Optimized CART cell therapy to remodel the tumor microenvironment and eliminate GI malignancies

Gastrointestinal (GI) cancers are rising in people < 50 and remain major causes of cancer mortality despite therapeutic advances. Chimeric antigen receptor T (CART) cell therapy has transformed treatment of hematological cancers. However, its efficacy in GI cancers has been hindered by antigen heterogeneity and immunosuppressive cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME.) Here we aimed to 1) study CART-CAF interactions and 2) engineer CART cells capable of eliminating CAFs to overcome their inhibitory role and enhance their antitumor activity in GI cancers. We first analyzed primary CAFs from 20 GI cancer patients. CAFs showed high fibroblast activation protein (FAP) expression and significantly suppressed T cells proliferation (26-77%, p≤0.0001). We next generated CART cells capable of safely eliminating both GI cancer cells and CAFs. To target cancer cells, we developed a novel nanobody (VHH)-based CAR against Claudin18.2, a GI cancer specific target. To avoid off-tumor toxicity in healthy FAP+ tissues, we incorporated an inducible FAP-CAR using the synNotch platform, enabling conditional targeting upon engagement with Claudin18.2 + cells, restricting FAP targeting within the TME. VHH Claudin18.2 CART cells showed superior specificity and efficacy vs scFv-based CART cells (hu8E85 or zolbetuximab). In Claudin18.2+ SNU-C2B xenografts, tumors were 92mm 3 (VHH) vs 48mm 3 (huE85) vs 211mm 3 (zolbetuximab) on day 23 post-CART, median survival undefined vs 17 days vs 23 days respectively; p=0005. In Claudin18.2+ LS411N xenografts, tumors were 315mm 3 vs 106mm 3 vs 382 mm 3 respectively at 23 days post-CART, median survival undefined vs 19 days vs undefined respectively; p=0004. VHH Claudin18.2 CART cells showed no off-tumor effects, while hu8E85 CART induced significant toxicity. We next established a co-engraftment model. NSG mice received 1x10 6 SNU-C2B and 6x10 6 CAFs from a GI cancer patient. At 500 mm 3 tumors, mice received 5x10 6 VHH Claudin18.2 or inducible Claudin18.2/FAP CART. Dual-targeting CART cells showed greater antitumor response (728mm 3 vs 320mm 3 , p=0.0011 at day 20 post-CART), expansion (61 vs 2,350 CART/µl of blood, p=0.0037), and tumor infiltration (8.71 x 10 5 vs 1.03 x 10 6 CART/g of tumor, p<0.0001). This was also observed in gastric, colon, and pancreatic PDX models, with no observable off-tumor cytotoxic effects with inducible dual-targeting Claudin18.2/FAP CART. Our findings demonstrate that our novel VHH-based Claudin18.2 CART cells are highly specific and effective against GI malignancies, and that inducible Claudin18.2/FAP dual-targeting CART cells safely remodel the TME into an immune-permissive state. This approach represents a mechanistically innovative and translationally relevant strategy to overcome CAF-mediated immunosuppression, enhance CART cell efficacy, and improve clinical outcomes in refractory solid tumors.