WO2022063314A1 - 靶向PD-1或PD-L1和TGF-β的双功能蛋白及其医药用途 - Google Patents

靶向PD-1或PD-L1和TGF-β的双功能蛋白及其医药用途 Download PDF

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WO2022063314A1
WO2022063314A1 PCT/CN2021/121198 CN2021121198W WO2022063314A1 WO 2022063314 A1 WO2022063314 A1 WO 2022063314A1 CN 2021121198 W CN2021121198 W CN 2021121198W WO 2022063314 A1 WO2022063314 A1 WO 2022063314A1
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seq
amino acid
bifunctional protein
acid sequence
antibody
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PCT/CN2021/121198
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French (fr)
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冯辉
刘洪川
姚盛
何丽
周岳华
张静
刘丹丹
吴纯
刘辉
赵强
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Priority to CN202180066168.0A priority Critical patent/CN116323658A/zh
Publication of WO2022063314A1 publication Critical patent/WO2022063314A1/zh

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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to the field of medical biotechnology. Specifically, the present invention relates to bifunctional proteins targeting programmed death protein-1 (PD-1)/programmed death protein ligand-1 (PD-L1) and transforming growth factor-beta (TGF-beta), encoding
  • PD-1 programmed death protein-1
  • PD-L1 programmed death protein ligand-1
  • TGF-beta transforming growth factor-beta
  • the polynucleotides of the bifunctional protein of the present invention, vectors and host cells for expressing the bifunctional protein of the present invention, and the bifunctional protein of the present invention are related to PD-1 or PD-L1 activity and TGF- Use in diseases associated with beta family activity.
  • Immune checkpoints are a class of inhibitory signaling molecules in the immune system that avoid tissue damage by regulating the persistence and strength of immune responses in peripheral tissues, and are involved in maintaining tolerance to self-antigens. Studies have found that one of the reasons why tumor cells can escape the immune system and proliferate out of control is the use of the inhibitory signaling pathway of immune checkpoints, thereby inhibiting the activity of T lymphocytes, making T lymphocytes unable to effectively kill tumors. .
  • PD-1 Programmed death 1
  • PD-1 is a member of the CD28 superfamily.
  • PD-1 is an important immune checkpoint protein and is currently an important target for tumor immunotherapy.
  • PD-1 is expressed in activated T cells, B cells and myeloid cells, and it has two ligands, programmed death ligand 1 (PD-L1) and programmed death ligand-2 (PD-L2).
  • PD-L1 interacts with the receptor PD-1 on T cells and plays an important role in the negative regulation of immune responses.
  • the expression of PD-L1 protein can be detected in many human tumor tissues.
  • the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells.
  • the expressed PD-L1 is beneficial to the occurrence and growth of tumors and induces anti-tumor effects.
  • PD-1/PD-L1 pathway inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, and restore the activity of T cells, thereby enhancing the immune response. Therefore, PD-1/PD- L1-targeted immune regulation has important implications for tumor suppression.
  • PD-L1 Programmed death-ligand 1
  • CD274 cluster of differentiation 274
  • B7 homologous protein 1 B7homolog1, B7-H1
  • the tumor necrosis factor superfamily is a type I transmembrane glycoprotein composed of 290 amino acid residues, including an IgV-like domain, an IgC-like domain, a transmembrane hydrophobic domain, and an intracellular tail of 30 amino acids.
  • the complete molecular weight is 40kDa1.
  • PD-L1 mRNA is expressed in almost all tissues, but PD-L1 protein is only continuously expressed in a small number of tissues, including liver, lung, tonsil, and immune amnesty tissues such as eye and placenta.
  • PD-L1 is also expressed in activated T cells, B cells, monocytes, dendritic cells, macrophages, etc.
  • the receptor of PD-L1 is PD-1, which is mainly expressed on the surface of immune cells such as CD4+ T cells, CD8+ T cells, NKT cells, B cells and activated monocytes.
  • TGF- ⁇ Transforming growth factor- ⁇ belongs to the TGF- ⁇ superfamily that regulates cell growth and differentiation.
  • TGF-beta signals through a heterotetrameric receptor complex consisting of two type I and two type II transmembrane serine/threonine kinase receptors.
  • Transforming growth factor-beta is a pleiotropic cytokine with immunomodulatory properties such as restriction and termination of inflammatory and allergic immune responses.
  • TGF[beta] has been implicated in inflammatory, malignant, infectious and autoimmune diseases, as well as in osteoporosis and fibrosis, including cirrhosis and systemic sclerosis.
  • persistently high levels of TGF ⁇ in tumors are associated with increased immune tolerance, angiogenesis, metastasis, and tumor extracellular matrix deposition, all of which may drive cancer development and resistance to therapy.
  • blocking the PD-1/PD-L1 pathway by targeting PD-1 or PD-L1 antibodies is used to block negative regulatory signals and restore the activity of T cells, thereby enhancing the immune response and improving the immune system. Effectively improve the effect of inhibiting the occurrence and development of tumors.
  • the present invention provides a bifunctional protein targeting programmed death protein-1 (PD-1)/programmed death protein ligand 1 (PD-L1) and transforming growth factor-beta (TGF-beta) and its use in the treatment, Use in the prevention and/or diagnosis of diseases associated with PD-1 activity and TGF-beta activity.
  • PD-1 programmed death protein-1
  • P-L1 programmed death protein ligand 1
  • TGF-beta transforming growth factor-beta
  • the bifunctional proteins provided by the present invention can be used as a stand-alone therapy or in combination with other therapies and/or other anticancer agents, for example, in the treatment of cancer.
  • the invention provides a bifunctional protein targeting PD-1 or PD-L1 and TGF-beta, comprising
  • TGF- ⁇ RII extracellular domain ECD
  • the antigen-binding fragment of the antibody that blocks the PD-1/PD-L1 pathway of the present invention is an antigen-binding fragment of an anti-PD-1 or PD-L1 antibody.
  • the antigen-binding fragment of the anti-PD-1 antibody of the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.
  • the antigen-binding fragment of the anti-PD-1 antibody of the present invention comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:7 and a light chain whose amino acid sequence is shown in SEQ ID NO:8
  • the variable region, or the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:7 such as the light chain variable region shown in SEQ ID NO:8 has at least 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the antigen-binding fragment of the anti-PD-L1 antibody of the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively.
  • the antigen-binding fragment of the anti-PD-L1 antibody of the present invention comprises at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 24 , 96%, 97%, 98%, 99% or 100% sequence identity heavy chain variable region, and with the amino acid sequence shown in SEQ ID NO: 25 has at least 90%, 91%, 92%, 93%, Light chain variable regions of 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the antigen-binding fragment of the anti-PD-L1 antibody of the present invention comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:24 and a light chain whose amino acid sequence is shown in SEQ ID NO:25 variable region.
  • the antigen-binding fragment of the anti-PD-1 or PD-L1 antibody of the present invention is Fab, Fab', F(ab')2, Fv, scFv or sdAb.
  • the immunoglobulin Fc domain of the present invention is the Fc domain of human IgG1, IgG2, IgG3 or IgG4; preferably the Fc domain of human IgG4; preferably, the Fc domain of IgG4 comprises 1, 2, 3, 4 or 5 amino acid differences, preferably S228P amino acid substitution in the Fc domain; more preferably, a C-terminal K and G amino acid deletion in the Fc domain.
  • the immunoglobulin Fc domain of the present invention comprises the amino acid sequence shown in SEQ ID NO: 9, or has at least 90%, 91%, 92% with the amino acid sequence shown in SEQ ID NO: 9 , 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of Fc domains.
  • the bifunctional protein of the present invention wherein the immunoglobulin Fc domain comprises at least 90%, 91%, 92%, 93%, 94% with the amino acid sequence shown in SEQ ID NO:9 Fc domains of %, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the immunoglobulin Fc domain of the present invention comprises the amino acid sequence shown in SEQ ID NO:9.
  • the TGF- ⁇ RII extracellular domain of the present invention has the amino acid sequence shown in SEQ ID NO: 10, or at least 90%, 91%, 92% with the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequences of %, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the TGF- ⁇ RII extracellular domain of the present invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequences of %, 97%, 99% or 100% sequence identity.
  • the TGF- ⁇ RII extracellular domain of the present invention comprises the amino acid sequence shown in SEQ ID NO:10.
  • the bifunctional protein of the invention is in the order of (i), (ii) and (iii); (iii), (i) and (ii); or ( The sequence of iii), (ii) and (i) is operatively linked; preferably, said (i) and (ii) are linked by a hinge region, and (i) and (iii) and (ii) and (iii) are connected by a linker peptide, the amino acid sequence of the linker peptide contains or consists of glycine (G) residue and serine (S) residue, and is 2-31 amino acid residues in length; preferably each is (GGGGS)nG independently, wherein n is independently selected from 3, 4, 5 or 6; preferably 4 or 5.
  • the bifunctional proteins of the present invention are operably linked in the order of (i), (ii) and (iii) from the N-terminus to the C-terminus, preferably, between said (i) and (ii) Connected by the hinge region, (ii) and (iii) are connected by a linker peptide, the amino acid sequence of the linker peptide contains or consists of glycine residues and serine residues, and has a length of 2-31 amino acid residues; preferably each independently is (GGGGS)nG, wherein n is each independently selected from 3, 4, 5 or 6; preferably 4 or 5.
  • the bifunctional proteins of the invention comprise an anti-PD-1 or PD-L1 antibody and one TGF- ⁇ RIIECD operably linked to the C-terminus of each of the two heavy chains of the antibody.
  • the antibody of the invention is an IgG1, IgG2, IgG3 or IgG4 class antibody, preferably an IgG4 class antibody; more preferably, the IgG4 class antibody comprises 1, 2, 3, 4 or 1 in the Fc domain 5 amino acid difference, preferably, with S228P amino acid substitution in the Fc domain; further preferably, with two amino acid deletions of C-terminal K and G in the Fc domain.
  • the bifunctional protein of the present invention wherein the IgG4 comprises 1, 2, 3, 4 or 5 amino acid differences in the Fc domain, preferably, has the S228P amino acid substitution in the Fc domain (according to the EU numbering system); further preferably, there is a C-terminal K and G two amino acid deletion (according to the EU numbering system) in the Fc domain.
  • the anti-PD-1 antibody of the present invention has the heavy chain amino acid sequence shown in SEQ ID NO: 11 and the light chain amino acid sequence shown in SEQ ID NO: 12, or the same as SEQ ID NO:
  • the amino acid sequences shown in 11 and/or 12 have amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the anti-PD-L1 antibody of the present invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 26 or 31 , 97%, 98%, 99% or 100% sequence identity of heavy chain amino acids, and have at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence shown in SEQ ID NO: 27 , 96%, 97%, 98%, 99% or 100% sequence identity of light chain amino acids.
  • the anti-PD-L1 antibody of the present invention comprises the heavy chain amino acid sequence shown in SEQ ID NO: 26 or 31 and the light chain amino acid sequence shown in SEQ ID NO: 27.
  • the bifunctional protein of the present invention comprises the first subunit of the bifunctional protein whose amino acid sequence is shown in SEQ ID NO: 13, and the first subunit of the bifunctional protein whose amino acid sequence is shown in SEQ ID NO: 12. two subunits.
  • the bifunctional protein of the present invention comprises a bifunctional protein first subunit whose amino acid sequence is shown in SEQ ID NO:28, and a bifunctional second subunit whose amino acid sequence is shown in SEQ ID NO:27 subunit.
  • the bifunctional proteins of the present invention have one or more of the following properties:
  • the present invention provides a polynucleotide encoding a bifunctional protein as described herein or an antigen-binding fragment thereof.
  • the polynucleotide has the nucleotide sequence of the first subunit as set forth in SEQ ID NO:16 and/or the nucleotide sequence of the second subunit as set forth in SEQ ID NO:17.
  • the present invention provides a polynucleotide encoding a bifunctional protein as described herein or an antigen-binding fragment thereof.
  • the polynucleotide has the nucleotide sequence of the first subunit as set forth in SEQ ID NO:29 and/or the nucleotide sequence of the second subunit as set forth in SEQ ID NO:30.
  • the present invention provides an expression vector comprising the polynucleotide as described herein, preferably, the expression vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising a polynucleotide or an expression vector as described herein, preferably, the host cell is a mammalian cell.
  • the present invention provides a method of making a bifunctional protein as described herein, the method comprising expressing the bifunctional protein in a host cell as described herein under conditions suitable for expression of the bifunctional protein the bifunctional protein, and the expressed bifunctional protein is recovered from the host cell.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a medicament as described herein
  • a composition in the manufacture of a medicament for the treatment and/or prevention of a disease or condition associated with PD-1, PD-L1 or TGF- ⁇ activity preferably the disease or condition is cancer, more preferably, the The cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer and rectal cancer.
  • the present invention provides a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, and a pharmaceutical composition as described herein , which is used for the treatment and/or prevention of a disease or condition associated with PD-1, PD-L1 or TGF- ⁇ activity, preferably the disease or condition is cancer, more preferably the cancer is selected from melanoma, kidney cancer , prostate, breast, colon, lung, bone, pancreatic, skin, head and neck, uterine, ovarian and rectal cancers.
  • the present invention provides a method of treating and/or preventing a disease or disorder associated with PD-1, PD-L1 or TGF-beta activity, comprising administering to a subject in need thereof, as described herein
  • the present invention also provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof as described herein conjugated to a therapeutic or diagnostic agent.
  • the present invention provides a combination product comprising an antibody or antigen-binding fragment thereof as described herein, an immunoconjugate as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agent.
  • the present invention provides a kit comprising a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein.
  • the present invention provides a method of detecting the presence of PD-1, PD-L1 or TGF-beta in a sample using a bifunctional protein as described herein.
  • the bifunctional protein of the present invention has strong affinity with PD-1 or PD-L1 protein, has good thermal stability, and can significantly promote the release of IFN ⁇ and inhibit the growth of tumors.
  • Figure 1 Schematic representation of the molecular structure of a bifunctional protein targeting PD-1 and TGF- ⁇ .
  • Figure 2 Binding of a bifunctional protein targeting PD-1 and TGF- ⁇ to human TGF- ⁇ 1 by ELISA.
  • Figure 3 Binding of a bifunctional protein targeting PD-1 and TGF- ⁇ to human TGF- ⁇ 2 by ELISA.
  • Figure 4 Binding of bifunctional proteins targeting PD-1 and TGF- ⁇ to human TGF- ⁇ 3 by ELISA.
  • Figure 5 Binding of a bifunctional protein targeting PD-1 and TGF- ⁇ to human PD-1 by ELISA.
  • Reporter gene assay detects the blocking effect of PD-1/PD-L1 pathway by bifunctional proteins targeting PD-1 and TGF- ⁇ .
  • Figure 7 Reporter gene assay to detect the blocking effect of bifunctional protein targeting PD-1 and TGF- ⁇ on TGF ⁇ pathway.
  • Figure 8 Endocytosis experiments of bifunctional proteins targeting PD-1 and TGF-beta.
  • Figure 9 In vitro mixed lymphocyte reaction of bifunctional proteins targeting PD-1 and TGF-beta.
  • 9a The effect of bifunctional protein targeting PD-1 and TGF- ⁇ on IL2 cytokine release levels after T cell activation;
  • 9b Bifunctional protein targeting PD-1 and TGF- ⁇ on IFN ⁇ cells after T cell activation The effect of factor release levels.
  • Figure 10 Inhibition of human melanoma A375 tumor growth in mice by a bifunctional protein targeting PD-1 and TGF-beta.
  • Figure 11 Schematic representation of the molecular structure of a bifunctional protein targeting PD-L1 and TGF-beta.
  • Figure 12 Binding of bifunctional proteins targeting PD-L1 and TGF-beta to human TGF-beta1 by ELISA.
  • Figure 13 Binding of a bifunctional protein targeting PD-L1 and TGF-beta to human TGF-beta2 by ELISA.
  • Figure 14 Binding of bifunctional proteins targeting PD-L1 and TGF-beta to human TGF-beta3 by ELISA.
  • FIG. 15 ELISA detects the binding of bifunctional proteins targeting PD-L1 and TGF- ⁇ to human PD-L1.
  • Figure 16 Reporter gene assay detects PD-1/PD-L1 pathway blocking effect of bifunctional proteins targeting PD-L1 and TGF- ⁇ .
  • Figure 17 Reporter gene assay detects the blocking effect of bifunctional protein targeting PD-L1 and TGF- ⁇ on TGF ⁇ pathway.
  • Figure 18 Endocytosis experiments of bifunctional proteins targeting PD-L1 and TGF-beta.
  • Figure 19 In vitro mixed lymphocyte reaction of bifunctional proteins targeting PD-L1 and TGF-beta.
  • 19a Effect of bifunctional protein targeting PD-L1 and TGF- ⁇ on IL2 cytokine release levels after T cell activation;
  • 19b Bifunctional protein targeting PD-L1 and TGF- ⁇ on IFN ⁇ cells after T cell activation The effect of factor release levels.
  • PD-1 Programmed death receptor-1
  • PD-1 is mainly expressed on previously activated T cells in vivo and binds two ligands, PD-L1 and PD-L2.
  • the term "PD-1” as used herein includes human PD-1 (hPD-1), variants, isoforms and species homologues of hPD-1, and analogs that share at least one epitope with hPD-1 .
  • P-L1 Programmed cell death protein ligand 1
  • CD274 cluster of differentiation274
  • B7 homologous protein 1 B7homolog1, B7-H1
  • PD-L1 belongs to the tumor necrosis factor superfamily .
  • the term "PD-L1” as used herein includes human PD-L1 (hPD-L1), variants, isoforms and species homologues of hPD-L1, and analogs that share at least one epitope with hPD-L1 .
  • TGF-beta Transforming growth factor-beta
  • TGF-beta belongs to the TGF-beta superfamily that regulates cell growth and differentiation.
  • TGF-beta signals through a heterotetrameric receptor complex consisting of two type I and two type II transmembrane serine/threonine kinase receptors.
  • type I and type II receptors are transmembrane proteins composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity constitute.
  • Type I receptors are essential for signaling, and type II receptors are necessary for binding ligands and expression of type I receptors.
  • Type I and type II receptors form stable complexes upon ligand binding, resulting in phosphorylation of type I receptors by type II receptors.
  • TGF ⁇ has three mammalian isoforms, TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3, each with distinct functions in the body. Binding of TGF ⁇ to TGF ⁇ RII is a key step in initiating activation of the TGF ⁇ signaling pathway, resulting in phosphorylation of SMAD2 and translocation of the activated SMAD2/SMAD4 complex to the nucleus to regulate gene expression.
  • percent (%) amino acid sequence identity or simply “identity” is defined as the maximum percent sequence identity obtained when amino acid sequences are aligned (and where necessary gaps are introduced), and no conservative substitutions are considered to be Following the portion of sequence identity, the percentage of amino acid residues in the candidate amino acid sequence that are identical to those in the reference amino acid sequence.
  • Sequence alignments to determine percent amino acid sequence identity can be performed using various methods in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
  • immune response refers to the action by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines and complement) by these cells or by the liver, which results in selective Damage, destroy or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of growth factors to receptors, that results in the transmission of signals from one part of a cell to another of the cell. part.
  • delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions leading to signal transduction.
  • the penultimate process usually involves nuclear events that lead to changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological signature” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, neutralization or antagonism of PD in vivo or in vitro -1 capacity for activity, IC50, in vivo stability of the antibody and immunogenic properties of the antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (ie, generally with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention of The ability of proteins to be expressed at high levels in mammalian cells.
  • antibody refers to any form of antibody that possesses the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric and camelized single domain antibodies.
  • isolated antibody refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media .
  • isolated does not mean the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include large numbers of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.
  • full-length antibody refers to an immunoglobulin molecule comprising four peptide chains in nature: two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (full length) 25kDa) are connected to each other by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and spaced by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • antibody-binding fragment of an antibody (“parent antibody”) includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding or variable region (eg, one or more CDRs) of the parent antibody that retains the parental At least some binding specificity of an antibody.
  • antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
  • a binding fragment or derivative typically retains at least 10% of its antigen-binding activity when the antigen-binding activity is expressed on a molar basis.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of an antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • single-chain Fv or "scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides typically also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • Fc or “Fc region” or “Fc fragment” or “Fc domain” are used herein to define the 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” contains two or three constant domains, a CH2 domain, a CH3 domain and an optional CH4 domain.
  • an immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) derived from the two heavy chains of antibodies of the IgG, IgA and IgD classes; or The second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of both heavy chains of IgM and IgE class antibodies.
  • the Fc region herein may comprise a hinge region; the amino acid sequence of a human IgG1 immunoglobulin Fc domain is SEQ ID NO:32, and the amino acid sequence of a human IgG4 immunoglobulin Fc domain is SEQ ID NO:33.
  • flankinge region refers to a proline-rich polypeptide chain located between CH1 and CH2 in an antibody, which is easy to stretch and bend.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • immunoglobulin refers to a protein having the structure of a naturally occurring antibody.
  • immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons composed of two light and two heavy chains joined by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a variable domain (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), also known as the heavy chain constant region.
