Background: ITIL-306 is an autologous tumor-infiltrating lymphocyte (TIL) therapy that integrates T-cell receptor (TCR)-specific antigen recognition (Signal 1) with robust costimulation via the novel CoStAR transgene upon engagement with FOLR1 (Signal 2; Sukumaran, et al. JITC. 2021;9:198). Here, we assessed IL-2 independent effector function by anti-FOLR1 CoStAR T cells in vitro and evaluated activity in a novel, more representative murine solid tumor model. Methods: For in vitro studies, healthy donor T cells were manufactured to express anti-FOLR1 CoStAR or left nontransduced (NTD). Product cells were stimulated with a single (Day [D]0; ±IL-2) or serial (D0, 7, 14, 21; no IL-2) addition of target cells expressing membrane-anchored OKT3 (Signal 1) and FOLR1 (Signal 2). T-cell activation and proliferation were measured. For in vivo studies, the carcinoembryonic antigen (CEA)-positive (Signal 1) H508 cell line was engineered to express FOLR1 (Signal 2) and injected into 6 mice/group (D–21). NTD or healthy donor T cells expressing HLA-A*02-restricted anti-CEA TCR only, anti-FOLR1 CoStAR only, or dual TCR+CoStAR were given intravenously (D0), and compared with PBS control. Tumor growth, survival, and T-cell expansion were assessed in 2 donors to D96. Results: After single and serial stimulation, only anti-FOLR1 CoStAR T cells showed sustained proliferation without exogenous IL-2. PD-1 positivity was low ( < 10%) on anti-FOLR1 T cells up to D24. In vivo, T-cell persistence on D14 was increased in dual TCR+CoStAR vs anti-CEA TCR only mice (P<.001) and all other groups (P<.01-.001). Tumor growth up to D58 was significantly lower in dual TCR+CoStAR ( <.5 cm³) vs all other groups (each > 2 cm³; P<.0001). Survival was significantly longer in dual TCR+CoStAR (P<.01) vs all other groups; only dual TCR+CoStAR mice were alive at D58, with 5/6 alive at D96. The second donor showed similar tumor control irrespective of exogenous IL-2 in responder mice, with > 50% alive at D96, and robust T-cell expansion in dual TCR+CoStAR mice. Conclusions: When combined with TCR-specific binding, CoStAR significantly enhanced T-cell proliferation, persistence, and antitumor activity in vivo vs TCR alone, resulting in tumor control and prolonged survival. Effects were not observed with CoStAR alone, underscoring that signaling through CoStAR alone does not induce T-cell effector function. The sustained proliferation of anti-FOLR1 CoStAR T cells without exogenous IL-2 support in vitro and in vivo supports a clinical TIL regimen free of high-dose IL-2. These results suggest that CoStAR may improve the clinical performance and benefit/risk profile of TILs, whereby fewer toxicities are expected with the removal of post-TIL IL-2 support. This will be explored in an upcoming first-in-human clinical study with ITIL-306.