WO2023186079A1 - Cd80蛋白变体及cd80融合蛋白 - Google Patents

Cd80蛋白变体及cd80融合蛋白 Download PDF

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WO2023186079A1
WO2023186079A1 PCT/CN2023/085423 CN2023085423W WO2023186079A1 WO 2023186079 A1 WO2023186079 A1 WO 2023186079A1 CN 2023085423 W CN2023085423 W CN 2023085423W WO 2023186079 A1 WO2023186079 A1 WO 2023186079A1
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protein
amino acid
protein variant
cancer
fusion protein
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PCT/CN2023/085423
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French (fr)
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WO2023186079A9 (zh
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吕明
丁晓然
缪仕伟
谈彬
陶俊
崔航
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杭州尚健生物技术有限公司
尚健单抗（北京）生物技术有限公司
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Publication of WO2023186079A1 publication Critical patent/WO2023186079A1/zh
Publication of WO2023186079A9 publication Critical patent/WO2023186079A9/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present application relates to the field of biomedicine, specifically to a CD80 protein variant and a fusion protein containing the CD80 variant.
  • CD80 also known as B7, B7.1 or BB1 belongs to the immunoglobulin superfamily and is mainly expressed on activated B lymphocytes, activated T lymphocytes, macrophages, etc.
  • the receptors for CD80 include CD28, CD152 (CTLA4) and PD-L1.
  • CTLA4 CD152
  • PD-L1 and CD80 can inhibit the functions of PD-1 and CTLA-4 respectively; the combination of CD28 and CD80 can produce a costimulation signals to enhance T cell activation, function and survival in response to cognate antigens.
  • CD80 maintains the balance between T cell activation and inhibition, and exerts its attack effect on tumor cells.
  • the present application provides a fusion protein containing CD80, which can combine with PD-L1, CTLA-4, and CD28 to activate the activity of T lymphocytes and inhibit the growth and/or proliferation of tumors or tumor cells.
  • the present application also relates to nucleic acids encoding the fusion proteins, vectors containing the fusion proteins, cells containing the nucleic acids or the vectors, methods for preparing the fusion proteins and applications of the fusion proteins.
  • the application provides a CD80 protein variant comprising a functionally active fragment of the CD80 protein or a variant thereof that has one or more amino acids compared to the corresponding functionally active fragment of wild-type CD80. Mutation of the site, and the variant has one or more of the following properties:
  • the PD-L1 is human PD-L1.
  • the CTLA-4 is human CTLA-4.
  • the CD28 is human CD28.
  • the functionally active fragment of the CD80 protein comprises the extracellular domain (ECD) of the CD80 protein or a fragment thereof.
  • ECD extracellular domain
  • the fragment of the extracellular domain of the CD80 protein comprises the IgV domain of the CD80 protein.
  • the CD80 protein variant comprises an amino acid mutation at one or more amino acid residues selected from the group consisting of: N55, N64, N152, N173, N177, N192 and N198.
  • the CD80 protein variant comprises mutations at any one of the following amino acid positions:
  • the CD80 protein variant comprises an amino acid mutation at one or more amino acid residues selected from the group consisting of: N55A/Q, N64A, N152A, N173Q, N177A, N192A and N198A/Q.
  • the CD80 protein variant comprises any set of amino acid mutations selected from the following group:
  • the CD80 protein variant includes at least one first type of amino acid mutation, and the first type of amino acid mutation can maintain or enhance the stability of the CD80 protein variant.
  • the CD80 protein variant comprises a Type I amino acid mutation at one or more of the following amino acid positions: N55, N64, N152, N192, and N198.
  • the CD80 protein variant comprises a first type of amino acid mutation at any set of amino acid positions selected from the following group:
  • the CD80 protein variant comprises one or more of the following Type I amino acid mutations: N55A/Q, N64A, N152A, N192A, and N198A/Q.
  • the CD80 protein variant comprises any group of first-class amino acid mutations selected from the following group:
  • the CD80 protein variant includes at least one second type of amino acid mutation, and the second type of amino acid mutation can maintain the binding activity of the CD80 protein variant.
  • the CD80 protein variant comprises a second type of amino acid mutation at one or more of the following amino acid positions: N152; N192 and N198.
  • the CD80 protein variant comprises one or more of the following Type II amino acid mutations: N152A; N192A and N198A/Q.
  • the CD80 protein variant comprises a second type of amino acid mutation at positions N55 and/or N64.
  • the CD80 protein variant comprises a second type of amino acid mutation of N55A/Q and/or N64A.
  • the CD80 protein variant includes at least one third type of amino acid mutation that can maintain the affinity of the CD80 protein variant.
  • the CD80 protein variant comprises a third type of amino acid mutation at one or more of the following amino acid positions: N55, N64, N152, and N198.
  • the CD80 protein variant comprises one or more of the following Type III amino acid mutations: N55A/Q, N64A, N152A and N198A.
  • the CD80 protein variant comprises a third type of amino acid mutation at position N192.
  • the CD80 protein variant comprises a third type of amino acid mutation of N192A.
  • the CD80 protein variant includes at least one Class IV amino acid mutation, which mutation can enhance the binding of the CD80 protein variant to CD28 and/or PD-L1.
  • the CD80 protein variant comprises a fourth type of amino acid mutation at positions N173 and/or N177.
  • the CD80 protein variant comprises a Class IV amino acid mutation of N173Q and/or N177A.
  • the numbering of the amino acid positions in the CD80 protein variant is determined with reference to the position numbering of the amino acids in SEQ ID NO:1.
  • the CD80 protein variant comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19 , the amino acid sequence shown in any one of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
  • the CD80 protein variant is capable of binding to PD-L1, CD28 and/or CTLA-4.
  • the CD80 protein variant has a sialic acid content of greater than or equal to about 0.7.
  • the present application provides a fusion protein comprising the CD80 protein variant.
  • the fusion protein further comprises an immunoglobulin Fc region or a variant thereof.
  • the CD80 protein variant and the immunoglobulin Fc region in the fusion protein are directly or indirectly connected.
  • the immunoglobulin Fc region in the fusion protein includes the Fc region of IgG.
  • the IgG in the fusion protein is selected from the group consisting of IgG1 and IgG4.
  • the CD80 protein variant in the fusion protein is located at the N-terminus or C-terminus of the immunoglobulin Fc region.
  • the immunoglobulin Fc region in the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 26.
  • the fusion protein includes the amino acid sequence shown in any one of SEQ ID NOs: 27-37.
  • the present application also provides a polypeptide comprising the CD80 protein variant or the fusion protein.
  • the present application provides isolated one or more nucleic acid molecules encoding a CD80 protein variant described herein, a fusion protein described herein, or a polypeptide described herein.
  • the present application provides a vector comprising the nucleic acid molecule described in the present application.
  • the present application provides a host cell comprising the nucleic acid molecule described in the present application or the vector described in the present application.
  • the present application provides a method for preparing the CD80 protein variant described in the present application or the fusion protein described in the present application.
  • the method includes culturing the CD80 protein variant described in the present application under conditions that allow the expression of the fusion protein. described cells.
  • the present application provides a pharmaceutical composition, which includes the CD80 variant protein described in the present application, the fusion protein described in the present application, the polypeptide described in the present application, the nucleic acid molecule described in the present application, and the The vector described in the application and/or the host cell described in the application, and optionally a pharmaceutically acceptable carrier.
  • the present application provides a pharmaceutical combination comprising the CD80 protein variant, and an immune checkpoint inhibitor.
  • the present application provides a pharmaceutical combination comprising the fusion protein and an immune checkpoint inhibitor.
  • the present application provides a pharmaceutical combination comprising the polypeptide, and an immune checkpoint inhibitor.
  • the immune checkpoint includes PD-1, PD-L1, GITR, and/or CTLA-4.
  • the immune checkpoint includes one or more selected from the group consisting of: PD-1 antibody, PD-L1 Antibodies, CTLA-4 antibodies and GITR antibodies.
  • the present application provides a method of modulating the immune response of a subject, the method comprising administering the CD80 protein variant, the fusion protein or the polypeptide described in the present application.
  • the method of modulating an immune response in a subject includes increasing the immune response.
  • the CD80 protein variant, the fusion protein, or the polypeptide exhibits an increase compared to a wild-type CD80 protein or fragment thereof.
  • the CD80 protein variant, the fusion protein, or the polypeptide exhibits an increase compared to a wild-type CD80 protein or fragment thereof. Binding affinity for CD28.
  • the CD80 protein variant, the fusion protein, or the polypeptide exhibits enhanced activity compared to a wild-type CD80 protein or fragment thereof.
  • the activity of activated T lymphocytes is not limited to a wild-type CD80 protein or fragment thereof.
  • the present application provides the CD80 protein variant, the CD80 fusion protein, the polypeptide, the nucleic acid molecule, the vector, the host cell, the Pharmaceutical compositions, or the use of said pharmaceutical combinations in the preparation of the prevention and/or treatment of diseases and/or conditions.
  • the disease and/or disorder includes tumors.
  • the tumors include solid tumors and/or hematological tumors.
  • the tumor includes a tumor responsive to proliferation of central memory T cells.
  • the tumors in the application include: malignant melanoma, breast cancer, gastric cancer, kidney cancer, non-small cell lung cancer, colon cancer, rectal cancer, head and neck squamous cell carcinoma, liver cancer, Kidney cancer, mesothelioma bladder cancer, pancreatic cancer, ovarian cancer, endometrial cancer and lymphoma.
  • the application provides a method for preventing and/or treating diseases and/or disorders, the method comprising administering the CD80 protein variant, the fusion protein, the polypeptide to a subject in need thereof , the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition or the pharmaceutical combination.
  • the disease and/or disorder includes tumors.
  • the tumors include solid tumors and/or hematological tumors.
  • the tumor includes a tumor responsive to proliferation of central memory T cells.
  • the present application provides the CD80 protein variant, the fusion protein, the polypeptide, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition or the pharmaceutical combination, wherein For the prevention and/or treatment of diseases diseases and/or conditions.
  • the disease and/or disorder includes tumors.
  • the tumors include solid tumors and/or hematological tumors.
  • the tumor includes a tumor responsive to proliferation of central memory T cells.
  • the present application provides a method for detecting a CD80 binding ligand in a biological sample, the method comprising contacting the biological sample with a binding reagent of the CD80 protein variant or the fusion protein described in the present application.
  • the binding partner in the method includes PD-L1, CD28 and/or CTLA-4.
  • the present application also provides a method for detecting the frequency and/or proliferation of central memory T cells in a subject, the method comprising administering the CD80 protein variant, the fusion protein, and the polypeptide.
  • the central memory T cells are CD95+ and CD28+ cells.
  • the central memory T cells are CD4+ central memory T cells and/or CD8+ central memory T cells.
  • the application also provides a kit comprising the CD80 protein variant, the fusion protein, the polypeptide, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition or The drug combination.
  • the kit is used to detect central memory T cell frequency and/or proliferation in a subject.
  • the kit is used to detect the presence and/or amount of CD80 binding ligand in a biological sample.
  • the present application also provides the CD80 protein variant, the fusion protein, the polypeptide, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition or the pharmaceutical combination, It is used to enhance the proliferation or frequency of central memory T cells.
  • Figure 1 shows the structural diagram of the CD80 fusion protein described in this application.
  • Figures 2A-2B show the detection results of the activity of the CD80 fusion protein in the present application in binding to human PD-L1 protein.
  • Figures 3A-3D show the detection results of the activity of the CD80 fusion protein binding to human CTLA-4 protein described in the present application.
  • Figures 4A-4C show the detection results of the activity of the CD80 fusion protein binding to human CD28 protein described in the present application.
  • Figure 5 shows the detection results of the activity of the CD80 fusion protein in the present application in binding to human CTLA-4 positive cells.
  • Figure 6 shows the detection results of the activity of the CD80 fusion protein in the present application in binding to human PD-L1 positive cells.
  • Figure 7 shows the detection results of the activity of the CD80 fusion protein in the present application in binding to human CD28-positive cells.
  • Figure 8 shows the results of testing the activity of the CD80 fusion protein described in the present application in activating T lymphocytes under aAPC/T mixed cell culture conditions.
  • Figures 9A-9C show the detection of CD28 activation activity of the CD80 fusion protein described in the present application.
  • Figure 10 shows the anti-tumor activity results of the CD80 fusion protein described in this application in the CT26 subcutaneous implantation model.
  • Figure 11 shows the anti-tumor effect results of the CD80 fusion protein described in the present application in combination with Treg inhibitors in the CT26 subcutaneous implantation model.
  • Figure 12 shows the anti-tumor effect of the CD80 fusion protein described in this application in combination with a PD-1 inhibitor in the CT26 subcutaneous implantation model.
  • Figure 13 shows the anti-tumor effect results of the CD80 fusion protein described in the present application in combination with a CTLA-4 inhibitor in the CT26 subcutaneous implantation model.
  • Figures 14A-14B show the results of cytokine release induced by the CD80 fusion protein described in this application.
  • Figure 15 shows the results of charge heterogeneity analysis of the CD80 fusion protein described in this application.
  • Figure 16 shows the anti-tumor effect of the CD80 fusion protein described in this application with different degrees of sialic acid modification.
  • CD80 protein variant refers to a protein with an altered amino acid sequence relative to wild-type CD80.
  • the wild-type CD80 usually refers to the naturally occurring CD80 obtained from nature.
  • the wild-type CD80 may be human CD80.
  • the variant generally refers to the substitution, deletion and/or addition of one or more amino acids to the sequence compared to the wild type.
  • the CD80 protein variant may be the extracellular domain of the CD80 protein (ECD); further, the CD80 protein variant can be a protein with one or more amino acid residue mutations based on ECD; in some cases, the CD80 protein variant can be the IgV domain of the CD80 protein; further , CD80 protein variants can be proteins in which one or more amino acid residues are mutated based on the IgV domain.
  • the sequence number of the human CD80 in the GenBank database may be NP_005182.1.
  • extracellular domain (ECD) of the CD80 protein generally refers to the polypeptide fragment located on the surface of the cell membrane expressing the CD80 protein.
  • the CD80 protein is a transmembrane glycoprotein, including two Ig-like extracellular domains, a transmembrane helix segment and a short cytoplasmic tail.
  • the Ig-like ectodomain includes individual V-type and C2-type domains.
  • the extracellular domain of the CD80 protein may include all or part of the polypeptide fragments of two Ig-like extracellular domains.
  • the extracellular domain of the CD80 protein may refer to the extracellular domain of the human CD80 protein.
  • the extracellular domain of the CD80 protein described in this application may comprise the amino acids shown in SEQ ID NO: 1 Sequence of all or part of a protein domain.
  • the term "IgV domain of CD80 protein” generally refers to the polypeptide fragment of the V-shaped domain located on the surface of the cell membrane expressing the CD80 protein.
  • the IgV domain of the CD80 protein may include all or part of the polypeptide fragment of the V-type domain.
  • the IgV domain of the CD80 protein may refer to the IgV domain of the human CD80 protein.
  • the IgV domain of the CD80 protein described in this application may include the entire amino acid sequence shown in SEQ ID NO:2 or part of a protein domain.
  • PD-L1 and PDL1 are used interchangeably and generally refer to programmed cell death ligand 1, which may also be referred to as the surface antigen cluster of differentiation 274 (CD274) or B7 homologous protein 1 ( B7-H1), PDCD1L1, PDCD1LG1, and PDL1, belong to the tumor necrosis factor superfamily.
  • PD-L1 may be complete PD-L1 and its fragments, may be functional variants, isoforms, species homologs, derivatives, analogs of PD-L1, and may have at least one property related to PD-L1 Analogs of common epitopes.
  • PD-L1 can be a type I transmembrane glycoprotein composed of 290 amino acid residues, including an IgV-like region, an IgC-like region, a transmembrane hydrophobic region and a 30-amino acid intracellular tail.
  • the PD-L1 may be human PD-L1.
  • CTLA-4" generally refers to cytotoxic T-lymphocyte antigen-4, also known as CTLA4, surface antigen cluster of differentiation 152 (CD152), a leukocyte differentiation antigen shared with CD28 B7 molecular ligand, an immunosuppressive receptor belonging to the CD28 family.
  • CTLA-4 contains an extracellular V-shaped domain, a transmembrane domain, and a cytoplasmic tail.
  • CTLA-4" may be complete CTLA-4 and fragments thereof, may be functional variants, isoforms, species homologs, derivatives, analogs of CTLA-4, and may have at least one property related to CTLA-4 Analogs of common epitopes.
  • the CTLA-4 may be human CTLA-4.
  • CD28 generally refers to the surface antigen cluster of differentiation 28, which is a costimulatory molecule expressed on the surface of T lymphocytes and a receptor for CD80 and CD86 proteins that mediates costimulation of T cells and promotes their survival. multiply and produce Cytokines.
  • CD28 can be complete CD28 and fragments thereof, and can be functional variants, isoforms, species homologs, derivatives, analogs of CD28, and analogs having at least one epitope in common with CD28.
  • the CD28 may be human CD28.
  • amino acid mutation generally refers to amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct.
  • an amino acid mutation may be the replacement of at least one existing amino acid residue by another different amino acid residue (replacement amino acid residue), and the replacement amino acid residue may be a "naturally occurring amino acid residue” , and is selected from the group consisting of: alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), Isoleucine (ile, I), leucine (leu, L)), lysine (lys, K), methionine (met, M), phen
  • the amino acid position is based on the amino acid sequence of the extracellular domain of SEQ ID NO: 1 (human CD80 (UniProtKB/Swiss-Prot: P33681.1 sequence), that is, UniProtKB/Swiss-Prot: P33681.1
  • SEQ ID NO: 1 human CD80 (UniProtKB/Swiss-Prot: P33681.1 sequence)
  • the amino acid sequence shown in the sequence was used as the basis for confirmation. That is, the number of the first amino acid in the amino acid sequence shown in SEQ ID NO:1 is the first amino acid.
  • the extracellular domain of the CD80 protein may comprise amino acids 35-243 of the UniProtKB/Swiss-Prot:P33681.1 sequence.
  • the IgV domain of the CD80 protein may comprise amino acids 35-140 of the UniProtKB/Swiss-Prot:P33681.1 sequence.
  • the term "type 1 amino acid mutations" generally refers to amino acid mutations that have an impact on the stability of the CD80 protein variant.
  • the stability of the CD80 protein variant refers to the ability of the CD80 protein variant to maintain approximately the same structure and function as the pre-mutation CD80 protein.
  • the impact may be the maintenance, reduction or improvement of the stability of the CD80 protein variant.
  • the CD80 protein variant may be an amino acid sequence mutation based on human CD80 protein.
  • the first type of amino acid mutation may include one or more amino acid mutations in all or part of the AA35-AA242 fragment of human CD80.
