Bifunctional fusion protein with anticancer activity

Abstract

The invention relates to the technical field of biology, in particular to a bifunctional fusion protein with anticancer activity and a preparation method and application thereof. The invention provides a fusion protein. The fusion protein comprises an anti-PD-L1 single-domain antibody fragment and an antagonistic VEGF (Vascular Endothelial Growth Factor) fragment. The bifunctional fusion protein with anticancer activity provided by the invention can organically combine the functions of blocking PD-L1 / PD-1 interaction of the anti-PD-L1 monoclonal antibody and reducing microvascular growth and inhibiting metastatic diseases of the anti-VEGF monoclonal antibody together 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] The present invention relates to the field of biotechnology, and in particular to a bifunctional fusion protein with anti-cancer activity, a preparation method thereof, and uses thereof. Background Art

[0002] One of the differences between tumor cells and normal cells is the high metabolic demand for their growth. Tumor cells rely on blood vessels to provide nutrients and oxygen, process metabolic products, and promote the formation of new blood vessels on existing blood vessels. Among the angiogenic factors secreted by tumors, human vascular endothelial growth factor (VEGF), especially VEGF-A, is an important factor leading 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 many tumors. For example, bevacizumab (trade name Bevacizumab is a humanized anti-VEGF monoclonal antibody that binds to VEGF and prevents its interaction with VEGF receptors (Flt-1 and KDR) on the surface of endothelial cells. Currently, bevacizumab is FDA-approved 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, resulting in ineffective treatment. Immunotherapy, on the other hand, focuses on activating the immune system and truly changing the immune status of the tumor microenvironment. For example, the first immune checkpoint inhibitor targeting CTLA-4 was approved for marketing by the FDA in 2011. After that, five antibody drugs targeting PD-1 / PD-L1 were launched in the past three years, pushing the research of immune checkpoints into a hot topic. However, although anti-PD-1 / PD-L1 monoclonal antibodies are highly effective and broad-spectrum in tumor treatment, they also have obvious shortcomings, such as limited efficacy (<30%), drug resistance, toxic side effects, etc.

[0004] Given the synergistic biological mechanisms of signaling pathways associated with some targets, combination therapy can often achieve effects not achievable with a single agent. For example, Roche's anti-PD-L1 monoclonal antibody (atezolizumab) and bevacizumab for the treatment of hepatocellular carcinoma (HCC) achieved dual endpoints (significantly prolonged OS and PFS) in a Phase III clinical trial (IMbrave 150) and received FDA approval for marketing in 2020. Furthermore, the triple combination therapy of atezolizumab + bevacizumab + chemotherapy (paclitaxel + carboplatin) significantly prolonged progression-free survival (PFS) in a Phase III clinical trial (IMpower 150) for non-small cell lung cancer (NSCLC) and received FDA approval for marketing in 2018. However, a wealth of data also indicates that while combination therapy improves efficacy, it is often accompanied by increased toxicity. Therefore, finding highly effective and low-toxic treatments 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 above-mentioned shortcomings of the prior art, the object of the present invention is to provide a bifunctional fusion protein with anti-cancer activity and a preparation method and use thereof, so as to solve the problems in the prior art.

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

[0007] Another aspect of the present invention provides an isolated polynucleotide encoding the above-mentioned fusion protein.

[0008] Another aspect of the present invention provides a construct comprising the isolated polynucleotide described above.

[0009] Another aspect of the present invention provides an expression system, which contains the above-mentioned construct or the above-mentioned polynucleotide integrated into the genome.

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

[0011] Another aspect of the present invention provides use of the above-mentioned fusion protein or the culture of the above-mentioned expression system in preparing a drug.

[0012] Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned fusion protein or the culture of the above-mentioned expression system. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1It is a schematic diagram showing the tumor-suppressing effect of the bifunctional fusion protein in Example 4 of the present invention in M-NSG mice. DETAILED DESCRIPTION

[0014] After extensive exploration and research, the inventors of the present invention unexpectedly discovered a fusion protein molecule that can reduce tumor neovascularization and relieve immunosuppression by blocking the PD-L1 / PD-1 interaction and the VEGF signaling pathway, and has excellent tumor inhibitory effects. The present invention was completed on this basis.

