Anti-pancreatic cancer vaccine and its pharmaceutical use

Abstract

An anti-tumor fusion protein, which can inhibit MUC1-positive tumor cell growth and can inhibit pancreatic cancer tumor cell growth, has broad application prospects for the prevention and / or treatment of pancreatic cancer.

Description

[Technical Field]

[0001] This application claims priority from a prior application bearing patent application number 202111301004X and entitled "Anti-pancreatic cancer vaccine and its medical use," filed with the State Intellectual Property Office of China on November 4, 2021. The above prior application is incorporated herein by reference in its entirety.

[0002] The present invention relates to the application of fusion proteins, and in particular to the use of fusion proteins containing human MUC1 in the manufacture of medicaments for preventing and treating pancreatic cancer. [Background technology]

[0003] Pancreatic cancer has been dubbed the "king of cancer," with a five-year survival rate of less than 5%. Early symptoms are often inconspicuous, easily overlooked, and by the time a diagnosis is made, the disease has typically reached an advanced stage. The area around the pancreas is rich in blood vessels, nerves, and vital organs, such as the kidneys and liver, making surgery difficult. Furthermore, because the disease is typically detected late, only 10–15% of early-stage patients are suitable for surgical treatment. Most patients can be treated with gemcitabine alone, often in combination with carboplatin or capecitabine, but these treatments only achieve slightly better results than no treatment. Patients with advanced or metastatic pancreatic cancer generally survive for only a few months. As pancreatic cancer progresses, it exploits immune, vascular, and connective tissue damage repair responses in the surrounding tissue matrix to create a favorable tumor microenvironment that promotes tumor growth. While immune-mediated monitoring therapy is effective in treating many types of cancer, pancreatic cancer exhibits a very poor therapeutic response.

[0004] Tumor cell vaccines can activate the immune system and confront cancer cells. Tumor vaccines induce tumor-specific immune responses, activating the immune system to attack cancer cells bearing specific antigens. Among many immunogens, mucin MUC1 is desirable. MUC1 was originally discovered to protect and lubricate epithelia. It was subsequently found to play an important role in all stages of tumor development, including cell signaling and the transformation of malignant cells into tumor spread. MUC1 is a highly glycosylated type I transmembrane protein with a highly glycosylated extracellular domain extending 200–500 nanometers from the cell surface. Its total length is divided into an extracellular segment, a transmembrane segment, and an intracellular segment. The extracellular segment consists of a proline, threonine, and serine-rich (PTS) domain and an SEA domain. The PTS domain, also known as the variable tandem repeat (VNTR) region, is encoded by a highly polymorphic exon, which encodes multiple repeats (i.e., 20–21 amino acids), while the intracellular region (CT) of MUC1 is highly conserved. Overexpression of MUC1 is commonly associated with colon, breast, ovarian, lung, and pancreatic cancers. MUC1 has been shown to be present in various adenocarcinomas. In studies of primary liver cancer patients, the proportion of patients with high MUC1 expression was as high as 68%. At the same time, the recurrence rate after surgery was also the highest, positively correlated with MUC1 expression intensity. MUC1 is also overexpressed in some hematologic malignancies. Not only are MUC1 expression levels different between tumor and normal tissues, but MUC1 glycosylation also differs between tumor and normal tissues. Underglycosylated MUC1 is overexpressed throughout the entire tumor cell surface, reducing tumor cell adhesion and facilitating tumor metastasis. In already established tumors, high amounts of MUC1 can suppress the killing of tumor cells by NK cells, as well as suppress the proliferation of cytotoxic lymphocytes (CTLs) and induce apoptosis of CTLs.

[0005] Vaccination is an important method for treating cancer, providing long-term protection to the host with minimal side effects. While MUC1 is theoretically a promising vaccine antigen, its clinical efficacy in human clinical trials has not been ideal. Many of the antibodies produced by vaccines are unable to bind effectively to tumor cells and are therefore unable to effectively protect the body from tumors.

