(47d) Improved Protein Switches for Cancer-Activated Enzyme Prodrug Therapy

Prevailing approaches for developing cancer protein therapeutics focus on creating proteins that therapeutically modulate a cancer marker’s function.  Such an approach limits the therapeutic mechanism to those that naturally arise from modulation of the cancer marker and precludes the use of cancer markers for which therapeutic modulation is not feasible.  Furthermore, many potential protein therapies lack the desired cancer targeting.  The ability to link recognition of any cancer marker with activation of any desired therapeutic function would enormously expand the number of possible protein therapeutics. 

We have previously engineered a switchable prodrug-activating enzyme that selectively kills human cancer cells that accumulate the cancer marker hypoxia-inducible factor 1α (HIF-1α).  This HIF-1α-activated enzyme switch (Haps59) was created by fusing the prodrug-converting enzyme yeast cytosine deaminase (yCD) and the CH1 domain of the p300 protein, which binds HIF-1α. Haps59 autonomously increases its ability to convert the prodrug 5-fluorcytosine (5FC) into the chemotherapeutic 5-fluorouracil (5FU) in a HIF-1α-dependent manner, rendering colon and breast cancer cells more susceptible to the prodrug. However, the difference in 5FC sensitivity between the presence and absence of HIF-1α was not as large as desired.  Using circular permutation as well as random and cassette mutagenesis followed by a two-tiered genetic selection for improved switches, we have identified new HIF-1α-activated enzymes that confer increased 5FC sensitivity in the presence of HIF-1α and reduced 5FC sensitivity in the absence of HIF-1α.

Our strategy offers a platform for the development of inherently selective protein therapeutics for cancer and other diseases.  By introducing protein-level regulation into cancer prodrug therapies, our approach skirts the problematic selective transduction requirement currently limiting enzyme-prodrug therapies. In addition, our approach is complementary to both transcriptional and transductional targeting and might be combined with these approaches to afford a double or triple layer of specificity: at the gene delivery level, at the transcription level and at the protein level.