil is administered in an amount of 50 to 70 mg / kg / time.
  • the application amount of 5-fluorouracil is 50 to 60 mg / kg / time.
  • the oxaliplatin or its prodrug or its active metabolite is administered at a dose of 50 to 300 mg / m 2 / time. In a preferred example, oxaliplatin is administered at 100 to 200 mg / m 2 / time. In a more preferred example, oxaliplatin is administered at 130 mg / m 2 / times.
  • the oxaliplatin or its prodrug or its active metabolite is administered at a dose of 1 to 10 mg / kg / time. In a preferred example, oxaliplatin is administered at 1 to 5 mg / kg / time.
  • the antibody that specifically recognizes CLDN18 is a humanized antibody or a chimeric antibody.
  • the antibody that specifically recognizes CLDN18 comprises:
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 34 HCDR2 shown in the amino acid sequence of SEQ ID NO: 35 or 42
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 36 and amino acid sequence of SEQ ID NO: 37
  • LCDR2 shown in the amino acid sequence of SEQ ID NO: 39
  • amino acid sequence of SEQ ID NO: 39 or
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 25 LCDR1, LCDR2 shown in the amino acid sequence of SEQ ID NO: 26
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 28 HCDR2 shown in the amino acid sequence of SEQ ID NO: 29, HCDR3 shown in the amino acid sequence of SEQ ID NO: 30, and HCDR3 shown in the amino acid sequence of SEQ ID NO: 31 LCDR1, LCDR2 shown in the amino acid sequence of SEQ ID NO: 32, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 33; or
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 43 HCDR2 shown in the amino acid sequence of SEQ ID NO: 44
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 46 LCDR1, LCDR2 shown in the amino acid sequence of SEQ ID NO: 47
  • LCDR3 shown in the amino acid sequence of SEQ ID NO: 48 or
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 49 HCDR2 shown in the amino acid sequence of SEQ ID NO: 50
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 51 and amino acid sequence of SEQ ID NO: 52
  • LCDR3 shown in the amino acid sequence of SEQ ID NO: 54 LCDR1, LCDR3 shown in the amino acid sequence of SEQ ID NO: 53
  • LCDR3 shown in the amino acid sequence of SEQ ID NO: 54 LCDR1, LCDR3 shown in the amino acid sequence of SEQ ID NO: 53
  • the antibody that specifically recognizes CLDN18 includes:
  • HCDR1 shown in the amino acid sequence of SEQ ID NO: 34 HCDR2 shown in the amino acid sequence of SEQ ID NO: 35 or 42
  • HCDR3 shown in the amino acid sequence of SEQ ID NO: 36 and amino acid sequence of SEQ ID NO: 37
  • LCDR2 shown in the amino acid sequence of SEQ ID NO: 39.
  • the antibody that specifically recognizes CLDN18 contains:
  • the antibody that specifically recognizes CLDN18 contains:
  • the antibody that specifically recognizes CLDN18 contains:
  • the antibody that specifically recognizes CLDN18 is selected from:
  • the CLDN18-positive tumors include: gastric cancer, pancreatic cancer, esophageal cancer, and lung cancer.
  • the taxane drug is selected from paclitaxel, albumin-bound paclitaxel, and docetaxel; preferably, it is albumin-bound paclitaxel.
  • a pharmaceutical composition for treating CLDN18-positive tumors wherein the pharmaceutical composition includes an antibody that specifically recognizes CLDN18 and a chemotherapeutic drug, and the chemotherapeutic drug is as described above 5-fluorouracil or its prodrug or its active metabolite, oxaliplatin or its prodrug or its active metabolite; or 5-fluorouracil or its prodrug or its active metabolite, oxaliplatin or its prodrug Drugs or their active metabolites, and a taxane drugs; the antibody that specifically recognizes CLDN18 is an antibody as described above.
  • kits combination or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating a CLDN18-positive tumor.
  • the above-mentioned kit combination or pharmaceutical composition is provided for treating CLDN18-positive tumors.
  • FIG. 1 is a tumor volume statistical chart of a BALB / c nude mouse bearing a human gastric cancer PDX-GA0006 model after being treated with different treatment schemes;
  • FIG. 2 is a statistical chart of body weight of BALB / c nude mice bearing human gastric cancer PDX-GA0006 model after being treated with different treatment schemes;
  • FIG. 3 is a tumor photograph of a BALB / c nude mouse bearing a human gastric cancer PDX-GA0006 model after being treated with different treatment schemes;
  • FIG. 4 is a statistical graph of tumor weights of BALB / c nude mice bearing human gastric cancer PDX-GA0006 model after being treated with different treatment schemes. Note: Compared with the solvent group, * indicates P ⁇ 0.05, ** indicates P ⁇ 0.01, and *** indicates P ⁇ 0.001;
  • FIG. 5 shows the effect of different doses of antibodies on antitumor effects
  • Figure 6 shows the effect of different doses of antibodies on mouse body weight.
  • the present inventors After intensive research and repeated experiments, the present inventors have obtained a method for treating tumors by combining antibodies targeting CLDN18.2, 5-fluoropyrimidine, and oxaliplatin.
  • the tumors are tumors that express the dense protein 18A2, especially gastric cancer. , Pancreatic cancer, esophageal cancer, lung cancer treatment.
  • the antibody of the present invention is an antibody that specifically binds to an epitope present on cladin 18A2 (CLDN18A2), and includes a polyclonal antibody and a monoclonal antibody, preferably a monoclonal antibody.
  • Monoclonal antibodies encompassed by the invention include IgA, IgG1-4, IgE, IgM, and IgD antibodies. In one embodiment, the antibody is an IgG1 antibody.
  • the antibody may also be an IgG3 antibody, such as an IgG3 ⁇ or IgG3 ⁇ isotype; it may also be an IgG4 antibody, such as an IgG4 ⁇ or IgG4 ⁇ isotype; or it may be IgA1 or IgA2
  • the antibody may be an IgM antibody.
  • the CLD18A2 antibody is administered at a dose of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 / kg / time; 1, 2, 3, per week 4, 5, 6, or 7 times; injections for 1 week, or continuous injections for 2, 3, 4, 5, or 6 weeks.
  • the CLD18A2 antibody is administered at a dose of 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1050, 1100, 1150, 1200 mg / m 2 / times; 1, 2, 3, 4, 5 or 6 times per week; 1 week injection, or 2, 3, 4, 5 or 6 consecutive injections week.
  • CLDN18.2 includes isoforms, mammalian (e.g., human) CLDN18.2, posttranslationally modified variants of human CLDN18.2, isoforms and interspecies homologs, and includes at least one Of the common epitope.
  • the amino acid sequence of CLDN18.2 e.g., human CLDN18.2
  • CLDN18A2 is known in the art, as shown in the NCBI database.
  • CLDN18.2 and “cell-linkin 18.2” are used interchangeably herein, and the terms “CLDN18A1", “CLDN18.1”, and “cell-linkin 18.1” are interchangeable.
  • oxaliplatin herein refers to the compound [(1R, 2R) -cyclohexane-1,2-diamine] ((2-)-O, O ') platinate.
  • the oxaliplatin is injected 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg / time per injection, 1, 2, 3, 4, 5, 6, or 7 injections for 1 week, or continuous injections for 2, 3, 4, 5, or 6 weeks.
  • the oxaliplatin is injected 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg / m 2 / times, 1, 2 , 3, 4, 5, 6 or 7 times per week, 1 week injection, or continuous 2, 3 injections , 4, 5 or 6 weeks.
  • the term "5-fluoropyrimidine” herein refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.
  • the 5-fluorouracil or prodrug or active metabolite thereof is administered in an amount of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 mg / kg / time, 1, 2, 3, 4, 5, 6, or 7 times per week, 1 week injection, or 2, 3, 4 continuous injections , 5 or 6 weeks; in certain embodiments, the 5-fluorouracil or prodrug or active metabolite thereof is administered in an amount of 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900 mg /
  • the prodrug of 5-fluorouracil is capecitabine.
  • the capecitabine or its prodrug or active metabolite is administered in an amount of 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660 , 665, 670, 675, 680, 685, 690, 695, 700 mg / m 2 / times, 1, 2, 3, 4, 5, 6, or 7 times per week, 1 week injection, or continuous injection of 2, 3 , 4, 5 or 6 weeks.
  • epirubicin refers to the compound 10-[(3-amino-2,3,6-trideoxy-A-L-alpha-hexylpyranosyl) oxy] -7,8, 9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -methoxy- (8S-CIS) -tetrabenzo-5,12-dione.
  • antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains or antigen-binding fragments thereof interconnected by disulfide bonds.
  • antibody also includes all recombinant forms of antibodies, particularly antibodies described herein, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, as well as antibody-binding antibody fragments and derivatives described below.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are scattered in more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (such as effector cells) and the first component of the classical complement system (C1q)
  • the term "monoclonal antibody” as used herein refers to a preparation of antibody molecules consisting of a single molecule. Monoclonal antibodies show single binding specificity and affinity for a particular epitope. In one embodiment, the monoclonal antibody is produced by a hybridoma that includes B cells derived from a non-human animal (e.g., mouse) fused with biochemical cells.
  • the antibody described herein may be a recombinant antibody, and the "recombinant antibody” includes all antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) an animal (such as a mouse) from an immunoglobulin gene to a transgene or transchromosome ) Or an antibody isolated from a hybridoma prepared therefrom, (b) an antibody isolated from a host cell (eg, a transfected tumor) transformed to express the antibody, (c) an antibody isolated from a recombinant combinatorial antibody library, And (d) antibodies produced, expressed, produced or isolated by any other means involving splicing of immunoglobulin gene sequences into DNA sequences.
  • recombinant antibody includes all antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) an animal (such as a mouse) from an immunoglobulin gene to a transgene or transchromosome ) Or an antibody isolated from a hybridoma prepared therefrom, (b) an
  • humanized antibody herein refers to a CDR sequence in which a germline derived from another mammalian species, such as a mouse, is grafted onto a human framework sequence. Additional framework region modifications can also be made in human framework sequences and CDR sequences derived from germlines of another mammalian species.
  • chimeric antibody herein refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, the variable region sequence is derived from a mouse antibody and the constant region sequence source is Antibodies from human antibodies.
  • Fab or "Fab region” herein includes polypeptides comprising VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region isolated, or this region located in the context of a full-length antibody or antibody fragment.
  • Fc or "Fc region” herein includes a polypeptide comprising an antibody constant region other than the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. For IgA and IgM, the Fc may include a J chain.
