Bispecific fusion proteins

Abstract

overview 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 that specifically binds to PD-L1, the antibody comprises two antibody light chains and two antibody heavy chains, the antibody light chain and the antibody heavy chain are linked by a disulfide bond, and the second binding domain comprises one Ig-like structural domain of VEGFR1 and one Ig-like structural domain of VEGFR2, and the N-terminus of the Ig-like structural domain of VEGFR1 or the N-terminus of the Ig-like structural domain of VEGFR2 is directly or indirectly linked to the C-terminus of the antibody heavy chain, respectively. Also relates to a pharmaceutical composition comprising the bispecific fusion protein and its use in pharmaceutical preparations, wherein the bispecific fusion protein can effectively and safely kill tumor cells.

Description

[Technical field]

[0001] This application relates to the biopharmaceutical field, and specifically to bispecific fusion proteins. [Background technology]

[0002] Currently, tumor diseases are becoming increasingly serious, and the immune checkpoints PD-1 / PD-L1 and VEGF have become important targets for tumor treatment. PD-L1 is overexpressed in a variety of malignancies and is often associated with poor prognosis. Therapeutic targeting of PD-1, and other molecules that signal through interactions with PD-1 (e.g., PD-L1 and PD-L2), is an exciting area of ​​tumor therapy. VEGF is a potent and specific mitogen for vascular endothelial cells, and can promote all aspects of the angiogenesis process. Each VEGF receptor (VEGFR) has a different role. VEGFR- plays a role in regulating endothelial growth, differentiation, and permeability, while VEGFR-1 is involved in regulating endothelial cell migration and aggregation and inhibits signaling through VEGFR-2. Binding of VEGF to VEGFR leads to dimerization and autophosphorylation of VEGFR, which transmits signals through multiple intracellular pathways and ultimately plays a role. There is a need for new fusion proteins that can effectively inhibit tumor growth. Summary of the Invention

[0003] The present application provides bispecific fusion proteins and corresponding polynucleotides, vectors, cells, manufacturing methods, pharmaceutical compositions and uses. The bispecific fusion proteins described herein have at least one of the following properties: 1) specifically bind to PD-L1, 2) inhibit the binding of PD-1 to PD-L1, 3) specifically bind to VEGF, 4) inhibit the binding of VEGF to VEGFR, 5) suppress tumor growth (in vivo), 6) activate immune cells (e.g., T cells), 7) promote cytokine secretion (e.g., interferon and / or cytokines) of immune cells (e.g., T cells), and / or 8) inhibit tumor neovascularization and promote vascular normalization.

[0004] In one aspect, the application provides a bispecific fusion protein comprising a first binding domain and a second binding domain, wherein the first binding domain comprises an antibody that specifically binds to PD-L1, the antibody comprising two antibody light chains and two antibody heavy chains, the antibody light chains and the antibody heavy chains being linked by a disulfide bond, and the second binding domain comprises one VEGFR1 Ig-like structural domain and one VEGFR2 Ig-like structural domain, and the N-terminus of the VEGFR1 Ig-like structural domain or the N-terminus of the VEGFR2 Ig-like structural domain is linked directly or indirectly to the C-terminus of the antibody heavy chain, respectively. In some embodiments, the first binding domain specifically binds to human PD-L1. In some embodiments, the second binding domain specifically binds to the human VEGF family.

[0005] In some embodiments, the second binding domain specifically binds to a protein selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D and PLGF. In some embodiments, the Ig-like structural domain of VEGFR1 comprises the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the Ig-like structural domain of VEGFR2 comprises the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the Ig-like structural domain of VEGFR1 and the Ig-like structural domain of VEGFR2 are directly linked. In some embodiments, the second binding domain comprises the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the indirect link is via a linker. In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the antibody light chains each comprise a light chain variable region comprising LCDR1-3, and the LCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs:4 and 10. In some embodiments, the LCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs:5 and 11. In some embodiments, the LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs:6 and 12. In some embodiments, the LCDR1-3 comprises an amino acid sequence selected from any one of the following groups: (1) said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; (2) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:10, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:11, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the antibody light chains each comprise a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs:16-17. In some embodiments, the antibody light chains each comprise a light chain constant region, wherein the light chain constant region is derived from a light chain constant region of a protein selected from the group consisting of Igκ and Igλ. In some embodiments, the antibody heavy chains each comprise a heavy chain variable region comprising HCDR1-3, wherein HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs:7 and 13. In some embodiments, the HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs:8 and 14. In some embodiments, the HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs:9 and 15. In some embodiments, the HCDR1-3 comprises an amino acid sequence selected from the following group: (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:7, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:8, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:9; (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:13, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:14, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the antibody heavy chains each comprise a heavy chain constant region, and the heavy chain constant regions comprise an amino acid sequence set forth in any one of SEQ ID NOs:18-19. In some embodiments, the antibody heavy chains each comprise an Fc region. In some embodiments, the Fc region is derived from the Fc of a protein selected from the group consisting of IgG1 and IgG4. In some embodiments, the Fc region comprises an amino acid mutation at an amino acid position selected from the group consisting of N298, D357, and L359. In some embodiments, the Fc region comprises an amino acid mutation selected from the group consisting of N298A, D357E and L359M. In some embodiments, the Fc region comprises an amino acid sequence set forth in any one of SEQ ID NOs:20-22. In some embodiments, the bispecific fusion protein is a multimer consisting of two copies of a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the antibody light chain and the second polypeptide chain comprises, in N-terminal order, the antibody heavy chain, the linker, and the second binding domain. In some embodiments, the first polypeptide chain comprises an amino acid sequence set forth in any one of SEQ ID NOs:23-24. In some embodiments, the second polypeptide chain comprises an amino acid sequence set forth in any one of SEQ ID NOs:25-27. In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:23 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:25; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:24 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:26; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:23 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:27. In another aspect, the present application provides a polynucleotide encoding a bispecific fusion protein described herein.

[0006] In another aspect, the present application provides a vector comprising a polynucleotide described herein. In another aspect, the present application provides a cell comprising the vector described herein. In another aspect, the present application provides a method for producing a bispecific fusion protein described herein, comprising culturing a cell described herein under conditions suitable for expression of a bispecific fusion protein described herein. In another aspect, the present application provides a pharmaceutical composition comprising a bispecific fusion protein described herein and, optionally, a pharma- ceutically acceptable carrier. In another aspect, the present application provides a drug molecule comprising a bispecific fusion protein described herein. In another aspect, the present application provides the use of the bispecific fusion protein, the polynucleotide, the vector, the cell, the pharmaceutical composition and / or the pharmaceutical molecule in the manufacture of a medicament for the treatment of a disease, wherein the disease comprises a tumor. In some embodiments, the disease includes solid and non-solid tumors. In some embodiments, the disease comprises a PD-L1 positive tumor. In some embodiments, the tumor comprises lung cancer, colorectal cancer, cervical cancer, liver cancer, gastric cancer and / or renal cancer. In another aspect, the present application provides a method for inhibiting vascular growth, comprising administering an effective amount of the bispecific fusion protein, the polynucleotide, the vector, the cell, the drug composition and / or the drug molecule. In another aspect, the present application provides a method for inhibiting activity of a VEGF receptor ligand, comprising administering an effective amount of the bispecific fusion protein, the polynucleotide, the vector, the cell, the drug composition and / or the drug molecule. In another aspect, the present application provides a method for inhibiting the activity of PD-L1, comprising administering an effective amount of the bispecific fusion protein, the polynucleotide, the vector, the cell, the drug composition and / or the drug molecule. Those skilled in the art can easily ascertain other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the contents of the present application will enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention of the present application. Therefore, the accompanying drawings and the description of the present application are merely illustrative and not restrictive. [Brief description of the drawings]

