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WO2019080872A1 - 阻断pd-1/pd-l1信号传导途径且活化t细胞的融合蛋白及其用途 - Google Patents

阻断pd-1/pd-l1信号传导途径且活化t细胞的融合蛋白及其用途

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Publication number
WO2019080872A1
WO2019080872A1 PCT/CN2018/111652 CN2018111652W WO2019080872A1 WO 2019080872 A1 WO2019080872 A1 WO 2019080872A1 CN 2018111652 W CN2018111652 W CN 2018111652W WO 2019080872 A1 WO2019080872 A1 WO 2019080872A1
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Prior art keywords
fusion protein
antibody
seq
cancer
domain
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PCT/CN2018/111652
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English (en)
French (fr)
Inventor
胡品良
邹敬
洪伟东
何芸
白洁
宋凌云
杨文第
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北京比洋生物技术有限公司
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Publication of WO2019080872A1 publication Critical patent/WO2019080872A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention generally relates to the field of medical biotechnology.
  • the invention relates to the inclusion of (i) a derivative of death-1 (PD-1) antibody and/or a programmed death-1 ligand (PD) -L1)) an antigen-binding fragment of an antibody; (ii) an immunoglobulin Fc domain; and (iii) a fusion protein of the CD80 extracellular domain (ECD), a polynucleotide encoding the fusion protein, comprising the multinuclear A vector for a nucleotide, a host cell comprising the polynucleotide or vector, and the fusion protein is treated, prevented and/or diagnosed in an individual to be activated by the PD-1/PD-L1 signaling pathway and T cell function is regulated Use in inhibiting related diseases.
  • PD-1 derivative of death-1
  • PD programmed death-1 ligand
  • L1 programmed death-1 ligand
  • Immune checkpoint is a type of inhibitory signaling molecule present in the immune system that prevents tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and is involved in maintaining tolerance to autoantigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264).
  • PD-1 Programmed death protein-1
  • PD-1 is an important immunological checkpoint protein and is currently an important target for tumor immunotherapy. PD-1 was first discovered in 1992, and its gene cloning and expression indicated that PD-1 activation can induce programmed cell death of T cells. PD-1 protein was found on activated T cells, B cells and myeloid cells. PD-1 is also inducibly expressed in macrophages, dendritic cells, and monocytes. There is no PD-1 expression on the resting lymphocyte surface.
  • PD-1 is a 55kDa type I transmembrane protein with a cytoplasmic region containing an immunoreceptor tyrosine inhibitory motif and homology to CD28 and CTLA-4.
  • Two cell surface glycoprotein ligands of PD-1 have been identified, Programmed Death Protein Ligand 1 (PD-L1) and Programmed Death Protein Ligand 2 (PD-L2).
  • Ligand expression of PD-1 has been found on many cancer cells, including human lung cancer, ovarian cancer, colon cancer, and various myeloma.
  • PD-1 ligands are highly expressed on the surface of various epithelial cancers, hematological cancers, and other malignant tumor cells.
  • PD-1 ligands such as PD-L1
  • PD-L1 Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD -L1blockade, PNAS, 2002, 99(19): 12293-7).
  • PD-1 functions to limit T cell activation, inhibit T cell proliferation, and increase tolerance to antigen.
  • Upregulation of PD-1 expression on activated lymphocytes can lead to inhibition of acquired or innate immune responses, resulting in tumor infiltrating lymphocytes (including T lymphocytes), although they have tumor antigen specificity, Since the ligand of PD-1 on tumor cells binds to PD-1 on tumor infiltrating lymphocytes to produce a signal that inhibits the activation of tumor infiltrating lymphocytes, tumor cells can escape the killing of tumor cells by the immune system.
  • antibodies that inhibit the binding of PD-1 to PD-1 mainly include anti-PD-1 monoclonal antibodies and anti-PD-L1 monoclonal antibodies, but also products for PD-L2.
  • the more mature anti-PD-1 antibodies are the Nivolumab of BMS, and the Pembrolizumab of Merck.
  • Nawu monoclonal antibody (trade name) ) is a fully humanized IgG4 antibody molecule
  • pemizumab (trade name) ) is a humanized IgG4 antibody molecule.
  • the anti-PD-1 monoclonal antibody binds to PD-1 on T lymphocytes and inhibits the binding of PD-1 to its ligands PD-L1 and PD-L2, thereby promoting T lymphocyte activation, proliferation and immunity.
  • Activated cytokines such as IL-2, and relieve the inhibition of PD-1 on immune surveillance of T lymphocytes with anti-tumor activity.
  • NAV monoclonal antibodies currently approved by the US Food and Drug Administration include: melanoma, non-small cell lung cancer, kidney cancer, head and neck cancer, etc.; indications for pemimab include: head and neck cancer, non- Small cell lung cancer, melanoma, etc.
  • atezolizumab developed by Roche avelumab developed by Merck KGaA and Pfizer, and durvalumab developed by AstraZeneca also showed therapeutic effects on tumors.
  • anti-PD-1 antibodies and anti-PD-L1 antibodies have therapeutic effects on tumors, their average therapeutic efficiency is only about 20%, and the five-year survival rate of lung cancer is only 16%. A significant proportion of cancer patients still have no response to treatment with anti-PD-1 antibodies and anti-PD-L1 antibodies. Therefore, how to improve the effectiveness of cancer treatment is still a difficult problem that needs to be solved in the field of cancer treatment.
  • T cell activation requires dual signal stimulation: the first signal consists of the T cell antigen receptor (TCR) and the antigenic peptide MHC (major histocompatibility complex) molecular complex on antigen presenting cells (APC).
  • TCR T cell antigen receptor
  • APC antigen presenting cells
  • the binding provides that the second signal is provided by the co-stimulatory molecule on the APC in combination with the corresponding ligand on the T cell.
  • the binding of CD28 molecules on the surface of T cells to the corresponding ligands of CDA and CD86 on the APC surface plays an important role.
  • low or no expression of CD80 and CD86 in the tumor microenvironment is one of the important mechanisms for tumor immune evasion.
  • Both CD80 and CD86 are transmembrane glycoproteins and are members of the immunoglobulin superfamily (IgSF).
  • the mature CD80 molecule consists of 254 amino acids with an extracellular domain (ECD) of 208 amino acids, a transmembrane domain of 25 amino acids, and an intracellular domain of 21 amino acids.
  • ECD extracellular domain
  • transmembrane domain is 20 amino acids
  • intracellular domain is 61 amino acids.
  • the extracellular domain of CD80 and CD86 comprises the immunoglobulin V (IgV) region and the immunoglobulin C (IgC) region, and CD80 and CD86 bind to their ligands CD28 and CTLA-4 via the immunoglobulin V (IgV) region.
  • CD80 and CD86 In the case of binding of CD80 and CD86 to CD28, CD80 and CD86 have important regulatory roles for antigen-induced T cell activation, proliferation and effector function production, and are positive regulators; whereas in the case of CD80 and CD86 binding to CTLA-4, CD80 Downregulation of immune response with CD86 is a negative regulator. Therefore, CD80 and CD86 are synergistic stimulating factors for T lymphocyte activation, and play an important role in autoimmune monitoring, humoral immune response, and transplantation response.
  • CD80 is also able to block PD-1/PD-L1 interaction by binding to PD-L1, thereby participating in the activation of the immune system.
  • CD80 protein the effect of CD80 protein on T cell response is complex, and its effects are unpredictable before actual testing (Salomon, B et al., Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation .Annual review of immunology, 2001, 19: 225-252).
  • the inventors have developed a set of fusion proteins that interfere with, inhibit or block the PD-1/PD-L1 signaling pathway and activate T cells, which are capable of inhibiting PD-1/PD-L1 signaling, through intensive research. Routes and ability to induce T cell activation, thereby enabling treatment, prevention, and/or diagnosis of diseases associated with activation of PD-1/PD-L1 signaling pathways and inhibition of T cell function in individuals, particularly Enhance the tumor immune response in individuals who are unresponsive or weakly responding to PD-1 antibodies and/or PD-L1 antibody treatment.
  • the present invention discloses a novel fusion protein that blocks the PD-1/PD-L1 signaling pathway and activates T cells, a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, and the same Host cells of a polynucleotide or vector, and the use of the fusion protein in a subject for the treatment, prevention and/or diagnosis of a disease associated with activation of the PD-1/PD-L1 signaling pathway and inhibition of T cell function.
  • the invention provides a novel fusion protein that inhibits the PD-1/PD-L1 signaling pathway and is capable of inducing T cell activation, comprising (i) derived from anti-PD-1 An antigen-binding fragment of an antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) a CD80 extracellular domain (ECD).
  • the (i) of the fusion protein is a Fab, Fab', F(ab') 2 , Fv, single-chain Fv derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody .
  • the (ii) of the fusion protein is a human immunoglobulin Fc domain.
  • the (iii) of the fusion protein comprises human CD80 ECD.
  • the (i) antigen-binding fragment contained in the fusion protein may be derived from any anti-PD-1 antibody as long as it is an antibody capable of inhibiting or reducing the binding of PD-1 to its ligand, including those known in the art. PD-1 antibody and anti-PD-1 antibody developed in the future.
  • the antigen-binding fragment comprises SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, 15/16, All 6 heavy chain CDRs and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequences of 17/18, 19/20, 21/22, 23/24 and 25/26
  • the antigen-binding fragment comprises SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, 15/16, 17/18 a pair of heavy chain variable region sequence/light chain variable region sequences of 19/20, 21/22, 23/24, and 25/26, or variable with the paired heavy chain variable region sequence/light chain a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; more preferably, the antigen is bound
  • the fragment comprises a heavy chain variable region and a light chain variable region of an anti-PD-1 antibody selected from the group consisting
  • the (i) antigen-binding fragment contained in the fusion protein may also be derived from any anti-PD-L1 antibody as long as it is capable of inhibiting or reducing PD-L1 binding to its receptor (for example, with PD-1 or CD80 (B7-1)
  • the antibodies may be combined with either, including anti-PD-L1 antibodies known in the art and anti-PD-L1 antibodies developed in the future.
  • the antigen-binding fragment comprises all of the contents of the pair of heavy chain variable region sequences/light chain variable region sequences selected from the group consisting of SEQ ID NOs: 27/28, 29/30, and 31/32 6 heavy chain CDRs and light chain CDRs.
  • the antigen-binding fragment comprises or is paired with a pair of heavy chain variable region sequence/light chain variable region sequences selected from the group consisting of SEQ ID NOs: 27/28, 29/30 and 31/32
  • the chain variable region sequence/light chain variable region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity
  • the antigen-binding fragment is Fab, Fab', F(ab') 2 , Fv, single-chain Fv selected from the group consisting of atezolizumab, avelumab and durvalumab.
  • the light chain constant region type in the antigen-binding fragment may be a kappa type or a lambda type, preferably a kappa type.
  • the kappa type light chain constant region amino acid sequence of an exemplary anti-PD-1 antibody is shown in SEQ ID NO:33.
  • the lambda-type light chain constant region amino acid sequence of an exemplary anti-PD-1 antibody is shown in SEQ ID NO:34.
  • the (ii) immunoglobulin Fc domain contained in the fusion protein may be any immunoglobulin Fc domain, in particular, the (ii) is a human immunoglobulin Fc domain.
  • the immunoglobulin Fc domain is the Fc domain of an IgG class antibody, particularly an Fc domain of an IgG 1 subclass, an IgG 2 subclass, an IgG 4 subclass of antibodies.
  • the immunoglobulin Fc domain comprised in the fusion protein of the invention is the Fc domain of an IgG 4 subclass antibody, in particular the Fc domain of a human IgG 4 subclass antibody.
  • the IgG 4 subclass antibody comprises an amino acid substitution at position S228 (EU numbering) in the Fc region, in particular amino acid substitution S228P.
  • the heavy chain constant region amino acid sequence of an exemplary IgG 1 subclass anti-PD-1 antibody is shown in SEQ ID NO:35.
  • the heavy chain constant region amino acid sequence of an exemplary IgG 2 subclass anti-PD-1 antibody is shown in SEQ ID NO:36.
  • the heavy chain constant region amino acid sequence of an exemplary IgG 4 subclass anti-PD-1 antibody is shown in SEQ ID NO:37.
  • the Fc domain amino acid sequence of an exemplary IgG 1 subclass anti-PD-1 antibody is shown in SEQ ID NO:38.
  • the Fc domain amino acid sequence of an exemplary IgG 2 subclass anti-PD-1 antibody is shown in SEQ ID NO:39.
  • the Fc domain amino acid sequence of an exemplary IgG 4 subclass anti-PD-1 antibody is shown in SEQ ID NO:40.
  • the (ii) immunoglobulin Fc domain comprised in the fusion protein is at least 90%, 91%, 92%, 93%, 94 with the amino acid sequence set forth in SEQ ID NO: 38, 39 or 40 %, 95%, 96%, 97%, 98%, 99% or more Fc domains of sequence identity.
  • the (iii) CD80 ECD contained in the fusion protein is part of the extracellular domain of CD80.
  • the CD80 ECD comprises a CD80 immunoglobulin V (IgV) region (CD80-IgV).
  • the CD80 ECD comprises a CD80 immunoglobulin V region and a C region (CD80-IgVIgC).
  • the CD80 ECD is human CD80 ECD, preferably the CD80 ECD comprises human CD80 IgV.
  • the CD80-IgV has the amino acid sequence set forth in SEQ ID NO: 41 or at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 41 Amino acid sequence of 95%, 96%, 97%, 98%, 99% or more identity.
  • the CD80-IgVIgC has the amino acid sequence set forth in SEQ ID NO: 42 or at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 42 Amino acid sequence of 95%, 96%, 97%, 98%, 99% or more identity.
  • the fusion protein further comprises a peptide linker between (i), (ii) and/or (iii); preferably, the peptide linker comprises one or more amino acids, more preferably It comprises at least 5 amino acids, most preferably a peptide linker selected from the group consisting of SEQ ID NOs: 43-71.
  • the fusion protein is in the order of (i), (ii), and (iii) from the N-terminus to the C-terminus; (iii), (i), and (ii); or (iii), (ii) Effectively connected to the order of (i).
  • the fusion protein comprises
  • an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-PD-1 and PD-L1 bispecific antibody a CD80 ECD operably linked at the N-terminus of each heavy chain of the two heavy chains of the antibody
  • a CD80ECD operably linked at the N-terminus of each of the two light chains of the antibody
  • CD80ECD a dimeric form of an immunoglobulin Fc domain operably linked at the C-terminus of CD80 ECD; and an anti-PD operably linked at the C-terminus of the immunoglobulin Fc domain of the dimeric form -1 antibody and/or an antigen-binding fragment of an anti-PD-L1 antibody;
  • the anti-PD-1 antibody is selected from the group consisting of navobizumab, pidilizumab, and pemizumab; the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.
  • the antibody is an IgG class antibody, in particular an IgG 1 subclass, an IgG 2 subclass, an IgG 4 subclass antibody, more particularly an IgG 4 subclass antibody; more preferably, the IgG 4 subclass
  • the antibody comprises an amino acid substitution at position S228 of the Fc domain, more preferably an amino acid substitution S228P; further preferably, the light chain of the antibody is of a kappa type or a lambda type, preferably a kappa type.
  • the fusion protein is a fusion protein comprising the first subunit of the fusion protein of SEQ ID NO: 77 and the second subunit of the fusion protein of SEQ ID NO: 79, hereinafter referred to as fusion protein BY31. 2. It comprises an anti-PD-1 antibody (IgG4, kappa, S228P) and CD80IgV operably linked to the C-terminus of the antibody heavy chain by a peptide linker.
  • IgG4, kappa, S228P anti-PD-1 antibody
  • CD80IgV operably linked to the C-terminus of the antibody heavy chain by a peptide linker.
  • the fusion protein is a fusion protein comprising the first subunit of the fusion protein of SEQ ID NO: 81 and the second subunit of the fusion protein of SEQ ID NO: 83, hereinafter referred to as fusion protein BY31. 3. It comprises an anti-PD-1 antibody (IgG2, ⁇ ) and CD80IgVIgC operably linked to the C-terminus of the antibody heavy chain by a peptide linker.
  • the fusion protein is a fusion protein comprising the first subunit of the fusion protein of SEQ ID NO: 85 and the second subunit of the fusion protein of SEQ ID NO: 87, hereinafter referred to as fusion protein BY31. 7. It comprises an anti-PD-1 antibody (IgG4, kappa, S228P) and a CD80 IgV operably linked to each light chain of the antibody and the N-terminus of each heavy chain by a peptide linker.
  • IgG4, kappa, S228P anti-PD-1 antibody
  • CD80 IgV operably linked to each light chain of the antibody and the N-terminus of each heavy chain by a peptide linker.
  • the fusion protein is a fusion protein comprising the first subunit of the fusion protein of SEQ ID NO: 89 and the second subunit of the fusion protein of SEQ ID NO: 91, hereinafter referred to as fusion protein BY31. 14.
  • the first subunit of the fusion protein comprises an operably linked CD80 IgV, IgG4 CH2 and CH3 domain, a peptide linker, an anti-PD-1 antibody light chain from the N-terminus to the C-terminus, and the second subunit of the fusion protein from N
  • the terminal to C-terminus comprises a heavy chain variable region and a CH1 domain of an anti-PD-1 antibody Fab fragment, and the second subunit and the anti-PD-1 antibody light chain portion of the first subunit of the fusion protein BY31.14 are passed Disulfide bond.
  • the fusion protein is a fusion protein comprising the first subunit of the fusion protein of SEQ ID NO: 93 and the second subunit of the fusion protein of SEQ ID NO: 95, hereinafter referred to as fusion protein BY31.
  • the first subunit of the fusion protein comprises a variable region and a constant region of an anti-PD-1 antibody light chain from the N-terminus to the C-terminus
  • the second subunit of the fusion protein comprising an effective linkage from the N-terminus to the C-terminus CD80IgV, IgG4CH2 and CH3 domains, peptide linkers, heavy chain variable region of anti-PD-1 antibody Fab fragment and CH1 domain, heavy chain variable region of PD-1 antibody Fab fragment in said second subunit
  • the CH1 domain is linked to the first subunit by a disulfide bond.
  • the fusion protein comprises
  • an anti-PD-L1 antibody selected from the group consisting of atezolizumab, avelumab and durvalumab; and operably linked at the C-terminus of each of the two heavy chains of said anti-PD-L1 antibody (optionally, by peptide a CD80 extracellular domain (ECD) operably linked to the adaptor;
  • an anti-PD-L1 antibody selected from the group consisting of atezolizumab, avelumab and durvalumab, operably linked at the N-terminus of each of the two heavy chains of said anti-PD-L1 antibody (optionally, via a peptide linker)
  • CD80ECD a dimeric form of an immunoglobulin Fc domain operably linked at the C-terminus of CD80 ECD; and an anti-PD operably linked at the C-terminus of the immunoglobulin Fc domain of the dimeric form -L1 antibody antigen-binding fragment of atezolizumab, avelumab or durvalumab.
  • the CD80 ECD is CD80IgV or CD80IgVIgC.
  • the fusion protein specifically targets the PD-1/PD-L1 signaling pathway and activates T cells.
  • the fusion protein of the present invention not only binds PD-1 and/or PD-L1 with high affinity, but also binds CD28 constitutively expressed on the surface of T cells with high affinity.
