WO2024183791A1 - 包含抗ctla4-抗pd-1双特异性抗体的药物组合及其用途 - Google Patents

包含抗ctla4-抗pd-1双特异性抗体的药物组合及其用途 Download PDF

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WO2024183791A1
WO2024183791A1 PCT/CN2024/080526 CN2024080526W WO2024183791A1 WO 2024183791 A1 WO2024183791 A1 WO 2024183791A1 CN 2024080526 W CN2024080526 W CN 2024080526W WO 2024183791 A1 WO2024183791 A1 WO 2024183791A1
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seq
variable region
amino acid
chain variable
acid sequence
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夏瑜
王忠民
李百勇
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Akeso Pharmaceuticals Inc
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Akeso Pharmaceuticals Inc
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Priority to AU2024232344A priority Critical patent/AU2024232344A1/en
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Priority to EP24766505.2A priority patent/EP4678186A1/en
Priority to JP2025552130A priority patent/JP2026507907A/ja
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    • 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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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • 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
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    • A—HUMAN NECESSITIES
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    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
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    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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 present invention belongs to the field of tumor treatment and molecular immunology, and relates to a drug combination comprising an anti-CTLA4-anti-PD-1 bispecific antibody and its use. Specifically, the present invention relates to a drug combination comprising an anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine and paclitaxel (e.g., albumin paclitaxel) and its use.
  • paclitaxel e.g., albumin paclitaxel
  • the transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 gene family and is expressed in activated T cells, B cells, and myeloid cells.
  • PDL1 is expressed in a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is only expressed in antigen presenting cells such as dendritic cells and macrophages.
  • the PD-1/PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune escape.
  • PD-1 is mainly expressed in immune cells such as T cells, and the ligand PDL1 of PD-1 is highly expressed in many human tumor tissues.
  • Blocking the PD-1/PDL1 signaling pathway can activate suppressed T cells and attack cancer cells. Blocking the PD-1/PDL1 signal can promote the proliferation of tumor antigen-specific T cells, play a role in killing tumor cells, and inhibit local tumor growth (Julie R et al., 2012, N Engl J Med. 366: 2455-2465).
  • Cytotoxic T lymphocyte associated antigen 4 CTLA4 (also referred to as CTLA4) has a very similar relationship with CD28 molecules in gene structure, chromosome location, sequence homology and gene expression. Both are receptors for the costimulatory molecule B7 and are mainly expressed on the surface of activated T cells. After binding to B7, CTLA4 can inhibit the activation of mouse and human T cells and play a negative regulatory role in T cell activation.
  • CTLA4 antibodies (or anti-CTLA4 monoclonal antibodies) or CTLA4 ligands can prevent CTLA4 from binding to its natural ligand, thereby blocking the conduction of CTLA4 negative regulatory signals to T cells and enhancing the responsiveness of T cells to various antigens.
  • CTLA4 monoclonal antibodies are in clinical trials or approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN., 2013, Cancer Immun. 13:5.).
  • Interleukin 2 is produced by T cells. It is a growth factor that regulates T cell subsets and an important factor in regulating immune responses. It can promote the proliferation of activated B cells and participate in antibody response, hematopoiesis and tumor surveillance. Recombinant human IL-2 has been approved by the US FDA for the treatment of malignant tumors (including melanoma, renal tumors, etc.), and is currently undergoing clinical research on the treatment of chronic viral infections (Chavez, A.R., et al., 2009, Ann N Y Acad Sci, 1182: p.14-27). CTLA4 and CTLA4 antibodies are important factors affecting the function of T cells, and they intervene in the body's immune microenvironment. In in vitro and in vivo experiments, CTLA4 antibodies can specifically relieve CTLA4's immune suppression on the body, activate T cells, and induce IL-2 production. They have broad application prospects in gene therapy for anti-tumor and parasitic diseases.
  • CTLA4 antibodies can have a specific therapeutic effect on the disease and exert a high efficacy, supplementing the deficiencies of traditional medications and thus opening up a new avenue for gene therapy.
  • Bispecific antibodies also known as bifunctional antibodies, are specific drugs that target two different antigens at the same time. They can be produced by immunoprecipitation and purification. In addition, they can also be obtained through genetic engineering. Genetic engineering methods have certain advantages because they have corresponding flexibility in terms of binding site optimization, synthetic form considerations, and yield. At present, more than 45 forms of bispecific antibodies have been proven (Dafne Müller, Kontermann R E. 2010, BioDrugs, 24 (2): 89-98). Many bispecific antibodies that have been developed are in the form of IgG-scFv, namely the Morrison model (Coloma MJ, Morrison SL. 1997, Nat Biotechnol. 15: 159-163).
  • Gemcitabine is a commonly used first-line chemotherapy drug for the treatment of many solid tumors such as non-small cell lung cancer, pancreatic cancer, breast cancer, bladder cancer, etc. It can also be used to treat malignant lymphoma, ovarian cancer, and nasopharyngeal carcinoma.
  • Glycoside anti-metabolism anti-tumor drugs inhibit the synthesis of deoxynucleic acid, interfere with cell DNA replication and transcription, and thus inhibit the proliferation and metastasis of tumor cells.
  • the effect of gemcitabine alone in treating cancer is limited. The most significant disadvantage is its short half-life. It must be continuously administered intravenously in large doses to maintain effective concentrations, which is bound to cause more serious toxic side effects and significantly reduce the quality of life of patients.
  • the chemical structure of gemcitabine is shown in Formula I below.
  • Gemcitabine hydrochloride is commonly used clinically, and its structural formula is shown in Formula II below.
  • Paclitaxel is a natural plant-based anti-tumor drug. It is a monomeric diterpenoid compound extracted from the bark of Pacific yew by the National Cancer Institute (NCI) of the United States in the late 1960s. It is hailed as one of the three major achievements in anti-tumor drugs in the 1990s and is widely used in the treatment of cancers such as ovarian cancer, breast cancer, colorectal cancer, non-small cell lung cancer, or carboxygenic malignancies (Anil K. Singla, et al. (2002) Int. J. Pharm. 235: 179-192). The structural formula of paclitaxel is shown in Formula III below.
  • Albumin-bound paclitaxel (also referred to as albumin paclitaxel in the present invention) is a new type of chemotherapy drug formed by combining paclitaxel with albumin. Compared with traditional solvent-based paclitaxel, it has better efficacy, good solubility, and is more convenient in clinical application. Albumin-bound paclitaxel can increase the drug concentration outside the tumor through the albumin receptor (Gp60) transcellular pathway and the cysteine-rich acidic secretory protein (SPARC) pathway in the extracellular stroma of tumor cells, thereby improving the anti-tumor activity.
  • Gp60 albumin receptor
  • SPARC cysteine-rich acidic secretory protein
  • the inventors creatively combined anti-CTLA4-anti-PD-1 antibodies with gemcitabine and albumin-paclitaxel appropriately, and the resulting drug combination has a good anti-tumor effect.
  • One aspect of the present invention relates to a pharmaceutical combination comprising an anti-CTLA4-anti-PD-1 bispecific antibody and gemcitabine or a pharmaceutically acceptable salt thereof;
  • the drug combination further comprises paclitaxel.
  • the drug combination wherein the amount of the anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof and/or paclitaxel is an effective amount for treating or preventing the indications of the present invention.
  • the drug combination consists of an anti-CTLA4-anti-PD-1 bispecific antibody and gemcitabine or a pharmaceutically acceptable salt thereof.
  • the drug combination consists of an anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
  • the drug combination consists of an anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and paclitaxel.
  • the drug combination consists of an anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, paclitaxel, and one or more pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable salt of gemcitabine is gemcitabine hydrochloride.
  • the paclitaxel in the drug combination, is nab-paclitaxel.
  • the drug combination consists of an anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and albumin-paclitaxel.
  • the drug combination consists of an anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, nab-paclitaxel, and one or more pharmaceutically acceptable excipients.
  • the albumin paclitaxel wherein the mass ratio of paclitaxel to albumin is (1:15) to (5:1), (1:15) to (3:1), (1:15) to (2:1), (1:10) to (3:1), (1:10) to (2:1), 1:(5-15), 1:(8-12), 1:8, 1:9, 1:10, 1:11 or 1:12.
  • the unit dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 100 mg-1000 mg, 200 mg-800 mg, 200 mg-500 mg, 300 mg-600 mg, 400 mg-500 mg or 450 mg.
  • the drug combination wherein the single administration dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 0.1-100 mg per kilogram of body weight, preferably 1-15 mg, 1-12 mg, 1-10 mg Or 6-10mg.
  • the unit dose of gemcitabine or its pharmaceutically acceptable salt is 100-2000 mg, 500-1500 mg, 600-1300 mg, 800-1200 mg, 800-1000 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg or 1500 mg.
  • the drug combination wherein the single administration dose of gemcitabine or its pharmaceutically acceptable salt, calculated as the mass (mg) of gemcitabine or its pharmaceutically acceptable salt/body surface area (m 2 ) of the subject, is 500-1500 mg/m 2 , 600-1300 mg/m 2 , 800-1250 mg/m 2 , 800-1000 mg/m 2 , 800 mg/m 2 , 850 mg/m 2 , 900 mg/m 2 , 950 mg/m 2 , 1000 mg/m 2 , 1050 mg/m 2 , 1100 mg/m 2 , 1150 mg/m 2 , 1200 mg/m 2 or 1250 mg/m 2 .
  • the unit dose of nab paclitaxel (calculated by the mass of nab paclitaxel) is 200-2000 mg, 500-1500 mg, 800-1200 mg, 900-1100 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mg.
  • the unit dose of paclitaxel (calculated by the mass of paclitaxel) is 20-300 mg, 50-250 mg, 80-200 mg, 100-180 mg, 120-180 mg, 125-175 mg, 135-175 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg or 175 mg.
  • the pharmaceutical combination wherein the single administration dose of nab-paclitaxel, calculated by the mass (mg) of nab-paclitaxel/body surface area (m 2 ) of the subject, is 80-300 mg/m 2 , 100-280 mg/m 2 , 120-260 mg/m 2 , 120-200 mg/m 2 , 120-180 mg/m 2 , 125-175 mg/m 2 , 125 mg/m 2 , 130 mg/m 2 , 135 mg/m 2 , 140 mg/m 2 , 145 mg/m 2 , 150 mg/m 2 , 155 mg/m 2 , 160 mg/m 2 , 165 mg/m 2 , 170 mg/m 2 , 175 mg/m 2 or 260 mg/m 2 .
  • the drug combination wherein the single administration dose of paclitaxel, calculated by the mass (mg) of paclitaxel/body surface area (m 2 ) of the subject, is 20-300 mg/m 2 , 50-250 mg/m 2 , 80-200 mg/m 2 , 100-180 mg/m 2 , 120-180 mg/m 2 , 125-175 mg/m 2 , 135-175 mg/m 2 , 125 mg/m 2 , 130 mg/m 2 , 135 mg/m 2 , 140 mg/m 2 , 145 mg/m 2 , 150 mg/m 2 , 155 mg/m 2 , 160 mg/m 2 , 165 mg/m 2 , 170 mg/m 2 or 175 mg / m 2 .
  • the drug combination is a fixed combination, for example, a pharmaceutical composition
  • the drug combination is a non-fixed combination, for example, the anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and paclitaxel in the non-fixed combination are each present in an independent pharmaceutical composition; or
  • Gemcitabine or a pharmaceutically acceptable salt thereof and paclitaxel are a fixed combination, for example, a pharmaceutical composition; the anti-CTLA4-anti-PD-1 bispecific antibody is present in an independent pharmaceutical composition.
  • the pharmaceutical composition in the pharmaceutical combination, is independently a solid pharmaceutical composition or a liquid pharmaceutical composition.
  • the pharmaceutical combination wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, such as carriers and/or excipients.
  • the pharmaceutical combination wherein the pharmaceutical composition does not contain other active pharmaceutical ingredients except anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof and paclitaxel.
