Annals of Hematology (2021) 100:1733–1742 https://doi.org/10.1007/s00277-021-04536-6 ORIGINAL ARTICLE Pretreatment with ibrutinib reduces cytokine secretion and limits the risk of obinutuzumab-induced infusion-related reactions in patients with CLL: analysis from the iLLUMINATE study Richard Greil 1 & Alessandra Tedeschi 2 & Carol Moreno 3 & Bertrand Anz 4 & Loree Larratt 5 & Martin Simkovic 6 & Devinder Gill 7 & John G. Gribben 8 & Ian W. Flinn 9 & Zhengyuan Wang 10 & Leo W. K. Cheung 10 & Aaron N. Nguyen 10 & Cathy Zhou 11 & Lori Styles 12 & Fatih Demirkan 13 Received: 15 April 2020 / Accepted: 16 April 2021 / Published online: 20 May 2021 # The Author(s) 2021 Abstract Anti-CD20 antibody treatments, such as obinutuzumab, have been associated with infusion-related reactions (IRRs). In the phase 3 iLLUMINATE study of ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab in first-line chronic lymphocytic leukemia/small lymphocytic lymphoma, IRRs were substantially reduced with ibrutinib-obinutuzumab versus chlorambucil- obinutuzumab. We prospectively analyzed inflammatory cytokines to evaluate the impact of ibrutinib on circulating cytokine levels following obinutuzumab infusion. In iLLUMINATE, ibrutinib or chlorambucil was given approximately 30–120 min before the first obinutuzumab infusion. Cytokines evaluated were IFNγ, IL-6, IL-8, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, and TNFα. Changes in peak cytokine levels from baseline (immediately before obinutuzumab) to post-obinutuzumab infusion were compared between arms and between patients with versus without IRRs using Wilcoxon rank sum test. Of 228 treated patients, 95 on ibrutinib-obinutuzumab (15 with IRRs, 80 without) and 88 on chlorambucil-obinutuzumab (45 with IRRs, 43 without) with cytokine data were included. Irrespective of IRR occurrence, median increase in cytokines was lower with ibrutinib- obinutuzumab versus chlorambucil-obinutuzumab for all cytokines (P < 0.01) except MIP-1β. Across treatment arms, post- obinutuzumab median increase in all cytokines except MIP-1β was greater in patients with versus without IRRs (P < 0.001). IL-6 and IL-8 elevations were associated with IRRs in both treatment arms. Among patients with IRRs, those receiving ibrutinib- obinutuzumab had lower post-obinutuzumab increases in IL-6, IL-8, IL-10, and MCP-1 (P < 0.04) than patients receiving chlorambucil-obinutuzumab. For patients in the ibrutinib-treatment arm, we observed a reduction in both the rate of clinically apparent IRRs and the levels of IRR-related cytokines and chemokines. This observation supports an immunomodulatory mechanism of action for ibrutinib. Clinical Trial Registration: NCT02264574 * Richard Greil 7 Department of Clinical Hematology, Princess Alexandra Hospital, r.greil@salk.at Brisbane, Queensland, Australia 8 1 Centre for Haemato-Oncology, Queen Mary University of London, IIIrd Medical Department Paracelsus Medical University, Salzburg Barts Cancer Institute, London, UK Cancer Research Institute-CCCIT, Cancer Cluster Salzburg, Salzburg, Austria 9 Center for Blood Cancers, Medical Oncology, Sarah Cannon 2 Department of Hematology, ASST Grande Ospedale Metropolitano Research Institute/Tennessee Oncology, Nashville, TN, USA Niguarda, Milano, Italy 3 10 Department of Hematology, Hospital de la Santa Creu i Sant Pau, Translational Medicine, Pharmacyclics LLC, an AbbVie Company, Autonomous University of Barcelona, Barcelona, Spain Sunnyvale, CA, USA 4 Department of Medical Oncology, Tennessee Oncology, 11 Biostatistics, Pharmacyclics LLC, an AbbVie Company, Chattanooga, TN, USA Sunnyvale, CA, USA 5 Department of Clinical Hematology, University of Alberta