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Background Adoptive T cell therapy with expanded tumor-infiltrating lymphocytes (TILs) and PD-1 targeting immune checkpoint inhibitors has shown the potential to induce long-lasting responses in metastatic melanoma (MM), although such responses remain limited. Genetic editing of TILs, such as PD-1 knockout, offers a promising strategy to enhance antitumor efficacy by rendering these cells resistant to tumor-driven immunosuppression. We previously established that non-viral delivery of CRISPR-Cas9 can efficiently knockout PD-1 in human TILs. 1 Here, we present initial data from the first-in-human trial employing CRISPR-Cas9 to knockout PD-1 in TILs from patients with metastatic melanoma (NCT06783270).Methods The CRISPR-TIL infusion products were phenotypically characterized by flow cytometry. Antitumor reactivity was assessed by measuring the TCR-mediated recognition of autologous tumor antigens through the expression of IFN-γ, TNF-α, CD137, and CD107a following co-culture. Additionally, the cytotoxic capacity of an infusion product was evaluated using a real-time target cell killing assay.Results Four patients received CRISPR-edited TIL infusion products. The infusion products had an efficient PD-1 knockout and maintained their cellular composition, with CD3 + subsets predominantly comprising effector memory T cells. The infusion product from patient 02 was evaluated for antitumor reactivity and found to contain tumor-reactive TILs, as demonstrated by cytokine production and the upregulation of CD107a and CD137 when co-cultured with autologous tumor cells. Cytotoxicity assays confirmed robust killing of autologous tumor cells and sustained growth control during a 72-hour co-culture period.Conclusions These preliminary data from the first-in-human trial of CRISPR-edited TILs demonstrate the feasibility of non-viral PD-1 knockout with high editing efficiency. Functional assays indicate that gene-edited TILs retain potent antitumor activity. Further investigation is warranted to evaluate clinical efficacy and durability of response.Trial Registration 2023-510417-25-01 (EU Trial (CTIS) Number) NCT06783270 - clinicaltrials.govReference Chamberlain CA, Bennett EP, Kverneland AH, Svane IM, Donia M, Met Ö. Highly efficient PD-1-targeted CRISPR-Cas9 for tumor-infiltrating lymphocyte-based adoptive T cell therapy. Mol Ther Oncolytics. 2022 Jan 10;24:417–428.Ethics Approval The Danish Medicines Agency and The Danish National Committee on Health Research Ethics approve the study. All participants gave oral and written informed consent before taking part 2023-510417-25-01 (EU Trial (CTIS) Number).