The programmed cell death ligand 1 (PD-L1) participates in an immune checkpoint system involved in preventing autoimmunity. PD-L1 is expressed on tumor cells, tumor-associated macrophages, and other cells in the tumor microenvironment. Anti-PD-L1 antibodies are active against a variety of cancers, and combined anti-PD-L1 therapy with external beam radiotherapy has been shown to increase therapeutic efficacy. PD-L1 expression status is an important indicator of prognosis and therapy responsiveness, but methods to precisely capture the dynamics of PD-L1 expression in the tumor microenvironment are still limited. In this study, we developed a murine anti-PD-L1 antibody conjugated to the radioactive isotope Indium-111 (111In) for imaging and biodistribution studies in an immune-intact mouse model of breast cancer. The distribution of 111In-DTPA-anti-PD-L1 in tumors as well as the spleen, liver, thymus, heart, and lungs peaked 72 hours after injection. Co-injection of labeled and 100-fold unlabeled antibody significantly reduced spleen uptake at 24 hours, indicating that an excess of unlabeled antibody effectively blocked PD-L1 sites in the spleen, thus shifting the concentration of 111In-DTPA-anti-PD-L1 into the blood stream and potentially increasing tumor uptake. Clearance of 111In-DTPA-anti-PD-L1 from all organs occurred at 144 hours. Moreover, dosimetry calculations revealed that radionuclide-labeled anti-PD-L1 antibody yielded tolerable projected marrow doses, further supporting its use for radiopharmaceutical therapy. Taken together, these studies demonstrate the feasibility of using anti-PD-L1 antibody for radionuclide imaging and radioimmunotherapy, and highlight a new opportunity to optimize and monitor the efficacy of immune checkpoint inhibition therapy.

Alpha-particle emitters have a high linear energy transfer and short range, offering the potential for treating micrometastases while sparing normal tissues. We developed a urea-based, 211 At-labeled small molecule targeting prostate-specific membrane antigen (PSMA) for the treatment of micrometastases due to prostate cancer (PC). Methods: PSMA-targeted (2S)-2-(3-(1-carboxy-5-(4-211 At-astatobenzamido) pentyl)ureido)-pentanedioic acid ( 211 At-6) was synthesized. Cellular uptake and clonogenic survival were tested in PSMA-positive (PSMA1) PC3 PIP and PSMA-negative (PSMA−) PC3 flu human PC cells after 211 At-6 treatment. The antitumor efficacy of 211 At-6 was evaluated in mice bearing PSMA1 PC3 PIP and PSMA-PC3 flu flank xenografts at a 740-kBq dose and in mice bearing PSMA1, luciferase-expressing PC3-ML micrometastases. Biodistribution was determined in mice bearing PSMA1 PC3 PIP and PSMA-PC3 flu flank xenografts. Suborgan distribution was evaluated using α-camera images, and microscale dosimetry was modeled. Longterm toxicity was assessed in mice for 12 mo. Results: 211 At-6 treatment resulted in PSMA-specific cellular uptake and decreased clonogenic survival in PSMA1 PC3 PIP cells and caused significant tumor growth delay in PSMA1 PC3 PIP flank tumors. Significantly improved survival was achieved in the newly developed PSMA1 micrometastatic PC model. Biodistribution showed uptake of 211 At-6 in PSMA1 PC3 PIP tumors and in kidneys. Microscale kidney dosimetry based on α-camera images and a nephron model revealed hot spots in the proximal renal tubules. Long-term toxicity studies confirmed that the dose-limiting toxicity was late radiation nephropathy. Conclusion: PSMA-targeted 211 At-6 α-particle radiotherapy yielded significantly improved survival in mice bearing PC micrometastases after systemic administration. 211 At-6 also showed uptake in renal proximal tubules resulting in late nephrotoxicity, highlighting the importance of long-term toxicity studies and microscale dosimetry.