  • the structure of the fusion protein designed by the invention fully ensures the suitable physical spatial distance of the fusion protein to bind to the target, and the fusion protein of the structure does not affect the fusion protein and the target after specifically binding to one of the targets. Specific binding of other molecules in it.
  • the invention further provides a polynucleotide encoding a fusion protein of the invention, a vector comprising a polynucleotide encoding a fusion protein of the invention, preferably an expression vector, most preferably a glutamine synthetase expression vector having a double expression cassette.
  • the invention provides a host cell comprising a polynucleotide or vector of the invention.
  • the host cell is a CHO, HEK293 or NSO cell.
  • the invention also provides a method for producing a fusion protein of the invention comprising the steps of (i) cultivating a host cell of the invention under conditions suitable for expression of a fusion protein of the invention, and (ii) recovering the fusion protein of the invention .
  • the invention provides diagnostic kits and pharmaceutical compositions comprising the fusion proteins of the invention. Further, there is also provided the use of a fusion protein, diagnostic kit or pharmaceutical composition of the invention for the treatment, prevention and/or diagnosis of activation of a PD-1/PD-L1 signaling pathway and inhibition of T cell function Related diseases, particularly for the treatment, prevention and/or diagnosis of cancerous diseases (eg, solid tumors and soft tissue tumors), most particularly for the treatment, prevention and/or diagnosis of melanoma, breast cancer, colon cancer, Esophageal cancer, gastrointestinal stromal tumor (GIST), renal cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, head and neck cancer, stomach cancer, blood Learn about malignant diseases (for example, lymphoma).
  • cancerous diseases eg, solid tumors and soft tissue tumors
  • GIST gastrointestinal stromal tumor
  • renal cancer eg, renal cell carcinoma
  • liver cancer non-small cell lung cancer (NS
  • FIG. 1A, B and C provide schematic diagrams of fusion proteins of the invention, wherein Figure 1A illustrates a schematic representation of the structure of a fusion protein comprising an antigen-binding fragment of an antibody, an immunoglobulin Fc domain and a CD80 ECD from the N-terminus to the C-terminus; A schematic diagram of the structure of a fusion protein comprising a CD80 ECD, an antigen-binding fragment of an antibody, and an immunoglobulin Fc domain from the N-terminus to the C-terminus is illustrated; FIG. 1C illustrates the inclusion of CD80 ECD, an immunoglobulin Fc domain from the N-terminus to the C-terminus and Schematic diagram of the structure of the fusion protein of the antigen-binding fragment of the antibody.
  • Figure 2 shows the results of the fusion protein of the present invention prepared and purified in Example 2 by SDS-PAGE and staining with Coomassie blue in the presence of a reducing agent (5 mM 1,4-dithiothreitol).
  • Lane 1 Protein Molecular Weight Standard Marker
  • Lane 2 Antibody BY18.1
  • Lane 3 Fusion Protein BY31.2
  • Lane 4 Fusion Protein BY31.3
  • Lane 5 Fusion Protein BY31.7
  • Lane 6 Fusion Protein BY31 .14
  • Lane 7 Fusion protein BY31.15.
  • Figure 3A is a graph showing the binding curve of the fusion protein BY31.2 of the present invention prepared and purified in Example 2 to human CD28;
  • Figure 3B exemplifying the fusion protein BY31.2 of the present invention prepared and purified in Example 2 and human PD -L1 binding curve;
  • Figure 3C illustrates the binding curve of the fusion protein BY31.2 of the present invention prepared and purified in Example 2 to human CTLA-4.
  • Figure 4 shows the effect of the fusion protein BY31.2 of the present invention on the body weight of experimental animals in the animal model of Example 6.
  • Figure 5 shows the in vivo anti-tumor effect of the fusion protein BY31.2 of the present invention in the animal model of Example 6.
  • Figure 6 shows the effect of the fusion protein BY31.2 of the present invention on the body weight of experimental animals in the animal model of Example 7.
  • Figure 7 shows a schematic diagram comparing the in vivo anti-tumor effect of the fusion protein BY31.2 of the present invention with anti-PD-L1 mAb Avelumab and anti-PD-1 mAb Opdivo in the animal model of Example 7.
  • the present invention provides fusion proteins and pharmaceutical compositions that interfere with, inhibit or block the PD-1/PD-L1 signaling pathway and activate T cells.
  • the invention also provides a method for producing the fusion protein, and the fusion protein treating, preventing and/or diagnosing a disease associated with activation of a PD-1/PD-L1 signaling pathway and inhibition of T cell function in an individual Use in.
  • PD-1 pathway refers to any intracellular signaling pathway initiated by binding to PD-1, including but not limited to intracellular signaling pathways triggered by PD-1 binding to PD-L1, or PD-1 and The intracellular signaling pathway triggered by PD-L2 binding, or the intracellular signaling pathway triggered by the binding of PD-1 to both PD-L1 and PD-L2.
  • PD-L1 pathway refers to any intracellular signaling pathway initiated by binding to PD-L1, including but not limited to, intracellular signaling pathways triggered by PD-L1 binding to PD-1, or PD-L1 and Intracellular signaling pathway triggered by binding of CD80 (B7-1), or intracellular signaling pathway triggered by binding of PD-L1 to both PD-1 and CD80 (B7-1).
  • interfering means interfering with the interaction between PD-1 and PD-L1; / or (ii) inhibition of at least one biological function of the PD-1/PD-L1 signaling pathway.
  • the "interference”, “inhibition” or “blocking” of the PD-1/PD-L1 signaling pathway resulting from the specific binding of the fusion protein of the invention to PD-1 and/or PD-L1 does not need to be completely disrupted, Inhibit or block.
  • the term "specifically binds” means selective for binding of an antigen or molecule of interest and may be distinguished from unwanted or non-specific interactions.
  • the specific binding can be by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Analysis of agalacto- IgG in rheumatoid arthritis using surface plasmon resonance, Glyco J., 2000, 17, 323-329).
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • affinity molecule X for its partner Y can generally dissociation constant (K D) is represented by the solution, the dissociation constant is the ratio of the dissociation rate constant and association rate constant (k off, respectively, and k on) of.
  • K D dissociation constant
  • association rate constant k off, respectively, and k on
  • Affinity can be measured by common methods known in the art.
  • One specific method for measuring affinity is surface plasmon resonance (SPR).
  • antibody is used herein in its broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), so long as they exhibit the desired antigen binding activity.
  • An antibody can be an intact antibody of any type and subtype (eg, IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA2) (eg, having two full-length light chains and two full-length weights) chain).
  • full antibody full length antibody
  • complete antibody complete antibody
  • intact antibody refers to an antibody having a structure that is substantially similar in structure to the native antibody.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in the antibody molecule in their naturally occurring conformation, which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the lesser of the two types of polypeptide chains present in the antibody molecule in their naturally occurring conformation.
  • the kappa light chain and the lambda light chain refer to two major antibody light chain types.
  • bispecific antibody is an artificial hybrid antibody having two different heavy/light chain pairs and having two different binding sites.
  • Bispecific antibodies can be prepared by a variety of methods, including hybridoma fusion or ligation of Fab' fragments.
  • antigen-binding fragment of an antibody is a portion or portion of an antibody or antibody chain that is less than an intact or fully antibody or antibody chain, which is capable of binding to an antigen or to an intact antibody (ie, from an antigen-binding fragment) Intact antibodies) compete for binding to antigen.
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, single chain Fv.
  • the Fab fragment is a monovalent fragment consisting of the V L , V H , C L and CH1 domains, for example, a Fab fragment can be obtained by papain digestion of a complete antibody. Furthermore, digestion of the complete antibody by pepsin digestion under the disulfide bond of the hinge region produces F(ab') 2 , which is a dimer of Fab' and is a bivalent fragment. F(ab') 2 can be reduced under neutral conditions by disrupting the disulfide bond in the hinge region, thus converting the F(ab') 2 dimer to a Fab' monomer.
  • the Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, see: Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)).
  • the Fv fragment consisting of V L and V H domains of a single arm of an antibody composition.
  • the two domains V L and V H Fv fragment encoded by separate genes but the use of recombinant methods, they may be able to pass these two domains synthetic linker produced as a single protein chain is connected, in the The VL region and the VH region in a single protein chain are paired to form a single-chain Fv.
  • the antibody fragment can be obtained by chemical methods, recombinant DNA methods or protease digestion.
  • immunoglobulin refers to a protein having the structure of a naturally occurring antibody.
  • an IgG-like immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a variable region (VH), also known as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2) And CH3), also known as the heavy chain constant region.
  • VH variable region
  • CH1 variable heavy chain domain
  • CH3 constant domains
  • each immunoglobulin light chain has a variable region (VL), also known as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) A domain, also known as a light chain constant region.
  • VL variable region
  • CL constant light chain
  • the heavy chain of immunoglobulin can belong to one of five categories, called ⁇ (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG) or ⁇ (IgM), some of which can be further divided into sub- Classes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG 3 ), ⁇ 4 (IgG 4 ), ⁇ 1 (IgA 1 ), and ⁇ 2 (IgA 2 ).
  • the light chain of an immunoglobulin can be divided into one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain. Immunoglobulins consist essentially of two Fab molecules and one Fc domain joined by an immunoglobulin hinge region.
  • Fc domain or "Fc region” is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” comprises two or three constant domains, a CH2 domain, a CH3 domain, and an optional CH4 domain.
  • the immunoglobulin Fc domain comprises second and third constant domains (CH2 domain and CH3 domain) derived from two heavy chains of IgG, IgA and IgD class antibodies; or a source comprising The second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of the two heavy chains of the IgM and IgE class antibodies.
  • amino acid residue numbering in the Fc region or heavy chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system (also known as the EU index) described in 1991 is numbered.
  • Human immunoglobulin is an immunoglobulin that possesses an amino acid sequence corresponding to an immunoglobulin produced by a human or human cell or from a human immunoglobulin library or other sequence encoding human immunoglobulin. Source derived.
  • the “percent identity (%)" of the amino acid sequence means that the candidate sequence is aligned with the specific amino acid sequence shown in the present specification and, if necessary, the vacancy is introduced to achieve the maximum percent sequence identity, and no consideration is given.
  • a “signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein that promotes secretion of the protein out of the cell.
  • the mature form of the extracellular protein has no signal sequence that is cleaved during the secretory process.
  • N-terminus refers to the last amino acid at the N-terminus
  • C-terminus refers to the last amino acid at the C-terminus
  • fusion refers to the direct attachment of two or more components by peptide bonds or by one or more peptide linkers.
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom.
  • a host cell is any type of cellular system that can be used to produce a fusion protein of the invention.
  • Host cells include cultured cells, as well as transgenic animals, transgenic plants, or cultured plant tissues or cells within animal tissues.
  • mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse).
  • domesticated animals eg, cows, sheep, cats, dogs, and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits eg, mice and large mouse.
  • rodents eg, mice and large mouse.
  • the individual is a human.
  • treatment refers to the clinical intervention intended to alter the natural course of the disease in an individual being treated. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or mitigating the disease state, and alleviating or improving the prognosis.
  • the fusion proteins of the invention are used to delay disease progression or to slow the progression of the disease.
  • anti-tumor effect refers to a biological effect that can be exhibited by a variety of means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid tumors and liquid tumors.
  • the present invention provides a novel fusion protein comprising (i) an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD).
  • the (i), (ii), and/or (iii) are operably linked by a peptide linker.
  • a fusion protein of the invention is a heterotetrameric glycoprotein consisting of a disulfide-bonded fusion protein first subunit and a fusion protein second subunit.
  • the fusion protein of the invention blocks the PD-1/PD-L1 signaling pathway and activated T cells at the immunological checkpoint.
  • the immunological checkpoint PD-1 pathway blocked by this fusion protein is the signal transduction pathway mediated by PD-1 binding to its ligand.
  • the PD-L1 pathway blocked by this fusion protein is a signaling pathway mediated by the binding of PD-L1 to its receptor. Activation of T cells by this fusion protein is achieved by blocking the immunosuppressive PD-1/PD-L1 pathway and positive regulation of T cells by CD80 ECD.
  • the fusion protein of the invention binds to PD-1 or PD-L1 at a dissociation constant (K D ) of 10 -8 M or less, for example, 10 -9 M to 10 -12 M; Specific binding to CD28 molecule at a dissociation constant (K D ) of 10 -8 M or less, for example, 10 -9 M to 10 -12 M,
  • An antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody contained in the fusion protein of the present invention is capable of specifically binding to PD-1 and/or PD-L1; or with an intact anti-PD-1 antibody and/or Or anti-PD-L1 antibodies compete for binding to PD-1 and/or PD-L1.
  • the antigen binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, single chain Fv.
  • the antigen-binding fragment derived from the anti-PD-1 antibody and/or the anti-PD-L1 antibody contained in the fusion protein of the present invention enables the fusion protein of the present invention to have a high affinity, for example, 10 -8 M or less, preferably in K D 10 -9 M to 10 -12 M of PD-1 and / or specifically binds to PD-L1, PD-1 and thereby blocking binding to a ligand PD-L1 / PD-L2 mediated Signaling pathways and/or blocking signaling pathways mediated by PD-L1 binding to receptor PD-1/CD80 (B7-1).
  • a high affinity for example, 10 -8 M or less, preferably in K D 10 -9 M to 10 -12 M of PD-1 and / or specifically binds to PD-L1, PD-1 and thereby blocking binding to a ligand PD-L1 / PD-L2 mediated Signaling pathways and/or blocking signaling pathways mediated by PD-L1 binding to receptor
  • the antigen-binding fragment of the fusion protein of the invention comprises a sequence substantially identical to the amino acid sequence set forth in Table 1A and/or Table 1B, for example, as shown in Table 1A and/or Table 1B.
  • the heavy chain variable region sequence/light chain variable region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity Sexual sequence.
  • Table 1A Examples of heavy chain variable region and light chain variable region sequences in antigen binding fragments of anti-PD-1 antibodies
  • the antigen-binding fragment of the fusion protein of the invention comprises a SEQ ID NO: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, Complementation of all six heavy chains contained in the paired heavy chain variable region sequence/light chain variable region sequences of 15/16, 17/18, 19/20, 21/22, 23/24 and 25/26 Region (CDR) and light chain CDRs.
  • the antigen-binding fragment of the fusion protein of the invention comprises a pair of heavy chain variable region sequence/light chain variable region sequences selected from the group consisting of SEQ ID NOs: 27/28, 29/30 and 31/32 All 6 heavy chain CDRs and light chain CDRs contained.
  • CDRs in the amino acid sequences of heavy chain variable regions and light chain variable regions are known in the art and can be used to identify particular heavy chain variable regions and/or light chains disclosed herein.
  • the CDRs in the amino acid sequence of the variable region are exemplary well-known techniques that can be used to identify CDR boundaries include, for example, Kabat definition, Chothia definition, and AbM definition. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins., J. Mol. Biol. 273:927 -948 (1997); and Martin AC et al, Modeling antibody hypervariable loops: a combined algorithm, Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989).
  • the anti-PD-1 antibody or the anti-PD-L1 antibody derived from the antigen-binding fragment in the fusion protein of the present invention may be classified into a kappa type or a lambda type, preferably a kappa type, based on the amino acid sequence of the light chain constant region thereof.
  • amino acid sequences of the light chain constant region of the anti-PD-1 antibody are provided herein below in Table 2.
  • the amino acid sequence of the anti-PD-1 antibody or the anti-PD-L1 antibody derived from the antigen-binding fragment of the fusion protein of the present invention based on the heavy chain constant region thereof is preferably an IgG class antibody, particularly an IgG 1 subclass, an IgG 2 subunit.
  • the IgG 4 subclass anti-PD-1 antibody or anti-PD-L1 antibody comprises an amino acid substitution preventing the occurrence of arm-exchange at the S228 position in the Fc region, in particular amino acid substitution S228P.
  • amino acid sequences of the heavy chain constant region of the anti-PD-1 antibody are provided herein below in Table 3.
  • immunoglobulin Fc domain in a fusion protein of the invention comprises all amino acid residues of a naturally occurring immunoglobulin Fc domain or a portion of an amino acid residue comprising a naturally occurring immunoglobulin Fc domain.
  • the immunoglobulin Fc domain provides advantageous pharmacokinetic properties to the fusion proteins of the invention, including but not limited to long serum half-life.
  • the immunoglobulin Fc domain also makes it possible to purify the fusion protein of the present invention by, for example, protein A affinity chromatography.
  • the immunoglobulin Fc domain is typically a dimeric molecule that can be produced by papain digestion or trypsin digestion of intact (full length) immunoglobulin or can be produced recombinantly, comprising a CH2 domain, a CH3 domain, and optionally CH4 domain.
  • the IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain.
  • the immunoglobulin Fc domain has the amino acid sequence set forth in SEQ ID NOs: 38-40 of Table 4 or has at least 90%, 91%, 92% of the amino acid sequence set forth in SEQ ID NOs: 38-40. Amino acid sequence of 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the IgG Fc region may also comprise a peptide sequence obtained after additional sequence modification of SEQ ID NO: 38-40, for example, for SEQ ID NO: 38-40
  • the amino acid residue in the amino acid residue is subjected to one or more amino acid substitutions, deletions or derivatization of the obtained peptide sequence.
  • an amino acid substitution in particular an amino acid substitution S228P, is prevented at the S228 position in the IgG Fc region to prevent arm-exchange.
  • the "extracellular domain of CD80 (ECD)" in a fusion protein of the invention comprises all amino acid residues of a naturally occurring CD80 ECD or a portion of an amino acid residue comprising a naturally occurring CD80 ECD.
  • the CD80 ECD comprises CD80 IgV
  • the CD80 ECD comprises human CD80 IgV
  • the CD80 ECD has the amino acid sequence set forth in SEQ ID NO: 41 or 42 of Table 5.
  • the CD80 ECD may also comprise a peptide sequence obtained after additional sequence modification of SEQ ID NOS: 41 and 42, for example, in SEQ ID NOS: 41 and 42
  • the amino acid residue is subjected to one or more conservative substitutions, deletions or derivatization of the peptide sequence as long as it has substantially the same activity or function as the unmodified peptide.
  • the modified peptide will retain the activity or function associated with the unmodified peptide.
  • the modified peptide typically has an amino acid sequence substantially homologous to the amino acid sequence of the unmodified sequence, for example, having at least 90%, 91%, 92%, 93%, and the amino acid sequence set forth in SEQ ID NO: 41 or 42. Amino acid sequence of 94%, 95%, 96%, 97%, 98%, 99% or more identity.
  • the fusion protein of the present invention will (i) an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) CD80ECD optionally operably linked
  • a "peptide linker” is a peptide of one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein.
  • the peptide linker comprises at least 5 amino acids, preferably comprising selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 43); AKTTPKLEEGEFSEARV (SEQ ID NO: 44); AKTTPKLGG (SEQ ID NO: 45); SAKTTPKLGG ( SEQ ID NO: 46); SAKTTP (SEQ ID NO: 47); RADAAP (SEQ ID NO: 48); RADAAPTVS (SEQ ID NO: 49); RADAAAAGGPGS (SEQ ID NO: 50); RADAAAA (SEQ ID NO: 51) SAKTTPKLEEGEFSEARV (SEQ ID NO: 52); ADAAP (SEQ ID NO: 53); DAAPTVSIFPP (SEQ ID NO: 54); TVAAP (SEQ ID NO: 55); TVAAPSVFIFPP (SEQ ID NO: 56); QPKAAP (SEQ) ID NO: 57); QPKAAPSVTLFPP (SEQ ID NO: 58); AKTTPP (SEQ ID NO
  • fusion proteins comprising (i) an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) a CD80 ECD, in any order, including But not limited to the order of (i), (ii), and (iii) of the fusion protein from the N-terminus to the C-terminus; (iii), (i), and (ii); or (iii), (ii), and The order of i) is effectively connected.
