טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSaid Louie
SubjectCrystallization of MHC-Peptide - TCRL Antibody
Trimeric Complex
DepartmentDepartment of Biology
Supervisor Professor Yoram Reiter
Full Thesis textFull thesis text - English Version


Abstract

Clinical studies have been facing challenges in the utilization of antibodies in immunotherapeutic modalities for cancer and autoimmune diseases therapy. We offer a new set of next generation antibodies with T cell receptor-like specificity (TCRL) that can restrictively recognize MHC molecules loaded with an antigenic peptide. TCRL Abs can be utilized for a variety of applications and different modes of action for cancer immunotherapy and immunomodulation of autoimmune responses in autoimmune diseases. The high affinity and unique specificity of TCRL Abs towards their target presented as a peptide in the MHC mechanism, has driven interest in revealing their three-dimensional binding structure. This study has overcome several challenges to optimize the stable molecular structure of the HLA-A2-Tyrosinase complex, in terms of flexibility, solubility and the quantity of yield after the different chromatography methods. Additional challenge was to optimize the production homogeneity of the TCRL Ab Fab by applying different expression systems to overcome heterogeneity caused by the post translational modifications of the eukaryotic system machinery. We utilized different Fab folding systems, directed mutagenesis for obtaining an ancestral-like amino-acid sequence of the Fd-heavy chain of the Fab, that proved to be unsuccessful. Ultimately, utilization of a prokaryotic-originated Fab for obtaining a pure, stable and crystallized trimeric complex of HLA-A2-Tyrosinase-TCRL-Fab has proved successful. The trimeric complex yielded several crystals that resulted in X-ray data collection of a candidate space group at a moderate resolution. Moreover, abundant crystals have been reproduced with condition optimization and with the use of micro-seeding method and additives for optimized crystallization, for an enhanced data collection in a more advanced X-ray beamline at the synchrotron of ESRF, Grenoble. An overall study of the structure of several TCRL Abs different affinities will provide us vital data for understanding and providing a structural basis of a next-generation Abs family for immunotherapy of cancer and autoimmune diseases.