Tunneling CARs: Increasing CAR T-cell tumor infiltration through the overexpression of MMP-7 and Osteopontin-b

Abstract Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy against hematologic malignancies but has struggled to achieve comparable success in solid tumors. A key obstacle in solid tumors is the extracellular matrix (ECM), which impedes CAR T-cell infiltration. In clinical trials, neuroblastoma (NB) has shown responsiveness to GD2-directed CAR T-cell therapy, however, the failure of GD2.CAR T cells to effectively clear bulky disease - characterized by dense ECM - highlights the critical challenge of infiltration. In this study, we demonstrate that GD2.CAR T cells exhibit a unique infiltration-restriction compared to other CAR T cells and endogenous T cells. A separate analysis of clinical datasets identified MMP7 and SPP1 (which encodes osteopontin; OPN) as candidate genes to improve the infiltration of GD2.CAR T cells as these were upregulated in tumor-infiltrating leukocytes. MMP-7 and OPN overexpression enhanced CAR T-cell extravasation and interstitial movement in ECM-dense environments in vitro. Overexpression of either OPN or MMP-7 significantly improved tumor infiltration in a xenograft model of NB. This resulted in improved tumor control and a survival extension in OPN-GD2.CAR T-cell treated mice compared to unmodified GD2.CAR T cells. OPN overexpression did not increase off-target infiltration into healthy tissues or promote tumor metastasis, highlighting its potential for safe therapeutic application. Our study provides a framework for further exploration of gene modifications to improve CAR T-cell infiltration in solid tumors and identifies OPN as a candidate to explore in this regard.