  • the drug combination, wherein the anti-CTLA4-anti-PD-1 bispecific antibody comprises:
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is a single-chain antibody; wherein the immunoglobulin, its heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:27-29, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:30-32, respectively; and the single-chain antibody, its heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:33-35, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:36-38, respectively;
  • the first protein functional region is a single-chain antibody
  • the second protein functional region is an immunoglobulin; wherein the single-chain antibody, its heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:27-29, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:30-32; and the immunoglobulin, its heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:33-35, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:36-38;
  • the immunoglobulins are all human IgG1 subtype; and according to the EU numbering system, the heavy chain constant region of the immunoglobulin is mutated at any two or three of the 234th, 235th and 237th positions, and after the mutation, the bispecific antibody binds to Fc ⁇ RIIIa and/or C1q
  • the affinity constant is lower than that before mutation; preferably, the affinity constant is measured by Fortebio Octet molecular interaction instrument.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein, after the above-mentioned mutation occurs, the affinity constant of the bispecific antibody to Fc ⁇ RIIIa, Fc ⁇ RI, Fc ⁇ RIIa_H131, Fc ⁇ RIIIa_V158 and/or Fc ⁇ RIIb is reduced compared with before the mutation; preferably, the affinity constant is measured by Fortebio Octet molecular interaction instrument.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the heavy chain constant region of the immunoglobulin comprises the following mutations according to the EU numbering system:
  • the letters before the site represent the amino acid before mutation
  • the letters after the site represent the amino acid after mutation
  • the drug combination, wherein the anti-CTLA4-anti-PD-1 bispecific antibody comprises:
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is a single-chain antibody; wherein the immunoglobulin, its heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:27-29, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:30-32, respectively; and the single-chain antibody, its heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:33-35, and its light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:36-38, respectively;
  • the first protein functional region is a single-chain antibody
  • the second protein functional region is an immunoglobulin; wherein the single-chain antibody, the heavy chain variable region comprises HCDR1- HCDR3, whose light chain variable region comprises LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NOs:30-32, respectively; and the immunoglobulin, whose heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences as shown in SEQ ID NOs:33-35, respectively, and whose light chain variable region comprises LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NOs:36-38, respectively;
  • the immunoglobulins are all of human IgG1 subtype; and according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises one of the following mutation combinations:
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin further comprises one or more mutations selected from the following:
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody is in the form of IgG-scFv, i.e., Morrison mode.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 18; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 41 and SEQ ID NO: 43; and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 42 and SEQ ID NO: 44;
  • the drug combination, wherein the anti-CTLA4-anti-PD-1 bispecific antibody is selected from any one of the following (1)-(20):
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14 and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 8;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14 and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 12;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: The amino acid sequence is shown in SEQ ID NO:8;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 16;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 20;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:8; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:16;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 12
  • amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 14
  • amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 14
  • amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:
  • the amino acid sequence is shown in SEQ ID NO: 16;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 12
  • amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 18
  • amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 20;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14
  • amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16
  • amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 43
  • amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 44;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 42;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20; and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 44;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 42; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: The amino acid sequence is shown in SEQ ID NO: 16;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:44; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:16;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:42; and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO:20;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:44; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:20.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody:
  • amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:40, and the amino acid sequence of its light chain is shown in SEQ ID NO:24.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the first protein functional region is directly connected to the second protein functional region or is connected through a connecting fragment; and/or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or is connected through a connecting fragment.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the connecting fragment is a polypeptide shown in SEQ ID NO:45 (GGGGS), or a polypeptide composed of multiple (e.g., 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO:45 connected in series.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the first protein functional region and the second protein functional region are independently 1, 2 or More than 2 persons.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the first protein functional region is 1, and the second protein functional region is 2.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin.
  • the immunoglobulin consists of two heavy chains, one immunoglobulin molecule is connected to two single-chain antibody molecules.
  • the two single-chain antibody molecules are the same.
  • the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through the aforementioned connecting fragment to form an amide bond.
  • the constant region of the immunoglobulin is humanized.
  • the heavy chain constant region adopts Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody binds to the CTLA4 protein and/or the PD-1 protein with a KD of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less.
  • the anti-CTLA4-anti-PD-1 bispecific antibody is a monoclonal antibody.
  • the anti-CTLA4-anti-PD-1 bispecific antibody is a humanized antibody.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the single-chain antibody is respectively connected to the C-termini of the two heavy chains of the immunoglobulin.
  • the drug combination, wherein the anti-CTLA4-anti-PD-1 bispecific antibody comprises:
  • the number of the first protein functional region is 1, and the number of the second protein functional region is 2;
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is a single-chain antibody
  • amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:40, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO:24;
  • amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 43, and the single-chain The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:44;
  • the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin;
  • the first protein functional region is connected to the second protein functional region via a first connecting fragment; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a second connecting fragment; the first connecting fragment and the second connecting fragment are the same or different;
  • amino acid sequences of the first connecting fragment and the second connecting fragment are independently selected from SEQ ID NO: 25 and SEQ ID NO: 26;
  • amino acid sequences of the first connecting fragment and the second connecting fragment are as shown in SEQ ID NO:26.
  • Another aspect of the present invention relates to a medicine kit product, which comprises the drug combination described in any one of the present invention and a product instruction sheet.
  • the medicine kit product comprises a first product, a second product and a third product which are independently packaged.
  • the first product comprises the anti-CTLA4-anti-PD-1 bispecific antibody described in any one of the present invention
  • the second product comprises gemcitabine or a pharmaceutically acceptable salt thereof
  • the third product comprises nab-paclitaxel
  • the first product, the second product and the third product further independently contain one or more pharmaceutically acceptable excipients;
  • the medicine kit product further comprises a product instruction sheet.
  • the drug kit product wherein the unit dose of the first product, calculated according to the mass of the anti-CTLA4-anti-PD-1 bispecific antibody therein, is 100 mg-1000 mg, 200 mg-800 mg, 200 mg-500 mg, 300 mg-600 mg, 400 mg-500 mg or 450 mg.
  • Another aspect of the present invention relates to the use of the drug combination described in any one of the present invention in the preparation of a drug for treating or preventing tumors;
  • the tumor is selected from one or more of pancreatic cancer, melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma;
  • the pancreatic cancer is selected from pancreatic ductal adenocarcinoma and pancreatic adenosquamous carcinoma;
  • the pancreatic cancer is advanced or metastatic pancreatic cancer
  • the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer;
  • the non-small cell lung cancer is intermediate or advanced non-small cell lung cancer
  • the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma.
  • the pharmaceutical combination according to any one of the present invention is used for treating or preventing tumors
  • the tumor is selected from one or more of pancreatic cancer, melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma;
  • the pancreatic cancer is selected from pancreatic ductal adenocarcinoma and pancreatic adenosquamous carcinoma;
  • the pancreatic cancer is advanced or metastatic pancreatic cancer
  • the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer;
  • the non-small cell lung cancer is intermediate or advanced non-small cell lung cancer
  • Another aspect of the present invention relates to a method for treating or preventing tumors, comprising the step of administering an effective amount of the drug combination described in any one of the present invention to a subject in need;
  • the tumor is selected from one or more of pancreatic cancer, melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma;
  • the pancreatic cancer is selected from pancreatic ductal adenocarcinoma and pancreatic adenosquamous carcinoma;
  • the pancreatic cancer is advanced or metastatic pancreatic cancer
  • the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer;
  • the non-small cell lung cancer is intermediate or advanced non-small cell lung cancer
  • the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma.
  • the method for treating or preventing tumors wherein the step of administering an effective amount of an anti-CTLA4-anti-PD-1 bispecific antibody to a subject in need is before or after surgical treatment, and/or before or after radiotherapy.
  • the method for treating or preventing tumors wherein:
  • the single dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 0.1-100 mg per kg body weight, preferably 1-15 mg, 1-12 mg, 1-10 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg) or 6-10 mg; alternatively, the single dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 10-1000 mg per subject (e.g.
  • the drug is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;
  • the administration is by intravenous drip or intravenous injection.
  • the anti-CTLA4-anti-PD-1 bispecific antibody is administered in a cycle of 2 weeks (14 days) or 3 weeks (21 days), and the anti-CTLA4-anti-PD-1 bispecific antibody is preferably administered intravenously on the first day (D1) of each cycle.
  • the anti-CTLA4-anti-PD-1 bispecific antibody is administered once every two weeks (q2w) or once every three weeks (q3w).
  • the single administration dose of gemcitabine or its pharmaceutically acceptable salt is 500-1500 mg/m 2 , 600-1300 mg/m 2 , 800-1250 mg/m 2 , 800-1000 mg/m 2 , 800 mg/m 2 , 850 mg/m 2 , 900 mg/m 2 , 950 mg/m 2 , 1000 mg/m 2 , 1050 mg/m 2 , 1100 mg/m 2 , 1150 mg/m 2 , 1200 mg/m 2 or 1250 mg/m 2 ;
  • the drug is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week or 2 weeks;
  • the administration is by intravenous drip or intravenous injection.
  • gemcitabine or its pharmaceutically acceptable salt is administered once a week for three consecutive weeks, followed by a one-week break as a course of treatment; it can also be administered once a week for two consecutive weeks, followed by a one-week break as a course of treatment.
  • the treatment is continued for 2-4 courses.
  • the single administration dose of nab-paclitaxel is (m 2 ) 80-300 mg/m 2 , 100-280 mg/m 2 , 120-260 mg/m 2 , 120-200 mg/m 2 , 120-180 mg/m 2 , 125-175 mg/m 2 , 125 mg/m 2 , 130 mg/m 2 , 135 mg/m 2 , 140 mg/m 2 , 145 mg/m 2 , 150 mg/m 2 , 155 mg/m 2 , 160 mg/m 2 , 165 mg/m 2 , 170 mg/m 2 , 175 mg/m 2 or 260 mg/m 2 .
  • the single administration dose of paclitaxel is 20-300 mg/m 2 , 50-250 mg/m 2 , 80-200 mg/m 2 , 100-180 mg/m 2 , 120-180 mg/m 2 , 125-175 mg/m 2 , 135-175 mg/m 2 , 125 mg/m 2 , 130 mg/m 2 , 135 mg/m 2 , 140 mg/m 2 , 145 mg/m 2 , 150 mg/m 2 , 155 mg/m 2 , 160 mg/m 2 , 165 mg/m 2 , 170 mg/m 2 or 175 mg/m 2 .
  • the drug is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks or 4 weeks;
  • the administration is by intravenous drip or intravenous injection.
  • paclitaxel eg, nab-paclitaxel
  • the method for treating or preventing tumors comprises a first stage and a second stage, wherein in the first stage, anti-CTLA4-anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and paclitaxel (e.g., nab-paclitaxel) are administered.
  • paclitaxel e.g., nab-paclitaxel
  • the method for treating or preventing tumors wherein an anti-CTLA4-anti-PD-1 bispecific antibody and gemcitabine or a pharmaceutically acceptable salt thereof are administered in the second stage.
  • the method for treating or preventing tumors wherein an anti-CTLA4-anti-PD-1 bispecific antibody is administered in the second stage of treatment.
  • the first stage is a combined drug treatment stage, which includes 1-10 courses of treatment, 2-8 courses of treatment or 4-6 courses of treatment, each course of treatment is 2-4 weeks, preferably 4 weeks.
  • the second stage is the maintenance treatment stage.
  • the dosage and frequency of each active ingredient can be determined by referring to the above description, the drug instructions or according to the doctor's advice.
  • Antibody therapeutics especially monoclonal antibodies (mAB) have achieved good results in the treatment of many diseases.
  • the traditional experimental method to obtain these therapeutic antibodies is to immunize animals with antigens, obtain antibodies targeting antigens in the immunized animals, or improve antibodies with low affinity to antigens through affinity maturation.
  • variable regions of the light chain and heavy chain determine the binding of the antigen; the variable region of each chain contains three highly variable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; they were named by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md).
