The ITK Inhibitor Cpi-818 Blocks Activation of T Cells from Autoimmune Lymphoproliferative Syndrome (ALPS) Patients and Is Active in a Murine Model of ALPS

Introduction

We present preclinical evidence confirming the expression of ITK in double negative (DN) T cells of ALPS patients and demonstrate that CPI-818 inhibits the TCR-induced activation of these cells ex vivo. Furthermore, CPI-818 effectively suppressed the accumulation of DN T cells in the MRL/lpr mice, leading to dramatic improvements in lymphadenopathy, splenomegaly and other pathologies associated with lymphocyte accumulation due to Fas-deficiency.

ALPS is a disorder of lymphocyte apoptosis resulting in childhood onset lymphadenopathy, multilineage cytopenias, hypersplenism and autoimmune peripheral destruction. Most ALPS cases are associated with a loss of function mutation in the Fas gene resulting in immune dysregulation such as a marked elevation of abnormal TCR+CD4-CD8-double negative (DN) T cells. ALPS patients are currently treated with immunosuppressive and cytotoxic agents, hence targeted, less toxic treatments are imperative. The critical role of Fas protein in maintaining lymphocyte homeostasis and peripheral immune tolerance to prevent autoimmunity was initially discovered with studies in Fas-deficient MRL/LpJ-Tnfrsf6lpr (MRL/lpr) mice. These mice have dramatically elevated levels of DN T cells and develop many clinical manifestations similar to those observed in ALPS patients. Thus, MRL/lpr mice have been a useful model to assess compounds and therapeutic strategies for the treatment of ALPS. Interleukin-2-inducible T cell kinase (ITK) is a tyrosine kinase critical to T cell receptor (TCR) signal transduction, which modulates T cell proliferation and differentiation. We sought to test whether the effect of selective inhibition of TCR signaling through ITK inhibition could be a strategy to suppress the DN T cell expansion and disease manifestation in MRL/lpr mice. CPI-818 is an orally bioavailable, covalent inhibitor of ITK that potently inhibits TCR signal transduction. It is currently being evaluated in a phase 1/1b clinical trial in T-cell lymphoma (NCT03952078) where it has been well tolerated and resulted in anti-tumor activity.

Methods

PBMC samples were obtained from healthy donors or ALPS patients collected under NIAID IRB-approved protocol 93-I-0063. Cells were cultured with or without anti-CD3/CD28 activation. The impact of CPI-818 on viability, expression of ITK, and expression of the surface markers CD 25 and CD69 was assessed by flow cytometry. For in vivo studies, MRL/lpr mice received a control diet or a CPI-818 formulated diet (300 mg/kg/day). Lymph nodes were calipered weekly over the course of 22 weeks of disease development. Spleens, lungs and kidneys were harvested at 22 weeks to assess the impact of CPI-818 treatment on T cell subsets and histology.

Results

Ex vivo studies with PBMC preparations from normal donors and ALPS patients confirmed the expression of ITK in CD4+, CD8+, and DN T cells from ALPS patients. When these T cells were stimulated with anti-CD3/CD28, CPI-818 potently inhibited the upregulation of the activation markers CD25 and CD69 in DN T cells assessed by flow cytometry. In companion experiments, CPI-818 was not cytotoxic to DN T cells when activated through the TCR signaling pathway. Treatment of MRL/lpr mice with CPI-818 prevented the onset or, in a therapeutic design, led to the regression of lymphadenopathy and splenomegaly comparable to the treatment effects observed with cyclophosphamide. Unlike the effects seen with the cytotoxic agent cyclophosphamide which suppressed all T cells, as well as B, NK and myeloid cells in the spleen, CPI-818 selectively depleted DN T cells (see Figure). CPI-818 treatment also resulted in decreased levels of DN T cells in the kidneys and lungs of these mice without impacting the level of normal CD4 and CD8 T cells. CPI-818 limited the increased proteinuria measured in the control animals during the 22-week treatment window. Histological assessment of the kidney revealed reduced inflammatory damage in CPI-818 treated animals.

Conclusions

The selective, covalent ITK inhibitor, CPI-818, significantly reduced DN T cell numbers, lymphadenopathy and other disease end points in the MRL/lpr murine model of ALPS. This suggests that the dysregulated growth of Fas-deficient DN T cells requires functional TCR signaling. Further, our data suggests that blocking TCR signaling by targeting ITK may be an effective strategy for treating adult and pediatric ALPS.