The broad long-term objective is to determine the genetic origins of unique tumor antigens, which can serve as highly effective targets for tumor rejection. In particular, the goal is to determine whether these unique antigens are encoded by tumor-specific mutations or by normal genes that are present but silent in healthy cells, and whether they are products of known mutant oncogenes or suppressor genes. The first two specific Aims are to determine the genetic origins of a unique CD4+ T cell-recognized antigen and a unique CD8+ T cell-recognized antigen from UV-induced murine tumors.
The third Aim i s to determine the genetic origin of a unique monoclonal antibody-defined antigen expressed by a spontaneous murine tumor. For the first Aim, our uniquely tumor-specific CD4+ T cell hybridoma which produces IL-2 on interaction with specific antigen will be used to screen HPLC fractions and thus to purify the antigen. Alternatively, we will screen a cDNA library that produces peptides fused to protein A for positive bacterial colonies producing the antigen, again using antigen-specific IL-2 release from the hybridoma as indicator. For the second Aim, an antigen that elicits a highly restricted CTL response will be studied with """"""""subtractive"""""""" HPLC mass spectroscopy using the parental tumor and two closely related tumor cell variants: an H-2 K molecule-negative variant to establish the signal to noise ratio and a K molecule-positive, antigen-negative variant to determine which peptides are irrelevant in the sensitizing HPLC fraction. This will be done by comparing the profiles of peptides that have been """"""""fingerprinted"""""""" by unique mass to charge ratio. This subtractive approach should require relatively few peptides to be sequenced and synthesized in order to determine which have sensitizing activity. For the third Aim, specifically immunoprecipitated antigen will be separated by SDS-PAGE electrophoresis, and partially sequenced. Sequences will be entered into GENBANK to search for identity with known sequences or will be used to design genetic probes for cloning the relevant gene. While the unique antigens recognized by CD4+ or by CD8+ T cells have different but important roles in tumor rejection, determining the genetic origin of an antibody-recognized antigen on a spontaneous tumor is important because this spontaneous tumor may represent a model for common human tumors. Resolving the molecular origins of unique antigens on such tumors may suggest new approaches to make these antigens targets for tumor destruction.
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