title: 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.
abstract: 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 leukocyte antigen (HLA)-A2 restricted and melanoma reactive. These observations have culminated in an array of clinical trials of MART-1 immunization using recombinant viruses or MART-1 immunodominant peptides. Polynucleotide immunization is a promising alternative to recombinant viral vaccines that allows delivery of the full-length cDNA encoding all potential peptide epitopes in a vector that is uncompromised by anti-viral immunity. In preparation for a phase I clinical trial of MART-1 polynucleotide immunization in patients with resected melanoma who were at significant risk for recurrence, the authors constructed a plasmid DNA encoding the MART-1 cDNA under transcriptional regulatory control of the cytomegalovirus immediate early promoter-enhancer and partially deleted intron A. This plasmid directs high-level MART-1 expression in transduced myoblasts and maturing myocytes diffusely throughout the cytoplasm. Immunization of mice with this construct by intramuscular injection elicited MART-1-specific immune responses in all animals. Previous 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 electrophoresis 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 reports using different expression systems for recombinant MART-1. This protein preparation functioned well in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MART-1 antibody responses in a mouse model; and a panel of healthy donor human sera showed minimal binding to ELISA plates coated with the protein, supporting its utility in monitoring human anti-MART-1 antibody responses. The glutathione-S-transferase fusion method yielded approximately 200 micrograms MART-1 per 2-L bacterial culture, enough to coat 100 ELISA plates.
authors.affiliation: Department of Medicine, University of Alabama at Birmingham 35294-3300, USA.
authors.forename: S W
authors.initials: SW
authors.lastname: Lee
authors.forename: H
authors.initials: H
authors.lastname: Li
authors.forename: T V
authors.initials: TV
authors.lastname: Strong
authors.forename: S E
authors.initials: SE
authors.lastname: Moore
authors.forename: R M
authors.initials: RM
authors.lastname: Conry
mesh_terms: D000818:Animals; D000917:Antibody Formation; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000302:isolation & purification*; D019496:Cancer Vaccines / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D007150:Immunohistochemistry; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D019444:Vaccines, DNA / Q000276:immunology*
chemical_list: D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D058965:MART-1 Antigen; C548667:MLANA protein, human; C548668:Mlana protein, mouse; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D019444:Vaccines, DNA