WO2022218383A1 - 抗体在抗肿瘤治疗中的用途 - Google Patents

抗体在抗肿瘤治疗中的用途 Download PDF

Info

Publication number
WO2022218383A1
WO2022218383A1 PCT/CN2022/086870 CN2022086870W WO2022218383A1 WO 2022218383 A1 WO2022218383 A1 WO 2022218383A1 CN 2022086870 W CN2022086870 W CN 2022086870W WO 2022218383 A1 WO2022218383 A1 WO 2022218383A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
variant
variable region
amino acid
acid sequence
Prior art date
Application number
PCT/CN2022/086870
Other languages
English (en)
French (fr)
Inventor
夏瑜
王忠民
李百勇
Original Assignee
康方药业有限公司
康融东方（广东）医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 康方药业有限公司, 康融东方（广东）医药有限公司 filed Critical 康方药业有限公司
Priority to EP22787605.9A priority Critical patent/EP4324854A1/en
Priority to JP2023562813A priority patent/JP2024515618A/ja
Priority to IL307494A priority patent/IL307494A/en
Priority to AU2022258365A priority patent/AU2022258365A1/en
Priority to BR112023021177A priority patent/BR112023021177A2/pt
Priority to CA3213734A priority patent/CA3213734A1/en
Priority to KR1020237038950A priority patent/KR20230171456A/ko
Publication of WO2022218383A1 publication Critical patent/WO2022218383A1/zh

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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 [IGs], 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen

