WO2023160647A1 - 包含抗ctla4-抗pd-1双特异性抗体和西奥罗尼的药物组合 - Google Patents

包含抗ctla4-抗pd-1双特异性抗体和西奥罗尼的药物组合 Download PDF

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WO2023160647A1
WO2023160647A1 PCT/CN2023/078103 CN2023078103W WO2023160647A1 WO 2023160647 A1 WO2023160647 A1 WO 2023160647A1 CN 2023078103 W CN2023078103 W CN 2023078103W WO 2023160647 A1 WO2023160647 A1 WO 2023160647A1
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seq
amino acid
variable region
acid sequence
chain variable
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French (fr)
Inventor
夏瑜
鲁先平
李百勇
宁志强
王忠民
潘德思
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Shenzhen Chipscreen Biosciences Co Ltd
Akeso Pharmaceuticals Inc
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Shenzhen Chipscreen Biosciences Co Ltd
Akeso Pharmaceuticals Inc
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Priority to JP2024550141A priority Critical patent/JP2025506805A/ja
Priority to KR1020247031947A priority patent/KR20240153590A/ko
Priority to EP23759269.6A priority patent/EP4483877A4/en
Priority to CA3253289A priority patent/CA3253289A1/en
Priority to AU2023225857A priority patent/AU2023225857A1/en
Priority to IL315159A priority patent/IL315159A/en
Application filed by Shenzhen Chipscreen Biosciences Co Ltd, Akeso Pharmaceuticals Inc filed Critical Shenzhen Chipscreen Biosciences Co Ltd
Priority to MX2024010334A priority patent/MX2024010334A/es
Publication of WO2023160647A1 publication Critical patent/WO2023160647A1/zh
Priority to US18/840,378 priority patent/US20250188164A1/en
Anticipated expiration legal-status Critical
Priority to ZA2024/06651A priority patent/ZA202406651B/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • 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
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—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
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of tumor therapy and molecular immunology, and relates to a drug combination comprising an anti-CTLA4-anti-PD-1 bispecific antibody and cioronib and its application.
  • the present invention relates to a pharmaceutical combination comprising a mutated anti-CTLA4-anti-PD-1 bispecific antibody and cioronib or its pharmaceutically acceptable salt or its crystal form and its use.
  • 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.
  • the ligands PDL1 (Programmed cell death 1 ligand 1, also referred to as PDL-1) and PDL2 (Programmed cell death 1 ligand 2, also referred to as PDL-2) of PD-1 belong to the B7 superfamily. Cells are expressed, including T cells, B cells and endothelial cells and epithelial cells, 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.
  • the expression of PD-1 is mainly in immune cells such as T cells, and the ligand of PD-1, PDL1, is mainly highly expressed in many human tumor tissues.
  • Blocking the PD-1/PDL1 signaling pathway can activate suppressed T cells to attack cancer cells. Blocking PD-1/PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, play a role in killing tumor cells, and then inhibit local tumor growth (Julie R et al., 2012, N Engl J Med.366:2455–2465 ).
  • An effective method is to regulate the expression of PD-1 by injecting anti-PD-1 antibody in vivo.
  • Cytotoxic T lymphocyte associated antigen-4 (CTLA4 for short) has a very similar relationship with CD28 molecule in gene structure, chromosomal location, sequence homology and gene expression, both of which are co-stimulatory Receptor for molecule B7, mainly expressed on the surface of activated T cells.
  • CTLA4 and B7 can inhibit the activation of mouse and human T cells and play a negative regulatory role in T cell activation.
  • CTLA4 antibody or anti-CTLA4 monoclonal antibody
  • CTLA4 ligand can prevent CTLA4 from binding to its natural ligand, thereby blocking CTLA4's transduction of T cell negative regulatory signals and enhancing T cell reactivity to various antigens, in this regard
  • CTLA4 monoclonal antibodies are currently 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 and is a growth factor that regulates T cell subsets. It is also an important factor that regulates immune responses. It can promote the proliferation of activated B cells and participate in antibody responses, hematopoiesis and tumor surveillance. Recombinant human IL-2 has been approved by the U.S. FDA for the treatment of malignant tumors (including melanoma, kidney tumors, etc.), and is undergoing clinical research for the treatment of chronic viral infections (Chavez, A.R., et al., 2009, Ann N Y Acad Sci, 1182: p.14-27).
  • CTLA4 and CTLA4 antibody as an important factor affecting the function of T cells, interfere with the immune microenvironment of the body. In vitro and in vivo experiments, CTLA4 antibody can specifically relieve CTLA4's immune suppression on the body, activate T cells, and induce IL-2 production. It has broad application prospects in gene therapy for anti-tumor and parasite diseases.
  • Antibody to CTLA4 can produce specific therapeutic effect on diseases, and exert a high curative effect, complement the deficiency of traditional medicine, thus opening up a new way of gene therapy.
  • Bispecific Antibody also known as bifunctional antibody
  • Bispecific Antibody is a specific drug that targets two different antigens at the same time, which can be produced by immuno-sorting and purification. In addition, it can also be obtained through genetic engineering. Genetic engineering methods have corresponding flexibility in terms of binding site optimization, synthetic form considerations, and yields, so they have certain advantages. Currently, more than 45 forms of it have been proven (Dafne Müller, Kontermann R E. 2010, BioDrugs, 24(2):89-98). A variety of bispecific antibodies that have been developed are in the form of IgG-scFv, that is, the Morrison model (Coloma MJ, Morrison SL.1997, Nat Biotechnol.15:159-163).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fab segment of the antibody binds to the epitope of virus-infected cells or tumor cells, and its Fc segment interacts with killer cells (NK cells) , macrophages, etc.) on the surface of Fc receptors (Fc Receptor, FcR) binding, mediating killer cells directly kill target cells.
  • NK cells killer cells
  • Fc Receptor, FcR Fc Receptor
  • CDC complement dependent cytotoxicity
  • complement-dependent cytotoxicity means that when antibodies After specifically binding to the corresponding antigen on the surface of the cell membrane, a complex is formed to activate the complement system, and then MAC is formed on the surface of the target cell, resulting in the lysis of the target cell.
  • Complement can lead to the lysis of various bacteria and other pathogenic organism cells, and is an important defense mechanism for the body to resist pathogenic organism infection.
  • Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells for recognizing the Fc region of antibodies to mediate immune responses. After the Fab region of the antibody recognizes the antigen, the Fc region of the antibody binds to the Fc receptor on the immune cell (such as a killer cell) to initiate the response function of the immune cell, such as phagocytosis and ADCC.
  • Fc receptors are mainly divided into three types: Fc ⁇ R, Fc ⁇ R and Fc ⁇ R, and Fc ⁇ R can be further divided into Fc ⁇ RI (also known as CD64), Fc ⁇ RII (also known as CD32), Fc ⁇ RIII (also known as CD16) and FcRn (also known as Neonatal Fc receptor) four subtypes.
  • Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII are closely related to ADCC effect.
  • Fc ⁇ RIII is the most important molecule that mediates ADCC. There are two subtypes of Fc ⁇ RIIIa and Fc ⁇ RIIIb with high homology in different cell types.
  • Fc ⁇ RIIIa In the Fc ⁇ RIIIa population, there is a high-affinity Fc ⁇ RIIIa subtype caused by a single nuclear stem polymorphism (SNP) site Types, respectively called Fc ⁇ RIIIa_V158 and low affinity Fc ⁇ RIIIa_F158 two subtypes.
  • SNP nuclear stem polymorphism
  • Fc ⁇ RI has a high affinity to the Fc region of IgG and participates in the ADCC process;
  • Fc ⁇ RII has three subtypes: Fc ⁇ RIIa, Fc ⁇ RIIb and Fc ⁇ RIIc (also known as CD32a, CD32b, CD32c, respectively), among which Fc ⁇ RIIa has ADCC activity;
  • Fc ⁇ RIIa exists in the human population The two subtypes caused by single nucleotide mutations are called Fc ⁇ RIIa_H131 and Fc ⁇ RIIa_R131 respectively;
  • Fc ⁇ RIIb is an inhibitory receptor, and Fc ⁇ RIIb is a typical inhibitory Fc ⁇ R that can inhibit the nearby ITAM pathway.
  • the Fc segment will bind to Fc ⁇ RIIb on the same cell, negatively regulate the activation of B cells, and reduce the secretion of antibodies and cytokines (Hogarth PM, Pietersz GA.2012, NATURE REVIEWS DRUG DISCOVERY, 11( 4): 311-331).
  • the IgG family consists of four members, IgG1, IgG2, IgG3 and IgG4, and there are amino acid differences in the fragment crystallizable (Fc) region of their heavy chain constant regions, resulting in their different affinities to Fc ⁇ Rs.
  • IgG1 is the most subtype in the human body, and it is also the most used subtype in monoclonal antibody drugs. IgG1 can bind to various Fc ⁇ Rs and can trigger ADCC and CDC effects.
  • IgG2 has the weakest affinity for Fc ⁇ Rs, but IgG2 can still trigger monocyte-mediated ADCC by binding to Fc ⁇ RIIa.
  • IgG3 has the strongest binding ability to Fc ⁇ Rs, can trigger ADCC, and has a stronger CDC effect than IgG1.
  • IgG4 molecules bind weakly to Fc ⁇ Rs other than Fc ⁇ RI, and IgG4 molecules are less likely to cause CDC and NK cell-mediated ADCC; however, IgG4 subtype antibodies can mediate ADCP effects by binding to Fc ⁇ RI, targeting immune The ADCP effect of antibody drugs in cells may cause immune cell damage and have negative effects on drug pharmacology.
  • Chemotherapy drugs are currently divided into the following nine categories (Hejie et al., Clinical Oncology, Beijing: People's Health Publishing House, 2016: 230-237).
  • the first category is drugs that directly bind to DNA and prevent DNA replication, including various Kangarizing agents, mitomycin and bleomycin, dacarbazine, platinum drugs such as cisplatin and carboplatin, camptothecin drugs and their derivatives;
  • the second category is drugs that prevent nucleic acid biosynthesis.
  • Drugs mainly affect the enzyme system of tumor cells, hindering the synthesis of DNA and RNA precursors, thereby inhibiting the formation of DNA or RNA, mainly including methotrexate, fluorouracil, 6-mercaptopurine, hydroxyurea and cytarabine, etc.
  • Mainly acting on cells in S phase it belongs to anti-metabolism chemotherapeutic drugs and cycle-specific anticancer drugs; the third type is chemotherapeutic drugs that affect transcription. Transcription of the genetic information on DNA to mRNA dependent on DNA, resulting in damage to the function of the template, transcription blockage, mainly including.
  • the fourth category is drugs that affect tubulin and mitosis, mainly including botanical drugs such as vinblastine, podophyllotoxins, and paclitaxel;
  • the fifth category is drugs that affect ribosome function and prevent protein synthesis. Represented by alkali herbal drugs, it can inhibit the initial stage of protein synthesis, decompose ribosomes and release nascent peptide chains, but cannot prevent the combination of mRNA and tRNA with ribosomes.
  • Such drugs can make nuclear DNA and cytoplasmic RNA Reduce, depolymerize polyribosomes, and possess mitosis;
  • the sixth category is drugs that affect cell membranes.
  • Plant lectins such as concanavalin (Con-A) and phytohemagglutinin (PHA) can interact with glycoproteins on cell membranes Receptor binding, thereby affecting the DNA synthesis of tumor cells and preventing tumor cell division;
  • the seventh category is drugs that induce apoptosis (apoptosis), such as arsenic trioxide;
  • the eighth category is hormones that mainly treat tumors by regulating endocrine rice, Including estrogens, anti-estrogens, progestogens, androgens, anti-androgens, adrenocortical hormones, anti-adrenocorticoids (including chlorophenyl dichloroethane and aminoglutethimide);
  • ninth The category is anti-tumor target therapy, including monoclonal antibodies, epidermal growth factor signaling inhibitors (such as targeted drugs for receptor tyrosine kinase pathway), ubiquitin proteasome inhibitors, angiogenesis inhibitors, etc
  • Chiauranib is a multi-target multi-pathway selective kinase inhibitor developed by Shenzhen Microchip Biotechnology Co., Ltd.
