Abstract
BACKGROUND & AIMS: Immune checkpoint inhibitor (ICI) therapy has significantly improved the treatment of solid tumors such as hepatocellular carcinoma (HCC); however, most patients fail to respond. Here, we examined whether co-administration of the tumor-penetrating internalizing (i)RGD peptide, which selectively increases tumor vascular permeability in a neuropilin-1-dependent manner, enhances intratumoral delivery and therapeutic efficacy of αPD-L1 in mouse models of HCC. METHODS: αPD-L1, with or without iRGD, was administered intravenously to mice bearing endogenous HCCs (TGFα/c-myc and diethylnitrosamine [DEN]/carbon tetrachloride [CCl(4)] models). Tumor growth was monitored by MRI. Immune cell composition and activation were analyzed by flow cytometry. RNA sequencing was performed on whole tumors and isolated intratumoral CD45(+) immune cells. RESULTS: While αPD-L1 monotherapy had minimal impact on tumor progression, combination treatment with iRGD significantly improved therapeutic efficacy, resulting in markedly reduced tumor growth (mean difference -198.2%, p <0.0001 in TGFα/c-myc and -88.8%, p = 0.0159 in DEN/CCl(4)-induced HCC mice) and increased objective response rates from 0 to 33% (90% confidence interval 14.6-58.6) in TGFα/c-myc mice and to 80% (90% confidence interval 39.6-95.8) in DEN/CCl(4)-induced HCC mice. Flow cytometry revealed reduced PD-1(high) CD8(+) T cells and enhanced expression of activation markers (Ki67, CD44, IFN-γ) in the combination group. RNA sequencing of CD45(+) cells and whole-tumor transcriptomes indicated decreased immunosuppression and increased vascular permeability in mice receiving the combination therapy. Immunoblot analysis showed enhanced accumulation of αPD-L1 in tumors following iRGD co-administration. CONCLUSIONS: iRGD co-administration significantly improves the therapeutic efficacy of αPD-L1 in HCC mouse models by increasing intratumoral αPD-L1 delivery and more effectively alleviating the immunosuppressive tumor microenvironment. This non-conjugated, systemic approach holds strong translational potential to enhance ICI responses in patients with HCC. IMPACT AND IMPLICATIONS: Immune checkpoint inhibitors (ICIs) show limited efficacy in hepatocellular carcinoma (HCC), to which poor intratumoral drug delivery is likely to contribute. This study demonstrates that intravenous co-administration of the tumor-penetrating peptide iRGD with anti-PD-L1 significantly improves antibody distribution and therapeutic response without increasing toxicity in preclinical HCC models. These findings are highly relevant for clinicians and researchers aiming to enhance ICI effectiveness in solid tumors. Given the ongoing clinical evaluation of iRGD, this simple, non-conjugated strategy offers a feasible and rapidly translatable approach to overcome a key limitation of current ICI therapy and improve outcomes for patients with HCC.