The investigators overall objective is to identify tumor-specific determinants that elicit MHC restricted CTL responses. To test this hypothesis he will follow two complementary approaches.
His specific aims are: 1) to clone and sequence the tumor- specific antigen(s) in ovarian carcinoma: (a) to construct on a cosmid vector a genomic DNA library from an appropriate ovarian carcinoma tumor cell clone expressing tumor-specific antigen(s); (b) to express the cosmid library into appropriate recipient cells; (c) to identify recipient cells expressing tumor- specific antigen(s) by their ability to stimulate autologous tumor specific TIL T cell clones to proliferate and/or produce cytokines and to confirm such expression using a cytotoxic assay; (d) to clone and sequence the tumor- specific antigen(s) from the recipient cells; (e) to employ a retroviral cDNA library approach, in the event that the cosmid library/transfection method does not allow for expression of tumor-specific antigen(s); and (f) to identify the peptides of the tumor- specific antigens recognized in association with MHC by autologous tumor- specific CTL. 2) To isolate and identify tumor- specific peptides from purified HLA class I from ovarian carcinoma tumor cell lines: (a) to isolate HLA-bound peptides from purified HLA class I from ovarian tumor cell lines, susceptible to lysis by MHC-restricted ovarian tumor-specific CTL lines/clones; (b) to determine the amino acid sequence of these peptides and whether they have been derived from known self proteins; (c) to determine if these peptides are tumor-specific by determining whether they render target cells, expressing the appropriate class I, susceptible to lysis by MHC-restricted ovarian tumor-specific CTL; and (d) in the event that the sequences of these tumor-specific peptides do not match to the tumor- specific antigen sequences obtained from the DNA cloning, to synthesize oligodeoxynucleotides corresponding to the sequence of the peptides and to clone the tumor-specific antigen(s) from an appropriate library.
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