טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZafir-Lavie Inbal
SubjectGlioblastoma Multiforme Microenvironment: Immune Response
and Antibody Mediated Targeting
DepartmentDepartment of Biology
Supervisors Professor Yoram Reiter
Professor Menashe Zaaroor
Full Thesis textFull thesis text - English Version


Abstract

Glioblastoma Multiforme (GBM) is a common neoplasm of the central nervous system, exhibiting   poor prognosis of 14 months and no treatment progression in the past two decades. Despite advances in molecular biology and immunology research, the immunoprofile of GBM is not well characterized. Melanoma and GBM share embryonic antigens.  However, in contrast to GBM, anti melanoma T-cell response and melanoma associated differentiation antigens presentation are well characterized.

In this study, with the use of antibodies bearing T-cell receptor specificity, we characterized HLA-A2 restricted melanoma associated peptides presentation on GBM cells and tumor samples. We studied four of the most characterized melanoma associated antigens: MART-1, Tyrosinase, Gp100 and MAGE A1. We showed that similarly to melanoma, and opposed to what is known from cytotoxic T-cells assays, the Tyrosinase derived peptide is presented in high levels in GBM cells and tumor specimen.  We have also detected high presentation levels of HLA-A2/ MAGE-A1 complexes on GBM and melanoma cells. The HLA-A2/MART-1 presentation levels on GBM cell line was significantly low when comparing to melanoma cells, and the HLA-A2- Gp100 was presented in low levels both in GBM and melanoma. Similar data was obtained for GBM tumor specimen.

In addition, we developed new immunotherapeutic approach which delivers HLA-A2/immunogenic peptide towards GBM cells. We designed two independent chimeric proteins which consist of targeting domain and effector domain. In the first approach, we utilized the targeting abilities of an antibody fragment, fused to HLA-A2 complex. In the second approach, we used the specific properties of scorpion venom derived peptide Chlorotoxin for targeting, which was fused to HLA-A2 directly linked via linker to the immunogenic CMV pp65 derived peptide. In these two approaches, antibody and peptide based targeting of HLA-A2/immunogenic peptide complexes, we successfully demonstrated the specific targeting abilities and the potent initiation of CTL mediated lysis of the described fusion proteins.

Our results present direct analysis of HLA-A2 restricted peptides derived from melanoma associated differentiation antigen on the surface of GBM cell lines and tumor samples. This data sheds light on the immunological profile of GBM, and may create new targets for immunotherapy. In addition, we demonstrated the construction, expression, refolding and in vitro activity of antibody and peptide based approaches for targeting HLA-A2/peptides to tumor cells. We have shown the ability of the two chimeric proteins to target and mediate efficient lysis of Glioblastoma cells.