  • CDRs complementarity determining regions
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is shown in As shown in SEQ ID NO:16.
  • amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:
  • amino acid sequences of the three CDR regions of its light chain variable region are as follows:
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:20.
  • amino acid sequences of the three CDR regions of its heavy chain variable region are identical to those of 14C12.
  • amino acid sequences of the three CDR regions of its light chain variable region are identical to those of 14C12.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4;
  • amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:
  • amino acid sequences of the three CDR regions of its light chain variable region are as follows:
  • LCDR1 TGAVTTSNF(SEQ ID NO:36)
  • LCDR3 ALWYSNHWV(SEQ ID NO:38)
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8;
  • amino acid sequences of the three CDR regions of its heavy chain variable region are identical to those of 4G10.
  • amino acid sequences of the three CDR regions of its light chain variable region are identical to those of 4G10.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12;
  • amino acid sequences of the three CDR regions of its heavy chain variable region are identical to those of 4G10.
  • amino acid sequences of the three CDR regions of its light chain variable region are identical to those of 4G10.
  • amino acid sequences of the 9 CDR regions involved in the heavy chain variable region are as follows:
  • amino acid sequences of the three CDR regions involved in its light chain variable region are as follows:
  • amino acid sequence of the 9 CDR regions involved in the heavy chain variable region is the same as that of BiAb001(M).
  • amino acid sequences of the three CDR regions involved in its light chain variable region are identical to those of BiAb001(M).
  • amino acid sequences of the three CDR regions involved in its light chain variable region are identical to those of BiAb001(M).
  • the antibody BiAb004 (hG1TM) of the present invention introduces amino acid mutations in the non-variable region of BiAb004 (M). According to the EU numbering system, amino acid mutations were introduced at positions 234, 235 and 237:
  • BiAb004 (hG1TM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) in the hinge region of its heavy chain.
  • CTLA4 protein Cytotoxic T-Lymphocyte associated Antigen 4
  • CTLA4 protein includes but is not limited to the full length of CTLA4 protein, or the extracellular fragment CTLA4ECD of CTLA4 or a fragment containing CTLA4ECD; it also includes fusion proteins of CTLA4ECD, such as fragments fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • Fc protein fragment mFc or hFc
  • the term "CTLA4 protein” shall include all such sequences, including its natural or artificial variants.
  • CTLA4 protein when describing a sequence fragment of CTLA4 protein, it also includes the corresponding sequence fragments in its natural or artificial variants.
  • PD-1 protein when referring to the amino acid sequence of PD-1 protein, it includes but is not limited to the full length of PD-1 protein (NCBI GenBank: NM_005018), or the extracellular fragment PD-1ECD of PD-1 or a fragment containing PD-1ECD; it also includes fusion proteins of PD-1ECD, such as fragments fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • Fc protein fragment mFc or hFc
  • the B7 is B7-1 and/or B7-2; its specific protein sequence is a known sequence in the prior art, and reference may be made to the sequence disclosed in existing literature or GenBank.
  • B7-1 CD80, NCBI Gene ID: 941
  • B7-2 CD86, NCBI Gene ID: 942
  • EC50 refers to the concentration for 50% of the maximum effect.
  • the maximal effect refers to the concentration that can cause 50% of the maximum effect.
  • the term "antibody” refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • a heavy chain can be understood as a polypeptide chain of larger molecular weight in an antibody
  • a light chain refers to a polypeptide chain of smaller molecular weight in an antibody.
  • Light chains can be classified as kappa and lambda light chains.
  • Heavy chains can generally be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively.
  • variable and constant regions are connected by a "amino acid region of about 12 or more amino acids, and the heavy chain also contains a "amino acid region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of three 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 light chain constant region is composed of one domain, CL .
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementary determining regions
  • FRs framework regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions ( VH and VL ) of each heavy chain/light chain pair form the antibody binding site, respectively.
  • the heavy chain can also contain more than 3 CDRs, such as 6, 9, or 12.
  • the heavy chain in the bifunctional antibody of the present invention, can be the C-terminus of the heavy chain of an IgG antibody connected to the scFv of another antibody, in which case the heavy chain contains 9 CDRs.
  • antibody is not limited to any particular method for producing antibodies.
  • the antibodies includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • the antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Antibody antigen-binding fragments can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and screened for specificity in the same manner as for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • the terms "monoclonal antibody” and “monoclonal antibody” refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of completely homologous antibody molecules except for possible spontaneous mutations. The same antibody molecule.
  • Monoclonal antibodies have high specificity for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, and usually contain at least 2 or more different antibodies, which usually recognize different epitopes on an antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, for example, US Patent 4,816,567).
  • humanized antibody refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also referred to as an "antigenic determinant” in the art.
  • An epitope or antigenic determinant is usually composed of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and usually has specific three-dimensional structural characteristics and specific charge characteristics.
  • an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear” or “conformational”. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • the term “isolated” or “isolated” refers to that obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated.
  • isolated or “isolated” does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • E. coli expression system refers to an expression system consisting of E. coli (strain) and a vector, wherein the E. coli (strain) is derived from a commercially available strain, such as but not limited to: GI698, ER2566,BL21(DE3),B834(DE3),BLR(DE3).
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • plasmids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as SV40
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed.
  • an antibody that specifically binds to an antigen means that the antibody binds to the antigen with an affinity ( KD ) of less than about 10-5 M, such as less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less.
  • affinity KD
  • targeting refers to specific binding.
  • KD refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen.
  • an antibody binds to an antigen with a dissociation equilibrium constant ( KD ) of less than about 10-5 M, e.g., less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less, e.g., as measured in a BIACORE instrument using surface plasmon resonance (SPR), or measured by a Fortebio Octet molecular interaction instrument.
  • SPR surface plasmon resonance
  • amino acids are generally represented by single letter and For example, alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • adjuvant refers to a non-specific immunopotentiator that, when delivered into the body together with an antigen or in advance, can enhance the body's immune response to the antigen or change the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc.
  • Freund's adjuvant is the most commonly used adjuvant in animal experiments.
  • Aluminum hydroxide adjuvants are used more in clinical trials.
  • 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., a disease associated with CTLA4 binding to B7 or excessive CTLA4 activity, such as a tumor
  • 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 a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art.
  • 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 condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.
  • a “recurrent” cancer is one that grows back in the original site or at a distant site after responding to initial treatment (such as surgery).
  • a “locally recurrent” cancer is one that appears after treatment in the same location as a previously treated cancer.
  • Metalstatic cancer is cancer that has spread from one part of the body (such as the lungs) to another part of the body.
  • the "first” e.g., the first protein functional region, the first connecting fragment or the first product
  • the "second” e.g., the second protein functional region, the second connecting fragment or the second product
  • “about” or “approximately” means floating within the range of 10%, 20% or 30% of the modified numerical value or physical quantity, for example, about 100 minutes or about 100 minutes can be 90 minutes to 110 minutes, 80 minutes to 120 minutes or 70 minutes to 130 minutes.
  • the present invention achieves one or more of the following technical effects (1) to (6):
  • the drug combination of the present invention has a good effect in treating or preventing tumors.
  • the anti-CTLA4-anti-PD-1 bispecific antibody has a synergistic effect with gemcitabine and/or paclitaxel (e.g., nab-paclitaxel), achieving a synergistic effect in treating or preventing tumors, which is better than the anti-CTLA4-anti-PD-1 bispecific antibody alone, or the combination of other anti-PD-1 monoclonal antibodies (e.g., Opdivo) + anti-CTLA4 monoclonal antibodies (e.g., Yervoy) + gemcitabine and/or paclitaxel (e.g., nab-paclitaxel); wherein the tumors include but are not limited to: pancreatic cancer, melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma or
  • the present invention may be beneficial in reducing the dosage and/or effective concentration of gemcitabine.
  • the present invention may be beneficial in reducing the toxic and side effects of gemcitabine.
  • the present invention may be beneficial for reducing the dosage and/or effective concentration of paclitaxel.
  • the present invention may be beneficial for reducing the toxic and side effects of paclitaxel.
  • Figure 1 Anti-CTLA4-anti-PD-1 bispecific antibody combined with gemcitabine and nab-paclitaxel promotes INF- ⁇ secretion in a mixed lymphocyte reaction system.
  • Human peripheral blood mononuclear cells were isolated and prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd.
  • Raji-PDL1 is a cell expressing human PD-L1 constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. based on the human B cell line Raji cells through transfection.
  • Ficoll-Paque TM PLUS (or Ficoll-Paque PLUS) was purchased from Cytiva.
  • RPMI 1640 medium, DMEM medium, Trypsin-EDTA (0.25%) phenol red, and Blasticidin were all from Gibco.
  • Staphylococcus aureus enterotoxin antigen is from Toxin technology, product number: BT202.
  • FBS was from Excell bio.
  • Gemcitabine (abbreviated as Gem) was purchased from MCE, product number: HY-17026.
  • Albumin paclitaxel Abraxane (abbreviated as Abra), was purchased from MCE, product number: HY-P99974.
  • amino acid sequences of the heavy and light chains of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3, as well as the encoding nucleic acid sequences are the same as those of 4G10, 4G10H1L1 and 4G10H3L3 in Chinese patent publication CN 106967172A, respectively.
  • amino acid sequence it encodes is: (124aa)
  • amino acid sequence it encodes is: (126aa)
  • amino acid sequence it encodes is: (115aa)
  • amino acid sequence it encodes is: (109aa)
  • amino acid sequences of the heavy and light chains of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as the encoding nucleic acid sequences are respectively the same as those of 14C12 and 14C12H1L1 in Chinese patent publication CN 106967172A.
  • amino acid sequence it encodes is: (118aa)
  • amino acid sequence it encodes is: (107aa)
  • amino acid sequence it encodes is: (118aa)
  • amino acid sequence it encodes is: (107aa)
  • amino acid sequence it encodes is: (448aa)
  • amino acid sequence it encodes is: (214aa)
  • the structural model of the bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) belongs to the Morrison model (IgG-scFv), that is, the C-termini of the two heavy chains of an IgG antibody are connected to the scFv of another antibody through a connecting fragment, and the design composition of the heavy chain and the light chain is shown in Table A below.
  • the amino acid sequence of Linker1 is (GGGGS)3 (SEQ ID NO: 25)
  • Linker2 is (GGGGS)4 (SEQ ID NO: 26)
  • 4G10H1V(M), 4G10L1V(M), 4G10H3V(M), 4G10L3V(M) in the scFv fragments of BiAb001(M), BiAb002(M), BiAb003(M), and BiAb004(M) antibodies are obtained by modifying the framework regions of 4G10H1V, 4G10L1V, 4G10H3V, and 4G10L3V, respectively. Mutations were performed to effectively optimize the structure of the antibody and improve its effectiveness.
  • BiAb004(M) is also referred to as BiAb004(hG1WT) in the examples of the present invention.
  • the above-mentioned BiAb004(M) is a "wild type", which uses Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region.
  • BiAb004 (hG1WT) obtained in Preparation Example 3
  • the present inventors introduced a point mutation from leucine to alanine (L234A) at position 234 of its heavy chain and a point mutation from leucine to alanine (L234A) at position 235 of its heavy chain.
  • a point mutation from glycine to alanine (L235A) was introduced at position 237, and a point mutation from glycine to alanine (G237A) was introduced at position 237 to obtain BiAb004 (hG1TM).
  • Example 1 Mixed Lymphocyte Reaction (MLR) detection of anti-CTLA4- Biological activity of anti-PD-1 bispecific antibody combined with gemcitabine and paclitaxel in promoting IFN- ⁇ secretion
  • PBMC treatment Normal human PBMCs were isolated according to the instructions of the separation medium Ficoll-Paque TM Plus. Two days before the experiment, PBMCs were resuscitated and cultured in complete medium at 37°C, 5% carbon dioxide incubator; 2 hours later, when the PBMCs were restored, SEB (Staphylococcus aureus enterotoxin antigen, final concentration 0.5 ⁇ g/mL) was added for stimulation for two days.
