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

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.