  • the Fc includes the immunoglobulin domains C ⁇ 2 and C ⁇ 3 and the hinge between C ⁇ 1 and C ⁇ 2.
  • the human IgG heavy chain Fc region is generally defined as containing residues C226 or P230 at its carboxy terminus, where numbering is according to Kabat's EU index.
  • Fc is defined herein as including residue P232 to its carboxy terminus, where numbering is according to the EU index in Kabat.
  • Fc can refer to this region isolated, or to a region in an Fc polypeptide, such as an antibody, in the environment.
  • the term “hinge” or “hinge region” or “antibody hinge region” herein includes a flexible polypeptide comprising an amino acid between the first and second constant domains of an antibody. Structurally, the IgGCH1 domain starts at EU220 and the IgGCH2 domain starts at residue EU237.
  • the antibody hinge is defined herein as including positions 221 (D221 of IgG1) to 231 (A231 of IgG1), where numbering is according to Kabat's EU index.
  • variant includes antibody sequences that differ from the parent antibody sequence due to at least one amino acid modification compared to the parent antibody.
  • the variant antibody sequences herein preferably have at least about 80%, preferably at least about 90%, and more preferably at least about 95% amino acid sequence identity to the parent antibody sequence.
  • An antibody variant may refer to the antibody itself, a composition comprising the parent antibody, or an amino acid sequence encoding it.
  • amino acid modification herein includes amino acid substitutions, insertions and / or deletions in a polypeptide sequence.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in the parent polypeptide sequence with another amino acid.
  • substitution of R94K means that the arginine at position 94 is replaced by a lysine, which is a variant of the heavy chain variable framework region.
  • 94K means that the 94 position is replaced with lysine.
  • multiple substitutions are usually separated by slashes.
  • R94K / L78V refers to a double variant comprising the substitutions R94K and L78V.
  • amino acid insertion or “insertion” means the addition of an amino acid at a particular position in the parent polypeptide sequence.
  • insert -94 indicates an insert at 94 bits.
  • amino acid deletion or “deletion” means the removal of an amino acid at a particular position in a parent polypeptide sequence.
  • R94- indicates deletion of arginine at position 94.
  • full-length antibody includes structures that make up the natural biological form of the antibody, including variable and constant regions.
  • the full-length IgG antibodies are tetramers, consisting of two identical pairs of immunoglobulin chains, each pair having a light chain and a heavy chain, Each light chain contains immunoglobulin domains VL and CL, and each heavy chain contains immunoglobulin domains VH, CH1 (C ⁇ 1), CH2 (C ⁇ 2), and CH3 (C ⁇ 3).
  • IgG antibodies may consist of only two heavy chains, each heavy chain comprising a variable domain linked to the Fc region.
  • Antibody fragments include, but are not limited to: (i) Fab fragments consisting of VL, VH, CL and CH1 domains, including Fab 'and Fab'-SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragment consisting of VL and VH domains of a single antibody; (iv) dAb fragment consisting of a single variable region; (v) F (ab ') 2 fragment, a bivalent fragment containing 2 linked Fab fragments; (vi) single-chain Fv molecule antigen binding site; (vii) bispecific single-chain Fv dimer; (viii) "dibody” or “tribody”, a multivalent or multispecific fragment constructed by gene fusion And (ix) scFv genetically fused to the same or different antibodies.
  • Antibodies are classified into isotypes based on constant region gene assays.
  • Human constant light chains are divided into ⁇ (C ⁇ ) and ⁇ (C ⁇ ) light chains.
  • the heavy chain is divided into ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ and defines the isotypes of antibodies, IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgGs are commonly used for therapeutic purposes. In humans, this category includes the subclasses IgG1, IgG2, IgG3, and IgG4. In mice, this category includes the subclasses IgG1, IgG2a, IgG2b, and IgG3.
  • IgM has subclasses, including but not limited to IgM1 and IgM2.
  • IgA has several subclasses, including but not limited to IgA1 and IgA2.
  • isotype means any class or subclass of immunoglobulin defined by the chemical and antigenic characteristics of the constant region.
  • the known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • enhancing ADCC effector function may mean enhanced potency or enhanced efficacy.
  • Tier as used in the experimental context means the concentration (half-large effective concentration) of the antibody when observing a specific therapeutic efficacy EC50.
  • Effectiveacy as used in the experimental context means the large possible effector function of an antibody at a saturated level.
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • complement protein components recognize antibodies bound on a target cell, which then results in the lysis of the target cell.
  • effector function includes a biochemical event caused by the interaction of an Fc region of an antibody with an Fc receptor or ligand. Effector functions include Fc ⁇ R-mediated effector functions, such as ADCC and ADCP, and complement-mediated effector functions, such as CDC.
  • body surface area (m2) 0.0061 ⁇ height (cm) + 0.0128 ⁇ weight (kg)-0.1529.
  • Solution: 0.0061 ⁇ 168 + 0.0128 ⁇ 55-0.1529 1.576m2
  • the heavy and light chain variable region sequences of the antibodies listed in the present invention can each bind human dense protein 18A2, and the heavy and light chain variable region sequences can be "mixed and matched" to produce the anti-human dense protein 18A2 of the present invention. Binding molecule.
  • the invention also provides variants of antibodies or fragments thereof that bind human avidin 18A2. Accordingly, the invention also includes antibodies or fragments thereof having a heavy or light chain variable region sequence that is at least 80% of the heavy and / or light chain variable region sequence of an antibody specifically disclosed herein Amino acid sequence identity.
  • the amino acid sequence identity is at least 85%, more preferably at least 90%, preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98%, especially 99%, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% and 100%.
  • sequence identity can be determined by standard methods commonly used to compare amino acid position similarities of two polypeptides.
  • a computer program such as BLAST or FASTA is used to align the two amino acids for a good match (along the full length of one or two sequences or along a predetermined portion of one or two sequences).
  • the program provides default open penalties and default gap penalties.
  • a scoring matrix such as PAM250 can be used in conjunction with a computer program. For example, percent identity can be calculated as: the total number of identical matches multiplied by 100, divided by the total length of the longer sequence in the match span and the number of empty spaces poured into the longer sequence in order to compare the two sequences.
  • framework sequences can be used to engineer variable regions to produce variant antibodies.
  • the variant antibodies of the present invention include variants in which framework region residues in VH and / or VK are modified, for example, to improve the characteristics of the antibody.
  • framework region modifications are made to reduce the immunogenicity of the antibody.
  • one approach is to "backmutate" one or more framework region residues to the corresponding murine sequence, or "backmutate” one or more framework region residues to the corresponding germline sequence.
  • the present invention provides a humanized antibody or fragment thereof that binds human avidin 18A2, wherein the framework region of at least one heavy chain variable region of the humanized antibody or fragment thereof comprises At least one amino acid modification of the corresponding framework region of the heavy chain variable region.
  • the amino acid modification is an amino acid substitution.
  • no more than five, preferably no more than four, more preferably no more than three, even more preferably no more than two, and preferably no more than one amino acid modification are performed in the framework region.
  • the invention also provides humanized antibodies and fragments thereof that bind human dense protein 18A2, which also include human heavy and / or light chain constant domains.
  • the human heavy chain constant region can be selected from the group of human immunoglobulins consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE, and human heavy chain constant region IgG, especially IgG1 is preferred.
  • the human light chain constant region may be selected from the group of human immunoglobulins composed of K or lambda constant regions, and human K constant regions are preferred.
  • the humanized antibody or fragment thereof comprises a human IgG1 heavy chain constant domain and a human light chain K constant domain.
  • the present invention also provides a humanized antibody or fragment thereof that binds human avidin 18A2, which includes a human heavy chain and / or light chain constant region, wherein the human heavy chain constant region includes CH1 of human IgG1, a hinge region of human IgG1, and Isotype variants of the Fc region of human IgG3.
  • a preferred humanized antibody comprising an isotype variant is a full-length antibody.
  • the sequence includes a heavy chain variable region sequence comprising SEQ ID NO: 12, 14, 15, 55, or 57 and a sequence comprising SEQ ID NO: 11, 13, 56, or 58. The sequence of the light chain variable region is shown.
  • a combination of the heavy chain variable region shown in SEQIDNO: 12 and the light chain variable region shown in SEQIDNO: 11 is preferred; or the sequence of the heavy chain variable region shown in SEQIDNO: 14 or 15 and the sequence shown in SEQIDNO: 13 are preferred.
  • Effector functions are usually complement-dependent cytotoxicity (CDC) and / or C1q binding and / or antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody binding affinity to Fc ⁇ receptors, preferably complement-dependent cells Toxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC).
  • CDC complement-dependent cells Toxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the binding affinities of CDC, C1q binding, ADCC, and antibodies to the Fc ⁇ receptor are measured by standard in vitro assays known in the art and commercially available.
  • ADCC is measured by a lactate dehydrogenase (LDH) release assay
  • CDC is measured by an assay administered to a cell.
  • Standard assays to assess the binding capacity of antibodies such as human claudin 18A2 are known in the art and include, for example, ELISA, Western blot, and flow cytometry analysis. Suitable assays are described in detail in the examples.
  • 293T cells stably transfected with CLD18A2 AGS cells stably transfected with CLD18A2, NCI-N87 cells stably transfected with CLD18A2, BGC cells stably transfected with CLD18A2, and flow cytometric analysis Determine EC50.
  • the antibody targeting CLD18A2 selected in this example is a humanized antibody, which has a heavy chain variable region shown in SEQ ID NO: 15 and a light chain variable region shown in SEQ ID NO: 13 and a nucleoside of the light chain.
  • the acid sequence is shown in SEQ ID NO: 60
  • the heavy chain nucleotide sequence is shown in SEQ ID NO: 59.
  • Amplification of the light and heavy chain variable regions was performed according to the "step-out PCR" method described by Matz et al. (Nucleic Acids Research, 1999, Vol. 27, No. 6).
  • the nucleotides of the light chain (sequence shown in SEQ ID NO: 60) and the nucleotides of the heavy chain (sequence shown in SEQ ID NO: 59) were cloned to true by standard methods known to those skilled in the art.
  • Nuclear expression vector. 293F cells in the logarithmic growth phase were transiently transfected with 293fectin TM Transfection reagent (Invitrogen, 12347-019) or polyethyleneimine (PEI) (Sigma-Aldrich, 408727).
  • the above eukaryotic expression vector uses the vector pH or vector pK used by CN101602808B.
  • mice 2 ⁇ 2 ⁇ 2mm PDX tumors of gastric cancer were inoculated on BALB / c mice (D0 on the day of inoculation), and the tumors were inoculated subcutaneously for D27 days.
