Chemically induced tumors of inbred rodents display individually distinct antigens, which elicit specific tumor rejection in syngeneic hosts. It has been difficult to obtain serological probes for these antigens and their biochemical purification has not been possible due to the cumbersome transplantation assays. Recently, a 96,000 dalton cell surface glycoprotein, gp96, has been isolated from two antigenically distinct BALB/c mouse sarcomas, CMS5 and Meth A. Purified gp96 molecules elicit rejection of the tumor from which they are derived, but not of another tumor. Hyperimmunized rabbit sera to Meth A gp96 identify gp96 molecules in Meth A as well as CMS5 and other tumors and normal cells. Amino terminal sequencing of gp96 from Meth A and CMS5 shows and identical stretch of 14 amino acid residues. On basis of these results, it is postulated that individual antigens of chemically induced tumors are products of a single hypermutable gene or a large polymorphic gp96 family.
The aim of the present proposal is to translate these results from the realm of tumor transplantation tests and protein biochemistry to an understanding of the molecular basis of diversity of gp96 antigens in chemically induced mouse sarcomas. Specifically, the objectives of the present proposal are: 1. Obtain amino terminal and internal amino acid sequences of the gp96 molecules; use the amino acid sequence to derive an oligonucleotide probe to isolate cDNA and genomic clones containing the genes encoding these molecules. 2. Construct cDNA libraries in expression vectors and screen them with the rabbit anti-gp96 antiserum. 3. Sequence genomic and cDNA clones encoding gp96 from various tumors and normal cells to understand the molecular basis of diversity of these antigens. 4. Express the genes encoding gp96 in expression vectors and demonstrate tumor rejection activity of the expressed product. 5. Construct synthetic peptides spanning the variable regions of gp96 molecules and use them in tumor transplantation tests in order to define the immunogenic portions of these molecules.
