T cell receptor repertoire dynamics and newly identified functional regulators of autologous tumor-infiltrating lymphocyte therapy in advanced solid tumors.

Tumor-infiltrating lymphocyte (TIL) therapy has demonstrated clinical potential in melanoma; however, its applicability across diverse solid tumors in Asian patients remains unclear. Identification of molecular targets to enhance the antitumor activity of TIL is urgently needed. In a phase I investigator-initiated trial, we evaluated the safety, feasibility, and preliminary efficacy of autologous TIL therapy in twelve patients with advanced melanoma, cervical, lung, or head and neck cancers. Twelve patients who had progressed after standard therapies were enrolled between August 2022 and December 2024. Patients received lymphodepletion with cyclophosphamide (30 mg/kg) for 2 days, followed by fludarabine (25 mg/m²) for 5 days, approximately 24 h before intravenous infusion of autologous TIL. High-dose interleukin-2 (IL-2) was administered for 6-12 days to support T-cell survival and expansion. After a 28-day safety observation period, tumor assessments were performed every 6 weeks for the first 6 months and thereafter every 12 weeks during long-term follow-up, according to RECIST 1.1 criteria. T cell receptor (TCR) sequencing was conducted to evaluate in vivo persistence of infused TIL. Differential gene expression analysis was performed using bulk RNA sequencing on TIL from responders and non-responders. Target gene knockout in TIL was achieved using CRISPR/Cas9 technology to assess enhancement of antitumor function. The most common adverse events were fever, anemia, nausea, hypertension, and hyponatremia. The objective response rate (ORR) was 33.3% (4/12), including one complete response (CR) and three partial responses (PR), with a disease control rate (DCR) of 75% (9/12). Infused TIL persisted in peripheral blood and induced a reversal in the peripheral CD4 + T to CD8 + T cell ratio. TCR sequencing revealed dynamic clonal remodeling in responders. Transcriptomic profiling identified ASS1 and CEP20 as genes negatively associated with therapeutic efficacy. CRISPR/Cas9-mediated knockout of ASS1 or CEP20 enhanced TIL memory phenotypes, cytokine production, and tumor cytotoxicity both in vitro and in vivo. This study demonstrates the efficacy of TIL therapy in diverse solid tumors among Asian patients, reveals dynamic TCR clonal remodeling in responders, and identifies ASS1 and CEP20 knockout as strategies to potentiate antitumor activity. These findings provide mechanistic insights that may guide improvement of TIL therapy for broader clinical application. NCT, NCT05366478. Registered 01 August 2022, http://www. gov/NCT05366478 .