Integrating Protein Engineering and Genomics for Cancer Therapy

Abstract

We have developed a broadly applicable platform that harnesses the power of protein engineering and genetic screening to produce efficacious protein-drug combinations for cancer therapy. For proof-of-concept, we implemented this strategy to engineer targeted pro-apoptotic drug combinations that overcome cancer resistance to protein agonists of death receptor 5 (DR5), a key upregulated marker in colorectal cancer (CRC). Over the past decade, various DR5 agonists have shown poor clinical efficacy, including both engineered antibodies and TRAIL, the natural ligand for this receptor. Comprehensive studies suggest that there are three major obstacles to success of these agents: 1) potency, 2) delivery, and 3) resistance. We have systematically addressed these challenges by engineering a sustained-release formulation of a highly potent, hexavalent death receptor 5 agonist (DRA), and administering the agonist as a sustained release depot, in combination with rationally nominated targeted drugs that overcome intrinsic resistance to DRAs. To address the need for sustained delivery of therapeutic proteins, we developed injectable depots of DRAs recombinantly fused to thermally responsive elastin-like polypeptide (ELP) biopolymers. The bioactive ELP-DRA fusions undergo temperature-driven phase transition upon subcutaneous injection in vivo, resulting in the formation of a gel-like depot suitable for sustained drug delivery. A single 30 mg/kg injection of the gel-like ELP-DRA depot induced significant tumor regression in Colo205 mouse xenografts. To pinpoint the genetic drivers of CRC resistance to the DRA, we used a gain-of-function ORF screen and a CRISPR/Cas9 knockout screen. The screens identified genes that confer sensitivity to the DRA in resistant CRC cell lines. Over twenty small molecule drugs targeting pathways and proteins identified from the screens were then tested in combination with the DRA to identify highly synergistic combinations using cytotoxicity assays. Clonogenic, time-to-progression, and cell viability assays showed that pharmacological blockade of XIAP, Bcl-XL, and CDK4/6 strongly enhances antitumor activity of DRA in established human CRC cell lines and patient-derived CRC cells. In vivo tumor regression studies demonstrated the potent anti-tumor efficacy of combining inhibitors of XIAP and Bcl-XL with the sustained release formulation of ELP-DRA.By addressing both delivery and resistance issues with our protein engineering and genomics platform, we have overcome the key obstacles to DRA translation as a successful drug in the clinic. Our rational approach elegantly provides optimal protein-small molecule drug combinations that elicit a robust anticancer response, exhibit minimal toxicity, and combat drug resistance.