Identification of HPV type 16 E7 epitopes restricted to major HLA type for generation of cervix cancer-specific cytotoxic T lymphocyte

Abstract

Cervix cancer is the second most frequent carcinoma in women worldwide. The high-risk HPV type 16 E7 is a viral oncoprotein expressed in HPV type 16-infected cervix cancer cells, is known as an inducer of cervical carcinoma by inhibition of the tumor suppressor, Rb, and maintains the transformed state and immortal growth of malignant tumors. Subsequently, HPV type 16 E7 has been considered a promising target for CTL-based adoptive immunotherapy. In spite of that, very few HPV type 16 E7-derived epitopes have been reported only in the HLA-A*0201 allele. Herein, for the identification of other major types of HLA-A alleles, HLA-A*2402 and HLA-A*3303-restricted HPV type 16 E7 epitopes, in vitro sensitization and expansion of CTLs were performed with synthetic E7 peptides, the expression of CTL activation markers and examined cytolytic activity were quantified using flow cytometry and 51Cr release assay. Based on these results, it can be suggested that CD8+ T lymphocytes stimulated with HPV E761-69 (A24 9mer peptide 1) and E767-76 (A24 10mer peptide 20) for HLA-A*2402 subjects and E750-59 (A33 10mer peptide 22) and E752-61 (A33 10mer peptide 24) for HLA-A*3303 subjects exhibit an anti-tumor effect. In addition, for identification of the most effective E7 epitopes, binding affinity of E7 peptides to MHC molecules were estimated and new peptides were synthesized with modified amino acid compositions based on E761-69 (A24 9mer peptide 1) and E767-76 (A24 10mer peptide 20) for HLA-A*2402 subjects. Consequently, modified E7 peptide 3 and modified E7 peptide 7 were identified as newly synthesized E7 epitopes, and significant suppression of tumor growth was demonstrated when transplanted with E761-69 (A24 9mer peptide 1), E767-76 (A24 10mer peptide 20), modified E7 peptide 3, and modified E7 peptide 7-sensitized CD8+ T lymphocyte in the established mouse cervix cancer model. This study verified that CD8+ T lymphocytes stimulated with identified and newly synthesized E7 peptides have anti-tumor potency in HLA-A*2402 subjects.In conclusion, novel HPV type 16 E7 epitopes have been identified for HLA-A*2402 and HLA-A*3303 subjects and these epitopes-sensitized PBMC as well as CTLs have cytolytic activity and anti-tumor effect against cervix cancer. Our study also proposes that transplantation of HPV E7-specific CD8+ T lymphocytes has the potential to improve anti-cancer therapy in patients with HPV type 16-associated cervix cancer