The Journal of Experimental Medicine
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Published online 2 January 2001.
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© The Rockefeller University Press, 0022-1007/2001/1/73/ $5.00
The Journal of Experimental Medicine, Volume 193, Number 1, January 1, 2001 73-88

Original Article

Efficient Identification of Novel HLA-A*0201–presented Cytotoxic T Lymphocyte Epitopes in the Widely Expressed Tumor Antigen PRAME by Proteasome-mediated Digestion Analysis

Jan H. Kesslera, Nico J. Beekmana, Sandra A. Bres-Vloemansa, Pauline Verdijka, Peter A. van Veelena, Antoinette M. Kloosterman-Joostena, Debby C.J. Vissersa, George J.A. ten Boscha, Michel G.D. Kestera, Alice Sijtsb, Jan Wouter Drijfhouta, Ferry Ossendorpa, Rienk Offringaa, and Cornelis J.M. Meliefa
a Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
b Institute of Biochemistry, Charité, Humboldt University, D-10117 Berlin, Germany

Correspondence to: Jan H. Kessler, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Building 1: E3-Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Tel:31-71-526-1671 Fax:31-71-521-6751 E-mail:kesslerj{at}worldonline.nl.

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, PRA100–108; SLYSFPEPEA, PRA142–151; ALYVDSLFFL, PRA300–309; and SLLQHLIGL, PRA425–433) 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.

Key Words: antigen presentation, antigen processing, cytotoxic T lymphocyte induction, human histocompatibility leukocyte antigen class I binding, tumor immunotherapy


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