Characterizing the granzyme-perforin pathway and its utility as a cell-to-cell delivery system for cellular therapeutics

• Main Author: Woodsworth, Daniel
• Language: English
• Published: University of British Columbia 2017
• Online Access: http://hdl.handle.net/2429/62073

Alongside small molecules and biologics, cell-based therapies are emerging as a third class of medical therapy. Additional sensors, actuators and control circuits would greatly expand the range of function and application of cellular therapeutics. To this end, a cell-to-cell delivery module has been developed by investigating and re-engineering the granzyme-perforin pathway of cytotoxic lymphocytes. A computational biophysical model of this process was developed and implemented using a spatial stochastic simulation algorithm, which indicated that hindered diffusion in the immune synapse is critical to ensure reliable granzyme internalization and that large amounts of granzyme escape the synapse, but should not have toxic effects due to rapid spatiotemporal dilution. Additionally, these results indicated that passive diffusion is sufficient for granzyme entry into the target cell, which motivated efforts to use granzyme as a molecular chaperone to transfer exogenous payloads from effector to target cells. Using a fluorescent protein payload, the subcellular localization of several granzyme B derived chaperones was characterized using fluorescence microscopy, and then their capacity to transfer the payload to target cells was evaluated in co-culture experiments. The results indicated that the motifs in granzyme B that are required for lytic granule loading are only functional and contiguous in the folded protein. Additionally, these experiments demonstrated that full length granzyme B is a suitable chaperone for delivering protein payloads to target cells via the granzyme-perforin pathway. Attempts were then made to use this system to deliver potent orthogonal toxins to apoptosis and lymphocyte resistant tumor cells. A range of granzyme B toxin fusion proteins were constructed, all of which retained toxic activity to varying degrees. To render target cells resistant to lymphocyte attack both small molecule and protein based inhibitors of apoptosis were tested in several cell lines, which delayed cell death, but did not stop it. Using effector target dose response curves, a moderate increase in target cell death was observed in cells targeted by lymphocytes expressing granzyme toxin fusion proteins, as compared to wild type lymphocytes, but the biological significance of this effect is uncertain. Approaches to improve this granzyme-perforin mediated delivery system and its therapeutic utility are discussed and explored. === Science, Faculty of === Graduate