Synthesis and Evaluation of 177Lu-DOTA-PD-L1-i and 225Ac-HEHA-PD-L1-i as Potential Radiopharmaceuticals for Tumor Microenvironment-Targeted Radiotherapy

Abstract

Current cancer therapies focus on reducing immunosuppression and remodeling the tumor microenvironment to inhibit metastasis, cancer progression, and therapeutic resistance. Programmed death receptor 1 (PD-1) is expressed on immune T cells and is one of the so-called checkpoint proteins that can suppress or stop the immune response. To evade the immune system, cancer cells overexpress a PD-1 inhibitor protein (PD-L1), which binds to the surface of T cells to activate signaling pathways that induce immune suppression. This research aimed to synthesize PD-L1 inhibitory peptides (PD-L1-i) labeled with lutetium-177 (¹⁷⁷Lu-DOTA-PD-L1-i) and actinium-225 (²²⁵Ac-HEHA-PD-L1-i) and to preclinically evaluate their potential as radiopharmaceuticals for targeted radiotherapy at the tumor microenvironment level. Using PD-L1-i peptide as starting material, conjugation with HEHA-benzene-SCN and DOTA-benzene-SCN was performed to yield DOTA-PD-L1-i and HEHA-PD-L1-I, which were characterized by FT-IR, UV-vis spectroscopy, and HPLC. After labeling the conjugates with ²²⁵Ac and ¹⁷⁷Lu, cellular uptake in HCC827 cancer cells (PD-L1 positive), conjugate specificity evaluation by immunofluorescence, radiotracer effect on cell viability, biodistribution, biokinetics, and assessment of radiation absorbed dose in mice with in duced lung micrometastases were performed. ²²⁵Ac-HEHA-PD-L1-i and ¹⁷⁷Lu-DOTA-PD-L1-i, obtained with radiochemical purities of 95 ± 3% and 98.5 ± 0.5%, respectively, showed in vitro and in vivo specific recognition for the PD-L1 protein in lung cancer cells and high uptake in HCC287 lung micrometastases (>30% ID). The biokinetic profiles of ¹⁷⁷Lu-DOTA-PD-L1-i and ²²⁵Ac-DOTA-PD-L1-i showed rapid blood clearance with renal and hepatobiliary elimination and no accumulation in normal tissues. ²²⁵Ac-DOTA-PD-L1-i produced a radiation dose of 5.15 mGy/MBq to lung micrometastases. In the case of ¹⁷⁷Lu-DOTA-PD-L1-i, the radiation dose delivered to the lung micrometastases was ten times (43 mGy/MBq) that delivered to the kidneys (4.20 mGy/MBq) and fifty times that delivered to the liver (0.85 mGy/MBq). Therefore, the radiotherapeutic PD-L1-i ligands of ²²⁵Ac and ¹⁷⁷Lu developed in this research could be combined with immunotherapy to enhance the therapeutic effect in various types of cancer.

Keywords: PD-L1 radioinhibitors; actinium-225; lutetium-177; targeted radiotherapy.