A dual-function XCL1-CD40L mRNA adjuvant potentiates cancer vaccines by coordinating DC recruitment and maturation

Abstract

Despite the significant promise of mRNA tumor vaccines in treating solid tumors, their clinical response rate is constrained by the low effectiveness of antigen presentation resulting from the immune-suppressive tumor microenvironment (TME). Conventional type 1 dendritic cells (cDC1) are pivotal in antigen cross-presentation; yet, they frequently exhibit insufficient immune-activating communication within solid malignancies. This investigation comprehensively examined the functional attributes of cDC1 by TCGA cohorts and single-cell transcriptomic data. Guided by these bioinformatic insights, we engineered and synthesized a dual-functional mRNA adjuvant encoding the mXCL1-mCD40L fusion protein. We assessed the biological functions and anti-tumor efficacy of this adjuvant in combination with Glypican-3 (GPC3) and HPV-associated mRNA vaccines via protein expression, structure prediction, in vivo antigen uptake and presentation assays, antigen-presenting cell activation assays, antigen-specific T cell activation tests, and therapeutic studies in tumor-bearing mouse models. A high cDC1 signature score was associated with improved overall survival across LIHC and SKCM TCGA cohorts. Compared to normal sample, cDC1s in HCC and melanoma retained their antigen-presentation machinery but received reduced activating ligand inputs (XCL1-XCR1 signaling in HCC and CD40-CD40L signaling in melanoma). AlphaFold 3 showed that the mXCL1-mCD40L fusion protein can maintain structural integrity without spatial steric hindrance. The fusion protein was confirmed to be secreted and to retain both XCL1-dependent chemotactic activity and CD40-dependent dendritic-cell activation. mXCL1-mCD40L mRNA-LNP activated macrophages and dendritic cells in vitro , upregulating immune-related gene expression as confirmed by qPCR. In vivo , the dual-functional adjuvant significantly enhanced the anti-tumor effect of the GPC3 vaccine, outperformed co-delivery of separate mXCL1 and mCD40L mRNA-LNP, and induced stronger specific IFN-γ producing T cells. Meanwhile, this adjuvant also boosted the anti-tumor efficacy of HPV-associated mRNA vaccines. Mechanistically, this adjuvant increased tumor-antigen acquisition and cross-presentation by CD103 + DCs and promoted DC maturation in draining lymph nodes, accompanied by an increased proportion of peripheral CD8 + T cells. By enhancing the recruitment and maturation of cDC1s, the dual-function mXCL1-mCD40L mRNA adjuvant boosts the efficacy of GPC3 and HPV-associated mRNA vaccines, supporting its potential as an antigen-flexible adjuvant platform against solid tumors.