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WO2021185337A1 - 一种双特异性融合蛋白及其应用 - Google Patents

一种双特异性融合蛋白及其应用 Download PDF

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WO2021185337A1
WO2021185337A1 PCT/CN2021/081659 CN2021081659W WO2021185337A1 WO 2021185337 A1 WO2021185337 A1 WO 2021185337A1 CN 2021081659 W CN2021081659 W CN 2021081659W WO 2021185337 A1 WO2021185337 A1 WO 2021185337A1
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amino acid
acid sequence
fusion protein
binding domain
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房健民
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Remegen Co Ltd
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Remegen Co Ltd
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Priority to KR1020227022059A priority Critical patent/KR20220107257A/ko
Priority to CN202180005265.9A priority patent/CN114466868A/zh
Priority to US17/594,768 priority patent/US20220204626A1/en
Priority to CA3137211A priority patent/CA3137211A1/en
Priority to AU2021237513A priority patent/AU2021237513B2/en
Priority to JP2022539415A priority patent/JP7489468B2/ja
Priority to EP21770764.5A priority patent/EP4056596A4/en
Publication of WO2021185337A1 publication Critical patent/WO2021185337A1/zh
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    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

Definitions

  • the present invention relates to a bispecific fusion protein with a novel structure and its application.
  • the present invention also relates to polynucleotides, nucleic acid constructs and host cells for encoding or preparing the fusion protein.
  • BsAbs Bispecific antibody
  • dual targeting strategies have become one of the effective methods for the treatment of tumors and immune diseases (Kontermann R. Dual targeting strategies with bispecific antibodies[C]//MAbs.Taylor& Francis, 2012, 4(2):182-197.).
  • Bispecific antibodies can specifically bind to two different antigens or epitopes (Carter P. Bispecific human IgG by design[J]. Journal of immunological methods, 2001,248(1-2): 7-15.). As early as the 1980s, Morrison et al. first prepared the first true anti-dextran and dansyl tetravalent double chain antibody by connecting single-chain antibodies with different specificities using flexible peptides and then fusion expression. Specific antibodies (Coloma M J, Morrison S L. Design and production of novel tetravalent bispecific antibodies [J]. Nature biotechnology, 1997, 15(2): 159.). However, due to the bottleneck of antibody preparation technology and the insufficiency of basic research on signal pathways, the development of bispecific antibodies has been hindered.
  • bispecific antibodies In the development of bispecific antibodies, the choice of molecular structure is very important. Different bispecific antibody designs have their own advantages and disadvantages, but the design of bispecific antibodies for clinical treatment purposes must solve the following problems: First, ensure the correct coupling or pairing of two pairs (or more) of different light chains and heavy chains ; Second, maintain the independence of each binding domain of each monoclonal antibody, and when binding different epitopes at the same time, there will be no steric interference between each other; third, antibody molecules should be easy to express in mammalian cells. No complicated protein modification process is required; fourthly, it has good druggability, such as high thermal stability, high chemical stability, high solubility, not easy to polymerize, low viscosity, high expression level, suitable half-life, etc. (Spiess C ,Zhai Q, Carter P J. Alternative molecular formats and therapeutic applications for bispecific antibodies[J]. Molecular immunology, 2015, 67(2): 95-106.).
  • bispecific antibodies can be divided into many types. For example, they can be divided into symmetrical and asymmetrical structures according to the symmetry of the structure. According to the presence or absence of the Fc region, they can be classified as containing Fc. The bispecific antibody of the region and the bispecific antibody without the Fc region, as well as the configuration according to the number of antigen binding regions, are classified into bivalent, trivalent, tetravalent or more valent configurations, etc.
  • bispecific antibodies have developed dozens of structures (Spiess C, Zhai Q, Carter P J. Alternative molecular formats and therapeutic applications for bispecific antibodies[J]. Molecular immunoology, 2015, 67(2): 95-106.
  • bispecific antibodies have diverse structures, roughly divided into 5 categories, including IgG-like bispecific antibodies, IgG-like bispecific antibodies with additional functional regions, and bispecific antibodies with different antigen-binding fragments in series , Fusion protein type bispecific antibody, chemically coupled bispecific antibody.
  • IgG-like bispecific antibodies mainly uses recombinant DNA technology for structural modification.
  • the constructed IgG-like bispecific antibodies have complete crystallizable fragments (ie Fc fragments), which can bind to different antigens while still retaining Fc fragment mediated ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-mediated cytotoxicity, complement-dependent cytotoxicity) and other functions, while this type of antibody also retains The characteristics of prolonging the half-life of antibodies in vivo by combining with neonatal Fc receptors (FcRn) (Ridgway J B, Presta L G, Carter P.' Knobs-into-holes' engineering of antibody CH3domains for heavy chain heterodimerization[ J].Protein Engineering, Design and Selection,1996,9(7):617-621.); IgG-like bispecific antibodies with additional functional regions are generally based on traditional IgG antibodies by means of fusion proteins, Add other specific antigen-binding fragments (that is, additional functional regions, such as single domain antibodies, single chain antibodies, etc.) on the heavy chain and/or light
  • Monoclonal antibody therapeutics with up to five specificities functional enhancement through fusion of target-specific peptides[C]//MAbs.Taylor&Francis,2013,5(2):208-218.); bispecific antibodies with different antigen-binding fragments in series are passed
  • the connecting peptide is obtained by connecting different Fabs, single-chain antibodies, single-domain antibodies, or antigen-binding fragments in a certain sequence (Stork R, Müller D, Kontermann R EA novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an album-binding domain from streptococcal protein G[J].Protein Engineering, Design &Selection,2007,20(11):569-576.); fusion protein type bispecific antibodies combine different specificities through linkers such as peptide fragments The antibody/antibody fragment (IgG, Fab, scFv, etc.) is connected to form a protein molecule that
  • Tumor antigens include Claudin 18.2, HER-2, Mesothelin, BCMA, SSTR2, GPRC5D, PSMA, FCRH5, CD33, CD123, CD20, A33, CEA, CD28, DLL3, EGFR, VEGFR, VEGFR2, VEGF-A, Nectin-4, FGFR, C-met, RANKL, PDGF, PDGFR, PDGFR ⁇ , DLL4, Ang-1, Ang-2, etc.
  • MEDI5752 A novel bispecific antibody that preferentially targets CTLA-4 on PD-1 expressing T-cells.Cancer Res.78(13),Supplement.Abstract 2776:(2018).Hedvat,M.etal.Simultaneous checkpoint-checkpoint or checkpoint-costimulatory receptortargeting with bispecific antibodies promotes enhanced human T cell activation[abstract P664].
  • SITC Society for Immunotherapy of Cancer
  • Aran F. Labrijn, etpipeal. Bispecific Antibodies a mechanism review of the Drug Review, NatureRecovery volume 18, pages585–608 (2019).
  • bispecific antibodies are still in the clinical or preclinical research stage. Only three bispecific antibodies have been approved for the market. They are Trion Pharma’s Catumaxomab (delisted due to commercial factors in 2017) and Amgen’s Blinatumomab And Roche's Emicizumab. Catumaxomab is an IgG-like bispecific antibody in the form of Triomab.
  • Blinatumomab is a bispecific antibody without Fc region and adopts the BiTE (bispecific T cell engager) structure.
  • Emicizumab is a modified humanized bispecific IgG4 monoclonal antibody that can bind factor IXa and factor X.
  • the antibody uses "Knobs-into-Holes (KiH)" for Fc design, “Common Light Chain-IgG (CLC-IgG)” for LC design, and "Multidimensional Optimization” for variable region optimization.
  • KiH Knobs-into-Holes
  • CLC-IgG Common Light Chain-IgG
  • Multidimensional Optimization for variable region optimization.
  • bispecific antibodies/platforms and 3 marketed drugs research and development experience due to the complex structure of bispecific antibodies and diverse functions, there are more problems and problems in their development than general antibodies. challenge.
  • IgG-like bispecific antibodies face the problem of heavy chain-heavy chain or heavy chain-light chain mismatches during the preparation process.
  • the most typical one is Catumaxomab.
  • Catumaxomab is the first bispecific antibody approved for marketing, At the same time, it also has very obvious limitations, which are mainly reflected in the complex production process of Triomab antibody and the immunogenicity problem that heterologous antibodies are relatively easy to produce.
  • bispecific antibodies have many problems, such as low expression, poor stability, complex production processes, and significantly higher research and development costs than monoclonal antibodies, all of which limit the development of bispecific antibodies. Therefore, there is an urgent need to develop bispecific antibodies with new structures to provide more clinical options.
  • the present invention provides a bispecific fusion protein with a novel structure, which belongs to an IgG-like bispecific fusion protein containing additional functional regions. Specifically, the fusion protein is inserted into the second binding domain through an optional peptide linker in the hinge region of a full-length immunoglobulin G (IgG), and the Fab region of the IgG is the first of the fusion protein.
  • IgG immunoglobulin G
  • Binding domain the heavy chain of the IgG after the insertion of the second binding domain is the heavy chain of the fusion protein, and the light chain of the IgG is the light chain of the fusion protein; the second binding domain is selected From human receptors, ligands, fragments of the extracellular region of the receptors or ligands, binding domain fragments, or combinations of fragments; wherein the receptors or ligands are dimerized or combined in the natural signal pathway
  • the multimerization structure activates or inhibits the receptor or ligand of the signaling pathway, and the second binding domain and the first binding domain target different targets.
  • Hibody structural fusion protein or Hibody Hibody structural fusion protein
  • the first binding domain is targeted to bind immune checkpoint molecules or tumor antigens.
  • the immune checkpoint molecules include PD-1 (Programmed cell death protein 1), PD-L1 (Programmed cell death 1 ligand 1), CTLA-4 (Cytotoxic T lymphocyte-associated antigen-4), LAG-3 (Lymphocyte activation gene-3), FGL1 (Fibrinogen-like protein 1), TIM-3 (T cell immunoglobulin-3), Galectin-9, TIGIT (T-cell immunoreceptor with Ig and ITIM domains), CD155, CD47;
  • the tumor antigens include Claudin 18.2, Her-2 (Human epidermal growth factor receptor-2), Mesothelin, BCMA (B Cell Maturation Antigen), SSTR2 (Somatostatin receptor 2), GPRC5D (G-protein coupled receptor family C SMA member) (Prostate specific membrane antigen), FcRH5 (Fc receptor-like protein 5), CD33, CD123, CD20, A33, CEA (Carcino-embryonic antigen), CD28, DLL3 (Delta-like protein 3
  • the second binding domain targets and binds human TGF- ⁇ (Transforming growth factor- ⁇ ), CTLA-4, VEGF, LAG3, CD27, 4-1BB (CD137), OX40 (CD134), CD47, FGL1 (Fibrinogen Like Protein 1), TLT-2 (Trem-like transcript 2), CD28, HGF (Hepatocyte growth factor), CSF1 (Colony-stimulating factor 1), CXCL1 (CXC chemokine ligand 1), CXCL2 (CXC chemokine) ), CXCL3(CXCchemokine ligand 3), CXCL5(CXCchemokineligand5), CXCL6(CXCchemokineligand 6), CXCL7(CXCchemokineligand7), CXCL8(CXCchemokineCligand),XCL8(CXCchemokineCligand9, XCLchemokineCligand9, XCL (CXC chemokine ligand 10),
  • the receptor or ligand is human TGF- ⁇ receptor (TGF- ⁇ R), CD80, CD86, VEGFR, VEGF-trap, FGL1, CD70, 4-1BBL, OX40L, SIRP ⁇ (Signal regulatory protein ⁇ ), B7-H3 (CD276), C-met, CSF1R (Colony-stimulating factor 1 receptor), CXCR2 (CXC chemokine receptor 2), CXCR3 (CXC chemokine receptor 3), CXCR4 (CXC chemokine receptor-induced (GlucocRL) TNF receptor ligand), EGFR, ICOS (Inducible co-stimulator).
  • both CD80 and CD86 are transmembrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF).
  • the mature CD80 molecule consists of 254 amino acids, including 208 amino acids in the extracellular domain (ECD), 25 amino acids in the transmembrane domain, and 21 amino acids in the intracellular domain.
  • the mature CD86 molecule is composed of 303 amino acids, of which the extracellular domain is 222 amino acids, the transmembrane domain is 20 amino acids, and the intracellular domain is 61 amino acids.
  • the extracellular domains of CD80 and CD86 include immunoglobulin V (IgV) and immunoglobulin C (IgC) regions.
  • CD80 and CD86 bind to their ligands CD28 and CTLA-4 through the immunoglobulin V (IgV) region.
  • IgV immunoglobulin V
  • CD80 and CD86 play an important regulatory role in antigen-induced T cell activation, proliferation and effector function production, and are positive regulators; while in the case of CD80 and CD86 binding to CTLA-4, CD80 And CD86 down-regulates the immune response and is a negative regulator. Therefore, CD80 and CD86 are co-stimulatory factors when T lymphocytes are activated, and they play an important role in autoimmune monitoring, humoral immune response and transplantation response.
  • CD28 ligand CD80 as an activating factor to activate T cells is different from using agonistic anti-CD28 antibodies to activate T cells. It does not trigger a severe cytokine storm, thereby greatly reducing the possibility of endangering the life of the patient.
  • the CD80 ECD is selected from human CD80 (such as human CD80 of SEQ ID NO: 134) or human CD80 ECD from CD80 isotype 2 or isotype 3 (SEQ ID NO: 135 and 136).
  • the CD80ECD includes a CD80 immunoglobulin V (IgV) region (CD80-IgV, SEQ ID NO: 133).
  • the CD80 ECD comprises human CD80 immunoglobulin V region and C region (CD80-IgVIgC, SEQ ID NO: 32).
  • the CD80 ECD is human CD80 ECD, and in one embodiment, the CD80 ECD comprises human CD80 IgV.
  • CD80-IgV (SEQ ID NO: 133):
  • CD80-IgVIgC (SEQ ID NO: 32):
  • CD80 isotype 1 (SEQ ID NO: 134):
  • CD80 isotype 2 (SEQ ID NO: 135):
  • CD80 isotype 3 (SEQ ID NO: 136):
  • first binding domain and the second binding domain are selected from the following combinations:
  • the first binding domain targets PD-L1
  • the second binding domain targets human TGF- ⁇ , VEGF, FGL1, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, EGF or ICOSL; or
  • the first binding domain targets PD-1
  • the second binding domain targets human CTLA-4, VEGF, HGF, EGF, CD28, LAG3, CD27, 4-1BB or OX40; or
  • the first binding domain targets CTLA-4, and the second binding domain targets human CD28, VEGF, HGF, EGF, LAG3, CD27, 4-1BB or OX40; or
  • the first binding domain targets LAG-3
  • the second binding domain targets human CD28, VEGF, HGF, EGF, CTLA-4, CD27, 4-1BB or OX40; or
  • the first binding domain targets FGL1, and the second binding domain targets human TGF- ⁇ , VEGF, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, EGF or ICOSL; or
  • the first binding domain targets TIM-3, and the second binding domain targets human VEGF, HGF, EGF, LAG3, CD27, 4-1BB or OX40; or
  • the first binding domain targets Galectin-9
  • the second binding domain targets human TGF- ⁇ , VEGF, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, EGF or ICOSL; or
  • the first binding domain targets TIGIT
  • the second binding domain targets human TGF- ⁇ , HGF, EGF or VEGF; or
  • the first binding domain targets CD155
  • the second binding domain targets human TGF- ⁇ , VEGF, HGF, EGF, CD47 or CD155; or
  • the first binding domain targets Claudin 18.2, HER-2, mesothelin, BCMA, SSTR2, GPRC5D, PSMA, FCRH5, CD33, CD123, CD20, A33, CEA, CD28, DLL3, EGFR, VEGFR, VEGFR2, VEGF-A, Nectin-4, FGFR, C-met, RANKL, PDGF, PDGFR, PDGFR ⁇ , DLL-4, Ang-1, Ang-2, the second binding domain targets human CTLA-4 , TGF- ⁇ , VEGF, FGL1, LAG3, 4-1BB, OX40, CD27, CD28, CD47, CD155, HGF, CSF1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, EGF Or ICOSL.
  • first binding domain, the receptor, and the ligand are selected from the following combinations:
  • the first binding domain targets PD-L1, and the receptor and ligand are selected from the group consisting of TGF- ⁇ RII, VEGFR, LAG-3, SIRP ⁇ , TIGIT, C-MET, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or ICOS; or
  • the first binding domain targets PD-1, and the receptor and ligand are selected from human CD80, CD86, VEGFR, c-MET, EGFR, FGL1, CD70, 4-1BBL or OX40L; or
  • the first binding domain targets CTLA-4, and the receptor and ligand are selected from human CD80, CD86, VEGFR, c-MET, EGFR, FGL1, CD70, 4-1BBL or OX40L; or
  • the first binding domain targets LAG-3 are selected from human VEGFR, c-MET, EGFR, CD80, CD86, CD70, 4-1BBL or OX40L; or
  • the first binding domain targets FGL1, and the receptor and ligand are selected from human TGF- ⁇ RII, VEGFR, SIRP ⁇ , TIGIT, c-MET, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or ICOS ;or
  • the first binding domain targets TIM-3, and the receptor and ligand are selected from human VEGFR, c-MET, EGFR, FGL1, CD70, 4-1BBL or OX40L; or
  • the first binding domain targets Galectin-9 are selected from the group consisting of human TGF- ⁇ RII, VEGFR, SIRP ⁇ , TIGIT, c-MET, CSF1R, CXCR2, CXCR3, CXCR4, EGFR Or ICOS; or
  • the first binding domain targets TIGIT and the receptor and ligand are selected from human TGF- ⁇ RII, c-MET, EGFR or VEGFR; or
  • the first binding domain targets CD155 are selected from human TGF- ⁇ RII, VEGFR, c-MET, EGFR, SIRP ⁇ or TIGIT; or
  • the first binding domain targets Claudin 18.2, HER-2, mesothelin, BCMA, SSTR2, GPRC5D, PSMA, FCRH5, CD33, CD123, CD20, A33, CEA, CD28, DLL3, EGFR, VEGFR, VEGFR2, Nectin-4, FGFR, c-MET, RANKL, PDGF, PDGFR, PDGFR ⁇ , DLL4, Ang-1 or Ang-2, the receptor and ligand are selected from human CTLA-4, CD80, CD86, TGF- ⁇ RII, VEGFR, FGL1, LAG3, 4-1BB, OX40, CD27, CD28, CD70, c-MET, SIRP ⁇ , TIGIT, CSF1R, CXCR2, CXCR3, CXCR4, EGFR or ICOS.