[0015] The first aspect of the present invention provides a fusion protein comprising an anti-PD-L1 single-domain antibody fragment and an antagonist VEGF fragment. In the above-mentioned fusion protein, the anti-PD-L1 single-domain antibody fragment can generally be used to block the PD-L1 / PD-1 interaction, increase the expression of IFN-γ and / or IL-2 in T lymphocytes, and thus inhibit tumor growth. The variable region of the antagonist VEGF fragment can bind to VEGF, thereby blocking the formation of tumor angiogenesis and "starving" cancer cells. The antagonist VEGF fragment can also generally include an Fc portion, which can bind to the FcRn receptor, thereby extending the half-life in vivo, and can also bind to effector cells expressing Fc receptors to kill cancer cells.

[0016] The fusion protein provided by the present invention may include an anti-PD-L1 single domain antibody fragment. The above-mentioned anti-PD-L1 single domain antibody fragment may generally be a polypeptide or protein fragment that can specifically bind to PD-L1. In the anti-PD-L1 single domain antibody fragment, the corresponding antibody light chain is usually missing, and only the fragment corresponding to the heavy chain variable region is present. The binding properties of the anti-PD-L1 single domain antibody fragment can generally be determined by the three complementarity determining regions (CDRs) it includes. The CDR region can be arranged in order with the framework region (FR), and the FR region is generally not directly involved in the binding reaction. These CDRs can form a loop structure, and the β-folds formed by the FRs therebetween are close to each other in the spatial structure, forming the antigen binding site of the antibody. For example, the complementarity determining region (CDR) of the above-mentioned anti-PD-L1 single domain antibody fragment may include an amino acid sequence such as CDR1 shown in one of SEQ ID NOs. 1 to 5, CDR2 shown in one of SEQ ID NOs. 6 to 9, and CDR3 shown in one of SEQ ID NOs. 10 to 15.

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

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

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

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

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

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

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

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

[0025] The amino acid sequence is FR1 shown in SEQ ID NO.38, FR2 shown in SEQ ID NO.39, FR3 shown in SEQ ID NO.42; FR4 shown in SEQ ID NO.45, or

[0026] The amino acid sequence is FR1 shown in SEQ ID NO.38, FR2 shown in SEQ ID NO.40, FR3 shown in SEQ ID NO.43; FR4 shown in SEQ ID NO.45, or

[0027] The amino acid sequence is FR1 shown in SEQ ID NO.38, FR2 shown in SEQ ID NO.41, FR3 shown in SEQ ID NO.43; FR4 shown in SEQ ID NO.45, or

[0028] The amino acid sequence is FR1 shown in SEQ ID NO.38, FR2 shown in SEQ ID NO.41, FR3 shown in SEQ ID NO.44; and FR4 shown in SEQ ID NO.45.

[0029] In another specific embodiment of the present invention, the anti-PD-L1 single-domain antibody fragment may include: a) a polypeptide fragment having an amino acid sequence as shown in one of SEQ ID Nos. 16 to 21; or, b) a polypeptide fragment having an amino acid sequence having a sequence identity of more than 80% with one of SEQ ID Nos. 16 to 21 and having the function of the polypeptide fragment specified in a). Specifically, the polypeptide fragment in the above b) specifically refers to: an amino acid sequence as shown in one of SEQ ID Nos. 16 to 21, obtained by substitution, deletion or addition of one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or a polypeptide fragment obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the functions of a polypeptide fragment as shown in one of SEQ ID Nos. 16 to 21, for example, the ability to specifically bind to PD-L1, the ability to block the PD-L1 / PD-1 interaction, thereby blocking the PD-L1 / PD1 pathway, the ability to increase the expression of IFN-γ and / or IL-2 in T lymphocytes, or the ability to inhibit 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 greater identity to one of SEQ ID Nos. 16 to 21. The anti-PD-L1 single-domain antibody fragment may typically be derived from alpaca (Vicugna pacos), for example, its CDR region may be derived from alpaca. The anti-PD-L1 single-domain antibody fragment may typically be humanized, for example, its framework region may be derived from human.

