IL-2mRNA multi-pathway delivery method for immunotherapy of solid tumors

Abstract

The invention relates to the technical field of solid tumor immunotherapy, in particular to an IL-2mRNA multichannel delivery method for solid tumor immunotherapy, which comprises the following steps: designing a DNA template, using a plasmid containing an IL-2 gene sequence such as human IL-2cDNA, adding a T7 promoter, a 5'untranslated region UTR and a 3 'UTR at two ends to enhance translation efficiency, carrying out in-vitro transcription through T7 RNA polymerase, and carrying out in-vitro transcription through T7 RNA polymerase. And adding a 5 '-end cap analogue such as CleanCapAG to realize co-transcription cap adding, and then preparing lipid nanoparticle LNP encapsulation and carrying out surface modification. According to the IL-2mRNA multi-channel delivery method for solid tumor immunotherapy, through multi-channel delivery design, targeting optimization and combined treatment cooperation, the toxicity problem of a traditional IL-2 therapy and the drug resistance of MHC-I defective tumors are remarkably solved, meanwhile, efficient conversion is achieved through existing equipment, IL-2 expression can be accurately regulated and controlled, the immune microenvironment can be remodeled, and the immunotherapy effect is good. Meanwhile, the system is compatible with various treatment means such as chemotherapy, radiotherapy and immune checkpoint inhibitors.

Description

Technical Field

[0001] The present invention relates to the technical field of solid tumor immunotherapy, and in particular to a multi-pathway delivery method of IL-2 mRNA for solid tumor immunotherapy. Background Art

[0002] Interleukin-2 (IL-2) is a cytokine secreted by T lymphocytes. It mainly participates in anti-tumor, anti-infection and autoimmune regulation by regulating the proliferation and activity of immune cells such as T cells and NK cells. It is a treatment for late-stage tumors.

[0003] The main problems of existing IL-2 therapies include systemic toxicity and limited effectiveness in solid tumors. Traditional IL-2 therapies require large doses, leading to serious side effects such as capillary leak syndrome, and are not effective against MHC-I-deficient tumors because such tumors lead to desertification of the immune microenvironment, and the delivery efficiency and targeting are insufficient.

[0004] To solve the above problems, we made improvements and proposed a multi-pathway delivery method of IL-2 mRNA for solid tumor immunotherapy. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a multi-channel delivery method of IL-2 mRNA for solid tumor immunotherapy, comprising the following steps:

[0007] S1. Design a DNA template using a plasmid containing an IL-2 gene sequence, such as human IL-2 cDNA. Add a T7 promoter, 5' untranslated region (UTR), and 3' untranslated region (UTR) at both ends to enhance translation efficiency. Perform in vitro transcription using T7 RNA polymerase. Add a 5' end cap analog, such as CleanCapAG, for co-transcriptional capping. Then, formulate and encapsulate the protein in lipid nanoparticles (LNPs) and perform surface modification.

[0008] S2. LNP-IL-2 mRNA was administered via tail vein injection or peripheral vein at a dose of 0.5-1 mg mRNA / kg body weight, followed by intratumoral injection of naked mRNA and electroporation;

[0009] S3. LNPs accumulate in tumor blood vessels through the EPR effect. Surface CD8 antibodies bind to tumor-infiltrating T cells, releasing mRNA. The local electric field of electroporation opens the cell membrane, enhancing the rate of mRNA transfection within the tumor.

[0010] S4. T cells express IL-2, expand and activate NK cells and CTLs, reshape the immunosuppressive microenvironment, and inhibit tumor growth;

[0011] S5. Detect and evaluate the targeting effect.

[0012] As a preferred technical solution of the present invention, during the design of the DNA template, the length of the Poly-A tail is designed to be 100-150 adenylate residues to ensure mRNA stability. After the in vitro transcription reaction, the residual DNA template is digested with DNaseI, purified and modified using a magnetic bead purification method, and an RNA cleaning and concentration kit is used to remove unreacted nucleotides and enzymes. The purity and integrity of the mRNA are verified by high-performance liquid chromatography to ensure the absence of degraded fragments.

