Ibrutinib Treatment Improves T-Cell Proliferative Ability and Effector Function in Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL) Patients
Authors: Solman Isabelle G. , Taylor Marlene , You Hana , O'Brien Susan M. , Mulligan Stephen P. , Byrd John C. , Dean James P. , James Danelle F. , Mongan Ann 
Published: October 2019

Abstract
Background: CLL is a B-cell malignancy characterized by profound immune dysregulation, including dysfunctional T cells. Ibrutinib (ibr), a first-in-class, once-daily BTK inhibitor, is approved in the US for the treatment of CLL/SLL, and has been reported to additionally inhibit ITK in T cells. Previous studies showed that ibr has a positive impact on T-cell compartments by decreasing abnormally elevated regulatory T cells and pseudo-exhausted effector T cells, while preserving naive T-cell counts post 1 year of treatment (Solman ASH Lymphoma 2016; Solman ASCO 2017). Moreover, lower infection rates have been reported following 6 months of ibr treatment, suggesting that ibr might restore T-cell functions over time (Long JCI 2017). To further assess the effects of ibr on T-cell function, we evaluated the ability of cells to proliferate, degranulate, and release cytokines upon stimulation.
Methods: Samples for this study were derived from patients (pts) with relapsed/refractory CLL enrolled in the RESONATE™ trial (Byrd NEJM 2014). In the 2 treatment arms, ibr and ofatumumab (ofa), we compared responders (PR) and non-responders (SD) per investigator assessment at week (wk) 24 - end of ofa treatment - to isolate the treatment effect on T cells. In total, we selected 19 pts wk1 through wk48 from ibr arm (420 mg once daily; 12 PR & 7 SD at wk24) and 21 pts wk1 through wk24 from ofa arm (300 mg once followed by weekly 2000 mg; 10 PR & 11 SD at wk24). Also included in the study for reference were 18 untreated, age-matched healthy donors. Cryopreserved PBMCs were immunophenotyped before and after 4-day in vitro stimulation with anti-CD3/CD28. T-cell proliferation was measured with CFSE staining, while secreted lytic proteins and cytokines were measured by bead-based immunoassays. Unless specified otherwise, median changes at wk24 relative to baseline (wk1) are reported.
Results: Compared to reference range (ref) of healthy donors, all CLL samples were found to have higher percentage of apoptotic T cells. We further evaluated the fraction of apoptotic T cells in each treatment arm. In the treated CLL patients, a larger decrease in the apoptotic fraction was observed in vivo with ibr (46%) compared to ofa (24%). To evaluate how well T cells were able to respond to stimulation, we assessed T-cell death post in vitro stimulation. In vitro, both treatments were found to reduce cell death, with ofa exerting an earlier effect on apoptosis (57% for ofa vs 17% for ibr at wk24, down to 37% at wk48), and ibr having a larger effect on non-apoptotic cell death (53% for ibr at wk12-48 vs no change for ofa). T-cell proliferative ability improved significantly with ibr (+28%, P=0.001), while it decreased with ofa (-49%, P=0.003). In both arms, T-cell proliferation was higher in pts with PR compared to SD (+39% vs +6% for ibr and -20% vs -96% for ofa), and for CD8+ cells compared to CD4+ (14% for ibr and 52% for ofa). We confirmed degranulation impairment in CLL pts at baseline with a lower ability to release perforin, granzyme-A, granzyme-B, and granulysin upon stimulation. Lytic protein secretion was improved by both treatments, with a greater overall effect observed in ibr (5-fold) vs ofa pts (3-fold) at wk24. In particular, granulysin increased up to 11-fold at wk48 with ibr treatment, leading to full restoration according to ref. Relative to healthy donors, CLL pts at baseline secreted less IL-4, IL-6, IL-10, and IL-17. Each of these cytokines was increased by 3-4-fold and maintained near ref through treatment with ibr, while maximum 2-fold improvement was observed in ofa pts. IL-2 and TNF-α were elevated in CLL pts, and their secretion remained high with both treatments. Further analyses showed that degranulation and cytokine secretion changes were likely not a direct result of cell counts or disease resolution.
Conclusions: Together, these data suggest ibr has a positive impact on T-cell proliferation and effector functions including degranulation and cytokine release. T-cell proliferative ability was found to be associated with treatment response, but improvement was significantly higher in ibr-treated pts, indicating a unique immune reconstitution effect of ibr beyond the effect of disease resolution. Improvement in effector T-cell function at wk24 and beyond may lead to lower infection rates in later cycles of therapy and/or support an adaptive anti-CLL immune response.

Disclosures
Solman: AbbVie: Equity Ownership; Pharmacyclics LLC, an AbbVie company: Employment, Other: TRAVEL, ACCOMMODATIONS, EXPENSES. Taylor:AbbVie: Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, accomodations, expenses. You:Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, accomodations, expenses; AbbVie: Equity Ownership. O'Brien:Acerta: Research Funding; Regeneron: Research Funding; Kite Pharma: Research Funding; Vaniam Group LLC: Consultancy; TG Therapeutics: Consultancy, Research Funding; Janssen: Consultancy; Amgen: Consultancy; Pharmacyclics: Consultancy, Research Funding; Celgene: Consultancy; Abbvie: Consultancy; Pfizer: Consultancy, Research Funding; Aptose Biosciences Inc.: Consultancy; GlaxoSmithKline: Consultancy; Astellas: Consultancy; Alexion: Consultancy; Sunesis: Consultancy, Research Funding; Gilead: Consultancy, Research Funding. Dean:CTI BioPharma Corp.: Employment, Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. James:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership, Other: Spouse's employment and stocks, Patents & Royalties: AbbVie Patent Applications. Mongan:Thermo Fisher Scientific: Patents & Royalties: Patents; AbbVie: Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, accommodations, expenses.