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
T. Flad et al., 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, CANCER RES, 58(24), 1998, pp. 5803-5811
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.