An mRNA vaccine targeting enpp3 and its application in immunotherapy of pancreatic cancer

Abstract

The application discloses an mRNA vaccine targeting ENPP3 and application thereof in immunotherapy of pancreatic cancer, and belongs to the technical field of tumor immunotherapy. The application first takes ENPP3 as an mRNA vaccine target point, the mRNA is codon-optimized and delivered through lipid nanoparticles, can effectively induce T cell and dendritic cell immune responses, promote M1 type polarization of macrophages, reduce the level of immunosuppressive cells, significantly improve the immunosuppressive microenvironment of pancreatic cancer, and inhibit tumor growth. In-vivo experiments prove that the vaccine has a significant anti-tumor effect and high biological safety, and can be used alone or in combination with an immune checkpoint inhibitor for treatment of pancreatic ductal adenocarcinoma, thereby providing a new strategy for immunotherapy of pancreatic cancer.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy and nucleic acid drug technology, specifically relating to a messenger RNA (mRNA) vaccine encoding ENPP3 and its application in the immunotherapy of pancreatic ductal adenocarcinoma. Background Technology

[0002] Malignant tumors remain one of the major diseases threatening human life and health. With the development of tumor immunology and nucleic acid drug technology, tumor vaccines have gradually become an important research direction in tumor treatment due to their high specificity, safety, and good tolerability. Among them, vaccines constructed based on messenger RNA (mRNA) platforms show broad application prospects in the field of tumor immunotherapy due to their flexible design, short preparation cycle, ease of large-scale production, and good biosafety. mRNA vaccines deliver exogenous mRNA encoding specific antigens into the body's cells, where they are translated into corresponding antigen proteins, thereby activating the body's immune system and inducing a specific immune response. Compared with traditional tumor vaccines, mRNA vaccines have several advantages, including no need for genome integration, high expression efficiency, rapid industrial production, controllable degradation, and strong immunogenicity. In addition, mRNA vaccines can simultaneously activate innate and adaptive immunity, further activating T cell-mediated anti-tumor immune responses by promoting the uptake and presentation of tumor-associated antigens by antigen-presenting cells (APCs), thereby improving the tumor immune microenvironment. Currently, mRNA technology has been widely used in the treatment of various diseases, including cardiovascular diseases, inherited metabolic diseases, infectious diseases, and tumors. In recent years, research on mRNA vaccines targeting malignant tumors such as prostate cancer, melanoma, and glioma has been continuously advancing, showing good potential for clinical translation.

[0003] Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant digestive system tumor with a very poor prognosis, characterized by insidious onset, rapid progression, and difficulty in early diagnosis. The complex immunosuppressive tumor microenvironment and significant immune escape capabilities are key reasons for the low response rate of PDAC to current immunotherapies. Currently, for resectable PDAC patients, surgery combined with adjuvant chemotherapy remains the primary treatment option; while for locally advanced or metastatic PDAC patients, chemotherapy remains the first-line treatment. Since gemcitabine (GEM) was approved for PDAC treatment in 1997, it has long been used as a first-line chemotherapy drug for advanced pancreatic cancer. However, clinical data show that the overall survival (OS) of metastatic PDAC patients receiving GEM monotherapy is only about 7.4 months, indicating limited efficacy. To improve treatment outcomes, the FOLFIRINOX regimen and combination therapy with gemcitabine and nab-paclitaxel have been further developed in clinical practice. Although the above-mentioned regimens improved overall survival (OS) and progression-free survival (PFS) compared to GEM monotherapy, severe adverse reactions significantly limited their clinical application. Therefore, developing a precise, effective, and less toxic novel treatment strategy is of great clinical significance.

[0004] During transcriptomic analysis of plasma from pancreatic cancer patients, the inventors identified a significantly upregulated differentially expressed molecule—ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3). ENPP3 is a membrane-bound extracellular enzyme involved in extracellular nucleotide metabolism and adenosine-related immune regulation pathways, potentially playing a crucial role in the formation of the tumor immune microenvironment. Analysis of the Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases revealed high expression of ENPP3 in various solid tumors. Further studies have shown that ENPP3 exhibits a polar distribution in normal epithelial tissues, while displaying abnormal expression and disordered spatial distribution in malignant tumor tissues, suggesting its potential involvement in tumor progression and immune escape. In addition, studies have reported that ENPP3 is significantly overexpressed in liver cancer, kidney cancer and colorectal cancer, and antibody-drug conjugates targeting ENPP3 have shown good antitumor activity and safety in various xenograft tumor models.