[0006] Our research has shown that MUC1 can be bound to proteins on bacteria or viruses to generate T cell responses against cancer cells, resulting in anti-tumor effects. Currently, neoantigen vaccines require high-throughput sequencing and bioinformatics screening, which requires significant time and resources, making it difficult to benefit patients. The development of therapeutic cancer vaccines with relatively low development costs and higher efficacy is desirable. The present invention discloses a method for gene optimization of a recombinant protein that can be used to produce inexpensive therapeutic cancer vaccines, is suitable for production in E. coli, and suppresses the growth of pancreatic cancer cells. Summary of the Invention

[0007] In order to provide a more effective biological preparation for preventing and treating pancreatic cancer, the present invention provides the use of a fusion protein in the manufacture of an anti-tumor drug, especially a pancreatic cancer drug. The present invention is realized by the following technical solutions:

[0008] The use of the fusion protein in the manufacture of a medicament for preventing and / or treating tumors.

[0009] According to the present invention, the tumor is a MUC1-positive tumor, for example a MUC1-expressing adenocarcinoma or a MUC1-expressing hematological tumor, and more preferably the tumor is pancreatic cancer.

[0010] The present invention further provides the use of the fusion protein in the manufacture of a medicament for preventing and / or treating pancreatic cancer.

[0011] According to the invention, the fusion protein comprises the maltose binding protein MBP and / or the protein MUC1-N.

[0012] According to the invention, the fusion protein consists of a tandem of the maltose binding protein MBP and / or the protein MUC1-N.

[0013] Furthermore, the nucleotide sequence of the MUC1-N gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the MBP gene is shown in SEQ ID NO. 2.

[0014] Furthermore, the amino acid sequence of the above fusion protein is shown in SEQ ID NO.3.

[0015] The present invention further provides a method for inhibiting tumor cell growth in pancreatic cancer, comprising administering a fusion protein according to the present invention.

[0016] The present invention provides a pharmaceutical composition comprising the fusion protein of the present invention.

[0017] According to the present invention, the drug is used to treat tumors, preferably, the tumors are MUC1-positive tumors, such as MUC1-expressing adenocarcinomas or MUC1-expressing hematological tumors, more preferably pancreatic cancer.

[0018] According to the present invention, the drug is used in the treatment of breast cancer.

[0019] According to the invention, the fusion protein comprises the maltose binding protein MBP and / or the protein MUC1-N.

[0020] According to the present invention, the fusion protein consists of a tandem of the maltose binding protein MBP and / or the mucin MUC1-N.

[0021] Furthermore, the nucleotide sequence of the MUC1-N gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the MBP gene is shown in SEQ ID NO. 2.

[0022] More preferably, the amino acid sequence of said fusion protein is shown in SEQ ID NO.3.

[0023] The present invention further provides an application of the fusion protein in supplements, cosmetics, foods or food additives, characterized in that the fusion protein comprises maltose-binding protein MBP and / or protein MUC1-N.

[0024] According to the present invention, the above supplements, cosmetics, foods or food additives can be used to inhibit tumors.

[0025] Preferably, the tumor is a MUC1-positive tumor, such as a MUC1-expressing adenocarcinoma or a MUC1-expressing hematological tumor, more preferably a pancreatic cancer.

[0026] According to a preferred embodiment of the present application, the fusion protein consists of the tandem of the maltose binding protein MBP and the protein MUC1-N.

[0027] Furthermore, the nucleotide sequence of the MUC1-N gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the MBP gene is shown in SEQ ID NO. 2.

[0028] More preferably, the amino acid sequence of said fusion protein is shown in SEQ ID NO.3.

[0029] The present invention further provides the use of the MBP gene protein and / or the MUC1-N gene protein in the manufacture of a drug for preventing and / or treating tumors, characterized in that the nucleotide sequence of the MUC1-N gene is set forth in SEQ ID NO. 1 and the MBP gene is set forth in SEQ ID NO. 2.

[0030] According to the present invention, the cancers include all MUC1-expressing cancers, including MUC1-expressing adenocarcinomas or MUC1-expressing hematological tumors, and more preferably pancreatic cancer.

[0031] The present invention further provides the use of the MBP gene protein and / or the MUC1-N gene protein in the manufacture of a drug for preventing and / or treating pancreatic cancer, characterized in that the nucleotide sequence of the MUC1-N gene is set forth in SEQ ID NO. 1 and the nucleotide sequence of the MBP gene is set forth in SEQ ID NO. 2.