[0030] Herein, sequence identity refers to the percentage of identical residues in the compared sequences. The sequence identity of two or more sequences can be calculated using software well known in the art, such as those available from NCBI.

[0031] The fusion proteins provided herein may include VEGF antagonist fragments. These VEGF antagonist fragments may generally be polypeptides or protein fragments capable of antagonizing VEGF. For example, these VEGF antagonist fragments may be monoclonal antibodies. In another example, these VEGF antagonist fragments may be bevacizumab.

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

[0033] c) a polypeptide fragment whose amino acid sequence is shown in one of SEQ ID Nos. 22 to 23;

[0034] d) A polypeptide fragment having an amino acid sequence with more than 80% sequence identity with one of SEQ ID Nos. 22 to 23 and having the function of the polypeptide fragment defined in c). Specifically, the amino acid sequence in d) above specifically refers to an amino acid sequence as set forth in any one of SEQ ID Nos. 22-23, obtained by substitution, deletion, or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or a polypeptide fragment obtained by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and / or C-terminus, and having the functions of a polypeptide fragment as set forth in any one of SEQ ID Nos. 22-23, for example, the function of specifically antagonizing VEGF, or the function of an Fc portion that binds to an FcRn receptor, thereby prolonging the in vivo half-life, and also binding to effector cells expressing Fc receptors to kill cancer cells. The amino acid sequence in d) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or greater identity to one of SEQ ID Nos. 22-23. The antagonist VEGF fragment may typically be derived from mouse (Mus musculus), for example, its CDR region may be derived from mouse. The antagonist VEGF fragment may typically be humanized, for example, its framework region may be derived from human.

[0035] The fusion protein provided by the present invention may further include a connecting peptide fragment. The fusion protein may generally include a plurality of connecting peptide fragments, and connecting peptide fragments may be provided between at least some of the domains or each domain. For example, a connecting peptide may be provided between an anti-PD-L1 single domain antibody fragment and an antagonist VEGF fragment. The connecting peptide fragment may generally be a flexible polypeptide rich in G, S and / or A (mainly composed of glycine (G), serine (S) and / or alanine (A)) of suitable length, so that adjacent protein domains can move freely relative to each other. For example, the amino acid sequence of the connecting peptide fragment may include sequences such as (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGS)nG, (GGGGA)nA, (GGGGG)nA, etc., wherein n is selected from an integer between 1-10. In a specific embodiment of the present invention, the length of the amino acid sequence of the connecting 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] In the fusion protein provided by the present invention, the fusion protein can be linear. For example, the fusion protein can include an anti-PD-L1 single domain antibody fragment and an antagonist VEGF fragment in sequence from the N-terminus to the C-terminus. The fusion protein can also have a structure similar to that of 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 antagonist VEGF fragment, or the anti-PD-L1 single domain antibody fragment can be located at the N-terminus of the light chain of the antagonist VEGF fragment. In a specific embodiment of the present invention, the amino acid sequence of the fusion protein can include a sequence shown in one of SEQ ID NOs. 22-29. For example, the amino acid sequence of the fusion protein can include a sequence shown in SEQ ID NOs. 22 and 24, a sequence shown in SEQ ID NOs. 22 and 25, a sequence shown in SEQ ID NOs. 22 and 26, a sequence shown in SEQ ID NOs. 27 and 23, a sequence shown in SEQ ID NOs. 28 and 23, or a sequence shown in SEQ ID NOs. 29 and 23.

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

[0038] The third aspect of the present application provides a construct comprising the isolated polynucleotide provided by the second aspect of the present application. Suitable methods for constructing the construct should be known to those skilled in the art. For example, the construct can be constructed by in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. More specifically, the construct can be constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector. The expression vector in the present application 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 adenovirus, retrovirus or other vectors. Generally, a suitable vector can contain a replication origin that functions in at least one organism, a promoter sequence, convenient restriction sites, and one or more selectable markers. For example, the promoters can include but are not limited to the lac or trp promoters of E. coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the methanol oxidase promoter of Pichia pastoris and other known promoters that control expression of genes in prokaryotic or eukaryotic cells or viruses. Marker genes can be used to provide phenotypic traits for selection of transformed host cells, for example, can include but are not limited to dihydrofolate reductase for eukaryotic cell culture, neomycin resistance and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc. When the polynucleotide is expressed, an enhancer sequence can also be included in the expression vector, which will enhance transcription if inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, which act on promoters to enhance transcription of genes.

