P165 Guselkumab binding to CD64+ IL-23–producing myeloid cells enhances potency for neutralizing IL-23 signaling

Abstract

Abstract Background IL-23 is implicated in the pathogenesis of inflammatory bowel disease (IBD) and myeloid cells that express FcγRI (CD64) have been identified as the primary cellular source of IL-23 in inflamed IBD gut tissue. Guselkumab (GUS) and risankizumab (RZB) are monoclonal antibodies (mAbs) specifically directed against the IL-23p19 subunit. GUS is a fully human IgG1 mAb with a native Fc region while RZB is a humanized IgG1 mAb with a mutated Fc region. Here, we evaluated CD64 and IL-23 expression in IBD patient gut biopsies, binding of GUS and RZB to CD64, and the functional consequences of CD64 binding by IL-23p19 subunit mAbs, in in vitro assays. Methods IL23A and FCGR1A (CD64) expression was analyzed from bulk and single-cell RNAseq datasets. Binding of mAbs to IFNγ-primed human monocytes, as well as binding to IL-23–secreting inflammatory monocytes and capture of endogenously secreted IL-23, were assessed by flow cytometry. Internalization of IL-23, GUS, and RZB within CD64+ macrophages was evaluated using live cell confocal imaging. Potency of GUS and RZB for inhibiting IL-23 signaling was determined in a co-culture of THP-1 (a CD64+ monocyte cell line activated to produce IL-23) and an IL-23 reporter cell line (measuring biologically active IL-23). Results Analysis of RNAseq datasets showed that FCGR1A, IL23A, and IL12B were significantly increased in inflamed versus non-inflamed IBD gut biopsies and that IL23A was predominantly expressed by FCGR1A-expressing myeloid cells. In in vitro assays GUS, but not RZB, showed Fc-mediated binding to CD64 on IFNγ-primed monocytes. CD64-bound GUS simultaneously captured IL-23 secreted from the same cells. GUS, but not RZB, bound to the surface of CD64+ macrophages and mediated internalization of IL-23 to low pH intracellular compartments. GUS and RZB demonstrated similar potency for inhibiting signaling by IL-23 present in THP-1–conditioned media. However, in a co-culture of IL-23–producing THP-1 cells with an IL-23–responsive reporter cell line, GUS demonstrated enhanced potency compared to RZB for inhibition of IL-23 signaling. Conclusion Our transcriptomic analysis supported previous observations of CD64+ myeloid cells as a key source of IL-23 production in inflamed IBD gut tissue. GUS binding to CD64 on IL-23–producing cells likely contributed to the enhanced functional potency of GUS compared to RZB for inhibition of IL-23 signaling in the co-culture assay. These in vitro data support a hypothesis for optimal enrichment of GUS in inflamed tissues where CD64+ IL-23–producing myeloid cells are increased and in proximity to IL-23–responsive lymphoid cells, enabling GUS to more potently neutralize IL-23 by targeting IL-23 at its source of production.