Introduction:
Immune checkpoint inhibitors such as anti-CTLA-4 enhance anti-tumor immunity but are often limited by immune-related adverse events (irAEs) caused by non-specific T cell activation. Virus-like particles (VLPs) presenting tumor antigens offer a strategy to selectively prime tumor-specific T cells, potentially enhancing therapeutic efficacy while minimizing off-target toxicity.

Methods:
We investigated a lymphatic-targeted immunotherapy approach in a B16F10 melanoma mouse model. Mice received VLPs carrying the tumor-associated antigen PMEL via intradermal vaccination to a non-tumor-draining lymph node (non-tdLN), followed by αCTLA-4 checkpoint blockade delivered either systemically (intravenous) or regionally (intradermally to the same non-tdLN). Single-cell RNA and TCR sequencing were used to characterize immune cell phenotypes and T cell clonal dynamics in the tumor microenvironment. Functional assays and histopathological analysis assessed tumor-specific activity and irAE profiles.

Results:
Regional αCTLA-4 delivery synergized with VLP vaccination to promote infiltration and clonal expansion of non-exhausted CD8⁺ effector and CD4⁺ Th1 T cells while reducing regulatory T cells. VLPs alone induced PMEL-specific CD8⁺ T cells, and regional αCTLA-4 further amplified this response. Functional assays confirmed increased tumor-specific T cell activation with minimal bystander T cell infiltration into normal tissues. Additionally, αCTLA-4 promoted macrophage activation, upregulating interferon signaling and T cell costimulation pathways.

Conclusions:
Lymphatic-targeted combination therapy with VLP vaccination and regional αCTLA-4 delivery drives potent, tumor-specific immune responses while minimizing systemic toxicity. This approach offers a promising strategy to improve the efficacy and safety of cancer immunotherapy.