  • the fusion protein comprises an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-PD-1 and PD-L1 bispecific antibody from the N-terminus to the C-terminus; and two of the antibodies A CD80 ECD operably linked to the C-terminus of each heavy chain in the heavy chain.
  • the fusion protein comprises an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-PD-1 and PD-L1 bispecific antibody; each of the two heavy chains of the antibody A CD80 ECD operably linked at the N-terminus of the strand; and a CD80 ECD operably linked at the N-terminus of each of the two light chains of the antibody.
  • the fusion protein comprises a CD80 ECD from the N-terminus to the C-terminus; a dimeric form of an immunoglobulin Fc domain operably linked at the C-terminus of the CD80 ECD; and immunization in the dimeric form
  • the C-terminus of the globulin Fc domain is operably linked to an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody.
  • the fusion proteins of the invention can be obtained, for example, by solid peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production.
  • a polynucleotide encoding a first subunit of the fusion protein and/or a polynucleotide encoding a second subunit of the fusion protein is isolated and inserted into one or more vectors for further propagation in a host cell Cloning and/or expression.
  • the polynucleotide can be easily isolated and sequenced using conventional methods.
  • a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided.
  • Expression vectors can be constructed using methods well known to those of skill in the art.
  • Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • a glutamine synthetase high expression vector having a dual expression cassette is used.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to accomplish this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection, or other conventional techniques.
  • a host cell comprising one or more polynucleotides of the invention.
  • a host cell comprising an expression vector of the invention.
  • the term "host cell” refers to any type of cellular system that can be engineered to produce a fusion protein of the invention.
  • Host cells suitable for replicating and supporting expression of the fusion proteins of the invention are well known in the art. Such cells can be transfected or transduced with a particular expression vector, as desired, and a large number of cells containing the vector can be cultured for inoculating large scale fermenters to obtain a sufficient amount of the fusion protein of the invention for clinical use.
  • Suitable host cells include prokaryotic microorganisms such as E.
  • coli eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like.
  • CHO Chinese hamster ovary cells
  • Mammalian cell lines suitable for suspension culture can be used.
  • Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63, and Sp2/0.
  • the host cell is a CHO, HEK293 or NSO cell.
  • a method of producing a fusion protein of the invention comprising culturing a host cell as provided herein under conditions suitable for expression of the fusion protein, the host cell comprising the encoding The polynucleotide of the fusion protein is ligated and the fusion protein is recovered from the host cell (or host cell culture medium).
  • the fusion protein prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art.
  • the purity of the fusion protein of the present invention can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high performance liquid chromatography, and the like.
  • the physical/chemical properties and/or biological activities of the fusion proteins provided herein can be identified, screened or characterized by a variety of assays known in the art.
  • compositions for example, pharmaceutical compositions comprising a fusion protein as described herein formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the pharmaceutical compositions of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).
  • compositions of the invention may be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (for example, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories.
  • liquid solutions for example, injectable solutions and infusible solutions
  • dispersions or suspensions for example, liposomes, and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • a common preferred composition is in the form of an injectable solution or an infusible solution.
  • a preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection.
  • the fusion protein is administered by intravenous infusion or injection.
  • the fusion protein is administered by intramuscular, intraperitoneal or subcutaneous injection.
  • parenteral administration and “parenteral administration” as used herein mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.
  • compositions should generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, dispersions, liposomes or lyophilized forms.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the fusion protein) in a suitable amount in a suitable solvent, followed by filter sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle containing base dispersion medium and other ingredients.
  • a coating agent such as lecithin or the like can be used.
  • the proper fluidity of the solution can be maintained by the use of surfactants.
  • Prolonged absorption of the injectable compositions can be brought about by the inclusion in the compositions of the compositions which delay the absorption, such as the monostearate and gelatin.
  • the fusion proteins of the invention can be administered orally, for example, orally with an inert diluent or an edible carrier.
  • the fusion proteins of the invention may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets or incorporated directly into the subject's diet.
  • the compound can be incorporated with excipients and in ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, glutinous rice papers It is used in the form of a wafer or the like.
  • Therapeutic compositions can also be administered using medical devices known in the art.
  • compositions of the invention may comprise a "therapeutically effective amount” or a “prophylactically effective amount” of a fusion protein of the invention.
  • “Therapeutically effective amount” means an amount effective to achieve the desired therapeutic result at the desired dosage and for the period of time required.
  • the therapeutically effective amount can vary depending on various factors such as the disease state, the age, sex, and weight of the individual.
  • a therapeutically effective amount is any amount that is toxic or detrimental to a therapeutically beneficial effect.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (eg, a tumor growth rate) of at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%.
  • the ability of the fusion proteins of the invention to inhibit measurable parameters e.g., tumor volume
  • prophylactically effective amount is meant an amount effective to achieve the desired prophylactic result at the desired dosage and for the period of time required. Generally, a prophylactically effective amount is less than a therapeutically effective amount because the prophylactic dose is administered to the subject prior to the earlier stage of the disease or at an earlier stage of the disease.
  • Kits comprising the fusion proteins described herein are also within the scope of the invention.
  • the kit may contain one or more additional elements including, for example, instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
  • the fusion proteins disclosed herein have diagnostic and therapeutic and prophylactic uses in vitro and in vivo.
  • these molecules can be administered to cultured cells in vitro or ex vivo or to a subject, eg, a human subject, to treat, prevent, and/or diagnose a variety of PD-1/PD-L1 signaling pathways.
  • the invention provides for the detection of a biological sample, such as serum, semen or urine or a tissue biopsy sample (eg, from a hyperproliferative or cancerous lesion) in the presence of PD-1, PD-L1, CD28, or CTLA, in vitro or in vivo.
  • a biological sample such as serum, semen or urine or a tissue biopsy sample (eg, from a hyperproliferative or cancerous lesion) in the presence of PD-1, PD-L1, CD28, or CTLA, in vitro or in vivo.
  • the diagnostic method comprises: (i) contacting a sample (and optionally a control sample) with a fusion protein as described herein or administering the fusion protein to a subject and (ii) allowing the interaction to occur.
  • the formation of a complex between the fusion protein and the sample (and optionally the control sample) is detected. Formation of the complex indicates the presence of PD-1, PD-L1, CD28 or CTLA-4 and may indicate the suitability or
  • PD-1 or PD-L1, CD28, CTLA-4 is detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after the treatment interval.
  • Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR-technology (eg, RT-PCR) or in vivo imaging techniques.
  • fusion proteins used in in vivo and in vitro assays are labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound conjugates.
  • Suitable detectable materials include a variety of biologically active enzymes, prosthetic groups, fluorescent materials, luminescent materials, paramagnetic (eg, nuclear magnetic resonance) materials, and radioactive materials.
  • the level and/or distribution of PD-1, PD-L1, CD28, or CTLA-4 is determined in vivo, eg, in a non-invasive manner (eg, by using a suitable imaging technique (eg, positron emission) Tomography (PET) scan) detection of a detectably labeled fusion protein of the invention.
  • a PET reagent eg, 18 F-fluorodeoxyglucose (FDG)
  • FDG F-fluorodeoxyglucose
  • the invention provides a diagnostic kit comprising the fusion protein described herein and instructions for use.
  • the invention relates to the use of a fusion protein in vivo for the treatment or prevention of a disease in need of enhancing T cell activation and immune response in a subject, thereby inhibiting or reducing the growth or emergence, metastasis or associated disease of a related disease such as a cancerous tumor relapse.
  • the fusion protein can be used alone to inhibit the growth of cancerous tumors or prevent their appearance.
  • the fusion protein can be administered in combination with other cancer therapeutic/preventive agents.
  • the combination can be administered in any order or simultaneously.
  • the invention provides a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of a fusion protein described herein.
  • the invention provides a method of preventing the appearance or metastasis or recurrence of tumor cells in a subject, the method comprising administering to the subject a prophylactically effective amount of a fusion protein described herein.
  • cancers treated and/or prevented with a fusion protein include, but are not limited to, solid tumors, hematological cancers (eg, leukemias, lymphomas, myeloma, eg, multiple myeloma), and metastatic lesions.
  • the cancer is a solid tumor.
  • solid tumors include malignant tumors, for example, sarcomas and carcinomas of multiple organ systems, such as invasive lungs, breasts, ovaries, lymphoid, gastrointestinal (eg, colon), anal, genital, and genitourinary tract (eg, Kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (eg, brain, nerve or glial cells), head and neck, skin (eg, melanoma), nasopharynx (eg, differentiated or undifferentiated) Metastatic or locally recurrent nasopharyngeal carcinoma) and those of the pancreas, as well as adenocarcinomas, including malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, and esophageal cancer. Cancer can be in early, intermediate or advanced stages or metastatic cancer.
  • the cancer is selected from the group consisting of melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC) ), ovarian cancer, pancreatic cancer, prostate cancer, head and neck cancer, stomach cancer, hematological malignancies (eg, lymphoma).
  • GIST gastrointestinal stromal tumor
  • renal cancer eg, renal cell carcinoma
  • liver cancer eg, non-small cell lung cancer (NSCLC)
  • ovarian cancer pancreatic cancer
  • prostate cancer head and neck cancer
  • stomach cancer hematological malignancies
  • Example 1 Construction of a high-efficiency expression vector for glutamine synthetase containing a gene of interest
  • nucleotide sequences suitable for expression in Chinese hamster ovarian cancer cells were optimized and commissioned by Shanghai Jierui Biotechnology Co., Ltd. Engineering Co., Ltd. synthesized the nucleotide sequence.
  • the anti-PD-1 antibody produced after expression of the nucleotide sequence is referred to herein as antibody BY18.1.
  • the light chain (BY18.1L) nucleotide sequence of the anti-PD-1 antibody BY18.1 is shown in SEQ ID NO: 72; the light chain (BY18.1L) amino acid sequence of the anti-PD-1 antibody BY18.1 is SEQ ID: NO: 73.
  • the heavy chain (BY18.1H) nucleotide sequence of PD-1 antibody BY18.1 is shown in SEQ ID NO: 74; the heavy chain (BY18.1H) amino acid sequence of anti-PD-1 antibody BY18.1 is SEQ ID NO :75 is shown.
  • the BY18.1L coding nucleotide was digested with XhoI-EcoRI, and the glutamine synthetase high expression vector with double expression cassette (patent authorization number: CN104195173B, obtained from Beijing Biyang Biotechnology Co., Ltd.) was used for XhoI.
  • .1L coding nucleotides of glutamine synthetase high expression vector with double expression cassette were digested with XbaI-SalI, and then ligated into the BY18.1H coding nucleotide double-digested by XbaI-SalI
  • the expression vector was expressed and verified by sequencing, and the anti-PD-1 antibody BY18.1 was obtained.
  • the BY18.1L coding nucleotide can also be ligated into a glutamine synthetase high expression vector having a double expression cassette into which a BY18.1H coding nucleotide has been introduced, and the antibody BY18.1 is expressed and obtained.
  • the heavy chain variable region and light chain variable region sequences of the anti-PD-1 antibody in Table 1A the light chain constant region sequence of the antibody in Table 2, the heavy chain constant region sequence of the antibody in Table 3, and the CD80 cell in Table 5
  • the sequence of the outer domain, and the peptide linker sequence of SEQ ID NO: 43-71 were optimized to be suitable for expression in Chinese hamster ovarian cancer cells (CHO), and were commissioned by Shanghai Jierui Bioengineering Co., Ltd.
  • the first subunit (BY31.2L) nucleotide sequence of the fusion protein BY31.2 ( ⁇ , IgG4) is shown in SEQ ID NO: 76; the first subunit of the fusion protein BY31.2 ( ⁇ , IgG4) (BY31) .2L)
  • the amino acid sequence is set forth in SEQ ID NO:77.
  • the second subunit (BY31.2H) nucleotide sequence of the fusion protein BY31.2 ( ⁇ , IgG4) is shown in SEQ ID NO: 78; the second subunit of the fusion protein BY31.2 ( ⁇ , IgG4) (BY31) .2H)
  • the amino acid sequence is set forth in SEQ ID NO:79.
  • the first subunit (BY31.3L) nucleotide sequence of the fusion protein BY31.3 ( ⁇ , IgG2) is shown in SEQ ID NO: 80; the first subunit of the fusion protein BY31.3 ( ⁇ , IgG2) (BY31) .3L)
  • the amino acid sequence is set forth in SEQ ID NO:81.
  • the second subunit (BY31.3H) nucleotide sequence of the fusion protein BY31.3 ( ⁇ , IgG2) is shown in SEQ ID NO: 82; the second subunit of the fusion protein BY31.3 ( ⁇ , IgG2) (BY31) .3H)
  • the amino acid sequence is set forth in SEQ ID NO:83.
  • the first subunit (BY31.7L) nucleotide sequence of the fusion protein BY31.7 is represented by SEQ ID NO: 84; the first subunit (BY31.7L) amino acid sequence of the fusion protein BY31.7 is SEQ ID NO: 85 is shown.
  • the second subunit (BY31.7H) nucleotide sequence of the fusion protein BY31.7 is represented by SEQ ID NO: 86; the second subunit (BY31.7H) amino acid sequence of the fusion protein BY31.7 is SEQ ID NO: 87 is shown.
  • the nucleotide sequence of the first subunit of the fusion protein BY31.14 (the linker of the PD-1 antibody light chain to the C-terminus of the CD80-Fc fusion, ie, BY31.14L) is shown in SEQ ID NO: 88; the fusion protein BY31
  • the amino acid sequence of the first subunit of .14 (the linker of the PD-1 antibody light chain to the C-terminus of the CD80-Fc fusion, BY31.14L) is set forth in SEQ ID NO:89.
  • the second subunit of the fusion protein BY31.14 (the heavy chain variable region and the CH1 domain of the PD-1 antibody Fab fragment, ie, BY31.14H, and the PD-1 antibody in the first subunit of the fusion protein BY31.14)
  • the light chain portion is linked by a disulfide bond) the nucleotide sequence is set forth in SEQ ID NO: 90;
  • the second subunit of the fusion protein BY31.14 (BY31.14H) has the amino acid sequence set forth in SEQ ID NO:91.
  • the nucleotide sequence of the first subunit of the fusion protein BY31.15 (BY31.15L, ie, the PD-1 antibody light chain) is shown in SEQ ID NO: 92; the first subunit of the fusion protein BY31.15 (BY31.
  • the 15 L) amino acid sequence is set forth in SEQ ID NO:93.
  • the nucleotide sequence of the second subunit of the fusion protein BY31.15 (BY31.15H, ie, the C-terminus of the CD80-Fc fusion and the linker of the heavy chain variable region and the CH1 domain of the PD-1 antibody Fab fragment) is as SEQ ID NO: 94; the second subunit of the fusion protein BY31.15 (BY31.15H, the heavy chain variable region and the CH1 domain of the PD-1 antibody Fab fragment in the second subunit of the fusion protein BY31.15
  • the amino acid sequence is linked to the first subunit of the fusion protein BY31.15 by a disulfide bond as shown in SEQ ID NO:95.
  • Example 1 (1) the BY31.2L, BY31.3L, BY31.7L, BY31.14L and BY31.15L coding nucleotides were ligated to the double expression cassette by XhoI-EcoRI double digestion.
  • High-efficiency expression vector of glutamine synthetase (patent authorization number: CN104195173B, obtained from Beijing Biyang Biotechnology Co., Ltd.); BY31.2H, BY31.3H, BY31.7H, BY31 are further digested by XbaI-SalI.
  • the 14H and BY31.15H coding nucleotides are each cloned into a glutamine synthetase high expression vector having a double expression cassette to which a coding nucleotide of another subunit of the corresponding fusion protein has been ligated; or vice versa.
  • the recombinant vector was sequenced and verified for expression.
  • the expressed fusion proteins were designated as fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15, respectively.
  • PEI Polyethylenimine
  • transient expression gave the antibody BY18.1 as a control.
  • the fusion protein present in the culture supernatant collected in the above Example 2 (1) was purified using a HiTrap MabSelect SuRe 1 ml column (GE Healthcare Life Sciences product, catalog number: 11-0034-93) equilibrated with a pH 7.4 PBS solution. Briefly, a HiTrap MabSelect SuRe 1 ml column was equilibrated with a pH 7.4 PBS solution at a volume of 10 bed volumes at a flow rate of 0.5 ml/min; the culture supernatant collected in the above Example 2 (1) was filtered through a 0.45 ⁇ m filter.
  • the sample was loaded onto a HiTrap MabSelect SuRe 1 ml column equilibrated with a pH 7.4 PBS solution; after loading the supernatant, the column was first washed with a pH 7.4 PBS solution at a flow rate of 0.5 ml/min for 5-10 bed volumes, and This was followed by elution with 100 mM citrate buffer (pH 4.0) at a flow rate of 0.5 ml/min. The elution peak was collected and the protein of interest was present in the elution peak.
  • the purity and molecular weight of the fusion protein were analyzed by SDS-PAGE and staining with Coomassie blue in the presence of a reducing agent (5 mM 1,4-dithiothreitol). The result is shown in Figure 2.
  • the predicted values of the molecular weight theory and the actual measured values are shown in Table 6. Because of the glycosylation of proteins in eukaryotic expression systems, the actual measured molecular weight is slightly higher than the theoretical prediction.
  • Recombinant human CD28 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 50103-M08H), recombinant human PD-L1 (Beijing Baipusis Biotechnology Co., Ltd., catalog number: PD1-H5229), and reorganization Human CTLA-4 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11159-H08H) was diluted to 0.5 ⁇ g/ml, 0.25 ⁇ g/ml and 1.0 ⁇ g/ml and coated with 96-well ELISA plate (Corning, article number :42592).
  • the fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 purified in the above Example 2 (2) were diluted to 2000 ⁇ g/ml, and then serially diluted 3 times, and 15-16 samples were diluted. Gradient, double hole detection for each concentration gradient. 50 ⁇ l of the diluted sample was separately added to the above-mentioned recombinant human CD28 or recombinant human PD-L1-coated 96-well plate, and incubated at 37 ° C for 2 hours.
  • the ELISA results showed that the fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 of the present invention can specifically bind to recombinant human PD-L1 and recombinant human CD28; Recombinant human CTLA-4.
  • the binding curves of the fusion protein BY31.2 to recombinant human CD28, recombinant human PD-L1 and recombinant human CTLA-4 were specifically shown in Figures 3A, 3B and 3C, respectively.
  • the novel fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 constructed by the present invention are capable of specifically binding to human PD-L1, human CD28 and human CTLA- 4.
  • the fusion protein BY31.3 has better binding ability to human CD28, CTLA-4 and PD-L1 than the fusion protein BY31.2 and the fusion protein BY31.7, which may be due to the IgC region in CD80ECD versus CD80 and CD28, CTLA-
  • the combination of 4 and PD-L1 has a certain stabilizing effect.
  • the fusion proteins BY31.14 and BY31.15 bind better to human PD-L1, human CD28 and human CTLA-4 than BY31.2, BY31.3 and BY31.7. It may be that BY31.2, BY31.3 and BY31.7 place the CD80 extracellular domain (ECD) on the N-terminus or C-terminus of the full-length antibody, which has an effect on the binding of human PD-L1, human CD28 and human CTLA-4, respectively. .