  • SEB Staphylococcus aureus enterotoxin antigen, final concentration 0.5 ⁇ g/mL
  • BxPC-3 treatment Two days before the experiment, human pancreatic cancer cells BxPC-3 (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were routinely collected and centrifuged at 170 ⁇ g for 5 min. 3*10 6 BxPC-3 cells were inoculated into culture dishes and divided into 4 groups according to the following experimental design:
  • Raji-PDL1 treatment On the day of the experiment, Raji-PDL1 cells were collected, centrifuged at 110 x g for 5 min, and resuspended in The cells were counted (resuspended in assay medium 1640 + 10% FBS), the cell density was adjusted to 2 million/mL, and MMC (Mito-mycin C, final concentration of 2 ⁇ g/mL, purchased from Stressmarq, catalog number: SIH-246-10MG) was added and incubated at 37°C, 5% carbon dioxide incubator for 1 hour.
  • MMC Mito-mycin C, final concentration of 2 ⁇ g/mL, purchased from Stressmarq, catalog number: SIH-246-10MG
  • BiAb004(hG1TM) group, gemcitabine+BiAb004(hG1TM) group, and gemcitabine+nab-paclitaxel+BiAb004(hG1TM) group were added with BiAb004(hG1TM) at a final concentration of 0.3/3/30 nM, respectively;
  • the gemcitabine + Opdivo + Yervoy group and the gemcitabine + nab-paclitaxel + Opdivo + Yervoy group were added with final concentrations of 0.3/3/30 nM Opdivo and 0.3/3/30 nM Yervoy, respectively;
  • the final volume of each system was 200 ⁇ L and cultured for 3 days.

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Abstract

一种包含抗CTLA4-抗PD-1双特异性抗体的药物组合物，包含抗CTLA4-抗PD-1双特异性抗体、吉西他滨以及可选的紫杉醇(例如白蛋白紫杉醇)。所述药物组合物具有良好的抗肿瘤效果。

Description

包含抗CTLA4-抗PD-1双特异性抗体的药物组合及其用途
相关申请的交叉引用
本申请是以CN申请号为202310213266.3，申请日为2023年3月7日的申请为基础，并主张其优先权，该CN申请的全部内容在此作为整体引入本申请中。
技术领域
本发明属于肿瘤治疗和分子免疫学领域，涉及一种包含抗CTLA4-抗PD-1双特异性抗体的药物组合及其用途。具体地，本发明涉及包含抗CTLA4-抗PD-1双特异性抗体、吉西他滨和紫杉醇(例如白蛋白紫杉醇)的药物组合及其用途。
背景技术
跨膜受体PD-1(程序性细胞死亡-1)是CD28基因家族成员之一，在活化的T细胞，B细胞以及骨髓系细胞都有表达。PD-1的配体PDL1(Programmed cell death 1 ligand 1，亦简称为PDL-1)和PDL2(Programmed cell death 1ligand 2，亦简称为PDL-2)均属于B7超家族，其中PDL1在多种细胞都有表达，包括T细胞，B细胞以及内皮细胞和上皮细胞，PDL2则仅表达于抗原呈递细胞如树突状细胞和巨噬细胞。
PD-1/PDL1信号通路在调节免疫耐受、微生物感染及肿瘤免疫逃逸中发挥重要作用。PD-1的表达主要在T细胞等免疫细胞，而PD-1的配体PDL1主要在许多人类肿瘤组织呈高表达。阻断PD-1/PDL1信号通路可使被抑制的T细胞激活，进而攻击癌细胞。阻断PD-1/PDL1信号可以促进肿瘤抗原特异性T细胞的增殖，发挥杀伤肿瘤细胞的作用，进而抑制局部肿瘤生长(Julie R et al.,2012，N Engl J Med.366:2455-2465)。另外，高表达PDL1的肿瘤伴随着很难被检测到的癌症(Hamanishi et al.,2007,Proc.Natl.Acad.Sci.USA 104:3360-5)。一种实施有效的方法是通过体内注射抗PD-1抗体对PD-1的表达进行调控。由于PD-1抗体的广谱抗肿瘤前景和惊人的药效，业界普遍认为针对PD-1通路的抗体将带来治疗多种肿瘤治疗的突破性的进展：用于治疗非小细胞性肺癌，肾细胞癌，卵巢癌，黑色素瘤(Homet M.B.,Parisi G.,et al.,2015,Semin Oncol.42(3):466-473)，白血病以及贫血病(Held SA,Heine A,et al.,2013,Curr Cancer Drug Targets.13(7):768-74)。
细胞毒性T淋巴细胞相关抗原-4(cytotoxic T lymphocyte associated antigen 4， 亦简称为CTLA4)与CD28分子在基因结构、染色体定位、序列的同源性及基因表达具有十分相近的关系，都是共刺激分子B7的受体，主要表达于被激活T细胞表面。CTLA4与B7结合后能抑制小鼠和人T细胞的激活，在T细胞活化中起负调节作用。
CTLA4抗体(或抗CTLA4单克隆抗体)或CTLA4配体可以阻止CTLA4与其天然配体结合，从而封闭CTLA4对T细胞负性调节信号的传导，增强T细胞对各种抗原的反应性，在这方面体内与体外研究结果基本一致。目前已有CTLA4单克隆抗体处于临床试验阶段或被批准用于治疗前列腺癌，膀胱癌，结肠直肠癌，胃肠道癌，肝癌，恶性黑色素瘤等(Grosso JF.,Jure-Kunkel MN.,2013,Cancer Immun.13:5.)。
白细胞介素2(IL-2)由T细胞产生，是调节T细胞亚群的生长因子，也是调控免疫应答的重要因子，并可促进活化B细胞增殖，参与抗体反应、造血和肿瘤监视。重组的人IL-2已经被美国FDA批准用于治疗恶性肿瘤(包括黑色素瘤、肾肿瘤等)，同时正在进行治疗慢性病毒感染的临床研究(Chavez,A.R.,et al.,2009,Ann N Y Acad Sci,1182:p.14-27)。CTLA4及CTLA4抗体作为T细胞功能状况的重要影响因素，通过干预机体免疫微环境。体外和体内试验实验中，CTLA4抗体可特异地解除CTLA4对机体免疫抑制，激活T细胞，诱导IL-2产生，在抗肿瘤及寄生虫等疾病的基因治疗中有广泛应用前景。
CTLA4抗体可对疾病产生特异性治疗作用，并发挥较高疗效，补充传统用药的不足，由此开辟基因治疗的新途径。
双特异性抗体(Bispecific Antibody)亦称为双功能抗体，是同时靶向两种不同抗原的特异性药物，其可通过免疫分选纯化生产。另外，也可通过基因工程获得。基因工程方法在结合位点优化，合成形式的考量以及产量等方面都具有相应的灵活性，所以具有一定的优势。目前，其存在形式已被证明有超过45种(Dafne Müller,Kontermann R E.2010,BioDrugs,24(2):89-98)。目前已开发的多种双特异性抗体为IgG-scFv形式即Morrison模式(Coloma MJ,Morrison SL.1997,Nat Biotechnol.15:159-163)，由于这种类似于天然存在的IgG形式，其在抗体工程、表达和纯化上所具有的优势，已被证明是双功能抗体的其中一种理想存在形式(Miller BR,Demarest SJ,et al.,2010,Protein Eng Des Sel；23:549-57；Fitzgerald J,Lugovskoy A.2011.MAbs；3:299-309)。
吉西他滨是治疗许多实体瘤如非小细胞肺癌、胰腺癌、乳腺癌、膀胱癌等常用的一线化疗药物，也可用于恶性淋巴瘤、卵巢癌、鼻咽癌的治疗。该药物是一种二氟核 苷类抗代谢抗肿瘤药物，通过抑制脱氧核酸的合成，干扰细胞DNA复制和转录，从而抑制肿瘤细胞的增殖和转移。然而，吉西他滨单药治疗癌症效果有限，最显著的不足是半衰期短，必须大剂量持续静脉给药才能维持有效浓度，如此势必造成较严重的毒副作用，明显降低患者的生活质量。吉西他滨化学结构式如下面的式I所示。临床上常用的是盐酸吉西他滨，其结构式如下面的式II所示。
紫杉醇(Paclitaxel)是一种天然植物类抗肿瘤新药，于20世纪60年代末由美国国立癌症研究所(NCI)从太平洋红豆杉的树皮中提取的单体双萜类化合物，被誉为20世纪90年代抗肿瘤药的三大成就之一，广泛应用于卵巢癌、乳腺癌、直肠癌、非小细胞肺癌、或卡波络式恶性肿瘤等癌症的治疗(Anil K.Singla,et al.(2002)Int.J.Pharm.235:179-192)。紫杉醇的结构式如下面的式III所示。
白蛋白结合型紫杉醇(本发明中也简称为白蛋白紫杉醇)是紫杉醇与白蛋白结合而成的新型化疗药物，与传统溶剂型紫杉醇相比，疗效更好，并具有良好的可溶性，临床应用上也更加方便。白蛋白结合型紫杉醇可通过白蛋白受体(Gp60)穿胞途径及结合于肿瘤细胞外间质的富含半胱氨酸的酸性分泌蛋白(SPARC)途径来提高肿瘤外药物浓度，从而提高了抗肿瘤活性。有研究报道，紫杉醇与白蛋白之间的结合以静电作用和疏水作用为主(陈东晓,张娟梅,李建业等.紫杉醇白蛋白纳米粒的研究现状与进展[J].河南大学学报(医学版),2017,36(04):296-300.DOI:10.15991/j.cnki.41-1361/r.2017.04.020.)。
由于化药单药疗效有限，且大剂量使用毒副作用和耐药性出现，使得很多肿瘤患者在接受化疗药物治疗后疾病仍无法得到长期控制，因此开发更加有效的治疗手段或者抗体与化疗药物联合治疗方案，对于降低耐药的产生，改善药物体内代谢动力学作用，提高药物的治疗效果，和/或减少药物的副作用，具有巨大的临床意义。
发明内容
本发明人经过深入的研究和创造性的劳动，创造性地将抗CTLA4-抗PD-1抗体与吉西他滨、白蛋白紫杉醇适当地联用，得到的药物组合具有良好的抗肿瘤效果。
由此提供了下述发明：
本发明的一个方面涉及一种药物组合，其包含抗CTLA4-抗PD-1双特异性抗体、以及吉西他滨或其可药用盐；
优选地，所述药物组合还包含紫杉醇。
在本发明的一些实施方式中，所述的药物组合，其中，抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐和/或紫杉醇的量为治疗或预防本发明的适应症的有效量。
在本发明的一些实施方式中，所述的药物组合，其由抗CTLA4-抗PD-1双特异性抗体、以及吉西他滨或其可药用盐组成。
在本发明的一些实施方式中，所述的药物组合，其由抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐以及一种或多种药学上可接受的辅料组成。
在本发明的一些实施方式中，所述的药物组合，其由抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐以及紫杉醇组成。
在本发明的一些实施方式中，所述的药物组合，其由抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐、紫杉醇以及一种或多种药学上可接受的辅料组成。
在本发明的一些实施方式中，所述的药物组合，其中，所述吉西他滨的可药用盐为盐酸吉西他滨。
在本发明的一些实施方式中，所述的药物组合，其中，所述紫杉醇为白蛋白紫杉醇。
在本发明的一些实施方式中，所述的药物组合，其由抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐以及白蛋白紫杉醇组成。
在本发明的一些实施方式中，所述的药物组合，其由抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐、白蛋白紫杉醇以及一种或多种药学上可接受的辅料组成。
在本发明的一些实施方式中，其中，所述的白蛋白紫杉醇，其中紫杉醇与白蛋白的质量比为(1：15)至(5：1)、(1：15)至(3：1)、(1：15)至(2：1)、(1：10)至(3：1)、(1：10)至(2：1)、1：(5-15)、1：(8-12)、1：8、1：9、1：10、1：11或1：12。
在本发明的一些实施方式中，所述的药物组合，其中，所述抗CTLA4-抗PD-1双特异性抗体的单位剂量为100mg-1000mg、200mg-800mg、200mg-500mg、300mg-600mg、400mg-500mg或者450mg。
在本发明的一些实施方式中，所述的药物组合，其中，所述抗CTLA4-抗PD-1双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选1-15mg、1-12mg、1-10mg 或6-10mg。
在本发明的一些实施方式中，所述的药物组合，其中，吉西他滨或其可药用盐的单位剂量为100-2000mg、500-1500mg、600-1300mg、800-1200mg、800-1000mg、800mg、900mg、1000mg、1100mg、1200mg或1500mg。
在本发明的一些实施方式中，所述的药物组合，其中，按吉西他滨或其可药用盐的质量(mg)/受试者的体表面积(m2)计算，吉西他滨或其可药用盐的单次给药剂量为500-1500mg/m2、600-1300mg/m2、800-1250mg/m2、800-1000mg/m2、800mg/m2、850mg/m2、900mg/m2、950mg/m2、1000mg/m2、1050mg/m2、1100mg/m2、1150mg/m2、1200mg/m2或1250mg/m2。
在本发明的一些实施方式中，所述的药物组合，其中，白蛋白紫杉醇(按白蛋白紫杉醇的质量计算)的单位剂量为200-2000mg、500-1500mg、800-1200mg、900-1100mg、800mg、900mg、1000mg、1100mg、或1200mg。
在本发明的一些实施方式中，所述的药物组合，其中，紫杉醇(按紫杉醇的质量计算)的单位剂量为20-300mg、50-250mg、80-200mg、100-180mg、120-180mg、125-175mg、135-175mg、125mg、130mg、135mg、140mg、145mg、150mg、155mg、160mg、165mg、170mg或175mg。
在本发明的一些实施方式中，所述的药物组合，其中，按白蛋白紫杉醇的质量(mg)/受试者的体表面积(m2)计算，白蛋白紫杉醇的单次给药剂量为80-300mg/m2、100-280mg/m2、120-260mg/m2、120-200mg/m2、120-180mg/m2、125-175mg/m2、125mg/m2、130mg/m2、135mg/m2、140mg/m2、145mg/m2、150mg/m2、155mg/m2、160mg/m2、165mg/m2、170mg/m2、175mg/m2或260mg/m2。
在本发明的一些实施方式中，所述的药物组合，其中，按紫杉醇的质量(mg)/受试者的体表面积(m2)计算，紫杉醇的单次给药剂量为20-300mg/m2、50-250mg/m2、80-200mg/m2、100-180mg/m2、120-180mg/m2、125-175mg/m2、135-175mg/m2、125mg/m2、130mg/m2、135mg/m2、140mg/m2、145mg/m2、150mg/m2、155mg/m2、160mg/m2、165mg/m2、170mg/m2或175mg/m2。
在本发明的一些实施方式中，所述的药物组合，其中，
所述药物组合为固定组合，例如为药物组合物；