[0005] However, there are currently no systematic research reports on ENPP3 as a target for mRNA vaccines for pancreatic cancer immunotherapy. Therefore, developing an mRNA vaccine targeting ENPP3 is of great significance for improving the efficacy of PDAC immunotherapy, reshaping the tumor immune microenvironment, and enhancing patient survival benefits. Summary of the Invention

[0006] The purpose of this invention is to provide an mRNA vaccine targeting ENPP3 and its application in pancreatic cancer immunotherapy, thereby addressing the problems of limited target selection, insufficient immune activation, and the difficulty in reversing the immunosuppressive microenvironment in existing technologies. To achieve the above objective, this invention solves at least one of the following technical problems: (1) The lack of therapeutic targets for pancreatic cancer with immunomodulatory functions; (2) The problem that existing mRNA vaccines are unable to effectively activate anti-tumor cellular immune responses; (3) The problem that the tumor immunosuppressive microenvironment is difficult to remodel; (4) The problem that existing immunotherapy has limited efficacy against pancreatic cancer.

[0007] The objective of this invention can be achieved through the following technical solutions: Step 1: Target screening and validation: Through plasma transcriptome sequencing of pancreatic cancer patients, TCGA database analysis, and clinical sample ELISA validation, ENPP3, which is specifically highly expressed, was screened as a target for mRNA vaccines. Step 2: mRNA optimization design: Codon optimization of the human ENPP3 coding sequence, introduction of N1-methylpseudouridine modification to reduce immunogenicity, and addition of efficient 5'UTR and 3'UTR sequences to improve translation efficiency; Step 3: Construction of LNP delivery system: Using a lipid composition of SM-102 / cholesterol / DSPC / DMG-PEG2000, an ENPP3-LNP vaccine with uniform particle size and high encapsulation efficiency was prepared by microfluidic technology; Step 4: Immunotherapy Strategy: ENPP3-LNP is administered intravenously and taken up by antigen-presenting cells in the liver and spleen, translating and expressing the ENPP3 antigen, activating dendritic cell maturation, and subsequently inducing specific CD8+. + T cell response; simultaneously promotes macrophage M1 polarization, reduces the proportion of regulatory T cells, and reverses the immunosuppressive microenvironment.

[0008] Furthermore, mRNA can induce specific immune responses in T cells and dendritic cells.

[0009] Furthermore, mRNA can promote macrophage polarization towards the M1 type, increasing the M1 / M2 macrophage ratio in tumor tissue.

[0010] Furthermore, mRNA is optimized with human or mouse codons to improve in vivo translation efficiency.

[0011] Furthermore, mRNA is delivered via lipid nanoparticles (LNPs); the lipid nanoparticles contain SM-102, cholesterol, DSPC, and DMG-PEG2000.

[0012] Furthermore, the lipid nanoparticles have a particle size of 60–80 nm.

[0013] Furthermore, vaccines are used in combination with immune checkpoint inhibitors.

[0014] Furthermore, immune checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

[0015] Furthermore, vaccines can reduce the proportion of regulatory T cells in tumor tissues and improve the tumor immunosuppressive microenvironment.

[0016] Furthermore, the vaccine can inhibit the growth, proliferation, and metastasis of pancreatic ductal adenocarcinoma.

[0017] The beneficial effects of this invention are: This study marks the first time ENPP3 has been used as a target for an mRNA vaccine in the treatment of pancreatic cancer, filling a gap in the application of this target in nucleic acid vaccines. It significantly inhibits the growth of pancreatic ductal adenocarcinoma, induces tumor regression, effectively activates innate and adaptive immunity, increases the proportion of dendritic cells, enhances the M1 / M2 macrophage ratio, reduces the level of immunosuppressive cells, and thoroughly improves the immunosuppressive microenvironment of pancreatic cancer. It causes no significant damage to the heart, liver, spleen, lungs, or kidneys in mice, maintains normal serum biochemical indicators, and has no significant toxic side effects. The mRNA vaccine features flexible design, a short in vitro transcription cycle, and is easy to scale up for production, making it suitable for clinical translation. It can be used in combination with immune checkpoint inhibitors to further enhance the efficacy of pancreatic cancer immunotherapy. Attached Figure Description

[0018] Figure 1 A: Immunohistochemical detection of ENPP3 expression level in pancreatic cancer tissue.