  • VH variable domain
  • CH1 and CH3 constant domains
  • each immunoglobulin light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain, also known as the light chain constant region.
  • VL variable region
  • CL constant light chain
  • the heavy chains of immunoglobulins can be assigned to one of 5 classes, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or mu (IgM), some of which can be further divided into subclasses Classes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), ⁇ 4 (IgG4), ⁇ 1 (IgA1) and ⁇ 2 (IgA2).
  • the light chains of immunoglobulins can be divided into one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains.
  • a "human immunoglobulin” is an immunoglobulin that possesses an amino acid sequence corresponding to an immunoglobulin produced by a human or human cell or derived from a non-human immunoglobulin utilizing a human immunoglobulin library or other sequences encoding human immunoglobulins source derived.
  • bivalent antibody contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
  • diabody refers to a small antibody fragment with two antigen-binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH).
  • VL light chain variable domain
  • VH-VL or VL-VH Variable domain
  • chimeric antibody is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domains are obtained from rodent or the like antibodies (“parental antibodies”), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibody induces induction in human subjects as compared to the parental rodent antibody The likelihood of an adverse immune response is low.
  • humanized antibody refers to a form of antibody that contains sequences from human and non-human (eg, mouse, rat) antibodies.
  • humanized antibodies comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • FR hypervariable loops Framework
  • a humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, “mouse antibody” refers to an antibody comprising only mouse immunoglobulin sequences. Alternatively, fully human antibodies may contain rat sugar chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Likewise, “rat antibody” refers to an antibody comprising only rat immunoglobulin sequences.
  • PD-1 antibody refers to binding to the PD-1 receptor, blocking the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T, B, NK cells) and preferably also blocking Any chemical compound or biomolecule that blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells.
  • Alternative nouns or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7-H1, B7H1, B7-4, CD274 and B7-H; and for PD-L2 PDCD1L2, PDL2, B7-DC and CD273.
  • the PD-1 antibody blocks the binding of human PD-L1 to human PD-1, and preferably blocks both human PD-L1 and PD-L2 from binding to human PD1 binding.
  • the human PD-1 amino acid sequence can be found at NCBI locus number: NP_005009.
  • Human PD-L1 and PD-L2 amino acid sequences can be found at NCBI locus numbers: NP_054862 and NP_079515, respectively.
  • anti-PD-1 antibody when referring to an "anti-PD-1 antibody”, unless otherwise specified or described, the term includes antigen-binding fragments thereof.
  • PD-L1 antibody refers to any chemical compound that binds to the PD-L1 receptor and blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T, B, NK cells) or Biomolecules.
  • Alternative nouns or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7-H1, B7H1, B7-4, CD274 and B7-H; and for PD-L2 PDCD1L2, PDL2, B7-DC and CD273.
  • the PD-L1 antibody blocks the binding of human PD-1 to human PD-L1.
  • anti-PD-L1 antibody when referring to an "anti-PD-L1 antibody”, unless otherwise specified or described, the term includes antigen-binding fragments thereof.
  • an “isotype” antibody refers to the class of antibody provided by the heavy chain constant region genes (eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4). Isotypes also include modified forms of one of these species, wherein modifications have been made to alter Fc function, eg, to enhance or reduce effector function or binding to Fc receptors.
  • heavy chain constant region genes eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4
  • epitope refers to the region of an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein.
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively).
  • KD equilibrium dissociation constant
  • kdis and kon association rate constant
  • Affinity can be measured by common methods known in the art.
  • One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • does not bind to a protein or cell means that it does not bind to a protein or cell, or does not bind to it with high affinity, i.e. binds the protein or cell with a KD of 1.0 x 10 -6 M or higher, more preferably 1.0 x 10 - 5 M or higher, more preferably 1.0 ⁇ 10 ⁇ 4 M or higher, 1.0 ⁇ 10 ⁇ 3 M or higher, more preferably 1.0 ⁇ 10 ⁇ 2 M or higher.
  • high affinity for IgG antibodies means a KD for antigen of 1.0x10-6 M or less, preferably 5.0x10-8 M or less, more preferably 1.0x10-8 M or lower, 5.0 ⁇ 10 ⁇ 9 M or lower, more preferably 1.0 ⁇ 10 ⁇ 9 M or lower.
  • high affinity binding may vary.
  • “high affinity” binding of an IgM subtype refers to a KD of 10-6 M or lower, preferably 10-7 M or lower, more preferably 10-8 M or lower.
  • antibody-dependent cytotoxicity refers to cell-mediated immune defense in which immune system effector cells actively associate cell membrane surface antigens with antibodies, such as Claudin18. 2 Antibodies that bind to target cells such as cancer cells are lysed.
  • CDC complement-dependent cytotoxicity
  • IgG and IgM antibodies that, when bound to surface antigens, initiate the canonical complement pathway, including formation of membrane attack complexes and lysis of target cells.
  • the antibodies of the present invention when bound to Claudin 18.2, trigger CDC against cancer cells.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless expressly limited, the term includes nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, in Kariko et al. Human US Patent No. 8,278,036, which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods of synthesizing such mRNA molecules, and methods for delivering therapeutic proteins in vivo).
  • nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complements thereof, as well as sequences explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construct refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), derived from Any source, capable of integrating with the genome or replicating autonomously, constitutes a polynucleotide molecule in which one or more polynucleotide molecules have been functionally linked (ie, operably linked).
  • the recombinant construct will typically comprise a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Expression of the nucleic acids of the invention can be directed using both heterologous and non-heterologous (ie, endogenous) promoters.
  • Vector refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell).
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors After introduction into the host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell.
  • Expression vectors contain one or more phenotypic selectable markers and origins of replication to ensure maintenance of the vector and to provide for amplification within the host if desired.
  • Activation can have the same meaning, eg, activation, stimulation, or treatment of a cell or receptor with a ligand, unless the context otherwise or clearly dictates.
  • Ligand includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules such as peptidomimetics of cytokines and peptidomimetics of antibodies.
  • Activation can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.
  • a “response/response”, eg, the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (eg, concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment), wherein changes Related to activation, stimulation, or processing, or to internal mechanisms such as genetic programming.
  • the terms “treating” or “treating” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (ie, slowing or arresting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treating” or “treating” refers to alleviating or ameliorating at least one physical parameter, including those physical parameters that may not be discernible by a patient. In another embodiment, “treating” or “treating” refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of physical parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
  • cyno or “cynomolgus monkey” refers to a cynomolgus monkey.
  • Administration "in combination with” one or more other therapeutic agents includes simultaneous (co) administration and sequential administration in any order.
  • a “therapeutically effective amount,” “therapeutically effective dose,” and “effective amount” means that an antibody or antigen-binding fragment or bifunctional protein of the invention is effective when administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents.
  • a therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • the therapeutically effective dose refers to that ingredient only.
  • a therapeutically effective dose refers to the combined amount of active ingredients that elicits a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter of at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%.
  • Cancer and “cancerous” refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Benign and malignant cancers as well as dormant tumors or micrometastases are included in this definition. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, cancer of the stomach or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, Cancer of salivary gland, kidney or kidney, liver, prostate, vulva, thyroid, liver, and various types of head and neck cancer, and B-cell lymphomas (including low-grade/follicular non-HoJ King's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small anucleate NHL, bulky disease (NHL), small lymph
  • the present invention provides bifunctional proteins targeting PD-1 or PD-L1 and TGF-beta, comprising
  • TGF- ⁇ RII extracellular domain ECD
  • the antigen-binding fragment of the antibody that blocks the PD-1/PD-L1 pathway of the present invention is an antigen-binding fragment of an anti-PD-1 or PD-L1 antibody.
  • the bifunctional protein of the invention is in the order of (i), (ii) and (iii); (iii), (i) and (ii); or ( The sequence of iii), (ii) and (i) is operatively linked; preferably, said (i) and (ii) are linked by a hinge region, and (i) and (iii) and (ii) and (iii) are connected by a linking peptide, the amino acid sequence of the linking peptide contains or consists of glycine residues and serine residues, and is 2-31 amino acid residues in length; preferably each independently is (GGGGS)nG , wherein n is independently selected from 3, 4, 5 or 6; preferably 4 or 5.
  • the bifunctional proteins of the present invention are operably linked in the order of (i), (ii) and (iii) from the N-terminus to the C-terminus, preferably, between said (i) and (ii) Connected by the hinge region, (ii) and (iii) are connected by a linker peptide, the amino acid sequence of the linker peptide contains or consists of glycine residues and serine residues, and has a length of 2-31 amino acid residues; preferably each independently is (GGGGS)nG, wherein n is each independently selected from 3, 4, 5 or 6; preferably 4 or 5.
  • the bifunctional proteins of the invention comprise an anti-PD-1 or PD-L1 antibody and one TGF- ⁇ RIIECD operably linked to the C-terminus of each of the two heavy chains of the antibody.
  • the bifunctional protein of the present invention is a heterotetrameric glycoprotein consisting of two bifunctional protein first subunits and two bifunctional protein second subunits that are disulfide-bonded.
  • the present invention adopts the monoclonal antibody targeting PD-1 or PD-L1 as the molecular part of the bifunctional protein that blocks the PD-1/PD-L1 pathway, so as to block the negative regulatory signal and restore the activity of T cells, thereby Enhance immune response.
  • the present invention adopts the extracellular domain of TGF- ⁇ RII as the part of the immunomodulatory molecule in the bifunctional protein to weaken the immune tolerance of cancer cells. Inhibiting the PD-1/PD-L1 pathway on the basis of targeting and neutralizing TGF- ⁇ in the tumor microenvironment can restore the activity of T cells, enhance the immune response, and more effectively inhibit the occurrence and development of tumors.
  • the bifunctional protein of the invention binds to PD-1 or PD-L1 with a dissociation constant (KD) of 10-8 M or less, eg, 10-9 M to 10-12 M; and Binds specifically to TGF ⁇ 1/TGF ⁇ 2/TGF ⁇ 3 molecules with a dissociation constant (KD) of 10 ⁇ 8 M or less, eg, 10 ⁇ 9 M to 10 ⁇ 12 M, respectively.
  • KD dissociation constant
  • the bifunctional protein of the present invention comprises an anti-PD-1 antibody and/or an antigen-binding fragment of an anti-PD-1 antibody, which can specifically bind to PD-1; or with an intact anti-PD-1 antibody and/or an antigen-binding fragment thereof Competes for binding to PD-1.
  • the "anti-PD-1 antibody or antigen-binding fragment thereof" of the present invention may include any anti-PD-1 antibody or antigen-binding fragment thereof described in the art.
  • the anti-PD-1 antibody may be a commercially available or a PD-1 antibody that has been disclosed in the literature. Including but not limited to, such as PD-1 antibody nivolumab, pembrolizumab, toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Cemiplimab, etc.
  • Antibodies can be monoclonal, chimeric, humanized, or fully human.
  • Antigen-binding fragments include Fab fragments with antigen-binding activity, Fab' fragments, F(ab')2 fragments, and antibody-binding Fv fragments and ScFv fragments.
  • the antigen-binding fragment of the anti-PD-1 antibody in the bifunctional protein of the present invention comprises a light chain with the heavy chain variable region shown in SEQ ID NO:7 and the amino acid sequence shown in SEQ ID NO:8
  • a chain variable region has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the antigen-binding fragment of the anti-PD-1 antibody of the present invention comprises Fab, Fab', F(ab')2, Fv, scFv or sdAb.
  • CDRs in the amino acid sequences of heavy and light chain variable regions are known in the art and can be used to identify specific heavy chain variable regions and/or light chains disclosed herein CDRs in the amino acid sequence of the variable region.
  • Exemplary well-known techniques that can be used to identify CDR boundaries include, for example, Kabat delineation, Chothia delineation, IMGT delineation, and AbM delineation. See, eg, Kabat, Sequences of Proteins of Immunoglobulins., National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins., J. Mol. Biol.
  • amino acid sequence of the antigen-binding fragment of the anti-PD-1 antibody constructed by the bifunctional protein JS-TZO3 of the present invention is as follows (the CDR boundary adopts the Kabat scheme):
  • Anti-PD-L1 antibodies and/or antigen-binding fragments of anti-PD-L1 antibodies are provided.
  • the bifunctional protein of the present invention comprises an anti-PD-L1 antibody and/or an antigen-binding fragment of an anti-PD-L1 antibody, which can specifically bind to PD-L1; or is combined with an intact anti-PD-L1 antibody and/or its antigen-binding fragment Competes for binding to PD-L1.
  • anti-PD-L1 antibody or antigen-binding fragment thereof may include any anti-PD-L1 antibody or antigen-binding fragment thereof described in the art.
  • Anti-PD-L1 antibodies may be commercially available or PD-L1 antibodies that have been disclosed in the literature.
  • the antibodies of the present invention may be monoclonal, chimeric, humanized, or fully human.
  • Antigen-binding fragments include Fab fragments with antigen-binding activity, Fab' fragments, F(ab')2 fragments, and antibody-binding Fv fragments and ScFv fragments.
  • the antigen-binding fragment of the anti-PD-L1 antibody of the present invention comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively.
  • the antigen-binding fragment of the anti-PD-L1 antibody of the present invention comprises at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 24 , 96%, 97%, 98%, 99% or 100% sequence identity heavy chain variable region, and with the amino acid sequence shown in SEQ ID NO: 25 has at least 90%, 91%, 92%, 93%, Light chain variable regions of 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the antigen-binding fragment of the anti-PD-L1 antibody of the present invention comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:24 and a light chain whose amino acid sequence is shown in SEQ ID NO:25 variable region.
  • the antigen-binding fragment of the anti-PD-L1 antibody of the present invention comprises Fab, Fab', F(ab')2, Fv, scFv or sdAb.
  • amino acid sequence of the antigen-binding fragment of the anti-PD-L1 antibody constructed by the bifunctional protein JS-TZO4 of the present invention is as follows (the CDR boundary adopts the IMGT scheme):
  • Amino acid sequence of heavy chain variable region of anti-PD-L1 antibody (SEQ ID NO:24)
  • Amino acid sequence of light chain variable region of anti-PD-L1 antibody (SEQ ID NO:25)
  • immunoglobulin Fc domain in the bifunctional protein of the invention comprises all or a portion of the amino acid residues of a naturally occurring immunoglobulin Fc domain.
  • the immunoglobulin Fc domain provides favorable pharmacokinetic properties for the bifunctional proteins of the invention, including, but not limited to, long serum half-life.
  • Immunoglobulin Fc domains are typically dimeric molecules that can be produced by papain or trypsin digestion of intact (full-length) immunoglobulins 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence set forth in SEQ ID NO:9 Amino acid sequences of %, 99% or more sequence identity.
  • the immunoglobulin Fc domain has the amino acid sequence set forth in SEQ ID NO:9.
  • the immunoglobulin Fc domain of the invention is the Fc domain of human IgGl, IgG2, IgG3 or IgG4; preferably the Fc domain of human IgG4.
  • the immunoglobulin Fc domain of the present invention further comprises a peptide sequence obtained by subjecting its amino acid sequence to one or more amino acid substitutions, deletions or derivatizations.
  • an amino acid substitution at position S228 in the IgG Fc domain is included that prevents the exchange of arms, particularly the amino acid substitution S228P.
  • a terminal amino acid deletion to prevent C-terminal cleavage is included in the IgG Fc domain, preferably two amino acid deletions of the C-terminal K and G.
  • amino acid sequence of the immunoglobulin Fc domain used for the construction of the bifunctional proteins JS-TZO3 and JS-TZO4 of the present invention can be selected from the following: (SEQ ID NO: 9)
  • the anti-PD-1 antibody of the present invention and the amino acid sequence shown in SEQ ID NO: 11 and/or SEQ ID NO: 12 have at least 90%, 91%, 92%, 93%, 94%, Amino acid sequences of 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the anti-PD-1 antibody of the present invention has the heavy chain amino acid sequence shown in SEQ ID NO: 11, and the light chain amino acid sequence shown in SEQ ID NO: 12.
  • the PD-1 antibody heavy chain sequence of the present invention for the construction of bifunctional protein JS-TZO3 can be selected from the following: (SEQ ID NO: 11)
  • the PD-1 antibody light chain sequence of the present invention for the construction of bifunctional protein JS-TZO3 can be selected from the following: (SEQ ID NO: 12)
  • the anti-PD-L1 antibody of the present invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 26 or 31 , 97%, 98%, 99% or 100% sequence identity heavy chain amino acids, and have at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence shown in SEQ ID NO: 27 , 96%, 97%, 98%, 99% or 100% sequence identity of light chain amino acids.
  • the anti-PD-L1 antibody of the present invention comprises the heavy chain amino acid sequence shown in SEQ ID NO: 26 or 31 and the light chain amino acid sequence shown in SEQ ID NO: 27.
  • the PD-L1 antibody heavy chain sequence of the present invention for the construction of bifunctional protein JS-TZO4 can be selected from the following: (SEQ ID NO:31)
  • the PD-L1 antibody light chain sequence of the present invention for the construction of bifunctional protein JS-TZO4 can be selected from the following: (SEQ ID NO:27)
  • the "extracellular domain (ECD) of TGF- ⁇ RII" in the bifunctional protein of the present invention comprises all amino acid residues of naturally occurring TGF- ⁇ RIIIECD or a part of amino acid residues of naturally occurring TGF- ⁇ RIIIECD.
  • the TGF- ⁇ RII extracellular domain of the present invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequences of %, 97%, 98%, 99% or more identity.
  • the TGF- ⁇ RII extracellular domain of the present invention has the amino acid sequence shown in SEQ ID NO:10.
  • the TGF- ⁇ RIIECD may also comprise peptide sequences obtained after additional sequence modifications to SEQ ID NO:10, such as amino acid residues in SEQ ID NO:10.
  • the peptide sequence obtained after one or more conservative substitutions, deletions or derivatizations is sufficient 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.
  • Modified peptides typically have amino acid sequences that are substantially homologous to the amino acid sequence of the unmodified sequence.
  • the amino acid sequence of the TGF- ⁇ RII extracellular domain constructed by the bifunctional protein JS-TZO3 or JS-TZO4 of the present invention is as follows:
  • TGF- ⁇ RII extracellular domain (1-136 polypeptide, SEQ ID NO: 10):
  • the present invention uses the linking peptide (G4S) 4G to link the PD1 or PD-L1 antibody heavy chain portion and the TGF ⁇ RII extracellular domain into a bifunctional protein.
  • the bifunctional protein of the present invention comprises the first subunit of the bifunctional protein whose amino acid sequence is shown in SEQ ID NO: 13, and the first subunit of the bifunctional protein whose amino acid sequence is shown in SEQ ID NO: 12. two subunits.
  • amino acid sequence of the first subunit of the bifunctional protein JS-TZO3 of the present invention is as follows: (heavy chain, SEQ ID NO: 13):
  • the amino acid sequence of the second subunit of the bifunctional protein JS-TZO3 of the present invention is identical to the light chain sequence of the PD-1 antibody, which is the amino acid sequence shown in SEQ ID NO: 12.
  • the bifunctional protein of the present invention comprises the first subunit of the bifunctional protein whose amino acid sequence is shown in SEQ ID NO:28, and the first subunit of the bifunctional protein whose amino acid sequence is shown in SEQ ID NO:27. two subunits.
  • amino acid sequence of the first subunit of the bifunctional protein JS-TZO4 of the present invention is as follows: (heavy chain, SEQ ID NO: 28):
  • the amino acid sequence of the second subunit of the bifunctional protein JS-TZO4 of the present invention is identical to the light chain sequence of the PD-L1 antibody, which is the amino acid sequence shown in SEQ ID NO: 27.
  • amino acid changes include amino acid deletions, insertions or substitutions.
  • the bifunctional proteins of the invention include those that have been mutated by amino acid deletion, insertion or substitution, but still have at least about 90%, 91%, 92%, 93%, 94%, Those bifunctional proteins with amino acid sequences of 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the polynucleotides encoding the antibodies of the invention include those that have been mutated by nucleotide deletions, insertions, or substitutions, but still have at least about 60% of the coding regions corresponding to the CDRs depicted in the sequences described above, Polynucleotides of 70%, 80%, 90%, 95% or 100% identity.
  • the bifunctional proteins provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl
  • the present invention provides a polynucleotide encoding a bifunctional protein as described herein.
  • the polynucleotide may comprise a polynucleotide encoding the amino acid sequence of the light chain variable region and/or heavy chain variable region of an antibody, or an amino acid encoding the first and/or second subunit of a bifunctional protein sequence of polynucleotides.
  • the polynucleotides of the present invention comprise a first subunit nucleotide sequence as set forth in SEQ ID NO:16 and/or a second subunit nucleoside as set forth in SEQ ID NO:17 acid sequence.
  • nucleotide sequence of the first subunit of the bifunctional protein JS-TZO3 of the present invention is as follows: (SEQ ID NO: 16):
  • nucleotide sequence of the second subunit of the bifunctional protein JS-TZO3 of the present invention is as follows: (SEQ ID NO: 17):
  • the polynucleotides of the present invention comprise a first subunit nucleotide sequence as set forth in SEQ ID NO:29 and/or a second subunit nucleoside as set forth in SEQ ID NO:30 acid sequence.