  • the one or more amino acid mutations may include one or more amino acid mutations selected from the group consisting of Amino acid mutations at any of the following positions: N55, N64, N152, N192 and N198.
  • the term "second type of amino acid mutations" generally refers to amino acid mutations that affect the binding activity of the CD80 protein variant. The effect may be maintenance, reduction or improvement of the binding activity of the CD80 protein variant.
  • the CD80 protein variant may be an amino acid sequence mutation based on human CD80 protein.
  • the second type of amino acid mutation may include one or more amino acid mutations in all or part of the AA35-AA242 fragment of human CD80.
  • the one or more amino acid mutations may include any one selected from the following group Amino acid mutations at positions: N55, N64, N152, N192 and N198.
  • binding activity generally refers to the ability of two binding sites to bind to each other.
  • the binding site may be a region on a binding molecule, which may include a protein, an enzyme substrate, a second Messenger, hormone or allosteric modulator.
  • binding activity may be the ability of a CD80 protein variant to bind to its ligand, and the binding may be reversible (transient and non-covalent), covalently reversible or irreversible.
  • the term "type 3 amino acid mutations" generally refers to amino acid mutations that have an impact on the affinity of the CD80 protein variant. The effect may be maintenance, reduction, or improvement of affinity for the CD80 protein variant.
  • the CD80 protein variant may be an amino acid sequence mutation based on human CD80 protein.
  • the third type of amino acid mutation can include one or more amino acid mutations in all or part of the AA35-AA242 fragment of human CD80.
  • the one or more amino acid mutations can include selected from Amino acid mutations at any of the following positions: N55, N64, N152, N192 and N198.
  • affinity generally refers to the specific affinity ability that can cause the interaction of two or more substances. In some cases, it can be the affinity ability of a receptor and a ligand.
  • the term “category 4 amino acid mutations” generally refers to amino acid mutations that affect the binding of the CD80 protein variant to CD28 and/or PD-L1. The effect may be maintenance, reduction or improvement of the binding of the CD80 protein variant to CD28 and/or PD-L1.
  • the CD80 protein variant may be an amino acid sequence mutation based on human CD80 protein.
  • the third type of amino acid mutation can include one or more amino acid mutations in all or part of the AA35-AA242 fragment of human CD80.
  • the one or more amino acid mutations can include selected from Amino acid mutations at any of the following positions: N173 and/or N177.
  • fusion protein generally refers to a macromolecule in which two or more different protein molecules are linked together through chemical and/or genetic fusion. Fusion proteins can be the recombinant expression products of two or more genes obtained through DNA recombinant technology, or they can be a group of proteins that mediate the fusion of two or more cell plasma membranes. In this application, the fusion protein can be a protein molecule with the above two parts of the domain that connects the target gene to the immunoglobulin Fc region or its variant fragment gene at the gene level and is expressed in eukaryotic or prokaryotic cells.
  • immunoglobulin Fc region generally refers to the crystallizable segment of an immunoglobulin, which may include the CH2 and CH3 domains of immunoglobulins IgG, IgA, and IgD or fragments thereof, and may include those of IgM and IgE. CH2, CH3 and CH4 domains or fragments thereof.
  • the IgG may include IgGl, IgG2, IgG3 or IgG4.
  • polypeptide or amino acid residues
  • polypeptide or protein
  • amino acid polymers in which one or more amino acid residues are synthetic chemical mimics of their corresponding natural amino acids. It can also be used to refer to natural amino acid polymers, those containing modified residues, and non-natural amino acid polymers. Amino acid polymers.
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into cells (eg, host cells) and/or between cells.
  • the vectors may include vectors primarily used for insertion of DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and vectors primarily used for expression of transcription and/or translation of DNA or RNA.
  • the vectors also include vectors having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell.
  • the vector can produce the desired expression product by culturing appropriate cells containing the vector.
  • the vector may be a plasmid.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a patient, which may be a human patient.
  • the pharmaceutical composition described in the present application may comprise the CD80 variant protein described in the present application, the fusion protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and/or the present application.
  • the cells described in the application and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also contain one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations. Acceptable ingredients of the composition are preferably non-toxic to the recipient at the doses and concentrations used.
  • Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • drug combination generally refers to the use of two or more drugs together.
  • different pharmaceutical components can be mixed or placed separately.
  • different pharmaceutical components can be placed in the same container or in different containers.
  • the different pharmaceutical components in the pharmaceutical combination can be administered simultaneously or separately.
  • the different pharmaceutical components can be administered sequentially.
  • different pharmaceutical components may be administered in the same manner or in different manners.
  • pharmaceutically acceptable carrier generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc. that are compatible with drug administration and are generally safe, non-toxic and Toxic and neither biologically nor otherwise undesirable.
  • tumor generally refers to a neoplasm or solid lesion formed by abnormal cell growth.
  • a tumor may be a solid tumor or a non-solid tumor.
  • the tumor may include a tumor that is responsive to proliferation of central memory T cells.
  • the tumor may include tumors containing cells that are surface positive for CTLA-4.
  • proteins, polypeptides and/or amino acid sequences involved should also be understood to include at least the following range: variants or homologues that have the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and/or polypeptide (eg, a CD80 protein variant) .
  • the functional variant may comprise one that has been replaced by at least 1, such as 1-30, 1-20 or 1-10, such as 1, 2, 3, 4 or 5 amino acids. , deletion and/or insertion of proteins or polypeptides with amino acid changes.
  • the functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (eg, substitution, deletion, or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen-binding ability) of the protein or polypeptide prior to the alteration.
  • the substitutions may be conservative substitutions.
  • the homolog may be at least about 85% (e.g., at least about 85%, about 90%) identical to the amino acid sequence of the protein and/or the polypeptide (e.g., a CD80 protein variant). , about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology to a protein or polypeptide.
  • homology generally refers to similarity, similarity or association between two or more sequences.
  • Perfect sequence homology can be calculated by comparing the two sequences to be aligned in the comparison window and determining the presence of identical nucleic acid bases (for example, A, T, C, G, I) in the two sequences.
  • FASTA and BLAST A description of the FASTA algorithm can be found in WR Pearson and DJ Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and DJ Lipman and WRPearson, "Fast and Sensitive Protein Similarity Search," Science, 227: 1435-1441, 1989.
  • a description of the BLAST algorithm can be found in "A basic local alignment search tool" by S. Altschul, W. Gish, W. Miller, EW Myers, and D. Lipman, Journal of Molecular Biology, 215: 403-410, 1990.
  • the term “comprising” generally means including, encompassing, containing or encompassing. In some cases, it also means “for” or “composed of”.
  • the term "about” generally refers to a variation within the range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a CD80 protein variant, which may comprise a functionally active fragment of the CD80 protein or a variant thereof that has one or more amino acids compared to the corresponding functionally active fragment of wild-type CD80. Site mutations.
  • the CD80 protein variant may have one or more of the following properties: 1) binds to PD-L1; 2) binds to CTLA-4; 3) binds to CD28; 4 ) activates the activity of T lymphocytes; and 5) inhibits the growth and/or proliferation of tumors or tumor cells.
  • the CD80 protein variant can bind to both PD-L1 and CD28 proteins.
  • the CD80 variant can simultaneously bind to PD-L1, CD28 and CTLA-4 proteins.
  • the PD-L1 may be human PD-L1.
  • the CD28 may be human CD28.
  • the CTLA-4 may be human CTLA-4.
  • the CD80 protein may be human CD80 protein.
  • the CD80 protein variant may comprise a functionally active fragment of CD80 or a variant thereof.
  • the functionally active fragment of CD80 may comprise the CD80 protein extracellular domain or a fragment thereof.
  • the fragment of the extracellular domain of the CD80 protein may comprise the IgV domain of CD80.
  • the IgV domain of wild-type CD80 protein may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the extracellular domain of wild-type CD80 protein may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the variant of the IgV domain of the CD80 protein may comprise one or more amino acid mutations.
  • the variant of the IgV domain of the CD80 protein may include the following amino acid sequence: VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNX 1 LSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKA.
  • the variant of the IgV domain of the CD80 protein may comprise at least an amino acid substitution at Xi .
  • the IgV domain variant of the CD80 protein may comprise at least an amino acid substitution at X 1 .
  • the amino acid at X 1 can be substituted with A.
  • the extracellular domain variant of the CD80 protein may comprise one or more amino acid mutations.
  • the extracellular domain variant of the CD80 protein may include the following amino acid sequence: VIHVTKEVKEVATLSCGHX 1 VSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKX 2 RTIFDITNX 3 LSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAIX 4 TTVSQD PETELYAVSSSKLDFX 5 MTTX 6 HSFMCLIKYGHLRVX 7 QTFNWX 8 TTKQEHFPDNL.
  • the extracellular domain variant of the CD80 protein may comprise at least amino acid substitutions at X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 and/or X 8 .
  • the CD80 protein variant may comprise an amino acid mutation at one or more positions among X1 , X2 , X3 , X4 , X5 , X6 , X7 and X8 .
  • the amino acid at X 1 can be substituted for A; the amino acid at X 2 can be substituted for A or Q; the amino acid at X 3 can be substituted for A; the amino acid at X 4 can be substituted for A;
  • the amino acid at X can be substituted as A or Q; the amino acid at X 6 can be substituted as A; the amino acid at X 7 can be substituted as A; the amino acid at X 8 can be substituted as A or Q.
  • the CD80 protein variant may comprise a first type of amino acid mutation.
  • the first type of amino acid mutation can maintain the stability of the CD80 protein variant.
  • the CD80 protein variant comprising the first type of amino acid mutation is compared with the extracellular domain of the CD80 protein shown in SEQ ID NO: 1.
  • the extracellular domain variant of the CD80 protein can be At least amino acid substitutions at X 2 , X 3 , X 4 , X 7 and/or X 8 are included, wherein the amino acid at The amino acid at X 7 can be substituted as A; the amino acid at X 8 can be substituted as A or Q.
  • the first type of amino acid mutation may include an amino acid mutation at position N64.
  • the first type of amino acid mutations may include amino acid mutations at positions N55 and N64.
  • the first type of amino acid mutations may include amino acid mutations at positions N55, N64 and N152.
  • the first type of amino acid mutations may include amino acid mutations at positions N55, N64 and N192.
  • the first type of amino acid mutations may include amino acid mutations at positions N55, N64 and N198.
  • the first type of amino acid mutations may include amino acid mutations at positions N55, N64, N152 and N198.
  • the first type of amino acid mutations may include amino acid mutations at positions N55, N64, N152 and N192.
  • the first type of amino acid mutation may comprise the amino acid mutation of N64A.
  • the first type of amino acid mutations may include amino acid mutations of N55A and N64A.
  • the first type of amino acid mutations may include the amino acid mutations of N55A, N64A, and N152A.
  • the first type of amino acid mutations may include amino acid mutations of N55A, N64A, and N192A.
  • the first type of amino acid mutations may include the amino acid mutations of N55A, N64A, and N198A.
  • the first type of amino acid mutations may include amino acid mutations of N55A, N64A, N152A, and N198A.
  • the first type of amino acid mutations may include amino acid mutations of N55A, N64A, N152A and N192A.
  • the first type of amino acid mutations may include amino acid mutations of N55Q, N64A, N152A, and N198Q.
  • the CD80 variant may comprise a second type of amino acid mutation.
  • the second type of amino acid mutation can maintain the binding activity of the CD80 protein variant.
  • the CD80 protein variant comprising the second type of amino acid mutation is compared with the extracellular domain of the CD80 protein shown in SEQ ID NO: 1.
  • the extracellular domain variant of the CD80 protein can At least amino acid substitutions at X 4 , X 7 and/or X 8 are included, wherein the amino acid at X 4 may be substituted as A; the amino acid at A and Q.
  • the second type of amino acid mutations may include mutations at one or more amino acid positions of N152, N192, and N198.
  • the second type of amino acid mutations may comprise one or more of N152A; N192A and N198A/Q.
  • the second type of amino acid mutation may also include amino acid mutations at positions N55 and/or N64.
  • the second type of amino acid mutations may also include amino acid mutations of N55A/Q and/or N64A.
  • the CD80 protein variant may comprise a third type of amino acid mutation.
  • the third type of amino acid mutation is capable of maintaining the affinity of the CD80 protein variant.
  • the CD80 protein variant comprising the third type of amino acid mutation is compared with the extracellular domain of the CD80 protein shown in SEQ ID NO: 1.
  • the extracellular domain variant of the CD80 protein can be Comprising at least amino acid substitutions at X 2 , X 3 , X 4 and/or X 8 , wherein the amino acid at The amino acid can be substituted with A; the amino acid at X 8 can be substituted with A.
  • the third type of amino acid mutations may include amino acid mutations at one or more of the following positions: N55, N64, N152, and N198.
  • the third type of amino acid mutations may include one or more of the following amino acid mutations: N55A/Q, N64A, N152A, and N198A.
  • the third type of amino acid mutation may also include an amino acid mutation at position N192.
  • the third type of amino acid mutation may also include the amino acid mutation of N192A.
  • the CD80 protein variant may comprise a fourth type of amino acid mutation.
  • the fourth type of amino acid mutation can enhance the binding of CD80 protein variants to CD28 and/or PD-L1.
  • the CD80 protein variant comprising the fourth type of amino acid mutation is compared with the extracellular domain of the CD80 protein shown in SEQ ID NO: 1.
  • the extracellular domain variant of the CD80 protein can be At least amino acid substitutions at X 5 and/or X 6 are included, wherein the amino acid at X 5 can be substituted for Q; the amino acid at X 6 can be substituted for A.
  • the fourth type of amino acid mutation may include amino acid mutations at positions N173 and/or N177.
  • the fourth type of amino acid mutations may include amino acid mutations of N173Q and/or N177A.
  • the CD80 protein variant may comprise N64 amino acid mutations.
  • the CD80 protein variant may include amino acid mutations at positions N64 and N55.
  • the CD80 protein variant may include amino acid mutations at positions N55, N64 and N152.
  • the CD80 protein variant may include amino acid mutations at positions N55, N64 and N192.
  • the CD80 protein variant may include amino acid mutations at positions N55, N64 and N198. In the present application, compared with the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise amino acid mutations at positions N55, N64, N152 and N198. In the present application, compared with the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise amino acid mutations at positions N55, N64, N152 and N192. In the present application, compared with the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise amino acid mutations at positions N55, N64, N152, N173, N177 and N198.
  • the CD80 protein variant may comprise amino acid mutations at positions N55, N64, N152, N173 and N198. In the present application, compared with the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise amino acid mutations at positions N55, N64, N152, N177 and N198.
  • the CD80 protein variant may comprise an amino acid mutation of N64A.
  • the CD80 protein variant may comprise amino acid mutations of N55A and N64A.
  • the CD80 protein variant may comprise amino acid mutations of N55A, N64A and N152A.
  • the CD80 protein variant may comprise amino acid mutations of N55A, N64A and N192A.
  • the CD80 protein variant may comprise amino acid mutations of N55A, N64A and N198A.
  • the CD80 protein variant may comprise amino acid mutations of N55A, N64A, N152A and N198A.
  • the CD80 protein variant may comprise amino acid mutations of N55A, N64A, N152A and N192A.
  • the CD80 protein variant may comprise amino acid mutations of N55Q, N64A, N152 and N198Q.
  • the CD80 protein variant may comprise amino acid mutations of N55Q, N64A, N152A, N173Q, N177A and N198Q.
  • the CD80 protein variant may comprise amino acid mutations of N55Q, N64A, N152A, N173Q and N198Q.
  • the CD80 protein variant may comprise amino acid mutations of N55Q, N64A, N152A, N177A and N198Q.
  • the CD80 protein variant may comprise SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, Any of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 The amino acid sequence shown in the item.
  • the sialic acid content of the CD80 protein variant may be greater than or equal to about 0.7, greater than or equal to about 0.8, greater than or equal to about 0.9, greater than or equal to about 1.0, greater than or equal to about 1.1, greater than or equal to about 1.2, or greater than about 1.3 , greater than or equal to about 1.4, greater than or equal to about 1.5, greater than or equal to about 2, greater than or equal to about 3, greater than or equal to about 4, greater than or equal to about 5, greater than or equal to about 6, greater than or equal to about 7, greater than or equal to about 8, greater than or equal to about 9 5.
  • the sialic acid content of the CD80 protein variant may be greater than or equal to about 0.7 and less than or equal to about 15, greater than or equal to about 0.8 and less than or equal to about 15, greater than or equal to about 0.9 and less than or equal to about 15, greater than or equal to about 1.0 and less than or equal to about 15, greater than or equal to about 1.1 and less than or equal to about 15, greater than or equal to about 1.2 and less than or equal to about 15, greater than or equal to about 1.3 and less than or equal to about 15, greater than or equal to about 1.4 and less than or equal to about 15, greater than or equal to about 1.5 and about 15 or less, about 2 or more and about 15 or less, about 3 or more and about 15 or less, about 4 or more and about 15 or less, about 5 or more and about 15 or less, about 6 or more and about 15 or less, about 7 or more and about 15 or less, about 8 or more and about 15 or less, about 9 or more and about 15 or less, about 10 or more and about 15 or less, about 11 or more and
  • the present application provides a fusion protein comprising a CD80 protein variant.
  • the fusion protein may also contain other functional fragments.
  • the fusion protein may include the CD80 protein variant and an immunoglobulin Fc region or a variant thereof.
  • the immunoglobulin Fc region may comprise the Fc region of an IgG, the IgG may be human IgG, and the human IgG may be human IgG1 and/or human IgG4.
  • the variant may be all or part of the amino acid fragment of the immunoglobulin Fc region.
  • the immunoglobulin Fc region may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 25 or SEQ ID NO: 26.
  • the IgG1 Fc region may comprise the amino acid sequence shown in SEQ ID NO: 25.
  • the IgG4 Fc region may comprise the amino acid sequence shown in SEQ ID NO: 26.
  • the CD80 protein variant and the immunoglobulin Fc region may be directly or indirectly linked.
  • Indirect connection as described in this application includes connection through a polypeptide linker.
  • the polypeptide linker may include an amino acid sequence consisting of one or more amino acid residues, and the amino acid residues may include proline or cysteine, and the linker may connect two binding fragments. , the binding fragments may include the same or different fragments.
  • the linker can connect the CD80 protein variant and the immunoglobulin White Fc area.
  • the linker may comprise the amino acid sequence of (GGGGS)n, where n may be any positive integer from 1 to 10.
  • n can be 1-4.
  • the linker may comprise the amino acid sequence of (GGGGS) 2 .
  • the linker may comprise the amino acid sequence of (GGGGS) 3 .
  • the linker may comprise the amino acid sequence of (GGGGS) 4 .
  • the fusion protein may comprise a first polypeptide chain or a second polypeptide chain.