  • fragments of the receptors and ligands include fragments of the extracellular region of the receptors or ligands, and fragments of the binding domains.
  • the first binding domain is targeted to bind PD-L1; the second binding domain is a human TGF- ⁇ RII fragment; or the first binding domain is targeted to bind PD-1, and the second binding
  • the domain includes human CD80 ECD, CD80 IgV region, human CD80 IgVIgC, VEGFR1 ECD, VEGFR2 ECD, or the combination of the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2; or the first binding domain targets binding Claudin 18.2 or HER-2, EGFR
  • the second binding domain is selected from the group consisting of human CD80 ECD, CD80 IgV region, human CD80 IgVIgC, VEGFR1 ECD, VEGFR2 ECD or the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2 Regional combination.
  • the second binding domain is selected from:
  • Human TGF- ⁇ RII comprising the sequence shown in SEQ ID NO: 31, 131, or 132 or containing more than 80%, 85%, 90%, or more than the sequence shown in SEQ ID NO: 31, 131 or 132. 95%, 96%, 97%, 98%, 99% sequence or 1, 2, 3, 4, 5, 6 compared with the sequence shown in SEQ ID NO: 31, 131 or 132 , 7, 8, 9, or 10 amino acid substitutions or deletions of the amino acid sequence; or
  • the C-terminus or/and the N-terminus of the second binding domain has a peptide linker, and the peptide linker consists of 2-30 amino acids.
  • peptide linker may be:
  • TVAAPSVFIFPP SEQ ID NO: 93
  • AKTTPPSVTPLAP SEQ ID NO: 97
  • AKTTAP SEQ ID NO: 98
  • n is equal to 1, 2, 3 or 4;
  • the (GGGGS)n is GGGGS (SEQ ID NO: 120);
  • the (GGGGS)n is GGGGSGGGGS (SEQ ID NO: 121) or (GGGGS) 2 ;
  • the (GGGGS)n is GGGGSGGGGSGGGGS (SEQ ID NO: 122) or (GGGGS) 3 ;
  • the (GGGGS)n is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 123) or (GGGGS) 4 .
  • the size of the inserted second binding domain does not exceed 235, 240, 250, 260, 270, 280, 290 or 300 amino acids.
  • the insertion site of the second binding domain is located in the front part of the hinge region, and the insertion site does not affect the formation of the disulfide bond of the immunoglobulin.
  • the middle front part of the hinge area usually refers to the part before 231A.
  • part of the amino acids in the hinge region before and after the insertion site are replaced or deleted, for example, the hinge region contains D221G and/or C220V mutations.
  • the IgG is selected from mammalian IgG, humanized IgG and human IgG.
  • the mammal includes mouse and rat; preferably, the IgG is IgG1, IgG2, IgG3, and IgG4.
  • the Fc region of the fusion protein is aglycosylated or deglycosylated or has reduced fucosylation or afucosylation.
  • the IgG is Atezolizumab, Avelumab, Durvalumab, Nivolumab, Pembrolizumab, Cemiplimab, Ipilimumab.
  • the IgG is Atezolizumab, the heavy chain amino acid sequence of the IgG is shown in SEQ ID NO: 106, and the light chain amino acid sequence of the IgG is shown in SEQ ID NO: 107; or the IgG is Avelumab, The heavy chain amino acid sequence of the IgG is shown in SEQ ID NO: 108, and the light chain amino acid sequence of the IgG is shown in SEQ ID NO: 109; or the IgG is Durvalumab, and the heavy chain amino acid sequence of the IgG is shown in SEQ ID NO: 110, the light chain amino acid sequence of the IgG is shown in SEQ ID NO: 111; or the IgG is Nivolumab, and the heavy chain amino acid sequence of the IgG is shown in SEQ ID NO
  • the present invention provides a Hibody structural fusion protein with a first binding domain targeting human PD-L1, the CDR of the heavy chain variable region in the Fab of the IgG and/or the light chain variable region
  • the CDR of the antibody has the same CDR sequence as the antibody defined by the following sequence, or 1-2 amino acid substitutions are made on the CDR of the antibody defined by the following sequence, and the antibody defined by the sequence is:
  • variable region of the heavy chain is shown in SEQ ID NO: 66; and/or
  • the CDRs of the heavy chain variable region and/or the CDRs of the light chain variable region in the Fab of the IgG are as follows:
  • the CDR1 amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 1-5 and the amino acid sequence of SEQ ID NO: 1-5 with 1 or 2 amino acid substitutions;
  • the CDR2 amino acid sequence is selected from SEQ ID NO: 6-10 and the amino acid sequence of SEQ ID NO: 6-10 after 1 or 2 amino acid substitutions;
  • the CDR3 amino acid sequence is selected from SEQ ID NO: 11-15 and SEQ ID NO: 11-15 is 1 or 2 The amino acid sequence after the amino acid substitution; and/or
  • the CDR1 amino acid sequence of the light chain variable region is selected from SEQ ID NO: 16-20 and the amino acid sequence of SEQ ID NO: 16-20 with 1 or 2 amino acid substitutions; the CDR2 amino acid sequence is selected from SEQ ID NO: 21-25 and the amino acid sequence of SEQ ID NO: 21-25 after 1 or 2 amino acid substitutions; the CDR3 amino acid sequence is selected from SEQ ID NO: 26-30 and SEQ ID NO: 26-30 is 1 or 2 The amino acid sequence after the amino acid substitution.
  • the complementarity determining regions CDR 1-3 of the corresponding heavy chain and light chain variable regions are shown in Table 2.
  • the fusion protein contains the following CDR sequences,
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, 6, 11 or the amino acid sequence of SEQ ID NO: 1, 6, 11 with 1 or 2 amino acid substitutions; and/or
  • the CDR 1-3 amino acid sequence of the light chain variable region is SEQ ID NO: 16, 21, 26 or the amino acid sequence of SEQ ID NO: 16, 21, 26 after 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, 7, 12 or the amino acid sequence of SEQ ID NO: 2, 7, 12 with 1 or 2 amino acid substitutions; and/or
  • the CDR 1-3 amino acid sequence of the light chain variable region is SEQ ID NO: 17, 22, 27 or the amino acid sequence of SEQ ID NO: 17, 22, 27 after 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 3, 8, 13 or the amino acid sequence of SEQ ID NO: 3, 8, 13 after 1 or 2 amino acid substitutions; and/or
  • the amino acid sequence of the CDR 1-3 of the light chain variable region is SEQ ID NO: 18, 23, 28 or the amino acid sequence of SEQ ID NO: 18, 23, 28 after 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 4, 9, 14 or the amino acid sequence of SEQ ID NO: 4, 9, 14 after 1 or 2 amino acid substitutions; and/or
  • the CDR 1-3 amino acid sequence of the light chain variable region is SEQ ID NO: 19, 24, 29 or the amino acid sequence of SEQ ID NO: 19, 24, 29 after 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 5, 10, 15 or the amino acid sequence of SEQ ID NO: 5, 10, 15 after 1 or 2 amino acid substitutions; and/or
  • the CDR 1-3 amino acid sequence of the light chain variable region is SEQ ID NO: 20, 25, 30 or the amino acid sequence of SEQ ID NO: 20, 25, 30 with 1 or 2 amino acid substitutions.
  • amino acid sequence of the CDR 1-3 of the heavy chain variable region in the heavy chain of the fusion protein is SEQ ID NO: 3, 8, 13; and/or the amino acid sequence of the CDR 1-3 of the light chain variable region is SEQ ID NO: 18, 23, 28.
  • sequences of the heavy chain variable region and the light chain variable region of the fusion protein are as follows,
  • variable region of the heavy chain is shown in SEQ ID NO: 66, or has the same CDR 1-3 as SEQ ID NO: 66 and is more than 80% or 85% identical to SEQ ID NO: 66 , 90%, 95%, 96%, 97%, 98%, 99% sequence; and/or
  • variable region of the light chain is shown in SEQ ID NO: 67, or has the same CDR 1-3 as SEQ ID NO: 67 and the identity is greater than 80%, 85% compared with SEQ ID NO: 67 , 90%, 95%, 96%, 97%, 98%, 99% sequence.
  • amino acid sequences of the heavy chain and light chain of the fusion protein are shown below,
  • the heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 72 or is more than 90%, 95%, 96%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 72 sequence;
  • the light chain amino acid sequence of the fusion protein is as shown in SEQ ID NO: 73 or is more than 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 73. the sequence of.
  • amino acid sequences of the heavy chain and light chain of the fusion protein are as follows:
  • the heavy chain amino acid sequence of the fusion protein has 1-15 amino acid site mutations or a substituted peptide linker
  • the light chain amino acid sequence of the fusion protein has 1-10 amino acid site mutations.
  • the present invention provides a Hibody structural fusion protein with a first binding domain targeting human PD-1, wherein the CDR of the heavy chain variable region in the Fab and/or the light chain variable region
  • the CDR has the same CDR sequence as the antibody defined by the following sequence or 1-2 amino acid substitutions are made on the CDR of the antibody defined by the following sequence.
  • the antibody defined by the sequence is:
  • variable region of the heavy chain is shown in SEQ ID NO: 64; and/or
  • the CDRs of the heavy chain variable region of the Fab in the fusion protein and/or the CDRs of the light chain variable region of the fusion protein are as follows:
  • the CDR1 amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 34-38 and the amino acid sequence of SEQ ID NO: 34-38 after 1 or 2 amino acid substitutions;
  • the CDR2 amino acid sequence is selected from SEQ ID NO: 39-43 and the amino acid sequence of SEQ ID NO: 39-43 after 1 or 2 amino acid substitutions;
  • the CDR3 amino acid sequence is selected from SEQ ID NO: 44-48 and SEQ ID NO: 44-48 is 1 or 2 The amino acid sequence after the amino acid substitution; and/or
  • the CDR1 amino acid sequence of the light chain variable region is selected from SEQ ID NO: 49-53 and SEQ ID NO: 49-53 with 1 or 2 amino acid substitutions;
  • the CDR2 amino acid sequence is selected from SEQ ID NO: 54-58 and the amino acid sequence of SEQ ID NO: 54-58 with 1 or 2 amino acid substitutions;
  • the CDR3 amino acid sequence is selected from SEQ ID NO: 59-63 and SEQ ID NO: 59-63 with 1 or 2 The amino acid sequence after the amino acid substitution.
  • the complementarity determining regions CDR 1-3 of the corresponding heavy chain and light chain variable regions are shown in Table 3.
  • CDR1-3 of the variable region of the fusion protein are defined as follows,
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 34, 39, 44 or the amino acid sequence of SEQ ID NO: 34, 39, 44 after 1 or 2 amino acid substitutions; and/or
  • the amino acid sequence of the CDR 1-3 of the light chain variable region is SEQ ID NO: 49, 54, 59 or the amino acid sequence of SEQ ID NO: 49, 54, 59 after 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 35, 40, 45 or the amino acid sequence of SEQ ID NO: 35, 40, 45 after 1 or 2 amino acid substitutions; and/or
  • the amino acid sequence of the CDR 1-3 of the light chain variable region is SEQ ID NO: 50, 55, 60 or the amino acid sequence of SEQ ID NO: 50, 55, 60 after 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 36, 41, 46 or the amino acid sequence of SEQ ID NO: 36, 41, 46 after 1 or 2 amino acid substitutions; and/or
  • the CDR 1-3 amino acid sequence of the light chain variable region is SEQ ID NO: 51, 56, 61 or the amino acid sequence of SEQ ID NO: 51, 56, 61 after 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 37, 42, 47 or the amino acid sequence of SEQ ID NO: 37, 42, 47 after 1 or 2 amino acid substitutions; and/or
  • the amino acid sequence of the CDR 1-3 of the light chain variable region is SEQ ID NO: 52, 57, 62 or the amino acid sequence of SEQ ID NO: 52, 57, 62 after 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 38, 43, 48 or the amino acid sequence of SEQ ID NO: 38, 43, 48 after 1 or 2 amino acid substitutions; and/or
  • the CDR 1-3 amino acid sequence of the light chain variable region is SEQ ID NO: 53, 58, 63 or the amino acid sequence of SEQ ID NO: 53, 58, 63 with 1 or 2 amino acid substitutions.
  • variable region CDR1-3 of the fusion protein is defined as follows: the amino acid sequence of the heavy chain variable region CDR1-3 is SEQ ID NO: 36, 41, 46; and/or the light chain variable region CDR1
  • the amino acid sequence of -3 is SEQ ID NO: 51, 56, 61.
  • amino acid sequences of the heavy chain variable region and the light chain variable region of the fusion protein are as follows:
  • the sequence of the heavy chain variable region is shown in SEQ ID NO: 64, or it has the same CDR 1-3 as SEQ ID NO: 64 and the identity is greater than 80%, 85% compared with SEQ ID NO: 64 , 90%, 95%, 96%, 97%, 98%, 99% sequence; and/or
  • variable region of the light chain is shown in SEQ ID NO: 65, or has the same CDR 1-3 as SEQ ID NO: 65 and the identity is greater than 80%, 85% compared with SEQ ID NO: 65 , 90%, 95%, 96%, 97%, 98%, 99% sequence.
  • heavy chain and light chain amino acid sequences of the fusion protein are defined as follows:
  • the heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 68 or is more than 90%, 95%, 96%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 68 sequence;
  • amino acid sequence of the light chain of the fusion protein is shown in SEQ ID NO: 69 or the identity is greater than 90%, 95%, 96%, 97%, 98%, 99% compared with SEQ ID NO: 69. the sequence of.
  • amino acid sequences of the heavy chain and light chain of the fusion protein are as follows:
  • the heavy chain amino acid sequence of the fusion protein has 1-15 amino acid site mutations or a substituted peptide linker
  • the light chain amino acid sequence of the fusion protein has 1-10 amino acid site mutations.
  • heavy chain and light chain amino acid sequences of the fusion protein are defined as follows:
  • the heavy chain amino acid sequence of the fusion protein is shown in SEQ ID NO: 70 or is more than 90%, 95%, 96%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 70 sequence;
  • the light chain amino acid sequence of the fusion protein is as shown in SEQ ID NO: 71 or is more than 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 71. the sequence of.
  • amino acid sequences of the heavy chain and light chain of the fusion protein are as follows:
  • the heavy chain amino acid sequence of the fusion protein has 1-15 amino acid site mutations or a substituted peptide linker
  • the light chain amino acid sequence of the fusion protein has 1-10 amino acid site mutations.
  • the present invention also provides an isolated polynucleotide, which encodes the fusion protein as described above.
  • the present invention also provides a nucleic acid construct comprising the polynucleotide as described above, and preferably the nucleic acid construct is a vector.
  • the present invention also provides a host cell comprising the polynucleotide or nucleic acid construct or vector as described above.
  • the cell is a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammalian cell, an E. coli cell or CHO cells, NS0 cells, Sp2/0 cells, BHK cells.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein as described above and a pharmaceutically acceptable carrier.
  • the present invention also provides a method for treating tumors or cancers, which includes the step of administering the above-mentioned fusion protein or the pharmaceutical composition to a subject in need of treatment or relief.
  • the present invention also provides the use of the above-mentioned fusion protein, polynucleotide, nucleic acid construct or carrier and pharmaceutical composition in the preparation of a medicine for treating or preventing tumor or cancer.
  • the tumor or cancer includes solid tumors or non-solid tumors.
  • the present invention also provides a diagnostic kit, which comprises the fusion protein as described above.
  • the present invention also provides a method for producing a fusion protein, the method comprising: culturing the host cell under conditions that allow the expression of the nucleic acid construct as described above, and recovering the produced fusion protein from the culture.
  • the Hibody structural fusion protein provided by the present invention because the second binding domain is inserted in the hinge region, has the same expression and production advantages as IgG, and can be seamlessly connected to the existing IgG expression platform and purification platform, greatly improving The subsequent development cost is reduced.
  • the bispecific antibody constructed by the Hibody platform has a higher expression level without the problem of light and heavy chain mismatches, which also greatly reduces the antibody Cost of production.
  • the receptor or ligand selected as the second binding domain will form a dimerization or multimerization structure in the natural signaling pathway to activate or inhibit the signaling pathway, and when it is inserted into the hinge region, its The spatial positional relationship in the hinge region promotes the formation of the dimerization of the corresponding receptor or ligand or its fragments, thereby imitating the natural signal pathway to obtain a more significantly improved binding activity, thereby making the fusion protein relative to the use of soluble
  • the activation or inhibition ability of receptors or ligands or binding fragments has been greatly improved.
  • the target of receptor inhibition or receptor activation can be flexibly selected, which has an effective synergistic effect. Compared with the two target drugs being administered alone or the double-dose of the prior art Specific antibodies have a more significant disease treatment effect and can effectively treat or control the disease process, especially in various tumors and cancers.
  • Figure 1 Schematic diagram of Hibody structure fusion protein.
  • Figure 2 shows the tumor inhibition curve of Hib-PDV and its control group on mouse colon cancer model.
  • Figure 3 shows the inhibition curve of Hib-PDC and its control group on mouse colon cancer.
  • Fig. 4 is a comparison chart of Hib-PLT and its control group at a concentration of 10-100 nM to promote cell secretion of IFN- ⁇ factor.
  • amino acids involved in the present invention can be represented by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission (IUPAC-IUB Biochemical Nomenclature Commission). Likewise, nucleotides can be represented by their generally accepted one-letter codes.