[0007] Particular features of the invention are set forth in the appended claims. A better understanding of the features and advantages of the invention can be obtained by reference to the exemplary embodiments described in detail herein and the accompanying drawings, the brief description of which is set forth below. [Figure 1] Structure of the bispecific fusion proteins described herein. [Diagram 2] Specific binding of the bispecific fusion proteins described herein to human PD-L1. [Diagram 3] Specific binding of the bispecific fusion proteins described herein to VEGF165. [Figure 4] Specific and simultaneous binding of the bispecific fusion proteins described herein to human PD-L1 and VEGF165. [Diagram 5] Inhibition of the interaction of the bispecific fusion proteins described herein with PD-1 and PD-L1. [Figure 6] Inhibition of the interaction of the bispecific fusion proteins described herein with VEGF and VEGFR. [Figure 7] Inhibition of the interaction of the bispecific fusion proteins described herein with VEGF165 and VEGFR2. [Figure 8] Inhibition of the interaction of the bispecific fusion proteins described herein with VEGF121 and VEGFR2. [Figure 9] Inhibition of the interaction of the bispecific fusion proteins described herein with VEGF C and VEGFR2. [Figure 10] Inhibition of the bispecific fusion proteins described herein on HUVEC cell proliferation. [Figure 11] Activation of lymphocytes for IFN-γ secretion by bispecific fusion proteins described herein. [Figure 12] Activation of lymphocytes to secrete IL-2 by the bispecific fusion proteins described herein. [Figure 13]ADCC activity assay of the bispecific fusion proteins described herein. [Figure 14] Inhibition of mouse colon cancer growth by the bispecific fusion proteins described herein.

[0008] Specific embodiments The embodiments of the present application are described in the following specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed herein. Term definition As used herein, the term "bispecific" generally refers to the ability of the fusion proteins described herein to interact with two different ligands. As used herein, the bispecific fusion proteins are capable of specifically binding to PD-L1 and are also capable of specifically binding to VEGF. As used herein, the term "specific binding" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that allows the presence of the target to be determined in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to that target with higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets. In one embodiment, the extent of binding of the antibody to a non-related target is less than about 10% of the binding of the antibody to the target, as measured by radioimmunoassay (RIA). For example, as used herein, the bispecific fusion protein has a binding affinity of <1×10 -7 The PD-L1 antibody can bind to PD-L1 and VEGF with a dissociation constant (KD) of <1×10 -7 It can bind to PD-L1 with a dissociation constant (KD) of less than or equal to M. In some embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins of different species. In another embodiment, specific binding can include, but does not require, exclusive binding. As used herein, the term "first binding domain" generally refers to a binding domain that can specifically bind to PD-L1. For example, the first binding domain may comprise an antibody that specifically binds to PD-L1. In this application, the term "second binding domain" generally refers to a binding domain that can specifically bind to VEGF.For example, the second binding domain can include one Ig-like structural domain of VEGFR1 and one Ig-like structural domain of VEGFR2. In this application, the term "PD-L1" generally refers to programmed death ligand 1 protein, its functional variants and / or its functional fragments. PD-L1 is also called cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). The PD-L1 can be a protein encoded by the CD274 gene. PD-L1 binds to its receptor, such as programmed cell death protein 1 (PD-1), which can be expressed on 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. Science, 2001; 291:319-22). Complex formation between PD-L1 and PD-1 can exert immunosuppressive effects by suppressing T cell proliferation and production of the cytokines IL-2 and IFN-γ (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-l: PD-L inhibitory pathway affects both CD4(+)and CD8(+) T cells and is overcome by IL-2. Eur J. Immunol. 2002, 32:634-643). The term "PD-L1" can include any naturally occurring PD-L1 of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length," unprocessed PD-L1 and any form of PD-L1 produced by processing of cells. PD-L1 can exist as a transmembrane or soluble protein, and the term also includes naturally occurring variants of PD-L1, such as splice variants and allelic variants.The basic structure of PD-L1 may include four domains: an extracellular Ig-like V-type domain, an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. The sequence of PD-L1 is known in the art. For example, information about the human PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 29126. Also, for example, information about the mouse PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 60533. Also, for example, information about the cynomolgus monkey PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 102145573. An exemplary full-length human PD-L1 protein amino acid sequence can be found under NCBI Accession No. NP_054862 or UniProt Accession No. Q9NZQ7. The amino acid sequence of an exemplary full-length mouse PD-L1 protein can be found under NCBI Accession No. NP_068693 or UniProt Accession No. Q9EP73.The amino acid sequence of an exemplary full-length cynomolgus monkey PD-L1 protein can be found under NCBI Accession No. XP_005581836 or UniProt Accession No. G7PSE7.

[0009] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, including a polypeptide that contains an antigen-binding site, whether generated in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units and one more polypeptide called the J chain, and contain 10 antigen-binding sites, whereas IgA antibodies contain two to five basic four-chain units, which combine with the J chain to form a multivalent bond. For IgG, the four-chain unit is usually about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isoform. 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) in the α and γ chains, and four CH domains in the μ and ε isoforms. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain at the 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 an interface between the light and heavy chain variable domains. A pair of VH and VL together forms a single antigen-binding site. The structure and properties of different classes of antibodies are described, for example, in Basic and Clinical Immunology, 8 thEdition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and chapter 6. L chains from any vertebrate can be classified into one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domain. Immunoglobulins can be classified into different classes or isoforms depending on the amino acid sequence of their heavy (CH) constant domain. There are five types of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. γ and α are further subclassified based on relatively minor differences in CH sequence and function, e.g., IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and Igκ1 are expressed in humans. In this application, the term "CDR" refers to the region of the variable domain of an antibody that usually has a highly variable sequence and / or forms a structurally defined loop. Antibodies usually contain six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). In natural antibodies, HCDR3 and LCDR3 represent the majority of the diversity of the six CDRs, and HCDR3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al, Immunity 13:37-45(2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25(Lo, ed., Human Press, Totowa, NJ,2003). In the present application, the term "VEGF" generally refers to vascular endothelial growth factor. VEGF may be involved in the regulation of normal and abnormal angiogenesis, as well as angiogenesis associated with tumors and intraocular lesions (see, for example, Ferrara, N. and Davis-Smyth, T., Endocr.Rev.l8, 1997, 4-25). VEGF can play an important regulatory role in angiogenesis during embryonic vasculogenesis 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. In the present application, the VEGF can be human VEGF.

[0010] In this application, the term "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 mosaic protein. The extramembrane portion of VEGFR consists of about 750 amino acid residues and consists of seven Ig-like structural domains structurally similar to immunoglobulins. The second Ig structural domain in the extramembrane region of VEGFR1 is the region that binds to ligands. The different spliceosomes of VEGFR1 inhibit the binding of VEGF to VEGFR2 by competitively binding to VEGF (e.g., it can bind to VEGF-A, VEGF-B). In this application, the term "VEGFR2" generally refers to vascular endothelial growth factor receptor 2. The third Ig structural domain of VEGFR2 may play a role in the specificity of binding to ligands. VEGFR2 can bind to VEGF-A, VEGF-E. In this application, the term "Ig-like structural domain" generally refers to a structure in the extracellular region of a VEGFR that may be involved in binding to VEGF. For example, the first Ig-like structural domain of the extracellular region of VEGFR2 is the site required for binding to VEGF, the second and third Ig-like structural domains are the main sites for tightly binding to VEGF, the receptor forms a homodimer active form via the fourth Ig-like structural domain, and the fifth to seventh Ig-like structural domains are not closely related to VEGF binding. For example, the extracellular region of VEGFR1 has seven Ig-like structural domains that may be involved in binding to VEGF and promoting angiogenesis.