  • the polypeptide fragment of the first polypeptide chain from N-terminus to C-terminus can be: CD80 protein variant-linker-immunoglobulin Fc region, and the CD80 protein variant can be connected to the linker.
  • the linker can be connected to the N-terminus of the immunoglobulin Fc region.
  • polypeptide fragments of the second polypeptide chain from N-terminus to C-terminus can be: CD80 protein variant-immunoglobulin Fc region, and the CD80 protein variant can be the same as the immunoglobulin Fc region.
  • the N end of the area is connected.
  • the fusion protein may comprise two identical first polypeptide chains, or may comprise two identical second polypeptide chains.
  • the first polypeptide chain or the second polypeptide chain may include SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ The CD80 protein variant sequence shown in any one of ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
  • the first polypeptide chain or the second polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 27-37.
  • the fusion protein includes the CD80 protein variant, as well as other protein molecules/polypeptide molecules.
  • the other protein molecules/polypeptide molecules do not affect the function of the CD80 protein variant.
  • the present application also provides isolated one or more nucleic acid molecules, which can encode the CD80 protein variants described in the present application, and fusion proteins thereof.
  • each nucleic acid molecule in the one or more nucleic acid molecules may encode the complete CD80 protein variant and its fusion protein, or may encode a part thereof.
  • the nucleic acid molecules described herein can be isolated. For example, it can be produced or synthesized by: (1) amplification in vitro, such as by polymerase chain reaction (PCR) amplification; (2) production by clonal recombination; (3) purification , such as through enzyme digestion and gel electrophoresis fractionation, or (4) synthetic, such as through chemical synthesis.
  • the isolated nucleic acid can be produced by polymerase chain reaction (PCR) amplification.
  • PCR polymerase chain reaction
  • nucleic acids encoding the CD80 protein variants, and fusion proteins thereof can be prepared by various methods known in the art.
  • the present application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • One or more such nucleic acid molecules may be included in each vector.
  • other genes may also be included in the vector, such as allowing The vector's marker gene is allowed to be selected in an appropriate host cell and under appropriate conditions.
  • the vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain an origin of replication site.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage or other vectors commonly used in, for example, genetic engineering.
  • the present application provides a host cell that may comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein.
  • each or each host cell may comprise one or more nucleic acid molecules or vectors described herein.
  • each or each host cell may comprise multiple (eg, 2 or more) or multiple (eg, 2 or more) nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into the host cells, such as prokaryotic cells (eg, bacterial cells), CHO cells, NS/0 cells, HEK293 cells, or other eukaryotic cells, such as cells from plants, Fungi or yeast cells, etc.
  • the vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.
  • the present application provides methods for preparing CD80 protein variants and fusion proteins thereof described in the present application.
  • the method may include culturing the host cells described herein under conditions that allow expression of the CD80 protein variant, and its fusion protein. For example, by using appropriate culture medium, appropriate temperature and culture time, etc., these methods are understood by those of ordinary skill in the art.
  • the methods may further include the step of harvesting (eg, isolating and/or purifying) the CD80 protein variants described herein, and fusion proteins thereof.
  • harvesting eg, isolating and/or purifying
  • protein G-agarose, protein A-agarose or metal chelation can be used for affinity chromatography
  • the CD80 protein described in the present application can also be purified and separated by gel electrophoresis and/or high performance liquid chromatography. variants, and their fusion proteins.
  • the present application provides a pharmaceutical composition, which may comprise the CD80 protein variant and its fusion protein described in the present application, the nucleic acid molecule, the vector, the host cell, and Optionally a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or non-ionic surfaces Active agents, etc.
  • the pharmaceutical composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration Administer or via subcutaneous depot.
  • the pharmaceutical composition can be used to inhibit tumor growth.
  • the pharmaceutical composition of the present application can inhibit or delay the development or progression of the disease, can reduce the size of the tumor (even substantially eliminate the tumor), and/or can alleviate and/or stabilize the disease state.
  • the pharmaceutical composition described in the present application may comprise a prophylactically and/or therapeutically effective amount of the CD80 protein variant, and its fusion protein.
  • the prophylactically and/or therapeutically effective amount is that amount required to prevent and/or treat (at least in part) the disease or condition and/or any complications thereof in a subject suffering from or at risk of developing it.
  • the present application provides a pharmaceutical combination, which may include the CD80 protein variant, and an immune checkpoint inhibitor.
  • the present application provides a pharmaceutical combination, which may include the fusion protein, and an immune checkpoint inhibitor.
  • the immune checkpoint may include PD-1, PD-L1, GITR and/or CTLA-4.
  • the immune checkpoint inhibitor may include one or more selected from the group consisting of PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, and GITR antibodies.
  • the PD-1 antibody can be selected from: nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, Geno monoclonal antibodies.
  • the PD-L1 antibody can be selected from: durvalumab, atezolizumab, envolizumab, adebelimab, sugemalimab, Tate Rizumab.
  • the CTLA-4 antibody can be selected from: ipilimumab, tremelimumab.
  • the GITR antibody can be selected from: MK-4166, TRX-518, INCAGN01876, BMS-986156, AMG228, IBI102, ASP-1951.
  • antibodies are only examples. Such antibodies can be used in this application as long as they can function as inhibitors of corresponding immune checkpoints.
  • the CD80 protein variant/fusion protein may be present in the same container as the immune checkpoint inhibitor.
  • the CD80 protein variant/fusion protein and the immune checkpoint inhibitor may be present in different containers.
  • the CD80 protein variant/fusion protein and the immune checkpoint inhibitor can be administered simultaneously.
  • Simultaneous administration may involve mixing the components together or applying them separately. It can be administered in the same way, for example, applied to the same vein or other blood vessels, or it can be administered in different ways, for example, intravenous administration and intratumoral administration at the same time.
  • the CD80 protein variant/fusion protein and the immune checkpoint inhibitor can be administered sequentially.
  • the order of administration may be to administer the CD80 protein variant/fusion protein first, and then administer the immune checkpoint inhibitor; it may also be to administer the immune checkpoint inhibitor first, and then administer the CD80 protein variant/fusion protein. protein.
  • They can be administered in the same way or in different ways.
  • Each component can be applied once or in multiple portions.
  • sequential administration can be at any time interval, including minutes, hours, days, weeks, months, or years.
  • sequential administration means at least 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months , administered at intervals of one of 5 or 6 months.
  • the present application provides the use of the CD80 protein variant, and its fusion protein in the preparation of medicaments for preventing or treating diseases or conditions.
  • the present application provides the CD80 protein variants, and fusion proteins thereof, which are prepared to prevent or treat diseases or disorders.
  • the present application provides a method for preventing or treating tumors, which includes administering the CD80 protein variant described in the present application, and its fusion protein to a subject in need thereof.
  • Colorectal cancer may also be included.
  • the tumor may include solid tumors.
  • the tumor may include non-solid tumors.
  • the tumor may include one or more selected from the group consisting of: malignant melanoma, breast cancer, gastric cancer, renal cancer, non-small cell lung cancer, colon cancer, rectal cancer, head and neck squamous cell carcinoma cell carcinoma, liver cancer, kidney cancer, mesothelioma bladder cancer, pancreatic cancer, ovarian cancer, endometrial cancer and lymphoma.
  • the CD80 fusion protein is obtained by connecting the CD80 extracellular domain or its variant to the N-terminus of the Fc fragment of human IgG1 (amino acid sequence number: SEQ ID NO: 25), in which the CD80 extracellular domain or its variant includes : Wild-type CD80ECD (SEQ ID NO:1, extracellular domain of wild-type CD80), M12 (SEQ ID NO:6), M13 (SEQ ID NO:7), M14 (SEQ ID NO:8), M15 ( SEQ ID NO:9), M16 (SEQ ID NO:10), M18 (SEQ ID NO:11), M19 (SEQ ID NO:12), M20 (SEQ ID NO:13), M21 (SEQ ID NO:14 ), M22(SEQ ID NO:15), M23(SEQ ID NO:16), M24(SEQ ID NO:17), M25(SEQ ID NO:18), M26(SEQ ID NO:19), M27(SEQ ID NO:20), M28 (SEQ ID NO:21), M29 (S
  • CD80 extracellular domain or its variant was connected to the N-terminus of the Fc fragment of human IgG1 (amino acid sequence number: SEQ ID NO: 25) to obtain CD80 fusion proteins, which were named: CD80-WT, CD80M12, and CD80M13. , CD80M14, CD80M15, CD80M16, CD80M18, CD80M19, CD80M20, CD80M21, CD80M22, CD80M23, CD80M24, CD80M25, CD80M26, CD80M27, CD80M28, CD80M29, CD80M30, CD80M31, CD80-IgV, CD80M09, CD80M10, CD80M11.
  • CD80 fusion protein The specific structure of CD80 fusion protein is shown in Figure 1.
  • CD80M12, CD80M11, CD80M14, CD80M15, CD80M16, CD80M26, CD80M27, CD80M28, CD80M29, CD80M30, CD80M31 are stably expressed with moderate expression levels; they have good binding activity with PD-L1, CTLA-4 and/or CD28, and can be combined with PD-L1, CTLA-4 and/or CD28 maintain affinity.
  • CTLA-4 Human CTLA-4/CD152 Protein, ACROBiosystems
  • PBST 10% fetal calf serum was added and blocked at 37°C for 1 hour; CD80 fusion proteins of different concentrations were added.
  • the results are shown in Figures 3A-3C.
  • the results in Figure 3A show that except for CD80M09, CD80M10, CD80M11, CD80M19, and CD80M20, which have significantly weaker binding than wild-type CD80-WT, the binding activity of the remaining variants to CTLA-4 Comparable to CD80WT.
  • CD28 Human CD28, Sino Biological was coated on the ELISA strip at 4°C overnight; after washing with PBST, Add 10% fetal bovine serum and block at 37°C for 1 hour; add different concentrations of CD80 fusion protein and react at 37°C for 1 hour; after washing with PBST, add horseradish peroxidase-labeled goat anti-human IgG Fc secondary antibody (Goat anti-human IgG Fc antibody, horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen), react at 37°C for 30 minutes; wash 5 times with PBST; add 100 ⁇ L TMB (eBioscience) to each well, place at room temperature (20 ⁇ 5°C) away from light 1 to 2 minutes; then add 100 ⁇ L of 2N H 2 SO 4 stop solution to each well to terminate the substrate reaction, read the OD value at 450 nm with a microplate reader, and analyze the binding ability of the CD80 fusion protein to CD28.
  • PBST horseradish peroxid
  • CHOK1 cells (CHOK1-CTLA-4) that overexpress human CTLA-4, and add 5 ⁇ 10 5 cells per tube to a 1.5mL EP tube; add different concentrations of CD80 fusion protein, and incubate on ice in the dark for 30 minutes; After washing with FACS washing solution, add PE fluorescently labeled Goat Anti-Human IgG Fc Secondary Antibody (Invitrogen) and incubate on ice in the dark for 30 minutes; wash twice with FACS washing solution; add 400 ⁇ L 1 to each tube The cells were fixed with % paraformaldehyde fixative (Solarbio), mixed and put on a machine to detect the positive rate of PE fluorescence, and the binding ability of CD80 fusion protein to CTLA-4 positive cells was analyzed.
  • % paraformaldehyde fixative Solarbio
  • CHOK1 cells (CHOK1-PD-L1) that overexpress human PD-L1, and add 5 ⁇ 10 5 cells per tube to a 1.5mL EP tube; add different concentrations of CD80 fusion protein, and incubate on ice in the dark for 30 minutes; After washing with FACS washing solution, add PE fluorescently labeled Goat Anti-Human IgG Fc Secondary Antibody (Invitrogen) and incubate on ice in the dark for 30 minutes; wash twice with FACS washing solution; add 400 ⁇ L 1 to each tube The cells were fixed with % paraformaldehyde fixative (Solarbio), mixed and put on a machine to detect the positive rate of PE fluorescence, and the binding ability of CD80 fusion protein to PD-L1 positive cells was analyzed.
  • % paraformaldehyde fixative Solarbio
  • Collect CHOK1 cells overexpressing human CD28 (CHOK1-CD28), add 5 ⁇ 10 5 cells per tube to a 1.5mL EP tube; add CD80 fusion proteins of different concentrations, and incubate on ice in the dark for 30 minutes; wash with FACS wash solution Then, add PE fluorescently labeled goat anti-human IgG Fc secondary antibody (Goat Anti-Human IgG Fc Secondary Antibody, Invitrogen), incubate on ice for 30 minutes in the dark; wash twice with FACS washing solution; add 400 ⁇ L of 1% paraformaldehyde fixative (Solarbio) to each tube to fix the cells, mix and use the machine to detect the positive rate of PE fluorescence, and analyze the CD80 fusion protein Binding ability to CD28-positive cells.
  • PE fluorescently labeled goat anti-human IgG Fc secondary antibody Goat Anti-Human IgG Fc Secondary Antibody, Invitrogen
  • Example 4 Using aAPC/T mixed cell culture to detect the activity of CD80 fusion protein in activating T lymphocytes
  • CD80 fusion protein CD80-Fc and IgG1-Fc proteins were used as controls, 293T cells overexpressing OKT3scFv and CD64 (293T-OKT3-CD64) were used as antigen-presenting cells (artificial APC, aAPC), and CD4+ T lymphocytes were used as For effector cells, the experimental system of mixed culture of aAPC cells and CD4+ T lymphocytes was used to evaluate the activity of CD80 fusion protein in activating T lymphocytes.
  • FCGR2B/TCR Activator/CHO Collect FCGR2B/TCR Activator/CHO (FCGR2B/CHO for short) cells and add them to a white transparent bottom 96-well plate (96well white/Clear bottom plate, Thermo) at 1.5 ⁇ 10 4 cells/well; add different concentrations to the cells Fusion protein; add CD28 Effector Reporter (referred to as CD28 effector) effector cells at 2.5 ⁇ 10 4 cells/well, place the 96-well plate in a 37°C 5% CO 2 incubator and culture for 5.5 hours; add 120 ⁇ L fluorescein to each well Enzyme detection reagent (Bright-Lumi TM Firefly Luciferase Reporter Gene Detection Kit, Beyotime), after incubation at room temperature in the dark for 10 minutes, use a multifunctional microplate reader to detect the relative fluorescence intensity value of chemiluminescence, and analyze the expression of the fusion protein in FCGR2B/ CD28 activation activity mediated in CHO cells
  • Figures 9A-9C show that all fusion proteins have CD28 activating activity.
  • the activity of CD80M28 is stronger than that of CD80-WT, CD80M12, CD80M14, CD80M16, CD80M26, CD80M30, and CD80M31.
  • the activity is close to that of CD80-WT.
  • Example 6 Using CT26 subcutaneous implantation model to evaluate the anti-tumor activity of CD80 fusion protein
  • mice 5 ⁇ 10 5 CT26 cells were subcutaneously inoculated on the right back of experimental mice, and the cells were resuspended in PBS (0.1ml/mouse). Observe the tumor growth regularly. When the tumor grows to an average volume of about 76 mm, the mice will be randomly divided into groups for administration according to the tumor size and weight of the mice. Divide into 5 groups, with 6 mice in each group. The G1 group was CD80M28, 2mg/kg, the G2 group was CD80M28, 0.5mg/kg, the G3 group was CD80M28, 0.1mg/kg, the G4 group was CD80-WTG1, 0.5mg/kg, and the G5 group was the vehicle control group. Medicine, 3 times a week, 3 times in total.
  • mice in the vehicle control group were euthanized because the tumor volume exceeded 3000 mm 3 , so the data on Day 22 were used as the basis for analysis.
  • the average tumor volume of the vehicle control group (G5) on Day 22 was 2189.79mm3, and the average tumor volume of the CD80M282mg/kg TIW administration group on Day22 was 865.57mm3 .
  • the relative tumor inhibition rate (TGI) was 60.47%, which was significantly different from the control group. (p ⁇ 0.05).
  • the average tumor volume of the CD80M28 0.5mg/kg TIW administration group was 877.01mm 3 on Day 22, and the relative tumor inhibition rate (TGI) was 59.95%, which was significantly different from the control group (p ⁇ 0.05).
  • the average tumor volume of the CD80M28 0.1 mg/kg TIW administration group on Day 22 was 1507.25 mm 3 , and the relative tumor inhibition rate (TGI) was 31.17%. There was no significant difference compared with the control group.
  • the average tumor volume of the CD80-WTG1 0.5 mg/kg TIW administration group on Day 22 was 1337.30 mm3, and the relative tumor inhibition rate (TGI) was 38.93%. There was no significant difference compared with the control group.
  • the mice in each group had normal weight, no abnormal symptoms, and were in good general condition.
  • CD80M28 inhibits tumor growth in a dose-dependent manner, and its activity is significantly better than wild-type CD80-WT at the same dose.
  • Example 7 Using the CT26 subcutaneous implantation model to evaluate the anti-tumor effect of the combination of CD80 fusion protein and Treg inhibitors
  • the anti-GITR antibody disclosed in the WO2017096189A1 document was used in combination with the fusion protein of the present application to study the synergistic effect of the two.
  • Mouse colorectal cancer cell CT26 was inoculated subcutaneously into the right front flank of female PD1/GITR double humanized mice. When the tumor grew to about 80 mm, it was administered in groups . There were 6 groups in total, 5 animals in each group, respectively.
  • G1 vehicle group
  • G2 CD80M28 (3mg/kg) group
  • G3 CD80-WT (3mg/kg) group
  • G4 anti-GITR antibody (10mg/kg) group
  • G5 CD80M28+anti-GITR antibody (3+10mg/ kg) group
  • G6 CD80-WT + anti-GITR antibody (3 + 10 mg/kg) group
  • CD80-WT and CD80M28 were administered intravenously, three times a week, and the anti-GITR antibody was administered intraperitoneally, twice a week. times, administered for two weeks.
  • the tumor volume and body weight were measured every week, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.
  • the tumor growth inhibition rate TGI TV (%) was calculated and statistically analyzed.
  • the results are shown in Figure 11.
  • the tumor growth inhibition rates of CD80M28, CD80-WT, anti-GITR antibody, CD80M28+anti-GITR antibody, and CD80-WT+anti-GITR antibody were 86.43%, 64.57%, 69.98%, 89.06%, and 80.80% respectively.
  • the tumor volume of each treatment group was significantly lower than that of the vehicle control group (p ⁇ 0.05), showing significant anti-tumor effect and effectively inhibiting tumor growth.
  • the tumor volume of the CD80M28 + anti-GITR antibody combination treatment group was the smallest among all treatment groups, and the tumor inhibitory activity of the combination treatment group was better than that of the single-drug group.