  • ECD extracellular domain
  • Example 4 Determination of the affinity of the fusion protein of the present invention for PD-1 using Biacore T100
  • an anti-IgG antibody (GE Healthcare Life Sciences, catalog number: BR-1008-39) was covalently immobilized on a CM5 chip by amide coupling.
  • the CM5 chip was activated with 60 ⁇ l of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 60 ⁇ l of N-hydroxysuccinimide (NHS), followed by 5 ⁇ l of anti- IgG antibody plus 95 ⁇ l of dilution buffer HBST (0.1 M HEPES, 1.5 M NaCl, pH 7.4, plus 0.005% Tween 20) was filtered through a 0.2 um filter, and the anti-IgG antibody was covalently immobilized on the CM5 chip by amide coupling. On top, a capture system of approximately 9,000-14,000 resonance units (RU) is produced. The CM5 chip was blocked with 120 ⁇ l of ethanolamine.
  • the fusion protein of the present invention prepared in Example 2 and the antibody BY18.1 were each diluted to 5 ⁇ g/ml, and the dilution was injected at a flow rate of 10 ⁇ L/min for 2 minutes to prepare 1600 RU of the fusion protein of the present invention prepared in Example 2.
  • antibody BY18.1 was non-covalently captured on the surface of the CM5 chip by the respective Fc regions. The resulting complex was stabilized by cross-linking with EDC/NHS to avoid baseline drift during measurement and regeneration.
  • the antigen PD-1 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 10377-H08H) was prepared to have the following concentration gradients: 7 nM, 22 nm, 66 nM, 200 nM, 600 nM. Binding was measured by injecting each concentration for 180 seconds at a flow rate of 30 ⁇ l/min with a dissociation time of 600 seconds. The surface was regenerated by washing with a 3 M MgCl 2 solution at a flow rate of 10 ⁇ L/min for 30 seconds. Data analysis was performed using BIA evaluation software (BIAevaluation 4.1 software from GE Healthcare Biosciences AB, Sweden), and the affinity data shown in Table 8 below was obtained.
  • the fusion proteins BY31.2, BY31.3 and BY31.7 of the present invention were all capable of binding PD-1 with high affinity, and the affinity (KD) reached 2.37 ⁇ 10 -9 M, 1.01 ⁇ , respectively. 10 -9 M and 4.15 ⁇ 10 -9 M.
  • Antibody BY18.1 binds PD-1 with a KD value of 2.94 x 10 -9 M.
  • the ability of the fusion proteins BY31.14 and BY31.15 to bind PD-1 was weaker than BY31.2, BY31.3 and BY31.7. Possibly because the antigen-binding fragments in the fusion proteins BY31.14 and BY31.15 are Fab structures located at the C-terminus, and the binding effect is reduced compared with the whole antibody.
  • the fusion protein BY31.14 binds PD-1 better than the fusion protein BY31.15, and the KD value can reach the level of 10 -9 M.
  • Example 5 Effect of fusion protein of the present invention on secretion of IL-2 and IFN- ⁇ in mixed lymphocyte reaction (MLR)
  • CD4 + T lymphocytes and dendritic cells were obtained from Beijing Shihe Biotechnology Co., Ltd., and the CD4 + T lymphocytes and dendritic cells (DC) were derived from different healthy individuals.
  • CD4 + T lymphocytes and dendritic cells were plated in 96-well cell culture plates at 1 ⁇ 10 5 cells/well and 1 ⁇ 10 4 cells/well, respectively. The experiment was divided into 6 groups, namely blank control group, BY18.1 group, BY31.2 group, BY31.3 group, BY31.7 group and BY31.14 group, with 6 duplicate wells in each group. Except for the blank control group, the other groups were separately added with antibodies or fusion proteins in the amounts shown in Table 9. Finally, 1640 medium containing 10% fetal calf serum was added to make a final volume of 200 ⁇ l. Incubate at 37 ° C, 5% CO 2 .
  • Example 6 In vivo anti-tumor effect of the fusion protein of the present invention in a humanized mouse model of B-hPD-1
  • This example investigated the in vivo anti-tumor effect of the fusion protein of the present invention using the B-hPD-1 humanized mouse model.
  • B-hPD-1 humanized mouse (obtained from Beijing Baiao Saitu Gene Biotechnology Co., Ltd., product number: B-CM-001) is a type of human PD-1 (hPD-1) knocked into C57BL/6 Mice obtained after the mouse genome. Human PD-1 + cells can be detected in B-hPD-1 humanized mice.
  • 5 ⁇ 10 5 MC38 murine colon cancer cells obtained from ATCC, USA
  • 0.1 mL DMEM medium were inoculated to approximately 18 g, 6-week-old female B-hPD-1 humanized mouse right anterior flank Under the skin.
  • the tumor grew up in the mouse.
  • the tumor-bearing mice were randomly divided into groups of 6 and 3 groups, respectively: PBS solvent control group, fusion protein BY31.2 group (6.0 mg/kg) and antibody BY18.1.
  • the dose (5 mg/kg) of each administration group was based on the dose of the antibody BY18.1 group, and the doses applied to the fusion protein BY31.2 group and the antibody BY18.1 group were equivalent in molar amount.
  • the time of the first administration was set to day 0. All groups were administered intraperitoneally (ip), once every three days, six times in a row, and the experiment was terminated three days after the last administration. Tumor volume and mouse body weight were measured twice a week, and mouse body weight and tumor volume were recorded. At the end of the experiment, the animals were euthanized, the tumor weighed stripping, pictures, calculate tumor growth inhibition rate (T umor G rowth I nhibition% ). The formula used to calculate TGI% is: [1 - (mean of tumor volume change in the drug-administered group / mean change in tumor volume of the PBS solvent control group)] x 100%. The experiment was carried out in Beijing Baiao Saitu Gene Biotechnology Co., Ltd.
  • the mean tumor volume ⁇ standard error of the antibody BY18.1 as a drug control was 739 ⁇ 128, and the TGI% was 46.9%, indicating that the antibody BY18.1 as a drug control specifically binds to B-hPD-1.
  • the hPD-1 + cells in humanized mice exert an anti-tumor effect in vivo.
  • navobizumab is a specific monoclonal antibody directed against human PD-1 molecules that blocks human PD by specifically binding to human PD-1 molecules. -1 molecule-mediated inhibitory biological effects, thereby exerting an anti-tumor effect on human patients expressing PD-1.
  • the effect of the fusion protein BY31.2 on tumor growth was not significantly different from that of the antibody-controlled antibody BY18.1, probably because only the anti-PD-1 antibody moiety was able to function in the fusion protein BY31.2.
  • the human CD80ECD in the fusion protein BY31.2 does not bind to murine CD28, murine CTLA-4 and murine PD-L1 of B-hPD-1 humanized mice; in addition, it may be due to the molecular weight of the fusion protein BY31.2 Greater than the control antibody BY18.1, when administered in an equivalent molar amount, the amount of fusion protein BY31.2 infiltrated into the tumor tissue is also small (the hypertonic environment inside the tumor tissue).
  • Example 7 In vivo anti-tumor effect of the fusion protein of the present invention in a human CD34 + HSC transplanted NSG mouse model
  • This example was inoculated by MDA-MB-231 human triple negative breast cancer cell line (ie, breast cancer cell line negative for estrogen receptor, progesterone receptor and HER-2, obtained from ATCC, USA).
  • MDA-MB-231 human triple negative breast cancer cell line ie, breast cancer cell line negative for estrogen receptor, progesterone receptor and HER-2, obtained from ATCC, USA.
  • the in vivo anti-tumor effect of the fusion protein of the present invention was investigated in a human CD34 + HSC transplanted NSG mouse model.
  • Human CD34 + HSC-transplanted NSG mice are a type of transplanted human CD34 + hematopoietic stem cells (HSC) to immunodeficient NOD/SCID/IL2r- ⁇ null (NSG)
  • HSC hematopoietic stem cells
  • NSG mice are reconstructed from the human immune system to obtain a mouse model that is humanized in vivo immunity.
  • a human tumor cell line ie, a MDA-MB-231 human triple negative breast cancer cell line
  • a human tumor cell line ie, a MDA-MB-231 human triple negative breast cancer cell line
  • a human CD34 + HSC transplanted NSG mouse model ie, a human CD34 + HSC transplanted NSG mouse model
  • Both are humanized mice.
  • Such a mouse which has both a human immune system and a human tumor tissue, can realistically simulate the interaction between the human immune system and the tumor, and is an ideal animal model for evaluating the effectiveness and safety of tumor immunotherapy. .
  • MDA-MB-231 cells were seeded to a body weight of approximately 23 g in an amount of 5 x 10 6 MDA-MB-231 human triple negative breast cancer cells per mouse, and 28-32 weeks old human CD34 + HSC successfully transplanted NSG In female mice, the inoculation site was the right lower back subcutaneous.
  • the tumor-bearing mice were randomly divided into 5 groups of 4 groups, respectively: PBS solvent control group, fusion protein BY31.2 group (5.8 mg/kg), anti-PD-L1 Monoclonal Avelumab group (10mg/kg, prepared by Beijing Shangjian Biotechnology Co., Ltd.) and anti-PD-1 monoclonal antibody Opdivo group (10mg/kg, prepared by Yiqiao Shenzhou Biotechnology Co., Ltd., batch number: MB09MA1201), including fusion protein BY31
  • the molar amount of application of the .2 group was 1/2 application molar amount of the anti-PD-1 mAb Opdivo group and the anti-PD-L1 mAb Avelumab group.
  • the time of the first administration was set to day 0. All groups were administered intraperitoneally, once every 5 days, and continuously for 4 times. Tumor volume and mouse body weight were measured 3 times per week, and mouse body weight and tumor volume were recorded. At the end of the experiment, the animals were euthanized, the tumors were weighed and photographed, and the tumor growth inhibition rate (TGI%) was calculated. The experiment was carried out at Beijing Edmer (IDMO) Biotechnology Co., Ltd.
  • Figure 6 shows the change in body weight of the animals after administration.