[0011] In this application, the term "PLGF" generally refers to placental growth factor, which may be encoded by the PGF gene. The PLGF may be a member of the VEGF subfamily. The PLGF may be expressed in human umbilical vein endothelial cells (HUVE) and placenta. The PLGF may play a role in the proliferation and differentiation of trophoblast cells. The PLGF may be involved in angiogenesis.

[0012] In the present application, the term "directly connected" is used in contrast to the term "indirectly connected", and the term "directly connected" generally refers to a direct connection. For example, the direct connection is a case where the substances are directly connected without a spacer between them. The spacer can be a linker. For example, the linker can be a peptide linker. The term "indirect connection" generally refers to a situation where the substances are not directly connected between them. For example, the indirect connection may be a case where the substances are connected through a spacer. For example, the N-terminus of the Ig-like structural domain of VEGFR1 described in the present application or the N-terminus of the Ig-like structural domain of VEGFR2 described in the present application can be linked to the C-terminus of the antibody heavy chain, respectively. For example, one Ig-like structural domain of VEGFR1 described in the present application can be directly linked to one Ig-like structural domain of VEGFR2.

[0013] In the present application, the term "amino acid mutation" generally refers to the replacement of at least one existing amino acid residue with another different amino acid residue. The replaced amino acid residue may be a "naturally occurring amino acid residue", for example, 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 (Tyr) and valine (Val). The substituted amino acid residue may also be an amino acid residue that exists in a non-natural form, such as, for example, n-leucine, ornithine, n-valine, homoserine, aib and other amino acid residue analogs. In the present application, the amino acid substitution may be a non-conservative substitution. The non-conservative substitution may include changing an amino acid residue in a target protein or polypeptide in a non-conservative manner, such as changing an amino acid residue with a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue with a different side chain size or a different property (e.g., hydrophobicity). In the present application, the amino acid substitution may be a conservative substitution. The conservative substitution may include changing an amino acid residue in a target protein or polypeptide in a conservative manner, such as changing an amino acid residue with a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue with the same or similar size or the same or similar property (e.g., still hydrophilicity). These conservative substitutions usually do not significantly affect the structure or function of the resulting protein.

[0014] In this application, the term "multimer" generally refers to a molecule formed by the combination of monomers. For example, the multimer may be a multimeric protein that includes at least two structurally identical or structurally different components. For example, the multimer may be a molecule of two or more polypeptide chains that are linked by covalent or non-covalent association or covalent or non-covalent interaction. For example, the multimer may be a dimer or a tetramer.

[0015] As used herein, the term "first polypeptide chain" generally refers to the light chain of a bispecific fusion protein described herein that targets a PD-L1 antibody. As used herein, the term "second polypeptide chain" generally refers to a polypeptide comprising a heavy chain of a bispecific fusion protein described herein that targets a PD-L1 antibody, a linker described herein, and a second binding domain described herein that targets VEGF. For example, the second polypeptide chain may consist, in order from the N-terminus, of a heavy chain of a bispecific fusion protein described herein that targets a PD-L1 antibody, a linker described herein, and a second binding domain described herein that targets VEGF.

[0016] In this application, the terms "first" and "second" have no distinction in terms of order, and the "second" is merely a term used to indicate content different from the "first". As used herein, the term "nucleic acid molecule" generally refers to nucleotides of any length (e.g., nucleotides that can be in isolated form), deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized. In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and in which the protein can be expressed. A vector expresses a gene in a host cell by transformation, transformation or transfection into the host cell. For example, a vector can include a plasmid, a bacteriophage, a coplasmid, an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a phage such as a lambda phage or an M13 phage, an animal virus, etc. Types of animal viruses used as vectors are reverse transcriptase viruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and human papilloma viruses (in the case of SV 40). A vector contains various expression control elements, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. A vector can also contain a replication origin. Vectors may also contain components that aid in cell entry, including but not limited to viral particles, liposomes, protein shells, and the like.

[0017] In this application, the term "cell" generally refers to an individual cell, cell line, or cell culture that can be or has been a recipient of a subject plasmid or vector, including a nucleic acid molecule described herein or a vector described herein. A cell can include the progeny of a single cell. Due to spontaneous, accidental, or deliberate mutations, the progeny may not necessarily be identical (morphological or genomic of the total DNA cross-complement) to the primordial mother cell. A cell can include a cell that has been transfected in vitro with a vector described herein. A cell can be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell. For example, COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, human non-small cell lung cancer A549 cells, human squamous skin cancer A431 cells, clear cell renal cell carcinoma 786-O cells, human pancreatic adenocarcinoma MIA PaCa-2 cells, erythroleukemia K562 cells, acute T-cell leukemia Jurkat cells, human breast cancer MCF-7 cells, human breast cancer MDA-MB-231 cells, human breast cancer MDA-MB-468 cells, human breast cancer SKBR3 cells, human ovarian cancer SKOV3 cells, lymphoma U-937 cells, lymphoma Raji cells, human myeloma U266 cells, or human multiple myeloma RPMI8226 cells. In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are HEK293 cells.

[0018] As used herein, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, e.g., a human patient. For example, a pharmaceutical composition described herein may include a bispecific fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and optionally, a pharmaceutical acceptable adjuvant. In addition, the pharmaceutical composition may also include a suitable formulation of one or more (pharmaceutical effective) support agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. As used herein, acceptable components of the pharmaceutical composition may be non-toxic to a recipient at the dosages and concentrations. The formats of the pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0019] As used herein, the term "pharmaceutical acceptable adjuvant" generally refers to any solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with drug administration and are generally safe, non-toxic, and not biologically or otherwise undesirable. In the present application, the term "drug molecule" generally refers to a molecule that has a desired biological effect. Drugs can be prophylactic and therapeutic. Drug molecules include, but are not limited to, proteins, including protein molecules, including but not limited to peptides, polypeptides, post-translationally modified proteins, fusion proteins, antibodies, and the like. For example, in the present application, the drug molecule can include one or more other amino acids in addition to the bispecific fusion proteins described herein. For example, the drug molecule can include other structures in addition to the bispecific fusion proteins described herein. In the present application, the drug molecule can also include small molecule structures including inorganic or organic compounds. In the present application, 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 (e.g., antisense, RNAi, etc.), intronic sequences, triple-helical nucleic acid molecules, and aptamers.

[0020] In this application, the term "comprising" generally refers to the inclusion of features explicitly specified without the exclusion of other elements. As used herein, the term "about" generally refers to a variation of 0.5% to 10% above the specified value, for example, a variation of 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%, or 10%.