  • Example 8 Using CT26 subcutaneous implantation model to evaluate the anti-tumor effect of the combination of fusion protein and anti-PD-1 antibody
  • the anti-m-PD-1 antibody used in the study was purchased from Bioxcell Company, product number BP0146.
  • Mouse colorectal cancer cell CT26 was inoculated subcutaneously into the right front flank of female Balb/c mice. On the 6th day after inoculation, the average tumor volume was approximately 70.9mm 3 .
  • the tumor-bearing mice were divided into four groups using random block method. 4 groups, including Group 1 PBS group (hereinafter referred to as Group 1), Group 2 CD80M28 (0.4mg/kg) group (hereinafter referred to as Group 2), Group 3 anti-mPD-1 antibody (5mg/kg) group (hereinafter referred to as Group 3) ) and Group 4 anti-mPD-1 antibody (5 mg/kg) + CD80M28 (0.4 mg/kg) group (hereinafter referred to as Group 4), with 6 animals in each group.
  • Group 1 PBS group hereinafter referred to as Group 1
  • Group 2 CD80M28 (0.4mg/kg) group hereinafter referred to as Group 2
  • Group 3 anti-mPD-1 antibody hereinafter referred to as Group 3
  • the administration volume of each group was 10 mL/kg.
  • Group 1 and Group 2 were administered via the tail vein for 1 week, three times a week;
  • Group 3 was administered intraperitoneally, and administered continuously for 2 weeks, every week.
  • Administration was given 2 times for a total of 4 times;
  • Group 4 adopted combined administration: intraperitoneal injection of anti-mPD-1 antibody (5 mg/kg) for 2 consecutive weeks, 2 times weekly administration, and tail vein injection of CD80M28 ( 0.4mg/kg), 3 times a week, 7 times in total.
  • the results are shown in Figure 12.
  • the results showed that on the 18th day after the start of administration, the tumor volume in Group 1 was 2264.23 ⁇ 514.76mm 3 .
  • the tumor volume of Group 3 was 1109.87 ⁇ 224.72mm 3 and the tumor volume inhibition rate (TGI) was 52.64%.
  • the tumor volume of Group 2 was 821.79 ⁇ 387.96mm 3
  • the tumor volume inhibition rate (TGI) was 65.76%.
  • the anti-tumor activity of the combination group was significantly better than the anti-tumor activity of each single-drug group, and showed a significant synergistic effect.
  • Example 9 Using MC38 subcutaneous implantation model to evaluate the anti-tumor effect of the combination of fusion protein and anti-CTLA-4 antibody
  • the anti-m-CTLA-4 antibody used in the study was purchased from Bioxcell Company, product number BP0164, and mouse colorectal cancer cell MC38 was inoculated into females.
  • the average tumor volume was approximately 78 mm 3 on the 7th day after vaccination under the skin of the right anterior flank of C57BL/6 mice, using random blocks.
  • the tumor-bearing mice were divided into 4 groups, including Group 1 PBS group (hereinafter referred to as Group 1), Group 2 CD80M28 (0.5mg/kg) group (hereinafter referred to as Group 2), and Group 3 CTLA-4 (4mg/kg) group.
  • Group 3 Group 3 and Group 4CTLA-4 (5mg/kg) + CD80M28 (0.4mg/kg) group (hereinafter referred to as Group 4), with 6 animals in each group.
  • CD80M28 is administered intravenously once, and CTLA-4 antibody is administered intraperitoneally twice a week, for a total of three administrations.
  • Example 10 CD80 fusion protein induces cytokine release
  • CD80 fusion protein was evaluated by ELISA.
  • CD80 fusion protein Different concentrations of CD80 fusion protein, as well as CD3 antibody hOKT3 (Human CD3 (humanized OKT3) Antibody-Human IgG4 (GMP-grade), Sino Biological), CD28 antibody TGN1412 (self-produced) and IgG1-Fc protein (Human IgG1 Fc Protein , Tag Free, ACROBiosystems) was added to a 96-well cell culture plate and air-dried at room temperature overnight; after washing twice with physiological saline, 1 ⁇ 10 5 healthy donor PBMC cells (Schbio Biotech) were added to each well and incubated at 37°C.
  • CD3 antibody hOKT3 Human CD3 (humanized OKT3) Antibody-Human IgG4 (GMP-grade), Sino Biological)
  • CD28 antibody TGN1412 self-produced
  • IgG1-Fc protein Human IgG1 Fc Protein , Tag Free, ACROBiosystems
  • TNF- ⁇ ELISA detection kit (HUMAN TNF- ⁇ ELISA KIT, ExCell Bio) and IL-6 ELISA detection kit (Human IL-6 Precoated ELISA Kit, DAKEWE) were used to detect the TNF- ⁇ and IL-6 levels were analyzed to analyze the ability of CD80 fusion protein to induce PBMC to release cytokines.
  • CD80M26 and CD80M28 perform charge heterogeneity analysis of CD80 variant fusion proteins, rinse the capillary at room temperature, and prepare mixed standard sample solution and detection sample solution.
  • the preparation method of CD80M28 solution is: 1% MC50uL, 8.6mM urea 50Ul, 200mM IDA, 10ul, 500mM arginine 7uL, 1mg/mL CD80M28, 25uL, ampholytes 3-10, 1.6uL, ampholytes 5-8, 6.4uL.
  • the preparation method of CD80-WT, CD80M26 solution is: 1% MC 50uL, 8.6mM urea 50Ul, 200mM IDA, 10ul, 500mM arginine 7uL, 1mg/mL CD80-WT, CD80M26, 25uL, ampholyte 3-10, 1.2uL , amphoteric electrolytes 5-8, 10.8uL injection pressure is selected to 2bar, injection time is set to 100s, electrophoresis is performed, and chromatograms that achieve separation due to different charge heterogeneity are collected. Acid-base variants were quantified using the area normalization method.
  • Example 12 Using HPLC-FLD to detect and analyze the sialic acid ratio of CD80M28 samples derived from different cell clones
  • Sialic acid content was detected and analyzed by HPLC-FLD. Change the protein test sample with PBS 5 times and measure the protein concentration. Then dilute the protein to 1 mg/mL. Add 4M acetic acid to the protein test sample in a 1:1 ratio, mix well, and incubate at 80°C for 2 hours. In the above system, add 50 ⁇ L DMB solution and incubate at 50°C for 2.5 hours. A standard curve was drawn using two common sialic acid Neu5Ac(NA)/Neu5Gc(NG) quantitative standards with different concentrations in glycoproteins, and quantitative analysis of Neu5Ac(NA)/Neu5Gc(NG) was performed.
  • Example 13 Using CT26 subcutaneous implantation model to evaluate the anti-tumor effect of CD80M28 with different degrees of sialic acid modification
  • Mouse colorectal cancer cell CT26 was inoculated subcutaneously into the right front flank of female BABL/C mice. A total of 20 mice were inoculated. When the tumor grew to about 80 mm, they were divided into 7 groups with 6 mice in each group. , each administration group is: G1 group vehicle control (PBS), G2 group CD80M28-15, G3 group CD80M28-1, G4 group CD80M28-12, G5 group CD80M28-99, G6 group CD80M28-5, G7 group CD80- WT was administered every three days for a total of 3 administrations.
  • PBS group vehicle control
  • G2 group CD80M28-15 G3 group CD80M28-1
  • G4 group CD80M28-12 G5 group CD80M28-99
  • G6 group CD80M28-5 G7 group CD80- WT was administered every three days for a total of 3 administrations.
  • the average tumor volume of the vehicle control group at the end of the experiment was 2906.96mm 3
  • the average tumor volume of the CD80M28-15 treatment group was 1979.69mm 3 at the end of the experiment
  • the relative tumor inhibition rate (TGI) was 31.90%
  • the CD80M28-1 treatment group was at the end of the experiment.
  • the average tumor volume of the experiment was 2536.42mm 3 , and the relative tumor inhibition rate (TGI) was 12.75%; the average tumor volume of the CD80M28-12 treatment group at the end of the experiment was 1683.28mm 3 , and the relative tumor inhibition rate (TGI) was 42.09%; CD80M28 The average tumor volume of the -9 treatment group at the end of the experiment was 1612.37mm 3 , and the relative tumor inhibition rate (TGI) was 44.53%; the average tumor volume of the CD80M28-5 treatment group at the end of the experiment was 1940.70mm 3 , and the relative tumor inhibition rate was 44.53%. (TGI) was 33.24%; the average tumor volume of the CD80-WT treatment group at the end of the experiment was 2805.13mm 3 , and the relative tumor inhibition rate (TGI) was 3.50%.
  • the results are shown in Figure 16.
  • the results show that the tumor inhibitory effect of CD80M28 with different sialic acid contents is better than that of wild-type CD80-WT.
  • the sialic acid content is greater than 0.76
  • the CD80 fusion protein has a more significant tumor inhibitory rate.
  • the tumor inhibition rate of CD80 fusion protein is significantly improved. This shows that improving the sialic acid content can help improve the tumor suppressive effect of CD80 fusion protein.
  • the method of administration is intravenous infusion, and the infusion time is 4 minutes.

Abstract

提供了一种CD80蛋白变体，以及包含所述CD80蛋白变体的融合蛋白，其能够与PD-L1、CTLA-4、CD28相结合，激活T淋巴细胞的活性，抑制肿瘤或肿瘤细胞的生长和/或增殖。还提供了编码所述融合蛋白的核酸、包含所述融合蛋白的载体、包含所述核酸或所述载体的细胞、制备所述融合蛋白的方法以及所述融合蛋白的应用。

Description

CD80蛋白变体及CD80融合蛋白 技术领域
本申请涉及生物医药领域，具体的涉及一种CD80蛋白变体及包含CD80变体的融合蛋白。
背景技术
CD80又称为B7、B7.1或BB1，属于免疫球蛋白超家族，主要表达于活化B淋巴细胞、活化T淋巴细胞、巨噬细胞等。CD80的受体有CD28、CD152(CTLA4)和PD-L1，PD-L1与CD80的顺式相互作用，可以分别抑制PD-1和CTLA-4的功能；CD28与CD80结合可以产生一种共刺激信号，增强T细胞对同源抗原的激活、功能和存活。CD80通过这些机制，维持T细胞活化与抑制之间的平衡，发挥对肿瘤细胞的攻击作用。
目前针对这些机制开发了靶向CTLA4、PD-L1的单抗或双抗，但在肿瘤微环境中可能由于缺少足够的T细胞共刺激作用，造成患者应答率低且产生耐药性，鉴于CD80配体在免疫调节中的重要作用，靶向CD80配体在癌症和自身免疫中一直是一个有吸引力的策略。
发明内容
本申请提供了一种包含CD80的融合蛋白，其能够与PD-L1、CTLA-4、CD28相结合，激活T淋巴细胞的活性，抑制肿瘤或肿瘤细胞的生长和/或增殖。本申请还涉及编码所述融合蛋白的核酸、包含所述融合蛋白的载体、包含所述核酸或所述载体的细胞、制备所述融合蛋白的方法以及所述融合蛋白的应用。
