WO2025185599A1 - Mrna tumor vaccine encoding membrane-bound il-12 cytokine adjuvant, and use thereof - Google Patents

Abstract

The present invention relates to an mRNA tumor vaccine encoding a membrane-bound IL-12 cytokine adjuvant, and the use thereof. By means of the mRNA tumor vaccine, the toxicity of IL-12 is reduced and the therapeutic effect of the mRNA tumor vaccine is improved. The mRNA contains three parts: a sequence (IL12) encoding IL-12, a sequence (MD) encoding a membrane domain, and a sequence (Ag) encoding a tumor antigen.

Description

mRNA tumor vaccine encoding membrane-bound IL-12 cytokine adjuvant and its use

Cross-reference to related applications

This application claims the benefit of priority to Chinese National Application No. 202410245327.9, filed on March 4, 2024, which is incorporated herein by reference in its entirety.

Technical Field

The present invention belongs to the field of biomedicine technology, and specifically relates to an mRNA tumor vaccine encoding a membrane-bound IL-12 cytokine adjuvant and uses thereof.

Background Art

Cancer is a major threat to human health. Abnormal cell proliferation or differentiation leads to dysfunction, unregulated cell growth, and local tissue invasion and metastasis. Cancer cells, due to genetic mutations, express specific neoantigens that can be recognized and killed by immune T lymphocytes, forming the cornerstone of tumor immunotherapy.

Insufficient numbers of cytotoxic T lymphocytes (CTLs) targeting immunogenic tumors are a major challenge for immunotherapy (Kalbasi and Ribas, 2020). Traditional vaccines, such as protein-based vaccines combined with alum, often fail to generate robust CTL responses against viral infections or tumors; while stronger adjuvants can enhance CTL responses, they are often accompanied by severe toxicity (Awate et al., 2013; Coffman et al., 2010). Antigen generation and adjuvant selection are crucial for vaccine success. Recently developed mRNA vaccine platforms offer a promising strategy for generating more potent antigens and effective adjuvants for the prevention of viral infections and the treatment of cancer (Barbier et al., 2022; Beck et al., 2021; Chaudhary et al., 2021). To increase antigen expression, most clinical mRNA vaccines use methylated bases to prevent innate immune induction and reduce toxicity, although this may also limit the adjuvant effect of mRNA (Andries et al., 2015; Kariko et al., 2005). Lipid nanoparticles (LNPs) for mRNA delivery can act as built-in adjuvants, inducing the production of proinflammatory cytokines such as IL-1β and IL-6, thereby promoting antigen-specific CD4+ follicular helper T cell (Tfh) and B cell responses (Alameh et al., 2021; Tahtinen et al., 2022). However, cytokines such as IL-6 may inhibit the differentiation of naive T cells into effector cells, which are crucial in anti-tumor immune responses (Huseni et al., 2023). Therefore, selecting the right adjuvant for mRNA cancer vaccines is crucial to ensure their efficacy and safety. For the induction of tumor-specific killer T cells, it is important to create a Th1 immune microenvironment dominated by IL-12 and TNF-α. However, IL-12 itself is a highly toxic cytokine. Directly injecting IL-12 protein as an adjuvant with RNA vaccine or expressing free IL-12 with mRNA will lead to serious treatment-related side effects. Therefore, how to create an immune microenvironment that is conducive to the differentiation of cytotoxic T cells for the T cell activation process of mRNA vaccine and reduce the side effects of the immune process on the body is crucial to improving the efficacy of mRNA tumor vaccines.

Recent studies have found that intramuscularly injected mRNA-LNPs are primarily distributed to the draining lymph nodes and spleen, where they are captured by macrophages and dendritic cells and express proteins (Kimberly, J.H. et al. Mol Ther Nucleic Acids. 2023 Nov 24;35(1):102083.). Draining lymph nodes and spleen are important organs for tumor vaccines to trigger adaptive immune responses. Therefore, specifically delivering IL-12 to the draining lymph nodes and spleen to prevent IL-12 from being released into peripheral tissues may improve the immune effect of tumor mRNA vaccines while reducing the peripheral toxic side effects caused by IL-12.

Summary of the Invention

In view of this, the purpose of the present invention is to provide an mRNA tumor vaccine encoding a membrane-bound IL-12 cytokine adjuvant, using RNA technology to simultaneously express IL-12 and tumor antigens on antigen-presenting cells in peripheral lymphoid tissues, and use membrane-bound IL-12 as an adjuvant to improve the therapeutic effect of conventional mRNA tumor vaccines; compared with directly expressing free IL-12 protein, the cell membrane-anchored form of IL-12 protein can limit IL-12 to the surface of antigen-presenting cells in peripheral lymphoid tissues, while not affecting its adjuvant effect and avoiding the release of IL-12 into peripheral tissues to cause peripheral toxicity.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

In one aspect, the present invention provides an mRNA tumor vaccine encoding a membrane-type IL-12 cytokine adjuvant, wherein the mRNA comprises three parts: a sequence encoding IL-12 (IL12), a sequence encoding a membrane domain (MD), and a sequence encoding a tumor antigen (Ag).

According to a specific embodiment of the present invention, the sequence encoding IL-12 (IL12) and the sequence encoding the membrane domain (MD) are located on one mRNA, and the sequence encoding the tumor antigen (Ag) is located on another mRNA; or the sequence encoding IL-12 (IL12), the sequence encoding the membrane domain (MD) and the sequence encoding the tumor antigen (Ag) are located on the same mRNA, and the arrangement order of the three parts is IL12-L1-MD-L2-Ag, or Ag-L2-IL12-L1-MD; wherein L1 is a glycine/serine (Glycine/Serine, Gly/Ser) polypeptide linker, L2 is an IRES sequence or a Glycine/Serine or a 2A polypeptide linker, and L2 is preferably a 2A polypeptide linker.

According to a specific embodiment of the present invention, the tumor antigen includes a tumor neoantigen derived from a mutation, a tumor-specific antigen derived from a virus, or a tumor-related antigen that is highly expressed.

According to a specific embodiment of the present invention, the mutation-derived tumor neoantigens include KARS G12C, KRASG12D, EGFRvIII or BRAF ^V600E ; the viral-derived tumor-specific antigens include HPV E6/E7, endogenous retroviral antigen hERT, LMP1 or LMP2; the tumor-related antigens highly expressed include WT1, MAGE-A3, GP100, NY-ESO-I, HER2/Neu, Claudin18.2, Mesothelin or MUC.

According to a specific embodiment of the present invention, the sequence encoding the IL-12 cytokine comprises an ORF encoding an IL-12B polypeptide and an IL-12A polypeptide, wherein IL-12B and IL-12A are connected by a polypeptide linker, the linker comprises a Gly/Ser linker, and the Gly/Ser linker comprises (GnS)m, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; the linker is preferably a (G4S) ₃ linker.

