Ragon Institute of MGH, MIT and Harvard Cambridge, MA, United States
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Thomas Guy1, Hang Liu2, Vinay Mahajan2, Cory Perugino3, Zachary Wallace4, Shauna quinn5, Allen Poma6, Debra Zach7, John Stone8 and Shiv Pillai9, 1Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, 2Ragon Institute, Cambridge, MA, 3Massachusetts General Hospital, Boston, MA, 4Massachusetts General Hospital, Newton, MA, 5Zenas Biopharma, New York, NY, 6Zenas Biosciences, New York, NY, 7Exagen, Vista, CA, 8Massachusetts General Hospital Rheumatology Unit, Harvard Medical School, Concord, MA, 9Harvard Medical School, Boston, MA
Background/Purpose: Obexelimab is a bifunctional, non-cytolytic, humanized monoclonal antibody that binds CD19 and ligates FcγRIIb with high affinity. Data from a phase 2 clinical trial of patients with IgG4‑related disease (IgG4-RD), a chronic immune-mediated fibro-inflammatory disease, demonstrated rapid, and sustained clinical improvement, including complete remission in most patients following treatment with obexelimab. Although not shown to drive rapid apoptotic death of B cells, obexelimab treatment does result in an approximate 50% reduction in circulating B cell numbers, as well as near complete reduction in circulating plasmablasts. We have shown that obexelimab dampens the activation of BTK in B cells, easily ascribable to the loss of PIP3 that would result from the predicted activation by FcgRIIb of the SH2-domain containing inositol phosphatase SHIP-1. Here, we aimed to examine the changes in gene expression induced by obexelimab in B cells from treated patients with IgG4-RD to better understand its mechanism of action.
Methods: Peripheral blood was collected from patients with IgG4-RD before and after treatment with obexelimab (two days after the eighth dose). PBMCs were isolated using standard density gradient centrifugation. Naïve (CD19+IgD+CD27-) and memory (CD19+IgD-CD27+) B cell populations were FACS purified and used for subsequent ATAC-sequencing (N = 3 with 3 technical replicates for each sample) and RNA-sequencing (N = 16).
Results: We observed many changes in gene expression and chromatin accessibility attributed to treatment with obexelimab in naïve B cells. RNA sequencing identified 775 genes upregulated and 808 genes downregulated in post- compared to pre-obexelimab treated B cells. ATAC sequencing analysis identified 1040 increased and 708 decreased differentially accessible genes in post- versus pre-obexelimab treated B cells. Gene ontology biological process analysis of ATAC sequencing were consistent with altered cytoskeleton organization, chemotaxis and migration. In addition, nucleotide and purine metabolic processes were impacted after treatment. Furthermore, KEGG pathway analysis demonstrated key components of actin cytoskeleton regulation associated with altered phosphoinositide regulation. Gene ontology biological process analysis of RNA-seq data aligned with ATAC-seq analysis, confirming alteration of cell-cell adhesion and cytoskeletal pathways.
Conclusion: Collectively, these chromatin accessibility and transcriptromic data suggest that reciprocal changes in PIP3 and PI(3,4)P2 dependent events induced by obexelimab on B cells alter B cell activation and migration. Paired with the previous published data demonstrating attenuation of BCR signalling, obexelimab not only reduces PIP3-dependent BCR-induced BTK activation in B cells from IgG4-RD patients but could also enhance PI(3,4 )P2 phosphoinositide-mediated B cell migration, possibly causing sequestration of B cells. Together, these two mechanisms may eliminate the potential of pathogenic, self-reactive B cells to respond to self-antigen, migrate out of secondary lymphoid organs, and infiltrate inflamed tissues.