  • the complementarity determining region (CDR) determination or numbering method of the antibody variable domain includes IMGT, Kabat (such as the sequence of proteins of interest in immunology by Kabat et al.), which are well-known in the art. Immunological Interest), 5th edition, Public Health Service, National Institutes of Health, Bethesda MD (1991), Chothia, AbM and Contact method.
  • the "identity”, “identity” or “similarity” between two nucleic acid or amino acid sequences refers to the two to be compared obtained after the best alignment (optimal alignment).
  • the percentage of identical nucleotides or identical amino acid residues between sequences is purely statistical and the differences between the two sequences are randomly distributed and cover their entire length.
  • Sequence comparisons between two nucleic acid or amino acid sequences are usually performed by comparing these sequences after they are matched in an optimal manner, and the comparison can be performed through segments or through a "comparison window".
  • the optimal alignment used to compare sequences can also be passed through the local homology algorithm of Smith and Waterman (1981) [Ad.App.Math.
  • the immunoglobulin referred to in the present invention refers to an animal protein with antibody activity. It is composed of two identical light chains and two identical heavy chains. It is an important class of immune effector molecules.
  • the protein produced by the lymphocytes of the immune system of higher animals can be transformed into antibodies by the induction of antigens. Due to different structures, it can be divided into 5 types: IgG, IgA, IgM, IgD and IgE.
  • the immunoglobulin involved in the present invention is IgG.
  • the receptor involved in the present invention is a type of receptor that exists in the cell membrane or in the cell, and can bind to a specific signal molecule outside the cell to activate a series of biochemical reactions in the cell, so that the cell responds to external stimuli.
  • the biologically active substances that bind to the receptor are collectively referred to as ligands.
  • the binding of receptors and ligands will cause molecular conformational changes, which will cause cellular responses, such as mediating intercellular signal transduction, intercellular adhesion, endocytosis and other processes.
  • the bispecific fusion protein of the present invention is based on the immunoglobulin structure with a second binding domain inserted in its hinge region. Therefore, the bispecific fusion protein of the present invention has two binding domains.
  • Hibody bispecific fusion proteins Hib-PLT, Hib-PDC, Hib-PDV;
  • Control protein 1-3 constructed according to the bifunctional molecular structure in WO2015/118175;
  • 3Other control proteins TGF- ⁇ -R-His, VEGF-R-His, CD80-His, PD-L1 control antibodies.
  • nucleic acid sequence and amino acid sequence of the fusion protein Hib-PLT heavy chain and light chain are as follows:
  • Nucleic acid sequence of fusion protein Hib-PLT heavy chain (SEQ ID NO: 78):
  • Fusion protein Hib-PLT heavy chain amino acid sequence (SEQ ID NO: 72):
  • the bolded framed fragment is the hinge region sequence, italicized is the peptide linker, and the underlined bolded sequence is the TGF- ⁇ RII amino acid sequence.
  • Nucleic acid sequence of fusion protein Hib-PLT light chain (SEQ ID NO: 79):
  • Amino acid sequence of fusion protein Hib-PLT light chain (SEQ ID NO: 73):
  • nucleic acid sequence and amino acid sequence of the heavy chain and light chain of the fusion protein Hib-PDC are as follows:
  • Nucleic acid sequence of fusion protein Hib-PDC heavy chain (SEQ ID NO: 74):
  • the heavy chain amino acid sequence of the fusion protein Hib-PDC (SEQ ID NO: 68):
  • the peptide linker is in italics, and the underlined and bold sequence is the CD80 amino acid sequence.
  • Nucleic acid sequence of fusion protein Hib-PDC light chain (SEQ ID NO: 75):
  • Amino acid sequence of fusion protein Hib-PDC light chain (SEQ ID NO: 69):
  • nucleic acid sequence and amino acid sequence of the fusion protein Hib-PDV heavy chain and light chain are as follows:
  • Nucleic acid sequence of fusion protein Hib-PDV heavy chain (SEQ ID NO: 76):
  • Fusion protein Hib-PDV heavy chain amino acid sequence (SEQ ID NO: 70):
  • the peptide linker is in italics, and the underlined and bold sequence is the VEGF amino acid sequence.
  • Fusion protein Hib-PDV light chain nucleic acid sequence (SEQ ID NO: 77):
  • Amino acid sequence of fusion protein Hib-PDV light chain (SEQ ID NO: 71):
  • control protein 1 The heavy chain amino acid sequence of control protein 1 (SEQ ID NO: 124):
  • the peptide linker is in italics, and the underlined and bold sequence is the amino acid sequence of TGF- ⁇ RII.
  • the heavy chain amino acid sequence of the control protein 2 (SEQ ID NO: 126):
  • the peptide linker is in italics, and the underlined and bold sequence is the CD80 amino acid sequence.
  • control protein 3 The heavy chain amino acid sequence of control protein 3 (SEQ ID NO: 128):
  • the peptide linker is in italics, and the underlined and bold sequence is the VEGFR amino acid sequence.
  • TGF- ⁇ -R-His is a fragment of the TGF- ⁇ RII extracellular region of His tag, wherein the TGF-
  • VEGF-R-His is a combination fragment of the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2, wherein the second extracellular region of VEGFR1 and the third extracellular region of VEGFR2 Amino acid sequence of the combined fragment (SEQ ID NO: 130):
  • CD80-His is the CD80 fragment of His tag, wherein the amino acid sequence of the CD80 fragment (SEQ ID NO: 32):
  • the CHO host cells were resuscitated, the resuscitation culture volume was 10 mL, and the culture conditions were 37.0°C, 200 rpm, 8% CO 2 , and 80% humidity. After resuscitation, the cell density was 2.82 ⁇ 10 5 cells/mL, and the viability was 95.41%.
  • the density of viable cells was 2.22 ⁇ 10 6 cells/mL, and the viability rate was 98.32%.
  • the cells were rejoined at 5 ⁇ 10 5 cells/mL for cell rejoining operation. After 48 hours of continuous culture, the viable cell density was 2 ⁇ 10 6 cells/mL, and the viability was 98.68%.
  • the cells were spread into a 6-well plate at 5 ⁇ 10 5 cells/well with a culture volume of 2 mL/well, and placed in a carbon dioxide incubator (cultivation conditions 37° C., 8% CO 2 ) for overnight culture for adherent transfection the next day.
  • the transfection reagent is LTX Reagent (Invitrogen, catalog number 15338-100), contains LTX and Plus reagents.
  • Transfection operation change the medium in the six-well plate to prepare the transfection complex: LTX: 9 ⁇ l/well + plasmid: 2.5 ⁇ g/well + plus: 2.5 ⁇ l/well, according to each well 250 ⁇ l was added to the six-well plate.
  • the cells in each well of the 6-well plate were exchanged 5 hours after cell transfection. After 48 hours of transfection, the supernatant was taken, and the antibody content of the supernatant was detected by the double antigen ELISA method.
  • the cells in the six-well plate were digested and collected into a cell culture tube.
  • the cell density was 3 ⁇ 10 5 cells/mL for suspension adaptation and growth.
  • the Hib-PLT-expressing cells pool Perform batch culture with the control protein 1 expressing cell pool, and feed Hib-PDC, control protein 2, Hib-PDV, and control protein 3 into fed-batch culture.
  • Batch culture culture in a TPP cell culture tube at a cell density of 3 ⁇ 10 5 cells/mL, with a volume of 30 mL, culture conditions 37.0°C, 200 rpm, 8% CO 2 , and 80% humidity. The cell viability and metabolism are monitored every day, and the culture is terminated until the viability is less than 60%, and the cell supernatant after the culture is taken for concentration detection.
  • Fed-feed culture culture in a TPP cell culture tube at a cell density of 3 ⁇ 10 5 cells/mL, with a volume of 30 mL, and culture conditions at 37.0°C, 200 rpm, 8% CO 2 , and 80% humidity.
  • the cells were given concentrated medium, during which the Glc was controlled at 2-6g/L. End the culture until the viability is less than 60%, and take the cell supernatant after the end of the culture for concentration detection.
  • Table 6 shows the expression results of Hibody bispecific fusion proteins Hib-PLT, Hib-PDC, Hib-PDV and the corresponding control proteins.
  • the results show that the expression of Hib-PLT increases relative to the corresponding control protein.
  • the relative value of the increase in expression of Hib-PDC increased by 33.43%; compared with the corresponding control protein, the relative value of the increase in expression of Hib-PDV increased by 25.6%
  • the expression level of the Hibody bispecific fusion protein provided by the present invention is significantly better than the expression level of the corresponding control protein, and the bispecific antibody provided by the present invention has unexpected technical effects.
  • the relative value of increase (the expression of the bispecific fusion protein provided by the present invention-the expression of the corresponding control protein)/the expression of the corresponding control protein.
  • Coating antigen Dilute TGF- ⁇ 1 to 2 ⁇ g/ml with coating buffer, add 100 ⁇ l per well to a 96-well plate, and let stand overnight at 2-8°C;
  • each concentration point of the sample is as follows, 100 ⁇ l of each concentration point sample is added to a 96-well plate in turn, each concentration point is paralleled with 2 replicate wells, and incubated at 37°C for 2 hours;
  • ⁇ Hib-PLT sample well After Goat-anti-Human-Fc-HRP is diluted 1:5000, 100 ⁇ l per well, incubate at 37°C for 2 hours;
  • ⁇ TGF- ⁇ -R-His well After Anti-His-HRP is diluted 1:5000, 100 ⁇ l per well, incubate at 37°C for 2 hours;
  • TMB color development solution, 100 ⁇ l each well develops color for 2 minutes;
  • Stop color development 2M H 2 SO 4 is the stop solution, 50 ⁇ l per well to stop color development;
  • Reading MD microplate reader reads the absorbance value at 450/655nm respectively, taking the concentration of the sample to be tested as the abscissa and the absorbance value as the ordinate to make a 4-PL curve, and the software automatically gives the EC50 value.
  • Coating antigen Dilute VEGF to 1 ⁇ g/ml with coating buffer, add 100 ⁇ l per well to a 96-well plate, and let stand overnight at 2 ⁇ 8°C;
  • each concentration point of the sample is as follows, 100 ⁇ l of each concentration point sample is added to a 96-well plate in turn, each concentration point is paralleled with 2 replicate wells, and incubated at 37°C for 2 hours;
  • ⁇ Hib-PDV sample well Goat-anti-Human-Fc-HRP diluted 1:5000, 100 ⁇ l per well, incubate at 37°C for 2 hours;
  • ⁇ VEGFR-His well Anti-His-HRP diluted 1:5000, 100 ⁇ l per well, incubate at 37°C for 2 hours
  • TMB color development solution, 100 ⁇ l each well develops color for 2 minutes;
  • Stop color development 2M H2SO4 is the stop solution, 50 ⁇ l per well to stop color development;
  • Reading MD microplate reader reads the absorbance value at 450/655nm respectively, taking the concentration of the sample to be tested as the abscissa and the absorbance value as the ordinate to make a 4-PL curve, and the software automatically gives the EC50 value.
  • Coating antigen Reconstitute CTLA-4Protein with 1 mL of sterilized water at a concentration of 200ug/mL. Dilute to 20 ⁇ g/ml with coating buffer, add 100 ⁇ l per well to 96-well plate, and let stand overnight at 2 ⁇ 8°C;
  • Stop color development 2M H 2 SO 4 is the stop solution, 50 ⁇ l per well to stop color development;
  • Reading MD microplate reader reads the absorbance value at 450/655nm respectively, taking the concentration of the sample to be tested as the abscissa and the absorbance value as the ordinate, making a 4-PL curve, and the software automatically gives the EC50 value (see table 7).
  • Detection group EC50 value Hib-PLT 0.164nM TGF- ⁇ -R-His 11.60nM Hib-PDV 0.061nM VEGFR-His 2154nM Hib-PDC 24.36nM CD80-His 50.69nM
  • the binding capacity of Hib-PLT is compared with TGF- ⁇ receptor monomer: the EC50 value of Hib-PLT is 0.164 nM, the EC50 value of TGF- ⁇ receptor monomer is 11.60 nM, and the binding activity of Hib-PLT is excellent.
  • Hib-PDV compared with VEGF receptor monomer The EC50 value of Hib-PDV is 0.061 nM, the EC50 value of VEGF receptor monomer is 2154 nM, and Hib-PDV binding activity It is about 35,311 times better than VEGF receptor monomer;
  • Hib-PDC is compared with CD80 monomer: C50 value of Hib-PDC is 24.36 nM, EC50 value of CD80 monomer is 50.69 nM, Hib-PDC binding activity is better than CD80 monomer
  • the body is about 2 times. That is, the binding activity of Hib-PLT, Hib-PDV, and Hib-PDC constructed with the bi-antibody model provided by the present invention is much better than the binding activity of their corresponding monomers.
  • Example 4 The effect of Hib-PDV on colon cancer MC38 cells transplanted subcutaneously in C57BL/6J-hPD-1 mice
  • mice from Shanghai Southern Model Biotechnology Co., Ltd.
  • 0.1 mL of colon cancer MC38 cell suspension (1 ⁇ 10 6 cells/mouse) was subcutaneously inoculated into the right abdomen of mice.
  • the tumor When the tumor grows to about 100mm 3 , they are randomly grouped according to the size of the tumor and divided into Vehicle (PBS) group, Hib-PDV (1.25mg/kg) group, Hib-PDV (2.5mg/kg) group and Hib-PDV (5mg/kg) group, a total of 4 groups, each group of 8 tumor-bearing mice, intraperitoneal administration, once a week, a total of 3 administrations, during the administration period of 2 times a week to measure the length and shortness of the tumor Diameter and weight.
  • the tumor inhibition rate TGI RTV
  • Table 8 the tumor inhibition rate
  • Table 8 The dosage regimen for the study of Hib-PDV's tumor inhibitory activity on mouse colon cancer
  • Dosage (mg/kg) Route of administration Dosing frequency
  • Dosing volume Vehicle - i.p. QW ⁇ 3 10mL/kg 2 Hib-PDV 5 i.p. QW ⁇ 3 10mL/kg 3 Hib-PDV 2.5 i.p. QW ⁇ 3 10mL/kg 4 Hib-PDV 1.25 i.p. QW ⁇ 3 10mL/kg
  • TGI RTV (%) (1-T RTV /C RTV ) ⁇ 100%
  • T RTV represents the RTV of the administration group or the positive control group
  • C RTV represents the RTV of the negative control group.
  • Hib-PDV's tumor inhibitory effects on mouse colon cancer model are shown in Table 9 and Figure 2.
  • Table 9 is Hib-PDV's tumor volume and tumor inhibition rate in mouse colon cancer model
  • Figure 2 shows Tumor growth curve after medication.
  • Experimental results show that Hib-PDV has a significant inhibitory effect on mouse colon cancer model tumors.
  • mice Thirty-two 7-9 weeks old, humanized C57BL/6J humanized PD-1 transgenic female mice (from the southern model) were subcutaneously implanted with 0.1 mL of transgenic hPD-L1MC38 cell suspension ( 1 ⁇ 10 6 cells/piece).
  • the tumor When the tumor grows to about 100mm 3 , they are randomly grouped according to the size of the tumor and divided into Vehicle (PBS) group, Hib-PDC (1.25mg/kg) group, Hib-PDC (2.5mg/kg) group, Hib-PDC (5mg/kg)
  • PBS Vehicle
  • Hib-PDC 1.25mg/kg
  • Hib-PDC 2.5mg/kg
  • Hib-PDC 5mg/kg
  • a total of 4 groups 8 tumor-bearing mice in each group, intraperitoneal administration, once a week, a total of 3 administrations, during the administration period, the tumor’s long diameter, short diameter and body weight were measured twice a week .
  • TGI RTV tumor inhibition rate
  • Table 10 The dosage regimen for the study of Hib-PDC's tumor inhibitory activity on mouse colon cancer
  • Dosage (mg/kg) Mode of administration Dosing volume
  • Dosing cycle 1 Vehicle(PBS) - i.p. 10ml/kg QW ⁇ 3 times 2 Hib-PDC 1.25 i.p. 10ml/kg QW ⁇ 3 times 3 Hib-PDC 2.5 i.p. 10ml/kg QW ⁇ 3 times 4 Hib-PDC 5 i.p. 10ml/kg QW ⁇ 3 times
  • TGI RTV (%) (1-T RTV /C RTV ) ⁇ 100%
  • T RTV represents the RTV of the administration group or the positive control group
  • C RTV represents the RTV of the negative control group.
  • Table 11 shows the tumor volume and tumor inhibition rate of Hib-PDC on mouse colon cancer models.
  • Figure 3 shows the administration After the tumor growth curve.
  • the test results show that Hib-PDC has a significant inhibitory effect on subcutaneously transplanted tumors of colorectal cancer in mice.
  • Example 6 The effect of Hib-PLT on the secretion of cytokines in the mixed reaction of allogeneic lymphocytes
  • ELISA method was used to detect the concentration of cytokine IFN- ⁇ in the cell supernatant to evaluate the effect of Hib-PLT in the activation of CD4 + T cells.