  • nucleotide sequence of the first subunit of the bifunctional protein JS-TZO4 of the present invention is as follows: (SEQ ID NO: 29):
  • nucleotide sequence of the second subunit of the bifunctional protein JS-TZO4 of the present invention is as follows: (SEQ ID NO:30):
  • the present invention provides an expression vector comprising the polynucleotide as described herein, preferably, the vector is a eukaryotic expression vector.
  • the polynucleotides as described herein are contained in one or more expression vectors.
  • the present invention provides a host cell comprising a polynucleotide as described herein or an expression vector as described herein, preferably the host cell is a eukaryotic cell, more preferably a mammalian cell .
  • the present invention provides a method for making a bifunctional protein as described herein, the method comprising expressing said host cell as described herein under conditions suitable for expression of said antibody antibody, and recover the expressed bifunctional protein from the host cell.
  • the invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese Hamster Ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, Monkey Kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
  • ATCC American Type Culture Collection
  • the present invention provides a method of making a bifunctional protein of the present invention, wherein the method comprises, when introducing an expression vector into a mammalian host cell, by culturing the host cell for a period of time sufficient to allow the bifunctional protein
  • the bifunctional protein is produced by expression in the host cell or, more preferably, by secretion of the bifunctional protein into the medium in which the host cell is grown.
  • Bifunctional proteins can be recovered from the culture medium using standard protein purification methods.
  • bifunctional proteins expressed by different cell lines or in transgenic animals are glycosylated differently from each other.
  • all bifunctional proteins encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibody.
  • afucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo, and are unlikely to be immunogenic , because their carbohydrate structure is a normal component of native human serum IgG.
  • compositions and pharmaceutical preparations are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, or a host cell as described herein, and a pharmacy an acceptable carrier or excipient.
  • bifunctional proteins or pharmaceutical compositions thereof provided by the present invention may incorporate suitable carriers, excipients and other agents in the formulation for co-administration, thereby providing improved transfer, delivery, tolerance, and the like.
  • composition refers to a formulation that allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered.
  • the compounds described herein can be prepared by mixing a bifunctional protein of the invention of the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)).
  • the pharmaceutical preparation of the bifunctional protein is preferably in the form of an aqueous solution or a lyophilized preparation.
  • compositions or formulations of the present invention may also contain one or more other active ingredients required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other .
  • the other active ingredient is a chemotherapeutic agent, an immune checkpoint inhibitor, a growth inhibitory agent, an antibiotic, or various anti-tumor or anti-cancer agents known, in a suitable amount effective for the intended use exist in combination.
  • compositions of the present invention further comprise compositions of polynucleotides encoding bifunctional proteins.
  • the present invention provides a pharmaceutical combination comprising a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a the described pharmaceutical composition, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or the pharmaceutical compositions described herein.
  • the present invention provides a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a medicament as described herein
  • a composition in the manufacture of a medicament for the treatment and/or prevention of a disease or condition associated with PD-1, PD-L1 or TGF- ⁇ activity preferably the disease or condition is cancer, more preferably, the The cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer and rectal cancer.
  • the present invention provides a bifunctional protein as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, and a pharmaceutical composition as described herein , which is used for the treatment and/or prevention of a disease or condition associated with PD-1, PD-L1 or TGF- ⁇ activity, preferably the disease or condition is cancer, more preferably the cancer is selected from melanoma, kidney cancer , prostate, breast, colon, lung, bone, pancreatic, skin, head and neck, uterine, ovarian and rectal cancers.
  • the present invention provides a method of treating and/or preventing a disease or disorder associated with PD-1, PD-L1 or TGF-beta activity, comprising administering to a subject in need thereof, as described herein
  • the cancer or tumor described herein can be selected from melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer cancer and rectal cancer.
  • modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (eg, in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration Wait.
  • the present invention provides for co-administration of a therapeutically effective amount of one or more therapies (eg, therapeutic modalities and/or other therapeutic agents) to a subject.
  • the therapy includes surgery and/or radiation therapy.
  • the methods or uses provided herein further comprise administering to an individual one or more therapies (eg, therapeutic modalities and/or other therapeutic agents).
  • therapies eg, therapeutic modalities and/or other therapeutic agents.
  • Antibodies of the invention can be used alone or in combination with other therapeutic agents in therapy. For example, it can be co-administered with at least one additional therapeutic agent.
  • PD-1/PD-L1 antibody, LAG-3 antibody and/or CTLA-4 antibody for example, PD-1/PD-L1 antibody, LAG-3 antibody and/or CTLA-4 antibody.
  • the present invention provides a method of detecting the presence of PD-1, PD-L1 or TGF-beta in a sample using a bifunctional protein as described herein.
  • detection includes quantitative or qualitative detection.
  • the sample is a biological sample.
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by their combination with other methods, and equivalent substitutions known to those skilled in the art
  • preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • the present invention adopts the following abbreviations:
  • ECD stands for extracellular domain
  • PEI stands for polyethyleneimine
  • BSA stands for bovine serum albumin
  • PBS stands for Phosphate Buffered Saline
  • FBS stands for fetal bovine serum
  • TMB stands for 3,3',5,5'-tetramethylbenzidine
  • PBST stands for Phosphate Buffered Saline with 0.02% Tween.
  • Nanjing GenScript to synthesize the nucleotide sequence encoding anti-human PD-1 antibody (the heavy chain amino acid sequence is shown in SEQ ID NO: 11, and the light chain amino acid sequence is shown in SEQ ID NO: 12), and restricted by BSPQI
  • the gene synthesis fragment 1A6H2 was ligated to the vector HXT4s by endonuclease to obtain the vector HXT4s-1A6H2.
  • Nanjing GenScript was entrusted to synthesize the nucleotide sequence encoding the extracellular domain (ECD) of TGF- ⁇ RII (its amino acid sequence is shown in SEQ ID NO: 10).
  • the nucleotide sequence of the linking peptide (G 4 S) 4 G) is obtained by means of techniques in the art.
  • the primers TGF ⁇ R2-G4SBSPQIFV2 and TGF ⁇ R2-BSPQIRV2 were synthesized, and the human TGF- ⁇ RII ECD gene fragment was obtained by PCR amplification.
  • the TGF- ⁇ RII ECD gene fragment was connected to the vector HXT4s-1A6H2 by BSPQI restriction endonuclease, and the expression vector HXT4s- 1A6H2-TGF ⁇ RII.
  • the expression vector HXT4s-1A6H2-TGF ⁇ RII expresses a bifunctional protein JS-TZO3, the amino acid sequence of the second subunit of the bifunctional protein JS-TZO3 is shown in SEQ ID NO:12, and the amino acid sequence of the first subunit is shown in SEQ ID NO:13 Show.
  • the nucleotide sequence (SEQ ID NO: 14) of the primer TGF ⁇ R2-G4SBSPQIFV2 in the 5'-3' order is as follows:
  • the nucleotide sequence (SEQ ID NO: 15) of the primer TGF ⁇ R2-BSPQIRV2 in 5'-3' order is as follows:
  • the 293F cells stored in liquid nitrogen (FreeStyle TM 293-F Cells, Thermofisher) were recovered and added to an expression medium (Freestyle TM 293 expression medium, Gibco) for culture. After several generations of culture, the 293F cells were diluted to (1.5-2.0) ⁇ 10 6 cells/mL one day before transfection. During transfection, 1/10 of the transfection volume of the above medium was used to incubate the expression vector HXT4s-1A6H2-TGF ⁇ RII and transfection reagent (PEI), the dosage of expression vector is 0.5-2 ⁇ g/mL, and the dosage of transfection reagent is 3-14 ⁇ g/mL. The incubation time is 20-30min.
  • the transfection mixture is added to the above-mentioned 293F cells treated one day in advance, and the mixture is shaken while adding.
  • the transfected shake flask was placed in a carbon dioxide shaker for incubation at 36.5° C., 120 rpm, and 7% CO 2 . After 7 days, the supernatant was collected by centrifugation for purification.
  • AKTA AVANT instrument for purification, use protein A affinity packing (MabSelect SuRe TM , GE Healthcare) pre-packed purification column, the above supernatant is centrifuged and filtered through a 0.22 ⁇ m filter before loading, washed with PBS, lemon Acid buffer was used for elution, and after elution was completed, Tris buffer was neutralized to obtain bifunctional protein JS-TZO3.
  • the prepared bifunctional protein JS-TZO3 can be used in the following examples.
  • Example 2 ELISA detects the binding of bifunctional protein JS-TZO3 to human TGF- ⁇
  • ELISA method was used to detect the binding of the bifunctional protein JS-TZO3 of the present invention to human TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3.
  • HRP horseradish peroxidase
  • the bifunctional protein JS-TZO3 of the present invention has strong binding to TGF- ⁇ 1 and TGF- ⁇ 3, and relatively weak binding to TGF- ⁇ 2.
  • Biacore detects the affinity of bifunctional protein JS-TZO3 and TGF- ⁇
  • the affinity of the bifunctional protein JS-TZO3 of the present invention with human TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3 was detected by Biacore T200 instrument (GE Healthcare Life Sciences).
  • the method for determining the binding affinity of the bifunctional protein of the present invention to human TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3 is as follows: use a Protein A chip (GE Healthcare Life Sciences) to capture 4 ⁇ g/ml of the bifunctional protein, and then flow over the surface of the chip Serial dilutions of human TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3 (nearshore).
  • the dilution gradient of TGF- ⁇ 1 was 1.14nM, 0.76nM, 0.5057nM, 0.3378nM, 0.2252nM, 0.1501nM, of which 1.14nM was repeated injection.
  • the dilution gradient of TGF- ⁇ 2 was 1.8nM, 1.2nM, 0.8nM, 0.5333nM, 0.3556nM, 0.2370nM, of which 1.2nM was repeated injection.
  • the dilution gradient of TGF- ⁇ 3 was 1.2nM, 0.8nM, 0.5333nM, 0.3556nM, 0.2370nM, of which 1.2nM was repeated injection.
  • association and dissociation signals were measured using a Biacore T200 system (GE Healthcare) to obtain association and dissociation kinetic curves. Affinity KD values were determined by fitting binding dissociation curves using the software Biacore T200 Evaluation Software 3.0.
  • the bifunctional protein JS-TZO3 of the present invention has strong affinity with TGF ⁇ 1 and TGF ⁇ 3, and has a slightly weaker binding affinity with TGF ⁇ 2.
  • Example 4 ELISA detects the binding of bifunctional protein JS-TZO3 to PD-1
  • the binding of the bifunctional protein JS-TZO3 of the present invention to PD-1 was detected by ELISA.
  • HRP horseradish peroxidase
  • the bifunctional protein JS-TZO3 of the present invention has strong binding to PD-1, which is equivalent to the positive control antibody Toripalimab.
  • the affinity between the bifunctional protein JS-TZO3 of the present invention and the human PD-1 protein was detected using Biacore T200 instrument (GE Healthcare Life Sciences).
  • the method for determining the binding affinity of the bifunctional protein of the present invention to PD-1 is as follows: 40 ⁇ g/ml goat anti-human IgG-Fc fragment antibody (Jackson Immuno Research) is coupled to a CM5 chip (GE Healthcare Life Sciences, item number BR-1005-30 ) surface for capturing antibodies. 5 ⁇ g/ml of bifunctional protein was then captured on the surface of the CM5 chip.
  • the human PD-1 protein (Junshi Bio) was serially diluted (140nM, 70nM, 35nM, 17.5nM, 8.75nM, 4.375nM, of which 140nM was repeated injection), and the human PD-1 was injected and flowed through CM5 chip, binding and dissociation signals were measured using a Biacore T200 system (GE Healthcare). Affinity KD values were determined by fitting binding dissociation curves using the software Biacore T200Evaluation Software 3.0.
  • the bifunctional protein JS-TZO3 of the present invention has strong affinity with PD-1 protein.
  • Example 6 Detection of PD-1/PD-L1 pathway blocking test by bifunctional protein JS-TZO3 by reporter gene method
  • the PD-1/PD-L1 reporter gene system constructed by Promaga was used to detect PD in the bifunctional protein of the present invention.
  • CHO/PD-L1 cells (Promega) were taken, digested and resuspended in F-12 Nutrient Mixture complete medium, and the cell density was adjusted to 5 ⁇ 10 5 cells/ml using complete medium according to the cell count results.
  • Transfer to the sample addition tank add 100 ⁇ l/well to a 96-well plate using a multi-channel pipette, and place it in a 37°C, 5% CO 2 incubator for 16-20 hours; prepare Jurkat/PD-1 ( Promega) cell suspension, resuspend the cells in assay medium (RPMI 1640 Medium+2% FBS) according to the cell count results, and adjust the cell density to 2.5 ⁇ 10 6 cells/ml; add CHO/PD-L1 cells Remove the cell culture plate from the incubator, and use a multichannel pipette to remove 100 ⁇ l of culture medium per well.
  • the bifunctional protein JS-TZO3, the positive control PD-1 monoclonal antibody (Toripalimab, Junshi Biotechnology) and the negative control antibody (Anti-KLH IgG4) were added in a gradient dilution (initial concentration of 1650 nM) according to 40 ⁇ l/well. Then, the above Jurkat/PD-1 cell suspension was transferred to the sample addition tank, 40 ⁇ l/well was added to the cell culture plate, and placed in a 37° C., 5% CO 2 incubator for 5-6 h. During protein incubation, One-Glo reagent (Promega, Cat. No. E6130) was removed and allowed to return to room temperature.
  • both the bifunctional protein JS-TZO3 of the present invention and the anti-PD-1 monoclonal antibody Toripalimab can effectively block the interaction of PD-1/PD-L1 and promote the enhancement of NFAT signal.
  • the EC 50 values are respectively 11.01nM and 10.4nM, there is a significant dose-dependent effect of drug concentration, and the activity levels of the two are similar.
  • Example 7 Detection of TGF ⁇ pathway blocking test by bifunctional protein JS-TZO3 by reporter gene method
  • 4T1-SBE cells (Junshi Bio) were cultured in 1640 complete medium (Gibco, 11875-093) containing 10% FBS and passaged every 2 days. On the first day of the experiment, the cells were seeded at a density of 100,000 cells per well in a 96-well plate (Costar, 3917) and cultured at 37°C and 5% CO 2 for 16-20 hours. The next day, the medium in the cell culture plate was discarded, and 50 ⁇ l/well of bifunctional protein and positive control G4Fc TGF ⁇ R2ecd (TGF ⁇ Trap, Junshi Bio) were added at a gradient dilution (initial concentration of 1650nM).
  • both the bifunctional protein JS-TZO3 of the present invention and G4Fc TGF ⁇ R2ecd can inhibit the activation of pSMAD3 induced by TGF ⁇ 1 in a dose-dependent manner.
  • the endocytosis activity of the bifunctional protein JS-TZO3 of the present invention was detected by antibody endocytosis assay at the in vitro level.
  • CypHer 5 Mono NHS Ester dye GE, PA15401
  • Endocytosis experiment (co-incubation of antibody and target cells): (At this step, attention should be paid to the temperature, antibody endocytosis can be observed at 37 °C, and antibody endocytosis can be inhibited at 4 °C) Take a 96-well ultra-low adsorption U bottom plate, and spread the cells on 96 In the well plate, add 50 ⁇ l of cell suspension to each well with a multi-channel pipette (the number of cells in each well is 1 ⁇ 10 5 cells/well); Functional protein, PD-1 mAb (Toripalimab) and negative control (Anti-KLH IgG4) were incubated with cells for 4 h (37°C) in a CO 2 incubator.
  • the 96-well cell plate was taken out, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded; then the cells were resuspended with staining buffer (PBS+1%BSA), and centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the operation was repeated twice to wash off.
  • staining buffer PBS+1%BSA
  • 150ul of the above staining buffer was added to each well of a 96-well cell plate, and FACS was used for on-machine detection and data analysis was performed with FlowJo software.
  • both the bifunctional protein JS-TZO3 of the present invention and the anti-PD-1 monoclonal antibody (Toripalimab) have strong endocytosis activities, and the effect is dose-dependent.
  • Example 9 In vitro mixed lymphocyte reaction of bifunctional protein JS-TZO3
  • the present invention studies the release level of IL2 and IFN ⁇ cytokines after the activation of T cells by the bifunctional protein JS-TZO3 of the present invention by using the mixed lymphocyte reaction experiment at the in vitro level.
  • PBMCs Commercial human PBMCs (SAILY BIO, cat#SLB-HP050B) were incubated with serum-containing medium at 37°C for 2h, and the suspended cells were washed away, and then 10ng/ml of GM-CSF (Sino Biological, cat#GMP) was used respectively.
  • GM-CSF Seo Biological, cat#GMP
  • the bifunctional protein JS-TZO3 and PD-1 monoclonal antibody of the present invention and the combination group of PD-1 monoclonal antibody and TGF ⁇ Trap can promote the release of IL-2, but the difference is different. Not obvious (Fig. 9a).
  • the bifunctional protein JS-TZO3 of the present invention can significantly promote the release of IFN ⁇ , and the activity is significantly higher than that of the PD-1 monoclonal antibody combined with TGF ⁇ Trap group and the PD-1 monoclonal antibody group (Fig. 9b).
  • Example 10 Inhibitory effect of bifunctional protein JS-TZO3 on human melanoma A375 tumor growth in mice
  • mice 18 6-7 week old female NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were taken, and the mice were subcutaneously inoculated with pre-mixed 4.5 x 10 6 (pcs/0.1 mL) A375 cells (ATCC CRL) on the right back. -1619) and 1 x 10 6 (cells/0.1 mL) of activated T cells (#250PBMC donor, Crown Biotech (Taicang) Co., Ltd.) in suspension (0.2 ml/cell). Dosing started 1 hour after cell seeding. The day of tumor cell inoculation was defined as day 0. According to body weight, they were divided into 3 groups with 6 animals in each group, namely:
  • TGF ⁇ Trap control group (G4Fc TGF ⁇ R2ecd), 10mg/kg;
  • PD-1 monoclonal antibody (Toripalimab) control group 10mg/kg;
  • Bifunctional protein (JS-TZO3) treatment group 10mg/kg.
  • TGI% tumor inhibition rate
  • the mean tumor volume of the TGF ⁇ Trap control group (G4Fc TGF ⁇ R2ecd) was 2009 ⁇ 217 mm 3 on day 31 after the start of dosing.
  • the mean tumor volumes of the PD-1 monoclonal antibody (Toripalimab) control group and the bifunctional protein (JS-TZO3) group were 579 ⁇ 217 mm 3 and 404 ⁇ 123 mm 3 , respectively.
  • the tumor inhibition rates were 71.17%, respectively. and 79.89%.
  • the humanized antibody JS-TZO3 which dual-targets PD-1 and TGF ⁇ R2ecd, can significantly inhibit the growth of human melanoma A375 cells subcutaneously transplanted at a dose level of 10 mg/kg, and is better than PD-1 monoclonal antibody (Toripalimab) and TGF ⁇ Trap control (G4Fc TGF ⁇ R2ecd).
  • Example 11 Stability evaluation of bifunctional protein JS-TZO3 of the present invention
  • the thermal stability of the bifunctional protein JS-TZO3 of the present invention was detected. Using QPCR-7500, in pH 5.2 buffer system (20 mM acetic acid-sodium acetate/50 mM sodium chloride/150 mM mannose), the stability of bifunctional proteins at different temperatures was investigated.
  • the sample was replaced with the above-mentioned buffer, and the concentration of the sample was controlled to be about 10 mg/ml, and the detection was performed by QPCR-7500.
  • the results are shown in Table 3.
  • the bifunctional protein JS-TZO3 of the present invention exhibits good thermal stability.
  • Tm thermal transition temperature
  • bifunctional protein JS-TZO3 Put the bifunctional protein in JS-TZO3 and change it to a buffer system of pH 5.2 (20mM acetic acid-sodium acetate/50mM sodium chloride/150mM mannitol, control the sample concentration at about 10mg/ml, and dispense a certain sample volume (200 ⁇ l/tube). ) place 40 °C of incubators, investigate the stability of 0W, 1W, 2W and 4W, and send samples to detect according to the time point.Testing result shows that bifunctional protein JS-TZO of the present invention has good thermal stability, specifically sees Table 4 and Table 5 .
  • NA means not detected.
  • the human antibody heavy chain IgG4 constant region gene fragment was synthesized, and the IgG4 constant region gene fragment was ligated to the pCDNA3.1 vector by EcoRI and NotI restriction enzymes to obtain the vector HXT4s.
  • Nanjing GenScript was entrusted to synthesize the nucleotide sequence encoding the anti-human PD-L1 antibody, and the gene synthesis fragment 9B1HC2 was linked to the vector HXT4s through BSPQI restriction endonuclease to obtain the vector HXT4s-9B1HC2.
  • Nanjing GenScript was commissioned to synthesize the nucleotide sequence encoding the extracellular domain (ECD) of TGF- ⁇ RII.
  • the nucleotide sequence of the linking peptide ((G 4 S) 4 G) is obtained by means of techniques in the art.
