ADVANCES IN THE DEVELOPMENT OF HIF-1α-ACTIVATED PROTEIN SWITCHES FOR ENZYME PRODRUG THERAPY
Embargo until
2015-05-01
Date
2014-03-19
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Publisher
Johns Hopkins University
Abstract
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 a variety of mutagenesis methods, followed by a two-tiered genetic selection for improved switches, we have identified new HIF-1α-activated enzymes that confer E. coli with modest increases in HIF-1α-dependent 5FC toxicity. However, the current bottleneck in further translation of HIF-1α-activated protein switches is screening potential switch candidates in mammalian cells. To accommodate higher throughput, we explored the use of Flp recombinase-mediated isogenic integration. While initial results in this system are promising, these experiments also brought to light the disadvantageous promiscuous binding activity of the CH1 domain, which we further confirmed in E. coli. This promiscuous binding and subsequent off-target activation needs to be examined under normal physiological conditions to pinpoint off-target activity in these potential therapeutics. With relevant aberrant activators identified, further directed evolution can be used to improve the cancer specificity of HIF-1α-activated protein switches.
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Keywords
Enzyme Prodrug Therapy, Protein Engineering, Cancer Therapy