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Background Bladder Cancer is the fourth most common cancer in men and a leading cause of cancer death among men and women. Bladder cancer patients with recurrent and/or locally advanced tumors have a poor prognosis often requiring radical cystectomy, a potentially high risk and quality of life changing operation. While the landscape of therapies for non-muscle invasive bladder cancer (NMIBC) has improved over the last 5 years current therapies have achieved responses around 50%. 1–4 Tumor-specific neoantigens have been employed in cancer immunotherapies including vaccines and adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL). The purpose of this study is to identify and enrich for neoantigen-reactive TIL from bladder tumors as a potential cell-based therapy for patients with bladder cancer.Methods Tumors and PBMC samples were collected from 8 bladder cancer patients at Moffitt Cancer Center under an IRB approved study (Pro00037380). TIL’s were expanded ex vivo from whole tumor fragments in media containing high dose IL2 (pre-REP). Neoantigens were identified using whole exome and RNA sequencing. Peptides were pulsed onto patient-derived B-cells (24 hrs) and subsequently co-cultured with bulk and neoantigen-reactive TILs for 12 hours. TIL were sorted based on expression of OX40 and 4-1BB. Sorted TIL were expanded through the rapid expansion protocol (REP). Neoantigen enriched TIL were analyzed for neoantigen peptide reactivity by flow cytometry for 4-1BB/OX40 upregulation and cytokine release and degranulation via ELLA platform.Results TIL’s and B cells expanded in 8/8 (100%) of the available samples. Tumor-specific neoantigens were predicted and long peptides (25aa), designed to bind to autologous HLA molecules, were synthesized (ranging from 42 to 200 peptides) for individual tumor samples. After co-culture of TIL with peptide-pulsed B cells, upregulation of OX40/4-1BB as well as IFNg, granzyme B, and TNFa expression was measured in all 8 samples. The OX40/4-1BB upregulation ranged between 2-65% in individual samples. Additionally following sorting of OX40/4-1BB positive TIL and REP, reactivity against pooled neoantigen peptides was measured in 5/8.Conclusions Neoantigen-reactive TIL can be isolated from the tumors of bladder cancer patients. This study raises the potential for a future clinical trial to evaluate the efficacy of TIL therapy in patients with bladder cancer.References Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J. Clin. 2019;69:7–34.Lamm DL, Blumenstein BA, Crissman JD, et al. Maintenance bacillus Calmette-Guerin immunotherapy for recurrent TA, T1 and carcinoma in situ transitional cell carcinoma of the bladder: a randomized southwest oncology group study. J. Urol. 2000;163:1124–1129.Garcia JA, Dreicer R. Systemic chemotherapy for advanced bladder cancer: update and controversies. J. Clin. Oncol. 2006;24:5545–5551.Choueiri TK, Ross RW, Jacobus S, et al. Double-blind, randomized trial of docetaxel plus vandetanib versus docetaxel plus placebo in platinum-pretreated metastatic urothelial cancer. J. Clin. Oncol. 2012;30:507–512.