  • the body of the fusion protein BY31.2 of the present invention was compared with the body of the PBS solvent control group, and no significant difference was observed (P>0.05), indicating that the fusion protein BY31.2 of the present invention did not have an animal. Obvious toxicity.
  • Figure 7 shows the change in tumor volume of animals over time after administration.
  • the mean tumor volume ⁇ standard error of the PBS solvent control group was 1287.11 ⁇ 184.71 mm 3
  • the anti-PD-L1 monoclonal antibody Avelumab group was 964.25 ⁇ 20.01 mm 3
  • the anti-PD-1 monoclonal antibody Opdivo group was 1354.62 ⁇ 126.65 mm. 3
  • the fusion protein BY31.2 group was 773.14 ⁇ 310.66mm 3 .
  • the tumor inhibition rate (TGI%) of the anti-PD-L1 monoclonal antibody Avelumab group was 25.08%, and the TGI% of the anti-PD-1 monoclonal antibody Opdivo group was -5.24%, compared with the PBS solvent control group.
  • the TGI% is 39.93%.
  • the molar dose of the fusion protein BY31.2 of the present invention is less than the molar dose of the anti-PD-L1 monoclonal antibody and the anti-PD-1 monoclonal antibody in the prior art. In the case of half, tumor growth was also significantly inhibited (P ⁇ 0.05).
  • the tumor volume of one mouse was reduced to only 60 mm 3 on the 25th day, while the tumor volume of the PBS solvent control group was as high as 1287.11 on the 25th day. ⁇ 184.71 mm 3 , indicating that the mouse had a complete response to the tumor (CR, complete response). No complete response mice were observed in the anti-PD-L1 mAb Avelumab group and the anti-PD-1 mAb Opdivo group.
  • the anti-PD-1 mAb Opdivo group had no anti-tumor effect at all in this mouse model.
  • Anti-PD-L1 monoclonal antibody Avelumab has a certain tumor suppressive effect in this mouse model (the tumor volume of the PBS solvent control group is 1287.11 ⁇ 184.71 mm 3 , and the tumor volume of the anti-PD-L1 monoclonal antibody Avemulab group is 964.25 ⁇ 20.01. Mm 3 ), however, the anti-tumor effect of the fusion protein BY31.2 of the present invention was not significant (the tumor volume of the fusion protein BY31.2 group was 773.14 ⁇ 310.66 mm 3 ). This indicates that the fusion protein BY31.2 of the present invention is capable of producing a very significant antitumor effect in a mouse model that mimics the process of interaction between the human immune system and the tumor.

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Abstract

本发明提供了一种阻断PD-1/PD-L1信号传导途径且活化T细胞的融合蛋白，其包含(i)衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)。本发明还提供了编码所述融合蛋白的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、以及所述融合蛋白在个体中治疗、预防和/或诊断与PD-1/PD-L1信号传导途径被活化和T细胞功能受到抑制相关的疾病中的用途。

Description

阻断PD-1/PD-L1信号传导途径且活化T细胞的融合蛋白及其用途 技术领域
本发明总体上涉及医药生物技术领域。具体地，本发明涉及包含(i)衍生自抗程序性死亡蛋白-1(programmed death-1(PD-1))抗体和/或抗程序性死亡蛋白配体1(programmed death-1 ligand(PD-L1))抗体的抗原结合片段；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)的融合蛋白、编码所述融合蛋白的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、以及所述融合蛋白在个体中治疗、预防和/或诊断与PD-1/PD-L1信号传导途径被活化和T细胞功能受到抑制相关的疾病中的用途。
背景技术
免疫检查点(immune checkpoint)是免疫系统中存在的一类抑制性信号分子，通过调节外周组织中免疫反应的持续性和强度避免组织损伤，并参与维持对于自身抗原的耐受(Pardoll DM.,The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer,2012,12(4):252-264)。研究发现，肿瘤细胞能够逃避体内免疫系统而失控增殖的原因之一是利用了免疫检查点的抑制性信号通路，由此抑制了T淋巴细胞活性，使得T淋巴细胞不能有效发挥对肿瘤的杀伤效应(Yao S,Zhu Y和Chen L.，Advances in targeting cell surface signaling molecules for immune modulation.Nat Rev Drug Discov,2013,12(2):130-146)。
程序性死亡蛋白-1(PD-1)是一种重要的免疫检查点蛋白，目前也是肿瘤免疫治疗的一个重要靶标。PD-1在1992年首次被发现，对其基因的克隆和表达表明PD-1活化后能够诱导T细胞程序性死亡。在活化的T细胞、B细胞和髓样细胞上均发现存在PD-1蛋白。PD-1还诱导性地表达于巨噬细胞、树突状细胞以及单核细胞。在静息的淋巴细胞表面无PD-1表达。
PD-1是一种55kDa I型跨膜蛋白，其胞浆区含有一个免疫受体酪氨酸抑制基序，与CD28和CTLA-4具有同源性。已鉴定到PD-1的两种细胞表面糖蛋白配体，分别为程序性死亡蛋白配体1(PD-L1)和程序性死亡蛋白配体2(PD-L2)。已经在许多癌细胞上发现了PD-1的配体表达，包括人肺癌、卵巢癌、结肠癌和多种骨髓瘤。另外，在各类上皮癌、血液癌和其他恶性肿瘤细胞表面上高表达PD-1的配体。在肿瘤患者中，PD-1的配体如PD-L1的表达经常与癌的预后不 良相关(Iwai Y等人，Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1blockade，PNAS，2002，99(19):12293-7)。
PD-1与PD-1的配体的结合对于调节T淋巴细胞活性和维持外周免疫耐受发挥重要作用。PD-1与PD-1的配体结合后可导致T细胞凋亡、免疫无应答、T细胞“耗竭”和分泌IL-10等。因此，PD-1发挥限制T细胞活化、抑制T细胞增殖和提高对抗原的耐受性的功能。活化的淋巴细胞表面PD-1的表达上调能够导致对获得性或者固有的免疫反应的抑制，由此导致了(包括T淋巴细胞在内的)肿瘤浸润性淋巴细胞虽然具有肿瘤抗原特异性，但由于肿瘤细胞上PD-1的配体与肿瘤浸润性淋巴细胞上的PD-1结合产生了抑制肿瘤浸润性淋巴细胞活化的信号，从而肿瘤细胞能够逃避免疫系统对肿瘤细胞的杀伤。
研究表明，这些表达PD-1的肿瘤浸润性淋巴细胞是功能障碍型淋巴细胞，所述淋巴细胞的生物学功能可以通过阻断PD-1与PD-1的配体结合的抗体恢复。目前，抑制PD-1与PD-1的配体结合的抗体主要包括抗PD-1单克隆抗体和抗PD-L1单克隆抗体，但也有针对PD-L2的产品。
当前，研究比较成熟的抗PD-1抗体有百时美施贵宝(BMS)公司的纳武单抗(Nivolumab)和默克(Merck)公司的派姆单抗(Pembrolizumab)。纳武单抗(商品名
Figure PCTCN2018111652-appb-000001
)为完全人源化的IgG4抗体分子，派姆单抗(商品名
Figure PCTCN2018111652-appb-000002
)为人源化IgG4抗体分子。所述抗PD-1单克隆抗体与T淋巴细胞上的PD-1结合后能够抑制PD-1与其配体PD-L1和PD-L2的结合，由此促进T淋巴细胞活化、增殖和产生免疫活化型细胞因子如IL-2，并解除PD-1对具有抗肿瘤活性的T淋巴细胞免疫监视的抑制。美国食品药品监督管理局目前批准的纳武单抗适应症包括：黑素瘤、非小细胞肺癌、肾癌、头颈部肿瘤等；派姆单抗的适应症包括：头颈部肿瘤、非小细胞肺癌、黑素瘤等。关于抗PD-L1抗体，罗氏(Roche)研发的atezolizumab、德国默克(Merck KGaA)和美国辉瑞(Pfizer)合作开发的avelumab、阿斯利康研发的durvalumab也显示了对肿瘤的治疗效果。
虽然抗PD-1抗体、抗PD-L1抗体对肿瘤具有治疗效果，但它们平均的治疗有效率仅为20％左右，肺癌的五年生存率仅16％。仍有相当一部分的肿瘤患者对使用抗PD-1抗体、抗PD-L1抗体的治疗无应答。因此，如何提高肿瘤治疗的有效性仍是目前肿瘤治疗领域迫切需要解决的一个难题。
另一方面，T细胞的激活需要双信号刺激：第一信号由T细胞抗原受体(TCR) 与抗原提呈细胞(APC)上的抗原肽MHC(主要组织相容性复合体)分子复合物结合提供，第二信号由APC上的共刺激分子与T细胞上的相应配体结合提供。在参与T细胞激活的共刺激分子中，T细胞表面CD28分子与APC表面相应配体CD80和CD86的结合具有重要作用。但是，在肿瘤微环境中CD80和CD86低表达或不表达，这也是造成肿瘤免疫逃避的重要机制之一。
CD80和CD86均是跨膜糖蛋白，属免疫球蛋白超家族(IgSF)成员。成熟的CD80分子由254个氨基酸组成，其中胞外结构域(ECD)208个氨基酸、跨膜结构域25个氨基酸和胞内结构域21个氨基酸。类似地，成熟CD86分子由303个氨基酸组成，其中胞外结构域222个氨基酸、跨膜结构域20个氨基酸和胞内结构域61个氨基酸。CD80和CD86的胞外结构域包含免疫球蛋白V(IgV)区和免疫球蛋白C(IgC)区，CD80和CD86通过免疫球蛋白V(IgV)区与其配体CD28和CTLA-4结合。在CD80和CD86与CD28结合的情形，CD80和CD86对于抗原诱导T细胞活化、增殖和效应功能的产生具有重要的调节作用，是正调节因子；而在CD80和CD86与CTLA-4结合的情形，CD80和CD86下调免疫应答，是负性调节因子。因此，CD80和CD86是T淋巴细胞活化时的协同刺激因子，在自身免疫监控、体液免疫应答及移植反应中发挥重要作用。
此外，CD80还能够通过与PD-L1结合来阻断PD-1/PD-L1相互作用，从而参与免疫系统的激活。
综上所述可见，CD80蛋白质对T细胞应答的影响是复杂的，其影响在实际的测试之前是无法预测的(Salomon,B等人，Complexities of CD28/B7:CTLA-4costimulatory pathways in autoimmunity and transplantation.Annual review of immunology，2001，19:225-252)。
由于PD-1/PD-L1信号传导途径被活化和T细胞功能受到抑制都促成了肿瘤的发生和发展，因此，存在研发具有干扰、抑制或阻断PD-1/PD-L1信号传导途径且活化T细胞这两种活性的蛋白的需要。
本发明人通过锐意研究，开发了一组干扰、抑制或阻断PD-1/PD-L1信号传导途径且活化T细胞的融合蛋白，所述融合蛋白能够抑制PD-1/PD-L1信号传导途径并能够诱导T细胞活化，由此能够用于在个体中治疗、预防和/或诊断与PD-1/PD-L1信号传导途径被活化和T细胞功能受到抑制相关的疾病，特别地能够用于在对PD-1抗体和/或PD-L1抗体治疗无反应或弱反应的个体中提高肿瘤免疫应答。
发明概述
本发明公开了一种新型的阻断PD-1/PD-L1信号传导途径且活化T细胞的融合蛋白、编码所述融合蛋白的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、以及所述融合蛋白在个体中治疗、预防和/或诊断与PD-1/PD-L1信号传导途径被活化和T细胞功能受到抑制相关的疾病中的用途。
因此，在一个方面，本发明提供了一种新型的融合蛋白，所述融合蛋白抑制PD-1/PD-L1信号传导途径并能够诱导T细胞活化，其包含(i)衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)。在一个实施方案中，所述融合蛋白的所述(i)是衍生自抗PD-1抗体和/或抗PD-L1抗体的Fab、Fab'、F(ab') 2、Fv、单链Fv。在一个实施方案中，所述融合蛋白的所述(ii)是人免疫球蛋白Fc结构域。在一个实施方案中，所述融合蛋白的所述(iii)包含人CD80ECD。
所述融合蛋白中包含的(i)抗原结合片段可以衍生自任何抗PD-1抗体，只要是能够抑制或减少PD-1与其配体结合的抗体即可，包括现有技术中已知的抗PD-1抗体和将来研发出的抗PD-1抗体。在一个实施方案中，所述抗原结合片段包含选自SEQ ID NO：1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24和25/26的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR，优选地，所述抗原结合片段包含选自SEQ ID NO：1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24和25/26的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；更优选地，所述抗原结合片段包含选自纳武单抗、pidilizumab和派姆单抗的抗PD-1抗体的重链可变区和轻链可变区，特别地，所述抗原结合片段是选自纳武单抗、pidilizumab和派姆单抗的Fab、Fab'、F(ab') 2、Fv、单链Fv。
所述融合蛋白中包含的(i)抗原结合片段也可以衍生自任何抗PD-L1抗体，只要是能够抑制或减少PD-L1与其受体结合(例如与PD-1或CD80(B7-1)或与这两者结合)的抗体即可，包括现有技术中已知的抗PD-L1抗体和将来研发出的抗PD-L1抗体。在一个实施方案中，所述抗原结合片段包含选自SEQ ID NO： 27/28、29/30和31/32的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR。优选地，所述抗原结合片段包含选自SEQ ID NO：27/28、29/30和31/32的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；更优选地，所述抗原结合片段是选自atezolizumab、avelumab和durvalumab的Fab、Fab'、F(ab') 2、Fv、单链Fv。
在一个实施方案中，所述抗原结合片段中的轻链恒定区型别可以是κ型或λ型，优选地是κ型。SEQ ID NO:33中显示了示例性抗PD-1抗体的κ型轻链恒定区氨基酸序列。SEQ ID NO:34中显示了示例性抗PD-1抗体的λ型轻链恒定区氨基酸序列。
所述融合蛋白中包含的(ii)免疫球蛋白Fc结构域可以是任何免疫球蛋白Fc结构域，特别地，所述(ii)是人免疫球蛋白Fc结构域。在一个实施方案中，所述免疫球蛋白Fc结构域是IgG类抗体的Fc结构域，特别地是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体的Fc结构域。在一个优选的实施方案中，包含于本发明融合蛋白中的所述免疫球蛋白Fc结构域是IgG 4亚类抗体的Fc结构域，特别地是人IgG 4亚类抗体的Fc结构域。在一个实施方案中，所述IgG 4亚类抗体在Fc区中第S228位置(EU编号)处包含氨基酸置换，特别是氨基酸置换S228P。SEQ ID NO:35中显示了示例性IgG 1亚类抗PD-1抗体的重链恒定区氨基酸序列。SEQ ID NO:36中显示了示例性IgG 2亚类抗PD-1抗体的重链恒定区氨基酸序列。SEQ ID NO:37中显示了示例性IgG 4亚类抗PD-1抗体的重链恒定区氨基酸序列。SEQ ID NO:38中显示了示例性IgG 1亚类抗PD-1抗体的Fc结构域氨基酸序列。SEQ ID NO:39中显示了示例性IgG 2亚类抗PD-1抗体的Fc结构域氨基酸序列。SEQ ID NO:40中显示了示例性IgG 4亚类抗PD-1抗体的Fc结构域氨基酸序列。在一些实施方案中，融合蛋白中包含的(ii)免疫球蛋白Fc结构域是与SEQ ID NO：38、39或40所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的Fc结构域。
在一个实施方案中，所述融合蛋白中包含的(iii)CD80ECD是CD80的胞外结构域的一部分。在一个实施方案中，所述CD80ECD包含CD80免疫球蛋白V(IgV)区(CD80-IgV)。在一个实施方案中，所述CD80ECD包含CD80免疫球蛋白V区和C区(CD80-IgVIgC)。在一个实施方案中，所述CD80ECD是 人CD80ECD，优选地所述CD80ECD包含人CD80IgV。在一个具体实施方案中，所述CD80-IgV具有SEQ ID NO：41所示的氨基酸序列，或与SEQ ID NO：41的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。在一个实施方案中，所述CD80-IgVIgC具有SEQ ID NO：42所示的氨基酸序列，或与SEQ ID NO：42的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
在一个实施方案中，所述融合蛋白还包含所述(i)、(ii)和/或(iii)之间的肽接头；优选地，所述肽接头包含一个或多个氨基酸，更优选地包含至少5个氨基酸，最优选地包含选自SEQ ID NO：43－71的肽接头。
在一个实施方案中，所述融合蛋白从N端至C端以(i)、(ii)和(iii)的顺序；(iii)、(i)和(ii)的顺序；或者(iii)、(ii)和(i)的顺序有效连接。
在一个实施方案中，所述融合蛋白包含
(a)抗PD-1抗体、抗PD-L1抗体或者抗PD-1和PD-L1双特异性抗体；和在所述抗体的两条重链中的每一重链的C端有效连接的一个CD80ECD；
(b)抗PD-1抗体、抗PD-L1抗体或者抗PD-1和PD-L1双特异性抗体；在所述抗体的两条重链中的每一重链的N端有效连接的一个CD80ECD；和在所述抗体的两条轻链中的每一轻链的N端有效连接的一个CD80ECD；或者
(c)CD80ECD；在CD80ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段；
优选地，所述抗PD-1抗体选自纳武单抗、pidilizumab和派姆单抗；所述抗PD-L1抗体选自atezolizumab、avelumab和durvalumab。优选地，所述抗体是IgG类抗体，特别地是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体，更特别地是IgG 4亚类抗体；还优选地，所述IgG 4亚类抗体在Fc结构域中第S228位置处包含氨基酸置换，更优选地是氨基酸置换S228P；进一步优选地，所述抗体的轻链型别为κ型或λ型，优选地为κ型。
在一个具体实施方案中，所述融合蛋白是包含SEQ ID NO:77的融合蛋白第一亚基和SEQ ID NO:79的融合蛋白第二亚基的融合蛋白，下文中称为融合蛋白BY31.2，其包含抗PD-1抗体(IgG4，κ，S228P)和通过肽接头与抗体重链C端有效连接的CD80IgV。
在一个具体实施方案中，所述融合蛋白是包含SEQ ID NO:81的融合蛋白第一亚基和SEQ ID NO:83的融合蛋白第二亚基的融合蛋白，下文中称为融合蛋白BY31.3，其包含抗PD-1抗体(IgG2，κ)和通过肽接头与抗体重链C端有效连接的CD80IgVIgC。
在一个具体实施方案中，所述融合蛋白是包含SEQ ID NO:85的融合蛋白第一亚基和SEQ ID NO:87的融合蛋白第二亚基的融合蛋白，下文中称为融合蛋白BY31.7，其包含抗PD-1抗体(IgG4，κ，S228P)和通过肽接头与抗体每一轻链和每一重链的N端有效连接的CD80IgV。
在一个具体实施方案中，所述融合蛋白是包含SEQ ID NO:89的融合蛋白第一亚基和SEQ ID NO:91的融合蛋白第二亚基的融合蛋白，下文中称为融合蛋白BY31.14，其中所述融合蛋白第一亚基从N端至C端包含有效连接的CD80IgV、IgG4CH2和CH3结构域、肽接头、抗PD-1抗体轻链，所述融合蛋白第二亚基从N端至C端包含抗PD-1抗体Fab片段的重链可变区和CH1结构域，所述第二亚基与融合蛋白BY31.14第一亚基中的抗PD-1抗体轻链部分通过二硫键连接。