According to a specific embodiment of the present invention, the membrane domain may include a transmembrane domain, preferably a transmembrane domain plus an intracellular domain, the transmembrane domain includes the CD80 transmembrane domain, the CD86 transmembrane domain, the CD4 transmembrane domain, the CD8 transmembrane domain, the VEGFR transmembrane domain, and the PDGF-RB transmembrane domain, preferably the CD80 transmembrane domain; the intracellular domain includes the CD80 intracellular domain, the CD86 intracellular domain, the CD4 intracellular domain, the CD8 intracellular domain, the VEGFR intracellular domain, the PDGF-RB intracellular domain, and a truncation of the PDGF-RB intracellular domain, and the intracellular domain and the transmembrane domain may come from the same gene combination or different gene combinations; preferably, a combination of the CD80 transmembrane domain plus the CD80 intracellular domain.

According to a specific embodiment of the present invention, the mRNA tumor vaccine further comprises a nucleic acid vector loaded with mRNA, and the nucleic acid vector comprises liposomes, LPP or LPX, preferably LNP nanoliposomes.

In another aspect, the present invention further provides a pharmaceutical composition comprising the mRNA tumor vaccine of the present invention and a second anticancer agent, wherein the second anticancer agent comprises an immune checkpoint inhibitor and/or a bispecific T cell engager (BiTE). Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In yet another aspect, the present invention further provides the use of the mRNA tumor vaccine or pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing tumors. In yet another aspect, the present invention further provides the use of the mRNA tumor vaccine or pharmaceutical composition of the present invention in treating or preventing tumors. In yet another aspect, the present invention further provides a method for treating or preventing tumors in a subject, the method comprising administering the mRNA tumor vaccine or pharmaceutical composition of the present invention to the subject.

According to a specific embodiment of the present invention, the tumor is selected from B-cell lymphoma, bronchial cancer, prostate cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, head and neck cancer, cervical cancer, uterine or endometrial cancer, oral cancer, laryngeal cancer, salivary gland cancer, thymic cancer, adrenal cancer, osteosarcoma, chondrosarcoma, adipose cancer, testicular cancer, malignant fibrous histiocytoma, colorectal cancer, melanoma, gastric cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, bile duct cancer, small intestine cancer or appendix cancer, squamous cell carcinoma, breast cancer and ovarian cancer.

Beneficial effects

In the present invention, we have successfully improved the therapeutic effect of existing mRNA tumor vaccines by adding a cell membrane-anchored IL-12 sequence to mRNA. Compared with directly expressing free IL-12 protein, the cell membrane-anchored form of IL-12 protein can limit IL-12 to the surface of antigen-presenting cells in peripheral lymphoid tissue, greatly reducing the release of IL-12 protein into the peripheral blood without affecting the adjuvant effect of IL-12, and significantly reducing the systemic toxicity produced by IL-12 protein. In various types of tumor vaccines, the addition of membrane-anchored IL-12 sequences can significantly improve the therapeutic effect of mRNA vaccines. Therefore, we believe that mRNA tumor vaccines based on membrane-anchored IL-12 have the potential to become a new generation of tumor therapeutic vaccines and achieve better therapeutic effects in a variety of clinical tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments.

Figure 1 shows that IL-12 can act as an adjuvant to enhance the antitumor efficacy of mRNA vaccines. (A-E) C57BL/6J mice (n = 4-6) were subcutaneously inoculated with 4× ^10⁵ B16F10-OVA cells and received 5 μg of OVA-encoding mRNA via intramuscular injection on days 8 and 12 after tumor inoculation. Six hours after vaccination, 2.5 μg of IL2-Fc (C), 5 μg of IL15-Fc (D), or 0.25 μg of IL12-Fc protein (E) mixed with sodium alginate was injected intramuscularly into the same site. (B) Peripheral blood was collected on day 9 after the first vaccination, and the number of OVA-specific T cells was enumerated by flow cytometry. (C-E) Tumor growth curves for each treatment are shown. Data are presented as mean ± standard error of variance (SEM) and are from two independent experiments. P values were determined by two-way analysis of variance with Bonfferoni's multiple comparison test (C-E). **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2 shows the construction of RNA tumor vaccines adjuvanted with free or membrane-anchored IL12. (A) Schematic diagram of the structures of the free sIL12-OVA and membrane-anchored mtIL12-OVA mRNA vaccines. (B) DC2.4 cells were transfected with CTRL mRNA (top), mtEGFP-OVA RNA (center), or mtIL12-OVA mRNA (bottom). 24 hours after transfection, cells were harvested and stained with antibodies against IL12p40 and OVA peptide-MHC complexes, and the expression of membrane IL12 or OVA-pMHC complexes was analyzed by flow cytometry. (C) DC2.4 cells were transfected with mtIL12-OVA mRNA or sIL12-OVA mRNA. 24 hours after transfection, cell culture fluid was harvested to measure the concentration of secreted free IL12.

Figure 3 shows that RNA tumor vaccines adjuvanted with membrane-anchored IL12 exhibit similar antitumor efficacy but lower peripheral toxicity compared to vaccines adjuvanted with free IL12. (A, B) C57BL/6J mice (n=6) were inoculated with 4×10 ⁵ B16F10-OVA cells and injected intramuscularly with 2.5 μg of mRNA-LNP vaccines encoding mtEGFP-OVA, mtIL12-OVA, or sIL12-OVA on days 8 and 12 post-inoculation. Tumor growth curves, mouse survival curves (A), and body weight changes (B) were recorded. (C) C57BL/6J mice (n=4) were injected intramuscularly with 2.5 μg of mRNA encoding mtIL12-OVA or sIL12-OVA. Serum was collected 24 and 48 hours after injection for measurement of proinflammatory cytokine concentrations.

Figure 4 shows that the mtIL12 mRNA vaccine specifically activates antigen-specific T cells but does not affect bystander T cells or NK cells. (A) C57/B6J mice (n = 4) were intramuscularly injected with 2.5 μg of mRNA encoding mtIL12-OVA or sIL12-OVA. 24 hours after vaccination, dLNs were dissected, and cell subsets expressing IL12p40 on their surface were analyzed by flow cytometry. (B) C57BL/6J mice (n = 4-5) were adoptively transferred with 5 × 10 ⁵ OT1 T cells and vaccinated one day later with 5 μg of mRNA vaccine encoding mtEGFP-OVA, mtIL12-OVA, or sIL12-OVA. 42 hours after immunization, 50 μL of BFA were injected intraperitoneally. 48 hours after immunization, draining lymph nodes (dLNs) were dissected and digested, and IFN-γ+ cells were analyzed by flow cytometry. (C) C57BL/6J mice received 5 μg of mtIL12 mRNA or 5 μg of sIL12 mRNA by intramuscular injection. 36 hours later, peripheral blood was collected and blocked with BFA solution for 6 hours, and the proportion of IFNγ-positive cell subsets was analyzed by flow cytometry.

Figure 5 shows that the mtIL12-OVA mRNA vaccine has a superior anti-tumor effect compared to OVA mRNA or mtIL12 mRNA alone. C57BL/6J mice (n=6) were inoculated with 4×10 ⁵ B16F10-OVA cells. Tumor-bearing mice were injected intramuscularly with 2.5 μg of mRNA encoding mtEGFP-OVA, mtIL12-OVA, or mtIL12 on days 8 and 12, and tumor growth curves were recorded.