[0019] Figure 1 B: TCGA database analysis of ENPP3 expression levels in pancreatic cancer tissues.

[0020] Figure 1 C: ELISA was used to detect the expression level of ENPP3 in the plasma of pancreatic cancer patients.

[0021] Figure 1 D: Quantitative PCR was used to detect the expression level of ENPP3 in the plasma of pancreatic cancer patients.

[0022] Figure 1 E: Expression level of ENPP3 in different pancreatic cancer cell lines.

[0023] Figure 1 F: Distribution of immune cells in tissues with high ENPP3 expression.

[0024] Figure 1 G: Correlation analysis of ENPP3 with immune cells Figure 2 A: Schematic diagram of ENPP3 encoding sequence.

[0025] Figure 2 B: Capillary electrophoresis analysis of the integrity and purity of ENPP3 mRNA.

[0026] Figure 3 A: Live mouse image.

[0027] Figure 3 B: Quantitative analysis of tumor bioluminescence intensity (BL intensity).

[0028] Figure 3 C: Representative image of an ex vivo tumor.

[0029] Figure 3 D: Statistical analysis of tumor volume.

[0030] Figure 3 E: Statistical analysis of mouse weight.

[0031] Figure 3 F: Statistical analysis of tumor volume before and after ENPP3-LNP treatment.

[0032] Figure 3 G: Statistical analysis of tumor weight.

[0033] Figure 3 H: Serum cytokine levels (IL-6, IL-12, IL-1β, TNF-α) were measured using chemiluminescent immunoassay.

[0034] Figure 4 A: Flow cytometry analysis of the proportion of dendritic cells.

[0035] Figure 4 B: Quantitative analysis of the proportion of dendritic cells.

[0036] Figure 4 C: Flow cytometry detection of CD4 + Foxp3 in T cells + Cell ratio.

[0037] Figure 4 D:CD4 + FoxP3+ Quantitative analysis of cell proportions.

[0038] Figure 4 E: Flow cytometry analysis of the proportion of M1 macrophages.

[0039] Figure 4 F: Flow cytometry analysis of the proportion of M2 macrophages.

[0040] Figure 4 G: Analysis of the proportion and ratio of M1 / M2 macrophages.

[0041] Figure 5 A: Immunofluorescence detection of CD8+ / CD4 in tumor tissue + T cell ratio.

[0042] Figure 5 B: CD8+ / CD4 ratio in tumor tissue + Quantitative analysis of T cell proportion and fluorescence intensity Figure 5 C: Immunofluorescence detection of CD8+ / CD4 in spleen tissue + T cell ratio.

[0043] Figure 5 D: CD8+ / CD4 in spleen tissue + Quantitative analysis of T cell proportion and fluorescence intensity Figure 5 E: Immunofluorescence assay to detect the proportion of macrophages in tumor tissue.

[0044] Figure 5 F: Quantitative analysis of macrophage proportion and fluorescence intensity in tumor tissue Figure 6 A: Indices of various organs in mice.

[0045] Figure 6 BF: Graph showing the results of serum biochemical indicators (ALT, AST, LDH, BUN, CREA).

