WO2022262832A1 - 双特异性融合蛋白 - Google Patents

双特异性融合蛋白 Download PDF

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WO2022262832A1
WO2022262832A1 PCT/CN2022/099322 CN2022099322W WO2022262832A1 WO 2022262832 A1 WO2022262832 A1 WO 2022262832A1 CN 2022099322 W CN2022099322 W CN 2022099322W WO 2022262832 A1 WO2022262832 A1 WO 2022262832A1
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amino acid
fusion protein
acid sequence
seq
bispecific fusion
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PCT/CN2022/099322
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English (en)
French (fr)
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吕明
丁晓然
缪仕伟
谈彬
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杭州尚健生物技术有限公司
尚健单抗（北京）生物技术有限公司
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Application filed by 杭州尚健生物技术有限公司, 尚健单抗（北京）生物技术有限公司 filed Critical 杭州尚健生物技术有限公司
Priority to JP2023577295A priority Critical patent/JP2024523029A/ja
Priority to CA3223047A priority patent/CA3223047A1/en
Priority to EP22824305.1A priority patent/EP4357366A1/en
Priority to CN202280004996.6A priority patent/CN115708412A/zh
Publication of WO2022262832A1 publication Critical patent/WO2022262832A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • This application relates to the field of biomedicine, in particular to a bispecific fusion protein.
  • PD-L1 is overexpressed in a variety of malignancies and is often associated with poor prognosis.
  • VEGF is a potent and specific mitogen of vascular endothelial cells, which can promote all aspects of the angiogenesis process.
  • Each VEGF receptor (VEGFR) acts differently.
  • VEGFR-2 plays a role in regulating endothelial growth, differentiation, and permeability; while VEGFR-1 is related to regulating the movement and aggregation of endothelial cells, and inhibits signal transduction through VEGFR-2.
  • VEGF combines with VEGFR to dimerize and autophosphorylate VEGFR, transmit signals through multiple intracellular pathways, and finally play a role.
  • the application provides a bispecific fusion protein and its corresponding polynucleotide, carrier, cell, preparation method, pharmaceutical composition and application.
  • the bispecific fusion protein described in this application has at least one of the following properties: 1) specifically binds to PD-L1; 2) blocks the combination of PD-1 and PD-L1; 3) specifically binds to VEGF; 4) blocks VEGF binds to VEGFR; 5) inhibits tumor growth (in vivo); 6) activates immune cells (eg, T cells); 7) promotes immune cells (eg, T cells) to secrete cytokines (eg, interferons and/or cytokines); and /or, 8) Inhibit tumor angiogenesis and promote normalization of blood vessels.
  • the present application provides a bispecific fusion protein comprising a first binding domain and a second binding domain, wherein: the first binding domain comprises an antibody specifically binding to PD-L1, wherein the antibody comprises Two antibody light chains and two antibody heavy chains, the antibody light chains and the antibody heavy chains are linked by disulfide bonds; the second binding domain comprises an Ig-like domain of VEGFR1 and an Ig-like structure of VEGFR2 domain; wherein the N-terminus of the Ig-like domain of VEGFR1 or the N-terminus of the Ig-like domain of VEGFR2 is directly or indirectly linked to the C-terminus of the antibody heavy chain, respectively.
  • the first binding domain specifically binds human PD-L1.
  • the second binding domain specifically binds the human VEGF family.
  • the second binding domain specifically binds a protein selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PLGF.
  • the Ig-like domain of VEGFR1 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the Ig-like domain of VEGFR2 comprises the amino acid sequence shown in SEQ ID NO:2.
  • the Ig-like domain of VEGFR1 and the Ig-like domain of VEGFR2 are directly linked.
  • the second binding domain comprises the amino acid sequence shown in SEQ ID NO: 28.
  • the indirect linkage is through a linker.
  • the linker comprises the amino acid sequence shown in SEQ ID NO:3.
  • the light chain of the antibody comprises a light chain variable region, wherein the light chain variable region comprises LCDR1-3, wherein LCDR1 comprises the amino acids shown in any one of SEQ ID NO: 4, 10 sequence.
  • the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:5,11.
  • the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:6,12.
  • said LCDR1-3 comprises an amino acid sequence selected from the group consisting of:
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:11
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.
  • the antibody light chain comprises a light chain variable region, wherein the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 16-17.
  • the antibody light chain comprises a light chain constant region, wherein the light chain constant region is derived from a light chain constant region selected from the group consisting of: Ig ⁇ and Ig ⁇ .
  • the antibody heavy chain comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1-3, wherein the HCDR1 comprises any one of SEQ ID NO: 7, 13 amino acid sequence.
  • the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:8,14.
  • the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:9,15.
  • the HCDR1-3 comprises an amino acid sequence selected from the group consisting of:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:7
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:13
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:14
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:15.
  • the antibody heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NOs: 18-19.
  • the antibody heavy chain comprises an Fc region.
  • the Fc region is derived from an Fc selected from the following histones: IgGl and IgG4.
  • the Fc region comprises an amino acid mutation at an amino acid position selected from the group consisting of: N298, D357, and L359.
  • the Fc region comprises an amino acid mutation selected from the group consisting of N298A, D357E, and L359M.
  • the Fc region comprises the amino acid sequence shown in any one of SEQ ID NO: 20-22.
  • the bispecific fusion protein is a multimer composed of two copies of the first polypeptide chain and the second polypeptide chain, wherein the first polypeptide chain comprises the antibody light chain, wherein the second polypeptide chain sequentially comprises the antibody heavy chain, the linker and the second binding domain from the N-terminus.
  • the first polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO: 23-24.
  • the second polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO: 25-27.
  • the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 23; the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 25; or, wherein the The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 24; the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 26; or, wherein the first polypeptide chain comprises SEQ ID NO : the amino acid sequence shown in 23; the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 27.
  • the present application provides a polynucleotide encoding the bispecific fusion protein described in the present application.
  • the present application provides a vector comprising the polynucleotide described in the present application.
  • the present application provides a cell comprising the vector described in the present application.
  • the present application provides a method for preparing the bispecific fusion protein described in the present application, which includes culturing the cells described in the present application under conditions suitable for expressing the bispecific fusion protein described in the present application.
  • the present application provides a pharmaceutical composition, which comprises the bispecific fusion protein described in the present application, and optionally a pharmaceutically acceptable carrier.
  • the present application provides a drug molecule comprising the bispecific fusion protein described in the present application.
  • the present application provides a bispecific fusion protein, the polynucleotide, the carrier, the cell, the pharmaceutical composition and/or the pharmaceutical molecule described in the present application for the preparation and treatment of diseases.
  • the disease comprises a tumor.
  • the disease includes solid tumors and non-solid tumors.
  • the disease comprises a PD-L1 positive tumor.
  • the disease comprises lung cancer, colorectal cancer, cervical cancer, liver cancer, gastric cancer, and/or kidney cancer.
  • the present application provides a method for inhibiting angiogenesis, which comprises administering an effective amount of the bispecific fusion protein described in the present application, the polynucleotide, the carrier, the cell, the A pharmaceutical composition and/or said drug molecule.
  • the present application provides a method for inhibiting the activity of VEGF receptor ligands, which comprises administering an effective amount of the bispecific fusion protein described in the present application, the polynucleotide, the carrier, the cell , said pharmaceutical composition and/or said drug molecule.
  • the present application provides a method for inhibiting PD-L1 activity, which comprises administering an effective amount of the bispecific fusion protein described in the present application, the polynucleotide, the carrier, the cell, the The pharmaceutical composition and/or the pharmaceutical molecule described above.
  • Figure 1 shows the structure of the bispecific fusion protein described in this application.
  • Figure 2 shows that the bispecific fusion protein described in this application specifically binds to human PD-L1.
  • FIG. 3 shows that the bispecific fusion protein described in this application specifically binds to VEGF165.
  • Figure 4 shows that the bispecific fusion protein described in this application specifically binds to human PD-L1 and VEGF165 at the same time.
  • Figure 5 shows that the bispecific fusion protein described in this application blocks the interaction between PD-1 and PD-L1.
  • Figure 6 shows that the bispecific fusion protein described in this application blocks the interaction between VEGF and VEGFR.
  • Figure 7 shows that the bispecific fusion protein described in this application blocks the interaction between VEGF165 and VEGFR2.
  • Figure 8 shows that the bispecific fusion protein described in this application blocks the interaction between VEGF121 and VEGFR2.
  • Figure 9 shows that the bispecific fusion protein described in the present application blocks the interaction between VEGFR2 and VEGFR2.
  • Figure 10 shows that the bispecific fusion protein described in this application inhibits the proliferation of HUVEC cells.
  • Figure 11 shows that the bispecific fusion protein described in this application activates lymphocytes to secrete IFN- ⁇ .
  • Figure 12 shows that the bispecific fusion protein described in this application activates lymphocytes to secrete IL-2.
  • Figure 13 shows the detection of ADCC activity of the bispecific fusion protein described in this application.
  • Figure 14 shows that the bispecific fusion protein described in this application inhibits the growth of colon cancer in mice.
  • bispecific generally refers to the ability of a fusion protein as described herein to interact with two different ligands.
  • the bispecific fusion protein can specifically bind both PD-L1 and VEGF.
  • the term "specific binding” generally refers to a measurable and reproducible interaction, such as between a target and an antibody, which can be determined in the presence of a heterogeneous population of molecules, including biomolecules. The presence.
  • an antibody that specifically binds a target is one that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets.
  • the extent to which the antibody binds an unrelated target is less than about 10% of the binding of the antibody to the target, eg, as measured by radioimmunoassay (RIA).
  • the bispecific fusion protein can bind to PD-L1 and VEGF with a dissociation constant (KD) of ⁇ 1 ⁇ 10 ⁇ 7 M or lower.
  • the PD-L1 antibody can bind to PD-L1 with a dissociation constant (KD) of ⁇ 1 ⁇ 10 ⁇ 7 M or lower.
  • KD dissociation constant
  • an antibody specifically binds an epitope on a protein that is conserved among proteins of different species.
  • specific binding can include, but does not require exclusive binding.
  • first binding domain generally refers to a binding domain that can specifically bind PD-L1.
  • the first binding domain can comprise an antibody that specifically binds PD-L1.
  • the term "second binding domain” generally refers to a binding domain that can specifically bind VEGF.
  • the second binding domain may comprise an Ig-like domain of VEGFR1 and an Ig-like domain of VEGFR2.
  • the term "PD-L1" generally refers to the programmed death-ligand 1 protein, its functional variants and/or its functional fragments. PD-L1 is also known as cluster of differentiation 274 (CD274)) or B7 homolog 1 (B7-H1). The PD-L1 may be a protein encoded by the CD274 gene. PD-L1 binds its receptors, such as programmed death 1 (PD-1), which can be expressed in activated T cells, B cells and macrophages (see Ishida et al., 1992 EMBO J, 11 :3887-3395; Okazaki et al., Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.
  • PD-1 programmed death 1
  • the complexation of PD-L1 and PD-1 can play an immunosuppressive role by inhibiting T cell proliferation and producing cytokines IL-2 and IFN- ⁇ (see Freeman et al., Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation, J.Exp.Med.2000,192:1027-1034; Carter et al.,PD-1:PD-L inhibitory pathway affects both CD4(+)and CD8(+)T cells and is overcome by IL-2.Eur.J.Immunol.2002,32:634–643).
  • PD-L1 can encompass any native PD-L1 of any vertebrate origin, which can include mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats) ).
  • the term encompasses "full length", unprocessed PD-L1 as well as any form of PD-L1 that results from processing in the cell.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • the term may also encompass naturally occurring variants of PD-L1, such as splice variants or allelic variants.
  • the basic structure of PD-L1 can include four structural domains: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain.
  • PD-L1 sequences are known in the art. Information on the human PD-L1 gene, including the genomic DNA sequence, can be found, for example, under NCBI Gene ID No. 29126. For another example, information on the mouse PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 60533. For another example, information on the cynomolgus monkey PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 102145573.
  • amino acid sequence of an exemplary full-length human PD-L1 protein can be found under NCBI Accession No. NP_054862 or UniProt Accession No. Q9NZQ7.
  • Exemplary full-length mouse PD-L1 protein sequences can be found under NCBI accession number NP_068693 or Uniprot accession number Q9EP73.
  • An exemplary full-length cynomolgus monkey PD-L1 protein sequence can be found under NCBI Accession No. XP_005581836 or Uniprot Accession No. G7PSE7.
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 that can be combined with the J chain to form a multivalent A basic 4-chain unit for combinations.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site.
  • immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
  • the gamma and alpha classes are further divided into subclasses based on relatively minor differences in CH sequence and function, eg, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl and IgKl.
  • CDR generally refers to regions of antibody variable domains, the sequences of which are highly variable and/or form structurally defined loops.
  • antibodies typically comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • HCDR3 and LCDR3 display most of the diversity of the six CDRs, and HCDR3 in particular is thought to play a unique role in conferring the fine specificity of antibodies.
  • VEGF generally refers to vascular endothelial growth factor.
  • VEGF can be involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumors and intraocular disorders (see Ferrara, N. and Davis-Smyth, T., Endocr. Rev. 18, 1997, 4-25, etc.) .
  • VEGF may have important regulatory functions in neovascularization during embryonic angiogenesis and in angiogenesis during adulthood. VEGF can promote tumor growth.
  • VEGF is a highly conserved homodimeric glycoprotein.
  • VEGF can have six isoforms: VEGF-A, VEGF-B (including VEGF-B167 and VEGF-B186), VEGF-C, VEGF-D and VEGF-E.
  • the VEGF may be human VEGF.
  • VEGFR1 generally refers to vascular endothelial growth factor receptor 1.
  • VEGFR1 is a type of VEGFR.
  • VEGFR belongs to the receptor tyrosine kinase superfamily and is a membrane-embedded protein.
  • the extramembrane part of VEGFR has about 750 amino acid residues and consists of seven Ig-like domains similar in structure to immunoglobulins.
