Neoantigens, unique tumor-specific mutations presented on MHC class I and/or II, are promising targets for cancer immunotherapy. However, current vaccination strategies using synthetic long peptides (SLPs) often predominantly induce CD4⁺ T cell responses, limiting antitumor efficacy. The authors hypothesized that enhanced delivery of neoantigen peptides to secondary lymphoid tissues (specifically the spleen) would improve both CD4⁺ and CD8⁺ T cell responses and antitumor activity. This hypothesis is grounded in the understanding that optimal CD8⁺ T cell responses require both CD4⁺ and CD8⁺ T cell activation within the spleen and lymph nodes, where antigen-presenting cells (APCs), primarily dendritic cells (DCs), can effectively process and present antigens. Furthermore, studies indicate that CD4⁺ T cell help is crucial for generating effective cytotoxic T lymphocytes (CTLs) that kill tumor cells. The research aimed to develop a peptide-based cancer vaccine platform that effectively elicits both CD4⁺ and CD8⁺ T cell responses by improving peptide delivery to the spleen using SLP-containing cationic lipoplexes (SLP-Lpx). The use of KRAS G12D mutations as a model neoantigen provided a relevant and frequently occurring target for this investigation. The overall goal was to improve the therapeutic efficacy of neoantigen-derived cancer vaccines by optimizing antigen delivery and T cell activation.

The introduction reviews existing literature on neoantigen vaccines and their limitations. It highlights the challenges in eliciting sufficient tumor antigen-specific effector T cells to inhibit tumor progression, noting that existing peptide-based vaccines often induce primarily CD4⁺ T cell responses, despite targeting MHC class I alleles. The review cites research demonstrating the importance of both CD4⁺ and CD8⁺ T cell activation in secondary lymphoid organs for robust antitumor responses. The crucial role of CD4⁺ T cell help in enhancing CD8⁺ T cell responses and the importance of targeting neoantigens to secondary lymphoid organs are discussed. The studies of Shreiner et al. and others concerning the synergistic effects of MHC class I and II antigens and the role of IL-15 in stimulating potent CD8⁺ T cell responses are mentioned to further support the rationale for a vaccine design that elicits both CD4⁺ and CD8⁺ T cell responses. The limited success of previous clinical trials using SLPs is also addressed, setting the stage for the proposed novel approach.

The study employed several key methodologies. Initially, IFN-γ ELISpot screens were performed to identify immunogenic T cell epitopes within KRAS G12 variants (G12D/C/V). This involved immunizing mice with peptides and CpG adjuvant and then assessing IFN-γ production by splenocytes upon re-stimulation with overlapping peptides. To enhance peptide delivery, SLP-Lpx were created by formulating peptides with cationic liposomes. The biodistribution of liposomes (rhodamine-labeled) was evaluated in vivo, confirming their enhanced delivery to the spleen compared to anionic or neutral liposomes. The uptake of Cy5-labeled peptides by splenic myeloid cells (macrophages, DCs) was assessed using flow cytometry to determine the efficiency of the liposomal delivery system. The immunogenicity of SLP-Lpx was then evaluated by assessing CD4⁺ and CD8⁺ T cell responses using ELISpot and flow cytometry. In vivo efficacy studies were conducted using mouse models of KRAS-driven lung adenocarcinoma (GEM model) and syngeneic tumor models (MC38, CT26). Tumor growth was monitored, and tumor-infiltrating lymphocytes (TILs) were analyzed for markers of T cell exhaustion (PD-1). Combination therapy with SLP-Lpx and anti-PD-1 checkpoint blockade was also evaluated in the syngeneic models. Detailed procedures for liposome preparation, peptide synthesis, tissue preparation, flow cytometry, ELISpot assays, in silico prediction of MHC binding, bioluminescence imaging, and statistical analysis are provided in the methods section. The specific peptides used in the study are listed, including KRAS WT, G12C, G12D, G12V variants and other neoantigens from MC38 and CT26 cells.

The key findings demonstrate the superior immunogenicity of the SLP-Lpx vaccine compared to naked peptides. Immunization with naked G12D peptides and CpG predominantly elicited CD4⁺ T cell responses with limited tumor growth inhibition. In contrast, SLP-Lpx vaccination stimulated both CD4⁺ and CD8⁺ T cell responses and significantly suppressed tumor growth in a CD8⁺ T cell-dependent manner. The enhanced efficacy of SLP-Lpx was attributed to improved delivery of peptides to splenic myeloid cells, leading to increased antigen presentation. Flow cytometry revealed a significant increase in the uptake of Cy5-labeled peptides by splenic macrophages and DCs when delivered via SLP-Lpx. In the GEM model of KRAS-G12D-driven lung adenocarcinoma, G12D23→Lpx vaccination significantly reduced tumor volume and increased T cell infiltration. In syngeneic tumor models (MC38 and CT26), prophylactic and therapeutic vaccination with SLP-Lpx inhibited tumor growth. The effect was more pronounced with the mutant KRAS G12D peptide than with WT KRAS. Furthermore, the use of multiple neoantigens in SLP-Lpx (Neo-Lpx) further enhanced tumor growth suppression, particularly when combined with anti-PD-1 checkpoint blockade. This combination therapy resulted in partial or complete responses in a significant proportion of animals. Analysis of TILs revealed that the Neo-Lpx vaccine alone increased PD-1 expression on tumor-reactive T cells, suggesting T cell exhaustion; however, the addition of anti-PD-1 treatment significantly improved therapeutic efficacy.

The results confirm the hypothesis that targeted delivery of neoantigen peptides to lymphoid tissues enhances the immunogenicity of cancer vaccines. The superior performance of SLP-Lpx compared to naked peptides highlights the importance of optimizing antigen delivery for effective T cell activation. The CD8⁺ T cell-dependent tumor growth suppression demonstrates the critical role of CTLs in mediating antitumor activity. The study also shows that combining the SLP-Lpx vaccine with checkpoint inhibitors can overcome T cell exhaustion and significantly enhance antitumor effects, resulting in much greater tumor regression. This combinatorial approach addresses the limitations of single-agent therapies. The findings suggest that SLP-Lpx vaccines hold significant promise as a novel cancer immunotherapy strategy. The modular nature of the platform also allows for customization to different tumor types by incorporating diverse neoantigens.

This study demonstrates that a nanoparticle vaccine delivering neoantigen peptides to lymphoid tissues via cationic lipoplexes elicits robust CD4⁺ and CD8⁺ T cell responses leading to significant tumor growth inhibition. The combination of this vaccine with a checkpoint inhibitor further enhances antitumor effects. The ease of manufacturing and modification makes this a promising approach for personalized cancer immunotherapy. Further research should focus on clinical translation, optimizing the vaccine formulation, and exploring combinations with other immunotherapies.

The study was conducted in mouse models, and the findings may not directly translate to humans. While the KRAS G12D mutation is a frequent target, the results might not generalize to all types of cancer or all neoantigens. The use of a limited number of neoantigens in some experiments might not fully capture the complexity of tumor-specific antigens. Further studies are needed to evaluate the long-term efficacy and safety of this vaccine approach in humans.