A bifunctional fusion protein having anticancer activity

Abstract

The present application relates to the technical field of biotechnology, in particular to a bifunctional fusion protein with anticancer activity and a preparation method and application thereof. The present application provides a fusion protein, which comprises an anti-PD-L1 single-domain antibody fragment and an antagonistic VEGF fragment. The bifunctional fusion protein with anticancer activity provided by the present application can organically combine the functions of blocking PD-L1 / PD-1 interaction by the anti-PD-L1 monoclonal antibody, reducing microvessel growth and inhibiting metastatic diseases by the anti-VEGF monoclonal antibody in one antibody fusion protein molecule, so that the bifunctional fusion protein can be used for treating tumors and has a good industrialization prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a bifunctional fusion protein with anticancer activity, its preparation method, and its uses. Background Technology

[0002] One of the differences between tumor cells and normal cells lies in their high metabolic demands during growth. Tumor cells rely on blood vessels for nutrients and oxygen, process metabolic waste products, and promote angiogenesis on existing blood vessels. Among the pro-angiogenic factors secreted by tumors, human vascular endothelial growth factor (VEGF), particularly VEGF-A, is a key factor contributing to tumor angiogenesis (Josep Garcia et al., 2020, Cancer Treatment Reviews 86: 1-2). Therefore, inhibition of the VEGF signaling pathway can limit the progression of various tumors. For example, bevacizumab (trade name...) Bevacizumab is a humanized anti-VEGF monoclonal antibody that binds to VEGF and prevents VEGF from interacting with VEGF receptors (Flt-1 and KDR) on the surface of endothelial cells. Currently, bevacizumab is approved by the FDA for the treatment of metastatic colorectal cancer, advanced, metastatic, or recurrent non-small cell lung cancer, and recurrent glioblastoma.

[0003] However, targeted therapy is often accompanied by mutations in cancer cells, leading to treatment ineffectiveness. Immunotherapy, on the other hand, focuses on activating the immune system and truly altering the immune status of the tumor microenvironment. For example, the first immune checkpoint inhibitor targeting CTLA-4 was approved by the FDA in 2011. In the three years that followed, five antibody drugs targeting PD-1 / PD-L1 were launched, pushing immune checkpoint research into the spotlight. However, while anti-PD-1 / PD-L1 monoclonal antibody drugs have shown high efficacy and broad-spectrum activity in cancer treatment, they also have significant limitations, such as limited efficacy (<30%), drug resistance, and toxic side effects.

[0004] Given the synergistic effects of signaling pathways targeting certain targets in biological mechanisms, combination therapy often achieves results that monotherapy cannot. For example, Roche's use of the anti-PD-L1 monoclonal antibody (Atezolizumab) and Bevacizumab to treat hepatocellular carcinoma (HCC) achieved dual endpoints (significantly prolonged overall survival (OS) and progression-free survival (PFS)) in a phase III clinical trial (IMbrave 150), and was approved by the FDA in 2020. Furthermore, the triple therapy of Atezolizumab + Bevacizumab + chemotherapy (paclitaxel + carboplatin) significantly prolonged progression-free survival (PFS) in a phase III clinical trial of non-small cell lung cancer (NSCLC) (IMpower 150), and was approved by the FDA in 2018. However, a large amount of data also indicates that while combination therapy improves efficacy, it often comes with increased toxicity. Therefore, finding highly effective and low-toxicity treatment methods remains a core issue that needs to be addressed in clinical immunotherapy, such as the development of single-molecule bispecific antibodies. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a bifunctional fusion protein with anticancer activity, its preparation method and uses, in order to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides a fusion protein comprising an anti-PD-L1 single-domain antibody fragment and an antagonistic VEGF fragment.

[0007] In another aspect, the present invention provides an isolated polynucleotide encoding the aforementioned fusion protein.

[0008] In another aspect, the present invention provides a construct containing the isolated polynucleotides described above.

[0009] In another aspect, the present invention provides an expression system containing the above-described construct or genome in which exogenous polynucleotides described above are integrated.

[0010] Another aspect of the present invention provides a method for preparing the above-described fusion protein, comprising: culturing the above-described expression system under suitable conditions to express the fusion protein, and separating and purifying the fusion protein to provide the fusion protein.

[0011] In another aspect, the present invention provides the use of the above-described fusion protein or the culture of the above-described expression system in the preparation of a drug.

[0012] Another aspect of the present invention provides a pharmaceutical composition comprising the above-described fusion protein or a culture of the above-described expression system. Attached Figure Description

[0013] Figure 1This diagram illustrates the tumor-suppressing effect of the bifunctional fusion protein in M-NSG mice in Example 4 of this invention. Detailed Implementation

[0014] Through extensive research, the inventors of this invention unexpectedly discovered a fusion protein molecule that can reduce tumor angiogenesis and relieve immunosuppression by blocking PD-L1 / PD-1 interaction and blocking the VEGF signaling pathway, thus exhibiting excellent tumor suppression effects. Based on this discovery, the invention was completed.

[0015] The first aspect of this invention provides a fusion protein comprising an anti-PD-L1 single-domain antibody fragment and an antagonistic VEGF fragment. In the aforementioned fusion protein, the anti-PD-L1 single-domain antibody fragment can typically be used to block PD-L1 / PD-1 interaction, increase the expression of IFN-γ and / or IL-2 in T lymphocytes, thereby inhibiting tumor growth. The variable region of the antagonistic VEGF fragment can bind to VEGF, thereby blocking tumor angiogenesis and "starving" cancer cells. The antagonistic VEGF fragment may also typically include an Fc portion, which can bind to FcRn receptors, thereby prolonging its in vivo half-life, and can also bind to effector cells expressing Fc receptors to kill cancer cells.

[0016] The fusion protein provided by this invention may include an anti-PD-L1 single-domain antibody fragment. This anti-PD-L1 single-domain antibody fragment is typically a polypeptide or protein fragment capable of specifically binding to PD-L1. The anti-PD-L1 single-domain antibody fragment usually lacks the corresponding antibody light chain, containing only the fragment corresponding to the variable region of the heavy chain. The binding characteristics of the anti-PD-L1 single-domain antibody fragment are typically determined by its three complementarity determining regions (CDRs). CDRs can be arranged in an orderly manner with framework regions (FRs), which typically do not directly participate in the binding reaction. These CDRs can form a ring structure, with the β-sheets formed by the FRs between them spatially close to each other, constituting the antigen-binding site of the antibody. For example, the complementarity determining regions (CDRs) of the aforementioned anti-PD-L1 single-domain antibody fragment may include CDR1 as shown in one of SEQ ID NO. 1–5, CDR2 as shown in one of SEQ ID NO. 6–9, and CDR3 as shown in one of SEQ ID NO. 10–15.

[0017] In a specific embodiment of the present invention, the complementarity-determining region of the anti-PD-L1 single-domain antibody fragment includes: CDR1 as shown in SEQ ID NO.1, CDR2 as shown in SEQ ID NO.6, and CDR3 as shown in SEQ ID NO.10.

[0018] In another specific embodiment of the present invention, the complementarity-determining region of the anti-PD-L1 single-domain antibody fragment includes: CDR1 as shown in SEQ ID NO.2, CDR2 as shown in SEQ ID NO.7, and CDR3 as shown in SEQ ID NO.11.