  • the second Ig domain in the extramembrane region of VEGFR1 is the ligand-binding region.
  • Different splice forms of VEGFRl can compete for binding to VEGF (eg, can bind VEGF-A, VEGF-B), thereby preventing VEGF from binding to VEGFR2.
  • VEGFR2 generally refers to vascular endothelial growth factor receptor 2.
  • the third Ig domain of VEGFR2 may contribute to the specificity of binding to ligand.
  • VEGFR2 can bind to VEGF-A and VEGF-E.
  • Ig-like domain generally refers to a structure in the extracellular region of VEGFR, which may be responsible for binding to VEGF.
  • the first Ig-like domain in the extracellular region of VEGFR2 is the necessary site for binding to VEGF
  • the second-third Ig-like domain is the main site for tight binding with VEGF
  • the receptor passes through the fourth Ig-like domain.
  • the active form forms a homodimer
  • the 5th-7th Ig-like domains are not closely related to VEGF binding.
  • the extracellular region of VEGFR1 has seven Ig-like domains that may be responsible for binding to VEGF and promoting angiogenesis.
  • PLGF placental growth factor
  • PGF placental growth factor
  • the PLGF may be a member of the VEGF subfamily.
  • the PLGF can be expressed in human umbilical vein endothelial cells (HUVE) and placenta.
  • HUVE human umbilical vein endothelial cells
  • the PLGF may play a role in the growth and differentiation of trophoblasts.
  • the PLGF may be associated with angiogenesis.
  • the term “directly connected” is opposite to the term “indirectly connected”, and the term “directly connected” generally refers to a direct connection.
  • the direct connection may be a case where substances are directly connected without a spacer.
  • the spacer may be a linker.
  • the linker can be a peptide linker.
  • the term “indirectly linked” generally refers to the situation where substances are not directly linked.
  • the indirect connection may be through a spacer.
  • the N-terminus of the Ig-like domain of VEGFR1 or the N-terminus of the Ig-like domain of VEGFR2 in the present application may be connected to the C-terminus of the heavy chain of the antibody respectively.
  • an Ig-like domain of VEGFR1 described in this application can be directly linked to an Ig-like domain of VEGFR2.
  • amino acid mutation generally refers to the replacement of at least one existing amino acid residue with another different amino acid residue.
  • the substituted amino acid residues may be "naturally occurring amino acid residues", for example, may be alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp) , cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile): leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine amino acid (Tyr) and valine (Val).
  • the amino acid residues to be replaced may also be amino acid residues that do not exist naturally, for example, they may be norleucine, ornithine, norvaline, homoserine, aib and other amino acid residue analogs.
  • the amino acid substitutions may be non-conservative substitutions.
  • the non-conservative substitution may include changing amino acid residues in the target protein or polypeptide in a non-conservative manner, such as changing amino acid residues with a certain side chain size or a certain characteristic (for example, hydrophilicity) to have different Amino acid residues with side chain size or different properties (eg, hydrophobicity).
  • the amino acid substitutions may also be conservative substitutions.
  • the conservative substitution may include changing amino acid residues in the target protein or polypeptide in a conservative manner, such as changing amino acid residues with a certain side chain size or a certain characteristic (for example, hydrophilicity) to have the same or similar Amino acid residues with side chain size or the same or similar properties (eg, still hydrophilic).
  • a certain side chain size or a certain characteristic for example, hydrophilicity
  • Such conservative substitutions generally do not substantially affect the structure or function of the resulting protein.
  • the term "polymer” generally refers to a molecule formed by combining monomers.
  • the multimer may be a multimeric protein comprising at least two structurally identical or structurally different components.
  • the multimer can be a molecule of two or more polypeptide chains that are covalently or non-covalently associated or bound by covalent or non-covalent interactions.
  • the multimer may be a dimer or a tetramer.
  • first polypeptide chain generally refers to the light chain of the PD-L1-targeting antibody in the bispecific fusion protein described in the present application.
  • second polypeptide chain generally refers to the heavy chain comprising the PD-L1 targeting antibody in the bispecific fusion protein described in this application, the linker described in this application and the polypeptide described in this application.
  • a polypeptide targeting the second binding domain of VEGF can be sequentially composed from the N-terminus of the heavy chain of the PD-L1-targeting antibody in the bispecific fusion protein described in the application, the linker described in the application, and the VEGF-targeting antibody described in the application. of the second binding domain.
  • nucleic acid molecule generally refers to nucleotides of any length (for example, nucleotides that may be in isolated form), deoxyribonucleotides or ribonucleotides, or isolated from their natural environment or Synthetic analogues.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell.
  • vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 Phages and animal viruses, etc.
  • Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as SV40
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • Vectors may also
  • the term "cell” generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes the nucleic acid molecules described herein or the nucleic acid molecules described herein.
  • Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • Cells may include cells transfected in vitro with the vectors described herein.
  • the cells can be bacterial cells (e.g., E.
  • the cells are mammalian cells.
  • the cells are mammalian cells.
  • the cells are mammalian cells.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a patient, for example, may be administered to a human patient.
  • the pharmaceutical composition described in the present application may comprise the bispecific fusion protein described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and/or the cell described in the present application, and Optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations.
  • the acceptable ingredients of the pharmaceutical compositions may be nontoxic to recipients at the dosages and concentrations employed.
  • the form of the pharmaceutical composition of the present application may not be limited to liquid, frozen and lyophilized compositions.
  • pharmaceutically acceptable adjuvant generally refers to any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents, etc. that are compatible with drug administration, usually safe, non-toxic , and is neither biologically nor otherwise undesirable.
  • drug molecule generally refers to a molecule having the desired biological effect. Medications can be preventative or therapeutic.
  • Drug molecules may include but are not limited to: protein molecules, including but not limited to peptides, polypeptides, proteins, including post-translationally modified proteins, fusion proteins, antibodies, etc., for example, in this application, the drug molecules in addition to the In addition to the bispecific fusion protein, it may also contain other one or more amino acids.
  • the drug molecule may also contain other structures besides the bispecific fusion protein described in this application.
  • the drug molecules may also include small molecular structures, including inorganic or organic compounds.
  • the drug molecule can also include nucleic acid molecules, including but not limited to double-stranded or single-stranded DNA, or double-stranded or single-stranded RNA (such as antisense (molecule), RNAi, etc.), intron sequences, Triple helix nucleic acid molecule and aptamer.
  • nucleic acid molecules including but not limited to double-stranded or single-stranded DNA, or double-stranded or single-stranded RNA (such as antisense (molecule), RNAi, etc.), intron sequences, Triple helix nucleic acid molecule and aptamer.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a bispecific fusion protein comprising a first binding domain and a second binding domain, wherein: the first binding domain comprises an antibody specifically binding to PD-L1, wherein the antibody comprises Two antibody light chains and two antibody heavy chains, the antibody light chains and the antibody heavy chains are linked by disulfide bonds; the second binding domain comprises an Ig-like domain of VEGFR1 and an Ig-like structure of VEGFR2 domain; wherein the N-terminus of the Ig-like domain of VEGFR1 or the N-terminus of the Ig-like domain of VEGFR2 is directly or indirectly linked to the C-terminus of the antibody heavy chain, respectively.
  • the first binding domain can specifically bind to human PD-L1.
  • the second binding domain can specifically bind human VEGF family.
  • the second binding domain may specifically bind a protein selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D and PLGF.
  • the Ig-like domain of VEGFR1 may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the Ig-like domain of VEGFR2 may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the Ig-like domain of VEGFR1 may be directly connected to the Ig-like domain of VEGFR2.
  • the second binding domain may comprise the Ig-like domain of VEGFR1 and the Ig-like domain of VEGFR2 sequentially from the N-terminus.
  • the second binding domain may consist of the Ig-like domain of VEGFR1 and the Ig-like domain of VEGFR2 sequentially from the N-terminus.
  • the second binding domain may comprise the amino acid sequence shown in SEQ ID NO: 28.
  • the indirect connection may be connected through a linker.
  • the N-terminal of the Ig-like domain of VEGFR1 in the second binding domain can be connected to the C-terminal of the antibody heavy chain through the linker.
  • the N-terminal of the Ig-like domain of VEGFR2 in the second binding domain can be connected to the C-terminal of the antibody heavy chain through the linker.
  • the linker may comprise the amino acid sequence shown in SEQ ID NO: 3.
  • any known PD-L1 antibody has a light chain variable region and a heavy chain variable region that can specifically bind to PD-L1, it can be used as all antibodies in the first binding domain. said antibody.
  • the light chain of the antibody comprises a light chain variable region, wherein the light chain variable region may comprise LCDR1-3, wherein LCDR1 may comprise amino acids shown in SEQ ID NO: 4 or SEQ ID NO: 10 sequence.
  • LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 11.
  • LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 12.
  • the light chain of the antibody comprises a light chain variable region, wherein the light chain variable region may comprise LCDR1-3, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 Can comprise the amino acid sequence shown in SEQ ID NO: 5; And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 6; Alternatively, the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 10; The LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11; and said LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the light chain of the antibody may comprise a light chain variable region, wherein the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17.
  • the antibody light chain may comprise a light chain constant region, wherein the light chain constant region may be derived from a light chain constant region selected from the following histones: Ig ⁇ and Ig ⁇ .
  • the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24.
  • the antibody heavy chain may comprise a heavy chain variable region, wherein the heavy chain variable region may comprise HCDR1-3, wherein the HCDR1 may comprise SEQ ID NO: 7 or SEQ ID NO: 13 The amino acid sequence shown.
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 14.
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 15.
  • the antibody heavy chain may comprise a heavy chain variable region, wherein the heavy chain variable region may comprise HCDR1-3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9; or, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13; The HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 14; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 15.
  • the antibody heavy chain may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 19.
  • the light chain variable region of the antibody may comprise the amino acid sequence shown in SEQ ID NO: 16, and the heavy chain variable region of the antibody may comprise the amino acid sequence shown in SEQ ID NO: 18;
  • the light chain variable region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 17, and the heavy chain variable region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 19.
  • the antibody heavy chain may comprise an Fc region.
  • the Fc region may be derived from Fc selected from the following histones: IgG1 and IgG4.
  • the Fc region may comprise amino acid mutations at amino acid positions selected from the group consisting of N298, D357 and L359.
  • the amino acid mutation at N298 in the Fc region may include N298A, that is, amino acid N at position 82 in the amino acid sequence shown in SEQ ID NO: 20 is mutated to amino acid A.
  • the amino acid mutation at D357 in the Fc region may include D357E, that is, amino acid D at position 141 in the amino acid sequence shown in SEQ ID NO: 20 is mutated to amino acid E.
  • the amino acid mutation at L359 in the Fc region may include L359M, that is, amino acid L at position 143 in the amino acid sequence shown in SEQ ID NO: 20 is mutated to amino acid M.
  • the Fc region may comprise amino acid mutations selected from the group consisting of N298A, D357E and L359M.
  • the Fc region may have amino acid mutations, and the amino acid mutations may consist of N298A, D357E and L359M.
  • the Fc region may comprise the amino acid sequence shown in any one of SEQ ID NO: 20-22.
  • the bispecific fusion protein may be a multimer composed of two copies of the first polypeptide chain and the second polypeptide chain, wherein the first polypeptide chain may contain the antibody light chain, wherein the second polypeptide chain may sequentially comprise the antibody heavy chain, the linker and the second binding domain from the N-terminus.
  • said first polypeptide chain may consist of said antibody light chain.
  • the antibody heavy chain in the first polypeptide chain and the second polypeptide chain can be linked by a covalent bond (such as a disulfide bond); the first polypeptide chain and the second polypeptide chain
  • the antibody heavy chains in the peptide chains may be linked by non-covalent bonds.
  • the two first polypeptide chains may comprise the same amino acid sequence.
  • the two first polypeptide chains may have the same amino acid sequence.
  • the two second polypeptide chains may comprise the same amino acid sequence.
  • two said second polypeptide chains may have the same amino acid sequence.
  • the two first polypeptide chains can be arbitrarily linked to the two second polypeptide chains respectively through covalent bonds and/or non-covalent bonds, so as to form the bispecific fusion protein described in this application.
  • the first polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NO: 23-24.
  • the second polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NO: 25-27.
  • the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO: 23; and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO: 25; or, wherein the The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 24; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 26; or, wherein the first polypeptide chain comprises SEQ ID NO: the amino acid sequence shown in 23; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 28.
  • the amino acid sequence of the first polypeptide chain may be shown in SEQ ID NO: 23; and the amino acid sequence of the second polypeptide chain may be shown in SEQ ID NO: 25.
  • the amino acid sequence of the first polypeptide chain may be shown in SEQ ID NO: 24; and the amino acid sequence of the second polypeptide chain may be shown in SEQ ID NO: 26.
  • the amino acid sequence of the first polypeptide chain may be as shown in SEQ ID NO: 23; and the amino acid sequence of the second polypeptide chain may be as shown in SEQ ID NO: 28.
  • the present application provides a polynucleotide encoding the bispecific fusion protein described in the present application.
  • the polynucleotides described herein may be isolated. For example, it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified (iv) synthetic, for example by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques. Recombinant DNA and molecular cloning techniques include those discussed by Maniatis, T., Fritsch, E.F., Sambrook, J.: Molecular Cloning. A Laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY, 1982 and by T.J.
  • nucleic acids can be prepared from genomic DNA fragments, cDNA and RNA, all of which can be extracted directly from cells or produced recombinantly by various amplification methods including, but not limited to, PCR and RT-PCR.
  • a polynucleotide described herein may comprise more than one different nucleotide sequence.
  • the polynucleotides described herein may encode more than one different component of the bispecific fusion proteins described herein.
  • the polynucleotide may encode the first polypeptide chain described herein, and/or the polynucleotide may encode the second polypeptide chain described herein.
