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With the approval of the antibody-drug conjugate enfortumab vedotin (EV), NECTIN4 has emerged as a bona fide therapeutic target in urothelial carcinoma (UC). Here, we report the development of a NECTIN4-directed chimeric antigen receptor (CAR) T cell, which exhibits reactivity across cells expressing a range of endogenous NECTIN4, with enhanced activity in high expressors. We demonstrate that the PPARγ pathway, critical for luminal differentiation, transcriptionally controls NECTIN4 , and that the PPARγ agonist rosiglitazone primes and augments NECTIN4 expression, thereby increasing sensitivity to NECTIN4-CAR T cell-mediated killing. NECTIN4-CAR T cells have potent anti-tumor activity even against EV resistant cells, which largely retain NECTIN4 expression, including in a post-EV biopsy cohort. Our results elucidate a therapeutically actionable mechanism that UC cells use to control NECTIN4 expression and suggest therapeutic approaches that leverage PPARγ agonists for rational combinations with NECTIN4-targeting agents in UC, as well as future potential treatment options for EV-refractory patients. Subject terms: Bladder cancer, Cancer immunotherapy, Cancer therapeutic resistance, Oncology, Bladder cancer

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for their biological differences. Here we show using single cell analysis that HVs harbor a tumor cell state characterized by expression of MUC16 (CA125), MUC4 , and KRT24 . This cell state is enriched in metastases, predicted to be highly resistant to chemotherapy, and linked with poor survival. We also find enriched expression of TM4SF1 , a transmembrane protein, in HV tumor cells. Chimeric antigen receptor (CAR) T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression-dependent manner, highlighting its potential as a therapeutic target. Subject terms: Bladder cancer, Tumour biomarkers, Targeted therapies

SummaryHere, we show that a NOT gated cell therapy (Tmod) can exploit antigens such as epidermal growth factor receptor (EGFR) and human leukocyte antigen-E (HLA-E) which are widely expressed on cancer cells. Noncancerous cells—despite high expression of these antigens—are protected from cytotoxicity by the action of an inhibitory receptor (“blocker”) via a mechanism that involves blocker modulation of CAR surface expression. The blocker is triggered by the product of a polymorphic HLA allele (e.g., HLA-A∗02) deleted in a significant subset of solid tumors via loss of heterozygosity. Moreover, Tmod constructs that target mouse homologs of EGFR or HLA-E for activation, and a mouse-equivalent of HLA-A∗02 for inhibition, protect mice from toxicity caused by the CAR alone. The blocker also controls graft vs. host response in allogeneic T cells in vitro, consistent with the use of Tmod cells for off-the-shelf therapy without additional gene-editing. Graphical abstract Highlights•The Tmod NOT gate can target widely expressed antigens such as EGFR and HLA-E•Tmod works in mice, targeting mouse homologs of EGFR and HLA to kill tumors•Ligand-dependent CAR removal may facilitate Tmod localization to the tumor site•Tmod inherently resists GvHD without gene edits, a benefit for allogeneic products Natural sciences; Biological sciences; Immunology; Systems biology; Cancer systems biology