[0039] The fourth aspect of the present invention provides an expression system comprising the construct provided in the third aspect of the present invention or the polynucleotide provided in the second aspect of the present invention integrated into its genome, thereby expressing the aforementioned fusion protein. The aforementioned expression system can be a host cell. Any cell suitable for expression by 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, plant cells; insect cells of Drosophila S2 or Sf9; and 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, and can include, for example, microinjection, gene gun techniques, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like.

[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 select 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 to provide the fusion protein.

[0041] A sixth aspect of the present invention provides use of the fusion protein provided in the first aspect of the present invention and the culture of the expression system provided in the fourth aspect of the present invention in the preparation of a medicament. The medicament can be a medicament for treating a tumor, for example, a cancer or solid tumor, 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 in the early, middle, or late stages, such as metastatic cancer.

[0042] A seventh aspect of the present invention provides a pharmaceutical composition comprising the fusion protein provided in the first aspect of the present invention or a culture of the expression system provided in the fourth aspect of the present invention. The fusion protein or culture in the pharmaceutical composition is generally present in a therapeutically effective amount. As used herein, a "therapeutically effective amount" generally refers to an amount that, after an appropriate administration period, results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or the prevention of impairment or disability resulting from the pain of the disease. The ability to inhibit tumor growth can be evaluated in animal model systems that are predictive of efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be measured in vitro using assays known to those skilled in the art. A therapeutically effective amount of the fusion protein or pharmaceutical composition is generally capable of reducing tumor size or otherwise alleviating symptoms in a subject. A person skilled in the art can select an appropriate therapeutically effective amount based on the specific circumstances, for example, the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected. The prescription of treatment (e.g., dosage determination, 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 delivery site, the method of administration, and other factors.

[0043] The pharmaceutical compositions provided herein may further include a pharmaceutically acceptable carrier. Such carriers may include various excipients and diluents, which are not essential active ingredients and are not excessively toxic upon administration. Suitable carriers are well known to those skilled in the art. For example, a comprehensive 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 by the first aspect of the present invention, the culture of the expression system provided by the fourth aspect of the present invention, or the pharmaceutical composition provided by the seventh aspect of the present invention.

[0045] As used herein, the term "treatment" encompasses preventive, curative, or palliative treatments that result in the desired pharmaceutical and / or physiological effect. Preferably, the effect is a reduction in one or more symptoms of a disease, complete elimination of the disease, or the retardation or delay of the onset of the disease and / or a reduction in the risk of disease progression or worsening.

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

[0047] In the present invention, the above-mentioned fusion protein, culture product of the expression system, 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 above-mentioned bifunctional fusion protein with anti-cancer activity, culture product of the expression system, or pharmaceutical composition can be combined with at least one other anti-tumor drug. For another example, the above-mentioned bifunctional fusion protein with anti-cancer activity, culture product of the expression system, or pharmaceutical composition can be combined with antibodies targeting other tumor-specific antigens.

[0048] The bifunctional fusion protein with anti-cancer activity provided by the present invention can organically combine the functions of anti-PD-L1 monoclonal antibody to block PD-L1 / PD-1 interaction and anti-VEGF monoclonal antibody to reduce microvascular growth and inhibit metastatic disease in a single antibody fusion protein molecule, thereby being used to treat tumors and having good industrial prospects.

