More Than a BTK Inhibitor; Ibrutinib Profoundly Impacts the Function of Cell Mediated Immunity
Authors: Sharpe Chia , Davis Joanne , Mason Kylie D. , Godfrey Dale I , Uldrich Adam , Tam Constantine S. , Koldej Rachel , Ritchie David 
Published: October 2019

Abstract
Background: Chronic lymphocytic leukaemia (CLL) is associated with profound immune dysfunction, which is often exacerbated by CLL therapies. This study aimed to understand the impact of multiple BTK inhibitors on the function of cytotoxic T cells. BTK, along with ITK, TXK, BMX and TEC is a member of the Tec family of nonreceptor tyrosine kinases which are integral to the downstream signalling of many immune receptors. Critically, BTK and ITK are downstream of the B cell and T cell receptors. Whilst identified as a selective BTK inhibitor, ibrutinib inhibits all five members of the Tec family at clinically meaningful doses (Long et al, JCI, 2017). More selective BTK inhibitors have been developed, including acalabrutinib and zanubrutinib, which have less affinity for ITK but variable inhibition for other Tec family members. Functional impairment of NK cells by ibrutinib is well established (Kohrt et al, Blood 2014), however the effects on T cell function remains unclear. It has been suggested that inhibiting ITK results in skewing of CD4 T cells toward a T helper 1 phenotype whilst CD8 T cell function is preserved due to redundant kinase signalling (Dubovsky et al, Blood, 2013). This is yet to be demonstrated in patients and little is known about the impact of BTK inhibitors on cytotoxic T cell populations.
Methods: Peripheral blood mononuclear cells were isolated from CLL patients receiving either ibrutinib or zanubrutinib and from treatment naive CLL patients or age matched healthy donors. T cell proliferation was measured using CellTrace Violet, cultures of whole PBMC were stimulated with CD3/CD28 beads and 20 IU/mL IL-2 and treated with 1uM ibrutinib or zanubrutinib or acalabrutinib or vehicle control for 7 days, n=7. CD8 T cell and NKT cell degranulation in response to CD3/CD28 stimulation was assessed using CD107a mobilisation and IFN𝛾 production over 4 hours, n=6 and n=7. Whole PBMC from patients treated with either ibrutinib or zanubrutinib were stimulated for 24 hours using CD3/CD28 beads, supernatants were analysed using a BD™ Cytometric Bead Array.
Results: In vitro treatment with ibrutinib significantly impairs the function of cytotoxic T cells. Both CD4 and CD8 T cells cultured in the presence of ibrutinib had significantly decreased proliferation, whilst zanubrutinib and acalabrutinib did not significantly impact T cell proliferation (Figure 1, p=0.0002 and p<0.0001). Furthermore, CD8 T cells from CLL patients and healthy donors had significant abrogation of degranulation and cytokine production when treated with Ibrutinib but not the more selective BTK inhibitors. Similarly, ibrutinib treated healthy donor NKT cells showed significantly diminished degranulation and IFN𝛾 production (Figure 2, p =0.0004 and p=0.0003). Finally, PBMC isolated from patients after treatment with ibrutinib had muted cytokine production including IL-2 (p= 0.003), IL-17A (p=0.023), TNF (p=0.031) and IL-10 (p=0.016) as compared to PBMC isolated before ibrutinib treatment. However there was no significant change in Th1 or Th2 cytokines.
Discussion: Together these results highlight the impact of Ibrutinib on cell mediated cytotoxicity. In both in vitro and ex vivo functional assays ibrutinib, but not more selective BTK inhibitors perturbed proper T cell function. Whilst CD4 T cell proliferation was suppressed Th1 skewing was not observed in ibrutinib treated patients. Furthermore. CD8 T cells had profoundly impaired responses to TCR stimulation. Understanding how BTK inhibitors alter the function of cytotoxic cells is essential for the combination of these therapies with immunotherapies and may inform the use of these therapies in the context of adoptive cellular therapies and transplantation.

Disclosures
Tam: Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.