  • the nucleotide sequence encoding the anti-human PD-L1 antibody, the nucleotide sequence encoding the extracellular region of TGF- ⁇ RII, and the nucleotide sequence of the adaptor protein fragment ((G 4 S) 4 G) were obtained by means of techniques in the art.
  • the primers TGF ⁇ R2-G4SBSPQIFV2 and TGF ⁇ R2-BSPQIRV2 were synthesized, and the human TGF ⁇ R2 was used as a template for PCR amplification to obtain the human TGF- ⁇ RII ECD gene fragment, and the TGF- ⁇ RII ECD gene fragment was connected to the vector HXT4s-9B1HC2 by BSPQI restriction endonuclease, The expression vector HXT4s-9B1HC2-TGF ⁇ RII was obtained.
  • the nucleotide sequence (SEQ ID NO: 14) of primer TGF ⁇ R2-G4SBSPQIFV2 in 5'-3' order is as follows:
  • the nucleotide sequence (SEQ ID NO: 15) of primer TGF ⁇ R2-BSPQIRV2 in 5'-3' order is as follows:
  • the amino acid sequence of the anti-human PD-L1 antibody used for the construction of the bifunctional protein JS-TZO4 is shown in SEQ ID NO: 31 (heavy chain) and SEQ ID NO: 27 (light chain); the amino acid sequence of the extracellular region of TGF- ⁇ RII As shown in SEQ ID NO: 10; expression vector HXT4s-1A6H2-TGF ⁇ RII expresses bifunctional protein JS-TZO4, the second subunit amino acid sequence of this bifunctional protein JS-TZO4 is as SEQ ID NO:27, and its encoding nucleic acid is as SEQ ID NO: 27 Shown in ID NO: 30; its first subunit amino acid sequence is shown in SEQ ID NO: 28, and its encoding nucleic acid is shown in SEQ ID NO: 29.
  • the 293F cells stored in liquid nitrogen (FreeStyle TM 293-F Cells, Thermofisher) were recovered and added to an expression medium (Freestyle TM 293 expression medium, Gibco) for culture. After several generations of culture, the 293F cells were diluted to (1.5-2.0) ⁇ 10 6 cells/mL one day before transfection. During transfection, 1/10 of the transfection volume of the above medium was used to incubate the expression vector HXT4s-9B1HC2-TGF ⁇ RII and transfection reagent (PEI), the dosage of expression vector is 0.5-2 ⁇ g/mL, and the dosage of transfection reagent is 3-14 ⁇ g/mL. The incubation time is 20-30min.
  • the transfection mixture is added to the above-mentioned 293F cells treated one day in advance, and the mixture is shaken while adding.
  • the transfected shake flask was placed in a carbon dioxide shaker for incubation at 36.5° C., 120 rpm, and 7% CO 2 . After 7 days, the supernatant was collected by centrifugation for purification.
  • AKTA AVANT instrument for purification, use protein A affinity packing (MabSelect SuRe TM , GE Healthcare) pre-packed purification column, the above supernatant is centrifuged and filtered through a 0.22 ⁇ m filter before loading, washed with PBS, lemon Acid buffer was used for elution, and after elution was completed, Tris buffer was neutralized to obtain bifunctional protein JS-TZO4.
  • the prepared bifunctional protein JS-TZO4 can be used in the following examples.
  • Example 13 ELISA detects the binding of bifunctional protein JS-TZO4 to human TGF- ⁇
  • the binding of the bifunctional protein JS-TZO4 of the present invention to human TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3 was detected by ELISA method.
  • HRP horseradish peroxidase
  • the bifunctional protein JS-TZO4 of the present invention has strong binding to TGF- ⁇ 1 and TGF- ⁇ 3, and relatively weak binding to TGF- ⁇ 2.
  • Example 14 ELISA detects the binding of bifunctional protein JS-TZO4 to PD-L1
  • bifunctional protein JS-TZO4 The binding of bifunctional protein JS-TZO4 to PD-L1 was detected by ELISA.
  • HRP horseradish peroxidase
  • Positive control anti-PD-L1 monoclonal antibody from Junshi Biotechnology (patent application number PCT/CN2018/076669), the heavy chain is shown in SEQ ID NO:26, and the light chain is shown in SEQ ID NO:27.
  • the bifunctional protein JS-TZO4 of the present invention has strong binding to PD-L1, which is equivalent to that of the positive control antibody.
  • Example 15 Detection of PD-1/PD-L1 pathway blocking test by bifunctional protein JS-TZO4 by reporter gene method
  • the PD-1/PD-L1 reporter gene system constructed by Promaga was used to detect the cells at the PD-L1 end of the bifunctional protein. biological activity.
  • CHO/PD-L1 cells (Promega) were taken, digested and resuspended in F-12 Nutrient Mixture complete medium, and the cell density was adjusted to 5 ⁇ 10 5 cells using complete medium according to cell count results /ml, transfer the cell suspension to the sample addition tank, add 100 ⁇ l/well to a 96-well plate using a multi-channel pipette, and place it at 37°C in a 5% CO 2 incubator for 16-20 hours; the next day Prepare Jurkat/PD-1 (Promega) cell suspension, resuspend cells in assay medium based on cell count results, and adjust cell density to 2.5 ⁇ 10 6 cells/ml; add CHO/PD-L1 cells to cell culture The plate was taken out from the incubator, and 100 ⁇ l of culture medium was taken out from each well using a multi-channel pipette, and 40 ⁇ l/well was added to the bifunctional protein JS-TZO4 (initial concentration of 16.5 nM, 3-fold gradient dilution of 10 concentration
  • Positive control PD-L1 monoclonal antibody from Junshi Biotechnology (patent application number PCT/CN2018/076669), the heavy chain is shown in SEQ ID NO:26, and the light chain is shown in SEQ ID NO:27.
  • both the bifunctional protein JS-TZO4 of the present invention and the anti-PD-L1 monoclonal antibody can effectively block the interaction of PD-1/PD-L1, promote the enhancement of NFAT signal, and have significant drug concentration and dosage Depending on the effect, the activity levels of the two are comparable.
  • Example 16 Reporter gene method to detect bifunctional protein JS-TZO4 blocking test of TGF ⁇ pathway
  • Smad3-binding element SBE
  • 4T1-SBE cells (Junshi Bio) were cultured in 1640 complete medium (Gibco, 11875-093) containing 10% FBS and passaged every 2 days. On the first day of the experiment, the cells were seeded at a density of 100,000 cells per well in a 96-well plate (Costar, 3917) and cultured at 37°C and 5% CO 2 for 16-20 hours. The next day, the medium in the cell culture plate was discarded, and the bifunctional protein JS-TZO4 and the positive control G4Fc TGF ⁇ R2ecd ( TGF ⁇ Trap, Junshi Biotechnology). Cells were further cultured for 0.5 to 1 hour at 37°C and 5% CO 2 .
  • both the bifunctional protein JS-TZO4 of the present invention and G4Fc TGF ⁇ R2ecd can inhibit the activation of pSMAD3 induced by TGF ⁇ 1 in a dose-dependent manner.
  • Antibody endocytosis assay was used to detect the endocytic activity of bifunctional protein JS-TZO4 in vitro.
  • CypHer 5Mono NHS Ester dye-labeled samples to be tested the samples to be tested (bifunctional protein JS-TZO4, anti-PD-L1 mAb and Negative control anti-KLH IgG4) concentrations were adjusted to 1.0 mg/ml. Calculate the mass of the sample to be tested, add CypHer 5Mono NHS Ester dye (GE, PA15401), and mix for 1 h at room temperature in the dark. Transfer the reacted sample to be tested and the dye mixture to a 0.5ml ultrafiltration tube (30KD), centrifuge for 10 minutes, discard the waste liquid, add Label buffer to the ultrafiltration tube, and continue centrifugation for 4 times as above. the excess dye is washed away.
  • Endocytosis experiment (co-incubation of the sample to be tested and target cells): (At this step, attention should be paid to the temperature. Antibody endocytosis occurs at 37°C, and antibody endocytosis can be inhibited at 4°C) Take a 96-well ultra-low adsorption U bottom plate, and place the cells on the bottom.
  • a multi-channel pipette to add 50 ⁇ l of cell suspension to each well (the number of cells in each well is 1 ⁇ 10 5 cells/well); JS-TZO4: starting concentration of 55.56nM, 3-fold serial dilution for 12 concentration points; anti-PD-L1 mAb and Anti-KLH IgG4: starting concentration of 66.7nM, 3-fold serial dilution for 12 concentration points) and cells Incubate for 4 h in a CO2 incubator.
  • the 96-well cell plate was taken out, centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded; then the cells were resuspended with staining buffer (PBS+1%BSA), and centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the operation was repeated twice to wash off.
  • staining buffer PBS+1%BSA
  • 150 ⁇ l of staining buffer was added to each well of a 96-well cell plate, which was detected by FACS and analyzed by FlowJo software.
  • Positive control PD-L1 monoclonal antibody Junshi Biotechnology (patent application number PCT/CN2018/076669), the heavy chain is shown in SEQ ID NO:26, and the light chain is shown in SEQ ID NO:27.
  • both the bifunctional protein JS-TZO4 of the present invention and the anti-PD-L1 monoclonal antibody have strong endocytosis activities, and the effect is dose-dependent.
  • Example 18 In vitro mixed lymphocyte reaction of bifunctional protein JS-TZO4
  • the present invention studies the release level of IL2 and IFN ⁇ cytokines after the activation of T cells by the bifunctional protein JS-TZO4 by using the mixed lymphocyte reaction experiment at the in vitro level.
  • PBMCs Commercial human PBMCs (SAILY BIO, cat#SLB-HP050B) were incubated with serum-containing medium at 37°C for 2h, and the suspended cells were washed away, and then 10ng/ml of GM-CSF (Sino Biological, cat#GMP) was used respectively.
  • GM-CSF Seo Biological, cat#GMP
  • Biological control group PD-L1 monoclonal antibody group and PD-L1 monoclonal antibody combined with TGF ⁇ Trap group (PD-L1 antibody + TGF ⁇ Trap control group) were treated with a constant concentration (100ng/ml) of TGF ⁇ 1 recombinant protein (novoprotein, cat# CA59) as well as mDC cells (10,000 cells per well) and purified CD4 T cells (100,000 cells per well) were co-incubated for 5 days in a CO incubator at 37°C.
  • TGF ⁇ 1 recombinant protein novoprotein, cat# CA59
  • mDC cells 10,000 cells per well
  • CD4 T cells 100,000 cells per well
  • PD-L1 monoclonal antibody Junshi Biotechnology (patent application number PCT/CN2018/076669), the heavy chain is shown in SEQ ID NO:26, and the light chain is shown in SEQ ID NO:27.
  • the bifunctional protein JS-TZO4, PD-L1 monoclonal antibody and PD-L1 monoclonal antibody of the present invention in combination with TGF ⁇ Trap can all promote the release of IL-2, but the difference is Not obvious (Fig. 19a).
  • the bifunctional protein JS-TZO4 of the present invention can significantly promote the release of IFN ⁇ , and the activity is comparable to the PD-L1 monoclonal antibody combined with TGF ⁇ Trap group and the PD-L1 monoclonal antibody group (Fig. 19b).
  • Example 19 Stability evaluation of bifunctional protein JS-TZO4 of the present invention
  • the sample was replaced with the above-mentioned buffer, and the concentration of the sample was controlled to be about 10 mg/ml, and the detection was performed by QPCR-7500. See Table 6 for details.
  • the bifunctional protein JS-TZO4 of the present invention exhibits good thermal stability.
  • Tm thermal transition temperature
  • Stability was assessed by the following parameters: (a) SEC-HPLC (size exclusion chromatography) to measure the content of antibody monomers, aggregates or fragments; (b) CE-SDS (sodium dodecyl sulfate capillary electrophoresis) detection The molecular weight of the antibody; (c) ELISA and reporter gene method to detect the biological activity of the antibody.
  • SEC-HPLC size exclusion chromatography
  • NA means not detected.

Abstract