[0032] Beneficial effects of the present invention: The present invention has demonstrated through animal experiments that the fusion protein and gene of the present invention have significant inhibitory effects on the growth of pancreatic cancer cells at different doses, and have significant preventive and therapeutic effects on pancreatic cancer. [Brief explanation of the drawings]

[0033] [Figure 1] Gel graph after protein expression and purification. [Figure 2] This is an experiment on the inhibition of pancreatic cancer cell proliferation by MBP-Muc1-N. [Figure 3] This is an experiment on the inhibition of pancreatic cancer cell proliferation by MBP-Muc1-N. [Figure 4-1] These are the results of a trial to treat pan-02 pancreatic cancer using a single-agent double-adjuvant vaccine. [Figure 4-2] Continued from Figure 4-1. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention will be described below with reference to examples, which are well known to those skilled in the art. The following examples do not limit the scope of the present invention, and any modifications or variations made based on the examples of the present invention are included in the scope of the present invention.

[0035] Example 1. Construction and expression of fusion proteins 1. Genetic optimization The nucleotide sequence of the optimized MUC1-N protein is shown in SEQ ID NO. 1:

[0036] [Table 1] The nucleotide sequence of the optimized MBP protein is shown in SEQ ID NO. 2:

[0037] [Table 2] Synthesis of optimized gene sequence of MUC1-N fusion MBP To achieve sequential tandem expression of MBP and Mucl-N, the resulting fusion protein sequence is shown in SEQ ID NO. 3: .

[0038] Tandem synthesis was performed using the gene sequences for MBP and Muc1-N fusion protein. To achieve this, we first synthesized oligonucleotide sequences 1a_1, 1a_2, 1a_3, 1a_4, 1a_5, 1a_6, 1a_7, 1a_8, 1a_9, 1a_10, 1a_11, 1a_12, 1a_13, 1a_14, 1a_15, 1a_16, 1a_17, 1a_18, 1a_19, 1a_20, 1a_21, 1a_22, 1a_23, 1a_24, 1a_25, 1a_26, 1a_27, 1a_28, 1a_29, and 1a_30. We then synthesized sequences 1b_1, 1b_2, 1b_3, and 1b_4. Gene amplification was performed using sequences 1-seq2 and 1-R to obtain the optimized MUC1-N fusion MBP gene sequence.

[0039] [Table 3] [Table 4] 2. Recombinant protein expression and purification An NcoI enzyme site was added to the 5' PCR primer for the fusion gene, and an EcoI enzyme site was added to the 3' PCR primer. The amplified gene was double-digested and inserted into a similarly double-digested pET26b(+) E. coli expression vector. After screening of resistant bacterial culture plates and selection of monoclonal clones, the clones were grown in kanamycin-resistant medium and operon expression was induced with IPTG. Both the unoptimized and optimized sequences were used in the experiments. Finally, the resulting whole bacterial culture was pretreated at 95°C in an SDS-containing buffer and analyzed by 5%-12% polyacrylamide gel electrophoresis. The results showed that the expression level of the unoptimized MBP-Muc1-N sequence accounted for only 2% of the total protein, whereas the expression level of the optimized MBP-Muc1-N sequence accounted for 51% of the total protein, a 25.5-fold increase. After affinity column purification, the protein expressed by the unoptimized gene could only be loaded and observed when concentrated 10-fold, with the yield of purified protein after concentration being 0.8 mg per 100 mL of culture, whereas the yield of the optimized sequence MBP-Muc1-N was 9.6 mg, a 12-fold increase.

[0040] Example 2: Activity test of MBP-Muc1-N fusion protein 1.Material Experimental reagents: MBP-MUC1-N fusion protein was recombinantly prepared using the method described in this application. Pan-02 pancreatic cancer cells were purchased from the National Experimental Cell Resource Center, and injectable saline was purchased from Beijing Tiantan Biological Products Co., Ltd.

[0041] Experimental animals: C57 BL / 6J mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0042] 2. Method (1) Each group consisted of six C57BL / 6J males, 6-8 weeks old. The number of Pan-02 pancreatic cancer cells was 1.75 × 10 5Each mouse was inoculated subcutaneously into the right axilla, and 50 μg of MBP-Muc1-N was administered in a single dose. Seven days after inoculation, when the visible tumor was approximately 5 mm, the first MBP-Muc1-N injection was administered into the leg muscle. On day 50 after inoculation, a second MBP-Muc1-N injection was administered, followed by a third and fourth MBP-Muc1-N injection on days 54 and 57 after inoculation.

