טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDenkberg-Frenkel Galit
SubjectStudy of Antigen Presentation by Tumor Cells and Development
of Novel Targeting Agents Using Recombinant
Antibodies with T-cell Receptor-like
Specificity
DepartmentDepartment of Biology
Supervisor Professor Yoram Reiter


Abstract

Soluble recombinant MHC-peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this report, soluble recombinant single-chain human MHC-peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA-A2 in the presence of tumor associated peptides.

The single-chain MHC-peptide complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA-A2-restricted peptides. We also generated single-chain MHC-peptide tetramers which were biologically functional.

In this research we used recombinant single-chain MHC for two main experimental strategies:

i)        For the molecular characterization of CTL immune responses, using single-chain MHC tetramers. We were able to show that CD8 is recruited and participates directly in TCR-peptide-MHC interactions before the MHC-peptide complex has stably bound to the TCR.

ii)       For the selection and characterization of recombinant antibodies with TCR-like specificity. We were able to isolate high affinity recombinant antibodies with TCR-like specificity from both immune and naïve phage libraries. These antibodies directed toward the melanoma tumor-associated antigen-gp100, and were able to recognize the native MHC-peptide complexes expressed on the surface of APC, and cancer melanoma cells. Using this TCR-like antibodies we could visualize directly the melanoma epitope in situ on melanoma cells by immunohistochemistry. Moreover, when fused to a very potent cytotoxic effector molecule in the form of a truncated bacterial toxin, it was able to specifically kill antigen-presenting cells in a peptide-dependent manner and melanoma cells with TCR-like specificity. We used also these recombinant antibodies with TCR-like specificity to show that modifications at anchor residues can dramatically influence the conformation of the MHC peptide groove and thus may have a profound effect on TCR interactions. Our results may have important implications in designing modifications in peptides for cancer immunotherapy.