Definitions

  • the invention belongs to the field of molecular immunology, in particular to the application of a type of anti-vascular endothelial growth factor receptor VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody combined with anti-PD-1/CTLA-4 bispecific antibody in the treatment of tumors .
  • Tumor is a major disease endangering human health, and it is urgent to develop effective treatment methods and drugs.
  • Vascular endothelial growth factor receptor 2 (vascular endothelial growth factor receptor 2, VEGFR2) is a growth factor receptor that specifically acts on vascular endothelial cells, also known as KDR or FLK1.
  • VEGFRs There are five main types of VEGFRs: VEGFR-1, VEGFR-2, VEGFR-3, NP-1 and NP-2, among which VEGFR-1 and VEGFR2 mainly exist in vascular endothelial cells, and VEGFR3 mainly exists in lymphatic endothelial cells .
  • NP-1 and NP-2 are not only expressed in endothelial cells, but also in some tumor cells. However, the biological activities of VEGF to promote the proliferation of vascular endothelial cells are mainly realized by combining with VEGFR2.
  • the formation of new blood vessels plays an important role in a variety of human diseases, such as retinopathy, arthritis, endometriosis and so on.
  • the growth of tumors is usually accompanied by the formation of new blood vessels.
  • J. Folkman proposed that the growth and metastasis of tumors could be inhibited by blocking the formation of tumor blood vessels.
  • the development, metastasis and prognosis of malignant tumors are related to vascular endothelial growth factor and its receptor family. Therefore, anti-tumor therapy targeting VEGF and its receptors has attracted attention again.
  • VEGFR-2 is the most widely studied.
  • PD-1 (PD-1) is a key immune checkpoint receptor expressed by activated T and B lymphocytes and mediates immunosuppression, and its ligands include at least PD-L1 and PD-L2.
  • Anti-PD-1 antibodies specifically bind to programmed death-1 (PD-1) and block inhibition of the PD-1/PD-1 ligand pathway (Topalian et al. (2012a) N Engl J Med 366:2443-54).
  • PD-L1 Programmed death-ligand 1
  • CD274 also known as CD274 or B7-H1
  • CD274 also known as CD274 or B7-H1
  • PD-1 a 40kDa type 1 transmembrane protein encoded by the CD274 gene and is a ligand of PD-1. It is widely distributed on leukocytes and non-hematopoietic cells in lymphoid tissues, and is also widely distributed in various cancer cells. It is highly expressed on the surface of various tumor cells, and the malignancy and poor prognosis of tumors are closely related to the expression level of PD-L1. . Both PD-L1 and PD-1 belong to the immunoglobulin superfamily and both have two extracellular Ig domains.
  • PD-L1 programmed death receptor-1
  • B7-1 B7-1
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITSM Immunoreceptor tyrosine-based switching motif
  • PD-1 recruits the tyrosine phosphatase SHP-2 to the ITSM motif within its cytoplasmic tail, resulting in effector molecules (such as CD3 ⁇ , PKC ⁇ , and CD3 ⁇ ) involved in the CD3+ T cell signaling cascade.
  • effector molecules such as CD3 ⁇ , PKC ⁇ , and CD3 ⁇
  • PD-1/PD-L1 is an important specific immune checkpoint, the formation of PD-1/PD-L1 complex transmits inhibitory signals and negatively regulates T cell immune responses; it inhibits TCR-mediated T cell activation, cellular Factor production and T cell proliferation (Fife et al (2011) Nature Immunology 10:1185-1193); induction of exhaustion or anergy among cognate antigen-specific T cells (Hofmeyer et al (2011) Journal of Biomedicine and Biotechnology 2011: 1-9); promotes the differentiation of Th1 cells into Foxp3+ regulatory T cells (Armanath et al. (2011) Science TransMed 3:1-13; Francisco et al. (2009) J.Exp.Med.206:3015-3029); and induces effects Apoptosis of T cells.
  • Disruption of the PD-L1 gene results in an upregulated T cell response and the generation of autoreactive T cells (Latchman et al. (2004) PNAS 101:10691-10696).
  • Antibody blockade of PD-1 or PD-L1 results in increased anti-tumor immunity (Iwai et al. (2002) PNAS 99: 12293-12297).
  • CTLA-4 is also one of the representative specific immune checkpoint proteins.
  • CTLA-4 is the first clinical target, and anti-CTLA-4 antibody can promote T cell activation and improve its anti-tumor effect.
  • Ipilimumab is the first CTLA-4 immune checkpoint inhibitor that has been shown to improve overall survival in previously treated metastatic melanoma patients.
  • Current studies have shown that CTLA-4 inhibits the response of T cells mainly in two ways: one is to reduce TCR (T cell receptor ) and CD28 signals. Another is to reduce the expression levels of CD80 and CD86 in antigen presenting cells (APCs) or to remove them from APCs by transendocytosis, thus reducing the involvement of CD28 in T cell activation.
  • TCR T cell receptor
  • CD28 signals Another is to reduce the expression levels of CD80 and CD86 in antigen presenting cells (APCs) or to remove them from APCs by transendocytosis, thus reducing the involvement of CD28 in T cell activation.
  • APCs anti
  • CTLA-4 also mediates dendritic cell binding to CD80/CD86 and induces the expression of the tryptophan-degrading enzyme IDO, resulting in the inhibition of TCR.
  • CTLA-4 antibody reduces Tregs and activates TCR by binding CTLA-4
  • PD-1 antibody and CTLA-4 cytotoxic T lymphocyte-associated protein 4
  • CTLA-4 cytotoxic T lymphocyte-associated protein 4
  • Bevacizumab is an anti-angiogenic targeted drug that binds to vascular endothelial growth factor (VEGF) to block the growth of blood vessels and inhibit tumor metastasis. It is a broad-spectrum anticancer drug. Since its launch, bevacizumab has mainly been approved for the treatment of various types of cancer, such as brain cancer (such as invasive brain cancer, such as glioblastoma), kidney cancer, lung cancer (such as metastatic non-squamous non-squamous Small cell lung cancer), colon cancer, rectal cancer, cervical cancer (such as metastatic cervical cancer), endometrial cancer, ovarian cancer (such as advanced ovarian cancer) or fallopian tube cancer, kidney cancer, etc.
  • VEGF vascular endothelial growth factor
  • the following invention is provided.
  • the present invention relates to:
  • Anti-CTLA4-anti-PD-1 bispecific antibody preferably for the treatment of tumors, preferably said anti-CTLA4-anti-PD-1 bispecific antibody is administered in combination with component A, wherein said component A is selected from anti-VEGFR2 monoclonal antibody or antigen-binding fragment thereof, anti-VEGF monoclonal antibody or antigen-binding fragment thereof, or a combination thereof,
  • 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 first protein functional region is a single-chain antibody
  • the second protein functional region is an immunoglobulin protein
  • Described immunoglobulin it comprises HCDR1-HCDR3 in the variable region of heavy chain shown in SEQ ID NO:14 (preferably HCDR1-HCDR3 shown in SEQ ID NOs:27-29 respectively), and SEQ ID NO : LCDR1-LCDR3 in the light chain variable region shown in 16 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 30-32 respectively); and described single-chain antibody, which comprises SEQ ID NO: 2 HCDR1-HCDR3 in the variable region of the heavy chain shown (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 33-35, respectively) and LCDR1-LCDR3 in the variable region of the light chain shown in SEQ ID NO: 4 ( Preferably LCDR1-LCDR3 as shown in SEQ ID NOs: 36-38);
  • Described immunoglobulin it comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO:2 (preferably HCDR1-HCDR3 shown in SEQ ID NOs:33-35 respectively) and SEQ ID NO: LCDR1-LCDR3 in the light chain variable region shown in 4 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 36-38 respectively); and described single-chain antibody, which comprises SEQ ID NO: shown in 14 HCDR1-HCDR3 in the variable region of the heavy chain (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 27-29, respectively), and LCDR1-LCDR3 in the variable region of the light chain shown in SEQ ID NO: 16 ( Preferably LCDR1-LCDR3 as shown in SEQ ID NOs: 30-32);
  • the anti-VEGFR2 monoclonal antibody comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 50 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 53-55, respectively) and SEQ ID NOs: 53-55.
  • LCDR1-LCDR3 in the light chain variable region shown in NO:52 preferably LCDR1-LCDR3 shown in SEQ ID NOs:56-58, respectively; or
  • the anti-VEGF monoclonal antibody comprises HCDR1-HCDR3 (preferably HCDR1-HCDR3 respectively shown in SEQ ID NOs: 63-65) and SEQ ID NOs: HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 60 LCDR1-LCDR3 in the light chain variable region shown in NO:62 (preferably LCDR1-LCDR3 shown in SEQ ID NOs:66-68, respectively).
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 14, SEQ ID NO: 18 or a variant thereof; and the amino acid sequence of the light chain variable region of the immunoglobulin selected from SEQ ID NO: 16, SEQ ID NO: 20 or variants thereof; 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, SEQ ID NO:43 or a variant thereof; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:8, ID NO: 12, SEQ ID NO: 42, SEQ ID NO: 44 or variants thereof;
  • 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, SEQ ID NO:43 or a variant thereof; And the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42, SEQ ID NO:44 or a variant thereof and, the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO: 14, SEQ ID NO: 18 or a variant thereof; and the amino acid sequence of the light chain variable region of the single chain antibody selected from SEQ ID NO: 16, SEQ ID NO: 20 or variants thereof;
  • the amino acid sequence of the heavy chain variable region of the anti-VEGFR2 monoclonal antibody is the sequence shown in SEQ ID NO: 50 or a variant thereof; and the amino acid sequence of the light chain variable region of the anti-VEGFR2 monoclonal antibody
  • the sequence is the sequence shown in SEQ ID NO: 52 or a variant thereof;
  • the amino acid sequence of the heavy chain variable region of the anti-VEGF monoclonal antibody is the sequence shown in SEQ ID NO: 60 or a variant thereof and the amino acid sequence of the light chain variable region of the anti-VEGF monoclonal antibody is the sequence shown in SEQ ID NO: 62 or a variant thereof.
  • the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 2, wherein the bispecific antibody is selected from any one of (1) to (20) below :
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 2 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 4 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 6 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 8 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 10 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 12 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 2 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 4 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 6 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 8 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 10 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 12 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 4 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 14 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 16 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 4 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 18 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 20 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 8 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 14 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 16 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 8 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 18 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 20 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence 10 shown in SEQ ID NO: or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 12 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 14 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 16 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 12 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 18 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 20 or a variant thereof;
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is SEQ ID The sequence shown in NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 41 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 42 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 43 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 44 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 41 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 42 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 43 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 44 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 42 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 14 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 16 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 44 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 14 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 16 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 42 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 18 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 20 or a variant thereof;
  • the amino acid sequence of the variable region of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID The sequence shown in NO: 44 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the single-chain antibody has the sequence shown in SEQ ID NO: 18 or a variant thereof.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 20 or a variant thereof,
  • the variant has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% with the corresponding sequence %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology.
  • the amino acid sequence of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 40 or a variant thereof
  • the amino acid sequence of the light chain thereof is the sequence shown in SEQ ID NO: 24 or a variant thereof, wherein the The variant sequence has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% with the corresponding sequence , 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology.
  • the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 4, wherein the first protein functional region is directly connected to the second protein functional region or is connected by a linker fragment and/or the variable region of the heavy chain of the single-chain antibody is directly linked to the variable region of the light chain of the single-chain antibody or is linked by a linking fragment.
  • the immunoglobulin is of human IgG1 subtype
  • the heavy chain constant region of the immunoglobulin has an A mutation, and the A mutation is selected from one of the following mutation combinations:
  • the heavy chain constant region of the immunoglobulin has a B mutation selected from one or more of the following mutations:
  • the heavy chain constant region of the immunoglobulin has a combination of A and B mutations according to the EU numbering system
  • the heavy chain constant region of the anti-VEGFR2 monoclonal antibody or the anti-VEGF monoclonal antibody is Ig gamma-1 chain C region, ACCESSION P01857, and the light chain constant region is: Ig kappa chain C region, ACCESSION: P01834.
  • the bispecific antibody includes:
  • the first protein functional region is 1, and the second protein functional region is 2;
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is a single-chain antibody
  • the amino acid sequence of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 40 or a variant thereof, and the amino acid sequence of the light chain thereof is the sequence shown in SEQ ID NO: 24 or a variant thereof, wherein the The variant and the corresponding sequence have at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology;
  • the amino acid sequence of the variable region of the heavy chain of the single-chain antibody is the sequence shown in SEQ ID NO:43 or a variant thereof, and the amino acid sequence of the variable region of the light chain of the single-chain antibody is SEQ ID NO:44
  • the single-chain antibody is attached to the C-terminus of the heavy chain of the immunoglobulin;
  • the first protein functional region and the second protein functional region are connected by a first connecting fragment; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second connection Fragment ligation; the first ligated fragment and the second ligated fragment are the same or different;
  • amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO:25 and SEQ ID NO:26;
  • amino acid sequences of the first connecting fragment and the second connecting fragment are as shown in SEQ ID NO: 26,
  • the light chain sequence of the anti-PD-1/CTLA-4 bispecific antibody is shown in SEQ ID NO:72, and the heavy chain sequence is shown in SEQ ID NO:70,
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab/c, complementarity determining region (CDR) fragments, single chain antibodies (eg, scFv), dual valent antibody or domain antibody.
  • a pharmaceutical composition for treating tumors comprising an effective amount of the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1-10 and any one of items 1-10 Component A as defined in item , preferably the pharmaceutical composition is in solid form or liquid form, preferably, the mass ratio of anti-CTLA4-anti-PD-1 bispecific antibody and component A is selected from (1:5) -(5:1), eg: 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1; optionally, all
  • the pharmaceutical composition further comprises one or more chemotherapeutic drugs (preferably, the chemotherapeutic drugs are alkylating agents, antimetabolites, antibiotics, botanical drugs and/or hormone drugs, platinum drugs (such as cisplatin, carboplatin) , oxaliplatin), doxorubicin, cyclophosphamide, paclitaxel, nab-paclitaxel, liposomal paclitaxel, docetaxe
  • the medicament or the kit further comprises the component A defined in any one of items 1-10, optionally, the medicament or the kit further comprises one or more medicaments for treating tumors (preferably the medicament is Chemotherapeutic or growth inhibitory agents (eg, alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes , antimetabolites, topoisomerase inhibitors, cytotoxic or antitumor antibiotics, proteasome inhibitors, antimicrotubules, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine acid kin
  • Chemotherapeutic or growth inhibitory agents eg, alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase
  • a method for preventing and/or treating tumors comprising administering to a subject a therapeutically effective amount of the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1-10, in combination with Administration of component A as defined in any one of items 1 to 10, more preferably, further combined administration of one or more drugs for the treatment of tumors (preferably the drugs are chemotherapeutic agents or growth inhibitors (such as alkylating agents) , Anthracyclines, Antihormones, Aromatase Inhibitors, Antiandrogens, Protein Kinase Inhibitors, Lipid Kinase Inhibitors, Antisense Oligonucleotides, Ribozymes, Antimetabolites, Topoisomerase Inhibitors , cytotoxic or antitumor antibiotics, proteasome inhibitors, anti-microtubule agents, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine
  • a kit for preventing and/or treating tumors comprising (a) a first pharmaceutical composition comprising the anti-CTLA4-anti-PD-1 dual according to any one of items 1-10 a specific antibody as an active ingredient; and (b) a second pharmaceutical composition comprising, as an active ingredient, component A as defined in any one of items 1 to 10; and optionally (c) one or more for Drugs for the treatment of tumors (preferably the drugs are chemotherapeutic agents or growth inhibitors (such as alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors) , antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic or antitumor antibiotics, proteasome inhibitors, antimicrotubules, EGFR antagonists, VEGF antagonists, angiopoietins 2
  • cisplatin carboplatin) , oxaliplatin
  • doxorubicin cyclophosphamide
  • paclitaxel such as nab-paclitaxel, liposomal paclitaxel, docetaxel
  • etoposide gemcitabine, pemetrexed, capecitabine
  • Laparib rucaparib, niraparib, tarazoparib, fluzoparib, vinblastines, tamoxifen, megestrol, goserelin, asparaginase and/ or fluorouracil antineoplastic drugs.
  • a unit preparation preferably for the treatment of tumors (especially malignant tumors), wherein the unit preparation comprises: 1-1000 mg (preferably 10-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg) or 200 mg) of the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1-10, 1-1000 mg (preferably 10-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150- 250 mg or 200 mg) of component A as defined in any one of items 1 to 10 and optionally one or more drugs for the treatment of tumors (preferably the drugs are chemotherapeutic agents or growth inhibitors such as alkylating agents , Anthracyclines, Antihormones, Aromatase Inhibitors, Antiandrogens, Protein Kinase Inhibitors, Lipid Kinase Inhibitors, Antisense Oligonucleotides, Ribozymes, Antimetabolites, Topoisomerase Inhibitors , cytotoxic or antitumor
  • a method for preventing or treating cancer or tumor wherein one or more unit formulations of item 15 are administered to a subject in need, preferably, anti-CTLA4-anti-PD in the unit formulation -1 Bispecific antibody, component A and chemotherapeutics are administered separately.
  • a single pharmaceutical dosage unit preferably for the treatment of tumors (especially malignant tumors), comprising 0.1-10000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100mg) of the anti-CTLA4 anti-PD-1 bispecific antibody of any one of items 1-10, 0.1-10000mg (preferably 1-1000mg, preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg, 200 mg or 100 mg) of component A as defined in any one of items 1 to 10.
  • the tumor is selected from one or more of the following:
  • Cervical cancer eg metastatic cervical cancer
  • lung cancer such as non-small cell lung cancer (eg squamous non-small cell lung cancer or non-squamous non-small cell lung cancer), esophageal cancer, esophageal squamous cell carcinoma, ileocecal adenocarcinoma, ampullary gland Carcinoma, small cell lung cancer, gastric cancer (eg, advanced gastric cancer, gastric adenocarcinoma or gastroesophageal junction adenocarcinoma), kidney cancer, kidney cancer, endometrial cancer, adrenocortical cancer, prostate cancer, thyroid cancer, peritoneal cancer, adenocarcinoma, pancreas Carcinoma, glioma, head and neck cancer, bone cancer, testicular cancer, leukemia, myeloma, lymphoma, sarcoma, mesothelioma, hepatocellular carcinoma, colon cancer,
  • the anti-CTLA4-anti-PD-1 bispecific antibody of any one of items 1 to 10, the pharmaceutical composition of item 11, the application of item 12, the method of item 13, the method of item 14 The kit, the unit preparation described in item 15, or the method described in item 16, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, component A and/or the chemotherapeutic agent are suitable for In the form of intravenous injection or intravenous drip, preferably in liquid form.
