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
Sw. Lee et al., 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, J IMMUNOTH, 23(3), 2000, pp. 379-386
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.