JAK1/2 inhibition with ruxolitinb potentiates ex vivo activity of epcoritamab  in primary CLL cells Free

Introduction Epcoritamab,a bispecific antibody targeting CD20 and CD3, has demonstrated promising clinical activity in chronic lymphocytic leukemia(CLL) in an early-phase clinical trial(Danilov et al., ASH, 2024);however, its efficacy appears to be suboptimal compared to its performance in diffuse large B-cell lymphoma (DLBCL),where it is now FDA approved.This limitation may be due in part to the highly immunosuppressive tumor microenvironment in CLL.Ruxolitinib,a selective JAK1/2 inhibitor, has shown promising immunomodulatory effects in various hematologic malignancies by improving immunosuppressive microenvironments and reversing T-cell exhaustion.Given these mechanistic insights, here we we report on the ex vivo combination of epcoritamab and ruxolitinib in primary treatment-naïve CLL patient samples to evaluate its potential to enhance the therapeutic activity of CD20&CD3 bispecific antibody therapy in this disease.

Methods Viably frozen peripheral blood mononuclear cells(PBMCs) from twenty-five treatment-naïve patients with CLL were cultured in vitro under four experimental conditions:DMSO (control), epcoritamab alone(5ug/ml),ruxolitinib alone(1uM), and an epcoritamab(5ug/ml) plus ruxolitinib(1uM) combination.Culture supernatants were subjected to cytokine profiling using a multiplex membrane-based antibody array capable of detecting 80 cytokines(RayBiotech).CD19+ B cells were isolated for transcriptomic profiling via poly(A)-enriched RNA sequencing(mRNA-Seq) followed by differential expression analysis(BGI Americas Corporation).For functional validation,the top differentially expressed cytokines were applied individually to CLL PBMCs.Flow cytometry was used to assess the viability of CD19+CD5+ tumor cells(annexinV-Draq7-) and the expression of T-cell exhaustion markers(Mean Fluorescence Intensity, MFI).Protein expression changes were examined through Western blot analysis.

ResultsIn vitro studies demonstrated that the combination of ruxolitinib and epcoritamab induced significantly greater cytotoxic effects against primary CLL tumor cells(relative tumor cell viability:1.06 vs 0.71,p=0.0005) compared to epcoritamab monotherapy.The activity of the combination did not differ significantly between IGHV mutated and unmutated patient samples(p>0.05).RNA sequencing-based enrichment analysis revealed that epcoritamab monotherapy activated both the PD-1/PD-L1 immune checkpoint pathway and the JAK-STAT signaling pathway.Given this latter finding,we next explored the addition of ruxolitinib to epcoritamab,and found that this combination reduced the upregulation of PD-L1(relative MFI:1.55vs1.07,p=0.0202) on CD19+CD5+ CLL tumor cells and attenuating epcoritamab-induced increases in the protein levels of p-STAT1,p-STAT3,p-STAT5, and MCL1 in CLL PBMCs. Comprehensive cytokine profiling identified thirteen cytokines(IFN-γ, IL-5, IL-8, IL-10, IL-13, GM-CSF, CCL2, CCL4, CCL5, CXCL9, CXCL10, TNF-α, and TNF-β) that were significantly elevated following epcoritamab alone.Subsequent treatment of CLL PBMCs with each of these thirteen cytokines individually demonstrated that only IFN-γ uniquely enhanced CLL tumor cell viability(relative tumor cell viability was 1.42, p=0.0005),whereas the other cytokines had no significant effect on CD19+CD5+ tumor cell viability.The combination of ruxolitinib and epcoritamab effectively suppressed this epcoritamab-induced cytokine release(all these 13 cytokines).Additionally,flow cytometry analysis revealed that epcoritamab alone led to increased expression of T-cell exhaustion markers(PD-1,LAG-3 and TIGIT) and these immunological effects were substantially reversed with the addition of ruxolitinib on both CD4+ (relative MFI of PD1:4.94vs2.78, p=0.04, relative MFI of LAG-3: 2.08vs1.20,p=0.0005, and relative MFI of TIGIT:3.54vs1.79,p=0.019) and CD8+ T(relative MFI of PD1: 1.59 vs 1.15,p=0.0015,relative MFI of LAG-3: 2.94 vs2.03,p=0.03, and relative MFI of TIGIT: 2.62 vs 1.48,p=0.0296) cell populations.

Conclusions The combination of ruxolitinib and epcoritamab demonstrated enhanced cytotoxic effects in primary CLL patient samples compared to either agent alone.Ruxolitinib appears to suppress JAK-STAT pathway activation in CLL cells,while simultaneously reducing IFN-γ production and reversing T-cell exhaustion in both CD4+ and CD8+ T cell populations.Collectively,these findings suggest that ruxolitinib plus epcoritamab is a promising combination to study in the clinic for patients with CLL.