Targeted radiopharmaceutical therapy (TRT) using α-particle radiation is a promising approach for treating both large and micrometastatic lesions. We developed prostate-specific membrane antigen (PSMA)-targeted low-molecular-weight agents for 212 Pb-based TRT of patients with prostate cancer (PC) by evaluating the matching γ-emitting surrogate, 203 Pb. Methods: Five rationally designed low-molecular-weight ligands (L1-L5) were synthesized using the lysine-urea-glutamate scaffold, and PSMA inhibition constants were determined. Tissue biodistribution and SPECT/CT imaging of 203 Pb-L1-203 Pb-L5 were performed on mice bearing PSMA(1) PC3 PIP and PSMA(−) PC3 flu flank xenografts. The absorbed radiation dose of the corresponding 212 Pb-labeled analogs was determined using the biodistribution data. Antitumor efficacy of 212 Pb-L2 was evaluated in PSMA(1) PC3 PIP and PSMA(−) PC3 flu tumor models and in the PSMA(1) luciferase-expressing micrometastatic model. 212 Pb-L2 was also evaluated for dose-escalated, long-term toxicity. Results: All new ligands were obtained in high yield and purity. PSMA inhibitory activities ranged from 0.10 to 17 nM. 203 Pb-L1-203 Pb-L5 were synthesized in high radiochemical yield and specific activity. Whole-body clearance of 203 Pb-L1-203 Pb-L5 was fast. The absorbed dose coefficients (mGy/kBq) of the tumor and kidneys were highest for 203 Pb-L5 (31.0, 15.2) and lowest for 203 Pb-L2 (8.0, 4.2). The tumor-to-kidney absorbed dose ratio was higher for 203 Pb-L3 (3.2) and 203 Pb-L4 (3.6) than for the other agents, but with lower tumor-to-blood ratios. PSMA(1) tumor lesions were visualized through SPECT/CT as early as 0.5 h after injection. A proof-of-concept therapy study with a single administration of 212 Pb-L2 demonstrated dose-dependent inhibition of tumor growth in the PSMA(1) flank tumor model. 212 Pb-L2 also demonstrated an increased survival benefit in the micrometastatic model compared with 177 Lu-PSMA-617. Long-term toxicity studies in healthy, immunocompetent CD-1 mice revealed kidney as the dose-limiting organ. Conclusion: 203 Pb-L1-203 Pb-L5 demonstrated favorable pharmacokinetics for 212 Pb-based TRT. The antitumor efficacy of 212 Pb-L2 supports the corresponding 203 Pb/ 212 Pb theranostic pair for PSMA-based α-particle TRT in advanced PC.

Programmed cell death ligand 1 (PD-L1) is part of an immune checkpoint system that is essential for preventing autoimmunity and cancer. Recent approaches in immunotherapy that target immune checkpoints have shown great promise in a variety of cancers, including metastatic melanoma. The use of targeted molecular imaging would help identify patients who will best respond to anti-PD-L1 treatment while potentially providing key information to limit immune-related adverse effects. Recently, we developed an antibody-based PD-L1-targeted SPECT agent-In-diethylenetriaminepentaacetic acid (DTPA)-anti-PD-L1-to identify PD-L1-positive tumors in vivo. To best use such PD-L1-targeted imaging agents, it is important, as a first step, to understand how the signal is affected by different parameters. We evaluated the impact of protein concentration on the distribution ofIn-DTPA-anti-PD-L1 in a murine model of aggressive melanoma. In-DTPA-anti-PD-L1 (dissociation constant, 0.6 ± 0.1 nM) demonstrated increased uptake in B16F10 tumors at protein concentrations equaling or exceeding 1 mg/kg at 24 h and 3 mg/kg at 72 h. At 24 h, the PD-L1-rich spleen and lungs demonstrated decreasing uptake with increasing protein concentration. At 72 h, uptake in the thymus was significantly increased at protein concentrations of 3 mg/kg or greater. Both time points demonstrated increased tracer amounts remaining in circulation as the amount of cold antibody was increased. These studies demonstrate that In-DTPA-anti-PD-L1 is capable of identifying tumors that overexpresses PD-L1 and monitoring the impact of PD-L1-rich organs on the distribution of anti-PD-L1 antibodies.

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