[0021] Detailed Description of the Invention In one aspect, the application provides a bispecific fusion protein comprising a first binding domain and a second binding domain, wherein the first binding domain comprises an antibody that specifically binds to PD-L1, the antibody comprising two antibody light chains and two antibody heavy chains, the antibody light chains and the antibody heavy chains being linked by a disulfide bond, and the second binding domain comprises one VEGFR1 Ig-like structural domain and one VEGFR2 Ig-like structural domain, and the N-terminus of the VEGFR1 Ig-like structural domain or the N-terminus of the VEGFR2 Ig-like structural domain is linked directly or indirectly to the C-terminus of the antibody heavy chain, respectively. In the present application, the first binding domain can specifically bind to human PD-L1. In the present application, the second binding domain is capable of specifically binding to the human VEGF family. In the present application, the second binding domain is capable of specifically binding to a protein selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D and PLGF. In the present application, the Ig-like structural domain of VEGFR1 may comprise the amino acid sequence shown in SEQ ID NO:1. In the present application, the Ig-like structural domain of VEGFR2 may comprise the amino acid sequence shown in SEQ ID NO:2. In the present application, the Ig-like structural domain of VEGFR1 can be directly linked to the Ig-like structural domain of VEGFR2. For example, the second binding domain can include, in order from the N-terminus, the Ig-like structural domain of VEGFR1 and the Ig-like structural domain of VEGFR2. For example, the second binding domain can consist, in order from the N-terminus, of the Ig-like structural domain of VEGFR1 and the Ig-like structural domain of VEGFR2. In the present application, the second binding domain can include the amino acid sequence shown in SEQ ID NO:28. In the present application, the indirect link may be linked by a linker.For example, in the bispecific fusion protein, the N-terminus of the Ig-like structural domain of VEGFR1 in the second binding domain may be linked to the C-terminus of the antibody heavy chain via the linker.For example, in the bispecific fusion protein, the N-terminus of the Ig-like structural domain of VEGFR2 in the second binding domain may be linked to the C-terminus of the antibody heavy chain via the linker. In the present application, the linker may comprise the amino acid sequence shown in SEQ ID NO:3. In the present application, any known PD-L1 antibody may be used as the antibody in the first binding domain, as long as it has a light chain variable region and a heavy chain variable region that can specifically bind to PD-L1. In the present application, the antibody light chains each comprise a light chain variable region, and the light chain variable region may comprise LCDR1-3, where LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10. In the present application, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:11. In the present application, LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:12. In the present application, the antibody light chains each comprise a light chain variable region, and the light chain variable region may comprise LCDR1-3, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:4, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:5, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:6, or the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:10, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:11, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:12. In the present application, the antibody light chains may each comprise a light chain variable region, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO:16 or SEQ ID NO:17. In the present application, the antibody light chains may each comprise a light chain constant region, which may be derived from a light chain constant region of a protein selected from the group consisting of Igκ and Igλ. In the present application, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO:23 or SEQ ID NO:24. In the present application, the antibody heavy chains may each comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3, where HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:13. In the present application, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:14. In the present application, HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:15.

[0022] In the present application, the antibody heavy chains may each comprise a heavy chain variable region, the heavy chain variable region may comprise HCDR1-3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:7, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:8, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:9, or the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:13, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:14, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:15. In the present application, the antibody heavy chains may each 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. In the present application, the light chain variable region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO:16 and the heavy chain variable region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO:18, or 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. In the present application, the antibody heavy chain may comprise an Fc region. In the present application, the Fc region may be derived from the Fc of a protein selected from the group consisting of IgG1 and IgG4. In the present application, the Fc region may comprise an amino acid mutation at an amino acid position selected from the group consisting of N298, D357 and L359. In the present application, the amino acid mutation at position N298 of the Fc region may comprise N298A, i.e., the amino acid N at position 82 in the amino acid sequence shown in SEQ ID NO:20 is mutated to the amino acid A. In the present application, the amino acid mutation at position D357 of the Fc region may comprise D357E, i.e., the amino acid D at position 141 in the amino acid sequence shown in SEQ ID NO:20 is mutated to the amino acid E. In the present application, the amino acid mutation at position L359 of the Fc region may comprise L359M, i.e., the amino acid L at position 143 in the amino acid sequence shown in SEQ ID NO:20 is mutated to the amino acid M. In the present application, the Fc region may comprise an amino acid mutation selected from the group consisting of N298A, D357E and L359M. In the present application, the Fc region may have an amino acid mutation, which may consist of N298A, D357E and L359M. In the present application, the Fc region may comprise an amino acid sequence set forth in any one of SEQ ID NOs:20-22. In the present application, the bispecific fusion protein may be a multimer consisting of two copies of a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain may comprise the antibody light chain and the second polypeptide chain may comprise, in order from the N-terminus, the antibody heavy chain, the linker, and the second binding domain. In the present application, the first polypeptide chain may consist of the antibody light chain. For example, the antibody heavy chain in the first polypeptide chain and the second polypeptide chain may be linked via a covalent bond (e.g., a disulfide bond), and the antibody heavy chain in the first polypeptide chain and the second polypeptide chain may be linked via a non-covalent bond. In the present application, the two first polypeptide chains may comprise the same amino acid sequence. For example, the two first polypeptide chains may comprise the same amino acid sequence. In the present application, the two second polypeptide chains may comprise the same amino acid sequence. For example, the two second polypeptide chains may comprise the same amino acid sequence. The two first polypeptide chains may be linked, optionally via covalent and / or non-covalent bonds, to two second polypeptide chains, respectively, to form the bispecific fusion protein described herein. In the present application, the first polypeptide chain may comprise an amino acid sequence set forth in any one of SEQ ID NOs:23-24, respectively. In the present application, the second polypeptide chain may comprise an amino acid sequence set forth in any one of SEQ ID NOs:25-27, respectively.

[0023] In the present application, the first polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:23 and the second polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:25, or the first polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:24 and the second polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:26, or the first polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:23 and the second polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:28. In the present application, the amino acid sequence of the first polypeptide chain may be set forth in SEQ ID NO:23 and the amino acid sequence of the second polypeptide chain may be set forth in SEQ ID NO:25. In the present application, the amino acid sequence of the first polypeptide chain may be set forth in SEQ ID NO:24 and the amino acid sequence of the second polypeptide chain may be set forth in SEQ ID NO:26. Or, in the present application, the amino acid sequence of the first polypeptide chain may be set forth in SEQ ID NO:23 and the amino acid sequence of the second polypeptide chain may be set forth in SEQ ID NO:28. In another aspect, the present application provides a polynucleotide encoding a bispecific fusion protein described herein.

[0024] The polynucleotides described herein may be isolated, for example, produced or synthesized (i) in vitro, e.g., by polymerase chain reaction (PCR) amplification, (ii) by clonal recombination, (iii) by purification, e.g., enzymatic digestion and stepwise separation by gel electrophoresis, or (iv) synthetically, e.g., chemically. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques. Recombinant DNA and molecular cloning techniques include those described in Maniatis, T., Fritsch, EF, Sambrook, J.: Molecular Cloning. A laboratory manual. Cold Spring Harbour Laboratory. Cold Spring Harbour, NY,1982; TJ Silhavy, ML Bennan and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984); and Ausubel, FM et al., Current Protocols in Molecular Biology, pub. By Greene Publishing Assoc, and Wiley-Interscience(1987). Briefly, the nucleic acid can be produced from genomic DNA fragments, cDNA and RNA, all of which can be extracted directly from cells or recombinantly produced by a variety of amplification methods, including but not limited to PCR and RT-PCR. The polynucleotides described herein may comprise at least two different nucleotide sequences. The polynucleotides described herein may encode at least two different components of the bispecific fusion proteins described herein. For example, the polynucleotides may encode a first polypeptide chain described herein, and / or the polynucleotides may encode a second polypeptide chain described herein. For example, the polynucleotides may encode the light chain of the PD-L1 antibody, may encode the heavy chain of the PD-L1 antibody, may encode the second binding domain, may encode the linker, may encode the Ig-like structural domain of the VEGFR1, and / or may encode the Ig-like structural domain of the VEGFR2.

