Peptide coacervate-mediated siRNA delivery for dual PD-1/PD-L1 blockade to enhance colorectal cancer immunotherapy.

Abstract

Immune checkpoint blockade targeting the PD-1/PD-L1 axis shows limited efficacy in microsatellite-stable (MSS) colorectal cancer (CRC), primarily due to an immunosuppressive tumor microenvironment (TME) and insufficient T-cell activation. Here, we report a peptide coacervate-mediated siRNA delivery platform that enables coordinated gene silencing of PD-1 in T-cells and PD-L1 in tumor cells to enhance CRC immunotherapy. HBpep-SP coacervates (HCs) were functionalized with anti-CD3 antibodies to generate targeted coacervates (THCs), enabling efficient T-cell-targeted delivery of PD-1 siRNA, robust PD-1 knockdown, and enhanced T-cell effector function, as indicated by increased IL-2 and IFN-γ production. In parallel, HCs efficiently delivered PD-L1 siRNA into CRC cells, achieving significant PD-L1 knockdown. Dual checkpoint silencing in a co-culture system of T-cells and CRC cells synergistically enhanced T-cell proliferation and activation, leading to increased tumor cell apoptosis. Importantly, in a murine MSS CRC model, intratumoral co-administration of siPD-1@THC and siPD-L1@HC simultaneously suppressed PD-1 and PD-L1 expression within the TME, increased intratumoral T-cell abundance, and elevated pro-inflammatory cytokine levels, resulting in restored antitumor immunity and significant tumor growth inhibition. Collectively, this peptide coacervate-based dual-checkpoint RNA interference strategy provides a promising approach for advancing T-cell-mediated immunotherapy in MSS colorectal cancer.