Abstract 4544: Multifaceted mechanism of action of DuoHexaBody-CD37 involves both complement- and Fc gamma receptor-mediated cytotoxicity in pre-clinical B-cell lymphoma models
Authors: Kil Laurens P. , Oostindie Simone C. , Strumane Kristin , van der Horst Hilma J. , van Kessel Berris , Overdijk Marije B. , Lingnau Andreas , Brandhorst Marcel , van den Brakel Jeroen , Lindorfer Margaret A. , Taylor Ronald P. , Chamuleau Martine E. , Mutis Tuna , Sasser A. Kate , Schuurman Janine , Parren Paul W. , Beurskens Frank J. , Breij Esther C. 
Published: September 2020

Abstract
CD37 is a tetraspanin expressed on mature B cells where it orchestrates plasma membrane organization, receptor signaling, cell migration and adhesion. As CD37 is abundantly expressed on many mature B cell-derived malignancies, it represents an attractive target for new antibody therapies. DuoHexaBody®-CD37 is a novel biparatopic bispecific CD37 antibody with an Fc domain engineered to enhance antibody hexamerization upon binding to CD37 on the plasma membrane. DuoHexaBody-CD37 was shown to induce potent complement-dependent cytotoxicity (CDC) of malignant B-cell lines and primary chronic lymphocytic leukemia and non-Hodgkin lymphoma patient samples (Oostindie et al., Blood 2018 132:4170; van der Horst et al., Blood 2018 132:4179).
Here, we demonstrate that the DuoHexaBody-CD37 mechanism of action also encompasses FcγR-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Daudi and Raji cells opsonized with DuoHexaBody-CD37 induced FcγRIIa and FcγRIIIa signaling in luciferase reporter assays. In agreement with efficient FcγR engagement, opsonization with DuoHexaBody-CD37 resulted in dose-dependent ADCC and ADCP by human healthy donor peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs), respectively. In a 4-hour Chromium-51 release assay, DuoHexaBody-CD37 induced ADCC in Daudi cells with PMBCs from 11 out of 12 tested donors (average EC50 of 9.87 [±9.98] ng/mL; maximum kill 20.4 [±9.2]%), and in Raji cells with 1 out of 3 donors. Image- and flow cytometry-based assays with MDMs illustrated that in presence of DuoHexaBody-CD37, Daudi cells were efficiently engulfed by effector cells from 3 different donors (average percentage phagocytic macrophages 29.7 ± 7.6%) resulting in target cell depletion (maximum depletion 75.9 [±18.0]%).
Potent anti-tumor activity of DuoHexaBody-CD37 in vivo was reported in a screening approach using B-cell lymphoma patient-derived xenograft (PDX) models with single-mouse treatment groups. Two weekly doses of 5 mg/kg DuoHexaBody-CD37 resulted in strong tumor growth inhibition (tumor stasis or tumor regression) in 3/9 models compared to untreated tumors. Follow-up cohort PDX studies with eight mice per group confirmed potent, dose-dependent anti-tumor activity of DuoHexaBody-CD37 at doses as low as 1 mg/kg.
In summary, DuoHexaBody-CD37 induces efficient tumor cell kill through CDC, ADCC and ADCP in vitro and shows potent anti-tumor activity in B-cell lymphoma PDX models in vivo. These data further strengthen the rationale for exploring the safety and efficacy of DuoHexaBody-CD37 in B-cell malignancy patients.
Citation Format: Laurens P. Kil, Simone C. Oostindie, Kristin Strumane, Hilma J. van der Horst, Berris van Kessel, Marije B. Overdijk, Andreas Lingnau, Marcel Brandhorst, Jeroen van den Brakel, Margaret A. Lindorfer, Ronald P. Taylor, Martine E. Chamuleau, Tuna Mutis, A. Kate Sasser, Janine Schuurman, Paul W. Parren, Frank J. Beurskens, Esther C. Breij. Multifaceted mechanism of action of DuoHexaBody-CD37 involves both complement- and Fc gamma receptor-mediated cytotoxicity in pre-clinical B-cell lymphoma models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4544.