在一个具体实施方案中，所述融合蛋白是包含SEQ ID NO:93的融合蛋白第一亚基和SEQ ID NO:95的融合蛋白第二亚基的融合蛋白，下文中称为融合蛋白BY31.15，其中所述融合蛋白第一亚基从N端至C端包含抗PD-1抗体轻链的可变区和恒定区，所述融合蛋白第二亚基从N端至C端包含有效连接的CD80IgV、IgG4CH2和CH3结构域、肽接头、抗PD-1抗体Fab片段的重链可变区和CH1结构域，所述第二亚基中的PD-1抗体Fab片段的重链可变区和CH1结构域与第一亚基通过二硫键连接。
在一个具体实施方案中，所述融合蛋白包含
(a)一个选自atezolizumab、avelumab和durvalumab的抗PD-L1抗体；和在所述抗PD-L1抗体的两条重链中的每一重链的C端有效连接的(任选地，通过肽接头有效连接的)一个CD80胞外结构域(ECD)；
(b)一个选自atezolizumab、avelumab和durvalumab的抗PD-L1抗体、在所述抗PD-L1抗体的两条重链中的每一重链的N端有效连接的(任选地，通过肽接头有效连接的)一个CD80ECD、和在所述抗PD-L1抗体的两条轻链中的每一轻链的N端有效连接的(任选地，通过肽接头有效连接的)一个CD80ECD；或者
(c)CD80ECD；在CD80ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的衍生自抗PD-L1抗体atezolizumab、avelumab或durvalumab的抗原结合片段。
优选地，所述CD80ECD是CD80IgV或CD80IgVIgC。
在一个实施方案中，所述融合蛋白特异性地靶向PD-1/PD-L1信号传导途径和活化T细胞。本发明的融合蛋白不仅能高亲和性结合PD-1和/或PD-L1，而且也能高亲和性地结合在T细胞表面上组成型表达的CD28。本发明所设计的融合蛋白的结构充分保证了该融合蛋白与其靶标结合的合适物理空间距离，这种结构的融合蛋白与所述靶标中的一种分子特异性结合后不影响该融合蛋白与靶标中其他分子的特异性结合。
本发明还提供了编码本发明融合蛋白的多核苷酸、包含编码本发明融合蛋白的多核苷酸的载体，优选地表达载体，最优选地具有双表达盒的谷氨酰胺合成酶表达载体。在另一个方面，本发明提供了包含本发明多核苷酸或载体的宿主细胞。在一个实施方案中，所述宿主细胞是CHO、HEK293或NSO细胞。本发明也提供了一种用于产生本发明融合蛋白的方法，包括步骤(i)在适于表达本发明融合蛋白的条件下培养本发明的宿主细胞，和(ii)回收本发明的融合蛋白。
在一个方面，本发明提供了包含本发明融合蛋白的诊断试剂盒和药物组合物。进一步地，还提供了本发明的融合蛋白、诊断试剂盒或药物组合物的用途，用于治疗、预防和/或诊断与PD-1/PD-L1信号传导途径被活化和T细胞功能受到抑制相关的疾病，特别地用于治疗、预防和/或诊断癌性疾病(例如，实体瘤和软组织瘤)，最特别地用于治疗、预防和/或诊断黑素瘤、乳腺癌、结肠癌、食管癌、胃肠道间质肿瘤(GIST)、肾癌(例如，肾细胞癌)、肝癌、非小细胞肺癌(NSCLC)、卵巢癌、胰腺癌、前列腺癌、头颈部肿瘤、胃癌、血液学恶性病(例如，淋巴瘤)。
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入本文作为参考。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1A、B和C提供了本发明融合蛋白的示意图，其中图1A例示了从N端至C端包含抗体的抗原结合片段、免疫球蛋白Fc结构域和CD80ECD的融合蛋白的结构示意图；图1B例示了从N端至C端包含CD80ECD、抗体的抗原结合片段和免疫球蛋白Fc结构域的融合蛋白的结构示意图；图1C例示了从N端至C端包含CD80ECD、免疫球蛋白Fc结构域和抗体的抗原结合片段的融合蛋白的结构示意图。
图2：显示了实施例2中制备并纯化的本发明融合蛋白在还原剂(5mM 1,4-二硫苏糖醇)存在下通过SDS-PAGE并用考马斯蓝染色后的结果。泳道1：蛋白分子量标准标志物；泳道2：抗体BY18.1；泳道3：融合蛋白BY31.2；泳道4：融合蛋白BY31.3；泳道5：融合蛋白BY31.7；泳道6：融合蛋白BY31.14；泳道7：融合蛋白BY31.15。
图3A：例示了实施例2中制备并纯化的本发明融合蛋白BY31.2与人CD28的结合曲线；图3B：例示了实施例2中制备并纯化的本发明融合蛋白BY31.2与人PD-L1的结合曲线；图3C：例示了实施例2中制备并纯化的本发明融合蛋白BY31.2与人CTLA-4的结合曲线。
图4：显示了在实施例6的动物模型中本发明的融合蛋白BY31.2对实验动物体重的影响。
图5：显示了在实施例6的动物模型中本发明的融合蛋白BY31.2的体内抗肿瘤作用。
图6：显示了在实施例7的动物模型中本发明的融合蛋白BY31.2对实验动物体重的影响。
图7：显示了在实施例7的动物模型中将本发明的融合蛋白BY31.2与抗PD-L1单抗Avelumab和抗PD-1单抗Opdivo的体内抗肿瘤作用进行比较的示意图。
发明详述
本发明提供了干扰、抑制或阻断PD-1/PD-L1信号传导途径和活化T细胞的融合蛋白和药物组合物。本发明还提供了用于产生该融合蛋白的方法，以及 该融合蛋白在个体中治疗、预防和/或诊断与PD-1/PD-L1信号传导途径被活化和T细胞功能受到抑制相关的疾病中的用途。
除非下文中另外定义，否则本说明书中的术语如本领域通常所用那样使用。
I.定义
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
“PD-1途径”是指任何通过与PD-1结合而引发的细胞内信号传导途径，包括但不限于PD-1与PD-L1结合而引发的细胞内信号传导途径、或PD-1与PD-L2结合而引发的细胞内信号传导途径、或者PD-1与PD-L1和PD-L2这两者结合而引发的细胞内信号传导途径。
“PD-L1途径”是指任何通过与PD-L1结合而引发的细胞内信号传导途径，包括但不限于PD-L1与PD-1结合而引发的细胞内信号传导途径、或PD-L1与CD80(B7-1)结合而引发的细胞内信号传导途径、或者PD-L1与PD-1和CD80(B7-1)这两者结合而引发的细胞内信号传导途径。
本文所用的“干扰”、“抑制”或“阻断”PD-1/PD-L1信号传导途径可以互换使用，是指(i)干扰PD-1和PD-L1之间的相互作用；和/或(ii)导致PD-1/PD-L1信号传导途径的至少一种生物学功能的抑制。由本发明融合蛋白与PD-1和/或PD-L1特异性结合后导致导致的“干扰”、“抑制”或“阻断”PD-1/PD-L1信号传导途径不需要是完全的干扰、抑制或阻断。
如本文所用，术语“特异性结合”意指对抗原或目的分子的结合具有选择性并且可以与不想要的或非特异的相互作用区别。所述特异性结合可以通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其他技术，例如表面等离子体共振(SPR)技术(在BIAcore仪上分析)(Liljeblad等人,Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance，Glyco J.,2000，17,323-329)测量。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由解离常数(K D)代表，解离常数是解离速率常数和缔合速率常数(分别是k off和k on)的比例。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是表面等离子体共振 法(SPR)。
术语“抗体”在本文中以最广意义使用并且包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)，只要它们显示出所需的抗原结合活性即可。抗体可以是任何型和亚型(例如，IgM、IgD、IgG1、IgG2、IgG3、IgG4、IgE、IgA1和IgA2)的完整抗体(例如，具有两个全长的轻链和两个全长的重链)。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指一种抗体，所述抗体具有基本上与天然抗体结构相似的结构。
术语“抗体重链”指以其天然存在构象在抗体分子中存在的两种类型多肽链中的较大者，其在正常情况下决定抗体所属的类别。
术语“抗体轻链”指以其天然存在构象在抗体分子中存在的两种类型多肽链中的较小者。κ轻链和λ轻链指两个主要的抗体轻链型别。
“双特异性抗体”是具有两个不同的重链/轻链对且具有两个不同的结合部位的人工杂合抗体。可以通过多种方法，包括杂交瘤融合或Fab’片段的连接制备双特异抗体。
术语抗体的“抗原结合片段”是比完整或完全抗体或抗体链更少的氨基酸残基的抗体或抗体链的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’) 2、Fv、单链Fv。所述Fab片段是一种由V L、V H、C L和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab') 2，其为Fab’的二聚体，是双价片段。F(ab') 2可以在中性条件下通过破坏铰链区中的二硫键而被还原，因此将F(ab') 2二聚体转化为Fab'单体。Fab'单体基本上是具有铰链区的Fab片段(其它抗体片段的更详细的描述请参见：基础免疫学(Fundamental Immunology),W.E.Paul编辑,Raven Press,N.Y.(1993))。所述Fv片段由抗体单臂的V L和V H结构域组成。另外，虽然Fv片段的两个结构域V L和V H由独立的基因编码，但是使用重组方法，可以将它们通过能够使这两个结构域作为单条蛋白链产生的合成性接头连接，在所述单条蛋白链中V L区和V H区配对以形成单链Fv。可以通过化学方法、重组DNA方法或蛋白酶消化法获得所述抗体片段。
术语“免疫球蛋白”指具有天然存在抗体的结构的蛋白质。例如，IgG类免疫 球蛋白是由二硫键结合的两条轻链和两条重链组成的约150,000道尔顿的异四聚体糖蛋白。从N端至C端，每条免疫球蛋白重链具有一个可变区(VH)，也称作可变重链域或重链可变结构域，随后是三个恒定结构域(CH1、CH2和CH3)，也称作重链恒定区。类似地，从N端至C端，每条免疫球蛋白轻链具有一个可变区(VL)，也称作可变轻链域或轻链可变结构域，随后一个恒定轻链(CL)结构域，也称作轻链恒定区。免疫球蛋白的重链可以归属5个类别之一，称作α(IgA)、δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM)，其中某些类别可以进一步划分成亚类，例如γ 1(IgG1)、γ 2(IgG2)、γ 3(IgG 3)、γ 4(IgG 4)、α 1(IgA 1)和α 2(IgA 2)。免疫球蛋白的轻链可以基于其恒定结构域的氨基酸序列而划分成两种型之一，称作κ和λ。免疫球蛋白基本上由借助免疫球蛋白铰链区连接的两个Fab分子和一个Fc结构域组成。
术语“Fc结构域”或“Fc区”在本文中用来定义免疫球蛋白重链的含有至少一部分恒定区的C端区域。该术语包括天然序列Fc区和变体Fc区。天然的免疫球蛋白“Fc结构域”包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。例如，在天然抗体中，免疫球蛋白Fc结构域包含源自IgG、IgA和IgD类抗体的两条重链的第二和第三恒定结构域(CH2结构域和CH3结构域)；或者包含源自IgM和IgE类抗体的两条重链的第二、第三和第四恒定结构域(CH2结构域、CH3结构域和CH4结构域)。除非本文中另外说明，否则Fc区或重链恒定区中的氨基酸残基编号根据如Kabat等人,Sequences of Proteins of Immunological Interes,第5版,Public Health Service,National Institutes of Health,Bethesda,MD,1991中所述的EU编号体系(也称作EU索引)进行编号。
“人免疫球蛋白”是这样一种免疫球蛋白，其拥有对应于人或人细胞产生的免疫球蛋白的氨基酸序列或从利用人免疫球蛋白库或其他编码人免疫球蛋白的序列的非人来源衍生。
氨基酸序列的“同一性百分数(％)”是指将候选序列与本说明书中所示的具体氨基酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后，并且不考虑任何保守置换作为序列同一性的一部分时，候选序列中与本说明书中所示的具体氨基酸序列的氨基酸残基相同的氨基酸残基百分数。
术语“有效连接”意指指定的各组分处于一种允许它们以预期的方式起作用的关系。
“信号序列”是连接至蛋白质的N-端部分的氨基酸的序列，其促进蛋白质分泌至细胞外。细胞外蛋白质的成熟形式没有信号序列，其在分泌过程期间被切除。
术语“N端”指N端的最末氨基酸，术语“C端”指C端的最末氨基酸。
术语“融合”指将两个或多个组分由肽键直接连接或借助一个或多个肽接头有效连接。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的子代。宿主细胞是可以用来产生本发明融合蛋白的任何类型的细胞系统。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。
术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体是人。
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的融合蛋白用来延缓疾病发展或用来减慢疾病的进展。
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。
II.融合蛋白
本发明提供了一种新型的融合蛋白，其包含(i)衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)。
在一些实施方案中，所述(i)、(ii)和/或(iii)任选地通过肽接头有效连接。
在一些实施方案中，本发明的融合蛋白是由二硫键键合的融合蛋白第一亚基和融合蛋白第二亚基组成的异四聚体糖蛋白。
本发明的融合蛋白阻断免疫检查点PD-1/PD-L1信号传导途径和活化T细胞。该融合蛋白阻断的免疫检查点PD-1途径是PD-1与其配体结合所介导的信 号传导途径。该融合蛋白阻断的PD-L1途径是PD-L1与其受体结合所介导的信号传导途径。该融合蛋白对T细胞的活化是通过阻断免疫抑制性PD-1/PD-L1通路且通过CD80ECD对T细胞的正调节作用实现的。
在一些实施方案中，本发明的融合蛋白以10 -8M或更小、例如以10 -9M至10 -12M的解离常数(K D)与PD-1或PD-L1结合；且以10 -8M或更小、例如以10 -9M至10 -12M的解离常数(K D)与CD28分子特异性结合，
－衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段
本发明融合蛋白中包含的衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段能够特异性结合PD-1和/或PD-L1；或者与完整抗PD-1抗体和/或抗PD-L1抗体竞争结合PD-1和/或PD-L1。所述抗原结合片段包括但不限于Fab、Fab’、F(ab’) 2、Fv、单链Fv。
本发明融合蛋白中包含的衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段使得本发明的融合蛋白能够以高的亲和力，例如以10 -8M或更小、优选地以10 -9M至10 -12M的K D与PD-1和/或PD-L1特异性结合，并由此阻断PD-1与配体PD-L1/PD-L2结合所介导的信号传导途径和/或阻断PD-L1与受体PD-1/CD80(B7-1)结合所介导的信号传导途径。
本文在下表1A中提供了本发明融合蛋白中包含的抗PD-1抗体的抗原结合片段中成对重链可变区(VH)和轻链可变区(VL)的例子。另外，本文在下表1B中提供了本发明融合蛋白中包含的抗PD-L1抗体的抗原结合片段中成对重链可变区(VH)和轻链可变区(VL)的例子。在一些实施方案中，本发明融合蛋白中的抗原结合片段包含与表1A和/或表1B中所示的氨基酸序列基本上同一的序列，例如，与表1A和/或表1B所示的成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
表1A.抗PD-1抗体的抗原结合片段中重链可变区和轻链可变区序列的例子
Figure PCTCN2018111652-appb-000003
Figure PCTCN2018111652-appb-000004
Figure PCTCN2018111652-appb-000005
表1B.抗PD-L1抗体的抗原结合片段中重链可变区和轻链可变区序列的例子
Figure PCTCN2018111652-appb-000006
在一个实施方案中，本发明融合蛋白中的抗原结合片段包含选自SEQ ID NO：1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24和25/26的成对重链可变区序列/轻链可变区序列中所含的全部6个重链互补决定区(CDR)与轻链CDR。在一个实施方案中，本发明融合蛋白中的抗原结合片段包含选自SEQ ID NO：27/28、29/30和31/32的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR。用于鉴定重链可变区与轻链可变区的氨基酸序列中的CDR的方法及技术为本领域中已知的，且可用于鉴定本文公开的特定重链可变区及/或轻链可变区的氨基酸序列中的CDR。可用于鉴定CDR边界的示例性公知技术包括例如Kabat界定法、Chothia界定法以及AbM界定法。参见，例如Kabat,Sequences of Proteins of Immunological Interest,National Institutes of Health,Bethesda,Md.(1991)；Al-Lazikani等人，Standard conformations for the canonical structures of immunoglobulins.，J.Mol.Biol. 273:927-948(1997)；以及Martin AC等人，Modeling antibody hypervariable loops:a combined algorithm，Proc.Natl.Acad.Sci.USA 86:9268-9272(1989)。
作为本发明融合蛋白中的抗原结合片段来源的抗PD-1抗体或抗PD-L1抗体可以基于其轻链恒定区的氨基酸序列而划分为κ型或λ型，优选为κ型。
本文在下表2中提供了抗PD-1抗体轻链恒定区的氨基酸序列的例子。
表2.抗PD-1抗体的轻链恒定区序列的例子
Figure PCTCN2018111652-appb-000007
作为本发明融合蛋白中的抗原结合片段来源的抗PD-1抗体或抗PD-L1抗体基于其重链恒定区的氨基酸序列优选地是IgG类抗体，特别地是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体，更特别地是IgG 4亚类抗体。优选地，所述IgG 4亚类抗PD-1抗体或抗PD-L1抗体在Fc区中第S228位置处包含防止发生臂交换(arm-exchange)的氨基酸置换，特别地是氨基酸置换S228P。
本文在下表3中提供了抗PD-1抗体重链恒定区的氨基酸序列的例子。
表3.抗PD-1抗体的重链恒定区序列的例子
Figure PCTCN2018111652-appb-000008
－免疫球蛋白Fc结构域
本发明融合蛋白中的“免疫球蛋白Fc结构域”包含天然存在的免疫球蛋白Fc结构域的全部氨基酸残基或包含天然存在的免疫球蛋白Fc结构域的一部分氨基酸残基。免疫球蛋白Fc结构域对本发明的融合蛋白提供有利的药代动力学特性，包括但不限于长血清半寿期。另外，免疫球蛋白Fc结构域还使得通过例如蛋白A亲和层析纯化本发明的融合蛋白成为可能。
免疫球蛋白Fc结构域通常是二聚体分子，可以通过木瓜蛋白酶消化或胰蛋白酶消化完整(全长)免疫球蛋白来产生或可以重组产生，其包含CH2结构域、CH3结构域和可选的CH4结构域。
在一个实施方案中，IgG Fc区包含IgG CH2结构域和IgG CH3结构域。优选地，免疫球蛋白Fc结构域具有表4中SEQ ID NO：38－40所示的氨基酸序列或者具有与SEQ ID NO：38－40所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
表4融合蛋白中的免疫球蛋白Fc结构域氨基酸序列的例子
Figure PCTCN2018111652-appb-000009
除了如SEQ ID NO：38－40所定义的序列之外，IgG Fc区还可以包含对SEQ ID NO：38－40进行额外的序列修饰后获得的肽序列，例如对SEQ ID NO：38－40中的氨基酸残基进行一个或多个氨基酸替换、缺失或衍生后获得的肽序列。在一个实施方案中，在IgG Fc区中第S228位置处包含防止发生臂交换(arm-exchange)的氨基酸置换，特别地是氨基酸置换S228P。
－CD80的胞外结构域(ECD)
本发明融合蛋白中的“CD80的胞外结构域(ECD)”包含天然存在的CD80ECD的全部氨基酸残基或包含天然存在的CD80ECD的一部分氨基酸残基。在一些实施方案中，所述CD80ECD包含CD80IgV，优选地，所述CD80ECD包含人CD80IgV，更优选地，所述CD80ECD具有表5中SEQ ID NO：41或42所示的氨基酸序列。
表5融合蛋白中的CD80ECD氨基酸序列的例子
Figure PCTCN2018111652-appb-000010
除了如SEQ ID NO：41和42所定义的序列之外，CD80ECD还可以包含对SEQ ID NO：41和42进行额外的序列修饰后获得的肽序列，例如对SEQ ID NO：41和42中的氨基酸残基进行一个或多个保守性替换、缺失或衍生后获得的肽序列，只要具有与未修饰的肽基本上相同的活性或功能即可。经修饰的肽将保留与未修饰肽相关的活性或功能。经修饰的肽通常具有与未修饰序列的氨基酸序列基本上同源的氨基酸序列，例如，与SEQ ID NO：41或42所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
－肽接头
本发明的融合蛋白中将(i)衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段；(ii)免疫球蛋白Fc结构域；和(iii)CD80ECD任选地有效连接的“肽接头”是一个或多个氨基酸、一般约2－20个氨基酸的肽。本领域已知或本文中描述了肽接头。