[0025] In another aspect, the present application provides a vector comprising a polynucleotide described herein. In the present application, the vector may comprise one or more of the polynucleotides described herein. For example, the vector may be used directly to express the bispecific fusion protein described herein. In the present application, the vector may be used to express any component of the bispecific fusion protein described herein. In the present application, at least two of the vectors may be used to express the bispecific fusion protein described herein. For example, the vector may express a first polypeptide chain described herein, and / or the vector may express a second polypeptide chain described herein. For example, the vector may express the light chain of the PD-L1 antibody, the heavy chain of the PD-L1 antibody, the linker, the Ig-like structural domain of the VEGFR1, and / or the Ig-like structural domain of the VEGFR2. In the present application, the vector may also include other genes, such as marker genes that select the vector under appropriate conditions in a suitable host cell. The vector may also include expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art, and for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a regulatable element. The specific structure of the expression control sequence may vary depending on the species or cell type function, but may usually include 5' non-transcribed sequences involved in transcription and translation initiation, such as TATA, capping sequences, CAAT sequences, and 5' and 3' non-translated sequences. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence operably linked to a nucleic acid for transcriptional control. The expression control sequence may further include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may be SP6, T3 and T7 polymerase promoters, human U6RNA promoter, CMV promoter and its artificial heterologous promoters (e.g. CMV), a part of which may be fused to a part of the promoter of other cellular protein (e.g. human GAPDH, glyceraldehyde 3-phosphate dehydrogenase) genes, which may or may not further contain introns. One or more nucleotides described in the present application may be operably linked to an expression control element. The vector may include a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0026] In another aspect, the present application provides a cell expressing or containing a bispecific fusion protein described herein, a polynucleotide described herein, or a vector described herein. The cell may be a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / 0 cell, a HEK293T cell, or a HEK293A cell, or other eukaryotic cell, such as a fungal or yeast cell. The vectors described herein may be introduced into the cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc. For example, the host cell may be COS, CHO, NSO, sf9, sf21, DH5a, BL21(DE3) or TG1. In another aspect, the present application provides a method for producing a bispecific fusion protein described herein, comprising culturing a cell described herein under conditions suitable for expression of a bispecific fusion protein described herein.

[0027] This can be done, for example, by using an appropriate medium, appropriate temperature and incubation time, etc., methods known to those skilled in the art. In certain embodiments, the method can further comprise a step of recovering (e.g., isolating and / or purifying) the bispecific fusion protein described herein. For example, the bispecific fusion protein described herein can be purified and isolated by affinity chromatography using protein G-agarose or protein A-agarose, or by gel electrophoresis and / or high performance liquid chromatography. In another aspect, the present application provides a pharmaceutical composition comprising a bispecific fusion protein described herein and a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and / or non-ionic surfactants. In the present application, the pharmaceutical composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous reservoir. For example, for oral administration, the pharmaceutical composition may be prepared as mini-tablets, tablets, capsules, elixirs, suspensions, syrups, or flakes. For injectable formulations, the pharmaceutical composition may be prepared as, for example, single-dose ampoules or unit-dosage forms in multi-dose containers. The pharmaceutical composition may also be prepared as solutions, suspensions, tablets, pills, capsules, and sustained-release formulations. The frequency and dosage of the drug composition can be determined by a number of related factors, including the type of disease to be treated, the route of administration, the age, sex, weight, and severity of disease of the patient, and the type of drug used as the active ingredient.The drug composition has excellent in vivo efficacy and concentration duration, so that the frequency and dosage of administration can be significantly reduced.

[0028] In another aspect, the present application provides a drug molecule comprising the bispecific fusion protein. In the present application, the drug molecule may have the properties and / or functions of the bispecific fusion protein. In the present application, the drug molecule may also have other properties and / or functions. For example, the drug molecule may also include other structures. For example, the drug molecule may also include one or more other amino acids. In another aspect, the present application provides the use of a bispecific fusion protein as described herein in the manufacture of a medicament for the treatment of a disease, wherein the disease comprises a tumor. The bispecific fusion proteins described herein and / or the pharmaceutical compositions described herein are used to treat diseases, said diseases including tumors. The present application provides a method of treating a disease, comprising administering to a subject in need thereof an effective amount of a bispecific fusion protein and / or a pharmaceutical composition described herein, the disease including a tumor.

[0029] In the present application, the tumor may be a solid tumor or a non-solid tumor. For example, the tumor may include various tumor types known to those skilled in the art, but is not limited to one or more specific tumor types. For example, the tumor may include lung cancer, colorectal cancer, cervical cancer, liver cancer, gastric cancer, and / or kidney cancer. For example, the tumor may include colorectal cancer. In the present application, the tumor may be a PD-L1 positive (e.g., PD-L1 overexpressing) tumor. In certain embodiments, the tumor may be PD-L1 negative. In the present application, the tumor may be associated with overexpression of VEGF (e.g., in blood vessels). In another aspect, the present application provides a method for inhibiting blood vessel (e.g., human) proliferation, comprising administering an effective amount of a bispecific fusion protein described herein and / or a pharmaceutical composition described herein. In another aspect, the present application provides a method for inhibiting the activity of a VEGF receptor ligand, comprising administering an effective amount of a bispecific fusion protein described herein and / or a pharmaceutical composition described herein.

[0030] For example, the VEGF receptor ligand activity may include the biological activity and / or function of VEGF and / or VEGFR itself, for example, the binding of VEGF to VEGFR. In another aspect, the present application provides a method for inhibiting the activity of PD-L1, comprising administering an effective amount of a bispecific fusion protein described herein and / or a pharmaceutical composition described herein.

[0031] For example, the PD-L1 activity may include the biological activity and / or function of PD-L1 and / or PD-1 itself, for example, the binding of PD-L1 to PD-1. The following examples are not intended to be limited by theory, and are only used to illustrate each technical solution of the present application, and are not used to limit the scope of the present application.

[0032] Working Example The protein codes according to the present application have the following meanings: SG1201: PD-L1 antibody 1, the amino acid sequence of whose light chain variable region is set forth in SEQ ID NO.16 and the amino acid sequence of whose heavy chain variable region is set forth in SEQ ID NO.18; SG1202: PD-L1 antibody 2, the amino acid sequence of whose light chain variable region is set forth in SEQ ID NO.17 and the amino acid sequence of whose heavy chain variable region is set forth in SEQ ID NO.19; SG1501: refers to a fusion protein of VEGFRs (the amino acid sequence of which is shown in SEQ ID NO. 28)-Fc (the amino acid sequence of which is shown in SEQ ID NO. 20); 12VF1: A bispecific fusion protein described herein, the amino acid sequence of the first polypeptide chain of which is set forth in SEQ ID NO.23 and the amino acid sequence of the second polypeptide chain of which is set forth in SEQ ID NO.25. 12VF2: A bispecific fusion protein described herein, the amino acid sequence of the first polypeptide chain of which is set forth in SEQ ID NO.24 and the amino acid sequence of the second polypeptide chain of which is set forth in SEQ ID NO.26. 12VF8: A bispecific fusion protein described herein, the amino acid sequence of the first polypeptide chain of which is set forth in SEQ ID NO.23 and the amino acid sequence of the second polypeptide chain of which is set forth in SEQ ID NO.27.

[0033] Example 1 Construction of a Fusion Protein With reference to the structure of the bispecific fusion protein shown in Figure 1, the sequence of PD-L1 antibody 1, a linker (its amino acid sequence is shown in SEQ ID NO.3) and VEGFRs were sequentially linked, in which the N-terminus of VEGFRs was linked to the C-terminus of the heavy chain of PD-L1 antibody 1 to obtain the bispecific fusion protein 12VF1. Based on 12VF1, the mutated IgG1 Fc (the amino acid sequence of which is shown in SEQ ID NO. 22) was replaced with an IgG4 Fc (the amino acid sequence of which is shown in SEQ ID NO. 21) to obtain the bispecific fusion protein 12VF8. With reference to the structure of the fusion protein shown in Figure 1, the sequence of PD-L1 antibody 2, a linker (its amino acid sequence is shown in SEQ ID NO.3) and VEGFRs were sequentially linked, where the N-terminus of VEGFRs was linked to the C-terminus of the heavy chain of PD-L1 antibody 2 to obtain the bispecific fusion protein 12VF2.

