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Immune responses contribute to the pathogenesis of vitiligo and target melanoma sometimes associated with vitiligo-like depigmentation in some melanoma patients. We analyzed the sera from patients with vitiligo and cutaneous melanoma for reactivity toward tyrosinase peptide sequences 1) endowed with low level of similarity to human proteome, and 2) potentially able to bind HLA-DR1 Ags. We report that the tyrosinase autoantigen was immunorecognized with the same molecular pattern by sera from vitiligo and melanoma patients. Five autoantigen peptides composed the immunodominant anti-tyrosinase response: aa95-104FMGFNCGNCK; aa175-182 LFVWMHYY; aa176-190FVWMHYYVSMDALLG; aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC. All of the five antigenic peptides were characterized by being (or containing) a sequence with low similarity level to the self proteome. Sera from healthy subjects were responsive to aa95-104FMGFNCGNCK, aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC, but did not react with the aa175-182LFVWMHYY and aa176-190FVWMHYYVSMDALLG peptide sequences containing the copper-binding His180 and the oculocutaneous albinism I-A variant position F176. Our results indicate a clear-cut link between peptide immunogenicity and low similarity level of the corresponding amino acid sequence, and are an example of a comparative analysis that might allow to comprehensively distinguish the epitopic peptide sequences within a disease from those associated to natural autoantibodies. In particular, these data, for the first time, delineate the linear B epitope pattern on tyrosinase autoantigen and provide definitive evidence of humoral immune responses against tyrosinase.

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-MAA781-789RATVWMLRL 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-discrimination principle is discussed., (Copyright 2004 Wiley-Liss, Inc.)

Willers J, Lucchese A, Mittelman A, Dummer R, Kanduc D. Definition of anti-tyrosinase MAb T311 linear determinant by proteome-based similarity analysis. Using non-self discrimination as a driving force in generating peptide immunogenicity, we have developed a computer-assisted proteomic analysis in order to identify the protein antigenic regions that have evoked humoral response. The purpose of this study was to further validate the computational analysis for melanoma-associated antigens and, at the same time, to assess the efficacy of the methodology in defining antigenic regions of autoantigens associated to autoimmune diseases. To achieve this two-fold objective, we have examined the enzyme tyrosinase, a protein that represents an important autoantigen in patients with vitiligo or melanoma. Here, we report that the antigenic linear determinant of the monoclonal antibody (Mab) T311 raised against the melanoma/vitiligo tyrosinase autoantigen is located in the low similarity 15-mer amino acid sequence tyrosinase 233–247 IPYWDWRDAEKCDIC , within the fragment 237–247. These data confirm non-similarity to the host proteome as a factor that participates in shaping peptide immune reactivity and may be a first step towards designing tyrosinase antigenic peptides to be used for (i) direct neutralization of harmful melanocytes-attacking autoantibodies in vitiligo, or (ii) production of antibodies against tyrosinase-positive melanomas. Moreover tyrosinase peptide antigens might be used as key tools in studying the boundaries between self-tolerance and autoimmunity phenomena. [ABSTRACT FROM AUTHOR]

An efficient strategy is presented for the identification of antigenic sequences in the context of given MHC molecules of interest. The proteomic analysis of the antigenic peptide repertoire is described and demonstrated by using high-molecular weight melanoma-associated antigen. The identification of the epitopic sequence of a monoclonal antibody raised against the 250 kDa tumor associated antigen was reached by using only seven short synthetic peptide fragments, instead of the 155 non-overlapping 15-mer peptides theoretically necessary as minimum screening library. The present result has been obtained by applying as driving criteria the analysis of the peptide affinity to MHC class II molecules and the non-self discrimination concept. [ABSTRACT FROM AUTHOR]

Following a discussion of the rationale underlying the selection of human melanoma to test the usefulness of anti-idiotypic monoclonal antibodies in the therapy of solid tumors, the development of the anti-idiotypic monoclonal antibody MoAb) MF11-30 is described. This antibody recognizes a private idiotope within the antigen-combining site of the immunizing antihuman high molecular weight melanoma-associated antigen MoAb 225.28. The results of a phase I clinical trial with the MoAb MF11-30 in patients with advanced melanoma are described. The lack of toxic effects and the minor responses in six patients suggest that these studies should be extended to a larger number of patients with an emphasis on the analysis of the mechanisms underlying the clinical response. [ABSTRACT FROM AUTHOR]