Upregulation of FcγRIIb on monocytes is necessary to promote the superagonist activity of TGN1412

Abstract

Key Points• TGN1412-induced T-cell activation following highdensity preculture of PBMCs is a consequence of FcgRIIb upregulation on monocytes.• In vivo, cytokine release syndrome may be due to the close association of FcgRIIbbearing cells with T cells in lymphoid tissues.The anti-CD28 superagonist antibody TGN1412 caused life-threatening cytokine release syndrome (CRS) in healthy volunteers, which had not been predicted by preclinical testing. T cells in fresh peripheral blood mononuclear cells (PBMCs) do not respond to soluble TGN1412 but do respond following high-density (HD) preculture. We show for the first time that this response is dependent on crystallizable fragment gamma receptor IIb (FcgRIIb) expression on monocytes. This was unexpected because, unlike B cells, circulating monocytes express little or no FcgRIIb. However, FcgRIIb expression is logarithmically increased on monocytes during HD preculture, and this upregulation is necessary and sufficient to explain TGN1412 potency after HD preculture. B-cell FcgRIIb expression is unchanged by HD preculture, but B cells can support TGN1412-mediated T-cell proliferation when added at a frequency higher than that in PBMCs. Although lowdensity (LD) precultured PBMCs do not respond to TGN1412, T cells from LD preculture are fully responsive when cocultured with FcgRIIb-expressing monocytes from HD preculture, which shows that they are fully able to respond to TGN1412-mediated activation. Our novel findings demonstrate that cross-linking by FcgRIIb is critical for the superagonist activity of TGN1412 after HD preculture, and this may contribute to CRS in humans because of the close association of FcgRIIb-bearing cells with T cells in lymphoid tissues. (Blood. 2015;125(1):102-110)

Development and Characterization of Monoclonal Antibodies Specific for Mouse and Human Fcγ Receptors

“…Although initially proposed to be related solely to CD28 expression on a particular subset of T cells and independent of hFcgR expression, it is now evident that hFcgR and in particular hFcgRIIB is critical for this response, at least in predictive in vitro assays (53,54) and our own observations (55). However, what is not yet resolved is where the hFcgR interaction occurs in vivo to cause the resulting, rapid, cytokine storm.…”

Antagonistic Human FcγRIIB (CD32B) Antibodies Have Anti-Tumor Activity and Overcome Resistance to Antibody Therapy In Vivo

“…The same lack of depletion of monocytes and neutrophils was seen in combination with rituximab ( Figures 4G-4I). Next, we used a recently developed in vitro cytokine release syndrome (CRS) assay (CRA) (Hussain et al, 2015) to assess the potential impact of 6G11 on human peripheral blood mononuclear cells (PBMCs) rendered sensitive to stimulation through high-density culture (Rö mer et al, 2011) and detected substantial levels of interferon gamma, tumor necrosis factor alpha and/or interleukin 8 following addition of several mAb specificities (CD3, CD28, or CD52) previously highlighted as eliciting CRS. Application of WT or N297Q 6G11 for 48 hr did not result in substantial cytokine release, unlike with 5003 lower doses of CD3 mAb ( Figure 4J).…”

Proteomics Profiling of CLL Versus Healthy B-cells Identifies Putative Therapeutic Targets and a Subtype-independent Signature of Spliceosome Dysregulation

“…Cells were stained with either the manufacturer's recommended concentration, or 10 μg/ml, of in-house antibody for 30 minutes in the dark at 4°C, washed and analysed by flow cytometry with a FACScalibur (BD Biosciences, Franklin Lakes, New Jersey) (35,36).…”