  • Cioronib can selectively inhibit multiple kinase targets such as Aurora B, CSF1R and VEGFR/PDGFR/c-Kit, thereby inhibiting tumor cell proliferation, enhancing anti-tumor immunity and inhibiting tumor angiogenesis, realizing the multi-pathway mechanism of anti-tumor efficacy.
  • SCLC small cell lung cancer
  • the inventors creatively combined the anti-CTLA4-anti-PD-1 antibody with cioronib, and the obtained drug combination has good anti-tumor effect.
  • One aspect of the present invention relates to a pharmaceutical combination comprising cioronib or a pharmaceutically acceptable salt thereof (such as hydrochloride) or a crystal form thereof (such as an unsolvated crystal form A, B or C), and at least one One (eg, one, two or three) anti-CTLA4-anti-PD-1 bispecific antibodies.
  • a pharmaceutical combination comprising cioronib or a pharmaceutically acceptable salt thereof (such as hydrochloride) or a crystal form thereof (such as an unsolvated crystal form A, B or C), and at least one One (eg, one, two or three) anti-CTLA4-anti-PD-1 bispecific antibodies.
  • the drug combination wherein, the mass ratio of Theocronib or its pharmaceutically acceptable salt or its crystal form to the anti-CTLA4-anti-PD-1 bispecific antibody It is 1:(1-1000), preferably 1:(5-500), more preferably 1:(10-100).
  • the drug combination wherein, the mass ratio of Theocronib or its pharmaceutically acceptable salt or its crystal form to the anti-CTLA4-anti-PD-1 bispecific antibody 1:(5-1000), preferably 1:(10-500), more preferably 1:(10-100), 1:(10-150), 1:(10-200), 1:(10 -250), 1:(10-300), 1:(10-350), 1:(10-400), or 1:(10-450).
  • the pharmaceutical combination wherein the unit dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg or 450mg.
  • the pharmaceutical combination wherein, the unit dose of ciorone or its pharmaceutically acceptable salt or its crystal form is 0.1mg-100mg, 0.5mg-50mg, 1mg-20mg , 2mg-15mg, 3mg-12mg, 4mg-8mg or 5mg.
  • the pharmaceutical combination is a fixed combination, such as a pharmaceutical composition
  • the pharmaceutical combination is a non-fixed combination, for example, ciorone or its pharmaceutically acceptable salt or its crystal form, and the anti-CTLA4-anti-PD-1 bispecific antibody in the non-fixed combination are each present in an independent pharmaceutical composition middle.
  • the pharmaceutical composition wherein the pharmaceutical composition 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 adjuvants, such as carriers and/or excipients.
  • the pharmaceutical combination wherein the pharmaceutical composition does not contain the anti-CTLA4-anti-PD-1 bispecific Antibodies to other active pharmaceutical ingredients.
  • the drug combination, wherein the anti-CTLA4-anti-PD-1 bispecific antibody includes:
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is a single-chain antibody
  • the heavy chain variable region of the immunoglobulin contains amino acid sequences such as SEQ ID NOs: 27- HCDR1-HCDR3 shown in 29, whose light chain variable region comprises amino acid sequences respectively LCDR1-LCDR3 shown in SEQ ID NOs:30-32
  • the single-chain antibody whose heavy chain variable region comprises an amino acid sequence HCDR1-HCDR3 as shown in SEQ ID NOs:33-35 respectively and the light chain variable region thereof 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
  • the heavy chain variable region of the immunoglobulin contains amino acid sequences such as SEQ ID NOs: 33- HCDR1-HCDR3 shown in 35 and its light chain variable region comprises amino acid sequences respectively LCDR1-LCDR3 shown in SEQ ID NOs: 36-38
  • the single-chain antibody, its heavy chain variable region comprises an amino acid sequence Respectively as SEQ ID NOs: 27-29
  • the light chain variable regions thereof include LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 30-32, respectively;
  • the immunoglobulins are all of human IgG1 subtype
  • the heavy chain constant region of the immunoglobulin is mutated at any 2 or 3 of the 234th, 235th and 237th positions, and After the mutation, the affinity constant of the bispecific antibody to Fc ⁇ RIIIa and/or C1q is lower than that before the mutation; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument.
  • the drug combination wherein, the anti-CTLA4-anti-PD-1 bispecific antibody, wherein, after the above mutation occurs, the bispecific antibody and Fc ⁇ RIIIa, Fc ⁇ RI , Fc ⁇ RIIa_H131, Fc ⁇ RIIIa_V158 and/or Fc ⁇ RIIb have lower affinity constants than those before the mutation; preferably, the affinity constants are measured by a Fortebio Octet molecular interaction instrument.
  • 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 has Mutate as follows:
  • the letter before the site indicates the amino acid before the mutation
  • the letter after the site indicates the amino acid after the mutation
  • the drug combination, wherein the anti-CTLA4-anti-PD-1 bispecific antibody includes:
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is a single-chain antibody
  • the heavy chain variable region of the immunoglobulin contains amino acid sequences such as SEQ ID NOs: 27- HCDR1-HCDR3 shown in 29, whose light chain variable region comprises amino acid sequences respectively LCDR1-LCDR3 shown in SEQ ID NOs:30-32
  • the single-chain antibody whose heavy chain variable region comprises an amino acid sequence Respectively as SEQ ID NOs: 33-35
  • the shown HCDR1-HCDR3 and its light chain variable region comprise the amino acid sequences of LCDR1-LCDR3 shown in SEQ ID NOs: 36-38, respectively;
  • the functional region of the first protein is a single-chain antibody
  • the functional region of the second protein is an immunoglobulin
  • the variable region of the heavy chain of the immunoglobulin contains amino acid sequences such as SEQ ID NOs: 33- HCDR1-HCDR3 shown in 35 and its light chain variable region comprises amino acid sequences respectively LCDR1-LCDR3 shown in SEQ ID NOs: 36-38
  • the single-chain antibody, its heavy chain variable region comprises an amino acid sequence HCDR1-HCDR3 shown in SEQ ID NOs: 27-29 respectively, the light chain variable region of which comprises LCDR1-LCDR3 shown in SEQ ID NOs: 30-32 respectively;
  • the immunoglobulins are all of human IgG1 subtype
  • the heavy chain constant region of the immunoglobulin has 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 is also have one or more mutations selected from:
  • the drug combination, wherein, the anti-CTLA4-anti-PD-1 bispecific antibody is in the form of IgG-scFv, namely Morrison pattern.
  • 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 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 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 SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41 and SEQ ID NO:41 and SEQ ID NO:20; ID NO:43; and the light of said single chain antibody
  • the amino acid sequence of the chain variable region 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 amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 41 and SEQ ID NO: 43; and the immunization
  • the amino acid sequence of the light chain variable region of the globulin 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; and, said single chain antibody
  • the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO:14 and SEQ ID NO:18; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO:16 and SEQ ID NO: 20.
  • the drug combination, wherein the anti-CTLA4-anti-PD-1 bispecific antibody is selected from any one of the following (1)-(20):
  • the 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 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;
  • the 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 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;
  • the 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 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 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 The amino acid sequence is shown in SEQ ID NO:4;
  • the 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 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;
  • the 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 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;
  • the 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 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;
  • the 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 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 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 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: 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 8; and, the 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 The amino acid sequence is shown in SEQ ID NO:20;
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 12; and, the 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;
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 12; and, the 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 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 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;
  • the 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 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;
  • the 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 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 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 The amino acid sequence is shown in SEQ ID NO:44;
  • the 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 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;
  • the 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 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;
  • the 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 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 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 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 the light chain is shown in SEQ ID NO:24.
  • the drug combination wherein, the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the immunoglobulin or antigen-binding fragment thereof is greater than about 10 ⁇ 7 M, e.g., an affinity constant greater than about 10 ⁇ 6 M, 10 ⁇ 5 M, 10 ⁇ 4 M, or 10 ⁇ 3 M or greater binds Fc ⁇ RIIIa_F158, Fc ⁇ RI, Fc ⁇ RIIa_H131, Fc ⁇ RIIIa_V158 and/or Fc ⁇ RIIb; preferably, the affinity constant Pass Measured by Fortebio Octet molecular interaction instrument;
  • the immunoglobulin or its antigen-binding fragment has no binding signal or the binding signal is less than 0.1nm with Fc ⁇ RIIIa_F158, Fc ⁇ RI, Fc ⁇ RIIa_H131, Fc ⁇ RIIIa_V158 and/or Fc ⁇ RIIb; preferably, the binding signal refers to the molecular interaction through Fortebio Octet The response value measured by the instrument.
  • the drug combination wherein, the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the immunoglobulin or antigen-binding fragment thereof has a concentration greater than about 10 ⁇ 9 M, for example, an affinity constant greater than about 10 ⁇ 8 M, 10 ⁇ 7 M, 10 ⁇ 6 M, or 10 ⁇ 5 M or greater binds to C1q; preferably, said affinity constant is measured by a Fortebio Octet molecular interaction instrument;
  • the immunoglobulin or its antigen-binding fragment has no binding signal with C1q or the binding signal is less than 0.1 nm; preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction instrument.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the functional domain of the first protein is directly related to the functional domain of the second protein connected or connected through a connecting fragment; and/or the heavy chain variable region of the single-chain antibody is directly connected with the light chain variable region of the single-chain antibody or connected through a connecting fragment.
  • the drug combination wherein, the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the connecting fragment is (GGGGS)n, n is a positive integer; preferably Preferably, n is 1, 2, 3, 4, 5 or 6.
  • the drug combination wherein, the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the first protein functional domain and the second protein functional domain are independently 1, 2 or more than 2.
  • the drug combination wherein, the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the first protein functional domain is one, and the second Two protein functional areas are 2.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein the single-chain antibody is linked to the heavy chain of immunoglobulin the C-terminus.
  • immunoglobulins consist of two heavy chains, one immunoglobulin molecule is linked to two single-chain antibody molecules.
  • the two single chain antibody molecules are identical.
  • the single-chain antibody forms an amide bond connection with the C-terminus of the heavy chain of the immunoglobulin through the aforementioned linking fragment.
  • the constant region of the immunoglobulin is humanized, for example, Ig gamma-1 chain C region, ACCESSION:P01857 was used for the heavy chain constant region; Ig kappa chain C region was used for the light chain constant region, ACCESSION:P01834.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody is less than about 10 -5 M, such as less than about 10 -6 M, A KD of 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or less binds CTLA4 protein and/or PD-1 protein.
  • the drug combination wherein the anti-CTLA4-anti-PD-1 bispecific antibody is a monoclonal antibody.
  • the pharmaceutical combination wherein 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 linked to the C-terminus of the heavy chain of the immunoglobulin .
  • the drug combination, wherein the anti-CTLA4-anti-PD-1 bispecific antibody includes:
  • the functional region of the first protein is immunoglobulin, and the functional region of the second protein 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 its light chain 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 amino acid sequence of the light chain variable region of the single-chain 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 through a first connecting segment; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody through a second Segment concatenation; said first concatenated fragment and said second concatenated fragment are the same or different;
  • amino acid sequences of the first connecting segment and the second connecting segment 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 both shown in SEQ ID NO:26.
  • Another aspect of the present invention relates to a pharmaceutical kit product, which comprises any one of the pharmaceutical combinations described in the present invention, and product instructions.
  • the medicine box product includes the first product and the second product in independent packaging
  • the first product comprises the anti-CTLA4-anti-PD-1 bispecific antibody described in any one of the present invention
  • the second product comprises cioronib or a pharmaceutically acceptable salt thereof (e.g. hydrochloride) or a crystalline form thereof (e.g. non-solvated crystalline form A, B or C);
  • a pharmaceutically acceptable salt thereof e.g. hydrochloride
  • a crystalline form thereof e.g. non-solvated crystalline form A, B or C
  • the first product and the second product also independently comprise one or more pharmaceutically acceptable excipients;
  • the kit product also includes product instructions.
  • the kit product wherein the unit dose of the first product is calculated according to the mass of the anti-CTLA4-anti-PD-1 bispecific antibody therein, 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg or 450mg.
