טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBassani Sternberg Michal
SubjectA Novel Methodology for Cancer Diagnosis Based on Analysis
of the Serum Soluble HLA Peptidome
DepartmentDepartment of Biology
Supervisor Professor Emeritus Arie Admon
Full Thesis textFull thesis text - English Version


Abstract

The human leukocyte antigen (HLA) molecules are normally membrane-bound peptide transporters that carry peptides, resulting from protein degradation within the cytoplasm, to the cell surface for surveillance by circulating T lymphocytes. Importantly, many types of cancer cells are known to release into the plasma large amounts of soluble HLA molecules (sHLA), presumably to counter the immune surveillance by circulating T cells. Since healthy cells shed only small amounts of sHLA molecules relative to the cancer cells, the sHLA molecules present in the plasma of cancer patient are mostly derived from the diseased cells. We describe in this thesis the development of a new simple and universal blood-based diagnosis paradigm for identifying multiple serum biomarkers, potentially useful for diagnosis of different diseases, to be based on the large-scale analyses of the sHLA peptides repertoires (sHLA-peptidomes) recovered by immunoaffinity from small volumes of blood samples. 

We first describe our methodology, which is based on a single-step immunoaffinity purification of the soluble HLA molecules from the plasma, followed by extraction of the bound peptides by adding a small volume of acid and collection of purified peptide pools. The released sHLA peptides are identified and quantified by capillary chromatography combined with tandem mass spectrometry. We first demonstrated that the plasma sHLA molecules are bound with their authentic peptide repertoires (sHLA peptidome), similar to the peptidome presented on the HLA molecules at the surface of the tumor cells. We next show that the peptides extracted from the plasma matched the consensus binding motifs of the HLA alleles of the blood samples donors. We validated our method for biomarkers discovery by the identification of a few thousand sHLA peptides, including many cancer related peptides present only among the sHLA peptidomes of the patients. From about five ml of plasma we purified with this approach a few micrograms of sHLA molecules, thus enabling many orders of magnitude enrichment of these biomarkers.

The new methodology forms the foundation for development of a simple and universal blood-based paradigm for diagnosis, prognosis and follow-up of different diseases, such as cancer and autoimmunity. Furthermore, it allows for the investigation of both the patterns of protein synthesis and their degradation within the cancer cells in the human body.