[0019] In another specific embodiment of the present invention, the complementarity-determining region of the anti-PD-L1 single-domain antibody fragment includes: CDR1 as shown in SEQ ID NO.3, CDR2 as shown in SEQ ID NO.7, and CDR3 as shown in SEQ ID NO.12.

[0020] In another specific embodiment of the present invention, the complementarity-determining region of the anti-PD-L1 single-domain antibody fragment includes: CDR1 as shown in SEQ ID NO.4, CDR2 as shown in SEQ ID NO.8, and CDR3 as shown in SEQ ID NO.13.

[0021] In another specific embodiment of the present invention, the complementarity-determining region of the anti-PD-L1 single-domain antibody fragment includes: CDR1 as shown in SEQ ID NO.2, CDR2 as shown in SEQ ID NO.7, and CDR3 as shown in SEQ ID NO.14.

[0022] In another specific embodiment of the present invention, the complementarity-determining region of the anti-PD-L1 single-domain antibody fragment includes: CDR1 as shown in SEQ ID NO.5, CDR2 as shown in SEQ ID NO.9, and CDR3 as shown in SEQ ID NO.15.

[0023] The aforementioned anti-PD-L1 single-domain antibody fragment may also include a framework region (FR). As mentioned above, the CDR region can be arranged sequentially with the FR region. For example, the anti-PD-L1 single-domain antibody fragment may sequentially include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. Furthermore, the framework region FR may include FR1 with the amino acid sequence shown in SEQ ID No. 38, FR2 with the amino acid sequence shown in any one of SEQ ID Nos. 39–41, FR3 with the amino acid sequence shown in any one of SEQ ID Nos. 42–44, and FR4 with the amino acid sequence shown in SEQ ID No. 45.

[0024] In a specific embodiment of the present invention, the frame region FR includes:

[0025] The amino acid sequences are as shown in SEQ ID NO. 38 (FR1), SEQ ID NO. 39 (FR2), SEQ ID NO. 42 (FR3), and SEQ ID NO. 45 (FR4), or...

[0026] The amino acid sequences are as shown in SEQ ID NO. 38 (FR1), SEQ ID NO. 40 (FR2), SEQ ID NO. 43 (FR3), and SEQ ID NO. 45 (FR4), or...

[0027] The amino acid sequences are as shown in SEQ ID NO. 38 (FR1), SEQ ID NO. 41 (FR2), SEQ ID NO. 43 (FR3), and SEQ ID NO. 45 (FR4), or...

[0028] The amino acid sequences are as shown in SEQ ID NO.38 (FR1), SEQ ID NO.41 (FR2), SEQ ID NO.44 (FR3), and SEQ ID NO.45 (FR4).

[0029] In another specific embodiment of the present invention, the anti-PD-L1 single-domain antibody fragment may include: a) a polypeptide fragment with an amino acid sequence as shown in any one of SEQ ID No. 16 to 21; or, b) a polypeptide fragment with an amino acid sequence having more than 80% sequence identity with any one of SEQ ID No. 16 to 21 and having the function of the polypeptide fragment defined in a). Specifically, the polypeptide fragment in b) above refers to a polypeptide fragment obtained by substituting, deleting, or adding one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) of the amino acid sequence shown in one of SEQ ID No. 16 to 21, or by adding one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) to the N-terminus and / or C-terminus, and having the function of a polypeptide fragment with an amino acid sequence shown in one of SEQ ID No. 16 to 21. For example, it may have the ability to specifically bind to PD-L1, or it may block the PD-L1 / PD-1 interaction, thereby blocking the PD-L1 / PD1 pathway, or it may have the function of increasing the expression of IFN-γ and / or IL-2 in T lymphocytes, or it may have the function of inhibiting tumor growth. The amino acid sequence of the anti-PD-L1 single-domain antibody fragment in b) above may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or higher identity with one of SEQ ID Nos. 16–21. The aforementioned anti-PD-L1 single-domain antibody fragment may typically be derived from alpacas (Vicugna pacos), for example, its CDR region may be derived from alpacas. The aforementioned anti-PD-L1 single-domain antibody fragment may typically be humanized, for example, its frame region may be derived from humans.

[0030] In this paper, sequence identity refers to the percentage of identical residues in sequences being compared. Sequence identity of two or more entries can be calculated using computational software known in the art, such as NCBI.

[0031] The fusion protein provided by this invention may include a VEGF-antagonizing fragment. This VEGF-antagonizing fragment is typically a polypeptide or protein fragment capable of antagonizing VEGF. For example, the VEGF-antagonizing fragment may be a monoclonal antibody. Another example is bevacizumab.

[0032] In a specific embodiment of the present invention, the VEGF antagonist fragment may include:

[0033] c) A polypeptide fragment with an amino acid sequence as shown in one of SEQ ID Nos. 22-23;

[0034] d) A polypeptide fragment whose amino acid sequence has more than 80% sequence identity with one of SEQ ID No. 22 to 23 and has the function of the polypeptide fragment defined in c). Specifically, the amino acid sequence in d) above refers to a polypeptide fragment that is obtained by substituting, deleting, or adding one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) of the amino acid sequence shown in SEQ ID No. 22 to 23, or by adding one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) to the N-terminus and / or C-terminus, and has the function of a polypeptide fragment with amino acids shown in SEQ ID No. 22 to 23. For example, it may have the function of specifically antagonizing VEGF, or the function of the Fc part that binds to the FcRn receptor, thereby prolonging the half-life in vivo, and also binding to effector cells expressing the Fc receptor to kill cancer cells. The amino acid sequence in d) has 80%, 85%, 90%, 93%, 95%, 97%, or 99% or higher identity with one of SEQ ID Nos. 22-23. The aforementioned antagonistic VEGF fragment is typically derived from mice (Mus musculus), for example, its CDR region may be derived from mice. The aforementioned antagonistic VEGF fragment is typically humanized, for example, its framework region may be derived from humans.

[0035] The fusion protein provided by this invention may further include linker peptide fragments. The fusion protein typically includes multiple linker peptide fragments, with linker peptide fragments present at least partially within or between the domains. For example, linker peptides may be present between anti-PD-L1 single-domain antibody fragments and VEGF antagonist fragments. These linker peptide fragments are typically flexible polypeptides of suitable length, rich in G, S, and / or A (mainly composed of glycine (G), serine (S), and / or alanine (A)), allowing adjacent protein domains to move freely relative to each other. For example, the amino acid sequence of the linker peptide fragment may include sequences such as (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGS)nG, (GGGGA)nA, (GGGGG)nA, etc., where n is selected from integers between 1 and 10. In a specific embodiment of the present invention, the length of the amino acid sequence of the linker peptide fragment can be 3-30, 3-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-22, 22-24, 24-26, 26-28, or 28-30.

[0036] The fusion protein provided by this invention can be linear. For example, the fusion protein can sequentially include an anti-PD-L1 single-domain antibody fragment and an antagonistic VEGF fragment from the N-terminus to the C-terminus. The fusion protein can also have a structure similar to a monoclonal antibody. For example, the anti-PD-L1 single-domain antibody fragment can be located at the N-terminus of the heavy chain of the antagonistic VEGF fragment, or the anti-PD-L1 single-domain antibody fragment can be located at the N-terminus of the light chain of the antagonistic VEGF fragment. In a specific embodiment of this invention, the amino acid sequence of the fusion protein can include the sequence shown in any one of SEQ ID NO. 22-29. For example, the amino acid sequence of the fusion protein can include the sequences shown in SEQ ID NO. 22 and SEQ ID NO. 24, SEQ ID NO. 22 and SEQ ID NO. 25, SEQ ID NO. 22 and SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 23, SEQ ID NO. 28 and SEQ ID NO. 23, and SEQ ID NO. 29 and SEQ ID NO. 23.