  • the single dose of anti-CTLA4 anti-PD-1 bispecific antibody is 0.1-100 mg per kg body weight, preferably 1-10 mg; alternatively, the single dose of anti-CTLA4-anti-PD-1 bispecific antibody is per kg body weight. 10-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg for each subject;
  • the single administration dose of component A is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or, the single administration dose of component A is 10-1000 mg per subject, preferably 50-500 mg, 100- 400mg, 150-300mg, 150-250mg, 200mg or 100mg,
  • it is administered twice daily to about once every other day, or once every 3, 4, 5, 6, 10, 1, 2, or 3 weeks;
  • the mode of administration is intravenous drip or intravenous injection.
  • the anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody is administered at 1 mg/kg, 2 mg/kg, 3 mg/kg, 5 mg/kg, 6 mg/kg kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, Doses were administered at 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg body weight.
  • the anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody is administered in one or more unified doses effective to treat the cancer.
  • the unitary dose is in the range of about 1 mg to about 1000 mg. In some embodiments, the unitary dose is selected from about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg.
  • the anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody, or anti-VEGF monoclonal antibody is administered in cycles of 2 weeks (14 days) or 3 weeks (21 days),
  • Anti-PD-1 and CTLA4 bispecific antibodies are preferably administered intravenously on the first day (D1) of each cycle. That is, the anti-PD-1 and CTLA4 bispecific antibodies are administered at a frequency of once every two weeks (q2w) or once every three weeks (q3w).
  • the present invention also provides anti-CTLA4-anti-PD-1 bispecific antibodies, anti-VEGFR2 monoclonal antibodies and anti-VEGF monoclonal antibodies in the preparation of anti-CTLA4-anti-PD-1 bispecific antibodies containing a fixed dose for the treatment of cancer , the use of the container of anti-VEGFR2 monoclonal antibody (or anti-VEGF monoclonal antibody).
  • the container is an ampoule.
  • the fixed dose is selected from about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 500 mg, or about 1000mg.
  • the article of manufacture further comprises a package insert instructing the user to administer the fixed dose of anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody to a cancer patient or drug instructions.
  • the article of manufacture comprises 1 or more ampoules containing about 50 mg, 100 mg, 200 mg, 300 mg, 350 mg, 400 mg, 500 mg or 600 mg of an anti-VEGFR2 monoclonal antibody or Anti-VEGF monoclonal antibody.
  • the article of manufacture is formulated with 50 mg/5 mL/vial, 100 mg/10 mL/vial, 200 mg/10 mL/vial, or 350 mg/35 mL/vial of anti-PD-1 and CTLA4 bispecific antibodies, anti-VEGFR2 monospecific antibodies It is packaged in the form of a solution of cloned antibody or anti-VEGF monoclonal antibody.
  • chemotherapeutic agent to be used can be determined by one of ordinary skill in the art.
  • platinum chemotherapy drugs taking cisplatin as an example, the single dose of cisplatin is calculated according to the Calvert formula:
  • Cisplatin dose (mg) area under the set curve, namely AUC (mg/ml/min) ⁇ [creatinine clearance (ml/min)+25], where AUC is 4-7, preferably 5 ;
  • the mode of administration is intravenous drip or intravenous injection.
  • the chemotherapeutic agent or growth inhibitory agent is selected from the group consisting of alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid Plasma kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic or antitumor antibiotics, proteasome inhibitors, antimicrotubules, EGFR antagonists, VEGF antagonists , Angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors and combinations thereof.
  • the targeted therapeutic agent is selected from the group consisting of B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, VEGF inhibitors, PARP inhibitors, bisphosphatidylinositol 3-kinase/mTOR inhibitors, and combinations thereof.
  • the antibody-drug conjugate comprises a drug selected from the group consisting of maytansine, monomethyl auristatin E, calicheamicin, esperamicin, and radioisotope chelate mixture.
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent is any amount of a drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes regression of disease, said Disease regression is evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of injury or disability caused by disease affliction.
  • the ability of therapeutic agents to promote disease regression can be assessed using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by in vitro assays The activity of the agent was determined.
  • a prophylactically effective amount of a drug means that when administered alone or in combination with an antineoplastic agent to a subject at risk of developing cancer (eg, a subject with a premalignant condition) or a subject at risk of cancer recurrence, The amount of any drug that inhibits the development or recurrence of cancer. In certain embodiments, a prophylactically effective amount completely prevents the occurrence or recurrence of cancer. “Inhibiting" the occurrence or recurrence of cancer means reducing the likelihood of occurrence or recurrence of cancer, or preventing the occurrence or recurrence of cancer altogether.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agent.
  • pH adjusting agents include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
  • a “recurrent” cancer is one that regenerates at the initial site or at a distant site in response to initial treatment (eg, surgery).
  • a “locally recurrent” cancer is one that, after treatment, develops in the same location as the previously treated cancer.
  • Metalstatic cancer refers to cancer that has spread from one part of the body (eg, the lungs) to another part of the body.
  • variable regions of light and heavy chains determine antigen binding: 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 Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volume 1-3, NIH Publication 91-3242, Bethesda Md ). Given the known sequences of antibody heavy and light chain variable regions, there are currently several methods for determining antibody CDR regions, including the Kabat, IMGT, Chothia and AbM numbering systems.
  • CDRs complementarity determining regions
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent is any amount of a drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes regression of disease, said Disease regression is evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of injury or disability caused by disease affliction.
  • the ability of therapeutic agents to promote disease regression can be assessed using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by in vitro assays The activity of the agent was determined.
  • a prophylactically effective amount of a drug means that when administered alone or in combination with an antineoplastic agent to a subject at risk of developing cancer (eg, a subject with a premalignant condition) or a subject at risk of cancer recurrence, The amount of any drug that inhibits the development or recurrence of cancer. In certain embodiments, a prophylactically effective amount completely prevents the occurrence or recurrence of cancer. “Inhibiting" the occurrence or recurrence of cancer means reducing the likelihood of occurrence or recurrence of cancer, or preventing the occurrence or recurrence of cancer altogether.
  • a "recurrent" cancer is one that regenerates at the original site or at a distant site in response to initial treatment (e.g., surgery).
  • a "locally recurrent" cancer is one that, after treatment, develops in the same location as the previously treated cancer.
  • Metalstatic cancer refers to cancer that has spread from one part of the body (eg, the lungs) to another part of the body.
  • single drug dosage unit refers to the amount of the anti-CTLA4 anti-PD-1 bispecific antibody of the present invention, comprising the anti-CTLA4 anti-PD-1 bispecific antibody of the present invention, to be administered to a subject (preferably per kg body weight of the subject) at the time of the dosing regimen.
  • the anti-VEGFR2 antibody or anti-VEGF antibody is administered in a single dosage form, such as an injection, eg, in an ampule.
  • dosing regimens include, for example, administration of a single drug dosage unit according to a dosing cycle of twice daily to about once every other day, or every 3, 4, 5, 6 days , 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.
  • first and second are intended to refer to Generational distinctions or clarity of presentation do not have typical sequential meanings.
  • FIG. 1 Mixed lymphocyte reaction MLR detection results of biological activity of anti-PD-1/CTLA-4 bispecific antibody Cadonilimab combined with anti-VEGFR2 antibody HpL3 to promote INF- ⁇ secretion.
  • Figure 2 The results of the mixed lymphocyte reaction MLR detection of the biological activity of cadonilimab combined with bevacizumab to stimulate the activation and secretion of IL-2 by PBMC.
  • the used isotype control antibody anti-HEL is an antibody targeting human anti-egg lysosome (HEL), and the variable region sequences of these antibodies are from Affinity maturation increases published by Acierno et al. the stability and plasticity of the Fv domain of anti-protein antibodies (Acierno et al. J Mol Biol. 2007; 374(1): 130-46.
  • the heavy chain variable region sequence is shown in SEQ ID No: 45, and the light The chain variable region sequence is shown in SEQ ID NO:47), the constant region fragment of hIgG1WT (ie anti-HEL) adopts the Ig gamma-1 chain C region, ACCESSION:P01857 as the heavy chain constant region (SEQ ID NO:46) , Ig kappa chain C region, ACCESSION:P01834 is the light chain constant region (SEQ ID NO:48); anti-HEL is prepared in the laboratory of Zhongshan Kangfang Biomedical Co., Ltd.
  • Bevacizumab anti-VEGF monoclonal antibody used in the examples is a commercial drug produced by Roche.
  • amino acid sequences of the heavy chain and light chain of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3, and the coding nucleic acid sequences are respectively the same as 4G10, 4G10H1L1 and 4G10H3L3 in Chinese Patent Publication CN106967172A.
  • 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 sequences, are respectively the same as 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A.
  • the structural patterns of the bifunctional antibodies CP001(M), CPb002(M), CP003(M) and CP004(M) belong to the Morrison pattern (IgG-scFv), that is, the C-termini of the two heavy chains of an IgG antibody are connected by The fragment is linked to an scFv fragment of another antibody, whose heavy and light chain design composition is shown in Table A below.
  • Table A Sequence design of CP001(M), CP002(M), CP003(M) and CP004(M)
  • 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 based on 4G10H1V, 4G10L1V, 4G10H3V, and 4G10L3V, respectively, mutating individual amino acids in its framework region, which effectively optimizes the structure of the antibody and improves its effectiveness.
  • CP004(M) is also referred to as CP004(hG1WT) in the examples of the present invention.
  • CP004 (M) is as " wild type ", it adopts Ig gamma-1 chain C region, ACCESSION:P01857 is as heavy chain constant region, Ig kappa chain C region, ACCESSION:P01834 is light chain constant region.
  • CP004 (hG1WT) obtained in Preparation Example 3
  • the present inventors introduced a point mutation (L234A) from leucine to alanine at the 234th position of its heavy chain, the 235th position A point mutation of leucine to alanine (L235A) was introduced, and a point mutation of glycine to alanine (G237A) was introduced at position 237 to obtain CP004 (hG1TM).
  • the structural pattern of the anti-PD-1/CTLA-4 bispecific antibody Cadonilimab belongs to the Morrison pattern (IgG-scFv), that is, the C of the two heavy chains of an immunoglobulin part (IgG) antibody Both ends are connected to the VH end of the scFv fragment of another antibody through a linker fragment, and its immunoglobulin part is based on the PD-1 antibody, and the scFv fragment is based on an anti-CTLA-4 antibody, and the middle is linked by a linker fragment.
  • the anti-PD-1/CTLA-4 bispecific antibody Cadonilimab antibody ie CP004 (hG1TM)
  • light chain sequence SEQ ID NO:72
  • heavy chain sequence SEQ ID NO:70
  • the DNA sequence of the heavy chain coding gene of the candidate monoclonal antibody, including the constant region and the variable region, and the DNA sequence of the light chain coding gene, including the constant region and the variable region, were cloned into pUC57simple (provided by GenScript) In the vector, pUC57simple-H and pUC57simple-L plasmids were obtained respectively.
  • the above plasmids were digested with enzymes (HindIII & EcoRI) respectively, and the heavy chain and light chain recovered by electrophoresis were subcloned into the expression vector pcDNA3.1 (purchased from Invitrogen Company), respectively, and the recombinant plasmid was extracted, according to pcDNA3.1-H+pcDNA3.1. 1-L was combined to co-transfect 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto a HiTrap MabSelect SuRe column, and purified using a protein purification liquid chromatography system (AKTA Purifier 10, GE).
  • AKTA Purifier 10 protein purification liquid chromatography system
  • the purified sample namely the antibody Cadonilimab
  • the target protein of the reduced protein sample was at 70kD and 30KD.
  • non-reduced protein samples (single antibody) target protein at 200kD. In agreement with the theoretical size, purified antibodies were obtained.
  • the amino acid sequence of the heavy chain variable region of HpL3 is shown in SEQ ID NO:50, the DNA sequence of its encoding gene is shown in SEQ ID NO:49, and the sequences of the heavy chain variable region CDR1-3 are respectively shown in SEQ ID NO:53 -55 shown.
  • amino acid sequence of the light chain variable region of HpL3 is shown in SEQ ID NO:52
  • DNA sequence of its encoding gene is shown in SEQ ID NO:51
  • sequences of the light chain variable region CDR1-3 are respectively shown in SEQ ID NO:56 -58 shown.
  • HpL3 uses Ig gamma-1 chain C region, ACCESSION:P01857 as the heavy chain constant region, Ig kappa chain C region, ACCESSION:P01834 as the light chain constant region
  • the DNA sequence of the heavy chain coding gene of the candidate monoclonal antibody, including the constant region and the variable region, and the DNA sequence of the light chain coding gene, including the constant region and the variable region, were cloned into pUC57simple (provided by GenScript) In the vector, pUC57simple-PCABH and pUC57simple-L3 plasmids were obtained respectively.
  • the above plasmids were digested with enzymes (HindIII&EcoRI) respectively, and the heavy chain and light chain recovered by electrophoresis were subcloned into the expression vector pcDNA3.1 (purchased from Invitrogen Company), and the recombinant plasmid was extracted.
  • Press pcDNA3.1-PCABH+pcDNA3.1. 1-L3 was combined to co-transfect 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto a HiTrap MabSelect SuRe column, and purified using a protein purification liquid chromatography system (AKTA Purifier 10, GE).
  • the purified sample namely antibody HpL3
  • the reduced protein electrophoresis loading buffer and the non-reduced protein electrophoresis loading buffer After boiling, SDS-PAGE electrophoresis was carried out for detection.
  • the target protein of the reduced protein sample was at 45kD and 30KD.
  • non-reduced protein samples (single antibody) target protein at 150kD. In accordance with the theoretical size, a purified monoclonal antibody was obtained.
  • Example 1 Mixed lymphocyte reaction MLR detection of biological activity of anti-PD-1/CTLA-4 bispecific antibody Cadonilimab combined with anti-VEGFR2 antibody HpL3 to promote INF- ⁇ secretion
  • the human PD-L1 overexpression lentiviral vector plenti6.3/V5-PDL1FL-BSD was infected with Raji cells after virus packaging, and drug-resistant cell lines were obtained after 10ug/ml BSD (Blasticidin, Blasticidin) was added and screened.
  • plenti6.3/V5-PDL1FL-BSD was obtained by synthesizing the gene PDL1FL (PDL1, Genebank ID: NP_054862.1) by the supplier Nanjing GenScript, and then linking it to plenti6.3/V5-BSD (Invitrogen product number k531520) by enzyme digestion .
  • PBMCs isolated from normal human peripheral blood
  • PBMCs were recovered and cultured in complete medium at 37°C in a 5% carbon dioxide incubator; technology, product number: BT202) for two days; on the day of the experiment, collect Raji-PDL1 cells, resuspend and count by centrifugation (resuspend with 1640+10% FBS), adjust the cell density to 2 ⁇ 10 6 /mL, add MMC (Mito- mycin C, final concentration of 2 ⁇ g/mL, mitomycin C, manufacturer: Stressmarq, product number: SIH-246) was treated at 37°C in a 5% carbon dioxide incubator for 1 hour; PBMCs after SEB stimulation for two days and after MMC treatment were collected Raji-PDL1 cells were washed twice with 1640 basal medium, resuspended in complete medium to count the cells, and seeded to 96 cells at 1 ⁇ 10 5 /well of PBMC and 1 ⁇ 10 5 /well of Raji-PDL1 (
  • cadonilimab and HpL3 antibodies can significantly induce PBMC to further secrete IFN- ⁇ , with a significant dose-dependent relationship.
  • the combination of cadonilimab and HpL3 has better potential to promote IFN- ⁇ secretion.
  • Example 2 Mixed lymphocyte reaction MLR to detect the biological activity of cadonilimab combined with bevacizumab in promoting PBMC activation and secretion of IL-2
  • PBMCs Two days before the experiment, PBMCs were resuscitated (isolated from peripheral blood voluntarily donated by healthy volunteers after informed consent), and cultured in complete medium at 37°C in a 5% carbon dioxide incubator; 0.5 ⁇ g/mL) (Toxin technology, product number: BT202) stimulated for two days; on the day of the experiment, Raji-PDL1 (Kangfang Bioconstruction) cells were collected, centrifuged, resuspended and counted (resuspended with 1640+10% FBS), and the cell density was adjusted.
  • Raji-PDL1 Karlfang Bioconstruction
  • MMC Mito-mycin C, the final concentration is 2 ⁇ g/mL
  • Mitomycin C manufacturer: Stressmarq, product number: SIH-246
  • PBMCs after SEB stimulation for two days and Raji-PDL1 cells after MMC treatment were collected, washed twice with 1640 basal medium, resuspended in analysis medium and counted, according to PBMC 1 ⁇ 10 5 /well, Raji-PDL1 1 ⁇ 10 5 /well (each 40 ⁇ L/well) was inoculated into a 96-well U-shaped plate (3799); Hela cells (purchased from Chinese Academy of Sciences) were collected by routine digestion, and 2 ⁇ 10 4 /well (40 ⁇ L/well) were inoculated into 96 Well U-shaped plate (3799); dosing according to the experimental design, and set up negative control and isotype control, co-culture for 3 days (the
  • the amino acid sequence of the heavy chain variable region of bevacizumab is shown in SEQ ID NO: 60, the DNA sequence of its encoding gene is shown in SEQ ID NO: 59, and the sequences of the heavy chain variable region CDR1-3 are shown in SEQ ID NO: 59 respectively. ID NO: 63-65.
  • the amino acid sequence of the light chain variable region of bevacizumab is shown in SEQ ID NO: 62, the DNA sequence of its encoding gene is shown in SEQ ID NO: 61, and the sequences of the light chain variable region CDR1-3 are respectively shown in SEQ ID NO: 61.
  • ID NO: 66-68 is the amino acid sequence of the heavy chain variable region of bevacizumab.
  • SEQ ID No. 1 Nucleic acid sequence of 4G10 heavy chain variable region
  • SEQ ID No.2 Amino acid sequence of 4G10 heavy chain variable region
  • SEQ ID No.3 Nucleic acid sequence of 4G10 light chain variable region
  • SEQ ID No. 4 Amino acid sequence of 4G10 light chain variable region
  • SEQ ID No. 5 Nucleic acid sequence of 4G10H1L1 heavy chain variable region
  • SEQ ID No. 6 Amino acid sequence of 4G10H1L1 heavy chain variable region
  • SEQ ID No.7 Nucleic acid sequence of 4G10H1L1 light chain variable region
  • SEQ ID No. 8 Amino acid sequence of 4G10H1L1 light chain variable region
  • SEQ ID No. 9 Nucleic acid sequence of 4G10H3L3 heavy chain variable region
  • SEQ ID No. 10 Amino acid sequence of 4G10H3L3 heavy chain variable region
  • SEQ ID No. 11 Nucleic acid sequence of 4G10H3L3 light chain variable region
  • SEQ ID No. 12 Amino acid sequence of 4G10H3L3 light chain variable region
  • SEQ ID No. 13 Nucleic acid sequence of 14C12 heavy chain variable region
  • SEQ ID No. 14 Amino acid sequence of 14C12 heavy chain variable region
  • SEQ ID No. 15 Nucleic acid sequence of 14C12 light chain variable region
  • SEQ ID No. 16 Amino acid sequence of 14C12 light chain variable region
  • SEQ ID No. 17 Nucleic acid sequence of 14C12H1L1 heavy chain variable region
  • SEQ ID No. 18 Amino acid sequence of 14C12H1L1 heavy chain variable region
  • SEQ ID No. 19 Nucleic acid sequence of 14C12H1L1 light chain variable region
  • SEQ ID No. 20 Amino acid sequence of 14C12H1L1 light chain variable region
  • SEQ ID No. 21 DNA sequence of 14C12H1L1 heavy chain (14C12H1) (1344bp)
  • SEQ ID No. 22 Amino acid sequence (448aa) encoded by the 14C12H1L1 heavy chain (14C12H1)
  • SEQ ID No. 23 DNA sequence of 14C12H1L1 light chain (14C12L1) (642 bp)
  • SEQ ID No. 24 Amino acid sequence (214aa) encoded by 14C12H1L1 light chain (14C12L1)
  • SEQ ID No. 32 LCDR3 of 14C12: LQYDEFPLT
  • SEQ ID No. 36 LCDR1 of 4G10: TGAVTTSNF
  • SEQ ID No. 38 LCDR3 of 4G10: ALWYSNHWV
  • SEQ ID No. 39 DNA sequence of the heavy chain of the immunoglobulin portion of CP004 (hG1TM), mutation sites are underlined
  • SEQ ID No. 40 Amino acid sequence of the heavy chain of the immunoglobulin moiety in CP004 (hG1TM), mutation sites are underlined
  • SEQ ID No.41 Amino acid sequence of 4G10H1V(M), the mutation site is underlined
  • SEQ ID No.42 Amino acid sequence of 4G10L1V(M), the mutation site is underlined
  • SEQ ID No.43 Amino acid sequence of 4G10H3V(M), the mutation site is underlined
  • SEQ ID No.44 Amino acid sequence of 4G10L3V(M), the mutation site is underlined
  • SEQ ID No:45 Heavy chain variable region sequence of hIgG
  • HCDR1 GFTFSSYS (SEQ ID NO: 53)
  • HCDR2 ISSSSSYI (SEQ ID NO: 54)
  • HCDR3 ARVTDAFDI (SEQ ID NO: 55)
  • LCDR1 QGLDNW (SEQ ID NO: 56)
  • LCDR2 DAS (SEQ ID NO: 57)
  • LCDR3 QQAKAFPPT (SEQ ID NO: 58)
  • VEGF Bevacizumab heavy chain variable region
  • amino acid sequences of the three CDR regions of the variable region of the heavy chain of Bevacizumab are as follows:
  • HCDR1 GYTFTNYG (SEQ ID NO: 63)
  • HCDR3 AKYPHYYGSSHWYFDV (SEQ ID NO:65)
  • amino acid sequences of the three CDR regions of the light chain variable region of Bevacizumab are as follows:

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Oncology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

涉及抗CTLA4-抗PD-1双特异性抗体，其用于治疗肿瘤，所述抗CTLA4-抗PD-1双特异性抗体与抗VEGFR2单克隆抗体或抗VEGF单克隆抗体联合施用，其中 (1)所述抗CTLA4-抗PD-1双特异性抗体包括：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；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。

Description

抗体在抗肿瘤治疗中的用途 技术领域
本发明属于分子免疫学领域，具体涉及一类抗血管内皮生长因子受体VEGFR2的单克隆抗体或抗VEGF的单克隆抗体联合抗PD-1/CTLA-4双特异性抗体在治疗肿瘤中的应用。
背景技术
肿瘤是危害人类健康的重大疾病，亟需开发有效的治疗手段和药物。
血管内皮生长因子受体2(vascular endothelial growth factor receptor 2，VEGFR2)是一种特异作用于血管内皮细胞的生长因子的受体，又称作KDR或FLK1。目前发现的VEGFR主要有5种：VEGFR-1、VEGFR-2、VEGFR-3、NP-1和NP-2，其中VEGFR-1和VEGFR2主要存在于血管内皮细胞，VEGFR3主要存在于淋巴管内皮细胞。NP-1和NP-2不仅在内皮细胞表达，还表达于某些肿瘤细胞内。但VEGF促血管内皮细胞增殖等生物活性的发挥主要是通过与VEGFR2结合实现。
新生血管的形成在人类的多种疾病中发挥重要作用，如视网膜病变、关节炎、子宫内膜异位症等。肿瘤的生长通常也会伴随着新生血管的形成，早在1971年，J.Folkman就提出可通过阻断肿瘤血管的生成来抑制肿瘤的生长及转移，近年来，研究发现，越来越多的恶性肿瘤其发展、转移、预后与血管内皮细胞生长因子及其受体家族相关。因此，以VEGF及其受体为靶点的抗肿瘤治疗再次受到关注，在受体家族中，以对VEGFR-2为靶点的研究最为广泛。
PD-1(PD-1)是一种由活化的T淋巴细胞和B淋巴细胞表达的关键免疫检查点受体并介导免疫抑制，其配体至少包括PD-L1和PD-L2。抗PD-1抗体特异性地结合程序性死亡受体-1(programmed death-1，PD-1)并阻断抑制PD-1/PD-1配体途径(Topalian等人(2012a)N Engl J Med 366：2443-54)。
PD-L1(Programmed death-ligand 1)又称为CD274或B7-H1，是由CD274基因编码的40kDa的1型跨膜蛋白，是PD-1的一个配体， PD-L1不仅在淋巴和非淋巴组织中的白细胞和非造血细胞上广泛分布，而且还在各种癌细胞中广泛分布，在多种肿瘤细胞表面高表达，而且肿瘤的恶性程度以及不良预后与PD-L1的表达水平密切相关。PD-L1和PD-1两者都属于免疫球蛋白超家族并且都有两个细胞外Ig结构域。PD-L1与程序性死亡受体-1(PD-1)和B7-1(CD80)的结合界面是在IgV样结构域上(Lin等(2008)PNAS 105：3011-3016)。PD-L1含有保守的短的细胞内尾区(约30个氨基酸)，PD-1含有两个基于细胞质酪氨酸的信号基序，即基于免疫受体酪氨酸的抑制基序(ITIM)和基于免疫受体酪氨酸的转换基序(ITSM)。在T细胞刺激之后，PD-1将酪氨酸磷酸酶SHP-2募集到其胞质尾区内的ITSM基序，导致参与CD3+T细胞信号传导级联的效应分子(诸如CD3ζ、PKCθ和ZAP70)的去磷酸化(Freeman等(2000)J Exp Med 192：1027-34；Latchman等(2001)Nat Immunol 2:261-8；Carter等(2002)Eur J Immunol32：634-43)。有临床数据表明PD-L1的高肿瘤表达与增加的肿瘤侵袭性和较差的预后相关联。
PD-1/PD-L1是一个重要的特异性免疫检查点，PD-1/PD-L1复合物的形成传输抑制信号并负调节T细胞免疫应答；它抑制TCR介导的T细胞活化、细胞因子产生和T细胞增殖(Fife等(2011)Nature Immunology 10：1185-1193)；诱导同源抗原特异性T细胞之中的衰竭或无反应性(Hofmeyer等(2011)Journal of Biomedicine and Biotechnology 2011：1-9)；促进Th1细胞分化成Foxp3+调节性T细胞(Armanath等(2011)Science TransMed 3：1-13；Francisco等(2009)J.Exp.Med.206：3015-3029)；并诱导效应T细胞的凋亡。PD-L1基因的破坏导致上调的T细胞应答和自身反应性T细胞的产生(Latchman等(2004)PNAS 101：10691-10696)。PD-1或PD-L1的抗体阻断导致增加的抗肿瘤免疫性(Iwai等(2002)PNAS 99：12293-12297)。
CTLA-4也是一个具有代表性的特异性免疫检查点蛋白之一。CTLA-4是首个应用于临床的靶点，抗CTLA-4抗体可促进T细胞活化，提高其抗肿瘤效果。Ipilimumab是首个被证实可以提高经治转移性黑色素瘤患者总生存率且获FDA批准上市的CTLA-4免疫检查点抑制剂。 目前的研究表明CTLA-4抑制T细胞的反应主要是通过两种途径：一是通过与CD28竞争性的结合B7或者招募磷酸酶到CTLA-4的胞内结构域部分从而降低TCR(T cell receptor)和CD28的信号。另一种是降低CD80和CD86在抗原呈递细胞(APC)的表达水平或者通过转胞吞作用(transendocytosis)将它们从APC移除，这样就减少了CD28参与进行T细胞激活。此外，CTLA-4还会介导树突细胞结合CD80/CD86并诱导色氨酸降解酶IDO的表达，从而导致TCR的抑制。CTLA-4抗体通过结合CTLA-4来减少Treg，激活TCR
PD-1抗体和CTLA-4(细胞毒性T淋巴细胞相关蛋白4)抗体药物的联合用药在多种肿瘤上显示出药效优于PD-1抗体单药。然而联合用药药效的提升始终伴随更高的毒性，应用受到了很大限制。
贝伐珠单抗(Bevacizumab)是一种抗血管生成的靶向药物，通过与血管内皮生长因子(VEGF)结合，阻碍血管的生长，抑制肿瘤的转移,是一种广谱抗癌药。上市以来，贝伐单抗主要被获批用于治疗各种类型的癌症，如脑癌(如侵袭性脑癌，例如胶质母细胞瘤)、肾癌、肺癌(如转移性非鳞状非小细胞肺癌)、结肠癌、直肠癌、宫颈癌(如转移性宫颈癌)、子宫内膜癌、卵巢癌(如晚期卵巢癌)或输卵管癌、肾癌等。
发明内容
本发明人发现抗CTLA4-抗PD-1双特异性抗体联合VEGFR2的单克隆抗体或联合抗VEGF的单克隆抗体能够有效地防治肿瘤，毒副作用较低。由此提供了下述发明。
具体地，本发明涉及：
1.抗CTLA4-抗PD-1双特异性抗体，其优选用于治疗肿瘤，优选所述抗CTLA4-抗PD-1双特异性抗体与组分A联合施用，其中所述组分A选自抗VEGFR2单克隆抗体或其抗原结合片段，抗VEGF单克隆抗体或其抗原结合片段，或其组合，
其中
(1)所述抗CTLA4-抗PD-1双特异性抗体包括：
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；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，其包含SEQ ID NO:14所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:27－29所示的HCDR1－HCDR3)，和SEQ ID NO:16所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3)；和所述的单链抗体，其包含SEQ ID NO:2所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:33－35所示的HCDR1－HCDR3)和SEQ ID NO:4所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3)；
或者，
所述的免疫球蛋白，其包含SEQ ID NO:2所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:33－35所示的HCDR1－HCDR3)和SEQ ID NO:4所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3)；和所述的单链抗体，其包含SEQ ID NO:14所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:27－29所示的HCDR1－HCDR3)，和SEQ ID NO:16所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3)；
(2)所述抗VEGFR2单克隆抗体包含SEQ ID NO:50所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:53-55所示的HCDR1－HCDR3)和SEQ ID NO:52所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:56-58所示的LCDR1－LCDR3)；或
(3)所述抗VEGF单克隆抗体包含SEQ ID NO:60所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:63-65所示的HCDR1－HCDR3)和SEQ ID NO:62所示的轻链可变区中的LCDR1-LCDR3(优 选分别如SEQ ID NOs:66-68所示的LCDR1－LCDR3)。
2.根据项目1所述的抗CTLA4-抗PD-1双特异性抗体，其中，
(1)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、SEQ ID NO:18或其变体；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、SEQ ID NO:20或其变体；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、SEQ ID NO:43或其变体；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、SEQ ID NO:44或其变体；
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、SEQ ID NO:43或其变体；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、SEQ ID NO:44或其变体；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、SEQ ID NO:18或其变体；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、SEQ ID NO:20或其变体；
(2)所述抗VEGFR2单克隆抗体的重链可变区的氨基酸序列为SEQ ID NO:50所示的序列或其变体；并且所述抗VEGFR2单克隆抗体的轻链可变区的氨基酸序列为SEQ ID NO:52所示的序列或其变体；或(3)所述抗VEGF单克隆抗体的重链可变区的氨基酸序列为SEQ ID NO:60所示的序列或其变体；并且所述抗VEGF单克隆抗体的轻链可变区的氨基酸序列如SEQ ID NO:62所示的序列或其变体。
3.根据项目1至2中任一项所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述双特异性抗体选自如下的(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为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:2所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:4所示的序列或其变 体；
(2)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:6所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:8所示的序列或其变体；
(3)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:10所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:12所示的序列或其变体；
(4)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:2所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:4所示的序列或其变体；
(5)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:6所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:8所示的序列或其变体；
(6)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:10所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:12所示的序列或其变 体；
(7)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:2所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:4所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；
(8)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:2所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:4所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；
(9)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:6所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:8所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；
(10)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:6所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:8所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；
(11)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:所示的序列10或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:12所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或 其变体；
(12)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:10所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:12所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；
(13)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:41所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:42所示的序列或其变体；
(14)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:43所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:44所示的序列或其变体；
(15)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:41所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:42所示的序列或其变体；
(16)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:43所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:44所示的序列或 其变体；
(17)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:41所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:42所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；
(18)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:43所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:44所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；
(19)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:41所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:42所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；
以及，
(20)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:43所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:44所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体，
其中所述变体与对应序列具有至少60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％同源性。
4.根据项目1至3中任一项所述的抗CTLA4-抗PD-1双特异性抗 体，其中，
所述免疫球蛋白的重链的氨基酸序列为SEQ ID NO:40所示的序列或其变体，并且其轻链的氨基酸序列为SEQ ID NO:24所示的序列或其变体，其中所述变体序列与对应序列具有至少60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％同源性。
5.根据项目1至4中任一项所述的抗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。
6.根据项目5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。
7.根据项目1至6中任一项所述的抗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。
8.根据项目1至7中任一项所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述单链抗体(优选重链可变区)连接在免疫球蛋白的重链的C末端。
9.根据项目1至8中任一项所述的抗CTLA4-抗PD-1双特异性抗体，其中，
所述免疫球蛋白为人IgG1亚型；
其中，按照EU编号系统，所述免疫球蛋白的重链恒定区具有A突变，所述A突变选自如下突变组合之一：
L234A和L235A；或者
L234A和G237A；或者
L235A和G237A；或者
L234A、L235A、G237A；或者
按照EU编号系统，所述免疫球蛋白的重链恒定区具有B突变，所述B突变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；
或者，按照EU编号系统，所述免疫球蛋白的重链恒定区具有A突变和B突变的组合；
或者
所述抗VEGFR2单克隆抗体或所述抗VEGF单克隆抗体的重链恒定区为Ig gamma-1 chain C region，ACCESSION P01857，轻链恒定区为:Ig kappa chain C region，ACCESSION:P01834。
10.根据项目1至9中任一项所述的抗CTLA4-抗PD-1双特异性抗体，
其中，所述双特异性抗体包括：
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为SEQ ID NO:40所示的序列或其变体，并且其轻链的氨基酸序列为SEQ ID NO:24所示的序列或其变体，其中所述变体与对应序列具有至少60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％同源性；
所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:43所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:44所示的序列或其变体，其中所述变体与对应序列具有至少60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％同源性；
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，
优选地，所述抗PD-1/CTLA-4双特异性抗体轻链序列如SEQ ID NO:72所示，重链序列如SEQ ID NO:70所示，
优选地，所述抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、Fab/c、互补决定区(CDR)片段、单链抗体(例如，scFv)、双价抗体或结构域抗体。
11.药物组合物，用于治疗肿瘤(特别是恶性肿瘤)，其包含有效量的项目1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体和项目1-10任一项中定义的组分A，，优选所述药物组合物为固体形式或液体形式，优选地，抗CTLA4-抗PD-1双特异性抗体和组分A的质量比选自(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1；任选地，所述药物组合物还包含一种或多种化疗药物(优选所述化疗药为烷化剂，抗代谢药物，抗生素，植物类药物和/或激素类药物，铂类药物(如顺铂、卡铂、奥沙利铂)，阿霉素类，环磷酰胺，紫杉醇，白蛋白紫杉醇，脂质体紫杉醇，多烯紫杉醇，依托泊苷，吉西他滨，培美曲塞，卡培他滨，奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利，长春碱类，他莫昔芬，甲地孕酮，戈舍瑞林，门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)。
