Heparanase‐Loaded CAR T Extracellular Vesicles Remodel the Colorectal Tumour Microenvironment and Boost T Cell Antitumor Immunity

Chimeric antigen receptor (CAR) T cell therapy has shown promise in solid tumours, but its efficacy is limited by dense extracellular matrix (ECM) that blocks T cell entry. We engineered mesothelin-targeted CAR T cells to express heparanase (HPSE) fused to the truncated hepatitis A virus pX domain (pX-Δ1-30), enabling surface display of HPSE on extracellular vesicles for localized, pH-dependent ECM degradation within the acidic tumour microenvironment while limiting systemic exposure. HPSE-pX-Δ1-30 CAR T (referred to as HPSE CAR T) cells penetrated ECM mimics nearly fourfold more effectively than standard CAR T cells. They also expressed more TNF-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL), and perforin, leading to stronger tumour killing in 2D and 3D colorectal cancer models. HPSE-pX-Δ1-30 CAR T-derived extracellular vesicles (EVs) retained CAR and chemokine receptors (CCR5/CCR7), carried apoptotic ligands, and were efficiently taken up by tumour cells and T cells. EV exposure promoted T cell proliferation, CCR5 expression, and central/stem-like memory formation while lowering PD-1 and CD57. In HCT116 xenografts, HPSE CAR T cells showed increased intratumoral infiltration, and EVs from these cells promoted infiltration of host T cells. Treatment reduced tumour burden, extended survival beyond 70 days, and did not cause systemic toxicity. These results highlight a dual strategy of ECM remodelling and immune modulation, offering a translational approach to overcome barriers to CAR T therapy in colorectal cancer.