Figure 6 shows that the antitumor effect of an RNA tumor vaccine containing a membrane-anchored IL12 adjuvant is dependent on CD8 ⁺ T cells. C57BL/6J mice (n=6) were inoculated with 4× ¹⁰⁵ B16F10-OVA cells and treated intramuscularly with 2.5 μg of mtIL12-OVA mRNA on days 8 and 12 after inoculation. Anti-NK1.1 (200 μg), anti-CD8 (200 μg), anti-CD4 (200 μg), and anti-CSF1R (500 μg) antibodies were administered one day before treatment and continued twice weekly for the next two weeks. Tumor growth curves (A) and survival curves (B) were recorded. (C) C57BL/6J mice received 2.5 μg of mtEGFP-OVA mRNA or 2.5 μg of mtIL12-OVA mRNA via intramuscular injection on days 0 and 5. OVA-specific T cells were measured in the draining lymph nodes on day 8 after the first vaccination and in the spleen on day 13 after the first vaccination.

Figure 7 shows that the mtIL12-based vaccine produces stronger effector T cells in the OVA antigen model. (A) B6 mice were immunized with 2.5 μg mtEGFP-OVA or mtIL12-OVA RNA intramuscularly on days 0 and 7, and blood was collected on day 14 for flow cytometry analysis. (B) Flow cytometry analysis showed that IL12 immunization promoted the differentiation of OVA-specific T cells from a precursor phenotype (Ly108+CD69-) to an effector phenotype (Ly108-CD69-). (C) Peripheral blood cells were incubated with the OT1 peptide (SIINFEKL) for 3 hours and then analyzed by flow cytometry. The results showed that OVA-specific T cells from mice immunized with the IL12 vaccine expressed higher levels of effector molecules (GZMB, IFN-gamma), higher expression of the effector T cell-specific transcription factor T-bet, and lower expression of the immunosuppressive molecule PD-1. (D) Equal numbers of OVA-specific T cells were isolated from mice immunized with mtEGFP-OVA or mtIL12-OVA and incubated with different concentrations of OT1 peptide (SIINFEKL). IFN-gamma concentrations in the culture supernatant were measured 4 hours later.

Figure 8. Demonstrates that cis-delivery of IL12 and antigen can generate more antigen-specific T cells. (A) B6 mice were intravenously injected with 2 μg mtEGFP-OVA, 2 μg mtEGFP-OVA + 1 μg mtIL12, and 1 μg mtEGFP-OVA + 1 μg mtIL12-OVA RNA-LNP on days 0 and 7, and blood was collected on day 14 for flow cytometric analysis. (B) Flow cytometric analysis shows that mice treated with the cis-delivery of IL12 and antigen vaccine (1 μg mtEGFP-OVA + 1 μg mtIL12-OVA) generated higher numbers of antigen-specific T cells in peripheral blood.

Figure 9 shows that membrane-bound IL12 adjuvanted mRNA vaccines overcome resistance to immune checkpoint blockade. (A) C57BL/6J mice (n=6) were subcutaneously inoculated with 4×105B16F10-OVA cells and received 2.5μg of mRNA encoding mtIL12-OVA on days 11 and 15 after tumor inoculation. Anti-CTLA4 (200μg) and anti-PD1 (200μg) were given on day 6 after the first vaccination and continued twice a week for two weeks. Tumor growth curves and mouse survival were recorded.

Figure 10 shows that the mtIL12-E7 vaccine inhibits the growth of HPV-associated TC-1 tumors. C57BL/6J mice (n=6) were vaccinated with 4×105 TC-1 cells. Tumor-bearing mice were treated with an intramuscular injection of 1 μg of mRNA encoding mtEGFP-E7, mtIL12-E7, or mtIL12 on day 14 and boosted with 2 μg of the same mRNA vaccine on day 20. Tumor growth curves and mouse survival (A) and body weight changes (B) were recorded. It can be seen that neither the control E7 vaccine nor mtIL12 alone failed to control tumor growth in mice bearing advanced TC-1 tumors. However, although TC-1 tumors continued to grow to approximately 500 mm3 after two mtIL12-E7 vaccinations, the tumors ultimately regressed, with approximately 80% of tumors successfully eradicated (A). Importantly, mice treated with the mtIL12-E7 mRNA vaccine did not experience significant weight loss compared to the untreated group (B).

Figure 11 shows that the mtIL12-based vaccine generates enhanced effector T cells in the E7 antigen model. (A) TC-1 tumor-bearing mice were intramuscularly injected with 2.5 μg mtEGFP-E7 on day 12 after tumor implantation. On day 18, they were immunized with either 2.5 μg mtEGFP-E7 or 2.5 μg mtIL12-E7. Tumors were harvested and analyzed by flow cytometry on day 22. (B) Flow cytometry analysis shows the gates and expression of selected surface markers for antigen-specific T cells. (C) Flow cytometry analysis shows that IL12 immunization promotes the intratumoral enrichment of E7-specific T cells. Compared to T cells immunized with mtGFP-E7, T cells immunized with mtIL12-E7 are more likely to remain in the effector state (LY108-CD69-) rather than the exhausted state (LY108-CD69+). They also express higher levels of the effector molecule GZMB and lower levels of the inhibitory marker PD1.

Figure 12 shows that mtIL12 adjuvant can be applied to the design of mRNA vaccines for endogenous retroviral-derived tumor neoantigens. P15E protein, derived from gp70 retroviral gene expression, has been detected in many cancers, including the MC38 colon adenocarcinoma cell line (Ye et al., 2020). 4×10 ⁵ MC38 tumor cells were subcutaneously injected into C57BL/6J mice on day 0. After tumor formation, mice were injected intramuscularly with 2.5 μg of mtIL12-p15E or control mtEGFP-p15E mRNA-LNP on days 12 and 16, and tumor growth curves were monitored twice a week. As shown in Figure 7, control P15E mRNA vaccine treatment barely inhibited tumor growth, while mtIL12-P15E treatment induced significant tumor regression and complete eradication (Figure 7).

Figure 13 shows that the mtIL12-based vaccine generates enhanced effector T cells in the p15E antigen model. (A) MC38 tumor-bearing mice were intramuscularly injected with 2.5 μg mtEGFP-p15E mRNA on day 12 after tumor inoculation. They were then immunized again with either 2.5 μg mtEGFP-p15E or 2.5 μg mtIL12-p15E mRNA on day 18. Peripheral blood was collected for flow cytometry analysis on day 21. (B) Flow cytometry analysis showed that mtIL12-p15E immunization generated more p15E-specific T cells. Furthermore, compared to T cells immunized with mtEGFP-p15E, these T cells expressed higher expression of the effector molecules GZMB and IFN-gamma, and lower expression of the inhibitory marker PD1.

DETAILED DESCRIPTION

The present invention provides an mRNA tumor vaccine encoding a membrane-type IL-12 cytokine adjuvant, wherein the mRNA comprises three parts: a sequence encoding IL-12 (IL12), a sequence encoding a membrane domain (MD), and a sequence encoding a tumor antigen (Ag).

