Dual CD40/CD28 co-stimulation reprograms the solid-tumor microenvironment to unleash CAR-T and endogenous anti-tumor immunity

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy against solid tumors, owing to poor T-cell persistence, an immunosuppressive tumor microenvironment (TME) and cancer immune evasion. Here we describe LACO-Stim (lymphocytes-APC co-stimulator), a chimeric co-stimulatory molecule composed of an agonistic anti-CD40 single-chain variable fragment (scFv) and the CD28 intracellular signaling domain, such that a single molecule activates CD40-expressing antigen-presenting cells while delivering CD28 co-stimulation to the T cell that bears it. Co-expressed with a 4-1BB-based CAR (LACO-CAR-T), it enhanced anti-tumor activity in vitro and achieved durable, complete in most mice, tumor regression rather than transient control in xenograft and immunocompetent models. In immunocompetent hosts, LACO-CAR-T activated dendritic cells, reprogrammed tumor macrophages toward M1-like, antigen-presenting states, and induced epitope spreading with endogenous CD8 T-cell responses against tumor neoantigens. Single-cell transcriptomics and RNA-velocity analysis revealed coordinated immune remodeling: T cells shifted toward a more stem-like, less terminally exhausted state, regulatory T cells declined in frequency and suppressive function, and myeloid cells acquired antigen-presentation programs while excessive inflammatory and suppressive signaling was restrained. LACO-Stim further synergized with anti-PD-1 blockade to control tumor growth. LACO-Stim thus enables CAR-T cells to coordinate intrinsic T-cell reinforcement with extrinsic immune reprogramming, converting an immunosuppressive TME into a de-inflamed (reduced inflammatory signaling), de-suppressed rather than merely armed niche that supports endogenous anti-tumor immunity.