⁶⁸Ga/¹⁶¹Tb-CP2 as a Paired Radiopharmaceutical Targeting KDM4A and the Preliminary Verification in NCI-H2228...

Objective: To address the unmet clinical need for Lysine (K)-specific demethylase 4A (KDM4A)-targeted therapies in nonsmall cell lung cancer (NSCLC), we developed a novel theranostic pair, ⁶⁸Ga/¹⁶¹Tb-CP2, leveraging the epigenetic regulator KDM4A and terbium-161's high linear energy transfer (LET) Auger electrons for precision therapy. Methods: The radiopharmaceutical was synthesized via a one-step chelation protocol. Radiochemical purity and stability were validated by radio-HPLC and radio-TLC. In vitro studies evaluated the nuclear targeting ability of ¹⁶¹Tb-CP2 by cellular uptake and cellular internalization, and quantified tumor cell proliferation by carboxyfluorescein diacetate, succinimidyl ester immunofluorescence and flow cytometry. In vivo studies used NCI-H2228 xenografts for ⁶⁸Ga-CP2 micro-PET/CT imaging and ¹⁶¹Tb-CP2 micro-SPECT/CT biodistribution analysis. The DNA damage was assessed by γ-H2AX immunohistochemistry. Results: We successfully synthesized a paired diagnostic and therapeutic radiopharmaceutical specifically targeting KDM4A⁶⁸Ga/¹⁶¹Tb-CP2. The preparation protocol is simple, with the radiochemical purity of the radiopharmaceutical exceeding 97%, and the quality control results meet the requirements for stability and specific activity. Cellular uptake and cellular internalization showed that tumor cells were able to efficiently uptake ¹⁶¹Tb-CP2 and transport ¹⁶¹Tb-CP2 into the nucleus to exert its killing effect. ¹⁶¹Tb-CP2 inhibited tumor cell proliferation (mean fluorescence intensity: 100.00 ± 0.35% vs 125.77 ± 1.93%, control vs 48 h group, P < 0.001) at the cellular level. The NCI-H2228-bearing NSCLC model demonstrated high tumor uptake of ⁶⁸Ga-CP2 (SUV_max of 0.26 ± 0.06). Micro-SPECT/CT revealed that ¹⁶¹Tb-CP2 is metabolized mainly through the kidney-bladder pathway, has moderate uptake (0.29 ± 0.01%ID/g at 12 h) and retention rates (0.34 ± 0.01%ID/g at 72 h) in tumors, and has a high target/nontarget ratio (3.4 ± 0.1 at 72 h). Immunohistochemistry confirmed the tumor-killing effect of ¹⁶¹Tb-CP2 and its safety in normal tissues. Conclusions: ⁶⁸Ga/¹⁶¹Tb-CP2 has transformative value for the evaluation and treatment of KDM4A-overexpressing tumors. This KDM4A-targeting radiopharmaceutical provides new directions for tumor research and new ideas for tumor management that use the Auger electron of Tb-161 to target genetic material.