  • the average tumor volume was 128mm 3 , and the model of gastric cancer PDX-GA0006 was obtained.
  • the tumor-forming mice were randomly divided into 6 groups, namely the vehicle group, the antibody group (Ab group), the EOF group, the EOF + Ab group, the OF group, and the OF + Ab group, and the general clinical symptoms of the animals were observed during the experiment.
  • the body weight and tumor diameter were measured twice, and on day D51, mice were euthanized.
  • the method of administration is as follows:
  • Vehicle group intraperitoneal injection, 3 times a week.
  • Ab group CLD18A2 antibody prepared in Example 1, 40 mg / kg, intraperitoneal injection, 3 times a week.
  • EOF group epirubicin hydrochloride (1.25 mg / kg) for intraperitoneal injection + oxaliplatin (3.25 mg / kg) for injection + fluorouracil injection (56.25 mg / kg), once a week; vehicle, Intraperitoneal injection, 3 times a week.
  • EOF + Ab group EOF (epirubicin hydrochloride (1.25mg / kg) + oxaliplatin for injection (3.25mg / kg) + fluorouracil injection (56.25mg / kg)) given once a week, CLD18A2 antibody (40mg / kg) 3 times a week.
  • OF group oxaliplatin for injection (3.25mg / kg) + fluorouracil injection (56.25mg / kg), once a week; vehicle, intraperitoneal injection, three times a week.
  • OF + Ab group OF (Oxaliplatin for injection (3.25mg / kg) + Fluorouracil injection (56.25mg / kg)) was administered once a week, and CLD18A2 antibody (40mg / kg) was administered three times a week.
  • Tumors were obtained after euthanasia of mice, and tumor weights were weighed. The results are shown in Figs. 3 and 4.
  • the tumor load was too large.
  • One mouse died at 51 days after tumor inoculation.
  • the tumor suppression rates of the groups were: Ab group, 1.21%; EOF group, 48.70%; EOF + Ab group, 69.46%; OF group, 71.64%; OF + Ab group, 96.86%, and tumor regression of 1 mouse among 5 mice in this group (* p ⁇ 0.05; ** p ⁇ 0.01 or *** p ⁇ 0.001, Oneway ANOVA).
  • Example 2 Referring to the operation of Example 2, the tumor-forming mice were given different doses of the antibodies, and the specific administration methods were as follows:
  • AB1 the CLD18A2 antibody prepared in Example 1 was administered at a dose of 40 mg / kg, intraperitoneally, 3 times a week for 2 weeks.
  • the CLD18A2 antibody prepared in Example 1 was administered at a dose of 70 mg / kg, intraperitoneally, 3 times a week for a total of 2 weeks.
  • AB3 the CLD18A2 antibody prepared in Example 1 was administered at a dose of 100 mg / kg, intraperitoneally, 3 times a week for a total of 2 weeks.
  • the tumor growth results are shown in Figure 5, which shows that the administration of the AB3 group has a better anti-tumor effect.
  • the weight change of the mice is shown in Figure 6, and there is no significant change in body weight, indicating that the administration of a large dose of CLD18A2 antibody To better antitumor effect, and the side effects are small.
  • an example of an antibody using the humanized antibody CLD18A2 is used.
  • Those skilled in the art may use other antibodies according to the teaching of the present application, such as those containing SEQ ID NO: 12.
  • the light chain variable region antibody shown in SEQ ID NO: 58 is also used.

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Abstract

一种治疗CLDN18阳性的肿瘤的方法，其给予有需要的个体特异性识别CLDN18的抗体及化疗药物，所述化疗药物为：5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物；或，5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物、及紫衫烷类药物。一种治疗CLDN18阳性的肿瘤的药盒组合。治疗方法和药盒组合与现有技术相比，其抑瘤率得到了极大地增强，具有更优的抗肿瘤效果。此外还能减少化疗药的使用，提高了用药的安全性和患者的耐受性。

Description

CLDN18的抗体和化疗药物的联合治疗
本申请要求申请日为2018年9月30日，申请号为201811159863.8，发明名称为“CLDN18.2的抗体和化疗药物的联合治疗”以及申请日为2018年10月19日，申请号为201811224605.3，发明名称为“CLDN18的抗体和化疗药物的联合治疗”的中国专利申请的优先权。所述在先申请的内容通过引用全文纳入本文。
技术领域
本发明属于生物医药领域，具体涉及一种CLDN18的抗体和化疗药物的联合治疗。
背景技术
胃癌在全世界范围内是发病率高的癌症之一，据世界卫生组织癌控项目的统计数据，全球每年死于癌症的患者高达700万，其中死于胃癌的患者占了70万例。
德国生物技术公司Ganymed报道了EOX(表柔比星、奥沙利铂和卡培他滨)的化疗药组合和靶向Claudin18.2的抗体连用治疗胃癌，EP2852408B也报道了采用EOF(表柔比星、5-氟嘧啶和奥沙利铂)的化疗药组合和靶向细胞连接密蛋白(Claudin或CLDN)18.2的抗体连用在小鼠体内治疗胃癌。
然而由于化疗药物通常具有较大的细胞毒性，患者耐受性差，使用多种化疗药带来了很大的临床应用风险。
发明内容
本发明的目的在于提供一种CLDN18的抗体和化疗药物的联合治疗，其用于有效治疗与表达CLD18A相关的疾病。
在本发明的第一方面，提供了一种治疗CLDN18阳性的肿瘤的方法，给予有需要的个体特异性识别CLDN18的抗体、5-氟尿嘧啶或其前药或其活性代谢物、及奥沙利铂或其前药或其活性代谢物；或者给予有需要的个体特异性识别CLDN18的抗体、5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物、及紫衫烷类药物。
在具体实施方式中，给予有需要的个体特异性识别CLDN18的抗体、5-氟尿嘧啶或其前药、及奥沙利铂或其前药；或者给予有需要的个体特异性识别CLDN18的抗体、5-氟尿嘧啶或其前药、奥沙利铂或其前药、及紫衫烷类药物。
在具体实施方式中，所述5-氟尿嘧啶的前药是卡培他滨。
在具体实施方式中，所述CLDN18为CLDN18.2。
在具体实施方式中，所述抗体特异性识别CLDN18.2，而不识别CLDN18.1。
在具体实施方式中，CLDN18的抗体、5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物给予时间不分先后。
在具体实施方式中，5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂、CLDN18的抗体 在同一天给予。
在具体实施方式中，所述CLDN18的抗体的施用剂量为500～1200mg/m 2/次。在一优选例中，为500-1000mg/m 2/次。在一优选例中，为500-900mg/m 2/次。在一优选例中，为600-800mg/m 2/次。
在具体实施方式中，所述CLDN18的抗体的施用剂量为20～300mg/kg/次。在一优选例中，为20～100mg/kg/次。在一优选例中，为20-50mg/kg/次。
在具体实施方式中，所述5-氟尿嘧啶或其前药或其活性代谢物的施用剂量为300～900mg/m 2/次。
在一具体实施方式中，给予5-氟尿嘧啶，施用量为300-800mg/m 2/次。
在一具体实施方式中，给予卡培他滨600-700mg/m 2/次。在一优选例中，为625mg/m 2/次。
在具体实施方式中，所述5-氟尿嘧啶或其前药或其活性代谢物的施用剂量为50～70mg/kg/次。在一优选例中，给予5-氟尿嘧啶，施用量为50～70mg/kg/次。在一更优选例中，5-氟尿嘧啶的施用量为50～60mg/kg/次。