According to a specific embodiment of the present invention, the sequence encoding IL-12 (IL12) and the sequence encoding the membrane domain (MD) are located on one mRNA, and the sequence encoding the tumor antigen (Ag) is located on another mRNA; or the sequence encoding IL-12 (IL12), the sequence encoding the membrane domain (MD) and the sequence encoding the tumor antigen (Ag) are located on the same mRNA, and the arrangement order of the three parts is IL12-L1-MD-L2-Ag, or Ag-L2-IL12-L1-MD; wherein L1 is a Glycine/Serine polypeptide linker, and L2 is an IRES sequence or a Glycine/Serine or 2A polypeptide linker.

Preferably, the sequence encoding IL-12 (IL12), the sequence encoding the membrane domain (MD), and the sequence encoding the tumor antigen (Ag) are located on the same mRNA. This is because the same mRNA can ensure that IL12, MD, and Ag are expressed simultaneously in the same cell, avoiding the situation where some cells only express IL-12-MD or Ag due to differences in the transfection efficiency of the two mRNAs. It can more accurately achieve the synchronous expression of the three, ensure their coordination in quantity and time, and facilitate the synergistic effect between them. In addition, it is possible to optimize the production process and reduce production costs. The synthesis, purification, and quality control costs of a single mRNA are significantly lower than the parallel production of two independent mRNAs. In practical applications such as gene therapy, only one mRNA needs to be delivered into the cell. Compared with delivering two mRNAs, the operation is simpler and the delivery efficiency is higher. It also reduces the problems of carrier capacity limitations and cell uptake differences caused by delivering multiple mRNAs, thereby improving the feasibility and operability of the treatment. A single molecular structure is also easier to ensure stability (such as avoiding differences in the degradation rates of the two mRNAs) and has higher consistency between batches.

The tumor antigens described in the present invention may include tumor neoantigens of mutation origin such as KARS G12C, KRAS G12D, EGFRvIII and BRAF ^V600E ; tumor-specific antigens of viral origin such as HPV E6/E7, endogenous retrovirus antigens (hERT), LMP1 and LMP2; tumor-related antigens highly expressed such as WT1, MAGE-A3, GP100, NY-ESO-I, HER2/Neu, Claudin18.2, Mesothelin and MUC, etc. The type of antigen can be changed according to the tumor-specific antigen. Ovalbumin (OVA) is a protein antigen that is widely used to induce cellular and humoral immune responses in cancer immunotherapy and is commonly used as a tumor model antigen in the art. In the embodiments of the present invention, OVA is used as an example of a tumor-specific antigen.

Interleukin-12 (IL-12) is a multipotent cytokine encoded by two independent genes, IL-12A (p35) and IL-12B (p40), which exist as an active heterodimer (p70) or a homodimer of p40 (p80). In the present invention, IL-12B and IL-12A are linked by a polypeptide linker comprising a Gly/Ser linker comprising (GnS)m, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In embodiments of the present invention, the two subunits of the IL-12 cytokine are linked by a (G4S) ₃ linker.

The membrane domain of the present invention can be a transmembrane domain plus an intracellular domain, and the transmembrane domain includes the CD80 transmembrane domain, the CD86 transmembrane domain, the CD4 transmembrane domain, the CD8 transmembrane domain, the VEGFR transmembrane domain, and the PDGF-RB transmembrane domain, preferably the CD80 transmembrane domain; the intracellular domain includes the CD80 intracellular domain, the CD86 intracellular domain, the CD4 intracellular domain, the CD8 intracellular domain, the VEGFR intracellular domain, the PDGF-RB intracellular domain, and a truncated PDGF-RB intracellular domain. The intracellular domain and the transmembrane domain can be derived from the same gene combination or different gene combinations. For example, the following transmembrane domain sequence and intracellular domain sequence can be added together, but the present invention is not limited thereto:

1. Human CD8A transmembrane domain, IYIWAPLAGTCGVLLLSLVITLYCY (SEQ ID NO: 85)

2. Human PDGF-RB transmembrane domain, VVISAILALVVLTIISLIILI (SEQ ID NO: 3)

3. Human CD80 transmembrane domain, LLPSWAITLISVNGIFVICCL (SEQ ID NO: 5)

4. Human CD86 transmembrane domain, WITAVLPTVIICVMVFCLILW (SEQ ID NO: 7)

5. Mouse CD80 transmembrane domain, TLVLFGAGAVITVVVIVVII (SEQ ID NO: 9)

6. Human CD8A intracellular domain (ICD) amino acid sequence, LYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 11)

7. Human PDGF-RB intracellular domain (ICD) amino acid sequence,

8. Truncated human PDGF-RB intracellular domain (ICD) amino acid sequence

9. Human CD80 intracellular domain (ICD) amino acid sequence

10. Human CD86 intracellular domain (ICD) amino acid sequence

11. Mouse CD80 intracellular domain (ICD) amino acid sequence

The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

Example

We sought to determine whether T cell-stimulating cytokines could rapidly expand antigen-primed CTLs and effectively enhance antitumor immune responses. Although IL-2 is essential for T cell activation and IL-15 is highly effective in expanding T cell numbers (Guo et al., 2021; Zou et al., 2024), we observed limited efficacy of these cytokines in enhancing antitumor activity in the context of mRNA vaccines. IL-12 can induce a Th1-type immune microenvironment and promote the differentiation of CD8+ T cells with tumor-killing potential (Hewitt et al., 2020; Tucker et al., 2020; Tugues et al., 2015; Zou et al., 2024). Notably, we observed that IL-12 was the most potent cytokine adjuvant for enhancing the antitumor effects of mRNA cancer vaccines. To optimize the stimulatory effects of IL-12 and minimize peripheral toxicity, we designed an mRNA vaccine expressing a tumor antigen and a membrane-bound IL-12 adjuvant. Our results revealed that IL-12 induces and expands an understudied subset of pre-effector T cells. We further explored how mtIL-12 could provide a robust antitumor response while minimizing toxicity.

Materials and methods

Mice and cell lines:

C57BL/6J, BALB/c, and C57BL/6J-Tg(TcraTcrb)1100Mjb/J (OT1 TCR transgenic) mice, 6–8 weeks old, were purchased from Vital River or Jackson Laboratory. Mice were housed in a SPF environment, and all animal experiments were conducted in accordance with the Regulations for Laboratory Animal Care of Tsinghua University.

MC38, B16F10, TC-1, and DC2.4 cell lines were purchased from the American Type Culture Collection (ATCC). Freestyle 293-F (R79007) was purchased from Invitrogen. The B16F10-OVA cell line was derived from a single-cell clone after lentiviral expression of OVA. All cell lines were routinely tested for mycoplasma contamination. MC38, B16F10, and TC-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C and 5% _CO₂ . DC2.4 cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, 0.1 mmol/L MEM non-essential amino acids, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in 5% CO2. 293-F cells were cultured in SMM 293-TI medium (M293TI; Sino Biological).

