The Ublituximab-Umbralisib (U2) Drug Regimen Potentiates the Activity of the Novel CD47-CD19 Bispecific Antibody, TG-1801, through the Activation of the G Protein-Coupled Receptor EBI2/GPR183
Authors: Normant Emmanuel , Ribeiro Marcelo Lima , Profitos-Peleja Nuria , Blecua Pedro , Reyes-Garau Diana , Santos Juliana Carvalho , Armengol Marc , Fernández-Serrano Miranda , Miskin Hari P. , Roue Gael 
Published: November 2021

Abstract
Introduction: TG-1801 is a novel, bispecific anti-CD47 and anti-CD19 fully human IgG1 antibody that targets CD47 selectively on CD19+ B-cells, sparing red blood cells or platelets and blocking the CD47-SIRPα macrophage checkpoint on mature B cells. TG-1801 is currently in clinical trial as a single agent or in combination with ublituximab, a glyco-engineered CD20 antibody, in B-cell non-Hodgkin lymphoma (B-NHL). The doublet therapy of ublituximab with the dual PI3Kδ/CK1e inhibitor umbralisib ("U2" regimen), provides a non-chemotherapy backbone regimen on which several novel multidrug combinations are being explored clinically. Here we explored in vitro and in vivo potential synergies between TG-1801 and ublituximab, umbralisib, and the U2 combination, in preclinical models of B-NHL.
Methods: A panel of n=12 human B-cell lymphoma cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells (BMSCs), M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 +/- U2 treatments was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). In vivo, drug efficacy was determined in a Raji xenograft model, by dosing tumor-bearing mice for 17 days with ublituximab (5mg/kg, qw) +/- umbralisib (150mg/kg, bid), the combination of both and/or TG-1801 (5mg/kg, qw).
Results: Here we show on a panel of lymphoma cells lines that the number of receptors per cell and the ratio CD47/CD19 do not impact TG-1801-mediated ADCC or phagocytosis (ADCP). In addition, we show that TG-1801 potentiated ublituximab-mediated ADCC and ADCP and exhibited a similar additive effect when added to U2 combination. In vivo, ublituximab alone displayed a tumor growth inhibition (TGI) of 88%, with 3/8 mice harboring a barely palpable tumor, while the umbralisib alone treatment arm showed a TGI of 50%, with 2/8 mice lacking detectable tumors. TG-1801 exhibited a 76% TGI with 1/8 tumor free-mouse. Most importantly, the combination of TG-1801 with umbralisib alone, ublituximab alone, and U2 achieved TGI of 85%, 93% and 93% respectively, with more tumor-free mice 35 days after the last dose in these three groups. Interestingly, this superior anti-tumor effect of the different TG-1801 combinations was associated with a higher infiltration of mouse macrophages within the tumors as assessed by F4/80 IHC labeling. RNA-seq analysis of the Raji xenografts and of n=4 representative in vitro B-NHL co-cultures treated with TG-1801, U2 or the triple combination uncovered the upregulation of the G-protein coupled receptor EBI2/GRP183 as a common event associated with the synergistic antitumor effects of TG-1801 and U2 in vitro and in vivo. A critical role of EBI2 in the regulation of macrophage activity, B cell migration and in the promotion of a pro-inflammatory phenotype was demonstrated upon exposure of the co-cultures with the EBI2 small molecule inhibitor NIBR189, which impaired the U2/TG-1801-evoked ADCP, B-cell cytoskeleton remodeling and inflammatory cytokine production.
Conclusion: The data presented here set the preclinical rationale and support a combination strategy of the novel CD47-CD19 bispecific antibody TG-1801 in B-NHL with other B-cell targeted mechanisms, including umbralisib and ublituximab.

Disclosures
Normant: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Miskin: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Roue: TG Therapeutics, Inc.: Research Funding.