[0043] (2) Each group consisted of six C57BL / 6J males, 6-8 weeks old. The number of Pan-02 pancreatic cancer cells was 1.0 × 10 6 Each mouse was inoculated subcutaneously into the right axilla, and 50 μg of MBP-Muc1-N was administered in a single dose. Seven days after inoculation, the visible tumor was approximately 5 mm. On day 17, a second MBP-Muc1-N injection was administered, followed by a third, fourth, fifth, and sixth MBP-Muc1-N injections on days 20, 24, 27, and 31.

[0044] 3. Detection results 3.1 In Method 1, as shown in Figure 2, the number of Pan-02 pancreatic cancer cells was 1.75 × 10 5 Seven days after inoculation, visible tumors were approximately 5 mm in size. The first MBP-Muc1-N injection was administered into the leg muscle. After this, visible tumors began to grow slowly. From day 50 after inoculation, tumors began to grow rapidly. A second MBP-Muc1-N injection was administered, followed by a third and fourth MBP-Muc1-N injection on days 54 and 57 after inoculation. After the third and fourth MBP-Muc1-N injections, visible tumor growth was significantly inhibited.

[0045] [Table 5] 3.2 In Method 2, as shown in Figure 3, on day 7 after inoculation, visible tumors were approximately 5 mm in size. Thereafter, the tumors grew slowly. Starting on day 17 after inoculation, a second injection of MBP-Muc1-N was administered, followed by a third, fourth, fifth, and sixth injections of MBP-Muc1-N on days 20, 24, 27, and 31 after inoculation. After the second, third, fourth, fifth, and sixth injections of MBP-Muc1-N, the growth of visible tumors was significantly inhibited.

[0046] [Table 6] As can be seen from the above, the present invention has demonstrated through animal experiments that the modified MBP-Muc1-N fusion protein has significant inhibitory effects on the growth of pancreatic cancer cells at different concentrations, and can effectively prevent and / or treat pancreatic cancer.

[0047] Example 3: Treatment of pan-02 pancreatic cancer with a single-adjuvant vaccine 1.Material Experimental reagents: The MBP-MUC1-N fusion protein expression strain was engineered using the method described in this application. Anti-PD-1 antibody was purchased from Bioxcell, pan-02 pancreatic cancer cells were purchased from the National Experimental Cell Resource Center, and aluminum hydroxide adjuvant was purchased from Heda Company.

[0048] Experimental animals: C57 BL / 6J mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0049] 2. Method 2.1 Production of MBP-MUC1-N fusion protein Using the method of the present application, a recombinant strain expressing MBP-MUC1-N fusion protein was fermented, the fermentation broth was collected, the cells were clarified and disrupted, and the supernatant amylose resin was collected by centrifugation. The MBP-MUC1-N fusion protein was affinity purified by elution with maltose. The buffer was then exchanged by dialysis, followed by application to Q-Sepharose and anion exchange purification using a high concentration of NaCl. The buffer was then exchanged with citrate buffer, applied to SP-Sepharose, and cation exchange purification using a high concentration of arginine. The purified product contained endotoxins, residual host DNA, and residual proteins at the original concentration. The buffer was then exchanged with 20 mM acetic acid-sodium acetate buffer and 150 mM arginine by dialysis.

[0050] 2.2 Immunization of mice The mice were weighed and randomly assigned to groups with 10 mice per group. Pan-02 pancreatic cancer cells were then injected at a dose of 1 × 10 6 The cells were diluted with PBS (cells / mouse) and inoculated into the right axilla of C57BL / J mice in a total volume of 100 μL per mouse. After tumors reached 5 mm in diameter (10 days), mice were divided into five groups: aluminum hydroxide control group (100 μL, intramuscular injection, twice weekly), MNRvax low-dose group (0.2 mg / kg, 100 μL, intramuscular injection, twice weekly), MNRvax medium-dose group (2 mg / kg, 100 μL, intramuscular injection, twice weekly), MNRvax high-dose group (8 mg / kg, 100 μL, subcutaneous injection, twice weekly), and immune checkpoint inhibitor anti-PD-1 antibody group (10 mg / kg, 100 μL, intraperitoneal injection, twice weekly).

[0051] Drug injections were performed on days 10, 13, 17, and 20 after tumor development, and tumor size was measured on days 10, 13, 17, 20, and 24. The tumor inhibition rate and tumor suppression rate for each group were calculated using the following formula: [tumor suppression rate = (mean tumor weight in the control group - mean tumor weight in the experimental group) / mean tumor weight in the control group × 100%]. [tumor cure rate = (number of tumor-free mice / 10 mice) × 100%].