提供了一种靶向PD-1或PD-L1和TGF-β的双功能蛋白及其医药用途。所述双功能蛋白包含(i)抗PD-1或PD-L1抗体的抗原结合片段；(ii)免疫球蛋白Fc结构域；和(iii)TGF-βRⅡ胞外结构域(ECD)，所述双功能蛋白与PD-1或PD-L1蛋白有较强的亲和力，具有良好的热稳定性，能够显著促进IFNγ的释放和抑制肿瘤的生长。

Description

靶向PD-1或PD-L1和TGF-β的双功能蛋白及其医药用途 技术领域
本发明涉及医药生物技术领域。具体地，本发明涉及靶向程序性死亡蛋白-1(PD-1)/程序性死亡蛋白配体-1(PD-L1)和转化生长因子-β(TGF-β)的双功能蛋白、编码本发明双功能蛋白的多核苷酸、用于表达本发明双功能蛋白的载体和宿主细胞、以及本发明双功能蛋白在治疗、预防和/或诊断与PD-1或PD-L1活性和TGF-β家族活性相关的疾病中的用途。
背景技术
免疫检查点(immune checkpoint)是免疫***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。研究发现，肿瘤细胞能够逃避体内免疫***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。
程序性死亡受体1(programmed death 1，PD-1)为CD28超家族成员。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表达于活化的T细胞，B细胞及髓系细胞，其有两个配体，即程序性死亡配体-1(programmed death ligand 1，PD-L1)和程序性死亡配体-2(PD-L2)。PD-L1与T细胞上的受体PD-1相互作用，在免疫应答的负调控方面发挥着重要作用。在许多人类肿瘤组织中均可检测到PD-L1蛋白的表达，肿瘤部位的微环境可诱导肿瘤细胞上的PD-L1的表达，表达的PD-L1有利于肿瘤的发生和生长，诱导抗肿瘤T细胞的凋亡。PD-1/PD-L1通路抑制剂可以阻断PD-1与PD-L1的结合，阻断负向调控信号，使T细胞恢复活性，从而增强免疫应答，因此，以PD-1/PD-L1为靶点的免疫调节对肿瘤抑制有重要的意义。
程序性细胞死亡蛋白配体1(Programmed death-ligand 1，PD-L1)，又可称为分化簇274(cluster of differentiation 274，CD274)或者B7同源蛋白1(B7homolog1，B7-H1)，属于肿瘤坏死因子超家族，是由290个氨基酸残基组成的I型跨膜糖蛋白，包含一个IgV样区、一个IgC样区、一个跨膜疏水区和一个30个氨基酸的胞内尾部，完整分子量为40kDa1。PD-L1mRNA在几乎所有组织中都有表达，但PD-L1蛋白只在少部分组织中持续表达，包括肝脏、肺脏、扁桃体以及免疫特赦组织如眼、胎盘等。PD-L1也表达于活化的T细胞，B细胞，单核细胞，树突状细胞，巨噬细胞等。PD-L1的受体为PD-1，主要表达于 CD4+T细胞、CD8+T细胞、NKT细胞、B细胞和活化的单核细胞等免疫细胞表面。
转化生长因子-β(transforming growth factor-β，TGF-β)属于调节细胞生长和分化的TGF-β超家族。TGF-β通过异源四聚体受体复合物传递信号，这个受体复合物是由两个I型和两个II型的跨膜丝氨酸/苏氨酸激酶受体组成。
转化生长因子-β(TGFβ)是一种多效性细胞因子，其具有免疫调控性质，例如炎性和过敏性免疫应答的限制和终止。TGFβ与炎性、恶性、感染性和自体免疫疾病以及骨质疏松症和纤维化包括肝硬化和***性硬化症具有一定联系。具体来说，肿瘤中持续高水平的TGFβ与免疫耐受、血管发生、转移和肿瘤细胞外基质沉积的增加相关，所有这些都可能驱动癌症发展和对疗法的抗性。
在免疫微环境中，通过靶向PD-1或PD-L1的抗体阻断PD-1/PD-L1通路，用于阻断负向调控信号，使T细胞恢复活性，从而增强免疫应答，更有效地提高抑治肿瘤发生和发展的效果。
尽管目前已有抗体/TGF-β受体融合蛋白公开，但一些融合蛋白仍存在不稳定或表达量不高的问题，仍需要在各方面包括稳定性和活性相比于已知抗体改善的新型的融合蛋白。
发明内容
本发明提供了靶向程序性死亡蛋白-1(PD-1)/程序性死亡蛋白配体1(PD-L1)和转化生长因子-β(TGF-β)的双功能蛋白以及其在治疗、预防和/或诊断与PD-1活性和TGF-β活性相关的疾病中的用途。本发明提供的双功能蛋白可作为独立的疗法或与其它疗法/或其他抗癌药剂联合，用于诸如癌症的治疗。
在一个方面，本发明提供了靶向PD-1或PD-L1和TGF-β的双功能蛋白，其包含
(i)阻断PD-1/PD-L1通路的抗体的抗原结合片段；
(ii)免疫球蛋白Fc结构域；和
(iii)TGF-βRⅡ胞外结构域(ECD)。
在一些实施方式中，本发明所述阻断PD-1/PD-L1通路的抗体的抗原结合片段为抗PD-1或PD-L1抗体的抗原结合片段。
在一些实施方式中，本发明所述抗PD-1抗体的抗原结合片段包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述抗PD-1抗体的抗原结合片段包含氨基酸序列如SEQ  ID NO:7所示的重链可变区和氨基酸序列如SEQ ID NO:8所示的轻链可变区，或与SEQ ID NO:7所示的重链可变区如SEQ ID NO:8所示的轻链可变区的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，本发明所述抗PD-L1抗体的抗原结合片段包含氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述抗PD-L1抗体的抗原结合片段包含与SEQ ID NO:24所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链可变区，和与SEQ ID NO:25所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链可变区。
在一些实施方式中，本发明所述抗PD-L1抗体的抗原结合片段包含氨基酸序列如SEQ ID NO:24所示的重链可变区和氨基酸序列如SEQ ID NO:25所示的轻链可变区。
在一些实施方式中，本发明所述抗PD-1或PD-L1抗体的抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
在一些实施方式中，本发明所述免疫球蛋白Fc结构域是人IgG1、IgG2、IgG3或IgG4的Fc结构域；优选为人IgG4的Fc结构域；优选地，所述IgG4的Fc结构域中包含1、2、3、4或5个氨基酸差异，优选为在Fc结构域中具有S228P氨基酸置换；进一步优选为在Fc结构域中具有C末端K和G两个氨基酸缺失。
在一些实施方式中，本发明所述免疫球蛋白Fc结构域包含如SEQ ID NO:9所示的氨基酸序列，或与SEQ ID NO:9所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的Fc结构域。
在一些实施方式中，本发明所述的双功能蛋白，其中所述免疫球蛋白Fc结构域包含与SEQ ID NO:9所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的Fc结构域。
在一些实施方式中，本发明所述免疫球蛋白Fc结构域包含如SEQ ID NO:9所示的氨基酸序列。
在一些实施方式中，本发明所述TGF-βRⅡ胞外结构域具有SEQ ID NO:10所示的氨基酸序列，或与SEQ ID NO:10所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，本发明所述TGF-βRⅡ胞外结构域包含与SEQ ID NO:10所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、99％或100％序列同一性的氨基酸序列。
在一些实施方式中，本发明所述TGF-βRⅡ胞外结构域包含如SEQ ID NO:10所示的氨基酸序列。
在一些实施方式中，本发明所述双功能蛋白从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；优选地，所述(i)和(ii)之间通过铰链区连接，(i)和(iii)以及(ii)和(iii)之间通过连接肽连接，所述连接肽的氨基酸序列含有甘氨酸(G)残基和丝氨酸(S)残基或由甘氨酸残基和丝氨酸残基组成，长度为2-31个氨基酸残基；优选各自独立为(GGGGS)nG，其中，n分别独立地选自3、4、5或6；优选为4或5。
在一些实施方式中，本发明所述双功能蛋白从N端至C端以(i)、(ii)和(iii)的顺序有效连接，优选地，所述(i)和(ii)之间通过铰链区连接，(ii)和(iii)之间通过连接肽连接，所述连接肽的氨基酸序列含有甘氨酸残基和丝氨酸残基或由甘氨酸残基和丝氨酸残基组成，长度为2-31个氨基酸残基；优选各自独立为(GGGGS)nG，其中，n分别独立地选自3、4、5或6；优选为4或5。
在一些实施方式中，本发明所述双功能蛋白包含抗PD-1或PD-L1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TGF-βRⅡECD。
在一些实施方式中，本发明所述抗体是IgG1、IgG2、IgG3或IgG4类抗体，优选IgG4类抗体；更优选地，所述IgG4类抗体在Fc结构域中包含1、2、3、4或5个氨基酸差异，优选地，在Fc结构域中具有S228P氨基酸置换；进一步优选地，在Fc结构域中具有C末端K和G两个氨基酸缺失。
在一些实施方式中，本发明所述的双功能蛋白，其中所述IgG4在Fc结构域中包含1、2、3、4或5个氨基酸差异，优选地，在Fc结构域中具有S228P氨基酸置换(根据EU编号***)；进一步优选地，在Fc结构域中具有C末端K和G两个氨基酸缺失(根据EU编号***)。
在一些实施方式中，本发明所述抗PD-1抗体具有如SEQ ID NO:11所示的重链氨基酸序列和如SEQ ID NO:12所示的轻链氨基酸序列，或与SEQ ID NO:11和/或12所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，本发明所述抗PD-L1抗体包含与SEQ ID NO:26或31所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％ 序列同一性的重链氨基酸，和与SEQ ID NO:27所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链氨基酸。
在一些实施方式中，本发明所述抗PD-L1抗体包含如SEQ ID NO:26或31所示的重链氨基酸序列和如SEQ ID NO:27所示的轻链氨基酸序列。
在一些实施方式中，本发明所述的双功能蛋白包含氨基酸序列如SEQ ID NO:13所示的双功能蛋白第一亚基，和氨基酸序列如SEQ ID NO:12所示的双功能蛋白第二亚基。
在一些实施方式中，本发明所述的双功能蛋白包含氨基酸序列如SEQ ID NO:28所示的双功能蛋白第一亚基，和氨基酸序列如SEQ ID NO:27所示的双功能第二亚基。
在一些实施方式中，本发明所述的双功能蛋白具有以下特性中的一种或多种：
(1)对TGF-β1及TGF-β3均有较强的结合；
(2)以高亲和力与PD-1或PD-L1结合；
(3)阻断PD-1/PD-L1的相互作用；
(4)抑制TGFβ1诱导的pSMAD3的活化；
(5)有较强的内吞活性；
(6)促进IL-2和/或IFNγ的释放。
在又一个方面，本发明提供了一种多核苷酸，其编码如本文所述的双功能蛋白或其抗原结合片段。优选地，所述多核苷酸具有如SEQ ID NO:16所示的第一亚基核苷酸序列和/或如SEQ ID NO:17所示的第二亚基核苷酸序列。
在又一个方面，本发明提供了多核苷酸，其编码如本文所述的双功能蛋白或其抗原结合片段。优选地，所述多核苷酸具有如SEQ ID NO:29所示的第一亚基核苷酸序列和/或如SEQ ID NO:30所示的第二亚基核苷酸序列。
在又一个方面，本发明提供了一种表达载体，其包含如本文所述的多核苷酸，优选地，所述表达载体为真核表达载体。
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸或表达载体，优选地，所述宿主细胞是哺乳动物细胞。
在又一个方面，本发明提供了一种制备如本文所述的双功能蛋白的方法，所述方法包括在适合于所述双功能蛋白表达的条件下在如本文所述的宿主细胞中表达所述双功能蛋白，并从所述宿主细胞回收所表达的双功能蛋白。
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的双功能蛋白、如本文所述的多核苷酸、如本文所述的表达载体、如本文所述的宿主细胞和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了如本文所述的双功能蛋白、如本文所述的多核苷酸、如 本文所述的表达载体、如本文所述的宿主细胞，或如本文所述的药物组合物在制备用于治疗和/或预防与PD-1、PD-L1或TGF-β活性相关的疾病或病症的药物中的用途，优选所述疾病或病症为癌症，更优选地，所述癌症选自黑素瘤、肾癌、***癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明提供了如本文所述的双功能蛋白、如本文所述的多核苷酸、如本文所述的表达载体、如本文所述的宿主细胞和如本文所述药物组合物，其用于治疗和/或预防与PD-1、PD-L1或TGF-β活性相关的疾病或病症，优选所述疾病或病症为癌症，更优选所述癌症选自黑素瘤、肾癌、***癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明提供了一种治疗和/或预防与PD-1、PD-L1或TGF-β活性相关的疾病或病症的方法，其包括向有需要的受试者施用如本文所述的双功能蛋白、如本文所述的多核苷酸、如本文所述的表达载体、如本文所述的宿主细胞，或如本文所述的药物组合物；优选所述疾病或病症为癌症，更优选所述癌症选自黑素瘤、肾癌、***癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明还提供了一种免疫缀合物，其包含与治疗剂或诊断剂缀合的本文所述抗体或其抗原结合片段。
在又一个方面，本发明提供了组合产品，其包含如本文所述的抗体或其抗原结合片段、本文所述的免疫缀合物或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的双功能蛋白、如本文所述的多核苷酸、如本文所述的表达载体、如本文所述的宿主细胞，或如本文所述的药物组合物。
在又一个方面，本发明提供了一种使用如本文所述的双功能蛋白检测PD-1、PD-L1或TGF-β在样品中的存在的方法。
本发明的双功能蛋白与PD-1或PD-L1蛋白有较强的亲和力，具有良好的热稳定性，能够显著促进IFNγ的释放和抑制肿瘤的生长。
附图说明
图1：靶向PD-1和TGF-β的双功能蛋白的分子结构示意图。
图2：ELISA检测靶向PD-1和TGF-β的双功能蛋白与人源TGF-β1的结合。
图3：ELISA检测靶向PD-1和TGF-β的双功能蛋白与人源TGF-β2的结合。
图4：ELISA检测靶向PD-1和TGF-β的双功能蛋白与人源TGF-β3的结合。
图5：ELISA检测靶向PD-1和TGF-β的双功能蛋白与人源PD-1的结合。
图6：报告基因法检测靶向PD-1和TGF-β的双功能蛋白对PD-1/PD-L1通路阻断作用。
图7：报告基因法检测靶向PD-1和TGF-β的双功能蛋白对TGFβ通路阻断作用。
图8：靶向PD-1和TGF-β的双功能蛋白的细胞内吞实验。
图9：靶向PD-1和TGF-β的双功能蛋白的体外混合淋巴细胞反应。9a：靶向PD-1和TGF-β的双功能蛋白对T细胞激活后IL2细胞因子释放水平的影响；9b：靶向PD-1和TGF-β的双功能蛋白对T细胞激活后IFNγ细胞因子释放水平的影响。
图10：靶向PD-1和TGF-β的双功能蛋白在小鼠体内对人黑色素瘤A375肿瘤生长的抑制作用。
图11：靶向PD-L1和TGF-β的双功能蛋白的分子结构示意图。
图12：ELISA检测靶向PD-L1和TGF-β的双功能蛋白与人源TGF-β1的结合。
图13：ELISA检测靶向PD-L1和TGF-β的双功能蛋白与人源TGF-β2的结合。
图14：ELISA检测靶向PD-L1和TGF-β的双功能蛋白与人源TGF-β3的结合。
图15：ELISA检测靶向PD-L1和TGF-β的双功能蛋白与人源PD-L1的结合。
图16：报告基因法检测靶向PD-L1和TGF-β的双功能蛋白对PD-1/PD-L1通路阻断作用。
图17：报告基因法检测靶向PD-L1和TGF-β的双功能蛋白对TGFβ通路阻断作用。
图18：靶向PD-L1和TGF-β的双功能蛋白的细胞内吞实验。
图19：靶向PD-L1和TGF-β的双功能蛋白的体外混合淋巴细胞反应。19a：靶向PD-L1和TGF-β的双功能蛋白对T细胞激活后IL2细胞因子释放水平的影响；19b：靶向PD-L1和TGF-β的双功能蛋白对T细胞激活后IFNγ细胞因子释放水平的影响。
具体实施方式
定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocolsin Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
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。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的组合。
“程序性死亡受体-1(PD-1)”是指属于CD28家族的免疫抑制性受体。PD-1主要在体内先前活化的T细胞上表达，并且结合两种配体PD-L1和PD-L2。本文使用的术语“PD-1”包括人PD-1(hPD-1)、hPD-1的变体、同种型和物种同源物，以及与hPD-1具有至少一个共同表位的类似物。
“程序性细胞死亡蛋白配体1(PD-L1)”，又可称为分化簇274(cluster of differentiation274，CD274)或者B7同源蛋白1(B7homolog1，B7-H1)，属于肿瘤坏死因子超家族。本文使用的术语“PD-L1”包括人PD-L1(hPD-L1)、hPD-L1的变体、同种型和物种同源物，以及与hPD-L1具有至少一个共同表位的类似物。
“转化生长因子-β(TGF-β)”属于调节细胞生长和分化的TGF-β超家族。TGF-β通过异源四聚体受体复合物传递信号，这个受体复合物是由两个I型和两个II型的跨膜丝氨酸/苏氨酸激酶受体组成。这些I型和II型受体是跨膜蛋白，其由具有富半胱氨酸区的配体结合细胞外结构域、跨膜结构域和具有预测的丝氨酸/苏氨酸特异性的细胞质结构域构成。I型受体对于信号传导至关重要，并且II型受体对于结合配体和I型受体的表达是必需的。I型和II型受体在配体结合后形成稳定的复合物，从而引起II型受体对I型受体的磷酸化。TGFβ具有三种哺乳动物同种型，TGFβ1、TGFβ2和TGFβ3，每一种在体内均具有不同的功能。TGFβ与TGFβRII的结合是启动TGFβ信号传导路径活化，引起SMAD2磷酸化以及使活化的SMAD2/SMAD4复合物向细胞核移位以调节基因表达的关键步骤。
术语“百分比(％)氨基酸序列同一性”或简称“同一性”定义为在将氨基酸序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守取代视为序列同一性的部分之后，候选氨基酸序列中的氨基酸残基与参比氨基酸序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
术语“免疫应答”是指由例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝产生可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入的病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。
术语“信号转导途径”或“信号转导活性”是指通常由蛋白质间相互作用诸如生长因子 对受体的结合启动的生化因果关系，所述关系导致信号从细胞的一部分传递至细胞的另一部分。一般地，传递包括引起信号转导的系列反应中的一种或多种蛋白质上的一个或多个酪氨酸、丝氨酸或苏氨酸残基的特定磷酸化。倒数第二过程通常包括细胞核事件，从而导致基因表达的变化。
术语“活性”或“生物活性”，或术语“生物性质”或“生物特征”此处可互换使用，包括但不限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗PD-1活性的能力、IC50、抗体的体内稳定性和抗体的免疫原性质。本领域公知的抗体的其它可鉴定的生物学性质或特征包括，例如，交叉反应性(即通常与靶定肽的非人同源物，或与其它蛋白质或组织的交叉反应性)，和保持哺乳动物细胞中蛋白质高表达水平的能力。使用本领域公知的技术观察、测定或评估前面提及的性质或特征，所述技术包括但不局限于ELISA、FACS或BIACORE等离子体共振分析、不受限制的体外或体内中和测定、受体结合、细胞因子或生长因子的产生和/或分泌、信号转导和不同来源(包括人类、灵长类或任何其它来源)的组织切片的免疫组织化学。
术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。
术语“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“全长抗体”，是指在天然存在时包含四条肽链的免疫球蛋白分子：两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端 至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫***的各种细胞(例如，效应细胞)和经典补体***的第一组分(Clq))的结合。
术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。术语“结合化合物”是指抗体及其结合片段两者。
术语“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。Fv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。
术语“Fc”或“Fc区”或“Fc片段”或“Fc结构域”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)。本文中的Fc区可包含铰链区；人IgG1免疫球蛋白Fc结构域的氨基酸序列为SEQ ID NO:32，人IgG4免疫球蛋白Fc结构域的氨基酸序列为SEQ ID NO:33。
术语“铰链区”是指抗体中位于CH1与CH2之间，富含脯氨酸，易伸展弯曲的多肽链。
术语“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。
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γ1(IgG1)、γ2(IgG2)、γ3(IgG3)、γ4(IgG4)、α1(IgA1)和α2(IgA2)。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。
“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。
术语“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。
术语“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
术语“全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
“PD-1抗体”是指结合PD-1受体，阻断表达于癌细胞上的PD-L1与表达于免疫细胞(T、B、NK细胞)上的PD-1结合且优选也能阻断表达于癌细胞上的PD-L2与表达于免疫细胞上的PD-1结合的任何化学化合物或生物分子。PD-1及其配体的替代名词或同义词包括：对于PD-1而言有PDCD1、PD1、CD279及SLEB2；对于PD-L1而言有PDCD1L1、PDL1、B7-H1、B7H1、B7-4、CD274及B7-H；且对于PD-L2而言有PDCD1L2、PDL2、B7-DC及CD273。在治疗人个体的任何本发明治疗方法、药物及用途中，PD-1抗体阻断人PD-L1与人PD-1的结合，且优选阻断人PD-L1和PD-L2二者与人PD1结合。人PD- 1氨基酸序列可见于NCBI基因座编号：NP_005009。人PD-L1及PD-L2氨基酸序列可分别见于NCBI基因座编号：NP_054862及NP_079515。
本文中，当提及“抗PD-1抗体”时，除非另有说明或描述，否则该术语包括其抗原结合片段。
“PD-L1抗体”是指结合PD-L1受体，阻断表达于癌细胞上的PD-L1与表达于免疫细胞(T、B、NK细胞)上的PD-1结合的任何化学化合物或生物分子。PD-1及其配体的替代名词或同义词包括：对于PD-1而言有PDCD1、PD1、CD279及SLEB2；对于PD-L1而言有PDCD1L1、PDL1、B7-H1、B7H1、B7-4、CD274及B7-H；且对于PD-L2而言有PDCD1L2、PDL2、B7-DC及CD273。在治疗人个体的任何本发明治疗方法、药物及用途中，PD-L1抗体阻断人PD-1与人PD-L1的结合。
本文中，当提及“抗PD-L1抗体”时，除非另有说明或描述，否则该术语包括其抗原结合片段。
“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
术语“表位”是指抗体所结合的抗原区域。表位可以由连续的氨基酸形成或者通过蛋白的三级折叠而并置的非连续氨基酸形成。
“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平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。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本文中的ForteBio动力学结合测定法。