  • the polynucleotide can encode the light chain of the PD-L1 antibody; can encode the heavy chain of the PD-L1 antibody; can encode the second binding domain; can encode the linker; can encode The Ig-like domain of VEGFR1; and/or, the Ig-like domain of VEGFR2 may be encoded.
  • the present application provides a vector comprising the polynucleotide described in the present application.
  • the vector may contain one or more polynucleotides described in the present application.
  • the vector can be directly used to express the bispecific fusion protein described in this application.
  • the vector can be used to express any component of the bispecific fusion protein described in the present application.
  • at least two of the vectors can be used to express the bispecific fusion protein described in the present application.
  • the vector can express the first polypeptide chain described herein, and/or, the vector can express the second polypeptide chain described herein.
  • the vector can express the light chain of the PD-L1 antibody, the heavy chain of the PD-L1 antibody, the linker, the Ig-like domain of the VEGFR1; and/or, the Ig of the VEGFR2 like domain.
  • the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host.
  • control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the expression control sequences are regulatable elements.
  • the specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally may include 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, Capping sequence, CAAT sequence, etc.
  • the 5' non-transcribed expression control sequence may comprise a promoter region which may comprise a promoter sequence for transcriptional control of the functionally linked nucleic acid.
  • the expression control sequences may also include enhancer sequences or upstream activator sequences.
  • suitable promoters can be promoters of SP6, T3 and T7 polymerases, human U6RNA promoters, CMV promoters and artificial hybrid promoters thereof (such as CMV), wherein certain parts of the promoters can be combined with Parts of the promoters of other cellular proteins (eg human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) genes are fused, which may or may not contain additional introns.
  • One or polynucleotides described herein may be operably linked to the expression control element.
  • Such vectors may include, plasmids, cosmids, viruses, phages, or other vectors commonly used, for example, in genetic engineering.
  • the vector is an expression vector.
  • the present application provides a cell comprising or expressing the bispecific fusion protein described in the present application, the polynucleotide described in the present application or the vector described in the present application.
  • the cells can be prokaryotic cells (eg, bacterial cells), CHO cells, NS/0 cells, HEK293T cells or HEK293A cells, or can be other eukaryotic cells, such as fungal or yeast cells.
  • the vectors described in this application can be introduced into the cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.
  • the host cell can be COS, CHO, NSO, sf9, sf21, DH5a, BL21(DE3) or TG1.
  • the present application provides a method for preparing the bispecific fusion protein described in the present application, which includes culturing the cells described in the present application under conditions suitable for expressing the bispecific fusion protein described in the present application.
  • the method may further comprise the step of harvesting (eg, isolating and/or purifying) the bispecific fusion protein described herein.
  • the bispecific fusion protein described herein can be used for affinity chromatography, and the bispecific fusion protein described in this application can also be purified and separated by gel electrophoresis and/or high performance liquid chromatography.
  • the present application provides a pharmaceutical composition, which comprises the bispecific fusion protein described in the present application, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or nonionic surfactants agent etc.
  • the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at tumor site, inhalation, rectal administration, vaginal administration, transdermal administration Administration or administration via a subcutaneous depot.
  • the pharmaceutical composition can be prepared as mini-tablets, capsules, elixirs, suspensions, syrups or wafers.
  • the pharmaceutical compositions can be presented in unit dosage form, eg, in unit dose form, eg, in ampoules, or in unit dosage form, eg, in multi-dose containers.
  • the pharmaceutical composition can also be prepared as solutions, suspensions, tablets, pills, capsules and depots.
  • the administration frequency and dosage of the pharmaceutical composition can be determined by a number of related factors including the type of disease to be treated, the route of administration, the patient's age, sex, body weight and severity of the disease and the active ingredient drug type. Since the pharmaceutical composition has excellent in vivo efficacy and duration of concentration, it can significantly reduce the administration frequency and dosage of the drug.
  • the present application provides a drug molecule comprising the bispecific fusion protein.
  • the drug molecule can have the properties and/or functions of the bispecific fusion protein.
  • the drug molecule may also have other properties and/or functions.
  • the drug molecule may also comprise other structures.
  • the drug molecule may also contain one or more other amino acids.
  • the present application provides an application of the bispecific fusion protein described in the present application in the preparation of a drug for treating diseases, wherein the diseases include tumors.
  • the bispecific fusion protein described in the present application, and/or the pharmaceutical composition described in the present application, are used for treating diseases, wherein the diseases include tumors.
  • the present application provides a method for treating a disease, which comprises administering an effective amount of the bispecific fusion protein and/or the pharmaceutical composition described in the present application to a subject in need, wherein the disease includes a tumor.
  • the tumor may be a solid tumor or a non-solid tumor.
  • the tumors may include various tumor types known to those skilled in the art, and are not limited to specific one or more tumor types.
  • the tumor may comprise lung cancer, colorectal cancer, cervical cancer, liver cancer, gastric cancer and/or kidney cancer.
  • the tumor can include colorectal cancer.
  • the tumor may be PD-L1 positive (eg, may be PD-L1 overexpressed) tumor. In certain instances, the tumor may also be PD-L1 negative. In the present application, the tumor may be associated with overexpression of VEGF (eg in blood vessels).
  • the present application provides a method for inhibiting the growth of blood vessels (such as human), which comprises administering an effective amount of the bispecific fusion protein described in the present application, and/or the pharmaceutical composition described in the present application.
  • the present application provides a method for inhibiting the activity of a VEGF receptor ligand, which comprises administering an effective amount of the bispecific fusion protein described in the present application, and/or the pharmaceutical composition described in the present application.
  • the VEGF receptor ligand activity may include the biological activity and/or function of VEGF and/or VEGFR itself.
  • binding of VEGF to VEGFR may be involved.
  • the present application provides a method for inhibiting PD-L1 activity, which comprises administering an effective amount of the bispecific fusion protein described in the present application, and/or the pharmaceutical composition described in the present application.
  • the PD-L1 activity may include the biological activity and/or function of PD-L1 and/or PD-1 itself.
  • the combination of PD-L1 and PD-1 can be involved.
  • SG1201 PD-L1 antibody 1
  • the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.16
  • the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.18;
  • SG1202 PD-L1 antibody 2, the amino acid sequence of its light chain variable region is shown in SEQ ID NO.17; the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.19;
  • VEGFRs (its amino acid sequence is shown in SEQ ID NO.28)-Fc (its amino acid sequence is shown in SEQ ID NO.20) fusion protein;
  • 12VF1 it is the bispecific fusion protein described in this application, wherein the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.23; and the amino acid sequence of its second polypeptide chain is shown in SEQ ID NO.25 Show;
  • 12VF2 It is the bispecific fusion protein described in this application, wherein the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.24; and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.26 Show;
  • 12VF8 It is the bispecific fusion protein described in this application, wherein the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.23; and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.27 Show.
  • Embodiment 1 constructs fusion protein
  • the sequence of PD-L1 antibody 1 the linker (its amino acid sequence is shown in SEQ ID NO.3) and VEGFRs are sequentially connected, wherein the N-terminal of VEGFRs and PD- The C-terminus of the heavy chain of L1 antibody 1 was linked to obtain the bispecific fusion protein 12VF1.
  • the mutated IgG1Fc (whose amino acid sequence is shown in SEQ ID NO.22) was replaced with IgG4Fc (whose amino acid sequence was shown in SEQ ID NO.21), and the bispecific fusion protein 12VF8 was obtained.
  • fusion protein structure shown in Figure 1 connect the PD-L1 antibody 2, the linker (the amino acid sequence of which is shown in SEQ ID NO.3) and VEGFRs in sequence, wherein the N-terminal of VEGFRs and the heavy weight of PD-L1 antibody 2 The C-terminals of the chains were connected to obtain the bispecific fusion protein 12VF2.
  • ELISA was used to evaluate the binding activity of bifunctional fusion protein and PD-L1.
  • PD-L1 human recombinant PD-L1 protein (ECD, His Tag), purchased from Sino Biological) was coated with ELISA strips, overnight at 4°C; after washing with PBST, 10% fetal bovine serum was added, and blocked for 1 hour at 37°C Add different concentrations of antibodies SG1201 and SG1202, the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 prepared in Example 1, and react at 37°C for 1 hour; after washing with PBST, add horseradish peroxidase-labeled goat anti-human IgG Fab Secondary antibody (Goat Anti-Human IgG (Fab') 2 (HRP), Abcam), react at 37°C for 30 minutes; wash 5 times with PBST; add 100 ⁇ L TMB (eBioscience) to each well, and store in the dark at room temperature (20 ⁇ 5°C) 1 to 2 minutes; then add 100 ⁇ L 2N H 2 SO 4 stop solution to each well to stop the substrate reaction, read the OD value at 450 nm
  • Figure 2 shows that the binding ability of the bispecific fusion protein 12VF1 to PD-L1 is slightly stronger than that of 12VF8, which is the same as that of the antibody SG1201.
  • Figure 2 shows that the bispecific fusion protein 12VF2 and the antibody SG1202 have comparable binding abilities to PD-L1.
  • VEGFRs fusion protein As a control, ELISA was used to evaluate the binding activity of bifunctional fusion protein and VEGF165.
  • VEGF165 Human VEGF165 Protein, His Tag, Acro Biosystems
  • PBST 10% fetal bovine serum was added, and blocked for 1 hour at 37°C; different concentrations of bispecific fusion proteins were added 12VF1, 12VF2, 12VF8 and SG1501 were reacted at 37°C for 1 hour; after washing with PBST, horseradish peroxidase-labeled goat anti-human IgG Fc secondary antibody (Goat anti-human IgG Fc antibody, horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen), react at 37°C for 30 minutes; wash 5 times with PBST; add 100 ⁇ L TMB (eBioscience) to each well, and place in the dark at room temperature (20 ⁇ 5°C) for 1-2 min; then add 100 ⁇ L 2N H 2 SO4 to each well to stop The substrate reaction was terminated with a microplate reader, and the OD value was
  • Figure 3 shows that the binding ability of bispecific fusion proteins 12VF1, 12VF2 and 12VF8 to VEGF165 is equivalent, slightly weaker than that of fusion protein SG1501.
  • the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 prepared in Example 1 were evaluated by ELISA for their biological activity of simultaneously binding to dual antigens.
  • PD-L1 human recombinant PD-L1 protein (ECD, His Tag), purchased from Sino Biological) was coated with ELISA strips, overnight at 4°C; after washing with PBST, 10% fetal bovine serum was added, and blocked for 1 hour at 37°C Add different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8, PD-L1 antibodies SG1201, SG1202, and VEGFRs fusion protein SG1501, and react at 37°C for 1 hour; after washing with PBST, add biotin-labeled VEGF165 (Biotinylated Human VEGF165 Protein, His, Avitag, Acro Biosystems), reacted at 37°C for 30 minutes; washed 5 times with PBST; added horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiaxuan Biosystems), reacted at 37°C for 30 minutes; PBST Wash 5 times; add 100 ⁇ L TMB (eBioscience) to
  • Figure 4 shows that the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 can simultaneously bind to PD-L1 and VEGF165, while the PD-L1 antibodies SG1201 and SG1202, and the VEGFRs fusion protein SG1501 cannot simultaneously bind to PD-L1 and VEGF165.
  • the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 were evaluated for their biological activity in blocking PD-1/PD-L1 interaction.
  • PD-L1-his human recombinant PD-L1 protein (ECD, His Tag), purchased from Sino Biological) was coated on an enzyme-linked plate, 1ug/ml, overnight at 4°C; after washing with PBST, 10% fetal bovine serum was added , blocked at 37°C for 1 hour; added different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8 and antibodies SG1201, SG1202, and reacted at 37°C for 1 hour; after washing with PBST, added biotin-labeled PD1 (Biotinylated Human PD-1/ PDCD1 Protein, Fc, Avitag TM , His Tag, Acro Biosystems) to a final concentration of 2 ⁇ g/ml, reacted at 37°C for 30 min; washed 5 times with PBST; added horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiaxuan Bioscience), incubate at 37°C for 30 minutes
  • the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 can competitively block the binding of PD-1 and PD-L1 like the PD-L1 antibodies SG1201 and SG1202, and have similar activities.
  • the IC 50 value of SG1201 is 0.3968nM
  • the IC 50 value of SG1202 is 0.4216nM
  • the IC 50 value of 12VF1 is 0.393nM
  • the IC 50 value of 12VF8 is 0.5002nM
  • the IC 50 value of 12VF2 is 0.4256nM.
  • VEGFR1 Human VEGF R1/Flt-1 Protein, His Tag, Acro Biosystems
  • PBST 10% fetal bovine serum
  • biotin-labeled VEGF165 Biotinylated Human VEGF165 Protein, His, Avitag, Acro Biosystems
  • FIG. 6 shows that the bispecific fusion proteins 12VF1, 12VF2, and 12VF8, like the fusion protein SG1501, can competitively block the binding of VEGF to its receptor VEGFR1.
  • the IC 50 value of 12VF1 is 3.749nM
  • the IC 50 value of 12VF2 is 5.049nM
  • the IC 50 value of 12VF8 is 2.182nM
  • the IC 50 value of SG1501 is 1.470nM.
  • bispecific fusion protein IMM25011 (see patent application US2020/0172623A1) as a control, evaluate the biological activity of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 prepared in Example 1 in blocking VEGF165/VEGFR2 interaction.
  • VEGF165 Protein, Human, Cynomolgus, Recombinant, HPLC-verified, Sino Biological
  • VEGF121 VEGF121 Protein, Human, Recombinant, Sino Biological
  • Figure 8 shows that the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 can all block the binding of VEGF121 to its receptor VEGFR2, and the activity is stronger than that of IMM25011.
  • the IC 50 value of 12VF1 is 3.536nM
  • the IC 50 value of 12VF2 is 3.291nM
  • the IC 50 value of 12VF8 is 2.955nM
  • the IC 50 value of IMM25011 is 5.281nM.
  • bispecific fusion protein IMM25011 evaluates the biological activity of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 prepared in Example 1 to block the VEGF C/VEGFR2 interaction.
