טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentCohen-Inbar Or
SubjectRecruitment of Immune Effector Cells against Astrocytoma
by MHC-chlrotoxin Chimeric Proteins
DepartmentDepartment of Medicine
Supervisors Professor Yoram Reiter
Professor Menashe Zaaroor


Abstract

Introduction: Glioblastoma Multiforme (GBM) is the most common malignant primary brain neoplasm having a mean survival of 12 months. The lack of an efficient immune response to the tumor and its microinvasive nature have been explained by its immunosupressive capabilities. The immunoprofile of GBM is not well characterized. Melanoma and GBM are both neuroectodermally originated tumors, sharing differentiation/other antigens, yet only the immune profile of melanoma has been well-characterized.

Aim: We designed a molecule that specifically binds MMP-2 expressed on GBM cells, and through its effector domain recruit elements of the immune system to mount an effective response. We characterized  HLA-A2  restricted  melanoma  associated  peptides  presentation  on GBM cell lines and solid tumor samples from GBM patients.

Methods: The targeting moiety is the small molecule Chlorotoxin, from the Israeli Yellow scorpion. The effector domain is a single chain HLA-A2 bound to a protein derived from the cytomegalovirus. RNA analysis, Western blotting, mass spectroscopy and  cytotoxic  lymphocytes  toxicity  assay  were  compared to immunohistochemistry  with  engineered  antibodies  bearing  T-cell receptor specificity, constructed in this work.

Results: The molecular construct was cloned and expressed in E.Coli. The protein product was isolated and purified, then folded in vitro. Different activity assays designed at proving retained activity of each domain were employed with success. The presentation of HLA-A2/MART-1 complexes on U-251MG GBM cell line was relatively low, moderate for HLA-A2/gp-100 and HLA-A2/MAGE-A1 and high for HLA-A2/Tyrosinase on cell lines and human  GBM  tumor samples. This was inconsistent with our results from cytolytic T-cell assay which may suggest  non-to-low  presentation  of  HLA-A2/Tyrosinase.

Conclusions: This molecular construct represents a new family of molecules which contain a non-antibody compact and highly specific targeting domain, combined with the ability to recruit different lymphocyte populations using HLA-molecules bearing a single, preselected, highly antigenic peptide derived from immunogenic viral or bacterial T cell epitopes. Our profiling sheds light on the immunological profile of GBM and its evolution and may create new targets for immunotherapy.