[0049] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0050] Before further describing the 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 terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0051] When the embodiments provide numerical ranges, it should be understood that unless otherwise specified 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 the present invention have the same meaning as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0052] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are well described in the literature, for example, in 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] The amino acid sequences of the bifunctional fusion proteins in Table 1 were converted into base sequences according to the codon bias of CHO cells, and a HindIII enzyme cutting site and a Kozak sequence (GCCACC) were introduced at the 5' end of the heavy chain and light chain coding sequences, respectively, and a stop codon and an EcoRI enzyme cutting site were introduced at the 3' end to obtain the full-length DNA by gene synthesis (Universal Biological Systems (Anhui) Co., Ltd.). The synthesized heavy chain and light chain coding genes were subjected to HindIII-HF (purchased from NEB, R3104V) and EcoRI-HF (purchased from NEB, R3101V) double enzyme digestion, respectively, and the gel was recovered with an agarose gel DNA / PCR product small volume recovery kit (purchased from Biomiga), and ligated with a pCDNA3.1(+) vector subjected to HindIII and EcoRI double enzyme digestion using T4 ligase (purchased from NEB, M0202V), transformed into Top10 competent cells, and cultured on LB ampicillin-resistant plates. The clones were picked and identified, and sequencing was confirmed to construct a heavy chain and light chain expression plasmid based on pCDNA3.1(+), respectively. The heavy chain and light chain expression plasmids were extracted using a de-toxified plasmid large extraction kit (purchased from Biomiga, BW-PD3511-02), and mixed at a ratio of 1:1. 1.0 mg of the mixed plasmid was diluted to 25 mL using Wayne293 expression medium (purchased from Zhongshan Kangsheng, A21501); 3.0 mg of PEI (linear, 25KD, Polysciences, Inc.) was diluted to 25 mL using Wayne293 expression medium and added to the plasmid solution, mixed, and incubated at room temperature for 30 minutes. The Hek293F cells in the 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 above plasmid-PEI mixture was added to the cell suspension, and after 7 days of culture in a 37°C, 5% CO2 incubator, the supernatant was centrifuged and used for subsequent protein purification.

[0056] Table 1 Bifunctional fusion protein amino acid sequences and coding sequences

[0057]

[0058] Example 2

[0059] Purification of bifunctional fusion proteins

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

[0061] The cell fermentation supernatant was adjusted to pH 7.0 and loaded onto a Protein A affinity chromatography column (Borglon Biotechnology Co., Ltd.). The column was equilibrated with 20 mM PB, 0.15 M NaCl (pH = 7.0) and eluted with 100% 0.1 M Gly-HCl (pH = 3.0). The eluate was pre-spiked with 10% 1 M Tris-HCl (pH = 8.5). The 100% eluate was diluted to a conductivity < 3 ms / cm. The supernatant was adjusted to pH 7.0 and loaded onto a DSP column (Borglon Biotechnology Co., Ltd.). Elution was performed at 15% and 100% (20 mM PB, 0.5 M NaCl, pH 7.0). The 15% elution fraction was the target protein. Protein concentration was determined using UV280.

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

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

[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°C. The results showed a purity greater than 95%.

[0065] Example 3

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

[0067] 3.1 Anti-PD-L1 in vitro activity detection:

[0068] CD5L-OKT3scFv-CD14 (GenBank: ADN42857.1) was synthesized and digested with HindIII-EcoRI (Takara) and inserted into the vector pCDNA3.1 to construct pCDNA3.1-antiCD3TM. The PD-L1 fragment was amplified with high fidelity using human PD-L1 (GenBank: NM_014143.2) as a template. A CMV promoter sequence was introduced at the 5' end of the sequence by overlapping PCR and recombinantly ligated into pCDNA3.1-antiCD3TM to construct pCDNA3.1-antiCD3TM-PDL1. CHO cells (Thermo) were transfected, followed by G418 selection for 10-14 days to generate the stable cell line CHO-antiCD3TM-PDL1.

[0069] The resulting fragment was amplified using human PD1 (GenBank: NP_005009.2) as a template and recombined with the PB513B1-dual-puro vector (Ubao Biotech) digested with HindIII-BamHI (Takara) to construct the plasmid pB-PD1. High-fidelity amplification was performed using pGL4.30 (Ubao Biotech) as a template. The resulting fragment was recovered and recombined with the pB-PD1 vector digested with SfiI-XbaI (Takara) to construct the pB-NFAT-Luc2p-PD1 plasmid. After successful plasmid construction, the plasmid was extracted using the Endotoxin-Removed Plasmid Maximizer Kit (Biomiga) and used for transfection into Jurkat cells (Stem Cell Bank, Chinese Academy of Sciences). Referring to the method described in patent CN107022571A, Jurkat cells were treated with 0.1 mg / ml poly-D-lysine to achieve a relatively adherent state. They were then transfected according to the instructions in the lipofectamine 3000 transfection kit (Invitrogen). On the third day, pressure selection was performed using RPMI1640 medium (Thermo) supplemented with 10% FBS and 2.5 μg / ml puromycin. Thereafter, medium was replenished at regular intervals, and the puromycin content was gradually increased to 4 μg / ml after cell viability recovered. This resulted in a monoclonal Jurkat-NFAT-Luc2p-PD1 cell line.