[0052] 3.Results [Table 7]

[0053] [Table 8] As can be seen from Tables 3 and 4, the low, medium, and high dose MBP-Muc1-N vaccine treatment groups significantly eliminated tumors compared to the control group. The tumors shrank significantly on days 20 and 24 after tumor inoculation, with a mean size of 0.359±0.079 mm, respectively. 3 to 0.286±0.062, 0.295±0.072, 0.291±0.083 mm 3 reduced to 0.408±0.091 mm 3 to 0.332±68, 0.327±0.091, 0.320±0.112 mm 3 The p-values ​​were 0.077, 0.036, 0.081, and 0.049, 0.062, 0.069, respectively, which was significant. On the other hand, the tumor size in the PD-1 antibody treatment group was 0.359±0.079 mm 3 to 0.301±0.075 mm 3 reduced to 0.408±0.091 mm 3 to 0.338±0.087 mm 3 This suggests that the efficacy of the MBP-Muc1-N vaccine is superior to that of the PD-1 antibody.

[0054] Example 4: Treatment of pan-02 pancreatic cancer with a single agent of the dual adjuvant vaccine 1.Material Experimental reagents: The recombinant MBP-MUC1-N fusion protein was prepared using the same method as described above. Aluminum hydroxide adjuvant was purchased from Heda Corporation. CpG1826 adjuvant (5'-TCCATGACGTTCCTGACGTT-3') and thio*CpG1826* adjuvant (5'-T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T-3') were purchased from Shanghai Bio-Engineering Corporation.

[0055] Experimental animals: C57 BL / 6J mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0056] 2. Method 2.1 Production of MBP-MUC1-N fusion protein The recombinant MBP-MUC1-N fusion protein was obtained through a three-step purification process, and the endotoxins, residual host DNA, and residual proteins in the purified product all met the requirements of the original solution.

[0057] 2.2 Immunization of mice The mice were weighed and randomly assigned to groups of six mice each. Pan-02 pancreatic cancer cells were then injected at a dose of 1 × 10 6 The cells were diluted with PBS at 100 μL per mouse and inoculated into the right axilla of C57BL / J mice. After tumors reached 5 mm in diameter (10 days), the mice were divided into six groups: PBS control (100 μL, intramuscular injection twice weekly), MNRvax (2 mg / kg, 100 μL, intramuscular injection twice weekly), CpG (10 μg / mouse, 100 μL, intramuscular injection twice weekly), *CpG* (10 μg / mouse, 100 μL, intramuscular injection twice weekly), MNRvax+CpG, and MNRvax+*CpG*.

[0058] Drug injections were performed on days 8, 21, 29, and 36 after tumor development, and tumor size was measured on day 43. The tumor inhibition rate and tumor suppression rate for each group were calculated using the following formula: [tumor suppression rate = (mean tumor weight in the control group - mean tumor weight in the experimental group) / mean tumor weight in the control group × 100%]. [tumor cure rate = (number of tumor-free mice / 10 mice) × 100%].

[0059] 3.Results

[0060] [Table 9] 4. Conclusion The recombinant MBP-MUC1-N fusion protein vaccine prepared in an acetic acid-sodium acetate buffer system had a relatively low inhibitory effect on tumors, while the addition of CpG1826 could improve this inhibitory effect.

[0061] Example 5. Study of the mechanism of action of vaccine-only treatment of pan-02 pancreatic cancer 1.Material Experimental reagents: Recombinant MBP-MUC1-N fusion protein was self-produced. Anti-PD-1 antibody was purchased from Bioxcell, pan-02 pancreatic cancer cells were purchased from the National Experimental Cell Resource Center, and aluminum hydroxide adjuvant was purchased from Heda Company. PE / DAZZLIE594-CD3 antibody (17A2 clone, rat IgG2b, κ), BV421-CD4 antibody (GK1.5 clone, rat IgG2b, κ), APC-FIRE75-CD8a (53-6.7 clone, rat IgG2a, κ), APC-CD335 (29A1.4 clone, rat IgG2a, κ), PE-CD19 (6D5 clone, rat IgG2a, κ), PE-CY7-F4 / 80 (BM8 clone, rat IgG2a, κ), FITC-CD45 (30-F11 clone, rat IgG2b, κ), and BV711-CD11c (N418 clone, Armenian hamster IgG) were purchased from Biolegend and labeled with eFluorescent. 506-L / D was purchased from Invitrogen, BB700CD11b (M1 / 70 clone, rat IgG2b, κ) was purchased from BD, and the mouse tumor dissociation reagent kit was purchased from Miltenyi.

