Identification of a new shared HLA-A2.1 restricted epitope from the melanoma antigen tyrosinase

Tyrosinase has many advantages as a target antigen for the immunotherapy of patients with melanoma because it is expressed in nearly all melanoma spec imens with a high degree of cellular homogeneity, and its distribution in n ormal tissues is limited to melanocytes. To broaden our ability to direct c ellular immune responses against this protein, we pursued an investigation to identify new shared human leukocyte antigen (HLA)A2.1 restricted epitope s from tyrosinase. Peptides were synthesized that fit a permissive HLA-A2.1 binding motif and did not span common sites of polymorphism. The binding a ffinity of each peptide to HLA-A2.1 relative to a standard peptide with int ermediate binding affinity was evaluated in a competitive inhibition assay. Twelve peptides were selected that had binding affinities within 80% of th at of the standard peptide, and these were used to stimulate peripheral blo od mononuclear cells (PBMC) in vitro from three HLA-A2.1(+) patients with m etastatic melanoma. Cytotoxic T lymphocytes that specifically recognized pe ptide-pulsed target cells as well as HLA-A2.1(+) tyrosinase(+) melanoma cel ls were raised from one patient with tyrosinase:8-17 (CLLWSFQTSA). To evalu ate further the immunogenicity of this peptide, PBMC from 23 HLA-A2.1(+) pa tients were stimulated in vitro with tyrosinase:8-17, Eleven bulk T-cell cu ltures demonstrated specific peptide recognition, and six of these also rec ognized HLA-A2.1(+) tyrosinase(+) melanoma cells. These data suggest that t yrosinase:8-17 may be clinically useful for the treatment of patients with melanoma.