Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis.
We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A(*)0201-presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved "reverse immunology" strategy. Next to motif-based HLA-A(*)0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A(*)0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA(100-108); SLYSFPEPEA, PRA(142-151); ALYVDSLFFL, PRA(300-309); and SLLQHLIGL, PRA(425-433)) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A(*)0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.
Kessler
J H
JH
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. kesslerj@worldonline.nl
Beekman
N J
NJ
Bres-Vloemans
S A
SA
Verdijk
P
P
van Veelen
P A
PA
Kloosterman-Joosten
A M
AM
Vissers
D C
DC
ten Bosch
G J
GJ
Kester
M G
MG
Sijts
A
A
Wouter Drijfhout
J
J
Ossendorp
F
F
Offringa
R
R
Melief
C J
CJ
D000595:Amino Acid Sequence; D017951:Antigen Presentation; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002461:Cell Line, Transformed; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D017931:DNA Primers / Q000235:genetics; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D015234:HLA-A Antigens / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D017931:DNA Primers; D000939:Epitopes; D015234:HLA-A Antigens; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex