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Background Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) targeting patient-specific neoantigens can mediate durable tumor regressions in metastatic epithelial cancers. In melanoma, frequency of neoantigen-reactive stem-like TIL (TIL SL) (CD39-CD69-) within infusion products correlate with better clinical outcomes. Antitumor TILSL could serve as an ideal cell therapy, since they self-renew, differentiate into effectors, lack inhibitory proteins of exhaustion, and persist in patients post-ACT. However, in most solid tumors, only a minority of antitumor neoantigen-TIL exist in TILSL state, while the vast majority are CD39+ terminally-differentiated, dysfunctional, exhausted (TILEX) states with poor proliferative, and antitumor effect. Therefore, a goal for enhancing TIL-ACT is to identify, or engineer stem-like antitumor T cells to enhance cell therapy response.Methods The transcription factor Krüppel-like factor 2 ( KLF2) is enriched in ACT response-associated stem-like neoantigen TILs in melanoma. We investigated the impact of KLF2-overexpression on patient-derived, neoantigen-reactive CD8+ TILs from multiple epithelial cancers. Flow cytometry, single-cell transcriptomics, and T cell receptor (TCR) clonotype tracking were used to analyze changes in T cell phenotype and clone frequency. In vivo antitumor efficacy of KLF2-engineered T cells was evaluated using human TP53-R175H TCR-transduced T cells in an ovarian cancer cell-line model in immunodeficient mice.Results KLF2 overexpression in dysfunctional neoantigen-reactive CD8+ TILEX showed a significant increase of the stem-like TILSL population (CD39-/CD69-, 3-80-fold, P=0.001, paired T-test) and decrease of the exhausted population (CD39+/CD69+, 1-16-fold, P=0.017, paired T-test). Single cell transcriptomic and TCR analysis of antitumor or neoantigen-reactive TIL from 6 patients demonstrated that KLF2-engineered selected TIL exhibited high enrichment of TILSL transcriptional signature and consistent reduction in TILEX transcriptional signature, even in monoclonal antitumor TIL. Single cell TCR-clonotype tracking studies of 40 selected TIL clones in two patients demonstrated that 11 out of 40 (27.5%) clones enriched exclusively in CD39+ states were detected in CD39- stem-like state after KLF2-expression. Lastly, human T cells transduced with KLF2 and TP53-R175H TCR exhibited significant tumor regression of human TYK-nu ovarian cancer in vivo, even in the very low cell dose of 6e5 cells/mouse, when compared to TP53-R175H TCR-transduced cells (P=0.025, 2way ANOVA, n=5 mice per group).Conclusions KLF2-overexpression in neoantigen-reactive human T cells results in a phenotypically distinct population, enriched with stem-like signature and exhibiting enhanced antitumor activity. These results could potentially improve KLF2-engineered neoantigen-reactive stem-like T cells for ACT targeting human cancer.