Fusion proteins comprising mesothelin, tumor vaccines, and methods of making and using the same

Abstract

This invention discloses a mesothelin-containing fusion protein, a tumor vaccine, its preparation method, and its applications. The amino acid sequence of the fusion protein is shown in SEQ ID NO:1, and the active ingredient of the tumor vaccine is the fusion protein. The tumor vaccine of this invention induced an effective tumor-specific cytotoxic T lymphocyte (CTL) response in mice, significantly enhancing antigen immunogenicity, exhibiting a highly efficient response, markedly inhibiting the growth of lung cancer cells, and prolonging the lifespan of mice. This tumor vaccine can be applied to various cancers that highly express MSLN and PD-L1, including mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, and triple-negative breast cancer, providing a novel immunotherapy approach for numerous solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically to the field of gene-engineered drugs and vaccine manufacturing technology, and more specifically, to a fusion protein containing mesothelin, a tumor vaccine prepared from said fusion protein, its preparation method and application. Background Technology

[0002] Lung cancer is the most common cancer worldwide, with its incidence rate rising annually and its mortality rate ranking first among malignant tumor-related deaths. The prognosis for lung cancer is relatively poor, with a 5-year survival rate ranging from 4% to 17%. According to national statistics in China, approximately 631,000 people die from lung cancer each year. Contrary to the declining incidence rates in some Western countries, the incidence rate in China continues to rise, placing a significant burden on society. Tumor immunotherapy is a treatment method that works by reshaping the immune system of cancer patients to kill tumor cells; it is another comprehensive treatment approach following surgery, chemotherapy, and radiotherapy.

[0003] Currently, major tumor immunotherapy regimens include therapeutic tumor vaccines, immune checkpoint inhibitors, adoptive cell immunotherapy, and oncolytic viruses. Although research on immune checkpoint inhibitors and CAR-T has yielded significant results, their efficacy against solid tumors remains limited. For example, while immune checkpoint inhibitors have been approved for many solid tumor indications, their therapeutic characteristics suggest they can only relieve the binding of T cells already located at the tumor margin or enhance their presentation; they cannot stimulate T cells to attack the tumor, and some patients do not exhibit an immune response. CAR-T therapy has shown good efficacy against hematologic malignancies targeting the CD19 antigen. In the second half of 2017, two CAR-T products were approved, including Novartis' Kymriah and Gilead's Yescarta, both for B-cell lymphoma. However, their efficacy in treating solid tumors still awaits further breakthroughs. Therapeutic tumor vaccines have become an important breakthrough in the treatment of solid tumors because they enable T cells to attack tumors with high specificity. The principle is to activate the patient's own immune system, use tumor cells or tumor antigens to induce specific cellular and humoral immune responses in the body, enhance the body's anti-cancer ability, and prevent the growth, spread and recurrence of tumors, so as to achieve the purpose of eliminating or controlling tumors.

[0004] Mesothelin (MSLN) is a cell surface-bound glycosylphosphatidylinositol (GPI)-anchored protein that is typically expressed only in mesothelial cells on the surface of body cavities and not in other normal tissues. Overexpression of MSLN has been reported in nearly 40% of solid tumors, such as mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, and triple-negative breast cancer. Studies have confirmed that MSLN plays an important role in tumor cell survival, invasion, metastasis, and angiogenesis. Therefore, MSLN has become an important target for developing novel anti-tumor immunotherapies. Over the years, various types of anti-MSLN therapies have been developed, including antibodies, antibody-drug conjugates (ADCs), cancer vaccines, and chimeric antigen receptor (CAR)-T cell immunotherapy. However, the weak immunogenicity and low response rate of tumor antigens remain a major challenge. Therefore, developing novel tumor vaccines targeting the tumor target protein MSLN is of great significance. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a vaccine that targets the tumor target protein MSLN, has strong antigenic immunogenicity, and has a high response efficiency.

[0006] The specific technical solutions for achieving the above-mentioned objectives are as follows.