12.项目1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体在制备用于预防和/或治疗肿瘤(特别是恶性肿瘤)的药物或试剂盒中的应用，优选所述药物或试剂盒还包含项目1-10任一项中定义的组分A， 任选地，所述药物或试剂盒还包含一种或多种用于治疗肿瘤的药物(优选所述药物为化疗剂或生长抑制剂(如烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、血管生成素2拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合)，靶向治疗剂(如B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、PARP抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合)、抗体-药物缀合物(如美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂)、表达嵌合抗原受体的T细胞、抗体或其抗原结合片段、血管生成抑制剂、抗肿瘤剂、癌症疫苗、佐剂及其组合、烷化剂、抗代谢药物、抗生素、植物类药物和/或激素类药物、铂类药物(如顺铂、卡铂、奥沙利铂)、阿霉素类、环磷酰胺、紫杉醇、白蛋白紫杉醇、脂质体紫杉醇、多烯紫杉醇、依托泊苷、吉西他滨、培美曲塞、卡培他滨、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、长春碱类、他莫昔芬、甲地孕酮、戈舍瑞林、门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)。
13.预防和/或治疗肿瘤(特别是恶性肿瘤)的方法，包括给受试者施用治疗有效量的项目1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体，联合施用项目1-10任一项中定义的组分A，更优选地，进一步联合施用一种或多种用于治疗肿瘤的药物(优选所述药物为化疗剂或生长抑制剂(如烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、血管生成素2拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合)、靶向治疗剂(如B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、PARP抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合)、抗体-药物缀合 物(如美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂)、表达嵌合抗原受体的T细胞、抗体或其抗原结合片段、血管生成抑制剂、抗肿瘤剂、癌症疫苗、佐剂及其组合，抗代谢药物，抗生素、植物类药物和/或激素类药物、铂类药物(如顺铂、卡铂、奥沙利铂)、阿霉素类、环磷酰胺、紫杉醇(如白蛋白紫杉醇、脂质体紫杉醇，多烯紫杉醇)、依托泊苷、吉西他滨、培美曲塞、卡培他滨、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、长春碱类、他莫昔芬、甲地孕酮、戈舍瑞林、门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)，优选地，所述抗CTLA4-抗PD-1双特异性抗体、所述组分A和肿瘤化疗药物同时或顺序施用。
14.试剂盒，用于预防和/或治疗肿瘤(特别是恶性肿瘤)，其含有(a)第一药物组合物，含有项目1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体作为活性成分；和(b)第二药物组合物，其中含有项目1-10任一项中定义的组分A作为活性成分；和任选地(c)一种或多种用于治疗肿瘤的药物(优选所述药物为化疗剂或生长抑制剂(如烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、血管生成素2拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合)、靶向治疗剂(如B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、PARP抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合)、抗体-药物缀合物(如美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂)、表达嵌合抗原受体的T细胞、抗体或其抗原结合片段、血管生成抑制剂、抗肿瘤剂、癌症疫苗、佐剂及其组合、烷化剂、抗代谢药物、抗生素、植物类药物和/或激素类药物、铂类药物(如顺铂、卡铂、奥沙利铂)、阿霉素类、环磷酰胺、紫杉醇(如白蛋白紫杉醇，脂质体紫杉醇，多烯紫杉醇)、依托泊苷、吉西他滨、培美曲塞、卡培他滨、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、长春碱类、他莫昔芬、甲地孕 酮、戈舍瑞林、门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)。
15.单位制剂，优选用于治疗肿瘤(特别是恶性肿瘤)，其中，所述单位制剂包含：1～1000mg(优选10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg)的项目1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体，1～1000mg(优选10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg)的项目1-10任一项中定义的组分A和任选地一种或多种用于治疗肿瘤的药物(优选所述药物为化疗剂或生长抑制剂(如烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、血管生成素2拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合)、靶向治疗剂(如B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、PARP抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合)、抗体-药物缀合物(如美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂)、表达嵌合抗原受体的T细胞、抗体或其抗原结合片段、血管生成抑制剂、抗肿瘤剂、癌症疫苗、佐剂及其组合、烷化剂、抗代谢药物、抗生素、植物类药物和/或激素类药物、铂类药物(如顺铂、卡铂、奥沙利铂)、阿霉素类、环磷酰胺、紫杉醇(如白蛋白紫杉醇、脂质体紫杉醇、多烯紫杉醇)、依托泊苷、吉西他滨、培美曲塞、卡培他滨、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、长春碱类、他莫昔芬、甲地孕酮、戈舍瑞林、门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)；其中，所述抗CTLA4-抗PD-1双特异性抗体、组分A和化疗药分别单独包装。
16.用于预防或治疗癌症或肿瘤的方法，其中，向有需要的受试者给予一份或多份项目15所述的单位制剂，优选地，所述单位制剂中的抗CTLA4-抗PD-1双特异性抗体，组分A和化疗药各自分开施用。
17.单次药物剂量单元，优选用于治疗肿瘤(特别是恶性肿瘤)，其包含0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、 150-300mg、150-250mg、200mg或100mg)的项目1-10任一项所述的抗CTLA4抗PD-1双特异性抗体，0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的项目1-10任一项中定义的组分A。
18.项目1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体，项目11所述的药物组合物，项目12所述的应用，项目13所述的方法，项目14所述的试剂盒，项目15所述的单位制剂，或，项目16所述的方法，其中
所述肿瘤选自如下的一种或多种：
宫颈癌(如转移性宫颈癌)、肺癌如非小细胞肺癌(例如鳞状非小细胞肺癌或非鳞状非小细胞肺癌)、食管癌、食管鳞癌、回盲部腺癌、壶腹部腺癌、小细胞肺癌、胃癌(例如晚期胃癌、胃腺癌或胃食管结合部腺癌)、肾癌、肾癌子宫内膜癌、肾上腺皮质癌、前列腺癌、甲状腺癌、腹膜癌、腺癌、胰腺癌、胶质瘤、头颈癌、骨癌、睾丸癌、白血病、骨髓瘤、淋巴瘤、肉瘤、间皮瘤、肝细胞癌、结肠癌、胆癌、胆管癌、直肠癌、结肠癌、子宫内膜癌、卵巢癌(如晚期卵巢癌)或输卵管癌、大细胞神经内分泌癌、尿路上皮癌(例如上尿路上皮癌或膀胱癌)、乳腺癌、三阴乳腺癌、外周T细胞淋巴瘤、黑色素瘤、鼻咽癌，以及高度微卫星不稳定型(MSI-H)或错配修复缺陷型(dMMR)实体瘤、脑癌(如侵袭性脑癌，例如胶质母细胞瘤)、卵巢癌、鳞状细胞癌、基底细胞癌、腺瘤、粘液性或浆液性囊腺癌、平滑肌肉瘤、横纹肌肉瘤、绒毛膜上皮癌、恶性葡萄胎、恶性支持细胞-间质细胞瘤、恶性颗粒细胞瘤、无性细胞瘤。
19.项目1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体，项目11所述的药物组合物，项目12所述的应用，项目13所述的方法，项目14所述的试剂盒，项目15所述的单位制剂，或，项目16所述的方法，其中所述抗CTLA4-抗PD-1双特异性抗体，组分A和/或所述化疗药为适于静脉注射或静脉滴注的形式，优选液体的形式。
20.根据项目13或16所述的方法，其中，所述给受试者施用有效量的抗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。
21.根据项目13或16所述的方法，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；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，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg；
组分A的单次给药剂量为每千克体重0.1-100mg，优选1-10mg；或者，组分A的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg，
优选地，每天两次至约每隔一天一次给药、或每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。
在一些实施方案中，所述抗CTLA4-抗PD-1双特异性抗体、抗VEGFR2单克隆抗体或抗VEGF单克隆抗体以1mg/kg、2mg/kg、3mg/kg、5mg/kg、6mg/kg、9mg/kg、10mg/kg，11mg/kg、12mg/kg、13mg/kg、14mg/kg、15mg/kg、16mg/kg、17mg/kg、18mg/kg、19mg/kg、20mg/kg、21mg/kg、22mg/kg、23mg/kg、24mg/kg、25mg/kg体重的剂量施用。
在一些实施方案中，所述抗CTLA4-抗PD-1双特异性抗体、抗VEGFR2单克隆抗体或抗VEGF单克隆抗体以能够有效治疗所述癌症的一个或多个统一剂量施用。在一些具体实施方式中，所述统一剂量在大约1mg至大约1000mg的范围内。在一些具体实施方式中，所述统一剂量选自大约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。
在一些实施方案中，抗CTLA4-抗PD-1双特异性抗体、抗VEGFR2单克隆抗体或抗VEGF单克隆抗体的施用治疗以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PD-1和CTLA4双特异性抗体。即，所述抗PD-1和CTLA4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。
本发明还提供抗CTLA4-抗PD-1双特异性抗体、抗VEGFR2单克隆抗体和抗VEGF单克隆抗体在制备用于治疗癌症的包括含有固定剂量的抗CTLA4-抗PD-1双特异性抗体、抗VEGFR2单克隆抗体(或抗VEGF单克隆抗体)的容器的制品中的用途。在一些具体实施方式中，所述容器是安瓿瓶。所述固定剂量选自大约10mg、大约20mg、大约30mg、大约40mg、大约50mg、大约60mg、大约100mg、大约150mg、大约200mg、大约250mg、大约300mg、大约350mg、大约400mg、大约500mg、或大约1000mg。在一些具体实施方式中，所述制品进一步包括指导使用者为癌症病人施用所述固定剂量的抗CTLA4-抗PD-1双特异性抗体、抗VEGFR2单克隆抗体或抗VEGF单克隆抗体的包装插页或药品说明书。在一些具体实施方式中，所述制品包括1个或1个以上的安瓿瓶，所述安瓿瓶含有大约50mg、100mg、200mg、300mg、350mg、400mg、500mg或600mg的、抗VEGFR2单克隆抗体或抗VEGF单克隆抗体。在一些具体实施方式中，所述制品以50mg/5mL/瓶、100mg/10mL/瓶、200mg/10mL/瓶、或350mg/35mL/瓶的抗PD-1和CTLA4双特异性抗体、抗VEGFR2单克隆抗体或抗VEGF单克隆抗体溶液的形式进行包装。
化疗药的用量是本领域的普通技术人员可以确定的。如对于铂类化疗药，以顺铂为例，顺铂的单次给药剂量按照Calvert公式计算：
顺铂剂量(mg)＝所设定的曲线下的面积即AUC(mg/ml/min)×[肌酐清除率(ml/min)+25]，其中，AUC取值为4-7，优选5；
每2-6周给药一次，优选每3周或每4周给药一次；
和/或
给药方式为静脉滴注或静脉注射。
在本发明的一个或多个实施方案中，所述化疗剂或生长抑制剂选自烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、血管生成素2拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合。
在本发明的一个或多个实施方案中，所述靶向治疗剂选自B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、PARP抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合。
在本发明的一个或多个实施方案中，所述抗体-药物缀合物包含选自下组的药物：美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂。
本发明中，一些表述具有如下含义：
药物或治疗剂的“治疗有效量”或“治疗上有效的剂量”是当单独使用或与另一种治疗剂联合使用时保护主体免于疾病发作或促进疾病消退的药物的任何量，所述疾病消退通过疾病征状的严重程度的降低、无疾病征状阶段的频率和持续时间的增加、或由疾病折磨引起的损伤或失能的预防来证明。使用熟练的从业人员已知的多种方法可以评价治疗剂的促进疾病消退的能力，诸如在临床试验期间在人主体中，在预测对于人类的效力的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。
药物的“预防有效量”是指，其为当单独地或与抗肿瘤剂联合施用给处于发生癌症的风险的主体(例如，具有恶化前病症的主体)或具有癌症复发的风险的主体时，抑制癌症的发生或复发的任何药物量。在某些实施方案中，预防有效量完全阻止癌症的发生或复发。“抑制”癌症的发生或复发是指减少癌症的发生或复发的可能性，或完全阻止癌症的发生或复发。
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。
“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。
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)。在已知抗体重链和轻链可变区序列的情况下，目前有几种确定抗体CDR区的方法，包括Kabat，IMGT，Chothia和AbM编号系统。然而，每种关于抗体或其变体的CDR的定义的应用都将在本文定义和使用的术语的范围内。如果给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定特定CDR，而不依赖于该序列自身之外的任何实验数据。
药物或治疗剂的“治疗有效量”或“治疗上有效的剂量”是当单独使用或与另一种治疗剂联合使用时保护主体免于疾病发作或促进疾病消退的药物的任何量，所述疾病消退通过疾病征状的严重程度的降低、无疾病征状阶段的频率和持续时间的增加、或由疾病折磨引起的损伤或失能的预防来证明。使用熟练的从业人员已知的多种方法可以评价治疗剂的促进疾病消退的能力，诸如在临床试验期间在人主体中，在预测对于人类的效力的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。
药物的“预防有效量”是指，其为当单独地或与抗肿瘤剂联合施用给处于发生癌症的风险的主体(例如，具有恶化前病症的主体)或具有癌症复发的风险的主体时，抑制癌症的发生或复发的任何药物量。在某些实施方案中，预防有效量完全阻止癌症的发生或复发。“抑制”癌症的发生或复发是指减少癌症的发生或复发的可能性，或完全阻止癌症的发生或复发。
“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。
术语“单次药物剂量单元”表示在给药方案时刻，(优选按照受试者每kg体重)待给药于受试者的包含本发明所述的抗CTLA4抗PD-1双特异性抗体、抗VEGFR2抗体或抗VEGF抗体的单次药物剂型，如注射剂，例如置于安瓿瓶中。在本发明的具体实施方案中，给药方案例如包括根据每天两次至约每隔一天一次的给药周期来给药单次药物剂量单元，或者每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次。
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)和“第二”(例如第二蛋白功能区、第二连接片段)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。
附图说明
图1.混合淋巴细胞反应MLR检测抗PD-1/CTLA-4双特异性抗体Cadonilimab与抗VEGFR2抗体HpL3联用促INF-γ分泌的生物活性检测结果。
图2.混合淋巴细胞反应MLR检测Cadonilimab联合贝伐珠单抗促PBMC活化分泌IL-2的生物活性检测结果。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在本发明的下述实施例中，所用到同型对照抗体anti-HEL为靶向人抗鸡蛋溶酶体(HEL)的抗体，这些抗体的可变区序列来自于Acierno 等人发表的Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies(Acierno等人.J Mol Biol.2007；374(1):130-46.其中重链可变区序列为SEQ ID No:45所示，轻链可变区序列如SEQ ID NO:47所示)，hIgG1WT(即anti-HEL)的恒定区片段采用Ig gamma-1 chain C region，ACCESSION:P01857作为重链恒定区(SEQ ID NO:46)，Ig kappa chain C region，ACCESSION:P01834为轻链恒定区(SEQ ID NO:48)；anti-HEL为在中山康方生物医药有限公司实验室制备而成。
实施例中所使用的贝伐珠单抗(Bevacizumab，抗VEGF单克隆抗体)为罗氏制药生产的商品化药物。
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CN106967172A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，SEQ ID NO:1)
其编码的氨基酸序列：(124aa，SEQ ID NO:2)
轻链可变区的核酸序列：(378bp，SEQ ID NO:3)
其编码的氨基酸序列：(126aa，SEQ ID NO:4)
(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，SEQ ID NO:5)
其编码的氨基酸序列：(115aa，SEQ ID NO:6)
轻链可变区(4G10L1V)的核酸序列：(327bp，SEQ ID NO:7)
其编码的氨基酸序列：(109aa，SEQ ID NO:8)
(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，SEQ ID NO:9)
其编码的重链可变区氨基酸序列(4G10H3V)：(115aa，SEQ ID  NO:10)
轻链可变区(4G10L3V)的核酸序列：(327bp，SEQ ID NO:11)
其编码的氨基酸序列(4G10L3V)：(109aa，SEQ ID NO:12)
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，SEQ ID NO:13)
其编码的氨基酸序列：(118aa，SEQ ID NO:14)
轻链可变区的核酸序列：(321bp，SEQ ID NO:15)
其编码的氨基酸序列：(107aa，SEQ ID NO:16)
(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，SEQ ID NO:17)
其编码的氨基酸序列：(118aa，SEQ ID NO:18)
轻链可变区的核酸序列：(321bp，SEQ ID NO:19)
其编码的氨基酸序列：(107aa，SEQ ID NO:20)
14C12H1L1重链(14C12H1)的DNA序列：(1344bp，
SEQ ID NO:21)
其编码的氨基酸序列：(448aa，SEQ ID NO:22)
14C12H1L1轻链(14C12L1)的DNA序列：(642bp，SEQ ID NO:23)
其编码的氨基酸序列：(214aa，SEQ ID NO:24)
制备例3：双功能抗体CP001(M)、CP002(M)、CP003(M)和CP004(M) 的序列设计
双功能抗体CP001(M)、CPb002(M)、CP003(M)和CP004(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：CP001(M)、CP002(M)、CP003(M)和CP004(M)的序列设计
Figure PCTCN2022086870-appb-000001
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，CP001(M)、CP002(M)、CP003(M)和CP004(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，SEQ ID NO:41)
