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Targeting guanylyl cyclase-C: colon cancer metastasis diagnostic signal amplification and subcellular delivery of immunotoxin therapeutic
Dissertation   Open access

Targeting guanylyl cyclase-C: colon cancer metastasis diagnostic signal amplification and subcellular delivery of immunotoxin therapeutic

Glen P. Marszalowicz
Doctor of Philosophy (Ph.D.), Drexel University
May 2014
DOI:
https://doi.org/10.17918/etd-6956
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Abstract

Medical sciences Cancer--Diagnosis Colon (Anatomy)--Cancer--Diagnosis
Colorectal cancer is the second deadliest cancer in the US due to mortality from eventual metastatic outgrowth. The National Cancer Institute estimates that approximately 136,000 men and women will be diagnosed with colorectal cancer in 2014 alone, with greater than a third dying from the disease during the same period. While primary tumors can be effectively treated with surgical resection if diagnosed prior to spreading from the primary site, the 5-year survival rate for cases with distant metastasis is a dismal 13% despite current therapeutic intervention, highlighting the unmet need for a more efficacious approach. Targeted cancer therapy holds much promise but few standout successes because the markers are broadly expressed in both healthy and diseased tissue. The transmembrane protein guanylyl cyclase C (GCC), however, provides the specificity for targeted therapy: it is selectively expressed in intestinal epithelium; absent in healthy extra-intestinal tissues; and expressed through the metastatic event. Here we investigated signal amplification via an avidin biotin complex (ABC) with an ST-biotin pretargeting moiety, a GCC exogenous ligand labeled with biotin, complexed with tetrameric avidin and tertiary detection with125I -labeled biotin. We demonstrated that while the ST-biotin delivery of avidin-125I-biotin to GCC fails, biotin-IgG is a tenable system in vitro, but with significantly diminished amplification in vivo. Simultaneously we demonstrated that the GCC monoclonal antibody (GCCmAb) generated for diagnostic pretargeting internalized in the acidic, reductive environment of the lysosomal compartment. Ricin, a two-part toxin derived from the castor oil plant Ricinus communis, which depurinates the ribosome halting protein synthesis, also travels through the endosomal-lysosomal pathway causing cytotoxicity. Leveraging these shared characteristics to provide the proof of concept for GCC-dependent intracellular payload delivery, we generated an immunotoxin (IT) linking the GCCmAb targeting moiety via a disulfide bond to the catalytic A chain of ricin toxin (RTA). Targeting GCC provides cellular specificity, while disulfide linkage endows subcellular release of RTA to act on the ribosome and inhibit protein synthesis. GCC-IT therapy reduces tumor burden in vivo fivefold versus controls, providing proof of concept for subcellular deliverable GCC therapeutics targeting colorectal cancer metastasis.

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