[0013] As a preferred technical solution of the present invention, during encapsulation, an ionizable cationic lipid such as DLin-MC3-DMA accounting for 50%, an auxiliary lipid DSPC accounting for 10%, a cholesterol accounting for 38.5% and a PEG lipid accounting for 1.5% are mixed and dissolved in ethanol. The thin film hydration method is used to rotary evaporate the lipid solution to form a thin film, and pH 4.0 citric acid buffer is added to hydrate to form liposomes. The liposomes are homogenized at 20,000 psi by a high-pressure microfluidizer to achieve an mRNA encapsulation efficiency of 90%. The CD8 monoclonal antibody is coupled to the LNP surface through a maleimide-thiol reaction. The terminal carboxyl group of the PEG lipid is first cross-linked with the amino group of the CD8 antibody, and then the unbound antibody is removed by ultrafiltration centrifugation to finally form a targeted LNP.

[0014] As a preferred technical solution of the present invention, LNP relies on the EPR effect, that is, the high permeability of tumor blood vessels to passively enrich in the tumor site, while the surface CD8 antibody actively binds to the CD8 receptor on the surface of tumor-infiltrating T cells to promote the endocytic release of mRNA.

[0015] As a preferred technical solution of the present invention, under ultrasound guidance, 50-100 μg of naked IL-2 mRNA is dissolved in physiological saline and injected directly into the core and edge areas of the tumor to ensure coverage of the main lesions; a square wave electroporator is used, with the parameters set to a voltage of 100-150 V / cm, a pulse duration of 5 ms, an interval of 1 s, and a total of 6 pulses. The electrode needle is inserted parallel to the tumor tissue, and after power is applied, the cell membrane pores are instantly opened, promoting the entry of mRNA into tumor cells and immune cells.

[0016] As a preferred technical solution of the present invention, LNP is retained in the tumor interstitium through the leakiness of tumor blood vessels, and activates the positive charge properties of ionizable lipids in the acidic microenvironment, promoting fusion with the cell membrane. After the CD8 antibody on the LNP surface binds to the T cell, it enters the cell through clathrin-mediated endocytosis, and the mRNA escapes the lysosome in the cell for degradation and is released into the cytoplasm.

[0017] As a preferred technical solution of the present invention, the electric field generated by electroporation temporarily forms nanoscale pores in the cell membrane lipid bilayer, allowing naked mRNA to diffuse directly into the cytoplasm, bypassing the endocytosis pathway and enhancing the rate of mRNA transfection within the tumor.

[0018] As a preferred technical solution of the present invention, IL-2 binds to the IL-2Rβγ receptor on the surface of T cells, activates the JAK-STAT5 pathway, promotes the proliferation of CD8+ T cells and secretes effector molecules such as IFN-γ and granzyme B, IL-2 activates the ADCC effect of NK cells, and at the same time promotes CTL to recognize and kill tumor antigens presented by MHC-I. Through local high concentrations of IL-2, it competitively inhibits the IL-2Rα-dependent immunosuppressive function of Treg, inducing IFN-γ to downregulate VEGF expression.

[0019] As a preferred technical solution of the present invention, Cy5 fluorescent dye is used to label LNP-encapsulated mRNA, and the distribution of LNP in tumors, livers, and spleens is observed in real time using a small animal in vivo imaging system such as PerkinElmer IVIS to evaluate the targeting efficiency. Tissue samples are collected 6, 24, and 48 hours after administration, and the signal ratio of the tumor / non-tumor area is calculated by fluorescence intensity. Tumor-infiltrating lymphocytes (TILs) are isolated and labeled with anti-CD8, CD4, CD25, and FoxP3 antibodies to analyze the proportion of T cell subsets and activation markers. Finally, ELISA is performed to measure the IL-2 and IFN-γ concentrations in serum and tumor homogenates to verify the local and systemic immune response levels.

