NON-SELF-DISCRIMINATION AS A DRIVING CONCEPT IN THE IDENTIFICATION OF AN IMMUNODOMINANT HMW-MAA EPITOPIC PEPTIDE SEQUENCE BY AUTOANTIBODIES FROM MELANOMA CANCER PATIENTS Reinhard DUMMER 1 , Abraham MITTELMAN 2 , Francesco P. FANIZZI 3 , Guglielmo LUCCHESE 4 , Jo ¨rg WILLERS 1 and Darja KANDUC 4 * 1 Department of Dermatology, University Hospital Zurich, Zurich, Switzerland 2 Department of Medicine/Oncology, New York Medical College, Valhalla, NY, USA 3 NMR Unit, CARSO Cancer Research Center, Valenzano, Italy 4 Department of Biochemistry and Molecular Biology, University of Bari, Bari, Italy We analyzed the sera of patients with melanoma to define the human humoral autoantibody profile towards HMW- MAA. Computational proteome scanning using the non-self- discrimination principle as a guide led to the individuation of the low-similarity HMW-MAA 781–789 RATVWMLRL peptide fragment as an immunodominant B-cell epitope. Linear B- cell determinant individuation was experimentally validated by dot blot immunoassay and NMR spectroscopy analysis. Regulation of physiologic self-reactivity by the non-self-dis- crimination principle is discussed. © 2004 Wiley-Liss, Inc. Key words: melanoma; antigenic epitope; immunogenic peptide; au- toantibody; non-self-discrimination The vast majority of melanomas develop from cutaneous intra- epidermal melanocytes either in normal skin or within melanocytic nevi and progress through radial and vertical growth phases. Cu- taneous melanoma accounts for about 10% of skin cancer cases but 75% of skin cancer deaths. The incidence of cutaneous melanoma is rising steadily worldwide, and the median survival of patients with distant metastases remains 1 year. 1,2 Adolescent cases are on the rise too: the incidence of melanoma increased by 85% among 15- to 19-year-olds from 1973 to 1996. 3 This situation demands efforts to develop new biologic thera- pies. 4 A promising approach is the use of specific immunotherapy with peptide-based vaccine strategies. Conditio sine qua non to this goal is the identification of immunogenic tumor antigens, including antigenic peptides able to evoke humoral/cellular re- sponses. 5–7 Melanoma presents a heterogeneous pattern of various tumor markers, including melanocytic antigens and cancer testis anti- gens. 8 Among them, HMW-MAA 9 is a melanoma marker of particular interest since (i) it is highly expressed at the surface of tumor cells, (ii) it has restricted distribution in normal tissues 10 –12 and (iii) the induction of specific humoral response to anti-idio- typic anti-HMW-MAA MAb increases survival in patients with advanced melanoma. 13,14 Given these premises, identification of the epitopic sequences of this proteoglycan MAA is of great interest for obtaining specific antibodies to activate the destruction of melanoma cells. However, the dimensions of the HMW-MAA molecule do not allow the definition of HMW-MAA conformational determinants and ham- per the exact individuation of the immunogenic linear peptide portions of this antigen through the usual epitope mapping meth- odology. We have used computational biology to exploring TAA epitopes recognized by MAbs or polyclonal antibodies. 15–18 By measuring the antigenic peptide similarity to the host’s proteome, we dem- onstrated that a low level of similarity is a necessary condition for a peptide to be immunogenic. Consequently, based on the assump- tion that antigenic motifs that are scarcely represented in human proteins may offer epitopic determinants unknown to the host, we searched for immunogenic epitopes of the human melanoma- associated chondroitin sulfate proteoglycan by, first, selecting for portions of the HMW-MAA not shared with the human proteome and, then, analyzing the low-similarity peptide reactivity pattern using sera from 26 melanoma patients. MATERIAL AND METHODS Computational analyses Similarity analysis was carried out on the human HMW-MAA (or MCSP) sequence (GenBank accession AAQ62842). 9 Analyses of sequence similarity to the human proteome were conducted using the PIR nonredundant reference protein database and peptide match program (http://pir.georgetown.edu/pirwww). 19 Peptides The synthetic peptides used in dot immunoassays are described in Table I. The 15 N-labeled peptides used in NMR spectroscopic anal- yses corresponded to the HMW-MAA 780 –789 QRATVWMLRL, HMW-MAA 1418 –1427 SAFSWRMVEE and HMW-MAA 1666 –1675 PFWEAHDTLE (with 15 N-labeled amino acid residues underlined) sequences. Chemical shift assignment in NMR spectroscopy experi- ments was controlled utilizing the following peptides (with 15 N- labeled amino acid residues underlined): HMW-MAA 780 –789 QRATVWMLRL and HMW-MAA 780 –789 QRATVWMLRL, HMW- MAA 1418 –1427 SAFSWRMVEE and HMW-MAA 1418-1427 SAF- SWRMVEE, HMW-MAA 1666 –1675 PFWEAHDTLE and HMW- MAA 1666-1675 PFWEAHDTLE. Peptides were synthesized by standard Fmoc solid-phase pep- tide synthesis (Primm, Milan, Italy). Peptide purity was 95%, as assessed by HPLC, and the molecular mass of purified peptides was confirmed by fast atom bombardment mass spectrometry. Peptides were dissolved in 0.9% NaCl, aliquoted and stored at –20°C. Abbreviations: Fmoc, N-(9-fluorenyl)methoxycarbonyl; HMW-MAA, high m.w. melanoma-associated antigen; HPR, horseradish peroxidase; MAb, monoclonal antibody; MCSP, melanoma chondroitin sulfate proteo- glycan; NMR, nuclear magnetic resonance; PBST, PBS/0.05% (v/v) Tween-20; PIR, Protein Information Resource; RT, room temperature; TAA, tumor-associated antigen; TSP, tetrasilyl propionate. Grant sponsor: European Community; Grant sponsor: Zalmin A. Arlin Cancer Fund; Grant sponsor: Swiss National Science Foundation; Grant number: 3100-055727.98/1; Grant sponsor: Gottfried and Julia Bangerter- Rhyner-Stiftung. *Correspondence to: Department of Biochemistry and Molecular Biol- ogy, University of Bari, Via Orabona 4, Bari 70125, Italy. Fax: +39-080-544-3317. E-mail: d.kanduc@biologia.uniba.it Received 19 January 2004 ; Revised 17 February 2004; Accepted 19 February 2004 DOI 10.1002/ijc.20310 Published online 20 May 2004 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 111, 720 –726 (2004) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer