Development of a polynucleotide vaccine from melanoma antigen recognized by T cells-1 and recombinant protein from melanoma antigen recognized by T cells-1 for melanoma vaccine clinical trials

MART-1, a melanoma antigen recognized by T cells-1, is a melanocyte lineage -differentiation antigen expressed only in melanocytes and melanoma cells. This protein is recognized by many T-lymphocyte lines that are human leukoc yte antigen (HLA)-A2 restricted and melanoma reactive. These observations h ave culminated in an array of clinical trials of MART-1 immunization using recombinant viruses or MART-1 immunodominant peptides. Polynucleotide immun ization is a promising alternative to recombinant viral vaccines that allow s delivery of the full-length cDNA encoding all potential peptide epitopes in a vector that is uncompromised by anti-viral immunity. In preparation fo r a phase I clinical trial of MART-1 polynucleotide immunization in patient s with resected melanoma who were at significant risk for recurrence, the a uthors constructed a plasmid DNA encoding the MART-1 cDNA under transcripti onal regulatory control of the cytomegalovirus immediate early promoter-enh ancer and partially deleted intron A. This plasmid directs high-level MART- 1 expression in transduced myoblasts and maturing myocytes diffusely throug hout the cytoplasm. Immunization of mice with this construct by intramuscul ar injection elicited MART-1-specific immune responses in all animals. Prev ious trials of MART-1 immunization have been unable to examine the humoral immune response to MART-1 because of a lack of sufficient, highly purified protein. We have produced and purified Escherichia coli recombinant MART-1 protein using a glutathione-S-transferase fusion protein expression system. Protein staining of a sodium dodecyl sulfate polyacrylamide gel electropho resis revealed a band of MART-1 protein at approximately 20 kD; and Western immunoblotting with an anti-MART-1 monoclonal antibody confirmed a doublet at approximately 20 kD. These findings are consistent with previous report s using different expression systems for recombinant MART-1. This protein p reparation functioned well in enzyme-linked immunosorbent assays (ELISAs) t o detect anti-MART-1 antibody responses in a mouse model; and a panel of he althy donor human sera showed minimal binding to ELISA plates coated with t he protein, supporting its utility in monitoring human anti-MART-1 antibody responses. The glutathione-S-transferase fusion method yielded approximate ly 200 mu g MART-1 per 2-L bacterial culture, enough to coat 100 ELISA plat es.