Reagents:

Anti-CD4 (clone GK1.5), anti-CD8 (clone 53-5.8), anti-mCSF1R (clone AFS98), anti-NK1.1 (clone PK136), anti-PD-L1 antibodies (10F.9G2), anti-mCTLA4 (clone 4F10), and anti-mIL12p75 (clone JR2-9A5) were purchased from Bio X Cell. IL2-Fc, IL15-Fc, and IL12-Fc were produced in-house. Cytokine coding sequences were fused to the human IgG1 Fc fragment via a GGGGS linker and cloned into the pEE12.4 vector. Protein-encoding plasmids were transfected into 293F cells, and the supernatant was purified by Protein-A affinity chromatography (GE Healthcare) according to established protocols.

Mouse tumor model establishment and treatment:

MC38 cells (4× ^10⁵ ), TC-1 cells (3× ^10⁵ ), and B16F10-OVA cells (2-5× ^10⁵ ) were inoculated subcutaneously on the right dorsum of mice. After tumor establishment, tumor length (a) and width (b) were measured three times weekly, and tumor volume was calculated as a*b*b/2. Mice were randomly grouped according to tumor size. At specific time points, mice were injected with 1-5 μg of mRNA-nanoliposome complexes encoding different coding regions, or with empty liposomes as a control. To exclude experiments targeting different cell subsets, mice were intraperitoneally injected with 200 μg of anti-CD8 antibody, 200 μg of anti-CD4 antibody, 200 μg of anti-NK1.1 antibody, or 500 μg of anti-CSFIR antibody every three days, starting one day before mRNA vaccination. Six hours after mRNA vaccination, 2 μg of IL2-Fc, 3.5 μg of IL15-Fc, and 0.25 μg of IL12-Fc were mixed with sodium alginate and injected intramuscularly. Starting on day 6 after the first mRNA vaccination, 200 μg of anti-PD-L1 antibody and 200 μg of anti-CTLA4 antibody were injected intraperitoneally every three days.

Mice were sacrificed when the length, width or height of the tumor exceeded 2 cm, or the tumor size exceeded 1500 mm ³ , or the body weight of tumor-bearing mice decreased by more than 20%.

Flow cytometric analysis

After digestion and centrifugation, cells were prepared into a cell suspension. Anti-FcgRIII/II (clone 2.4G2) was added to block nonspecific binding and incubated on ice for 15 minutes. The corresponding fluorescently conjugated antibodies were then added and incubated at 4°C in the dark for 30 minutes for staining. Fixable viability Dye eFluor™ 506 or Dye eFluor™ 780 was used to exclude dead cells. After staining, free antibodies were washed with PBS to remove free antibodies. Flow cytometry was performed, and data were analyzed using FlowJo software (Treestar).

Cytometric Bead Array (CBA) and ELISA analysis of serum and tissue samples

The CBAMouse Th1/Th2/Th17 Kit (BD Biosciences) was used according to the manufacturer's instructions to measure cytokine levels in mouse serum and tumor tissue homogenates. ELISA was performed using a 96-well microplate (Corning Costar). 2 μg/mL (100 μL/well) of capture antibody was added to the plate and adsorbed overnight at 4°C. The plate was washed with PBS and then blocked with blocking buffer (PBS containing 0.05% TWEEN-20 and 5% skim milk). Serum or tumor homogenate diluted in blocking buffer was then added and incubated at 37°C for 1.5 hours. After washing with PBST, alkaline phosphatase-labeled Goat Anti-Human IgG secondary antibody was added and incubated at 37°C for 50 minutes. After washing with PBST, 100 μL of p-Nitrophenyl Phosphate was added for color development and the plate was read at 405 nm using a SPECTROstar Nano (BMG LABTECH).

T cell culture in vitro and adoptive cell transfer:

To generate activated OT-1 cytotoxic T cells (CTLs) in vitro, splenocytes from mice were stimulated with 1 mM SIINFEKL peptide (New England Peptide) at a concentration of 108 cells/mL. After a 2-hour peptide pulse, the peptide was washed away, and splenocytes were cultured at a concentration of ¹⁰⁶ cells/mL in T cell culture medium supplemented with 5 ng/mL mouse recombinant IL-2 (mrIL-2) and 10 ng/mL mrIL-12 (R&D), or 5 ng/mL mrIL-2 alone, and supplemented daily. On day 4, 2 × ¹⁰⁶ Ficoll-separated, viable OT-1 cells were adoptively transferred into B16-OVA tumor-bearing mice via intravenous injection.

For in vivo cell transfer experiments, 10 ⁵ naive OT-1 cells were transferred into naive mice, and 24 hours after cell transfer, the mice were immunized with 10 μg of mtEGFP-OVA or mtIL12-OVA mRNA vaccine. Seven days after immunization, activated OT-1 cells were isolated from the spleen of the mice, purified, and 5 × 10 5 OT-1 cells were transferred intravenously into B16-OVA tumor-bearing mice.

mRNA production

DNA plasmid containing the complete sequence of mRNA and carrying poly-A sequence was digested to prepare in vitro transcription template. Co-transcription capping was performed using the T7 Co-transcription RNA Synthesis Kit (C3111). mRNA produced by in vitro transcription was decapped using the cellulose method and stored at -80°C for subsequent experiments.

mRNA nanoliposome packaging:

mRNA was dissolved in 100 mM sodium citrate buffer (pH 4.0). DSPC, cholesterol, DMG-PEG2000, and SM102 were dissolved in anhydrous ethanol at a molar ratio of 38.5:10:1.5:50. The LNP:RNA mass ratio was adjusted between 30:1 and 50:1. The RNA in sodium citrate and the LNP in anhydrous ethanol were then mixed at a volume ratio of 3:1 using a microfluidic system to prepare mRNA-LNP nanoliposomes. The mRNA nanoliposome solution was dialyzed against PBS buffer. The dialyzed mRNA nanoliposomes were suitable for in vitro and in vivo animal experiments.

Data Analysis

Before treatment, tumor-bearing mice were randomly assigned to different groups based on tumor volume. Data were analyzed using GraphPad Prism statistical software and presented as mean ± SEM. P values were calculated using two-way ANOVA for tumor curves and log-rank tests for mouse survival curves. All other data were analyzed using unpaired two-tailed t-tests. A p value < 0.05 indicated significant differences.

The results are shown in the following examples.

Example 1. IL-12 enhances the efficacy of mRNA tumor vaccines to achieve superior tumor control

Effective induction, activation, and differentiation of CD8+ T cells requires a third signal in addition to TCR-pMHC recognition and co-stimulatory signals (Tugues et al., 2015). Although current mRNA-LNP vaccines can expand a large number of tumor-specific CD8+ T cells (Figure 1A-B, Sequence S1 (SEQ ID NO:69)), they only partially inhibit tumor growth (Figure 1C-E). Such mRNA vaccines are known to induce high levels of IL-1β, IL-6, and type I interferon, thereby promoting immune activation (Li et al., 2022). However, these cytokines may not effectively activate CD8+ T cells for optimal tumor control. To identify key cytokine signals that can synergize with mRNA vaccines, we screened several T lymphocyte-stimulating cytokines, including IL2, IL15, and IL12, for tumor therapy. These cytokines are known to promote T cell proliferation, activation, or effector differentiation through different downstream transcription factors (Propper and Balkwill, 2022). To deliver these cytokines to the draining lymph nodes (dLN) to assist T cell priming, we co-injected specific Fc-fused cytokines with the mRNA vaccine intramuscularly into the same site of tumor-bearing mice (Figure 1A). Although IL-2 and IL-15 failed to further enhance the anti-tumor effect of the mRNA vaccine (Figure 1C-D), IL-12 significantly enhanced the efficacy of the vaccine, resulting in significant tumor inhibition (Figure 1E). These results prompted us to include IL-12 as an adjuvant in the mRNA vaccine to better activate antigen-specific T cells and improve tumor control.

