The Inhibitory Receptor FcgRIIB Is Overexpressed, and Its Ligation by Anti- FcgRIIB Antibodies Suppresses IgM Production and Induces Apoptosis in Waldenstrom's Macroglobulinemia.
Authors: Yang Guang , Gong Ping , Xu Lian , Hunter Zachary , Sun Jenny , Ciccarelli Bryan , Zhou Yangsheng , Liu Xia , Tseng Hsiuyi , Cao Yang , Manning Robert , Lewicki Megan , Hanzis Christina , Ioakimidis Thea , Sheehy Patricia , Patterson Christopher J , Treon Steven 
Published: October 2019

Abstract
Abstract 3736
Poster Board III-672

Introduction
Waldenstrom's macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by overproduction of a monoclonal IgM paraprotein which can produce morbidity including hyperviscosity, as well as autoimmune related neuropathy, hemolysis, and thrombocytopenia. Therefore approaches aimed at both suppressing IgM production, as well as selectively inducing apoptosis of WM cells represent an ideal treatment strategy for WM. FcgRIIB is an inhibitory receptor that is expressed on B-cells, and whose expression we recently identified as highly over-expressed in WM. Importantly, FcgRIIB possesses an immunoreceptor tyrosine-based inhibitory motif (ITIM), and which becomes phosphorylated at Tyr 292 upon activation, and is then followed by inhibition of BCR signaling and induction of apoptosis. We therefore validated the expression of this receptor in WM, and examined the impact of its ligation on tumor cell killing, IgM secretion and downstream signaling events in WM cells.

Patients and Methods
Bone marrow lymphoplasmacytic cells (LPC) from 12 WM patients which were sorted for CD19+ and CD138+, and BCWM.1 WM cells were subjected to real-time PCR and flow cytometric analysis. Cells were then subjected to co-culture with anti- FcgRIIB (AT10, 7.3) or control antibodies for 24-48 hours, and their effects on survival, IgM production and downstream signaling were assessed.

Results
Real-time PCR and flow-cytometric analysis demonstrated strong expression of FcgRIIB in WM patient bone marrow CD19+ and CD138+ cells, thus confirming our recent microarray results. Importantly, the expression of FcgRIIB in WM LPC correlated with the memory B-cell marker CD27. Anti-FcgRIIB antibody treatment dramatically reduced constitutive, and/or IL-6 induced IgM production in CD19+ and CD138+ sorted primary WM LPC, as well as CD32hiCD138hi BCWM.1 cells. This effect was observed in some experiments at an early time point that had not effected survival. Among primary CD138+ WM LPC and CD32hiCD138hi expressing BCWM.1 cells, treatment with anti-FcgRIIB antibodies for 48 hours led to increased apoptosis in 10 of 12 patients, as assessed by Annexin V and PI staining which occurred despite blockade with a pan-caspase inhibitor, and was even more pronounced when anti-FcgRIIB antibodies were cross-linked. Western blot analysis revealed that treatment of CD32hiCD138hi expressing BCWM.1 cells with cross-linked anti- FcgRIIB antibodies led to phosphorylation of Tyr 292 of the FcgRIIB ITIM which was not observed in the absence of cross-linking. Binding of FcgRIIB by both the AT10 and 7.3 antibodies resulted in dephosphorylation of Akt, which was further reduced in the presence of cross-linking. Coincident with the above, the pro-apoptotic molecule JNK also underwent phosphorylation in the presence of anti-FcgRIIB binding.

Conclusion
Taken together, these studies validate our previous microarray data by showing that the inhibitory receptor FcgRIIB is strongly expressed on LPC from WM patients, and ligation thereof leads to suppression of IgM production and induction of apoptosis thereby identifying FcgRIIB as a novel therapeutic target in WM.

Disclosures:
No relevant conflicts of interest to declare.