[0037] A second aspect of this invention provides an isolated polynucleotide encoding the fusion protein provided in the first aspect of this invention. The polynucleotide may be RNA, DNA, or cDNA, etc. Methods for providing the isolated polynucleotide should be known to those skilled in the art. For example, it can be prepared by automated DNA synthesis and / or recombinant DNA techniques, or it can be isolated from a suitable natural source.

[0038] A third aspect of this invention provides a construct containing the isolated polynucleotides provided in the second aspect of this invention. Suitable methods for constructing the construct are known to those skilled in the art. For example, the construct can be obtained by in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc., and more specifically, it can be constructed by inserting the isolated polynucleotides into the multiple cloning site of an expression vector. The expression vector in this invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. Generally, a suitable vector may contain at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers. For example, these promoters may include, but are not limited to, the lac or trp promoter of *E. coli*; the PL promoter of *λ* bacteriophage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the Pichia pastoris methanol oxidase promoter, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. Marker genes can be used to provide phenotypic traits for selecting host cells for transformation. Examples include, but are not limited to, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli. When the polynucleotide is expressed, the expression vector may also include an enhancer sequence. Insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription.

[0039] A fourth aspect of this invention provides an expression system containing a construct or genome provided in the third aspect of this invention with an integrated exogenous polynucleotide provided in the second aspect of this invention, thereby enabling the expression of the aforementioned fusion protein. The expression system can be a host cell; any cell suitable for expression via an expression vector can serve as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; a filamentous fungal cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: *Escherichia coli*, *Streptomyces*; bacterial cells of *Salmonella typhimurium*; fungal cells such as yeast, filamentous fungi, and plant cells; insect cells of *Drosophila S2* or *Sf9*; animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells. Methods for introducing the construct into host cells should be known to those skilled in the art; for example, methods such as microinjection, gene gun methods, electroporation, virus-mediated transformation, electron bombardment, and calcium phosphate precipitation can be used.

[0040] The fifth aspect of the present invention provides a method for preparing the fusion protein provided in the first aspect of the present invention. Those skilled in the art can choose a suitable method to prepare the fusion protein. For example, the preparation method may include: culturing the expression system provided in the fourth aspect of the present invention under suitable conditions to express the fusion protein, collecting the culture containing the fusion protein, and then separating and purifying it to provide the fusion protein.

[0041] The sixth aspect of this invention provides the use of the fusion protein provided in the first aspect of this invention and the culture of the expression system provided in the fourth aspect of this invention in the preparation of pharmaceuticals. The aforementioned pharmaceuticals can be drugs for treating tumors, for example, cancers or solid tumors, specifically lung cancer, melanoma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin's lymphoma, hematological malignancies, head and neck cancer, and nasopharyngeal carcinoma, etc. These cancers can be early, intermediate, or late stages, such as metastatic cancer.

[0042] A seventh aspect of this invention provides a pharmaceutical composition comprising a fusion protein provided in the first aspect of this invention or a culture of an expression system provided in the fourth aspect of this invention. In the above pharmaceutical composition, the content of the fusion protein or culture is typically a therapeutically effective amount. In this invention, a "therapeutically effective amount" generally refers to a dosage that, after an appropriate period of administration, results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of injury or disability caused by disease-related suffering. The ability to inhibit tumor growth can be evaluated in animal model systems used to predict the efficacy of treatment against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro using assays known to those skilled in the art. Therapeutically effective amounts of the fusion protein or pharmaceutical composition typically reduce tumor size or otherwise alleviate symptoms in the subject. Those skilled in the art can select an appropriate therapeutically effective amount based on the specific circumstances, such as the size of the subject, the severity of the subject's symptoms, and the chosen specific composition or route of administration. The prescription for treatment (e.g., determination of dosage, etc.) can be determined by a physician, typically considering factors including, but not limited to, the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors.

[0043] The pharmaceutical compositions provided by this invention may further include pharmaceutically acceptable carriers. These carriers may include various excipients and diluents that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers should be well known to those skilled in the art; for example, a thorough discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991).

[0044] The eighth aspect of the present invention provides a treatment method comprising: administering to an individual a therapeutically effective amount of the fusion protein provided in the first aspect of the present invention, a culture of the expression system provided in the fourth aspect of the present invention, or a pharmaceutical composition provided in the seventh aspect of the present invention.

[0045] In this invention, the term "treatment" includes preventative, curative, or palliative measures that can lead to desired pharmaceutical and / or physiological effects. Preferred effects mean that the treatment can medically reduce one or more symptoms of a disease or completely eliminate the disease, or prevent or delay the onset of the disease and / or reduce the risk of its development or worsening.

[0046] In this invention, "individual" generally includes humans, non-human primates, or other mammals (such as dogs, cats, horses, sheep, pigs, cattle, etc.) who can benefit from treatment using the preparation, kit, or combination of preparations.

[0047] In this invention, the aforementioned fusion protein, expression system culture, or pharmaceutical composition can be used as a single active ingredient or in combination with other agents for administration in combination therapy. For example, the aforementioned bifunctional fusion protein with anticancer activity, expression system culture, or pharmaceutical composition can be combined with at least one other antitumor drug. As another example, the aforementioned bifunctional fusion protein with anticancer activity, expression system culture, or pharmaceutical composition can be used in combination with antibodies targeting other tumor-specific antigens.

[0048] The bifunctional fusion protein with anticancer activity provided by this invention can organically combine the functions of anti-PD-L1 monoclonal antibody blocking PD-L1 / PD-1 interaction, anti-VEGF monoclonal antibody reducing microvascular growth and inhibiting metastatic diseases in a single antibody fusion protein molecule, thereby enabling its use in the treatment of tumors and showing good prospects for industrialization.

[0049] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0050] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0051] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0052] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; these series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATINSTRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0053] Example 1

[0054] Construction and recombinant expression of fusion proteins

[0055] Based on the codon preference of CHO cells, the amino acid sequences of the bifunctional fusion proteins in Table 1 were converted into base sequences. HindIII restriction sites and Kozak sequences (GCCACC) were introduced at the 5' end of the heavy and light chain coding sequences, respectively, and stop codons and EcoRI restriction sites were introduced at the 3' end. Full-length DNA was obtained through gene synthesis (General Biosystems (Anhui) Co., Ltd.). The synthesized heavy and light chain coding genes were double-digested with HindIII-HF (NEB, R3104V) and EcoRI-HF (NEB, R3101V), respectively, and recovered using an agarose gel DNA / PCR product mini-recovery kit (Biomiga). The recovered DNA was then ligated to pCDNA3.1(+) vectors that had also been double-digested with HindIII and EcoRI using T4 ligase (NEB, M0202V). The ligation was performed on Top10 competent cells and cultured on LB ampicillin-resistant plates. Clones were selected for identification and sequencing confirmation. Heavy and light chain expression plasmids based on pCDNA3.1(+) were constructed. The heavy and light chain expression plasmids were extracted using an endotoxin-free plasmid extraction kit (Biomiga, BW-PD3511-02) and mixed 1:1. 1.0 mg of the mixed plasmid was diluted to 25 mL with Wayne293 expression medium (Zhongshan Kangsheng, A21501). 3.0 mg of PEI (linear, 25 KD, Polysciences, Inc.) was diluted to 25 mL with Wayne293 expression medium and added to the plasmid solution. The mixture was incubated at room temperature for 30 minutes. Hek293F cells in logarithmic growth phase (viability >95%) were counted; centrifuged at 1100 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in 450 mL of Wayne293 expression medium. The plasmid-PEI mixture was added to the cell suspension and cultured at 37°C in a 5% CO2 shaker for 7 days. The supernatant was then collected by centrifugation for subsequent protein purification.

