טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSivan Slobodkin
SubjectAntigen-Specific Immunomodulation for Multiple Sclerosis
DepartmentDepartment of Biology
Supervisor Full Professor Reiter Yoram
Full Thesis textFull thesis text - English Version


Abstract

Multiple Sclerosis (MS) is an autoimmune disease of the CNS.  The disease is characterized by demyelination, axonal damage and a range of neurological symptoms.  The disease is triggered by presentation of myelin autoantigens on MHC class II molecules by antigen-presenting cells (APC).  Among the autoantigens is the Myelin Oligodendrocyte Glycoprotein (MOG).  The presentation of myelin antigens leads to activation of auto reactive CD4 T cells in the periphery.  After their activation, the T cells express cell surface activation markers that enable their extravasation across the blood-brain barrier. Upon entering the CNS, the CD4 T cells are reactivated by myelin epitopes presented by local APCs, which lead to local inflammation and activation of macrophages and microglia and the secretion of soluble mediators that cause myelin damage.  Evidence for the importance of CD4 T cells come from the animal model of MS, Experimental Autoimmune Encephalomyelitis (EAE), where adoptive transfer of auto reactive CD4 T cells was found to be sufficient to induce the disease.  In addition, the disease has strong genetic association to the MHC class II allele, HLA-DR2.


Previous work in our laboratory identified TCR-like antibodies (TCRL Abs) that bind to a MOG35-55 peptide in context with HLA-DR2.  TCRL Abs are recombinant antibodies that bind to a specific peptide antigen with T cell specificity; thus, they bind with peptide-specific, MHC-restricted manner to the MHC-peptide complex.

The goal of this study was to construct, produce, and characterize a TCRL Ab-based immunocytokine that is fused to an anti-inflammatory cytokine directed against the MOG35-55 peptide. The cytokine that was used to generate the TCRL-based immunocytokine was Interleukin-10 (IL-10) a strong anti-inflammatory cytokine which acts as a general suppressor of immune response. It is suggested that the TCRL-IL10 immunocytokine will reduce inflammation in a site specific manner and will minimize MS pathology.


The TCRL Ab-immunocytokine represents a valuable tool for significantly inhibiting MOG35-55 specific HLA-DR2 restricted T-cell response towards the MS-associated T cell epitopes.  These TCRL Abs can be used as a novel therapeutic delivery vehicles for IL-10 at the selective sites of the disease, to inhibit progression of the disease inflammation in MS.


Two forms of recombinant TCRL-immunocytokine were cloned in two different expression systems: i) a TCRL Fab fragment-IL10 fusion expressed in a prokaryotic system by in vitro refolding and ii) a TCRL Diabody fragment-IL10 fusion expressed in a eukaryotic system.  Both immunocytokines were tested for their ability to bind native DR2/ MOG35-55 complexes as presented by APCs and to bind recombinant DR2/ MOG35-55 complexes.  This experiment revealed that the diabody-based immunocytokine is superior and thus was used for further characterization.  The TCRL diabody-IL-10 fusion was biologically active and most significant were preliminary studies that demonstrate ability to reduce the symptoms of EAE in HLA-DRB1*1501-Tg mice disease. 

         

For future studies, the DR2/MOG35-55-specific TCRL-immunocytokine will be used and tested for two major research directions; to treat in vivo, in the Tg mouse model of MS (EAE) and to demonstrate a shift in the balance of the cytokines microenvironment toward an anti-inflammatory state indicating that the immunocytokine can induce antigen and site-specific immunomodulation.