  • the unit dose of the second product is calculated according to the mass of ciorone or its pharmaceutically acceptable salt or its crystal form , 0.1mg-100mg, 0.5mg-50mg, 0.5mg-10mg, 1mg-10mg, 2mg-8mg or 1mg-5mg or 5mg.
  • the unit dose of the second product is calculated according to the mass of ciorone or its pharmaceutically acceptable salt or its crystal form , 1mg-20mg, 2mg-15mg, 3mg-12mg, 4mg-8mg or 5mg.
  • 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 drugs for treating or preventing tumors;
  • the tumor is selected from melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrium One or more of carcinoma, lymphoma, pancreatic cancer and nasopharyngeal carcinoma;
  • the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and lung squamous cell carcinoma;
  • the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma;
  • the tumor is a solid tumor with MSI-H/dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with MSI-H/dMMR phenotype:
  • Colon rectal, endometrial, gastric, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.
  • MSI microsatellite instability
  • Microsatellite is a short tandem repeat sequence throughout the human genome, with single nucleotide, dinucleotide or high nucleotide repeats, and the number of repeats is 10-50 times.
  • MSI microsatellite instability high
  • MSI-L microsatellite instability low
  • MSS microsatellite stable
  • MSI DNA mismatch repair
  • MMR gene human mismatch repair gene
  • MSI gene can express the corresponding mismatch repair protein after transcription and translation. If the expression loss of any MMR protein can cause the defect of the mismatch repair function of the cell, the base mismatch in the DNA replication process Loss of repair function results in accumulation, leading to the occurrence of microsatellite instability (MSI).
  • MSI pathway About 15% of colorectal cancers are caused by the MSI pathway. It was first discovered in colorectal cancer, and can also occur in gastric cancer, endometrial cancer, adrenocortical tumors, etc. MSI-H/dMMR features are also found in adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.
  • MSI-H and dMMR represent the results of two different detection methods, dMMR and MSI-H are biologically consistent, called MSI-H/dMMR or MSI-high/dMMR, MSI-L and MSS are The phenotype of normal MMR (proficient mismatch repair, pMMR).
  • the dMMR detection method is based on the immunohistochemical protein detection of the four mismatched genes MSH2, MLH1, MSH6 and PMS2 based on tumor specimens (including surgical specimens and puncture specimens). If any one of the four proteins is missing, it is dMMR; If all four proteins are positively expressed, the tumor is pMMR, that is, the function of mismatch repair is complete.
  • MSI is a one-to-one paired detection of the length of repeated DNA sequences (microsatellite sequences) between tumor cells and somatic cells, and the lengths are compared.
  • MSI-H When the PCR method is used to detect 5 standard sites based on the US NCI standard, if two or more sites are inconsistent, it is defined as MSI-H, and if one site is inconsistent, it is called MSI- L (microsatellite low instability), if the 5 loci are consistent, it is MSS.
  • High-throughput sequencing High-throughput sequencing
  • NGS Next-generation sequencing technology
  • MSI-H When using more microsatellite loci, such as more than 5 or some other microsatellite loci, for PCR detection, usually ⁇ 30% of the loci are inconsistent, it is called MSI-H, and all loci are consistent, it is defined as MSS, between 0 and 30% is MSI-L.
  • the tumor is selected from melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrium One or more of carcinoma, lymphoma, pancreatic cancer and nasopharyngeal carcinoma;
  • the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and lung squamous cell carcinoma;
  • the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma;
  • the tumor is a solid tumor with MSI-H/dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with MSI-H/dMMR phenotype:
  • Colon rectal, endometrial, gastric, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.
  • Another aspect of the present invention relates to a method for treating or preventing tumors, comprising the step of administering an effective amount of any one of the drug combinations of the present invention to a subject in need;
  • the tumor is selected from melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrium One or more of carcinoma, lymphoma, pancreatic cancer and nasopharyngeal carcinoma;
  • the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and lung squamous cell carcinoma;
  • the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma;
  • the tumor is a solid tumor with MSI-H/dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with MSI-H/dMMR phenotype:
  • Colon rectal, endometrial, gastric, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.
  • 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 before or after surgery, and/or before or after radiation therapy.
  • the method for treating or preventing tumors wherein,
  • the single dose of anti-CTLA4-anti-PD-1 bispecific antibody is 0.1-100 mg per kg body weight, preferably 1-10 mg (such as 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg ); or, the single dose of anti-CTLA4-anti-PD-1 bispecific antibody is 10-1000 mg per subject (for example, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, About 400mg, about 450mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg or about 1000mg), preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg or 200mg;
  • it is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;
  • the administration method is intravenous drip or intravenous injection.
  • the administration of anti-CTLA4-anti-PD-1 bispecific antibody treatment takes 2 weeks (14 days) or 3 weeks (21 days) as a cycle, preferably administered intravenously on the first day (D1) of each cycle Anti-CTLA4-anti-PD-1 bispecific antibody.
  • the anti-CTLA4-anti-PD-1 bispecific antibody is administered at a frequency of once every two weeks (q2w) or once every three weeks (q3w).
  • the method for treating or preventing tumors wherein, the anti-CTLA4-anti-PD-1 bispecific antibody and ciocronib are administered simultaneously or sequentially.
  • Antibody therapeutics especially monoclonal antibodies (mABs), 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 immunized animals, or improve those antibodies with lower affinity to antigens through affinity maturation.
  • variable regions of the light and heavy chains determine the binding of antigens; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3 , the CDR of the light chain (L) comprises LCDR1, LCDR2, LCDR3; it is named by people such as Kabat, 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 SEQ ID NO: 16.
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 QDINTY (SEQ ID NO: 30)
  • 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 sequence of the three CDR regions of the heavy chain variable region is the same as that of 14C12.
  • amino acid sequence of the three CDR regions of the light chain variable region is the same as that 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 the heavy chain variable region are as follows:
  • amino acid sequences of the three CDR regions of the 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 sequence of the three CDR regions of the heavy chain variable region is the same as that of 4G10.
  • amino acid sequence of the three CDR regions of the light chain variable region is the same as that of 4G10.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12;
  • amino acid sequence of the three CDR regions of the heavy chain variable region is the same as that of 4G10.
  • amino acid sequence of the three CDR regions of the light chain variable region is the same as that of 4G10.
  • amino acid sequences of the nine CDR regions involved in the heavy chain variable region are as follows:
  • HCDR9 ALWYSNHWV (SEQ ID NO: 38)
  • amino acid sequences of the three CDR regions involved in the light chain variable region are as follows:
  • LCDR1 QDINTY (SEQ ID NO: 30)
  • 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 the light chain variable region are the same as those of BiAb001(M).
  • 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 the light chain variable region are the same as those of BiAb001(M).
  • 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 the light chain variable region are the same as those of BiAb001(M).
  • Antibody BiAb004(hG1TM) of the present invention introduces amino acid mutations in the non-variable region of BiAb004(M). Amino acid mutations were introduced at positions 234, 235 and 237 according to the EU numbering system:
  • a point mutation from leucine to alanine was introduced at position 234 of the hinge region of the heavy chain (L234A), a point mutation from leucine to alanine (L235A) was introduced at position 235, and A point mutation (G237A) from glycine to alanine was introduced at site 237 to obtain BiAb004(hG1TM).
  • CTLA4 protein Cytotoxic T-Lymphocyte associated Antigen 4
  • CTLA4ECD fragments fused to mouse or human IgG Fc protein fragments (mFc or hFc).
  • mFc or hFc fragments fused to mouse or human IgG Fc protein fragments
  • CTLA4 protein shall include all such sequences, including their natural or artificial variants.
  • sequence fragments of CTLA4 protein it also includes the corresponding sequence fragments in its natural or artificial variants.
  • the amino acid sequence of the PD-1 protein when referring to the amino acid sequence of the PD-1 protein, it includes but is not limited to the full length of the PD-1 protein (NCBI GenBank: NM_005018), or the extracellular fragment of PD-1 PD-1ECD or Fragments comprising PD-1ECD; fusion proteins of PD-1ECD are also included, for example, fragments fused with mouse or human IgG Fc protein fragments (mFc or hFc).
  • mutations or variations including but not limited to substitutions, deletions and/or additions
  • the term "additional protein” shall include all such sequences, including their natural or artificial variants.
  • the sequence fragment of PD-1 protein when describing the sequence fragment of PD-1 protein, it also includes the corresponding sequence fragment in its natural or artificial variant.
  • the B7 is B7-1 and/or B7-2; its specific protein sequence is a known sequence in the prior art, you can refer to the sequence disclosed in the existing literature or GenBank .
  • B7-1 CD80, NCBI Gene ID: 941
  • B7-2 CD86, NCBI Gene ID: 942).
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximal effect.
  • the term “antibody” refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain .
  • a heavy chain can be understood as a polypeptide chain with a larger molecular weight in an antibody
  • a light chain refers to a polypeptide chain with a smaller molecular weight in an antibody.
  • Light chains can be classified as kappa and lambda light chains.
  • Heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • variable and constant regions are joined by a "base region" of about 12 or more amino acids, with the heavy chain also comprising a "base region” of about 3 or more amino acids.
  • Each heavy chain consists of It consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of 3 domains ( CH1 , CH2 and CH3 ).
  • Each light chain is composed of a light chain variable region (V L ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus.
  • the variable regions ( VH and VL ) of each heavy chain/light chain pair form the antibody binding site, respectively.
  • the heavy chain may also comprise more than 3 CDRs, such as 6, 9, or 12.
  • the heavy chain in the diabody of the present invention, can be the scFv of another antibody linked to the C-terminus of the heavy chain of an IgG antibody. In this case, the heavy chain contains 9 CDRs.
  • antibody is not limited to any particular method of producing antibodies.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • Antigen-binding fragments of antibodies can be obtained from a given antibody using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and can be used in the same way as for the intact Antigen-binding fragments of antibodies are screened for specificity in the same manner as antibodies.
  • antibody includes not only whole antibodies but also antigen-binding fragments of antibodies.
  • the terms "monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, except for natural mutations that may occur spontaneously, A population of identical antibody molecules.
  • mAbs are highly specific for a single epitope on an antigen.
  • polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on antigens.
  • Monoclonal antibodies can usually be obtained using 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, U.S. Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody).
  • antibody or antibody tablet section wherein the donor antibody may be a non-human (eg, mouse, rat or rabbit) antibody having the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, so as 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 an antibody.
  • An “epitope” is also referred to in the art as an "antigenic determinant”.
  • Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrate or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear of” or “conformational”. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • isolated means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated or “isolated” do not exclude the admixture of artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substance.
  • Escherichia coli expression system refers to an expression system composed of Escherichia coli (bacterial strain) and vector, wherein Escherichia coli (bacterial strain) is derived from commercially available bacterial strains, such as but not limited to: GI698 , ER2566, BL21(DE3), B834(DE3), BLR(DE3).
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Can Animal viruses used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Tumor vacuolar virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • Tumor vacuolar virus 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 vector may also contain
  • the term "host cell” refers to cells that can be used to introduce vectors, 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 the reaction between an antibody and the antigen it is directed against.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Binds the antigen with an affinity (K D ) of 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or less.
  • affinity K D
  • the term “targeting” refers to specific binding.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • the antibody has a dissociation equilibrium constant (K D ) 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.
  • K D dissociation equilibrium constant
  • Bound antigen for example, as measured in a BIACORE instrument using surface plasmon resonance (SPR), or by a Fortebio Octet molecular interaction instrument.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agent.
  • pH regulators include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers Including but not limited to sodium chloride.
  • adjuvant refers to a non-specific immune enhancer, which can enhance the body's immune response to the antigen or change the type of immune response when it is delivered into the body together with the antigen or in advance.
  • adjuvants including but not limited to aluminum adjuvant (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium pumilus, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal experiments.
  • Aluminum hydroxide adjuvant is widely used in clinical trials.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease such as a disease associated with a combination of CTLA4 and B7 or an overactive CTLA4 such as a tumor
  • an effective amount for preventing a disease is sufficient to prevent, arrest, or delay a disease (such as a disease associated with a combination of CTLA4 and B7 or an overactive CTLA4 such as a tumor) ) of the occurrence of the amount
  • therapeutically effective amount 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 well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.
