A Cxcl10 mRNA-LNP adjuvant enhances immune checkpoint inhibitor responses in melanoma models

Immunotherapies have become standard of care in the treatment of melanoma; however, about 50% of patients are immunotherapy resistant. This can be caused by an immunosuppressive tumor microenvironment that has a low infiltration of immune cells, especially T cells. Tumors with high T cell infiltration demonstrate better responses to immune checkpoint therapy, making modulation of the tumor microenvironment to enhance T cell recruitment and activation a critical unmet need. In this study, we hypothesize that using an mRNA encoded “genetic adjuvant” can increase the infiltration of T cells into the tumor microenvironment and improve immunotherapy response. The chemokine CXCL10 plays a key role in T cell recruitment and activation, signaling via the CXCR3 receptor expressed by activated T cells. Consistent with this mechanism, analysis of melanoma patient serum samples showed significantly higher Cxcl10 levels in immunotherapy responders compared to non-responders. To directly enhance CXCL10, we developed a Cxcl10-encoded mRNA lipid nanoparticle (Cxcl10 LNP). A significant increase in CXCL10 levels in the sera and inguinal lymph nodes of C57BL/6J wild-type mice was observed post intramuscular administration of Cxcl10 LNP. Our data shows that intramuscular injection of Cxcl10 LNP significantly reduced the volume of established subcutaneous B16F10 murine melanoma tumors. Cxcl10 LNP treatment lead to a significant increase of CD8+ T cells within the tumor microenvironment at 48 hours assessed by flow cytometry and intravital imaging. Phenotypic profiling further revealed significant increases of TRP-2 tumor specific CD8+ T cells and CD107a+ cytotoxic effector CD8+ T cells 48hrs post intramuscular administration of Cxcl10 LNP. When combined with dual anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitor antibodies, Cxcl10 LNP improved long-term survival and significantly decreased subcutaneous tumor growth, with 20% of mice tumor-free at day 100. Moreover, in our spontaneous metastatic melanoma model, mice received Cxcl10 LNP 24h post primary tumor resection followed by dual anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitors for a total of 3 doses. Adjuvating immune checkpoint inhibitors with Cxcl10 LNP significant reduced lung and lymph node metastasis, with 80% of mice showing no detectable metastases. These findings demonstrate that using Cxcl10 LNP is an effective genetic adjuvant that enhances CD8+ T cell infiltration and cytotoxic function, resulting in improved efficacy of immune checkpoint inhibitors and reducing tumor burden and metastases.