[0056] Table 1. Amino acid sequence and coding sequence of the bifunctional fusion protein

[0057]

[0058] Example 2

[0059] Purification of bifunctional fusion proteins

[0060] 2.1 The anti-PD-L1 single-domain antibody is located at the N-terminus of the heavy chain of the anti-VEGF monoclonal antibody.

[0061] Cell fermentation supernatant was adjusted to pH 7.0 and loaded onto a Protein A affinity chromatography column (BorgLone Biotechnology Co., Ltd.). The equilibration buffer was 20 mM PB, 0.15 M NaCl (pH 7.0), and 100% 0.1 M Gly-HCl (pH 3.0). The eluent was pre-added with 10% 1 M Tris-HCl (pH 8.5). The 100% eluent was diluted to a conductivity <3 mS / cm, and the supernatant was adjusted to pH 7.0 and loaded onto a DSP chromatography column (BorgLone Biotechnology Co., Ltd.). Elution was performed at 15% and 100% concentrations (20 mM PB, 0.5 M NaCl, pH 7.0). The 15% eluent fraction was the target protein. Protein concentration was determined using the UV280 method.

[0062] 2.2 The anti-PD-L1 single-domain antibody is located at the N-terminus of the light chain of the anti-VEGF monoclonal antibody.

[0063] Cell fermentation supernatant was adjusted to pH 7.0 and loaded onto a Protein A affinity chromatography column (Borglon Biotechnology Co., Ltd.). The equilibration buffer was 20 mM PB, 0.15 M NaCl (pH 7.0), and elution was performed with 100% 0.1 M Gly-HCl (pH 3.0). The eluent was pre-added with 10% 1 M Tris-HCl (pH 8.5). The 100% eluent was diluted to a conductivity of 4 mS / cm and loaded onto a Super Q (TOSOH) chromatography column. The equilibration buffer was 20 mM Tris, pH 8.0, and the eluent was 500 mM NaCl + 20 mM Tris (pH 8.0). Elution was performed at 35% and 100% concentrations, respectively. Excess light chains were removed by flow-through to obtain the 35% eluent fraction, which was the target protein. Protein concentration was determined using the UV280 method.

[0064] Purity was determined using SEC-HPLC-UV analysis. Detector: Agilent 1100LC; Detection wavelength: 214 nm; Mobile phase: 150 mM pH 7.0 PB + 5% isopropanol; Column: Superdex 200 Increase 5 / 150 GL; Run time: 15 minutes; Column temperature: 25℃. Results showed purities greater than 95%.

[0065] Example 3

[0066] Identifying the function of bifunctional fusion proteins in vitro

[0067] 3.1 In vitro activity assay for anti-PD-L1:

[0068] CD5L-OKT3scFv-CD14 (GenBank: ADN42857.1) was synthesized, digested with HindIII-EcoRI (Takara), and inserted into the vector pCDNA3.1 to construct pCDNA3.1-antiCD3TM. Using human PD-L1 (GenBank: NM_014143.2) as a template, the PD-L1 fragment was amplified with high fidelity. A CMV promoter sequence was introduced at the 5' end of the fragment via overlap PCR, and the resulting ligation into pCDNA3.1-antiCD3TM constructed pCDNA3.1-antiCD3TM-PDL1. CHO cells (Thermo) were transfected with G418 and selected for 10–14 days to produce a stable cell line CHO-antiCD3TM-PDL1.

[0069] The fragment amplified using human PD1 (GenBank: NP_005009.2) as a template was recombinated and ligated into the PB513B1-dual-puro vector (Ubisoft Biotechnology) digested with HindIII-BamHI (Takara) to construct plasmid pB-PD1. High-fidelity amplification was performed using pGL4.30 (Ubisoft Biotechnology) as a template, and the resulting fragment was recovered and recombinated into the pB-PD1 vector digested with SfiI-XbaI (Takara) to construct plasmid pB-NFAT-Luc2p-PD1. After successful plasmid construction, the plasmid was extracted using an endotoxin-free plasmid extraction kit (Biomiga) for transfection of Jurkat cells (Chinese Academy of Sciences Stem Cell Bank). Following the method described in patent CN107022571A, Jurkat cells were treated to achieve a relatively adherent state using 0.1 mg / ml poly-D-lysine. Then, Jurkat cells were transfected according to the transfection instructions of the liposome transfection kit (Lipofectamine 3000; Invitrogen). On the third day, cells were selected under pressure using RPMI 1640 medium (Thermo) containing 10% FBS and 2.5 μg / ml puromycin. Subsequently, medium was added periodically, and the puromycin concentration was gradually increased to 4 μg / ml after cell viability recovered. The final result was a monoclonal Jurkat-NFAT-Luc2p-PD1 cell line.

[0070] CHO-antiCD3TM-PDL1 and Jurkat-NFAT-Luc2p-PD1 cells were collected and counted, and the cell density was adjusted to 4×10⁻⁶. 6Add 25 μl of the fusion protein sample prepared in Example 2 to each well of a 96-well plate at a concentration of 1% BSA. Dilute the sample serially with 1% BSA and add 50 μl to each well. After co-culturing at 37°C and 5% CO2 for 6 h, add 10 μl of luciferase substrate (Promega, E2620) to each well, shake for 2 min, and read the values. Follow the kit instructions.

[0071] 3.2 In vitro activity assay of VEGF antagonistic fragments:

[0072] HEK293 cells were seeded into 6-well cell culture plates, with 1.0 × 10⁶ cells per well. 6 Cells were cultured overnight at 37°C in a 5% CO2 incubator. The transfection system was prepared according to the instructions of the 3000 transfection reagent, containing 1.0 μg of pcDNA-KDR plasmid and 4 μg of pGL4.30 plasmid. Forty-eight hours after transfection, cells were scaled up to 10 cm cell culture dishes, and 200 μg / ml of G418 and 100 μg / ml of Hygromycin were added. Fresh pressurized medium was replaced every 3 days until a clear clonal cluster emerged. Cells were digested, seeded into 96-well cell culture plates, and after single clones emerged, they were stimulated with 0.1 μg / ml VEGF for 6 hours. Chemiluminescence was then detected, and clones with a strong signal response were selected for further scale-up. The final result was a single clone, HEK293-NFAT-KDR. HEK293-NFAT-KDR cells were seeded at a density of 40,000 cells / well and digested using Accutase. Digested cells were collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in analytical medium (DMEM + 5% FBS). Cell counts were performed, and the cell density was adjusted to 1.6 × 10⁶ cells / well. 6 / ml; seed 96-well cell culture plates, 25ul per well; prepare VEGF solution with analytical culture medium at a concentration of 60ng / ml; add to cell culture plates, 25ul per well; prepare fusion protein prepared in Example 2 with analytical culture medium, add to cell culture plates, 25ul per well; incubate at 37°C, 5% CO2 for 6h; add 10ul Bright-Glo luciferase assay reagent (Promega, E2620) to each well, shake for 2 minutes, transfer 80ul lysis buffer to an enzyme-labeled white plate, and read the value using an enzyme-labeled plate.

