The Regulation of FcγRIIB and its Effect on Antibodies Used in Cancer Therapy

Monoclonal antibody (mAb) therapy is revolutionising the way cancers are treated. However, a lack of therapeutic efficacy in some patients, the development of resistance, and occasionally severe side effects are obstacles in their continuing clinical development. One of the common requirements for efficacy is that the mAb Fc region binds to appropriate Fcγ receptors (FcγR) on the surface of immune cells. One particular FcγR shown to have a profound role in regulating the efficacy of mAb therapy is the inhibitory FcγR- FcγRIIB. Therefore, deciphering how FcγRIIB expression is regulated could provide a means to increase mAb efficacy. Previously it was found that culturing peripheral blood mononuclear cells (PBMCs) invitro at high density (HD; 1 x 107cells/ml) results in a profound upregulation of FcγRIIBon monocytes. This system was investigated to gain a better understanding of FcγRIIBregulation. De novo transcription and translation of FcγRIIB was observed in monocytes cultured at HD, and both isotypes of FcγRIIB – FcγRIIB1 and FcγRIIB2 were upregulated. To assess the transcriptional profile of HD-cultured monocytes, mRNA microarray experiments were performed, identifying genes modulated in HD monocytes. From these analyses, a panel of soluble factors, including cytokines, were identified as being differentially expressed in HD monocytes and were tested in vitro for their ability to elicit change sin monocytic FcγRs. IL-10 significantly upregulated expression of FcγRIIB and several other cytokines were able to prevent the upregulation of FcγRIIB in HD monocytes including IL-4, IL-13, IFN-γ, TNF-α and PGE2, suggesting FcγRIIB expression can be regulated by a diverse range of stimuli. Using petri net analyses, combinations of these soluble factors were identified as potential drivers of the changes in FcγRs observed on HD monocytes. Many were able to significantly downregulate expression of FcγRIIB onHD monocytes. However, none of these soluble factors individually or in combination were able to recreate the exclusive pattern of FcγR changes observed in HD monocytes. Using gene set enrichment analysis, a hypoxic gene signature was identified in HD monocytes. HD monocytes were subsequently found to upregulate the hypoxia-related proteins - HIF-1α, CAIX and GLUT1; and have a hypoxic level of <1%O2. To formally test this association, low density (LD) monocytes were cultured with a chemical inducer of hypoxia, dimethyloxaloylglycine (DMOG), or in a hypoxic chamber, both of which stimulated de novo transcription and translation of FcγRIIB, supporting the microarray data. To demonstrate causation, HIF-1α siRNA was used and shown to prevent the upregulation of FcγRIIB on monocytes induced by HD and DMOG providing further evidence for the hypoxia- and HIF-1α-mediated upregulation of FcγRIIB. There is no hypoxia response element in the FCGR2B promoter region suggesting HIF cannot mediate these changes directly. However, there is a binding site for AP-1, known to be upregulated under hypoxia. Therefore, a member of the AP-1 complex, Jun, was knocked down using siRNA. Jun siRNA was also shown to prevent HD and DMOG-stimulated upregulation ofFcγRIIB on monocytes which suggests a mechanism for hypoxia-stimulated upregulation of FcγRIIB.As one of the main effector functions of mAb therapy is antibody-dependent cellular phagocytosis (ADCP) via macrophages, macrophages were generated from monocytes and treated with DMOG. Similarly to monocytes, DMOG stimulated the upregulation ofFcγRIIB expression on macrophages, and these DMOG-stimulated macrophages had lower levels of ADCP. Together these data demonstrate that a hypoxic environment leads to an upregulation of FcγRIIB on monocytes and macrophages, in an AP-1-dependent manner, and as the tumour microenvironment is often hypoxic, this could limit the efficacy of direct targeting antibodies used in cancer therapy by inhibiting ADCP