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Abstract

提供了一种新型结构的双特异性融合蛋白，其在全长的IgG铰链区通过可选的肽接头插入第二结合结构域。该融合蛋白具备与IgG相同的表达和生产优势，不影响融合蛋白Fab区的结合活性并进一步提升了稳定性以及获得了更高的半衰期。另外第二结合结构域与靶向结合位点的结合活性相对于其对应可溶性天然结合片段的单体与相应靶点的结合活性相比，有了显著的提高。

Description

一种双特异性融合蛋白及其应用 技术领域
本发明涉及一种具有新型结构的双特异性融合蛋白及其应用，本发明还涉及编码或制备上述融合蛋白的多核苷酸、核酸构建体、宿主细胞。
背景技术
随着生物技术的发展，抗体作为成熟的治疗药物，在肿瘤和免疫疾病治疗领域发挥着重要的作用。目前，大部分已上市的抗体药物针对单一抗原，只能作用于一个靶分子。然而，复杂疾病在本质上往往是多因素的，涉及疾病介质的过度表达或相互协同作用，或不同受体的上调，包括其信号网络中分子相互作用。因此，调控多信号途径，可以提高治疗性抗体的疗效。使用双靶向策略的双特异性抗体(Bispecific antibody,BsAbs)治疗已成为肿瘤和免疫疾病治疗的有效手段之一(Kontermann R.Dual targeting strategies with bispecific antibodies[C]//MAbs.Taylor & Francis,2012,4(2):182-197.)。
双特异性抗体能够特异性结合两种不同的抗原或抗原表位(Carter P.Bispecific human IgG by design[J].Journal of immunological methods,2001,248(1-2):7-15.)。早在20世纪80年代，Morrison等首先通过将不同特异性的单链抗体使用柔性肽段连接后融合表达的方式制备了第一个真正意义上的抗葡聚糖和丹磺酰基的四价双特异性抗体(Coloma M J,Morrison S L.Design and production of novel tetravalent bispecific antibodies[J].Nature biotechnology,1997,15(2):159.)。但是由于抗体制备技术瓶颈和信号通路基础研究的不足，双特异性抗体的发展一直受到阻碍。随着对疾病发病机制的深入了解，同时治疗性单克隆抗体(monoclonal antibody，McAb)相关技术的飞速发展大大提高了抗体的构建、表达和纯化技术，使双特异性抗体发展具备了克服限制因素的技术和动力。目前双特异性或多特异性抗体发挥作用的机理主要有以下几种：(1)桥连细胞(Bridging cells(in trans))；(2)受体抑制或受体活化(Receptor inHibition(in-cis)or Receptor activation(in-cis))；(3)辅助因子模拟(Co-factor mimetic)；(4)背驮劫持法(PIgGyback approaches)(Aran F.Labrijn,et al.Bispecific antibodies:a mechanistic review of the pipeline,Nature Reviews Drug Discovery volume 18,pages585–608(2019))。
双特异性抗体开发中，对于分子结构形式的选择非常重要。不同的双特异性抗体设计各有利弊，但是以临床治疗为目的的双特异性抗体设计都要解决如下问题：第一，保证两对(或以上)不同轻链与重链的正确偶合或配对；第二，保持每个单克隆抗体各自结合域的独立性，同时结合不同表位的时候互相之间不会产生空间位阻的干扰；第三，抗体分子要易于用哺乳动物细胞进行表达，不需要复杂的蛋白修饰工艺；第四，有较好的成药性，如高热稳定性、高化学稳定性、高溶解性、不易聚合、低黏性、高表达量、合适的半衰期等(Spiess C,Zhai Q,Carter P J.Alternative molecular formats and therapeutic applications for bispecific antibodies[J].Molecular immunology,2015,67(2):95-106.)。
根据不同的分子结构形式(即组成部分以及构建方式)，双特异性抗体可以分为许多种类，例如根据结构左右对称性分为对称结构和不对称结构，根据有无Fc区可以分为含有Fc区的双特异性抗体和不含Fc区的双特异性抗体，以及根据抗原结合区域的数量构型分为两价、三价、四价或更多价的构型等。目前双特异性抗体已经发展出几十种结构(Spiess C,Zhai Q,Carter P J.Alternative molecular formats and therapeutic applications for bispecific antibodies[J].Molecular immunology,2015,67(2):95-106.)，这几十种双特异性抗体结构多样，大致细化为5类，包括IgG样双特异性抗体、含附加功能区的IgG样双特异性抗体、串联不同抗原结合片段的双特异性抗体、融合蛋白型双特异性抗体、化学偶联双特异性抗体。其中，IgG样双特异性抗体的构建主要是采用重组DNA技术进行结构改造，构建后的IgG样双特异性抗体具有完整的可结晶片段(即Fc片段)，在结合不同抗原的同时，仍保留了Fc片段介导的ADCC(抗体介导的细胞毒性效应、antibody-dependent cell-mediated cytotoxicity)和CDC(补体介导的细胞毒性效应、complement-dependent cytotoxicity)等功能，同时该类抗体也保留了通过与新生儿Fc受体(neonatal Fc receptor,FcRn)结合延长抗体体内半衰期的特点(Ridgway J B B,Presta L G,Carter P.‘Knobs-into-holes’engineering of antibody CH3domains for heavy chain heterodimerization[J].Protein Engineering,Design and Selection,1996,9(7):617-621.)；含附加功能区的IgG样双特异性抗体一般是在传统IgG抗体的基础上，通过融合蛋白的方式，在重链和/或轻链上增加其他特异性的抗原结合片段(即附加功能区，如单域抗体、单链抗体等)(LaFleur D,Abramyan D,Kanakaraj P,et al.Monoclonal antibody therapeutics with up to five specificities:functional enhancement through fusion of target-specific  peptides[C]//MAbs.Taylor & Francis,2013,5(2):208-218.)；串联不同抗原结合片段的双特异性抗体是通过连接肽将不同的Fab、单链抗体、单域抗体或抗原结合片段等以一定的排列顺序连接起来而获得的(Stork R,Müller D,Kontermann R E.A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G[J].Protein Engineering,Design & Selection,2007,20(11):569-576.)；融合蛋白型双特异性抗体通过多肽片段等连接子将不同特异性的抗体/抗体片段(IgG、Fab、scFv等)连接形成的可同时结合2个或以上抗原的蛋白分子；化学偶联双特异性抗体同融合蛋白型双特异性抗体类似，在化学偶联型双特异性抗体中，首先需分别制备2个抗体/抗体片段，之后通过化学键将两者偶联或将两者同时与载体蛋白偶联。
此外，随着信号通路基础研究和双抗发挥作用机理拓展，可供用于双特异性抗体选择的靶点及组合也越来越多，如PD-1、PD-L1、CTLA-4、LAG-3、FGL1、TIM-3、Galectin-9、TIGIT、CD155以及TGF-β受体(TGF-βR)、CD80、CD86、VEGFR、VEGF-trap、FGL1、CD70、4-1BBL、OX40L、SIRPα；以及肿瘤抗原包括Claudin 18.2、HER-2、Mesothelin、BCMA、SSTR2、GPRC5D、PSMA、FCRH5、CD33、CD123、CD20、A33、CEA、CD28、DLL3、EGFR、VEGFR、VEGFR2、VEGF-A、Nectin-4、FGFR、C-met、RANKL、PDGF、PDGFR、PDGFRα、DLL4、Ang-1、Ang-2等。通过免疫检查点靶点或肿瘤靶点与其他抵制癌细胞发展或激活T细胞相关靶点组合或是多个免疫检查点靶点组合使用，极大的提升了治疗肿瘤等恶性疾病的可能性。在早期，临床上将两个单独的检查点抗体联用提高疗效。例如，与单独使用ipilimumab治疗相比，用ipilimumab(抗CTLA4)+nivolumab(抗PD1)治疗黑色素瘤患者可提高生存率。目前，四种PD-1×CTLA4bsAb的安全性和早期疗效正在早期临床试验中进行评估。阻断两种免疫检查点抑制剂的概念也在临床上被用于其他靶标组合，如PD-1×LAG 3、PD-1×TIM3和PD-L1×CTLA 4。(Wolchok,J.D.et al.Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma，N.Engl.J.Med.377(14)：1345–1356,(2017)；Dovedi,S.J.et al.MEDI5752:A novel bispecific antibody that preferentially targets CTLA-4 on PD-1 expressing T-cells.Cancer Res.78(13),Supplement.Abstract 2776:(2018).Hedvat,M.et al.Simultaneous checkpoint—checkpoint or checkpoint—costimulatory receptortargeting with bispecific antibodies promotes enhanced human T cell activation[abstract P664].Presented at  the 2018 Society for Immunotherapyof Cancer(SITC)(2018)；Aran F.Labrijn,et al.Bispecific antibodies:a mechanistic review of the pipeline,Nature Reviews Drug Discovery volume 18,pages585–608(2019)。
目前，大多数双特异性抗体尚处于临床或临床前研究阶段，仅有三个双特异性抗体获批上市，它们分别是Trion Pharma公司的Catumaxomab(2017年因商业因素下市)，Amgen公司的Blinatumomab和罗氏的Emicizumab。Catumaxomab是一个Triomab结构形式的IgG样双特异性抗体，靶向作用于T细胞和上皮细胞黏附分子(EpCAM)过表达的肿瘤细胞，通过T细胞活化、抗体依赖的细胞介导的细胞毒作用(antibodydependent cell-mediated cytotoxicity，ADCC)、补体介导的细胞毒作用(complement-dependent cytotoxicity，CDC)来抑制肿瘤细胞，2009年被EMA批准用于标准治疗无效或不可行的EpCAM阳性肿瘤所致的恶性腹水的治疗，该药也是第一个被获批上市的双特异性抗体。Blinatumomab为不含Fc区双特异性抗体，采用BiTE(bispecific T cell engager)结构形式。Blinatumomab抗CD3部分结合T细胞，通过抗CD19部分结合恶性和正常B细胞，从而诱导T细胞介导的肿瘤细胞杀伤作用，2014年被FDA批准用于治疗费城染色体阴性前体B细胞急性淋巴性白血病。Emicizumab是一种经修饰的人源化双特异性IgG4单抗，可以结合因子IXa和因子X。该抗体采用“Knobs-into-Holes(KiH)”进行Fc设计、“Common Light Chain-IgG(CLC-IgG)”进行LC设计和“Multidimensional Optimization(多维优化)”进行可变区优化，2017年被FDA批准用于存在VIII因子抑制物的A型血友病的常规预防。
表1 已获批上市(含已下市)的双特异性抗体
Figure PCTCN2021081659-appb-000001
尽管目前已有几十种双特异性抗体/平台及3个已上市药物的研发经验，但由于双特异性抗体结构形式复杂，作用特点多样，其发展相对一般抗体而言存在更多的问题和挑战。例如IgG样双特异性抗体在制备过程中面临着重链-重链或者重链-轻链错配的问题，其中最为典型的就是 Catumaxomab，虽然Catumaxomab是第一个批准上市的双特异性抗体，但同时也具有非常明显的局限性，主要体现在Triomab抗体复杂的生产工艺以及异源抗体比较容易产生的免疫原性问题，2017年该药因商业因素下市；再比如串联不同抗原结合片段的双特异性抗体由于缺乏恒定区，特别是缺乏Fc片段，半衰期较短，而且其表达量和表达后的细胞内稳定性一般低于传统IgG(Stork R,Müller D,Kontermann R E.A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G[J].Protein Engineering,Design&Selection,2007,20(11):569-576.)。同时，多数的该类抗体在纯化过程中难以采用亲和纯化手段，因此其纯化工艺和成品中的杂质分析也较传统IgG复杂(Tan P H,Sandmaier B M,Stayton P S.Contributions of a highly conserved VH/VL hydrogen bonding interaction to scFv folding stability and refolding efficiency[J].Biophysical journal,1998,75(3):1473-1482.)，其中最典型的就是Blinatumomab，Blinatumomab由于没有Fc片段，分子量较小，在血液中的半衰期只有1.25±0.63h，远远比一个完整的抗体半衰期短，且需要连续给予4周的静脉注射才能达到有效剂量，并且试剂使用的时候需要额外配备连续输液装置，再加上生产时，稳定性差和聚集体容易形成，也影响产品的质量和增加生产成本。目前的双特异性抗体，存在表达量低、稳定性差、生产工艺复杂且研发成本明显高于单克隆抗体等多种问题，这些都局限了双特异性抗体的发展。因此，亟需开辟具有新结构的双特异性抗体，为临床提供更多的选择。
发明内容
针对上述问题，本发明提供了一种具有新型结构的双特异性融合蛋白，它属于含附加功能区的IgG样双特异性融合蛋白。具体的，所述的融合蛋白是在全长的免疫球蛋白G(IgG)的铰链区通过可选的肽接头插入第二结合结构域，所述IgG的Fab区为所述融合蛋白的第一结合结构域；插入第二结合结构域后的所述IgG的重链为所述融合蛋白的重链，所述IgG的轻链为所述融合蛋白的轻链；所述第二结合结构域选自人受体、配体、所述受体或配体的胞外区片段、结合结构域片段或片段组合；其中，所述受体或配体为在天然信号通路中会形成二聚化或多聚化结构以活化或抑制信号通路的受体或配体，且所述第二结合结构域与所述第一结合结构域靶向不同的靶点。我们将具有上述结构的双特异性融合蛋白命名为 Hibody(Hinge-insersion bispecific antibody)结构融合蛋白(下称Hibody结构融合蛋白或Hibody)。
进一步地，所述第一结合结构域靶向结合免疫检查点分子或肿瘤抗原。
进一步地，所述免疫检查点分子包括PD-1(Programmed cell death protein 1)、PD-L1(Programmed cell death 1 ligand 1)、CTLA-4(Cytotoxic T lymphocyte-associated antigen-4)、LAG-3(Lymphocyte activation gene-3)、FGL1(Fibrinogen-like protein 1)、TIM-3(T cell immunoglobulin-3)、Galectin-9、TIGIT(T-cell immunoreceptor with Ig and ITIM domains)、CD155、CD47；所述肿瘤抗原包括Claudin 18.2、Her-2(Human epidermalgrowth factor receptor-2)、Mesothelin、BCMA(B Cell Maturation Antigen)、SSTR2(Somatostatin receptor2)、GPRC5D(G-protein coupled receptor family Cgroup 5 member D)、PSMA(Prostate specific membrane antigen)、FcRH5(Fc receptor-like protein 5)、CD33、CD123、CD20、A33、CEA(Carcino-embryonic antigen)、CD28、DLL3(Delta-like protein 3)、EGFR(Epidermal growth factor receptor)、VEGFR(Vascular Endothelial Growth Factor Receptor)、VEGFR2(Vascular Endothelial Growth Factor Receptor 2)、VEGF-A(Vascular endothelial growth factor-A)、Nectin-4、FGFR(Fibroblast growth factor receptors)、C-met、RANKL(Receptor activator ofnuclear factor kappa-B ligand)、PDGF(Platelet Derived Growth Factor)、PDGFR(Platelet Derived Growth Factor Receptor)、PDGFRα(Platelet Derived Growth Factor Receptor α)、DLL4(Delta-like ligand 4)、Ang-1(Angiopoietin-1)、Ang-2(Angiopoietin-2)。
进一步地，所述第二结合结构域靶向结合人TGF-β(Transforming growth factor-β)、CTLA-4、VEGF、LAG3、CD27、4-1BB(CD137)、OX40(CD134)、CD47、FGL1(Fibrinogen Like Protein 1)、TLT-2(Trem-like transcript 2)、CD28、HGF(Hepatocyte growth factor)、CSF1(Colony-stimulating factor 1)、CXCL1(CXC chemokine ligand 1)、CXCL2(CXC chemokine ligand 2)、CXCL3(CXC chemokine ligand 3)、CXCL5(CXC chemokine ligand5)、CXCL6(CXC chemokine ligand 6)、CXCL7(CXC chemokine ligand 7)、CXCL8(CXC chemokine ligand 8)、CXCL9(CXC chemokine ligand 9)、CXCL10(CXC chemokine ligand 10)、CXCL12(CXC chemokine ligand 12)、GITR(Glucocorticoid-induced tumor necrosis factor receptor)、EGF(Epidermal Growth Factor)、ICOSL(Inducible co-stimulator ligand)。
进一步地，所述受体或配体为人TGF-β受体(TGF-βR)、CD80、CD86、VEGFR、VEGF-trap、FGL1、CD70、4-1BBL、OX40L、SIRPα(Signal regulatory protein α)、B7-H3(CD276)、C-met、CSF1R(Colony-stimulating factor 1 receptor)、CXCR2(CXC chemokine receptor 2)、CXCR3(CXC chemokine receptor 3)、CXCR4(CXC chemokine receptor 4)、GITRL(Glucocorticoid-induced TNF receptor ligand)、EGFR、ICOS(Inducible co-stimulator)。