[0007] In a first aspect, the present invention provides a fusion protein comprising mesothelin, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides a gene encoding the above-mentioned fusion protein comprising mesothelin, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0009] A third aspect of the present invention provides a method for preparing the above-described fusion protein containing mesothelin, the method comprising the following steps:

[0010] (1) Synthesize a fusion gene fragment containing the gene sequences of human MSLN, PDL1, PADRE and GMCSF; the nucleotide sequence of the fusion gene fragment is shown in SEQ ID NO:2;

[0011] (2) The fusion gene fragment and pET-21a plasmid vector from step (1) were digested with NdeI and XhoI, recovered by gel purification kit, and ligated to obtain the expression plasmid.

[0012] (3) The expression plasmid was transformed into the BL21(DE3) expression strain, and the target protein was obtained by IPTG induction. After purification and dialysis, the fusion protein was obtained.

[0013] In a fourth aspect, the present invention provides the use of the above-described fusion protein containing mesothelin in the preparation of a tumor vaccine.

[0014] In a fifth aspect, the present invention provides a tumor vaccine, wherein the active ingredient of the tumor vaccine is the aforementioned fusion protein containing mesothelin.

[0015] A sixth aspect of the present invention provides a method for preparing the above-mentioned tumor vaccine, comprising the following steps:

[0016] (1) After culturing dendritic cells for 4 to 5 days, add 80 μg / mL to 120 μg / mL of the above-mentioned fusion protein containing mesothelin to the culture medium overnight;

[0017] (2) Add bacterial lipopolysaccharide and continue culturing for 2-3 days to obtain the product.

[0018] A seventh aspect of the present invention provides the use of the above-described tumor vaccine in the preparation of a medicament for treating solid tumors.

[0019] After extensive research, the inventors of this invention selected a specific peptide region from the MSLN gene and combined it with the immune checkpoint PD-L1 as a target protein. Using GM-CSF as an adjuvant, and fusing PADRE and SPACE sequences at appropriate positions, they successfully expressed a fusion protein containing mesothelin using the pET-21a / HIS system. Loading this mesothelin-containing fusion protein into dendritic cells yielded a novel tumor vaccine that can significantly enhance antigen immunogenicity and provide a highly efficient response.

[0020] The tumor vaccine containing mesothelin as the active ingredient of this invention induced an effective tumor-specific cytotoxic T lymphocyte (CTL) response in mice, significantly inhibited the growth of lung cancer cells, and prolonged the lifespan of mice. This tumor vaccine can be applied to various cancers that highly express MSLN and PD-L1, including mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, and triple-negative breast cancer, providing a new immunotherapy for many solid tumors. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the pET-21a-MSLN-PDL1-GMCSF expression plasmid.

[0022] Figure 2 SDS-PAGE maps of the fusion proteins PDL1-GMCSF and MSLN-PDL1-GMCSF expressed by the expression plasmids, where M: marker; S△: supernatant of bacterial lysis induced by IPTG; P: precipitate after bacterial lysis without IPTG induction; P△: precipitate after bacterial lysis induced by IPTG induction.

[0023] Figure 3 The images are for Western blot analysis of the induced target fusion proteins PDL1-GMCSF and MSLN-PDL1-GMCSF.

[0024] Figure 4 This is an SDS-PAGE image of the lysis buffer and flow buffer of the fusion protein PDL1-GMCSF during the purification process.

[0025] Figure 5 The images show the SDS-PAGE spectra of the lysis buffer and flow buffer of the fusion protein MSLN-PDL1-GMCSF during the purification process.

[0026] Figure 6 This is a map of Western blot analysis (WB) of the dialysis-treated fusion proteins PDL1-GMCSF and MSLN-PDL1-GMCSF using His-tagged antibodies.

[0027] Figure 7 This diagram illustrates the process of inducing BMDC in mouse bone marrow cells.

[0028] Figure 8 For flow cytometry detection of CD11c in BMDC + and CD80 + Results of DC cells.