[0073] The results of the measurement of anti-PD-L1 and anti-VEGF activities of each bifunctional fusion protein are shown in Table 2. As can be seen from Table 2, there was no significant difference in the activities of each bifunctional fusion protein, and all of them showed good in vitro cell activity.

[0074] Table 2. In vitro cell activity of the bifunctional fusion protein

[0075]

[0076] Example 4

[0077] Tumor suppressive activity of bifunctional fusion protein in humanized mice

[0078] The in vivo efficacy of the bifunctional fusion protein of this invention was determined by modeling MDA-MB-231 (human breast cancer) cells in huPBMC-immune humanized mice (M-NSG mice). Female M-NSG mice aged 6-8 weeks were selected and inoculated with MDA-MB-231 cells (10*10E6 + 25% matrix gel). On day 7, PBMCs (5*10E6 / 0.2ml) were injected via the tail vein. Tumor volume and body weight were then observed, and mice with tumor volumes between 140-260 mm² were selected. 3 Mice were randomly divided into 6 groups of 7 mice each, based on tumor volume and body weight. Drug administration began on the day of grouping. Mice with tumors that were too large or too small were culled. Intraperitoneal injections were administered twice weekly: PBS, isotype control IgG1, positive control Avelumab (Merck), DAF-5a, DAF-6a, and combination therapy (see Table 3 for details), for approximately 3 weeks. Blood was collected from the orbital rim before grouping and at the end of the experiment. Animal body weight (measured twice weekly) and tumor volume (measured twice weekly) were recorded during the experiment. Results are as follows: Figure 1 As shown.

[0079] Table 3

[0080] Group Drug sample dose Group 1 Isotype control IgG1 15mg / kg Group 2 Positive control Avelumab 15mg / kg Group 3 DAF-5a 15mg / kg Group 4 DAF-6a 15mg / kg Group 5 PBS 5ul / kg Group 6 Combination therapy: Avelumab + Bevacizumab 15mg / kg each

