# Optimized Monothiol Thioredoxin Derivative (ORP100S) Protects In Vitro and In Vivo from Radiation and Chemotoxicity Without Promoting Tumor Proliferation

## TL;DR

A modified version of a natural protein, ORP100S, protects cells from radiation and chemotherapy damage without encouraging cancer growth.

## Contribution

ORP100S is a novel engineered thioredoxin variant that provides cell protection without promoting tumor proliferation.

## Key findings

- ORP100S shows improved pharmacokinetic and pharmacodynamic properties compared to native TRX.
- ORP100S protects hematopoietic stem cells from chemotherapy toxicity without promoting cancer cell survival.
- ORP100S modulates the KLF4-p53 pathway to selectively inhibit ferroptosis in non-cancer stem cells.

## Abstract

Human thioredoxin‐1 (TRX) is a target‐selective disulfide reductase with antioxidant, anti‐inflammatory, and regulatory functions that mitigates cellular stresses in various organ systems, providing a compelling rationale for therapeutic use as a broad‐spectrum cell protectant. However, clinical application of recombinant TRX (rhTRX) is constrained by rapid clearance and proliferative intracellular activity. To overcome these limitations, a rationally designed TRX variant, ORP100S, was engineered for enhanced stability, prolonged extracellular target engagement, and improved protective function, with development of novel single‐turnover insulin reduction and hybrid‐immunocapture LC‐MS assays. ORP100S demonstrates high‐yield expression in E. coli (16 g L−1) and exhibits significant in vivo mitigating effects when administered subcutaneously to rodents and non‐human primates exposed to otherwise‐lethal total‐body ionizing radiation. Compared to native TRX, ORP100S displays improved pharmacokinetic and pharmacodynamic properties without promoting murine or human cancer cell proliferation. Additionally, ORP100S protects hematopoietic stem/progenitor cells (HSPCs) from chemotherapy‐induced toxicity in vitro and in vivo synergistically with co‐administered granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Mechanistic studies revealed that ORP100S modulates the Kruppel‐like factor 4 (KLF4)‐p53 pathway to selectively inhibit ferroptosis in HSPCs but not cancer cells. These findings highlight the potential of ORP100S as a novel therapeutic agent for mitigating acute radiation injury and improving the safety and efficacy of chemotherapy without compromising antitumor activity.

Native thioredoxin‐1 (TRX) stimulates proliferation and rescues stem cells and cancer cells from multiple stressors by suppressing p53 and inhibiting ferroptosis via GPX4/SLC7A11 upregulation mediated by enhanced KLF4 expression and p53 promoter binding. In contrast, engineered monothiol TRX ORP100S inhibits p53 and ferroptosis selectively in non‐cancer stem cells, mitigating off‐target cell death without promoting cancer chemo/radiation resistance.