  • a “recurrent” cancer is one that regenerates at the original site or at a distant site after responding to initial treatment (eg, surgery).
  • a “locally recurrent” cancer is one that, after treatment, arises 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 term "completely eliminated” means that there is no binding signal or a very low binding signal detected by existing instruments (such as Fortebio Octet Molecular Interaction Instrument).
  • no binding signal or very low binding signal means that the binding signal (ie, response value or Response) is lower than 0.1 nm.
  • first such as the first protein functional region, the first connecting fragment or the first product
  • second such as the second protein functional region, the second connecting fragment Or the second product
  • “about” or “approximately” means fluctuating up and down within the range of 10%, 20% or 30% of the modified value or physical quantity, for example, about 100 minutes or about 100 minutes, It can be 90 minutes to 110 minutes, 80 minutes to 120 minutes or 70 minutes to 130 minutes.
  • the crystal forms of Theoroni include but are not limited to non-solvated crystal form A, non-solvated crystal form B, and non-solvated crystal form C.
  • the X-ray powder diffraction pattern of the non-solvated crystal form A has characteristic peaks at reflection angles 2 ⁇ of 6.88°, 9.26°, 12.74°, 13.82°, 18.58°, 20.86° and 25.72°;
  • the X-ray powder diffraction pattern of the non-solvated crystal form B has characteristic peaks at reflection angles 2 ⁇ of 4.88°, 9.68°, 12.74°, 14.52°, 17.72°, 24.30°, and 25.26°;
  • the X-ray powder diffraction pattern of the non-solvated crystal form C has characteristic peaks at reflection angles 2 ⁇ of 4.84°, 9.68°, 12.92°, 14.60°, 16.46°, 17.44°, 22.00°, and 25.28°.
  • the present invention has realized one or more in the technical effect described in following (1) to (2):
  • the drug combination of the present invention has a good effect of treating or preventing tumors.
  • the anti-CTLA4-anti-PD-1 bispecific antibody has a synergistic effect with Theoronib, achieving a synergistic effect in the treatment or prevention of tumors; wherein the tumors include but are not limited to: melanoma , kidney tumors, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, pancreatic cancer, or nasopharynx Cancer; preferably, the lung cancer is non-small cell lung cancer, small cell lung cancer or lung squamous cell carcinoma; preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma.
  • Figure 1 The detection result of the affinity constant between BiAb004(hG1TM) and Fc ⁇ RI.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 50nM, 25nM, 12.5nM, 6.25nM, 3.12nM, respectively.
  • Figure 2 The results of detection of the affinity constant of BiAb004(hG1WT) Fc ⁇ RI.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 50nM, 25nM, 12.5nM, 6.25nM, 3.12nM, respectively.
  • Figure 3 The results of detection of the affinity constant between BiAb004(hG1TM) and Fc ⁇ RIIIa_V158.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 500nM, 250nM, 125nM, 62.5nM, 31.25nM, respectively.
  • Figure 4 The results of detection of the affinity constant between BiAb004(hG1WT) and Fc ⁇ RIIIa_V158.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 500nM, 250nM, 125nM, 62.5nM, 31.25nM, respectively.
  • Figure 5 The results of detection of the affinity constant between BiAb004(hG1TM) and Fc ⁇ RIIIa_F158.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 500nM, 250nM, 125nM, 62.5nM, 31.25nM, respectively.
  • Fig. 6 A graph of the detection results of the affinity constant between BiAb004(hG1WT) and Fc ⁇ RIIIa_F158.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 500nM, 250nM, 125nM, 62.5nM, 31.25nM, respectively.
  • Fig. 7 The detection result of the affinity constant between BiAb004(hG1TM) and Fc ⁇ RIIa_H131.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 200nM, 100nM, 50nM, 25nM, and 12.5nM, respectively.
  • Figure 8 A graph of the detection results of the affinity constant between BiAb004(hG1WT) and Fc ⁇ RIIa_H131.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 200nM, 100nM, 50nM, 25nM, and 12.5nM, respectively.
  • Fig. 9 A graph showing the detection results of the affinity constant between BiAb004(hG1TM) and Fc ⁇ RIIa_R131.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 200nM, 100nM, 50nM, 25nM, and 12.5nM, respectively.
  • Fig. 10 A graph of the detection results of the affinity constant between BiAb004(hG1WT) and Fc ⁇ RIIa_R131.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 200nM, 100nM, 50nM, 25nM, and 12.5nM, respectively.
  • FIG. 11 A graph showing the detection results of the affinity constant between BiAb004(hG1TM) and Fc ⁇ RIIb.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 200nM, 100nM, 50nM, 25nM, and 12.5nM, respectively.
  • Fig. 12 A graph of detection results of affinity constants between BiAb004(hG1WT) and Fc ⁇ RIIb.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 200nM, 100nM, 50nM, 25nM, and 12.5nM, respectively.
  • Figure 13 A graph of the detection results of the affinity constant between BiAb004(hG1TM) and C1q.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 20nM, 10nM, 5nM, 2.5nM, 1.25nM, respectively.
  • Figure 14 A graph of the detection results of the affinity constant between BiAb004(hG1WT) and C1q.
  • concentration of antigen added to each pair of curves from top to bottom in the figure is 20nM, 10nM, 5nM, 2.5nM, 1.25nM respectively.
  • Figure 15 A graph of the detection results of the affinity constant between 5C10H2L2-IgG1mt and C1q.
  • concentration of antigen added to each pair of curves from top to bottom in the figure is 20nM, 10nM, 5nM, 2.5nM, 1.25nM respectively.
  • Figure 16 Detection of ADCC activity of BiAb004(hG1WT) and BiAb004(hG1TM) on 293T-CTLA4-PD1 cells expressing CTLA-4 and PD-1 antigens.
  • Figure 17 The results of IFN- ⁇ secretion induced by mixed lymphocytes induced by BiAb004(hG1TM) combined with cioronib.
  • Fig. 18 The result graph of the secretion of IL-2 induced by BiAb004(hG1TM) combined with Cioronib in mixed lymphocytes.
  • mice were purchased from Guangdong Medical Experimental Animal Center.
  • Human peripheral blood mononuclear cells were isolated and prepared in Zhongshan Kangfang Biomedical Co., Ltd., and were informed by the provider.
  • Raji-PDL1 is a cell expressing human PD-L1 constructed by Zhongshan Akeso Biomedical Co., Ltd. based on human B cell line Raji cells through transfection.
  • Ficoll-Paque TM PLUS (or Ficoll-Paque PLUS) was purchased from GE Healthcare.
  • Human IL-2ELISA Kit was purchased from Dakowei Biotechnology Co., Ltd.
  • RPMI 1640 medium, DMEM medium, Trypsin-EDTA (0.25%) phenol red, and Blasticidin were all from Gibco.
  • FBS comes from Excell bio.
  • the isotype control antibody is human anti-Hen Egg Lysozyme IgG (human anti-Hen Egg Lysozyme IgG, anti-HEL antibody, or human IgG, referred to as hIgG)
  • the sequence comes from Affinity maturation increases the stability and plasticity of the published by Acierno et al. Variable region sequence of the Fab F10.6.6 sequence in the Fv domain of anti-protein antibodies (Acierno et al. J Mol Biol. 2007; 374(1):130-146.).
  • Theoloni used is from Shenzhen Microchip Biotechnology Co., Ltd.
  • 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 coding nucleic acid sequences are identical to those of 4G10, 4G10H1L1 and 4G10H3L3 in Chinese Patent Publication CN 106967172A.
  • amino acid sequences of the heavy chain and light chain of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as the coding nucleic acid sequence are the same as 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A.
  • bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) belong to the Morrison pattern (IgG-scFv), that is, the C-terminals of the two heavy chains of an IgG antibody are linked by The fragments were linked to scFv fragments of another antibody whose heavy and light chains were designed as 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) is to mutate individual amino acids in the framework region on the basis of 4G10H1V, 4G10L1V, 4G10H3V, and 4G10L3V, which effectively optimizes the structure of the antibody and improves its effectiveness.
  • BiAb004(M) is also called BiAb004(hG1WT) in the embodiments of the present invention.
  • the above-mentioned BiAb004(M) is used as "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 inventor introduced a point mutation (L234A) from leucine to alanine at site 234 of its heavy chain, site 235 A point mutation from leucine to alanine (L235A) was introduced, and a point mutation from glycine to alanine (G237A) was introduced at the No. 237 site to obtain BiAb004 (hG1TM).
  • the Fc receptor Fc ⁇ RI also known as (CD64), can bind to the Fc end of an IgG antibody and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with Fc ⁇ RI to evaluate the potential ADCC and ADCP activities of each antibody.
  • the sample dilution buffer is a solution of PBS, 0.02% Tween-20, 0.1% BSA, and pH 7.4.
  • a solution of Fc ⁇ RI (purchased from Sinobio) at a concentration of 1 ⁇ g/mL was added to the HIS1K sensor for a fixation time of 50 s, so that Fc ⁇ RI was immobilized on the surface of the sensor.
  • the determination of the binding and dissociation parameters of the antibody to Fc ⁇ RI was carried out in a buffer solution, and the antibody concentration was 3.12-50 nM (two-fold serial dilution).
  • the sensor immobilized with antigen was equilibrated in the buffer solution for 60 s, and the binding time of Fc ⁇ RI immobilized on the sensor to each antibody was measured for 120 s; the dissociation time of Fc ⁇ RI and antibody was measured for 120 s.
  • the detection temperature is 30° C., and the frequency is 0.3 Hz.
  • Data were analyzed with a 1:1 model fit to obtain affinity constants for each antibody and FcyRI.
  • the Fc receptor Fc ⁇ RIIIa_V158 (also known as CD16a_V158) can bind to the Fc end of an IgG antibody to mediate the ADCC effect.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constant of BiAb004(hG1WT), and BiAb004(hG1TM) and Fc ⁇ RIIIa_V158 to evaluate the ADCC activity of each antibody.
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4.
  • 5 ⁇ g/mL Fc ⁇ RIIIa_V158 is immobilized on the HIS1K sensor
  • the time is 120s, the sensor is equilibrated in the buffer for 60s, Fc ⁇ RIIIa_V158 immobilized on the sensor binds to each antibody at a concentration of 31.25-500nM (two-fold dilution) for 60s, and the antibody dissociates in the buffer for 60s.
  • the sensor was regenerated using 10 mM glycine, pH 1.5, for 5 s, and repeated 4 times.
  • the detection temperature is 30° C., and the frequency is 0.3 Hz.
  • the data were analyzed with a 1:1 model fit to obtain affinity constants.
  • Fc receptor Fc ⁇ RIIIa_F158 (also known as CD16a_F158) can bind to the Fc end of IgG antibody to mediate ADCC.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with Fc ⁇ RIIIa_F158 to evaluate the ADCC activity of each antibody.
  • the Fortebio Octet molecular interaction instrument detects the affinity constant of BiAb004 (hG1WT) and BiAb004 (hG1TM) and Fc ⁇ RIIIa_F158.
  • the experimental method is as follows: the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4.
  • Fc ⁇ RIIIa_F158 was immobilized on the HIS1K sensor for 120s, the sensor was equilibrated in the buffer for 60s, and the Fc ⁇ RIIIa_F158 immobilized on the sensor was mixed with Each antibody is combined, the antibody concentration is 31.25-500nM (two-fold dilution), the time is 60s, and the antibody is dissociated in the buffer, the time is 60s.
  • the sensor was regenerated using 10 mM glycine, pH 1.5, for 5 s, and repeated 4 times. The detection temperature is 30° C., and the frequency is 0.3 Hz. The data were analyzed with a 1:1 model fit to obtain affinity constants.
  • Fc receptor Fc ⁇ RIIa_H131 also known as (CD32a_H131), can bind to the Fc end of IgG antibody and mediate ADCC.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with Fc ⁇ RIIa_H131 to evaluate the ADCC activity of each antibody.
