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CD137 (4-1BB) is a transmembrane costimulatory receptor on T and NK cells that enhances adaptive immune responses and is a critical mediator of antitumor immunity. CD137 signaling requires receptor clustering normally facilitated by the trimeric CD137 ligand (CD137L). Alternatively, CD137 signaling can be triggered either directly by agonistic monoclonal antibodies (mAbs) or indirectly via crosslinking of CD137 binding mAbs by Fcγ receptors on neighboring cells. The development of CD137 targeted agents for cancer therapy has been hampered by on-target off-tumor toxicity in the case of agonist, monospecific, bivalent mAbs or limited antitumor activity in the case of crosslinking mAbs. To address the issues of toxicity and efficacy a highly selective and potent CD137xPD-L1 bispecific antibody (bAb) was identified by applying an unbiased functional screening approach. Collections of common light chain Fabs recognizing CD137 and PD-L1 were produced based on antibody panels from immunized MeMo® mice. A large and diverse panel of CD137xPD-L1 bAbs was then produced by combining different CD137 and PD-L1 Fabs based on epitope and sequence diversity in the IgG1 Biclonics® format. The bAbs were screened for activity in reporter cell lines expressing the receptors. This unbiased combinatorial screening identified a CD137xPD-L1 bAb (MCLA-145) for which CD137 mediated activation is dependent on the presence of PD-L1 on a neighboring cell and, as such, the antibody acts in ‘trans’. Flow cytometry experiments demonstrated that MCLA-145 is fully cross-reactive to cynomolgus monkey CD137 and PD-L1. The CD137 Fab arm blocks the interaction of CD137 with CD137L as demonstrated in a competition assay by flow cytometry. The PD-L1 Fab arm blocks the interaction between PD-1 and PD-L1 as demonstrated in ELISA. Binding epitopes were mapped by shotgun mutagenesis using a flow-based screen. In addition, hydrogen-deuterium exchange experiments were performed to map the binding domain on CD137. Data show that MCLA-145 binds the ligand binding domain of CD137 domain (CRDII). The PD-L1 Fab arm binds PD-L1 in the PD-1 binding N-terminal V domain. Both epitope mapping data sets are consistent with the CD137 and PD-L1 ligand blocking activity of MCLA-145. Monovalent binding affinities were measured by surface plasma resonance (SPR) and radioactive iodine labeling and demonstrated affinities in the low nM (CD137) and subnanomolar (PD-L1) range. SPR experiments also confirmed that MCLA-145 was able to bind simultaneously to both CD137 and PD-L1 recombinant proteins. The unique binding properties of MCLA-145 may result in an increased therapeutic window by specifically activating CD137 expressing cells in the tumor niche where PD-L1 is expressed while simultaneously blocking inhibitory input from the PD-1/PD-L1 axis. Citation Format: Cecile A. Geuijen, Paul Tacken, Rinse Klooster, Horacio Nastri, Shaun Stewart, Jing Zhou, Steve Wang, Cheng-Yen Huang, Arjen Kramer, Linda Kaldenberg-Hendriks, John de Kruif, Renate den Blanken-Smit, Vanessa Zondag-van de Zande, Abdul Basmeleh, Willem Bartelink, Patrick Mayes, Gregory Hollis, Reid Huber, Mark Throsby. An unbiased screen identifies a CD137xPD-L1 bispecific IgG1 antibody with unique T cell activation and binding properties [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 541.

Background: MCLA-128 is an ADCC-enhanced humanized common light chain bispecific IgG1 antibody that targets the HER2:HER3 dimer with nanomolar affinity, potently inhibiting tumor growth in vitro and in vivo. MCLA-128 shows superior activity to the combination trastuzumab/ pertuzumab and HER3 targeting monoclonal antibodies and is currently being evaluated in a Phase I clinical trial. This study investigated the mechanism of action of MCLA-128. Methods: Phosphorylation of HER receptors and downstream signaling molecules was studied in vitro and in vivo on HER2-amplified cancer cell lines by Pathscan arrays, luminex beads and Western blot analysis. Inhibition of MCLA-128 cell growth in combination with tyrosine kinase inhibitors and small molecules targeting the MAPK and PI3 kinase/Akt pathway was determined by proliferation inhibition and high content imaging assays. The potential effect of MCLA-128 on primary cardiomyocytes in the presence of Doxorubicin was analyzed by measuring ATP. Binding of MCLA-128 to a panel of cell lines in comparison to HER2 and HER3 antibodies was determined by FACS. Results: In contrast to other HER2 and HER3 targeted agents, only MCLA-128 inhibited phosphorylation of HER3 and downstream Akt and ERK in HER2 amplified cell lines cultured with high concentrations of heregulin in vitro. In xenograft studies, growth inhibition of the trastuzumab-resistant cell line JIMT-1 by MCLA-128 was correlated with reduced HER2:HER3 dimerization and a profound inhibition of the PI3K pathway. Synergistic growth inhibition in vitro was observed when tyrosine kinase inhibitors or inhibitors of the PI3K pathway were added to HER2 amplified cancer cells in the presence of MCLA-128. MCLA-128 did not show any evidence of cardiotoxicity in vitro in contrast to trastuzumab. MCLA-128 binds and coats breast cancer cell lines with differing levels of HER2 expression more efficiently in comparison to monospecific HER2 or HER3 monoclonal antibodies. Conclusions: The unique simultaneous targeting of MCLA-128 to HER2 and HER3 on HER2-overexpressing breast cancer cells leads to severe impairment of PI3K signaling and reduced cell growth whereas proliferation of primary cardiomyocytes is unaffected. The enhanced coating effect of MCLA-128 also supports its ADCC activity. Citation Format: Cecile Geuijen, Eric Rovers, Tristan Gallenne, David Maussang-Detaille, Arjen Kramer, Nellie Nieuwenhuizen, Carina Clements, Katinka van Zoest, Roy Nijhuis, Therese Visser, Renate Den Blanken-Smit, Willem Bartelink, Vanessa Zondag-van der Zande, Linda Kaldenberg, Pieter-Fokko van Loo, Rob Roovers, Robert Doornbos, Leo Price, Stefan Braam, Setareh van Driel, Lex Bakker, Ton Logtenberg, John de Kruif, Mark Throsby. Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-261. doi:10.1158/1538-7445.AM2015-LB-261