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 radionuclide 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. Coinjection 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. Cancer Res; 76(2); 472–9. ©2015 AACR.

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—¹¹¹In-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. Methods: We evaluated the impact of protein concentration on the distribution of ¹¹¹In-DTPA-anti-PD-L1 in a murine model of aggressive melanoma. Results: ¹¹¹In-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. Conclusion: These studies demonstrate that ¹¹¹In-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.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein commonly found on the surface of late-stage and metastatic prostate cancer and a well-known imaging biomarker for staging and monitoring therapy. Although ¹¹¹In-labeled capropmab pendetide is the only approved agent available for PSMA imaging, its clinical use is limited because of its slow distribution and clearance that leads to challenging image interpretation. A small-molecule approach using radiolabeled urea-based PSMA inhibitors as imaging agents has shown promise for prostate cancer imaging. The motivation of this work is to explore phosphoramidates as a new class of potent PSMA inhibitors to develop more effective prostate cancer imaging agents with improved specificity and clearance properties. Methods: N-succinimidyl-4-¹⁸F-fluorobenzoate (¹⁸F-SFB) was conjugated to S-2-((2-(S-4-amino-4-carboxybutanamido)-S-2-carboxyethoxy)hydroxyphosphorylamino)-pentanedioic acid (Phosphoramidate (1)), yielding S-2-((2-(S-4-(4-¹⁸F-fluorobenzamido)-4-carboxybutanamido)-S-2-carboxyethoxy)hydroxyphosphorylamino)-pentanedioic acid (3). In vivo studies were conducted in mice bearing either LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors. PET images were acquired at 1 and 2 h with or without a preinjection of a nonradioactive version of the fluorophosphoramidate. Tissue distribution studies were performed at the end of the 2 h imaging sessions. Results: Phosphoramidate (1) and its fluorobenzamido conjugate (2) were potent inhibitors of PSMA (inhibitory concentration of 50% [IC₅₀], 14 and 0.68 nM, respectively). PSMA-mediated tumor accumulation was noted in the LNCaP versus the PC-3 tumor xenografts. The LNCaP tumor uptake was also blocked by the administration of nonradioactive (2) prior to imaging studies. With the exception of the kidneys, tumor-to-tissue and tumor-to-blood ratios were greater than 5:1 at 2 h. The strong kidney uptake may be due to the known PSMA expression in the mouse kidney, because significant reduction (>6-fold) in kidney activity was seen in mice injected with (2). Conclusion: ¹⁸F-labeled phosphoramidate (3) is a representative of a new class of PSMA targeting peptidomimetic molecules that shows great promise as imaging agents for detecting PSMA+ prostate tumors.