Lymphocytes from patients with
melanoma have been used to clone
melanoma associated
antigens which are, for the most part, nonmutated melanocyte tissue
differentiation antigens. To establish a mouse model for the use of these '
self' antigens as targets for anti-
tumor immune responses, we have employed the mouse homologues of the human
melanoma antigens Tyrosinase,
Tyrosinase Related Protein-1 (TRP-1), gp100, and MART-1. We sought to generate
antisera against these
proteins for use in the construction of experimental recombinant and synthetic anti-
cancer vaccines, and for use in
biologic studies. Using genes cloned from the B16 mouse
melanoma or from murine melanocytes, we immunized rabbits with plasmid DNAs coated onto microscopic
gold beads that were then delivered using a hand-held,
helium-driven 'gene gun'. This strategy enabled us to generate polyclonal rabbit sera containing
antibodies that specifically recognized each
antigen, as measured by immunostaining of vaccinia virus infected cells. The sera that we generated specifically for
TRP-1, gp100, and MART-1 recognized extracts of the spontaneous murine
melanoma, B16. The identities of the recognized
proteins was confirmed by Western blot analysis. The titers and specificities of these
antisera were determined using ELISA. Interestingly, serum samples generated against murine MART-1 and gp100 developed
antibodies that were cross-reactive with the corresponding human homologues. Recognition of human gp100 and murine
Tyrosinase appeared to be dependent upon conformational
epitopes since specificity was lost upon denaturation of the
antigens. These
antisera may be useful in the detection, purification and characterization of the mouse homologues of recently cloned human
tumor associated
antigens and may enable the establishment of an animal model of the immune consequences of vaccination against '
self antigens.