Background: Most patients (pts) with advanced (unresectable or metastatic) melanoma receiving front-line immune checkpoint inhibitor (ICI) therapy progress within a year (Robert Lancet Oncol 2019; Larkin NEJM 2019; Tawbi NEJM 2022). Early-line therapies are needed to improve the rate of deep and durable responses and increase the proportion of pts with long-term benefit. Lifileucel demonstrated an ORR of 31.4% and median DOR not reached (median 36.5 mo follow-up) in pts with post-ICI advanced melanoma (Sarnaik SITC 2022). Earlier-line treatment with lifileucel plus pembrolizumab (pembro) in pts with ICI-naïve advanced melanoma demonstrated an ORR of 67%, including a CR rate of 25% (Iovance Press Release, April 5, 2022; O’Malley JITC 2021). TILVANCE-301 will evaluate the efficacy and safety of lifileucel plus pembro compared with pembro alone in pts with untreated advanced melanoma. Methods: TILVANCE-301 (NCT05727904) is a phase 3, multicenter, randomized, open-label, parallel group, treatment study that will randomize ~670 pts (1:1) to either Arm A: lifileucel plus pembro (study intervention includes tumor tissue resection, pembro, nonmyeloablative lymphodepletion [NMA-LD], lifileucel infusion, an abbreviated course of high-dose IL-2, and thereafter, continued pembro) or Arm B: pembro alone. Pts in Arm B who receive pembro and experience confirmed progressive disease verified by blinded independent review committee (BIRC) have the option to receive lifileucel as the immediate next line of treatment. Eligible adults have histologically confirmed advanced melanoma (Stage IIIC, IIID, or IV); ECOG PS of 0 or 1; estimated life expectancy > 6 mo; ≥1 resectable lesion ~1.5 cm in diameter postresection to generate lifileucel and ≥1 measurable lesion (RECIST v1.1); and adequate hematologic parameters and organ function. Neoadjuvant or adjuvant treatment including ICI meeting protocol-specified criteria may be allowed. Exclusion criteria include prior therapy for metastatic disease; symptomatic untreated brain metastases; organ allograft or prior cell transfer therapy; uveal/ocular melanoma; chronic systemic steroid therapy; active systemic infections; cardiovascular, respiratory, or immune system illnesses; primary/acquired immunodeficiency; or other primary malignancy in the last 3 y. The dual primary efficacy endpoints are BIRC-assessed (RECIST v1.1) ORR and PFS. Key secondary efficacy endpoint is OS. Additional secondary efficacy endpoints include BIRC-assessed CR rate, DOR, and EFS; investigator-assessed ORR, PFS, CR rate, DOR, EFS, and PFS2; and safety as characterized by severity and seriousness of TEAEs, and relationship to study drug. The study will enroll globally. Clinical trial information: NCT05727904.