Direct identification of major histocompatibility complex class I-bound tumor-associated peptide antigens of a renal carcinoma cell line by a novel mass spectrometric method

Melanoma and renal cell carcinoma (RCC) are thought to be the most immunoge nic human tumors. Presently a series of tumor-specific peptides of melanoma is being tested in clinical trials with different immunotherapy protocols. In contrast, only one decameric peptide (SPSSNRIRNT) derived from one (ORF 2) of three possible open reading frames (ORFs) of a gene named RICE (Renal tumor AntiGEn) was shown to be the target for tumor-specific CTLs on renal carcinoma cells. One reason for the lack of identification of tumor antige ns on RCC compared with melanoma may be the difficulty in generating tumor- specific CTLs as screening instruments. Therefore, our approach was directl y to isolate and identify peptides bound to HLA class I molecules of the HL A-A2 and -B8 homozygous RCC line A-498. High performance liquid chromatogra phy-fractionated peptides fluted with acid from immunoaffinity-purified HLA class I-peptide complexes were sequenced and identified for the first time by the novel and highly sensitive mass spectrometric method matrix-assiste d laser desorption ionization-post source decay (MALDI-PSD) from minute amo unts of 100 fmol to 1.5 pmol of the fractionated peptide samples. Fourteen peptide sequences first deduced from interpretations of the mass spectra we re also shown to fulfill other reliability criteria such as matching the ma ss spectra of the respective synthetic peptides, Some peptides were identif ied to be derived from genes preferentially activated in malignant tissues or resulted from a possibly mutated gene. The most promising candidate for a CTL epitope is a decameric peptide (PASKKTD-PQK) derived from another pos sible ORF (ORF5) of the RAGE gene and probably presented in association wit h HLA-BS. This peptide was synthesized and used for the in vitro induction of CTLs that lysed the A-498 cells and another HLA-B8-positive RCC line sig nificantly more strongly than either other RAGE-positive but HLA-BS-negativ e RCC lines or K562 cells. Sensitive sequencing by MALDI-PSD thus may provi de a powerful method of identifying potentially tumor-specific and HLA-rest ricted antigens, even an native malignant cells and tissues.