[0029] Figure 9 Flowchart for DCs vaccine immunization in tumor-bearing mice.

[0030] Figure 10 To detect the frequency of IL-2 production in CD4+ T cells by intracellular staining and flow cytometry.

[0031] Figure 11 The frequency of IFN-γ production in CD4+ T cells was detected by intracellular staining and flow cytometry.

[0032] Figure 12 The frequency of GB production in CD8+ T cells was detected by intracellular staining and flow cytometry.

[0033] Figure 13 Perforin production in CD8+ T cells was detected by intracellular staining and flow cytometry.

[0034] The frequency.

[0035] Figure 14 H&E staining analysis of liver sections from immunized mice.

[0036] Figure 15 H&E staining analysis of kidney sections from immunized mice.

[0037] Figure 16 Tumor growth curves in mice after LLC inoculation, ****P<0.0001.

[0038] Figure 17 The survival curve of tumor-bearing mice after DCs vaccination (n=4), **P<0.01. Detailed Implementation

[0039] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0040] 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 to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0041] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional experimental methods, and all reagents and consumables used in the embodiments are commercially available products.

[0042] In some embodiments of the present invention, a fusion protein comprising mesothelin is disclosed, the amino acid sequence of which is shown in SEQ ID NO:1.

[0043] In other embodiments of the present invention, a gene encoding a fusion protein comprising mesothelin is disclosed, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0044] In other embodiments of the present invention, a method for preparing a fusion protein containing mesothelin is disclosed, the method comprising the following steps:

[0045] (1) Synthesize a fusion gene fragment containing gene sequences of human MSLN, PDL1 and GMCSF; the nucleotide sequence of the fusion gene fragment is shown in SEQ ID NO:2;

[0046] (2) The fusion gene fragment and pET-21a plasmid vector from step (1) were digested with NdeI and XhoI, recovered by gel purification kit, and ligated to obtain the expression plasmid.

[0047] (3) The expression plasmid was transformed into the BL21(DE3) expression strain, and the target protein was obtained by IPTG induction. The protein was purified and dialyzed to obtain the fusion protein.

[0048] In other embodiments of the present invention, the use of the above-described fusion protein containing mesothelin in the preparation of tumor vaccines is disclosed.

[0049] In other embodiments of the present invention, a tumor vaccine is disclosed, wherein the active ingredient of the tumor vaccine is the aforementioned fusion protein containing mesothelin.

[0050] In one embodiment, the tumor vaccine contains a mesothelin-containing fusion protein with a loading of 80 μg / mL to 120 μg / mL.

[0051] In one embodiment, the tumor vaccine contains a mesothelin-containing fusion protein with a loading of 90 μg / mL to 110 μg / mL.

[0052] In one embodiment, the tumor vaccine contains a mesothelin-containing fusion protein with a loading of 95 μg / mL to 105 μg / mL.

[0053] In other embodiments of the present invention, a method for preparing the above-mentioned tumor vaccine is disclosed, comprising the following steps:

[0054] (1) After culturing dendritic cells for 4-5 days, add the fusion protein containing mesothelin as described in claim 1 to the culture medium overnight;

[0055] (2) Add bacterial lipopolysaccharide and continue culturing for 2-3 days to obtain the product.

[0056] In one embodiment, the culture medium in step (1) is RPMI-1640 medium containing 18 ng / mL to 22 ng / mL GMCSF and 8 ng / mL to 12 ng / mL recombinant mouse IL-4.

[0057] In one embodiment, the concentration of the bacterial lipopolysaccharide is 0.8 ug / ml to 1.2 ug / ml.

[0058] In other embodiments of the present invention, the use of the above-described tumor vaccine in the preparation of medicaments for treating solid tumors is disclosed.

[0059] In one embodiment, the solid tumor is an MSLN-mutated cancer with high PD-L1 expression.

[0060] In one embodiment, the solid tumor is mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, or triple-negative breast cancer.

