Preclinical evaluation of ⁸⁹Zr-Df-radiolabeled bispecific anti-PD-L1/TGF-βRII fusion protein bintrafusp alfa

Introduction: Bintrafusp alfa (M7824) is a first in class bifunctional fusion protein allowing simultaneous binding of PD-L1 and trapping of transforming growth factor beta (TGF-β). In preparation of a clinical bioimaging trial, this study aimed to generate ⁸⁹Zr-labeled M7824 investigational drug product and controls, and to perform the in vitro and in vivo characterization of ⁸⁹Zr-Df-M7824 and ⁸⁹Zr-Df-control radioconjugates. Methods: Bintrafusp alfa (anti-PD-L1 human IgG1 antibody fused to TGF-β receptor II (TGF-βRII)), avelumab (anti-PD-L1 human IgG1 control antibody), isotype control (mutated inactive anti-PD-L1 IgG1 control antibody), and trap control (mutated inactive anti-PD-L1 human IgG1 fused to active TGF-βRII) were chelated with p-isothiocyanatobenzyl-desferrioxamine (Df). After radiolabeling with zirconium-89 (⁸⁹Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. In vivo biodistribution studies were performed at low protein dose (5 μg) to identify tissue distribution and ⁸⁹Zr-Df-bintrafusp alfa tumor uptake in a PD-L1/TGF-β positive murine breast cancer model (EMT-6). A combined biodistribution and imaging experiment was performed at increased protein dose (35 μg) with or without the presence of competing cold bintrafusp alfa (1 mg/kg). Results: Conditions for the Df-conjugation and ⁸⁹Zr-radiolabeling of M7824 and controls were established. PD-L1 binding and trapping of TGF-β ligands by M7824 was maintained after radiolabeling. At 5 µg ⁸⁹Zr-Df-M7824 (5.80 µCi) dose level, rapid blood clearance was observed with highest uptake seen in lung, spleen, liver and kidneys. Tissue distribution of 5 µg ⁸⁹Zr-Df-M7824 (5.80 µCi) was also compared to control antibodies (5 µg ⁸⁹Zr-Df-avelumab, 5 µg ⁸⁹Zr-Df-trap control, 5 µg ⁸⁹Zr-Df-Isotype control) on day 2 and 7 post injection. A combined PET imaging and biodistribution study showed that by increasing the amount of unlabeled protein, ⁸⁹Zr-Df-M7824 uptake in lung and spleen was reduced and tumor uptake of ⁸⁹Zr-Df-M7824 was increased. These results indicate that the biodistribution pattern of M7824 molecule is mainly driven by the PD-L1 moiety of the molecule, allowing targeted binding to PD-L1 in the tumor microenvironment and colocalized simultaneous inhibition of PD-L1 and TGF-β. Conclusion: We have been able to successfully establish the radiolabeling conditions of ⁸⁹Zr-labeled M7824. Our results demonstrate that ⁸⁹Zr-labeled M7824 is now suitable for use in a bioimaging clinical trial study of ⁸⁹Zr-M7824 PET scans in patients with advanced or metastatic NSCLC receiving M7824 alone or in combination with chemotherapy. Acknowledgements: This work was supported by funding from EMD Serono Research & Development Institute, Inc.; a business of Merck KGaA, Darmstadt, Germany; and the Australian Cancer Research Foundation for providing funds to purchase the nanoPET/MRI and nanoSPECT/CT imaging equipment. This research was also undertaken using the Solid Target Laboratory, an ANSTO-Austin-LICR Partnership. The support of the Operational Infrastructure Support Program of the Victorian State Government; and AMS (NHRMC Investigator Fellowship 11778378) is acknowledged.

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