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Background Chimeric antigen receptor (CAR)-T cells have shown underwhelming performance in the treatment of solid tumors. In contrast, tumor infiltrating lymphocytes (TIL) have an established track record of clinical efficacy, including an FDA-approved therapy for melanoma. Inefficient tumor homing and penetration by peripheral blood lymphocytes (PBL) are accepted as contributing factors to this difference. In the current study, we demonstrate a proof of concept for the generation of CAR-TIL, combining the tumor homing/penetration capacity of TIL with the HLA-independent reactivity of CAR-T cells.Methods We first mapped the biodistribution of murine TIL using a syngeneic tumor model. Briefly, 10 5 MC38 cells were injected subcutaneously in C57BL/6 donor (CD45.2) mice, and tumors were excised 15 days later for ex-vivo TIL expansion. 2 x 106 TIL were adoptively transferred to tumor-bearing recipient (CD45.1, n=5) mice, and CD45.2+ TIL were tracked 1, 3, 5, and 7 days post-transfer. We then generated human anti-CD19 CAR-TIL through retroviral transduction of melanoma TIL, and autologous CAR-T cells derived from PBL. Next, we compared the reactivity of CAR-TIL and CAR-T cells against autologous tumor cells (modified to express human CD19) based on cytokine secretion following coculture (ELLA assay). To evaluate tumor homing/penetration, we generated CD19-expressing patient derived xenografts in NSG mice, which were subsequently infused with 5 x 106 CAR-TIL or CAR-T cells (n=10). Untransduced PBL and untransduced TIL were used as controls. We harvested the tumors 25 days later and quantified CD3+ cells in tumor single-cell suspensions, by flow cytometry. T-test was used for comparisons between groups.Results CD45.2+ T cells were detected in tumor, spleen, bone marrow, and lymph nodes of recipient mice, as soon as 1 day post-transfer. By day 7, 98% of transferred T cells were found in the tumor, and had gradually acquired an effector-memory phenotype. Human melanoma TIL showed robust CAR expression (>80%) following transduction, using two independent donors. Upon activation by autologous CD19-expressing tumor cells, CAR-TIL secreted significantly higher levels of both IFNg (p=0.0001) and Granzyme B (p=0.0003) than CAR-T cells. Upon adoptive transfer to tumor-bearing mice, CAR-TIL abundance within tumors was 2-fold greater than that of CAR-T cells (p=0.004) or untransduced TIL (p=0.02).Conclusions We demonstrated the feasibility of CAR-TIL manufacture and documented their cytokine secretion capacity, and accumulation in tumors, relative to that of autologous CAR-T cells generated from PBL. Given these early results, we will further define the therapeutic potential of CAR-TIL and will evaluate their therapeutic value for clinical translation.Ethics Approval The study obtained ethics approval by the Institutional Review Board. Protocol ID for collection and use of de-identified human specimens: MCC50193 and MCC50232. Participants provided informed consent before taking part in the collection protocol.