[0081] In summary, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0082] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention. sequence list <110> Zhejiang Daer Biotechnology Co., Ltd. <120> A bifunctional fusion protein with anticancer activity <160> 45 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <400> 1 Gly Ser Ile Phe Ser Ala Asn Leu 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Gly Gly Thr Phe Ile Thr Tyr Ala 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <400> 3 Gly Arg Ala Phe Leu Thr Tyr Ala 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <400> 4 Gly Arg Pro Phe Ile Thr Tyr Ala 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <400> 5 Gly Arg Thr Phe Ser Thr His Ser 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <400> 6 Ile Ser Ser Arg Gly Ile Thr 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <400> 7 Ile Ser Trp Ser Gly Ser Ser Thr 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <400> 8 Ile Asn Trp Ser Gly Ser Ile Thr 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <400> 9 Ile Asn Trp Asn Gly Asp Ser Thr 1 5 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <400> 10 His Leu Phe His Thr Asp Gly Leu Gly Tyr 1 5 10 <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <400> 11 Ala Ala Lys Ile Ser Gly Ala Thr Arg Glu His Leu Met Thr Ser Tyr 1 5 10 15 Asp Tyr <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <400> 12 Ala Ala Lys Val Ser Gly Ala Thr Arg Asp His Leu Met Thr Thr Tyr 1 5 10 15 Asp Tyr <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <400> 13 Ala Ser Lys Arg Thr Ala Val Ala Pro Arg Ala Leu Asn Asp Tyr Asp 1 5 10 15 Phe <210> 14 <211> 18 <212> PRT <213> Artificial Sequence <400> 14 Ala Ala Lys Ile Ser Gly Ala Thr Arg Glu His Leu Arg Thr Ser Tyr 1 5 10 15 Asp Tyr <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <400> 15 Ala Ala Arg Pro Gly Ala Pro Ser Thr Ile Val Ala Met Leu Asp Tyr 1 5 10 15 <210> 16 <211> 116 <212> PRT <213> Artificial Sequence <400> 16 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ala Asn 20 25 30 Leu Ile Asp Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Val Ile Ser Ser Arg Gly Ile Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Leu Phe His Thr Asp Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 17 <211> 125 <212> PRT <213> Artificial Sequence <400> 17 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ile Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Gly Ile Ser Trp Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Ile Ser Gly Ala Thr Arg Glu His Leu Met Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 18 <211> 125 <212> PRT <213> Artificial Sequence <400> 18 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Leu Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Val Ser Gly Ala Thr Arg Asp His Leu Met Thr Thr Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 19 <211> 124 <212> PRT <213> Artificial Sequence <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Pro Phe Ile Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Ala Ile Asn Trp Ser Gly Ser Ile Thr Ala Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Lys Arg Thr Ala Val Ala Pro Arg Ala Leu Asn Asp Tyr Asp 100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 125 <212> PRT <213> Artificial Sequence <400> 20 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ile Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Gly Ile Ser Trp Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Ile Ser Gly Ala Thr Arg Glu His Leu Arg Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 21 <211> 123 <212> PRT <213> Artificial Sequence <400> 21 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr His 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Ala Ile Asn Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Pro Gly Ala Pro Ser Thr Ile Val Ala Met Leu Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 453 <212> PRT <213> Artificial Sequence <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 23 <211> 214 <212> PRT <213> Artificial Sequence <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 24 <211> 339 <212> PRT <213> Artificial Sequence <400> 24 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ile Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Gly Ile Ser Trp Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Ile Ser Gly Ala Thr Arg Glu His Leu Arg Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Ile Gln 115 120 125 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 130 135 140 Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile Tyr Phe Thr 165 170 175 Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 180 185 190 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 195 200 205 Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly 210 215 220 Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 225 230 235 240 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 245 250 255 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 260 265 270 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 275 280 285 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 290 295 300 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 305 310 315 320 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 325 330 335 Gly Glu Cys <210> 25 <211> 339 <212> PRT <213> Artificial Sequence <400> 25 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ile Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Gly Ile Ser Trp Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Ile Ser Gly Ala Thr Arg Glu His Leu Met Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Ile Gln 115 120 125 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 130 135 140 Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile Tyr Phe Thr 165 170 175 Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 180 185 190 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 195 200 205 Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly 210 215 220 Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 225 230 235 240 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 245 250 255 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 260 265 270 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 275 280 285 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 290 295 300 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 305 310 315 320 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 325 330 335 Gly Glu Cys <210> 26 <211> 337 <212> PRT <213> Artificial Sequence <400> 26 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr His 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Ala Ile Asn Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Pro Gly Ala Pro Ser Thr Ile Val Ala Met Leu Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile Tyr Phe Thr Ser Ser 165 170 175 Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly 210 215 220 Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 225 230 235 240 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 245 250 255 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 260 265 270 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 275 280 285 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 290 295 300 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 305 310 315 320 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 325 330 335 Cys <210> 27 <211> 578 <212> PRT <213> Artificial Sequence <400> 27 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ile Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Gly Ile Ser Trp Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Ile Ser Gly Ala Thr Arg Glu His Leu Arg Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Val Gln 115 120 125 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile 165 170 175 Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg 180 185 190 Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr 210 215 220 Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val Trp Gly Gln 225 230 235 240 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 245 250 255 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 260 265 270 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 275 280 285 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 290 295 300 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 305 310 315 320 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 325 330 335 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 340 345 350 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 355 360 365 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 370 375 380 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 385 390 395 400 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 405 410 415 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 420 425 430 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 435 440 445 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 450 455 460 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 465 470 475 480 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 485 490 495 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 500 505 510 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 515 520 525 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 530 535 540 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 545 550 555 560 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 565 570 575 Gly Lys <210> 28 <211> 578 <212> PRT <213> Artificial Sequence <400> 28 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ile Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Gly Ile Ser Trp Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Ile Ser Gly Ala Thr Arg Glu His Leu Met Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Val Gln 115 120 125 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile 165 170 175 Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg 180 185 190 Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr 210 215 220 Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val Trp Gly Gln 225 230 235 240 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 245 250 255 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 260 265 270 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 275 280 285 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 290 295 300 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 305 310 315 320 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 325 330 335 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 340 345 350 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 355 360 365 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 370 375 380 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 385 390 395 400 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 405 410 415 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 420 425 430 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 435 440 445 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 450 455 460 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 465 470 475 480 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 485 490 495 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 500 505 510 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 515 520 525 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 530 535 540 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 545 550 555 560 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 565 570 575 Gly Lys <210> 29 <211> 576 <212> PRT <213> Artificial Sequence <400> 29 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr His 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Ala Ile Asn Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Pro Gly Ala Pro Ser Thr Ile Val Ala Met Leu Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Val Gln Leu Val 115 120 125 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140 Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val 145 150 155 160 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr 165 170 175 Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr 180 185 190 Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser 195 200 205 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro His 210 215 220 Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 245 250 255 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 260 265 270 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 275 280 285 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 290 295 300 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 305 310 315 320 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 325 330 335 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 340 345 350 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 355 360 365 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 370 375 380 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 385 390 395 400 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 405 410 415 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 420 425 430 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 