一方面，本申请提供了一种CD80蛋白变体，其包含CD80蛋白的功能活性片段或其变体，所述变体与相应的野生型CD80的功能活性片段相比，具有一个或多个氨基酸位点的突变，且所述变体具有以下特性中的一种或多种：
a)与PD-L1相结合；
b)与CTLA-4相结合；
c)与CD28相结合；
d)激活T淋巴细胞的活性；以及
e)抑制肿瘤或肿瘤细胞的生长和/或增殖。
在某些实施方式中，所述PD-L1为人PD-L1。
在某些实施方式中，所述CTLA-4为人CTLA-4。
在某些实施方式中，所述CD28为人CD28。
在某些实施方式中，所述CD80蛋白的功能活性片段包含CD80蛋白的胞外结构域(ECD)或其片段。
在某些实施方式中，所述CD80蛋白的胞外结构域的片段包含CD80蛋白的IgV结构域。
在某些实施方式中，所述CD80蛋白变体与相应的野生型CD80蛋白胞外结构域或其片段相比，在选自下组的一个或多个氨基酸残基处包含氨基酸突变：N55、N64、N152、N173、N177、N192和N198。
在某些实施方式中，所述CD80蛋白变体包含选自下述的任一组氨基酸位置的突变：
1)N64；
2)N55和N64；
3)N55、N64和N152；
4)N55、N64和N192；
5)N55、N64和N198；
6)N55、N64、N152和N198；
7)N55、N64、N152和N192；
8)N55、N64、N152、N173、N177和N198；
9)N55、N64、N152、N173和N198；以及
10)N55、N64、N152、N177和N198。
在某些实施方式中，所述的CD80蛋白变体与相应的野生型CD80蛋白的功能活性片段相比，在选自下组的一个或多个氨基酸残基处包含氨基酸突变：N55A/Q、N64A、N152A、N173Q、N177A、N192A和N198A/Q。
在某些实施方式中，所述的CD80蛋白变体包含选自下组的任一组氨基酸突变：
1)N64A；
2)N55A和N64A；
3)N55A、N64A和N152A；
4)N55A、N64A和N192A；
5)N55A、N64A和N198A；
6)N55A、N64A、N152A和N198A；
7)N55A、N64A、N152A和N192A；
8)N55Q、N64A、N152A和N198Q；
9)N55Q、N64A、N152A、N173Q、N177A和N198Q；
10)N55Q、N64A、N152A、N173Q和N198Q；以及
11)N55Q、N64A、N152A、N177A和N198Q。
在某些实施方式中，所述的CD80蛋白变体包含至少一个第一类氨基酸突变，所述第一类氨基酸突变能够保持或增强所述CD80蛋白变体的稳定性。
在某些实施方式中，所述的CD80蛋白变体包含在以下一个或多个氨基酸位置处的第一类氨基酸突变：N55、N64、N152、N192和N198。
在某些实施方式中，所述的CD80蛋白变体包含选自下组的任一组氨基酸位置处的第一类氨基酸突变：
1)N64；
2)N55和N64；
3)N55、N64和N152；
4)N55、N64和N192；
5)N55、N64和N198；
6)N55、N64、N152和N198；以及
7)N55、N64、N152和N192。
在某些实施方式中，所述的CD80蛋白变体包含以下一个或多个第一类氨基酸突变：N55A/Q、N64A、N152A、N192A和N198A/Q。
在某些实施方式中，所述的CD80蛋白变体包含选自下组的任一组第一类氨基酸突变：
1)N64A；
2)N55A和N64A；
3)N55A、N64A和N152A；
4)N55A、N64A和N192A；
5)N55A、N64A和N198A；
6)N55A、N64A、N152A和N198A；
7)N55A、N64A、N152A和N192A；以及
8)N55Q、N64A、N152A和N198Q。
在某些实施方式中，所述的CD80蛋白变体包含至少一个第二类氨基酸突变，所述第二类氨基酸突变能够保持所述CD80蛋白变体的结合活性。
在某些实施方式中，所述的CD80蛋白变体包含在以下一个或多个氨基酸位置处的第二类氨基酸突变：N152；N192和N198。
在某些实施方式中，所述的CD80蛋白变体包含以下一个或多个第二类氨基酸突变：N152A；N192A和N198A/Q。
在某些实施方式中，所述的CD80蛋白变体包含N55和/或N64位的第二类氨基酸突变。
在某些实施方式中，所述的CD80蛋白变体包含N55A/Q和/或N64A的第二类氨基酸突变。
在某些实施方式中，所述的CD80蛋白变体包含至少一个第三类氨基酸突变，所述氨基酸突变能够保持所述CD80蛋白变体的亲和力。
在某些实施方式中，所述的CD80蛋白变体包含在以下一个或多个氨基酸位置处的第三类氨基酸突变：N55、N64、N152和N198。
在某些实施方式中，所述的CD80蛋白变体包含以下一个或多个第三类氨基酸突变：N55A/Q、N64A、N152A和N198A。
在某些实施方式中，所述的CD80蛋白变体包含N192位的第三类氨基酸突变。
在某些实施方式中，所述的CD80蛋白变体包含N192A的第三类氨基酸突变。
在某些实施方式中，所述的CD80蛋白变体包含至少一个第四类氨基酸突变，所述突变能够增强CD80蛋白变体与CD28和/或PD-L1的结合。
在某些实施方式中，所述的CD80蛋白变体包含N173和/或N177位的第四类氨基酸突变。
在某些实施方式中，所述的CD80蛋白变体包含N173Q和/或N177A的第四类氨基酸突变。
在某些实施方式中，所述的CD80蛋白变体中所述氨基酸位置的编号参考SEQ ID NO:1中氨基酸的位置编号确定。
在某些实施方式中，所述的CD80蛋白变体包含SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24中任一项所示的氨基酸序列。
在某些实施方式中，所述的CD80蛋白变体能够与PD-L1，CD28和/或CTLA-4相结合。
在某些实施方式中，所述的CD80蛋白变体的唾液酸含量大于等于约0.7。
另一方面，本申请提供了一种融合蛋白，其包含所述的CD80蛋白变体。
在某些实施方式中，所述融合蛋白还包含免疫球蛋白Fc区或其变体。
在某些实施方式中，所述的融合蛋白中所述CD80蛋白变体和所述免疫球蛋白Fc区直接或间接相连。
在某些实施方式中，所述的融合蛋白中所述免疫球蛋白Fc区包含IgG的Fc区。
在某些实施方式中，所述的融合蛋白中所述IgG选自下组：IgG1和IgG4。
在某些实施方式中，所述的融合蛋白中所述CD80蛋白变体位于所述免疫球蛋白Fc区的N端或C端。
在某些实施方式中，所述的融合蛋白中所述免疫球蛋白Fc区包含选自下组的氨基酸序列：SEQ ID NO:25和SEQ ID NO:26。
在某些实施方式中，所述的融合蛋白包含SEQ ID NO:27-37中任一项所示的氨基酸序列。
另一方面，本申请还提供了一种多肽，其包含所述的CD80蛋白变体、或所述的融合蛋白。
另一方面，本申请提供了分离的一种或多种核酸分子，其编码本申请所述的CD80蛋白变体、本申请所述的融合蛋白、或本申请所述的多肽。
另一方面，本申请提供了一种载体，其包含本申请所述的核酸分子。
另一方面，本申请提供了一种宿主细胞，其包含本申请所述的核酸分子或本申请所述的载体。
另一方面，本申请提供了一种制备本申请所述的CD80蛋白变体或本申请所述的融合蛋白的方法，所述方法包括在使得所述融合蛋白表达的条件下，培养本申请所述的细胞。
另一方面，本申请提供了一种药物组合物，其包含本申请所述的CD80变体蛋白、本申请所述的融合蛋白、本申请所述的多肽、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的宿主细胞，以及任选地药学上可接受的载剂。
另一方面，本申请提供了药物组合，其包含所述CD80蛋白变体，以及免疫检查点抑制剂。
另一方面，本申请提供了药物组合，其包含所述融合蛋白，以及免疫检查点抑制剂。
另一方面，本申请提供了药物组合，其包含所述多肽，以及免疫检查点抑制剂。
在某些实施方式中，所述免疫检查点包括PD-1、PD-L1、GITR和/或CTLA-4。
在某些实施方式中，所述免疫检查点包括选自下组中的一种或多种：PD-1抗体、PD-L1 抗体、CTLA-4抗体和GITR抗体。
另一方面，本申请提供了一种调节受试者的免疫反应的方法，所述方法包括施用本申请所述的CD80蛋白变体、所述的融合蛋白或所述的多肽。
在某些实施方式中，所述调节受试者的免疫反应的方法包括增加免疫反应。
在某些实施方式中，在所述调节受试者的免疫反应的方法中，所述CD80蛋白变体、所述融合蛋白或所述多肽与野生型CD80蛋白或其片段相比，表现出增加的对PD-L1的结合亲和力。
在某些实施方式中，在所述调节受试者的免疫反应的方法中，所述CD80蛋白变体、所述融合蛋白或所述多肽与野生型CD80蛋白或其片段相比，表现出增加的对CD28的结合亲和力。
在某些实施方式中，在所述调节受试者的免疫反应的方法中，所述CD80蛋白变体、所述融合蛋白或所述多肽与野生型CD80蛋白或其片段相比，表现出增强的激活T淋巴细胞的活性。
另一方面，本申请提供了一种所述的CD80蛋白变体、所述的CD80融合蛋白、所述的多肽、所述的核酸分子、所述的载体、所述的宿主细胞、所述的药物组合物、或所述的药物组合在制备预防和/或治疗疾病和/或病症中的应用。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液瘤。
在某些实施方式中，所述肿瘤包括对于中枢性记忆T细胞的增殖有响应的肿瘤。
在某些实施方式中，所述的应用中所述肿瘤包括：恶性黑色素瘤、乳腺癌、胃癌、肾癌、非小细胞肺癌、结肠癌、直肠癌、头颈部鳞状细胞癌、肝癌、肾癌、间皮瘤膀胱癌、胰腺癌、卵巢癌、子宫内膜癌和淋巴瘤。
另一方面，本申请提供了一种预防和/或治疗疾病和/或病症的方法，所述方法包括向有需要的受试者施用所述CD80蛋白变体、所述融合蛋白、所述多肽、所述核酸分子、所述载体、所述宿主细胞、所述药物组合物或所述药物组合。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液瘤。
在某些实施方式中，所述肿瘤包括对于中枢性记忆T细胞的增殖有响应的肿瘤。
另一方面，本申请提供了所述CD80蛋白变体、所述融合蛋白、所述多肽、所述核酸分子、所述载体、所述宿主细胞、所述药物组合物或所述药物组合，其用于预防和/或治疗疾 病和/或病症。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液瘤。
在某些实施方式中，所述肿瘤包括对于中枢性记忆T细胞的增殖有响应的肿瘤。
另一方面，本申请提供了一种检测生物样品中的CD80结合配体的方法，所述方法包括使生物样品与本申请所述的CD80蛋白变体或所述融合蛋白的结合试剂接触。
在某些实施方式中，所述的方法中所述结合配体包括PD-L1、CD28和/或CTLA-4。
另一方面，本申请还提供了检测受试者中中枢性记忆T细胞的频率和/或增殖的方法，所述方法包括施用所述CD80蛋白变体、所述融合蛋白、所述多肽。
在某些实施方式中，所述中枢性记忆T细胞是CD95+和CD28+细胞。
在某些实施方式中，所述中枢性记忆T细胞是CD4+中枢性记忆T细胞和/或CD8+中枢性记忆T细胞。
另一方面，本申请还提供了试剂盒，其包含所述CD80蛋白变体、所述融合蛋白、所述多肽、所述核酸分子、所述载体、所述宿主细胞、所述药物组合物或所述药物组合。
在某些实施方式中，所述试剂盒用于检测受试者中中枢性记忆T细胞频率和/或增殖。
在某些实施方式中，所述试剂盒用于检测生物样品中的CD80结合配体的存在和/或含量。
另一方面，本申请还提供了所述CD80蛋白变体、所述融合蛋白、所述多肽、所述核酸分子、所述载体、所述宿主细胞、所述药物组合物或所述药物组合，其用于增强中枢性记忆T细胞的增殖或频率。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是本申请所述CD80融合蛋白的结构图。
图2A-2B显示的是本申请所述CD80融合蛋白结合人PD-L1蛋白的活性检测结果。
图3A-3D显示的是本申请所述CD80融合蛋白结合人CTLA-4蛋白的活性检测结果。
图4A-4C显示的是本申请所述CD80融合蛋白结合人CD28蛋白的活性检测结果。
图5显示的是本申请所述CD80融合蛋白结合人CTLA-4阳性细胞的活性检测结果。
图6显示的是本申请所述CD80融合蛋白结合人PD-L1阳性细胞的活性检测结果。
图7显示的是本申请所述CD80融合蛋白结合人CD28阳性细胞的活性检测结果。
图8显示的是本申请所述CD80融合蛋白在aAPC/T混合细胞培养条件下检测其激活T淋巴细胞的活性结果。
图9A-9C显示的是本申请所述CD80融合蛋白的CD28激活活性检测。
图10显示的是本申请所述CD80融合蛋白在CT26皮下种植模型下的抗肿瘤活性结果。
图11显示的是本申请所述CD80融合蛋白在CT26皮下种植模型下与Treg抑制剂联合用药的抗肿瘤作用结果。
图12显示的是本申请所述CD80融合蛋白在CT26皮下种植模型下与PD-1抑制剂联合用药的抗肿瘤作用结果。
图13显示的是本申请所述CD80融合蛋白在CT26皮下种植模型下与CTLA-4抑制剂联合用药的抗肿瘤作用结果。
图14A-14B显示的是本申请所述CD80融合蛋白诱导细胞因子释放结果。
图15显示的是本申请所述CD80融合蛋白电荷异质性分析结果。
图16显示的是不同唾液酸修饰程度的本申请所述的CD80融合蛋白的抗肿瘤效果。
图17显示的是Sprague Dawley大鼠单次静脉输注CD80-WT-G1和CD80M28原液后各组平均血药浓度-时间变化曲线。(Mean+SD，n＝8)
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“CD80蛋白变体”是指相对于野生型CD80具有改变的氨基酸序列的蛋白。所述野生型CD80通常指从大自然中获得的，天然存在的CD80。在本申请中，所述野生型CD80可以是人CD80。所述变体通常指与野生型相比，对序列的一个或多个氨基酸的替代、缺失和/或添加。在某些情况下，CD80蛋白变体可以是CD80蛋白的胞外结构域 (ECD)；进一步地，CD80蛋白变体可以是在ECD基础上进行一个或多个氨基酸残基突变的蛋白；在某些情况下，CD80蛋白变体可以是CD80蛋白的IgV结构域；进一步地，CD80蛋白变体可以是在IgV结构域基础上进行一个或多个氨基酸残基突变的蛋白。在某些情况下，所述人CD80在GenBank数据库中的序列号可以为NP_005182.1。
在本申请中，术语“CD80蛋白的胞外结构域(ECD)”通常是指位于表达CD80蛋白的细胞膜表面的多肽片段。所述CD80蛋白是一种跨膜糖蛋白，包括两个Ig样胞外域、一个跨膜螺旋段和一个短的胞质尾。所述Ig样胞外域包括单个V型和C2型结构域。所述CD80蛋白的胞外结构域可以包括两个Ig样胞外域的全部或部分多肽片段。在本申请中，所述CD80蛋白的胞外结构域可以指人CD80蛋白的胞外结构域，例如本申请所述CD80蛋白的胞外结构域可以是包含SEQ ID NO:1中所示的氨基酸序列的全部或部分的蛋白质结构域。
在本申请中，术语“CD80蛋白的IgV结构域”通常是指位于表达CD80蛋白的细胞膜表面的V型结构域的多肽片段。所述CD80蛋白的IgV结构域可以包括V型结构域的全部或部分多肽片段。在本申请中，所述CD80蛋白的IgV结构域可以指人CD80蛋白的IgV结构域，例如本申请所述CD80蛋白的IgV结构域可以是包含SEQ ID NO:2中所示的氨基酸序列的全部或部分的蛋白质结构域。
在本申请中，术语“PD-L1”和“PDL1”可互换地使用，通常指细胞程序性死亡配体1，也可以称为表面抗原分化簇274(CD274)或B7同源蛋白1(B7-H1)、PDCD1L1、PDCD1LG1、PDL1，属于肿瘤坏死因子超家族。“PD-L1”可以是完整的PD-L1及其片段，可以是PD-L1的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与PD-L1共同表位的类似物。PD-L1可以是由290个氨基酸残基组成的I型跨膜糖蛋白，包含一个IgV样区、一个IgC样区、一个跨膜疏水区和一个30个氨基酸的胞内尾部。在本申请中，所述PD-L1可以是人PD-L1。
在本申请中，术语“CTLA-4”通常是指细胞毒性T-淋巴细胞抗原-4，也可以称为CTLA4、表面抗原分化簇152(CD152)，是一种白细胞分化抗原，与CD28共同享有B7分子配体，属于CD28家族的免疫抑制性受体。CTLA-4包含一个胞外V型结构域、一个跨膜结构域和一个胞质尾。“CTLA-4”可以是完整的CTLA-4及其片段，可以是CTLA-4的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与CTLA-4共同表位的类似物。在本申请中，所述CTLA-4可以是人CTLA-4。
在本申请中，术语“CD28”通常是指表面抗原分化簇28，是T淋巴细胞表面表达的共刺激分子，是CD80和CD86蛋白的受体，介导T细胞的共刺激并促进其存活、增殖以及产生 细胞因子。“CD28”可以是完整的CD28及其片段，可以是CD28的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与CD28共同表位的类似物。在本申请中，所述CD28可以是人CD28。
在本申请中，术语“氨基酸突变”通常是指氨基酸取代、缺失、插入和修饰。可以进行取代、缺失、插入和修饰的任意组合来实现最终构建体。在本申请中，氨基酸突变可以是至少一种已有氨基酸残基被另一种不同的氨基酸残基(替代氨基酸残基)置换，所述替代氨基酸残基可以是“天然存在的氨基酸残基”，并且选自由以下各项组成的组：丙氨酸(三字母代码：ala，单字母代码：A)、精氨酸(arg，R)、天冬酰胺(asn，N)、天冬氨酸(asp，D)、半胱氨酸(cys，C)、谷氨酰胺(gln，Q)、谷氨酸(glu，E)、甘氨酸(gly，G)、组氨酸(his，H)、异亮氨酸(ile，I)、亮氨酸(leu，L))、赖氨酸(lys，K)、蛋氨酸(met，M)、苯丙氨酸(phe，F)、脯氨酸(pro，P)、丝氨酸(ser，S)、苏氨酸(thr，T)、色氨酸(trp，W)、酪氨酸(tyr，Y)和缬氨酸(val，V)。
在本申请中，所述氨基酸位置是以SEQ ID NO:1(人CD80(UniProtKB/Swiss-Prot:P33681.1序列)的胞外结构域的氨基酸序列，即UniProtKB/Swiss-Prot:P33681.1序列的第35-243位)所示的氨基酸序列为基准进行确认。即，SEQ ID NO:1所示的氨基酸序列的第一个氨基酸的编号为第一位氨基酸。
在本申请中，所述CD80蛋白的胞外结构域可包含UniProtKB/Swiss-Prot:P33681.1序列的第35-243位氨基酸。
在本申请中，所述CD80蛋白的IgV结构域可包含UniProtKB/Swiss-Prot:P33681.1序列的第35-140位氨基酸。
在本申请中，术语“第一类氨基酸突变”通常是指对所述CD80蛋白变体稳定性有影响的氨基酸突变。所述CD80蛋白变体稳定性是指CD80蛋白变体的结构与功能与突变前的CD80蛋白保持近似一致的能力，所述影响可以是对CD80蛋白变体稳定性的保持、降低和提高。在本申请中，所述CD80蛋白变体可以是在人CD80蛋白基础上进行氨基酸序列突变。例如在本申请中第一类氨基酸突变可以包括在人CD80的AA35-AA242的全部或部分片段发生一个或多个氨基酸突变，在某些情况下，所述一个或多个氨基酸突变可以包含选自下组任一位置处的氨基酸突变：N55、N64、N152、N192和N198。
在本申请中，术语“第二类氨基酸突变”通常是指对所述CD80蛋白变体结合活性有影响的氨基酸突变。所述影响可以是对CD80蛋白变体结合活性的保持、降低和提高。在本申请中，所述CD80蛋白变体可以是在人CD80蛋白基础上进行氨基酸序列突变。例如在本申请 中第二类氨基酸突变可以包括在人CD80的AA35-AA242的全部或部分片段发生一个或多个氨基酸突变，在某些情况下，所述一个或多个氨基酸突变可以包含选自下组任一位置处的氨基酸突变：N55、N64、N152、N192和N198。
在本申请中，术语“结合活性”通常指两个结合位点相互结合的能力，所述结合位点可以是结合分子上的一个区域，所述结合分子可以包括蛋白质、酶底物、第二信使、激素或变构调节剂。在本申请中，结合活性可以是CD80蛋白变体与其配体结合的能力，结合可以是可逆的(瞬时和非共价)，也可以是共价可逆或不可逆的。
在本申请中，术语“第三类氨基酸突变”通常是指对所述CD80蛋白变体亲和力有影响的氨基酸突变。所述影响可以是对CD80蛋白变体亲和力的保持、降低和提高。在本申请中，所述CD80蛋白变体可以是在人CD80蛋白基础上进行氨基酸序列突变。例如在本申请中第三类氨基酸突变可以包括在人CD80的AA35-AA242的全部或部分片段发生一个或多个氨基酸突变，在某些情况下，所述一个或多个氨基酸突变可以包含选自下组任一位置处的氨基酸突变：N55、N64、N152、N192和N198。
在本申请中，术语“亲和力”通常是指能引起两种或两种以上物质相互作用的特异性亲和能力，在某些情况下，可以是受体与配体的亲和能力。
在本申请中，术语“第四类氨基酸突变”通常是指对所述CD80蛋白变体与CD28和/或PD-L1的结合有影响的氨基酸突变。所述影响可以是对CD80蛋白变体与CD28和/或PD-L1的结合的保持、降低和提高。在本申请中，所述CD80蛋白变体可以是在人CD80蛋白基础上进行氨基酸序列突变。例如在本申请中第三类氨基酸突变可以包括在人CD80的AA35-AA242的全部或部分片段发生一个或多个氨基酸突变，在某些情况下，所述一个或多个氨基酸突变可以包含选自下组任一位置处的氨基酸突变：N173和/或N177。