所述药物组合是非固定组合，例如所述非固定组合中的抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐和紫杉醇各自存在于独立的药物组合物中；或者
吉西他滨或其可药用盐和紫杉醇为固定组合，例如为药物组合物；所述抗CTLA4-抗PD-1双特异性抗体存在于独立的药物组合物中。
在本发明的一些实施方式中，所述的药物组合，其中，所述药物组合物独立地为固体药物组合物或液体药物组合物。
在本发明的一些实施方式中，所述的药物组合，其中，所述药物组合物还包含一种或多种药学上可接受的辅料，例如载体和/或赋形剂。
在本发明的一些实施方式中，所述的药物组合，其中，所述药物组合物不包含除了抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐以及紫杉醇之外的其它活性药物成分。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其包括：
靶向PD-1的第一蛋白功能区，和
靶向CTLA4的第二蛋白功能区；
其中：
所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；其中，所述的免疫球蛋白，其重链可变区包含氨基酸序列分别如SEQ ID NOs:27－29所示的HCDR1－HCDR3，其轻链可变区包含氨基酸序列分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3；和所述的单链抗体，其重链可变区包含氨基酸序列分别如SEQ ID NOs:33－35所示的HCDR1－HCDR3并且其轻链可变区包含氨基酸序列分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3；
或者，
所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白；其中，所述的单链抗体，其重链可变区包含氨基酸序列分别如SEQ ID NOs:27－29所示的HCDR1－HCDR3，其轻链可变区包含氨基酸序列分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3；和所述的免疫球蛋白，其重链可变区包含氨基酸序列分别如SEQ ID NOs:33－35所示的HCDR1－HCDR3并且其轻链可变区包含氨基酸序列分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3；
优选地，所述免疫球蛋白均为人IgG1亚型；并且按照EU编号系统(EU numbering system)，所述免疫球蛋白的重链恒定区在第234位点、235位点和237位点中的任意2个位点或3个位点发生突变，并且突变后，双特异性抗体与FcγRIIIa和/或C1q 的亲和力常数相比突变前降低；优选地，所述亲和力常数通过Fortebio Octet分子相互作用仪测得。
在本发明的一个或多个实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，发生上述突变后，双特异性抗体与FcγRIIIa、FcγRI、FcγRIIa_H131、FcγRIIIa_V158和/或FcγRIIb的亲和力常数相比突变前降低；优选地，所述亲和力常数通过Fortebio Octet分子相互作用仪测得。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区包含如下突变：
L234A和L235A；或者
L234A和G237A；或者
L235A和G237A；
或者
L234A、L235A、G237A。
本发明中，如果没有特别说明，位点之前的字母表示突变前的氨基酸，位点之后的字母表示突变后的氨基酸。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其包括：
靶向PD-1的第一蛋白功能区，和
靶向CTLA4的第二蛋白功能区；
其中：
所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；其中，所述的免疫球蛋白，其重链可变区包含氨基酸序列分别如SEQ ID NOs:27－29所示的HCDR1－HCDR3，其轻链可变区包含氨基酸序列分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3；和所述的单链抗体，其重链可变区包含氨基酸序列分别如SEQ ID NOs:33－35所示的HCDR1－HCDR3并且其轻链可变区包含氨基酸序列分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3；
或者，
所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白；其中，所述的单链抗体，其重链可变区包含氨基酸序列分别如SEQ ID NOs:27－29所示的HCDR1－ HCDR3，其轻链可变区包含氨基酸序列分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3；和所述的免疫球蛋白，其重链可变区包含氨基酸序列分别如SEQ ID NOs:33－35所示的HCDR1－HCDR3并且其轻链可变区包含氨基酸序列分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3；
优选地，所述免疫球蛋白均为人IgG1亚型；并且按照EU编号系统，所述免疫球蛋白的重链恒定区包含如下突变组合之一：
L234A和L235A；或者
L234A和G237A；或者
L235A和G237A；或者
L234A、L235A、G237A。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区还包含选自如下的一个或多个突变：
N297A、D265A、D270A、P238D、L328E、E233D、H268D、P271G、A330R、C226S、C229S、E233P、P331S、S267E、L328F、A330L、M252Y、S254T、T256E、N297Q、P238S、P238A、A327Q、A327G、P329A、K322A、T394D、G236R、G236A、L328R、A330S、P331S、H268A、E318A和K320A。
在本发明的一个或多个实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其为IgG-scFv形式即Morrison模式。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，
所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO:14和SEQ ID NO:18；并且所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO:16和SEQ ID NO:20；和，所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:41和SEQ ID NO:43；并且所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:42和SEQ ID NO:44；
或者，
所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:41和SEQ ID NO:43；并且所述免疫球蛋白的轻链可 变区的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:42和SEQ ID NO:44；和，所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:14和SEQ ID NO:18；并且所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO:16和SEQ ID NO:20。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其选自如下的(1)－(20)中的任一项：
(1)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
(2)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示；
(3)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:12所示；
(4)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
(5)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述单链抗体的轻链可变区的氨 基酸序列如SEQ ID NO:8所示；
(6)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:12所示；
(7)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:4所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
(8)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:4所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示；
(9)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:8所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
(10)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:8所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示；
(11)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:12所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨 基酸序列如SEQ ID NO:16所示；
(12)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:12所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示；
(13)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:41所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:42所示；
(14)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44所示；
(15)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:41所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:42所示；
(16)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44所示；
(17)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:41所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:42所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨 基酸序列如SEQ ID NO:16所示；
(18)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
(19)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:41所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:42所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示；
以及，
(20)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体：
所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:40所示，并且其轻链的氨基酸序列如SEQ ID NO:24所示。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述第一蛋白功能区与所述第二蛋白功能区直接连接或者通过连接片段连接；和/或所述单链抗体的重链可变区与所述单链抗体的轻链可变区直接连接或者通过连接片段连接。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述连接片段为SEQ ID NO:45(GGGGS)所示的多肽，或者为由多个(例如2、3、4、5或6个)SEQ ID NO:45所示多肽串联的多肽。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或 者2个以上。
在本发明的一个或多个实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述第一蛋白功能区为1个，和第二蛋白功能区为2个。
在本发明的一个或多个实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述单链抗体连接在免疫球蛋白的重链的C末端。由于免疫球蛋白由两条重链，因此，一个免疫球蛋白分子连接有两个单链抗体分子。优选地，两个单链抗体分子相同。优选地，所述单链抗体通过前述连接片段与免疫球蛋白的重链的C末端形成酰胺键连接。
在本发明的一个或多个实施方案中，所述免疫球蛋白的恒定区是人源化的，例如，重链恒定区均采用Ig gamma-1chain C region，ACCESSION:P01857；轻链恒定区均采用Ig kappa chain C region，ACCESSION:P01834。
在本发明的一个或多个实施方案中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10- 9M或10-10M或更小的KD结合CTLA4蛋白和/或PD-1蛋白。
在本发明的一个或多个实施方案中，所述的药物组合，其中，所述抗CTLA4-抗PD-1双特异性抗体为单克隆抗体。
在本发明的一个或多个实施方案中，所述的药物组合，其中，所述抗CTLA4-抗PD-1双特异性抗体为人源化抗体。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述单链抗体分别连接在免疫球蛋白的两条重链的C末端。
在本发明的一些实施方式中，所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其包括：
靶向PD-1的第一蛋白功能区，和
靶向CTLA4的第二蛋白功能区；
所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
其中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；
所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:40所示，并且其轻链的氨基酸序列如SEQ ID NO:24所示；
所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述单链 抗体的轻链可变区的氨基酸序列如SEQ ID NO:44所示；
所述单链抗体连接在免疫球蛋白的重链的C末端；
所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NO:25和SEQ ID NO:26；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO:26所示。
本发明的另一方面涉及一种药盒产品，其包含本发明中任一项所述的药物组合，以及产品说明书。
在本发明的一些实施方式中，所述的药盒产品，其包含独立包装的第一产品、第二产品和第三产品，
其中，
所述第一产品包含本发明中任一项所述的抗CTLA4-抗PD-1双特异性抗体；