  • VEGF C VEGF C Protein, Human, Recombinant (His Tag), Sino Biological
  • Figure 9 shows that the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 can all block the binding of VEGF C to its receptor VEGFR2, thereby effectively inhibiting angiogenesis, and the blocking activity of 12VF1, 12VF2, and 12VF8 resemblance.
  • IMM25011 could not block the binding of VEGF C to its receptor VEGFR2.
  • the bispecific fusion protein of the present application can not only block the binding of VEGF121 and VEGFR2, but also block the binding of VEGF C and its receptor VEGFR2, thus being more comprehensive
  • the regulatory effect of this signaling pathway can be fully exerted, and the effect of more completely blocking angiogenesis at the tumor site can be achieved.
  • the biological activities of the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 in inhibiting the proliferation of HUVEC cells were evaluated.
  • HUVEC cells were resuspended with the experimental medium and added to a 96-well cell culture plate, and incubated overnight in an incubator; VEGF165 ( Human VEGF165 Protein, Tag Free (MALS verified), Acro Biosystems) was diluted to 40ng/mL with the experimental medium, and then mixed with different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8 and fusion protein SG1501 in equal volumes, and pre-treated at 37°C.
  • VEGF165 Human VEGF165 Protein, Tag Free (MALS verified), Acro Biosystems
  • bispecific fusion proteins 12VF1, 12VF2 and 12VF8 can all inhibit the proliferation of HUVEC cells, slightly weaker than the fusion protein SG1501.
  • the biological activity of the bispecific fusion protein 12VF8 in activating T lymphocytes in mixed lymphocyte reactions was evaluated.
  • Monocytes were isolated from PBMCs of healthy donors with CD14 magnetic beads (CD14 microbeads, Miltenyi), and induced to differentiate into dendritic cells with a dendritic cell culture kit (ImmunoCultTM Dendritic Cell Culture Kit, Stemcell); CD4 + T Lymphocyte isolation kit (CD4+T Cell Isolation Kit, Miltenyi) was used to isolate CD4 + T lymphocytes from PBMC of another healthy donor, and the ratio of CD4 + T lymphocytes to dendritic cells was 5:1 , add to a 96-well cell culture plate, then add different concentrations of bispecific fusion protein 12VF8, mix well and culture in a 37°C incubator for 5 days; collect the cell supernatant, and use ELISA kits to detect cytokines IFN- ⁇ and IL The concentration of -2 was used to analyze the activity of the bispecific fusion protein 12VF8 in activating T lymphocytes in a mixed lymphocyte reaction.
  • the bispecific fusion protein 12VF8 like the PD-L1 antibody SG1201, can activate T cells in a dose-dependent manner and promote the release of IFN- ⁇ and IL-2 .
  • Embodiment 8 ADCC activity analysis
  • the bispecific fusion protein IMM25011 was used as the control, the PD-L1 overexpression cell line CHO-K1/PD-L1 was used as the target cell, and the Jurkat cells (Jurkat-ADCC cells for short) overexpressing the human Fc ⁇ RIIIa gene and NFAT fluorescent reporter gene were used as the effector Cells, the ADCC activity of the bispecific fusion proteins 12VF1 and 12VF8 prepared in Example 1 was evaluated by the reporter gene method.
  • CHO-K1/PD-L1 cells add 2 ⁇ 10 4 cells per well into 96-well cell culture plate; collect Jurkat-ADCC cells, add 1.2 ⁇ 10 5 cells per tube into 96-well cell culture plate; Add different concentrations of bispecific fusion proteins IMM25011, 12VF1, and 12VF8, and incubate in a 37°C incubator for 6 hours; add 100 ⁇ L of luciferase detection solution to each well, react for 10 minutes at room temperature in the dark, and read the chemiluminescence with a microplate reader.
  • ADCC activity of bispecific fusion proteins 12VF1 and 12VF8 was analyzed by relative fluorescence intensity value (RLU).
  • Example 9 Fusion protein suppresses tumor activity in vivo
  • Mouse colorectal cancer cell MC38 was inoculated subcutaneously in the right anterior flank of male C57BL/6J mice. A total of 36 mice were inoculated. When the tumors grew to about 58mm3, they were divided into 5 groups, with 6 mice in each group. Group 1: Administer PBS on the 17th day of the test (i.e.
  • Group 2 Administer SG1201 (2mg/kg), intraperitoneal injection medicine, administered twice a week for three weeks; group 3: administering SG1501 (1.4 mg/kg), intraperitoneal injection, administering twice a week, administering for three weeks; group 4: administering SG1201 ( 2mg/kg) and SG1501 (1.4mg/kg), administered by intraperitoneal injection, twice a week, for three weeks; Group 5: 12VF8 (2.7mg/kg) group, administered by intraperitoneal injection, weekly Administration was administered twice for three weeks (the results after administration corresponded to groups 1-5 in Fig. 14 in turn).
  • the tumor volume and body weight were measured every week, and the relationship between the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.
  • the serum of internally fixed mice in each group was collected 2 hours after the last administration.
  • the tumor-bearing mice were euthanized, the tumors were peeled off, weighed, and photographed.
  • the tumor growth inhibition rate TGITV (%) was calculated and statistically analyzed.
  • the tumor growth inhibition rates of Group 2, Group 3, Group 4 and Group 5 were 63%, 39%, 71%, and 83%, respectively, and the tumor volumes of each treatment group were significantly lower than those of the control group 1 (p ⁇ 0.05), showing significant anti-tumor effect, effectively inhibiting tumor growth, wherein the tumor volume treated by group 5 was the smallest in all treatment groups, significantly lower than group 3 (p ⁇ 0.05), and the anti-tumor activity was excellent In group 2 or group 3 administered with single drug, it is also better than group 4 administered with combination drug.
  • the tumor-bearing mice showed good tolerance to the treatment of Group 2-Group 5.
  • the mice in each group had normal body weight, no abnormal performance, and were in good general condition. It can be seen that 12VF8 has drug safety.

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Abstract

提供一种双特异性融合蛋白，其包含第一结合域和第二结合域，其中所述第一结合域包含特异性结合PD-L1的抗体，其中所述抗体包含两条抗体轻链和两条抗体重链，所述抗体轻链和所述抗体重链通过二硫键连接；所述第二结合域包含一个VEGFR1的Ig样结构域和一个VEGFR2的Ig样结构域；所述VEGFR1的Ig样结构域的N端或所述VEGFR2的Ig样结构域的N端分别与所述抗体重链的C端直接或间接连接。还涉及包含所述双特异性融合蛋白的药物组合物及其制药用途。其中所述双特异性融合蛋白可以有效安全地杀伤肿瘤细胞。

Description

双特异性融合蛋白 技术领域
本申请涉及生物医药领域，具体的涉及一种双特异性融合蛋白。
背景技术
目前，肿瘤疾病日益严重，免疫检查点PD-1/PD-L1和VEGF都已成为治疗肿瘤的重要靶点。
PD-L1在多种恶性肿瘤中过表达，并且经常与预后不良相关。PD-1以及通过与PD-1相互作用传递信号的其它分子(例如PD-L1和PD-L2)的治疗性靶向是肿瘤治疗的热门领域。
VEGF是血管内皮细胞强有力和特有的促有丝分裂原，可促进血管生成过程的所有环节。各VEGF受体(VEGFR)的作用有所不同。VEGFR-2起着调节内皮生长、分化、渗透性的作用；而VEGFR-1则与调节内皮细胞移动、聚集有关，并且通过VEGFR-2抑制信号的传导。VEGF与VEGFR结合，使VEGFR二聚体化、自我磷酸化，通过多个细胞内途径传递信号，最终发挥作用。
目前亟需可以有效抑制肿瘤生长的新型融合蛋白。
发明内容
本申请提供了一种双特异性融合蛋白及其相应的多核苷酸、载体、细胞、制备方法、药物组合物和用途。本申请所述的双特异性融合蛋白具有以下至少一种性质：1)特异性结合PD-L1；2)阻断PD-1与PD-L1结合；3)特异性结合VEGF；4)阻断VEGF与VEGFR结合；5)抑制(体内)肿瘤生长；6)激活免疫细胞(例如T细胞)；7)促进免疫细胞(例如T细胞)分泌细胞因子(例如干扰素和/或细胞因子)；和/或，8)抑制肿瘤新生血管发生、促进血管正常化。
一方面，本申请提供了一种双特异性融合蛋白，其包含第一结合域和第二结合域，其中：所述第一结合域包含特异性结合PD-L1的抗体，其中所述抗体包含两条抗体轻链和两条抗体重链，所述抗体轻链和所述抗体重链通过二硫键连接；所述第二结合域包含一个VEGFR1的Ig样结构域和一个VEGFR2的Ig样结构域；其中所述VEGFR1的Ig样结构域的N端或所述VEGFR2的Ig样结构域的N端分别与所述抗体重链的C端直接或间接连接。
在某些实施方式中，所述第一结合域特异性结合人PD-L1。
在某些实施方式中，所述第二结合域特异性结合人VEGF家族。
在某些实施方式中，所述第二结合域特异性结合选自下组的蛋白质：VEGF-A、VEGF-B、VEGF-C、VEGF-D和PLGF。
在某些实施方式中，所述VEGFR1的Ig样结构域包含SEQ ID NO：1所示的氨基酸序列。