在具体实施方式中，所述奥沙利铂或其前药或其活性代谢物的施用剂量为50～300mg/m 2/次。在一优选例中，给予奥沙利铂100～200mg/m 2/次。在一更优选例中，给予奥沙利铂130mg/m 2/次。
在具体实施方式中，所述奥沙利铂或其前药或其活性代谢物的施用剂量为1～10mg/kg/次。在一优选例中，给予奥沙利铂1～5mg/kg/次。
在具体实施方式中，所述特异性识别CLDN18的抗体为人源化的抗体或嵌合抗体。
在具体实施方式中，所述特异性识别CLDN18的抗体包含：
SEQ ID NO:16的氨基酸序列所示的HCDR1、SEQ ID NO:17、40、或41的氨基酸序列所示的HCDR2、SEQ ID NO:18的氨基酸序列所示的HCDR3，和SEQ ID NO:19的氨基酸序列所示的LCDR1、SEQ ID NO:20的氨基酸序列所示的LCDR2、SEQ ID NO:21的氨基酸序列所示的LCDR3；或者，
SEQ ID NO:34的氨基酸序列所示的HCDR1、SEQ ID NO:35或42的氨基酸序列所示的HCDR2、SEQ ID NO:36的氨基酸序列所示的HCDR3，和SEQ ID NO:37的氨基酸序列所示的LCDR1、SEQ ID NO:38的氨基酸序列所示的LCDR2、SEQ ID NO:39的氨基酸序列所示的LCDR3；或者，
SEQ ID NO:22的氨基酸序列所示的HCDR1、SEQ ID NO:23的氨基酸序列所示的HCDR2、SEQ ID NO:24的氨基酸序列所示的HCDR3，和SEQ ID NO:25的氨基酸序列所示的LCDR1、SEQ ID NO:26的氨基酸序列所示的LCDR2、SEQ ID NO:27的氨基酸序列所示的LCDR3；或者，
SEQ ID NO:28的氨基酸序列所示的HCDR1、SEQ ID NO:29的氨基酸序列所示的HCDR2、SEQ ID NO:30的氨基酸序列所示的HCDR3，和SEQ ID NO:31的氨基酸序列所示的LCDR1、SEQ ID NO:32的氨基酸序列所示的LCDR2、SEQ ID NO:33的氨基酸序列所示 的LCDR3；或者，
SEQ ID NO:43的氨基酸序列所示的HCDR1、SEQ ID NO:44的氨基酸序列所示的HCDR2、SEQ ID NO:45的氨基酸序列所示的HCDR3，和SEQ ID NO:46的氨基酸序列所示的LCDR1、SEQ ID NO:47的氨基酸序列所示的LCDR2、SEQ ID NO:48的氨基酸序列所示的LCDR3；或者，
SEQ ID NO:49的氨基酸序列所示的HCDR1、SEQ ID NO:50的氨基酸序列所示的HCDR2、SEQ ID NO:51的氨基酸序列所示的HCDR3，和SEQ ID NO:52的氨基酸序列所示的LCDR1、SEQ ID NO:53的氨基酸序列所示的LCDR2、SEQ ID NO:54的氨基酸序列所示的LCDR3。
在具体实施方式中，所述特异性识别CLDN18的抗体包含有：
SEQ ID NO:16的氨基酸序列所示的HCDR1、SEQ ID NO:17或41的氨基酸序列所示的HCDR2、SEQ ID NO:18的氨基酸序列所示的HCDR3，和SEQ ID NO:19的氨基酸序列所示的LCDR1、SEQ ID NO:20的氨基酸序列所示的LCDR2、SEQ ID NO:21的氨基酸序列所示的LCDR3；或者，
SEQ ID NO:34的氨基酸序列所示的HCDR1、SEQ ID NO:35或42的氨基酸序列所示的HCDR2、SEQ ID NO:36的氨基酸序列所示的HCDR3，和SEQ ID NO:37的氨基酸序列所示的LCDR1、SEQ ID NO:38的氨基酸序列所示的LCDR2、SEQ ID NO:39的氨基酸序列所示的LCDR3；或者
如SEQ ID NO:43的氨基酸序列所示的HCDR1、SEQ ID NO:44的氨基酸序列所示的HCDR2、SEQ ID NO:45的氨基酸序列所示的HCDR3，和SEQ ID NO:46的氨基酸序列所示的LCDR1、SEQ ID NO:47的氨基酸序列所示的LCDR2、SEQ ID NO:48的氨基酸序列所示的LCDR3。
在具体实施方式中，所述特异性识别CLDN18的抗体含有：
SEQ ID NO:2、9、或10的氨基酸序列所示的重链可变区和SEQ ID NO:1的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:8的氨基酸序列所示的重链可变区和SEQ ID NO:7的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:14或15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:4的氨基酸序列所示的重链可变区和SEQ ID NO:3的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:6的氨基酸序列所示的重链可变区和SEQ ID NO:5的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的 轻链可变区；或者，
SEQ ID NO:57的氨基酸序列所示的重链可变区和SEQ ID NO:58的氨基酸序列所示的轻链可变区；或者，
在具体实施例中，所述特异性识别CLDN18的抗体含有：
SEQ ID NO:2或10的氨基酸序列所示的重链可变区和SEQ ID NO:1的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:8的氨基酸序列所示的重链可变区和SEQ ID NO:7的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:14或15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区。
在一优选例中，所述特异性识别CLDN18的抗体含有：
SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或者，
SEQ ID NO:15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；
SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区。
在具体实施方式中，所述特异性识别CLDN18的抗体选自：
(a)具有SEQ ID NO:59所示的核苷酸序列编码的重链和SEQ ID NO:60所示的核苷酸序列编码的轻链；或，
(b)具有SEQ ID NO:64所示的重链氨基酸序列和SEQ ID NO:63所示的轻链氨基酸序列。
在具体实施方式中，所述CLDN18阳性的肿瘤包括：胃癌，胰腺癌，食管癌，肺癌。
在具体实施方式中，所述的紫衫烷类药物选自紫杉醇、白蛋白结合型紫杉醇、多西他赛；优选的，为白蛋白结合型紫杉醇。
在本发明的第二方面，提供了一种治疗CLDN18阳性的肿瘤的药盒组合，所述药盒组合包括药盒A和药盒B，所述药盒A包括特异性识别CLDN18的抗体，所述药盒B包括化疗药物，所述化疗药物为：5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物；或，5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物、及紫衫烷类药物。
在具体实施方式中，给予有需要的个体所述药盒A、药盒B，其中药盒B包括5-氟尿嘧啶或其前药、及奥沙利铂或其前药；或者给予有需要的个体药盒A、药盒B，其中药盒B 包括5-氟尿嘧啶或其前药、奥沙利铂或其前药、及紫衫烷类药物。
在具体实施方式中，所述5-氟尿嘧啶的前药是卡培他滨。
在具体实施方式中，所述CLDN18为CLDN18.2。
在具体实施方式中，所述抗体特异性识别CLDN18.2，而不识别CLDN18.1。
在具体实施方式中，药盒A与药盒B给予时间不分先后。
在具体实施方式中，含5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂的药盒B、含CLDN18的抗体的药盒A在同一天给予。
在具体实施方式的一所述药盒A中，所述CLDN18的抗体的施用剂量为500～1200mg/m 2/次。在一优选例中，为500-1000mg/m 2/次。在一优选例中，为500-900mg/m 2/次。在一优选例中，为600-800mg/m 2/次。
在具体实施方式的一所述药盒A中，所述CLDN18的抗体的施用剂量为20～300mg/kg/次。在一优选例中，为20～100mg/kg/次。在一优选例中，为20-50mg/kg/次。
在具体实施方式的一所述药盒B中，所述5-氟尿嘧啶或其前药或其活性代谢物的施用剂量为300～900mg/m 2/次。
在一具体实施方式中，给予5-氟尿嘧啶，施用量为300-800mg/m 2/次。
在一具体实施方式中，给予卡培他滨600-700mg/m 2/次。在一优选例中，为625mg/m 2/次。
在具体实施方式的一所述药盒B中，所述5-氟尿嘧啶或其前药或其活性代谢物的施用剂量为50～70mg/kg/次。在一优选例中，给予5-氟尿嘧啶，施用量为50～70mg/kg/次。在一更优选例中，5-氟尿嘧啶的施用量为50～60mg/kg/次。
在具体实施方式的一所述药盒B中，所述奥沙利铂或其前药或其活性代谢物的施用剂量为50～300mg/m 2/次。在一优选例中，给予奥沙利铂100～200mg/m 2/次。在一更优选例中，给予奥沙利铂130mg/m 2/次。
在具体实施方式的一所述药盒B中，所述奥沙利铂或其前药或其活性代谢物的施用剂量为1～10mg/kg/次。在一优选例中，给予奥沙利铂1～5mg/kg/次。
在具体实施方式的一所述药盒A中，特异性识别CLDN18的抗体为人源化的抗体或嵌合抗体。
在具体实施方式的一所述药盒A中，特异性识别CLDN18的抗体包含：
SEQ ID NO:16的氨基酸序列所示的HCDR1、SEQ ID NO:17、40、或41的氨基酸序列所示的HCDR2、SEQ ID NO:18的氨基酸序列所示的HCDR3，和SEQ ID NO:19的氨基酸序列所示的LCDR1、SEQ ID NO:20的氨基酸序列所示的LCDR2、SEQ ID NO:21的氨基酸序列所示的LCDR3；或者
SEQ ID NO:34的氨基酸序列所示的HCDR1、SEQ ID NO:35或42的氨基酸序列所示的HCDR2、SEQ ID NO:36的氨基酸序列所示的HCDR3，和SEQ ID NO:37的氨基酸序列所示的LCDR1、SEQ ID NO:38的氨基酸序列所示的LCDR2、SEQ ID NO:39的氨基酸序列所示的LCDR3；或者
SEQ ID NO:22的氨基酸序列所示的HCDR1、SEQ ID NO:23的氨基酸序列所示的HCDR2、SEQ ID NO:24的氨基酸序列所示的HCDR3，和SEQ ID NO:25的氨基酸序列所示的LCDR1、SEQ ID NO:26的氨基酸序列所示的LCDR2、SEQ ID NO:27的氨基酸序列所示的LCDR3；或者
SEQ ID NO:28的氨基酸序列所示的HCDR1、SEQ ID NO:29的氨基酸序列所示的HCDR2、SEQ ID NO:30的氨基酸序列所示的HCDR3，和SEQ ID NO:31的氨基酸序列所示的LCDR1、SEQ ID NO:32的氨基酸序列所示的LCDR2、SEQ ID NO:33的氨基酸序列所示的LCDR3；或者
SEQ ID NO:43的氨基酸序列所示的HCDR1、SEQ ID NO:44的氨基酸序列所示的HCDR2、SEQ ID NO:45的氨基酸序列所示的HCDR3，和SEQ ID NO:46的氨基酸序列所示的LCDR1、SEQ ID NO:47的氨基酸序列所示的LCDR2、SEQ ID NO:48的氨基酸序列所示的LCDR3；或者