[0061] In one embodiment, the solid tumor is a mesothelioma or lung cancer.

[0062] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0063] Example 1: Construction and identification of PET-21a-MSLN-PDL1-GMCSF expression plasmid

[0064] This embodiment constructed and identified the pET-21a-MSLN-PDL1-GMCSF expression plasmid. The specific steps included:

[0065] (1) A fusion protein consisting of human MSLN, PDL1 sequence, linker and human GM-CSF was designed. The amino acid sequence is shown in SEQ ID NO:1, which includes human MSLN (200aa, Human Mesothelin: GenBank: AAH03512.1), PDL1 (202aa, PDL1: GenBank: AF177937.1), PADRE (13aa), spacer (4aa), hGM-CSF (144aa, GenBank: M11734.1), ending with a 6 His-tag, and containing NdeI, BstBI and XhoI restriction sites. The gene fragment of the fusion protein (nucleotide sequence shown in SEQ ID NO:2) was synthesized by GenScript Biotech Inc.

[0066] SEQ ID NO:1 (576aa)

[0067] M EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQG YPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGY LCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVK FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYG GADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENH AKFVAAWTLKAAA GSNG SGSGMWLQSLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE LE HHHHHH

[0068] SEQ ID NO:2(1713bp)

[0069] CAT atgGAAGTGGAGAAGACAGCCTGTCCTTCAGGCAAGAAGGCCCGCGAGATAGACGAGAGCCTCAT CTTCTACAAGAAGTGGGAGCTGGAAGCCTGCGTGGATGCGGCCCTGCTGGCCACCCAGATGGACCGCGTGAACGCC ATCCCCTTCACCTACGAGCAGCTGGACGTCCTAAAGCATAAACTGGATGAGCTCTACCCACAAGGTTACCCCGAGT CTGTGATCCAGCACCTGGGCTACCTCTTCCTCAAGATGAGCCCTGAGGACATTCGCAAGTGGAATGTGACGTCCCT GGAGACCCTGAAGGCTTTGCTTGAAGTCAACAAAGGGCACGAAATGAGTCCTCAGGTGGCCACCCTGATCGACCGC TTTGTGAAGGGAAGGGGCCAGCTAGACAAAGACACCCTAGACACCCTGACCGCCTTCTACCCTGGGTACCTGTGCT CCCTCAGCCCCGAGGAGCTGAGCTCCGTGCCCCCCAGCAGCATCTGGGCGGTCAGGCCCCAGGACCTGGACACGTG TGACCCAAGGCAGCTGGACGTCCTCTATCCCAAGGCCCGCCTTGCTTTCCAGAACATGAACGGGTCCGAATACTTC GTGAAG TTTACTGTCACGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACATTGAATGCAAATTCCCAGTAGAAAAAACAAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTTCAATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATGGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGT GGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTCACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCAT GCCAAGTTCGTGGCCGCCTGGACCCTGAAGGCCGCCGCC gg TTCGAA cggcagcggcagcggc atgtggctgcagagcctgctgctcttgggcactgtggcctgcagcatctctgcacccgcccgctcgcccagccccagcacacagccctgggagcatgtgaatgccatc caggaggcccggcgtctcctgaacctgagtagagacactgctgctgctgagatgaatgaaacagtagaagtcatctcagaaatgtttgacctccaggagccgacctgccta cagacccgcctggagctgtacaagcagggcctgcggggcagcctcaccaagctcaagggccccttgaccatgatggccagccactacaaacagcactgccctccaacc ccggaaacttcctgtgcaacccagattatcacctttgaaagtttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgctggggagccagtccaggag CTCGAG caccaccaccaccac

[0070] (2) Using the NdeI and XhoI restriction sites downstream of the promoter of the pET-21a plasmid vector, insert the gene fragment of the fusion protein described in step (1) into the pET-21a plasmid vector.