[0020] The beneficial effects of the present invention are: the IL-2 mRNA multi-pathway delivery method for solid tumor immunotherapy significantly overcomes the toxicity problems of traditional IL-2 therapy and the drug resistance of MHC-I-deficient tumors through multi-pathway delivery design, targeted optimization and combined treatment synergy, while achieving efficient conversion using existing equipment. Its core advantage lies in the ability to accurately regulate IL-2 expression through local high concentration and low systemic exposure, which can reshape the immune microenvironment and reverse the "immune desert" state. It is also compatible with multiple treatment methods such as chemotherapy, radiotherapy, and immune checkpoint inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0022] Figure 1 is a diagram of the multi-pathway delivery method of IL-2 mRNA for solid tumor immunotherapy of the present invention;

[0023] Figure 2is a diagram of the multi-pathway delivery method of IL-2 mRNA for solid tumor immunotherapy of the present invention;

[0024] Figure 3 is a diagram of the multi-pathway delivery method of IL-2 mRNA for solid tumor immunotherapy of the present invention;

[0025] Figure 4 is a diagram of the multi-pathway delivery method of IL-2 mRNA for solid tumor immunotherapy of the present invention;

[0026] Figure 5 is a diagram of the multi-pathway delivery method of IL-2 mRNA for solid tumor immunotherapy of the present invention;

[0027] Figure 6 is a diagram of the multi-pathway delivery method of IL-2 mRNA for solid tumor immunotherapy of the present invention; DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0029] Example: Figures 1-6 As shown, the IL-2 mRNA multi-channel delivery method for solid tumor immunotherapy comprises the following steps:

[0030] S1. Design a DNA template using a plasmid containing an IL-2 gene sequence, such as human IL-2 cDNA. Add a T7 promoter, 5' untranslated region (UTR), and 3' untranslated region (UTR) at both ends to enhance translation efficiency. Perform in vitro transcription using T7 RNA polymerase. Add a 5' end cap analog, such as CleanCapAG, for co-transcriptional capping. Then, formulate and encapsulate the protein in lipid nanoparticles (LNPs) and perform surface modification.

[0031] S2. LNP-IL-2 mRNA was administered via tail vein injection or peripheral vein at a dose of 0.5-1 mg mRNA / kg body weight, followed by intratumoral injection of naked mRNA and electroporation;

[0032] S3. LNPs accumulate in tumor blood vessels through the EPR effect. Surface CD8 antibodies bind to tumor-infiltrating T cells, releasing mRNA. The local electric field of electroporation opens the cell membrane, enhancing the rate of mRNA transfection within the tumor.

[0033] S4. T cells express IL-2, expand and activate NK cells and CTLs, reshape the immunosuppressive microenvironment, and inhibit tumor growth;

[0034] S5. Detect and evaluate the targeting effect.

[0035] During the DNA template design process, the Poly-A tail length was designed to be 100-150 adenylate residues to ensure mRNA stability. After the in vitro transcription reaction, the residual DNA template was digested with DNaseI and purified and modified using magnetic bead purification. An RNA cleaning and concentration kit was used to remove unreacted nucleotides and enzymes, and the mRNA purity and integrity were verified by high-performance liquid chromatography to ensure the absence of degraded fragments.

[0036] During encapsulation, ionizable cationic lipids such as DLin-MC3-DMA (50%), auxiliary lipid DSPC (10%), cholesterol (38.5%), and PEG lipid (1.5%) were mixed and dissolved in ethanol. The thin film hydration method was used to rotary evaporate the lipid solution to form a thin film, and pH 4.0 citric acid buffer was added to hydrate it to form liposomes. The liposomes were homogenized at 20,000 psi using a high-pressure microfluidizer to achieve an mRNA encapsulation efficiency of 90%. The CD8 monoclonal antibody was coupled to the LNP surface through a maleimide-thiol reaction. The terminal carboxyl group of the PEG lipid was first cross-linked with the amino group of the CD8 antibody, and then the unbound antibody was removed by ultrafiltration centrifugation to finally form a targeted LNP.

[0037] LNP relies on the EPR effect, that is, the high permeability of tumor blood vessels passively enriched in the tumor site. At the same time, the surface CD8 antibody actively binds to the CD8 receptor on the surface of tumor-infiltrating T cells, promoting the endocytic release of mRNA.