SEQ ID NO:49的氨基酸序列所示的HCDR1、SEQ ID NO:50的氨基酸序列所示的HCDR2、SEQ ID NO:51的氨基酸序列所示的HCDR3，和SEQ ID NO:52的氨基酸序列所示的LCDR1、SEQ ID NO:53的氨基酸序列所示的LCDR2、SEQ ID NO:54的氨基酸序列所示的LCDR3。
在具体实施方式中，所述药盒A中，特异性识别CLDN18的抗体包含有：
SEQ ID NO:16的氨基酸序列所示的HCDR1、SEQ ID NO:17或41的氨基酸序列所示的HCDR2、SEQ ID NO:18的氨基酸序列所示的HCDR3，和SEQ ID NO:19的氨基酸序列所示的LCDR1、SEQ ID NO:20的氨基酸序列所示的LCDR2、SEQ ID NO:21的氨基酸序列所示的LCDR3；或者
SEQ ID NO:34的氨基酸序列所示的HCDR1、SEQ ID NO:35或42的氨基酸序列所示的HCDR2、SEQ ID NO:36的氨基酸序列所示的HCDR3，和SEQ ID NO:37的氨基酸序列所示的LCDR1、SEQ ID NO:38的氨基酸序列所示的LCDR2、SEQ ID NO:39的氨基酸序列所示的LCDR3。
在具体实施方式的一所述药盒A中，所述特异性识别CLDN18的抗体含有：
SEQ ID NO:2、9、或10的氨基酸序列所示的重链可变区和SEQ ID NO:1的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:8的氨基酸序列所示的重链可变区和SEQ ID NO:7的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:14或15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:4的氨基酸序列所示的重链可变区和SEQ ID NO:3的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:6的氨基酸序列所示的重链可变区和SEQ ID NO:5的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:57的氨基酸序列所示的重链可变区和SEQ ID NO:58的氨基酸序列所示的轻链可变区；或者
在具体实施例的一所述药盒A中，所述特异性识别CLDN18的抗体含有：
SEQ ID NO:2或10的氨基酸序列所示的重链可变区和SEQ ID NO:1的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:8的氨基酸序列所示的重链可变区和SEQ ID NO:7的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:14或15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区。
在一优选例的所述药盒A中，所述特异性识别CLDN18的抗体含有：
SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或者
SEQ ID NO:15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；
SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区。
在具体实施方式的一所述药盒A中，所述特异性识别CLDN18的抗体选自：
(a)具有SEQ ID NO:59所示的核苷酸序列编码的重链和SEQ ID NO:60所示的核苷酸序列编码的轻链；或，
(b)具有SEQ ID NO:64所示的重链氨基酸序列和SEQ ID NO:63所示的轻链氨基酸序列。
在具体实施方式中，所述CLDN18阳性的肿瘤包括：胃癌，胰腺癌，食管癌，肺癌。
在具体实施方式的一所述药盒B中，所述的紫衫烷类药物选自紫杉醇、白蛋白结合型紫杉醇、多西他赛；优选的，为白蛋白结合型紫杉醇。
在本发明的第三方面，提供了一种治疗CLDN18阳性的肿瘤的药物组合物，其中，所述药物组合物包括特异性识别CLDN18的抗体及化疗药物，所述化疗药物为如上所述所述的5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物；或，5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物、及紫衫烷类药物；所述特异 性识别CLDN18的抗体为如上所述的抗体。
在本发明的第四方面，提供了一种如上所述的药盒组合或如上所述的药物组合物在制备治疗CLDN18阳性的肿瘤的药物中的应用。
在本发明的第四方面，提供上述药盒组合或药物组合物，用于治疗CLDN18阳性肿瘤。
附图说明
图1是荷人胃癌PDX-GA0006模型的BALB/c裸鼠经不同的治疗方案治疗后的肿瘤体积统计图表；
图2是荷人胃癌PDX-GA0006模型的BALB/c裸鼠经不同的治疗方案治疗后的体重统计图表；
图3是荷人胃癌PDX-GA0006模型的BALB/c裸鼠经不同的治疗方案治疗后的肿瘤照片；
图4是荷人胃癌PDX-GA0006模型的BALB/c裸鼠经不同的治疗方案治疗后的肿瘤重量统计图表。注：与溶媒组比较，*表示P≤0.05，**表示P≤0.01，***表示P≤0.001；
图5显示了不同剂量的抗体对抗肿瘤作用的影响；
图6显示了不同剂量的抗体对小鼠体重的影响。
具体实施方式
本发明人经过深入研究和反复实验，获得靶向CLDN18.2的抗体、5-氟嘧啶、和奥沙利铂联合***的方法，所述的肿瘤是表达密蛋白18A2的肿瘤，特别是胃癌、胰腺癌、食道癌、肺癌的治疗。
本发明所述抗体是与密蛋白18A2(CLDN18A2)上存在的表位特异性结合的抗体，包含多克隆抗体和单克隆抗体，优选单克隆抗体。本发明涵盖的单克隆抗体包括IgA、IgG1-4、IgE、IgM和IgD抗体。在一个实施方案中，所述抗体为IgG1抗体。此外，在其它的一些可选方案中，所述抗体也可为IgG3抗体，如为IgG3К或IgG3λ同种型；也可为IgG4抗体，如为IgG4К或IgG4λ同种型；也可为IgA1或IgA2抗体或为IgM抗体。在某些实施方案中,所述CLD18A2抗体施用剂量为20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300/kg/次；每周1、2、3、4、5、6或7次；注射1周，或连续注射2、3、4、5或6周。在某些实施方案中,所述CLD18A2抗体施用剂量为500，525，550，575，600，625，650，675，700，725，750，775，800，825，850，875，900，925，950，975，1000，1050，1100，1150，1200mg/m 2/次；每周1、2、3、4、5或6次；注射1周，或连续注射2、3、4、5或6周。
为了更易于理解本发明，以下定义一些术语。
术语“CLDN18.2”包括同种型、哺乳动物(例如人)的CLDN18.2、人CLDN18.2的翻 译后修饰变体、同工型和种间同源物和包含至少一个与CLDN18.2的共同表位的类似物。CLDN18.2(例如人CLDN18.2)的氨基酸序列是本领域中己知的，如NCBI数据库显示。本文中的术语“CLDN18A2”、“CLDN18.2”和“细胞连接密蛋白18.2”可互换使用，术语“CLDN18A1”、“CLDN18.1”和“细胞连接密蛋白18.1”可互换使用。
本文中的术语“奥沙利铂”，指化合物[(1R,2R)-环己烷-1,2-二胺](乙二酸(2-)-O,O')铂。在某些实施方案中,所述奥沙利铂每次注射1、2、3、4、5、6、7、8、9、10mg/kg/次，每周1、2、3、4、5、6或7次，注射1周，或连续注射2、3、4、5或6周。在某些实施方案中,所述奥沙利铂每次注射50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300mg/m 2/次，每周1、2、3、4、5、6或7次，注射1周，或连续注射2、3、4、5或6周。
本文中的术语“5-氟嘧啶”，指化合物5-氟-1H-嘧啶-2,4-二酮。在某些实施方案中,所述5-氟尿嘧啶或其前药或其活性代谢物施用量为50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70mg/kg/次，每周1、2、3、4、5、6、或7次，注射1周，或连续注射2、3、4、5或6周；在某些实施方案中,所述5-氟尿嘧啶或其前药或其活性代谢物施用量为300、325、350、375、400、425、450、475、500、525、550、575、600、625、650、675、700、725、750、775、800、825、850、875、900mg/m 2/次，每周1、2、3、4、5、6、或7次，注射1周，或连续注射2、3、4、5或6周。在某些实施方案中,所述5-氟尿嘧啶的前药是卡培他滨。在某些实施方案中,所述卡培他滨或其前药或其活性代谢物施用量为600、605、610、615、620、625、630、635、640、645、650、655、660、665、670、675、680、685、690、695、700mg/m 2/次，每周1、2、3、4、5、6、或7次，注射1周，或连续注射2、3、4、5或6周。
本文中的术语“表柔比星”，指化合物10-[(3-氨基-2,3,6-三脱氧-Α-L-阿式-己吡喃糖基)氧]-7,8,9,10-四氢-6,8,11-三羟基-8-(羟乙酰基)-甲氧基-(8S-CIS)-并四苯-5,12-二酮。
术语“抗体”指包含通过二硫键互联的至少两条重(H)链和两条轻(L)链或其抗原结合片段的糖蛋白。术语“抗体”还包括抗体(特别是本文所述抗体)的所有重组形式，例如在原核细胞中表达的抗体，未糖基化的抗体以及与抗原结合的抗体片段和下文所述的衍生物。每条重链由重链可变区(本文缩写为VH)和重链恒定区组成。每条轻链由轻链可变区(本文缩写为VL)和轻链恒定区组成。VH和VL可进一步细分为称为互补性决定区(CDR)的高变区，他们散布在称为构架区(FR)的更保守区域中。每条VH和VL由三个CDR和四个FR组成，从氨基端至羧基端按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫***的多种细胞(如效应细胞)和经典补体***的第一成分(C1q)
本文使用的术语“单克隆抗体”指单分子组成的抗体分子制备物。单克隆抗体显示对特定表位的单结合特异性和亲和性。在一个实施方案中，单克隆抗体通过杂交瘤产生，所述杂交 瘤包括与用生化细胞融合的得自非人动物(例如小鼠)的B细胞。
本文所述的抗体可以为重组抗体，该“重组抗体”包括通过重组手段制备、表达、产生或分离的所有抗体，例如(a)从免疫球蛋白基因为转基因或转染色体的动物(如小鼠)或由其制备的杂交瘤中分离的抗体，(b)从经转化以表达抗体的宿主细胞(如转染瘤)中分离的抗体，(c)从重组的组合抗体文库中分离的抗体，和(d)通过涉及将免疫球蛋白基因序列剪接成DNA序列的任何其他手段制备、表达、产生或分离的抗体。
本文中的术语“人源化抗体”是指其中源自另一种哺乳动物物种(例如小鼠)的种系的CDR序列被移植到人的框架序列上。在人的框架序列和源自另一种哺乳动物物种的种系的CDR序列中也可以进行额外的框架区修饰。
本文中的术语“嵌合抗体”指这样的抗体，其中可变区序列源自一个物种，而恒定区序列源自另一个物种，例如可变区序列源自小鼠抗体，而恒定区序列源自人抗体的抗体。
本文中的术语“Fab”或“Fab区”包括包含VH、CH1、VL和CL免疫球蛋白结构域的多肽。Fab可以指分离的该区域，或者位于全长抗体或抗体片段环境中的该区域。本文中的术语“Fc”或“Fc区”包括包含除第一恒定区免疫球蛋白结构域以外的抗体恒定区的多肽。因而，Fc指IgA、IgD和IgG的后两个恒定区免疫球蛋白结构域，和IgE和IgM的后三个恒定区免疫球蛋白结构域，和这些结构域N端的柔性铰链。对于IgA和IgM，Fc可包括J链。对于IgG，Fc包括免疫球蛋白结构域Cγ2和Cγ3和在Cγ1和Cγ2之间的铰链。虽然Fc区的边界可以改变，但人IgG重链Fc区通常定义为在其羧基端包含残基C226或P230，其中编号是根据Kabat的EU索引。对于人IgG1，Fc在本文定义为包含残基P232至其羧基端，其中编号是根据Kabat中的EU索引。Fc可以指分离的该区域，或者位于Fc多肽，例如抗体，环境中的该区域。
