RNA sequencing of Isolated B cells from SM03 treatment on anti-IgM stimulated PBMC

SM03, an anti-CD22 monoclonal antibody was previously proposed to induce specific interaction of CD22, facilitating the CD22-mediated ‘self-recognition’ of autologous cells, thereby attenuating autoreactive BCR signaling. To explore potential downstream mechanisms and effects. Healthy volunteer PBMCs were stimulated with anti-IgM to mimic B-cell activation and treated with SM03 or IgG1 Isotype Control. B cells were Isolated and RNA-seq analysis was conducted, mRNA was purified using polyT oligo-attached magnetic beads, fragmented, and converted to cDNA using random hexamer priming. After end repair, A-tailing, adaptor ligation, size selection, and PCR amplification, libraries were quantified and assessed by bioanalyzer. Cluster generation followed standard Illumina procedures, and paired-end sequencing was performed on an Illumina platform. Raw sequencing reads underwent quality control to remove adapter sequences, low‑quality reads, and reads containing poly‑N, generating high‑quality clean data for analysis. Clean paired‑end reads were aligned to the reference genome using HISAT2, and gene‑level counts were obtained with feature Counts. Gene expression levels were quantified as FPKM values, followed by differential expression analysis using DESeq2 to identify significantly regulated genes. Functional enrichment of differentially expressed genes (DEGs) was performed using GO, KEGG, Reactome, Disease Ontology, and DisGeNET pathway frameworks, and complementary Gene Set Enrichment Analysis (GSEA) was conducted to evaluate coordinated pathway shifts. SM03‑treated samples were compared against IgG1 controls to identify transcriptional changes associated with B‑cell modulation. This analysis revealed SM03‑specific DEGs linked to reduced BCR signaling, altered checkpoint regulation, and attenuated autoreactive responses. GSEA further demonstrated downregulation of B‑cell activation pathways and autoimmune signatures, supporting that SM03 modulates B‑cell activation and immune crosstalk distinct from IgG1. Together, DEG and GSEA results highlight transcriptomic differences that reflect the immunomodulatory effects of SM03 on B cells.