所述第二产品包含吉西他滨或其可药用盐；
所述第三产品包含白蛋白紫杉醇；
优选地，所述第一产品、第二产品和第三产品还独立地包含一种或多种药学上可接受的辅料；
优选地，所述药盒产品还包含产品说明书。
在本发明的一个或多个实施方案中，所述的药盒产品，其中，所述第一产品的单位剂量，按照其中的所述抗CTLA4-抗PD-1双特异性抗体的质量计算，为100mg-1000mg、200mg-800mg、200mg-500mg、300mg-600mg、400mg-500mg或者450mg。
本发明的再一方面涉及本发明中任一项所述的药物组合在制备治疗或预防肿瘤的药物中的用途；
优选地，所述肿瘤选自胰腺癌、黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠癌、直肠癌、胃癌、肝癌、肺癌、卵巢癌、白血病、乳腺癌、间皮瘤、宫颈癌、子宫内膜癌、淋巴瘤和鼻咽癌和中的一种或多种；
优选的，所述胰腺癌选自胰腺导管腺癌和胰腺腺鳞癌；
优选地，所述胰腺癌为晚期或转移性胰腺癌；
优选地，所述肺癌选自非小细胞性肺癌、小细胞性肺癌和肺鳞癌中的一种或多种；
优选地，所述非小细胞性肺癌为中期或晚期非小细胞肺癌；
优选地，所述胃癌为胃腺癌或食管结合部腺癌。
根据本发明中任一项所述的药物组合，其用于治疗或预防肿瘤；
优选地，所述肿瘤选自胰腺癌、黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠癌、直肠癌、胃癌、肝癌、肺癌、卵巢癌、白血病、乳腺癌、间皮瘤、宫颈癌、子宫内膜癌、淋巴瘤和鼻咽癌和中的一种或多种；
优选的，所述胰腺癌选自胰腺导管腺癌和胰腺腺鳞癌；
优选地，所述胰腺癌为晚期或转移性胰腺癌；
优选地，所述肺癌选自非小细胞性肺癌、小细胞性肺癌和肺鳞癌中的一种或多种；
优选地，所述非小细胞性肺癌为中期或晚期非小细胞肺癌；
优选地，所述胃癌为胃腺癌或食管结合部腺癌。
本发明的再一方面涉及一种治疗或预防肿瘤的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的药物组合的步骤；
优选地，所述肿瘤选自胰腺癌、黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠癌、直肠癌、胃癌、肝癌、肺癌、卵巢癌、白血病、乳腺癌、间皮瘤、宫颈癌、子宫内膜癌、淋巴瘤和鼻咽癌和中的一种或多种；
优选的，所述胰腺癌选自胰腺导管腺癌和胰腺腺鳞癌；
优选地，所述胰腺癌为晚期或转移性胰腺癌；
优选地，所述肺癌选自非小细胞性肺癌、小细胞性肺癌和肺鳞癌中的一种或多种；
优选地，所述非小细胞性肺癌为中期或晚期非小细胞肺癌；
优选地，所述胃癌为胃腺癌或食管结合部腺癌。
在本发明的一个或多个实施方式中，所述的治疗或预防肿瘤的方法，其中，所述给予有需求的受试者以有效量的抗CTLA4-抗PD-1双特异性抗体的步骤为在手术治疗之前或之后，和/或在放射治疗之前或之后。
在本发明的一个或多个实施方式中，所述的治疗或预防肿瘤的方法，其中，
抗CTLA4-抗PD-1双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选1-15mg、1-12mg、1-10mg(例如1mg、2mg、3mg、4mg、5mg、6mg、7mg、8mg、9mg或10mg)或6-10mg；或者，抗CTLA4-抗PD-1双特异性抗体的单次给药剂量为每位受试者10-1000mg(例如大约100mg、大约150mg、大约200mg、大约250mg、大约300mg、大约350mg、大约400mg、大约450mg、大约500mg、大约600mg、大约700mg、大约800mg、大约900mg或大约1000mg)，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg；
优选地，每3天、4天、5天、6天、10天、1周、2周或3周给药一次；
优选地，给药方式为静脉滴注或静脉注射。
在一些方案中，抗CTLA4-抗PD-1双特异性抗体的施用治疗以2周(14天)或3周(21天)为一个周期，优选在每个周期第一天(D1)静脉给予抗CTLA4-抗PD-1双特异性抗体。例如，所述抗CTLA4-抗PD-1双特异性抗体以每两周一次(q2w)或者每三周一次(q3w)的频率施用。
按吉西他滨或其可药用盐的质量(mg)/受试者的体表面积(m2)计算，吉西他滨或其可药用盐的单次给药剂量为500-1500mg/m2、600-1300mg/m2、800-1250mg/m2、800-1000mg/m2、800mg/m2、850mg/m2、900mg/m2、950mg/m2、1000mg/m2、1050mg/m2、1100mg/m2、1150mg/m2、1200mg/m2或1250mg/m2；
优选地，每3天、4天、5天、6天、10天、1周或2周给药一次；
优选地，给药方式为静脉滴注或静脉注射。
在一些方案中，吉西他滨或其可药用盐的施用治疗以每周给药一次连续用药三周，休息一周作为一个疗程；也可以每周给药一次连续用药两周，休息一周作为一个疗程。优选地，连续治疗2-4个疗程。
按白蛋白紫杉醇的质量(mg)/受试者的体表面积(m2)计算，白蛋白紫杉醇的单次给药剂量为(m2)80-300mg/m2、100-280mg/m2、120-260mg/m2、120-200mg/m2、120-180mg/m2、125-175mg/m2、125mg/m2、130mg/m2、135mg/m2、140mg/m2、145mg/m2、150mg/m2、155mg/m2、160mg/m2、165mg/m2、170mg/m2、175mg/m2或260mg/m2。
按紫杉醇的质量(mg)/受试者的体表面积(m2)计算，紫杉醇的单次给药剂量为20-300mg/m2、50-250mg/m2、80-200mg/m2、100-180mg/m2、120-180mg/m2、125-175mg/m2、135-175mg/m2、125mg/m2、130mg/m2、135mg/m2、140mg/m2、145mg/m2、150mg/m2、155mg/m2、160mg/m2、165mg/m2、170mg/m2或175mg/m2。
优选地，每3天、4天、5天、6天、10天、1周、2周、3周或4周给药一次；
优选地，给药方式为静脉滴注或静脉注射。
在本发明的一个或多个实施方式中，所述的治疗或预防肿瘤的方法，其中，所述抗CTLA4-抗PD-1双特异性抗体和吉西他滨、紫杉醇(例如白蛋白紫杉醇)同时或顺序施用。
在本发明的一个或多个实施方式中，所述的治疗或预防肿瘤的方法，包括第一阶段和第二阶段，其中，在第一阶段施用抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐、以及紫杉醇(例如白蛋白紫杉醇)。
在本发明的一个或多个实施方式中，所述的治疗或预防肿瘤的方法，其中，在第二阶段施用抗CTLA4-抗PD-1双特异性抗体、以及吉西他滨或其可药用盐。
在本发明的一个或多个实施方式中，所述的治疗或预防肿瘤的方法，其中，在治疗的第二阶段施用抗CTLA4-抗PD-1双特异性抗体。
所述第一阶段为联合用药治疗阶段，包含1-10个疗程、2-8个疗程或者4-6个疗程，每个疗程为2-4周，优选4周。
所述第二阶段为维持治疗阶段。
在第一阶段和第二阶段中，各药效成分的给药量和给药频率等可以参考前面的描述、药物说明书或者根据医师的建议进行。
抗体治疗药物，特别是单克隆抗体(mAB)已在多种疾病的治疗中取得了良好的疗效。获取这些治疗性抗体的传统实验方法是采用抗原免疫动物，在免疫动物体内获取靶向抗原的抗体，或通过亲和力成熟的方法来改进那些与抗原的亲和力较低的抗体。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Sequences of Proteins of Immunological Interest,Fifth Edition(1991),第1-3卷,NIH Publication 91-3242,Bethesda Md)。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库分析下面的(1)-(9)项中的单克隆抗体序列的CDR区的氨基酸序列，结果如下：
(1)14C12
重链可变区的氨基酸序列如SEQ ID NO:14所示，轻链可变区的氨基酸序列如 SEQ ID NO:16所示。
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1:GFAFSSYD(SEQ ID NO:27)
HCDR2:ISGGGRYT(SEQ ID NO:28)
HCDR3:ANRYGEAWFAY(SEQ ID NO:29)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1:QDINTY(SEQ ID NO:30)
LCDR2:RAN(SEQ ID NO:31)
LCDR3:LQYDEFPLT(SEQ ID NO:32)
(2)14C12H1L1
重链可变区的氨基酸序列如SEQ ID NO:18所示，轻链可变区的氨基酸序列如SEQ ID NO:20所示。
其重链可变区的3个CDR区的氨基酸序列与14C12相同。
其轻链可变区的3个CDR区的氨基酸序列与14C12相同。
(3)4G10
重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:4所示；
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1:GYSFTGYT(SEQ ID NO:33)
HCDR2:INPYNNIT(SEQ ID NO:34)
HCDR3:ARLDYRSY(SEQ ID NO:35)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1:TGAVTTSNF(SEQ ID NO:36)
LCDR2:GTN(SEQ ID NO:37)
LCDR3:ALWYSNHWV(SEQ ID NO:38)
(4)4G10H1L1
重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示；
其重链可变区的3个CDR区的氨基酸序列与4G10相同。
其轻链可变区的3个CDR区的氨基酸序列与4G10相同。
(5)4G10H3L3
重链可变区的氨基酸序列如SEQ ID NO:10所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示；
其重链可变区的3个CDR区的氨基酸序列与4G10相同。
其轻链可变区的3个CDR区的氨基酸序列与4G10相同。
(6)BiAb001(M)
其重链可变区涉及的9个CDR区的氨基酸序列如下：
HCDR1:GFAFSSYD(SEQ ID NO:27)
HCDR2:ISGGGRYT(SEQ ID NO:28)
HCDR3:ANRYGEAWFAY(SEQ ID NO:29)
HCDR4:GYSFTGYT(SEQ ID NO:33)
HCDR5:INPYNNIT(SEQ ID NO:34)
HCDR6:ARLDYRSY(SEQ ID NO:35)
HCDR7:TGAVTTSNF(SEQ ID NO:36)
HCDR8:GTN(SEQ ID NO:37)
HCDR9:ALWYSNHWV(SEQ ID NO:38)
其轻链可变区涉及的3个CDR区的氨基酸序列如下：
LCDR1:QDINTY(SEQ ID NO:30)
LCDR2:RAN(SEQ ID NO:31)
LCDR3:LQYDEFPLT(SEQ ID NO:32)
(7)BiAb002(M)
其重链可变区涉及的9个CDR区的氨基酸序列与BiAb001(M)相同。
其轻链可变区涉及的3个CDR区的氨基酸序列与BiAb001(M)相同。
(8)BiAb003(M)
其重链可变区涉及的9个CDR区的氨基酸序列与BiAb001(M)相同。
其轻链可变区涉及的3个CDR区的氨基酸序列与BiAb001(M)相同。
(9)BiAb004(M)
其重链可变区涉及的9个CDR区的氨基酸序列与BiAb001(M)相同。
其轻链可变区涉及的3个CDR区的氨基酸序列与BiAb001(M)相同。
本发明的抗体BiAb004(hG1TM)在BiAb004(M)非可变区引入氨基酸突变。按 照EU编号系统在234、235和237位点引入氨基酸突变：
通过在其重链铰链区域第234号位点引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸到丙氨酸的点突变(L235A)，第237号位点引进了甘氨酸到丙氨酸的点突变(G237A)获得BiAb004(hG1TM)。
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、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。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，当提及CTLA4蛋白(Cytotoxic T-Lymphocyte associated Antigen 4)的氨基酸序列时，其包括但不限于CTLA4蛋白的全长，或者CTLA4的胞外片段CTLA4ECD或者包含CTLA4ECD的片段；还包括CTLA4ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在CTLA4蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“CTLA4蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述CTLA4蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，当提及PD-1蛋白的氨基酸序列时，其包括但不限于PD-1蛋白(NCBI GenBank:NM_005018)的全长，或者PD-1的胞外片段PD-1ECD或者包含PD-1ECD的片段；还包括PD-1ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PD-1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“PD-1蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述PD-1蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，如果没有特别说明，所述B7为B7-1和/或B7-2；其具体蛋白序列为现有技术中已知序列，可以参考现有文献或者GenBank中公开的序列。例如，B7-1(CD80,NCBI Gene ID:941)；B7-2(CD86,NCBI Gene ID:942)。
如本文中所使用的，术语EC50是指半数最大效应浓度(concentration for 50％of  maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。从一般意义上，重链可以理解为抗体中分子量较大的多肽链，轻链是指抗体中分子量较小的多肽链。轻链可分类为κ和λ轻链。重链通常可分类为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“基酸区连接，重链还包含大约3个或更多个氨基酸的“基酸区。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。抗体的恒定区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)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循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的定义。特别地，重链还可以包含3个以上CDR，例如6、9、或12个。例如在本发明的双功能抗体中，重链可以是IgG抗体的重链的C端连接另一个抗体的scFv，这种情况下重链含有9个CDR。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
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。