在某些实施方式中，所述VEGFR2的Ig样结构域包含SEQ ID NO：2所示的氨基酸序列。
在某些实施方式中，所述VEGFR1的Ig样结构域和VEGFR2的Ig样结构域直接连接。
在某些实施方式中，所述第二结合域包含SEQ ID NO：28所示的氨基酸序列。
在某些实施方式中，所述间接连接为通过连接子连接。
在某些实施方式中，所述连接子包含SEQ ID NO：3所示的氨基酸序列。
在某些实施方式中，所述抗体轻链包含轻链可变区，其中所述轻链可变区包含LCDR1-3，其中LCDR1包含SEQ ID NO：4、10中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR2包含SEQ ID NO：5、11中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR3包含SEQ ID NO：6、12中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR1-3包含选自下组的氨基酸序列：
(1)所述LCDR1包含SEQ ID NO:4所示的氨基酸序列，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:6所示的氨基酸序列；
(2)所述LCDR1包含SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述抗体轻链包含轻链可变区，其中所述轻链可变区包含SEQ ID NO：16-17中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体轻链包含轻链恒定区，其中轻链恒定区源自选自下组蛋白的轻链恒定区：Igκ和Igλ。
在某些实施方式中，所述抗体重链包含重链可变区，其中所述重链可变区包含HCDR1-3，其中所述HCDR1包含SEQ ID NO：7、13中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO：8、14中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR3包含SEQ ID NO：9、15中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR1-3包含选自下组的氨基酸序列：
(1)所述HCDR1包含SEQ ID NO:7所示的氨基酸序列，所述HCDR2包含SEQ ID NO:8所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:9所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2包含SEQ ID NO:14所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述抗体重链包含重链可变区，所述重链可变区包含SEQ ID NO：18-19中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体重链包含Fc区域。
在某些实施方式中，所述Fc区域源自选自下组蛋白的Fc：IgG1和IgG4。
在某些实施方式中，所述Fc区域在选自下组的氨基酸位置处包含氨基酸突变：N298、D357和L359。
在某些实施方式中，所述Fc区域包含选自下组的氨基酸突变：N298A、D357E和L359M。
在某些实施方式中，所述Fc区域包含SEQ ID NO：20-22中任一项所示的氨基酸序列。
在某些实施方式中，所述的双特异性融合蛋白为由两拷贝的第一多肽链和第二多肽链组成的多聚体，其中所述第一多肽链包含所述抗体轻链，其中所述第二多肽链自N端依次包含所述抗体重链、所述连接子和所述第二结合域。
在某些实施方式中，所述第一多肽链包含SEQ ID NO：23-24中任一项所示的氨基酸序列。
在某些实施方式中，所述第二多肽链包含SEQ ID NO：25-27中任一项所示的氨基酸序列。
在某些实施方式中，所述第一多肽链包含SEQ ID NO：23所示的氨基酸序列；所述第二多肽链包含SEQ ID NO：25所示的氨基酸序列；或者，其中所述第一多肽链包含SEQ ID NO：24所示的氨基酸序列；所述第二多肽链包含SEQ ID NO：26所示的氨基酸序列；或者，其中所述第一多肽链包含SEQ ID NO：23所示的氨基酸序列；所述第二多肽链包含SEQ ID NO：27所示的氨基酸序列。
另一方面，本申请提供一种多核苷酸，其编码本申请所述的双特异性融合蛋白。
另一方面，本申请提供一种载体，其包含本申请所述的多核苷酸。
另一方面，本申请提供一种细胞，其包含本申请所述的载体。
另一方面，本申请提供本申请所述的双特异性融合蛋白的制备方法，其包括在适于表达本申请所述的双特异性融合蛋白的情况下，培养本申请所述的细胞。
另一方面，本申请提供一种药物组合物，其包含本申请所述的双特异性融合蛋白，以及任选地药学上可接受的载体。
另一方面，本申请提供一种药物分子，其包含本申请所述的双特异性融合蛋白。
另一方面，本申请提供一种本申请所述双特异性融合蛋白、所述多核苷酸、所述载体、所述细胞、所述药物组合物和/或所述药物分子在制备治疗疾病的药物中的应用，其中所述疾病包括肿瘤。
在某些实施方式中，所述疾病包括实体瘤和非实体瘤。
在某些实施方式中，所述疾病包括PD-L1阳性肿瘤。
在某些实施方式中，所述疾病包括肺癌、结直肠癌、宫颈癌、肝癌、胃癌和/或肾癌。
另一方面，本申请提供了一种抑制血管生长的方法，其包括施用有效量的本申请所述的双特异性融合蛋白、所述多核苷酸、所述载体、所述细胞、所述的药物组合物和/或所述药物分子。
另一方面，本申请提供了一种抑制VEGF受体配体活性的方法，其包括施用有效量的本申请所述的双特异性融合蛋白、所述多核苷酸、所述载体、所述细胞、所述的药物组合物和/或所述药物分子。
另一方面，本申请提供了一种抑制PD-L1活性的方法，其包括施用有效量的本申请所述的双特异性融合蛋白、所述多核苷酸、所述载体、所述细胞、所述的药物组合物和/或所述药物分子。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是本申请所述双特异性融合蛋白的结构。
图2显示的是本申请所述双特异性融合蛋白特异性结合人PD-L1。
图3显示的是本申请所述双特异性融合蛋白特异性结合VEGF165。
图4显示的是本申请所述双特异性融合蛋白同时特异性结合人PD-L1和VEGF165。
图5显示的是本申请所述双特异性融合蛋白阻断PD-1和PD-L1的相互作用。
图6显示的是本申请所述双特异性融合蛋白阻断VEGF和VEGFR的相互作用。
图7显示的是本申请所述双特异性融合蛋白阻断VEGF165和VEGFR2的相互作用。
图8显示的是本申请所述双特异性融合蛋白阻断VEGF121和VEGFR2的相互作用。
图9显示的是本申请所述双特异性融合蛋白阻断VEGF C和VEGFR2的相互作用。
图10显示的是本申请所述双特异性融合蛋白抑制HUVEC细胞的增殖。
图11显示的是本申请所述双特异性融合蛋白激活淋巴细胞分泌IFN-γ。
图12显示的是本申请所述双特异性融合蛋白激活淋巴细胞分泌IL-2。
图13显示的是本申请所述双特异性融合蛋白的ADCC活性检测。
图14显示的是本申请所述双特异性融合蛋白抑制小鼠结肠癌生长。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“双特异性”通常是指本申请所述的融合蛋白可以与两个不同的配体相互作用的能力。在本申请中，所述双特异性融合蛋白既可以特异性结合PD-L1，又可以特异性结合VEGF。
在本申请中，术语“特异性结合”通常指可测量的和可再现的相互作用，比如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况可决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在一个实施方案中，抗体结合无关靶标的程度小于抗体对靶标的结合的约10％，例如通过放射免疫分析(RIA)测量的。例如，在本申请中，所述双特异性融合蛋白能够以<1x10 -7M或更低的解离常数(KD)与PD-L1和VEGF相结合。例如，在本申请中，所述PD-L1抗体能够以<1x10 -7M或更低的解离常数(KD)与PD-L1相结合。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在另一个实施方案中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“第一结合域”通常是指可以特异性结合PD-L1的结合域。例如，所述第一结合域可以包括特异性结合PD-L1的抗体。
在本申请中，术语“第二结合域”通常是指可以特异性结合VEGF的结合域。例如，所述第二结合域可以包含一个VEGFR1的Ig样结构域和一个VEGFR2的Ig样结构域。
在本申请中，术语“PD-L1”通常是指程序性死亡配体1蛋白、其功能变体和/或其功能片段。PD-L1也称为分化簇274(CD274))或B7同源物1(B7-H1)。所述PD-L1可以为由CD274基因编码的蛋白。PD-L1结合其受体，例如程序性死亡1(PD-1)，所述PD-1可以在活化的T细胞、B细胞和巨噬细胞中表达(参见Ishida et al.,1992EMBO J,11:3887-3395；Okazaki et al.,Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.Science,2001；291:319-22)。PD-L1和PD-1的络合可以通过抑制T细胞增殖和产生细胞因子IL-2和IFN-γ发挥免疫抑制作用(参见Freeman et al.,Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation,J.Exp.Med.2000,192:1027-1034；Carter et al.,PD-1:PD-L inhibitory pathway affects both CD4(+)and CD8(+)T cells and is overcome by IL-2.Eur.J.Immunol.2002,32:634–643)。术语“PD-L1”可以涵盖任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源可以包括哺乳动物，诸如灵长类(例如，人)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞中的加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。所述术语还可以涵盖天然存在的PD-L1的变体，例如剪接变体或等位基因变体。PD-L1的基本结构可以包括4个结构域：胞外Ig样V型结构域和Ig样C2型结构域、跨膜结构域以及细胞质结构域。PD-L1序列是本领域已知的。例如可在NCBI Gene ID No.29126下找到关于人PD-L1基因(包括基因组DNA序列)的信息。又例如可在NCBI Gene ID No.60533下找到关于小鼠PD-L1基因(包括基因组DNA序列)的信息。又例如可在NCBI Gene ID No.102145573下找到关于食蟹猴PD-L1基因(包括基因组DNA序列)的信息。示例性的全长人PD-L1蛋白的氨基酸序列可在NCBI登录号NP_054862或UniProt登录号Q9NZQ7下找到。示例性的全长小鼠PD-L1蛋白序列可在NCBI登录号NP_068693或Uniprot登录号Q9EP73下找到。示例性的全长食蟹猴PD-L1蛋白序列可在NCBI登录号XP_005581836或Uniprot登录号G7PSE7下找到。
在本申请中，术语“抗体”通常是指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互 连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton&Lange,Norwalk,Conn.,1994，第71页和第6章。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。取决于其重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。基于CH序列和功能方面的相对小的差异，将γ和α类进一步分成亚类，例如，人表达下述亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgK1。
在本申请中，术语“CDR”通常指抗体可变结构域的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在天然抗体中，HCDR3和LCDR3显示所述六个CDR的大多数多样性，并且特别地HCDR3被认为在赋予抗体的精细特异性方面起独特作用。参见，例如Xu et al,Immunity 13:37-45(2000)；Johnson and Wu,in Methods in Molecular Biology 248:1-25(Lo,ed.,Human Press,Totowa,N.J.,2003)。
在本申请中，术语“VEGF”通常是指血管内皮细胞生长因子(vascular endothelial growth factor)。VEGF可以涉及调节正常的和异常的血管发生及与肿瘤和眼内病症有关的新血管化(参见Ferrara,N.和Davis-Smyth,T.,Endocr.Rev.18，1997，4-25等)。VEGF可以在胚胎血管生成期间在新血管形成中和在成年期间在血管发生中具有重要的调节功能。VEGF可以促进肿瘤的生长。VEGF是高度保守的同源二聚体糖蛋白。VEGF可以有六个等型(isoforms)：VEGF-A、VEGF-B(包括VEGF-B167以及VEGF-B186)、VEGF-C、VEGF-D以及VEGF-E。在本申请中，所述VEGF可以为人VEGF。
在本申请中，术语“VEGFR1”通常是指血管内皮生长因子受体1。VEGFR1是VEGFR的一种。VEGFR属于受体酪氨酸激酶超家族，是一种膜镶嵌蛋白。VEGFR的膜外部分大约有750个氨基酸残基，由7个与免疫球蛋白结构类似的Ig样结构域组成。VEGFR1的膜外区中第二个Ig结构域是与配体结合的区域。VEGFR1的不同剪接体可以竞争性结合VEGF(例如， 可以结合VEGF-A、VEGF-B)，从而阻止VEGF与VEGFR2结合。
在本申请中，术语“VEGFR2”通常是指血管内皮生长因子受体2。VEGFR2的第三个Ig结构域可以对与配体的结合的专一性起作用。VEGFR2可以与VEGF-A、VEGF-E结合。
在本申请中，术语“Ig样结构域”通常是指VEGFR胞外区中的一个结构，其可以负责与VEGF结合。例如，VEGFR2胞外区中第1个Ig样结构域是与VEGF结合的必要部位,第2-3个Ig样结构域是与VEGF紧密结合的主要部位,受体通过第4个Ig样结构域形成同源二聚体的活性形式，而第5-第7个Ig样结构域与VEGF结合的关系不密切。例如，VEGFR1胞外区具有7个Ig样结构域，其可以负责与VEGF结合，并促进血管形成。
在本申请中，术语“PLGF”通常是指胎盘生长因子(Placental growth factor)，其可以由PGF基因编码。所述PLGF可以为VEGF亚家族成员。所述PLGF可以在人脐静脉内皮细胞(HUVE)和胎盘中表达。所述PLGF可以在滋养细胞的生长和分化中起作用。所述PLGF可以与血管生成相关。
在本申请中，术语“直接相连”与术语“间接相连”相对，术语“直接相连”通常是指直接连接。例如，所述直接相连可以为物质间没有间隔子而直接相连的情况。所述间隔子可以是连接子。例如，所述连接子可以为肽连接子。术语“间接相连”通常是指物质间不直接相连的情况。例如，所述间接相连可以为通过间隔子而连接的情况。例如，在本申请所述VEGFR1的Ig样结构域的N端或所述VEGFR2的Ig样结构域的N端可以分别与所述抗体重链的C端连接。例如，本申请所述的一个VEGFR1的Ig样结构域可以和一个VEGFR2的Ig样结构域直接连接。
在本申请中，术语“氨基酸突变”通常是指用另一种不同的氨基酸残基替换至少一个现有的氨基酸残基。所述替换的氨基酸残基可以是“天然存在的氨基酸残基”，例如，可以为丙氨酸(Ala)、精氨酸(Arg)、天冬酰胺(Asn)、天冬氨酸(Asp)、半胱氨酸(Cys)、谷氨酰胺(Gln)、谷氨酸(Glu)、甘氨酸(Gly)、组氨酸(His)、异亮氨酸(Ile)：亮氨酸(Leu)、赖氨酸(Lys)、甲硫氨酸(Met)、苯丙氨酸(Phe)、脯氨酸(Pro)、丝氨酸(Ser)、苏氨酸(Thr)、色氨酸(Trp)、酪氨酸(Tyr)和缬氨酸(Val)。所述替换的氨基酸残基也可以是非天然形式存在的氨基酸残基，例如可以为正亮氨酸、鸟氨酸、正缬氨酸、高丝氨酸、aib和其它氨基酸残基类似物。本申请中，所述的氨基酸取代可以为非保守取代。所述非保守取代可包括以非保守的形式改变目标蛋白或多肽中的氨基酸残基，例如将具有某种侧链大小或某种特性(例如，亲水性)的氨基酸残基变为具有不同侧链大小或不同特性(例如，疏水性)的氨基酸残基。本申请中，所述的氨基酸取代也可以为保守取代。所述保守取代可包括以保守的形式改 变目标蛋白或多肽中的氨基酸残基，例如将具有某种侧链大小或某种特性(例如，亲水性)的氨基酸残基变为具有相同或相似侧链大小或者相同或相似特性(例如，仍为亲水性)的氨基酸残基。这样的保守取代通常不会对所产生的蛋白质的结构或功能带来很大影响。