[0071] The primer sequences are as follows (where italics indicate restriction enzyme sites):

[0072] Upstream primer (SEQ ID NO:3): CATATGGTTTTTACTGTCACGGTTCCC

[0073] Downstream primer (SEQ ID NO:4): CTCGAGCACCACCACCACCACCACCAC

[0074] (3) The ligation solution of the pET-21a plasmid vector containing the gene fragment of the fusion protein was transformed into DH5a Escherichia coli, and 10 clones were selected for PCR identification and sequencing identification.

[0075] The structure of the constructed expression plasmid pET-21a-MSLN-PDL1-GMCSF is as follows: Figure 1 As shown.

[0076] Referring to the method of this embodiment, the expression plasmid pET-21a-PDL1-GMCSF was also constructed.

[0077] Example 2: Induced expression of fusion protein in pET-21a-MSLN-PDL1-GMCSF expression plasmid

[0078] Extract the correctly sequenced expression plasmids pET-21a-MSLN-PDL1-GMCSF and pET-21a-PDL1-GMCSF, and dissolve them in an appropriate amount of TE buffer for later use. Transform the plasmids into the BL21(DE3) expression strain to induce expression of the fusion protein.

[0079] Specifically, the steps include the following:

[0080] (1) Take out the E.coli BL21(DE3) competent cells (Shanghai Weidi) that were frozen at -80℃, place them on ice immediately, and transform them after thawing;

[0081] (2) Add 1 μL of plasmid to 100 μL of competent cells, mix gently, incubate on ice for 30 min, heat shock at 42℃ for 90 s, and immediately place on ice for 3 min.

[0082] (3) Add 500 μL of preheated LB liquid antibiotic-free medium at 37℃, shake at 37℃ and 200 rpm for 50 min; take 100-200 μL of bacterial culture and spread it on LB plates containing Amp (50 μg / mL), and incubate overnight at 37℃.

[0083] (4) Take a single colony and inoculate it into 5 mL of LB liquid medium (containing 50 μg / mL Amp), and culture it overnight at 37℃ with shaking at 200 r / min. Then inoculate it into 800 mL of LB liquid medium and culture it at 37℃ with shaking at 200 r / min for about 3 h until the logarithmic growth phase. Take 5 mL of the culture as a control (uninduced). Add IPTG to the remaining medium to a final concentration of 1 mM and culture it at 37℃ with shaking for 6 h. Collect the culture in a centrifuge tube.

[0084] (5) Take 5 mL of bacterial culture from each group into centrifuge tubes, centrifuge to remove the supernatant, and resuspend 1 mL of each in sonication buffer (PBS, 1% Triton X-100, 1 mM EDTA, pH 7.4). Then centrifuge at 15000g for 30 minutes and collect the supernatant and precipitate. Take an appropriate amount of bacterial culture from each group, add an equal volume of 2X SDS sample buffer mix, boil and heat for 10 min, and quickly cool on ice. Perform SDS-PAGE electrophoresis and Western blot analysis (WB).

[0085] The results are as follows Figure 2 and Figure 3As shown, the fusion protein was expressed as inclusion bodies with expected molecular weights of approximately 41.94 kDa (PDL1-GMCSF) and 65.41 kDa (MLSN-PDL1-GMCSF) after induction.

[0086] Example 3: Purification of MSLN-PDL1-GMCSF fusion protein

[0087] Under denaturing conditions with 8M urea solution, His-labeled fusion proteins PDL1-GMCSF and MLSN-PDL1-GMCSF were purified using a Ni-NTA column according to the kit instructions (Abbkine #KTP20010). Cell lysis, flow-through, and elution fractions were further analyzed by SDS-PAGE. The results are shown below. Figure 4 and Figure 5 As shown, after purification by Ni column, the content of impurities in the eluent was significantly reduced compared with the supernatant and through-flux, thus achieving the purpose of purification.