本文中的术语“铰链”或“铰链区”或“抗体铰链区”包括包含在抗体的第一和第二恒定结构域之间的氨基酸的柔性多肽。结构上，IgGCH1结构域中至于EU220位，IgGCH2结构域始于残基EU237位。因而，对于IgG，本文中抗体铰链定义为包括221(IgG1的D221)至231(IgG1的A231)位，其中编号是根据Kabat的EU索引。
本文中使用的术语“变体”包括由于相比亲本抗体至少一个氨基酸修饰，而不同于亲本抗体序列的抗体序列。本文中的变体抗体序列优选的具有与亲本抗体序列至少约80％，优选至少约90％，更优选至少约95％的氨基酸序列同一性。抗体变体可以指抗体本身，包含所述亲本抗体的组合物，或编码其地氨基酸序列。
本文中的术语“氨基酸修饰”包括在多肽序列中的氨基酸取代、***和/或缺失。本文中的“氨基酸取代”或“取代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。例如，取代R94K指94位的精氨酸被赖氨酸替换，此情况为重链可变框架区变体。对于前例，94K表示用赖氨酸取代94位。出于本文的目的，通常通过斜杠分隔多个取代。例如，R94K/L78V指包含取代R94K和L78V的双变体。本文中使用的“氨基酸***”或“***”意指在亲本多肽序列中的特定位置添加氨基酸。例如，***-94表示在94位的***。本文中使用的“氨基酸缺失”或“缺失”意指去除亲本多肽序列中特定位置上的氨基酸。例如R94-表示删除 94位的精氨酸。
本发明中讨论的所有免疫球蛋白重链恒定区位置，都根据Kabat的EU索引编号(Kabat等人，1991，sequences of proteins of immunological interest,第5版，United States Public Health Service,National Institutes of Health,Bethesda，通过引用全文整合)。“Kabat的EU索引”指人IgG1 EU抗体的残基编号，如Edelman等人，1969，Biochemistry 63：78-85所述。
本文中使用的术语“全长抗体”包括构成抗体的天然生物学形态的结构，包括可变区和恒定区。例如，在大部分哺乳动物包括人和小鼠中，IgG类的全长抗体是四聚体，由两对相同的成对免疫球蛋白链组成，每对都具有一条轻链和一条重链，每条轻链都包含免疫球蛋白结构域VL和CL，每条重链都包含免疫球蛋白结构域VH、CH1(Cγ1)、CH2(Cγ2)和CH3(Cγ3)。在一些哺乳动物中，例如骆驼和美洲驼，IgG抗体可以只由两条重链组成，每条重链都包含于Fc区连接的可变结构域。
抗体片段包括但不限于：(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点；(vii)双特异性单链Fv二聚体；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段；和(ix)与相同或不同抗体遗传融合的scFv。
根据恒定区基因测定，将抗体分类，也成为同种型。人的恒定轻链分为К(CК)和λ(Cλ)轻链。重链分为μ、δ、γ、α或ε，并分别定义抗体的同种型IgM、IgD、IgG、IgA和IgE。IgG类是常用于治疗目的的。在人中，该类别包括亚类IgG1、IgG2、IgG3和IgG4。在小鼠中，该类别包括亚类IgG1、IgG2a、IgG2b和IgG3。IgM具有亚类，包括但不限于IgM1和IgM2。IgA具有若干亚类，包括但不限于IgA1和IgA2。因而，本文中使用的“同种型”意指通过恒定区的化学和抗原特征定义的免疫球蛋白的任何类或亚类。已知的人免疫球蛋白同种型是IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgM1、IgM2、IgD和IgE。
本文中使用的术语“ADCC”或“抗体依赖性细胞介导的细胞毒性”包括细胞介导的反应，其中表达FcγR的非特异性细胞毒性细胞识别靶细胞上结合的抗体，之后导致靶细胞的裂解。在各个方面，增强ADCC效应子功能可以意指增强的效价或增强的功效。实验背景中使用的“效价”意指当观察特定的治疗功效EC50时抗体的浓度(半大有效浓度)。实验背景中使用的“功效”意指饱和水平的抗体的大可能的效应子功能。
本文中使用的术语“ADCP”或“抗体依赖性细胞介导的吞噬作用”包括细胞
介导的反应，其中表达FcγR的非特异性细胞毒性细胞识别靶细胞上结合的抗体，之后导致靶细胞的吞噬作用。
本文中使用的术语“CDC”或“补体依赖性细胞毒性”包括这样的反应，其中一种或多种补体蛋白质组分识别靶细胞上结合的抗体，之后导致靶细胞的裂解。
本文中使用的术语“效应子功能”包括由抗体Fc区与Fc受体或配体相互作用导致的生物化学事件。效应子功能包括FcγR介导的效应子功能，例如ADCC和ADCP，和补体介导的效应子功能，例如CDC。
在本文中，体表面积公式的选取可以采用许文生氏公式：体表面积(m2)＝0.0061×身高(cm)+0.0128×体重(kg)-0.1529。例：某人身高168cm，体重55kg，计算其体表面积，解：0.0061×168+0.0128×55-0.1529＝1.576m2男女体表面积计算公式分别为：S男＝0.0057×身高+0.0121×体重+0.0882，S女＝0.0073×身高+0.0127×体重-0.2106。
抗密蛋白18A2的抗体
本发明所列出的抗体的重链和轻链可变区序列各自可以结合人密蛋白18A2，可以“混合和匹配”重链和轻链可变区序列来产生本发明的抗人密蛋白18A2的结合分子。
本发明也提供了结合人密蛋白18A2的抗体或其片段的变体。因而，本发明也包括这样的抗体或其片段，所述抗体具有的重链或轻链可变区序列与本发明具体公开的抗体的重链和/或轻链可变区序列有至少80％的氨基酸序列同一性。优选的，所述氨基酸序列同一性是至少85％，更优选至少90％，优选至少95％，特别是96％，更特别97％，甚至更特别98％，特别99％，包括例如80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％和100％。本文中关于氨基酸序列的同一性或序列中与结合人密蛋白18A2的人源化抗体或其片段相同的氨基酸残基的百分比。因而，可以通过通常用于比较两条多肽的氨基酸位置相似性的标准方法，来确定序列同一性。使用计算机程序例如BLAST或FASTA，比对两条多肽各自氨基酸的佳匹配(沿一条或两条序列的全长或者沿一条或两条序列预定的部分)。程序提供了默认的开放罚分和默认的空位罚分，评分矩阵例如PAM250可与计算机程序结合使用。例如，百分比同一性可计算为：相同匹配的总数乘以100，再除以匹配跨度中较长序列的总长度和为了比对两条序列向较长序列中倒入的空位数。
在某些实施方案中，框架序列可用于改造可变区，产生变体抗体。本发明的变体抗体包括对VH和/或VK中的框架区残基进行了修饰的变体，例如改善抗体的特性。通常，进行此类框架区修饰来减少抗体的免疫原性。例如，一种方法是“回复突变”一个或多个框架区残基为相应的鼠序列，或者“回复突变”一个或多个框架区残基为相应的种系序列。
因而在另一个方面，本发明提供结合人密蛋白18A2的人源化抗体或其片段，其中所述人源化抗体或其片段的至少一个重链可变区的框架区包括来自相应鼠抗体的重链可变区的相应框架区的至少一个氨基酸修饰。优选的，所述氨基酸修饰是氨基酸取代。通常，在框架区内实施不超过5个，优选不超过4个，更优选不超过3个，甚至更优选不超过2个，优选不超过1个的氨基酸修饰。
本发明还提供了结合人密蛋白18A2的人源化抗体及其片段，其还包括人重链和/或轻链恒定结构域。人重链恒定区可选自IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgM1、IgM2、IgD和IgE构成的人免疫球蛋白的组，而人重链恒定区IgG，特别是IgG1是优选。人轻链恒定区可选自К或λ恒定区构成的人免疫球蛋白的组，而人К恒定区是优选的。在一些优选的实施方案中，所述人源化抗体或其片段包括人IgG1重链恒定结构域和人轻链K恒定结构域。本发明还提供了结合人密蛋白18A2的人源化抗体或其片段，其包括人重链和/或轻链恒定区，其中人重链恒定区包括人IgG1的CH1、人IgG1的铰链区和人IgG3的Fc区的同种 型变体。优选的包含同种型变体的人源化抗体是全长抗体。对于包括包含人IgG1的人源化抗体或其片段是包括包含SEQIDNO:12、14、15、55或57所示的重链可变区序列，和包含SEQIDNO:11、13、56、或58所示的的轻链可变区的序列。优选SEQIDNO:12所示的重链可变区和SEQIDNO:11所示的轻链可变区的组合；或者SEQIDNO:14或15的所示的重链可变区序列和SEQIDNO:13所示的轻链可变区序列的组合；或者SEQIDNO:55所示的重链可变区的序列和SEQIDNO:56所示的轻链可变区的序列的组合。
效应子功能通常是补体依赖性细胞毒性(CDC)和/或C1q结合和/或抗体依赖性细胞介导的细胞毒性(ADCC)和/或抗体对Fcγ受体的结合亲和力，优选补体依赖性细胞毒性(CDC)和/或抗体依赖性细胞介导的细胞毒性(ADCC)。通过标准的体外测定测量CDC、C1q结合、ADCC和抗体对Fcγ受体的结合亲和力，所述测定是本领域已知的和可商购的。通常，通过乳酸脱氢酶(LDH)释放测定测量ADCC，通过给予细胞的测定测量CDC。
评估抗体例如人密蛋白18A2的抗体的结合能力的标准测定是本领域已知的，包括例如ELISA、Western印迹和流式细胞仪分析。合适的测定详细描述在实施例中。为了评估结合，可以使用稳定转染了CLD18A2的293T细胞，稳定转染了CLD18A2的AGS细胞，稳定转染了CLD18A2的NCI-N87细胞，稳定转染了CLD18A2的BGC细胞，并应用流式细胞分析测定EC50。
以下将参考附图，对本发明的优选实施例进行详细描述。所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，属于本发明的保护范围。
实施例1抗体的制备
本实施例选择的靶向CLD18A2的抗体为人源化的抗体，具有SEQ ID NO：15所示的重链可变区和SEQ ID NO：13所示的轻链可变区，轻链的核苷酸序列如SEQ ID NO:60所示，重链核苷酸序列如SEQ ID NO:59所示。
轻链及重链可变区的扩增按照Matz等(Nucleic AcidsResearch，1999，笫27卷，No.6)描述的“step-outPCR”法进行。通过本领域技术人员已知的标准方法，将轻链的核苷酸(序列如SEQ ID NO:60所示)和重链的核苷酸(序列如SEQ ID NO:59所示)克隆至真核表达载体中。采用293fectin TMTransfection reagent(Invitrogen，12347-019)或者聚乙烯亚胺(PEI)(Sigma-Aldrich，408727)瞬时转染对数生长期的293F细胞。转染5-7天后收集培养上清通过Protein A进行亲和纯化，获得CLD18A2的抗体。通过SDS PAGE对得到的抗体进行定量和定性分析。上述真核表达载体采用CN101602808B所采用的载体pH或载体pK。
实施例2小鼠PDX模型皮下移植瘤的体内药效实验
将2×2×2mm大小的胃癌PDX瘤块接种于BALB/c小鼠上(接种日为D0)，皮下接种瘤块D27天，肿瘤体积平均为128mm 3，即得荷人胃癌PDX-GA0006模型，或简称PDX模型。将成瘤小鼠随机分为6组，分别是溶媒组、抗体组(Ab组)、EOF组、EOF+Ab组、OF组、OF+Ab组，实验过程中观察动物一般临床症状，每周进行2次体重和瘤径测量，D51天，对小鼠实施安乐死。给药方式如下：
