Overcoming resistance to bispecific antibodies through epigenetic priming in B-cell lymphoma

Abstract

Abstract CD20×CD3 bispecific antibodies (BsAbs) exhibit an impressive overall response rate in several B-cell lymphomas; however, progression-free survival declines over time, with BsAb resistance linked to reduced CD20 expression and T-cell exhaustion. Epigenetic drugs can modulate the tumor microenvironment: histone deacetylase inhibitors (HDACi) have been shown to regulate CD20 expression, while EZH2 inhibitors (EZH2i) prime circulating T cells. Our laboratory has shown that dual BEL+TAZ therapy cooperatively increases B cell CD20 expression and activates peripheral T cells. We hypothesize that dual BEL+TAZ therapy increases CD20 expression and enhances T-cell activity, thereby overcoming BsAb resistance in B-cell lymphoma. We previously reported that combination BEL+TAZ treatment significantly increases basal cell surface CD20 expression on various B-cell lymphoma cell lines and increases markers of activation in healthy T cells compared to BEL/TAZ monotherapy. To assess the effect of epigenetic priming on BsAb-resistant cells, a CD20low SU-DHL-4 B-cell lymphoma cell line was generated via co-culture with partially HLA-matched T cells and continuous exposure to the BsAb mosunetuzumab (MOSUN). CD20low B cells were isolated and exhibited a 71% reduction in cell surface CD20 expression compared to vehicle-treated CD20basal cells as assessed via flow cytometry (n=5, all data presented herein have P values < 0.05) and corroborated through immunofluorescent imaging (n=3). Isolated CD20low cells were treated with BEL, TAZ, or dual BEL+TAZ for six days and analyzed via flow. Monotherapy only induced marginal changes, while combination BEL+TAZ caused a 1.52-fold increase in CD20 levels compared to vehicle-treated controls (n=3). Transcription and alternate splicing of CD20 isoforms are associated with BsAb efficacy; transcription of the version 1 (V1) isoform results in insufficient cell surface CD20 levels and BsAb resistance, while version 3 (V3) promotes BsAb binding (Ang et al., Blood, 2023). CD20low cells treated with BEL+TAZ displayed a 0.87-fold decrease in the V1 isoform and a 1.64-fold increase in V3 compared to vehicle-treated controls as measured by qRT-PCR (n=2). BEL+TAZ or vehicle-treated CD20low luciferase+ SU-DHL-4 cells were co-cultured with healthy T cells, treated with MOSUN for 24 hours, and assessed for viability via luminescent assays. B-cell viability was significantly lower in cells primed with BEL+TAZ compared to their vehicle-treated counterparts (n=3). Ascitic fluid was collected from a patient diagnosed with follicular lymphoma (FL), and kappa light chain-restricted putative FL cells were isolated via cell sorting and treated with vehicle or BEL+TAZ in vitro for 6 days. Putative FL cells displayed a 1.4-fold increase in CD20 levels via flow following BEL+TAZ treatment (n=3). Isolated FL cells were primed with BEL+TAZ therapy for six days, co-cultured with autologous T cells, and treated with MOSUN for 24 hours in vitro. Co-cultures primed with BEL+TAZ before MOSUN exposure had 1.41-fold fewer viable B cells than those treated with MOSUN alone and displayed increased apoptosis via flow (n=3). Lymphoma spheroids were generated using isolated FL cells, pretreated with vehicle, BEL, TAZ, or BEL+TAZ for six days, then treated with MOSUN and co-cultured with autologous T cells for 24 hours. Spheroids with BEL+TAZ priming exhibited increased apoptosis through confocal microscopy compared to monotherapies or controls (n=2). To identify the transcriptional targets of BEL+TAZ therapy, CD20basal SU-DHL-4 B cells were treated for six days and analyzed using bulk RNA- and ATAC-seq (n=4). MS4A1 (CD20) gene expression increased following BEL+TAZ treatment, and various CD20-regulated pathways, such as B-cell receptor signaling and calcium ion transport pathways, were enriched in the treated samples. Dual BEL+TAZ therapy increased chromatin accessibility in the promoter region of MS4A1 and increased HOMER ATAC-seq enrichment of OCT2 and PU.1, transcription factors known to regulate CD20 expression, proposing potential targets of epigenetic therapy. Additional bulk RNA- and ATAC-seq experiments are underway to confirm the role of OCT2 and PU.1 in BEL+TAZ-treated CD20low cells. In conclusion, dual BEL+TAZ treatment increases CD20 expression on CD20low B cells and increases BsAb-directed cell kill, supporting dual HDAC and EZH2 inhibition as a potential means to overcome BsAb resistance in B-cell lymphomas.