Introduction: Graft-versus-host-disease (GVHD) is a severe complication in patients undergoing hematopoietic stem cell transplantation (HSCT) and one of the major causes of death in patients receiving HSCT despite immunosuppressant drug and intensive therapy. GVHD is a rare disease characterized by excessive activation of T and B cell pathways and drugs such as Ibrutinib and steroids are used to improve the survival rate of GVHD patients by controlling immune cell signaling, yet the use of these drugs is limited due to toxicity. It is necessary to develop new treatment agents for GVHD, because of the limitations of these existing treatment agents. Interleukin-2-inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are critical for T and B cells activation, and dysregulation of this process has been known to increase proinflammatory cytokines and cause various inflammatory diseases. Since ITK and BTK signaling pathway play a major role in T and B cell regulation in GVHD, we thought that ITK and BTK selective dual inhibition may act as a promising agent that have beneficial effects on GVHD. So, we developed a novel ITK and BTK selective dual inhibitor, DWP213388, to modulate both T and B cell pathways without any significant off-target effects, thus to ultimately improve survival rate in GVHD without any serious side effect.
Methods: Inhibition of ITK and BTK enzyme activity and selectivity against Cys-family kinase group were evaluated by biochemical activity assay. Target occupancy in human T and B cells was measured by quantifying unbound ITK or BTK in ELISA-based assay using biotinylated-DWP213388. To measure occupancy, mouse spleen and thymus were extracted after oral administration of DWP213388. Cellular assays for BCR or TCR stimulation-dependent activation and cytokine secretion were determined in human PBMC. In vitro mixed lymphocyte reaction (MLR) assay, CD4+ T cells were purified from spleens of B6 mice and were co-cultured with irradiated Balb/c splenic APCs as stimulators that were incubated in the presence or absence of DWP213388. In vivo MLR test, recipient BDF1 mouse was lethally irradiated and reconstituted by intravenous injection of splenocytes with B6. The efficacy of DWP213388 was investigated in mouse GVHD model; Recipient BDF1 mouse was lethally irradiated and reconstituted by intravenous injection of T cell depleted bone marrow (TCD-BM) and splenocytes with B6. In CMV study, DWP213388 was administered orally and an inoculum of 1 x 104 PFU/head MCMV in PBS was injected intraperitoneal. into each mouse on day 0. Five and ten days after the MCMV infection, the liver was removed. The NK cell and CD8 T cell was measured by FACs and virus titration was measured by RT-PCR.
Results: We developed a novel, potent dual target inhibitor, DWP213388, with ITK and BTK IC50 values of 1.0 nM and 0.4 nM, respectively. More importantly, this compound is highly selective against ITK and BTK, yet has low affinity toward EGFR. DWP213388 potently occupied both of its targets at low concentration, which shows successful inhibition of TCR/BCR-dependent CD69 expression. DWP213388 suppressed T and B cell subsets such as Th1, Th2, Th17 and plasma cells and inhibited T cell proliferation in both in-vitro and in-vivo MLR assays. In mouse GVHD model, DWP213388 at 10 mg/kg improved clinical symptoms and maintains 100% survival rate (vs Ibrutinib 85%). It showed 73% and 62% occupancy of BTK and ITK. To address any potential compromising immune response due to dual inhibition of T, B cell by DWP213388, we conducted a CMV study to evaluate the effects on infection. DWP213388 does not reduce the number of NK cells and CD8+ T cells, and it does not affect virus killing. Therefore, it has a low risk of infection. These results suggest that DWP213388 may serve as a next generation therapeutic agent for GVHD.
Conclusion: We developed a novel, highly potent, and selective dual inhibitor of ITK and BTK, DWP213388. We demonstrated that DWP213388 has potent pharmacological activities compared to Ibrutinib in various cellular models and a mouse GVHD model. DWP213388 intends to improve efficacy and safety by specifically inhibiting ITK and BTK without inhibiting EGFR, which is known to cause severe side effects. Therefore, DWP213388 may serve as a next generation therapeutic agent for GVHD.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.