IMMUNOPHENOTYPING OF MELANOMAS FOR TYROSINASE - IMPLICATIONS FOR VACCINE DEVELOPMENT

Tyrosinase (EC 1.14.18.1), the key enzyme in melanin synthesis, has been shown to be one of the targets for cytotoxic T-cell recognition in melanoma patients, To develop serological reagents useful for immunophenotyping melanoma for tyrosinase, human tyrosinase cDNA was expressed in an Escherichia coli expression vector, The purified recombinant tyrosinase was used to generate mouse monocional and rabbit polyclonal antibodies. The prototype monoclonal antibody, T311, recognized a cluster of protein moieties ranging from 70 to 80 kDa in tyrosinase mRNA-positive melanoma cell lines and melanoma specimens as well as in L cells transfected with tyrosinase cDNA, Untransfected L cells and L cells transfected with tyrosinase-related protein 1, TRP-1(gp75), were nonreactive. Immunohistochemical analysis of melanomas with T311 showed tyrosinase in melanotic and amelanotic variants, and tyrosinase expression correlated with the presence of tyrosinase mRNA. Melanocytes in skin stained with T311, whereas other normal tissues tested were negative, The expression pattern of three melanosome-associated proteins-tyrosinase, TRP-1(gp75), and gp100-in melanoma was also compared. Tyrosinase and gp100 are expressed in a higher percentage of melanomas than TRP-1(gp75), and the expression of these three antigens was discordant. Tyrosinase expression within individual tumor specimen is usually homogenous, distinctly different from the commonly observed heterogenous pattern of gp100 expression.