Use of an in vitro immunoselected tumor line to identify shared melanoma antigens recognized by HLA-A*0201-restricted T cells

An immunoselected melanoma cell line that had lost expression of the domina nt melanoma antigens MART-1 and gp100 was generated in an attempt to identi fy previously unknown tumor antigens, After repeated stimulation with the a utologous immunoselected tumor line, a number of HLA-A*0201-restricted T-ce ll clones were established from the peripheral blood of a single melanoma p atient. One T-cell clone (C-22) recognized 14 of 16 HLA-A2(+) melanoma cell lines, as well as HLA-A2(+) melanocytes but recognized neither HLA-A2(+) f ibroblasts nor autologous a cells. Screening of an autologous cDNA library resulted in the isolation of a transcript identical to an entry in the expr essed sequence tag database. Northern blot analysis revealed that this gene was expressed in most melanoma cell lines and melanocytes but not in norma l tissues. The peptide epitope (AMFGREFCYA) recognized by clone C-22 was id entified based on studies of the recognition of truncated cDNAs and the use of the consensus HLA-A*0201 binding motif, A second T-cell clone (C-29) wa s found to recognize a new tyrosinase-related protein 2 epitope (455-363; Y AIDLPVSV) in an HLA-A*0201-restricted manner. Together, these results provi de additional targets that can be used for the development of immunotherape utic protocols in HLA-A2(+) melanoma patients and demonstrate the utility o f immunoselected tumor lines for the identification of new melanoma antigen s.