Probody Therapeutic Design of ⁸⁹Zr-CX-072 Promotes Accumulation in PD-L1-Expressing Tumors Compared to Normal Murine Lymphoid Tissue.

Abstract

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 <i>in vivo</i> 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 (⁸⁹Zr). ⁸⁹Zr-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 <i>ex vivo</i> biodistribution. Intratumoral ⁸⁹Zr-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. PET imaging revealed ⁸⁹Zr-CX-072 accumulation in MDA-MB-231 tumors with 2.1-fold higher tumor-to-blood ratios at 6 days pi compared with ⁸⁹Zr-PbCtrl. Tumor tissue autoradiography showed high ⁸⁹Zr-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, ⁸⁹Zr-CX-072 uptake by lymphoid tissues of immune-competent mice bearing MC38 tumors was low compared with ⁸⁹Zr-CX-075, which lacks the Probody design. ⁸⁹Zr-CX-072 accumulates specifically in PD-L1-expressing tumors with limited uptake in murine peripheral lymphoid tissues. Our data may enable clinical evaluation of ⁸⁹Zr-CX-072 whole-body distribution as a tool to support CX-072 drug development (NCT03013491).