Theranostic Approach Using Radioiodinated Trimethoprim Targeting E. coli Dihydrofolate Reductase in Engineered Cells

Radiopharmaceutical therapy (RPT) has re-emerged as a potent approach for targeting tumors, particularly for the treatment of neuroendocrine tumors and prostate cancer expressing the somatostatin and prostate-specific membrane antigen receptors, respectively. In addition to endogenously expressed proteins specific for these cancers, RPT has been explored using synthetic, engineered expression of proteins in tumor tissues. An early example of this was the use of sodium iodide symporter delivered to tumors using viral vectors, which were subsequently treated with [¹³¹I]NaI. This approach is complicated by the natural uptake in human tissues that express sodium iodide symporter (e.g., the thyroid). However, given the rapid acceleration of gene and cell therapies, expansion and re-exploration of the synthetic, genetically engineered RPT paradigm is warranted. This is especially true for RPT, which can be coupled with PET companion imaging agents. Methods: Here, we developed a β-emitter radiotherapeutic probe, radioiodinated trimethoprim ([¹³¹I]I-TMP), and evaluated its therapeutic potential. In addition, we developed [^124/125I]I-TMP radiotracers for uptake and imaging studies. The selective cytotoxicity of [¹³¹I]I-TMP toward E. coli dihydrofolate reductase (eDHFR)-expressing cells was evaluated using time- and dose-dependent responses. Biodistribution was characterized in healthy mice followed by small-animal PET/CT studies using a tumor xenograft model and [¹²⁴I]I-TMP. Finally, the eDHFR synthetic RPT approach was applied in murine cancer models to evaluate its cytotoxicity in tumors. Results: Radioiodinated trimethoprim radiotracers ([^131/125/124I]I-TMP) exhibited selective uptake in eDHFR-positive tumors both in vitro and in vivo. Small-animal imaging with [¹²⁴I]I-TMP demonstrated specific retention in I45-eDHFR tumors with negligible background signals. A dose-dependent and time-dependent cytotoxic effect was observed selectively in eDHFR cell lines. Furthermore, targeted treatment with [¹³¹I]I-TMP led to a significant reduction in tumor volume expressing eDHFR compared with wild-type tumors or untreated controls. Conclusion: This synthetic RPT approach shows promise for future applications in targeted cancer therapies and genetic medicine, particularly in the realm of theranostic strategies that integrate trimethoprim-based companion imaging and radiotherapy for the treatment of cancer.