[0088] The purified protein solution was subjected to gradient dialysis to remove urea. Following the dialysis bag instructions (Solarbio #YA1071), the solution was dialyzed through 8M-6M-4M-2M-1M urea, and finally dialyzed into urea-free PBS. The dialysis solution was then concentrated by ultrafiltration and quantitative analysis using a BCA kit. Finally, Western blot analysis was performed to determine microbial binding. The specific steps are as follows:

[0089] a. Protein electrophoresis (12% SDS-PAGE) was performed at a rate of 50 μg per lane. After electrophoresis, the proteins were transferred to a nitrocellulose membrane using a GenScript eBlotL1 rapid wet transfer apparatus (Pall Corporation).

[0090] b. Seal the membrane with 5% skim milk powder at room temperature for 2 hours, then wash it 3 times with PBST.

[0091] c. Hybridize with anti-His monoclonal antibody at 4°C with shaking overnight. The next day, wash the membrane three times with PBST, then hybridize with horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody (Beyotime A0216) at room temperature for 1 hour. After washing three times with PBST, expose and analyze. The fusion proteins PDL1-GMCSF and MSLN-PDL1-GMCSF after dialysis show a band with a similar molecular weight, indicating successful protein purification. Figure 6 ).

[0092] Using the purification method described in this embodiment, approximately 10 mg of high-purity fusion protein MSLN-PDL1-GMCSF was obtained from 800 mL of bacterial culture for further functional identification.

[0093] Example 4: Obtaining a fusion protein vaccine (DC loaded with the fusion protein MSLN-PDL1-GMCSF)

[0094] Please refer to Figure 7 A diagram illustrating the process of inducing BMDC in mouse bone marrow cells, including the following steps:

[0095] 1. Isolate the femurs of C57BL / 6 mice. Use a 1 mL syringe to draw PBS to flush the mouse bone marrow (BM) from the femurs. Pass the bone marrow cell suspension through a 70 μm cell sieve, and then lyse the erythrocytes using erythrocyte lysis buffer (Biosharp #143190). After washing the cells with RPMI-1640, resuspend the bone marrow cells in RPMI-1640 supplemented with 10% FBS, mGMCSF (20 ng / mL), and recombinant mouse IL-4 (10 ng / mL; Peprotech), and culture at 37°C in 5% CO2. Every other day, replace half the medium with fresh medium containing 20 ng / mL rmGMCSF and 10 ng / mL rmIL-4.

[0096] 2. On day 5 of culture, purified and dialyzed fusion protein MSLN-PDL1-GMCSF (100ug / mL) / PDL1-GMCSF (100ug / mL, preparation method as per MSLN-PDL1-GMCSF) was added to the DC cell (dendritic cell) culture medium and cultured overnight. Then, 50ug / mL of MSLN-PDL1-GMCSF / PDL1-GMCSF was added and pulsed for 2 h to load the DC cells with the fusion protein. An equal volume of PBS was added to the DC cell culture medium as a control.

[0097] 3. Add bacterial lipopolysaccharide to a concentration of 1 μg / mL (LPS; Sigma) in the last 16 hours and continue culturing to stimulate DC maturation.

[0098] 4. After 7 days of culture, flow cytometry (FACS) analysis revealed that 75% of the cells expressed the characteristic DC-specific markers CD11c and CD80 (…). Figure 8 This yields DCs loaded with 100 ug / ml MSLN-PDL1-GMCSF (MSLN-PDL1-GMCSF-DCs vaccine), DCs loaded with 100 ug / ml PDL1-GMCSF (PDL1-GMCSF-DCs vaccine), and DCs loaded with PBS (PBS-DCs).

[0099] Example 5: Fusion protein vaccine induces IL-2 and IFN-γ secretion from mouse spleen cells.

[0100] To investigate whether fusion protein vaccines (DCs loaded with the fusion protein MSLN-PDL1-GMCSF, MSLN-PDL1-GMCSF-DCs vaccines) can induce an effective immune response, reference Figure 9 Tumor-bearing mice were immunized with DCs vaccine, and on day 0, LLC 4×10 was subcutaneously injected. 5 c57BL / 6 mice aged 6-8 weeks were intravenously injected with MSLN-PDL1-GMCSF-DCs and PBS-DCsDCs vaccines (1×10⁻⁶ cells / mouse) on days 7 and 14, respectively. 6 (cell / animal), immunized twice in total.

