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Determination of Cellularly Processed HLA-A2402-Restricted Novel CTL Epitopes Derived from Two Cancer Germ Line Genes, MAGE-A4 and SAGE

Authors' Affiliations: ¹ Second Department of Internal Medicine and ² Department of Otorhinolaryngology, Mie University School of Medicine, Mie, Japan; ³ Department of Hematology, Fujita Health University, School of Medicine, Aichi, Japan; ⁴ Departement-d'Immunologie, Unite d'Immunite Cellulaire Antivirale, Institute Pasteur, Paris, France; and ⁵ Ludwig Institute for Cancer Research, Brussels Branch, and Cellular Genetics Unit, Universite Catholique de Louvain, Brussels, Belgium

Requests for reprints: Hiroshi Shiku, Second Department of Internal Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie, Japan 514-8507. Phone: 81-59-231-5016; Fax: 81-59-231-5200; E-mail: shiku{at}clin.medic.mie-u.ac.jp.

Purpose: For identification of CTL epitopes useful for cancer vaccines, it is crucial to determine whether cognate epitopes are presented on the cell surface of target cancer cells through natural processing of endogenous proteins. For this purpose, we tried to use the cellular machinery of both mice and human to define naturally processed CTL epitopes derived from two "cancer germ line" genes, MAGE-A4 and SAGE.

Experimental Design: We vaccinated newly produced HLA-A2402 transgenic mice with DNA plasmids encoding target antigens. Following screening of synthesized peptides by splenic CD8⁺ T cells of vaccinated mice, we selected candidate epitopes bound to HLA-A2402. We then examined whether human CD8⁺ T cells sensitized with autologous CD4⁺ PHA blasts transduced by mRNA for the cognate antigens could react with these selected peptides in an HLA-A2402-restricted manner.

Results: After DNA vaccination, murine CD8⁺ T cells recognizing MAGE-A4_143-151 or SAGE_715-723 in an HLA-A2402-restricted manner became detectable. Human CTLs specific for these two peptides were generated after sensitization of HLA-A2402-positive CD8⁺ T cells with autologous CD4⁺ PHA blasts transduced with respective mRNA. CTL clones were cytotoxic toward tumor cell lines expressing HLA-A2402 and cognate genes. Taken together, these CTL epitopes defined in HLA-A24 transgenic mice are also processed and expressed with HLA-A2402 in human cells. The presence of SAGE_715-723-specific precursors was observed in HLA-A2402-positive healthy individuals.

Conclusions: Two novel HLA-A2402-restricted CTL epitopes, MAGE-A4_143-151 and SAGE_715-723, were identified. Our approach assisted by cellular machinery of both mice and human could be widely applicable to identify naturally processed CTL epitopes.

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