在本申请中，术语“融合蛋白”通常是指两个或多个不同的蛋白分子通过化学和/或基因融合的方法连成的一个大分子。融合蛋白可以是通过DNA重组技术得到的两个或多个基因重组后的表达产物，也可以是介导两个或多个细胞质膜融合的一组蛋白。在本申请中，融合蛋白可以是在基因水平将目的基因同免疫球蛋白Fc区或其变体片段基因相连，并在真核或原核细胞中表达出的具有上述两部分结构域的蛋白分子。
在本申请中，术语“免疫球蛋白Fc区”通常是指免疫球蛋白的可结晶段，可以包括免疫球蛋白IgG、IgA和IgD的CH2和CH3结构域或其片段，可以包括IgM和IgE的CH2、CH3和CH4结构域或其片段。例如，所述IgG可以包括IgG1，IgG2，IgG3或IgG4。
术语“多肽”、“肽”和“蛋白”在本文中可以互换，是指氨基酸残基的聚合物。这个术语可 用于指一个或多个氨基酸残基是其相应的天然氨基酸的人工合成化学模拟物的氨基酸聚合物，也可用于指天然的氨基酸聚合物，那些含修饰残基的氨基酸聚合物以及非天然的氨基酸聚合物。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到细胞(例如，宿主细胞)中和/或细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的细胞，所述载体可以产生期望的表达产物。在本申请中，所述载体可以为质粒。
在本申请中，术语“药物组合物”通常是指涉及适合施用于患者、可以是人患者的组合物。例如，本申请所述的药物组合物，其可以包含本申请所述的CD80变体蛋白、本申请所述的融合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药物组合”通常指将两个或多个药物搭配使用。在所述药物组合中，不同的药物组分可以混合放置，也可以分开放置。在所述药物组合中，不同的药物组分可以放置在同一容器中，也可以放置在不同容器中。所述药物组合中的不同药物组分可以同时施用，也可以分开施用，例如，不同的药物组分可以分先后施用。在所述药物组合中，不同的药物组分施用的方式可以以相同方式施用，也可以以不同方式施用。
在本申请中，术语“药学上可接受的载剂”通常是指与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂等，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在本申请中，术语“肿瘤”通常是指由异常细胞生长形成的赘生物或实体病变。在本申请中，肿瘤可以是实体瘤或非实体瘤。在某些情况下，肿瘤可以包括对于中枢性记忆T细胞的增殖有响应的肿瘤。在某些情况下，肿瘤可以包括含有对CTLA-4呈表面阳性的细胞的肿瘤。
在本申请中，涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，例如在所述蛋白质和/或所述多肽(例如，CD80蛋白变体)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述同源物可以为与所述蛋白质和/或所述多肽(例如，CD80蛋白变体)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，术语“包含”通常是指包括、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
CD80蛋白变体
一方面，本申请提供一种CD80蛋白变体，其可包含CD80蛋白的功能活性片段或其变体，所述变体与相应的野生型CD80的功能活性片段相比，具有一个或多个氨基酸位点的突变。
在本申请中，所述CD80蛋白变体可具有下述性质中的一种或多种：1)与PD-L1相结合；2)与CTLA-4相结合；3)与CD28相结合；4)激活T淋巴细胞的活性；以及5)抑制肿瘤或肿瘤细胞的生长和/或增殖。
例如，所述CD80蛋白变体能同时结合PD-L1与CD28蛋白。例如，所述CD80变体能同时结合PD-L1、CD28和CTLA-4蛋白。
在本申请中，所述PD-L1可以为人PD-L1。在本申请中，所述CD28可以为人CD28。在本申请中，所述CTLA-4可以为人CTLA-4。
在本申请中，所述CD80蛋白可以为人CD80蛋白。
在本申请中，所述CD80蛋白变体可包含CD80的功能活性片段或其变体。例如，所述CD80的功能活性片段可包含CD80蛋白胞外结构域或其片段。例如，所述CD80蛋白胞外结构域的片段可包含CD80的IgV结构域。
在本申请中，野生型CD80蛋白的IgV结构域可包含SEQ ID NO:2所示的氨基酸序列。
在本申请中，野生型CD80蛋白的胞外结构域可包含SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述CD80蛋白的IgV结构域的变体可以包含一个或多个氨基酸突变。
在本申请中，所述CD80蛋白的IgV结构域的变体可以包括以下氨基酸序列：VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNX1LSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKA。所述CD80蛋白的IgV结构域的变体可至少包含在X1处的氨基酸取代。
在本申请中，与包含SEQ ID NO:2所示的CD80蛋白的IgV结构域相比，所述CD80蛋白的IgV结构域变体可至少包含在X1处的氨基酸取代。例如，X1处的氨基酸可被取代为A。
在本申请中，所述CD80蛋白的胞外结构域变体可以包含一个或多个氨基酸突变。
在本申请中，所述CD80蛋白的胞外结构域变体可以包括以下氨基酸序列：VIHVTKEVKEVATLSCGHX1VSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKX2RTIFDITNX3LSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAIX4TTVSQDPETELYAVSSKLDFX5MTTX6HSFMCLIKYGHLRVX7QTFNWX8TTKQEHFPDNL。所述CD80蛋白的胞外结构域变体可至少包含在X1、X2、X3、X4、X5、X6、X7和/或X8处的氨基酸取代。
例如，所述CD80蛋白变体可包含在X1、X2、X3、X4、X5、X6、X7和X8中的一个或多个位置具有氨基酸突变。其中，X1处的氨基酸可被取代为A；X2处的氨基酸可被取代为A或Q；X3处的氨基酸可被取代为A；X4处的氨基酸可被取代为A；X5处的氨基酸可被取代为A或Q；X6处的氨基酸可被取代为A；X7处的氨基酸可被取代为A；X8处的氨基酸可被取代为A或Q。
在本申请中，所述CD80蛋白变体可以包含第一类氨基酸突变。例如，所述第一类氨基酸突变能够保持所述CD80蛋白变体的稳定性。
在本申请中，所述包含第一类氨基酸突变的CD80蛋白变体与包含SEQ ID NO:1所示的CD80蛋白的胞外结构域相比，所述CD80蛋白的胞外结构域变体可至少包含在X2、X3、X4、X7和/或X8处的氨基酸取代，其中X2处的氨基酸可被取代为A或Q；X3处的氨基酸可被取代为A；X4处的氨基酸可被取代为A；X7处的氨基酸可被取代为A；X8处的氨基酸可被取代为A或Q。
例如，所述第一类氨基酸突变可包含N64位的氨基酸突变。在本申请中，所述第一类氨基酸突变可包含N55和N64位的氨基酸突变。例如，所述第一类氨基酸突变可包含N55、N64和N152位的氨基酸突变。例如，所述第一类氨基酸突变可包含N55、N64和N192位的氨基酸突变。例如，所述第一类氨基酸突变可包含N55、N64和N198位的氨基酸突变。例如，所述第一类氨基酸突变可包含N55、N64、N152和N198位的氨基酸突变。例如，所述第一类氨基酸突变可包含N55、N64、N152和N192位的氨基酸突变。
例如，所述第一类氨基酸突变可包含N64A的氨基酸突变。例如，所述第一类氨基酸突变可包含N55A和N64A的氨基酸突变。例如，所述第一类氨基酸突变可包含N55A、N64A和N152A的氨基酸突变。例如，所述第一类氨基酸突变可包含N55A、N64A和N192A的氨基酸突变。例如，所述第一类氨基酸突变可包含N55A、N64A和N198A的氨基酸突变。例如，所述第一类氨基酸突变可包含N55A、N64A、N152A和N198A的氨基酸突变。例如， 所述第一类氨基酸突变可包含N55A、N64A、N152A和N192A的氨基酸突变。例如，所述第一类氨基酸突变可包含N55Q、N64A、N152A和N198Q的氨基酸突变。
在本申请中，所述CD80变体可包含第二类氨基酸突变。在本申请中，所述第二类氨基酸突变能够保持所述CD80蛋白变体的结合活性。
在本申请中，所述包含第二类氨基酸突变的CD80蛋白变体与包含SEQ ID NO:1所示的CD80蛋白的胞外结构域相比，所述CD80蛋白的胞外结构域变体可至少包含在X4、X7和/或X8处的氨基酸取代，其中X4处的氨基酸可被取代为A；X7处的氨基酸可被取代为A；X8处的氨基酸可被取代为A和Q。
例如，所述第二类氨基酸突变可包含N152、N192、N198中的一个或多个氨基酸位置处的突变。例如，所述第二类氨基酸突变可包含N152A；N192A和N198A/Q中的一个或多个氨基酸突变。例如，所述第二类氨基酸突变还可包含N55和/或N64位的氨基酸突变。例如，所述第二类氨基酸突变还可包含N55A/Q和/或N64A的氨基酸突变。
在本申请中，所述CD80蛋白变体可包含第三类氨基酸突变。在本申请中，所述第三类氨基酸突变能够保持所述CD80蛋白变体的亲和力。
在本申请中，所述包含第三类氨基酸突变的CD80蛋白变体与包含SEQ ID NO:1所示的CD80蛋白的胞外结构域相比，所述CD80蛋白的胞外结构域变体可至少包含在X2、X3、X4和/或X8处的氨基酸取代，其中X2处的氨基酸可被取代为A或Q；X3处的氨基酸可被取代为A；X4处的氨基酸可被取代为A；X8处的氨基酸可被取代为A。
例如，所述第三类氨基酸突变可包含在以下一个或多个位置处的氨基酸突变：N55、N64、N152和N198。例如，所述第三类氨基酸突变可包含以下一个或多个氨基酸突变：N55A/Q、N64A、N152A和N198A。例如，所述第三类氨基酸突变还可包含N192位的氨基酸突变。例如，所述第三类氨基酸突变还可包含N192A的氨基酸突变。
在本申请中，所述CD80蛋白变体可包含第四类氨基酸突变。在本申请中，所述第四类氨基酸突变能够增强CD80蛋白变体与CD28和/或PD-L1的结合。
在本申请中，所述包含第四类氨基酸突变的CD80蛋白变体与包含SEQ ID NO:1所示的CD80蛋白的胞外结构域相比，所述CD80蛋白的胞外结构域变体可至少包含在X5和/或X6处的氨基酸取代，其中X5处的氨基酸可被取代为Q；X6处的氨基酸可被取代为A。
例如，所述第四类氨基酸突变可包含N173和/或N177位的氨基酸突变。例如，所述第四类氨基酸突变可包含N173Q和/或N177A的氨基酸突变。
在本申请中，与SEQ ID NO:2所示的氨基酸序列相比，所述CD80蛋白变体可包含N64 位的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N64和N55位的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55、N64和N152位的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55、N64和N192位的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55、N64和N198位的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55、N64、N152和N198位的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55、N64、N152和N192位的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55、N64、N152、N173、N177和N198位的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55、N64、N152、N173和N198位的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55、N64、N152、N177和N198位的氨基酸突变。
在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N64A的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55A和N64A的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55A、N64A和N152A的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55A、N64A和N192A的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55A、N64A和N198A的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55A、N64A、N152A和N198A的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55A、N64A、N152A和N192A的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55Q、N64A、N152和N198Q的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55Q、N64A、N152A、N173Q、N177A和N198Q的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55Q、N64A、N152A、N173Q和N198Q的氨基酸突变。在本申请中，与SEQ ID NO:1所示的氨基酸序列相比，所述CD80蛋白变体可包含N55Q、N64A、N152A、N177A和N198Q的氨基酸突变。
在本申请中，所述CD80蛋白变体可包含SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:8、 SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24中任一项所示的氨基酸序列。
在本申请中，所述CD80蛋白变体的唾液酸含量可以大于等于约0.7、大于等于约0.8、大于等于约0.9、大于等于约1.0、大于等于约1.1、大于等于约1.2、大于等于约1.3、大于等于约1.4、大于等于约1.5、大于等于约2、大于等于约3、大于等于约4、大于等于约5、大于等于约6、大于等于约7、大于等于约8、大于等于约9 5、大于等于约10、大于等于约11、大于等于约12、大于等于约13、或大于等于约14。
在本申请中，所述CD80蛋白变体的唾液酸含量可以大于等于约0.7且小于等于约15、大于等于约0.8且小于等于约15、大于等于约0.9且小于等于约15、大于等于约1.0且小于等于约15、大于等于约1.1且小于等于约15、大于等于约1.2且小于等于约15、大于等于约1.3且小于等于约15、大于等于约1.4且小于等于约15、大于等于约1.5且小于等于约15、大于等于约2且小于等于约15、大于等于约3且小于等于约15、大于等于约4且小于等于约15、大于等于约5且小于等于约15、大于等于约6且小于等于约15、大于等于约7且小于等于约15、大于等于约8且小于等于约15、大于等于约9且小于等于约15、大于等于约10且小于等于约15、大于等于约11且小于等于约15、大于等于约12且小于等于约15、大于等于约13且小于等于约15、或大于等于约14且小于等于约15。
融合蛋白
另一方面，本申请提供了一种包含CD80蛋白变体的融合蛋白。在本申请中，所述融合蛋白还可包含其他功能性片段。例如，所述融合蛋白可以包括所述的CD80蛋白变体和免疫球蛋白Fc区或其变体。
在本申请中，所述免疫球蛋白Fc区可以包含IgG的Fc区，所述IgG可以是人IgG，所述人IgG可以是人IgG1和/或人IgG4。所述变体可以是免疫球蛋白Fc区的全部或部分氨基酸片段。例如，所述免疫球蛋白Fc区可以包含选自下组的氨基酸序列：SEQ ID NO:25或SEQ ID NO:26。例如，所述IgG1 Fc区可包含SEQ ID NO:25所示的氨基酸序列。例如，所述IgG4Fc区可包含SEQ ID NO:26所示的氨基酸序列。
在本申请中，所述CD80蛋白变体和所述免疫球蛋白Fc区可以直接或间接相连。本申请所述间接相连包括通过多肽连接子连接。
在本申请中，所述多肽连接子可以包括一个或多个氨基酸残基组成的氨基酸序列，所述氨基酸残基可以包括脯氨酸或半胱氨酸，所述连接子可以连接两个结合片段，所述结合片段可以包括相同或不同片段。例如，所述连接子可以连接所述CD80蛋白变体和所述免疫球蛋 白Fc区。例如，所述连接子可包含(GGGGS)n的氨基酸序列，其中，n可以为1-10中的任意正整数。例如，n可以为1-4。例如，所述连接子可包含(GGGGS)2的氨基酸序列。例如，所述连接子可包含(GGGGS)3的氨基酸序列。例如，所述连接子可包含(GGGGS)4的氨基酸序列。
在本申请中，所述融合蛋白可以包含第一条多肽链或第二条多肽链。
在本申请中，所述第一条多肽链的多肽片段从N端到C端可以依次为：CD80蛋白变体-连接子-免疫球蛋白Fc区，所述CD80蛋白变体可以与连接子相连，连接子可以与免疫球蛋白Fc区的N端相连。
在本申请中，所述第二条多肽链的多肽片段从N端到C端可以依次为：CD80蛋白变体-免疫球蛋白Fc区，所述CD80蛋白变体可以与所述免疫球蛋白Fc区的N端相连。
在本申请中，所述融合蛋白可以包含两条相同的所述第一条多肽链，或者可以包含两条相同的所述第二条多肽链。
例如，所述第一条多肽链或所述第二条多肽链可包含SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24中任一项所示的CD80蛋白变体序列。例如，所述第一条多肽链或所述第二条多肽链可包含SEQ ID NO:27-37中任一项所示的氨基酸序列。
在本申请中，所述融合蛋白包含所述CD80蛋白变体，以及其他蛋白分子/多肽分子。例如，所述其他蛋白分子/多肽分子不影响所述CD80蛋白变体的功能。
核酸、载体、宿主细胞和制备方法
另一方面，本申请还提供了分离的一种或多种核酸分子，所述一种或多种核酸分子可编码本申请所述的CD80蛋白变体、及其融合蛋白。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述CD80蛋白变体、及其融合蛋白，也可以编码其中的一部分。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(1)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的；(2)通过克隆重组产生的；(3)纯化的，例如通过酶切和凝胶电泳分级分离，或者(4)合成的，例如通过化学合成。例如，所述分离的核酸可以通过聚合酶链式反应(PCR)扩增产生。在本申请中，可以通过本领域已知的多种方法来制备编码所述CD80蛋白变体、及其融合蛋白的核酸。
另一方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允 许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。
另一方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如原核细胞(例如，细菌细胞)、CHO细胞、NS/0细胞、HEK293细胞，或者其他真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
另一方面，本申请提供了制备本申请所述的CD80蛋白变体、及其融合蛋白的方法。所述方法可包括，在使得所述CD80蛋白变体、及其融合蛋白表达的条件下，培养本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
在某些情形中，所述方法还可包括收获(例如分离和/或纯化)，本申请所述的CD80蛋白变体、及其融合蛋白的步骤。例如，可以采用蛋白G-琼脂糖、蛋白A-琼脂糖或金属螯合进行亲和层析，还可通过凝胶电泳和/或高效液相色谱等来纯化和分离本申请所述的CD80蛋白变体、及其融合蛋白。
药物组合物、药物组合、应用
另一方面，本申请提供了一种药物组合物，其可包含本申请所述的CD80蛋白变体、及其融合蛋白，所述的核酸分子，所述的载体，所述的宿主细胞，以及任选地药学上可接受的载剂。
所述药学上可接受的载剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
在本申请中，所述药物组合物可被配制用于口服给药，静脉内给药，肌肉内给药，在肿瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。所述药物组合物可以用于抑制肿瘤生长。例如，本申请的药物组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
