A Direct Mass Spectrometry Approach For Hpv T Cell Epitope Identification

To rationally design therapeutic cancer vaccines, it is important to know which T cell epitopes are present on cancer cells. Malignant transformation affects the cellular antigen processing machinery, thus not every epitope derived from intracellular proteins is necessarily presented by MHC molecules on the cancer cell surface. Up to now, T cell epitopes have mostly been defined by indirect methods, such as screening peptide libraries for MHC binders, and subsequent lymphocyte reactivity and cytotoxicity assays. However, all these assays with externally added peptides do not answer the question whether a candidate epitope is naturally processed and presented on the malignant cell. This can now be elucidated by using highly sensitive mass spectrometry approaches. For this study, we chose high-risk HPV-driven cancers as a model system. The induction and maintenance of the malignant phenotype in these tumors are dependent on two viral oncoproteins, E6 and E7, which are therefore present in all stages of HPV-driven cancers. We here aimed to assess the presence of T cell epitopes restricted by the major MHC class I supertypes, namely HLA-A1, A2, A3, A11, A24, B7, and B15, on HPV16-transformed cells. Prospective epitopes from the HPV16 E6 and E7 proteins for the above MHC supertypes were identified by high-throughput in silico epitope predictions, employing several web-based prediction algorithms. These peptides were tested for MHC binding in cellular binding assays on B-LCL of the respective HLA type. Finally, MHC-peptide complexes were immunoprecipitated from HPV16-transformed cells, and the presence of candidate epitopes was analyzed by nano-HPLC-MS2 and MS3 mass spectrometry. Binding of the majority of predicted peptides to the respective HLA allele was verified in the cellular assays. Several novel HPV16 MHC binders were identified. We optimized the immunoprecipitation protocol for epitope yield. Mass spectrometry analysis was successfully established and validated for HLA-A2, and is currently performed for the other supertypes. In conclusion, we show that ascertaining the actual cellular presentation of HPV T cell epitopes is feasible. As the included HLA supertypes have a cumulative population prevalence of >95%, the resulting set of peptides may be used for the formulation of a widely applicable therapeutic HPV vaccine, or for immunomonitoring purposes in other HPV immunotherapy studies. * These authors contributed equally to this study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3529. doi:1538-7445.AM2012-3529