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“单抗”和“单克隆抗体”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断，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。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Kohler et al.,Nature,256:495,1975)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，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)；和Clark,Immunol.Today 21:397 402(2000)。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“大肠杆菌表达系统”是指由大肠杆菌(菌株)与载体组成的表达系统，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：GI698, ER2566,BL21(DE3),B834(DE3),BLR(DE3)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，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。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。在本发明的一些实施方案中，术语“靶向”是指特异性结合。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合抗原，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测得，或通过Fortebio Octet分子相互作用仪测得。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；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“药学上可接受的辅料”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。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。
如本文中所使用的，术语“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
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CTLA4与B7结合或者CTLA4活性过高相关的疾病如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如CTLA4与B7结合或者CTLA4活性过高相关的疾病如肿瘤)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，以及同时施用的其他治疗等等。
“复发性”癌症是在对初始治疗(例如手术)产生应答后，在初始部位或远处部位再生的癌症。“局部复发性”癌症是在治疗后，在与先前治疗的癌症相同的位置出现的癌症。
“转移性”癌症是指从身体的一部分(例如肺部)扩散到身体的另一部分的癌症。
在本发明中，如果没有特别说明，所述“第一”(例如第一蛋白功能区、第一连接片段或者第一产品)和“第二”(例如第二蛋白功能区、第二连接片段或者第二产品)是为了指代上的区分或表述上的清楚，并不具有典型的次序上的含义。
本发明中，如果没有特别说明，“大约”或者“约”是指在所修饰的数值或者物理量的10％、20％或30％的范围内上下浮动，例如，大约100分钟或约100分钟，可以是 90分钟-110分钟、80分钟-120分钟或者70分钟-130分钟。
发明的有益效果
本发明实现了如下的(1)至(6)项中所述技术效果中的一项或多项：
(1)本发明的药物组合具有良好的治疗或预防肿瘤的效果。
(2)本发明的药物组合中，抗CTLA4-抗PD-1双特异性抗体与吉西他滨和/或紫杉醇(例如白蛋白紫杉醇)具有协同作用，取得了治疗或预防肿瘤协同效果，效果优于单用抗CTLA4-抗PD-1双特异性抗体，或者优于其它抗PD-1单抗(例如Opdivo)+抗CTLA4单抗(例如Yervoy)+吉西他滨和/或紫杉醇(例如白蛋白紫杉醇)的联用；其中所述肿瘤包括但不限于：胰腺癌、黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠癌、直肠癌、胃癌、肝癌、肺癌、卵巢癌、白血病、乳腺癌、间皮瘤、宫颈癌、子宫内膜癌、淋巴瘤或鼻咽癌。
(3)本发明可能有利于减少吉西他滨的用量和/或有效浓度。
(4)本发明可能有利于降低吉西他滨的毒副作用。
(5)本发明可能有利于减少紫杉醇的用量和/或有效浓度。
(6)本发明可能有利于降低紫杉醇的毒副作用。
附图说明
图1：抗CTLA4-抗PD-1双特异性抗体联用吉西他滨和白蛋白紫杉醇促进混合淋巴细胞反应体系中INF-γ分泌。
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。本领域技术人员将会理解，下面的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技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。
在本发明的下述实施例中：
人外周血单核细胞均为在中山康方生物医药有限公司分离制备。
Raji-PDL1为中山康方生物医药有限公司基于人B细胞系细胞Raji细胞通过转染构建的表达有人PD-L1的细胞。
Ficoll-PaqueTM PLUS(或Ficoll-Paque PLUS)购自Cytiva。
RPMI 1640培养基、DMEM培养基、Trypsin-EDTA(0.25％)phenol red、Blasticidin均来源于Gibco。
金黄色葡萄球菌肠毒素抗原(SEB)来源于Toxin technology，货号：BT202。
FBS来源于Excell bio。
吉西他滨，Gemcitabine(缩写为Gem)，购自MCE，货号：HY-17026。
白蛋白紫杉醇，Abraxane(缩写为Abra)，购自MCE，货号：HY-P99974。
制备例1：抗CTLA4的抗体的序列设计
抗CTLA4的抗体4G10及其人源化抗体4G10H1L1、4G10H3L3的重链和轻链的氨基酸序列、以及编码核酸序列分别与中国专利公开CN 106967172A中的4G10、4G10H1L1、4G10H3L3相同。
(1)4G10的重链可变区序列和轻链可变区序列
重链可变区的核酸序列：(372bp)
其编码的氨基酸序列：(124aa)
轻链可变区的核酸序列：(378bp)
其编码的氨基酸序列：(126aa)
(2)人源化单克隆抗体4G10H1L1的重链可变区序列和轻链可变区序列
重链可变区(4G10H1V)的核酸序列：(345bp)
其编码的氨基酸序列：(115aa)
轻链可变区(4G10L1V)的核酸序列：(327bp)

其编码的氨基酸序列：(109aa)
(3)人源化单克隆抗体4G10H3L3的重链可变区序列和轻链可变区序列
重链可变区(4G10H3V)的核酸序列：(345bp)
其编码的氨基酸序列(4G10H3V)：(115aa)
轻链可变区(4G10L3V)的核酸序列：(327bp)

其编码的氨基酸序列(4G10L3V)：(109aa)
制备例2：抗PD-1的抗体14C12及其人源化抗体14C12H1L1的序列设计
抗PD-1的抗体14C12及其人源化抗体14C12H1L1的重链和轻链的氨基酸序列、以及编码核酸序列分别与中国专利公开CN 106967172A中的14C12、14C12H1L1相同。
(1)14C12的重链可变区序列和轻链可变区序列
重链可变区的核酸序列：(354bp)
其编码的氨基酸序列：(118aa)
轻链可变区的核酸序列：(321bp)

其编码的氨基酸序列：(107aa)
(2)人源化单克隆抗体14C12H1L1的重链可变区序列和轻链可变区序列、重链序列和轻链序列
重链可变区的核酸序列：(354bp)
其编码的氨基酸序列：(118aa)
轻链可变区的核酸序列：(321bp)

其编码的氨基酸序列：(107aa)
14C12H1L1重链(14C12H1)的DNA序列：(1344bp)

其编码的氨基酸序列：(448aa)
14C12H1L1轻链(14C12L1)的DNA序列：(642bp)

其编码的氨基酸序列：(214aa)
制备例3：双功能抗体BiAb001(M)、BiAb002(M)、BiAb003(M)和BiAb004(M)的 序列设计
双功能抗体BiAb001(M)、BiAb002(M)、BiAb003(M)和BiAb004(M)的结构模式属于Morrison模式(IgG-scFv)，即在一个IgG抗体的两条重链的C端均通过连接片段连接另一个抗体的scFv，其重链和轻链的设计组成如下面的表A。
表A：BiAb001(M)、BiAb002(M)、BiAb003(M)和BiAb004(M)的序列设计
上面的表A中:
Linker1的氨基酸序列为(GGGGS)3(SEQ ID NO:25)
Linker2的氨基酸序列为(GGGGS)4(SEQ ID NO:26)
另外，上述的表A中，BiAb001(M)、BiAb002(M)、BiAb003(M)和BiAb004(M)抗体的scFv片段中的4G10H1V(M)、4G10L1V(M)、4G10H3V(M)、4G10L3V(M)是分别在4G10H1V、4G10L1V、4G10H3V、4G10L3V基础上对其骨架区中个别氨基酸进 行了突变，有效地优化了抗体的结构，提高了其有效性。
(1)4G10H1V(M)：(115aa，基于4G10H1V所做的氨基酸序列突变位点用下划线标出)
(2)4G10L1V(M)：(110aa，基于4G10L1V所做的氨基酸序列突变位点用下划线标出)
(3)4G10H3V(M)：(115aa，基于4G10H3V所做的氨基酸序列突变位点用下划线标出)
(4)4G10L3V(M)：(110aa，基于4G10L3V所做的氨基酸序列突变位点用下划线标出)
为了与下文中的突变后的抗体相区分，在本发明实施例中BiAb004(M)亦称为BiAb004(hG1WT)。上述的BiAb004(M)作为“野生型”，其采用Ig gamma-1chain C region，ACCESSION:P01857作为重链恒定区，Ig kappa chain C region，ACCESSION:P01834为轻链恒定区。
制备例4：基于人源化双功能抗体BiAb004的非可变区氨基酸突变设计
本发明人在制备例3所获得的BiAb004(hG1WT)的基础上，通过在其重链的第234号位点引进了亮氨酸到丙氨酸的点突变(L234A)，第235号位点引进了亮氨酸 到丙氨酸的点突变(L235A)，第237号位点引进了甘氨酸到丙氨酸的点突变(G237A)，获得了BiAb004(hG1TM)。
BiAb004(hG1TM)中的免疫球蛋白部分的重链的DNA序列：(1344bp，突变位点用下划线标出)
BiAb004(hG1TM)中的免疫球蛋白部分的重链的氨基酸序列：(448aa，突变位点用下划线标出)
BiAb004(hG1TM)和BiAb004(hG1WT)轻链的DNA序列相同，其编码的氨基酸序列也相同，具体序列见制备例3。
实施例1：混合淋巴细胞反应(Mixed Lymphocyte Reaction,MLR)检测抗CTLA4- 抗PD-1双特异性抗体联合吉西他滨和紫杉醇促IFN-γ分泌的生物活性
1.PBMC处理：按分离液Ficoll-PaqueTM Plus操作说明书分离获得正常人PBMC。实验前两天，复苏PBMC，完全培养基培养于37℃，5％二氧化碳培养箱；2h后，待PBMC状态恢复，添加SEB(金黄色葡萄球菌肠毒素抗原，终浓度0.5μg/mL)刺激两天。
2.BxPC-3处理：实验前两天，常规收集人胰腺癌细胞BxPC-3(购自中国科学院上海生命科学研究院)，170xg离心5min，将3*106个BxPC-3细胞接种至培养皿中，按以下实验设计分为4组：
①不加药(作为阴性对照)；
②加入吉西他滨至终浓度为100nM，处理48小时；
③加入白蛋白紫杉醇至终浓度为20ng/mL，处理24小时；
④加入吉西他滨至终浓度为100nM，处理24小时；然后加入白蛋白紫杉醇至终浓度为20ng/mL，继续处理24小时。
3.Raji-PDL1处理：实验当天，收集Raji-PDL1细胞，110xg离心5min，重悬计 数(用分析培养基1640+10％FBS重悬)，调整细胞密度至200万/mL，加入MMC(Mito-mycin C，终浓度为2μg/mL，购自Stressmarq，货号：SIH-246-10MG)于37℃，5％二氧化碳培养箱处理1小时。
4.收集SEB刺激两天后的PBMC、MMC处理后的Raji-PDL1细胞，用分析培养基洗涤两次，重悬计数；常规收集步骤2中不同化药处理/不处理的BxPC-3细胞，170xg离心5min，重悬计数；按PBMC 10万/孔、Raji-PDL1 10万/孔和BxPC-3 2万/孔接种到96孔U型板(3799)中，得到混合淋巴细胞体系。
5.按如下分组加入抗体：
BiAb004(hG1TM)组、吉西他滨+BiAb004(hG1TM)组以及吉西他滨+白蛋白紫杉醇+BiAb004(hG1TM)组分别加入体系终浓度为0.3/3/30nM的BiAb004(hG1TM)；
吉西他滨+Opdivo+Yervoy组和吉西他滨+白蛋白紫杉醇+Opdivo+Yervoy组分别加入体系终浓度为0.3/3/30nM的Opdivo和0.3/3/30nM的Yervoy；
阴性对照组(PBMC+Raji-PDL1+没有化药处理的BxPC-3)、单独吉西他滨(Gemcitabine)处理组、单独白蛋白紫杉醇(Abraxane)处理组以及吉西他滨+白蛋白紫杉醇(Gemcitabine+Abraxane)处理组不加入抗体；
各组体系终体积均为200μL，培养3天。
6.3天后，250xg离心5min(Beckman离心机)，收集细胞上清，采用达科为试剂盒检测IFN-γ含量。
结果如图1所示。
结果显示，在该混合淋巴细胞体系中，抗CTLA4-抗PD-1双特异性抗体BiAb004(hG1TM)+吉西他滨联用组促INF-γ的能力要强于BiAb004(hG1TM)单药组以及抗PD-1单克隆抗体Opdivo(购自BMS，批号ABS3203)+抗CTLA4单克隆抗体Yervoy(购自BMS，批号AAT3892)+吉西他滨联用组，并且BiAb004(hG1TM)+吉西他滨+白蛋白紫杉醇联用组促INF-γ的能力要强于抗PD-1单克隆抗体Opdivo+抗CTLA4单克隆抗体Yervoy+吉西他滨+白蛋白紫杉醇联用组。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (22)

  1. 一种药物组合，其包含抗CTLA4-抗PD-1双特异性抗体、以及吉西他滨或其可药用盐；
    优选地，所述药物组合还包含紫杉醇。
  2. 根据权利要求1所述的药物组合，其中，所述吉西他滨的可药用盐为盐酸吉西他滨。
  3. 根据权利要求1至2中任一权利要求所述的药物组合，其中，所述紫杉醇为白蛋白紫杉醇；
    优选地，紫杉醇与白蛋白的质量比为1：(5-15)、1：(8-12)、1：8、1：9、1：10、1：11或1：12。
  4. 根据权利要求1至3中任一权利要求所述的药物组合，其中，所述抗CTLA4-抗PD-1双特异性抗体的单位剂量为100mg-1000mg、200mg-800mg、200mg-500mg、300mg-600mg、400mg-500mg或者450mg。
  5. 根据权利要求1至4中任一权利要求所述的药物组合，其中，所述抗CTLA4-抗PD-1双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选1-15mg、1-12mg、1-10mg或6-10mg。
  6. 根据权利要求1至5中任一权利要求所述的药物组合，其中：
    按吉西他滨或其可药用盐的质量(mg)/受试者的体表面积(m2)计算，吉西他滨或其可药用盐的单次给药剂量为500-1500mg/m2、600-1300mg/m2、800-1250mg/m2、800-1000mg/m2、800mg/m2、850mg/m2、900mg/m2、950mg/m2、1000mg/m2、1050mg/m2、1100mg/m2、1150mg/m2、1200mg/m2或1250mg/m2；或者
    吉西他滨或其可药用盐的单位剂量为100-2000mg、500-1500mg、600-1300mg、800-1200mg、800-1000mg、800mg、900mg、1000mg、1100mg、1200mg或1500mg。
  7. 根据权利要求1至6中任一权利要求所述的药物组合，其中：
