PURPOSE: Generation of antigen-specific T cells from cancer patients employs large numbers of peripheral blood cells and/or tumor infiltrating cells to generate antigen-presenting and effector cells commonly requiring multiple rounds of re-stimulation ex vivo We used a novel paramagnetic, nanoparticle-based artificial antigen presenting cell (nano-aAPC) that combines anti-CD28 co-stimulatory and human MHC class I molecules that are loaded with antigenic peptides to rapidly expand tumor antigen-specific T cells from melanoma patients. EXPERIMENTAL DESIGN: Nano-aAPC expressing HLA-A*0201 molecules and costimulatory anti-CD28 antibody and loaded with MART-1 or gp100 class I restricted peptides were used to stimulate CD8 T cells purified from the peripheral blood of treatment-naive or PD-1 antibody-treated patients with stage IV melanoma. Expanded cells were re-stimulated with fresh peptide-pulsed nano-aAPC at day 7. Phenotype analysis and functional assays including cytokine release, cytolysis, and measurement of avidity were conducted. RESULTS: MART-1-specific CD8 T cells rapidly expanded up to 1000-fold by day 14 after exposure to peptide-pulsed nano-aAPC. Expanded T cells had a predominantly stem cell memory CD45RA(+)/CD62L(+)/CD95(+)phenotype, expressed ICOS, PD-1, Tim3, and LAG3 and lacked CD28. Cells from patients with melanoma were polyfunctional, highly avid, expressed IL-2, IFN-gamma, TNF-alpha and exhibited cytolytic activity against tumor cell lines. They expanded 2-3-fold after exposure to PD-1 antibody in vivo, and expressed a highly diverse TCR V beta repertoire. CONCLUSIONS: Peptide-pulsed nanoparticle aAPC rapidly expanded polyfunctional antigen-specific CD8 T cells with high avidity, potent lytic function and a stem-memory phenotype from melanoma patients.