[0034] Example 2 Assay for binding activity to dual antigens (1) The binding activity of the bifunctional fusion protein to PD-L1 was assessed by ELISA using a PD-L1 antibody as a control. PD-L1 (human recombinant PD-L1 protein (ECD, His Tag), purchased from Sino Biological) coated ELISA strips were left overnight at 4℃. After washing with PBST, 10% fetal bovine serum was added and sealed at 37℃ for 1h. Different concentrations of antibodies SG1201 and SG1202, bispecific fusion proteins 12VF1, 12VF2 and 12VF8 prepared in Example 1 were added and reacted at 37℃ for 1h. After washing with PBST, horseradish peroxidase-labeled goat anti-human IgG Fab secondary antibody (Goat Anti-Human IgG (Fab') 2 (HRP), Abeam) was added and reacted at 37℃ for 30min. Washed 5 times with PBST. 100μL TMB (eBioscience) was added to each well and left for 1-2min at room temperature (20±5℃) in the dark. Furthermore, 100 μL 2N H2SO4 stop solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm using a microplate reader to analyze the binding ability of the bifunctional fusion protein to PD-L1. The results are shown in Figure 2. Figure 2 shows that the binding ability of the bispecific fusion protein 12VF1 to PD-L1 is slightly stronger than that of 12VF8 and is equivalent to that of the antibody SG1201. Figure 2 shows that the binding ability of the bispecific fusion protein 12VF2 to PD-L1 is equivalent to that of the antibody SG1202. (2) The binding activity of the bifunctional fusion protein to VEGF165 was evaluated by ELISA using a VEGFRs fusion protein as a control. VEGF165 (Human VEGF165 Protein, His Tag, Acro Biosystems) coated ELISA strips were left overnight at 4°C. After washing with PBST, 10% fetal bovine serum was added and the strips were sealed at 37°C for 1h. Different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8 and SG1501 were added and reacted at 37°C for 1h. After washing with PBST, horseradish peroxidase-conjugated goat anti-human IgG Fc secondary antibody (Goat anti-human IgG Fc antibody, horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen) was added and reacted at 37°C for 30min. The strips were washed five times with PBST. 100μL TMB (eBioscience) was added to each well and the strips were left for 1-2min at room temperature (20±5°C) in the dark. Furthermore, 100 μL 2N H2SO4 stop solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm with a microplate reader to analyze the binding ability of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 to VEGF165. The results are shown in Figure 3. Figure 3 shows that the binding ability of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 to VEGF165 is comparable and slightly weaker than that of the fusion protein SG1501.

[0035] Example 3 Assay for simultaneous binding activity to dual antigens Using PD-L1 antibody and VEGFRs fusion protein as controls, the biological activities of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 prepared in Example 1 and the simultaneous binding of dual antigens were evaluated by ELISA. PD-L1 (human recombinant PD-L1 protein (ECD, His Tag), purchased from Sino Biological) coated ELISA strips were incubated overnight at 4℃. After washing with PBST, 10% fetal bovine serum was added and sealed at 37℃ for 1h. Different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8, PD-L1 antibodies SG1201, SG1202, and VEGFRs fusion protein SG1501 were added and incubated at 37℃ for 1h. After washing with PBST, biotinylated human VEGF165 protein (His, Avitag, Acro Biosystems) was added and incubated at 37℃ for 30min. The strips were washed five times with PBST. Horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiahui Biological) was added and incubated at 37℃ for 30min. The plates were washed five times with PBST. 100 μL TMB (eBioscience) was added to each well and left at room temperature (20 ± 5 °C) for 1 to 2 min in the dark. In addition, 100 μL 2N H2SO4 stop solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm using a microplate reader to analyze the simultaneous binding ability of the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 to PD-L1 and VEGF165. The results are shown in Figure 4. Figure 4 shows that the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 can bind to PD-L1 and VEGF165 simultaneously, while the PD-L1 antibodies SG1201 and SG1202 and the VEGFRs fusion protein SG1501 cannot bind to PD-L1 and VEGF165 simultaneously.

[0036] Example 4 Analysis of inhibitory activity against PD-1 / PD-L1 interaction The biological activity of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 to inhibit PD-1 / PD-L1 interaction was assessed using the PD-L1 antibodies SG1201 and SG1202 as controls. PD-L1-his (human recombinant PD-L1 protein (ECD, His Tag), purchased from Sino Biological) coated assay plate, lμg / ml, was incubated overnight at 4℃. After washing with PBST, 10% fetal bovine serum was added and sealed at 37℃ for 1 hour. Different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8 and antibodies SG1201 and SG1202 were added and reacted at 37℃ for 1 hour. After washing with PBST, biotinylated PD1 (Biotinylated Human PD-1 / PDCD1 Protein, Fc, Avitag) was added to the plate. TM , HisTag, Acro Biosystems) was added to a final concentration of 2μg / ml and incubated at 37℃ for 30min. The plate was washed five times with PBST. Horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiahui Biology) was added and incubated at 37℃ for 30min. The plate was washed five times with PBST, and 100μL TMB (eBioscience) was added to each well and incubated at room temperature (20±5℃) for 1-5min in the dark. The substrate reaction was terminated by adding 100μL 2N H2SO4 stop solution to each well, and the OD value was read at 450nm with a microplate reader to analyze the inhibitory effect of bispecific fusion proteins 12VF1, 12VF2 and 12VF8 on PD-1 / PD-L1. As can be seen from Figure 5, the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 can competitively inhibit the binding of PD-1 to PD-L1 with activity comparable to that of the PD-L1 antibodies SG1201 and SG1202. Here, the IC 50 The IC50 value was 0.3968nM, the IC50 value of SG1202 was 0.4216nM, the IC50 value of 12VF1 was 0.393nM, and the IC 50 The value is 0.5002nM, and the IC of 12VF2 50 The value is 0.4256nM.