[0038] Under ultrasound guidance, 50-100 μg of naked IL-2 mRNA was dissolved in physiological saline and injected directly into the core and edge areas of the tumor to ensure coverage of the main lesions; a square wave electroporator was used, with the parameters set to a voltage of 100-150 V / cm, a pulse duration of 5 ms, an interval of 1 s, and a total of 6 pulses. The electrode needle was inserted parallel to the tumor tissue, and the cell membrane pores were instantly opened after power was turned on, promoting the entry of mRNA into tumor cells and immune cells.

[0039] LNP is retained in the tumor interstitium through the leakiness of tumor blood vessels, and activates the positive charge properties of ionizable lipids in the acidic microenvironment, promoting fusion with the cell membrane. After the CD8 antibody on the LNP surface binds to the T cell, it enters the cell through clathrin-mediated endocytosis, and the mRNA escapes the lysosome in the cell for degradation and is released into the cytoplasm.

[0040] The electric field generated by electroporation temporarily forms nanoscale pores in the lipid bilayer of the cell membrane, allowing naked mRNA to diffuse directly into the cytoplasm, bypassing the endocytosis pathway and enhancing the rate of mRNA transfection within the tumor.

[0041] IL-2 binds to the IL-2Rβγ receptor on the surface of T cells, activates the JAK-STAT5 pathway, promotes the proliferation of CD8+ T cells and secretes effector molecules such as IFN-γ and granzyme B. IL-2 activates the ADCC effect of NK cells, while promoting CTL to recognize and kill tumor antigens presented by MHC-I. Through local high concentrations of IL-2, it competitively inhibits the IL-2Rα-dependent immunosuppressive function of Treg, inducing IFN-γ to downregulate VEGF expression.

[0042] LNP-encapsulated mRNA is labeled with Cy5 fluorescent dye, and the distribution of LNP in tumors, livers, and spleens is observed in real time using a small animal in vivo imaging system such as PerkinElmer IVIS to evaluate targeting efficiency. Tissue samples are collected 6, 24, and 48 hours after administration, and the signal ratio of tumor / non-tumor areas is calculated by fluorescence intensity. Tumor-infiltrating lymphocytes (TILs) are isolated and labeled with anti-CD8, CD4, CD25, and FoxP3 antibodies to analyze the proportion of T cell subsets and activation markers. Finally, ELISA tests are performed to measure the concentrations of IL-2 and IFN-γ in serum and tumor homogenates to verify the levels of local and systemic immune responses.

[0043] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A multi-channel delivery method for IL-2 mRNA for solid tumor immunotherapy, characterized in that: The following steps are involved: S1. Design a DNA template using a plasmid containing an IL-2 gene sequence, such as human IL-2 cDNA. Add a T7 promoter, 5' untranslated region (UTR), and 3' untranslated region (UTR) at both ends to enhance translation efficiency. Perform in vitro transcription using T7 RNA polymerase. Add a 5' end cap analog, such as CleanCapAG, for co-transcriptional capping. Then, formulate and encapsulate the protein in lipid nanoparticles (LNPs) and perform surface modification. S2. LNP-IL-2 mRNA was administered via tail vein injection or peripheral vein at a dose of 0.5-1 mg mRNA / kg body weight, followed by intratumoral injection of naked mRNA and electroporation; S3. LNPs accumulate in tumor blood vessels through the EPR effect. Surface CD8 antibodies bind to tumor-infiltrating T cells, releasing mRNA. The local electric field of electroporation opens the cell membrane, enhancing the rate of mRNA transfection within the tumor. S4. T cells express IL-2, expand and activate NK cells and CTLs, reshape the immunosuppressive microenvironment, and inhibit tumor growth; S5. Detect and evaluate the targeting effect.

2. The IL-2 mRNA multi-channel delivery method for solid tumor immunotherapy according to claim 1, characterized in that: During the DNA template design process, the Poly-A tail length was designed to be 100-150 adenylate residues to ensure mRNA stability. After the in vitro transcription reaction, the residual DNA template was digested with DNaseI and purified and modified using magnetic bead purification. An RNA cleaning and concentration kit was used to remove unreacted nucleotides and enzymes, and the mRNA purity and integrity were verified by high-performance liquid chromatography to ensure the absence of degraded fragments.

