Abstract 1532: A novel, monovalent tri-specific antibody-based molecule that simultaneously modulates PD-L1 and 4-1BB exhibits potent anti-tumoral activity in vivo
Authors: Gunde Tea , Brock Matthias , Warmuth Stefan , Simonin Alexandre , Hess Christian , Oswald Eva , Tietz Julia , Zeberer Julia , Mahler Dana , Muntwiler Simone , Küttner Benjamin , Wickihalder Belinda , Pölderl Antonia , Diem Dania , Beltrametti Teddy , Heiz Robin , Meyer Sebastian , Egan Timothy , Urech David 
Published: August 2019

Abstract
The combined immunomodulation of PD-L1/PD-1 and 4-1BB is considered a promising strategy to increase response rates among cancer patients who are eligible to receive PD-L1/PD-1 inhibitors. Unfortunately, encouraging pre-clinical results achieved with such regimens have not yet translated into durable clinical success, due to addition of 4-1BB-agonistic antibodies being either intolerable at effective doses or ineffective, despite tolerability, at all doses. To eliminate this safety/efficacy tradeoff, we engineered a novel, tri-specific immunomodulatory drug candidate. The molecule consists of three monovalent antibody Fvs - specific for PD-L1, serum albumin (SA) and 4-1BB - fused in a single chain (a PD-L1/4-1BB/SA tri-specific scDb-scFv). The monovalent and Fc-less structure of the molecule ensures that 4-1BB agonism is conditional upon drug-mediated formation of an immunological synapse between PD-L1+ cells and 4-1BB+ cells, thereby restricting costimulation of 4-1BB+ cells to the tumor microenvironment (TME). Therefore, the scDb-scFv molecule avoids the extratumoral costimulation of immune cells that is believed to cause the dose-limiting toxicities that arise from therapeutic 4-1BB agonism. Meanwhile, the αSA domain extends the molecule’s serum half-life and is expected to promote delivery to the TME. In the present study, we demonstrate that a novel PD-L1/4-1BB/SA tri-specific scDb-scFv potently blocks PD-L1/PD-1 signaling and elicits T cell costimulation solely in the presence of PD-L1+ cells. In in vitro experiments, the scDb-scFv molecule exhibits a greater capacity to costimulate T cells than combinations of clinical stage α4-1BB and αPD-L1/PD-1 IgGs. Moreover, in contrast to ADCC-enabled αPD-L1 IgGs, the Fc-less scDb-scFv spared CD11c+ monocytes from depletion. We also demonstrate that the carefully balanced relative affinity between the molecule’s component αPD-L1 and α4-1BB Fvs maximizes its pharmacological activity and avoids bell-shaped dose-response curves. Next, in vivo efficacy was demonstrated in two xenograft models - HCC827 NCSLC and HCC1954 breast carcinoma - using humanized mice. While equally effective at slowing tumor progression in vivo, the PD-L1/4-1BB/SA tri-specific scDb-scFv was better tolerated, and was more pro-proliferative vis-à-vis intratumoral CD8+ T cells, than combined αPD-L1 and α4-1BB IgGs. In PK/PD studies in cynomologus monkeys, we confirmed the tolerability, pharmacological activity and extended serum half-life of the scDb-scFv molecule. Finally, the molecule was produced from stable CHO cells at high titers (comparable to IgGs) and exhibits outstanding stability characteristics. In conclusion, our data confirm the successful development of a novel therapeutic that is designed to unlock the full potential of combined immunomodulation and overcome its present limitations.
Citation Format: Tea Gunde, Matthias Brock, Stefan Warmuth, Alexandre Simonin, Christian Hess, Eva Oswald, Julia Tietz, Julia Zeberer, Dana Mahler, Simone Muntwiler, Benjamin Küttner, Belinda Wickihalder, Antonia Pölderl, Dania Diem, Teddy Beltrametti, Robin Heiz, Sebastian Meyer, Timothy Egan, David Urech. A novel, monovalent tri-specific antibody-based molecule that simultaneously modulates PD-L1 and 4-1BB exhibits potent anti-tumoral activity in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1532.