Effectiveness of Neoantigen-Specific mRNA/DC Vaccines in Anticancer Immunotherapy

Academic Background

Cancer immunotherapy has emerged as a significant breakthrough in cancer treatment in recent years. Among the various approaches, dendritic cell (DC) vaccines have shown potential in extending the survival of some advanced cancer patients. However, despite their theoretical promise, DC vaccines face numerous challenges in practical applications, such as the complexity of the tumor immunosuppressive microenvironment, insufficient DC activation, inadequate antigen loading, and the low affinity of T cells for tumor-associated antigens (TAAs). These issues limit the efficacy of DC vaccines, particularly in China, where no DC vaccines have been approved yet.

In recent years, personalized anticancer vaccines based on neoantigens have become a new direction in cancer immunotherapy. Neoantigens, derived from mutated proteins in tumor cells, exhibit high tumor specificity and can effectively activate T cell immune responses. With technological advancements, especially in next-generation sequencing, the production of personalized mRNA anticancer vaccines has become feasible. However, the stability and translational efficiency of mRNA within cells remain critical challenges. Cell-penetrating peptides (CPPs) have emerged as a novel biological delivery system that can effectively deliver mRNA into cells and enhance its stability and translational efficiency.

This study aims to develop a neoantigen-based mRNA/DC vaccine and evaluate its effectiveness in anticancer immunotherapy. Using a mouse colon cancer (MC38) model, the research team investigated the immune and antitumor effects of the vaccine, providing experimental evidence for optimizing DC vaccine preparation and reducing costs.

Source of the Paper

This paper was co-authored by Wenli Zhang, Jiahao Guan, Wenwen Wang, Guo Chen, Li Fan, and Zifan Lu, affiliated with the Translational Medicine Center of Shaanxi Provincial People’s Hospital, the Medical Laboratory Center of Shaanxi Provincial People’s Hospital, the Department of Military Preventive Medicine at the Air Force Medical University, and the Traditional Chinese Medicine Department of Shouguang Hospital. The paper was published online on November 26, 2024, in the journal Genes & Immunity, with the DOI: https://doi.org/10.1038/s41435-024-00305-3.

Research Process and Results

1. Preparation of Neoantigen mRNA

The research team first screened six MHC class I neoantigens (DPAGT, REPS1, PTGFR, OLFR99, JAK1, and TRP53) from the mouse colon cancer MC38 cell line and designed a single open reading frame by concatenating these antigen sequences. Through in vitro transcription, mRNA encoding these neoantigens was prepared. To enhance the stability and translational efficiency of the mRNA, the team introduced β-globin sequences into the 5’ and 3’ untranslated regions (UTRs) of the mRNA.

2. Characterization of mRNA-RALA Complexes

RALA is a cell-penetrating peptide with an amphipathic α-helical structure that can encapsulate mRNA into nanocomplexes through electrostatic interactions. The team assessed the binding capacity of RALA to mRNA using gel electrophoresis and ζ-potential analysis. The results showed that as the ratio of RALA to mRNA increased, the ζ-potential of the complexes gradually increased, indicating that RALA could effectively bind and protect mRNA from degradation. Additionally, CCK-8 assays demonstrated that when the RALA-to-mRNA ratio was 10, the complex exhibited the lowest toxicity to DC2.4 cells and the highest transfection efficiency.

3. mRNA Uptake and Protein Expression in DCs Using RALA-Based Formulations

The team evaluated the transfection efficiency of mRNA-RALA complexes using the mouse DC2.4 cell line. The results showed that when the RALA-to-mRNA ratio was 10, the expression level of green fluorescent protein (GFP) in DC2.4 cells was significantly higher than in the control group, indicating that RALA-based formulations could effectively promote mRNA uptake and protein expression in DCs.

4. Neoantigen mRNA Complexes Promote DC Activation

To assess the impact of neoantigen mRNA complexes on DC activation, the team isolated DC precursor cells from mouse bone marrow and induced their maturation using GM-CSF, IL-4, and LPS. Western blot and qPCR results showed that the expression levels of CD80, CD86, and MHC II were significantly higher in mature DCs than in immature DCs. Furthermore, flow cytometry analysis revealed that the neoantigen mRNA-RALA complex significantly upregulated the expression of CD80, CD86, and MHC II in DCs, indicating that the complex could promote DC activation.

5. Delivery and Immune Enhancement Efficacy of Neoantigen mRNA/DC Vaccine

The team isolated tumor-infiltrating lymphocytes (TILs) from mouse MC38 tumor tissues and expanded them in vitro. ELISPOT and flow cytometry results demonstrated that the neoantigen mRNA/DC vaccine could significantly induce the proliferation of neoantigen-specific CD8⁺ IFN-γ⁺ T cells and CD8⁺ CD137⁺ T cells, indicating that the vaccine could activate neoantigen-specific T cell immune responses.

6. In Vivo Antitumor Effects of Neoantigen mRNA/DC Vaccine

The team evaluated the antitumor effects of the neoantigen mRNA/DC vaccine in a mouse MC38 colon cancer model. The results showed that the vaccine could significantly inhibit tumor growth, and the antitumor effect was more pronounced when combined with TILs. Additionally, flow cytometry and immunofluorescence staining revealed that the vaccine significantly increased the infiltration of CD8⁺ T cells into tumor tissues and promoted tumor cell apoptosis.

Conclusion and Significance

This study developed a neoantigen-based mRNA/DC vaccine and demonstrated its effectiveness in anticancer immunotherapy. The vaccine effectively activated T cell immune responses, significantly inhibited tumor growth, and exhibited enhanced antitumor effects when combined with TILs. The research provides experimental evidence for optimizing DC vaccine preparation and reducing costs, holding significant scientific and application value.

Research Highlights

1. Neoantigen Screening and mRNA Design: The team screened six MHC class I neoantigens from the mouse colon cancer MC38 cell line and designed a single mRNA encoding these antigens, enhancing the vaccine’s immunogenicity.
2. Development of RALA-Based Formulations: RALA, as a cell-penetrating peptide, effectively delivered mRNA into DCs and improved its stability and translational efficiency, offering new insights into mRNA vaccine preparation.
3. In Vitro and In Vivo Antitumor Efficacy Validation: Through in vitro and in vivo experiments, the team demonstrated that the neoantigen mRNA/DC vaccine could effectively activate T cell immune responses, significantly inhibit tumor growth, and exhibit enhanced antitumor effects when combined with TILs.

Additional Valuable Information

The team also explored the antitumor effects of non-neoantigen mRNA/DC vaccines, showing that the neoantigen mRNA/DC vaccine’s antitumor efficacy was significantly superior to that of non-neoantigen vaccines, further underscoring the importance of neoantigens in anticancer immunotherapy.

This study provides crucial experimental evidence for the development of personalized neoantigen-based anticancer vaccines, offering broad application prospects.