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

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.