[0101] Five days after the second vaccination, the spleens of immunized mice were isolated, digested into single-cell suspensions, and then plated in 96-well U-shaped plates for immunization again with 5 μg / mL MSLN-PDL1-GMCSF-DCs for 6 h. Cell surface staining and flow cytometry were used to detect and analyze mouse T cell proliferation. As expected, CD4+ and CD8+ T cells were significantly increased in mouse spleen cells.

[0102] T cells producing IL-2 and IFN-γ were detected and analyzed using intracellular staining and flow cytometry. For example... Figures 10 - 11 As shown, mouse CD4+ T cells immunized with the MSLN-PDL1-GMCSF-DCs vaccine produced IL-2 at a frequency 1.3 times higher than the PBS control group and IFN-γ at a frequency 3.2 times higher than the PBS control group, and 1.7 times higher than mice immunized with the PDL1-GMCSF-DCs vaccine. Similar results were observed in CD8+ T cells. These results clearly indicate that the MSLN-PDL1-GMCSF-DCs vaccine can promote the production of IFN-γ and IL-2 and may have good CTL induction ability. All data were collected on BDFACSVerse and analyzed using Flowjo software. The antibodies used were from BD Biosciences, including PE-A anti-mouse CD4, PerCP-Cy5.5 anti-mouse CD8a, PE-Cy7 anti-mouse IL-2, BV510 anti-mouse IFN-γ, and the FVS780 immobilized active dye for APC-Cy7.

[0103] Example 6: Fusion protein vaccine significantly enhances cytotoxic T cell response.

[0104] DCs enter lymphoid tissues and trigger peptide-responsive CTL responses, then release cytotoxic effector molecules such as perforin and granzyme, leading to the lysis of recognized tumor cells.

[0105] MSLN-PDL1-GMCSF-DCs, PDL1-GMCSF-DCs, and PBS-DCs were injected into mice via tail vein injection, and a second injection was given one week later, for a total of two immunizations (immunization method as in Example 5). Splenic T cells were obtained 5 days after the second immunization, and the effects of effector molecules perforin and granzyme B on cytotoxic T cell responses were then examined.

[0106] Intracellular staining and FACS were used to observe a 5.2-fold increase in the expression level of granzyme B (GB) in the target group in immune spleen cells compared to the PBS control group. Figure 12 Meanwhile, the expression level of perforin in the target group increased 10-fold compared to the PBS control group. Figure 13 This indicates that tumor cell lysis is initiated and a protective CTL response occurs.

[0107] Example 7: Fusion protein vaccine showed no significant liver or kidney toxicity.

[0108] To further verify whether the anti-tumor effect of the MSLN-PDL1-GMCSF-DCs vaccine could damage the liver and kidney cells of mice, we isolated the liver and kidney of mice immunized with MSLN-PDL1-GMCSF-DCs and PBS-DCs (immunization method as in Example 5).

[0109] Mouse livers and kidneys were placed in isopentane and rapidly frozen in liquid nitrogen. Tissue sections were prepared according to standard protocol. Frozen tissue (5 μm thick) was cut at -20°C and immediately transferred to a miniature slide preserved on dry ice and stored at -80°C. The slides were air-dried, fixed in formalin, and then embedded in paraffin. H&E staining was performed at the Pathology Center of Guangzhou Medical University.

[0110] H&E staining showed no positive markers detected in the cytoplasm of the liver and kidneys. Figures 14 - 15 This result indicates that the vaccine MSLN-PDL1-GMCSF-DCs has no hepatotoxicity or nephrotoxicity. Therefore, the effective antitumor CTL response induced by the DC-targeting MSLN-PDL1-GMCSF vaccine does not attack adjacent immune tissues.

