CAR T Cells Targeting O-Glycosylated Fibronectin Exhibit Potent Cytolytic Activity and Combine with Tumoral Toll-Like Receptor Agonism to Overcome Tumor Resistance.

Tumors remodel the extracellular matrix (ECM) and glycosylation, yielding epitopes with restricted or limited detectability in normal adult tissues. In this study, we evaluated the O-glycosylated type III connecting segment domain of fibronectin (Tn-FN) as a chimeric antigen receptor (CAR) T-cell target. FDC6-BBζ CAR T cells recognizing Tn-FN were benchmarked against extra domain B-FN-targeted L19-BBζ and Tn-MUC1-targeted 5E5-BBζ. FDC6-BBζ mediated robust, antigen-dependent activation and cytotoxicity, outperforming L19-BBζ and matching 5E5-BBζ in vitro and in NOD/SCID gamma xenografts of prostate cancer. FDC6-BBζ and 5E5-BBζ CAR T cells achieved durable tumor control with increased intratumoral CD3+ infiltration and reduced tumor-collagen overlap. Cytotoxicity required an intact tumor IFNγ receptor 1 (IFNγR1); L19-BBζ further depended on Fas, whereas FDC6-BBζ and 5E5-BBζ were less Fas-dependent. Tumoral Toll-like receptor (TLR) 2/6 or TLR4 agonism restored FDC6-BBζ killing of IFNγR1-deficient targets and induced broad inflammatory and stress-response programs. Pharmacologic perturbation implicated caspase-dependent mechanisms and a contribution from inflammasome-linked signaling, whereas ferroptosis blockade did not abrogate restored killing. These findings establish Tn-FN as a glycoform-restricted, ECM-derived CAR target and show that innate agonists can reprogram tumor state to overcome resistance from impaired IFNγ signaling.