    按白蛋白紫杉醇的质量(mg)/受试者的体表面积(m2)计算，白蛋白紫杉醇的单次给药剂量为80-300mg/m2、100-280mg/m2、120-260mg/m2、120-200mg/m2、120-180mg/m2、125-175mg/m2、125mg/m2、130mg/m2、135mg/m2、140mg/m2、145mg/m2、150mg/m2、155mg/m2、160mg/m2、165mg/m2、170mg/m2、175mg/m2或260mg/m2；或者
    白蛋白紫杉醇的单位剂量为200-2000mg、500-1500mg、800-1200mg、900-1100mg、800mg、900mg、1000mg、1100mg、或1200mg。
  8. 根据权利要求1至7中任一权利要求所述的药物组合，其中，
    所述药物组合为固定组合，例如为药物组合物；
    所述药物组合是非固定组合，例如所述非固定组合中的抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐和紫杉醇各自存在于独立的药物组合物中；或者
    吉西他滨或其可药用盐和紫杉醇为固定组合，例如为药物组合物；所述抗CTLA4-抗PD-1双特异性抗体存在于独立的药物组合物中。
  9. 根据权利要求8所述的药物组合，其中，所述药物组合物独立地为固体药物组合物或液体药物组合物；
    优选地，所述药物组合物还包含一种或多种药学上可接受的辅料；
    优选地，所述药物组合物不包含除了抗CTLA4-抗PD-1双特异性抗体、吉西他滨或其可药用盐以及紫杉醇之外的其它活性药物成分。
  10. 根据权利要求1至9中任一权利要求所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其包括：
    靶向PD-1的第一蛋白功能区，和
    靶向CTLA4的第二蛋白功能区；
    其中：
    所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；其中，所述的免疫球蛋白，其重链可变区包含氨基酸序列分别如SEQ ID NOs:27－29所示的HCDR1－HCDR3，其轻链可变区包含氨基酸序列分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3；和所述的单链抗体，其重链可变区包含氨基酸序列分别如SEQ ID NOs:33－35 所示的HCDR1－HCDR3并且其轻链可变区包含氨基酸序列分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3；
    或者，
    所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白；其中，所述的单链抗体，其重链可变区包含氨基酸序列分别如SEQ ID NOs:27－29所示的HCDR1－HCDR3，其轻链可变区包含氨基酸序列分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3；和所述的免疫球蛋白，其重链可变区包含氨基酸序列分别如SEQ ID NOs:33－35所示的HCDR1－HCDR3并且其轻链可变区包含氨基酸序列分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3；
    优选地，所述免疫球蛋白均为人IgG1亚型；并且按照EU编号系统，所述免疫球蛋白的重链恒定区包含如下突变组合之一：
    L234A和L235A；或者
    L234A和G237A；或者
    L235A和G237A；或者
    L234A、L235A、G237A。
  11. 根据权利要求1至10中任一权利要求所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区还包含选自如下的一个或多个突变：
    N297A、D265A、D270A、P238D、L328E、E233D、H268D、P271G、A330R、C226S、C229S、E233P、P331S、S267E、L328F、A330L、M252Y、S254T、T256E、N297Q、P238S、P238A、A327Q、A327G、P329A、K322A、T394D、G236R、G236A、L328R、A330S、P331S、H268A、E318A和K320A。
  12. 根据权利要求1至11中任一权利要求所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，
    所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO:14和SEQ ID NO:18；并且所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO:16和SEQ ID NO:20；和，所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:41和SEQ ID NO:43；并且所述单链抗体的轻 链可变区的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:42和SEQ ID NO:44；
    或者，
    所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:41和SEQ ID NO:43；并且所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:42和SEQ ID NO:44；和，所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:14和SEQ ID NO:18；并且所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO:16和SEQ ID NO:20。
  13. 根据权利要求1至12中任一权利要求所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其选自如下的(1)－(20)中的任一项：
    (1)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
    (2)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示；
    (3)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:12所示；
    (4)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重 链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
    (5)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示；
    (6)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:12所示；
    (7)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:4所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
    (8)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:4所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示；
    (9)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:8所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
    (10)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:6所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:8所示；和，所述单链抗体的重 链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示；
    (11)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:12所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
    (12)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:10所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:12所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示；
    (13)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:41所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:42所示；
    (14)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:16所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44所示；
    (15)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:41所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:42所示；
    (16)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:20所示；和，所述单链抗体的重 链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44所示；
    (17)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:41所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:42所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
    (18)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:14所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:16所示；
    (19)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:41所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:42所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示；
    以及，
    (20)
    所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:20所示。
  14. 根据权利要求1至13中任一权利要求所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体：
    所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:40所示，并且其轻链的氨基酸序列如SEQ ID NO:24所示。
  15. 根据权利要求1至14中任一权利要求所述的药物组合，其中，所述的抗 CTLA4-抗PD-1双特异性抗体，其中，所述第一蛋白功能区与所述第二蛋白功能区直接连接或者通过连接片段连接；和/或所述单链抗体的重链可变区与所述单链抗体的轻链可变区直接连接或者通过连接片段连接；
    优选地，所述连接片段为SEQ ID NO:45所示的多肽，或者为由多个(例如2、3、4、5或6个)SEQ ID NO:45所示多肽串联的多肽。
  16. 根据权利要求1至15中任一权利要求所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
  17. 根据权利要求1至16中任一权利要求所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述单链抗体分别连接在免疫球蛋白的两条重链的C末端。
  18. 根据权利要求1至17中任一权利要求所述的药物组合，其中，所述的抗CTLA4-抗PD-1双特异性抗体，其包括：
    靶向PD-1的第一蛋白功能区，和
    靶向CTLA4的第二蛋白功能区；
    所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
    其中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；
    所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:40所示，并且其轻链的氨基酸序列如SEQ ID NO:24所示；
    所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44所示；
    所述单链抗体连接在免疫球蛋白的重链的C末端；
    所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NO:25和SEQ ID NO:26；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO: 26所示。
  19. 一种药盒产品，其包含权利要求1至18中任一权利要求所述的药物组合，以及产品说明书。
  20. 权利要求1至18中任一权利要求所述的药物组合在制备治疗或预防肿瘤的药物中的用途；
    优选地，所述肿瘤选自胰腺癌、黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠癌、直肠癌、胃癌、肝癌、肺癌、卵巢癌、白血病、乳腺癌、间皮瘤、宫颈癌、子宫内膜癌、淋巴瘤和鼻咽癌和中的一种或多种；
    优选的，所述胰腺癌选自胰腺导管腺癌和胰腺腺鳞癌；
    优选地，所述胰腺癌为晚期或转移性胰腺癌；
    优选地，所述肺癌选自非小细胞性肺癌、小细胞性肺癌和肺鳞癌中的一种或多种；
    优选地，所述非小细胞性肺癌为中期或晚期非小细胞肺癌；
    优选地，所述胃癌为胃腺癌或食管结合部腺癌。
  21. 根据权利要求1至18中任一权利要求所述的药物组合，其用于治疗或预防肿瘤；
    优选地，所述肿瘤选自胰腺癌、黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠癌、直肠癌、胃癌、肝癌、肺癌、卵巢癌、白血病、乳腺癌、间皮瘤、宫颈癌、子宫内膜癌、淋巴瘤和鼻咽癌和中的一种或多种；
    优选的，所述胰腺癌选自胰腺导管腺癌和胰腺腺鳞癌；
    优选地，所述胰腺癌为晚期或转移性胰腺癌；
    优选地，所述肺癌选自非小细胞性肺癌、小细胞性肺癌和肺鳞癌中的一种或多种；
    优选地，所述非小细胞性肺癌为中期或晚期非小细胞肺癌；
    优选地，所述胃癌为胃腺癌或食管结合部腺癌。
  22. 一种治疗或预防肿瘤的方法，包括给予有需求的受试者以有效量的权利要求1至18中任一权利要求所述的药物组合的步骤；
    优选地，所述肿瘤选自胰腺癌、黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠癌、 直肠癌、胃癌、肝癌、肺癌、卵巢癌、白血病、乳腺癌、间皮瘤、宫颈癌、子宫内膜癌、淋巴瘤和鼻咽癌和中的一种或多种；
    优选的，所述胰腺癌选自胰腺导管腺癌和胰腺腺鳞癌；
    优选地，所述胰腺癌为晚期或转移性胰腺癌；
    优选地，所述肺癌选自非小细胞性肺癌、小细胞性肺癌和肺鳞癌中的一种或多种；
    优选地，所述非小细胞性肺癌为中期或晚期非小细胞肺癌；
    优选地，所述胃癌为胃腺癌或食管结合部腺癌。
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