[0037] Example 5 Analysis of inhibitory activity against VEGF / VEGFR interaction (1) The biological activities of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 prepared in Example 1 for inhibiting VEGF / VEGFR interaction were evaluated using the fusion protein SG1501 as a control. VEGFR1 (Human VEGF R1 / Flt-1 Protein, His Tag, Acro Biosystems) coated assay plate, 1μg / ml, was left overnight at 4℃. After washing with PBST, 10% fetal bovine serum was added and sealed at 37℃ for 1 hour. Different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8 and fusion protein SG1501 were added and reacted at 37℃ for 1 hour. After washing with PBST, biotinylated human VEGF165 protein (Biotinylated Human VEGF165 Protein, His, Avitag, Acro Biosystems) was added to a final concentration of 0.05μg / ml and incubated at 37℃ for 30min to react. Washed 5 times with PBST. Horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiayue Biology) was added and incubated at 37℃ for 30min. After washing five times with PBST, 100 μL TMB (eBioscience) was added to each well and left for 1-5 min at room temperature (20 ± 5 °C) in the dark. The substrate reaction was terminated by adding 100 μL 2N H2S04 stop solution to each well, and the OD value was read at 450 nm using a microplate reader to analyze the inhibitory effect of the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 on VEGF / VEGFR. The results are shown in Figure 6, which shows that the bispecific fusion proteins 12VF1, 12VF2, and 12VF8, as well as the fusion protein SG1501, can competitively inhibit the binding of VEGF to its receptor VEGFR1. Here, the IC 50 The value is 3.749nM, 12VF2 IC 50 The value is 5.049nM, IC of 12VF8 50 The value is 2.182nM, and the IC of SG150150 The value is 1.470nM. (2) The biological activity of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 prepared in Example 1 for inhibiting VEGF165 / VEGFR2 interaction was evaluated using the bispecific fusion protein IMM25011 (see patent application US2020 / 0172623A1) as a control. VEGF165 (VEGF165 Protein, Human, Cynomolgus, Recombinant, HPLC-verified, Sino Biological) coated assay plate, 1μg / ml, was left overnight at 4℃. After washing with PBST, 10% fetal bovine serum was added and sealed at 37℃ for 1h. Different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8 and IMM25011 were added and reacted at 37℃ for 1h. After washing with PBST, biotin-labeled VEGFR2 (VEGFR2 / KDR Protein, Human, Recombinant (His Tag), Biotinylated, Sino Biological) was added to a final concentration of 1μg / ml and incubated at 37℃ for 30min to react. Washed 5 times with PBST. Horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiahui Biology) was added and incubated at 37℃ for 30min. After washing five times with PBST, 100μL TMB (eBioscience) was added to each well and incubated at room temperature (20±5℃) for 1-5min in the dark. The substrate reaction was terminated by adding 100μL 2N H2SO4 stop solution to each well, and the OD value was read at 450nm with a microplate reader to analyze the inhibitory effect of bispecific fusion proteins 12VF1, 12VF2, and 12VF8 on VEGF165 / VEGFR2. The results are shown in Figure 7, which shows that the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 can all inhibit the binding of VEGF165 to its receptor VEGFR2, and their activity is stronger than that of IMM25011. Here, the IC 50 The value is 4.092nM, 12VF2 IC 50The value is 3.501nM, IC of 12VF8 50 The value is 3.422nM, IMM25011 IC 50 The value is 6.596nM. (3) The biological activities of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 prepared in Example 1 for inhibiting VEGF121 / VEGFR2 interaction were evaluated using the bispecific fusion protein IMM25011 as a control. VEGF121 (VEGF 121 Protein, Human, Recombinant, Sino Biological) coated assay plate, 1μg / ml, was left overnight at 4℃. After washing with PBST, 10% fetal bovine serum was added and sealed at 37℃ for 1h. Different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8 and IMM25011 were added and reacted at 37℃ for 1h. After washing with PBST, biotin-labeled VEGFR2 (VEGFR2 / KDR Protein, Human, Recombinant (HisTag), Biotinylated, Sino Biological) was added to a final concentration of 1μg / ml and reacted at 37℃ for 30min. Washed five times with PBST. Horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiahui Biological) was added and incubated at 37℃ for 30min. After washing five times with PBST, 100 μL TMB (eBioscience) was added to each well and incubated for 1-5 min at room temperature (20 ± 5°C) in the dark. The substrate reaction was terminated by adding 100 μL 2N H2SO4 stop solution to each well, and the OD value was read at 450 nm using a microplate reader to analyze the inhibitory effect of bispecific fusion proteins 12VF1, 12VF2, and 12VF8 on VEGF121 / VEGFR2. The results are shown in Figure 8, which shows that the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 can all inhibit the binding of VEGF121 to its receptor VEGFR2, and their activity is stronger than that of IMM25011. Here, the IC 50 The value is 3.536nM, 12VF2 IC 50The value is 3.291nM, IC of 12VF8 50 The value is 2.955nM, IMM25011 IC 50 The value is 5.281nM. (4) The biological activities of the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 prepared in Example 1 for inhibiting VEGFC / VEGFR2 interaction were evaluated using the bispecific fusion protein IMM25011 as a control. VEGFC (VEGF C Protein, Human, Recombinant (His Tag), Sino Biological) coated assay plate, 1μg / ml, was left overnight at 4℃. After washing with PBST, 10% fetal bovine serum was added and sealed at 37℃ for 1h. Bispecific fusion proteins 12VF1, 12VF2, 12VF8 and IMM25011 were added at 15μM each and reacted at 37℃ for 1h. After washing with PBST, biotin-labeled VEGFR2 (VEGFR2 / KDR Protein, Human, Recombinant (His Tag), Biotinylated, Sino Biological) was added to a final concentration of 20μg / ml and reacted at 37℃ for 30min. The plate was washed five times with PBST. Horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiahui Biological) was added and incubated at 37℃ for 30min. After washing five times with PBST, 100 μL TMB (eBioscience) was added to each well and incubated for 1-5 min at room temperature (20 ± 5°C) in the dark. The substrate reaction was terminated by adding 100 μL 2N H2SO4 stop solution to each well, and the OD value was read at 450 nm using a microplate reader to analyze the inhibitory effect of the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 on VEGFC / VEGFR2. The results are shown in Figure 9, which shows that the bispecific fusion proteins 12VF1, 12VF2, and 12VF8 can all effectively inhibit angiogenesis by inhibiting the binding of VEGFC to its receptor VEGFR2, and the inhibitory activities of 12VF1, 12VF2, and 12VF8 are comparable, whereas IMM25011 cannot inhibit the binding of VEGFC to its receptor VEGFR2. Therefore, compared with IMM25011, which can only inhibit the binding between VEGF121 and VEGFR2, the bispecific fusion protein of the present application can inhibit not only the binding between VEGF121 and VEGFR2, but also the binding between VEGF C and its receptor VEGFR2, thereby exerting a more comprehensive regulatory effect on this signaling pathway and playing a more complete role in inhibiting angiogenesis at tumor sites.

[0038] Example 6 Analysis of the inhibitory activity against HUVEC cell proliferation The biological activities of bispecific fusion proteins 12VF1, 12VF2, and 12VF8 to inhibit HUVEC cell proliferation were evaluated using the fusion protein SG1501 as a control. HUVEC cells were resuspended in experimental medium, placed in a 96-well cell culture plate, and cultured overnight in an incubator. VEGF 165 (ActiveMax™ Human VEGF 165 Protein, Tag Free (MALS verified), Acro Biosystems) was diluted to 40ng / mL in experimental medium, and mixed with different concentrations of bispecific fusion proteins 12VF1, 12VF2, 12VF8 and fusion protein SG1501 in equal amounts, pre-incubated at 37°C for 30min, and placed in a 96-well cell culture plate. After culturing in a 37°C incubator for 3 days, the 96-well plate was removed, equilibrated at room temperature for 10min, and 100μL of CellTiter-Lumi reagent (CellTiter-Lumi) was added to each well. TMLuminescent Cell Viability Assay Kit (Biyuntian) was added, gently shaken, and incubated in the dark for 10 min. The relative fluorescence intensity (RLU) values ​​of chemiluminescence were read using a microplate reader to analyze the HUVEC cell proliferation inhibitory activity of the bispecific fusion proteins 12VF1, 12VF2, and 12VF8. As can be seen from FIG. 10, the bispecific fusion proteins 12VF1, 12VF2 and 12VF8 can all inhibit HUVEC cell proliferation, but somewhat weaker than the fusion protein SG1501.

[0039] Example 7 Analysis of activity in T lymphocyte activation The biological activity of the bispecific fusion protein 12VF8 in activating T lymphocytes in a mixed lymphocyte reaction was assessed using the PD-L1 antibody SG1201 and an isotype control antibody as controls. Monocytes were isolated from PBMCs of healthy donors using CD14 microbeads (Miltenyi) and induced to differentiate into dendritic cells using an ImmunoCultTM Dendritic Cell Culture Kit (Stemcell). + T-lymphocyte isolation kit (CD4 + CD4 T Cell Isolation Kit (Miltenyi) was used to isolate CD4 T cells from PBMCs of another healthy donor. + Isolate CD4 T lymphocytes + T lymphocytes and dendritic cells were placed in a 96-well cell culture plate at a cell number ratio of 5:1, and different concentrations of bispecific fusion protein 12VF8 were added, mixed well, and then cultured in an incubator at 37°C for 5 days. The cell supernatants were collected and the concentrations of cytokines IFN-γ and IL-2 were measured by ELISA kit to analyze the activity of bispecific fusion protein 12VF8 in activating T lymphocytes in the mixed lymphocyte reaction. As can be seen from Figures 11-12, compared with the isotype control group, the bispecific fusion protein 12VF8 can activate T cells and promote the release of IFN-γ and IL-2 in a dose-dependent manner, similar to the PD-L1 antibody SG1201.