Example 2. Membrane-bound IL-12-based mRNA vaccine achieves superior anti-tumor efficacy with limited toxicity

To investigate the potential of IL-12 as an adjuvant for mRNA vaccines, we constructed an IL12-OVA fusion mRNA vaccine for treatment of an OVA-expressing B16F10-OVA tumor model. In this construct, the secreted form of IL-12 was linked to the OT1 epitope-encoding sequence via a 2A linker (sIL12-OVA) (Figure 2A, upper panel, sequence 19, i.e., SEQ ID NO: 67). For the control mRNA vaccine, the IL-12 coding sequence was replaced with the less immunogenic EGFP (Skelton et al., 2001). IL-12 is known to activate CD4, CD8, and NK cells and induce potent IFN-γ secretion (Guo et al., 2012). We hypothesized that systemic spread of the translated IL-12 protein following sIL12-OVA mRNA vaccination might contribute to the observed toxicity (Hewitt et al., 2020). To address this issue, we further developed a membrane-bound IL12 (mtIL12) adjuvanted mRNA vaccine (mtIL12-OVA) designed to restrict IL-12 to the surface of antigen-presenting cells in dLN or spleen. In this construct, IL-12 was fused to the transmembrane domain of the CD80 molecule and then linked to the tumor antigen via a 2A linker (Figure 2A, lower panel, sequence 10, i.e., SEQ ID NO: 58). In vitro transfection of DC2.4 cells confirmed that mtIL12-OVA mRNA transfection could simultaneously present the OT-1 peptide/MHC-I complex and the IL-12 cytokine on the cell surface (Figure 2B, sequence 10, i.e., SEQ ID NO: 58). In vitro transfection of sIL12-OVA mRNA induced IL-12 secretion into the culture supernatant (Figure 2C, sequence 19, i.e., SEQ ID NO: 67); while mtIL12-OVA mRNA transfection confirmed that IL-12 was anchored on the cell surface (Figure 2B, sequence 10, i.e., SEQ ID NO: 58), with almost no leakage into the supernatant (Figure 2B-C).

In B16-OVA tumor-bearing mice, sIL12-OVA vaccine significantly improved therapeutic efficacy and prolonged tumor control compared with the control mRNA vaccine (Figure 3A, sIL12-OVA sequence 19 (SEQ ID NO: 67), control sequence S1 (SEQ ID NO: 69)). However, sIL12-OVA mRNA vaccine caused a significant decrease in body weight in vaccinated mice (Figure 3B), indicating the presence of systemic toxicity. In vivo, mtIL12-OVA showed similar tumor growth inhibition and survival-prolonging effects as sIL12-OVA (Figure 3A, mtIL12-OVA sequence 10 (SEQ ID NO: 58), sIL12-OVA sequence 19 (SEQ ID NO: 67)). Notably, mtIL12-OVA administration did not exacerbate the toxicity associated with soluble IL12. As with the control OVA mRNA-treated mice, the body weight decreased briefly but recovered rapidly (Figure 3B). Compared with sIL12-OVA, mtIL12-OVA-induced serum IL-12, IFN-γ, and MCP-1 levels were significantly reduced (Figure 2C). IL-12 was only detected on the surface of monocytes, dendritic cells, and macrophages within dLNs (Figure 4A). These results indicate that membrane-bound IL12 effectively restricts IL-12 to the surface of APCs, preventing its release into peripheral tissues.

To further elucidate why mtIL12-adjuvanted vaccines can effectively separate toxic side effects from enhanced antitumor effects, we conducted experiments in which 1×106 OT-1 TCR transgenic CD8 T cells were sorted and transferred into wild-type (WT) mice, which were then immunized with control OVA, sIL12-OVA, or mtIL12-OVA mRNA vaccines (Figure 4B and 4C, sequences S1 (SEQ ID NO: 69), 10 (SEQ ID NO: 58), 19 (SEQ ID NO: 67)). Compared with the control OVA vaccine, sIL12-OVA and mtIL12-OVA vaccines induced a considerable number of antigen-specific IFN-γ-positive cells in the dLN (Figure 4B). However, the sIL12-OVA vaccine also induced higher numbers of IFN-γ-positive bystander CD8+ T cells, CD4+ T cells, and NK cells in dLN (Figure 4B) and peripheral blood (Figure 4C), which was not observed in the mtIL12-OVA group. This difference may explain the higher serum IFN-γ levels and peripheral toxicity in the sIL12-OVA group, which were not observed in the mtIL12-OVA group. Taken together, these results indicate that mRNA vaccines with membrane-bound IL-12 cytokine adjuvants can significantly enhance antitumor effects while minimizing adverse reactions.

In vivo, the therapeutic effect of mtIL12-OVA mRNA vaccine was superior to that of OVA-mRNA or mtIL12 mRNA alone (Figure 5, sequence S1 (SEQ ID NO: 69), sequence 19 (SEQ ID NO: 67), sequence S15 (SEQ ID NO: 83)), indicating that the simultaneous presentation of IL-12 cytokine and antigen synergistically enhanced the anti-tumor effect.

Example 3. mtIL12-based mRNA vaccines rely on CD8 T cells to achieve tumor control and induce unique pre-effector CD8 T cell subsets

IL-12-based therapies can act on multiple immune cell types, including CD4+ T cells, CD8+ T cells, NK cells, and macrophages, to optimize tumor control (Xue et al., 2022). To determine which cell subsets are essential for tumor control mediated by the mtIL12-OVA mRNA vaccine, we used antibodies to selectively delete specific immune cell subsets. Deletion of NK cells, CD4+ T cells, or macrophages did not affect the anti-tumor effect of the vaccine (Figure 6A and B, sequence 19 (SEQ ID NO:67)). In contrast, deletion of CD8+ T cells greatly impaired vaccine-mediated tumor suppression and mouse survival benefits, indicating that CD8+ T cell responses are critical for the anti-tumor effect of the vaccine.

We further explored the cellular and molecular mechanisms by which the mtIL12-OVA vaccine enhanced its anti-tumor effect. Compared with the control OVA mRNA vaccine, the mtIL12-OVA vaccine induced a higher proportion of antigen-specific CD8+ T cells in the draining lymph nodes and spleen after immunization (Figure 6C).