其中，CD80和CD86均是跨膜糖蛋白，属免疫球蛋白超家族(IgSF)成员。成熟的CD80分子由254个氨基酸组成，其中胞外结构域(ECD)208个氨基酸、跨膜结构域25个氨基酸和胞内结构域21个氨基酸。类似地，成熟CD86分子由303个氨基酸组成，其中胞外结构域222个氨基酸、跨膜结构域20个氨基酸和胞内结构域61个氨基酸。CD80和CD86的胞外结构域包含免疫球蛋白V(IgV)区和免疫球蛋白C(IgC)区，CD80和CD86通过免疫球蛋白V(IgV)区与其配体CD28和CTLA-4结合。在CD80和CD86与CD28结合的情形，CD80和CD86对于抗原诱导T细胞活化、增殖和效应功能的产生具有重要的调节作用，是正调节因子；而在CD80和CD86与CTLA-4结合的情形，CD80和CD86下调免疫应答，是负性调节因子。因此，CD80和CD86是T淋巴细胞活化时的协同刺激因子，在自身免疫监控、体液免疫应答及移植反应中发挥重要作用。采用CD28的配体CD80作为激活因子,活化T细胞，与采用激动性抗CD28抗体活化T细胞不同，其不会引发严重的细胞因子风暴，从而大大降低了危及患者生命情形的可能性。
在一些实施方案中，CD80ECD选自人CD80(诸如SEQ ID NO:134的人CD80)或来自CD80同种型2或同种型3的人CD80ECD(SEQ ID NO:135和136)。所述CD80ECD包含CD80免疫球蛋白V(IgV)区(CD80-IgV，SEQ ID NO:133)。在一个实施方案中，所述CD80ECD包含人CD80免疫球蛋白V区和C区(CD80-IgVIgC,SEQ ID NO:32)。在一个实施方案中，所述CD80ECD是人CD80ECD，在一个实施方案中，所述CD80ECD包含人CD80IgV。
CD80-IgV(SEQ ID NO：133):
Figure PCTCN2021081659-appb-000002
CD80-IgVIgC(SEQ ID NO：32)：
Figure PCTCN2021081659-appb-000003
Figure PCTCN2021081659-appb-000004
CD80同种型1(SEQ ID NO：134):
Figure PCTCN2021081659-appb-000005
CD80同种型2(SEQ ID NO：135):
Figure PCTCN2021081659-appb-000006
CD80同种型3(SEQ ID NO：136):
Figure PCTCN2021081659-appb-000007
进一步地，所述第一结合结构域和所述第二结合结构域选自以下组合：
(1)所述第一结合结构域靶向结合PD-L1，所述第二结合结构域靶向结合人TGF-β、VEGF、FGL1、CD47、CD155、HGF、CSF1、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL12、EGF或ICOSL；或
(2)所述第一结合结构域靶向结合PD-1，所述第二结合结构域靶向结合人CTLA-4、VEGF、HGF、EGF、CD28、LAG3、CD27、4-1BB或OX40；或
(3)所述第一结合结构域靶向结合CTLA-4，所述第二结合结构域靶向结合人CD28、VEGF、HGF、EGF、LAG3、CD27、4-1BB或OX40；或
(4)所述第一结合结构域靶向结合LAG-3，所述第二结合结构域靶向结合人CD28、VEGF、HGF、EGF、CTLA-4、CD27、4-1BB或OX40；或
(5)所述第一结合结构域靶向结合FGL1，所述第二结合结构域靶向结合人TGF-β、VEGF、CD47、CD155、HGF、CSF1、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL12、EGF或ICOSL；或
(6)所述第一结合结构域靶向结合TIM-3，所述第二结合结构域靶向结合人VEGF、HGF、EGF、LAG3、CD27、4-1BB或OX40；或
(7)所述第一结合结构域靶向结合Galectin-9，所述第二结合结构域靶向结合人TGF-β、VEGF、CD47、CD155、HGF、CSF1、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL12、EGF或ICOSL；或
(8)所述第一结合结构域靶向结合TIGIT，所述第二结合结构域靶向结合人TGF-β、HGF、EGF或VEGF；或
(9)所述第一结合结构域靶向结合CD155，所述第二结合结构域靶向结合人TGF-β、VEGF、HGF、EGF、CD47或CD155；或
(10)所述第一结合结构域靶向结合Claudin 18.2、HER-2、mesothelin、BCMA、SSTR2、GPRC5D、PSMA、FCRH5、CD33、CD123、CD20、A33、CEA、CD28、DLL3、EGFR、VEGFR、VEGFR2、VEGF-A、Nectin-4、FGFR、C-met、RANKL、PDGF、PDGFR、PDGFRα、DLL-4、Ang-1、Ang-2，所述第二结合结构域靶向结合人CTLA-4，TGF-β、VEGF、FGL1、LAG3、4-1BB、OX40、CD27、CD28、CD47、CD155、HGF、CSF1、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL12、EGF或ICOSL。
进一步地，所述第一结合结构域和所述受体、配体选自以下组合：
(1)所述第一结合结构域靶向结合PD-L1，所述受体、配体选自TGF-βRII、VEGFR、LAG-3、SIRPα、TIGIT、C-MET、CSF1R、CXCR2、CXCR3、CXCR4、EGFR或ICOS；或
(2)所述第一结合结构域靶向结合PD-1，所述受体、配体选自人CD80、CD86、VEGFR、c-MET、EGFR、FGL1、CD70、4-1BBL或OX40L；或
(3)所述第一结合结构域靶向结合CTLA-4，所述受体、配体选自人CD80、CD86、VEGFR、c-MET、EGFR、FGL1、CD70、4-1BBL或OX40L；或
(4)所述第一结合结构域靶向结合LAG-3，所述受体、配体选自人VEGFR、c-MET、EGFR、CD80、CD86、CD70、4-1BBL或OX40L；或
(5)所述第一结合结构域靶向结合FGL1，所述受体、配体选自人TGF-βRII、VEGFR、SIRPα、TIGIT、c-MET、CSF1R、CXCR2、CXCR3、CXCR4、EGFR或ICOS；或
(6)所述第一结合结构域靶向结合TIM-3，所述受体、配体选自人VEGFR、c-MET、EGFR、FGL1、CD70、4-1BBL或OX40L；或
(7)所述第一结合结构域靶向结合Galectin-9，所述受体、配体选自人TGF-βRII、VEGFR、SIRPα、TIGIT、c-MET、CSF1R、CXCR2、CXCR3、CXCR4、EGFR或ICOS；或
(8)所述第一结合结构域靶向结合TIGIT，所述受体、配体选自人TGF-βRII、c-MET、EGFR或VEGFR；或
(9)所述第一结合结构域靶向结合CD155，所述受体、配体选自人TGF-βRII、VEGFR、c-MET、EGFR、SIRPα或TIGIT；或
(10)所述第一结合结构域靶向结合Claudin 18.2、HER-2、mesothelin、BCMA、SSTR2、GPRC5D、PSMA、FCRH5、CD33、CD123、CD20、A33、CEA、CD28、DLL3、EGFR、VEGFR、VEGFR2、Nectin-4、FGFR、c-MET、RANKL、PDGF、PDGFR、PDGFRα、DLL4、Ang-1或Ang-2，所述受体、配体选自人CTLA-4，CD80，CD86，TGF-βRII、VEGFR、FGL1、LAG3、4-1BB、OX40、CD27、CD28、CD70、c-MET、SIRPα、TIGIT、CSF1R、CXCR2、CXCR3、CXCR4、EGFR或ICOS。
进一步地，所述受体、配体的片段包括受体或配体的胞外区片段、结合结构域片段。
进一步地，所述第一结合结构域靶向结合PD-L1；所述第二结合结构域为人TGF-βRII片段；或所述第一结合结构域靶向结合PD-1，所述第二结合结构域包含人CD80 ECD、CD80 IgV区、人CD80IgVIgC、VEGFR1 ECD、VEGFR2 ECD或VEGFR1的第2个细胞外区域和VEGFR2的第3个细胞外区域组合；或所述第一结合结构域靶向结合Claudin 18.2或HER-2、EGFR，所述第二结合结构域选自人CD80 ECD、CD80 IgV区、人CD80IgVIgC、VEGFR1ECD、VEGFR2 ECD或VEGFR1的第2个细胞外区域和VEGFR2的第3个细胞外区域组合。
进一步地，所述第二结合结构域选自：
(1)包含SEQ ID NO:31、131或132所示序列的人TGF-βRII或包含与SEQ ID NO:31、131或132所示序列相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列或与SEQ ID NO:31、131或132所示序列相比具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸替换或缺失后的氨基酸序列；或
(2)包含SEQ ID NO:32或133所示序列的CD80ECD或包含与SEQ ID NO:32或133所示序列相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列或与SEQ ID NO:32或133所示序列相比具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸替换或缺失后的氨基酸序列；或
(3)包含SEQ ID NO:33所示序列的VEGFR1的第2个细胞外区域和VEGFR2的第3个细胞外区域片段组合或包含与SEQ ID NO:33所示序列相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列或与SEQ ID NO:33所示序列相比具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸替换或缺失后的氨基酸序列。
进一步地，所述第二结合结构域的C端或/和N端带有肽接头，所述肽接头由2-30个氨基酸组成。
进一步地，所述肽接头可为：
(1)(GGGGS)n；或
(2)AKTTPKLEEGEFSEAR(SEQ ID NO：80)；或
(3)AKTTPKLEEGEFSEARV(SEQ ID NO：81)；或
(4)AKTTPKLGG(SEQ ID NO：82)；或
(5)SAKTTPKLGG(SEQ ID NO：83)；或
(6)SAKTTP(SEQ ID NO：84)；或
(7)RADAAP(SEQ ID NO：85)；或
(8)RADAAPTVS(SEQ ID NO：86)；或
(9)RADAAAAGGPGS(SEQ ID NO：87)；或
(10)RADAAAA(SEQ ID NO：88)；或
(11)SAKTTPKLEEGEFSEARV(SEQ ID NO：89)；或
(12)ADAAP(SEQ ID NO：90)；或
(13)DAAPTVSIFPP(SEQ ID NO：91)；或
(14)TVAAP(SEQ ID NO：92)；或
(15)TVAAPSVFIFPP(SEQ ID NO：93)；或
(16)QPKAAP(SEQ ID NO：94)；或
(17)QPKAAPSVTLFPP(SEQ ID NO：95)；或
(18)AKTTPP(SEQ ID NO：96)；或
(19)AKTTPPSVTPLAP(SEQ ID NO：97)；或
(20)AKTTAP(SEQ ID NO：98)；或
(21)AKTTAPSVYPLAP(SEQ ID NO：99)；或
(22)ASTKGP(SEQ ID NO：100)；或
(23)ASTKGPSVFPLAP(SEQ ID NO：101)；或
(24)GENKVEYAPALMALS(SEQ ID NO：102)；或
(25)GPAKELTPLKEAKVS(SEQ ID NO：103)；或
(26)GHEAAAVMQVQYPAS(SEQ ID NO：104)；或
(27)GGGGSGGGGSGGGGSA(SEQ ID NO：105)，
其中所述n等于1、2、3或4；
当n＝1时，所述的(GGGGS)n为GGGGS(SEQ ID NO：120)；
当n＝2时，所述的(GGGGS)n为GGGGSGGGGS(SEQ ID NO：121)或(GGGGS) 2；
当n＝3时，所述的(GGGGS)n为GGGGSGGGGSGGGGS(SEQ ID NO：122)或(GGGGS) 3；
当n＝4时，所述的(GGGGS)n为GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：123)或(GGGGS) 4。
进一步地，插入的所述第二结合结构域的大小不超过235、240、250、260、270、280、290或300个氨基酸。
进一步地，所述第二结合结构域的插入位点位于铰链区的中前部，插入位点不影响免疫球蛋白的二硫键的形成。其中，所述铰链区的中前部通常是指231A之前的部分。进一步地，所述插入位点前后铰链区的部分氨基酸被替换或缺失，例如，所述铰链区含有D221G和/或C220V突变。
进一步地，所述IgG选自哺乳动物IgG、人源化IgG和人IgG，所述哺乳动物包括小鼠、大鼠；优选的，所述IgG为IgG1、IgG2、IgG3、IgG4。
进一步地，所述的融合蛋白的Fc区是无糖基化的或脱糖基化的或具有降低的岩藻糖基化或是无岩藻糖基化的。
进一步地，所述IgG为Atezolizumab、Avelumab、Durvalumab、Nivolumab、Pembrolizumab、Cemiplimab、Ipilimumab。优选的，所述IgG为Atezolizumab，所述IgG的重链氨基酸序列如SEQ ID NO:106所示，所述IgG的轻链氨基酸序列如SEQ ID NO:107所示；或所述IgG为Avelumab，所述IgG的重链氨基酸序列如SEQ ID NO:108所示，所述IgG的轻链氨基酸序列如SEQ ID NO:109所示；或所述IgG为Durvalumab，所述IgG的重链氨基酸序列如SEQ ID NO:110所示，所述IgG的轻链氨基酸序列如SEQ ID NO:111所示；或所述IgG为Nivolumab，所述IgG的重链氨基酸序列如SEQ ID NO:112所示，所述IgG的轻链氨基酸序列如SEQ ID NO:113所示；或所述IgG为Pembrolizumab，所述IgG的重链氨基酸序列如SEQ ID NO:114所示，所述IgG的轻链氨基酸序列如SEQ ID NO:115所示；或所述IgG为Ipilimumab，所述IgG的重链氨基酸序列如SEQ ID NO:116所示，所述IgG的轻链氨基酸序列如SEQ ID NO:117所示；或所述IgG为Cemiplimab，所述IgG的重链氨基酸序列如SEQ ID NO:118所示，所述IgG的轻链氨基酸序列如SEQ ID NO:119所示。
具体的，本发明提供了一种具有第一结合结构域靶向人PD-L1的Hibody结构融合蛋白，所述IgG的Fab中重链可变区的CDR和/或所述轻链可变区的CDR与如下序列限定的抗体具有相同的CDR序列或在如下序列限定的抗体的CDR上进行1-2个氨基酸替换，所述序列限定的抗体为：
(1)重链可变区氨基酸序列为SEQ ID NO:66所示；和/或
(2)轻链可变区氨基酸序列为SEQ ID NO:67所示。
进一步地，所述IgG的Fab中重链可变区的CDR和/或所述轻链可变区的CDR如下所示：
(1)重链可变区CDR1氨基酸序列选自SEQ ID NO:1-5及对SEQ ID NO: 1-5进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列选自SEQ ID NO:6-10及对SEQ ID NO:6-10进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列选自SEQ ID NO:11-15及对SEQ ID NO:11-15进行1或2个氨基酸替换后的氨基酸序列；和/或
(2)轻链可变区CDR1氨基酸序列选自SEQ ID NO:16-20及对SEQ ID NO:16-20进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列选自SEQ ID NO:21-25及对SEQ ID NO:21-25进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列选自SEQ ID NO:26-30及对SEQ ID NO:26-30进行1或2个氨基酸替换后的氨基酸序列。
在特定实施方案中，根据采用不同的测定方法或系统认定，相应重链和轻链可变区的互补决定区CDR 1-3如表2所示。
表2 抗PD-L1抗体重链和轻链可变区CDR 1-3氨基酸序列
Figure PCTCN2021081659-appb-000008
Figure PCTCN2021081659-appb-000009
进一步地，所述融合蛋白含有以下CDR序列，
(1)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：1，6，11或对SEQ ID NO:1，6，11进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：16，21，26或对SEQ ID NO：16，21，26进行1或2个氨基酸替换后的氨基酸序列；或
(2)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：2，7，12或对SEQ ID NO:2，7，12进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：17，22，27或对SEQ ID NO：17，22，27进行1或2个氨基酸替换后的氨基酸序列；或
(3)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：3，8，13或对SEQ ID NO:3，8，13进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：18，23，28或对SEQ ID NO：18，23，28进行1或2个氨基酸替换后的氨基酸序列；或
(4)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：4，9，14或对SEQ ID NO:4，9，14进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：19，24，29或对SEQ ID NO：19，24，29进行1或2个氨基酸替换后的氨基酸序列；或
(5)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：5，10，15或对SEQ ID NO:5，10，15进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：20，25，30或对SEQ ID NO：20，25，30进行1或2个氨基酸替换后的氨基酸序列。
进一步地，所述融合蛋白的重链中重链可变区CDR 1-3氨基酸序列为 SEQ ID NO：3，8，13；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：18，23，28。
进一步地，所述融合蛋白的重链可变区和轻链可变区的序列如下所示，
(1)重链可变区的序列如SEQ ID NO:66所示，或与SEQ ID NO:66具有相同的CDR 1-3且与SEQ ID NO:66相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列；和/或
(2)轻链可变区的序列如SEQ ID NO:67所示，或与SEQ ID NO:67具有相同的CDR 1-3且与SEQ ID NO:67相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列。
更具体的，所述融合蛋白的重链和轻链的氨基酸序列如下所示，
(1)所述融合蛋白的重链氨基酸序列如SEQ ID NO:72所示或与SEQ ID NO:72相比同一性大于90％、95％、96％、97％、98％、99％的序列；
(2)所述融合蛋白的轻链氨基酸序列分别如SEQ ID NO:73所示或与SEQ ID NO:73相比同一性大于90％、95％、96％、97％、98％、99％的序列。
进一步地，所述融合蛋白的重链和轻链的氨基酸序列如下所示，
(1)所述融合蛋白的重链氨基酸序列与SEQ ID NO:72相比具有1-15个氨基酸位点突变或具有替换的肽接头；
(2)所述融合蛋白的轻链氨基酸序列与SEQ ID NO:73相比具有1-10个氨基酸位点突变。
具体的，本发明提供了一种第一结合结构域靶向人PD-1的Hibody结构融合蛋白，其中，所述Fab中重链可变区的CDR和/或所述轻链可变区的CDR与如下序列限定的抗体具有相同的CDR序列或在如下序列限定的抗体的CDR上进行1-2个氨基酸替换，所述序列限定的抗体为：
(1)重链可变区氨基酸序列为SEQ ID NO:64所示；和/或
(2)轻链可变区氨基酸序列为SEQ ID NO:65所示。
进一步地，融合蛋白中Fab中重链可变区的CDR和/或所述轻链可变区的CDR如下所示：
