Abstract New treatment approaches are warranted for patients with advanced melanoma refractory to immune checkpoint blockade (ICB) or BRAF-targeted therapy. We designed BNT221, a personalized, neoantigen-specific autologous T cell product derived from peripheral blood, and tested this in a 3 + 3 dose-finding study with two dose levels (DLs) in patients with locally advanced or metastatic melanoma, disease progression after ICB, measurable disease (Response Evaluation Criteria in Solid Tumors version 1.1) and, where appropriate, BRAF-targeted therapy. Primary and secondary objectives were evaluation of safety, highest tolerated dose and anti-tumor activity. We report here the non-pre-specified, final results of the completed monotherapy arm consisting of nine patients: three at DL1 (1 × 10 8 –1 × 10 9 cells) and six at DL2 (2 × 10 9 –1 × 10 10 cells). Drug products (DPs) were generated for all enrolled patients. BNT221 was well tolerated across both DLs, with no dose-limiting toxicities of grade 3 or higher attributed to the T cell product observed. Specifically, no cytokine release, immune effector cell-associated neurotoxicity or macrophage activation syndromes were reported. A dose of 5.0 × 10 8 –1.0 × 10 10 cells was identified for further study conduct. Six patients showed stable disease as best overall response, and tumor reductions (≤20%) were reported for four of these patients. In exploratory analyses, multiple mutant-specific CD4 + and CD8 + T cell responses were generated in each DP. These were cytotoxic, polyfunctional and expressed T cell receptors with broad functional avidities. Neoantigen-specific clonotypes were detected after treatment in blood and tumor. Our results provide key insights into this neoantigen-specific adoptive T cell therapy and demonstrate proof of concept for this new therapeutic approach. ClinicalTrials.gov registration: NCT04625205 .