在本申请中，术语“多聚体”通常是指由单体结合而形成的分子。例如，所述多聚体可以为包含至少2个结构相同或结构不同的成分的多聚蛋白质。例如，所述多聚体可以为两条或更多条以共价或非共价缔合或者通过共价或非共价相互作用结合的多肽链的分子。例如，所述多聚体可以为二聚体，可以为四聚体。
在本申请中，术语“第一多肽链”通常是指本申请所述双特异性融合蛋白中靶向PD-L1抗体的轻链。
在本申请中，术语“第二多肽链”通常是指包含本申请所述双特异性融合蛋白中靶向PD-L1抗体的重链、本申请所述的连接子和本申请所述的靶向VEGF的第二结合域的多肽。例如，所述第二多肽链可以自N端依次由本申请所述双特异性融合蛋白中靶向PD-L1抗体的重链、本申请所述的连接子和本申请所述的靶向VEGF的第二结合域组成。
在本申请中，所述“第一”、“第二”并没有顺序上的区别，所述“第二”仅作为与“第一”指代不同内容的称谓。
在本申请中，术语“核酸分子”通常指任何长度的核苷酸(例如，可以为分离形式的核苷酸)，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“细胞”通常指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本申请所述的核酸分子或本申请所述的载体。细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在 总DNA互补体的形态上或在基因组上)。细胞可包括用本申请所述的载体在体外转染的细胞。细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞、人非小细胞肺癌A549细胞、人皮肤鳞癌A431细胞、肾透明细胞腺癌786-O细胞、人胰腺癌MIA PaCa-2细胞、红白血病K562细胞、急性T细胞白血病Jurkat细胞、人乳腺癌MCF-7细胞、人乳腺癌MDA-MB-231细胞、人乳腺癌MDA-MB-468细胞、人乳腺癌SKBR3细胞、人卵巢癌SKOV3细胞、淋巴瘤U-937细胞、淋巴瘤Raji细胞、人骨髓瘤U266细胞或人多发性骨髓瘤RPMI8226细胞。在某些实施方案中，细胞为哺乳动物细胞。在某些实施方案中，哺乳动物细胞为HEK293细胞。
在本申请中，术语“药物组合物”通常指涉及适合施用于患者、例如可以为施用于人患者的组合物。例如，本申请所述的药物组合物，其可以包含本申请所述的双特异性融合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。在本申请中，所述药物组合物的可接受成分可以在所用剂量和浓度下对接受者无毒。本申请的药物组合物的形式可以不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的佐剂”通常指与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂等，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在本申请中，术语“药物分子”通常是指具有所需生物学效力的分子。药物可以是预防性或治疗性的。药物分子可以包括不限于：蛋白分子，包括但不限于肽，多肽，蛋白质，包括翻译后修饰的蛋白质、融合蛋白、抗体等，例如，在本申请中，所述药物分子中除了本申请所述的双特异性融合蛋白以外，还可包含其他的一个或多个氨基酸，例如，所述药物分子中除了本申请所述的双特异性融合蛋白以外，还可包含其他结构。在本申请中，所述药物分子还可包含小分子结构，包括无机或有机化合物。在本申请中，所述药物分子还可包含核酸分子，包括但不限于双链或单链DNA、或双链或单链RNA(如反义(分子)、RNAi等)、内含子序列、三螺旋核酸分子和适体。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、 6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
一方面，本申请提供了一种双特异性融合蛋白，其包含第一结合域和第二结合域，其中：所述第一结合域包含特异性结合PD-L1的抗体，其中所述抗体包含两条抗体轻链和两条抗体重链，所述抗体轻链和所述抗体重链通过二硫键连接；所述第二结合域包含一个VEGFR1的Ig样结构域和一个VEGFR2的Ig样结构域；其中所述VEGFR1的Ig样结构域的N端或所述VEGFR2的Ig样结构域的N端分别与所述抗体重链的C端直接或间接连接。
在本申请中，所述第一结合域可以特异性结合人PD-L1。
在本申请中，所述第二结合域可以特异性结合人VEGF家族。
在本申请中，所述第二结合域可以特异性结合选自下组的蛋白质：VEGF-A、VEGF-B、VEGF-C、VEGF-D和PLGF。
在本申请中，所述VEGFR1的Ig样结构域可以包含SEQ ID NO：1所示的氨基酸序列。
在本申请中，所述VEGFR2的Ig样结构域可以包含SEQ ID NO：2所示的氨基酸序列。
在本申请中，所述VEGFR1的Ig样结构域可以和VEGFR2的Ig样结构域直接连接。例如，所述第二结合域可以自N端依次包含所述VEGFR1的Ig样结构域和所述VEGFR2的Ig样结构域。例如，所述第二结合域可以自N端依次由所述VEGFR1的Ig样结构域和所述VEGFR2的Ig样结构域组成。在本申请中，所述第二结合域可以包含SEQ ID NO：28所示的氨基酸序列。
在本申请中，所述间接连接可以通过连接子连接。例如，在所述双特异性融合蛋白中，所述第二结合域中的所述VEGFR1的Ig样结构域的N端可以通过所述连接子与所述抗体重链的C端连接。例如，在所述双特异性融合蛋白中，所述第二结合域中的所述VEGFR2的Ig样结构域的N端可以通过所述连接子与所述抗体重链的C端连接。
在本申请中，所述连接子可以包含SEQ ID NO：3所示的氨基酸序列。
在本申请中，任何已知的PD-L1抗体只要具有可以特异性结合PD-L1的轻链可变区和重链可变区，都可以被用于作为所述第一结合域中的所述抗体。
在本申请中，所述抗体轻链包含轻链可变区，其中所述轻链可变区可以包含LCDR1-3，其中LCDR1可以包含SEQ ID NO：4或SEQ ID NO：10所示的氨基酸序列。
在本申请中，LCDR2可以包含SEQ ID NO：5或SEQ ID NO：11所示的氨基酸序列。
在本申请中，LCDR3可以包含SEQ ID NO：6或SEQ ID NO：12所示的氨基酸序列。
在本申请中，所述抗体轻链包含轻链可变区，其中所述轻链可变区可以包含LCDR1-3， 所述LCDR1可以包含SEQ ID NO：4所示的氨基酸序列；所述LCDR2可以包含SEQ ID NO：5所示的氨基酸序列；并且所述LCDR3可以包含SEQ ID NO：6所示的氨基酸序列；或者，所述LCDR1可以包含SEQ ID NO：10所示的氨基酸序列；所述LCDR2可以包含SEQ ID NO：11所示的氨基酸序列；并且所述LCDR3可以包含SEQ ID NO：12所示的氨基酸序列。
在本申请中，所述抗体轻链可以包含轻链可变区，其中所述轻链可变区可以包含SEQ ID NO：16或SEQ ID NO：17所示的氨基酸序列。
在本申请中，所述抗体轻链可以包含轻链恒定区，其中轻链恒定区可以源自选自下组蛋白的轻链恒定区：Igκ和Igλ。
在本申请中，所述抗体轻链可以包含SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列。
在本申请中，所述抗体重链可以包含重链可变区，其中所述重链可变区可以包含HCDR1-3，其中所述HCDR1可以包含SEQ ID NO：7或SEQ ID NO：13所示的氨基酸序列。
在本申请中，HCDR2可以包含SEQ ID NO：8或SEQ ID NO：14所示的氨基酸序列。
在本申请中，HCDR3可以包含SEQ ID NO：9或SEQ ID NO：15所示的氨基酸序列。
在本申请中，所述抗体重链可以包含重链可变区，其中所述重链可变区可以包含HCDR1-3，所述HCDR1可以包含SEQ ID NO：7所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO：8所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO：9所示的氨基酸序列；或者，所述HCDR1可以包含SEQ ID NO：13所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO：14所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO：15所示的氨基酸序列。
在本申请中，所述抗体重链可以包含重链可变区，所述重链可变区可以包含SEQ ID NO：18或SEQ ID NO：19所示的氨基酸序列。
在本申请中，所述抗体的轻链可变区可以包含SEQ ID NO：16所示的氨基酸序列，并且所述抗体的重链可变区可以包含SEQ ID NO：18所示的氨基酸序列；或者，所述抗体的轻链可变区可以包含SEQ ID NO：17所示的氨基酸序列，并且所述抗体的重链可变区可以包含SEQ ID NO：19所示的氨基酸序列。
在本申请中，所述抗体重链可以包含Fc区域。
在本申请中，所述Fc区域可以源自选自下组蛋白的Fc：IgG1和IgG4。
在本申请中，所述Fc区域可以在选自下组的氨基酸位置处包含氨基酸突变：N298、D357和L359。
在本申请中，所述Fc区域中N298处的氨基酸突变可以包括N298A，即SEQ ID NO：20 所示的氨基酸序列中第82位的氨基酸N突变为氨基酸A。在本申请中，所述Fc区域中D357处的氨基酸突变可以包括D357E，即SEQ ID NO：20所示的氨基酸序列中第141位的氨基酸D突变为氨基酸E。在本申请中，所述Fc区域中L359处的氨基酸突变可以包括L359M，即SEQ ID NO：20所示的氨基酸序列中第143位的氨基酸L突变为氨基酸M。
在本申请中，所述Fc区域可以包含选自下组的氨基酸突变：N298A、D357E和L359M。在本申请中，所述Fc区域可以具有氨基酸突变，所述氨基酸突变可以由N298A、D357E和L359M组成。
在本申请中，所述Fc区域可以包含SEQ ID NO：20-22中任一项所示的氨基酸序列。
在本申请中，所述的双特异性融合蛋白可以为由两拷贝的第一多肽链和第二多肽链组成的多聚体，其中所述第一多肽链可以包含所述抗体轻链，其中所述第二多肽链可以自N端依次包含所述抗体重链、所述连接子和所述第二结合域。
在本申请中，所述第一多肽链可以由所述抗体轻链组成。例如，所述第一多肽链和所述第二多肽链中的所述抗体重链可以通过共价键(例如二硫键)连接；所述第一多肽链和所述第二多肽链中的所述抗体重链可以通过非共价键连接。
在本申请中，两条所述第一多肽链可以包含相同的氨基酸序列。例如，两条所述第一多肽链可以具有相同的氨基酸序列。
在本申请中，两条所述第二多肽链可以包含相同的氨基酸序列。例如，两条所述第二多肽链可以具有相同的氨基酸序列。
两条所述第一多肽链可以任意地分别和两条所述第二多肽链通过共价键和/或非共价键连接，从而可以形成本申请所述的双特异性融合蛋白。
在本申请中，所述第一多肽链可以包含SEQ ID NO：23-24中任一项所示的氨基酸序列。
在本申请中，所述第二多肽链可以包含SEQ ID NO：25-27中任一项所示的氨基酸序列。
在本申请中，所述第一多肽链可以包含SEQ ID NO：23所示的氨基酸序列；且所述第二多肽链包含SEQ ID NO：25所示的氨基酸序列；或者，其中所述第一多肽链包含SEQ ID NO：24所示的氨基酸序列；且所述第二多肽链包含SEQ ID NO：26所示的氨基酸序列；或者，其中所述第一多肽链包含SEQ ID NO：23所示的氨基酸序列；且所述第二多肽链包含SEQ ID NO：28所示的氨基酸序列。
在本申请中，所述第一多肽链的氨基酸序列可以如SEQ ID NO：23所示；且所述第二多肽链的氨基酸序列可以如SEQ ID NO：25所示。在本申请中，所述第一多肽链的氨基酸序列可以如SEQ ID NO：24所示；且所述第二多肽链的氨基酸序列可以如SEQ ID NO：26所示。 或者，在本申请中，所述第一多肽链的氨基酸序列可以如SEQ ID NO：23所示；且所述第二多肽链的氨基酸序列可以如SEQ ID NO：28所示。
另一方面，本申请提供一种多核苷酸，其编码本申请所述的双特异性融合蛋白。
本申请所述的多核苷酸可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。重组DNA和分子克隆技术包括由Maniatis，T.，Fritsch，E.F.，Sambrook，J.：Molecular Cloning.A Iaboratory manual.Cold Spring Harbour Laboratory.Cold Spring Harbour，NY，1982和由T.J.Silhavy,M.L.Bennan和L.W.Enquist,Experiments with Gene Fusions,Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y.(1984)以及由Ausubel,F.M.等，Current Protocols in Molecular Biology,pub.by Greene Publishing Assoc.and Wiley-Interscience(1987)描述的那些技术。简而言之，可从基因组DNA片段、cDNA和RNA制备所述核酸，所有这些核酸可直接从细胞中提取或通过各种扩增方法(包括但不限于PCR和RT-PCR)重组产生。
本申请所述的多核苷酸可以包括一条以上不同的核苷酸序列。本申请所述的多核苷酸可以编码本申请所述双特异性融合蛋白的一个以上的不同的组分。例如，所述的多核苷酸可以编码本申请所述的第一多肽链，和/或，所述的多核苷酸可以编码本申请所述的第二多肽链。例如，所述的多核苷酸可以编码所述PD-L1抗体的轻链；可以编码所述PD-L1抗体的重链；可以编码所述第二结合域；可以编码所述连接子；可以编码所述VEGFR1的Ig样结构域；和/或，可以编码所述VEGFR2的Ig样结构域。
另一方面，本申请提供了一种载体，其包含本申请所述的多核苷酸。
在本申请中，所述载体中可包含一种或多种本申请所述的多核苷酸。例如，所述载体可以直接用于表达本申请所述的双特异性融合蛋白。在本申请中，所述载体可以用于表达本申请所述的双特异性融合蛋白中的任何组成部分。在本申请中，可以使用至少2个所述载体表达本申请所述的双特异性融合蛋白。例如，所述载体可以表达本申请所述的第一多肽链，和/或，所述载体可以表达本申请所述的第二多肽链。例如，所述载体可以表达所述PD-L1抗体的轻链，所述PD-L1抗体的重链，所述连接子，所述VEGFR1的Ig样结构域；和/或，所述VEGFR2的Ig样结构域。
在本申请中，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的 条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常可以包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可以为SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多核苷酸可以与所述表达控制元件可操作地连接。所述载体可以包括，质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。
另一方面，本申请提供了一种细胞，其包含或表达本申请所述的双特异性融合蛋白、本申请所述的多核苷酸或本申请所述的载体。
所述细胞可以为原核细胞(例如，细菌细胞)、CHO细胞、NS/0细胞、HEK293T细胞或HEK293A细胞，或者可以为其他真核细胞，例如真菌或酵母细胞。可通过本领域已知的方法将本申请所述的载体引入所述细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。例如，所述宿主细胞可以为COS、CHO、NSO、sf9、sf21、DH5a、BL21(DE3)或TG1。
另一方面，本申请提供本申请所述的双特异性融合蛋白的制备方法，其包括在适于表达本申请所述的双特异性融合蛋白的情况下，培养本申请所述的细胞。
例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。在某些情形中，所述方法还可包括收获(例如分离和/或纯化)本申请所述双特异性融合蛋白的步骤。例如，可以采用蛋白G-琼脂糖或蛋白A-琼脂糖进行亲和层析，还可通过凝胶电泳和/或高效液相色谱等来纯化和分离本申请所述的双特异性融合蛋白。