  • Fortebio Octet molecular interaction instrument detects the affinity constant of BiAb004(hG1WT) and BiAb004(hG1TM) with Fc ⁇ RIIa_H131.
  • the dilution buffer is PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH7.4.
  • 5 ⁇ g/mL Fc ⁇ RIIa_H131 is immobilized on the NTA sensor, the immobilization height is about 1.0nm, and the sensor is in PBS, 0.02% Tween- 20.
  • Fc receptor Fc ⁇ RIIa_R131 (also known as CD32a_R131) can bind to the Fc end of IgG antibody to mediate ADCC.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) with Fc ⁇ RIIa_R131 to evaluate the ADCC activity of each antibody.
  • the solidified product diluent is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4, analyte dilution buffer
  • the solution is PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH7.4.
  • 5 ⁇ g/mL Fc ⁇ RIIa_R131 is immobilized on the NTA sensor, and the fixed height is about 1.0nm.
  • the sensor is in PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, equilibrate in pH7.4 buffer for 300s to block the sensor, Fc ⁇ RIIa_R131 immobilized on the sensor is bound to the antibody, the antibody concentration is 12.5-200nM (two-fold serial dilution), time 60s, the antibody in the buffer Medium dissociation, time 60s.
  • the sensor uses 10mM Glycine, pH1.7 And 10mM nickel sulfate regeneration.
  • the detection temperature is 30° C., and the frequency is 0.6 Hz.
  • the data were analyzed with a 1:1 model fit to obtain affinity constants.
  • Fc receptor Fc ⁇ RIIb (also known as CD32b) can bind to the Fc end of IgG antibody, negatively regulate the function of immune cells, inhibit the activation and proliferation of immune cells, and inhibit the secretion of cytokines.
  • the Fortebio Octet molecular interaction instrument was used to detect the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) and Fc ⁇ RIIb to evaluate the binding ability of BiAb004(hG1WT) and BiAb004(hG1TM) to Fc receptors.
  • Fortebio Octet molecular interaction instrument detects the affinity constant of BiAb004 (hG1WT) and BiAb004 (hG1TM) and Fc ⁇ RIIb.
  • the dilution buffer is PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH7.4.
  • 5 ⁇ g/mL Fc ⁇ RIIb is immobilized on the NTA sensor, the immobilization height is about 1.0nm, and the sensor is in PBS, 0.02% Tween- 20.
  • Serum complement C1q can bind to the Fc end of IgG antibody and mediate the CDC effect.
  • the ability of a therapeutic monoclonal antibody to bind to C1q affects the safety and efficacy of that antibody.
  • the Fortebio Octet molecular interaction instrument detects the affinity constant of the corresponding antibody and C1q.
  • the experimental method is briefly described as follows: the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4. 50 ⁇ g/mL antibody is immobilized on the FAB2G sensor Above, the fixed height is about 2.0nm, the sensor is equilibrated in the buffer for 60s, the antibody immobilized on the sensor is combined with the antigen C1q, the antigen concentration is 1.25-20nM (two-fold serial dilution), the time is 60s, the antigen and antibody are in the buffer Dissociation, time 60s.
  • the sensor was regenerated using 10 mM glycine, pH 1.7, for 5 s, and repeated 4 times.
  • the vibration rate of the sample plate is 1000rpm
  • the detection temperature is 30 degrees
  • the detection frequency is 0.6Hz.
  • the data were analyzed with a 1:1 model fit to obtain affinity constants.
  • the data acquisition software is Fortebio Data Acquisition 7.0
  • the data analysis software is Fortebio Data Analysis 7.0.
  • the ADCC effect means that the effector immune cells with killing activity recognize the Fc segment of the antibody bound to the target cell antigen through the Fc receptor (FcR) expressed on its surface, and directly kill the target cell.
  • FcR Fc receptor
  • the inventors constructed 293T-CTLA4-PD1 cells expressing PD-1 and CTLA4 antigens and primary PBMC The culture system was used to detect the ADCC activity of the antibody.
  • A375 cells were collected by conventional digestion and centrifugation (purchased from ATCC, agented by Zhongyuan Company, product number: CRL-1619), spread on 10 cm plates at 2 ⁇ 10 6 cells/dish, and 1 dish was added with Theoroni (from Microchip Biotechnology). , batch number: 210604) to a final concentration of 15 ⁇ M, and a blank control group was set, and incubated at 37° C. in a 5% CO 2 incubator for 48 hours (medium: DMEM+10% FBS).
  • the detection results of IFN- ⁇ secretion after mixed culture of DC, PBMC and A375 cells are shown in Fig. 17 .
  • the results of IL-2 secretion detection after mixed culture of DC, PBMC and A375 cells are shown in FIG. 18 .
  • bifunctional antibody BiAb004(hG1TM) single-drug group, Theoronib single-drug group, and the bifunctional antibody BiAb004(hG1TM) combined with Theoronib group can effectively promote the IFN- ⁇ and IL-2 secretion.
  • the ability of bifunctional antibody BiAb004(hG1TM) combined with Theoronib to promote the secretion of IFN- ⁇ and IL-2 was significantly stronger than that of bifunctional antibody BiAb004(hG1TM) single drug group and Theoronib single drug group .

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Abstract

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双特异性抗体和西奥罗尼的药物组合。具体地，所述的药物组合，其包含西奥罗尼或其可药用盐或其晶型，以及至少一种抗CTLA4-抗PD-1双特异性抗体。本发明还涉及所述药物组合的用途。

Description

包含抗CTLA4-抗PD-1双特异性抗体和西奥罗尼的药物组合 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双特异性抗体和西奥罗尼的药物组合及其用途。具体地，本发明涉及一种包含突变的抗CTLA4-抗PD-1的双特异性抗体和西奥罗尼或其可药用盐或其晶型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 1 ligand 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 sociated 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)。
ADCC(antibody-dependent cell-mediated cytotoxicity)即抗体依赖的细胞介导的细胞毒性作用，是指抗体的Fab段结合病毒感染的细胞或肿瘤细胞的抗原表位，其Fc段与杀伤细胞(NK细胞、巨噬细胞等)表面的Fc受体(Fc Receptor,FcR)结合，介导杀伤细胞直接杀伤靶细胞。
CDC(complement dependent cytotoxicity)即补体依赖的细胞毒性，是指当抗体 与细胞膜表面相应抗原特异性结合后，形成复合物而激活补体系统，进而在靶细胞表面形成MAC，导致靶细胞溶解。补体能导致多种细菌和其他病原生物细胞的溶解，是机体抵抗病原生物感染的重要防御机制。
Fc受体是表达在特定免疫细胞表面，用于识别抗体Fc区域介导免疫应答的免疫球蛋白家族蛋白。抗体Fab区域识别抗原后，其抗体Fc区域与免疫细胞(如杀伤细胞)上的Fc受体结合，启动免疫细胞的应答功能，如吞噬作用及ADCC。
根据Fc受体识别抗体类型及表达细胞的不同，Fc受体主要分为FcγR、FcαR和FcεR三种类型，FcγR又可分为FcγRI(亦称为CD64)、FcγRII(亦称为CD32)、FcγRIII(亦称为CD16)和FcRn(亦称为Neonatal Fc receptor)四种亚型。其中FcγRI、FcγRII、FcγRIII与ADCC效应密切相关。FcγRIII是介导ADCC的最主要分子，在不同细胞类型中具有高度同源的FcγRIIIa和FcγRIIIb两种亚型，在FcγRIIIa人群中存在单核干多态性(SNP)位点引起的高亲和力FcγRIIIa亚型，分别称为FcγRIIIa_V158和低亲和力FcγRIIIa_F158两个亚型。FcγRI对IgG的Fc区域具有较高的亲和力，参与ADCC过程；FcγRII有FcγRIIa，FcγRIIb和FcγRIIc(亦分别称为CD32a，CD32b，CD32c)三个亚型，其中FcγRIIa具有ADCC活性；FcγRIIa在人群中存在由于单核苷酸突变导致的两种亚型，分别称为FcγRIIa_H131和FcγRIIa_R131；FcγRIIb为抑制性受体，FcγRIIb是典型的抑制性FcγR，可抑制附近的ITAM通路。例如，免疫复合物与BCR结合后，Fc段会结合同一细胞上的FcγRIIb，负性调节B细胞激活，减少抗体、细胞因子的分泌(Hogarth PM,Pietersz GA.2012,NATURE REVIEWS DRUG DISCOVERY,11(4):311-331)。
IgG家族包含四个成员，IgG1、IgG2、IgG3和IgG4，它们重链恒定区的可结晶片段(fragment crystallizable，Fc)区域存在氨基酸的差异，导致它们与FcγRs的亲和力各不相同。IgG1是人体内最多的亚型，也是单抗药物中用的最多的亚型，IgG1能够结合各种FcγRs，能够引发ADCC以及CDC效应。IgG2与FcγRs的亲和力最弱，但是IgG2仍能够通过与FcγRIIa的结合引发单核细胞介导的ADCC。IgG3与FcγRs的结合能力最强，能引发ADCC，且CDC效应比IgG1更强。IgG4分子与FcγRI之外的FcγRs结合较弱，IgG4分子引起CDC和NK细胞介导的ADCC的可能性较低；然而，IgG4亚型抗体可通过与FcγRI的结合介导ADCP效应，靶向于免疫细胞的抗体药物存在ADCP效应可能会导致免疫细胞损伤，具有影响药物药理学负面效应。目前，尚需要开发新的抗CTLA4-抗PD-1双特异性抗体，以减少或消除抗体介导的 ADCC、ADCP和/或CDC活性对抗CTLA4-抗PD-1双特异性抗体所结合的免疫细胞造成的损伤，提高抗体药物药效。