We further analyzed T cell differentiation stages by flow cytometry. We employed a Ly108- and CD69-based clustering strategy that can distinguish the differentiation trajectories of antigen-specific T cells in chronic infection or tumor models (Beltra et al., 2020). C57BL/6J mice were intramuscularly injected with 2.5 μg mtEGFP-OVA or mtIL12-OVA RNA on days 0 and 7, and blood was collected on day 14 for flow cytometry analysis (Figure 7A). The control OVA mRNA predominantly induced the Ly108+CD69- T cell subset (precursor state), while the mtIL12-OVA vaccine predominantly induced the Ly108-CD69- T cell subset (effector state) in peripheral blood (Figure 7B). Compared with the control OVA mRNA group, the proportion of granzyme B-positive or IFN-γ-positive OVA-specific CD8+ T cells in PBMCs was significantly higher in the mtIL12-OVA vaccine group (Figure 7C). In addition, the proportion of OVA-specific CD8+ T cells expressing the effector T cell transcription factor T-bet was higher in the mtIL12-OVA group, while the level of the inhibitory molecule PD-1 was lower (Figure 7C). To further verify the functional capacity of these cells, we isolated splenocytes from mice immunized with mtIL12-OVA or control OVA mRNA vaccines, stimulated them with different dilutions of SIINFEKL peptide, and analyzed IFN-γ secretion by ELISA. Baseline IFN-γ secretion was low in both groups, but after peptide stimulation, splenocytes from both groups secreted IFN-γ in a dose-dependent manner, and the amount detected in the mtIL12-OVA group was significantly higher (Figure 7D). These results indicate that peripheral antigen-specific T cells induced by the mtIL12-OVA vaccine are highly potent pre-effector T cells that exhibit higher sensitivity and stronger effector responses when they encounter antigen again, thereby achieving enhanced tumor suppression compared to traditional OVA mRNA vaccines.

Next, we analyzed whether a design in which mtIL12 and OVA antigens were delivered in tandem (mtIL12-OVA) had a better immune effect than a simple mixture of the two (OVA+mtIL12). B6 mice were intravenously injected with 2 μg mtEGFP-OVA, 2 μg mtEGFP-OVA + 1 μg mtIL12, or 1 μg mtEGFP-OVA + 1 μg mtIL12-OVA RNA-LNP on days 0 and 7. Blood was collected on day 14 for flow cytometry analysis (Figure 8A). Flow cytometry analysis showed that mice delivered with IL12 and antigen in cis (OVA+mtIL12-OVA) generated a higher proportion and number of antigen-specific T cells in peripheral blood (Figure 8B, Sequence S1 (SEQ ID NO: 69), Sequence 19 (SEQ ID NO: 67), Sequence S15 (SEQ ID NO: 83)).

Example 4. Membrane-bound IL12 adjuvanted mRNA vaccine overcomes immune checkpoint blockade resistance

Immune checkpoint blockade (ICB) therapy has shown promising results in the clinical setting (Hargadon et al., 2018); however, only a minority of patients achieve effective responses (Sharma et al., 2017). One mechanism of resistance is the lack of sufficient tumor-infiltrating lymphocytes (TILs) in “immunologically cold” tumors. We hypothesized that mtIL12-adjuvanted mRNA cancer vaccines could overcome resistance to ICB therapy by providing sufficient antigen-specific TILs to immune-cold tumors. To validate this, we treated B16-OVA tumor-bearing mice with either mtIL12-OVA (Figure 9A, sequence 19, SEQ ID NO:67) or ICB antibodies (anti-PD-L1 plus anti-CTLA4). Although ICB therapy alone had little effect on tumor growth and survival, the mtIL12-OVA vaccine significantly inhibited tumor growth. Furthermore, combining mtIL12-OVA with ICB therapy further improved tumor suppression and prolonged survival (Figure 9A).

Example 5. Vaccine design using membrane-bound IL12 adjuvant for E6/E7 antigens in HPV-related tumors

We further investigated whether the design of a membrane-bound IL12-adjuvanted mRNA vaccine could be extended to target clinically relevant tumor-specific antigens. The E6 and E7 oncoproteins are directly involved in HPV-induced carcinogenesis, both in preclinical and clinical settings. Despite extensive research into E6/E7-targeted therapeutic vaccines, progress has been limited (Grunwitz et al., 2019; Peng et al., 2021). We compared the therapeutic efficacy of a control E7 mRNA vaccine with that of an mtIL12-E7 vaccine in a TC-1 tumor mouse model that mimics HPV-associated cancer (Lin et al., 1996). In mice bearing advanced TC-1 tumors, neither the control E7 vaccine nor mtIL12 treatment alone failed to control tumor growth (Figure 10A, Sequence S15 (SEQ ID NO:83), Sequence S4 (SEQ ID NO:72), Sequence 18 (SEQ ID NO:66)). However, although TC-1 tumors continued to grow to approximately 500 mm ³ after two doses of mtIL12-E7 vaccination, they ultimately regressed, with approximately 80% of tumors successfully eradicated ( FIG10A ). Importantly, mice treated with the mtIL12-E7 mRNA vaccine did not experience significant weight loss compared to the untreated group ( FIG10B ).

TC-1 tumor-bearing mice were immunized with 2.5 μg mtEGFP-E7 on day 12 of tumor implantation and with either 2.5 μg mtEGFP-E7 or 2.5 μg mtIL12-E7 on day 18. Tumor tissue was harvested and analyzed by flow cytometry on day 22 (Figure 11A). The results showed that IL12 immunization promoted the intratumoral enrichment of E7-specific T cells. Compared with T cells immunized with mtGFP-E7, T cells immunized with mtIL12-E7 were more likely to remain in the effector state (LY108-CD69-) rather than the exhausted state (LY108-CD69+), and expressed higher levels of the effector molecule GZMB and lower levels of the inhibitory marker PD-1 (Figure 11C).

Example 6. Membrane-bound IL12 adjuvant is suitable for vaccine design against retroviral-derived tumor neoantigens

Endogenous retroviruses (ERVs) integrate into the host genome and are typically silent in healthy tissues, but their expression is reactivated under pathological conditions such as cancer (Cherkasova et al., 2013; Smith et al., 2018). These reactivated ERV proteins provide viable targets for cancer vaccine development. The p15E protein, derived from the gp70 retroviral gene, has been detected in many cancers, including the MC38 colon adenocarcinoma cell line (Ye et al., 2020). C57BL/6 mice were subcutaneously inoculated with 4 × 10 ⁵ MC38 tumors. Mice were injected intramuscularly with 2.5 μg of mtIL12-p15E or control mtEGFP-p15E mRNA-LNP on days 12 and 16 after tumor establishment, and tumor growth curves were monitored twice weekly. The results are shown in Figure 12. The control P15E mRNA vaccine treatment had minimal effect on tumor growth, while mtIL12-P15E treatment induced significant tumor regression and complete eradication (Figure 12, Sequence S5 (SEQ ID NO: 73), Sequence S9 (SEQ ID NO: 77)).

MC38 tumor-bearing mice were immunized with 2.5 μg of mtEGFP-p15E intramuscularly on day 12 after tumor implantation. On day 18, either 2.5 μg of mtEGFP-p15E or 2.5 μg of mtIL12-p15E was injected intramuscularly. Peripheral blood was collected on day 21 for flow cytometry analysis (Figure 13A). The results showed that compared to the mtEGFP-p15E mRNA vaccine, mtIL12-p15E immunization resulted in more p15E-specific T cells. Furthermore, compared to T cells immunized with mtGFP-p15E, T cells immunized with mtIL12-p15E expressed higher levels of the effector molecule IFN-gamma and lower levels of the immunosuppressive molecule PD-1.