术语“不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的KD为1.0×10 -6M或更高，更优选1.0×10 -5M或更高，更优选1.0×10 - 4M或更高、1.0×10 -3M或更高，更优选1.0×10 -2M或更高。
术语“高亲和性”对于IgG抗体而言，是指对于抗原的KD为1.0×10 -6M或更低，优选5.0×10 -8M或更低，更优选1.0×10 -8M或更低、5.0×10 -9M或更低，更优选1.0×10 -9M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指KD为10 -6M或更低，优选10 -7M或更低，更优选10 -8M或更低。
术语“抗体依赖的细胞毒性”、“抗体依赖的细胞介导的细胞毒性”或“ADCC”是指细胞介导的免疫防御，其中免疫***效应细胞主动地将细胞膜表面抗原与抗体，例如Claudin18.2抗体，结合的靶细胞例如癌细胞裂解。
术语“补体依赖的细胞毒性”或“CDC”是指IgG和IgM抗体的效应功能，当与表面抗 原结合时引发典型的补体途径，包括形成膜攻击复合体以及靶细胞裂解。本发明的抗体，与Claudin 18.2结合时，引发对癌细胞的CDC。
术语“核酸”或“多核苷酸”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语包括具有与参照核酸相似的结合性质并且以与天然存在的核苷酸相似的方式被代谢的含有已知的天然核苷酸的类似物的核酸(参见，属于Kariko等人的美国专利No.8,278,036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子取代可通过生成其中一个或多个选择的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代的序列来实现(Batzer等人，Nucleic Acid Res.19:5081(1991)；Ohtsuka等人，J.Biol.Chem.260:2605-2608(1985)；和Rossolini等人，Mol.Cell.Probes 8:91-98(1994))。
“构建体”是指任何重组多核苷酸分子(诸如质粒、粘粒、病毒、自主复制多核苷酸分子、噬菌体或线性或环状单链或双链DNA或RNA多核苷酸分子)，衍生自任何来源，能够与基因组整合或自主复制，构成如下多核苷酸分子，其中已经以功能操作的方式连接(即，可操作地连接)一或多个多核苷酸分子。重组构建体通常会包含可操作地连接至转录起始调节序列的本发明的多核苷酸，这些序列会导引多核苷酸在宿主细胞中的转录。可使用异源及非异源(即，内源)启动子两者导引本发明的核酸的表达。
“载体”是指任何重组多核苷酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。一种类型的载体为“质粒”，是指环状双链DNA环，可将额外DNA区段连接至该环中。另一类型的载体为病毒载体，其中可将额外DNA区段连接至病毒基因组中。某些载体能够在被引入到的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体及游离型哺乳动物载体)。在引入到宿主细胞中后，其他载体(例如，非游离型哺乳动物载体)整合至宿主细胞的基因组中，且因此与宿主基因组一起复制。此外，某些载体能够导引***作性连接的基因的表达。本文将此类载体称为“表达载体”。
本文所用术语“表达载体”是指能够在转化、转染或转导至宿主细胞中时复制及表达目的基因的核酸分子。表达载体包含一或多个表型选择标记及复制起点，以确保维护载体及以在需要的情况下于宿主内提供扩增。
用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环 境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。
如本文中所用，术语任何疾病或病症的“治疗”或“医治”在一个实施方式中是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在另一个实施方式中，“治疗”或“医治”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些物理参数。在另一个实施方式中，“治疗”或“医治”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。
“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴。
“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的连续施用。
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的抗体或其抗原结合片段或双功能蛋白当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％；通常至少20％；优选至少约30％；更优选至少40％，最优选至少50％。
“癌症”和“癌性”指或描述哺乳动物中特征通常为细胞生长不受调控的生理疾患。此定义中包括良性和恶性癌症以及休眠肿瘤或微转移。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤，和白血病。此类癌症的更具体例子包括鳞状细胞癌，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃的癌或胃癌(包括胃肠癌)，胰腺癌，成胶质细胞瘤，***，卵巢癌，肝癌，膀胱癌，肝瘤(hepatoma)，乳腺癌，结肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌或肾的癌，肝癌，***癌，外阴癌，甲状腺癌，肝的癌，及各种类型的头和颈癌，以及B细胞淋巴瘤(包括低级/滤泡性非何杰金氏淋巴瘤(NHL)，小淋巴细胞性(SL)NHL，中级/滤泡性NHL，中级弥漫性NHL，高级成免疫细胞性NHL，高级成淋巴细胞性NHL，高级小无核裂细胞性NHL，贮积病(bulky disease)NHL，套细胞淋巴瘤，AIDS相关淋巴瘤，和瓦尔登斯特伦氏(Waldenstrom)巨球蛋白血症)，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血 病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖。
双功能蛋白
本发明提供了靶向PD-1或PD-L1和TGF-β的双功能蛋白，其包含
(i)阻断PD-1/PD-L1通路的抗体的抗原结合片段；
(ii)免疫球蛋白Fc结构域；和
(iii)TGF-βRⅡ胞外结构域(ECD)。
在一些实施方式中，本发明所述阻断PD-1/PD-L1通路的抗体的抗原结合片段为抗PD-1或PD-L1抗体的抗原结合片段。
在一些实施方式中，本发明所述双功能蛋白从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；优选地，所述(i)和(ii)之间通过铰链区连接，(i)和(iii)以及(ii)和(iii)之间通过连接肽连接，所述连接肽的氨基酸序列含有甘氨酸残基和丝氨酸残基或由甘氨酸残基和丝氨酸残基组成，长度为2-31个氨基酸残基；优选各自独立为(GGGGS)nG，其中，n分别独立地选自3、4、5或6；优选为4或5。
在一些实施方式中，本发明所述双功能蛋白从N端至C端以(i)、(ii)和(iii)的顺序有效连接，优选地，所述(i)和(ii)之间通过铰链区连接，(ii)和(iii)之间通过连接肽连接，所述连接肽的氨基酸序列含有甘氨酸残基和丝氨酸残基或由甘氨酸残基和丝氨酸残基组成，长度为2-31个氨基酸残基；优选各自独立为(GGGGS)nG，其中，n分别独立地选自3、4、5或6；优选为4或5。
在一些实施方式中，本发明所述双功能蛋白包含抗PD-1或PD-L1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TGF-βRⅡECD。
在一些实施方案中，本发明所述双功能蛋白是由二硫键键合的两个双功能蛋白第一亚基和两个双功能蛋白第二亚基组成的异四聚体糖蛋白。
本发明采用靶向PD-1或PD-L1的单抗作为双功能蛋白中阻断PD-1/PD-L1通路的分子部分，用于阻断负向调控信号，使T细胞恢复活性，从而增强免疫应答。本发明采用TGF-βRII胞外结构域作为双功能蛋白中免疫调节分子部分，用于削弱癌细胞的免疫耐受性。在靶向并中和肿瘤微环境的TGF-β基础上抑制PD-1/PD-L1通路，可以使T细胞恢复活性，增强免疫应答，更有效地提高抑治肿瘤发生和发展。
在一些实施方案中，本发明的双功能蛋白以10 -8M或更小、例如以10 -9M至10 -12M的解离常数(KD)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(KD)分别与TGFβ1/TGFβ2/TGFβ3分子特异性结合。
抗PD-1抗体和/或抗PD-1抗体的抗原结合片段
本发明所述双功能蛋白包含抗PD-1抗体和/或抗PD-1抗体的抗原结合片段，其能够特异性结合PD-1；或者与完整抗PD-1抗体和/或其抗原结合片段竞争结合PD-1。
本发明所述的“抗PD-1抗体或其抗原结合片段”可包括本领域中所述的任何抗PD-1抗体或其抗原结合片段。抗PD-1抗体可以是市售可得的或己通过文献公开的PD-1抗体。包括但不限于，如PD-1抗体nivolumab、pembrolizumab、toripalimab、Sintilimab、Camrelizumab、Tislelizumab、Cemiplimab等。抗体可以是单克隆抗体、嵌合抗体、人源化抗体或全人抗体。抗原结合片段包括具有抗原结合活性的Fab片段，Fab'片段，F(ab')2片段，以及与抗体结合的Fv片段和ScFv片段。
在一些实施方式中，本发明所述抗PD-1抗体的抗原结合片段包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明双功能蛋白中的抗PD-1抗体的抗原结合片段包含与SEQ ID NO:7所示的重链可变区和氨基酸序列如SEQ ID NO:8所示的轻链可变区具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的氨基酸序列。
在一些实施方式中，本发明所述抗PD-1抗体的抗原结合片段包含氨基酸序列如SEQ ID NO:7所示的重链可变区和氨基酸序列如SEQ ID NO:8所示的轻链可变区。
在一些实施方式中，本发明所述抗PD-1抗体的抗原结合片段包含Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
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、IMGT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)；国际ImMunoGeneTics database(IMGT)(1999 Nucleic Acids Research,27,209-212)。
本发明用于双功能蛋白JS-TZO3构建的抗PD-1抗体的抗原结合片段氨基酸序列如下(CDR边界采用Kabat方案)：
PD-1抗体重链可变区的氨基酸序列：(SEQ ID NO:7)
Figure PCTCN2021121198-appb-000001
HCDR1      DYEMH                        SEQ ID NO:1
HCDR2      VIESETGGTAYNQKFKG            SEQ ID NO:2
HCDR3      EGITTVATTYYWYFDV             SEQ ID NO:3
PD-1抗体轻链可变区的氨基酸序列：(SEQ ID NO:8)
Figure PCTCN2021121198-appb-000002
LCDR1      RSSQSIVHSNGNTYLE               SEQ ID NO:4
LCDR2      KVSNRFS                        SEQ ID NO:5
LCDR3      FQGSHVPLT                      SEQ ID NO:6
抗PD-L1抗体和/或抗PD-L1抗体的抗原结合片段
本发明所述双功能蛋白包含抗PD-L1抗体和/或抗PD-L1抗体的抗原结合片段，其能够特异性结合PD-L1；或者与完整抗PD-L1抗体和/或其抗原结合片段竞争结合PD-L1。
本发明所述的“抗PD-L1抗体或其抗原结合片段”可包括本领域中所述的任何抗PD-L1抗体或其抗原结合片段。抗PD-L1抗体可以是市售可得的或己通过文献公开的PD-L1抗体。本发明抗体可以是单克隆抗体、嵌合抗体、人源化抗体或全人抗体。抗原结合片段包括具有抗原结合活性的Fab片段，Fab'片段，F(ab')2片段，以及与抗体结合的Fv片段和ScFv片段。
在一些实施方式中，本发明所述抗PD-L1抗体的抗原结合片段包含氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述抗PD-L1抗体的抗原结合片段包含与SEQ ID NO:24所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链可变区，和与SEQ ID NO:25所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链可变区。
在一些实施方式中，本发明所述抗PD-L1抗体的抗原结合片段包含氨基酸序列如SEQ ID NO:24所示的重链可变区和氨基酸序列如SEQ ID NO:25所示的轻链可变区。
在一些实施方式中，本发明所述抗PD-L1抗体的抗原结合片段包含Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
本发明用于双功能蛋白JS-TZO4构建的抗PD-L1抗体的抗原结合片段氨基酸序列如下(CDR边界采用IMGT方案)：
抗PD-L1抗体重链可变区的氨基酸序列：(SEQ ID NO:24)
Figure PCTCN2021121198-appb-000003
HCDR1      GDSITRGY                        SEQ ID NO:18
HCDR2      ISYTGST                         SEQ ID NO:19
HCDR3      ATSTGWLDPVDY                    SEQ ID NO:20
抗PD-L1抗体轻链可变区的氨基酸序列：(SEQ ID NO:25)
Figure PCTCN2021121198-appb-000004
LCDR1      QNVDTS                          SEQ ID NO:21
LCDR2      SAS                             SEQ ID NO:22
LCDR3      QQYYGYPFT                       SEQ ID NO:23
免疫球蛋白Fc结构域
本发明双功能蛋白中的“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结构域对本发明的双功能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。
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。
在一些实施方案中，IgG Fc区包含IgG CH2结构域和IgG CH3结构域。在一些实施方案中，免疫球蛋白Fc结构域具有与SEQ ID NO:9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氨基酸序列。在一些实施方案中，免疫球蛋白Fc结构域具有如SEQ ID NO:9所示的氨基酸序列。
在一些实施方式中，本发明所述免疫球蛋白Fc结构域是人IgG1、IgG2、IgG3或IgG4的Fc结构域；优选为人IgG4的Fc结构域。
在一些实施方式中，本发明所述免疫球蛋白Fc结构域还包含对其进行氨基酸序列进行一个或多个氨基酸替换、缺失或衍生后获得的肽序列。
在一些实施方案中，在IgG Fc结构域中第S228位置处包含防止发生臂交换的氨基酸置换，特别地是氨基酸置换S228P。
在一些实施方案中，在IgG Fc结构域中包含为防止C末端发生断裂的末位氨基酸缺失，优选为C末端K和G两个氨基酸缺失。
本发明用于双功能蛋白JS-TZO3和JS-TZO4构建的免疫球蛋白Fc结构域氨基酸序列可以选自如下：(SEQ ID NO:9)
Figure PCTCN2021121198-appb-000005
抗PD-1抗体
在一些实施方式中，本发明所述抗PD-1抗体与SEQ ID NO:11和/或SEQ ID NO:12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氨基酸序列。
在一些实施方式中，本发明所述抗PD-1抗体具有如SEQ ID NO:11所示的重链氨基酸序列，和如SEQ ID NO:12所示的轻链氨基酸序列。
本发明用于双功能蛋白JS-TZO3构建的PD-1抗体重链序列可以选自如下：(SEQ ID NO:11)
Figure PCTCN2021121198-appb-000006
本发明用于双功能蛋白JS-TZO3构建的PD-1抗体轻链序列可以选自如下：(SEQ ID NO:12)
Figure PCTCN2021121198-appb-000007
Figure PCTCN2021121198-appb-000008
抗PD-L1抗体
在一些实施方式中，本发明所述抗PD-L1抗体包含与SEQ ID NO:26或31所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链氨基酸，和与SEQ ID NO:27所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链氨基酸。
在一些实施方式中，本发明所述抗PD-L1抗体包含如SEQ ID NO:26或31所示的重链氨基酸序列和如SEQ ID NO:27所示的轻链氨基酸序列。
本发明用于双功能蛋白JS-TZO4构建的PD-L1抗体重链序列可以选自如下：(SEQ ID NO:31)
Figure PCTCN2021121198-appb-000009
本发明用于双功能蛋白JS-TZO4构建的PD-L1抗体轻链序列可以选自如下：(SEQ ID NO:27)
Figure PCTCN2021121198-appb-000010
TGF-βRⅡ胞外结构域
本发明双功能蛋白中的“TGF-βRⅡ的胞外结构域(ECD)”包含天然存在的TGF-βRⅡECD的全部氨基酸残基或包含天然存在的TGF-βRⅡECD的一部分氨基酸残基。
在一些实施方案中，本发明所述TGF-βRⅡ胞外结构域具有与SEQ ID NO:10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。
在一些实施方案中，本发明所述TGF-βRⅡ胞外结构域具有SEQ ID NO:10所示的氨基酸序列。
除了如SEQ ID NO:10所定义的序列之外，TGF-βRⅡECD还可以包含对SEQ ID NO:10进行额外的序列修饰后获得的肽序列，例如对SEQ ID NO:10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。
本发明用于双功能蛋白JS-TZO3或JS-TZO4构建的TGF-βRII胞外结构域的氨基酸序列如下：
TGF-βRII胞外结构域(1-136多肽，SEQ ID NO:10)：
Figure PCTCN2021121198-appb-000011
双功能蛋白
本发明使用连接肽(G4S)4G将PD1或PD-L1抗体重链部分和TGFβRII胞外结构域连接成双功能蛋白。
在一些实施方式中，本发明所述的双功能蛋白包含氨基酸序列如SEQ ID NO:13所示的双功能蛋白第一亚基，和氨基酸序列如SEQ ID NO:12所示的双功能蛋白第二亚基。
本发明双功能蛋白JS-TZO3第一亚基氨基酸序列如下：(重链，SEQ ID NO:13)：
Figure PCTCN2021121198-appb-000012
本发明双功能蛋白JS-TZO3第二亚基氨基酸与PD-1抗体轻链序列相同，其为SEQ ID NO:12所示的氨基酸序列。
在一些实施方式中，本发明所述的双功能蛋白包含氨基酸序列如SEQ ID NO:28所示的双功能蛋白第一亚基，和氨基酸序列如SEQ ID NO:27所示的双功能蛋白第二亚基。
本发明双功能蛋白JS-TZO4第一亚基氨基酸序列如下：(重链，SEQ ID NO:28)：
Figure PCTCN2021121198-appb-000013
Figure PCTCN2021121198-appb-000014
本发明双功能蛋白JS-TZO4第二亚基氨基酸与PD-L1抗体轻链序列相同，其为SEQ ID NO:27所示的氨基酸序列。
在一些实施方式中，氨基酸变化包括氨基酸缺失、***或置换。在一些实施方式中，本发明的双功能蛋白包括具有已通过氨基酸缺失、***或置换突变的，但仍与上述双功能蛋白有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些双功能蛋白。
在一些实施方式中，编码本发明抗体的多核苷酸包括已通过核苷酸缺失、***或置换突变的，但仍然与上文中所述的序列中描绘的CDR对应编码区具有至少约60％、70％、80％、90％、95％或100％同一性的多核苷酸。
在一些实施方式中，本文中所提供的双功能蛋白可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二烷、聚-1,3,6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
抗体表达
在又一个方面，本发明提供了一种多核苷酸，其编码如本文所述的双功能蛋白。所述多核苷酸可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列的多核苷酸，或包含编码双功能蛋白第一亚基和/或第二亚基的氨基酸序列的多核苷酸。
在一些实施方式中，本发明所述的多核苷酸包含如SEQ ID NO:16所示的第一亚基核苷酸序列和/或如SEQ ID NO:17所示的第二亚基核苷酸序列。
本发明双功能蛋白JS-TZO3第一亚基核苷酸序列如下：(SEQ ID NO:16)：
Figure PCTCN2021121198-appb-000015
本发明双功能蛋白JS-TZO3第二亚基核苷酸序列如下：(SEQ ID NO:17)：
Figure PCTCN2021121198-appb-000016
Figure PCTCN2021121198-appb-000017
在一些实施方式中，本发明所述的多核苷酸包含如SEQ ID NO:29所示的第一亚基核苷酸序列和/或如SEQ ID NO:30所示的第二亚基核苷酸序列。
本发明双功能蛋白JS-TZO4第一亚基核苷酸序列如下：(SEQ ID NO:29)：
Figure PCTCN2021121198-appb-000018
本发明双功能蛋白JS-TZO4第二亚基核苷酸序列如下：(SEQ ID NO:30)：
Figure PCTCN2021121198-appb-000019
Figure PCTCN2021121198-appb-000020
在又一个方面，本发明提供了一种表达载体，其包含如本文所述的多核苷酸，优选地，所述载体为真核表达载体。在一些实施方式中，如本文所述的多核苷酸包含在一个或多个表达载体中。
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸或如本文所述的表达载体，优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了一种用于制备如本文所述的双功能蛋白的方法，所述方法包括在适合于所述抗体表达的条件下在本文所述的宿主细胞中表达所述抗体，并从所述宿主细胞回收所表达的双功能蛋白。
本发明提供用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别优选的细胞系。
在一个实施方式中，本发明提供制备本发明双功能蛋白的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中时，通过将宿主细胞培养足够的一段时间，以允许双功能蛋白在宿主细胞中表达，或者更优选双功能蛋白分泌到宿主细胞生长的培养基中，来产生双功能蛋白。可采用标准蛋白质纯化方法从培养基中回收双功能蛋白。
很可能由不同细胞系表达或在转基因动物中表达的双功能蛋白彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有双功能蛋白是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。
药物组合物和药物制剂
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的双功能蛋白、本文所述的多核苷酸、本文所述的表达载体或本文所述的宿主细胞，和药学上可接受的载体或赋形剂。
应理解，本发明提供的双功能蛋白或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的双功能蛋白与一种或多种任选的药用辅料(Remington's Pharmaceutical Sciences，第16版，Osol,A.编辑(1980))混合来制备包含本文所述的双功能蛋白的药物制剂，优选地以水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。
在一些实施方式中，本发明的药物组合物还包含编码双功能蛋白的多核苷酸的组合物。
在又一个方面，本发明提供了一种药物组合，其包含如本文所述的双功能蛋白、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物。
医药用途