化疗药物目前主要分为下述九类(赫捷等，临床肿瘤学，北京：人民卫生出版社，2016：230-237)第一类为直接与DNA结合，阻止DNA复制的药物，包括各种炕化剂、丝裂霉素和博来霉素、达卡巴嗪、铂类药物如顺铂和卡铂、喜树碱类药物及其衍生物；第二类为阻止核酸生物合成的药物，这类药物主要影响瘤细胞的酶系，使DNA和RNA的前体物合成受阻，从而抑制DNA或RNA形成，主要包括甲氨蝶岭、氟尿嘧啶、6-巯基嘌呤、羟基脲和阿糖胞苷等，主要作用于S期细胞，属抗代谢类化疗药，为周期特异性抗癌药；第三类为影响转录的化疗药物，主要药理作用是插入DNA双螺旋与其形成非共价结合，从而干扰DNA上的遗传信息转录到依赖DNA的mRNA上，导致模报功能受到损害、转录受阻，主要包括。第四类为影晌微管蛋白和有丝分裂的药物主要包括长春碱类、鬼臼毒素类和紫杉醇类等植物药；第五类为影晌核糖体功能，阻止蛋白质合成的药物以二尖杉醋碱类植物药为代表，它能抑制蛋白质合成的起始阶段，使核糖体分解并释出新生肽链，但不能阻止mRNA和tRNA与核糖体的结合这类药可使核DNA和胞质RNA减少、多聚核糖体解聚，并拥制有丝分裂；第六类为影晌细胞膜的药物植物凝集素如刀豆素(Con-A)和植物凝集素(PHA)等可以和细胞膜上的糖蛋白受体结舍，从而影响瘤细胞的DNA合成，阻止瘤细胞分裂；第七类为诱导细胞凋亡(apoptosis)的药物，如三氧化二砷等；第八类为激素类主要通过调节内分泌米治疗肿瘤，包括雌激素类、抗雌激素类、孕激素类、雄激素类、抗雄激素类、肾上腺皮质激素类、抗肾上腺皮质激素类(包括氯苯二氯乙烷和氨鲁米特)；第九类为抗肿瘤靶点治疗，包括单克隆抗体、表皮生长因子信号传导抑制剂(如针对受体型酪氨酸激酶通路的靶向药物)、泛素蛋白酶体抑制剂、血管生成抑制剂等。
西奥罗尼(Chiauranib)是由深圳微芯生物科技股份有限公司开发的一种多靶点多通路选择性激酶抑制剂。西奥罗尼可选择性抑制Aurora B、CSF1R和VEGFR/PDGFR/c-Kit等多个激酶靶点，从而抑制肿瘤细胞增殖、增强抗肿瘤免疫以及抑制肿瘤血管生成，实现多通路机制的抗肿瘤药效。西奥罗尼在前期的临床试验中，针对多线治疗失败的小细胞肺癌(SCLC)患者，相比于历史对照数据，其单药治疗取得了十分积极的疗效结果。中国专利文献(公开号：CN 111728974 A)披露了西奥罗尼化合物的结构，为N-(2-氨基苯基)-6-(7-甲氧基喹啉-4-氧)-1-萘酰胺，其结构式如下面的式I所示。
然而，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，5年生存率仍然很低。因此，开发更低毒性、更有效的治疗手段或者联合给药治疗方案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抗体与西奥罗尼适当地联用，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，其包含西奥罗尼或其可药用盐(例如盐酸盐)或其晶型(例如非溶剂化晶型A、B或C)，以及至少一种(例如1种、2种或3种)抗CTLA4-抗PD-1双特异性抗体。
在本发明的一些实施方式中，所述的药物组合，其中，所述西奥罗尼或其可药用盐或其晶型与所述抗CTLA4-抗PD-1双特异性抗体的质量比为1：(1-1000)，优选为1：(5-500)，更优选为1：(10-100)。
在本发明的一些实施方式中，所述的药物组合，其中，所述西奥罗尼或其可药用盐或其晶型与所述抗CTLA4-抗PD-1双特异性抗体的质量比为1：(5-1000)，优选为1：(10-500)，更优选为1：(10-100)、1：(10-150)、1：(10-200)、1：(10-250)、1：(10-300)、1：(10-350)、1：(10-400)或1：(10-450)。
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，所述西奥罗尼或其可药用盐或其晶型的单位剂量为0.1mg-100mg、0.5mg-50mg、1mg-20mg、2mg-15mg、3mg-12mg、4mg-8mg或者5mg。
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；或者
所述药物组合是非固定组合，例如所述非固定组合中的西奥罗尼或其可药用盐或其晶型、抗CTLA4-抗PD-1双特异性抗体各自存在于独立的药物组合物中。
在本发明的一些实施方式中，所述的药物组合物，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西奥罗尼或其可药用盐或其晶型和抗CTLA4-抗PD-1双特异性抗体其它活性药物成分。
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；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: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；
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具有如下突变：
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；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: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；
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具有如下突变组合之一：
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具有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，所述免疫球蛋白或其抗原结合片段以大于大约10-7M，例如大于大约10-6M、10-5M、10-4M或10-3M或更大的亲和力常数结合FcγRIIIa_F158、FcγRI、FcγRIIa_H131、FcγRIIIa_V158和/或FcγRIIb；优选地，所述亲和力常数通 过Fortebio Octet分子相互作用仪测得；
优选地，所述免疫球蛋白或其抗原结合片段与FcγRIIIa_F158、FcγRI、FcγRIIa_H131、FcγRIIIa_V158和/或FcγRIIb没有结合信号或者结合信号小于0.1nm；优选地，所述结合信号是指通过Fortebio Octet分子相互作用仪测得的响应值。
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，所述免疫球蛋白或其抗原结合片段以大于大约10-9M，例如大于大约10-8M、10-7M、10-6M或10-5M或更大的亲和力常数结合C1q；优选地，所述亲和力常数通过Fortebio Octet分子相互作用仪测得；
优选地，所述免疫球蛋白或其抗原结合片段与C1q没有结合信号或者结合信号小于0.1nm；优选地，所述结合信号是指通过Fortebio Octet分子相互作用仪测得的响应值。
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(GGGGS)n，n为正整数；优选地，n为1、2、3、4、5或6。
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-1 chain 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，所述单链抗体连接在免疫球蛋白的重链的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和第二产品，
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；
所述第二产品包含西奥罗尼或其药学上可接受的盐(例如盐酸盐)或其晶型(例如非溶剂化晶型A、B或C)；
优选地，所述第一产品和所述第二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。
在本发明的一个或多个实施方案中，所述的药盒产品，其中，所述第二产品的单位剂量，按照其中的西奥罗尼或其可药用盐或其晶型的质量计算，为0.1mg-100mg、0.5mg-50mg、0.5mg-10mg、1mg-10mg、2mg-8mg或者1mg-5mg或者5mg。
在本发明的一个或多个实施方案中，所述的药盒产品，其中，所述第二产品的单位剂量，按照其中的西奥罗尼或其可药用盐或其晶型的质量计算，为1mg-20mg、2mg-15mg、3mg-12mg、4mg-8mg或者5mg。
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、胰腺癌和鼻咽癌中的一种或多种；
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，所述胃癌为胃腺癌或食管结合部腺癌；
优选地，所述肿瘤为具有MSI-H/dMMR表型的实体瘤；优选地，所述肿瘤选自具有MSI-H/dMMR表型的下述肿瘤中的一种或多种：
结肠癌、直肠癌、子宫内膜癌、胃癌、间皮瘤、肉瘤、肾上腺皮质癌、恶性黑色素瘤和卵巢生殖细胞肿瘤。
MSI(microsatellite instability)是微卫星不稳定性。微卫星(Microsatellite)是遍布于人类基因组中的短串联重复序列,有单核苷酸、双核苷酸或高位核苷酸的重复，重复次数10-50次。与正常细胞相比，某些异常组织细胞，如肿瘤，内的微卫星由于重复单位的插入或缺失而导致微卫星长度的改变，就叫做MSI。根据MSI不稳定性和程度，可分为微卫星高度不稳定型(MSI-H)、微卫星低度不稳定型(MSI-L)和微卫星稳定型(MSS)。造成MSI的主要原因是DNA错配修复(mismatch repair，MMR)功能缺陷。人类错配修复基因(MMR基因)经转录翻译后可表达相应的错配修复蛋白，如果任一MMR蛋白表达缺失可造成细胞的错配修复功能缺陷，则对DNA复制过程中的碱基错配丧失修复功能而造成累积，导致微卫星不稳定(MSI)的发生，约15％的结直肠癌是经由MSI途径引发的。最早发现于结直肠癌，也可发生于胃癌、子宫内膜癌、肾上腺皮质肿瘤等(Baretti M等人.Pharmacol Ther.2018；189:45‐62.)，后续研究在间皮瘤、肉瘤、肾上腺皮质癌、恶性黑色素瘤和卵巢生殖细胞肿瘤中亦发现MSI-H/dMMR特征。
MSI-H和dMMR代表两种不同检测方法所产生的结果，dMMR与MSI-H在生物学上具有一致性，称为MSI-H/dMMR或者MSI-high/dMMR，MSI-L和MSS则是MMR正常(proficient mismatch repair，pMMR)的表型。dMMR检测方法为基于肿瘤标本(包括手术标本、穿刺标本)进行MSH2、MLH1、MSH6以及PMS2四个错配基因的免疫组化蛋白检测，若四个蛋白里面任何一个蛋白表达缺失则为dMMR；如四个蛋白均为阳性表达则肿瘤为pMMR，即错配修复功能完整。MSI的检测为肿瘤细胞和体细胞重复DNA序列(微卫星序列)的长度进行一一配对检测，并比较长度。当使用PCR方法并基于美国NCI标准检测5个标准位点时，如果其中有两个或两个以上位点不一致则为不稳定，定义为MSI-H，如果一个位点不一致则称为MSI-L(微卫星低度不稳定)，如5个位点均一致则为MSS。高通量测序(High-throughput sequencing)(或称下一代测序，Next-generation sequencing technology，NGS)亦可作为检测微卫星不稳定性的方法。当选用更多的微卫星位点，如多于5个或者其他一些微卫星位点，进行PCR检测时，通常≥30％的位点不一致就叫MSI-H，所有位点都一致就定义为MSS，在0到30％之间是MSI-L。
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、胰腺癌和鼻咽癌中的一种或多种；
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，所述胃癌为胃腺癌或食管结合部腺癌；
优选地，所述肿瘤为具有MSI-H/dMMR表型的实体瘤；优选地，所述肿瘤选自具有MSI-H/dMMR表型的下述肿瘤中的一种或多种：
结肠癌、直肠癌、子宫内膜癌、胃癌、间皮瘤、肉瘤、肾上腺皮质癌、恶性黑色素瘤和卵巢生殖细胞肿瘤。
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、胰腺癌和鼻咽癌中的一种或多种；
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，所述胃癌为胃腺癌或食管结合部腺癌；
优选地，所述肿瘤为具有MSI-H/dMMR表型的实体瘤；优选地，所述肿瘤选自具有MSI-H/dMMR表型的下述肿瘤中的一种或多种：
结肠癌、直肠癌、子宫内膜癌、胃癌、间皮瘤、肉瘤、肾上腺皮质癌、恶性黑色素瘤和卵巢生殖细胞肿瘤。
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1-10mg(例如1mg、2mg、3mg、4mg、5mg、6mg、7mg、8mg、9mg或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。
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双特异性抗体和西奥罗尼同时或顺序施用。
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)-(13)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“添加)，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“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。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。
如本文中所使用的，术语“完全消除”是指通过现有的仪器设备(例如Fortebio Octet分子相互作用仪)检测显示没有结合信号或结合信号极低。在本发明的一个实施方案中，没有结合信号或结合信号极低是指结合信号(即响应值或Response)低于0.1nm。
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。
西奥罗尼的晶型包括但不限于非溶剂化晶型A、非溶剂化晶型B和非溶剂化晶型C，参照中国专利授权CN 107868044 B中的记载，其中：
所述非溶剂化晶型A的X-射线粉末衍射图在反射角2θ为6.88°、9.26°、12.74°、13.82°、18.58°、20.86°和25.72°处有特征峰；
所述非溶剂化晶型B的X-射线粉末衍射图在反射角2θ为4.88°、9.68°、12.74°、14.52°、17.72°、24.30°、25.26°处具有特征峰；
所述非溶剂化晶型C的X-射线粉末衍射图在反射角2θ为4.84°、9.68°、12.92°、14.60°、16.46°、17.44°、22.00°、25.28°处具有特征峰。
发明的有益效果
本发明实现了如下的(1)至(2)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双特异性抗体与西奥罗尼具有协同作用，取得了治疗或预防肿瘤协同效果；其中所述肿瘤包括但不限于：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、胰腺癌或鼻咽癌；优选地，所述肺癌为非小细胞性肺癌、小细胞性肺癌或肺鳞癌；优选地，所述胃癌为胃腺癌或食管结合部腺癌。
附图说明
图1：BiAb004(hG1TM)与FcγRI的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为50nM，25nM，12.5nM，6.25nM，3.12nM。
图2：BiAb004(hG1WT)FcγRI的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为50nM，25nM，12.5nM，6.25nM，3.12nM。
图3：BiAb004(hG1TM)与FcγRIIIa_V158的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为500nM，250nM，125nM，62.5nM，31.25nM。
图4：BiAb004(hG1WT)与FcγRIIIa_V158的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为500nM，250nM，125nM，62.5nM，31.25nM。
