|M.Sc Student||Said Louie|
|Subject||CTL Mediated Killing of Melanoma Cancer Cells by Class|
I MHC-TCR-like Ab Recombinant Fusion Molecule
|Department||Department of Biology||Supervisor||Professor Yoram Reiter|
|Full Thesis text|
The employment of T cells to kill tumor cells has been developed recently as an important approach for cancer immunotherapy. We and others have introduced a novel strategy for this that is based upon tumor-specific antibody-mediated targeting of MHC-class-I peptide complexes to the tumor which eventually induce potent CTL killing of the malignant cells. This approach consists of a fusion molecule that can bridge between antibody targeting capabilities against tumor cells, with the restricted and potent killing activities of CTLs. The novelty of this fusion molecule is its ability to recruit selected populations of CTLs towards tumor cells, by targeting an intracellular-derived antigen presented in the MHC-class-I mechanism. Recruited CTL populations are viral specific, because antigenic peptides originating from viruses (influenza, cytomegalovirus and Epstein Barr virus) are very immunogenic and can elicit strong immune responses. More importantly, a majority of most populations have been infected during their lifetime and possess efficient memory CTL activity.
In this research, an Ab with a TCR-Like specificity that targets an intracellular-derived antigen, replaces the cellular immune targeting of tumor cells through the recognition of a tumor associated MHC-class-I/peptide complex. The TCR-like Ab herein recognizes the unexpectedly over-presentation of Tyrosinase, a melanoma-associated Ag. This HLA-A2/peptide complex can be presented at very high density on the surface of melanoma due to the instability and aberrant intracellular processing of the Ag. We have isolated a high affinity recombinant TCR-like Fab that was found to recognize the melanoma associated Tyrosinase epitope in complex with HLA-A2 molecules in a highly specific manner. The Ab is termed MC1.
We used the MC1 Ab in two forms, a ScFv and a Fab fragment. These fragments of Ab can bind specifically to the HLA-A2/Tyrosinase complexes expressed on melanoma cells. This TCR-like antibody is designed now as a fusion molecule, encoded by a single gene, in which a single-chain β2m-HLA-A2 molecule carries a highly immunogenic viral peptide derived from CMV protein pp65 that is fused at its N-terminus. The fusion molecule possesses two functional domains, the anti-cancer moiety domain that specifically targets tumor cells, and the MHC-peptide effector domain that recruits specific populations of CTLs, depending on the antigenic peptide within the MHC groove. Eventually cmv-HLA-A2-MC1 will constitute a novel immunotherapeutic modality for targeting an intracellular-derived antigen of melanoma and inducing potent anti-tumor cellular activity towards it.
Results of this research show that cmv-HLA-ScFv molecules that were successfully expressed in the prokaryotic system do not refold correctly in the redox-shuffling in-vitro refolding method. While in the cmv-HLA-A2-Fab molecule variants, a sequence alteration in the heavy chains of the molecules has shown significant increase in the yield from protein, still the molecules were roughly purified. Only cmv-HLA-Light-ss-Heavy variant displayed specific binding ability and specific lysis on Tyrosinase-positive melanoma cells. Switching to a Eukaryotic system, production of the cmv-HLA-A2-ScFv molecule was yielded with high purity, and it displayed specific binding ability on the melanoma lines. We learned that some molecules demand a more sophisticated refolding mechanism for production which is achieved in Eukaryotic cells system.