在一些实施方案中，所述肽接头包含至少5个氨基酸，优选地包含选自AKTTPKLEEGEFSEAR(SEQ ID NO：43)；AKTTPKLEEGEFSEARV(SEQ ID NO：44)；AKTTPKLGG(SEQ ID NO：45)；SAKTTPKLGG(SEQ ID NO：46)；SAKTTP(SEQ ID NO：47)；RADAAP(SEQ ID NO：48)；RADAAPTVS(SEQ ID NO：49)；RADAAAAGGPGS(SEQ ID NO：50)；RADAAAA (SEQ ID NO：51)；SAKTTPKLEEGEFSEARV(SEQ ID NO：52)；ADAAP(SEQ ID NO：53)；DAAPTVSIFPP(SEQ ID NO：54)；TVAAP(SEQ ID NO：55)；TVAAPSVFIFPP(SEQ ID NO：56)；QPKAAP(SEQ ID NO：57)；QPKAAPSVTLFPP(SEQ ID NO：58)；AKTTPP(SEQ ID NO：59)；AKTTPPSVTPLAP(SEQ ID NO：60)；AKTTAP(SEQ ID NO：61)；AKTTAPSVYPLAP(SEQ ID NO：62)；ASTKGP(SEQ ID NO：63)；ASTKGPSVFPLAP(SEQ ID NO：64)；GGGGSGGGGSGGGGS(SEQ ID NO：65)；GENKVEYAPALMALS(SEQ ID NO：66)；GPAKELTPLKEAKVS(SEQ ID NO：67)；GHEAAAVMQVQYPAS(SEQ ID NO：68)；GGGGSGGGGSGGGGSA(SEQ ID NO：69)；GQGTKVEIKRGGSGGGGSG(SEQ ID NO：70)和GQGTLVTVSSGGGGSGGGGS(SEQ ID NO：71)的肽接头。
－融合蛋白
本文提供了按任意顺序包含(i)衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段；(ii)免疫球蛋白Fc结构域；和(iii)CD80ECD的融合蛋白，包括但不限于融合蛋白从N端至C端以(i)、(ii)和(iii)的顺序；(iii)、(i)和(ii)的顺序；或者(iii)、(ii)和(i)的顺序有效连接。
在一个实施方案中，所述融合蛋白从N端至C端包含抗PD-1抗体、抗PD-L1抗体或者抗PD-1和PD-L1双特异性抗体；和在所述抗体的两条重链中的每一重链的C端有效连接的一个CD80ECD。
在另一个实施方案中，所述融合蛋白包含抗PD-1抗体、抗PD-L1抗体或者抗PD-1和PD-L1双特异性抗体；在所述抗体的两条重链中的每一重链的N端有效连接的一个CD80ECD；和在所述抗体的两条轻链中的每一轻链的N端有效连接的一个CD80ECD。
在又一个实施方案中，所述融合蛋白从N端至C端包含CD80ECD；在CD80ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段。
III.本发明的融合蛋白的生产和纯化
本发明的融合蛋白可以例如通过固态肽合成(例如Merrifield固相合成)或重组生产获得。为了重组生产，将编码所述融合蛋白的第一亚基的多核苷酸和/或编码所述融合蛋白的第二亚基的多核苷酸分离并插入一个或多个载体中以便进一步在宿主细胞中克隆和/或表达。使用常规方法，可以轻易地分离所述多核苷酸并将其测序。在一个实施方案中，提供了包含本发明的一种或多种多核苷 酸的载体，优选地表达载体。
可以使用本领域技术人员熟知的方法来构建表达载体。表达载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个优选的实施方案中，使用了具有双表达盒的谷氨酰胺合成酶高效表达载体。
一旦已经制备了用于表达的包含本发明的一种或多种多核苷酸的表达载体，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质体的转染或其他常规技术。
在一个实施方案中，提供了包含一种或多种本发明多核苷酸的宿主细胞。在一些实施方案中，提供了包含本发明表达载体的宿主细胞。如本文所用，术语“宿主细胞”指可以工程化以产生本发明的融合蛋白的任何种类的细胞系统。适于复制和支持本发明的融合蛋白表达的宿主细胞是本领域熟知的。根据需要，这类细胞可以用特定表达载体转染或转导，并且可以培育大量含有载体的细胞用于接种大规模发酵器以获得足够量的本发明融合蛋白用于临床应用。合适的宿主细胞包括原核微生物，如大肠杆菌，真核微生物如丝状真菌或酵母，或各种真核细胞，如中国仓鼠卵巢细胞(CHO)、昆虫细胞等。可以使用适于悬浮培养的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK 293或293F细胞)、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、人肝脏细胞(Hep G2)、CHO细胞、NSO细胞、骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编著，Humana Press，Totowa，NJ)，第255-268页(2003)。在一个优选的实施方案中，所述宿主细胞是CHO、HEK293或NSO细胞。
本领域已知在这些宿主细胞系统中表达外源基因的标准技术。在一个实施方案中，提供了产生本发明的融合蛋白的方法，其中所述方法包括在适于表达所述融合蛋白的条件下培养如本文中提供的宿主细胞，所述宿主细胞包含编码所述融合蛋白的多核苷酸，并且从宿主细胞(或宿主细胞培养基)回收所述融合蛋白。
如本文所述制备的融合蛋白可以通过已知的现有技术如高效液相色谱、离 子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于如净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。
可以通过多种熟知分析方法中的任一种方法确定本发明的融合蛋白的纯度，所述熟知分析方法包括凝胶电泳、高效液相色谱等。可以通过本领域已知的多种测定法，鉴定、筛选或表征本文提供的融合蛋白的物理/化学特性和/或生物学活性。
IV.药物组合物和试剂盒
在另一个方面，本发明提供了组合物，例如，药物组合物，所述组合物包含与可药用载体配制在一起的本文所述的融合蛋白。如本文所用，“可药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。本发明的药物组合物适于静脉内、肌内、皮下、肠胃外、直肠、脊髓或表皮施用(例如，通过注射或输注)。
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、分散体剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。常见的优选组合物处于可注射用溶液剂或可输注溶液剂形式。优选的施用模式是肠胃外(例如，静脉内、皮下、腹腔(i.p.)、肌内)注射。在一个优选实施方案中，通过静脉内输注或注射施用融合蛋白。在另一个优选实施方案中，通过肌内、腹腔或皮下注射施用融合蛋白。
如本文所用的短语“肠胃外施用“和“肠胃外方式施用”意指除了肠施用和局部施用之外的施用模式，通常通过注射施用，并且包括但不限于静脉内、肌内、动脉内、皮内、腹腔、经气管、皮下注射和输注。
治疗性组合物一般应当是无菌的并且在制造和储存条件下稳定。可以将组合物配制为溶液、微乳液、分散体、脂质体或冻干形式。可以通过将活性化合物(即融合蛋白)以要求的量加入适宜的溶剂中，随后过滤消毒，制备无菌可注射溶液剂。通常，通过将所述活性化合物并入无菌溶媒中来制备分散体，所述无菌溶媒含有基础分散介质和其他成分。可以使用包衣剂如卵磷脂等。在分散体的情况下，可以通过使用表面活性剂来维持溶液剂的适宜流动性。可以通过在组合物中包含延迟吸收的物质例如单硬脂酸盐和明胶而引起可注射组合物的延长吸收。
在某些实施方案中，可以口服施用本发明的融合蛋白，例如随惰性稀释剂或可食用载体一起经口施用。本发明的融合蛋白也可以封闭在硬壳或软壳明胶胶囊中、压缩成片剂或直接掺入受试者的膳食中。对于口服治疗施用，所述化合物可以随赋形剂一起掺入并且以可摄取的片剂、颊用片剂、锭剂(troche)、胶囊剂、酏剂、混悬剂、糖浆剂、糯米纸囊剂(wafer)等形式使用。为了通过非肠胃外施用方法施用本发明的融合蛋白，可能需要将所述融合蛋白与防止其失活的材料包衣或随这种材料共施用。还可以用本领域已知的医疗装置施用治疗组合物。
本发明的药物组合物可以包含“治疗有效量”或“预防有效量”的本发明所述融合蛋白。“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。可以根据多种因素如疾病状态、个体的年龄、性别和重量等变动治疗有效量。治疗有效量是任何有毒或有害作用不及治疗有益作用的量。相对于未治疗的受试者，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约60％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价本发明的融合蛋白抑制可度量参数(例如，肿瘤体积)的能力。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在受试者中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量小于治疗有效量。
包含本文所述融合蛋白的试剂盒也处于本发明的范围内。试剂盒可以包含一个或多个其他要素，例如包括：使用说明书；其他试剂，例如标记物或用于偶联的试剂；可药用载体；和用于施用至受试者的装置或其他材料。
V.融合蛋白的用途
本文公开的融合蛋白具有体外和体内诊断用途以及治疗性和预防性用途。例如，可以将这些分子施用至体外或离体的培养细胞或施用至受试者，例如，人类受试者，以治疗、预防和/或诊断多种与PD-1/PD-L1信号传导途径被活化和T细胞功能受到抑制相关的疾病，例如癌症。
在一个方面，本发明提供了体外或体内检测生物样品，例如血清、精液或尿或组织活检样品(例如，来自过度增生性或癌性病灶)中存在PD-1、PD-L1、CD28或CTLA-4的诊断方法。该诊断方法包括：(i)在允许相互作用发生的条件下使样品(和任选地，对照样品)与如本文所述的融合蛋白接触或向受试者施用所 述融合蛋白和(ii)检测所述融合蛋白和样品(和任选地，对照样品)之间复合物的形成。复合物的形成表示存在PD-1、PD-L1、CD28或CTLA-4，并且可以显示本文所述治疗和/或预防的适用性或需求。
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测PD-1或PD-L1、CD28、CTLA-4。可以使用的检测方法包括免疫组织化学、免疫细胞化学、FACS、ELISA测定、PCR-技术(例如，RT-PCR)或体内成像技术。一般地，体内和体外检测方法中所用的融合蛋白直接或间接地用可检测物质标记以促进检测结合的或未结合的结合物。合适的可检测物质包括多种生物学活性酶、辅基、荧光物质、发光物质、顺磁(例如，核磁共振活性)物质和放射性物质。
在一些实施方案中，体内确定PD-1、PD-L1、CD28或CTLA-4的水平和/或分布，例如，以非侵入方式确定(例如，通过使用合适的成像技术(例如，正电子发射断层摄影术(PET)扫描)检测可检测物标记的本发明融合蛋白。在一个实施方案中，例如，通过检测用PET试剂(例如， 18F-氟脱氧葡萄糖(FDG))以可检测方式标记的本发明融合蛋白，体内测定PD-1、PD-L1、CD28或CTLA-4的水平和/或分布。
在一个实施方案中，本发明提供了包含本文所述融合蛋白和使用说明书的诊断试剂盒。
在另一个方面，本发明涉及使用融合蛋白体内用来治疗或预防需要在受试者中增强T细胞活化和免疫应答的疾病，从而抑制或减少相关疾病如癌性肿瘤的生长或出现、转移或复发。可以单独使用融合蛋白以抑制癌性肿瘤的生长或者预防其出现。备选地，融合蛋白可以与其他癌症治疗剂/预防剂组合施用。当本发明的融合蛋白与一种或多种其他药物组合施用时，这种组合可以按任何顺序施用或者同时施用。
因此，在一个实施方案中，本发明提供一种抑制受试者中肿瘤细胞生长的方法，所述方法包括向受试者施用治疗有效量的本文所述的融合蛋白。在另一个实施方案中，本发明提供一种防止受试者中肿瘤细胞出现或者转移或者复发的方法，所述方法包括向受试者施用预防有效量的本文所述的融合蛋白。
在一些实施方案中，用融合蛋白治疗和/或预防的癌包括但不限于实体瘤、血液学癌(例如，白血病、淋巴瘤、骨髓瘤，例如，多发性骨髓瘤)及转移性病灶。在一个实施方案中，癌是实体瘤。实体瘤的例子包括恶性肿瘤，例如，多个器 官系统的肉瘤和癌，如侵袭肺、乳房、卵巢、淋巴样、胃肠道的(例如，结肠)、肛门、生殖器和生殖泌尿道(例如，肾、膀胱上皮、膀胱细胞、前列腺)、咽、CNS(例如，脑、神经的或神经胶质细胞)、头和颈、皮肤(例如，黑素瘤)、鼻咽(例如，分化或未分化的转移性或局部复发性鼻咽癌)和胰的那些癌、以及腺癌，包括恶性肿瘤，如结肠癌、直肠癌、肾细胞癌、肝癌、非小细胞肺癌、小肠癌和食道癌。癌症可以处于早期、中期或晚期或是转移性癌。
在一些实施方案中，癌选自黑素瘤、乳腺癌、结肠癌、食管癌、胃肠道间质肿瘤(GIST)、肾癌(例如，肾细胞癌)、肝癌、非小细胞肺癌(NSCLC)、卵巢癌、胰腺癌、前列腺癌、头颈部肿瘤、胃癌、血液学恶性病(例如，淋巴瘤)。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
实施例1、包含目的基因的谷氨酰胺合成酶高效表达载体的构建
(1)作为对照的抗PD-1抗体BY18.1的编码核苷酸的合成及表达载体的构建
根据International Nonproprietary Name(INN)数据库中编号为9623的纳武单抗的氨基酸序列数据，优化为适合在中国仓鼠卵巢癌细胞(CHO)中表达的下述核苷酸序列，并委托上海捷瑞生物工程有限公司合成该核苷酸序列。所述核苷酸序列表达后产生的抗PD-1抗体在本文中表示为抗体BY18.1。
抗PD-1抗体BY18.1的轻链(BY18.1L)核苷酸序列如SEQ ID NO:72所示；抗PD-1抗体BY18.1的轻链(BY18.1L)氨基酸序列如SEQ ID NO:73所示。PD-1抗体BY18.1的重链(BY18.1H)核苷酸序列如SEQ ID NO:74所示；抗PD-1抗体BY18.1的重链(BY18.1H)氨基酸序列如SEQ ID NO:75所示。
在所述氨基酸序列中，“METDTLLLWVLLLWVPGSTG”为信号肽序列。
上海捷瑞生物工程有限公司合成了上述BY18.1L编码核苷酸序列和BY18.1H编码核苷酸序列。分别将BY18.1L编码核苷酸用XhoI-EcoRI双酶切，将具有双表达盒的谷氨酰胺合成酶高效表达载体(专利授权号：CN104195173B，获自北京比洋生物技术有限公司)用XhoI-EcoRI双酶切，再通过连接酶将经XhoI-EcoRI双酶切的BY18.1L编码核苷酸连接入经XhoI-EcoRI双酶切的具有双表达盒的谷氨酰胺合成酶高效表达载体，获得已导入了BY18.1L编码核苷酸的 具有双表达盒的谷氨酰胺合成酶高效表达载体；然后，分别将BY18.1H编码核苷酸用XbaI-SalI双酶切，将已导入了BY18.1L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体用XbaI-SalI双酶切，再通过连接酶将经XbaI-SalI双酶切的BY18.1H编码核苷酸连接入经XbaI-SalI双酶切的已导入了BY18.1L编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体，由此获得了已导入BY18.1L编码核苷酸和BY18.1H编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体，经测序验证正确后表达，获得抗PD-1抗体BY18.1。
备选地，也可以将BY18.1L编码核苷酸连接入已导入了BY18.1H编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体，表达并获得抗体BY18.1。
(2)包含抗PD-1抗体与CD80胞外结构域的融合蛋白的编码核苷酸的合成及表达载体的构建
根据表1A中抗PD-1抗体的重链可变区和轻链可变区序列、表2中抗体的轻链恒定区序列、表3中抗体的重链恒定区序列、表5中CD80胞外结构域的序列、以及SEQ ID NO：43－71的肽接头序列，优化为适合在中国仓鼠卵巢癌细胞(CHO)中表达的核苷酸序列，并委托上海捷瑞生物工程有限公司合成了SEQ ID NO:76－95中偶数编号所示的多核苷酸序列。
融合蛋白BY31.2(κ,IgG4)的第一亚基(BY31.2L)核苷酸序列如SEQ ID NO:76所示；融合蛋白BY31.2(κ,IgG4)的第一亚基(BY31.2L)氨基酸序列如SEQ ID NO:77所示。融合蛋白BY31.2(κ,IgG4)的第二亚基(BY31.2H)核苷酸序列如SEQ ID NO:78所示；融合蛋白BY31.2(κ,IgG4)的第二亚基(BY31.2H)氨基酸序列如SEQ ID NO:79所示。
融合蛋白BY31.3(κ,IgG2)的第一亚基(BY31.3L)核苷酸序列如SEQ ID NO:80所示；融合蛋白BY31.3(κ,IgG2)的第一亚基(BY31.3L)氨基酸序列如SEQ ID NO:81所示。融合蛋白BY31.3(κ,IgG2)的第二亚基(BY31.3H)核苷酸序列如SEQ ID NO:82所示；融合蛋白BY31.3(κ,IgG2)的第二亚基(BY31.3H)氨基酸序列如SEQ ID NO:83所示。
融合蛋白BY31.7的第一亚基(BY31.7L)核苷酸序列如SEQ ID NO:84所示；融合蛋白BY31.7的第一亚基(BY31.7L)氨基酸序列如SEQ ID NO:85所示。融合蛋白BY31.7的第二亚基(BY31.7H)核苷酸序列如SEQ ID NO:86所示；融合蛋白BY31.7的第二亚基(BY31.7H)氨基酸序列如SEQ ID NO:87所示。
融合蛋白BY31.14的第一亚基(PD-1抗体轻链与CD80-Fc融合物C端的连接物，即，BY31.14L)核苷酸序列如SEQ ID NO:88所示；融合蛋白BY31.14的第一亚基(PD-1抗体轻链与CD80-Fc融合物C端的连接物，BY31.14L)氨基酸序列如SEQ ID NO:89所示。融合蛋白BY31.14的第二亚基(PD-1抗体Fab片段的重链可变区和CH1结构域，即，BY31.14H，与融合蛋白BY31.14第一亚基中的PD-1抗体轻链部分通过二硫键连接)核苷酸序列如SEQ ID NO:90所示；融合蛋白BY31.14的第二亚基(BY31.14H)氨基酸序列如SEQ ID NO:91所示。
融合蛋白BY31.15的第一亚基(BY31.15L，即，PD-1抗体轻链)核苷酸序列如SEQ ID NO:92所示；融合蛋白BY31.15的第一亚基(BY31.15L)氨基酸序列如SEQ ID NO:93所示。融合蛋白BY31.15的第二亚基(BY31.15H，即，CD80-Fc融合物C端与PD-1抗体Fab片段的重链可变区和CH1结构域的连接物)核苷酸序列如SEQ ID NO:94所示；融合蛋白BY31.15的第二亚基(BY31.15H，融合蛋白BY31.15第二亚基中的PD-1抗体Fab片段的重链可变区和CH1结构域与融合蛋白BY31.15第一亚基通过二硫键连接)氨基酸序列如SEQ ID NO:95所示。
在所述氨基酸序列中，“METDTLLLWVLLLWVPGSTG”为信号肽。
使用上述实施例1(1)相同的方法，分别通过XhoI-EcoRI双酶切将BY31.2L、BY31.3L、BY31.7L、BY31.14L和BY31.15L编码核苷酸连接至具有双表达盒的谷氨酰胺合成酶高效表达载体(专利授权号：CN104195173B，获自北京比洋生物技术有限公司)；再通过XbaI-SalI双酶切将BY31.2H、BY31.3H、BY31.7H、BY31.14H和BY31.15H编码核苷酸分别克隆至已连接了相应的融合蛋白另一亚基的编码核苷酸的具有双表达盒的谷氨酰胺合成酶高效表达载体；或者反之亦然。将重组载体测序验证正确后用于表达。所表达的融合蛋白分别命名为融合蛋白BY31.2、BY31.3、BY31.7、BY31.14和BY31.15。
实施例2、融合蛋白的表达和纯化
(1)融合蛋白的瞬时表达
将293F(购自Invitrogen公司，目录号：11625-019)细胞悬浮培养于无血清CD 293培养液(购自Invitrogen公司，目录号：11913-019)中。转染前离心细胞培养物，获得细胞沉淀，用新鲜的无血清CD 293培养液悬浮细胞，将细胞浓度调整为1×10 6个细胞/ml。将细胞悬浮液置于摇瓶中。以100ml细胞悬浮液为 例，分别将实施例1制备的融合蛋白BY31.2、BY31.3、BY31.7、BY31.14和BY31.15的每一种重组表达载体质粒各DNA 250ug和聚乙烯亚胺(polyethylenimine(PEI))(Sigma，目录号：408727)500ug加入1ml无血清CD 293培养液中混匀，室温静置8分钟后，将PEI/DNA混悬液逐滴加入放置有100ml细胞悬浮液的摇瓶中。轻轻混匀，置于5％CO 2、37℃摇床培养(120转/分钟)。5天后收集培养上清。
根据同样的方法，瞬时表达产生作为对照的抗体BY18.1。
(2)表达蛋白的纯化
用pH 7.4PBS溶液平衡的HiTrap MabSelect SuRe 1ml柱(GE Healthcare Life Sciences产品,目录号：11-0034-93)纯化上述实施例2(1)收集的培养上清中存在的融合蛋白。简而言之，用pH 7.4的PBS溶液以10个柱床体积平衡HiTrap MabSelect SuRe 1ml柱，流速为0.5ml/分钟；将上述实施例2(1)收集的培养上清用0.45μm滤膜过滤后，载样至用pH 7.4PBS溶液平衡的HiTrap MabSelect SuRe 1ml柱；装载上清液后，将该柱首先用pH 7.4的PBS溶液以流速0.5ml/分钟洗涤5-10个柱床体积，并随后用100mM柠檬酸缓冲液(pH 4.0)以流速0.5ml/分钟洗脱。收集洗脱峰，目的蛋白存在于洗脱峰中。
在还原剂(5mM 1,4-二硫苏糖醇)存在下通过SDS-PAGE并用考马斯蓝染色，分析融合蛋白的纯度和分子量。结果如图2所示。分子量理论预测值与实际测定值见表6。因真核表达系统中存在对蛋白质的糖基化作用，故分子量实际测定值略高于理论预测值。
表6经纯化的表达蛋白的分子量大小
Figure PCTCN2018111652-appb-000011
Figure PCTCN2018111652-appb-000012
实施例3、使用ELISA方法检测特异性结合作用