435 440 445 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 450 455 460 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 465 470 475 480 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 485 490 495 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 500 505 510 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 515 520 525 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 530 535 540 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 545 550 555 560 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 565 570 575 <210> 30 <211> 1017 <212> DNA <213> Artificial Sequence <400> 30 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggtgg caccttcatc acctatgcca tgggctggtt ccgccaagct 120 ccagggaagg gcctggagtt tgtcgcaggt attagttgga gtggtagtag cacatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca acagcaagaa cacgctgtat 240 ctgcaaatga acagcctgcg ggctgaggac acggccgttt attactgtgc agccaaaatc 300 tcgggggcga ctagagagca cctaaggact tcgtatgact actggggcca ggggaccctg 360 gtcaccgtgt cttctgacat ccagatgacc cagagcccta gctctctgtc cgcctctgtg 420 ggcgacagag tgaccatcac atgttccgcc agccaggata tctctaacta cctgaattgg 480 tatcagcaga agccaggcaa ggctcccaag gtgctgatct actttacatc cagcctgcac 540 tctggagtgc catcccgctt ctctggttcc ggaagcggaa ccgactttac cctgacaatc 600 tcttccctgc aacctgagga tttcgccaca tactattgcc agcagtattc caccgtgcct 660 tggacatttg gccagggcac caaggtggag atcaagagga cagtggccgc tccatccgtg 720 ttcatctttc cccctagcga cgagcagctg aagagcggca ccgcctctgt ggtgtgcctg 780 ctgaacaatt tctacccccg ggaggctaag gtgcagtgga aggtggataa cgccctgcaa 840 tctggcaatt cccaggagag cgtgaccgag caggactcta aggattccac atatagcctg 900 agttctaccc tgacactgtc caaggctgac tacgagaagc ataaggtgta tgcctgcgag 960 gtgacccatc agggcctgtc cagccccgtg acaaagagct ttaaccgggg cgagtgt 1017 <210> 31 <211> 1017 <212> DNA <213> Artificial Sequence <400> 31 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggtgg caccttcatc acctatgcca tgggctggtt ccgccaagct 120 ccagggaagg gcctggagtt tgtcgcaggt attagttgga gtggtagtag cacatactat 180 gcagactccg tgaagggtcg attcaccatc tccagagaca acagcaagaa cacgctgtat 240 ctgcaaatga acagcctgcg ggctgaggac acggccgtct attactgtgc agccaaaatc 300 tcgggggcga ctagggagca ctaatgact tcgtatgact actggggcca ggggaccctg 360 gtcaccgtgt cttctgacat ccagatgacc cagagcccta gctctctgtc cgcctctgtg 420 ggcgacagag tgaccatcac atgttccgcc agccaggata tctctaacta cctgaattgg 480 tatcagcaga agccaggcaa ggctcccaag gtgctgatct actttacatc cagcctgcac 540 tctggagtgc catcccgctt ctctggttcc ggaagcggaa ccgactttac cctgacaatc 600 tcttccctgc aacctgagga tttcgccaca tactattgcc agcagtattc caccgtgcct 660 tggacatttg gccagggcac caaggtggag atcaagagga cagtggccgc tccatccgtg 720 ttcatctttc ccctagcga cgagcagctg aagagcggca ccgcctctgt ggtgtgcctg 780 ctgaacaatt tctacccccg ggaggctaag gtgcagtgga aggtggataa cgccctgcaa 840 tctggcaatt cccaggagag cgtgaccgag caggactcta aggattccac atatagcctg 900 agttctaccc tgacactgtc caaggctgac tacgagaagc ataaggtgta tgcctgcgag 960 gtgacccatc agggcctgtc cagccccgtg acaaagagct ttaaccgggg cgagtgt 1017 <210> 32 <211> 1011 <212> DNA <213> Artificial Sequence <400> 32 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggacg caccttcagt acacatagca tgggctggtt ccgccaggct 120 ccagggaagg gcctggagtt tgtagcagct attaactgga atggtgatag cacatattat 180 gcagactccg tgaagggccg attcaccatc tccagagaca acagcaagaa cacgctgtat 240 ctgcaaatga acagcctgcg ggctgaggac acggccgttt attactgtgc agcccgcccg 300 ggcgccccgt cgactatagt ggctatgttg gactactggg gccaggggac cctggtcacc 360 gtgtcttctg acatccagat gacccagagc cctagctctc tgtccgcctc tgtgggcgac 420 agagtgacca tcacatgttc cgccagccag gatatctcta actacctgaa ttggtatcag 480 cagaagccag gcaaggctcc caaggtgctg atctacttta catccagcct gcactctgga 540 gtgccatccc gcttctctgg ttccggaagc ggaaccgact ttaccctgac aatctcttcc 600 ctgcaacctg aggatttcgc cacatactat tgccagcagt attccaccgt gccttggaca 660 tttggccagg gcaccaaggt ggagatcaag aggacagtgg ccgctccatc cgtgttcatc 720 tttcccccta gcgacgagca gctgaagagc ggcaccgcct ctgtggtgtg cctgctgaac 780 aatttctacc cccgggaggc taaggtgcag tggaaggtgg ataacgccct gcaatctggc 840 aattcccagg agagcgtgac cgagcaggac tctaaggatt ccacatatag cctgagttct 900 accctgacac tgtccaaggc tgactacgag aagcataagg tgtatgcctg cgaggtgacc 960 catcagggcc tgtccagccc cgtgacaaag agctttaacc ggggcgagtg t 1011 <210> 33 <211> 1734 <212> DNA <213> Artificial Sequence <400> 33 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc tcctgtgcag cctctggtgg caccttcatc acctatgcca tgggctggtt ccgccaagct 120 ccagggaagg gcctggagtt tgtcgcaggt attagttgga gtggtagtag cacatactat gcagactccg tgaagggccg attcaccatc tccagagaca acagcaagaa cacgctgtat ctgcaaatga acagcctgcg ggctgaggac acggccgttt attactgtgc agccaaaatc 360. tcggggggcga ctaggagca cctaaggact tcgtatgact actggggcca ggggaccctg gtcaccgtgt cttctgaggt gcagctggtg gagtctggag gaggactggt gcagccagga 420 ggctctctga gactgtcctg cgccgctagc ggctacacct tcacaaacta tggcatgaat 480 tgggtgcgcc aggctccagg caagggcctg gagtgggtgg gctggatcaa cacctacaca 540 ggcgagccca cctatgccgc tgactttaag aggaggttca ccttctccct ggacacctcc 660. aagagcacag cctacctcca gatgaactcc ctgagggccg aggacaccgc cgtgtactat tgcgctaagt accccacta ctatggctcc agccattggt atttcgacgt gtggggcag 720 ggcaccctgg tgacagtgtc ttccgcctct accaagggac cttccgtgtt tcctctggct 780 ccaagctcta agtctacctc cggaggaaca gccgctctgg gatgtctggt gaaggactat 840 ttccctgagc cagtgaccgt gtcctggaac agcggcgccc tgacctccgg agtgcacaca 900 tttcctgctg tgctccagtc cagcggcctg tacagcctgt cttccgtggt gaccgtgcca 960 agctcttcc tgggcaccca gacatatatc tgcaacgtga atcacaagcc atccaacaca 1020 aaggtggaca agaaggtgga gcccaagtcc tgcgacaaaa ctcacacatg cccaccgtgc 1080 ccagcacctg aactcctggg tggaccgtca gtttctctct tccccccaaa acccaaggac 1140 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1200 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1260 aagccgcggg aggagcaga aatagcacg taccgtgtgg tcagcgtcct caccgtcctg 1320 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1380 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1440 accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1500 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1560 aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctatagcaag 1620 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1680 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taag 1734 <210> 34 <211> 1734 <212> DNA <213> Artificial Sequence <400> 34 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggtgg caccttcatc acctatgcca tgggctggtt ccgccaagct 120 ccagggaagg gcctggagtt tgtcgcaggt attagttgga gtggtagtag cacatactat 180 gcagactccg tgaagggtcg attcaccatc tccagagaca acagcaagaa cacgctgtat 240 ctgcaaatga acagcctgcg ggctgaggac acggccgtct attactgtgc agccaaaatc 300 tcgggggcga ctagggagca cctaatgact tcgtatgact actggggcca ggggaccctg 360 gtcaccgtgt cttctgaggt gcagctggtg gagtctggag gaggactggt gcagccagga 420 ggctctctga gactgtcctg cgccgctagc ggctacacct tcacaaacta tggcatgaat 480 tgggtgcgcc aggctccagg caagggcctg gagtgggtgg gctggatcaa cacctacaca 540 ggcgagccca cctatgccgc tgactttaag aggaggttca ccttctccct ggacacctcc 600 aagagcacag cctacctcca gatgaactcc ctgagggccg aggacaccgc cgtgtactat 660 tgcgctaagt acccccacta ctatggctcc agccattggt atttcgacgt gtggggacag 720 ggcaccctgg tgacagtgtc ttccgcctct accaagggac cttccgtgtt tcctctggct 780 ccaagctcta agtctacctc cggaggaaca gccgctctgg gatgtctggt gaaggactat 840 ttccctgagc cagtgaccgt gtcctggaac agcggcgccc tgacctccgg agtgcacaca 900 tttcctgctg tgctccagtc cagcggcctg tacagcctgt cttccgtggt gaccgtgcca 960 agctcttcc tgggcaccca gacatatatc tgcaacgtga atcacaagcc atccaacaca 1020 aaggtggaca agaaggtgga gcccaagtcc tgcgacaaaa ctcacacatg cccaccgtgc 1080 ccagcacctg aactcctggg tggaccgtca gtttctctct tccccccaaa acccaaggac 1140 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1200 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1260 aagccgcggg aggagcaga aatagcacg taccgtgtgg tcagcgtcct caccgtcctg 1320 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1380 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1440 accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1500 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1560 Your car ccacgctcc cgtgctggac tccgacggct ccttcttcct ctatagcaag 1620 ctcaccgtgg aagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1680 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taag 1734 <210> 35 <211> 1728 <212> DNA <213> Artificial Sequence <400> 35 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggacg caccttcagt acacatagca tgggctggtt ccgccaggct 120 ccagggaagg gcctggagtt tgtagcagct attaactgga atggtgatag cacatattat 180 gcagactccg tgaagggccg attcaccatc tccagagaca acagcaagaa cacgctgtat 240 ctgcaaatga acagcctgcg ggctgaggac acggccgttt attactgtgc agcccgcccg 300 ggcgccccgt cgactatagt ggctatgttg gactactggg gccaggggac cctggtcacc 360 gtgtcttctg aggtgcagct ggtggagtct ggaggaggac tggtgcagcc aggaggctct 420 ctgagactgt cctgcgccgc tagcggctac accttcacaa actatggcat gaattgggtg 480 cgccaggctc caggcaaggg cctggagtgg gtgggctgga tcaacaccta cacaggcgag 540 cccacctatg ccgctgactt taagaggagg ttcaccttct ccctggacac ctccaagagc 600 acagcctacc tccagatgaa ctccctgagg gccgaggaca ccgccgtgta ctattgcgct 660 aagtaccccc actactatgg ctccagccat tggtatttcg acgtgtgggg acagggcacc 720 ctggtgacag tgtcttccgc ctctaccaag ggaccttccg tgtttcctct ggctccaagc 780 tctaagtcta cctccggagg aacagccgct ctgggatgtc tggtgaagga ctatttccct 840 gagccagtga ccgtgtcctg gaacagcggc gccctgacct ccggagtgca cacatttcct 900 gctgtgctcc agtccagcgg cctgtacagc ctgtcttccg tggtgaccgt gccaagctct 960 tccctgggca cccagacata tatctgcaac gtgaatcaca agccatccaa cacaaaggtg 1020 gacaagaagg tggagcccaa gtcctgcgac aaaactcaca catgcccacc gtgcccagca 1080 cctgaactcc tgggtggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 1140 atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 1200 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1260 cgggaggagc agtacaatag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1320 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1380 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 1440 cccccatccc gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1500 ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1560 aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctatag caagctcacc 1620 gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 1680 ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaag 1728 <210> 36 <211> 1359 <212> DNA <213> Artificial Sequence <400> 36 gaggtgcagc tggtggagtc tggaggagga ctggtgcagc caggaggctc tctgagactg 60 tcctgcgccg ctagcggcta caccttcaca aactatggca tgaattgggt gcgccaggct 120 ccaggcaagg gcctggagtg ggtgggctgg atcaacacct acacaggcga gcccacctat 180 gccgctgact ttaagaggag gttcaccttc tcctggaca cctccaagag cacagcctac 240 ctccagatga actccctgag ggccgaggac accgccgtgt actattgcgc taagtacccc 300 cactactatg gctccagcca ttggtatttc gacgtgtggg gacagggcac cctggtgaca 360 gtgtcttccg cctctaccaa gggaccttcc gtgtttcctc tggctccaag ctctaagtct 420 acctccggag gaacagccgc tctgggatgt ctggtgaagg actatttccc tgagccagtg 480 accgtgtcct ggaacagcgg cgccctgacc tccggagtgc acacatttcc tgctgtgctc 540 cagtccagcg gcctgtacag cctgtctttcc gtggtgaccg tgccaagctc ttccctgggc 600 acccagacat atatctgcaa cgtgaatcac aagccatcca acacaaaggt ggacaagaag 660 gtggagccca agtcctgcga caaaactcac acatgcccac cgtgcccagc acctgaactc 720 ctgggtggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaata gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080 cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacgc agaagagcct ctccctgtct ccgggtaag 1359 <210> 37 <211> 642 <212> DNA <213> Artificial Sequence <400> 37 gacatccaga tgacccagag ccctagctct ctgtccgcct ctgtgggcga cagagtgacc 60 atcacatgtt ccgccagcca ggatatctct aactacctga attggtatca gcagaagcca 120 ggcaaggctc ccaaggtgct gatctacttt acatccagcc tgcactctgg agtgccatcc 180 cgcttctctg gttccggaag cggaaccgac tttaccctga caatctcttc cctgcaacct 240 gaggatttcg ccacatacta ttgccagcag tattccaccg tgccttggac atttggccag 300 ggcaccaagg tggagatcaa gaggacagtg gccgctccat ccgtgttcat ctttccccct 360 agcgacgagc agctgaagag cggcaccgcc tctgtggtgt gcctgctgaa caatttctac 420 ccccgggagg ctaaggtgca gtggaaggtg gataacgccc tgcaatctgg caattcccag 480 gagagcgtga ccgagcagga ctctaaggat tccacatata gcctgagttc taccctgaca 540 ctgtccaagg ctgactacga gaagcataag gtgtatgcct gcgaggtgac ccatcagggc 600 ctgtccagcc ccgtgacaaa gagctttaac cggggcgagt gt 642 <210> 38 <211> 25 <212> PRT <213> Artificial Sequence <400> 38 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <400> 39 Ile Asp Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val Ala 1 5 10 15 Val <210> 40 <211> 17 <212> PRT <213> Artificial Sequence <400> 40 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val Ala 1 5 10 15 Gly <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <400> 41 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val Ala 1 5 10 15 Ala <210> 42 <211> 38 <212> PRT <213> Artificial Sequence <400> 42 Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 43 <211> 38 <212> PRT <213> Artificial Sequence <400> 43 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 44 <211> 38 <212> PRT <213> Artificial Sequence <400> 44 Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 45 <211> 11 <212> PRT <213> Artificial Sequence <400> 45 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10