[0062] Experimental animals: C57 BL / 6J mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0063] 2. Method 2.1 Immunization of mice The mice were weighed and randomly assigned to groups of 10 mice each. Pan-02 pancreatic cancer cells were injected at 3 × 10 6The cells were diluted with PBS at 0.1 mL per mouse and inoculated into the right axilla of C57BL / J mice in a total volume of 100 μL per mouse. After tumors reached 5 mm in diameter (10 days), mice were divided into five groups: aluminum hydroxide control group (100 μL, intramuscular injection twice weekly), MNRvax low-dose group (0.2 mg / kg, 100 μL, intramuscular injection twice weekly), MNRvax medium-dose group (2 mg / kg, 100 μL, intramuscular injection twice weekly), MNRvax high-dose group (8 mg / kg, 100 μL, subcutaneous injection twice weekly), and immune checkpoint inhibitor anti-PD-1 antibody group (10 mg / kg, 100 μL, intraperitoneal injection twice weekly).

[0064] 2.2 Isolation of mouse samples 2.2.1 Isolation of mouse tumor samples Miltenyi's enzyme D, R, and A mixed enzyme solution was added to the tube, and a tumor mass measuring 3 mm in diameter was added. The tube was then attached to the cannula of a gentleMACS tissue processor. Using a gentleMACS Octo tissue processor equipped with a heating module, program 37C_m-TDK1 was run. The sample was resuspended and filtered through a 70 μm cell screen. The cell suspension was collected in a 50 mL centrifuge tube. The screen was washed with 20 mL of RPMI 1640. The cell suspension was centrifuged at 1500 rpm for 5 minutes and the supernatant was discarded. 20 mL of PBS was added to resuspend the cells, vortexed to mix uniformly, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in an appropriate volume of PBS, vortexed to mix uniformly, and 10 μL of the cell suspension was counted in a cell counter. The remaining cell suspension was then centrifuged at 1500 rpm for 5 minutes and the supernatant was discarded. Add an appropriate volume of PBS according to the counting result to resuspend the cells, vortex to mix evenly, and adjust the cell concentration to 2 × 10 7 / mL and prepared for use.

[0065] 2.2.2 Dissociation of mouse spleen / lymph node cells The collected spleens / lymph nodes were placed in a 6-well plate containing 3 mL of RPMI 1640 medium, and the spleens were compressed with a syringe to disaggregate them into a single-cell suspension. A cell screen was placed on top of a 15 mL tapered tube, and the cell suspension in the 6-well plate was passed through the screen to remove cell clumps and debris. The screen was washed with 5 mL of RPMI 1640, and the cell suspension was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 2 mL of red blood cell lysis solution, vortexed to mix evenly, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 10 mL of RPMI 1640, vortexed to mix evenly, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. An appropriate volume of FACS buffer was added to resuspend and count the cells, and then centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. Add an appropriate volume of FACS buffer depending on the counting result to resuspend the cells and adjust the cell concentration to 2 x 10 7 / mL and prepared for use.

[0066] 2.3 Staining of mouse tumor / blood samples After the experiment, tumor cells, spleen cells, lymph node cells, and blood cells were isolated and analyzed for immune cell subpopulations by flow cytometry using CD3, CD4, CD8, CD11, CD19, CD45, and CD335. CD45+ indicates leukocytes, CD45+CD19+ indicates B cells, CD45+CD19-CD3+ indicates T cells, CD45+CD19-CD3+CD8+ indicates cytotoxic T cells, CD45+CD19-CD3+CD4+ indicates helper T cells, CD45+CD19-CD3-CD11b+F4 / 80+ indicates macrophages, CD45+CD19-CD3-CD11c+ indicates stellate cells, and CD45+CD19-CD3-CD335+ indicates natural killer cells.