[0111] Example 8: Fusion protein vaccine can significantly inhibit tumor growth

[0112] A mouse lung cancer model was used to test the fusion protein vaccine. A lung cancer cell line, LLC, stably expressing human PD-L1 and MSLN, was obtained via lentiviral transfection.

[0113] Stable expression of PD-L1 and MSLN (4×10) 5LLC tumor cells were subcutaneously injected into mice (n=5). One week later, MSLN-PDL1-GMCSF-DCs, PDL1-GMCSF-DCs and PBS-DCs were injected into mice via tail vein. The mice were immunized twice, one week later (immunization method was the same as in Example 5).

[0114] Tumor growth was monitored every 5 days after vaccination, and tumor growth curves were calculated. Compared with the PBS control group, PDL1-GMCSF-DCs vaccine effectively delayed tumor growth, while MSLN-PDL1-GMCSF-DCs vaccine showed a more significant effect in delaying tumor growth. Figure 16 Notably, the MSLN-PDL1-GMCSF-DCs vaccine significantly improved the survival rate of tumor-bearing mice; 50% of the lung cancer-treated mice survived for at least 40 days, demonstrating a significant extension of survival. Figure 17 Therefore, these data suggest that this fusion protein vaccine may be a more effective therapeutic vaccine compared to traditional non-PD-L1 DC-targeting protein vaccines. This study also supports the potential benefits of developing novel cancer immunotherapies, which we believe may be applicable to pancreatic cancer, colon cancer, melanoma, breast cancer, lung cancer, and other solid tumors that highly express MSLN and PD-L1.

[0115] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0116] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A fusion protein comprising mesothelin, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

2. A gene encoding a fusion protein comprising mesothelin, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. A method for preparing a fusion protein containing mesothelin, characterized in that, The method includes the following steps: (1) Synthesize a fusion gene fragment containing gene sequences of human MSLN, PDL1 and GMCSF; the nucleotide sequence of the fusion gene fragment is shown in SEQ ID NO:2; (2) The fusion gene fragment and pET-21a plasmid vector from step (1) were digested with NdeI and XhoI, recovered by gel purification kit, and ligated to obtain the expression plasmid. (3) The expression plasmid was transformed into the BL21(DE3) expression strain, and the target protein was obtained by IPTG induction. The protein was purified and dialyzed to obtain the fusion protein.

4. The use of the mesothelin-containing fusion protein of claim 1 in the preparation of a tumor vaccine.

5. A tumor vaccine, characterized in that, The active ingredient of the tumor vaccine is the fusion protein containing mesothelin as described in claim 1.

6. The tumor vaccine according to claim 5, characterized in that, In the tumor vaccine, the loading of the fusion protein containing mesothelin is 80 μg / mL to 120 μg / mL, preferably 90 μg / mL to 110 μg / mL, and more preferably 95 μg / mL to 105 μg / mL.

7. A method for preparing a tumor vaccine according to claim 5 or 6, characterized in that, Includes the following steps: (1) After culturing dendritic cells for 4-5 days, add the fusion protein containing mesothelin as described in claim 1 to the culture medium overnight; (2) Add bacterial lipopolysaccharide and continue culturing for 2-3 days to obtain the product.

8. The method for preparing a tumor vaccine according to claim 7, characterized in that, The culture medium in step (1) is RPMI-1640 medium containing 18 ng / mL to 22 ng / mL GMCSF and 8 ng / mL to 12 ng / mL recombinant mouse IL-4; And / or, the concentration of the bacterial lipopolysaccharide is 0.8 ug / ml to 1.2 ug / ml.

9. The use of the tumor vaccine according to claim 5 or 6 in the preparation of a medicament for treating solid tumors, preferably, the solid tumor is a cancer with MSLN mutation and high expression of PD-L1.

10. The application according to claim 9, characterized in that, The solid tumors mentioned are mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, or triple-negative breast cancer.

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