(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，SEQ ID NO:42)
(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，SEQ ID NO:43)
(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，SEQ ID NO:44)
为了与下述的突变后的抗体相区分，在本发明实施例中CP004(M)亦称为CP004(hG1WT)。上述的CP004(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CP004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CP004(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CP004(hG1TM)。
CP004(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，SEQ ID NO:39)
CP004(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，SEQ ID NO:40)
CP004(hG1TM)和CP004(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。
制备例5：抗PD-1/CTLA-4双特异性抗体Cadonilimab的制备
抗PD-1/CTLA-4双特异性抗体Cadonilimab(即CP004(hG1TM))的结构模式属于Morrison模式(IgG-scFv)，即在一个免疫球蛋白部分(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片段的VH端，其免疫球蛋白部分基于PD-1抗体，scFv片段基于抗CTLA-4抗体，中间由连接片段链接。
本实施例所述的抗PD-1/CTLA-4双特异性抗体Cadonilimab抗体(即CP004(hG1TM))轻链序列(SEQ ID NO:72)和重链序列(SEQ ID NO:70)序列来源于WHO Drug Information，Proposed INN:List 124(WHO Drug Information,2020；34(4)：947-949)，制备方法如下述。
将候选单克隆抗体的重链编码基因的DNA序列，包括恒定区和可变区，和轻链编码基因的DNA序列，包括恒定区和可变区，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-H，pUC57simple-L质粒。
分别将以上质粒进行酶切(HindIII&EcoRI)，电泳回收得到的重链、 轻链分别亚克隆到表达载体pcDNA3.1中(购自Invitrogen公司)，提取重组质粒，按pcDNA3.1-H+pcDNA3.1-L进行组合共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，使用蛋白纯化液相色谱系统(AKTA Purifier 10，GE)进行纯化。
将纯化后的样品，即抗体Cadonilimab，加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液，煮沸后进行SDS-PAGE电泳检测，还原型蛋白样品目标蛋白在70kD和30KD处，非还原型蛋白样品(单个抗体)目标蛋白在200kD处。与理论大小符合，由此得到纯化的抗体。
制备例6：抗VEGFR2抗体HpL3的制备
本实施例所述的抗VEGFR2抗体HpL3的抗体轻链可变区和重链可变区序列及制备方法均引自申请号为CN201610573836.X的专利《一类抗血管内皮生长因子受体VEGFR2的单克隆抗体及其编码基因和应用》。
HpL3的重链可变区氨基酸序列如SEQ ID NO:50所示，其编码基因的DNA序列如SEQ ID NO:49所示，重链可变区CDR1-3的序列分别如SEQ ID NO:53-55所示。
HpL3的轻链可变区氨基酸序列如SEQ ID NO:52所示，其编码基因的DNA序列如SEQ ID NO:51所示，轻链可变区CDR1-3的序列分别如SEQ ID NO:56-58所示。
HpL3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
将候选单克隆抗体的重链编码基因的DNA序列，包括恒定区和可变区，和轻链编码基因的DNA序列，包括恒定区和可变区，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-PCABH，pUC57simple-L3质粒。
分别将以上质粒进行酶切(HindIII&EcoRI)，电泳回收得到的重链、轻链分别亚克隆到表达载体pcDNA3.1中(购自Invitrogen公司)，提取重组质粒，按pcDNA3.1-PCABH+pcDNA3.1-L3进行组合共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上 样至HiTrap MabSelect SuRe柱，使用蛋白纯化液相色谱系统(AKTA Purifier 10，GE)进行纯化。
将纯化后的样品，即抗体HpL3，加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液，煮沸后进行SDS-PAGE电泳检测，还原型蛋白样品目标蛋白在45kD和30KD处，非还原型蛋白样品(单个抗体)目标蛋白在150kD处。与理论大小符合，由此得到纯化的单克隆抗体。
实施例1.混合淋巴细胞反应MLR检测抗PD-1/CTLA-4双特异性抗体Cadonilimab与抗VEGFR2抗体HpL3联用促INF-γ分泌的生物活性
Raji-PDL1细胞的制备：
人PD-L1过表达慢病毒载体plenti6.3/V5-PDL1FL-BSD经病毒包装后感染Raji细胞，经10ug/ml BSD(杀稻瘟菌素，Blasticidin)加药筛选后，获得抗药性细胞株Raji-PDL1。plenti6.3/V5-PDL1FL-BSD由供应商南京金斯瑞合成基因PDL1FL(PDL1，Genebank ID:NP_054862.1)，然后通过酶切连接到plenti6.3/V5-BSD(Invitrogen货号k531520)上获得。
实验前两天，复苏PBMC(分离自正常人外周血)，完全培养基培养于37℃，5％二氧化碳培养箱；2h后，待PBMC状态恢复，添加SEB(终浓度0.5μg/mL)(Toxin technology，货号：BT202)刺激两天；实验当天，收集Raji-PDL1细胞，离心重悬计数(用1640+10％FBS重悬)，调整细胞密度至2×10 6/mL，加入MMC(Mito-mycin C，终浓度为2μg/mL，丝裂霉素C，厂家：Stressmarq，货号：SIH-246)于37℃，5％二氧化碳培养箱处理1小时；收集SEB刺激两天后的PBMC以及MMC处理后的Raji-PDL1细胞，用1640基础培养基洗涤两次，完全培养基重悬细胞计数，按PBMC 1×10 5/孔、Raji-PDL1 1×10 5/孔(各50μL/孔)接种到96孔U型板中(3799)；按实验设计加药，并设置空白对照和阴性对照，共培养3天(体系终体积为200μL)；3天后，250xg离心5min(Beckman离心机)，收集细胞上清，达科为试剂盒检测IFN-γ含量。
如图1所示，在PBMC、Raji-PDL1细胞混合培养体系中，加入Cadonilimab和HpL3抗体均能显著诱导PBMC进一步分泌IFN-γ，具有显著剂量依赖关系，促IFN-γ分泌活性水平方面，与Cadonilimab和HpL3抗体单药相比，Cadonilimab和HpL3联用具有更佳的促IFN-γ分泌潜能。
实施例2.混合淋巴细胞反应MLR检测Cadonilimab联合贝伐珠单抗促PBMC活化分泌IL-2的生物活性
实验前两天，复苏PBMC(分离自健康志愿者知情同意后自愿捐献的外周血)，完全培养基培养于37℃，5％二氧化碳培养箱；2h后，待PBMC状态恢复，添加SEB(终浓度0.5μg/mL)(Toxin technology，货号：BT202)刺激两天；实验当天，收集Raji-PDL1(康方生物构建)细胞，离心重悬计数(用1640+10％FBS重悬)，调整细胞密度至2×10 6/mL，加入MMC(Mito-mycin C，终浓度为2μg/mL)(丝裂霉素C，厂家：Stressmarq，货号：SIH-246)于37℃，5％二氧化碳培养箱处理1小时；收集SEB刺激两天后的PBMC以及MMC处理后的Raji-PDL1细胞，用1640基础培养基洗涤两次，分析培养基重悬细胞计数，按PBMC 1×10 5/孔、Raji-PDL1 1×10 5/孔(各40μL/孔)接种到96孔U型板中(3799)；常规消化收集Hela细胞(购自中国科学院)，按2×10 4/孔(40μL/孔)接种到96孔U型板中(3799)；按实验设计加药，并设置阴性对照和同型对照，共培养3天(体系终体积为200μL)；3天后，250xg离心5min(Beckman离心机)，收集细胞上清，达科为试剂盒检测IL-2含量。
如图2所示，在PBMC、Raji-PDL1、Hela(宫颈癌)细胞以及VEGF(批号：20180126-1，康方生物生产)的混合培养体系中，加入Cadonilimab和贝伐珠单抗均能显著诱导PBMC进一步分泌IL-2，具有显著剂量依赖关系，促IL-2分泌活性水平方面，与Cadonilimab和贝伐珠单抗单药相比，Cadonilimab和贝伐珠单抗联用具有更佳的促IL-2分泌潜能。
贝伐珠单抗的重链可变区氨基酸序列如SEQ ID NO:60所示，其编码基因的DNA序列如SEQ ID NO:59所示，重链可变区CDR1-3的序列分别如SEQ ID NO:63-65所示。贝伐珠单抗的轻链可变区氨基酸序列如SEQ ID NO:62所示，其编码基因的DNA序列如SEQ ID NO:61所示，轻链可变区CDR1-3的序列分别如SEQ ID NO:66-68所示。
序列表：
SEQ ID No.1：4G10重链可变区的核酸序列
Figure PCTCN2022086870-appb-000002
SEQ ID No.2：4G10重链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000003
SEQ ID No.3：4G10轻链可变区的核酸序列
Figure PCTCN2022086870-appb-000004
SEQ ID No.4：4G10轻链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000005
SEQ ID No.5：4G10H1L1重链可变区的核酸序列
Figure PCTCN2022086870-appb-000006
SEQ ID No.6：4G10H1L1重链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000007
Figure PCTCN2022086870-appb-000008
SEQ ID No.7：4G10H1L1轻链可变区的核酸序列
Figure PCTCN2022086870-appb-000009
SEQ ID No.8：4G10H1L1轻链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000010
SEQ ID No.9：4G10H3L3重链可变区的核酸序列
Figure PCTCN2022086870-appb-000011
SEQ ID No.10：4G10H3L3重链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000012
SEQ ID No.11：4G10H3L3轻链可变区的核酸序列
Figure PCTCN2022086870-appb-000013
SEQ ID No.12：4G10H3L3轻链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000014
SEQ ID No.13：14C12重链可变区的核酸序列
Figure PCTCN2022086870-appb-000015
SEQ ID No.14：14C12重链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000016
SEQ ID No.15：14C12轻链可变区的核酸序列
Figure PCTCN2022086870-appb-000017
SEQ ID No.16：14C12轻链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000018
SEQ ID No.17：14C12H1L1重链可变区的核酸序列
Figure PCTCN2022086870-appb-000019
SEQ ID No.18：14C12H1L1重链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000020
SEQ ID No.19：14C12H1L1轻链可变区的核酸序列
Figure PCTCN2022086870-appb-000021
SEQ ID No.20：14C12H1L1轻链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000022
SEQ ID No.21：14C12H1L1重链(14C12H1)的DNA序列(1344bp)
Figure PCTCN2022086870-appb-000023
SEQ ID No.22：14C12H1L1重链(14C12H1)编码的氨基酸序列(448aa)
Figure PCTCN2022086870-appb-000024
SEQ ID No.23：14C12H1L1轻链(14C12L1)的DNA序列(642bp)
Figure PCTCN2022086870-appb-000025
SEQ ID No.24：14C12H1L1轻链(14C12L1)编码的氨基酸序列(214aa)
Figure PCTCN2022086870-appb-000026
SEQ ID No.25：Linker1的氨基酸序列
Figure PCTCN2022086870-appb-000027
SEQ ID No.26：Linker2的氨基酸序列
Figure PCTCN2022086870-appb-000028
SEQ ID No.27：14C12的HCDR1：GFAFSSYD
SEQ ID No.28：14C12的HCDR2：ISGGGRYT
SEQ ID No.29：14C12的HCDR3：ANRYGEAWFAY
SEQ ID No.30：14C12的LCDR1：QDINTY
SEQ ID No.31：14C12的LCDR2：RAN
SEQ ID No.32：14C12的LCDR3：LQYDEFPLT
SEQ ID No.33：4G10的HCDR1：GYSFTGYT
SEQ ID No.34：4G10的HCDR2：INPYNNIT
SEQ ID No.35：4G10的HCDR3：ARLDYRSY
SEQ ID No.36：4G10的LCDR1：TGAVTTSNF
SEQ ID No.37：4G10的LCDR2：GTN
SEQ ID No.38：4G10的LCDR3：ALWYSNHWV
SEQ ID No.39：CP004(hG1TM)的免疫球蛋白部分的重链的DNA序列，突变位点用下划线标出
Figure PCTCN2022086870-appb-000029
Figure PCTCN2022086870-appb-000030
SEQ ID No.40：CP004(hG1TM)中的免疫球蛋白部分的重链的氨基酸序列，突变位点用下划线标出
Figure PCTCN2022086870-appb-000031
SEQ ID No.41：4G10H1V(M)的氨基酸序列，突变位点用下划线标出
Figure PCTCN2022086870-appb-000032
SEQ ID No.42：4G10L1V(M)的氨基酸序列，突变位点用下划线标出
Figure PCTCN2022086870-appb-000033
Figure PCTCN2022086870-appb-000034
SEQ ID No.43：4G10H3V(M)的氨基酸序列，突变位点用下划线标出
Figure PCTCN2022086870-appb-000035
SEQ ID No.44：4G10L3V(M)的氨基酸序列，突变位点用下划线标出
Figure PCTCN2022086870-appb-000036
SEQ ID No:45:hIgG的重链可变区序列
Figure PCTCN2022086870-appb-000037
SEQ ID No:46 hIgG的重链恒定区序列
Figure PCTCN2022086870-appb-000038
SEQ ID No:47 hIgG的轻链可变区序列
Figure PCTCN2022086870-appb-000039
SEQ ID No:48 hIgG的轻链恒定区序列
Figure PCTCN2022086870-appb-000040
SEQ ID No:49 HpL3重链可变区的核酸序列(VEGFR2)
GAAGTGCAGCTGGTGCAGAGCGGCGGGGGACTGGTGAAGCCCGGCGGGTCCCTGCGACTGTCTTGCGCCGCTAGT GGCTTCACCTTCAGCTCCTACTCTATGAACTGGGTGAGACAGGCCCCTGGAAAAGGCCTGGAGTGGGTCTCTAGT ATCTCAAG CTCCTCTAGTTACATCTACTATGCCGACAGCGTGAAGGGGCGGTTCACCATCTCAAGAGATAACGCTAAAAATAGTCTGTATCTGCAGATGAACAGCCTGAGGGCAGAAGACACTGCCGTGTACTATTGT GCTCGCGTCACCGACGCATTTGATATTTGGGGGCAGGGAACCATGGTGACAGTCTCAAGC(注：下划线表示CDR序列)
SEQ ID No:50 HpL3重链可变区的氨基酸序列
EVQLVQSGGGLVKPGGSLRLSCAAS GFTFSSYSMNWVRQAPGKGLEWVSS ISSSSSY IYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARVTDAFDIWGQGTMVTVSS(注：下划线表示CDR序列)
SEQ ID No:51 HpL3轻链可变区的核酸序列
GACATTCAGATGACTCAGAGCCCTTCTTCAGTGTCCGCCTCTATCGGCGACCGGGTCACCATTACATGCAGAGCTTCC CAGGGCCTCGATAACTGGCTGGGGTGGTACCAGCAGAAGCCTGGGAAAGCCCCAAAGCTGCTGATCTAC GACGCTTCCAATCTGGATACCGGAGTGCCATCTCGATTCAGTGGCTCAGGGAGCGGAACATACTTTACTCTGACCATCAGCTCCCTGCAGGCTGAGGACTTCGCAGTGTATTTTTGC CAGCAGG CAAAGGCCTTCCCTCCCACCTTTGGCGGGGGAACAAAAGTGGACATCAAG(注：下划线表示CDR序列)
SEQ ID No:52 HpL3轻链可变区的氨基酸序列
DIQMTQSPSSVSASIGDRVTITCRAS QGLDNWLGWYQQKPGKAPKLLIYDASNLDTGVPSRFSGSGSGTYFTLTISSLQAEDFAVYFC QQAKAFPPTFGGGTKVDIK(注：下划线表示CDR序列)
HpL3重链可变区的HCDR1-HCDR3
HCDR1：GFTFSSYS(SEQ ID NO：53)
HCDR2：ISSSSSYI(SEQ ID NO：54)
HCDR3：ARVTDAFDI(SEQ ID NO：55)
HpL3轻链可变区的LCDR1-LCDR3
LCDR1：QGLDNW(SEQ ID NO：56)
LCDR2：DAS(SEQ ID NO：57)
LCDR3：QQAKAFPPT(SEQ ID NO：58)
SEQ ID No:59 Bevacizumab重链可变区的核酸序列(VEGF)
Figure PCTCN2022086870-appb-000041
Figure PCTCN2022086870-appb-000042
SEQ ID No:60 Bevacizumab重链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000043
SEQ ID No:61 Bevacizumab轻链可变区的核酸序列
Figure PCTCN2022086870-appb-000044
SEQ ID No:62 Bevacizumab轻链可变区的氨基酸序列
Figure PCTCN2022086870-appb-000045
Bevacizumab重链可变区的3个CDR区的氨基酸序列如下：
HCDR1:GYTFTNYG(SEQ ID NO:63)
HCDR2:INTYTGEP(SEQ ID NO:64)
HCDR3:AKYPHYYGSSHWYFDV(SEQ ID NO:65)
Bevacizumab轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1:QDISNY(SEQ ID NO:66)
LCDR2:FTS(SEQ ID NO:67)
LCDR3:QQYSTVPWT(SEQ ID NO:68)
SEQ ID No.69 CP004(hG1TM)重链的核酸序列
Figure PCTCN2022086870-appb-000046
Figure PCTCN2022086870-appb-000047
SEQ ID No.70 CP004(hG1TM)重链的氨基酸序列(全长)
EVQLVESGGGLVQPGGSLRLSCAAS GFAFSSYDMSWVRQAPGKGLDWVAT IS GGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYC ANRYGEAWFA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGGGGSGGGGSGGGGSGGGGSQVQLVESGAEVKKPGASVKVSCKAS GYS FTGYTMNWVRQAPGQCLEWIGL INPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFC ARLDYRSYWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSS TGAVTTSNFPNWVQQKPGQAPRSLIG GTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYC ALWYSNHWVFGCGTKLTVLR(注：下划线表示CDR序列)
SEQ ID No.71 CP004(hG1TM)轻链的核酸序列
Figure PCTCN2022086870-appb-000048
SEQ ID No.72 CP004(hG1TM)轻链的氨基酸序列(全长)
Figure PCTCN2022086870-appb-000049