[0067] The resuspended tumor / spleen / lymph node / blood cells were vortexed to mix uniformly, and FcR Blocking Reagent was added. The mixture was incubated at 4°C in the dark for 10 minutes. All antibodies (including vital dyes) were prepared according to the antibody's recommended volume and thoroughly and uniformly mixed. The appropriate volume of the antibody mixture was added to the corresponding flow tube. FMO tubes, blank tubes, and single-positive tubes were also prepared as needed. The mixture was gently vortexed to thoroughly mix the antibody and cells, and incubated at 4°C in the dark for 30 minutes. 2 mL of red blood cell lysis solution was added to all flow tubes, vortexed to mix uniformly, and incubated at room temperature in the dark for 10 minutes. After incubation, the tubes were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. 2 mL of FACS buffer was added to all flow tubes, vortexed to mix uniformly, and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded. This procedure was repeated once. 50 μL of FACS buffer was added to resuspend the cells, vortexed to mix evenly, and prepared for loading.

[0068] 2.4 Data analysis All data generated by flow cytometry were analyzed using Kaluza software. To compare immune cell subpopulations in different treatment groups, we first verified the assumption of homogeneity of variance across all groups using the Bartlett test. If the p-value of the Bartlett test was 0.05 or greater, one-way analysis of variance was used to verify the equality of means across all groups. If the p-value of the one-way analysis of variance was less than 0.05, we performed pairwise comparisons across all groups using the Tukey HSD test or pairwise comparisons between each treatment group and the control group using Dunnett's t-test. If the p-value of the Bartlett test was less than 0.05, we performed pairwise comparisons across all groups using the Conover test or pairwise comparisons between each treatment group and the control group, calibrating the corresponding p-value based on the number of groups for multiple tests. All statistical analyses and graphic plots were performed in the R language environment. Unless otherwise stated, all tests were two-sided, and p values ​​less than 0.05 were considered statistically significant.

[0069] 3.Results Thirty-five days after cancer vaccine administration, the percentage of leukocytes in the tumor increased from 27.06 ± 4.61 to 34.44 ± 5.61 (p = 0.005) (Figure 4A). The ratio of CD8+ T cells in tumor tissue to total T cells increased from 25.99 ± 5.84% in the control group to 32.75 ± 9.74% (p < 0.01) (Figure 4B). The ratio of CD8+ T cells to total leukocytes increased from 2.24 ± 0.58% to 4.33 ± 2.95% (p < 0.05) (Figure 4C). Thirty-five days after cancer vaccine administration, the percentage of leukocytes in lymph nodes increased from 99.81 ± 0.12 to 99.93 ± 0.06 (p = 0.011) (Figure 4D). The ratio of CD4+ T cells to total T cells in lymph nodes increased from 29.85±5.96% in the control group to 37.75±4.71% (p<0.01) (Figure 4E). The ratio to total leukocytes increased from 19.22±3.73% to 26.70±3.3% (p<0.001) (Figure 4F). 35 days after cancer vaccine administration, the percentage of T cells in the spleen increased from 20.91±3.55 to 26.03±4.93 (p=0.017) (Figure 4G). The ratio of splenic CD4+ T cells to total T cells increased from 47.00±2.96% in the control group to 48.64±4.88% (p>0.05) (Figure 4H), and the ratio to total leukocytes increased from 9.80±1.69% to 12.63±2.66% (p<0.05) (Figure 4I).

[0070] 4. Conclusion After MNRVax vaccination, CD8+ T lymphocytes in pancreatic cancer cells significantly increased, directly killing cancer cells. Meanwhile, CD8+ T lymphocytes in lymph nodes and total T cells in the spleen significantly increased, suggesting that MNRVax vaccination can recruit immune cells into tumors.

[0071] Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention are intended to be included within the scope of the claims of the present invention.

Claims

1. 1. A method for producing a pharmaceutical composition for preventing and / or treating pancreatic cancer, comprising: The composition comprises a fusion protein, the fusion protein comprising the protein MBP and the protein MUC1-N, the amino acid sequence of the fusion protein being set forth in SEQ ID NO. 3; The fusion protein is expressed by linking the maltose binding protein MBP gene and the mucin MUC1-N gene in tandem; The method includes expressing the fusion protein in E. coli containing gene sequences for MBP and the MUC1-N fusion protein; The nucleotide sequence of the MUC1-N gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the MBP gene is shown in SEQ ID NO.

2.

2. The method described in claim 1, wherein the pharmaceutical composition comprises the fusion protein, an aluminum hydroxide adjuvant, and a CpG adjuvant.

3. 3. The method of claim 2, wherein the CpG adjuvant is partially or fully thio-modified.

4. 3. The method of claim 2, wherein the CpG adjuvant is CpG1826 or all thio-modified CpG1826.

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