(1)重链可变区CDR1氨基酸序列选自SEQ ID NO:34-38及对SEQ ID NO:34-38进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列选自 SEQ ID NO:39-43及对SEQ ID NO:39-43进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列选自SEQ ID NO:44-48及对SEQ ID NO:44-48进行1或2个氨基酸替换后的氨基酸序列；和/或
(2)轻链可变区CDR1氨基酸序列选自SEQ ID NO:49-53及对SEQ ID NO:49-53进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列选自SEQ ID NO:54-58及对SEQ ID NO:54-58进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列选自SEQ ID NO:59-63及对SEQ ID NO:59-63进行1或2个氨基酸替换后的氨基酸序列。
在特定实施方案中，根据采用不同的测定方法或系统认定，相应重链和轻链可变区的互补决定区CDR 1-3如表3所示。
表3 抗PD-1抗体重链和轻链可变区CDR 1-3氨基酸序列
Figure PCTCN2021081659-appb-000010
Figure PCTCN2021081659-appb-000011
进一步地，所述融合蛋白可变区的CDR1-3如下所定义，
(1)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：34，39，44或对SEQ ID NO:34，39，44进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：49，54，59或对SEQ ID NO：49，54，59进行1或2个氨基酸替换后的氨基酸序列；或
(2)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：35，40，45或对SEQ ID NO:35，40，45进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：50，55，60或对SEQ ID NO：50，55，60进行1或2个氨基酸替换后的氨基酸序列；或
(3)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：36，41，46或对SEQ ID NO:36，41，46进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：51，56，61或对SEQ ID NO：51，56，61进行1或2个氨基酸替换后的氨基酸序列；或
(4)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：37，42，47或对SEQ ID NO:37，42，47进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：52，57，62或对SEQ ID NO：52，57，62进行1或2个氨基酸替换后的氨基酸序列；或
(5)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：38，43，48或对SEQ ID NO:38，43，48进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：53，58，63或对SEQ ID NO：53，58，63进行1或2个氨基酸替换后的氨基酸序列。
进一步地，所述融合蛋白的可变区CDR1-3如下所定义：重链可变区 CDR 1-3氨基酸序列为SEQ ID NO：36，41，46；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：51，56，61。
进一步地，所述融合蛋白的重链可变区和轻链可变区的氨基酸序列如下所示，
(1)重链可变区的序列如SEQ ID NO:64所示，或与SEQ ID NO:64具有相同的CDR 1-3且与SEQ ID NO:64相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列；和/或
(2)轻链可变区的序列如SEQ ID NO:65所示，或与SEQ ID NO:65具有相同的CDR 1-3且与SEQ ID NO:65相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列。
更具体地，所述融合蛋白的重链和轻链氨基酸序列如下所定义：
(1)所述融合蛋白的重链氨基酸序列如SEQ ID NO:68所示或与SEQ ID NO:68相比同一性大于90％、95％、96％、97％、98％、99％的序列；
(2)所述融合蛋白的轻链氨基酸序列分别如SEQ ID NO:69所示或与SEQ ID NO:69相比同一性大于90％、95％、96％、97％、98％、99％的序列。
进一步地，所述融合蛋白的重链和轻链的氨基酸序列如下所示：
(1)所述融合蛋白的重链氨基酸序列与SEQ ID NO:68相比具有1-15个氨基酸位点突变或具有替换的肽接头；
(2)所述融合蛋白的轻链氨基酸序列与SEQ ID NO:69相比具有1-10个氨基酸位点突变。
更具体地，所述融合蛋白的重链和轻链氨基酸序列如下所定义：
(1)所述融合蛋白的重链氨基酸序列如SEQ ID NO:70所示或与SEQ ID NO:70相比同一性大于90％、95％、96％、97％、98％、99％的序列；
(2)所述融合蛋白的轻链氨基酸序列分别如SEQ ID NO:71所示或与SEQ ID NO:71相比同一性大于90％、95％、96％、97％、98％、99％的序列。
进一步地，所述融合蛋白的重链和轻链的氨基酸序列如下所示：
(1)所述融合蛋白的重链氨基酸序列与SEQ ID NO:70相比具有1-15个氨基酸位点突变或具有替换的肽接头；
(2)所述融合蛋白的轻链氨基酸序列与SEQ ID NO:71相比具有1-10个氨基酸位点突变。
本发明还提供了一种分离的多核苷酸，其编码如上所述的融合蛋白。
本发明还提供了一种核酸构建体，其包含如上所述的多核苷酸，优选的所述核酸构建体为载体。
本发明还提供了一种宿主细胞，其包含如上所述的多核苷酸或者核酸构建体或载体，优选的所述细胞是原核细胞、真核细胞、酵母细胞、哺乳动物细胞、大肠杆菌细胞或CHO细胞、NS0细胞、Sp2/0细胞、BHK细胞。
本发明还提供了一种药物组合物，包含如上所述的融合蛋白以及药学上可接受的载体。
本发明还提供了一种用于治疗肿瘤或癌症的方法，其包括对需要治疗或减轻的受试者施用如上所述的融合蛋白或所述药物组合物的步骤。
本发明还提供了如上所述的融合蛋白、多核苷酸、核酸构建体或者载体以及药物组合物在制备用于治疗或预防肿瘤或癌症的药物中的用途。所述的肿瘤或癌症包括实体瘤或非实体瘤。
本发明还提供了一种诊断试剂盒，其包含如上所述的融合蛋白。
本发明还提供了一种融合蛋白的生产方法，该方法包括：在允许如上所述的核酸构建体表达的条件下培养所述的宿主细胞，并从培养物中回收产生的融合蛋白。
本发明提供的Hibody结构融合蛋白，由于第二结合结构域插入在铰链区，因此具备与IgG相同的表达和生产优势，可以与现有的IgG表达平台以及纯化平台无缝接入，极大地了降低了后继开发成本，相对于现有的双特异抗体平台构建的双特异性抗体，Hibody平台构建的双特异性抗体表达量更高、且无轻重链错配的问题，也极大地降低了抗体生产成本。此外，我们惊奇的发现，在两条铰链区插入一定范围大小的第二结合结构域不仅不影响融合蛋白Fab区的结合活性以及蛋白的稳定性，且进一步提升了稳定性以及获得了更高的半衰期，更显著的是，第二结合结构域与靶向结合位点的结合活性相对于其对应可溶性天然结合片段的单体与相应靶点的结合活性相比，有了显著的提高，其原因可能在于由于选择的作为第二结合结构域的受体或配体其在天然信号通路中会形成二聚化或多聚化结构以活化或抑制信号通路，而当其插入到铰链区后，其在铰链区的空间位置关系促使形成了相应受体或配体或其片段的二聚化，从而通过模仿天然信号通路，获得了更显著提高的结合活性，进而使得该融合蛋白相对于使用可溶性的受体或配体或结合片段，其活化或抑制能力得到了极大的提 升。另外，通过两个结合结构域的合理组合，可以灵活选择受体抑制或受体活化的作用靶标，起到有效的协同作用效果，相对于两个靶点药物单独给药或现有技术的双特异性抗体，具有更为显著的疾病治疗效果，能够有效的治疗或控制疾病进程，尤其是在各种肿瘤、癌症中。
附图说明
图1 Hibody结构融合蛋白示意图。
图2为Hib-PDV及其对照组对小鼠结肠癌模型肿瘤抑制曲线。
图3为Hib-PDC及其对照组对小鼠结肠癌肿瘤抑制曲线。
图4为Hib-PLT及其对照组在10～100nM水平浓度促细胞分泌IFN-γ因子水平对照图。
具体实施方式
定义
除非另有定义，在此所使用的所有技术和科学术语具有与本发明涉及领域的普通技术人员通常所理解的相同的意义。专业人员具体可参考《细胞与分子生物学词典》(The Dictionary of Cell and Molecular Biology)，第三版，1999，学术出版社(Academic Press)；《生物医学与分子生物学简明词典》(the Concise Dictionary of Biomedicine and Molecular Biology)，Juo，Pei-Show，第二版，2002，CRC出版社；以及《生物化学与分子生物学牛津词典》(the Oxford Dictionary Of Biochemistry And Molecular Biology)，修订版，2000，牛津大学出版社(Oxford University Press)。
本发明涉及的氨基酸可以通过它们的通常己知的三字母符号或通过由IUPAC-IUB生物化学命名委员会(IUPAC-IUB Biochemical Nomenclature Commission)推荐的单字母符号表示。同样地，核苷酸可以通过它们的普遍公认的单字母代码表示。
在本发明中，抗体可变域的互补决定区(CDR)测定或编号方法包括本领域熟知的IMGT、Kabat(如在卡巴特(Kabat)等人的免疫学关注的蛋白质序列(Sequences of Proteins of Immunological Interest)，第5版，公共卫生服务(Public Health Service)，国家卫生研究所(National Institutes of Health)，马里兰州贝塞斯达(Bethesda MD.)(1991)中所阐述的)、Chothia、AbM以及Contact法。
对本发明来说，两种核酸或者氨基酸序列间的“一致性”、“同一性”或 “相似性”是指，在最佳比对(最优比对)后所获得的、待比较的两序列之间相同核苷酸或相同氨基酸残基的百分数，该百分数是纯粹统计学的并且两种序列间的差异随机分布并覆盖其全长。两种核酸或者氨基酸序列之间的序列比较通常是在以最优方式使它们匹配以后，通过比较这些序列而进行，所述比较能够通过区段或者通过“比较窗”实施。除了能够手工实施外，用于比较序列的最优比对，还能够通过Smith和Waterman(1981)[Ad.App.Math.2：482]的局部同源性算法、通过Neddleman和Wunsch的(1970)[J.MoI.Biol.48：443]局部同源性算法、通过Pearson和Lipman的(1988)[Proc.Natl.Acad.Sci.USA 85：2444)相似性搜索方法、通过使用这些算法的计算机软件实施(GAP、BESTFIT、FASTA和TFASTA in the Wisconsin Genetics Software Package，Genetics Computer Group，575Science Dr.，Madison，WI，或者通过BLAST N or BLAST P比较软件)。
如广义定义的那样，本发明涉及的免疫球蛋白(immunoglobulin)指具有抗体活性的动物蛋白，由两条相同的轻链和两条相同的重链所组成，是一类重要的免疫效应分子，由高等动物免疫系统淋巴细胞产生的蛋白质，经抗原的诱导可以转化为抗体。因结构不同可分为IgG、IgA、IgM、IgD和IgE 5种。优选的，本发明涉及的免疫球蛋白为IgG。
如广义定义的那样，本发明涉及的受体是一类存在于胞膜或胞内的，能与细胞外专一信号分子结合进而激活细胞内一系列生物化学反应，使细胞对外界刺激产生相应的效应的特殊蛋白质。与受体结合的生物活性物质统称为配体(ligand)。受体与配体结合即发生分子构象变化，从而引起细胞反应，如介导细胞间信号转导、细胞间黏合、胞吞等过程。
本发明涉及的双特异性融合蛋白是基于免疫球蛋白结构的基础上在其铰链区中插入有第二结合结构域，因此本发明涉及的双特异性融合蛋白具有两个结合结构域。
实施例
下面将通过实施例对本发明进行进一步的阐述，需要说明的是，以下实施例是对本发明进行进一步的阐述和解释，而不应被看作是对本发明的限制。
实施例1 蛋白的构建
本实施例采用本领域常规方法构建以下蛋白分子，并按照本领域常规 方法瞬时或稳定表达：
①三个Hibody双特异性融合蛋白：Hib-PLT、Hib-PDC、Hib-PDV；
②对照蛋白1-3：按照WO2015/118175中双功能分子结构构建；
③其他对照蛋白：TGF-β-R-His、VEGF-R-His、CD80-His、PD-L1对照抗体。
表4 融合蛋白靶点组合
Figure PCTCN2021081659-appb-000012
●融合蛋白Hib-PLT重链和轻链的核酸序列及氨基酸序列如下所示：
融合蛋白Hib-PLT重链核酸序列(SEQ ID NO：78)：
Figure PCTCN2021081659-appb-000013
Figure PCTCN2021081659-appb-000014
说明：下划线加粗序列为TGF-βRⅡ核酸序列。
融合蛋白Hib-PLT重链氨基酸序列(SEQ ID NO：72)：
Figure PCTCN2021081659-appb-000015
Figure PCTCN2021081659-appb-000016
说明：加粗加框片段为铰链区序列，斜体为肽接头，下划线加粗序列为TGF-βRⅡ氨基酸序列。
融合蛋白Hib-PLT轻链核酸序列(SEQ ID NO：79)：
Figure PCTCN2021081659-appb-000017
融合蛋白Hib-PLT轻链氨基酸序列(SEQ ID NO：73)：
Figure PCTCN2021081659-appb-000018
●融合蛋白Hib-PDC重链和轻链的核酸序列及氨基酸序列如下所示：
融合蛋白Hib-PDC重链核酸序列(SEQ ID NO：74)：
Figure PCTCN2021081659-appb-000019
Figure PCTCN2021081659-appb-000020
说明：下划线加粗序列为CD80核酸序列。
融合蛋白Hib-PDC重链氨基酸序列(SEQ ID NO：68)：
Figure PCTCN2021081659-appb-000021
Figure PCTCN2021081659-appb-000022
说明：斜体为肽接头，下划线加粗序列为CD80氨基酸序列。
融合蛋白Hib-PDC轻链核酸序列(SEQ ID NO：75)：
Figure PCTCN2021081659-appb-000023
融合蛋白Hib-PDC轻链氨基酸序列(SEQ ID NO：69)：
Figure PCTCN2021081659-appb-000024
●融合蛋白Hib-PDV重链和轻链的核酸序列及氨基酸序列如下所示：
融合蛋白Hib-PDV重链核酸序列(SEQ ID NO：76)：
Figure PCTCN2021081659-appb-000025
Figure PCTCN2021081659-appb-000026
说明：下划线加粗序列为VEGFR核酸序列。
融合蛋白Hib-PDV重链氨基酸序列(SEQ ID NO：70)：
Figure PCTCN2021081659-appb-000027
说明：斜体为肽接头，下划线加粗序列为VEGF氨基酸序列。
融合蛋白Hib-PDV轻链核酸序列(SEQ ID NO：77)：
Figure PCTCN2021081659-appb-000028
融合蛋白Hib-PDV轻链氨基酸序列(SEQ ID NO：71)：
Figure PCTCN2021081659-appb-000029
Figure PCTCN2021081659-appb-000030
●对照蛋白1的重链和轻链氨基酸序列
对照蛋白1的重链氨基酸序列(SEQ ID NO：124)：
Figure PCTCN2021081659-appb-000031
说明：斜体为肽接头，下划线加粗序列为TGF-βRII氨基酸序列。
对照蛋白1的轻链氨基酸序列(SEQ ID NO：125)：
Figure PCTCN2021081659-appb-000032
●对照蛋白2的重链和轻链氨基酸序列
对照蛋白2的重链氨基酸序列(SEQ ID NO：126)：
Figure PCTCN2021081659-appb-000033
Figure PCTCN2021081659-appb-000034
说明：斜体为肽接头，下划线加粗序列为CD80氨基酸序列。
对照蛋白2的轻链氨基酸序列(SEQ ID NO：127)：
Figure PCTCN2021081659-appb-000035
●对照蛋白3的重链和轻链氨基酸序列
对照蛋白3的重链氨基酸序列(SEQ ID NO：128)：
Figure PCTCN2021081659-appb-000036
说明：斜体为肽接头，下划线加粗序列为VEGFR氨基酸序列。
对照蛋白3的轻链氨基酸序列(SEQ ID NO：129)：
Figure PCTCN2021081659-appb-000037
Figure PCTCN2021081659-appb-000038
●TGF-β-R-His
TGF-β-R-His即His标签的TGF-βRⅡ胞外区片段，其中所述的TGF-
βRⅡ胞外区片段的氨基酸序列(SEQ ID NO：137)：
Figure PCTCN2021081659-appb-000039
●VEGF-R-His氨基酸序列
VEGF-R-His即His标签的VEGFR1的第2个细胞外区域和VEGFR2的第3个细胞外区域组合片段，其中所述的VEGFR1的第2个细胞外区域和VEGFR2的第3个细胞外区域组合片段的氨基酸序列(SEQ ID NO：130)：
Figure PCTCN2021081659-appb-000040
●CD80-His氨基酸序列
CD80-His即His标签的CD80片段，其中所述的CD80片段的氨基酸序列(SEQ ID NO：32)：
Figure PCTCN2021081659-appb-000041
实施例2 CHO表达
A.细胞复苏与扩增：
复苏CHO宿主细胞，复苏培养体积为10mL，培养条件为37.0℃，200rpm，8％CO 2，80％湿度。复苏后细胞密度2.82×10 5个/mL，活率95.41％。
细胞扩增培养72h后，活细胞密度为2.22×10 6个/mL，活率98.32％。
将细胞以5×10 5个/mL重接进行细胞重接操作，继续培养48小时后活细胞密度为2×10 6个/mL，活率98.68％。
将细胞按照5×10 5个/孔铺入6孔板，培养体积2mL/孔，置于二氧化碳培养箱(培养条件37℃，8％CO 2)过夜培养，用于次日贴壁转染。
B.转染
转染试剂为
Figure PCTCN2021081659-appb-000042
LTX Reagent(Invitrogen，货号15338-100)，内含LTX和Plus试剂。
转染操作：将六孔板中培养基进行换液操作，配制转染复合物：LTX：9μl/孔+质粒：2.5μg/孔+plus：2.5μl/孔，将转染复合物按照每孔250μl加入至六孔板中。细胞转染后5h对6孔板各孔细胞进行换液。转染完成48h后取上清，使用双抗原ELISA方法检测上清抗体含量。
C.批式培养与流加培养
转染完成48h后将六孔板细胞消化，收集进入细胞培养管，按照细胞密度为3×10 5个/mL进行悬浮适应生长，细胞活率回复至90％左右时将Hib-PLT表达细胞pool和对照蛋白1表达细胞pool进行批式培养，将Hib-PDC、对照蛋白2、Hib-PDV、对照蛋白3进行流加培养。
批式培养：按照3×10 5个/mL细胞密度培养在TPP细胞培养管中，体积为30mL，培养条件37.0℃，200rpm，8％CO 2，80％湿度。每天对细胞活率和代谢进行监测，至活率<60％结束培养，取结束培养后的细胞上清液进行浓度检测。
流加培养：按照3×10 5个/mL细胞密度培养在TPP细胞培养管中，体积为30mL，培养条件37.0℃，200rpm，8％CO 2，80％湿度。按照一定的流加策略(见表5)给予细胞浓缩培养基，期间保证Glc控制在2～6g/L。至活率<60％结束培养，取结束培养后的细胞上清液进行浓度检测。
表5 流加策略
Day0 3×10 5/ml，培养体积30ml
Day4、6、8、10、12 900μLCellBoost7A、90uLCellBoost7B
Day16 检测VCD、活率，留样结束