Claims (21)

  1. 抗CTLA4-抗PD-1双特异性抗体，其优选用于治疗肿瘤，优选所述抗CTLA4-抗PD-1双特异性抗体与组分A联合施用，所述组分A选自抗VEGFR2单克隆抗体或其抗原结合片段，抗VEGF单克隆抗体或其抗原结合片段，或其组合，
    其中
    (1)所述抗CTLA4-抗PD-1双特异性抗体包括：
    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；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，其包含SEQ ID NO:14所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:27－29所示的HCDR1－HCDR3)，和SEQ ID NO:16所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3)；和所述的单链抗体，其包含SEQ ID NO:2所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:33－35所示的HCDR1－HCDR3)和SEQ ID NO:4所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3)；
    或者，
    所述的免疫球蛋白，其包含SEQ ID NO:2所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:33－35所示的HCDR1－HCDR3)和SEQ ID NO:4所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:36－38所示的LCDR1－LCDR3)；和所述的单链抗体，其包含SEQ ID NO:14所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:27－29所示的HCDR1－HCDR3)，和SEQ  ID NO:16所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:30－32所示的LCDR1－LCDR3)；
    (2)所述抗VEGFR2单克隆抗体包含SEQ ID NO:50所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:53-55所示的HCDR1－HCDR3)和SEQ ID NO:52所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:56-58所示的LCDR1－LCDR3)；或
    (3)所述抗VEGF单克隆抗体包含SEQ ID NO:60所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:63-65所示的HCDR1－HCDR3)和SEQ ID NO:62所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:66-68所示的LCDR1－LCDR3)。
  2. 根据权利要求1所述的抗CTLA4-抗PD-1双特异性抗体，其中，
    (1)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、SEQ ID NO:18所示的序列或其变体；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、SEQ ID NO:20所示的序列或其变体；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、SEQ ID NO:43所示的序列或其变体；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、SEQ ID NO:44所示的序列或其变体；
    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、SEQ ID NO:43所示的序列或其变体；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、SEQ ID NO:44所示的序列或其变体；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、SEQ ID NO:18所示的序列或其变体；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、SEQ ID NO:20所示的序列或其变体；
    (2)所述抗VEGFR2单克隆抗体的重链可变区的氨基酸序列为 SEQ ID NO:50所示的序列或其变体；并且所述抗VEGFR2单克隆抗体的轻链可变区的氨基酸序列为SEQ ID NO:52所示的序列或其变体；或
    (3)所述抗VEGF单克隆抗体的重链可变区的氨基酸序列为SEQ ID NO:60所示的序列或其变体；并且所述抗VEGF单克隆抗体的轻链可变区的氨基酸序列为SEQ ID NO:62所示的序列或其变体，
    其中所述变体的序列与对应序列具有至少60％、70％、80％、81％、82％、83％、84％、85％、86％、87％，88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％同源性。
  3. 根据权利要求1至2中任一项所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述双特异性抗体选自如下的(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为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:2所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:4所示的序列或其变体；
    (2)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:6所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:8所示的序列或其变体；
    (3)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:10所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:12所示的序列或其变体；
    (4)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18 所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:2所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:4所示的序列或其变体；
    (5)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:6所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:8所示的序列或其变体；
    (6)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:10所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:12所示的序列或其变体；
    (7)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:2所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:4所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；
    (8)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:2所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:4所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；
    (9)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:6所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列 为SEQ ID NO:8所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；
    (10)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:6所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:8所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；
    (11)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:10所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:12所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；
    (12)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:10所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:12所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；
    (13)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:41所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:42所示的序列或其变体；
    (14)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸 序列为SEQ ID NO:16所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:43所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:44所示的序列或其变体；
    (15)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:41所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:42所示的序列或其变体；
    (16)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:43所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:44所示的序列或其变体；
    (17)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:41所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:42所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；
    (18)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:43所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:44所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:14所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:16所示的序列或其变体；
    (19)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:41所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸 序列为SEQ ID NO:42所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体；
    以及，
    (20)所述免疫球蛋白的重链可变区的氨基酸序列为SEQ ID NO:43所示的序列或其变体，并且所述免疫球蛋白的轻链可变区的氨基酸序列为SEQ ID NO:44所示的序列或其变体；和，所述单链抗体的重链可变区的氨基酸序列为SEQ ID NO:18所示的序列或其变体，并且所述单链抗体的轻链可变区的氨基酸序列为SEQ ID NO:20所示的序列或其变体，
    其中所述变体的序列与对应序列具有至少60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％同源性。
  4. 根据权利要求1至3中任一项所述的抗CTLA4-抗PD-1双特异性抗体，其中，
    所述免疫球蛋白的重链的氨基酸序列为SEQ ID NO:40所示的序列或其变体，并且其轻链的氨基酸序列为SEQ ID NO:24所示的序列或其变体，其中所述变体的序列与对应序列具有至少60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或至少99％同源性。
  5. 根据权利要求1至4中任一项所述的抗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。
  6. 根据权利要求5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。
  7. 根据权利要求1至6中任一项所述的抗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。
  8. 根据权利要求1至7中任一项所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述单链抗体(优选重链可变区)连接在免疫球蛋白的重链，优选连接在免疫球蛋白重链的C末端。
  9. 根据权利要求1至8中任一项所述的抗CTLA4-抗PD-1双特异性抗体，其中，
    所述免疫球蛋白为人IgG1亚型；
    其中，按照EU编号系统，所述免疫球蛋白的重链恒定区具有A突变，所述A突变选自如下突变组合之一：
    L234A和L235A；或者
    L234A和G237A；或者
    L235A和G237A；或者
    L234A、L235A、G237A；或者
    按照EU编号系统，所述免疫球蛋白的重链恒定区具有B突变，所述B突变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；或者
    或者，按照EU编号系统，所述免疫球蛋白的重链恒定区具有A突变和B突变的组合；
    所述抗VEGFR2单克隆抗体或所述抗VEGF单克隆抗体的重链恒定区为Ig gamma-1 chain C region，ACCESSION P01857，轻链恒定区为:Ig kappa chain C region，ACCESSION:P01834。
  10. 根据权利要求1至9中任一项所述的抗CTLA4-抗PD-1双特异性抗体，
    其中，所述双特异性抗体包括：
    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或与其具有至少60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％同源性的序列所示，并且其轻链的氨基酸序列如SEQ ID NO:24或与其具有至少60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％同源性的序列所示；
    所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％同源性的序列所示，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或与其具有至少60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％同源性的序列所示；
    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，
    优选地，所述抗PD-1/CTLA-4双特异性抗体轻链序列如SEQ ID NO:72所示，重链序列如SEQ ID NO:70所示，
    优选地，所述抗原结合片段选自Fab、Fab'、F(ab') 2、Fd、Fv、dAb、Fab/c、互补决定区(CDR)片段、单链抗体(例如，scFv)、双价抗体或 结构域抗体。
  11. 药物组合物，用于治疗肿瘤(特别是恶性肿瘤)，其包含有效量的权利要求1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体和权利要求1-10任一项中定义的组分A，优选地，抗CTLA4-抗PD-1双特异性抗体和组分A的质量比选自(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1；优选所述药物组合物为固体形式或液体形式，任选地，所述药物组合物还包含一种或多种化疗药物(优选所述化疗药为烷化剂，抗代谢药物，抗生素，植物类药物和/或激素类药物，铂类药物如顺铂、卡铂、奥沙利铂，阿霉素类，环磷酰胺，紫杉醇(如白蛋白紫杉醇，脂质体紫杉醇，多烯紫杉醇)，依托泊苷，吉西他滨，培美曲塞，卡培他滨，奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利，长春碱类，他莫昔芬，甲地孕酮，戈舍瑞林，门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)。
  12. 权利要求1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体在制备用于预防和/或治疗肿瘤(特别是恶性肿瘤)的药物或试剂盒中的应用，优选所述药物或试剂盒还包含权利要求1-10任一项中定义的组分A，任选地，所述药物或试剂盒还包含一种或多种用于治疗肿瘤的药物(优选所述药物为化疗剂或生长抑制剂(如烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、血管生成素2拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合)，靶向治疗剂(如B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、PARP抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合)、抗体-药物缀合物(如美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂)、表达嵌合抗原受体的T细胞、抗体或其抗原结合片段、血管生成抑制剂、抗肿瘤剂、癌症疫苗、佐剂及其组合、烷化剂、抗代谢药物、抗生素、植物类药物和/或激素类药物、铂类药物(如顺铂、卡铂、奥沙利铂)、阿霉素类、环磷酰胺、紫 杉醇、白蛋白紫杉醇、脂质体紫杉醇、多烯紫杉醇、依托泊苷、吉西他滨、培美曲塞、卡培他滨、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、长春碱类、他莫昔芬、甲地孕酮、戈舍瑞林、门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)。
  13. 预防和/或治疗肿瘤(特别是恶性肿瘤)的方法，包括给受试者施用治疗有效量的权利要求1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体，联合施用权利要求1-10任一项中定义的组分A，更优选地，进一步联合施用一种或多种用于治疗肿瘤的药物(优选所述药物为化疗剂或生长抑制剂(如烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、血管生成素2拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合)，靶向治疗剂(如B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、PARP抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合)、抗体-药物缀合物(如美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂)、表达嵌合抗原受体的T细胞、抗体或其抗原结合片段、血管生成抑制剂、抗肿瘤剂、癌症疫苗、佐剂及其组合、烷化剂，抗代谢药物、抗生素、植物类药物和/或激素类药物、铂类药物(如顺铂、卡铂、奥沙利铂)、阿霉素类、环磷酰胺、紫杉醇(如白蛋白紫杉醇、脂质体紫杉醇、多烯紫杉醇)、依托泊苷、吉西他滨、培美曲塞、卡培他滨、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、长春碱类、他莫昔芬、甲地孕酮、戈舍瑞林、门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)，优选地，所述抗CTLA4-抗PD-1双特异性抗体、所述组分A和肿瘤化疗药物同时或顺序施用。
  14. 试剂盒，用于预防和/或治疗肿瘤(特别是恶性肿瘤)，其含有(a)第一药物组合物，含有权利要求1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体作为活性成分；和(b)第二药物组合物，其中含有权利要求1-10任一项中定义的组分A作为活性成分；和任选地(c) 一种或多种用于治疗肿瘤的药物(优选所述药物为化疗剂或生长抑制剂(如烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、血管生成素2拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合)、靶向治疗剂(如B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、VEGF抑制剂、PARP抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合)、抗体-药物缀合物(如美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂)、表达嵌合抗原受体的T细胞、抗体或其抗原结合片段、血管生成抑制剂、抗肿瘤剂、癌症疫苗、佐剂及其组合、烷化剂、抗代谢药物、抗生素、植物类药物和/或激素类药物、铂类药物(如顺铂、卡铂、奥沙利铂)、阿霉素类、环磷酰胺、紫杉醇(如白蛋白紫杉醇，脂质体紫杉醇，多烯紫杉醇)、依托泊苷、吉西他滨、培美曲塞、卡培他滨、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、长春碱类、他莫昔芬、甲地孕酮、戈舍瑞林、门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)。
  15. 单位制剂，优选用于治疗肿瘤(特别是恶性肿瘤)，其中，所述单位制剂包含：1～10000mg(优选10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg)的权利要求1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体，1～10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的权利要求1-10任一项中定义的组分A和任选地一种或多种用于治疗肿瘤的药物(优选所述药物为化疗剂或生长抑制剂(如烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、VEGF拮抗剂、血管生成素2拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合)、靶向治疗剂(如B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制 剂、mTOR抑制剂、VEGF抑制剂、PARP抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合)、抗体-药物缀合物(如美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂)、表达嵌合抗原受体的T细胞、抗体或其抗原结合片段、血管生成抑制剂、抗肿瘤剂、癌症疫苗、佐剂及其组合、抗代谢药物、抗生素、植物类药物和/或激素类药物、铂类药物(如顺铂、卡铂、奥沙利铂)、阿霉素类、环磷酰胺、紫杉醇(如白蛋白紫杉醇、脂质体紫杉醇、多烯紫杉醇)、依托泊苷、吉西他滨、培美曲塞、卡培他滨、奥拉帕利、芦卡帕利、尼拉帕利、他拉唑帕利、氟唑帕利、长春碱类、他莫昔芬、甲地孕酮、戈舍瑞林、门冬酰胺酶和/或氟尿嘧啶类抗肿瘤药)；其中，所述抗CTLA4-抗PD-1双特异性抗体、组分A和化疗药分别单独包装。
  16. 用于预防或治疗癌症或肿瘤的方法，其中，向有需要的受试者给予一份或多份权利要求15所述的单位制剂，优选地，所述单位制剂中的抗CTLA4-抗PD-1双特异性抗体，所述组分A和化疗药各自分开施用。
  17. 单次药物剂量单元，优选用于治疗肿瘤(特别是恶性肿瘤)，其包含0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的权利要求1-10任一项所述的抗CTLA4抗PD-1双特异性抗体，0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的权利要求1-10任一项中定义的组分A。
  18. 权利要求1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体，权利要求11所述的药物组合物，权利要求12所述的应用，权利要求13所述的方法，权利要求14所述的试剂盒，权利要求15所述的单位制剂，或，权利要求16所述的方法，其中
    所述肿瘤选自如下的一种或多种：
    宫颈癌(如转移性宫颈癌)、肺癌如非小细胞肺癌(例如鳞状非小细胞肺癌或非鳞状非小细胞肺癌)、食管癌、食管鳞癌、回盲部腺癌、壶腹部腺癌、小细胞肺癌、胃癌(例如晚期胃癌、胃腺癌或胃食管结合部腺癌)、肾癌、肾癌子宫内膜癌、肾上腺皮质癌、前列腺癌、甲状腺癌、腹 膜癌、腺癌、胰腺癌、胶质瘤、头颈癌、骨癌、睾丸癌、白血病、骨髓瘤、淋巴瘤、肉瘤、间皮瘤、肝细胞癌、结肠癌、胆癌、胆管癌、直肠癌、结肠癌、子宫内膜癌、卵巢癌(如晚期卵巢癌)或输卵管癌、大细胞神经内分泌癌、尿路上皮癌(例如上尿路上皮癌或膀胱癌)、乳腺癌、三阴乳腺癌、外周T细胞淋巴瘤、黑色素瘤、鼻咽癌，以及高度微卫星不稳定型(MSI-H)或错配修复缺陷型(dMMR)实体瘤、脑癌(如侵袭性脑癌，例如胶质母细胞瘤)、卵巢癌、鳞状细胞癌、基底细胞癌、腺瘤、粘液性或浆液性囊腺癌、平滑肌肉瘤、横纹肌肉瘤、绒毛膜上皮癌、恶性葡萄胎、恶性支持细胞-间质细胞瘤、恶性颗粒细胞瘤、无性细胞瘤。
  19. 权利要求1-10任一项所述的抗CTLA4-抗PD-1双特异性抗体，权利要求11所述的药物组合物，权利要求12所述的应用，权利要求13所述的方法，权利要求14所述的试剂盒，权利要求15所述的单位制剂，或，权利要求16所述的方法，其中所述抗CTLA4-抗PD-1双特异性抗体，所述组分A和/或所述化疗药为适于静脉注射或静脉滴注的形式，优选液体的形式。
  20. 根据权利要求13或16所述的方法，其中，所述给受试者施用有效量的抗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。
  21. 根据权利要求13或16所述的方法，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；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，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg；
    组分A的单次给药剂量为每千克体重0.1-100mg，优选1-10mg；或者，组分A的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg，
    优选地，每天两次至约每隔一天一次给药，或每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次；
    优选地，给药方式为静脉滴注或静脉注射。
PCT/CN2022/086870 2021-04-14 2022-04-14 抗体在抗肿瘤治疗中的用途 WO2022218383A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP22787605.9A EP4324854A1 (en) 2021-04-14 2022-04-14 Use of antibody in anti-tumor treatment
JP2023562813A JP2024515618A (ja) 2021-04-14 2022-04-14 抗腫瘍処置における抗体の使用
IL307494A IL307494A (en) 2021-04-14 2022-04-14 Antibody use in antitumor therapy
AU2022258365A AU2022258365A1 (en) 2021-04-14 2022-04-14 Use of antibody in anti-tumor treatment
BR112023021177A BR112023021177A2 (pt) 2021-04-14 2022-04-14 Anticorpo, composição farmacêutica, uso do anticorpo, método para prevenir e/ou tratar um tumor, kit, formulação unitária, método para prevenir ou tratar um câncer ou tumor e unidade de dose única
CA3213734A CA3213734A1 (en) 2021-04-14 2022-04-14 Use of antibody in anti-tumor treatment
KR1020237038950A KR20230171456A (ko) 2021-04-14 2022-04-14 항-종양 치료에서의 항체의 용도