本申请所述的药物组合物可以包含预防和/或治疗有效量的所述CD80蛋白变体、及其融合蛋白。所述预防和/或治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的疾病或病症和/或其任何并发症而所需的剂量。
另一方面，本申请提供了药物组合，其可包含所述CD80蛋白变体，以及免疫检查点抑制剂。
另一方面，本申请提供了药物组合，其可包含所述融合蛋白，以及免疫检查点抑制剂。
在本申请中，所述免疫检查点可包括PD-1、PD-L1、GITR和/或CTLA-4。
在本申请中，所述免疫检查点抑制剂可包括选自下组中的一种或多种：PD-1抗体、PD-L1抗体、CTLA-4抗体、GITR抗体。
在本申请中，所述PD-1抗体可选自：纳武利尤单抗，帕博丽珠单抗，特瑞普利单抗，信迪利单抗，卡瑞利珠单抗，杰诺单抗。
在本申请中，所述PD-L1抗体可选自：度伐利尤单抗，阿替利珠单抗，恩沃利单抗，阿得贝利单抗，舒格利单抗，泰特利单抗。
在本申请中，所述CTLA-4抗体可选自：易普利姆玛，曲美木单抗。
在本申请中，所述GITR抗体可选自：MK-4166，TRX-518，INCAGN01876，BMS-986156，AMG228，IBI102，ASP-1951。
上述抗体举例仅为示例，此类抗体只要功能上能够实现相应免疫检查点的抑制剂的作用，都可以用于本申请。
在本申请的所述药物组合中，所述CD80蛋白变体/融合蛋白可与所述免疫检查点抑制剂存在于同一容器中。在本申请的所述药物组合中，所述CD80蛋白变体/融合蛋白可与所述免疫检查点抑制剂存在于不同容器中。
在本申请中，所述CD80蛋白变体/融合蛋白和所述免疫检查点抑制剂可同时施用。同时施用可以是各组分混合在一起施用，也可以是分开施用。可以以相同方式施用，例如施用于同一条静脉或其他血管，也可以是不同方式施用，例如静脉内施用和瘤内施用同时进行。
在本申请中，所述CD80蛋白变体/融合蛋白和所述免疫检查点抑制剂可先后施用。施用的顺序可以是先施用所述CD80蛋白变体/融合蛋白，再施用所述免疫检查点抑制剂；也可以是先施用所述免疫检查点抑制剂，再施用所述CD80蛋白变体/融合蛋白。可以以相同方式施用，也可以以不同方式施用。各组分可以一次施用，也可以分多次施用。在某些实施方案中，先后施用可以是以任何时间间隔施用，包括分钟、小时、天、周、月或年。在某些实施方案中，先后施用是指以至少2分钟、5分钟、10分钟、、30分钟、1小时、6小时、8小时、12 小时、24小时、36小时、48小时、3天、4天、5天、6天、1周、2周、3周、1个月、6周、2个月、3个月、4个月、5个月或6个月之一的时间间隔分开的施用。
另一方面，本申请提供了所述CD80蛋白变体、及其融合蛋白在制备预防或治疗疾病或病症的药物中的用途。
另一方面，本申请提供了所述CD80蛋白变体、及其融合蛋白，其制备预防或治疗疾病或病症。
另一方面，本申请提供了预防或治疗肿瘤的方法，其包括向有需要的受试者施用本申请所述的CD80蛋白变体、及其融合蛋白。在本申请中，。也可包括结直肠癌。
在本申请中，所述肿瘤可包括实体瘤。在本申请中，所述肿瘤可包括非实体瘤。在本申请中，所述肿瘤可包括选自下组中的一种或多种：恶性黑色素瘤、乳腺癌、胃癌、肾癌、非小细胞肺癌、结肠癌、直肠癌、头颈部鳞状细胞癌、肝癌、肾癌、间皮瘤膀胱癌、胰腺癌、卵巢癌、子宫内膜癌和淋巴瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的变体、融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1制备CD80融合蛋白
CD80融合蛋白是将CD80胞外结构域或其变体与人IgG1的Fc片段(氨基酸序列号为：SEQ ID NO:25)的N端连接得到的，其中CD80胞外结构域或其变体包括：野生型CD80ECD(SEQ ID NO:1，野生型CD80的胞外结构域)、M12(SEQ ID NO:6)、M13(SEQ ID NO:7)、M14(SEQ ID NO:8)、M15(SEQ ID NO:9)、M16(SEQ ID NO:10)、M18(SEQ ID NO:11)、M19(SEQ ID NO:12)、M20(SEQ ID NO:13)、M21(SEQ ID NO:14)、M22(SEQ ID NO:15)、M23(SEQ ID NO:16)、M24(SEQ ID NO:17)、M25(SEQ ID NO:18)、M26(SEQ ID NO:19)、M27(SEQ ID NO:20)、M28(SEQ ID NO:21)、M29(SEQ ID NO:22)、M30(SEQ ID NO:23)、M31(SEQ ID NO:24)。CD80蛋白IgV结构域或其变体包括：野生型CD80IgV(SEQ ID NO:2)、M09(SEQ ID NO:3)、M10(SEQ ID NO:4)、M11(SEQ ID NO:5)。
将上述CD80胞外结构域或其变体分别与人IgG1的Fc片段(氨基酸序列号为：SEQ ID NO:25)的N端连接得到CD80融合蛋白，分别命名为：CD80-WT、CD80M12、CD80M13、CD80M14、CD80M15、CD80M16、CD80M18、CD80M19、CD80M20、CD80M21、 CD80M22、CD80M23、CD80M24、CD80M25、CD80M26、CD80M27、CD80M28、CD80M29、CD80M30、CD80M31、CD80-IgV、CD80M09、CD80M10、CD80M11。
CD80融合蛋白的具体结构如图1所示。
其中，CD80M12、CD80M11、CD80M14、CD80M15、CD80M16、CD80M26、CD80M27、CD80M28、CD80M29、CD80M30、CD80M31稳定表达，表达量适中；与PD-L1、CTLA-4和/或CD28的结合活性较好，能够与PD-L1、CTLA-4和/或CD28保持亲和力。
实施例2 CD80融合蛋白结合人PD-L1，CTLA-4，CD28蛋白的活性检测
(1)以CD80-WT为对照，ELISA评价CD80融合蛋白与PD-L1的结合活性。
将PD-L1(Human PD-L1/B7-H1/CD274 Protein(His Tag)，Sino Biological)包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的CD80融合蛋白，37℃反应1小时；PBST洗涤后，加入辣根过氧化物酶标记的羊抗人IgG Fc二抗(Goat anti-human IgG Fc antibody，horseradish peroxidase(HRP)conjugate，affinity purified，Invitrogen)，37℃反应30分钟；PBST洗涤5次；每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μL 2N H2SO4终止液终止底物反应，酶标仪450nm处读取OD值，分析CD80融合蛋白与PD-L1的结合能力。
结果如图2A-2B所示，结果显示，在高浓度条件下，本申请所述的CD80蛋白变体与PD-L1的结合活性明显强于CD80-WT。
(2)以CD80-WT为对照，ELISA评价CD80融合蛋白与CTLA-4的结合活性。
将CTLA-4(Human CTLA-4/CD152 Protein，ACROBiosystems)包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的CD80融合蛋白，37℃反应1小时；PBST洗涤后，加入辣根过氧化物酶标记的羊抗人IgG Fc二抗(Goat anti-human IgG Fc antibody，horseradish peroxidase(HRP)conjugate，affinity purified，Invitrogen)，37℃反应30分钟；PBST洗涤5次；每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μL 2N H2SO4终止液终止底物反应，酶标仪450nm处读取OD值，分析CD80融合蛋白与CTLA-4的结合能力。
结果如图3A-3C所示，图3A的结果显示，除CD80M09，CD80M10，CD80M11，CD80M19，CD80M20与野生型CD80-WT相比，结合明显变弱以外，其余变体与CTLA-4的结合活性与CD80WT相当。
(3)以CD80-WT为对照，ELISA评价CD80融合蛋白与CD28的结合活性。
将CD28(Human CD28，Sino Biological)包被ELISA板条，4℃过夜；PBST洗涤后， 加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的CD80融合蛋白，37℃反应1小时；PBST洗涤后，加入辣根过氧化物酶标记的羊抗人IgG Fc二抗(Goat anti-human IgG Fc antibody，horseradish peroxidase(HRP)conjugate，affinity purified，Invitrogen)，37℃反应30分钟；PBST洗涤5次；每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μL 2N H2SO4终止液终止底物反应，酶标仪450nm处读取OD值，分析CD80融合蛋白与CD28的结合能力。
结果如图4A-4C所示，结果显示CD80M11、CD80M23，CD80M25，CD80M27，CD80M29，CD80M30，CD80M31与CD28的结合活性显著强于CD80-WT，其余融合蛋白的结合活性与CD80-WT相似。
实施例3 CD80融合蛋白结合人PD-L1，CTLA-4，CD28阳性细胞的活性检测
(1)以CD80-WT为对照，FACS评价CD80融合蛋白与CTLA-4阳性细胞的结合
收集过表达人CTLA-4的CHOK1细胞(CHOK1-CTLA-4)，按照每管5×105个细胞加至1.5mL EP管中；加入不同浓度的CD80融合蛋白，冰上避光孵育30min；FACS洗液洗涤后，加入PE荧光标记的羊抗人IgG Fc二抗(Goat Anti-Human IgG Fc Secondary Antibody，Invitrogen)，冰上避光孵育30min；FACS洗液洗涤2遍；每管加入400μL 1％多聚甲醛固定液(Solarbio)固定细胞，混匀后上机检测PE荧光的阳性率，分析CD80融合蛋白与CTLA-4阳性细胞的结合能力。
结果如图5所示，结果显示CD80M28与CD80-WT结合细胞表面CTLA-4的能力相似。
(2)以CD80-WT为对照，FACS评价CD80融合蛋白与PD-L1阳性细胞的结合
收集过表达人PD-L1的CHOK1细胞(CHOK1-PD-L1)，按照每管5×105个细胞加至1.5mL EP管中；加入不同浓度的CD80融合蛋白，冰上避光孵育30min；FACS洗液洗涤后，加入PE荧光标记的羊抗人IgG Fc二抗(Goat Anti-Human IgG Fc Secondary Antibody，Invitrogen)，冰上避光孵育30min；FACS洗液洗涤2遍；每管加入400μL 1％多聚甲醛固定液(Solarbio)固定细胞，混匀后上机检测PE荧光的阳性率，分析CD80融合蛋白与PD-L1阳性细胞的结合能力。
结果如图6所示，结果显示CD80M28与细胞表面PD-L1结合能力强于CD80-WT。
(3)以CD80-WT为对照，FACS评价CD80融合蛋白与CD28阳性细胞的结合
收集过表达人CD28的CHOK1细胞(CHOK1-CD28)，按照每管5×105个细胞加至1.5mL EP管中；加入不同浓度的CD80融合蛋白，冰上避光孵育30min；FACS洗液洗涤后，加入PE荧光标记的羊抗人IgG Fc二抗(Goat Anti-Human IgG Fc Secondary Antibody， Invitrogen)，冰上避光孵育30min；FACS洗液洗涤2遍；每管加入400μL 1％多聚甲醛固定液(Solarbio)固定细胞，混匀后上机检测PE荧光的阳性率，分析CD80融合蛋白与CD28阳性细胞的结合能力。
结果如图7所示，结果显示CD80M28与细胞表面CD28的结合能力略强于CD80-WT。
实施例4利用aAPC/T混合细胞培养检测CD80融合蛋白激活T淋巴细胞的活性
以CD80融合蛋白CD80-Fc和IgG1-Fc蛋白为对照，以过表达OKT3scFv及CD64的293T细胞(293T-OKT3-CD64)作为抗原递呈细胞(artificial APC，aAPC)，以CD4+T淋巴细胞作为效应细胞，利用aAPC细胞与CD4+T淋巴细胞混合培养的实验体系评价CD80融合蛋白激活T淋巴的活性。
收集293T-OKT3-CD64细胞，按照每孔2.5×104个细胞加入96孔细胞培养板中；将健康供体的PBMC细胞(MT-BIO)用CD4+T细胞分离试剂盒(CD4+T Cell Isolation Kit，human，Miltenyi Biotec)分离得到CD4+T淋巴细胞，按照每孔5×104个细胞加入96孔细胞培养板中；再加入1200nM的CD80融合蛋白，以及IgG1-Fc蛋白(Human IgG1 Fc Protein，Tag Free，ACROBiosystems)和CD80融合蛋白CD80-Fc(Human B7-1/CD80 Protein，Fc Tag，ACROBiosystems)，37℃培养箱中孵育24h后收集上清；用IL-2 ELISA检测试剂盒(HUMAN IL-2 ELISA KIT，ExCell Bio)检测细胞上清中的IL-2水平，分析CD80融合蛋白在aAPC/T细胞混合培养体系中激活T淋巴细胞的活性。
结果如图8显示CD80-WT、CD80M26、CD80M27、CD80M28能够激活APC/T混合细胞中的淋巴细胞，激活活性与CD80分子相似。
实施例5报告基因法评价融合蛋白的CD28信号通路激活活性
收集FCGR2B/TCR Activator/CHO(简称FCGR2B/CHO)细胞，按1.5×104个细胞/孔加入到白色透明底96孔板(96well white/Clear bottom plate,Thermo)中；向细胞中加入不同浓度的融合蛋白；按2.5×104个细胞/孔加入CD28 Effector Reporter(简称CD28 effector)效应细胞，将96孔板置于37℃5％CO2培养箱中培养5.5h；每孔加入120μL荧光素酶检测试剂(Bright-LumiTM萤火虫萤光素酶报告基因检测试剂盒，碧云天)，室温避光孵育10min后，多功能酶标仪检测化学发光的相对荧光强度值，分析融合蛋白在FCGR2B/CHO细胞+CD28effector细胞体系中介导的CD28激活活性。
结果如图9A-9C所示，图9A-9C显示融合蛋白均能具有CD28激活活性，CD80M28的活性强于CD80-WT，CD80M12、CD80M14、CD80M16、CD80M26、CD80M30、CD80M31 的活性与CD80-WT接近。
实施例6利用CT26皮下种植模型评价CD80融合蛋白的抗肿瘤活性
实验小鼠右侧背部皮下接种5×105CT26细胞，细胞重悬在PBS中(0.1ml/只)。定期观察肿瘤生长情况，待肿瘤生长至平均体积约76mm3时根据肿瘤大小和小鼠体重随机分组给药。分为5组，每组6只小鼠。G1组为CD80M28,2mg/kg，G2组为CD80M28,0.5mg/kg，G3组为CD80M28,0.1mg/kg，G4组为CD80-WTG1,0.5mg/kg，G5组为溶媒对照组，腹腔给药，每周3次，总共给药3次。
在Day22，溶媒对照组有小鼠因肿瘤体积超过3000mm3而安乐处死，因此以Day22的数据作为分析依据。溶媒对照组(G5)在Day22平均肿瘤体积为2189.79mm3，CD80M282mg/kg TIW给药组在Day22平均肿瘤体积为865.57mm3，相对肿瘤抑制率(TGI)为60.47％，相对对照组有显著性差异(p<0.05)。CD80M28 0.5mg/kg TIW给药组在Day22平均肿瘤体积为877.01mm3，相对肿瘤抑制率(TGI)为59.95％，相对对照组有显著性差异(p<0.05)。CD80M28 0.1mg/kg TIW给药组在Day22平均肿瘤体积为1507.25mm3，相对肿瘤抑制率(TGI)为31.17％，相对对照组没有显著性差异。CD80-WTG1 0.5mg/kg TIW给药组在Day22平均肿瘤体积为1337.30mm3，相对肿瘤抑制率(TGI)为38.93％，相对对照组没有显著性差异。各组小鼠体重正常，无异常表现，一般状态良好。
结果如图10所示，CD80M28对肿瘤的生长的抑制具有剂量依赖性，而且活性显著优于同等剂量条件下的野生型CD80-WT。
实施例7利用CT26皮下种植模型评价CD80融合蛋白与Treg抑制剂联合用药的抗肿瘤作用
以抗GITR抗体为例，探讨CD80融合蛋白与Treg抑制剂协同抗肿瘤作用。
用WO2017096189A1文件中公开的抗GITR抗体，联合本申请的融合蛋白，研究二者的协同作用。将小鼠结直肠癌细胞CT26接种于雌性PD1/GITR双人源化小鼠右侧前胁肋部皮下，在肿瘤生长至80mm3左右时分组给药，共6组，每组5只，分别为G1：溶媒组、G2：CD80M28(3mg/kg)组、G3：CD80-WT(3mg/kg)组、G4：抗GITR抗体(10mg/kg)组、G5：CD80M28+抗GITR抗体(3+10mg/kg)组、G6：CD80-WT+抗GITR抗体(3+10mg/kg)组，CD80-WT和CD80M28为静脉给药，每周给药三次，抗GITR抗体腹腔注射给药，每周给药两次，给药两周。每周测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系。计算肿瘤生长抑制率TGITV(％)并进行统计学分析。
结果如图11所示，CD80M28、CD80-WT、抗GITR抗体、CD80M28+抗GITR抗体、CD80-WT+抗GITR抗体的肿瘤生长抑制率分别为86.43％、64.57％、69.98％、89.06％、80.80％，各治疗组肿瘤体积均显著低于溶媒对照组(p<0.05)，显示出显著的抗肿瘤作用，有效地抑制了肿瘤生长。且CD80M28+抗GITR抗体联合治疗组肿瘤体积在所有治疗组中最小，联合治疗组抑制肿瘤活性优于单药组。
实施例8利用CT26皮下种植模型评价融合蛋白与抗PD-1抗体联合用药的抗肿瘤作用
研究本申请的CD80融合蛋白与抗m-PD-1抗体联合用药的协同作用，研究所用的抗m-PD-1抗体购自Bioxcell公司，货号BP0146。
将小鼠结直肠癌细胞CT26接种于雌性Balb/c小鼠右侧前胁肋部皮下，接种后第6天，肿瘤平均体积约为70.9mm3，采用随机区组法将荷瘤鼠分为4组，包括Group 1 PBS组(以下简称Group 1)、Group 2 CD80M28(0.4mg/kg)组(以下简称Group 2)、Group 3抗mPD-1抗体(5mg/kg)组(以下简称Group 3)和Group 4抗mPD-1抗体(5mg/kg)+CD80M28(0.4mg/kg)组(以下简称Group 4)，每组6只。各组给药体积均为10mL/kg，Group 1和Group 2采用尾静脉给药，连续给药1周，每周给药3次；Group 3采用腹腔给药，连续给药2周，每周给药2次，共给药4次；Group 4采用联合给药：连续2周腹腔注射抗mPD-1抗体(5mg/kg)，每周给药2次，以及连续1周尾静脉注射CD80M28(0.4mg/kg)，每周3次，共给药7次。
结果如图12所示，结果显示，给药开始后第18天，Group 1组肿瘤体积为2264.23±514.76mm3。Group 3组的肿瘤体积为1109.87±224.72mm3，肿瘤体积抑制率(TGI)为52.64％，与对照组肿瘤体积比较，无统计学差异(P＞0.05)；Group 2组的肿瘤体积为821.79±387.96mm3，肿瘤体积抑制率(TGI)为65.76％，与对照组肿瘤体积比较，有统计学差异(P＜0.05)；Group 4组的肿瘤体积为140.49±34.58mm3，肿瘤体积抑制率(TGI)为96.83％，与对照组肿瘤体积比较，有显著统计学差异(P＜0.01)。
联合用药组抗肿瘤活性显著优于各个单药组抗肿瘤活性，并且呈现显著的协同作用。
实施例9利用MC38皮下种植模型评价融合蛋白与抗CTLA-4抗体联合用药的抗肿瘤作用
研究本申请的CD80融合蛋白与抗m-CTLA-4抗体联合用药的协同作用，研究所用的抗m-CTLA-4抗体购自Bioxcell公司，货号BP0164，将小鼠结直肠癌细胞MC38接种于雌性C57BL/6小鼠右侧前胁肋部皮下，接种后第7天，肿瘤平均体积约为78mm3，采用随机区组 法将荷瘤鼠分为4组，包括Group 1 PBS组(以下简称Group 1)、Group 2 CD80M28(0.5mg/kg)组(以下简称Group 2)、Group 3 CTLA-4(4mg/kg)组(以下简称Group 3)和Group 4CTLA-4(5mg/kg)+CD80M28(0.4mg/kg)组(以下简称Group 4)，每组6只。CD80M28为静脉给药,给药一次，CTLA-4抗体为腹腔给药，一周两次，总共给药三次。
