⁸⁹Zr-radiopharmaceuticals to study whole-body distribution and response to antibody-based cancer immunotherapies

Purpose: Probody therapeutic CX-072 is a protease-activatable antibody that is cross-reactive with murine and human programmed death-ligand 1 (PD-L1). CX-072 can be activated in vivo by proteases present in the tumor microenvironment, thereby potentially reducing peripheral, anti–PD-L1-mediated toxicities. To study its targeting of PD-L1–expressing tissues, we radiolabeled CX-072 with the PET isotope zirconium-89 ( 89 Zr). Experimental Design: 89 Zr-labeled CX-072, nonspecific Probody control molecule (PbCtrl) and CX-072 parental antibody (CX-075) were injected in BALB/c nude mice bearing human MDA-MB-231 tumors or C57BL/6J mice bearing syngeneic MC38 tumors. Mice underwent serial PET imaging 1, 3, and 6 days after intravenous injection (pi), followed by ex vivo biodistribution. Intratumoral 89 Zr-CX-072 distribution was studied by autoradiography on tumor tissue sections, which were subsequently stained for PD-L1 by IHC. Activated CX-072 species in tissue lysates were detected by Western capillary electrophoresis. Results: PET imaging revealed 89 Zr-CX-072 accumulation in MDA-MB-231 tumors with 2.1-fold higher tumor-to-blood ratios at 6 days pi compared with 89 Zr-PbCtrl. Tumor tissue autoradiography showed high 89 Zr-CX-072 uptake in high PD-L1–expressing regions. Activated CX-072 species were detected in these tumors, with 5.3-fold lower levels found in the spleen. Furthermore, 89 Zr-CX-072 uptake by lymphoid tissues of immune-competent mice bearing MC38 tumors was low compared with 89 Zr-CX-075, which lacks the Probody design. Conclusions: 89 Zr-CX-072 accumulates specifically in PD-L1–expressing tumors with limited uptake in murine peripheral lymphoid tissues. Our data may enable clinical evaluation of 89 Zr-CX-072 whole-body distribution as a tool to support CX-072 drug development (NCT03013491). We performed a PET imaging study in murine models with 89 Zr-labeled CX-072 to reveal its whole-body distribution. Also, we compared 89 Zr-CX-072 targeting of tumor and lymphoid tissues in both an immune-compromised and an immune-competent setting. To enable clinical PET imaging of 89 Zr-CX-072 distribution to tumor and lymphoid tissues in patients, we characterized and developed a good manufacturing practice (GMP)–compliant tracer. These irAEs may be caused by immune checkpoint blockade in healthy tissues, and immune checkpoint–inhibiting antibodies with tumor-restricted activity are therefore of interest. Recently, imaging of 89 Zr-atezolizumab whole-body distribution in patients with cancer showed high uptake in healthy lymphoid tissues, including spleen, lymph nodes, and Waldeyer's ring. Our preclinical imaging study in mice reveals anti– PD-L1 Probody therapeutic CX-072 is preferentially activated in tumors, followed by PD-L1–mediated uptake, whereas accumulation in spleen and other PD-L1–expressing peripheral lymphoid tissues is limited. These findings demonstrate CX-072 may reduce anti–PD-L1-mediated toxicities in healthy tissues, thereby potentially expanding its use in combination therapies. We developed and characterized clinical grade 89 Zr-CX-072, which is currently studied in patients as part of a phase I/II clinical trial (NCT03013491) to support CX-072 drug development. internalization in MDA-MB-231 and MC38 cell lines 89 Zr-CX-075 was used for internalization experiments because it acts as a surrogate for potential internalization of 89 Zr-CX-072 after removal of its masking peptide by tumor-associated proteases. Internalization in MDA-MB-231 and MC38 cells was assessed by adding 50 ng 89 Zr-CX-075 (200 MBq/mg) to 1×10 6 cells. For control of binding without internalization, cells were incubated for 1 hour on ice. Cells were subsequently washed with ice-cold 1% human serum albumin (Sanquin) in PBS. Total PD-L1–bound 89 Zr-CX-075 was determined by measuring cell-associated activity in a calibrated well-type gamma counter (LKB instruments) followed by incubation for 1 and 2 hours at 37 °C, whereas controls were kept on ice. Cells were stripped of cell-surface–bound antibody using 0.05 mol/L glycine and 0.1 mol/L sodium chloride (pH 2.8) and subsequently counted for remaining cell-associated activity, ie, internalized PD-L1– bound 89 Zr-CX-075, in a calibrated well-type gamma counter. Internalization was expressed as percentage of total PD-L1–bound 89 Zr-CX-075, corrected for internalization in 4 °C controls. PD-L1–expressing xenografts limited uptake in lymphoid hypothesis that CX-072 anti–PD-L1-mediated toxicities in