[0040] Example 8 Analysis of ADCC activity The bispecific fusion protein IMM25011 was used as a control, the PD-L1-overexpressing cell line CHO-K1 / PD-L1 was used as a target cell, and Jurkat cells overexpressing the human FcγRIIIa gene and the NFAT fluorescent reporter gene (abbreviated as Jurkat-ADCC cells) were used as effector cells, and the ADCC activities of the bispecific fusion proteins 12VF1 and 12VF8 prepared in Example 1 were evaluated using the reporter gene method. Harvest CHO-K1 / PD-L1 cells and measure 2 × 10 per well. 4 1.2 x 10 cells per tube in a 96-well cell culture plate. Collect Jurkat-ADCC cells and place 1.2 x 10 cells per tube in a 96-well cell culture plate. 5 The cells were placed in a 96-well cell culture plate. Different concentrations of bispecific fusion proteins IMM25011, 12VF1 and 12VF8 were added and incubated in a 37℃ incubator for 6h. 100μL of luciferase detection solution was added per well and incubated at room temperature for 10min in the dark. The chemiluminescence relative fluorescence intensity (RLU) value was read using a microplate reader to analyze the ADCC activity of the bispecific fusion proteins 12VF1 and 12VF8. The results are shown in FIG. 13, which shows that the bispecific fusion protein IMM25011 has ADCC activity, whereas 12VF1 and 12VF8 do not. These results suggest that 12VF1 and 12VF8 are safer. Potential side effects can be reduced by avoiding the killing of other PD-L1-positive normal cells. PD-L1 is also expressed on many normal immune cells, including myeloid DCs, macrophages, lymphoid T effector cells, Treg cells, and NK cells, so 12VF1 and 12VF8 can avoid killing normal tissues in the absence of ADCC activity.

[0041] Example 9 In vivo tumor suppressor activity of the fusion protein The murine colorectal cancer MC38 model was used to evaluate the efficacy of the tumor suppressor activity of the bispecific fusion protein 12VF8. Mouse colorectal cancer cells MC38 were inoculated subcutaneously into the right flank of male C57BL / 6J mice, a total of 36 mice, and tumors grew to approximately 58 mm 3 When the mice reached 1000 mg / kg, they were divided into 5 groups, 6 mice per group. The details of each group are as follows: Group 1: PBS (i.e., as the vehicle group) was administered by intraperitoneal injection on day 17 of the study, twice a week for 3 weeks. Group 2: SG1201 (2 mg / kg) was administered by intraperitoneal injection twice a week for 3 weeks. Group 3: SG1501 (1.4 mg / kg) was administered by intraperitoneal injection twice a week for 3 weeks. Group 4: SG1201 (2 mg / kg) and SG1501 (1.4 mg / kg) were administered by intraperitoneal injection twice a week for 3 weeks. Group 5: 12VF8 (2.7 mg / kg) was administered by intraperitoneal injection twice a week for 3 weeks (the results after administration are shown in order for groups 1-5 in Figure 14). Tumor volume and body weight were measured every week, and the relationship between the weight and tumor volume of the tumor-bearing mice and the administration time was recorded. Two hours after the last administration, serum was collected from selected mice from each group. After the experiment, the tumor-bearing mice were euthanized, and the tumors were excised, weighed, and photographed. The tumor growth inhibition rate TGITV (%) was calculated and statistically analyzed. As a result, at the end of the experiment, the tumor growth inhibition rates of Groups 2, 3, 4 and 5 were 63%, 39%, 71% and 83%, respectively, and the tumor volumes of each treatment group were significantly lower than the control Group 1 (p<0.05), indicating a significant antitumor effect and effectively inhibiting tumor growth. Among them, the tumor volume of Group 5 was the smallest among all treatment groups and significantly lower than Group 3 (p<0.05). The tumor inhibition activity was superior to Groups 2 and 3, which were administered with a single drug, and to Group 4, which was administered with multiple drugs in combination. During the treatment period, the tumor-bearing mice showed good resistance to the treatment of Groups 2-5, and the mice in each group had normal body weight, no abnormal findings, and good general condition, suggesting the safety of administration of 12VF8.

[0042] The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the embodiments described herein will be apparent to those skilled in the art and remain within the scope of the appended claims and their equivalents.

Claims

1. A bispecific fusion protein comprising a first binding domain and a second binding domain, wherein the first binding domain comprises an antibody that specifically binds to PD-L1, the antibody comprises two antibody light chains and two antibody heavy chains, and the antibody light chain and the antibody heavy chain are linked by disulfide bonds, the second binding domain comprises one Ig-like domain of VEGFR1 and one Ig-like domain of VEGFR2, 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.

2. the Ig-like domain of VEGFR1 comprises the amino acid sequence shown in SEQ ID NO: 1, and the Ig-like domain of VEGFR2 comprises the amino acid sequence shown in SEQ ID NO: 2, the bispecific fusion protein according to claim 1.

3. the second binding domain comprises the amino acid sequence shown in SEQ ID NO: 28, the bispecific fusion protein according to claim 1.

4. the indirect linkage is effected via a linker, the bispecific fusion protein according to claim 1.

5. each of the antibody light chains comprises a light chain variable region, the light chain variable region comprises LCDR1-3, LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 4 and 10, LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 5 and 11, and LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 6 and 12, the bispecific fusion protein according to claim 1.

6. The amino acid sequences of the LCDR1-3 are selected from any one of the following groups, the bispecific fusion protein according to claim 5: (1) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 5, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 6, (2) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 10, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 11, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:

12.

7. The antibody light chain each includes a light chain variable region, and the light chain variable region includes the amino acid sequence shown in any one of SEQ ID NOs: 16-17, the bispecific fusion protein according to claim 1.

8. The antibody light chain each includes a light chain constant region, and the light chain constant region is derived from the light chain constant region of a protein selected from the group consisting of Igκ and Igλ, the bispecific fusion protein according to claim 1.

9. The antibody heavy chain each includes a heavy chain variable region, the heavy chain variable region includes HCDR1-3, the HCDR1 includes the amino acid sequence shown in any one of SEQ ID NOs: 7 and 13, The HCDR2 includes the amino acid sequence shown in any one of SEQ ID NOs: 8 and 14, and The HCDR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 9 and 15, the bispecific fusion protein according to claim 1.

10. The HCDR1-3 includes the amino acid sequence selected from any one of the following groups, the bispecific fusion protein according to claim 9. (1) The HCDR1 contains the amino acid sequence shown in SEQ ID NO: 7, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 8, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:

9. (2) The HCDR1 contains the amino acid sequence shown in SEQ ID NO: 13, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 14, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:

15.

11. The heavy chain of the antibody each contains a heavy chain constant region, and the heavy chain constant region contains the amino acid sequence shown in any one of SEQ ID NOs: 18-19. The bispecific fusion protein according to claim 1.

12. The heavy chain of the antibody each contains an Fc region. The bispecific fusion protein according to claim 1.

13. The Fc region contains the amino acid sequence shown in any one of SEQ ID NOs: 20-22. The bispecific fusion protein according to claim 12.

14. A polymer consisting of two copies of a first polypeptide chain and a second polypeptide chain. The bispecific fusion protein according to claim 4, wherein The first polypeptide chain contains the antibody light chain. The second polypeptide chain contains, in order from the N-terminus, the heavy chain of the antibody, the linker, and the second binding domain.

15. The bispecific fusion protein according to claim 14, wherein the first polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 23, the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 25, or The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 24, the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 26, or The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 23, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:

27.

16. A polynucleotide encoding the bispecific fusion protein according to Claim 1.

17. A drug composition comprising the bispecific fusion protein according to any one of Claims 1 to 15 and an optionally selected pharmaceutically acceptable carrier.

18. A drug composition for treating a disease, comprising the bispecific fusion protein according to any one of Claims 1 to 15 and / or the polynucleotide according to Claim 16, wherein the disease includes solid tumors and non-solid tumors.

19. The drug composition according to Claim 18, wherein the disease includes PD-L1 positive tumors.

20. The drug composition according to Claim 18, wherein the tumors include lung cancer, colorectal cancer, cervical cancer, liver cancer, gastric cancer and / or kidney cancer.

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