Anti-TGFβ peptide P144 augments CAR-T cell efficacy through neutrophil recruitment and TME remodeling in malignant mesothelioma

Abstract

Malignant mesothelioma is a rare, aggressive and highly desmoplastic cancer characterised by a profoundly immunosuppressive tumour microenvironment (TME). Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment of hematological malignancies, but its activity against solid tumours such as mesothelioma remains limited, in part through transforming growth factor-beta (TGF{beta})-mediated immunosuppression. To address this barrier, we engineered murine CAR-T cells that simultaneously target tumour antigen and deliver TGF{beta} blockade to the tumour. We first compared two anti-TGF{beta} peptides, the beta glycan-derived P144 and the phage-display-derived P17, in vitro; both attenuated TGF{beta}-induced epithelial-to-mesenchymal transition and reporter activity, but P144 suppressed canonical Smad3 phosphorylation more effectively and was selected for downstream work. Murine CAR constructs targeting either mesothelin (M11) or fibroblast activation protein (FAP) were modified to secrete P144, and the resulting products were assessed in co-culture assays and in the syngeneic AE17m model of mesothelioma. P144 secretion did not alter transduction efficiency, expansion or antigen-specific cytotoxicity, but markedly increased antigen-driven IFN{gamma} production, most notably at low effector-to-target ratios. In vivo, both M11-P144 and FAP-P144 CAR-T cells-controlled tumour growth better than their parental, peptide-free counterparts, although complete tumour resolution was not achieved. Flow cytometric profiling of the tumour immune infiltrate showed that P144 delivery increased intratumoral neutrophil frequency approximately three-fold, and neutrophils recovered from these tumours were substantially more cytotoxic ex vivo than those from control tumours. Antibody-mediated neutrophil depletion attenuated the additional benefit conferred by P144 while leaving parental CAR-T activity largely intact, indicating that neutrophils contribute functionally to the effect. Local, CAR-T-delivered TGF{beta} blockade therefore remodels the mesothelioma TME and engages a tumoricidal neutrophil response, offering a modular strategy for enhancing adoptive cell therapy in desmoplastic solid tumours.