表6给出了Hibody双特异性融合蛋白Hib-PLT、Hib-PDC、Hib-PDV及 相应对照蛋白的表达量结果，结果显示，Hib-PLT与相应对照蛋白相比，其表达量增加的相对值增加了52.85％；Hib-PDC与相应对照蛋白相比，其表达量增加的相对值增加了33.43％；Hib-PDV与相应对照蛋白相比，其表达量增加的相对值增加了25.6％，本发明提供的Hibody双特异性融合蛋白的表达量均显著优于相应对照蛋白的表达量，本发明提供的双特异性抗体具有意料不到的技术效果。
表6 双特异性抗体表达量
Figure PCTCN2021081659-appb-000043
注：增加相对值＝(本发明提供的双特异性融合蛋白的表达量-相应对照蛋白表达量)/相应对照蛋白表达量。
实施例3 结合活性测定
1.Hib-PLT结合活性测定：
(1)包被抗原：将TGF-β1用包被缓冲液稀释至2μg/ml，100μl每孔加入96孔板中，2～8℃静置过夜；
(2)洗板：以PBST为洗脱液，350μl每孔自动洗板3次，洗板完成后将板拍干；
(3)封板：以3％BSA-PBST为封闭液，300μl每孔，于37℃下孵育2小时；
(4)洗板：以PBST为洗脱液，350μl每孔自动洗板3次，洗板完成后将板拍干；
(5)加样品：样品各浓度点如下，将各浓度点样品依次以100μl每孔加入96孔板，每个浓度点平行2个复孔，37℃孵育2小时；
检测组 S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11
Hib-PLT 555.556 55.556 5.556 1.852 0.617 0.206 0.069 0.023 0.008 0.001 0.000
TGF-β-R-His 64516.000 6451.600 645.160 215.053 71.684 23.895 7.965 2.655 0.885 0.088 0.009
(6)洗板：以PBST为洗脱液，350μl每孔自动洗板3次，洗板完成后将板拍干；
(7)加二抗：
●Hib-PLT样品孔：Goat-anti-Human-Fc-HRP按1:5000稀释后，100μl每孔，37℃孵育2小时；
●TGF-β-R-His孔：Anti-His-HRP按1：5000稀释后，100μl每孔，37℃孵育2小时；
(8)洗板：以PBST为洗脱液，350μl每孔自动洗板5次，洗板完成后将板拍干；
(9)显色：TMB为显色液，100μl每孔显色2分钟；
(10)终止显色：2M H 2SO 4为终止液，50μl每孔终止显色；
(11)读数：MD酶标仪分别在450/655nm处读取吸光值，以待测样品浓度为横坐标，吸光值为纵坐标，作4-PL曲线，软件自动给出EC50值。
2.Hib-PDV结合活性测定：
(1)包被抗原：将VEGF用包被缓冲液稀释至1μg/ml，100μl每孔加入96孔板中，2～8℃静置过夜；
(2)洗板：以PBST为洗脱液，350μl每孔自动洗板3次，洗板完成后将板拍干；
(3)封板：以3％BSA-PBST为封闭液，300μl每孔，于37℃下孵育2小时；
(4)洗板：以PBST为洗脱液，350μl每孔自动洗板3次，洗板完成后将板拍干；
(5)加样品：样品各浓度点如下，将各浓度点样品依次以100μl每孔加入96孔板，每个浓度点平行2个复孔，37℃孵育2小时；
检测组 S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11
Hib-PDV 555.556 55.556 5.556 1.852 0.617 0.206 0.069 0.023 0.005 0.001 0.000
VEGFR-His 43478.300 4347.830 434.783 144.928 48.309 16.103 5.368 1.789 0.358 0.036 0.004
                                                       (单位：nM)
(6)洗板：以PBST为洗脱液，350μl每孔自动洗板3次，洗板完成后将 板拍干；
(7)加二抗：
●Hib-PDV样品孔：Goat-anti-Human-Fc-HRP按1:5000稀释后，100μl每孔，37℃孵育2小时；
●VEGFR-His孔：Anti-His-HRP按1：5000稀释后，100μl每孔，37℃孵育2小时
(8)洗板：以PBST为洗脱液，350μl每孔自动洗板5次，洗板完成后将板拍干；
(9)显色：TMB为显色液，100μl每孔显色2分钟；
(10)终止显色：2M H2SO4为终止液，50μl每孔终止显色；
(11)读数：MD酶标仪分别在450/655nm处读取吸光值，以待测样品浓度为横坐标，吸光值为纵坐标，作4-PL曲线，软件自动给出EC50值。
3.Hib-PDC结合活性测定：
(1)包被抗原：将CTLA-4Protein用1mL灭菌水复溶，浓度为200ug/mL。用包被缓冲液稀释至20μg/ml，100μl每孔加入96孔板中，2～8℃静置过夜；
(2)洗板：以PBST为洗脱液，350μl每孔自动洗板3次，洗板完成后将板拍干；
(3)封板：以3％BSA-PBST为封闭液，300μl每孔，于37℃下孵育2h±20min；
(4)洗板：以PBST为洗脱液，350μl每孔自动洗板3次，洗板完成后将板拍干；
(5)加样品：PBST稀释生物素化样品，样品各浓度点如下，将各浓度点样品依次以100μl每孔加入96孔板，每个浓度点平行2个复孔，空白孔为PBST，37℃孵育2h±20min；
检测组 S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11
Hib-PDC 1000000 100000 10000 3333.333 1111.111 370.370 123.457 41.152 13.717 1.372 0.137
CD80-His 1000000 100000 10000 3333.333 1111.111 370.370 123.457 41.152 13.717 1.372 0.137
                                                         (单位：ng/ml)
(6)洗板：以PBST为洗脱液，350μl每孔自动洗板3次，洗板完成后将板拍干；
(7)加二抗：
●Hib-PDC样品孔和CD80-His孔：Streptavidin-HRP按1：2000稀释后，100μl每孔，37℃孵育1h±10min；
(8)洗板：以PBST为洗脱液，350μl每孔自动洗板5次，洗板完成后将板拍干；
(9)显色：加入100μl/well TMB Double Slow substrate，室温避光显色6分钟；
(10)终止显色：2M H 2SO 4为终止液，50μl每孔终止显色；
(11)读数：MD酶标仪分别在450/655nm处读取吸光值，以待测样品浓度为横坐标，吸光值为纵坐标，作4-PL曲线，软件自动给出EC50值(见表7)。
表7 各检测组EC50值
检测组 EC50值
Hib-PLT 0.164nM
TGF-β-R-His 11.60nM
Hib-PDV 0.061nM
VEGFR-His 2154nM
Hib-PDC 24.36nM
CD80-His 50.69nM
经结合力测定，Hib-PLT与TGF-β受体单体相比较：Hib-PLT的EC50值为0.164nM，TGF-β受体单体的EC50值为11.60nM，Hib-PLT的结合活性优于TGF-β受体单体约70倍；Hib-PDV与VEGF受体单体相比较：Hib-PDV的EC50值为0.061nM，VEGF受体单体的EC50值为2154nM，Hib-PDV结合活性优于VEGF受体单体约35311倍；Hib-PDC与CD80单体相比较：Hib-PDC的C50值为24.36nM，CD80单体的EC50值为50.69nM，Hib-PDC结合活性优于CD80单体约2倍。即：以本发明提供的双抗模型构建的Hib-PLT、Hib-PDV、Hib-PDC的结合力活性均大幅优于其对应单体的结合活性。
实施例4 Hib-PDV对结肠癌MC38细胞在C57BL/6J-hPD-1小鼠皮下移植瘤疗效
取32只7周龄，C57BL/6J人源化PD-1转基因小鼠(来自上海南方模式生物科技股份有限公司)。在小鼠右侧腹皮下接种0.1mL结肠癌MC38细胞悬液(1×10 6cells/只)。待肿瘤生长至约100mm 3左右时，按照肿瘤体积大小随机分组，分为Vehicle(PBS)组，Hib-PDV(1.25mg/kg)组，Hib-PDV(2.5mg/kg)组与Hib-PDV(5mg/kg)组，共4组，每组8只荷瘤鼠，腹腔给药，每周给药1次，共给药3次，给药期间每周测量2次测量肿瘤长径、短径及体重。给药后第21天时计算肿瘤抑制率(TGI RTV)进行药效评价，如表8所示。
表8 Hib-PDV对小鼠结肠癌肿瘤抑制活性研究给药方案
组别 给药组 给药剂量(mg/kg) 给药途径 给药频次 给药体积
1 Vehicle -- i.p. QW×3 10mL/kg
2 Hib-PDV 5 i.p. QW×3 10mL/kg
3 Hib-PDV 2.5 i.p. QW×3 10mL/kg
4 Hib-PDV 1.25 i.p. QW×3 10mL/kg
计算公式：
肿瘤体积：TV＝D 1×D 2 2/2，其中D1、D2分别表示瘤长径、瘤短径；
相对肿瘤体积：RTV＝T VT/T V1，其中T V1为给药前肿瘤体积，T VT为每次测量时肿瘤体积；
相对肿瘤抑制率：TGI RTV(％)＝(1-T RTV/C RTV)×100％，T RTV表示给药组或阳性对照组RTV，C RTV表示阴性对照组RTV。
结果及结论：Hib-PDV对小鼠结肠癌模型肿瘤抑制作用分别如表9和图2所示，其中表9为Hib-PDV对小鼠结肠癌模型肿瘤体积以及肿瘤抑制率，图2为给药后的肿瘤生长曲线图。实验结果表明，Hib-PDV对小鼠结肠癌模型肿瘤有显著的抑制作用。
表9 Hib-PDV对小鼠结肠癌模型肿瘤体积以及肿瘤抑制率(mean±SEM)
Figure PCTCN2021081659-appb-000044
Figure PCTCN2021081659-appb-000045
实施例5 Hib-PDC对转hPD-L1MC38细胞在C57BL/6J-hPD-1小鼠皮下移植瘤药效研究
取32只7-9周龄，C57BL/6J人源化PD-1转基因雌性小鼠(来自南方模式)，在小鼠右前肢腋下靠背部皮下植入0.1mL转hPD-L1MC38细胞悬液(1×10 6cells/只)。待肿瘤生长至约100mm 3左右时，按照肿瘤体积大小随机分组，分为Vehicle(PBS)组，Hib-PDC(1.25mg/kg)组，Hib-PDC(2.5mg/kg)组，Hib-PDC(5mg/kg)共4组，每组8只荷瘤鼠，腹腔给药，每周给药1次，共给药3次，给药期间每周测量2次肿瘤长径、短径及体重。给药后第21天时计算肿瘤抑制率(TGI RTV)进行药效评价，如表10所示。
表10 Hib-PDC对小鼠结肠癌肿瘤抑制活性研究给药方案
组别 给药组 给药剂量(mg/kg) 给药方式 给药体积 给药周期
1 Vehicle(PBS) -- i.p. 10ml/kg QW×3次
2 Hib-PDC 1.25 i.p. 10ml/kg QW×3次
3 Hib-PDC 2.5 i.p. 10ml/kg QW×3次
4 Hib-PDC 5 i.p. 10ml/kg QW×3次
计算公式：
肿瘤体积：TV＝D1×D2 2/2，其中D1、D2分别表示瘤长径、瘤短径；
相对肿瘤体积：RTV＝T VT/T V1，其中T V1为给药前肿瘤体积，T VT为每次测量时肿瘤体积；
相对肿瘤抑制率：TGI RTV(％)＝(1-T RTV/C RTV)×100％，T RTV表示给药组或阳性对照组RTV，C RTV表示阴性对照组RTV。
结果及结论：Hib-PDC对小鼠结肠癌肿瘤抑制作用分别如表11和图3所示，其中表11为Hib-PDC对小鼠结肠癌模型肿瘤体积以及肿瘤抑制 率，图3为给药后肿瘤生长曲线。试验结果表明，Hib-PDC对小鼠结直肠癌皮下移植瘤具有显著的抑瘤作用。
表11 Hib-PDC对小鼠结肠癌模型肿瘤体积以及肿瘤抑制率(mean±SEM)
Figure PCTCN2021081659-appb-000046
实施例6 Hib-PLT对同种异体淋巴细胞混合反应中细胞因子分泌的影响
采用ELISA法检测细胞上清中细胞因子IFN-γ浓度，评价Hib-PLT在活化CD4 +T细胞中的作用。通过DC细胞体外快速成熟试剂盒(cat:CT-004，三一造血)体外诱导供体一的单核淋巴细胞为成熟树突状细胞，48h后收集细胞，稀释成2×10 5个/mL；磁珠富集供体二的CD4 +T细胞(EasySep TM Human CD4 +T Cell Enrichment Kit,StemCell,cat:19052)并将其稀释成1×10 6个/mL；将成熟树突状细胞和CD4 +T细胞按照体积比为1:1的比例混合，混合细胞悬液以每孔100μL的体积加入96孔板；Hib-PLT、PD-L1对照抗体+TGF-β Trap(联合用药)、PD-L1对照抗体、TGF-β Trap、人IgG1(Biolegend)以终浓度0.01～100nM加入含有混合细胞悬液的96孔板；置于37℃、5％CO 2培养箱中培养120h，2000rpm离心5min,收集细胞上清150～200μL；采用ELISA试剂盒(Human IL-2 Quantikine ELISA Kit,R&D,cat:S2050；Human IFN-gamma Quantikine ELISA Kit,R&D,cat:SIF50C)检测同种异体淋巴混合反应上清液中的IFN-γ因子。单因素方差分析组间差异。
结果及结论：如图4所示，Hib-PLT在10～100nM水平浓度，促细胞分泌IFN-γ因子水平与PD-L1对照抗体和TGF-β trap联合用药组相比显著升高(p＜0.05)。结果说明在10～100nM浓度水平下，Hib-PLT在混合淋巴反应中促进CD4 +T细胞活化的作用优于联合用药组。
本发明已通过各具体实施例作了举例说明。但是，本领域普通技术人员能够理解，本发明并不限于各具体实施方式，普通技术人员在本发明的 范文内可以作出各种改动或变型，并且在本说明书中各处提及的各个技术特征可以相互组合，而仍不背离本发明的精神和范围。这样的改动和变型均在本发明的范围之内。

Claims (45)

  1. 一种双特异性融合蛋白，所述融合蛋白的结构是在全长的免疫球蛋白G(IgG)的铰链区通过可选的肽接头插入第二结合结构域，所述IgG的Fab区为第一结合结构域；插入第二结合结构域后的所述IgG的重链为所述融合蛋白的重链，所述IgG的轻链为所述融合蛋白的轻链；所述第二结合结构域选自人受体、配体或所述受体、配体的片段和片段组合；其中，所述受体、配体为在天然信号通路中会形成二聚化或多聚化结构以活化或抑制信号通路的受体或配体，且所述第二结合结构域与所述第一结合结构域靶向不同的靶点。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述第一结合结构域靶向结合免疫检查点分子或肿瘤抗原。
  3. 根据权利要求2所述的融合蛋白，其特征在于，所述免疫检查点分子包括PD-1、PD-L1、CTLA-4、LAG-3、FGL1、TIM-3、Galectin-9、TIGIT、CD155、CD47；所述肿瘤抗原包括Claudin 18.2、HER-2、Mesothelin、BCMA、SSTR2、GPRC5D、PSMA、FCRH5、CD33、CD123、CD20、A33、CEA、CD28、DLL3、EGFR、VEGFR、VEGFR2、VEGF-A、Nectin-4、FGFR、C-met、RANKL、PDGF、PDGFR、PDGFRα、DLL4、Ang-1、Ang-2。
  4. 根据权利要求3所述的融合蛋白，其特征在于，所述第二结合结构域靶向结合人TGF-β、CTLA-4、VEGF、LAG3、CD27、4-1BB、OX40、CD47、FGL1、TLT-2、CD28、HGF、CSF1、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL12、GITR、EGF、ICOSL。
  5. 根据权利要求4所述的融合蛋白，其特征在于，所述受体或配体为人TGF-β受体(TGF-βR)、CD80、CD86、VEGFR、VEGF-trap、FGL1、CD70、4-1BBL、OX40L、SIRPα、B7-H3、C-met、CSF1R、CXCR2、CXCR3、CXCR4、GITRL、EGFR、ICOS。
  6. 根据权利要求4所述的融合蛋白，其特征在于，所述第一结合结构域和所述第二结合结构域选自以下组合：
    (1)所述第一结合结构域靶向结合PD-L1，所述第二结合结构域靶向结合人TGF-β、VEGF、FGL1、CD47、CD155、HGF、CSF1、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL12、EGF或ICOSL；或
    (2)所述第一结合结构域靶向结合PD-1，所述第二结合结构域靶向结合人CTLA-4、VEGF、HGF、EGF、CD28、LAG3、CD27、4-1BB或OX40； 或
    (3)所述第一结合结构域靶向结合CTLA-4，所述第二结合结构域靶向结合人CD28、VEGF、HGF、EGF、LAG3、CD27、4-1BB或OX40；或
    (4)所述第一结合结构域靶向结合LAG-3，所述第二结合结构域靶向结合人CD28、VEGF、HGF、EGF、CTLA-4、CD27、4-1BB或OX40；或
    (5)所述第一结合结构域靶向结合FGL1，所述第二结合结构域靶向结合人TGF-β、VEGF、CD47、CD155、HGF、CSF1、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL12、EGF或ICOSL；或
    (6)所述第一结合结构域靶向结合TIM-3，所述第二结合结构域靶向结合人VEGF、HGF、EGF、LAG3、CD27、4-1BB或OX40；或
    (7)所述第一结合结构域靶向结合Galectin-9，所述第二结合结构域靶向结合人TGF-β、VEGF、CD47、CD155、HGF、CSF1、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL12、EGF或ICOSL；或
    (8)所述第一结合结构域靶向结合TIGIT，所述第二结合结构域靶向结合人TGF-β、HGF、EGF或VEGF；或
    (9)所述第一结合结构域靶向结合CD155，所述第二结合结构域靶向结合人TGF-β、VEGF、HGF、EGF、CD47或CD155；或
    (10)所述第一结合结构域靶向结合Claudin 18.2、HER-2、mesothelin、BCMA、SSTR2、GPRC5D、PSMA、FCRH5、CD33、CD123、CD20、A33、CEA、CD28、DLL3、EGFR、VEGFR、VEGFR2、VEGF-A、Nectin-4、FGFR、C-met、RANKL、PDGF、PDGFR、PDGFRα、DLL-4、Ang-1、Ang-2，所述第二结合结构域靶向结合人CTLA-4，TGF-β、VEGF、FGL1、LAG3、4-1BB、OX40、CD27、CD28、CD47、CD155、HGF、CSF1、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL12、EGF或ICOSL。
  7. 根据权利要求6所述的融合蛋白，其特征在于，所述第一结合结构域和所述受体、配体选自以下组合：
    (1)所述第一结合结构域靶向结合PD-L1，所述受体、配体选自TGF-βRII、VEGFR、LAG-3、SIRPα、TIGIT、C-MET、CSF1R、CXCR2、CXCR3、CXCR4、EGFR或ICOS；或
    (2)所述第一结合结构域靶向结合PD-1，所述受体、配体选自人CD80、CD86、VEGFR、c-MET、EGFR、FGL1、CD70、4-1BBL或OX40L；或
    (3)所述第一结合结构域靶向结合CTLA-4，所述受体、配体选自人CD80、CD86、VEGFR、c-MET、EGFR、FGL1、CD70、4-1BBL或OX40L；或
    (4)所述第一结合结构域靶向结合LAG-3，所述受体、配体选自人VEGFR、c-MET、EGFR、CD80、CD86、CD70、4-1BBL或OX40L；或
    (5)所述第一结合结构域靶向结合FGL1，所述受体、配体选自人TGF-βRII、VEGFR、SIRPα、TIGIT、c-MET、CSF1R、CXCR2、CXCR3、CXCR4、EGFR或ICOS；或
    (6)所述第一结合结构域靶向结合TIM-3，所述受体、配体选自人VEGFR、c-MET、EGFR、FGL1、CD70、4-1BBL或OX40L；或
    (7)所述第一结合结构域靶向结合Galectin-9，所述受体、配体选自人TGF-βRII、VEGFR、SIRPα、TIGIT、c-MET、CSF1R、CXCR2、CXCR3、CXCR4、EGFR或ICOS；或
    (8)所述第一结合结构域靶向结合TIGIT，所述受体、配体选自人TGF-βRII、c-MET、EGFR或VEGFR；或
    (9)所述第一结合结构域靶向结合CD155，所述受体、配体选自人TGF-βRII、VEGFR、c-MET、EGFR、SIRPα或TIGIT；或