在又一个方面，本发明提供了如本文所述的双功能蛋白、如本文所述的多核苷酸、如本文所述的表达载体、如本文所述的宿主细胞，或如本文所述的药物组合物在制备用于治疗和/或预防与PD-1、PD-L1或TGF-β活性相关的疾病或病症的药物中的用途，优选所述疾病或病症为癌症，更优选地，所述癌症选自黑素瘤、肾癌、***癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明提供了如本文所述的双功能蛋白、如本文所述的多核苷酸、如本文所述的表达载体、如本文所述的宿主细胞和如本文所述药物组合物，其用于治疗和/或预防与PD-1、PD-L1或TGF-β活性相关的疾病或病症，优选所述疾病或病症为癌症， 更优选所述癌症选自黑素瘤、肾癌、***癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明提供了一种治疗和/或预防与PD-1、PD-L1或TGF-β活性相关的疾病或病症的方法，其包括向有需要的受试者施用如本文所述的双功能蛋白、如本文所述的多核苷酸、如本文所述的表达载体、如本文所述的宿主细胞，或如本文所述的药物组合物；优选所述疾病或病症为癌症，更优选所述癌症选自黑素瘤、肾癌、***癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在一些实施方式中，本文所述的癌症或肿瘤可以选自黑素瘤、肾癌、***癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在一些实施方式中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
在一些实施方式中，本发明提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。
在一些实施方式中，本发明提供的方法或用途还包括向个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体。例如，可以与至少一种另外的治疗剂共施用。例如，PD-1/PD-L1抗体、LAG-3抗体和/或CTLA-4抗体。
用于诊断和检测的方法
在又一个方面，本发明提供了一种使用如本文所述的双功能蛋白在检测PD-1、PD-L1或TGF-β在样品中的存在的方法。术语“检测”用于本文中时，包括定量或定性检测。在一些实施方式中，所述样品是生物样品。在某些实施方式中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方式中，生物样品包含细胞或组织。
本发明包括所叙述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他方法的结合所形成的实施方式以及本领域技术上人员所 熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明采用下述缩略词：
ECD代表胞外结构域；
PEI代表聚乙烯亚胺；
BSA代表牛血清白蛋白；
PBS代表磷酸缓冲盐溶液；
FBS代表胎牛血清；
TMB代表3,3',5,5'-四甲基联苯胺；
PBST代表添加0.02％吐温的磷酸盐缓冲液。
实施例
通过以下实施例对本发明进行说明，但并不旨在对本发明作出任何限制。本文已经详细描述了本发明，其中也公开了其具体实施方式。对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1、靶向PD-1和TGF-β的双功能蛋白JS-TZO3的制备
1.1双功能蛋白表达载体的构建
委托南京金斯瑞合成编码抗人PD-1抗体(重链氨基酸序列如SEQ ID NO:11所示，轻链氨基酸序列如SEQ ID NO:12所示)的核苷酸序列，并通过BSPQI限制性内切酶将基因合成片段1A6H2连接至载体HXT4s上，得到载体HXT4s-1A6H2。委托南京金斯瑞合成编码TGF-βRII胞外区(ECD)(其氨基酸序列如SEQ ID NO:10所示)的核苷酸序列。连接肽((G 4S) 4G)的核苷酸序列通过所属领域技术手段获得。合成引物TGFβR2-G4SBSPQIFV2和TGFβR2-BSPQIRV2，通过PCR扩增获得人TGF-βRII ECD基因片段，通过BSPQI限制性内切酶将该TGF-βRII ECD基因片段连接至载体HXT4s-1A6H2，得到表达载体HXT4s-1A6H2-TGFβRII。该表达载体HXT4s-1A6H2-TGFβRII表达双功能蛋白JS-TZO3，该双功能蛋白JS-TZO3的第二亚基氨基酸序列如SEQ ID NO:12，第一亚基氨基酸序列如SEQ ID NO:13所示。
其中，
该引物TGFβR2-G4SBSPQIFV2按5’-3’顺序的核苷酸序列(SEQ ID NO:14)如下：
Figure PCTCN2021121198-appb-000021
该引物TGFβR2-BSPQIRV2按5’-3’顺序的核苷酸序列(SEQ ID NO:15)如下：
Figure PCTCN2021121198-appb-000022
1.2双功能蛋白的表达和纯化
取液氮保存的293F细胞(FreeStyle TM 293-F Cells，Thermofisher)进行复苏，将其加入表达培养基(Freestyle TM 293 expression medium,Gibco)中进行培养。培养数代后，转染前一天将293F细胞稀释至(1.5-2.0)×10 6个细胞/mL，转染时，先取1/10转染体积的上述培养基孵育表达载体HXT4s-1A6H2-TGFβRII和转染试剂(PEI)，表达载体用量为0.5-2μg/mL，转染试剂用量为3-14μg/mL。孵育时间为20-30min，孵育完成后将转染混合物加入提前一天处理好的上述293F细胞中，边加边摇匀。将完成转染的摇瓶放入二氧化碳摇床进行培养，条件为36.5℃，120rpm，7％CO 2。7天以后，离心收集上清液进行纯化。使用AKTA AVANT仪器进行纯化，使用蛋白A亲和填料(MabSelect SuRe TM,GE Healthcare)预装的纯化柱，将上述上清液离心完成后经0.22μm滤器过滤后上样，PBS进行洗杂，柠檬酸缓冲液进行洗脱，洗脱完成后Tris缓冲液中和，得到双功能蛋白JS-TZO3。制备的双功能蛋白JS-TZO3可用于下述各实施例中。
JS-TZO3双功能蛋白分子结构示意图如图1所示。
实施例2：ELISA检测双功能蛋白JS-TZO3与人源TGF-β的结合
1、测试目的
使用ELISA方法检测本发明双功能蛋白JS-TZO3与人源TGF-β1、TGF-β2、TGF-β3的结合。
2、测试过程及结果
a.以0.5μg/ml浓度的重组人源TGF-β1(Novoprotein，CA59)为抗原按100μl/孔包被96孔板，37℃孵育1.5小时。
b.300μl 1×PBST洗涤4次，加入200μl 2％BSA，37℃封闭1.5小时。
c.300μl 1×PBST洗涤4次，加入梯度稀释的双功能蛋白JS-TZO3和阴性对照抗体(Anti-KLH IgG4)(起始浓度100μg/ml，2.5倍梯度稀释12个浓度点)，37℃孵育1小时。
d.300μl 1×PBST洗涤4次。
e.每孔加入100μl辣根过氧化物酶(HRP)偶联的鼠抗人抗体IgG4Fc(1∶5000)(Southern Biotech，9200-05)，37℃孵育1小时。
f.每孔加入100μl 0.1mg/ml的TMB，37℃孵育12分钟后加入100μl 2M盐酸溶液终止反应。
g.酶标仪上检测450nm/620nm测吸收值，Graphpad Prism5分析数据。
如图2、3和4所示，本发明双功能蛋白JS-TZO3与TGF-β1及TGF-β3均有较强的结合，与TGF-β2结合相对较弱。
实施例3：Biacore检测双功能蛋白JS-TZO3与TGF-β的亲和力
1、测试目的
使用Biacore T200仪器(GE医疗生命科学)检测本发明双功能蛋白JS-TZO3与人源TGF-β1、TGF-β2、TGF-β3的亲和力。
2、测试过程及结果
本发明双功能蛋白与人源TGF-β1、TGF-β2、TGF-β3结合亲和力的测定方法如下：用Protein A芯片(GE医疗生命科学)捕获4μg/ml双功能蛋白，然后在芯片表面流过梯度稀释的人源TGF-β1、TGF-β2、TGF-β3(近岸生物)。TGF-β1的稀释梯度为1.14nM，0.76nM，0.5057nM，0.3378nM，0.2252nM，0.1501nM，其中1.14nM为重复进样。TGF-β2的稀释梯度为1.8nM，1.2nM，0.8nM，0.5333nM，0.3556nM，0.2370nM，其中1.2nM为重复进样。TGF-β3的稀释梯度为1.2nM，0.8nM，0.5333nM，0.3556nM，0.2370nM，其中1.2nM为重复进样。各抗原梯度进样后，使用Biacore T200***(GE Healthcare)测量结合与解离信号，从而获得结合和解离动力学曲线。使用软件Biacore T200 Evaluation Software 3.0拟合结合解离曲线来确定亲和力KD值。
如表1所示，本发明双功能蛋白JS-TZO3与TGFβ1、TGFβ3有较强的亲和力，与TGFβ2结合的亲和力略弱。
表1：双功能蛋白JS-TZO3与TGF-β的亲和力
Figure PCTCN2021121198-appb-000023
实施例4：ELISA检测双功能蛋白JS-TZO3与PD-1的结合
1、测试目的：
ELISA检测本发明双功能蛋白JS-TZO3与PD-1的结合。
2、测试过程
a.以人源PD-1(0.3μg/ml浓度，君实生物)为抗原按100μl/孔包被96孔板，37℃孵育1.5小时。
b.300μl 1×PBST洗涤4次，加入200μl 2％BSA，37℃封闭1.5小时。
c.300μl 1×PBST洗涤4次，加入梯度稀释的双功能蛋白JS-TZO3、阳性对照PD-1单抗(Toripalimab，君实生物)和阴性对照抗体(Anti-KLH IgG4)(起始浓度1μg/ml，2.5倍梯度稀释12个浓度点)，37℃孵育1小时。
d.300μl 1×PBST洗涤4次。
e.每孔加入100μl辣根过氧化物酶(HRP)偶联的鼠抗人抗体IgG4Fc(1∶5000)(Southern Biotech，9200-05)，37℃孵育1小时。
f.每孔加入100μl 0.1mg/ml TMB，室温孵育30分钟后加入100μl 2M盐酸溶液终止反应。
g.酶标仪上检测450nm/620nm测吸收值，Graphpad Prism5分析数据。
结果如图5所示，本发明双功能蛋白JS-TZO3与PD-1有较强的结合，与阳性对照抗体Toripalimab相当。
实施例5：Biacore检测双功能蛋白JS-TZO3与PD-1的亲和力
1、测试目的
使用Biacore T200仪器(GE医疗生命科学)检测本发明双功能蛋白JS-TZO3与人源PD-1蛋白的亲和力。
2、测试过程及结果
本发明双功能蛋白与PD-1结合亲和力的测定方法如下：将40μg/ml羊抗人IgG-Fc片段抗体(Jackson Immuno Research)偶联在CM5芯片(GE Healthcare Life Sciences，货号BR-1005-30)表面，用于捕获抗体。之后在CM5芯片表面捕获5μg/ml双功能蛋白。将人源PD-1蛋白(君实生物)进行梯度稀释(140nM，70nM，35nM，17.5nM，8.75nM，4.375nM,其中140nM为重复进样)，将人源PD-1进样并流经CM5芯片，使用Biacore T200***(GE Healthcare)测量结合与解离信号。使用软件Biacore T200Evaluation Software 3.0拟合结合解离曲线来确定亲和力KD值。
如表2所示，本发明双功能蛋白JS-TZO3与PD-1蛋白有较强的亲和力。
表2：双功能蛋白JS-TZO3与PD-1的亲和力
Figure PCTCN2021121198-appb-000024
实施例6：报告基因法检测双功能蛋白JS-TZO3对PD-1/PD-L1通路阻断试验
1、测试目的：
为了研究本发明双功能蛋白JS-TZO3对PD-1/PD-L1信号通路的阻断作用，采用来自Promaga公司构建的PD-1/PD-L1报告基因***来检测本发明双功能蛋白中PD-1端的细胞生物学活性。
2、测试过程
取CHO/PD-L1细胞(Promega)，消化并用F-12营养混合物完全培养基重悬细胞，根 据细胞计数结果使用完全培养基调整细胞密度至5×10 5个细胞/ml，将细胞悬液转移至加样槽中，使用多道移液器以100μl/孔加入到96孔板中，放置于37℃，5％CO 2培养箱培养16～20h；第二天制备Jurkat/PD-1(Promega)细胞悬液，根据细胞计数结果使用分析培养基(RPMI 1640 Medium+2％FBS)重悬细胞，并调整细胞密度至2.5×10 6个细胞/ml；将加入CHO/PD-L1细胞的细胞培养板从培养箱中取出，使用多道移液器每孔取出100μl培养液。按照40μl/孔加入梯度稀释(起始浓度为1650nM)的双功能蛋白JS-TZO3、阳性对照PD-1单抗(Toripalimab，君实生物)和阴性对照抗体(Anti-KLH IgG4)。然后将上述Jurkat/PD-1细胞悬液转移至加样槽中，以40μl/孔加入到细胞培养板中，置于37℃，5％CO 2培养箱培养5～6h。在蛋白孵育期间，将One-Glo试剂(Promega，货号E6130)取出使其温度恢复至室温。取出细胞培养板，置于室温放置5～10min，然后每孔加入40μl One-Glo试剂(promega，E6130)，置于混匀器上混匀5～10min，然后使用M1000 pro多功能酶标仪(Tecan)读取发光信号值。
3、结果
如图6所示，本发明双功能蛋白JS-TZO3和抗PD-1单抗Toripalimab均能够有效地阻断PD-1/PD-L1的相互作用，促进NFAT信号的增强，EC 50值分别为11.01nM和10.4nM，有显著的药物浓度剂量依赖效应，二者活性水平相当。
实施例7：报告基因法检测双功能蛋白JS-TZO3对TGFβ通路阻断试验
1、测试目的
该实验通过4T1细胞表达带荧光素酶报告基因的Smad3结合原件(SBE)来研究本发明双功能蛋白JS-TZO3对TGF-β1诱导Smad3活化的抑制作用。
2、测试过程
4T1-SBE细胞(君实生物)使用含有10％FBS的1640完全培养基(Gibco，11875-093)培养，每2天传代一次。实验第一天以每孔100,000个细胞的密度接种于96孔板(Costar，3917)，在37℃、5％CO 2条件下培养16～20小时。第二天，弃去细胞培养板中的培养基，按照50μl/孔加入梯度稀释(起始浓度为1650nM)的双功能蛋白及阳性对照G4Fc TGFβR2ecd(TGFβTrap，君实生物)。细胞在37℃、5％CO 2条件下继续培养0.5～1小时。每孔加入50μL重组人源化TGFβ1(Novoprotein，货号CA59)。细胞在37℃、5％CO 2条件下继续培养5～6h。然后每孔加入50μl萤光素酶底物ONE-Glo  TM试剂(promega，E6130)，置于混匀器上混匀5～10min，然后使用M1000 pro多功能酶标仪(Tecan)读取发光信号值。待测样品的IC 50值使用数据处理软件Graphpad Prism7.0计算得到。
3、结果
如图7所示，本发明双功能蛋白JS-TZO3与G4Fc TGFβR2ecd(TGFβTrap)均能够以剂量依赖性形式抑制TGFβ1诱导的pSMAD3的活化。
实施例8：双功能蛋白JS-TZO3的细胞内吞试验
1、测试目的
在体外水平利用抗体内吞实验来检测本发明双功能蛋白JS-TZO3的内吞活性。
2、测试过程
CypHer 5 Mono NHS Ester染料标记待测样品：用Label buffer(标记缓冲液，PBS:NaCO 3＝9:1，pH:8.3)将待测双功能蛋白JS-TZO3浓度调整成1.0mg/ml。计算双功能蛋白质量，加入CypHer 5 Mono NHS Ester染料(GE，PA15401)，室温避光混匀1h。将反应后的双功能蛋白JS-TZO3与染料混合液转移至0.5ml超滤管(30KD)中，离心10min，弃去废液，再向超滤管中补充Label buffer，继续同上离心4次，将未结合的多余染料洗去。
取CHO/PD-L1细胞(Promega)，消化并用F-12营养混合物完全培养基重悬细胞，取293F JS1 1E4细胞(君实生物)，消化并用1640完全培养基重悬细胞，根据细胞计数结果使用1640完全培养基调整细胞密度至2×10 6个细胞/ml。
内吞实验(抗体与靶细胞共孵育)：(此步骤需注意温度，37℃有抗体内吞现象，4℃能抑制抗体内吞现象)取96孔超低吸附U底板，将细胞铺于96孔板中，用多通道移液枪每孔加入50μl的细胞悬浮液(每孔的细胞个数为1×10 5个/孔)；然后将不同浓度(起始浓度为100μg/ml)的双功能蛋白、PD-1单抗(Toripalimab)和阴性对照(Anti-KLH IgG4)与细胞在CO 2培养箱中共孵育4h(37℃)。孵育结束后取出96孔细胞板，1000rpm离心5分钟，弃上清；然后用染色缓冲液(PBS+1％BSA)重悬细胞，并1000rpm离心5分钟，弃上清，重复两次操作洗去未结合的多余双功能蛋白，在96孔细胞板中每孔加入150ul的上述染色缓冲液，FACS上机检测并用FlowJo软件进行数据分析。
3、结果
如图8所示，本发明双功能蛋白JS-TZO3和抗PD-1单抗(Toripalimab)均有很强的内吞活性，且呈剂量依赖效应。阴性对照Anti-KLH IgG4没有内吞活性。
实施例9：双功能蛋白JS-TZO3的体外混合淋巴细胞反应
1、测试目的
本发明在体外水平利用混合淋巴细胞反应实验研究了本发明双功能蛋白JS-TZO3对T细胞激活后IL2和IFNγ细胞因子的释放水平。
2、测试过程
将商品化人PBMC(SAILY BIO,cat#SLB-HP050B)用含血清培养基在37℃静置孵育2h后洗去悬浮细胞，然后分别用10ng/ml的GM-CSF(Sino Biological,cat#GMP-11846-HNAE)和10ng/ml的IL-4(Sino Biological,cat#GMP-10015-HNAH)刺激贴壁细胞5天，随后再用5ng/ml的IL-1β(Sino Biological,cat#10139-HNAE)、10ng/ml的IL-6(Sino Biological,cat#GMP-10395-HNAE)、10ng/ml的TNF-a(Sino Biological,cat#GMP-10602-HNAE)及和1μmol/L的PGE2刺激贴壁细胞2天，来诱导成熟树突状细胞(mDC)。用CD4T MicroBeads,Human(Miltenyi Biotec,cat#130-045-101)纯化商品化人PBMC(SAILY BIO,cat#SLB-HP050B,lot#190100)得到CD4T细胞。
在96孔圆底板中将不同浓度(0.15nM-150μM,10倍稀释)的双功能蛋白JS-TZO3组、阴性对照抗体(anti-KLH IgG4)+TGFβTrap(君实生物)对照组、PD-1单抗(Toripalimab)组以及PD-1单抗与TGFβTrap联用组(PD-1抗体+TGFβTrap对照组)分别与100ng/ml的TGFβ1重组蛋白(novoprotein,cat#CA59)以及mDC细胞(每孔10000个细胞)和上述纯化的CD4T细胞(每孔100000个细胞)在37℃的CO 2培养箱中共孵育5天。在共孵育第3天从96孔板中每孔收集部分反应上清用BD CBA human IL2Flex set(BD,cat#558270)检测IL-2释放，并在共孵育第5天用BD CBA human IFNγFlex set(BD,cat#558269)检测上清中的IFNγ含量。
3、结果
图9结果可以看出，在混合淋巴细胞反应中，本发明双功能蛋白JS-TZO3及PD-1单抗和PD-1单抗与TGFβTrap联用组均能促进IL-2的释放，但差别不明显(图9a)。本发明双功能蛋白JS-TZO3能够显著促进IFNγ的释放，活性显著高于PD-1单抗与TGFβTrap联用组和PD-1单抗组(图9b)。
实施例10：双功能蛋白JS-TZO3在小鼠体内对人黑色素瘤A375肿瘤生长的抑制作用
1、测试目的
评价本发明双功能蛋白JS-TZO3在人黑色素瘤A375 Mixeno皮下移植模型中的抗肿瘤作用。
2、测试过程
取18只6-7周龄雌性NCG小鼠(江苏集萃药康生物科技有限公司)，小鼠于右侧背部皮下接种预混合好的4.5 x 10 6(个/0.1mL)A375细胞(ATCC CRL-1619)和1 x 10 6(个/0.1mL)活化的T细胞(#250PBMC捐献者，中美冠科生物技术(太仓)有限公司)的 混悬液(0.2ml/只)。细胞接种后的1小时开始给药。肿瘤细胞接种当天定义为第0天。根据体重平均分为3组，每组6只动物，分别为：
TGFβTrap对照组(G4Fc TGFβR2ecd)，10mg/kg；
PD-1单抗(Toripalimab)对照组，10mg/kg；
双功能蛋白(JS-TZO3)治疗组，10mg/kg。
所有组给药途径均为腹腔注射，每周给药2次，连续给药6周，末次给药3天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI％(TGI＝1-T/C(％)。T/C％为肿瘤增值率，即在某一时间点，治疗组和对照组肿瘤体积或瘤重的百分比值。T和C分别为治疗组和对照组在某一特定时间点的肿瘤体积(TV)。
如图10所示，在开始给药后第31天，TGFβTrap对照组(G4Fc TGFβR2ecd)平均肿瘤体积为2009±217mm 3。PD-1单抗(Toripalimab)对照组和双功能蛋白(JS-TZO3)组的平均肿瘤体积分别为579±217mm 3和404±123mm 3，与TGFβTrap对照组相比，肿瘤抑制率分别为71.17％和79.89％。表明双靶向PD-1和TGFβR2ecd的人源化抗体JS-TZO3在10mg/kg的剂量水平可以显著抑制人黑色素瘤A375细胞皮下移植瘤的生长，且优于PD-1单抗(Toripalimab)和TGFβTrap对照(G4Fc TGFβR2ecd)。
实施例11：本发明双功能蛋白JS-TZO3稳定性评价
11.1：本发明双功能蛋白JS-TZO3的热稳定性
1、测试目的
检测本发明双功能蛋白JS-TZO3的热稳定性。利用QPCR-7500，在pH 5.2的缓冲体系中(20mM醋酸-醋酸钠/50mM氯化钠/150mM甘露)，考察不同温度下双功能蛋白的稳定性。
2、测试过程及结果
将样品置换到上述缓冲液中，控制样品浓度在10mg/ml左右，利用QPCR-7500进行检测。结果见表3，在上述缓冲液中，本发明双功能蛋白JS-TZO3表现了良好的热稳定性。
表3：双功能蛋白JS-TZO3的热稳定性
样品 Tm1(℃) Tm2(℃)
双功能蛋白JS-TZO3 65.7 76.8
注：Tm:热转化温度。
11.2：本发明双功能蛋白JS-TZO3的高温稳定性
1、测试目的
高温条件下双功能蛋白JS-TZO3稳定性研究。
2、测试过程及结果
将双功能蛋白置JS-TZO3换到pH 5.2的缓冲体系中(20mM醋酸-醋酸钠/50mM氯化钠/150mM甘露醇，控制样品浓度在10mg/ml左右，分装一定样品体积(200μl/管)放置40℃恒温箱，考察0W、1W、2W和4W稳定性，根据时间点送样检测。检测结果显示本发明双功能蛋白JS-TZO3具有良好的热稳定性，具体见表4和表5。
表4：双功能蛋白JS-TZO3的高温稳定性
Figure PCTCN2021121198-appb-000025
表5：双功能蛋白JS-TZO3的高温稳定性
Figure PCTCN2021121198-appb-000026
注：NA表示未检测。
实施例12、靶向PD-L1和TGF-β的双功能蛋白JS-TZO4的制备
12.1双功能蛋白表达载体的构建
合成人抗体重链IgG4恒定区基因片段，并通过EcoRI和NotI限制性内切酶将IgG4恒定区基因片段连接至pCDNA3.1载体，获得载体HXT4s。委托南京金斯瑞合成编码抗人PD-L1抗体的核苷酸序列，并通过BSPQI限制性内切酶将基因合成片段9B1HC2连接至载体HXT4s上，得到载体HXT4s-9B1HC2。委托南京金斯瑞合成编码TGF-βRII胞外区(ECD)的核苷酸序列。连接肽((G 4S) 4G)的核苷酸序列通过所属领域技术手段获得。编码抗人PD-L1抗体的核苷酸序列、编码TGF-βRII胞外区的核苷酸序列、接头蛋白片段((G 4S) 4G)的核苷酸序列通过所属领域技术手段获得。
合成引物TGFβR2-G4SBSPQIFV2和TGFβR2-BSPQIRV2，以人TGFβR2为模板进行PCR扩增获得人TGF-βRII ECD基因片段，通过BSPQI限制性内切酶将该TGF-βRII ECD基因片段连接至载体HXT4s-9B1HC2，得到表达载体HXT4s-9B1HC2- TGFβRII。
其中，
引物TGFβR2-G4SBSPQIFV2按5’-3’顺序的核苷酸序列(SEQ ID NO:14)如下：
Figure PCTCN2021121198-appb-000027
引物TGFβR2-BSPQIRV2按5’-3’顺序的核苷酸序列(SEQ ID NO:15)如下：
Figure PCTCN2021121198-appb-000028
用于双功能蛋白JS-TZO4构建的抗人PD-L1抗体的氨基酸序列如SEQ ID NO:31(重链)和SEQ ID NO:27(轻链)所示；TGF-βRII胞外区氨基酸序列如SEQ ID NO:10所示；表达载体HXT4s-1A6H2-TGFβRII表达双功能蛋白JS-TZO4，该双功能蛋白JS-TZO4的第二亚基氨基酸序列如SEQ ID NO:27，其编码核酸如SEQ ID NO:30所示；其第一亚基氨基酸序列如SEQ ID NO:28所示，其编码核酸如SEQ ID NO:29所示。
12.2双功能蛋白的表达和纯化
取液氮保存的293F细胞(FreeStyle TM 293-F Cells，Thermofisher)进行复苏，将其加入表达培养基(Freestyle TM 293 expression medium,Gibco)中进行培养。培养数代后，转染前一天将293F细胞稀释至(1.5-2.0)×10 6个细胞/mL，转染时，先取1/10转染体积的上述培养基孵育表达载体HXT4s-9B1HC2-TGFβRII和转染试剂(PEI)，表达载体用量为0.5-2μg/mL，转染试剂用量为3-14μg/mL。孵育时间为20-30min，孵育完成后将转染混合物加入提前一天处理好的上述293F细胞中，边加边摇匀。将完成转染的摇瓶放入二氧化碳摇床进行培养，条件为36.5℃，120rpm，7％CO 2。7天以后，离心收集上清液进行纯化。使用AKTA AVANT仪器进行纯化，使用蛋白A亲和填料(MabSelect SuRe TM,GE Healthcare)预装的纯化柱，将上述上清液离心完成后经0.22μm滤器过滤后上样，PBS进行洗杂，柠檬酸缓冲液进行洗脱，洗脱完成后Tris缓冲液中和，得到双功能蛋白JS-TZO4。制备的双功能蛋白JS-TZO4可用于下述各实施例中。