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110404963.8 2021-04-14
CN202110404963 2021-04-14

Publications (1)

Publication Number Publication Date
WO2022218383A1 true WO2022218383A1 (zh) 2022-10-20

Family

ID=83574345

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/086870 WO2022218383A1 (zh) 2021-04-14 2022-04-14 抗体在抗肿瘤治疗中的用途

Country Status (9)

Country Link
EP (1) EP4324854A1 (zh)
JP (1) JP2024515618A (zh)
KR (1) KR20230171456A (zh)
CN (1) CN115197325A (zh)
AU (1) AU2022258365A1 (zh)
BR (1) BR112023021177A2 (zh)
CA (1) CA3213734A1 (zh)
IL (1) IL307494A (zh)
WO (1) WO2022218383A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104974253A (zh) * 2014-04-01 2015-10-14 上海中信国健药业股份有限公司 抗ctla-4/pd-1双特异性抗体、其制备方法及应用
CN106188296A (zh) * 2016-07-19 2016-12-07 中山康方生物医药有限公司 一类抗血管内皮生长因子受体vegfr2的单克隆抗体及其编码基因和应用
CN106967172A (zh) 2016-08-23 2017-07-21 中山康方生物医药有限公司 抗ctla4‑抗pd‑1 双功能抗体、其药物组合物及其用途
WO2019179421A1 (en) * 2018-03-19 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel bispecific pd-1/ctla-4 antibody molecules
WO2020185722A2 (en) * 2019-03-13 2020-09-17 Merck Sharp & Dohme Corp. Anti-cancer combination therapies comprising ctla-4 and pd-1 blocking agents
CN112300286A (zh) * 2019-08-02 2021-02-02 康方药业有限公司 抗ctla4-抗pd-1双特异性抗体及其用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104974253A (zh) * 2014-04-01 2015-10-14 上海中信国健药业股份有限公司 抗ctla-4/pd-1双特异性抗体、其制备方法及应用
CN106188296A (zh) * 2016-07-19 2016-12-07 中山康方生物医药有限公司 一类抗血管内皮生长因子受体vegfr2的单克隆抗体及其编码基因和应用
CN106967172A (zh) 2016-08-23 2017-07-21 中山康方生物医药有限公司 抗ctla4‑抗pd‑1 双功能抗体、其药物组合物及其用途
WO2019179421A1 (en) * 2018-03-19 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel bispecific pd-1/ctla-4 antibody molecules
WO2020185722A2 (en) * 2019-03-13 2020-09-17 Merck Sharp & Dohme Corp. Anti-cancer combination therapies comprising ctla-4 and pd-1 blocking agents
CN112300286A (zh) * 2019-08-02 2021-02-02 康方药业有限公司 抗ctla4-抗pd-1双特异性抗体及其用途

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1995, MACK PUBLISHING COMPANY
ACIERNO ET AL.: "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies", J MOL BIOL., vol. 374, no. 1, 2007, pages 130 - 46, XP022310713, DOI: 10.1016/j.jmb.2007.09.005
ARMANATH ET AL., SCIENCE TRANSMED, vol. 3, 2011, pages 1 - 13
CARTER ET AL., EUR J IMMUNOL, vol. 32, 2002, pages 634 - 43
FIFE ET AL., NATURE IMMUNOLOGY, vol. 10, 2011, pages 1185 - 1193
FRANCISCO ET AL., J.EXP.MED., vol. 206, 2009, pages 3015 - 3029
FREEMAN ET AL., J EXP MED, vol. 192, 2000, pages 1027 - 34
HOFMEYER ET AL., JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY, 2011, pages 1 - 9
IWAI ET AL., PNAS, vol. 99, 2002, pages 12293 - 12297
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", vol. 1-3, 1991, NIH PUBLICATION
LATCHMAN ET AL., NAT IMMUNOL, vol. 2, 2001, pages 261 - 8
LATCHMAN ET AL., PNAS, vol. 101, 2004, pages 10691 - 10696
LIN ET AL., PNAS, vol. 105, 2008, pages 3011 - 3016
SONG LEI, ZHOU RENGUI, LI XIANGYONG, PAN DEJIAN: "Combination of bevacizumab and dual immunotherapy for extensive-disease small-cell lung cancer: a case report", IMMUNOTHERAPY, vol. 13, no. 16, 1 November 2021 (2021-11-01), GB , pages 1309 - 1315, XP055976804, ISSN: 1750-743X, DOI: 10.2217/imt-2021-0025 *
TOPALIAN ET AL., N ENGL J MED, vol. 366, 2012, pages 2443 - 54
WHO DRUG INFORMATION, vol. 34, no. 4, 2020, pages 947 - 949
WORLD HEALTH ORGANIZATION: "cadonilimabum", WHO DRUG INFORMATION, vol. 34, no. 4, 31 December 2020 (2020-12-31), CH , pages 947 - 949, XP009540392, ISSN: 1010-9609 *

Also Published As

Publication number Publication date
IL307494A (en) 2023-12-01
JP2024515618A (ja) 2024-04-10
EP4324854A1 (en) 2024-02-21
CA3213734A1 (en) 2022-10-20
BR112023021177A2 (pt) 2023-12-19
CN115197325A (zh) 2022-10-18
KR20230171456A (ko) 2023-12-20
AU2022258365A1 (en) 2023-11-30

Similar Documents

Publication Publication Date Title
US20180289790A1 (en) Anti-icos agonist antibodies and uses thereof
JP2023126946A (ja) 抗ｌａｇ－３抗体、ｐｄ－１経路阻害剤および免疫療法剤の組み合わせを含む組成物
JP2022533418A (ja) 抗ｒｏｒ１／抗ｃｄ３二重特異性結合分子
EP4273170A1 (en) Pharmaceutical composition comprising anti-tigit antibody and anti-pd-1-anti-vegfa bispecific antibody, and use
JP2021511372A (ja) Ｔｉｍ３に対する抗体で癌を処置する方法
WO2022243378A1 (en) Uses of anti-icos antibodies
JP2023103352A (ja) 抗原結合タンパク質
WO2022218383A1 (zh) 抗体在抗肿瘤治疗中的用途
JP2022513729A (ja) ヒト化及び親和性成熟抗ｃｅａｃａｍ１抗体
US20240043531A1 (en) Anti-cd47 monoclonal antibody and use thereof
US20210340252A1 (en) Anti-tim-3 antibodies and uses thereof
CN115485302A (zh) 具有增强的激动剂活性的针对cd40的抗体
US20240166747A1 (en) Antigen binding proteins and combinations thereof
WO2022254227A1 (en) Treatment of pd-l1 negative or low expressing cancer with anti-icos antibodies
WO2023222854A1 (en) Uses of anti-icos antibodies
WO2020119792A1 (en) Humanized antibodies against ox40, method for preparing the same, and use thereof
EP4340943A1 (en) Uses of anti-icos antibodies
KR20240038043A (ko) 약학적 조성물 및 용도
WO2020119793A1 (en) Humanized antibodies against ox40, method for preparing the same, and use thereof
EA045980B1 (ru) Антитела против рецептора полиовируса (pvr) и их применение

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22787605

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3213734

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 307494

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 2301006630

Country of ref document: TH

WWE Wipo information: entry into national phase

Ref document number: 2023562813

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 18286755

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: MX/A/2023/012182

Country of ref document: MX

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112023021177

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 202392856

Country of ref document: EA

ENP Entry into the national phase

Ref document number: 20237038950

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2022258365

Country of ref document: AU

Ref document number: 1020237038950

Country of ref document: KR

Ref document number: 805434

Country of ref document: NZ

Ref document number: AU2022258365

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2022787605

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022787605

Country of ref document: EP

Effective date: 20231114

ENP Entry into the national phase

Ref document number: 2022258365

Country of ref document: AU

Date of ref document: 20220414

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112023021177

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20231011