Claims

1. A fusion protein comprising an anti-PD-L1 single-domain antibody and an antagonistic VEGF antibody, wherein the anti-PD-L1 single-domain antibody is located at the N-terminus of the heavy chain of the antagonistic VEGF antibody; And / or, the anti-PD-L1 single-domain antibody is located at the N-terminus of the light chain of the VEGF antagonistic antibody; The complementarity-determining region of the anti-PD-L1 single-domain antibody includes: The amino acid sequences are as shown in SEQ ID NO.2 (CDR1), SEQ ID NO.7 (CDR2), and SEQ ID NO.11 (CDR3); or The amino acid sequences are as shown in SEQ ID NO.2 (CDR1), SEQ ID NO.7 (CDR2), and SEQ ID NO.14 (CDR3); or The amino acid sequences are as shown in SEQ ID NO.5 (CDR1), SEQ ID NO.9 (CDR2), and SEQ ID NO.15 (CDR3); The VEGF antagonist antibody includes: The amino acid sequences are as shown in SEQ ID No. 22 for the heavy chain and as shown in SEQ ID No. 23 for the light chain; or, The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 of the VEGF antagonistic antibody are as shown in SEQ ID No. 22, namely, CDR1, CDR2, and CDR3 of the heavy chain. The amino acid sequences of the light chains CDR1, CDR2, and CDR3 of the VEGF antagonistic antibody are as shown in SEQ ID No.

23.

2. The fusion protein as described in claim 1, characterized in that, The anti-PD-L1 single-domain antibody also includes a framework region. When the complementarity-determining region of the anti-PD-L1 single-domain antibody includes: amino acid sequences as shown in SEQ ID NO.2 (CDR1), SEQ ID NO.7 (CDR2), and SEQ ID NO.11 (CDR3), The frame region FR includes: FR1 as shown in SEQ ID NO.38, FR2 as shown in SEQ ID NO.40, FR3 as shown in SEQ ID NO.43, and FR4 as shown in SEQ ID NO.45; When the complementarity-determining region of the anti-PD-L1 single-domain antibody includes: amino acid sequences as shown in SEQ ID NO.2 (CDR1), SEQ ID NO.7 (CDR2), and SEQ ID NO.14 (CDR3); or amino acid sequences as shown in SEQ ID NO.5 (CDR1), SEQ ID NO.9 (CDR2), and SEQ ID NO.15 (CDR3), The frame region FR includes: FR1 as shown in SEQ ID NO.38, FR2 as shown in SEQ ID NO.41, FR3 as shown in SEQ ID NO.43, and FR4 as shown in SEQ ID NO.

45.

3. The fusion protein as described in claim 1, characterized in that, The anti-PD-L1 single-domain antibody includes: A polypeptide with an amino acid sequence as shown in one of SEQ ID No. 17, SEQ ID No. 20, or SEQ ID No. 21; And / or, the anti-PD-L1 single-domain antibody is derived from alpacas; And / or, the anti-PD-L1 single-domain antibody is humanized.

4. The fusion protein as described in claim 1, characterized in that, The fusion protein also includes linker peptide fragments.

5. The fusion protein as described in claim 4, characterized in that, The linker peptide fragment is rich in G, S and / or A.

6. The fusion protein as described in claim 5, characterized in that, The linker peptide fragment is selected from a flexible polypeptide chain composed of G-glycine and / or S-serine and / or A-alanine, and the length of the linker peptide fragment is 3 to 30 amino acids.

7. The fusion protein as described in claim 1, characterized in that, The amino acid sequence of the fusion protein includes the sequences shown in SEQ ID NO. 22 and SEQ ID NO. 24, SEQ ID NO. 22 and SEQ ID NO. 25, SEQ ID NO. 22 and SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 23, SEQ ID NO. 28 and SEQ ID NO. 23, and SEQ ID NO. 29 and SEQ ID NO.

23.

8. An isolated polynucleotide encoding the fusion protein as claimed in any one of claims 1 to 7.

9. A construct comprising the isolated polynucleotide as described in claim 8.

10. An expression system comprising an exogenous polynucleotide as described in claim 8 integrated into a construct or genome as described in claim 9.

11. A method for preparing the fusion protein according to any one of claims 1-7, comprising: The expression system as described in claim 10 is cultured under suitable conditions to express the fusion protein, and the fusion protein is isolated and purified to provide the fusion protein.

12. Use of the fusion protein of any one of claims 1-7, or a culture of the expression system of claim 10, in the preparation of a medicament; wherein the medicament is selected from those used to treat solid tumors.

13. A pharmaceutical composition comprising a fusion protein as claimed in any one of claims 1-7, or a culture of an expression system as claimed in claim 10.

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