Antibody Coengagement Of Fc?RIIB And CD19 (XmAb5871) Induces Specific Chromatin And Gene Expression Changes Linked To The Key B Lineage Transcription Factors In IgG4-related Disease

AIM: To characterize the effects of XmAb5871,a humanized anti-CD19 monoclonal antibody with an Fc engineered to bind to human Fc?RIIb with>400 higher affinity than native IgG1,treatment on B cells activation pathway and the related changes in chromatin accessibility and gene expression in IgG4-related disease(IgG4-RD).Methods: In a longitudinal study of a cohort of 20 IgG4-RD patients who meet the enrollment requirements,their peripheral blood mononuclear cells(PBMCs)were collected both before and after XmAb5871 treatment.Phospho-flow cytometry was performed to analyze the expression of phospho-BTK(p BTK)and phospho-SYK(p SYK)on naive B cells,memory B cells,double negative B cells and un-switched memory B cells.We further detected the changes on the negative feedback pathways through chromatin acceptability alteration,DNA methylation status and the genes linked to signaling pathways through assay for transposase-accessible chromatin using sequencing(ATAC-seq),cleavage under targets and release using nuclease(CUT & RUN),whole genome bisulfite sequencing(WGBS)and RNA sequencing(RNA-seq)on sorted naive B cells.Finally,gene enrichment analysis(PGSEA)was performed on the transcription factor target database ENCODE,the gene ontology database GO,and the Kyoto Encyclopedia of Genes and Genome database KEGG to compare ATAC-seq data and RNA-Seq data for physiological and biological functions of altered genes,further exploring the extensive inhibitory effect of XmAb5871 on B cells..Results : The expression of p BTK was decreased in all four different B cell populations(CD3-CD20+Ig D+CD27-: naive B cells;CD3-CD20+Ig D-CD27+: memory B cell;CD3-CD20+Ig D+CD27+: un-switched memory B cells;CD3-CD20+Ig D-CD27-:double negative B cells)after initiation of XmAb5871 treatment,but no evidence for decrease in p SYK.We examined approximately 7000 differentially accessible peaks on ATAC-seq and the distance of differentially accessible ATAC seq peaks are about 10 Kb,mapping to the expected enrichment for transcriptional start sites,enhancers.Then the enhancers were verified by CUT&RUN,a cutting edge approach to Ch Ip-seq and used antibodies to H3K27 acetylation(H3K27Ac)in CUT&RUN.WGBS was conducted on the differentially accessible peaks seen on ATAC-seq without Cp G methylation being significantly altered at these sites.808 downregulated genes and 775 upregulated genes were identified after XmAb5871 therapy by RNA-seq.Analysis of known interactors of some chromatin modifying enzymes in the ENCODE dataset supported that XmAb5871 treated patients exhibit a reduction in the expression of the transcriptional targets of EBF,E2 A,PAX-5,EBF1,WRNIP1,etc.and an increase in the expression of the transcriptional targets of ELF1,CHD2,UBTF,etc.We performed parametric gene set enrichment analysis(PGSEA)of the ATAC-seq and RNA-seq data based on genes ranked by their chromatin accessibility and compared GO and KEGG pathway datasets.As for ATAC-seq data,the top hits of GO Biological Process datasets correspond well with signatures linked to enhanced cytoskeletal organization and separately to diminished metabolism.Among the enriched KEGG pathways,the very top hits included diminished oxidative phosphorylation and enhanced regulation of actin cytoskeleton.As for RNA-seq data,top hits of KEGG pathways are cell-cell adhesion via plasma-membrane adhesion molecules,negative regulation of mitotic nuclear division,negative regulation of nuclear division,negative regulation of chromosome separation and regulation of dendrite morphogenesis etc.Conclusions : XmAb5871 downregulates BTK phosphorylation in an Fc ?RIIb-dependent manner in B cells.Notably the inhibitory effects are seen in na(?)ve B cells at level of BCR signaling,chromatin accessibility,and gene expression but not Cp G methylation.Genes with drug-induced alterations in accessibility and expression are highly enriched for targets of a specific set of transcription factors.XmAb5871 therapeutic improvement correlates with pathways linked to altered BCR signaling,metabolism and cytoskeletal biology which are linked to changes in the levels of phosphoinositide species in B cells,which may prevent the differentiation of na(?)ve B cells into auto-antigen specific,disease causing activated B cells that infiltrate tissues and drive the disease process in IgG4-RD.