THE PEPTIDE RECOGNIZED BY HLA-A68.2-RESTRICTED, SQUAMOUS-CELL CARCINOMA OF THE LUNG-SPECIFIC CYTOTOXIC T-LYMPHOCYTES IS DERIVED FROM A MUTATED ELONGATION-FACTOR-2 GENE

The identification of naturally processed tumor peptides that can stim ulate a tumor-specific, CTL response is crucial to the development of a vaccine-based, immunotherapeutic approach to cancer treatment. One t ype of cancer in which a tumor-specific, CTL response has been observe d is squamous cell carcinoma of the lung. In the system investigated h ere, the tumor-specific CTLs are HLA-A68.2 restricted. Immunoaffinity chromatography was used to isolate the HLA-A68.2 molecules from the tu mor cell line, and peptide was eluted with acid from the HLA-A68.2 mol ecules and subjected to three rounds of separation by reversed phase-h igh performance liquid chromatography (RP-HPLC). To determine which fr actions contained the peptide recognized by the tumor-specific CTLs, a n aliquot of each RP-HPLC fraction was added to the autologous, B-lymp hoblastoid cell line, and the cells were then tested as targets for tu mor-specific CTLs. After the third round of RP-HPLC, mass spectrometry was used to sequence individual peptide candidates, and a peptide wit h a m/z of 497 was identified as the active peptide. Collision-activat ed dissociation of m/z 497 allowed identification of the peptide seque nce as ETVSEQSNV. With the exception of a single amino acid difference (glutamic acid versus glutamine as the sixth position in the peptide) , this peptide is identical to residues 581 to 589 of elongation facto r 2. The PCR was used to amplify the elongation factor 2 gene in both the tumor cells and the autologous B cell line, and DNA sequencing of the products revealed the presence of a heterozygous mutation in the t umor cells that accounts for the difference between the two peptide se quences. Although a similar analysis did not reveal the presence of th e mutation in three additional lung cell carcinomas, this does not rul e out the possibility that a survey of a larger population of tumor ce lls would reveal the presence of the mutation at a low frequency. Thes e results demonstrate the utility of this approach for identifying tum or-specific antigens that are the targets of a CTL response.