Massively Parallel Protein Design to Develop Enzymes for Next‐Generation Chemotherapy

Abstract

Most chemotherapy targets dividing cells, rather than just tumor cells. This causes the failure of chemotherapy, as dosing limited by side-effects leads to drug resistance and relapse. One strategy to overcome this, referred to as Directed Enzyme Prodrug Therapy (DEPT), involves administration of an inactive prodrug that is activated by a foreign enzyme at the tumor site. Enzyme targeting can be achieved using mechanisms that include tumor-directed antibodies or viruses. Clinical trials have demonstrated the effectiveness of DEPT, but were hampered by the complex dosing schemes needed to ensure that the enzyme-antibody complex had cleared circulation and was entirely localized the tumor before the prodrug administered. Otherwise, the circulating enzyme would activate the circulating prodrug, resulting in systemic toxicity. We are employing a massively-parallel computationally-guided design approach using Rosetta, validated with a novel selection system and deep sequencing, to overcome this shortcoming in a model DEPT system, in which the bacterial enzyme, carboxypeptidase G2 (CPG2), is used to activate nitrogen mustard prodrugs. We have designed a “pro-enzyme” form of CPG2 that is activated by tumor-overexpressed proteases at the tumor site through removal of the designed “pro-domain.” The pro-enzyme will be dormant in circulation, thereby avoiding activation of the prodrug by circulating enzyme. Proteolytic processing at the tumor site by tumor overexpressed proteases removes the inhibitory “pro-domain,” restoring the activity exclusively in the tumor microenvironment. In addition to the design approach, the structural and biochemical characterization of our designed enzyme, in comparison to the native form, will be discussed. In addition, data showing the pro-enzyme's effectiveness in cancer cell culture will be shown. Support or Funding Information BJY is supported by fellowships from the Canadian Institutes of Health Research (CIHR) and the Mistletoe Research Foundation. Summary of the general experimental strategy for the design of a stimulus-responsive enzyme. The designed “pro-domain” is shown in green. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.