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Identification of Peptide Superagonists for a Self-K-ras-Reactive CD4⁺ T Cell Clone Using Combinatorial Peptide Libraries and Mass Spectrometry¹

Yoshihiko Tanaka^*,, Hideki Ohyama, Michio Ogawa, Yasuharu Nishimura^* and Sho Matsushita^2,*

^* Division of Immunogenetics, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; Department of Surgery II, Kumamoto University School of Medicine, Kumamoto, Japan; and Department of Periodontology and Endodontology, Okayama University Dental School, Okayama, Japan

The proliferative responses of a human CD4⁺ T cell clone 29.15.2, reactive with a self-K-ras-derived peptide (³EYKLVVVGAGGVGKSALT²⁰), were tested using a set of X9 combinatorial peptide libraries containing the flanking residues (EYKLVXXXXXXXXXSALT, where X indicates random amino acids). Certain peptide libraries, such as EYKLVXXXXXXMXXSALT and EYKLVXXXXXXXHXSALT, stimulated a marked proliferation of 29.15.2. However, no combinations of substitutions tested, such as EYKLVXXXXXXMHXSALT, exhibited additive effects. We subsequently synthesized peptides with degenerate sequences (a mixture of 480 species), where each position is composed of the wild-type (wt) residue or of amino acids that induced the proliferation of 29.15.2, in positional scanning. Interestingly, one fraction of degenerate peptides, separated by reverse-phase HPLC, stimulated much higher proliferation than did the wt; in addition, the retention time of this fraction was distinct from that of the wt. Mass spectrometry analysis of this fraction and flanking fractions identified five peptide species that exhibit strong signals in a manner that parallels the antigenic activity. Finally, 17 candidate peptide sequences were deduced from mass spectrometry and hydrophobicity scoring results, of which two peptides (EYKLVVVGAGGMLKSALT and EYKLVVVGAGGMIKSALT) did induce 52- and 61-fold stronger proliferation, respectively, compared with the wt. These findings indicate that: 1) synthetic peptides that carry "the best" residue substitution at each position of combinatorial peptide libraries do not always exhibit superagonism, and 2) such a drawback can be overcome with the use of mass spectrometry. This approach provides new perspectives for the accurate and efficient identification of peptide superagonists.

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