[0070] Take CHO-antiCD3TM-PDL1 and Jurkat-NFAT-Luc2p-PD1 cells and count them, and adjust the cell density to 4×10 6 / ml, add 25 μl to each cell per well of a 96-well plate; serially dilute the fusion protein sample prepared in Example 2 with 1% BSA and add 50 μl to the cells; incubate at 37°C, 5% CO2 for 6 hours, then add 10 μl of luciferase substrate (Promega, E2620) to each well, shake on a shaker for 2 minutes, and read the results. Follow the kit instructions.

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

[0072] HEK293 cells were plated in 6-well cell culture plates, with 1.0 × 10 cells per well. 6 The cells were cultured overnight in a 37°C, 5% CO2 incubator. The transfection system was prepared according to the instructions of the 3000 transfection reagent, including 1.0 μg of pcDNA-KDR plasmid and 4 μg of pGL4.30 plasmid. 48 hours after transfection, cells were expanded to 10 cm cell culture dishes and supplemented with 200 μg / ml G418 and 100 μg / ml Hygromycin. Fresh pressurized culture medium was replaced every 3 days until obvious clones grew. The cells were digested and plated on 96-well cell culture plates. After a single clone grew, 0.1 μg / ml VEGF was used to stimulate the cells for 6 hours and then the chemiluminescence was detected. The clones with obvious signal response were selected and further expanded and cultured. Finally, a single clone of HEK293-NFAT-KDR was obtained. HEK293-NFAT-KDR cells were plated at a density of 40,000 per well and digested with Accutase. The digested cells were collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in assay culture medium (DMEM + 5% FBS). The cells were counted and the cell density was adjusted to 1.6×10 6 / ml; plate a 96-well cell culture plate, 25ul per well; use analytical culture medium to prepare a VEGF solution at a concentration of 60ng / ml; add 25ul to the cell culture plate, 25ul per well; use analytical culture medium to prepare the fusion protein prepared in Example 2, add 25ul to the cell culture plate, 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 of the lysate to an enzyme-labeled white plate, and read the results with a microplate reader.

[0073] The measurement results of the anti-PD-L1 activity and anti-VEGF activity of each bifunctional fusion protein are shown in Table 2. As shown in Table 2, there is no significant difference in the activity of each bifunctional fusion protein, and all show good in vitro cell activity.

[0074] Table 2 In vitro cell activity of bifunctional fusion proteins

[0075]

[0076]

[0077] Example 4

[0078] Tumor suppressor activity of bifunctional fusion protein in humanized mice

[0079] The in vivo efficacy of the bifunctional fusion protein of the present invention was determined by modeling MDA-MB-231 (human breast cancer) cells in huPBMC immune system 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% Matrigel). On the 7th day, PBMC (5*10E6 / 0.2ml) was injected into the tail vein. Tumor volume and body weight were then observed. Mice with tumor volumes of 140-260 mm were selected. 3 Mice between 1 and 2 weeks old were randomly divided into 6 groups based on their tumor volume and body weight, with 7 mice in each group, and medication began on the day of grouping. Tumor-bearing mice with tumors that were too large / too small were eliminated. Twice a week, intraperitoneal injections of: PBS, isotype control IgG1, positive control Avelumab (Merck), DAF-5a, DAF-6a and combined administration (see Table 3 for details of the regimen) were given for a total of about 3 weeks. Blood was collected from the eye sockets before grouping and before the end of the experiment. During the experiment, the weight of the animals (measured twice a week) and the tumor volume (measured twice a week) were measured. The results are shown in the figure below. Figure 1 shown.

[0080] Table 3

[0081] Group Dosing samples dose Group 1 Isotype control IgG1 15 mg / kg Group 2 Positive control Avelumab 15 mg / kg Group 3 DAF-5a 15 mg / kg Group 4 DAF-6a 15 mg / kg Group 5 PBS 5ul / kg Group 6 Combination therapy: Avelumab + Bevacizumab 15 mg / kg each

[0082] In summary, the present invention effectively overcomes various shortcomings of the prior art and has high industrial utilization value.

[0083] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may 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 one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A fusion protein comprising an anti-PD-L1 single-domain antibody fragment and an antagonist VEGF fragment, wherein the complementarity determining region of the anti-PD-L1 single-domain antibody fragment comprises: The amino acid sequences are CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 6, and CDR3 shown in SEQ ID NO. 10; or The amino acid sequence is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 7, and CDR3 shown in SEQ ID NO. 11; or The amino acid sequence is CDR1 shown in SEQ ID NO.3, CDR2 shown in SEQ ID NO.7, and CDR3 shown in SEQ ID NO.12; or The amino acid sequence is CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.13; or The amino acid sequence is CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 7, and CDR3 shown in SEQ ID NO. 14; or The amino acid sequence is CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.9, and CDR3 shown in SEQ ID NO.

15.

2. The fusion protein according to claim 1, wherein The anti-PD-L1 single domain antibody fragment comprises: a) a polypeptide fragment whose amino acid sequence is represented by one of SEQ ID Nos. 16 to 21; or, b) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to one of SEQ ID Nos. 16 to 21 and having the function of the polypeptide fragment defined in a); and / or, the anti-PD-L1 single domain antibody fragment is derived from alpaca; And / or, the anti-PD-L1 single domain antibody fragment is humanized.

3. The fusion protein according to claim 1, wherein The antagonistic VEGF fragment comprises: c) a polypeptide fragment whose amino acid sequence is shown in one of SEQ ID No. 22 to 23; or d) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity with one of SEQ ID Nos. 22 to 23 and having the function of the polypeptide fragment defined in c); or e) the antigen-binding region of bevacizumab; and / or, the antagonistic VEGF fragment is of mouse origin; And / or, the antagonist VEGF fragment is humanized.

4. The fusion protein according to claim 1, wherein The fusion protein further includes a connecting peptide fragment. Preferably, the connecting peptide fragment is rich in G, S and / or A. More preferably, the connecting peptide is selected from a flexible polypeptide chain consisting of G glycine and / or S serine and / or A alanine, and the length of the connecting peptide is 3 to 30 amino acids.

5. The fusion protein according to any one of claims 1 to 4, characterized in that: The fusion protein includes an anti-PD-L1 single domain antibody fragment and an antagonistic VEGF fragment from the N-terminus to the C-terminus; and / or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the heavy chain of the antagonist VEGF fragment; And / or, the anti-PD-L1 single domain antibody fragment is located at the N-terminus of the light chain of the antagonist VEGF fragment.

6. The fusion protein according to claim 1, wherein The amino acid sequence of the fusion protein includes the sequence shown in one of SEQ ID NOs. 22-29; Or, the amino acid sequence of the fusion protein includes the sequence shown in SEQ ID NO.22 and SEQ ID NO.24, the sequence shown in SEQ ID NO.22 and SEQ ID NO.25, the sequence shown in SEQ ID NO.22 and SEQ ID NO.26, the sequence shown in SEQ ID NO.27 and SEQ ID NO.23, the sequence shown in SEQ ID NO.28 and SEQ ID NO.23, and the sequence shown in SEQ ID NO.29 and SEQ ID NO.

23.

7. An isolated polynucleotide or an expression construct comprising the polynucleotide, characterized in that The polynucleotide encodes the fusion protein according to any one of claims 1 to 6.

8. An expression system comprising the construct according to claim 7 or the exogenous polynucleotide according to claim 7 integrated into the genome.

9. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 6, or a culture of the expression system according to claim 8.

10. Use of the fusion protein according to any one of claims 1 to 6, the culture of the expression system according to claim 8, or the pharmaceutical composition according to claim 9 in preparing a drug, preferably, the drug is a drug for treating tumors.

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