GENERATION OF POLYCLONAL RABBIT ANTISERA TO MOUSE MELANOMA-ASSOCIATEDANTIGENS USING GENE GUN IMMUNIZATION

Lymphocytes from patients with melanoma have been used to clone melano ma associated antigens which are, for the most part, nonmutated melano cyte tissue differentiation antigens. To establish a mouse model for t he use of these 'self' antigens as targets for anti-tumor immune respo nses, we have employed the mouse homologues of the human melanoma anti gens Tyrosinase, Tyrosinase Related Protein-1 (TRP-1), gp100, and MART -1. We sought to generate antisera against these proteins for use in t he 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 w ith plasmid DNAs coated onto microscopic gold beads that were then del ivered using a hand-held, helium-driven 'gene gun'. This strategy enab led us to generate polyclonal rabbit sera containing antibodies that s pecifically 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 m urine melanoma, B16. The identities of the recognized proteins was con firmed by Western blot analysis. The titers and specificities of these antisera were determined using ELISA. Interestingly, serum samples ge nerated against murine MART-1 and gp100 developed antibodies that were cross-reactive with the corresponding human homologues. Recognition o f human gp100 and murine Tyrosinase appeared to be dependent upon conf ormational epitopes since specificity was lost upon denaturation of th e antigens. These antisera may be useful in the detection, purificatio n and characterization of the mouse homologues of recently cloned huma n tumor associated antigens and may enable the establishment of an ani mal model of the immune consequences of vaccination against 'self' ant igens. (C) 1998 Elsevier Science B.V. All rights reserved.