另一方面，本申请提供一种药物组合物，其包含本申请所述的双特异性融合蛋白，以及药学上可接受的载体。
所述药学上可接受的载体可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
在本申请中，所述药物组合物可被配制用于口服给药，静脉内给药，肌肉内给药，在肿 瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。例如，对于口服给药，药物组合物可以被制备成小片，片剂，胶囊，酏剂，混悬液，糖浆或薄片。对于注射制剂，药物组合物可以被制备成例如一次剂量的剂型的安瓿瓶，或者例如多剂量容器的单元型剂型。药物组合物还可以被制备成溶液，悬浮液，药片，药丸，胶囊和长效制剂。
所述药物组合物的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型，给药途径，病人年龄，性别，体重和疾病的严重程度以及作为活性成分的药物类型。由于所述药物组合物具有优良的体内功效和浓度的持续时间，它可以显著地减少所述药物的给药频率和剂量。
另一方面，本申请提供一种药物分子，其包含所述双特异性融合蛋白。在本申请中，所述药物分子能够具有所述双特异性融合蛋白的性质和/或功能。在本申请中，所述药物分子还可具有其他性质和/或功能。例如，所述药物分子还可包含其他结构。例如，所述药物分子还可包含其他一个或多个氨基酸。
另一方面，本申请提供一种本申请所述双特异性融合蛋白在制备治疗疾病的药物中的应用，其中所述疾病包括肿瘤。
本申请所述的双特异性融合蛋白，和/或本申请所述的药物组合物，其用于治疗疾病，其中所述疾病包括肿瘤。
本申请提供了一种治疗疾病的方法，其包括向有需要的受试者施用有效量的双特异性融合蛋白，和/或本申请所述的药物组合物，其中所述疾病包括肿瘤。
在本申请中，所述肿瘤可以为实体瘤，也可以为非实体瘤。例如，所述肿瘤可以包括本领域技术人员所知晓的各种瘤种，并不限于特定的一种或多种瘤种。例如，所述肿瘤可以包括肺癌、结直肠癌、宫颈癌、肝癌、胃癌和/或肾癌。例如，所述肿瘤可以包括结直肠癌。
在本申请中，所述肿瘤可以为PD-L1阳性(例如，可以为PD-L1过表达)的肿瘤。在某些情况系，所述肿瘤也可以为PD-L1阴性。在本申请中，所述肿瘤可以与VEGF(例如在血管中)的过表达相关。
另一方面，本申请提供了一种抑制血管(例如人)生长的方法，其包括施用有效量的本申请所述的双特异性融合蛋白，和/或本申请所述的药物组合物。
另一方面，本申请提供了一种抑制VEGF受体配体活性的方法，其包括施用有效量的本申请所述的双特异性融合蛋白，和/或本申请所述的药物组合物。
例如，所述VEGF受体配体活性可以包括VEGF和/或VEGFR本身的生物学活性和/或功能。例如，可以包括VEGF与VEGFR的结合。
另一方面，本申请提供了一种抑制PD-L1活性的方法，其包括施用有效量的本申请所述的双特异性融合蛋白，和/或本申请所述的药物组合物。
例如，所述PD-L1活性可以包括PD-L1和/或PD-1本身的生物学活性和/或功能。例如，可以包括PD-L1与PD-1的结合。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
本申请涉及的蛋白质的代号有如下的含义：
SG1201：PD-L1抗体1，轻链可变区的氨基酸序列如SEQ ID NO.16所示；其重链可变区的氨基酸序列如SEQ ID NO.18所示；
SG1202：PD-L1抗体2，其轻链可变区的氨基酸序列如SEQ ID NO.17所示；其重链可变区的氨基酸序列如SEQ ID NO.19所示；
SG1501：VEGFRs(其氨基酸序列如SEQ ID NO.28所示)-Fc(其氨基酸序列如SEQ ID NO.20所示)融合蛋白；
12VF1:其为本申请所述的双特异性融合蛋白，其中第一多肽链的氨基酸序列如SEQ ID NO.23所示；且其第二多肽链的氨基酸序列如SEQ ID NO.25所示；
12VF2：其为本申请所述的双特异性融合蛋白，其中第一多肽链的氨基酸序列如SEQ ID NO.24所示；且其第二多肽链的氨基酸序列如SEQ ID NO.26所示；
12VF8：其为本申请所述的双特异性融合蛋白，其中第一多肽链的氨基酸序列如SEQ ID NO.23所示；且其第二多肽链的氨基酸序列如SEQ ID NO.27所示。
实施例1构建融合蛋白
参照如图1所示的双特异性融合蛋白的结构，依次连接PD-L1抗体1序列、连接子(其氨基酸序列如SEQ ID NO.3所示)和VEGFRs，其中VEGFRs的N端和PD-L1抗体1的重链的C端连接，得到双特异性融合蛋白12VF1。
在12VF1基础上将突变的IgG1Fc(其氨基酸序列如SEQ ID NO.22所示)替换成IgG4Fc(其氨基酸序列如SEQ ID NO.21所示)，获得双特异性融合蛋白12VF8。
参照如图1所示的融合蛋白结构，依次连接PD-L1抗体2、连接子(其氨基酸序列如SEQ ID NO.3所示)和VEGFRs，其中VEGFRs的N端和PD-L1抗体2的重链的C端连接，获得双特异性融合蛋白12VF2。
实施例2与双抗原结合的活性检测
(1)以PD-L1抗体为对照，ELISA评价双功能融合蛋白和PD-L1的结合活性。
将PD-L1(人重组PD-L1蛋白(ECD,His Tag)，购自Sino Biological)包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的抗体SG1201和SG1202，实施例1制备的双特异性融合蛋白12VF1、12VF2和12VF8，37℃反应1小时；PBST洗涤后，加入辣根过氧化物酶标记的羊抗人IgG Fab二抗(Goat Anti-Human IgG(Fab')2(HRP)，Abcam)，37℃反应30分钟；PBST洗涤5次；每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1～2分钟；随后每孔加入100μL 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析双功能融合蛋白与PD-L1的结合能力。
结果如图2所示。图2显示双特异性融合蛋白12VF1与PD-L1的结合能力略强于12VF8，同抗体SG1201。图2显示双特异性融合蛋白12VF2和抗体SG1202与PD-L1的结合能力相当。
(2)以VEGFRs融合蛋白为对照，ELISA评价双功能融合蛋白和VEGF165的结合活性。
将VEGF165(Human VEGF165 Protein,His Tag，Acro Biosystems)包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的双特异性融合蛋白12VF1、12VF2、12VF8和SG1501，37℃反应1小时；PBST洗涤后，加入辣根过氧化物酶标记的羊抗人IgG Fc二抗(Goat anti-human IgG Fc antibody,horseradish peroxidase(HRP)conjugate,affinity purified，Invitrogen)，37℃反应30分钟；PBST洗涤5次；每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μL 2N H 2SO4终止液终止底物反应，酶标仪450nm处读取OD值，分析双特异性融合蛋白12VF1、12VF2和12VF8与VEGF165结合能力。
结果如图3所示。图3显示双特异性融合蛋白12VF1、12VF2和12VF8与VEGF165的结合能力相当，略弱于融合蛋白SG1501。
实施例3同时与双抗原结合的活性检测
以PD-L1抗体和VEGFRs融合蛋白为对照，ELISA评价实施例1制备的双特异性融合蛋白12VF1、12VF2和12VF8同时结合双抗原的生物学活性。
将PD-L1(人重组PD-L1蛋白(ECD,His Tag)，购自Sino Biological)包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；分别加入不同浓度的双特异性融合蛋白12VF1、12VF2、12VF8，PD-L1抗体SG1201、SG1202，和VEGFRs融合蛋白 SG1501，37℃反应1小时；PBST洗涤后，加入生物素标记VEGF165(Biotinylated Human VEGF165 Protein,His,Avitag，Acro Biosystems)，37℃反应30分钟；PBST洗涤5次；加入辣根过氧化物酶标记的亲和素(Streptavidin-HRP，嘉暄生物)，37℃反应30分钟；PBST洗涤5次；每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μL 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析双特异性融合蛋白12VF1、12VF2和12VF8与PD-L1和VEGF165同时结合的能力。
结果如图4所示。图4显示双特异性融合蛋白12VF1、12VF2和12VF8可与PD-L1和VEGF165同时结合，PD-L1抗体SG1201和SG1202，VEGFRs融合蛋白SG1501不能同时结合PD-L1及VEGF165。
实施例4阻断PD-1/PD-L1相互作用的活性分析
以PD-L1抗体SG1201、SG1202为对照，评价双特异性融合蛋白12VF1、12VF2和12VF8阻断PD-1/PD-L1相互作用的生物学活性。
将PD-L1-his(人重组PD-L1蛋白(ECD,His Tag)，购自Sino Biological)包被酶联板，1ug/ml，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；分别加入不同浓度的双特异性融合蛋白12VF1、12VF2、12VF8和抗体SG1201、SG1202，37℃反应1小时；PBST洗涤后，加入生物素标记PD1(Biotinylated Human PD-1/PDCD1 Protein,Fc,Avitag TM,His Tag，Acro Biosystems)至终浓度2μg/ml，37℃反应30min；PBST洗涤5次；加入辣根过氧化物酶标记的亲和素(Streptavidin-HRP，嘉暄生物)，37℃孵育30分钟；PBST洗5遍，每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1-5min；每孔加入100μL 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析双特异性融合蛋白12VF1、12VF2和12VF8对PD-1/PD-L1的阻断作用。
从图5中可以看出，双特异性融合蛋白12VF1、12VF2、12VF8与PD-L1抗体SG1201，SG1202一样可以竞争性阻断PD-1与PD-L1的结合，且活性相似。其中SG1201的IC 50值为0.3968nM，SG1202的IC50值为0.4216nM，12VF1的IC50值为0.393nM，12VF8的IC 50值为0.5002nM，12VF2的IC 50值为0.4256nM。
实施例5阻断VEGF/VEGFR相互作用的活性分析
(1)以融合蛋白SG1501为对照，评价实施例1制备的双特异性融合蛋白12VF1、12VF2和12VF8阻断VEGF/VEGFR相互作用的生物学活性。
将VEGFR1(Human VEGF R1/Flt-1 Protein,His Tag，Acro Biosystems)包被酶联板， 1ug/ml，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；分别加入不同浓度的双特异性融合蛋白12VF1、12VF2、12VF8和融合蛋白SG1501，37℃反应1小时；PBST洗涤后，加入生物素标记VEGF165(Biotinylated Human VEGF165 Protein,His,Avitag，Acro Biosystems)至终浓度0.05μg/ml，37℃反应30min；PBST洗涤5次；加入辣根过氧化物酶标记的亲和素(Streptavidin-HRP，嘉暄生物)，37℃孵育30分钟；PBST洗5遍，每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1-5min；每孔加入100μL 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析双特异性融合蛋白12VF1、12VF2、12VF8对VEGF/VEGFR的阻断作用。
结果如图6所示，图6显示双特异性融合蛋白12VF1、12VF2、12VF8与融合蛋白SG1501一样可以竞争性阻断VEGF与其受体VEGFR1的结合。其中12VF1的IC 50值为3.749nM，12VF2的IC 50值为5.049nM，12VF8的IC 50值为2.182nM，SG1501的IC 50值为1.470nM。
(2)以双特异性融合蛋白IMM25011(参见专利申请US2020/0172623A1)为对照，评价实施例1制备的双特异性融合蛋白12VF1、12VF2和12VF8阻断VEGF165/VEGFR2相互作用的生物学活性。
将VEGF165(VEGF165 Protein,Human,Cynomolgus,Recombinant,HPLC-verified，Sino Biological)包被酶联板，1μg/ml，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；分别加入不同浓度的双特异性融合蛋白12VF1、12VF2、12VF8和IMM25011，37℃反应1小时；PBST洗涤后，加入生物素标记VEGFR2(VEGFR2/KDR Protein,Human,Recombinant(His Tag),Biotinylated，Sino Biological)至终浓度1μg/ml，37℃反应30min；PBST洗涤5次；加入辣根过氧化物酶标记的亲和素(Streptavidin-HRP，嘉暄生物)，37℃孵育30分钟；PBST洗5遍，每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1-5min；每孔加入100μL 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析双特异性融合蛋白12VF1、12VF2、12VF8对VEGF165/VEGFR2的阻断作用。
结果如图7所示，图7显示双特异性融合蛋白12VF1、12VF2、12VF8均可以阻断VEGF165与其受体VEGFR2的结合，且活性均强于IMM25011。其中12VF1的IC 50值为4.092nM，12VF2的IC 50值为3.501nM，12VF8的IC 50值为3.422nM，IMM25011的IC 50值为6.596nM。
(3)以双特异性融合蛋白IMM25011为对照，评价实施例1制备的双特异性融合蛋白12VF1、12VF2和12VF8阻断VEGF121/VEGFR2相互作用的生物学活性。
将VEGF121(VEGF121 Protein,Human,Recombinant，Sino Biological)包被酶联板，1μg/ml，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；分别加入不同浓度的双特 异性融合蛋白12VF1、12VF2、12VF8和IMM25011，37℃反应1小时；PBST洗涤后，加入生物素标记VEGFR2(VEGFR2/KDR Protein,Human,Recombinant(His Tag),Biotinylated，Sino Biological)至终浓度1μg/ml，37℃反应30min；PBST洗涤5次；加入辣根过氧化物酶标记的亲和素(Streptavidin-HRP，嘉暄生物)，37℃孵育30分钟；PBST洗5遍，每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1-5min；每孔加入100μL 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析双特异性融合蛋白12VF1、12VF2、12VF8对VEGF121/VEGFR2的阻断作用。
结果如图8所示，图8显示双特异性融合蛋白12VF1、12VF2、12VF8均可以阻断VEGF121与其受体VEGFR2的结合，且活性均强于IMM25011。其中12VF1的IC 50值为3.536nM，12VF2的IC 50值为3.291nM，12VF8的IC 50值为2.955nM，IMM25011的IC 50值为5.281nM。
(4)以双特异性融合蛋白IMM25011为对照，评价实施例1制备的双特异性融合蛋白12VF1、12VF2和12VF8阻断VEGF C/VEGFR2相互作用的生物学活性。