3. The multi-channel delivery method of IL-2 mRNA for solid tumor immunotherapy according to claim 1, characterized in that: During encapsulation, ionizable cationic lipids such as DLin-MC3-DMA (50%), auxiliary lipid DSPC (10%), cholesterol (38.5%), and PEG lipid (1.5%) were mixed and dissolved in ethanol. The thin film hydration method was used to rotary evaporate the lipid solution to form a thin film, and pH 4.0 citric acid buffer was added to hydrate it to form liposomes. The liposomes were homogenized at 20,000 psi using a high-pressure microfluidizer to achieve an mRNA encapsulation efficiency of 90%. The CD8 monoclonal antibody was coupled to the LNP surface through a maleimide-thiol reaction. The terminal carboxyl group of the PEG lipid was first cross-linked with the amino group of the CD8 antibody, and then the unbound antibody was removed by ultrafiltration centrifugation to finally form a targeted LNP.

4. The multi-channel delivery method of IL-2 mRNA for solid tumor immunotherapy according to claim 1, characterized in that: LNP relies on the EPR effect, that is, the high permeability of tumor blood vessels passively enriched in the tumor site. At the same time, the surface CD8 antibody actively binds to the CD8 receptor on the surface of tumor-infiltrating T cells, promoting the endocytic release of mRNA.

5. The multi-channel delivery method of IL-2 mRNA for solid tumor immunotherapy according to claim 1, characterized in that: Under ultrasound guidance, 50-100 μg of naked IL-2 mRNA was dissolved in physiological saline and injected directly into the core and edge areas of the tumor to ensure coverage of the main lesions; a square wave electroporator was used, with the parameters set to a voltage of 100-150 V / cm, a pulse duration of 5 ms, an interval of 1 s, and a total of 6 pulses. The electrode needle was inserted parallel to the tumor tissue, and the cell membrane pores were instantly opened after power was turned on, promoting the entry of mRNA into tumor cells and immune cells.

6. The multi-channel delivery method of IL-2 mRNA for solid tumor immunotherapy according to claim 1, characterized in that: LNP is retained in the tumor interstitium through the leakiness of tumor blood vessels, and activates the positive charge properties of ionizable lipids in the acidic microenvironment, promoting fusion with the cell membrane. After the CD8 antibody on the LNP surface binds to the T cell, it enters the cell through clathrin-mediated endocytosis, and the mRNA escapes the lysosome in the cell for degradation and is released into the cytoplasm.

7. The multi-channel delivery method of IL-2 mRNA for solid tumor immunotherapy according to claim 1, characterized in that: The electric field generated by electroporation temporarily forms nanoscale pores in the lipid bilayer of the cell membrane, allowing naked mRNA to diffuse directly into the cytoplasm, bypassing the endocytosis pathway and enhancing the rate of mRNA transfection within the tumor.

8. The multi-channel delivery method of IL-2 mRNA for solid tumor immunotherapy according to claim 1, characterized in that: IL-2 binds to the IL-2Rβγ receptor on the surface of T cells, activates the JAK-STAT5 pathway, promotes the proliferation of CD8+ T cells and secretes effector molecules such as IFN-γ and granzyme B. IL-2 activates the ADCC effect of NK cells, while promoting CTL to recognize and kill tumor antigens presented by MHC-I. Through local high concentrations of IL-2, it competitively inhibits the IL-2Rα-dependent immunosuppressive function of Treg, inducing IFN-γ to downregulate VEGF expression.

9. The multi-channel delivery method of IL-2 mRNA for solid tumor immunotherapy according to claim 1, characterized in that: LNP-encapsulated mRNA is labeled with Cy5 fluorescent dye, and the distribution of LNP in tumors, livers, and spleens is observed in real time using a small animal in vivo imaging system such as PerkinElmer IVIS to evaluate targeting efficiency. Tissue samples are collected 6, 24, and 48 hours after administration, and the signal ratio of tumor / non-tumor areas is calculated by fluorescence intensity. Tumor-infiltrating lymphocytes (TILs) are isolated and labeled with anti-CD8, CD4, CD25, and FoxP3 antibodies to analyze the proportion of T cell subsets and activation markers. Finally, ELISA tests are performed to measure the concentrations of IL-2 and IFN-γ in serum and tumor homogenates to verify the levels of local and systemic immune responses.

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