溶媒组：腹腔注射，每周3次。
Ab组：实施例1制备的CLD18A2抗体，40mg/kg，腹腔注射，每周3次。
EOF组：腹腔注射用盐酸表柔比星(1.25mg/kg)+注射用奥沙利铂(3.25mg/kg)+氟尿嘧啶注射液(56.25mg/kg)联合用药，每周1次；溶媒，腹腔注射，每周3次。
EOF+Ab组：EOF(盐酸表柔比星(1.25mg/kg)+注射用奥沙利铂(3.25mg/kg)+氟尿嘧啶注射液(56.25mg/kg))每周给予1次，CLD18A2抗体(40mg/kg)每周3次。
OF组：注射用奥沙利铂(3.25mg/kg)+氟尿嘧啶注射液(56.25mg/kg)联合用药，每周1次；溶媒，腹腔注射，每周3次。
OF+Ab组：OF(注射用奥沙利铂(3.25mg/kg)+氟尿嘧啶注射液(56.25mg/kg))每周给予1次，CLD18A2抗体(40mg/kg)每周3次。
肿瘤体积增长结果如图1所示，OF+Ab组与EOF+Ab组相比，表柔比星未使用时，抗肿瘤作用大大增强。小鼠体重情况如图2所示，OF+Ab组的小鼠体重略优于EOF+Ab组。
小鼠安乐死后获取肿瘤，称量肿瘤重量。结果如图3、4所示，溶媒组小鼠肿瘤负荷太大，肿瘤接种51天时小鼠死亡1只，相对于溶媒组，各组抑瘤率分别为：Ab组，1.21％；EOF组，48.70％；EOF+Ab组，69.46％；OF组，71.64％；OF+Ab组，96.86％，且本组5只小鼠中有1只小鼠出现肿瘤消退(*p<0.05；**p<0.01或***p<0.001，One wayANOVA)。
实施例3抗体的给药量分析
参照实施例2的操作，分别给予成瘤小鼠不同剂量的抗体，给药方式具体如下：
1、AB1，给予实施例1制备的CLD18A2抗体，给药剂量40mg/kg，腹腔注射，每周3次，共2周。
2、AB2，给予实施例1制备的CLD18A2抗体，给药剂量70mg/kg，腹腔注射，每周3次，共2周。
3、AB3，给予实施例1制备的CLD18A2抗体，给药剂量100mg/kg，腹腔注射，每周3次，共2周。
肿瘤体积增长结果如图5所示，显示AB3组的给药具有更优的抗肿瘤效果，小鼠的体重变化图见图6所示，体重无显著性变化，说明给予大剂量的CLD18A2抗体起到更优的抗肿瘤作用，且副作用不大。
在上述实施例中，示例性的，采用了人源化的抗体CLD18A2的一种抗体，本领域技术人员可以依据本申请的教导，采用其他的抗体，如含有SEQ ID NO:12所示的重链可变区和SEQ ID NO:11所示的轻链可变区的抗体、或者含有SEQ ID NO:15所示的重链可变区和SEQ ID NO:13所示的轻链可变区的抗体、或者含有SEQ ID NO:55所示的重链可变区和SEQ ID NO:56所示的轻链可变区的抗体，或者含有SEQ ID NO:57所示的重链可变区和SEQ ID NO:58所示的轻链可变区的抗体。
本发明中涉及的序列如下表所示：
Figure PCTCN2019109214-appb-000001
Figure PCTCN2019109214-appb-000002
Figure PCTCN2019109214-appb-000003
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (37)

  1. 治疗CLDN18阳性的肿瘤的方法，其特征在于，给予有需要的个体特异性识别CLDN18的抗体及化疗药物，所述化疗药物为：
    5-氟尿嘧啶或其前药或其活性代谢物、及奥沙利铂或其前药或其活性代谢物；或，5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物、及紫衫烷类药物；
    优选地，所述化疗药物为：
    5-氟尿嘧啶或其前药、及奥沙利铂或其前药；或，5-氟尿嘧啶或其前药、奥沙利铂或其前药、及紫衫烷类药物。
    更优选地，所述5-氟尿嘧啶的前药是卡培他滨。
  2. 根据权利要求1所述的方法，其特征在于，所述抗体特异性识别CLDN18.2；优选地，所述抗体不识别CLDN18.1。
  3. 根据权利要求1或2所述的方法，其特征在于，所述化疗药物和所述CLDN18的抗体的给予时间不分先后。
  4. 根据权利要求3所述的方法，其特征在于，先给予所述化疗药物，再给予所述CLDN18的抗体。
  5. 根据权利要求4所述的方法，其特征在于，所述化疗药物和所述CLDN18的抗体在同一天给予；或，给予所述化疗药物至少一天后，再给予所述CLDN18的抗体。
  6. 根据权利要求1-5任一项所述的方法，其特征在于，所述CLDN18的抗体的施用剂量为500～1200mg/m 2/次；优选地，为500-1000mg/m 2/次；优选的，为500-900mg/m 2/次；更优选地，为600-800mg/m 2/次。
  7. 根据权利要求1-5任一项所述的方法，其特征在于，所述CLDN18的抗体的施用剂量为20～300mg/kg/次；优选地，为20～100mg/kg/次；更优选地，为20-50mg/kg/次。
  8. 根据权利要求1-5任一项所述的方法，其特征在于，所述5-氟尿嘧啶或其前药或其活性代谢物的施用剂量为300～900mg/m 2/次；
    优选地，给予5-氟尿嘧啶，施用量为300-800mg/m 2/次；或，给予卡培他滨600-700mg/m 2/次，更优选为625mg/m 2/次。
  9. 根据权利要求1-5任一项所述的方法，其特征在于，所述5-氟尿嘧啶或其前药或其活性代谢物的施用剂量为50～70mg/kg/次；
    优选地，给予5-氟尿嘧啶，施用量为50～70mg/kg/次；
    更优选地，5-氟尿嘧啶的施用量为50～60mg/kg/次。
  10. 根据权利要求1-5任一项所述的方法，其特征在于，所述奥沙利铂或其前药或其活性代谢物的施用剂量为50～300mg/m 2/次；
    优选地，给予奥沙利铂100～200mg/m 2/次；更优选地，给予奥沙利铂130mg/m 2/次。
  11. 根据权利要求1-5任一项所述的方法，其特征在于，所述奥沙利铂或其前药或其活性代谢物的施用剂量为1～10mg/kg/次；优选地，给予奥沙利铂1～5mg/kg/次。
  12. 根据权利要求1-5任一项所述的方法，其特征在于，所述抗体为人源化的抗体或嵌合抗体。
  13. 根据权利要求1-5任一项所述的方法，其特征在于，所述特异性识别CLDN18的抗体包含：
    如SEQ ID NO:16的氨基酸序列所示的HCDR1、SEQ ID NO:17、40或41的氨基酸序列所示的HCDR2、SEQ ID NO:18的氨基酸序列所示的HCDR3，和SEQ ID NO:19的氨基酸序列所示的LCDR1、SEQ ID NO:20的氨基酸序列所示的LCDR2、SEQ ID NO:21的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:34的氨基酸序列所示的HCDR1、SEQ ID NO:35或42的氨基酸序列所示的HCDR2、SEQ ID NO:36的氨基酸序列所示的HCDR3，和SEQ ID NO:37的氨基酸序列所示的LCDR1、SEQ ID NO:38的氨基酸序列所示的LCDR2、SEQ ID NO:39的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:22的氨基酸序列所示的HCDR1、SEQ ID NO:23的氨基酸序列所示的HCDR2、SEQ ID NO:24的氨基酸序列所示的HCDR3，和SEQ ID NO:25的氨基酸序列所示的LCDR1、SEQ ID NO:26的氨基酸序列所示的LCDR2、SEQ ID NO:27的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:28的氨基酸序列所示的HCDR1、SEQ ID NO:29的氨基酸序列所示的HCDR2、SEQ ID NO:30的氨基酸序列所示的HCDR3，和SEQ ID NO:31的氨基酸序列所示的LCDR1、SEQ ID NO:32的氨基酸序列所示的LCDR2、SEQ ID NO:33的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:43的氨基酸序列所示的HCDR1、SEQ ID NO:44的氨基酸序列所示的HCDR2、SEQ ID NO:45的氨基酸序列所示的HCDR3，和SEQ ID NO:46的氨基酸序列所示的LCDR1、SEQ ID NO:47的氨基酸序列所示的LCDR2、SEQ ID NO:48的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:49的氨基酸序列所示的HCDR1、SEQ ID NO:50的氨基酸序列所示的HCDR2、SEQ ID NO:51的氨基酸序列所示的HCDR3，和SEQ ID NO:52的氨基酸序列所示的LCDR1、SEQ ID NO:53的氨基酸序列所示的LCDR2、SEQ ID NO:54的氨基酸序列所示的LCDR3；
    优选地，所述特异性识别CLDN18的抗体包含有：
    SEQ ID NO:16的氨基酸序列所示的HCDR1、SEQ ID NO:17或41的氨基酸序列所示的HCDR2、SEQ ID NO:18的氨基酸序列所示的HCDR3，和SEQ ID NO:19的氨基酸序列所示的LCDR1、SEQ ID NO:20的氨基酸序列所示的LCDR2、SEQ ID NO:21的氨基酸序列所示的LCDR3；或者
    SEQ ID NO:34的氨基酸序列所示的HCDR1、SEQ ID NO:35或42的氨基酸序列所示的HCDR2、SEQ ID NO:36的氨基酸序列所示的HCDR3，和SEQ ID NO:37的氨基酸序列所示的LCDR1、SEQ ID NO:38的氨基酸序列所示的LCDR2、SEQ ID NO:39的氨基酸序列所示 的LCDR3；或者
    如SEQ ID NO:43的氨基酸序列所示的HCDR1、SEQ ID NO:44的氨基酸序列所示的HCDR2、SEQ ID NO:45的氨基酸序列所示的HCDR3，和SEQ ID NO:46的氨基酸序列所示的LCDR1、SEQ ID NO:47的氨基酸序列所示的LCDR2、SEQ ID NO:48的氨基酸序列所示的LCDR3。
  14. 根据权利要求13所述的方法，其特征在于，所述特异性识别CLDN18的抗体包含：
    如SEQ ID NO:2、9或10的氨基酸序列所示的重链可变区和SEQ ID NO:1的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:8的氨基酸序列所示的重链可变区和SEQ ID NO:7的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:14或15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:4的氨基酸序列所示的重链可变区和SEQ ID NO:3的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:6的氨基酸序列所示的重链可变区和SEQ ID NO:5的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:57的氨基酸序列所示的重链可变区和SEQ ID NO:58的氨基酸序列所示的轻链可变区；
    优选地，所述特异性识别CLDN18的抗体包含：
    如SEQ ID NO:2或10的氨基酸序列所示的重链可变区和SEQ ID NO:1的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:8的氨基酸序列所示的重链可变区和SEQ ID NO:7的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:14或15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区。
