|M.Sc Student||Hadar Liat|
|Subject||Development of a Cancer Vaccine Based on the Identification|
of Cancer Peptides Presented on Dendritic-Like
|Department||Department of Biology||Supervisor||Professor Emeritus Arie Admon|
|Full Thesis text|
Cancer specific peptides are sought after as candidates for development of cancer vaccines. The immune system uses the Major Histocompatibility Complex (MHC) to gain information on the cell health, by screening peptides presented on the cell surface.
In our lab a peptide purification system based on soluble MHC (sMHC) class 1 was developed. This method was based on transfecting truncated HLA constructs into cell lines. The peptides were separated from sHLA and analyzed by capillary RP-HPLC combined with ESI ion trap mass spectrometry and bioinformatics methods.
We suggest a new method for identifying HLA peptides derived from any of protein and cross presented to the immune sytem. For that purpose, we chose HL-60 cells (human myeloid leukemia). HL-60 as surrogate model for professional antigen presenting cells (APC) that in recent studies were shown to differentiate into dendritic like cells after incubation with calcium ionophore (CI) and interferon γ (IFNγ). After acquiring dendritic characteristics the cells should be able to uptake and digest any protein including Tumor Associated Antigens (TAA) and present the resulting peptides in the context of both MHC I and II.
We established that after the treatment with CI and IFNγ the treated cells undergo morphological changes: namely, they develop motile dendritic processes that parallel mature dendritic cells. In addition FACS analysis showed difference of expression of CD40, MHC II, and MHC I as a result of the treatment.
We continued by investigating the cells ability for uptake non-human florescent proteins. FACS analysis indicated a fine rise in the cells florescence as a result of the protein uptake.
We transfected the HL-60 cells with the sHLA-B7 construct and obtained a few peptides of interest for cancer vaccine. However, using HL-60 cells for this purpose, proved to be a very expensive an inefficient process.
We repeated the protein uptake experiments with the C1RsB7cells in the same manner and FACS analysis showed that the cells express phagocytic activity. The next step was to evaluate the cells ability to process proteins. We used non-human proteins to ascertain that the recovering peptides could not originate from the human cell lines. The scores of the peptides at mass spectrometry analysis were low and therefore the results were inconclusive. We received peptides from both cell lines: a few of them should be further evaluated for their potential as cancer vaccine candidates.