A34 Identification of Th1 Epitopes in Lung Non-Small Cell Lung Cancer Antigens to Develop a Multiantigen Vaccine

Abstract

Non-small cell lung cancer (NSCLC) represents 85% of all lung cancer cases and it is highly smoking related. The goal of this project is to develop a vaccine for lung cancer prevention in current or past smokers by identifying immunogenic proteins in lung cancer that are able to induce a potent inflammatory Th1 response. Lung cancer has one of the highest mutation rates of all types of cancer, but driver mutations that could be targeted for a vaccine for lung cancer prevention are unknown. Gene expression profiling of bronchial biopsy specimens from smokers has shown that changes in gene expression in histologically normal epithelia can discern people with and without lung cancer. Many of these changes are proteins aberrantly upregulated, but not mutated. We have used quantitative mass-spectroscopy analysis to identify proteins overexpressed in NSCLC cell lines compared with normal lung epithelial cells. Five NSCLC cell lines (three squamous cell carcinoma and two adenocarcinoma) and two normal lung epithelial cell lines were included in the analysis. A total of 14,219 peptides, corresponding to 2,875 proteins, were identified. We selected for further analysis those proteins identified with >95% confidence and at least 3 peptides per protein, and overexpressed in three or more NSCLC cell lines. We considered that a protein is overexpressed if [expression in the NSCLC cell line/ expression in the normal cell line]>1.5. A total of 154 antigens met our criteria. Candidate antigens were investigated by siRNA screening to identify those genes with a function in maintaining cell tumor growth. If a gene is required for tumor cell proliferation, knocking down the gene by siRNA should decrease cell survival and proliferation. We looked at both viability and apoptosis by caspase 3/7 activation after siRNA knockdown. We selected those antigens for which: [(mean of viability in NSCLC cell line) / (mean of viability in the normal lung cell line)] < 0.75 with a p-value of 0.1. We identified 14 candidates that are overexpressed in lung cancer and necessary for tumor cell survival. We have prioritized those proteins that have been previously described to play a role in lung cancer invasion, proliferation, metastasis, or survival. We selected 5 candidates to move forward: FKBP3, PARP1, RAN, S100A6, and SART3. An effective anticancer immune response needs to elicit a strong inflammatory Th1 response and avoid a Th2 response that promotes tumor tolerance. We used web-based modeling to predict epitopes that preferentially elicit a Th1 response, and assessed the presence of Th1 and Th2 responses via IFN-g (Th1) and IL10 (Th2). Six to seven epitopes (15-20 mer peptides) per antigen were evaluated by IFN-g and IL10 ELISPOT. Th1 epitopes identified in NSCLC antigens are the base for a preventive vaccine for NSCLC. The efficacy of the multiantigen Th1 vaccine to prevent lung cancer is currently under evaluation in the NTCU-induced lung cancer mouse model.