Abstract LB170: AND-gate dual-targeting EGFR and PD-L1 with a split prodrug converter enzyme for cancer treatment
Authors: Mengrou Lu, Yunhui Xing, Keng-Jung Lee, Xi Ren, Justin R. Pritchard, Boyle C. Cheng, Hongzhang He, Siyang Zheng
Journal: Cancer Research (2024)

Abstract Conventional cancer chemotherapies, while effective, often encounter "on-target, off-tissue" cytotoxicity linked to target prevalence. Although antibody-drug conjugates (ADCs) offer improved targeting, they face limitations in drug payload capacity. Therefore, a potent chemotherapy without off-tissue effects or payload limitations is in crucial need. To address the demand, we developed an AND-gate-based dual-targeting approach that minimizes off-tissue cytotoxicity with tune-able payload, exploiting a pair of split human simplex virus thymidine kinase (HSV-TK), which converts the prodrug ganciclovir (GCV) into a cytotoxic form with high specificity. We first successfully split a pair of HSV-TK (Split TK) to exhibit the possibility of an AND-gate drug activation system. HSV-TK was split into N-fragment (nTK) and C-fragment (cTK) with a 20 amino acid overlap, transduced into HEK293T cells, and treated with ganciclovir for 72 hours. Cells expressing one TK half showed IC50 &amp;gt;1000 µM, while co-expression of cTK and nTK reduced IC50 to ~3 µM, which is comparable to full-length TK. Successful splitting of HSV-TK demonstrated the feasibility of dual-targeting. We then validated this AND-gate targeting in cancer treatment. Split TK[HH1] was assessed in treatment of triple-negative breast cancer (TNBC) cells. TNBC lacks the overexpression of common breast cancer targets, which poses a significant challenge in treatment strategies. We designed a specific split TK pair to treat MDA-MD-231 TNBC, with MCF-7 cells as a control. MDA-MB-231 has upregulated epidermal growth factor receptor (EGFR) and programmed death-ligand 1(PD-L1) expression, while MCF-7 is high in EGFR but low in PD-L1. To enable a split TK pairs that selectively reconstituted HSV-TK activity in MDA-MB-231 cell, we choose epidermal growth factor (EGF) as the targeting ligand for EGFR, and PD-1 extracellular domain for PD-L1. We fused nTK to EGF (nTK-EGF), and the extracellular domain of programmed cell death protein 1 (PD-1) to cTK (PD-1-cTK). Fusion proteins were expressed in SHuffle E. coli, purified, and introduced into MDA-MB-231 and MCF-7 cells, together with 100 µM GCV. After 7 days of treatment, individual nTK-EGF and PD-1-cTK treatments with GCV showed minimal cytotoxicity. However, combined treatment of nTK-EGF and PD-1-cTK significantly decreased cell viability in MDA-MB-231 by ~20%, while MCF-7 remained healthy. In summary, we have successfully constructed and validated an AND-gate-based dual-targeting approach that minimizes off-tissue cytotoxicity with tune-able payload for cancer treatment. This versatile AND-gate dual-targeting system has the great potential of a paradigm-shifting strategy of cancer treatment, particularly for the cancers that cannot be treated by targeting a single antigen. The distinct cytotoxicity based on differential antigen expression introduces a novel cancer therapy category to overcome the targeting difficulties of triple-negative breast cancer (TNBC) and is potentially generalizable to other cancers. Citation Format: Mengrou Lu, Yunhui Xing, Keng-Jung Lee, Xi Ren, Justin R. Pritchard, Boyle C. Cheng, Hongzhang He, Siyang Zheng. AND-gate dual-targeting EGFR and PD-L1 with a split prodrug converter enzyme for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB170.