图5：BiAb004(hG1TM)与FcγRIIIa_F158的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为500nM，250nM，125nM，62.5nM，31.25nM。
图6：BiAb004(hG1WT)与FcγRIIIa_F158的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为500nM，250nM，125nM，62.5nM，31.25nM。
图7：BiAb004(hG1TM)与FcγRIIa_H131的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为200nM，100nM，50nM，25nM，12.5nM。
图8：BiAb004(hG1WT)与FcγRIIa_H131的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为200nM，100nM，50nM，25nM，12.5nM。
图9：BiAb004(hG1TM)与FcγRIIa_R131的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为200nM，100nM，50nM，25nM，12.5nM。
图10：BiAb004(hG1WT)与FcγRIIa_R131的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为200nM，100nM，50nM，25nM，12.5nM。
图11：BiAb004(hG1TM)与FcγRIIb的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为200nM，100nM，50nM，25nM，12.5nM。
图12：BiAb004(hG1WT)与FcγRIIb的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为200nM，100nM，50nM，25nM，12.5nM。
图13：BiAb004(hG1TM)与C1q的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为20nM，10nM，5nM，2.5nM，1.25nM。
图14：BiAb004(hG1WT)与C1q的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗原的浓度分别为20nM，10nM，5nM，2.5nM，1.25nM。
图15：5C10H2L2-IgG1mt与C1q的亲和力常数检测结果图。图中从上到下的每对曲线中加入抗原的浓度分别为20nM、10nM、5nM、2.5nM、1.25nM。
图16：BiAb004(hG1WT)和BiAb004(hG1TM)对表达CTLA-4和PD-1抗原的293T-CTLA4-PD1细胞的ADCC活性检测。
图17：BiAb004(hG1TM)联用西奥罗尼诱导混合淋巴细胞分泌IFN-γ结果图。
图18：BiAb004(hG1TM)联用西奥罗尼诱导混合淋巴细胞分泌IL-2结果图。
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：
BALB/c小鼠购自广东省医学实验动物中心。
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，且经提供者知情同意。
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GE Healthcare。
Human IL-2ELISA Kit购自达科为生物技术股份有限公司。
RPMI 1640培养基、DMEM培养基、Trypsin-EDTA(0.25％)phenol red、Blasticidin均来源于Gibco。
FBS来源于Excell bio。
同型对照抗体为人抗鸡蛋溶酶体抗体(human anti-Hen Egg Lysozyme IgG，即anti-HEL抗体，或者human IgG，简称hIgG)的序列来自于Acierno等人发表的Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies中Fab F10.6.6序列的可变区序列(Acierno等人.J Mol Biol.2007；374(1):130-146.)。
在本发明的下述实施例中，使用的西奥罗尼来自深圳微芯生物科技股份有限公司。
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片段，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-1 chain 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。
实验例1:BiAb004(hG1WT)和BiAb004(hG1TM)的受体FcγRI亲和力检测
Fc受体FcγRI又名(CD64)可与IgG抗体的Fc端结合，参与抗体依赖细胞介导的细胞毒作用(ADCC)。治疗性单克隆抗体与Fc受体结合的能力影响到该抗体的安全性和有效性。
本实验使用Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRI的亲和力常数，以评价各抗体的潜在的ADCC、ADCP活性。
Fortebio Octet分子相互作用仪检测相应抗体与FcγRI的亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4的溶液。HIS1K传感器上加入浓度为1μg/mL的FcγRI(购自Sinobio)溶液，固定时间50s，以使得FcγRI固定于传感器表面。抗体与FcγRI的结合和解离参数的测定均在缓冲液中进行，抗体浓度为3.12－50nM(两倍梯度稀释)。固定有抗原的传感器在缓冲液中平衡60s后测定固定在传感器上的FcγRI与各抗体的结合，时间120s；FcγRI与抗体的解离测定时间为120s。检测温度为30℃，频率为0.3Hz。数据以1:1模型拟合分析，以得到各抗体和FcγRI的亲和力常数。
FcγRI与BiAb004(hG1TM)和BiAb004(hG1WT)的亲和力常数测定结果如下面的表1以及图1、图2所示。
表1:BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRI结合的动力学参数
结果显示，BiAb004(hG1WT)可以与FcγRI结合，亲和力常数为5.92E-09M；而BiAb004(hG1TM)由于与FcγRI没有结合信号或信号极低，故未能获得相应数据，认为其不与FcγRI结合。
结果表明，相对于BiAb004(hG1WT)，BiAb004(hG1TM)被有效的消除了其与FcγRI的结合活性。
实验例2：FcγRIIIa_V158与BiAb004(hG1WT)和BiAb004(hG1TM)的亲和力常 数测定
Fc受体FcγRIIIa_V158(又名CD16a_V158)，可与IgG抗体的Fc端结合，介导ADCC效应。
实验中使用Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)，和BiAb004(hG1TM)与FcγRIIIa_V158的亲和力常数，以评价各抗体的ADCC活性。
Fortebio Octet分子相互作用仪检测相应抗体与FcγRIIIa_V158的亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。5μg/mL的FcγRIIIa_V158固定在HIS1K传感器上，时间120s，传感器在缓冲液中平衡60s， 固定在传感器上的FcγRIIIa_V158与各抗体结合，抗体浓度为31.25－500nM(两倍稀释)，时间60s，抗体在缓冲液中解离，时间60s。传感器使用10mM甘氨酸，pH1.5再生，时间5s，重复4次。检测温度为30℃，频率为0.3Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRIIIa_V158与BiAb004(hG1TM)和BiAb004(hG1WT)的亲和力常数测定结果如下面的表2以及图3、图4所示。
表2：BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIIa_V158结合的动力学参数
结果显示，BiAb004(hG1WT)可以与FcγRI_V158结合，亲和力常数为1.77E-07M；而BiAb004(hG1TM)由于与FcγRIIIa_V158没有结合信号或信号极低，故未能获得相应数据，认为其不与FcγRIIIa_V158结合。
结果表明，相对于BiAb004(hG1WT)，BiAb004(hG1TM)被有效的消除了其与FcγR IIIa_V158的结合活性。
实验例3：FcγRIIIa_F158与BiAb004(hG1WT)和BiAb004(hG1TM)的亲和力常 数测定
Fc受体FcγRIIIa_F158(又名CD16a_F158)可与IgG抗体的Fc端结合，介导ADCC。
本实验使用Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)，和BiAb004(hG1TM)与FcγRIIIa_F158的亲和力常数，以评价各抗体的ADCC活性。
Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIIa_F158的亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。5μg/mL的FcγRIIIa_F158固定在HIS1K传感器上，时间120s，传感器在缓冲液中平衡60s，固定在传感器上的FcγRIIIa_F158与 各抗体结合，抗体浓度为31.25-500nM(两倍稀释)，时间60s，抗体在缓冲液中解离，时间60s。传感器使用10mM甘氨酸，pH1.5再生，时间5s，重复4次。检测温度为30℃，频率为0.3Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRIIIa_F158与BiAb004(hG1TM)和BiAb004(hG1WT)的亲和力常数测定结果如下面表3以及图5、图6所示。
表3：BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIIa_F158结合的动力学参数
结果显示，BiAb004(hG1WT)可以与FcγRIIIa_F158结合，亲和力常数为2.21E-07M；而BiAb004(hG1TM)由于与FcγRIIIa_F158没有结合信号或信号极低没有结合，故未能获得相应数据，认为其不与FcγRIII_F158结合。
结果表明，相对于BiAb004(hG1WT)，BiAb004(hG1TM)被有效的消除了其与FcγR IIIa_F158的结合活性。
实验例4：FcγRIIa_H131与BiAb004(hG1WT)和BiAb004(hG1TM)的亲和力常数 测定
Fc受体FcγRIIa_H131又名(CD32a_H131)可与IgG抗体的Fc端结合，介导ADCC。
本实验使用Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIa_H131的亲和力常数，以评价各抗体的ADCC活性。
Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIa_H131的亲和力常数实验方法简述如下：固化物稀释液为PBS，0.02％Tween-20，0.1％BSA，pH7.4，分析物稀释缓冲液为PBS，0.02％Tween-20，0.02％casein，0.1％BSA，pH7.4。5μg/mL的FcγRIIa_H131固定在NTA传感器上，固定高度约为1.0nm，传感器在PBS，0.02％Tween-20，0.02％casein，0.1％BSA，pH7.4缓冲液中平衡300s以封闭传感器，固定在传感器上的FcγRIIa_H131与抗体结合，抗体浓度为 12.5-200nM(两倍梯度稀释)，时间60s，抗体在缓冲液中解离，时间60s。传感器使用10mM Glycine，pH1.7和10mM硫酸镍再生。检测温度为30℃，频率为0.6Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRIIa_H131与BiAb004(hG1TM)和BiAb004(hG1WT)的亲和力常数测定结果如下面的表4以及图7、图8所示。
表4：BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIa_H131结合的动力学参数
结果显示，BiAb004(hG1WT)可以与FcγRIIa_H131结合，亲和力常数为2.22E-08M；而BiAb004(hG1TM)由于与FcγRIIa_H131没有结合信号或信号极低，故未能获得相应数据，认为其不与FcγRIIa_H131结合。
结果表明，相对于BiAb004(hG1WT)，BiAb004(hG1TM)被有效的消除了其与FcγRIIa_H131的结合活性。
实验例5：FcγRIIa_R131与BiAb004(hG1WT)和BiAb004(hG1TM)的亲和力常数 测定
Fc受体FcγRIIa_R131(又名CD32a_R131)可与IgG抗体的Fc端结合，介导ADCC。
本实验使用Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIa_R131的亲和力常数，以评价各抗体的ADCC活性。
Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)和BiAb004(hG1TM)的亲和力常数实验方法简述如下：固化物稀释液为PBS，0.02％Tween-20，0.1％BSA，pH7.4，分析物稀释缓冲液为PBS，0.02％Tween-20，0.02％casein，0.1％BSA，pH7.4。5μg/mL的FcγRIIa_R131固定在NTA传感器上，固定高度约为1.0nm，传感器在PBS，0.02％Tween-20，0.02％casein，0.1％BSA，pH7.4缓冲液中平衡300s以封闭传感器，固定在传感器上的FcγRIIa_R131与抗体结合，抗体浓度为12.5-200nM(两倍梯度稀释)，时间60s，抗体在缓冲液中解离，时间60s。传感器使用10mM Glycine，pH1.7 和10mM硫酸镍再生。检测温度为30℃，频率为0.6Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRIIa_R131与BiAb004(hG1TM)和BiAb004(hG1WT)的亲和力常数测定结果如下面的表5以及图9、图10所示。
表5:BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIa_R131结合的动力学参数
结果显示，BiAb004(hG1WT)和BiAb004(hG1TM))均可以与FcγRIIa_R131结合，亲和力常数分别为1.43E-08M，4.20E-08M。
结果表明，BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIa_R131均有结合活性。但图9和图10的结果显示，BiAb004(hG1WT)的结合信号高度较高，亲和力也强于BiAb004(hG1TM)。
实验例6：FcγRIIb与BiAb004(hG1WT)和BiAb004(hG1TM)的亲和力常数测定
Fc受体FcγRIIb(又名CD32b)可与IgG抗体的Fc端结合，负性调控免疫细胞的功能，抑制免疫细胞的激活、增殖，抑制细胞因子的分泌等。
本实验使用Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIb的亲和力常数，以评价BiAb004(hG1WT)和BiAb004(hG1TM)与Fc受体的结合能力。
Fortebio Octet分子相互作用仪检测BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIb的亲和力常数实验方法简述如下：固化物稀释液为PBS，0.02％Tween-20，0.1％BSA，pH7.4，分析物稀释缓冲液为PBS，0.02％Tween-20，0.02％casein，0.1％BSA，pH7.4。5μg/mL的FcγRIIb固定在NTA传感器上，固定高度约为1.0nm，传感器在PBS，0.02％Tween-20，0.02％casein，0.1％BSA，pH7.4缓冲液中平衡300s以封闭传感器，固定在传感器上的FcγRIIb与抗体结合，抗体浓度为12.5-200nM(两倍梯度稀释)，时间60s，抗体在缓冲液中解离，时间60s。传感器使用10mM Glycine，pH1.7 和10mM硫酸镍再生。检测温度为30℃，频率为0.6Hz。数据以1:1模型拟合分析，得到亲和力常数。