将VEGF C(VEGF C Protein,Human,Recombinant(His Tag)，Sino Biological)包被酶联板，1μg/ml，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；分别加入15μM的双特异性融合蛋白12VF1、12VF2、12VF8和IMM25011，37℃反应1小时；PBST洗涤后，加入生物素标记VEGFR2(VEGFR2/KDR Protein,Human,Recombinant(His Tag),Biotinylated，Sino Biological)至终浓度20μg/ml，37℃反应30min；PBST洗涤5次；加入辣根过氧化物酶标记的亲和素(Streptavidin-HRP，嘉暄生物)，37℃孵育30分钟；PBST洗5遍，每孔加入100μL TMB(eBioscience)，室温(20±5℃)避光放置1-5min；每孔加入100μL 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析双特异性融合蛋白12VF1、12VF2、12VF8对VEGF C/VEGFR2的阻断作用。
结果如图9所示，图9显示双特异性融合蛋白12VF1、12VF2、12VF8均可以阻断VEGF C与其受体VEGFR2的结合，从而能够有效抑制血管生成，且12VF1、12VF2、12VF8的阻断活性相似。而IMM25011不能阻断VEGF C与其受体VEGFR2的结合。
因此，相比于IMM25011仅能阻断VEGF121与VEGFR2的结合，本申请的双特异性融合蛋白能够既阻断VEGF121与VEGFR2的结合，又能够阻断VEGF C与其受体VEGFR2的结合，从而更加全面地发挥该信号通路的调节效果，起到更加完全地阻断肿瘤部位血管生成的效果。
实施例6抑制HUVEC细胞增殖的活性分析
以融合蛋白SG1501为对照，评价双特异性融合蛋白12VF1、12VF2、12VF8抑制HUVEC 细胞增殖的生物学活性。
将HUVEC细胞用实验培养基重悬后加入96孔细胞培养板中，温箱过夜培养；将VEGF165(
Figure PCTCN2022099322-appb-000001
Human VEGF165 Protein,Tag Free(MALS verified)，Acro Biosystems)用实验培养基稀释至40ng/mL，再与不同浓度的双特异性融合蛋白12VF1、12VF2、12VF8和融合蛋白SG1501等体积混合，37℃预孵育30min，加入96孔细胞培养板中；37℃温箱培养3天后，取出96孔板室温平衡10min，每孔加入100μL CellTiter-Lumi试剂(CellTiter-Lumi TM Luminescent Cell Viability Assay Kit，碧云天)，轻摇混匀，避光反应10min后，用酶标仪读取化学发光的相对荧光强度值(RLU)，分析双特异性融合蛋白12VF1、12VF2和12VF8抑制HUVEC细胞增殖的活性。
从图10中可以看出，双特异性融合蛋白12VF1、12VF2和12VF8均可以抑制HUVEC细胞增殖，略弱于融合蛋白SG1501。
实施例7激活T淋巴细胞的活性分析
以PD-L1抗体SG1201和同型对照抗体为对照，评价双特异性融合蛋白12VF8在混合淋巴细胞反应中激活T淋巴细胞的生物学活性。
用CD14磁珠(CD14 microbeads，Miltenyi)从健康捐赠人的PBMC中分离出单核细胞，并用树突细胞培养试剂盒(ImmunoCultTM Dendritic Cell Culture Kit，Stemcell)诱导分化为树突细胞；用CD4 +T淋巴细胞分离试剂盒(CD4+T Cell Isolation Kit，Miltenyi)从另一个健康捐赠人的PBMC中分离出CD4 +T淋巴细胞，将CD4 +T淋巴细胞与树突细胞按照细胞数5:1的比例，加入96孔细胞培养板，再加入不同浓度的双特异性融合蛋白12VF8，混匀后放37℃培养箱中培养5天；收集细胞上清，使用ELISA试剂盒检测细胞因子IFN-γ和IL-2的浓度，分析双特异性融合蛋白12VF8在混合淋巴细胞反应中激活T淋巴细胞的活性。
从图11-12中可以看出，与同型对照组相比，双特异性融合蛋白12VF8与PD-L1抗体SG1201一样可以以剂量依赖的方式激活T细胞，促进IFN-γ和IL-2的释放。
实施例8 ADCC活性分析
以双特异性融合蛋白IMM25011为对照，PD-L1过表达细胞株CHO-K1/PD-L1为靶细胞，过表达人FcγRIIIa基因及NFAT荧光报告基因的Jurkat细胞(简称Jurkat-ADCC细胞)为效应细胞，用报告基因法评价实施例1制备的双特异性融合蛋白12VF1和12VF8的ADCC活性。
收集CHO-K1/PD-L1细胞，按照每孔2×10 4个细胞加入96孔细胞培养板中；收集Jurkat- ADCC细胞，按照每管1.2×10 5个细胞加入96孔细胞培养板中；加入不同浓度的双特异性融合蛋白IMM25011、12VF1和12VF8，37℃培养箱中孵育6小时；每孔加入100μL荧光素酶检测溶液，室温避光反应10min后，用酶标仪读取化学发光的相对荧光强度值(RLU)，分析双特异性融合蛋白12VF1和12VF8的ADCC活性。
结果如图13所示，图13显示双特异性融合蛋白IMM25011具有ADCC活性，而12VF1和12VF8均无ADCC活性。
上述结果表明，12VF1和12VF8的安全性更好。避免对PD-L1阳性的其他正常细胞的杀伤，从而降低了潜在的毒副作用。由于PD-L1在很多正常免疫细胞也有表达，如髓系的DC、巨噬细胞和淋巴系的T效应细胞、Treg以及NK细胞。12VF1和12VF8在没有ADCC活性的情况下，可避免对于正常组织的杀伤。
实施例9融合蛋白体内抑制肿瘤活性
小鼠结直肠癌MC38模型，评价双特异性融合蛋白12VF8抑制肿瘤活性的效果。
将小鼠结直肠癌细胞MC38接种于雄性C57BL/6J小鼠右侧前胁肋部皮下，共接种36只，在肿瘤生长至58mm 3左右时分组给药，共5组，每组6只，分别为组1：在试验第17天施用PBS(即作为Vehicle组)，腹腔注射给药，每周给药二次，给药三周；组2：施用SG1201(2mg/kg)，腹腔注射给药，每周给药二次，给药三周；组3：施用SG1501(1.4mg/kg)，腹腔注射给药，每周给药二次，给药三周；组4：同时施用SG1201(2mg/kg)以及SG1501(1.4mg/kg)，腹腔注射给药，每周给药二次，给药三周；组5：施用12VF8(2.7mg/kg)组，腹腔注射给药，每周给药二次，给药三周(施用后的结果依次对应图14的组1-5)。每周测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系。末次给药后2小时采集各组内固定小鼠的血清。实验结束时，将荷瘤鼠安乐死，剥离肿瘤称重、拍照。计算肿瘤生长抑制率TGITV(％)并进行统计学分析。
结果显示，实验结束时，组2、组3、组4和组5的肿瘤生长抑制率分别为63％、39％、71％、83％，各治疗组肿瘤体积均显著低于对照的组1(p<0.05)，显示出显著的抗肿瘤作用，有效地抑制了肿瘤生长，其中组5处理的肿瘤体积在所有治疗组中最小，显著低于组3(p<0.05)，抑制肿瘤活性优于施用单药的组2或组3，也优于施用联合用药的组4。治疗期间，荷瘤鼠对组2-组5的处理均表现出很好的耐受性，各组小鼠体重正常，无异常表现，一般状态良好。可见12VF8具有给药安全性。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本 申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (43)

  1. 双特异性融合蛋白，其包含第一结合域和第二结合域，其中：
    所述第一结合域包含特异性结合PD-L1的抗体，其中所述抗体包含两条抗体轻链和两条抗体重链，所述抗体轻链和所述抗体重链通过二硫键连接；
    所述第二结合域包含一个VEGFR1的Ig样结构域和一个VEGFR2的Ig样结构域；
    其中所述VEGFR1的Ig样结构域的N端或所述VEGFR2的Ig样结构域的N端分别与所述抗体重链的C端直接或间接连接。
  2. 根据权利要求1所述的双特异性融合蛋白，其中所述第一结合域特异性结合人PD-L1。
  3. 根据权利要求1-2中任一项所述的双特异性融合蛋白，其中所述第二结合域特异性结合人VEGF家族。
  4. 根据权利要求1-3中任一项所述的双特异性融合蛋白，其中所述第二结合域特异性结合选自下组的蛋白质：VEGF-A、VEGF-B、VEGF-C、VEGF-D和PLGF。
  5. 根据权利要求1-4中任一项所述的双特异性融合蛋白，其中所述VEGFR1的Ig样结构域包含SEQ ID NO：1所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的双特异性融合蛋白，其中所述VEGFR2的Ig样结构域包含SEQ ID NO：2所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的双特异性融合蛋白，其中所述VEGFR1的Ig样结构域和VEGFR2的Ig样结构域直接连接。
  8. 根据权利要求1-7中任一项所述的双特异性融合蛋白，其中所述第二结合域包含SEQ ID NO：28所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的双特异性融合蛋白，其中所述间接连接为通过连接子连接。
  10. 根据权利要求9所述的双特异性融合蛋白，其中所述连接子包含SEQ ID NO：3所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的双特异性融合蛋白，其中所述抗体轻链包含轻链可变区，其中所述轻链可变区包含LCDR1-3，其中LCDR1包含SEQ ID NO：4、10中任一项所示的氨基酸序列。
  12. 根据权利要求11所述的双特异性融合蛋白，其中LCDR2包含SEQ ID NO：5、11中任一项所示的氨基酸序列。
  13. 根据权利要求11-12中任一项所述的双特异性融合蛋白，其中LCDR3包含SEQ ID NO：6、12中任一项所示的氨基酸序列。
  14. 根据权利要求11-13中任一项所述的双特异性融合蛋白，其中所述LCDR1-3的氨基酸序 列选自下述任一组：
    (1)所述LCDR1包含SEQ ID NO:4所示的氨基酸序列，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:6所示的氨基酸序列；
    (2)所述LCDR1包含SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:12所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的双特异性融合蛋白，其中所述抗体轻链包含轻链可变区，其中所述轻链可变区包含SEQ ID NO：16-17中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的双特异性融合蛋白，其中所述抗体轻链包含轻链恒定区，其中轻链恒定区源自选自下组蛋白的轻链恒定区：Igκ和Igλ。
  17. 根据权利要求1-16中任一项所述的双特异性融合蛋白，其中所述抗体重链包含重链可变区，其中所述重链可变区包含HCDR1-3，其中所述HCDR1包含SEQ ID NO：7、13中任一项所示的氨基酸序列。
  18. 根据权利要求17所述的双特异性融合蛋白，其中HCDR2包含SEQ ID NO：8、14中任一项所示的氨基酸序列。
  19. 根据权利要求17-18中任一项所述的双特异性融合蛋白，其中HCDR3包含SEQ ID NO：9、15中任一项所示的氨基酸序列。
  20. 根据权利要求17-19中任一项所述的双特异性融合蛋白，其中所述HCDR1-3包含选自下组的氨基酸序列：
    (1)所述HCDR1包含SEQ ID NO:7所示的氨基酸序列，所述HCDR2包含SEQ ID NO:8所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:9所示的氨基酸序列；
    (2)所述HCDR1包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2包含SEQ ID NO:14所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:15所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的双特异性融合蛋白，其中所述抗体重链包含重链可变区，所述重链可变区包含SEQ ID NO：18-19中任一项所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的双特异性融合蛋白，其中所述抗体重链包含Fc区域。
  23. 根据权利要求22所述的双特异性融合蛋白，其中所述Fc区域源自选自下组蛋白的Fc：IgG1和IgG4。
  24. 根据权利要求22-23中任一项所述的双特异性融合蛋白，所述Fc区域在选自下组的氨基酸位置处包含氨基酸突变：N298、D357和L359。
  25. 根据权利要求22-24中任一项所述的双特异性融合蛋白，所述Fc区域包含选自下组的氨基酸突变：N298A、D357E和L359M。
  26. 根据权利要求22-25中任一项所述的双特异性融合蛋白，其中所述Fc区域包含SEQ ID NO：20-22中任一项所示的氨基酸序列。
  27. 根据权利要求9-26中任一项所述的双特异性融合蛋白，其为由两拷贝的第一多肽链和第二多肽链组成的多聚体，
    其中所述第一多肽链包含所述抗体轻链，
    其中所述第二多肽链自N端依次包含所述抗体重链、所述连接子和所述第二结合域。
  28. 根据权利要求27所述的双特异性融合蛋白，其中所述第一多肽链包含SEQ ID NO：23-24中任一项所示的氨基酸序列。
  29. 根据权利要求27-28中任一项所述的双特异性融合蛋白，其中所述第二多肽链包含SEQ ID NO：25-27中任一项所示的氨基酸序列。
  30. 根据权利要求27-29中任一项所述的双特异性融合蛋白，其中所述第一多肽链包含SEQ ID NO：23所示的氨基酸序列；所述第二多肽链包含SEQ ID NO：25所示的氨基酸序列；或者，
    其中所述第一多肽链包含SEQ ID NO：24所示的氨基酸序列；所述第二多肽链包含SEQ ID NO：26所示的氨基酸序列；或者，
    其中所述第一多肽链包含SEQ ID NO：23所示的氨基酸序列；所述第二多肽链包含SEQ ID NO：27所示的氨基酸序列。
  31. 多核苷酸，其编码权利要求1-30中任一项所述的双特异性融合蛋白。
  32. 载体，其包含权利要求31所述的多核苷酸。
  33. 细胞，其包含权利要求32所述的载体。
  34. 权利要求1-30中任一项所述的双特异性融合蛋白的制备方法，其包括在适于表达权利要求1-30中任一项所述的双特异性融合蛋白的情况下，培养权利要求33所述的细胞。
  35. 药物组合物，其包含权利要求1-30中任一项所述的双特异性融合蛋白，以及任选地药学上可接受的载体。
  36. 药物分子，其包含权利要求1-30中任一项所述的双特异性融合蛋白。
  37. 权利要求1-30中任一项所述的双特异性融合蛋白、权利要求31所述的多核苷酸、权利要求32所述的载体、权利要求33所述的细胞、权利要求35所述的药物组合物、和/或权利要求36所述的药物分子在制备治疗疾病的药物中的应用，其中所述疾病包括肿瘤。
  38. 根据权利要求37所述的应用，其中所述疾病包括实体瘤和非实体瘤。
  39. 根据权利要求37-38中任一项所述的应用，其中所述疾病包括PD-L1阳性肿瘤。
  40. 根据权利要求37-39中任一项所述的应用，其中所述肿瘤包括肺癌、结直肠癌、宫颈癌、 肝癌、胃癌和/或肾癌。
  41. 一种抑制血管生长的方法，其包括施用有效量的权利要求1-30中任一项所述的双特异性融合蛋白、权利要求31所述的多核苷酸、权利要求32所述的载体、权利要求33所述的细胞、权利要求35所述的药物组合物和/或权利要求36所述的药物分子。
  42. 一种抑制VEGF受体配体活性的方法，其包括施用有效量的权利要求1-30中任一项所述的双特异性融合蛋白、权利要求31所述的多核苷酸、权利要求32所述的载体、权利要求33所述的细胞、权利要求35所述的药物组合物和/或权利要求36所述的药物分子。
  43. 一种抑制PD-L1活性的方法，其包括施用有效量的权利要求1-30中任一项所述的双特异性融合蛋白、权利要求31所述的多核苷酸、权利要求32所述的载体、权利要求33所述的细胞、权利要求35所述的药物组合物和/或权利要求36所述的药物分子。
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