    (10)所述第一结合结构域靶向结合Claudin 18.2、HER-2、mesothelin、BCMA、SSTR2、GPRC5D、PSMA、FCRH5、CD33、CD123、CD20、A33、CEA、CD28、DLL3、EGFR、VEGFR、VEGFR2、Nectin-4、FGFR、c-MET、RANKL、PDGF、PDGFR、PDGFRα、DLL4、Ang-1或Ang-2，所述受体、配体选自人CTLA-4，CD80，CD86，TGF-βRII、VEGFR、FGL1、LAG3、4-1BB、OX40、CD27、CD28、CD70、c-MET、SIRPα、TIGIT、CSF1R、CXCR2、CXCR3、CXCR4、EGFR或ICOS。
  8. 根据权利要求1-7中任一项所述的融合蛋白，其特征在于，所述受体、配体的片段包括受体或配体的胞外区片段、结合结构域片段。
  9. 根据权利要求7所述的融合蛋白，其特征在于，所述第一结合结构域靶向结合PD-L1；所述第二结合结构域为人TGF-βRII片段；或所述第一结合结构域靶向结合PD-1，所述第二结合结构域选自人CD80 ECD、CD80 IgV区、人CD80IgVIgC、VEGFR1 ECD、VEGFR2 ECD或VEGFR1的第2个细胞外 区域和VEGFR2的第3个细胞外区域组合；或所述第一结合结构域靶向结合Claudin 18.2或HER-2、EGFR，所述第二结合结构域选自人CD80 ECD、CD80 IgV区、人CD80IgVIgC、VEGFR1 ECD、VEGFR2 ECD或VEGFR1的第2个细胞外区域和VEGFR2的第3个细胞外区域组合。
  10. 根据权利要求9所述的融合蛋白，其特征在于，所述第二结合结构域选自：
    (1)包含SEQ ID NO:31、131或132所示序列的人TGF-βRII或包含与SEQ ID NO:31、131或132所示序列相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列或与SEQ ID NO:31、131或132所示序列相比具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸替换或缺失后的氨基酸序列；或
    (2)包含SEQ ID NO:32或133所示序列的CD80 ECD或包含与SEQ ID NO:32或133所示序列相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列或与SEQ ID NO:32或133所示序列相比具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸替换或缺失后的氨基酸序列；或
    (3)包含SEQ ID NO:33所示序列的VEGFR1的第2个细胞外区域和VEGFR2的第3个细胞外区域片段组合或包含与SEQ ID NO:33所示序列相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列或与SEQ ID NO:33所示序列相比具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸替换或缺失后的氨基酸序列。
  11. 根据权利要求9或10所述的融合蛋白，其特征在于，所述第一结合结构域靶向结合PD-L1，所述IgG的Fab中重链可变区的CDR和/或所述轻链可变区的CDR与如下序列限定的抗体具有相同的CDR序列或在如下序列限定的抗体的CDR上进行1-2个氨基酸替换，所述序列限定的抗体为：
    (1)重链可变区氨基酸序列为SEQ ID NO:66所示；和/或
    (2)轻链可变区氨基酸序列为SEQ ID NO:67所示。
  12. 根据权利要求11所述的融合蛋白，其特征在于，所述IgG的Fab中重链可变区的CDR和/或所述轻链可变区的CDR如下所示：
    (1)重链可变区CDR1氨基酸序列选自SEQ ID NO:1-5及对SEQ ID NO:1-5进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列选自SEQ  ID NO:6-10及对SEQ ID NO:6-10进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列选自SEQ ID NO:11-15及对SEQ ID NO:11-15进行1或2个氨基酸替换后的氨基酸序列；和/或
    (2)轻链可变区CDR1氨基酸序列选自SEQ ID NO:16-20及对SEQ ID NO:16-20进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列选自SEQ ID NO:21-25及对SEQ ID NO:21-25进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列选自SEQ ID NO:26-30及对SEQ ID NO:26-30进行1或2个氨基酸替换后的氨基酸序列。
  13. 根据权利要求12所述的融合蛋白，其特征在于：
    (1)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：1，6，11或对SEQ ID NO:1，6，11进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：16，21，26或对SEQ ID NO：16，21，26进行1或2个氨基酸替换后的氨基酸序列；或
    (2)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：2，7，12或对SEQ ID NO:2，7，12进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：17，22，27或对SEQ ID NO：17，22，27进行1或2个氨基酸替换后的氨基酸序列；或
    (3)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：3，8，13或对SEQ ID NO:3，8，13进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：18，23，28或对SEQ ID NO：18，23，28进行1或2个氨基酸替换后的氨基酸序列；或
    (4)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：4，9，14或对SEQ ID NO:4，9，14进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：19，24，29或对SEQ ID NO：19，24，29进行1或2个氨基酸替换后的氨基酸序列；或
    (5)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：5，10，15或对SEQ ID NO:5，10，15进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：20，25，30或对SEQ ID NO：20，25，30进行1或2个氨基酸替换后的氨基酸序列。
  14. 根据权利要求13所述的融合蛋白，其特征在于，重链可变区CDR 1-3氨基酸序列为SEQ ID NO：3，8，13；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：18，23，28。
  15. 根据权利要求14所述的融合蛋白，其特征在于：
    (1)重链可变区的序列如SEQ ID NO:66所示，或与SEQ ID NO:66具有相同的CDR 1-3且与SEQ ID NO:66相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列；和/或
    (2)轻链可变区的序列如SEQ ID NO:67所示，或与SEQ ID NO:67具有相同的CDR 1-3且与SEQ ID NO:67相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列。
  16. 根据权利要求15所述的融合蛋白，其特征在于：
    (1)所述融合蛋白的重链氨基酸序列如SEQ ID NO:72所示或与SEQ ID NO:72相比同一性大于90％、95％、96％、97％、98％、99％的序列；
    (2)所述融合蛋白的轻链氨基酸序列分别如SEQ ID NO:73所示或与SEQ ID NO:73相比同一性大于90％、95％、96％、97％、98％、99％的序列。
  17. 根据权利要求16所述的融合蛋白，其特征在于：
    (1)所述融合蛋白的重链氨基酸序列与SEQ ID NO:72相比具有1-15个氨基酸位点突变或具有替换的肽接头；
    (2)所述融合蛋白的轻链氨基酸序列与SEQ ID NO:73相比具有1-10个氨基酸位点突变。
  18. 根据权利要求9所述的融合蛋白，其特征在于，所述第一结合结构域靶向结合PD-1，所述IgG的Fab中重链可变区的CDR和/或所述轻链可变区的CDR与如下序列限定的抗体具有相同的CDR序列或在如下序列限定的抗体的CDR上进行1-2个氨基酸替换，所述序列限定的抗体为：
    (1)重链可变区氨基酸序列为SEQ ID NO:64所示；和/或
    (2)轻链可变区氨基酸序列为SEQ ID NO:65所示。
  19. 根据权利要求18所述的融合蛋白，其特征在于，所述IgG的Fab中重链可变区的CDR和/或所述轻链可变区的CDR如下所示：
    (1)重链可变区CDR1氨基酸序列选自SEQ ID NO:34-38及对SEQ ID NO:34-38进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列选自SEQ ID NO:39-43及对SEQ ID NO:39-43进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列选自SEQ ID NO:44-48及对SEQ ID NO:44-48进行1或2个氨基酸替换后的氨基酸序列；和/或
    (2)轻链可变区CDR1氨基酸序列选自SEQ ID NO:49-53及对SEQ ID NO:49-53进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列选自SEQ ID NO:54-58及对SEQ ID NO:54-58进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列选自SEQ ID NO:59-63及对SEQ ID NO:59-63进行1或2个氨基酸替换后的氨基酸序列。
  20. 根据权利要求19所述的融合蛋白，其特征在于:
    (1)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：34，39，44或对SEQ ID NO:34，39，44进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：49，54，59或对SEQ ID NO：49，54，59进行1或2个氨基酸替换后的氨基酸序列；或
    (2)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：35，40，45或对SEQ ID NO:35，40，45进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：50，55，60或对SEQ ID NO：50，55，60进行1或2个氨基酸替换后的氨基酸序列；或
    (3)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：36，41，46或对SEQ ID NO:36，41，46进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：51，56，61或对SEQ ID NO：51，56，61进行1或2个氨基酸替换后的氨基酸序列；或
    (4)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：37，42，47或对SEQ ID NO:37，42，47进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：52，57，62或对SEQ ID NO：52，57，62进行1或2个氨基酸替换后的氨基酸序列；或
    (5)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：38，43，48或对SEQ ID NO:38，43，48进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：53，58，63或对SEQ ID NO：53，58，63进行1或2个氨基酸替换后的氨基酸序列。
  21. 根据权利要求20所述的融合蛋白，其特征在于，重链可变区CDR 1-3氨基酸序列为SEQ ID NO：36，41，46；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：51，56，61。
  22. 根据权利要求21所述的融合蛋白，其特征在于：
    (1)重链可变区的序列如SEQ ID NO:64所示，或与SEQ ID NO:64具有 相同的CDR 1-3且与SEQ ID NO:64相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列；和/或
    (2)轻链可变区的序列如SEQ ID NO:65所示，或与SEQ ID NO:65具有相同的CDR 1-3且与SEQ ID NO:65相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列。
  23. 根据权利要求22所述的融合蛋白，其特征在于：
    (1)所述融合蛋白的重链氨基酸序列如SEQ ID NO:68所示或与SEQ ID NO:68相比同一性大于90％、95％、96％、97％、98％、99％的序列；
    (2)所述融合蛋白的轻链氨基酸序列分别如SEQ ID NO:69所示或与SEQ ID NO:69相比同一性大于90％、95％、96％、97％、98％、99％的序列。
  24. 根据权利要求23所述的融合蛋白，其特征在于：
    (1)所述融合蛋白的重链氨基酸序列与SEQ ID NO:68相比具有1-15个氨基酸位点突变或具有替换的肽接头；
    (2)所述融合蛋白的轻链氨基酸序列与SEQ ID NO:69相比具有1-10个氨基酸位点突变。
  25. 根据权利要求22所述的融合蛋白，其特征在于：
    (1)所述融合蛋白的重链氨基酸序列如SEQ ID NO:70所示或与SEQ ID NO:70相比同一性大于90％、95％、96％、97％、98％、99％的序列；
    (2)所述融合蛋白的轻链氨基酸序列分别如SEQ ID NO:71所示或与SEQ ID NO:71相比同一性大于90％、95％、96％、97％、98％、99％的序列。
  26. 根据权利要求25所述的融合蛋白，其特征在于：
    (1)所述融合蛋白的重链氨基酸序列与SEQ ID NO:70相比具有1-15个氨基酸位点突变或具有替换的肽接头；
    (2)所述融合蛋白的轻链氨基酸序列与SEQ ID NO:71相比具有1-10个氨基酸位点突变。
  27. 根据权利要求1-10中任一项所述的融合蛋白，其特征在于：所述IgG为Atezolizumab、Avelumab、Durvalumab、Nivolumab、Pembrolizumab、Cemiplimab、Ipilimumab。
  28. 根据权利要求1-27中任一项所述的融合蛋白，其特征在于，所述第二结合结构域的C端或/和N端带有肽接头，所述肽接头由2-30个氨基酸组 成。
  29. 根据权利要求28所述的融合蛋白，其特征在于，所述肽接头为：
    (1)(GGGGS)n；或
    (2)AKTTPKLEEGEFSEAR(SEQ ID NO：80)；或
    (3)AKTTPKLEEGEFSEARV(SEQ ID NO：81)；或
    (4)AKTTPKLGG(SEQ ID NO：82)；或
    (5)SAKTTPKLGG(SEQ ID NO：83)；或
    (6)SAKTTP(SEQ ID NO：84)；或
    (7)RADAAP(SEQ ID NO：85)；或
    (8)RADAAPTVS(SEQ ID NO：86)；或
    (9)RADAAAAGGPGS(SEQ ID NO：87)；或
    (10)RADAAAA(SEQ ID NO：88)；或
    (11)SAKTTPKLEEGEFSEARV(SEQ ID NO：89)；或
    (12)ADAAP(SEQ ID NO：90)；或
    (13)DAAPTVSIFPP(SEQ ID NO：91)；或
    (14)TVAAP(SEQ ID NO：92)；或
    (15)TVAAPSVFIFPP(SEQ ID NO：93)；或
    (16)QPKAAP(SEQ ID NO：94)；或
    (17)QPKAAPSVTLFPP(SEQ ID NO：95)；或
    (18)AKTTPP(SEQ ID NO：96)；或
    (19)AKTTPPSVTPLAP(SEQ ID NO：97)；或
    (20)AKTTAP(SEQ ID NO：98)；或
    (21)AKTTAPSVYPLAP(SEQ ID NO：99)；或
    (22)ASTKGP(SEQ ID NO：100)；或
    (23)ASTKGPSVFPLAP(SEQ ID NO：101)；或
    (24)GENKVEYAPALMALS(SEQ ID NO：102)；或
    (25)GPAKELTPLKEAKVS(SEQ ID NO：103)；或
    (26)GHEAAAVMQVQYPAS(SEQ ID NO：104)；或
    (27)GGGGSGGGGSGGGGSA(SEQ ID NO：105)，
    其中所述n等于1、2、3或4。
  30. 根据权利要求1-29中任一项所述的融合蛋白，其特征在于，插入的所述第二结合结构域的大小不超过300个氨基酸。
  31. 根据权利要求1-30中任一项所述的融合蛋白，其特征在于，所述第二结合结构域的插入位点位于铰链区的中前部，插入位点不影响免疫球蛋白的二硫键的形成。
  32. 根据权利要求31所述的融合蛋白，其特征在于，所述铰链区的中前部是指231A之前的部分。
  33. 根据权利要求1-32中任一项所述的融合蛋白，其特征在于，所述插入位点前后铰链区的部分氨基酸被替换或缺失。
  34. 根据权利要求33所述的融合蛋白，其特征在于，所述铰链区含有D221G和/或C220V突变。
  35. 根据权利要求1-34中任一项所述的融合蛋白，其特征在于，所述IgG选自哺乳动物IgG、人源化IgG和人IgG，所述哺乳动物包括小鼠、大鼠、兔；优选的，所述IgG为IgG1、IgG2、IgG3、IgG4。
  36. 根据权利要求1-35中任一项所述的融合蛋白，其特征在于，该融合蛋白的Fc区是无糖基化的或脱糖基化的或具有降低的岩藻糖基化或是无岩藻糖基化的。
  37. 分离的多核苷酸，其编码权利要求1-36中任一项所述的融合蛋白。
  38. 一种核酸构建体，其包含权利要求37所述的多核苷酸，优选所述核酸构建体为载体。
  39. 一种宿主细胞，其包含权利要求37所述的多核苷酸或者权利要求38所述的核酸构建体或所述载体，优选的所述细胞是原核细胞、真核细胞、酵母细胞、哺乳动物细胞、大肠杆菌细胞或CHO细胞、NS0细胞、Sp2/0细胞、BHK细胞。
  40. 药物组合物，包含权利要求1-36中任一项所述的融合蛋白以及药学上可接受的载体。
  41. 用于治疗肿瘤或癌症的方法，其包括对需要治疗或减轻的受试者施用 权利要求1-36中任一项的融合蛋白或权利要求40的药物组合物的步骤。
  42. 权利要求1-36中任一项所述的融合蛋白、权利要求37所述的多核苷酸、权利要求38所述的核酸构建体或者所述载体、或权利要求40所述的药物组合物在制备用于治疗或预防肿瘤或癌症的药物中的用途。
  43. 根据权利要求42所述的用途，其特征在于，所述的肿瘤或癌症包括实体瘤或非实体瘤。
  44. 诊断试剂盒，其包含权利要求1-36中任一项所述的融合蛋白。
  45. 权利要求1至36中任一项的融合蛋白的生产方法，该方法包括：在允许权利要求38所述的核酸构建体表达的条件下培养权利要求39所述的宿主细胞，并从培养物中回收产生的融合蛋白。
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CA3137211A1 (en) 2021-09-23
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EP4056596A1 (en) 2022-09-14
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