FcγRIIb与BiAb004(hG1TM)和BiAb004(hG1WT)的亲和力常数测定结果如下面的表6以及图11、图12所示。
表6:BiAb004(hG1WT)和BiAb004(hG1TM)与FcγRIIb结合的动力学参数
结果显示，BiAb004(hG1WT)可以与FcγRIIb结合，亲和力常数分别为5.61E-08M；而BiAb004(hG1TM)由于与FcγRIIb没有结合信号或信号极低，故未能获得相应数据，认为其不与FcγRIIb结合。
结果表明，相对于BiAb004(hG1WT)，BiAb004(hG1TM)被有效的消除了其与FcγRIIb的结合活性。
实验例7：C1q与BiAb004(hG1WT)和BiAb004(hG1TM)的亲和力测定
血清补体C1q可与IgG抗体的Fc端结合，介导CDC效应。治疗性单克隆抗体与C1q结合的能力影响到该抗体的安全性和有效性。
本实验使用Fortebio Octet分子相互作用仪检测了BiAb004(hG1WT)和BiAb004(hG1TM)与C1q的亲和力常数，以评价各抗体的CDC活性。实验例中采用抗PDL1抗体5C10H2L2-IgG1mt作为对照抗体，其制备方法参照PCT公开WO2017148424A1。
Fortebio Octet分子相互作用仪检测相应抗体与C1q的亲和力常数实验方法简述如下：样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。50μg/mL的抗体固定在FAB2G传感器上，固定高度约为2.0nm，传感器在缓冲液中平衡60s，固定在传感器上的抗体与抗原C1q结合,抗原浓度为1.25-20nM(两倍梯度稀释)，时间60s，抗原抗体在缓冲液中解离，时间60s。传感器使用10mM甘氨酸，pH1.7再生，时间5s，重复4次。样品板震动速率为1000rpm，检测温度为30度，检测频率为 0.6Hz。数据以1:1模型拟合分析，得到亲和力常数。数据采集软件为Fortebio Data Acquisition 7.0，数据分析软件为Fortebio Data Analysis 7.0。
C1q与BiAb004(hG1TM)、BiAb004(hG1WT)以及5C10H2L2-IgG1mt的亲和力常数测定结果如下面的表7以及图13、图14、图15所示。
表7：BiAb004(hG1WT)、BiAb004(hG1TM)和5C10H2L2-IgG1mt与C1q结合的动力学参数
结果显示BiAb004(hG1WT))可以与C1q结合，亲和力常数为2.53E-09M；而BiAb004(hG1TM)由于与C1q没有结合信号或信号极低，故未能获得相应数据，认为其不与C1q结合。
结果表明，相对于BiAb004(hG1WT)，BiAb004(hG1TM)被有效的消除了其与C1q的结合活性。
实验例8：BiAb004(hG1WT)和BiAb004(hG1TM)对表达CTLA4和PD-1抗原的 293T-CTLA4-PD1细胞的ADCC活性检测
ADCC效应是指具有杀伤活性的效应免疫细胞通过其表面表达的Fc受体(FcR)识别结合于靶细胞抗原上抗体的Fc段，直接杀伤靶细胞。为检测抗CTLA4-抗PD-1的双功能抗体BiAb004(hG1WT)和BiAb004(hG1TM)的ADCC效应，发明人构建了表达有PD-1和CTLA4抗原的293T-CTLA4-PD1细胞和原代PBMC共培养体系以检测抗体的ADCC活性。
BiAb004(hG1WT)和BiAb004(hG1TM))对表达CTLA4和PD-1抗原的293T-CTLA4-PD1细胞的ADCC活性检测，方法具体如下：
按Ficoll外周血单个核细胞分离操作说明书分离获得正常人PBMC，分离后的PBMC用RPMI-1640完全培养基重悬，AO/PI染色计数，冻存。实验前一天，复苏 PBMC，计细胞数及活率，于37℃、5％CO2的二氧化碳培养箱中孵育过夜；实验当天，收集293T-CTLA4-PD1细胞、PBMC，800rpm或1200rpm离心5min后，再用RPMI-1640(含1％FBS)(以下称培养基)重悬细胞，洗涤细胞2次；计细胞数及活率，用培养基将细胞浓度调整到合适范围，根据试验设计，在96孔板中加入100μL 293T-CTLA4-PD1细胞3.0E+04/孔；加入50μL抗体室温预孵育1小时；1小时后加入50μl PBMC，9.0E+05/孔，充分混匀；37℃、5％CO2的二氧化碳培养箱中孵育4hrs。250xg离心5min；小心吸取100μL细胞上清(注意不要吸到板底细胞)于新的96孔平底微量板中，根据Cytotoxicity Detection Kit说明书每孔加入100μL现配的反应液，室温避光孵育30min。分别于490nm和650nm处测量OD值，根据ADCC(％)＝(实验组-阴性对照组)/(靶细胞LDH最大释放-靶细胞LDH自发释放)×100％计算各组ADCC百分数。
BiAb004(hG1WT)和BiAb004(hG1TM)对表达CTLA4和PD-1抗原的293T-CTLA4-PD1细胞的ADCC活性用ADCC百分数表示，结果如图16所示。
结果显示，在PBMC与293T-CTLA4-PD1混合培养体系中，在同等剂量水平下，BiAb004(hG1TM)所诱导的ADCC百分数显著低于BiAb004(hG1WT)。
结果表明，BiAb004(hG1TM)无ADCC活性。
实验例9：抗CTLA4-抗PD-1双特异性抗体联合西奥罗尼在淋巴细胞-肿瘤细胞 混合反应体系中的活性评价
新鲜分离的PBMC(来自健康捐献者)贴壁过夜后收集单核细胞，加入GM-CSF(2000U/mL，来源于peprotech，货号300-03)、IL-4(2000U/mL，来源于peprotech，货号200-04)，培养3天后进行诱导，iDC组半量换液加入GM-CSF(2000U/mL)、IL4(2000U/mL)，mDC组半量换液后加入GM-CSF(2000U/mL)、IFN-γ(50ng/mL，来源于Sino Biological，货号11725-HNAS-100)、LPS(100ng/mL，来源于SIGMA，货号L6529)，培养箱培养2天后收集细胞，进行冻存备用。
常规消化离心收集A375细胞(购自ATCC，由中原公司代理，货号：CRL-1619)，按照2×106个细胞/皿铺于10cm平皿，其中1皿加入西奥罗尼(来自微芯生物，批号：210604)至终浓度为15μM，并设置空白对照组，于37℃、5％CO2培养箱孵育48h(培养基：DMEM+10％FBS)。提前1晚复苏与iDC/mDC来自同一供者(自体)或者不同供者(异体)的PBMC(来自健康捐献者)，提前2h复苏iDC(未成熟的树突状细 胞)和mDC(成熟的树突状细胞)，计数后按照DC：PBMC＝1:10的比例铺于96孔平底板；收集未处理及15μM西奥罗尼处理48h后的A375细胞，3×104/50μL/孔加入相应的分组孔，加入BiAb004(hG1TM)，300nM/50μL/孔加入相应的分组孔；于培养箱中孵育3-5天；分别在第3天和第5天时收集细胞上清，采用ELISA试剂盒检测IFN-γ(购自达科为公司，货号：Cat：1110202)和IL-2(购自达科为公司，货号：Cat：1110202)的分泌量。
DC、PBMC和A375细胞混合培养后的IFN-γ分泌检测结果如图17所示。DC、PBMC和A375细胞混合培养后的IL-2分泌检测结果如图18所示。
由图可见，双功能抗体BiAb004(hG1TM)单药组、西奥罗尼单药组以及双功能抗体BiAb004(hG1TM)联用西奥罗尼组均能有效地促进反应体系中的IFN-γ和IL-2分泌。其中，双功能抗体BiAb004(hG1TM)联用西奥罗尼组促IFN-γ和IL-2分泌的能力分别均显著强于双功能抗体BiAb004(hG1TM)单药组、西奥罗尼单药组。
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 (26)

  1. 一种药物组合，其包含西奥罗尼或其可药用盐(例如盐酸盐)或其晶型(例如非溶剂化晶型A、B或C)，以及至少一种(例如1种、2种或3种)抗CTLA4-抗PD-1双特异性抗体。
  2. 根据权利要求1所述的药物组合，其中，所述西奥罗尼或其可药用盐或其晶型与所述抗CTLA4-抗PD-1双特异性抗体的质量比为1：(1-1000)，优选为1：(5-500)，更优选为1：(10-100)。
  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。
  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，所述西奥罗尼或其可药用盐或其晶型的单位剂量为0.1mg-100mg、0.5mg-50mg、1mg-20mg、2mg-15mg、3mg-12mg、4mg-8mg或者5mg。
  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，
    所述药物组合为固定组合，例如为药物组合物；或者
    所述药物组合是非固定组合，例如所述非固定组合中的西奥罗尼或其可药用盐或其晶型、抗CTLA4-抗PD-1双特异性抗体各自存在于独立的药物组合物中。
  6. 根据权利要求5所述的药物组合物，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。
  7. 根据权利要求5至6中任一权利要求所述的药物组合，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。
  8. 根据权利要求5至7中任一权利要求所述的药物组合，其中，所述药物组合物不包含除了西奥罗尼或其可药用盐或其晶型和抗CTLA4-抗PD-1双特异性抗体其它 活性药物成分。
  9. 根据权利要求1至8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；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: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；
    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。
  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，其中，按照EU编号系统，所述免疫球蛋白的重链恒定区具有如下突变：
    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；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: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；
    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具有如下突变组合之一：
    L234A和L235A；或者
    L234A和G237A；或者
    L235A和G237A；或者
    L234A、L235A、G237A。
  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，按照EU编号系统，所述免疫球蛋白的重链恒定区还具有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。
  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。
  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，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。
  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：
    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。
  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，所述免疫球蛋白或其抗原结合片段以大于大约10-7M，例如大于大约10-6M、10-5M、10-4M或10-3M或更大的亲和力常数结合FcγRIIIa_F158、FcγRI、FcγRIIa_H131、FcγRIIIa_V158和/或FcγRIIb；优选地，所述亲和力常数通过Fortebio Octet分子相互作用仪测得；
    优选地，所述免疫球蛋白或其抗原结合片段与FcγRIIIa_F158、FcγRI、FcγRIIa_H131、FcγRIIIa_V158和/或FcγRIIb没有结合信号或者结合信号小于0.1nm；优选地，所述结合信号是指通过Fortebio Octet分子相互作用仪测得的响应值。
  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，所述免疫球蛋白或其抗原结合片段以大于大约10-9M，例如大于大约10-8M、10-7M、10-6M或10-5M或更大的亲和力常数结合C1q；优选地，所述亲和力常数通过Fortebio Octet分子相互作用仪测得；
    优选地，所述免疫球蛋白或其抗原结合片段与C1q没有结合信号或者结合信号小于0.1nm；优选地，所述结合信号是指通过Fortebio Octet分子相互作用仪测得的响应值。
  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。
  19. 根据权利要求1至18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，所述连接片段为(GGGGS)n，n为正整数；优选地，n为1、2、3、4、5或6。
  20. 根据权利要求1至19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。
  21. 根据权利要求1至20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，所述单链抗体连接在免疫球蛋白的重链的C末端。
  22. 根据权利要求1至21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。
  23. 一种药盒产品，其包含权利要求1至22中任一权利要求所述的药物组合，以及产品说明书。
  24. 权利要求1至22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、胰腺癌和鼻咽癌中的一种或多种；
    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，所述胃癌为胃腺癌或食管结合部腺癌；
    优选地，所述肿瘤为具有MSI-H/dMMR表型的实体瘤；优选地，所述肿瘤选自具有MSI-H/dMMR表型的下述肿瘤中的一种或多种：
    结肠癌、直肠癌、子宫内膜癌、胃癌、间皮瘤、肉瘤、肾上腺皮质癌、恶性黑色素瘤和卵巢生殖细胞肿瘤。
  25. 根据权利要求1至22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、胰腺癌和鼻咽癌中的一种或多种；
    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，所述胃癌为胃腺癌或食管结合部腺癌；
    优选地，所述肿瘤为具有MSI-H/dMMR表型的实体瘤；优选地，所述肿瘤选自具有MSI-H/dMMR表型的下述肿瘤中的一种或多种：
    结肠癌、直肠癌、子宫内膜癌、胃癌、间皮瘤、肉瘤、肾上腺皮质癌、恶性黑色素瘤和卵巢生殖细胞肿瘤。
  26. 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22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、胰腺癌和鼻咽癌中的一种或多种；
    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，所述胃癌为胃腺癌或食管结合部腺癌；
    优选地，所述肿瘤为具有MSI-H/dMMR表型的实体瘤；优选地，所述肿瘤选自具有MSI-H/dMMR表型的下述肿瘤中的一种或多种：
    结肠癌、直肠癌、子宫内膜癌、胃癌、间皮瘤、肉瘤、肾上腺皮质癌、恶性黑色素瘤和卵巢生殖细胞肿瘤。
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