Enhancing Immunotherapeutic Response in Colorectal Cancer with a Neuropilin 1-Targeting Tumor-Penetrating Peptide.

Immune checkpoint inhibitors (ICI) provide durable responses in a subset of colorectal cancers, yet benefit is largely confined to microsatellite instability-high (MSI-H) tumors. We investigated whether the tumor-penetrating peptide iRGD enhances anti-programmed cell death protein 1 (PD-1) treatment efficacy and examined the clinical relevance of neuropilin 1 (NRP1) expression. Syngeneic mouse models (MSI-H MC38 and microsatellite-stable CT26) were treated with an anti-PD-1 antibody and iRGD. Tumor cell and stromal NRP1 contributions were compared using NRP1-knockout colorectal cancer cell lines. NRP1 expression was analyzed using immunohistochemistry in a consecutive cohort of 110 patients with stage III colorectal cancer who underwent curative surgery. iRGD and anti-PD-1 antibody combination therapy enhanced antitumor effects in both colorectal cancer models, accompanied by increased intratumoral CD8+ T-cell abundance and improvements in their function. This effect persisted in tumors lacking tumor cell NRP1, suggesting that stromal NRP1 played a key role in mediating iRGD activity. In pathologic specimens from patients with colorectal cancer, widespread NRP1 expression was noted across tumor cells, the stromal compartment, and the vasculature, suggesting potential applicability of iRGD-based therapy in human colorectal cancer. Notably, stromal NRP1 expression was associated with poor overall and disease-free survival. Collectively, these findings position stromal NRP1 as a prognostic marker and an actionable entry point for iRGD-based ICI strategies, providing a strong rationale for their clinical development to extend the benefits of ICIs to broader colorectal cancer populations. SIGNIFICANCE: We demonstrated that iRGD enhanced anti-PD-1 therapy efficacy in colorectal cancer models, improving intratumoral permeability and antitumor immune responses. NRP1 is broadly expressed across tumor compartments, and stromal, particularly endothelial, NRP1 is a key mediator of iRGD activity, associated with poor prognosis, representing a potential therapeutic entry point.