Together, these experiments demonstrate that mtIL12-based vaccine strategies are suitable for cancer therapy targeting a variety of clinically relevant epitopes.

discuss:

Cytokines act as a tertiary signal during T cell activation. The self-adjuvant effect of mRNA-LNPs primarily induces the production of proinflammatory cytokines, including IL-1β, IL-6, and type I interferons (Li et al., 2022; Tahtinen et al., 2022), which may not optimally support antigen-specific CD8+ T cell responses. After screening multiple T cell-stimulating cytokines, we found that IL-12 was the most effective cytokine in enhancing the antitumor effect of mRNA vaccines.

Many studies have focused on local delivery of IL-12 to the tumor microenvironment (TME) to enhance antitumor immune responses while mitigating systemic toxicity (Nguyen et al., 2020). Current clinical approaches involve intratumoral transfection of plasmids or mRNA encoding IL-12 (Hewitt et al., 2020; Telli et al., 2021). However, these local delivery methods face significant challenges, especially for deep-seated solid tumors, and there is still the risk of IL-12 spreading into the circulation, leading to toxic reactions and a limited therapeutic window. Our studies show that local injection of mRNA encoding soluble IL-12 leads to a substantial leakage of IL-12 into the serum, which subsequently triggers an IFN-γ response, a key proinflammatory cytokine closely associated with IL-12-related toxicity. By anchoring IL-12 to the surface of transfected cells via its transmembrane domain, we successfully decoupled the therapeutic benefits of IL-12 from its systemic toxicity.

T cell dysfunction is characterized by established mechanisms by which tumor cells evade T cell-mediated cytotoxicity (Zebley et al., 2024). Studies have shown that naive tumor-specific T cells rapidly acquire a dysfunctional phenotype within hours of entering the TME (Philip et al., 2017; Rudloff et al., 2023). In our studies, conventional mRNA vaccines expanded a large number of progenitor-like antigen-specific T cells, but these cells exhibited limited antitumor activity. We speculate that similar mechanisms may hinder the differentiation of progenitor T cells generated by conventional mRNA vaccines into functional effector cells. Introducing IL-12 signaling during the priming phase of mRNA vaccines facilitates the differentiation of antigen-specific T cells into a pre-effector phenotype. Interestingly, these pre-effector CD8+ T cells secrete few effector cytokines in circulation but exhibit increased sensitivity and stronger effector responses upon re-encountering tumor antigens, which may help explain their limited peripheral toxicity but enhanced antitumor efficacy. These cells also express lower levels of inhibitory PD-1 and display resistance to the suppressive TME, enhancing their antitumor capacity.

In conclusion, the use of membrane-bound IL-12 as an adjuvant significantly enhanced the therapeutic efficacy of mRNA tumor vaccines while minimizing toxicity. This strategy is applicable to a variety of clinically relevant tumor antigens and has considerable potential for clinical translation.

The sequence involved in the present invention is as follows:

Sequence information:

Claims (10)

A mRNA tumor vaccine encoding a membrane-type IL-12 cytokine adjuvant, wherein the mRNA comprises three parts: a sequence encoding IL-12 (IL12), a sequence encoding a membrane domain (MD), and a sequence encoding a tumor antigen (Ag). The mRNA tumor vaccine according to claim 1, wherein The IL-12 encoding sequence (IL12) and the membrane domain encoding sequence (MD) are located on one mRNA, and the tumor antigen encoding sequence (Ag) is located on another mRNA; or The sequence encoding IL-12 (IL12), the sequence encoding the membrane domain (MD), and the sequence encoding the tumor antigen (Ag) are located on the same mRNA, and the arrangement order of the three parts is IL12-L1-MD-L2-Ag; or Ag-L2-IL12-L1-MD; Wherein L1 is a Glycine/Serine polypeptide linker, L2 is an IRES sequence or a Glycine/Serine polypeptide linker or a 2A polypeptide linker, and L2 is preferably a 2A polypeptide linker. The mRNA tumor vaccine according to claim 1 or 2, wherein the tumor antigen Ag includes a tumor neoantigen derived from a mutation, a tumor-specific antigen derived from a virus, or a tumor-related antigen that is highly expressed. The mRNA tumor vaccine according to claim 3, wherein the mutation-derived tumor neoantigens include KARS G12C, KRASG12D, EGFRvIII or BRAF ^V600E ; the viral-derived tumor-specific antigens include HPV E6/E7, endogenous retroviral antigen hERT, LMP1 or LMP2; the tumor-related antigens highly expressed include WT1, MAGE-A3, GP100, NY-ESO-I, HER2/Neu, Claudin18.2, Mesothelin or MUC. The mRNA tumor vaccine according to claim 1 or 2, wherein the sequence encoding the IL-12 cytokine comprises an ORF encoding an IL-12B polypeptide and an IL-12A polypeptide, wherein IL-12B and IL-12A are connected by a polypeptide linker, the linker comprising a Gly/Ser linker, the Gly/Ser linker comprising (GnS) _m , wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; the linker is preferably a (G4S) ₃ linker. The mRNA tumor vaccine according to claim 1 or 2, wherein the membrane domain comprises a transmembrane domain, preferably a transmembrane domain plus an intracellular domain, the transmembrane domain includes a CD80 transmembrane domain, a CD86 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a VEGFR transmembrane domain, and a PDGF-RB transmembrane domain, preferably a CD80 transmembrane domain; the intracellular domain includes a CD80 intracellular domain, a CD86 intracellular domain, a CD4 intracellular domain, a CD8 intracellular domain, a VEGFR intracellular domain, a PDGF-RB intracellular domain, and a PDGF-RB intracellular domain truncation, wherein the membrane domain is preferably a combination of a CD80 transmembrane domain plus a CD80 intracellular domain. The mRNA tumor vaccine according to claim 1 or 2, wherein the mRNA tumor vaccine further comprises a nucleic acid vector loaded with mRNA, wherein the nucleic acid vector comprises a liposome, LPP or LPX, preferably an LNP nanoliposome. A pharmaceutical composition comprising the mRNA tumor vaccine according to any one of claims 1 to 7 and a second anticancer agent, wherein the second anticancer agent comprises an immune checkpoint inhibitor and/or a bispecific T cell engager (BiTE). Use of the mRNA tumor vaccine according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating or preventing tumors. The use according to claim 9, wherein the tumor comprises B-cell lymphoma, bronchial cancer, prostate cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, head and neck cancer, oral cancer, laryngeal cancer, salivary gland cancer, thymic cancer, adrenal cancer, osteosarcoma, chondrosarcoma, adipose cancer, testicular cancer, malignant fibrous histiocytoma, colorectal cancer, melanoma, gastric cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, bile duct cancer, small intestine cancer or appendix cancer, squamous cell carcinoma, breast cancer and ovarian cancer.