JS-TZO4双功能蛋白分子结构示意图如图11所示。
实施例13：ELISA检测双功能蛋白JS-TZO4与人源TGF-β的结合
1、测试目的
使用ELISA方法检测本发明双功能蛋白JS-TZO4与人源TGF-β1、TGF-β2、TGF-β3的结合。
2、测试过程及结果
a.以0.5μg/ml浓度的重组人源TGF-β1(Novoprotein，CA59)为抗原按100μl/孔包被96孔板，37℃孵育1.5小时。
b.300μl 1×PBST洗涤4次，加入200μl 2％BSA，37℃封闭1.5小时。
c.300μl 1×PBST洗涤4次，分别加入梯度稀释的双功能蛋白JS-TZO4和阴性对照抗体(Anti-KLH IgG4)(起始浓度100μg/ml，2.5倍梯度稀释12个浓度点)，37℃孵育1小时。
d.300μl 1×PBST洗涤4次。
e.每孔加入100μl辣根过氧化物酶(HRP)偶联的鼠抗人抗体IgG4Fc(1∶5000)(Southern Biotech，9200-05)，37℃孵育1小时。
f.每孔加入100μl 0.1mg/ml的TMB，37℃孵育12分钟后加入100μl 2M盐酸溶液终止反应。
g.酶标仪上检测450nm/620nm测吸收值，Graphpad Prism5分析数据。
如图12、13和14所示，本发明双功能蛋白JS-TZO4与TGF-β1及TGF-β3均有较强的结合，与TGF-β2结合相对较弱。
实施例14：ELISA检测双功能蛋白JS-TZO4与PD-L1的结合
1、测试目的：
ELISA检测双功能蛋白JS-TZO4与PD-L1的结合。
2、测试过程
a.以人源PD-L1(0.3μg/ml浓度，君实生物)为抗原按100μl/孔包被96孔板，37℃孵育1.5小时。
b.300μl 1×PBST洗涤4次，加入200μl 2％BSA，37℃封闭1.5小时。
c.300μl 1×PBST洗涤4次，分别加入梯度稀释的双功能蛋白JS-TZO4、阳性对照抗PD-L1单抗和阴性对照抗体(Anti-KLH IgG4)(起始浓度1μg/ml，2.5倍梯度稀释12个浓度点)，37℃孵育1小时。
d.300μl 1×PBST洗涤4次。
e.每孔加入100μl辣根过氧化物酶(HRP)偶联的鼠抗人抗体IgG4Fc(1∶5000)(Southern Biotech，9200-05)，37℃孵育1小时。
f.每孔加入100μl 0.1mg/ml TMB，室温孵育30分钟后加入100μl 2M盐酸溶液终止反应。
g.酶标仪上检测450nm/620nm测吸收值，Graphpad Prism5分析数据。
阳性对照抗PD-L1单抗：来自君实生物(专利申请号PCT/CN2018/076669)，重链如SEQ ID NO:26所示，轻链如SEQ ID NO:27所示。
结果如图15所示，本发明双功能蛋白JS-TZO4与PD-L1有较强的结合，与阳性对照抗体相当。
实施例15：报告基因法检测双功能蛋白JS-TZO4对PD-1/PD-L1通路阻断试验
1、测试目的：
为了研究双功能蛋白JS-TZO4对PD-1/PD-L1信号通路的阻断作用，采用来自Promaga公司构建的PD-1/PD-L1报告基因***来检测双功能蛋白中PD-L1端的细胞生物学活性。
2、测试过程
取CHO/PD-L1细胞(Promega)，消化并用F1-2营养混合物(F-12 Nutrient Mixture)完全培养基重悬细胞，根据细胞计数结果使用完全培养基调整细胞密度至5×10 5个细胞/ml，将细胞悬液转移至加样槽中，使用多道移液器以100μl/孔加入到96孔板中，放置于37℃，5％CO 2培养箱培养16～20h；第二天制备Jurkat/PD-1(Promega)细胞悬液，根据细胞计数结果使用分析培养基重悬细胞，并调整细胞密度至2.5×10 6个细胞/ml；将加入CHO/PD-L1细胞的细胞培养板从培养箱中取出，使用多道移液器每孔取出100μl培养液，按照40μl/孔分别加入梯度稀释的双功能蛋白JS-TZO4(起始浓度为16.5nM，3倍梯度稀释10个浓度点)、阳性对照PD-L1单抗(起始浓度为18.33nM，3倍梯度稀释10个浓度点)和阴性对照抗体(Anti-KLH IgG4；起始浓度为18.33nM，3倍梯度稀释10个浓度点)，然后将上述Jurkat/PD-1细胞悬液转移至加样槽中，以40μl/孔加入到细胞培养板中，置于37℃，5％CO 2培养箱培养5～6h。在蛋白孵育期间，将One-Glo试剂(Promega，E6130)取出使其温度恢复至室温。取出细胞培养板，置于室温放置5～10min，然后每孔加入40μl萤光素酶底物ONE-Glo  TM试剂(promega，E6130)，置于混匀器上混匀5～10min，然后使用M1000 pro多功能酶标仪(Tecan)读取发光信号值。
阳性对照PD-L1单抗：来自君实生物(专利申请号PCT/CN2018/076669)，重链如SEQ ID NO:26所示，轻链如SEQ ID NO:27所示。
3、结果
如图16所示，本发明双功能蛋白JS-TZO4和抗PD-L1单抗均能够有效地阻断PD-1/PD-L1的相互作用，促进NFAT信号的增强，有显著的药物浓度剂量依赖效应，二者活性水平相当。
实施例16：报告基因法检测双功能蛋白JS-TZO4对TGFβ通路阻断试验
1、测试目的
通过4T1细胞表达带荧光素酶报告基因的Smad3结合原件(SBE)来研究双功能蛋白JS-TZO4对TGF-β1诱导Smad3活化的抑制作用。
2、测试过程
4T1-SBE细胞(君实生物)使用含有10％FBS的1640完全培养基(Gibco，11875-093)培养，每2天传代一次。实验第一天以每孔100,000个细胞的密度接种于96孔板(Costar，3917)，在37℃、5％CO 2条件下培养16～20小时。第二天，弃去细胞培养板中的培养基，按照50μl/孔加入梯度稀释(起始浓度为1nM，4倍梯度稀释9个浓度点)的双功能蛋白JS-TZO4及阳性对照G4Fc TGFβR2ecd(TGFβTrap，君实生物)。细胞在37℃、5％CO 2条件下继续培养0.5～1小时。每孔加入50μl重组人源化TGFβ1。细胞在37℃、5％CO 2条件下继续培养5～6h。然后每孔加入50μl萤光素酶底物ONE-Glo  TM试剂(promega，E6130)，置于混匀器上混匀5～10min，然后使用M1000 pro多功能酶标仪(Tecan)读取发光信号值。待测样品的IC50值使用数据处理软件Graphpad Prism7.0计算得到。
3、结果
如图17所示，本发明双功能蛋白JS-TZO4与G4Fc TGFβR2ecd(TGFβTrap)均能够以剂量依赖性形式抑制TGFβ1诱导的pSMAD3的活化。
实施例17：双功能蛋白JS-TZO4的细胞内吞试验
1测试目的
在体外水平利用抗体内吞实验来检测双功能蛋白JS-TZO4的内吞活性。
2、测试过程
CypHer 5Mono NHS Ester染料标记待测样品：用Label buffer(标记缓冲液，PBS:NaCO 3＝9:1，pH:8.3)将待测样品(双功能蛋白JS-TZO4、抗PD-L1单抗和阴性对照抗-KLH IgG4)浓度分别调整成1.0mg/ml。计算待测样品质量，加入CypHer 5Mono NHS Ester染料(GE，PA15401)，室温避光混匀1h。将反应后的待测样品与染料混合液转移至0.5ml超滤管(30KD)中，离心10min，弃去废液，再向超滤管中补充Label buffer，继续同上离心4次，将未结合的多余染料洗去。
取CHO/PD-L1细胞(Promega)，消化并用F-12 Nutrient Mixture完全培养基重悬细胞，取293F JS1 1E4细胞，消化并用1640完全培养基重悬细胞，根据细胞计数结果使用1640完全培养基调整细胞密度至2×10 6个细胞/ml。
内吞实验(待测样品与靶细胞共孵育):(此步骤需注意温度，37℃有抗体内吞现象， 4℃能抑制抗体内吞现象)取96孔超低吸附U底板，将细胞铺于96孔板中，用多通道移液枪每孔加入50μl的细胞悬浮液(每孔的细胞个数为1×10 5个细胞/孔)；然后将不同浓度的待测样品(双功能蛋白JS-TZO4：起始浓度为55.56nM，3倍梯度稀释12个浓度点；抗PD-L1单抗和Anti-KLH IgG4：起始浓度为66.7nM，3倍梯度稀释12个浓度点)与细胞在CO 2培养箱中共孵育4h。孵育结束后取出96孔细胞板，1000rpm离心5分钟，弃上清；然后用染色缓冲液(PBS+1％BSA)重悬细胞，并1000rpm离心5分钟，弃上清，重复两次操作洗去未结合的多余样品,在96孔细胞板中每孔加入150μl的染色缓冲液，FACS上机检测并用FlowJo软件进行数据分析。
阳性对照PD-L1单抗：君实生物(专利申请号PCT/CN2018/076669)，重链如SEQ ID NO:26所示，轻链如SEQ ID NO:27所示。
3、结果
如图18所示，本发明双功能蛋白JS-TZO4和抗PD-L1单抗均有很强的内吞活性，且呈剂量依赖效应。阴性对照Anti-KLH IgG4没有内吞活性。
实施例18：双功能蛋白JS-TZO4的体外混合淋巴细胞反应
1、测试目的
本发明在体外水平利用混合淋巴细胞反应实验研究了双功能蛋白JS-TZO4对T细胞激活后IL2和IFNγ细胞因子的释放水平。
2、测试过程
将商品化人PBMC(SAILY BIO,cat#SLB-HP050B)用含血清培养基在37℃静置孵育2h后洗去悬浮细胞，然后分别用10ng/ml的GM-CSF(Sino Biological,cat#GMP-11846-HNAE)和10ng/ml的IL-4(Sino Biological,cat#GMP-10015-HNAH)刺激贴壁细胞5天，随后再用5ng/ml的IL-1β(Sino Biological,cat#10139-HNAE)、10ng/ml的IL-6(Sino Biological,cat#GMP-10395-HNAE)、10ng/ml的TNF-α(Sino Biological,cat#GMP-10602-HNAE)及和1μmol/L的PGE2刺激贴壁细胞2天，来诱导成熟树突状细胞(mDC)。用CD4T MicroBeads,Human(Miltenyi Biotec,cat#130-045-101)纯化商品化人PBMC(SAILY BIO,cat#SLB-HP050B,lot#190100)得到CD4T细胞。
在96孔圆底板中将不同浓度(0.15nM-150μM,10倍稀释)的待测样品(空白对照组、双功能蛋白JS-TZO4组、阴性对照抗体(抗-KLH IgG4)+TGFβTrap(君实生物)对照组、PD-L1单抗组以及PD-L1单抗与TGFβTrap联用组(PD-L1抗体+TGFβTrap对照组)分别与恒定浓度(100ng/ml)的TGFβ1重组蛋白(novoprotein,cat#CA59)以及mDC细胞(每孔10000个细胞)和纯化的CD4T细胞(每孔100000个细胞)在37℃的CO 2培养 箱中共孵育5天。在共孵育第3天从96孔板中每孔收集部分反应上清用BD CBA human IL2Flex set(BD,cat#558270)检测IL-2释放，并在共孵育第5天用BD CBA human IFNγFlex set(BD,cat#558269)检测上清中的IFNγ含量。
PD-L1单抗：君实生物(专利申请号PCT/CN2018/076669)，重链如SEQ ID NO:26所示，轻链如SEQ ID NO:27所示。
3、结果
从图19可以看出，在混合淋巴细胞反应中，本发明双功能蛋白JS-TZO4、PD-L1单抗和PD-L1单抗与TGFβTrap联用组均能促进IL-2的释放，但差别不明显(图19a)。本发明双功能蛋白JS-TZO4能够显著促进IFNγ的释放，活性与PD-L1单抗与TGFβTrap联用组和PD-L1单抗组相当(图19b)。
实施例19：本发明双功能蛋白JS-TZO4稳定性评价
8.1：本发明双功能蛋白JS-TZO4的热稳定性
1、测试目的
用于检测双功能蛋白JS-TZO4的热稳定性。利用QPCR-7500，检测不同抗体的热稳定性，在pH 5.2的缓冲液中(20mM醋酸盐(醋酸-醋酸钠)/50mM氯化钠/150mM甘露)，考察不同温度下的稳定性。
2、测试过程及结果
将样品置换到上述缓冲液中，控制样品浓度在10mg/ml左右，利用QPCR-7500进行检测。具体见表6，在上述缓冲液中，本发明双功能蛋白JS-TZO4表现了良好的热稳定性。
表6：双功能蛋白JS-TZO4的热稳定性
样品 Tm1(℃) Tm2(℃)
双功能蛋白JS-TZO4 65.4 76.9
注：Tm:热转化温度。
8.2：本发明双功能蛋白JS-TZO4的高温稳定性
1、测试目的
高温条件下双功能蛋白JS-TZO4稳定性研究。
2、测试过程及结果
将样品置换到pH 5.2的缓冲体系中(20mM醋酸-醋酸钠/50mM氯化钠/150mM甘露醇)，控制样品浓度在10mg/ml左右，分装一定样品体积(200μl/管)放置40℃恒温箱考察0W、1W、2W和4W稳定性，到时间点送样检测。通过以下参数评估稳定性：(a) SEC-HPLC(尺寸排阻色谱法)测量抗体单体、聚体或片段的含量；(b)CE-SDS(十二烷基硫酸钠毛细管电泳)法检测抗体的分子量；(c)ELISA法和报告基因法检测抗体生物学活性。
结果显示本发明双功能蛋白JS-TZO4具有良好的热稳定性，具体见表7和表8。
表7：双功能蛋白JS-TZO4的高温稳定性
Figure PCTCN2021121198-appb-000029
表8：双功能蛋白JS-TZO4的高温稳定性
Figure PCTCN2021121198-appb-000030
注：NA表示未检测。

Claims (20)

  1. 靶向PD-1或PD-L1和TGF-β的双功能蛋白，其包含
    (i)抗PD-1或PD-L1抗体的抗原结合片段；
    (ii)免疫球蛋白Fc结构域；和
    (iii)TGF-βRⅡ胞外结构域(ECD)。
  2. 如权利要求1所述的双功能蛋白，其中所述抗PD-1抗体的抗原结合片段包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；优选地，所述抗PD-1抗体的抗原结合片段包含氨基酸序列如SEQ ID NO:7所示的重链可变区和氨基酸序列如SEQ ID NO:8所示的轻链可变区，或与SEQ ID NO:7所示重链可变区和SEQ ID NO:8所示轻链可变区的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  3. 如权利要求1所述的双功能蛋白，其中所述抗PD-L1抗体的抗原结合片段包含氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23所示的LCDR1、LCDR2和LCDR3；优选地，所述抗PD-L1抗体的抗原结合片段包含与SEQ ID NO:24所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链可变区，和与SEQ ID NO:25所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链可变区；进一步优选地，所述抗PD-L1抗体的抗原结合片段包含氨基酸序列如SEQ ID NO:24所示的重链可变区和氨基酸序列如SEQ ID NO:25所示的轻链可变区。
  4. 如权利要求1-3中任一项所述的双功能蛋白，其中所述抗PD-1或PD-L1抗体的抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
  5. 如权利要求1-3中任一项所述的双功能蛋白，其中所述免疫球蛋白Fc结构域是人IgG1、IgG2、IgG3或IgG4的Fc结构域；优选为人IgG4的Fc结构域；优选地，所述IgG4的Fc结构域中包含1、2、3、4或5个氨基酸差异，优选为在Fc结构域中具有S228P氨基酸置换；进一步优选为在Fc结构域中具有C末端K和G两个氨基酸缺失。
  6. 如权利要求5所述的双功能蛋白，其中所述免疫球蛋白Fc结构域包含与SEQ ID NO:9所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98 ％、99％或100％序列同一性的Fc结构域；优选地，所述免疫球蛋白Fc结构域包含如SEQ ID NO:9所示的氨基酸序列。
  7. 如权利要求1-6中任一项所述的双功能蛋白，其中所述TGF-βRⅡ胞外结构域具有SEQ ID NO:10所示的氨基酸序列，或与SEQ ID NO:10所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  8. 如权利要求1-7中任一项所述的双功能蛋白，其中所述双功能蛋白从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；优选地，所述(i)和(ii)之间通过铰链区连接，(i)和(iii)以及(ii)和(iii)之间通过连接肽连接，所述连接肽的氨基酸序列含有甘氨酸残基和丝氨酸残基或由甘氨酸残基和丝氨酸残基组成，长度为2-31个氨基酸残基；优选各自独立为(GGGGS)nG，其中，n分别独立地选自3、4、5或6。
  9. 如权利要求1-8中任一项所述的双功能蛋白，其包含抗PD-1或PD-L1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TGF-βRⅡECD。
  10. 如权利要求9中所述的双功能蛋白，其中所述抗PD-1抗体具有如SEQ ID NO:11所示的重链氨基酸序列和如SEQ ID NO:12所示的轻链氨基酸序列，或与SEQ ID NO:11和/或SEQ ID NO:12所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  11. 如权利要求9中所述的双功能蛋白，其中所述抗PD-L1抗体包含与SEQ ID NO:31所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链氨基酸序列，和与SEQ ID NO:27所示氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链氨基酸序列；进一步优选地，所述抗PD-L1抗体包含如SEQ ID NO:31所示的重链氨基酸序列和如SEQ ID NO:27所示的轻链氨基酸序列。
  12. 如权利要求1-11中任一项所述的双功能蛋白，其包含氨基酸序列如SEQ ID NO:13所示的双功能蛋白第一亚基，和氨基酸序列如SEQ ID NO:12所示的双功能第二亚基；或包含氨基酸序列如SEQ ID NO:28所示的双功能蛋白第一亚基，和氨基酸序列如SEQ ID NO:27所示的双功能第二亚基。
  13. 一种多核苷酸，其编码权利要求1-12中任一项所述的双功能蛋白或其抗原结合片段；优选地，所述多核苷酸包含如SEQ ID NO:16所示第一亚基的核苷酸序列和/或如SEQ ID NO:17所示第二亚基的核苷酸序列；优选地，所述多核苷酸包含如SEQ ID NO:29所示第一亚基的核苷酸序列和/或如SEQ ID NO:30所示第二亚基的核苷酸序列。
  14. 一种表达载体，其包含如权利要求13所述的多核苷酸，优选地，所述表达载体为真核表达载体。
  15. 一种宿主细胞，其包含如权利要求13所述的多核苷酸或如权利要求14所述的表达载体，优选地，所述宿主细胞是哺乳动物细胞。
  16. 一种制备如权利要求1-12中任一项所述的双功能蛋白的方法，所述方法包括在适合于所述双功能蛋白表达的条件下在权利要求15所述的宿主细胞中表达所述双功能蛋白，并从所述宿主细胞回收所表达的双功能蛋白。
  17. 一种药物组合物，其包含权利要求1-12中任一项所述的双功能蛋白、权利要求13所述的多核苷酸、权利要求14所述的表达载体、权利要求15所述的宿主细胞，和药学上可接受的载体或赋形剂。
  18. 如权利要求1-12中任一项所述的双功能蛋白、权利要求13所述的多核苷酸、权利要求14所述的表达载体、权利要求15所述的宿主细胞，或权利要求17所述的药物组合物在制备用于治疗和/或预防与PD-1、PD-L1或TGF-β活性相关的疾病或病症的药物中的用途，优选所述疾病或病症为癌症，更优选地，所述癌症选自黑素瘤、肾癌、***癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
  19. 一种试剂盒，其包括如权利要求1-12中任一项所述的双功能蛋白、权利要求13所述的多核苷酸、权利要求14所述的表达载体、权利要求15所述的宿主细胞，或权利要求17所述的药物组合物。
  20. 一种使用如权利要求1-12中任一项所述的双功能蛋白检测PD-1、PD-L1或TGF-β在样品中的存在的方法。
PCT/CN2021/121198 2020-09-28 2021-09-28 靶向PD-1或PD-L1和TGF-β的双功能蛋白及其医药用途 WO2022063314A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104250302A (zh) * 2013-06-26 2014-12-31 上海君实生物医药科技有限公司 抗pd-1抗体及其应用
CN108456251A (zh) * 2017-02-21 2018-08-28 上海君实生物医药科技股份有限公司 抗pd-l1抗体及其应用
WO2018205985A1 (zh) * 2017-05-12 2018-11-15 江苏恒瑞医药股份有限公司 含有TGF-β受体的融合蛋白及其医药用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104250302A (zh) * 2013-06-26 2014-12-31 上海君实生物医药科技有限公司 抗pd-1抗体及其应用
CN108456251A (zh) * 2017-02-21 2018-08-28 上海君实生物医药科技股份有限公司 抗pd-l1抗体及其应用
WO2018205985A1 (zh) * 2017-05-12 2018-11-15 江苏恒瑞医药股份有限公司 含有TGF-β受体的融合蛋白及其医药用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GRENGA ITALIA, DONAHUE RENEE N., GARGULAK MORGAN L., LEPONE LAUREN M., ROSELLI MARIO, BILUSIC MARIJO, SCHLOM JEFFREY: "Anti-PD-L1/TGFβR2 (M7824) Fusion Protein Induces Immunogenic Modulation of Human Urothelial Carcinoma Cell Lines, Rendering Them More Susceptible to Immune-mediated Recognition and Lysis", UROLOGIC ONCOLOGY: SEMINARS AND ORIGINAL INVESTIGATIONS, vol. 36, no. 3, 1 March 2018 (2018-03-01), AMSTERDAM, NL, pages 1 - 21, XP055786925, ISSN: 1078-1439, DOI: 10.1016/j.urolonc.2017.09.027 *
HANNE LIND, SOFIA R GAMEIRO, CAROLINE JOCHEMS, RENEE N. DONAHUE, JULIUS STRAUSS, JAMES L GULLEY, CLAUDIA PALENA, JEFFREY SCHLOM: "Dual targeting of TGF-β and PD-L1 via a bifunctional anti-PD-L1/TGF-βRII agent: status of preclinical and clinical advances", JOURNAL FOR IMMUNOTHERAPY OF CANCER, vol. 8, no. 1, 19 December 2019 (2019-12-19), pages 1 - 10, XP055686662, DOI: 10.1136/jitc-2019-000433 *
KARIN M. KNUDSON, KRISTIN C. HICKS, XIAOLING LUO, JIN-QIU CHEN, JEFFREY SCHLOM, SOFIA R. GAMEIRO: "M7824, a novel bifunctional anti-PD-L1/TGFβ Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine", ONCOIMMUNOLOGY, vol. 7, no. 5, 4 May 2018 (2018-05-04), pages 1 - 14, XP055665440, ISSN: 2162-402X, DOI: 10.1080/2162402X.2018.1426519 *
RAJANI RAVI, NOONAN KIMBERLY A., PHAM VUI, BEDI RISHI, ZHAVORONKOV ALEX, OZEROV IVAN V., MAKAREV EUGENE, V. ARTEMOV ARTEM, WYSOCKI: "Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFβ enhance the efficacy of cancer immunotherapy", NATURE COMMUNICATIONS, vol. 9, no. 741, 21 February 2018 (2018-02-21), pages 1 - 14, XP055548010, DOI: 10.1038/s41467-017-02696-6 *

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