    更优地，所述抗体含有：
    如SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区。
  15. 根据权利要求14所述的方法，其特征在于，所述的抗体选自：
    (a)具有SEQ ID NO:59所示的核苷酸序列编码的重链和SEQ ID NO:60所示的核苷酸序列编码的轻链；或，
    (b)具有SEQ ID NO:64所示的重链氨基酸序列和SEQ ID NO:63所示的轻链氨基酸序列。
  16. 根据权利要求1所述的方法，其特征在于，所述的紫衫烷类药物选自紫杉醇、白蛋白结合型紫杉醇和多西他赛；优选的，为白蛋白结合型紫杉醇。
  17. 根据权利要求1-16任一项所述的方法，其特征在于，所述CLDN18阳性的肿瘤包括：胃癌、胰腺癌、食管癌和肺癌；优选的，为胃癌。
  18. 一种治疗CLDN18阳性的肿瘤的药盒组合，其特征在于，所述药盒组合包括药盒A和药盒B，所述药盒A包括特异性识别CLDN18的抗体，所述药盒B包括化疗药物，所述化疗药物为：5-氟尿嘧啶或其前药或其活性代谢物、及奥沙利铂或其前药或其活性代谢物；或，5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物、及紫衫烷类药物；
    优选地，所述化疗药物为：
    5-氟尿嘧啶或其前药、及奥沙利铂或其前药；或，5-氟尿嘧啶或其前药、奥沙利铂或其前药、及紫衫烷类药物。
    更优选地，所述5-氟尿嘧啶的前药是卡培他滨。
  19. 根据权利要求18所述的药盒组合，其特征在于，所述药盒A的特异性识别CLDN18的抗体特异性识别CLDN18.2；优选地，所述特异性识别CLDN18的抗体不识别CLDN18.1。
  20. 根据权利要求18或19所述的药盒组合，其特征在于，所述药盒A和所述药盒B的施用时间不分先后。
  21. 根据权利要求20所述的药盒组合，其特征在于，先施用所述药盒B，再施用所述药盒A。
  22. 根据权利要求21所述的药盒组合，其特征在于，所述药盒A和所述药盒B在同一天施用；或，施用所述药盒B至少一天后，再施用所述药盒A。
  23. 根据权利要求18-22任一项所述的药盒组合，其特征在于，所述药盒A的施用剂量为500～1200mg/m 2/次；优选地，为500-1000mg/m 2/次；优选的，为500-900mg/m 2/次；更优选地，为600-800mg/m 2/次。
  24. 根据权利要求18-22任一项所述的药盒组合，其特征在于，所述药盒A的施用剂量为20～300mg/kg/次；优选地，为20～100mg/kg/次；更优选地，为20-50mg/kg/次。
  25. 根据权利要求18-22任一项所述的药盒组合，其特征在于，所述5-氟尿嘧啶或其前 药或其活性代谢物的施用剂量为300～900mg/m 2/次；
    优选地，施用5-氟尿嘧啶，施用剂量为300-800mg/m 2/次；或，施用卡培他滨，施用剂量为600-700mg/m 2/次，更优选为625mg/m 2/次。
  26. 根据权利要求18-22任一项所述的药盒组合，其特征在于，所述5-氟尿嘧啶或其前药或其活性代谢物的施用剂量为50～70mg/kg/次；
    优选地，施用5-氟尿嘧啶，施用剂量为50～70mg/kg/次；
    更优选地，施用5-氟尿嘧啶，施用剂量为50～60mg/kg/次。
  27. 根据权利要求18-22任一项所述的药盒组合，其特征在于，所述奥沙利铂或其前药或其活性代谢物的施用剂量为50～300mg/m 2/次；
    优选地，施用奥沙利铂，施用剂量为100～200mg/m 2/次；更优选地，施用奥沙利铂，施用剂量为130mg/m 2/次。
  28. 根据权利要求18-22任一项所述的药盒组合，其特征在于，所述奥沙利铂或其前药或其活性代谢物的施用剂量为1～10mg/kg/次；优选地，施用奥沙利铂，施用剂量为1～5mg/kg/次。
  29. 根据权利要求18-22任一项所述的药盒组合，其特征在于，所述特异性识别CLDN18的抗体为人源化的抗体或嵌合抗体。
  30. 根据权利要求18-22任一项所述的药盒组合，其特征在于，所述特异性识别CLDN18的抗体包含有：
    如SEQ ID NO:16的氨基酸序列所示的HCDR1、SEQ ID NO:17、40或41的氨基酸序列所示的HCDR2、SEQ ID NO:18的氨基酸序列所示的HCDR3，和SEQ ID NO:19的氨基酸序列所示的LCDR1、SEQ ID NO:20的氨基酸序列所示的LCDR2、SEQ ID NO:21的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:34的氨基酸序列所示的HCDR1、SEQ ID NO:35或42的氨基酸序列所示的HCDR2、SEQ ID NO:36的氨基酸序列所示的HCDR3，和SEQ ID NO:37的氨基酸序列所示的LCDR1、SEQ ID NO:38的氨基酸序列所示的LCDR2、SEQ ID NO:39的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:22的氨基酸序列所示的HCDR1、SEQ ID NO:23的氨基酸序列所示的HCDR2、SEQ ID NO:24的氨基酸序列所示的HCDR3，和SEQ ID NO:25的氨基酸序列所示的LCDR1、SEQ ID NO:26的氨基酸序列所示的LCDR2、SEQ ID NO:27的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:28的氨基酸序列所示的HCDR1、SEQ ID NO:29的氨基酸序列所示的HCDR2、SEQ ID NO:30的氨基酸序列所示的HCDR3，和SEQ ID NO:31的氨基酸序列所示的LCDR1、SEQ ID NO:32的氨基酸序列所示的LCDR2、SEQ ID NO:33的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:43的氨基酸序列所示的HCDR1、SEQ ID NO:44的氨基酸序列所示的HCDR2、SEQ ID NO:45的氨基酸序列所示的HCDR3，和SEQ ID NO:46的氨基酸序列所示 的LCDR1、SEQ ID NO:47的氨基酸序列所示的LCDR2、SEQ ID NO:48的氨基酸序列所示的LCDR3；或，
    如SEQ ID NO:49的氨基酸序列所示的HCDR1、SEQ ID NO:50的氨基酸序列所示的HCDR2、SEQ ID NO:51的氨基酸序列所示的HCDR3，和SEQ ID NO:52的氨基酸序列所示的LCDR1、SEQ ID NO:53的氨基酸序列所示的LCDR2、SEQ ID NO:54的氨基酸序列所示的LCDR3；
    优选地，所述特异性识别CLDN18的抗体包含有：
    SEQ ID NO:16的氨基酸序列所示的HCDR1、SEQ ID NO:17或41的氨基酸序列所示的HCDR2、SEQ ID NO:18的氨基酸序列所示的HCDR3，和SEQ ID NO:19的氨基酸序列所示的LCDR1、SEQ ID NO:20的氨基酸序列所示的LCDR2、SEQ ID NO:21的氨基酸序列所示的LCDR3；或者
    SEQ ID NO:34的氨基酸序列所示的HCDR1、SEQ ID NO:35或42的氨基酸序列所示的HCDR2、SEQ ID NO:36的氨基酸序列所示的HCDR3，和SEQ ID NO:37的氨基酸序列所示的LCDR1、SEQ ID NO:38的氨基酸序列所示的LCDR2、SEQ ID NO:39的氨基酸序列所示的LCDR3。
  31. 根据权利要求30所述的药盒组合，其特征在于，所述特异性识别CLDN18的抗体含有：
    如SEQ ID NO:2、9或10的氨基酸序列所示的重链可变区和SEQ ID NO:1的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:8的氨基酸序列所示的重链可变区和SEQ ID NO:7的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:14或15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:4的氨基酸序列所示的重链可变区和SEQ ID NO:3的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:6的氨基酸序列所示的重链可变区和SEQ ID NO:5的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区；或，
    如SEQ ID NO:57的氨基酸序列所示的重链可变区和SEQ ID NO:58的氨基酸序列所示的轻链可变区；
    优选地，所述特异性识别CLDN18的抗体包含：
    如SEQ ID NO:2或10的氨基酸序列所示的重链可变区和SEQ ID NO:1的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:8的氨基酸序列所示的重链可变区和SEQ ID NO:7的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:14或15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区。
    更优地，所述抗体含有：
    如SEQ ID NO:12的氨基酸序列所示的重链可变区和SEQ ID NO:11的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:15的氨基酸序列所示的重链可变区和SEQ ID NO:13的氨基酸序列所示的轻链可变区；或
    如SEQ ID NO:55的氨基酸序列所示的重链可变区和SEQ ID NO:56的氨基酸序列所示的轻链可变区。
  32. 根据权利要求31所述的药盒组合，其特征在于，所述的抗体选自：
    (a)具有SEQ ID NO:59所示的核苷酸序列编码的重链和SEQ ID NO:60所示的核苷酸序列编码的轻链；或，
    (b)具有SEQ ID NO:64的氨基酸序列所示的重链和SEQ ID NO:63的氨基酸序列所示的轻链。
  33. 根据权利要求18所述的药盒组合，其特征在于，所述药盒B的紫衫烷类药物选自紫杉醇、白蛋白结合型紫杉醇和多西他赛；优选的，为白蛋白结合型紫杉醇。
  34. 根据权利要求18-33任一项所述的药盒组合，其特征在于，所述CLDN18阳性的肿瘤包括：胃癌、胰腺癌、食管癌和肺癌。
  35. 一种治疗CLDN18阳性的肿瘤的药物组合物，其特征在于，所述药物组合物包括特异性识别CLDN18的抗体及化疗药物，所述化疗药物为前述任一项所述的5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物；或，5-氟尿嘧啶或其前药或其活性代谢物、奥沙利铂或其前药或其活性代谢物、及紫衫烷类药物；所述特异性识别CLDN18的抗体为前述任一项所述的抗体。
  36. 根据权利要求18-34任一项所述的药盒组合或权利要求35所述的药物组合物在制备治疗CLDN18阳性的肿瘤的药物中的应用。
  37. 根据权利要求18-34任一项所述的药盒组合或权利要求35所述的药物组合物，用于治疗CLDN18阳性肿瘤。
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CN112888458A (zh) 2021-06-01

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