Generating cytotoxic t lymphocytes against cervical carcinoma with lentivirus transduced antigen presenting cells

Human Papillomavirus (HPV), the cause of cervical cancer, infects and immortalizes keratinocytes. Since the infected cells present viral antigens but lack co-stimulatory molecules for inducing immune responses, alternative antigen presenting cells (APCs) are needed. The 98aa HPV-E7 oncoprotein represents an attractive immunotherapy target, as it is overexpressed and vital for immortalization. Recent publications report abrogated transforming ability and destabilization of E7 following C58G and C91G mutations in ‘CXXC’ zinc finger motifs at positions 58–61 and 91–94. We have made 7 mutant E7 proteins including C94V mutation. This disrupts the zinc finger motifs, but preserves the T cell receptor region of the immunogenic 86–94 nonamer and increases its HLA-0201 binding 14-fold, based on anchor position affinities. The expression and stability of the wild type and mutant E7 (m-E7) proteins is being determined in transfected homozygous HLA-A0201 Epstein-Barr Virus transformed B-cells (LCLs). The mutant with peak E7 peptide processing and presentation will be determined using a HLA-A0201 specific T-cell clone recognizing E711–20. Based on transduction efficiencies of GFP with various gene transfer methods, we have designed a T-cell stimulation scheme in which m-E7 transduced autologous dendritic cells (DCs) and LCLs are used as priming and restimulating APCs, respectively. Using VSV-G pseudotyped recombinant human lent-virus, we detected >40% transduction efficiency in CD34+ derived DCs and >60% in LCLs. We believe that m-E7 specific cytotoxic T lymphocytes can be generated ex vivo with lentivirus modified patient-derived DCs and LCLs and that these CTLs will provide a potent treatment for cervical carcinoma.