ORIGINAL ARTICLE Ashley John Knights ® Angeliki Zaniou ® Robert C. Rees Graham Pawelec ® Ludmila Mu¨ller Prediction of an HLA-DR-binding peptide derived from Wilms’ tumour 1 protein and demonstration of in vitro immunogenicity of WT1(124–138)-pulsed dendritic cells generated according to an optimised protocol Received: 22 November 2001 / Accepted: 30 January 2002 / Published online: 26 April 2002 Ó Springer-Verlag 2002 Abstract The Wilms’ tumour 1 (WT1) protein is over- expressed in several types of cancer including leukaemias and might therefore constitute a novel target for im- munotherapy. Recently, human leucocyte antigen (HLA) class I-binding WT1 peptides have been identi- fied and shown to stimulate CD8 + T cells in vitro. For maximal CD8 cell efficacy, CD4 + helper T cells re- sponding to major histocompatibility complex (MHC) class II-binding epitopes are required. Here, we report that scanning the WT1 protein sequence using an evi- dence-based predictive computer algorithm (SYFPEI- THI) yielded a peptide WT1(124–138) predicted to bind the HLA-DRB1*0401 molecule with high affinity. Moreover, synthetic WT1(124–138)-peptide-pulsed den- dritic cells (DC), generated according to a protocol optimised in the present study, sensitised T cells in vitro to proliferate and secrete interferon-c (IFN-c) when rechallenged with specific peptide-pulsed DC, but not with peripheral blood mononuclear cells (PBMC). These results suggest that the WT1 protein may yield epitopes immunogenic to CD4 as well as CD8 T cells, and therefore constitute a novel potential target for specific immunotherapy. Keywords Cancer immunotherapy ® CD4 + T cell ® Leukaemia ® MHC class II-binding peptide ® Wilms’ tumour 1 Introduction In recent years, the role played by CD4 + T helper (Th) cells, predominantly of Th1 polarisation, in anti-tumour immunity has become more apparent. Consequently, it is now widely accepted that for the optimal implemen- tation of an immunotherapeutic approach to cancer therapy, both CD4 helper and CD8 cytotoxic cells should be targeted. One such line of approach has been to sensitise these cells with synthetically produced pep- tides derived from predicted tumour epitopes. Though several groups have reported on the successful genera- tion of both CD8 + and CD4 + T cell lines with dem- onstrated specificity and on their implementation in tumour recognition [10, 22, 24, 27, 34], the majority of these studies have been conducted with major histo- compatibility complex (MHC) class I-restricted peptides and hence have targeted only CD8 + cytotoxic T lym- phocytes (CTL). In the currently available literature, the majority of CD4 + cell-based studies have utilised pep- tides derived from well-characterised tumour models, such as that described by Stassar et al. [29] and tumour- associated proteins, such as the bcr–abl fusion protein that is common in chronic myelocytic leukaemia (CML) [38]. Other potential CML targets include epitopes of the proteinase 3 antigen, a highly expressed serine protease found in a variety of leukaemic cells and used to gen- erate reactive CTL [17]; and the recently reported CML 66, a broadly immunogenic tumour antigen identified by Serex in a CML patient [37]. A further protein associ- ated with CML, as with several other leukaemias, is the Wilms’ tumour protein. This protein has been demon- strated to be highly over-expressed [3, 31, 32] in several leukaemia models, and has been reported as having a possible role in leukaemogenesis [36], and hence pro- vides another potential target for use in immunotherapy. Human leucocyte antigen (HLA) class I peptides from this protein have been utilised to sensitise in vitro CD8 + T lymphocytes that demonstrated the ability to mediate Cancer Immunol Immunother (2002) 51: 271–281 DOI 10.1007/s00262-002-0278-2 A.J. Knights and A. Zaniou contributed equally to this work A.J. Knights ® G. Pawelec (&) ® L. Mu¨ ller University of Tu¨bingen, Section for Transplantation Immunology and Immunohaematology, Second Department of Internal Medicine, Zentrum fu¨r Medizinische Forschung ZMF, Waldho¨rnlestrasse 22, 72072 Tu¨bingen, Germany E-mail: graham.pawelec@uni-tuebingen.de Tel.: +49-7071-2982805 Fax: +49-7071-295755 A. Zaniou ® R.C. Rees Department of Life Sciences, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, UK