[0046] Figure 6 G: HE staining of major organs. Detailed Implementation

[0047] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0048] Example 1: An mRNA vaccine targeting ENPP3 and its application in pancreatic cancer immunotherapy, comprising the following steps: Screening and validation of ENPP3 as a vaccine target RNA sequencing analysis was performed on plasma samples from pancreatic cancer patients and healthy controls to screen for differentially expressed genes that were significantly upregulated in pancreatic cancer samples. Further screening of these differentially expressed genes for candidate proteins with secretion potential revealed that ENPP3 was significantly highly expressed in pancreatic cancer tissues. Figure 1 A). Consistent with the above results, TCGA database analysis revealed high expression of ENPP3 in pancreatic cancer tumor tissues. Figure 1 B). Further analysis using ELISA and quantitative PCR revealed significantly elevated ENPP3 levels in the blood of pancreatic cancer patients. Figure 1 C). Furthermore, ENPP3 expression was detected in various pancreatic cancer cell lines, and the results showed that its expression level was significantly higher than that of normal controls (C). Figure 1 D). Finally, we assessed the relationship between ENPP3 expression and the tumor immune microenvironment. The results showed that in tissues with high ENPP3 expression, the infiltration levels of various immune cells (including CD8+ T cells, dendritic cells, B cells, and cytotoxic cells) were significantly increased. Figure 1 F). Correlation analysis further showed that ENPP3 expression was significantly positively correlated with the degree of infiltration of various immune cells (F). Figure 1 (G) suggests that ENPP3 may be involved in the regulation of the immune microenvironment in pancreatic cancer. In summary, ENPP3 is significantly overexpressed in pancreatic cancer, exhibits secretory characteristics, and is closely related to immune cell infiltration, suggesting its potential as a candidate target for mRNA vaccines.

[0049] Example 2: An mRNA vaccine targeting ENPP3 and its application in pancreatic cancer immunotherapy, comprising the following steps: Construction of ENPP3 mRNA and its delivery system The ENPP3 coding sequence was codon optimized, an expression vector was constructed, and the complete mRNA was obtained by in vitro transcription using T7 RNA polymerase. Figure 2 A). The integrity and purity of the mRNA were detected by capillary electrophoresis. The results showed that the prepared mRNA had good integrity and high purity. Figure 2B), with a concentration of 1.11 mg / mL and a pH of 6.79. The mRNA and lipid components were mixed in a molar ratio of 50:38.5:10:1.5 (SM-102:cholesterol:DSPC:DMG-PEG2000) to prepare lipid nanoparticles (LNPs), forming the ENPP3_LNP vaccine. The obtained nanoparticles had a particle size of approximately 78.37 nm, while the control LNPs had a particle size of approximately 60.3 nm, indicating that the constructed system possesses good nanoscale properties.

[0050] Example 3: An mRNA vaccine targeting ENPP3 and its application in pancreatic cancer immunotherapy, comprising the following steps: Antitumor effects of ENPP3 mRNA vaccine A subcutaneous pancreatic cancer xenograft model was established in C57BL / 6 mice, and the mice were treated with ENPP3_LNP vaccine via tail vein injection. Tumor growth was monitored by in vivo imaging. Results showed that, compared with the control group, the tumor signal in the ENPP3_LNP treatment group was significantly reduced (…). Figure 3 A-3B). Further observation of ex vivo tumors showed that the tumor volume and weight in the ENPP3_LNP group were significantly lower than those in the control group (A-3B). Figure 3 (C-3G), suggesting that the vaccine can significantly inhibit tumor growth and induce tumor regression. To assess the systemic immune response, serum cytokine levels were measured in mice after treatment. Compared with the blank LNP control group, ENPP3-LNP administration induced increased expression levels of IL-6, IL-12, IL-1β, and TNF-α, indicating that the innate immune signaling pathway was activated in vivo.

[0051] Example 4: An mRNA vaccine targeting ENPP3 and its application in pancreatic cancer immunotherapy, comprising the following steps: ENPP3 mRNA vaccine induces immune response Flow cytometry analysis was performed on tumor tissue and spleen. Results showed: CD4 + Foxp3 in T cells + The proportion of regulatory T cells changed ( Figure 4 A-4B); the proportion of dendritic cells increased significantly ( Figure 4 C-4D); The proportion of M1 macrophages increased, the proportion of M2 macrophages decreased, and the M1 / M2 ratio increased significantly ( Figure 4 E-4G). The above results indicate that the ENPP3 mRNA vaccine can regulate the composition of immune cells, promote immune activation, and improve the immunosuppressive microenvironment.

[0052] Example 5: An mRNA vaccine targeting ENPP3 and its application in pancreatic cancer immunotherapy, comprising the following steps: ENPP3 mRNA vaccine reshapes the tumor immune microenvironment To spatially validate these immune alterations, immunofluorescence analysis was performed on tumor tissue. ENPP3-LNP treatment increased CD4 count. + and CD8 + The proportion of T cells ( Figure 5 AD provides spatial confirmation of enhanced adaptive immune infiltration. Furthermore, the tumor exhibits increased CD86 staining and decreased ARG1 expression, consistent with the transformation into M1-type pro-inflammatory macrophages. Figure 5 E, F). The above results indicate that ENPP3-LNPs remodel the tumor immune microenvironment by regulating T cell subsets and polarizing macrophages toward an immune-stimulated phenotype.

[0053] Example 6: An mRNA vaccine targeting ENPP3 and its application in pancreatic cancer immunotherapy, comprising the following steps: Safety assessment A systematic evaluation of the major organs (heart, liver, spleen, lung, and kidney) of experimental mice was performed. The results showed no significant differences in organ indices among the groups. Figure 6 A); Serum biochemical indicators (ALT, AST, LDH, BUN, and CREA) showed no significant abnormalities. Figure 6 B-6F); HE staining results showed that all tissue structures were normal, and no obvious pathological damage was observed (B-6F). Figure 6 G). The above results indicate that the mRNA vaccine described in this invention has good biocompatibility.

[0054] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the invention.

Claims

1. The use of an mRNA encoding ENPP3 protein in the preparation of a vaccine for the treatment of pancreatic ductal adenocarcinoma, characterized in that, Includes the following steps: Step 1: Target screening and validation: Through plasma transcriptome sequencing of pancreatic cancer patients, TCGA database analysis, and validation using clinical sample ELISA and quantitative PCR, ENPP3, which is specifically highly expressed, was screened as a target for mRNA vaccines. Step 2: mRNA optimization design: Codon optimization of the human ENPP3 coding sequence, introduction of N1-methylpseudouridine modification to reduce immunogenicity, and addition of efficient 5'UTR and 3'UTR sequences to improve translation efficiency; Step 3: Construction of LNP delivery system: Using a lipid composition of SM-102 / cholesterol / DSPC / DMG-PEG2000, an ENPP3-LNP vaccine with uniform particle size and high encapsulation efficiency was prepared by microfluidic technology; Step 4: Immunotherapy Strategy: ENPP3-LNP is administered intravenously and taken up by antigen-presenting cells in the liver and spleen, translating and expressing the ENPP3 antigen, activating dendritic cell maturation, and subsequently inducing specific CD8+. + T cell response; simultaneously promotes macrophage M1 polarization, reduces the proportion of regulatory T cells, and reverses the immunosuppressive microenvironment.

2. The application according to claim 1, characterized in that, The mRNA can induce specific immune responses in T cells and dendritic cells.

3. The application according to claim 1, characterized in that, The mRNA can promote macrophage polarization towards the M1 type and increase the M1 / M2 macrophage ratio in tumor tissue.

4. The application according to claim 1, characterized in that, The mRNA is optimized with human or mouse codons to improve in vivo translation efficiency.

5. The application according to claim 1, characterized in that, The mRNA is delivered via lipid nanoparticles (LNPs); the lipid nanoparticles contain SM-102, cholesterol, DSPC, and DMG-PEG2000.

6. The application according to claim 5, characterized in that, The lipid nanoparticles have a particle size of 60–80 nm.

7. The application according to claim 1, characterized in that, The vaccine is used in combination with immune checkpoint inhibitors; the immune checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

8. The application according to claim 1, characterized in that, The vaccine can reduce the proportion of regulatory T cells in tumor tissue and improve the tumor immunosuppressive microenvironment.

9. The application according to claim 1, characterized in that, The vaccine can inhibit the growth, proliferation, and metastasis of pancreatic ductal adenocarcinoma.

10. An mRNA vaccine for treating pancreatic ductal adenocarcinoma, characterized in that, The invention comprises mRNA encoding the ENPP3 protein and a pharmaceutically acceptable delivery vector, wherein the delivery vector is a lipid nanoparticle, and the mRNA is a codon-optimized ENPP3 coding sequence.

Images