分别将重组人CD28(北京义翘神州生物技术有限公司产品，目录号：50103-M08H)、重组人PD-L1(北京百普赛斯生物科技有限公司，目录号：PD1-H5229)、和重组人CTLA-4(北京义翘神州生物技术有限公司产品，目录号：11159-H08H)稀释至0.5μg/ml、0.25μg/ml和1.0μg/ml并包被96孔ELISA板(Corning公司，货号：42592)。将上述实施例2(2)纯化的融合蛋白BY31.2、BY31.3、BY31.7、BY31.14和BY31.15稀释至2000μg/ml，然后进行3倍系列稀释，共稀释15-16个梯度，对每个浓度梯度进行复孔检测。将稀释样品50μl分别加入上述经重组人CD28或重组人PD-L1包被的96孔板中，37℃孵育2小时。洗涤3次后，加入辣根过氧化物酶标记的山羊抗人二级抗体(北京中衫金桥公司产品，产品号：ZDR-5301)，37℃孵育1小时。洗涤3次后，加入3,3',5,5'-四甲基联苯胺(TMB)底物显色液(北京康为世纪生物科技有限公司，产品号：CW0050)50μl/孔。10分钟后，加入2N的H 2SO 4终止显色。使用ELISA读数仪在450nm处测定每孔的吸光度OD值。
ELISA结果显示，本发明的融合蛋白BY31.2、BY31.3、BY31.7、BY31.14和BY31.15既能特异性地结合重组人PD-L1和重组人CD28；也能特异性地结合重组人CTLA-4。图3A、图3B和图3C中分别给出了融合蛋白BY31.2与重组人CD28、重组人PD-L1和重组人CTLA-4特异性结合的结合曲线。
应用GraphPadPrism5软件，将融合蛋白BY31.2、BY31.3、BY31.7、BY31.14和BY31.15的蛋白质浓度对吸光度OD值作图，并且拟合数据以产生融合蛋白介导的特异性结合作用的半数最大有效浓度EC 50值。结果如下表7所示。
表7本发明的融合蛋白对人PD-L1、人CD28和人CTLA-4的结合作用
Figure PCTCN2018111652-appb-000013
Figure PCTCN2018111652-appb-000014
根据表7的结果可见，本发明所构建的新型融合蛋白BY31.2、BY31.3、BY31.7、BY31.14和BY31.15均能够特异性结合人PD-L1、人CD28和人CTLA-4。融合蛋白BY31.3与人CD28、CTLA-4和PD-L1的结合能力均好于融合蛋白BY31.2和融合蛋白BY31.7，这可能是因为CD80ECD中的IgC区对CD80与CD28、CTLA-4和PD-L1的结合有一定的稳定作用。
融合蛋白BY31.14和BY31.15与人PD-L1、人CD28和人CTLA-4的结合能力好于BY31.2、BY31.3和BY31.7。可能是BY31.2、BY31.3和BY31.7将CD80胞外结构域(ECD)置于全长抗体N端或C端对分别结合人PD-L1、人CD28和人CTLA-4有一定影响。
另外，同样地，使用ELISA方法检测了抗体BY18.1与抗原PD-1(北京义翘神州生物技术有限公司产品，目录号：10377-H08H)特异性结合作用的EC 50值，为3.154μg/ml。
实施例4、使用Biacore T100测定本发明的融合蛋白对PD-1的亲和力
在
Figure PCTCN2018111652-appb-000015
T100仪器(GE Healthcare Biosciences AB，瑞典)上于25℃进行表面等离子体共振测量。
首先，通过酰胺偶联将抗IgG抗体(GE Healthcare Life Sciences，目录号：BR-1008-39)共价固定在CM5芯片上。使用60μl N-乙基-N'-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和60μl N-羟基琥珀酰亚胺(NHS)活化CM5芯片，然后将5μl抗IgG抗体加95μl稀释缓冲液HBST(0.1M HEPES,1.5M NaCl，pH7.4，加0.005％吐温20)经0.2um滤膜过滤后，通过酰胺偶联将抗IgG抗体共价固定在CM5芯片上，产生约9000-14000共振单位(RU)的捕获系统。使用120μl乙醇胺封闭CM5芯片。
然后，将实施例2制备的本发明的融合蛋白和抗体BY18.1分别稀释为5μg/ml，以流速10μL/分钟注射该稀释液2分钟，将1600RU的实施例2制备的本发明的融合蛋白和抗体BY18.1通过各自的Fc区非共价地捕获到CM5芯片表面上。通过与EDC/NHS交联来稳定所得的复合物，以避免在测量和再生期间的基线漂移。
将抗原PD-1(北京义翘神州生物技术有限公司产品，目录号：10377-H08H) 配制为如下浓度梯度：7nM、22nm、66nM、200nM、600nM。通过以流速30μl/分钟注射每个浓度180秒，解离时间600秒，测量结合。通过用3M MgCl 2溶液以流速10μL/分钟洗涤30秒使表面再生。使用BIA评价软件(BIAevaluation 4.1 software，来自GE Healthcare Biosciences AB，瑞典)进行数据分析，获得下表8所示的亲和力数据。
表8各蛋白质与PD-1的结合
蛋白质名称 ka(1/Ms) kd(1/s) KD(M)
融合蛋白BY31.2 6.66E+05 1.58E-03 2.37E-09
融合蛋白BY31.3 5.62E+05 5.66E-04 1.01E-09
融合蛋白BY31.7 8.56E+05 3.55E-03 4.15E-09
融合蛋白BY31.14 5.04E+05 3.55E-03 7.04E-09
融合蛋白BY31.15 1.56E+04 8.18E-04 5.24E-08
抗体BY18.1 1.76E+05 5.18E-04 2.94E-09
根据表8所示的数据可见，本发明的融合蛋白BY31.2、BY31.3和BY31.7均能够以高亲和力结合PD-1，亲和力(KD)分别达到2.37×10 -9M、1.01×10 -9M和4.15×10 -9M。抗体BY18.1以2.94×10 -9M的KD值结合PD-1。
融合蛋白BY31.14和BY31.15结合PD-1的能力弱于BY31.2、BY31.3和BY31.7。可能是由于融合蛋白BY31.14和BY31.15中的抗原结合片段是位于C端的Fab结构，与全抗体比较结合作用有一定减弱。融合蛋白BY31.14结合PD-1强于融合蛋白BY31.15，KD值能达到10 -9M水平。
实施例5、混合淋巴细胞反应(MLR)中本发明融合蛋白对IL-2和IFN-γ分泌的影响
自北京时合生物科技有限公司获得CD4 +T淋巴细胞和树突状细胞(DC)，所述CD4 +T淋巴细胞和树突状细胞(DC)来源于不同的健康人。将CD4 +T淋巴细胞和树突状细胞(DC)分别按1×10 5个细胞/孔和1×10 4个细胞/孔铺种在96孔细胞培养板中。实验分为6组，分别为空白对照组、BY18.1组、BY31.2组、BY31.3组、BY31.7组和BY31.14组，每组6个复孔。除空白对照组外，其他组按表9所示的量分别加入抗体或融合蛋白。最后补加含10％胎牛血清的1640培养基，使终体积均为200μl。37℃，5％CO 2培养。
培养5天后，与对照组相比较，各实验组成团细胞较多，并有相当部分的梭状和贴壁细胞出现。取培养上清，分别用依科赛生物科技有限公司的IL-2试剂盒(货号EH002-96)和IFN-γ试剂盒(货号：EH008-96ELISA)检测各组IL-2和IFN-γ表达水平。与抗体BY18.1组相比较，BY31.2组、BY31.3组、BY31.7 组和BY31.14组均显著提高了IL-2和IFN-γ的表达水平(P<0.01)，其中以BY31.14组上清中IL-2和IFN-γ表达量最高。
表9各融合蛋白对IL-2和IFN-γ分泌的影响
抗体或蛋白质名称 μg/ml IL-2(pg/ml) IFN-γ(pg/ml)
抗体BY18.1 5.0 485±59.4 9552±754
融合蛋白BY31.2 6.0 775±143.6 26431±812
融合蛋白BY31.7 6.9 903±186.5 21651±1890
融合蛋白BY31.14 6.0 1896±266.2 36378±1531
空白对照 0 55.4±10.6 63.5±4.8
实施例6、本发明的融合蛋白在B-hPD-1人源化小鼠模型中的体内抗肿瘤作用
本实施例使用B-hPD-1人源化小鼠模型研究了本发明融合蛋白的体内抗肿瘤作用。
B-hPD-1人源化小鼠(获自北京百奥赛图基因生物技术有限公司，产品编号：B-CM-001)是一种将人PD-1(hPD-1)敲入C57BL/6小鼠基因组后获得的小鼠。在B-hPD-1人源化小鼠中能够检测到人PD-1 +细胞。
将0.1mL DMEM培养基中的5×10 5个MC38鼠结肠癌细胞(获自ATCC，美国)接种于约18g，6周龄雌性B-hPD-1人源化小鼠右侧前胁肋部皮下。肿瘤在所述小鼠体内长大。待肿瘤体积达到约108mm 3时将荷瘤小鼠随机分组，每组6只，共3组，分别为：PBS溶剂对照组、融合蛋白BY31.2组(6.0mg/kg)和抗体BY18.1组(5mg/kg)，各给药组剂量以抗体BY18.1组的剂量为标准，对融合蛋白BY31.2组和抗体BY18.1组而言所施用的剂量在摩尔量上是等同的。将第一次给药的时间设定为第0天。所有组给药途径均为腹腔(i.p.)注射，每三天给药1次，连续给药6次，末次给药3天后结束实验。每周测量肿瘤体积及小鼠体重2次，记录小鼠体重和肿瘤体积。实验结束时，将动物安乐死，剥取肿瘤称重、拍照，计算肿瘤生长抑制率( Tumor  Growth  Inhibition％)。计算TGI％使用的公式是：[1－(给药组肿瘤体积变化的均值/PBS溶剂对照组肿瘤体积变化的均值)]x 100％。该实验在北京百奥赛图基因生物技术有限公司实施。
整个实验过程中，所有动物精神状态良好，无动物死亡。实验结束时(首次给药后的第21天)，各组动物体重平均约为19g。将本发明的融合蛋白BY31.2组以及作为药物对照的抗体BY18.1组与PBS溶剂对照组动物进行体重比较，没 有显著性差异(P>0.05)，表明动物对本发明的融合蛋白BY31.2耐受良好(图4)。
实验结束时，PBS溶剂对照组平均肿瘤体积±标准误为1386±170mm 3,融合蛋白BY31.2组平均肿瘤体积±标准误为953±166mm 3，融合蛋白BY31.2组的肿瘤生长抑制率(TGI％)为31.2％，表明了融合蛋白BY31.2具有体内抗肿瘤的技术效果(图5)。
另外，实验结束时，作为药物对照的抗体BY18.1组平均肿瘤体积±标准误为739±128，TGI％为46.9％，表明作为药物对照的抗体BY18.1通过特异性结合B-hPD-1人源化小鼠中的hPD-1 +细胞发挥体内抗肿瘤作用。这与现有技术中的报导是一致的，即，纳武单抗是一种针对人PD-1分子的特异性单克隆抗体，其通过与人PD-1分子特异性结合来阻断人PD-1分子所介导的抑制性生物学效应，由此，对于表达PD-1的人类患者发挥抗肿瘤作用。
融合蛋白BY31.2抑制肿瘤生长的作用与作为药物对照的抗体BY18.1相比较没有显著性差异，这可能是因为在融合蛋白BY31.2中仅抗PD-1抗体部分能够发挥作用，而在融合蛋白BY31.2中的人CD80ECD不与B-hPD-1人源化小鼠的鼠CD28、鼠CTLA-4和鼠PD-L1相结合；另外，还可能是因为融合蛋白BY31.2的分子量大于对照抗体BY18.1，在以等同摩尔量施用时，融合蛋白BY31.2渗入肿瘤组织中的量较少也有关(肿瘤组织内部为高渗环境)。
实施例7、本发明融合蛋白在人CD34 +HSC移植的NSG小鼠模型中的的体内抗肿瘤作用
本实施例通过将MDA-MB-231人三阴性乳腺癌细胞系(即，雌激素受体、孕激素受体和HER-2均为阴性的乳腺癌细胞系，获自ATCC，美国)接种至人CD34 +HSC移植的NSG小鼠模型研究了本发明融合蛋白的体内抗肿瘤作用。
人CD34 +HSC移植的NSG小鼠(获自北京艾德摩生物技术有限公司)是一种将人的CD34 +造血干细胞(HSC)移植到免疫缺陷的NOD/SCID/IL2r-γ null(NSG)小鼠体内，使NSG小鼠重建人免疫系统从而获得的一种在机体免疫方面人源化的小鼠模型。
本实施例在人CD34 +HSC移植的NSG小鼠模型的基础上接种人源的肿瘤细胞系，即，MDA-MB-231人三阴性乳腺癌细胞系，最终获得了在肿瘤方面和在免疫方面均是人源化的小鼠。这样的小鼠由于同时具有人的免疫系统以及人源肿瘤组织，因此，能够真实模拟人免疫系统与肿瘤相互作用的过程，是用于评 价对肿瘤免疫疗法的有效性和安全性的理想动物模型。
按5×10 6个MDA-MB-231人三阴性乳腺癌细胞/只小鼠的量将MDA-MB-231细胞接种至体重约23g，28－32周龄的人CD34 +HSC移植成功的NSG雌性小鼠，接种部位为背部右后皮下。当肿瘤体积达到大约150mm 3，将荷瘤小鼠随机分组，每组5只，共4组，分别为：PBS溶剂对照组、融合蛋白BY31.2组(5.8mg/kg)、抗PD-L1单抗Avelumab组(10mg/kg，北京尚健生物技术有限公司制备)和抗PD-1单抗Opdivo组(10mg/kg，义翘神州生物技术有限公司制备，批号:MB09MA1201)，其中融合蛋白BY31.2组的施用摩尔量为抗PD-1单抗Opdivo组、抗PD-L1单抗Avelumab组的1/2施用摩尔量。将第一次给药的时间设定为第0天。所有组给药途径均为腹腔注射，每5天给药1次，连续给药4次。每周测量肿瘤体积及小鼠体重3次，记录小鼠体重和肿瘤体积。实验结束时，将动物安乐死，剥取肿瘤称重、拍照，计算肿瘤生长抑制率(TGI％)。该实验在北京艾德默(IDMO)生物技术有限公司实施。
图6显示了给药后动物的体重变化。在第25天时，将本发明的融合蛋白BY31.2组与PBS溶剂对照组的动物进行体重比较，没有观察到显著性差异(P>0.05)，表明本发明的融合蛋白BY31.2对动物没有明显毒性。
图7显示了给药后动物的肿瘤体积随时间的变化情况。在第25天时，PBS溶剂对照组平均肿瘤体积±标准误为1287.11±184.71mm 3，抗PD-L1单抗Avelumab组为964.25±20.01mm 3，抗PD-1单抗Opdivo组为1354.62±126.65mm 3，融合蛋白BY31.2组为773.14±310.66mm 3。
相对于PBS溶剂对照组，抗PD-L1单抗Avelumab组的肿瘤抑制率(TGI％)为25.08％，抗PD-1单抗Opdivo组的TGI％为-5.24％，融合蛋白BY31.2组的TGI％为39.93％。
由以上实验结果可见，与PBS溶剂对照组比较，本发明的融合蛋白BY31.2在使用的摩尔剂量相对于现有技术中的抗PD-L1单抗、抗PD-1单抗的摩尔剂量减半的情况下，也能够显著地抑制肿瘤生长(P<0.05)。
另外，出乎意料的是，在融合蛋白BY31.2组中，有一只小鼠的肿瘤体积在第25天时，缩小到只有60mm 3，而PBS溶剂对照组的肿瘤体积在第25天时已高达1287.11±184.71mm 3，表明了这只小鼠对肿瘤产生了完全应答(CR,complete response)。在抗PD-L1单抗Avelumab组和抗PD-1单抗Opdivo组中，均没有观察到完全应答的小鼠。
自北京艾德默(IDMO)生物技术有限公司开发出这样的同时具有人的免疫系统以及人源肿瘤组织的小鼠实施抗肿瘤药的药效实验以来，对受试的抗肿瘤药观察到肿瘤完全应答小鼠是非常少有的情形。本发明的融合蛋白在使用的摩尔剂量相对于现有技术中的抗PD-L1单抗、抗PD-1单抗的摩尔剂量减半的情况下，仍然能够观察到对肿瘤的完全应答，由此，获得了意料不到的技术效果。
抗PD-1单抗Opdivo组在此小鼠模型中完全没有抗肿瘤效果。抗PD-L1单抗Avelumab在此小鼠模型中虽然具有一定的肿瘤抑制作用(PBS溶剂对照组的肿瘤体积为1287.11±184.71mm 3，抗PD-L1单抗Avelumab组的肿瘤体积为964.25±20.01mm 3)，但是，没有本发明的融合蛋白BY31.2的抗肿瘤效果显著(融合蛋白BY31.2组的肿瘤体积为773.14±310.66mm 3)。这表明了，本发明的融合蛋白BY31.2在真实模拟人免疫系统与肿瘤相互作用过程的小鼠模型中能够产生非常显著的抗肿瘤效果。

Claims (18)

  1. 一种阻断PD-1/PD-L1信号传导途径且活化T细胞的融合蛋白，其包含(i)衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段；(ii)免疫球蛋白Fc结构域；和(iii)CD80胞外结构域(ECD)。
  2. 根据权利要求1所述的融合蛋白，其中所述(i)是衍生自抗PD-1抗体和/或抗PD-L1抗体的Fab、Fab'、F(ab') 2、Fv、单链Fv；优选地，所述(i)包含选自SEQ ID NO：1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24和25/26的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR，优选地，所述(i)包含抗PD-1抗体的选自SEQ ID NO：1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24和25/26的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；更优选地，所述(i)包含选自纳武单抗(Nivolumab)、pidilizumab和派姆单抗(Pembrolizumab)的抗PD-1抗体的重链可变区和轻链可变区；和/或所述(i)包含抗PD-L1抗体的选自SEQ ID NO：27/28、29/30和31/32的成对重链可变区序列/轻链可变区序列中所含的全部6个重链CDR与轻链CDR；优选地，所述(i)包含选自SEQ ID NO：27/28、29/30和31/32的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
  3. 根据权利要求1或2所述的融合蛋白，其中所述(ii)是人免疫球蛋白Fc结构域；优选地，所述(ii)是人IgG1、IgG2、IgG3或IgG4的Fc结构域；更优选地，所述(ii)包含SEQ ID NO：38、39或40所示氨基酸序列的Fc结构域，或者包含与SEQ ID NO：38、39或40所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的Fc结构域。
  4. 根据权利要求1－3中任一项所述的融合蛋白，其中所述(iii)包含人CD80ECD；优选地，所述(iii)包含人CD80IgV或人CD80IgVIgC；更优选地，所述(iii)具有SEQ ID NO：41或42所示的氨基酸序列或与SEQ ID NO：41或42所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的氨基酸序列。
  5. 根据权利要求1－4中任一项所述的融合蛋白，其还包含所述(i)、(ii)和/或(iii)之间的肽接头；优选地，所述肽接头包含一个或多个氨基酸，更优选地包含至少5个氨基酸，最优选地包含选自SEQ ID NO：43－71的肽接头。
  6. 根据权利要求1－5中任一项所述的融合蛋白，其中所述融合蛋白从N端至C端以(i)、(ii)和(iii)的顺序；(iii)、(i)和(ii)的顺序；或者(iii)、(ii)和(i)的顺序有效连接。
  7. 根据权利要求6所述的融合蛋白，其包含
    (a)抗PD-1抗体、抗PD-L1抗体或者抗PD-1和PD-L1双特异性抗体；和在所述抗体的两条重链中的每一重链的C端有效连接的一个CD80ECD；
    (b)抗PD-1抗体、抗PD-L1抗体或者抗PD-1和PD-L1双特异性抗体；在所述抗体的两条重链中的每一重链的N端有效连接的一个CD80ECD；和在所述抗体的两条轻链中的每一轻链的N端有效连接的一个CD80ECD；或者
    (c)CD80ECD；在CD80ECD的C端有效连接的二聚体形式的免疫球蛋白Fc结构域；和在所述二聚体形式的免疫球蛋白Fc结构域的C端有效连接的衍生自抗PD-1抗体和/或抗PD-L1抗体的抗原结合片段；
    优选地，所述抗体是IgG类抗体，特别地是IgG 1亚类、IgG 2亚类、IgG 4亚类抗体，更特别地是IgG 4亚类抗体；还优选地，所述IgG 4亚类抗体在Fc结构域中第S228位置处包含氨基酸置换，更优选地是氨基酸置换S228P；进一步优选地，所述抗体的轻链型别为κ型或λ型，优选地为κ型。
  8. 根据权利要求1－7中任一项所述的融合蛋白，其中所述抗PD-1抗体选自纳武单抗、pidilizumab和派姆单抗；所述抗PD-L1抗体选自atezolizumab、avelumab和durvalumab。
  9. 根据权利要求1－8中任一项所述的融合蛋白，其选自
    (1)包含SEQ ID NO:77的融合蛋白第一亚基和SEQ ID NO:79的融合蛋白第二亚基的融合蛋白；
    (2)包含SEQ ID NO:81的融合蛋白第一亚基和SEQ ID NO:83的融合蛋白第二亚基的融合蛋白；
    (3)包含SEQ ID NO:85的融合蛋白第一亚基和SEQ ID NO:87的融合蛋白第二亚基的融合蛋白；
    (4)包含SEQ ID NO:89的融合蛋白第一亚基和SEQ ID NO:91的融合蛋白第二亚基的融合蛋白；
    (5)包含SEQ ID NO:93的融合蛋白第一亚基和SEQ ID NO:95的融合蛋白第二亚基的融合蛋白。
  10. 多核苷酸，其编码权利要求1－9中任一项所述的融合蛋白。
  11. 载体，优选地表达载体，最优选地具有双表达盒的谷氨酰胺合成酶表达载体，所述载体包含权利要求10所述的多核苷酸。
  12. 宿主细胞，其包含权利要求10所述的多核苷酸或权利要求11所述的载体，优选地，所述宿主细胞是CHO、HEK293或NSO细胞。
  13. 用于产生权利要求1－9中任一项所述的融合蛋白的方法，所述方法包括步骤(i)在适于表达所述融合蛋白的条件下培养权利要求12所述的宿主细胞，和(ii)回收所述融合蛋白。
  14. 药物组合物，其包含权利要求1－9中任一项所述的融合蛋白和可药用载体。
  15. 权利要求1－9中任一项所述的融合蛋白、权利要求14所述的药物组合物的用途，用于制备在个体中治疗或预防与PD-1活性、PD-L1活性和CD28活性相关的疾病的药物，优选地，所述疾病是癌性疾病(例如，实体瘤和软组织瘤)，更优选地是黑素瘤、乳腺癌、结肠癌、食管癌、胃肠道间质肿瘤(GIST)、肾癌(例如，肾细胞癌)、肝癌、非小细胞肺癌(NSCLC)、卵巢癌、胰腺癌、前列腺癌、头颈部肿瘤、胃癌、血液学恶性病(例如，淋巴瘤)；特别地，所述疾病是结肠癌或三阴性乳腺癌；优选地，其中所述个体是哺乳动物，更优选地是人。
  16. 诊断试剂盒，其包含权利要求1－9中任一项所述的融合蛋白和任选地标记物或用于偶联的试剂。
  17. 根据权利要求16所述的诊断试剂盒，其包含用正电子发射断层摄影术可检测的标记物标记的权利要求1－9中任一项所述的融合蛋白，优选地，所述标记物是 18F-氟脱氧葡萄糖。
  18. 权利要求16或17所述的诊断试剂盒的用途，用于制备在个体中诊断与PD-1活性、PD-L1活性和CD28活性相关的疾病的试剂，优选地，所述疾病是癌性疾病(例如，实体瘤和软组织瘤)，更优选地是黑素瘤、乳腺癌、结肠癌、食管癌、胃肠道间质肿瘤(GIST)、肾癌(例如，肾细胞癌)、肝癌、非小细胞肺癌(NSCLC)、卵巢癌、胰腺癌、前列腺癌、头颈部肿瘤、胃癌、血液学恶性病(例如，淋巴瘤)；优选地，其中所述个体是哺乳动物，更优选地是人。
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