The hypothesis of my thesis project was that targeting the extracellular matrix of a solid tumor for a local generation of a cytotoxic drug could inhibit tumor growth and progression. To test this hypothesis of the matrix attachment therapy (MAT), a gene therapy and recombinant protein therapy were pursued. For the gene therapy, I cloned a mammalian expression vector containing the extracellular domain of CD44 (sCD44), yeast cytosine deaminase (CD), and E.coli uracil phosphoribosyltransferase (UPRT) genes to transfect tumor cells in vitro. The sCD44::CD::UPRT fusion gene construct was designed to secrete the expressed fusion protein and generate cytotoxic drugs to cause cell deaths in the presence of the prodrug 5-fluorocytosine (5-FC). This gene therapy strategy did not result in a potent cytotoxic effect on cells that expressed the fusion gene due to low expression of the fusion genes in transfected cells.

For the protein therapy, a recombinant fusion protein containing TSG-6 Link (Link) and the CD enzyme was expressed in E.coli and purified to characterize the functions of the LinkCD fusion protein. LinkCD exhibits K_m of 0.33 mM and V_max of 14 μM/min/μg for the conversion of 5-FC to 5-FU. LinkCD can bind to a hyaluronan oligomer (12-mer) at a K_D of 55 μM at pH 7.4 and a K_D of 5.32 μM at pH 6.0 measured using surface plasmon resonance (SPR). To evaluate the anti-tumor effect of LinkCD/5-FC combination therapy in vivo, mice received intratumoral injections of LinkCD on days 11 and 14 after C26 tumor implantation and drinking water containing 10 mg/mL of 5-FC starting on day 11. Animals that received LinkCD/5-FC treatment showed significant tumor size reduction and increased survival compared to the CD/5-FC treatment group. Other treatment groups that were unable to produce 5-FU had no effect on the tumor growth despite receiving the fusion protein that contained the Link domain. The results strongly suggest that a treatment regime consisting of a fusion protein containing the Link domain, the active CD enzyme, and the prodrug 5-FC are required to produce an anti-tumor effect. Intravenous administration of LinkCD to animals tumored with C26 that received 5-FC in the drinking water did not result in a therapeutic effect, possibly due to a short circulation time of LinkCD. I created chimeras of the CD44 Link domain in silico that could potentially be used to increase the circulation time of LinkCD in the blood. Thus, MAT is a promising alternative to antibody-directed prodrug enzyme therapy approach for cancer treatment.