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Background Tumor Infiltrating Lymphocyte (TIL) therapy is an autologous treatment for solid tumors involving isolation and ex vivo expansion of polyclonal T lymphocytes from tumor. TIL comprise tumor-reactive and bystander TCR clonotypes. The clinical activity of TIL is thought to be partly driven by intrinsic functionality of T cells and absolute number of tumor-reactive T cells (TRT) present within the infused drug product. KSQ-004EX, a CRISPR/Cas9 engineered TIL (eTIL Ò ) whereby SOCS1 and Regnase-1 are inactivated, drives strong anti-tumor activity in pre-clinical models and is being evaluated in a clinical trial for treatment of solid tumors (NCT06598371). Here, we interrogated the impact of SOCS1 and Regnase-1 inactivation on TRT present within KSQ-004EX.Methods Non-clinical KSQ-004EX lots, single-edited eTIL, and unedited TIL controls were manufactured. The frequency and functionality of TRT within eTIL was evaluated by co-culture with donor-matched autologous tumor cell lines in acute and serial re-stimulation assays. To evaluate the impact of SOCS1 and Regnase-1 inactivation on TRT function at single cell resolution, scRNA/TCRseq was performed on TIL within tumor starting material, on eTIL following manufacture, and upon co-culture with autologous tumor. We trained and applied a clone-level TRT classifier to identify candidate TRT clones in tumor starting material and tracked candidate clones to eTIL where their TRT status was validated upon tumor co-culture.Results scRNA/TCRseq analysis confirmed that many putative TRT clones identified in tumor starting material using our TRT classifier were also present in eTIL and exhibited functional reactivity against autologous tumor. SOCS1 and Regnase-1 edit-specific gene expression differences in tumor-reactive eTIL were observed after autologous tumor co-culture. Specifically, tumor-reactive TNFRSF9 (4-1BB)-expressing KSQ-004EX cells showed induction of GZMB, IFNG, CCL3, and BATF transcripts relative to unedited TIL controls, demonstrating the biological impact of SOCS1 and Regnase-1 inactivation. Elevated MHC Class I-dependent production of IFNγ and CCL3 was observed. In autologous tumor chronic re-stimulation assays, KSQ-004EX TRT clonotypes demonstrated resistance to exhaustion relative to single-edited and unedited TIL by retaining polyfunctionality, enhanced proliferation, and sustained control of tumor growth.Conclusions We identified and functionally assessed at single cell resolution TRT clonotypes present within KSQ-004EX and developed a clone-level TRT classifier able to identify candidate TRT clones within tumors. In KSQ-004EX, the dual-inactivation of SOCS1 and Regnase-1 in TRT clonotypes enhances their anti-tumor functionality and prevents their exhaustion upon acute and chronic engagement with autologous tumor. These data support continued evaluation of KSQ-004EX in patients with solid tumors.