|M.Sc Student||Kalaora Shelly|
|Subject||Identification of Unique HLA Peptides Presented on SCLC|
|Department||Department of Biology||Supervisor||Professor Emeritus Arie Admon|
|Full Thesis text|
Small cell lung carcinoma (SCLC) represents 13% of the newly diagnosed lung cancers, yet it is the more deadly of the different lung cancers. Treatment of SCLC remains challenging because of its rapid growth, early dissemination and development of drug resistance during the course of the disease. Thus, most SCLC patients face a grim prognosis, which calls for development of more specific and effective treatments, such as immunotherapy.
Our research focuses on the large-scale discovery of peptides presented by the HLA molecules on SCLC cells, while looking for peptides with potential to serve as targets for immunotherapy. Since the expression of HLA class I molecules on SCLC cells is significantly lower than on normal lung cells (due to the attempt of the SCLC cells to escape immune surveillance), we used two independent approaches to increase the level of expression of HLA molecules by SCLC cells in culture: First, treatment of the cells with interferon gamma (IFNγ), which increases the expression levels of the membranal HLA many folds. Second, we used recombinant expression of selected HLA alleles and induced the cells to express them as soluble HLA molecules (sHLA). The membranal HLA molecules were purified after solubilizing the IFNγ treated SCLC cells with mild detergents. The sHLA molecules were recovered from the growth medium of the transfected SCLC cells. Both approaches facilitate the recovery of large number of HLA peptides.
After immunoaffinity purification of the membranal or the soluble HLA molecules, the bound peptides were extracted and identified by capillary chromatography and tandem mass-spectrometry. In total, we were able to identify 13,544 different HLA peptides from three SCLC cells lines (NCI-H526, NCI-H69 and NCI-H82). We identified 12,932 peptides from the membrane HLA class I peptides and 1,122 peptides from the soluble HLA class I peptides. In addition, we identified 1,097 HLA class II peptides that were (unexpectedly) expressed by the NCI-H526 cells.
Tumor antigens potentially useful for vaccination were selected according to a number of criteria: 1) Antigens not expressed in normal tissues, such as tumor testis antigens; 2) Antigens that can elicit immune response. 3) Antigens encoded by genes known to be preferentially expressed in SCLC tumor cells. Using such databases and bioinformatics analyses we selected 52 such putative cancer vaccine candidate HLA peptides for further testing as SCLC immunotherapeutics.