在实验Day17时，Anti-mCTLA-4 4mg/kg(G3)及CD80M28+Anti-mCTLA-4(G4)联合用药组均有动物皮下肿物消失。至实验Day24，Anti-mCTLA-4 4mg/kg(G3)组有两只动物皮下肿瘤消失，肿瘤消除率为33.3％；CD80M28+Anti-mCTLA-4(G4)联合用药组有五只动物皮下肿瘤消失，肿瘤消除率为83.3％，活性优于单药组。结果参见图13。
实施例10 CD80融合蛋白诱导细胞因子释放
以CD3抗体hOKT3和CD28抗体TGN1412为对照，ELISA方法评价CD80融合蛋白诱导PBMC释放细胞因子的能力。
将不同浓度的CD80融合蛋白，以及CD3抗体hOKT3(Human CD3(humanized OKT3)Antibody-Human IgG4(GMP-grade)，Sino Biological)，CD28抗体TGN1412(自产)和IgG1-Fc蛋白(Human IgG1 Fc Protein，Tag Free，ACROBiosystems)加至96孔细胞培养板，室温过夜风干；用生理盐水清洗2遍后，向每孔加入1×105个健康供体的PBMC细胞(Schbio Biotech)，37℃培养箱中孵育24h后收集上清；用TNF-αELISA检测试剂盒(HUMAN TNF-αELISA KIT，ExCell Bio)和IL-6 ELISA检测试剂盒(Human IL-6 Precoated ELISA Kit，DAKEWE)分别检测上清中的TNF-α和IL-6水平，分析CD80融合蛋白诱导PBMC释放细胞因子的能力。
结果如图14A-14B，结果显示CD80-WT和CD80M28不刺激细胞因子释放，因此有良好的安全性。
实施例11 CD80融合蛋白电荷异质性分析
以CD80M26，CD80M28为例，进行CD80变体融合蛋白电荷异质性分析，在室温下冲洗毛细管，配置混合标准样品溶液和检测样品溶液，CD80M28溶液配制方式为，1％MC50uL，8.6mM尿素50Ul，200mM IDA，10ul，500mM精氨酸7uL，1mg/mL CD80M28，25uL，两性电解质3-10，1.6uL，两性电解质5-8，6.4uL。CD80-WT，CD80M26溶液配制方式为，1％MC 50uL，8.6mM尿素50Ul，200mM IDA，10ul，500mM精氨酸7uL，1mg/mL CD80-WT，CD80M26，25uL，两性电解质3-10，1.2uL，两性电解质5-8，10.8uL进样压力选择2bar，进样时间设置为100s，进行电泳，采集因电荷异质性不同而实现分离的色谱图。 使用面积归一法对酸碱变异体进行定量分析。
结果如图15，结果显示CD80M26，CD80M28的电荷异质性明显小于CD80-WT。电荷异质性的蛋白质质量控制更均一，成药性更好。
实施例12采用HPLC-FLD检测分析不同细胞克隆来源CD80M28样品的唾液酸比例
以CD80M28为例，检测唾液酸比例的影响。唾液酸含量采用HPLC-FLD进行检测分析。将蛋白质供试品用PBS换液5次后测定蛋白浓度，再将蛋白稀释到1mg/mL，按1：1比例将4M乙酸加入到蛋白质供试品中，混匀，80℃孵育2小时。在上述体系中，加入50μL DMB溶液，50℃孵育2.5小时。以糖蛋白中常见的两种唾液酸Neu5Ac(NA)/Neu5Gc(NG)定量标准品配置不同浓度绘制标准曲线，进行Neu5Ac(NA)/Neu5Gc(NG)定量分析。
实施例13利用CT26皮下种植模型评价不同唾液酸修饰程度的CD80M28的抗肿瘤效果
将小鼠结直肠癌细胞CT26接种于雌性BABL/C小鼠右侧前胁肋部皮下，共接种20只，在肿瘤生长至80mm3左右时分组给药，分为7组，每组6只，各给药组分别为，G1组溶媒对照(PBS)，G2组CD80M28-15，G3组CD80M28-1，G4组CD80M28-12，G5组CD80M28-99，G6组CD80M28-5，G7组CD80-WT，每三天给药一次，总共给药3次。
溶媒对照组在结束实验时平均肿瘤体积为2906.96mm3，CD80M28-15治疗组在结束实验的平均肿瘤体积为1979.69mm3，相对肿瘤抑制率(TGI)为31.90％；CD80M28-1治疗组在结束实验的平均肿瘤体积为2536.42mm3，相对肿瘤抑制率(TGI)为12.75％；CD80M28-12治疗组在结束实验的平均肿瘤体积为1683.28mm3，相对肿瘤抑制率(TGI)为42.09％；CD80M28-9治疗组在结束实验的平均肿瘤体积为1612.37mm3，相对肿瘤抑制率(TGI)为44.53％；CD80M28-5治疗组在结束实验的平均肿瘤体积为1940.70mm3，相对肿瘤抑制率 (TGI)为33.24％；CD80-WT治疗组在结束实验的平均肿瘤体积为2805.13mm3，相对肿瘤抑制率(TGI)为3.50％。
结果如图16所示，结果显示，不同唾液酸含量的CD80M28的肿瘤抑制效果均优于野生型CD80-WT，当唾液酸含量大于0.76时，CD80融合蛋白对肿瘤抑制率更为显著。特别是唾液酸含量大于1时，CD80融合蛋白对肿瘤抑制率显著提高。说明改善唾液酸含量有助于提高CD80融合蛋白的肿瘤抑制作用。
实施例14药代动力学动物实验
Sprague Dawley大鼠单次静脉输注给予CD80-WT-G1(CD80-WT)和CD80M28原液后，通过检测血清中受试物的浓度，比较两个受试物的药代动力学特征，为后续试验提供数据支持。
分析方法：
定量范围：50.0～3200ng/mL
试验设计：表1
注：给药方式为静脉输注，输注时间为4min。
样品采集：
给药前0min、给药后5min、30min、2h、4h、8h、24h、48h、72h、96h、120h、168h、240h、336h共计14个时间点，224个样品。
实验结果：表2
Sprague Dawley大鼠单次静脉输注CD80-WT-G1(CD80-WT)和CD80M28原液后各组平均药代动力学参数(Mean±SD，n＝8)


注：Tmax的均值用中位数或中位数和范围(最小值，最大值)表示；采用SPSS软件进行独立样本t
检验分析，P>0.05：组间无统计学差异，P＜0.05：组间有统计学差异。
Sprague Dawley大鼠分别单次静脉输注2mg/kg CD80-WT-G1和2mg/kg CD80M28原液后，与CD80-WT-G1相比，CD80M28的Cmax、AUC(0-t)和AUCinf分别为1.44、26.3和23.8倍，CL为0.0396倍，MRT为2.60倍。采用SPSS软件进行t检验分析，结果显示CD80-WT-G1和CD80M28剂量组间除Kel、t1/2和Tmax无统计学差异，其余参数Cmax、AUC(0-t)、AUCinf、AUCextr、Vd、CL和MRT有显著统计学差异。Sprague Dawley大鼠单次静脉输注CD80-WT-G1和CD80M28原液后各组平均血药浓度-时间变化曲线如图17所示，结果提示CD80M28在大鼠体内暴露量更高，清除更慢，体内持续暴露时间更长。

Claims (70)

  1. CD80蛋白变体，其包含CD80蛋白的功能活性片段或其变体，所述变体与相应的野生型CD80的功能活性片段相比，具有一个或多个氨基酸位点的突变，且所述变体具有以下特性中的一种或多种：
    a)与PD-L1相结合；
    b)与CTLA-4相结合；
    c)与CD28相结合；
    d)激活T淋巴细胞的活性；以及
    e)抑制肿瘤或肿瘤细胞的生长和/或增殖。
  2. 根据权利要求1所述的CD80蛋白变体，其中所述PD-L1为人PD-L1，其中所述CTLA-4为人CTLA-4，其中所述CD28为人CD28。
  3. 根据权利要求1或2所述的CD80蛋白变体，其中所述CD80蛋白的功能活性片段包含CD80蛋白的胞外结构域(ECD)或其片段。
  4. 根据权利要求3所述的CD80蛋白变体，其中所述CD80蛋白的胞外结构域的片段包括CD80蛋白的IgV结构域。
  5. 根据权利要求1-4中任一项所述的CD80蛋白变体，与相应的野生型CD80蛋白胞外结构域或其片段相比，在选自下组的一个或多个氨基酸残基处包含氨基酸突变：N55、N64、N152、N173、N177、N192和N198。
  6. 根据权利要求1-5中任一项所述的CD80蛋白变体，其包含选自下述的任一组氨基酸位置的突变：
    1)N64；
    2)N55和N64；
    3)N55、N64和N152；
    4)N55、N64和N192；
    5)N55、N64和N198；
    6)N55、N64、N152和N198；
    7)N55、N64、N152和N192；
    8)N55、N64、N152、N173、N177和N198；
    9)N55、N64、N152、N173和N198；以及
    10)N55、N64、N152、N177和N198。
  7. 根据权利要求1-6中任一项所述的CD80蛋白变体，其包含选自下组的一个或多个氨基酸突变：N55A/Q、N64A、N152A、N173Q、N177A、N192A和N198A/Q。
  8. 根据权利要求1-7中任一项所述的CD80蛋白变体，其包含选自下组的任一组氨基酸突变：
    1)N64A；
    2)N55A和N64A；
    3)N55A、N64A和N152A；
    4)N55A、N64A和N192A；
    5)N55A、N64A和N198A；
    6)N55A、N64A、N152A和N198A；
    7)N55A、N64A、N152A和N192A；
    8)N55Q、N64A、N152A和N198Q；
    9)N55Q、N64A、N152A、N173Q、N177A和N198Q；
    10)N55Q、N64A、N152A、N173Q和N198Q；以及
    11)N55Q、N64A、N152A、N177A和N198Q。
  9. 根据权利要求1-8中任一项所述的CD80蛋白变体，其包含至少一个第一类氨基酸突变，所述第一类氨基酸突变能够保持或增强所述CD80蛋白变体的稳定性。
  10. 根据权利要求9所述的CD80蛋白变体，其包含在以下一个或多个氨基酸位置处的第一类氨基酸突变：N55、N64、N152、N192和N198。
  11. 根据权利要求9-10中任一项所述的CD80蛋白变体，其包含选自下组的任一组氨基酸位置处的第一类氨基酸突变：
    1)N64；
    2)N55和N64；
    3)N55、N64和N152；
    4)N55、N64和N192；
    5)N55、N64和N198；
    6)N55、N64、N152和N198；以及
    7)N55、N64、N152和N192。
  12. 根据权利要求9-11中任一项所述的CD80蛋白变体，其包含以下一个或多个第一类氨基酸突变：N55A/Q、N64A、N152A、N192A和N198A/Q。
  13. 根据权利要求9-12中任一项所述的蛋白变体，其包含选自下组的任一组第一类氨基酸突变：
    1)N64A；
    2)N55A和N64A；
    3)N55A、N64A和N152A；
    4)N55A、N64A和N192A；
    5)N55A、N64A和N198A；
    6)N55A、N64A、N152A和N198A；
    7)N55A、N64A、N152A和N192A；以及
    8)N55Q、N64A、N152A和N198Q。
  14. 根据权利要求1-13中任一项所述的CD80蛋白变体，其包含至少一个第二类氨基酸突变，所述第二类氨基酸突变能够保持所述CD80蛋白变体的结合活性。
  15. 根据权利要求14所述的CD80蛋白变体，其包含在以下一个或多个位置处的第二类氨基酸突变：N152；N192和N198。
  16. 根据权利要求14-15中任一项所述的CD80蛋白变体，其包含以下一个或多个第二类氨基酸突变：N152A；N192A和N198A/Q。
  17. 根据权利要求14-16中任一项所述的CD80蛋白变体，其包含N55和/或N64位的第二类氨基酸突变。
  18. 根据权利要求14-17中任一项所述的CD80蛋白变体，其包含N55A/Q和/或N64A的第二类氨基酸突变。
  19. 根据权利要求1-18中任一项所述的CD80蛋白变体，其包含至少一个第三类氨基酸突变，所述氨基酸突变能够保持所述CD80蛋白变体的亲和力。
  20. 根据权利要求19所述的CD80蛋白变体，其包含在以下一个或多个位置处的第三类氨基酸突变：N55、N64、N152和N198。
  21. 根据权利要求19-20中任一项所述的CD80蛋白变体，其包含以下一个或多个第三类氨基酸突变：N55A/Q、N64A、N152A和N198A。
  22. 根据权利要求19-21中任一项所述的CD80蛋白变体，其包含N192位的第三类氨基酸突变。
  23. 根据权利要求19-22中任一项所述的CD80蛋白变体，其包含N192A的第三类氨基酸突变。
  24. 根据权利要求1-23中任一项所述的CD80蛋白变体，其包含至少一个第四类氨基酸突变，所述突变能够增强CD80蛋白变体与CD28和/或PD-L1的结合。
  25. 根据权利要求24所述的CD80蛋白变体，其包含N173和/或N177位的第四类氨基酸突变。
  26. 根据权利要求24-25中任一项所述的CD80蛋白变体，其包含N173Q和/或N177A的第四类氨基酸突变。
  27. 根据权利要求1-26中任一项所述的CD80蛋白变体，其中所述氨基酸位置的编号参考SEQ ID NO:1中氨基酸的位置编号确定。
  28. 根据权利要求1-27中任一项所述的CD80蛋白变体，其包含SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24中任一项所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的CD80蛋白变体，其唾液酸含量大于等于约0.7。
  30. 融合蛋白，其包含权利要求1-29中任一项所述的CD80蛋白变体。
  31. 根据权利要求30所述的融合蛋白，其还包含免疫球蛋白Fc区或其变体。
  32. 根据权利要求31所述的融合蛋白，其中所述CD80蛋白变体和所述免疫球蛋白Fc区直接或间接相连。
  33. 根据权利要求31-32中任一项所述的融合蛋白，其中所述免疫球蛋白Fc区包含IgG的Fc区。
  34. 根据权利要求33所述的融合蛋白，其中所述IgG选自下组：IgG1和IgG4。
  35. 根据权利要求31-34中任一项所述的融合蛋白，其中所述CD80蛋白变体位于所述免疫球蛋白Fc区的N端或C端。
  36. 根据权利要求31-35中任一项所述的融合蛋白，其中所述免疫球蛋白Fc区包含选自下组的氨基酸序列：SEQ ID NO:25和SEQ ID NO:26。
  37. 根据权利要求30-36中任一项所述的融合蛋白，其包含SEQ ID NO:27-37中任一项所示的氨基酸序列。
  38. 多肽，其包含权利要求1-29中任一项所述的CD80蛋白变体、或权利要求30-37中任一项所述的融合蛋白。
  39. 分离的一种或多种核酸分子，其编码权利要求1-29中任一项所述的CD80蛋白变体、权利要求30-37中任一项所述的融合蛋白、或权利要求38所述的多肽。
  40. 载体，其包含权利要求39所述的核酸分子。
  41. 宿主细胞，其包含权利要求39所述的核酸分子或权利要求40所述的载体。
  42. 制备权利要求1-29中任一项所述的CD80蛋白变体或权利要求30-37中任一项所述的融合蛋白的方法，所述方法包括在使得所述融合蛋白表达的条件下，培养权利要求41所述的细胞。
  43. 药物组合物，其包含权利要求1-29中任一项所述的CD80蛋白变体、权利要求30-37中任一项所述的融合蛋白、权利要求38所述的多肽、权利要求39所述的核酸分子、权利要求40所述的载体和/或权利要求41所述的宿主细胞，以及任选地药学上可接受的载剂。
  44. 药物组合，其包含权利要求1-19中任一项所述的CD80蛋白变体、权利要求30-37中任一项所述的融合蛋白或权利要求38所述的多肽，以及免疫检查点抑制剂。
  45. 根据权利要求44所述的药物组合，其中所述免疫检查点包含PD-1、PD-L1、GITR和/或CTLA-4。
  46. 根据权利要求44-45中任一项所述的药物组合，其中所述免疫检查点抑制剂包括选自下组中的一种或多种：PD-1抗体、PD-L1抗体、CTLA-4抗体和GITR抗体。
  47. 一种调节受试者的免疫反应的方法，所述方法包括施用权利要求1-29中任一项所述的CD80蛋白变体、权利要求30-37中任一项所述的融合蛋白或权利要求38所述的多肽。
  48. 根据权利要求41所述的方法，其中所述调节受试者的免疫反应的方法包括增加免疫反应。
  49. 根据权利要求47-48中任一项所述的方法，其中与野生型CD80蛋白或其片段相比，所述CD80蛋白变体表现出增加的对PD-L1的结合亲和力。
  50. 根据权利要求47-49中任一项所述的方法，其中与野生型CD80蛋白或其片段相比，所述CD80蛋白变体表现出增加的对CD28的结合亲和力。
  51. 根据权利要求47-50中任一项所述的方法，其中与野生型CD80蛋白或其片段相比，所述CD80蛋白变体表现出增强的激活T淋巴细胞的活性。
  52. 权利要求1-29中任一项所述的CD80蛋白变体、权利要求30-37中任一项所述的融合蛋白、权利要求38所述的多肽、权利要求39所述的核酸分子、权利要求40所述的载体、权利要求41所述的宿主细胞、权利要求43所述的药物组合物、或权利要求44-46中任一项所述的药物组合在制备预防和/或治疗疾病和/或病症的药物中的应用。
  53. 根据权利要求52所述的应用，其中所述疾病和/或病症包括肿瘤。
  54. 根据权利要求53所述的应用，其中所述肿瘤包括实体瘤和/或血液瘤。
  55. 根据权利要求53-54中任一项所述的应用，其中所述肿瘤包括对于中枢性记忆T细胞的增殖有响应的肿瘤。
  56. 根据权利要求53-55中任一项所述的应用，其中所述肿瘤选自下组中的一种或多种：恶性黑色素瘤、乳腺癌、胃癌、肾癌、非小细胞肺癌、结肠癌、直肠癌、头颈部鳞状细胞癌、肝癌、肾癌、间皮瘤膀胱癌、胰腺癌、卵巢癌、子宫内膜癌和淋巴瘤。
  57. 一种预防和/或治疗疾病和/或病症的方法，所述方法包括向有需要的受试者施用权利要求1-29中任一项所述的CD80蛋白变体、权利要求30-37中任一项所述的融合蛋白、权利要求38所述的多肽、权利要求39所述的核酸分子、权利要求40所述的载体、权利要求41所述的宿主细胞、权利要求43所述的药物组合物、或权利要求44-46中任一项所述的药物组合。
  58. 根据权利要求57所述的方法，其中所述疾病和/或病症包括肿瘤。
  59. 根据权利要求58所述的方法，其中所述肿瘤包括实体瘤和/或血液瘤。
  60. 根据权利要求58-59中任一项所述的方法，其中所述肿瘤包括对于中枢性记忆T细胞的增殖有响应的肿瘤。
  61. 根据权利要求58-60中任一项所述的方法，其中所述肿瘤选自下组中的一种或多种：恶性黑色素瘤、乳腺癌、胃癌、肾癌、非小细胞肺癌、结肠癌、直肠癌、头颈部鳞状细胞癌、肝癌、肾癌、间皮瘤膀胱癌、胰腺癌、卵巢癌、子宫内膜癌和淋巴瘤。
  62. 权利要求1-29中任一项所述的CD80蛋白变体、权利要求30-37中任一项所述的融合蛋白、权利要求38所述的多肽、权利要求39所述的核酸分子、权利要求40所述的载体、权利要求41所述的宿主细胞、权利要求43所述的药物组合物、或权利要求44-46中任一项所述的药物组合，其用于预防和/或治疗疾病和/或病症。
  63. 根据权利要求62所述的CD80蛋白变体、融合蛋白、核酸分子、载体、宿主细胞、药物组合物或药物组合，其中所述疾病和/或病症包括肿瘤。
  64. 根据权利要求63所述的CD80蛋白变体、融合蛋白、核酸分子、载体、宿主细胞、药物组合物或药物组合，其中所述肿瘤包括实体瘤和/或血液瘤。
  65. 根据权利要求63-64中任一项所述的CD80蛋白变体、融合蛋白、核酸分子、载体、宿主细胞、药物组合物或药物组合，其中所述肿瘤包括对于中枢性记忆T细胞的增殖有响应的肿瘤。
  66. 根据权利要求63-65中任一项所述的CD80蛋白变体、融合蛋白、核酸分子、载体、宿主细胞、药物组合物或药物组合，其中所述肿瘤选自下组中的一种或多种：恶性黑色素瘤、乳腺癌、胃癌、肾癌、非小细胞肺癌、结肠癌、直肠癌、头颈部鳞状细胞癌、肝癌、肾癌、间皮瘤膀胱癌、胰腺癌、卵巢癌、子宫内膜癌和淋巴瘤。
  67. 检测受试者中中枢性记忆T细胞的频率和/或增殖的方法，所述方法包括施用权利要求1-29中任一项所述的CD80蛋白变体、权利要求30-37中任一项所述的融合蛋白、或权利要求38所述的多肽。
  68. 根据权利要求67所述的方法，其中所述中枢性记忆T细胞是CD95+和CD28+细胞。
  69. 根据权利要求67所述的方法，其中所述中枢性记忆T细胞是CD4+中枢性记忆T细胞和/或CD8+中枢性记忆T细胞。
  70. 权利要求1-29中任一项所述的CD80蛋白变体、权利要求30-37中任一项所述的融合蛋白、权利要求38所述的多肽、权利要求39所述的核酸分子、权利要求40所述的载体、权利要求41所述的宿主细胞、权利要求43所述的药物组合物、或权利要求44-46中任一项所述的药物组合，其用于增强中枢性记忆T细胞的增殖或频率。
PCT/CN2023/085423 2022-04-02 2023-03-31 Cd80蛋白变体及cd80融合蛋白 WO2023186079A1 (zh)

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