Final Report Summary - THERAVAC (Development of a therapeutic HPV vaccine via target epitope identification by mass spectrometry)
The overall aim of this project is to develop a therapeutic cancer vaccine against malignancies induced by the human papillomavirus (HPV). Our strategy is to focus the immune response to epitopes that have been proven to be present on the surface of cancer target cells by a direct detection method, employing a specialized mass spectrometry (MS) approach.
The aims of the project were:
• Prediction of possible HPV-16 derived epitopes
• Experimental testing of these predicted peptides for binding to the respective HLA molecule
• Setting up a collection of HPV-16 positive cell lines
• Establishing the MS-based epitope detection method at the host institution
• Analyzing cell lines and fresh tumor samples for cell surface epitope presentation
• Conducting functional immunological assays with selected epitopes, to select suitable vaccine candidates
• Investigate effects of HPV on mechanisms of antigen processing and presentation
All of the above aims, with the exception of the analysis of fresh tumor material, have been met. Epitope predictions have been performed with 10 different prediction algorithms, and around 500 peptides have been experimentally tested for HLA binding to date. This data not only identified multiple new HPV-derived binding peptides, but also allows us to perform evaluations of the performance of the servers for specific HLA molecules and peptide lengths, and to formulate recommendations for other groups utilizing these algorithms.
We have set up a comprehensive cell line collection, and subjected every cell line to state-of-the art analysis for HLA type and HPV-16 variants, but also to authentication and regular contamination control. All the HLA types studied in this project are represented within the cell line collection, which to our knowledge is currently the largest HPV-16-positive cell line collection in the field.
Our MS-based epitope detection approach is based on HLA-specific immunoprecipitation of peptides from cells of interest. The immunoprecipitation protocol was established and optimized for further MS processing of the resulting sample. The MS detection approach was first developed together with the respective core facility of the host institution, but we subsequently succeeded in raising additional third-party funds for purchasing our own instrument, and are now independent of core facility instrument availability. During the MS analysis of cell lines, we found that HPV-derived epitopes were of lower abundance than expected. This necessitated extensive optimizations of the sample preparation and MS detection protocols. As we did not want to perform these experiments on valuable tumor samples, we stayed with the analysis of cell lines, until able to reduce the required number of input cells enough to expect reliable results from small biopsy samples.
Functional immunological assays, such as proliferation assays upon specific stimulation, cytokine production assays, ELISpots, and cytotoxicity assays have been established and conducted for promising epitope candidates. As planned, this analysis was not only done for HLA-A2-restricted epitopes, but extended to another important HLA class-I supertype, HLA-A24, and for multiple HLA-DR class-II molecules. Several peptides could be denoted to be promising vaccine candidates.
A systematic screen of HPV effects on components of the antigen processing machinery on the mRNA and protein level resulted in the identification of one consistently upregulated protease. We are currently investigating its effects on the HPV epitope repertoire. To this end, we employ the functional assays as well as the MS methodology described above.
Our work resulted in a peer-reviewed publication, another one is in the final stages of preparation. Moreover, we contributed as co-authors to two more papers, and wrote two reviews.
Regarding the prospect of the research career development and integration of the fellow, she succeeded in accruing funding for her independent group until the end of 2020. This funding comes through the German Center for Infection Research (DZIF), and includes the transformation of the current research group into a professorship upon successful evaluation. It was agreed between the DKFZ and the DZIF to combine the planned evaluations, which will take place in the end of 2016. Thus, the prospect to attain a stable long-term position has improved from a tenure option at the DKFZ to a true tenure track-position. The fellow’s integration into the host institution is still reflected by numerous collaborations within the center, but also by her being the elected representative of the Research Topic “Infection and Cancer” in the DKFZ Scientific Council, one of the two elected speakers of the Junior Group Leaders, and even serving on the DKFZ Board of Trustees for a year (2015-2016).
The aims of the project were:
• Prediction of possible HPV-16 derived epitopes
• Experimental testing of these predicted peptides for binding to the respective HLA molecule
• Setting up a collection of HPV-16 positive cell lines
• Establishing the MS-based epitope detection method at the host institution
• Analyzing cell lines and fresh tumor samples for cell surface epitope presentation
• Conducting functional immunological assays with selected epitopes, to select suitable vaccine candidates
• Investigate effects of HPV on mechanisms of antigen processing and presentation
All of the above aims, with the exception of the analysis of fresh tumor material, have been met. Epitope predictions have been performed with 10 different prediction algorithms, and around 500 peptides have been experimentally tested for HLA binding to date. This data not only identified multiple new HPV-derived binding peptides, but also allows us to perform evaluations of the performance of the servers for specific HLA molecules and peptide lengths, and to formulate recommendations for other groups utilizing these algorithms.
We have set up a comprehensive cell line collection, and subjected every cell line to state-of-the art analysis for HLA type and HPV-16 variants, but also to authentication and regular contamination control. All the HLA types studied in this project are represented within the cell line collection, which to our knowledge is currently the largest HPV-16-positive cell line collection in the field.
Our MS-based epitope detection approach is based on HLA-specific immunoprecipitation of peptides from cells of interest. The immunoprecipitation protocol was established and optimized for further MS processing of the resulting sample. The MS detection approach was first developed together with the respective core facility of the host institution, but we subsequently succeeded in raising additional third-party funds for purchasing our own instrument, and are now independent of core facility instrument availability. During the MS analysis of cell lines, we found that HPV-derived epitopes were of lower abundance than expected. This necessitated extensive optimizations of the sample preparation and MS detection protocols. As we did not want to perform these experiments on valuable tumor samples, we stayed with the analysis of cell lines, until able to reduce the required number of input cells enough to expect reliable results from small biopsy samples.
Functional immunological assays, such as proliferation assays upon specific stimulation, cytokine production assays, ELISpots, and cytotoxicity assays have been established and conducted for promising epitope candidates. As planned, this analysis was not only done for HLA-A2-restricted epitopes, but extended to another important HLA class-I supertype, HLA-A24, and for multiple HLA-DR class-II molecules. Several peptides could be denoted to be promising vaccine candidates.
A systematic screen of HPV effects on components of the antigen processing machinery on the mRNA and protein level resulted in the identification of one consistently upregulated protease. We are currently investigating its effects on the HPV epitope repertoire. To this end, we employ the functional assays as well as the MS methodology described above.
Our work resulted in a peer-reviewed publication, another one is in the final stages of preparation. Moreover, we contributed as co-authors to two more papers, and wrote two reviews.
Regarding the prospect of the research career development and integration of the fellow, she succeeded in accruing funding for her independent group until the end of 2020. This funding comes through the German Center for Infection Research (DZIF), and includes the transformation of the current research group into a professorship upon successful evaluation. It was agreed between the DKFZ and the DZIF to combine the planned evaluations, which will take place in the end of 2016. Thus, the prospect to attain a stable long-term position has improved from a tenure option at the DKFZ to a true tenure track-position. The fellow’s integration into the host institution is still reflected by numerous collaborations within the center, but also by her being the elected representative of the Research Topic “Infection and Cancer” in the DKFZ Scientific Council, one of the two elected speakers of the Junior Group Leaders, and even serving on the DKFZ Board of Trustees for a year (2015-2016).