Hospital, Edmonton, Alberta, Canada 12 Clinical Science, Pharmacyclics LLC, an AbbVie Company, 6 Department of Internal Medicine – Hematology, University Hospital Sunnyvale, CA, USA and Medical School Hradec Králové, Hradec 13 Králové, Czech Republic Department of Hematology, Dokuz Eylül University, Izmir, Turkey 1734 Ann Hematol (2021) 100:1733–1742 Keywords Infusion-related reactions . Ibrutinib . Obinutuzumab . Cytokines Introduction compared with those treated with chlorambucil- obinutuzumab (any grade: 25% versus 58%; grade ≥ 3 or Established therapies for the first-line treatment of chronic serious: 3% versus 9%, respectively) [1]. This finding sup- lymphocytic leukemia (CLL) include novel targeted agents, ported the notion that the administration of ibrutinib prior to such as ibrutinib, and chemoimmunotherapy regimens. obinutuzumab infusion reduced the risk of IRRs. To investi- Typically, chemoimmunotherapy regimens contain anti- gate the role of ibrutinib in IRRs, we prospectively analyzed CD20 monoclonal antibodies (mAbs), such as rituximab or patient samples from iLLUMINATE for circulating cytokines obinutuzumab. Anti-CD20 mAbs have demonstrated efficacy and chemokines thought to be associated with IRRs. when used alone or in combination for patients with previous- ly untreated and relapsed/refractory CLL [1–3], with greater efficacy observed for obinutuzumab compared with rituximab Methods when combined with chlorambucil in first-line CLL [3]. Ibrutinib is the only once-daily oral inhibitor of Bruton’s ty- Study design and population rosine kinase (BTK) approved for treatment of CLL in the United States and European Union that has demonstrated sig- iLLUMINATE (PCYC-1130; NCT02264574) is a phase 3, nificant progression-free survival (PFS) and overall survival international, randomized open-label study of ibrutinib plus (OS) benefit versus established therapies (including obinutuzumab versus chlorambucil plus obinutuzumab in chemoimmunotherapy regimens) in randomized phase 3 stud- first-line CLL/SLL. Eligible patients were aged ≥ 18 years ies in first-line CLL/small lymphocytic lymphoma (SLL) [2, with previously untreated, active CLL/SLL and were aged ≥ 4, 5]. 65 years or < 65 years with coexisting conditions (Cumulative Despite their efficacy, the administration of mAbs can re- Illness Rating Scale score > 6, creatinine clearance < 70 mL/ sult in adverse events including infusion-related reactions min, presence of del(17p) by fluorescent in situ hybridization, (IRRs). IRRs are potentially serious complications that occur or TP53 mutation). Informed consent was obtained from all rapidly post-infusion and arise from an immune-mediated re- participants included in the study. The study design has been action to the infusion of a drug, notably including mAbs [6]. previously described [1]. The severity of IRRs ranges from mild reactions involving flu- like symptoms to rare fatal events. Procedures The rate and severity of IRRs vary depending on the spe- cific treatment. For instance, obinutuzumab is generally asso- Patients were randomly assigned (1:1) to receive ciated with more frequent and more severe IRRs than rituxi- ibrutinib-obinutuzumab or chlorambucil-obinutuzumab. mab as observed in the CLL11 study of patients with previ- In the ibrutinib-obinutuzumab arm, patients received ously untreated CLL (any grade: 66% versus 38%; grade ≥ 3: 420-mg ibrutinib once daily until progressive disease or 20% versus 4%, respectively) [3] and in the GALLIUM study unacceptable toxicity in combination with obinutuzumab of patients with follicular lymphoma (any grade: 68% versus (100 mg on day 1, 900 mg on day 2, 1000 mg on days 8 59%; grade ≥ 3: 12% versus 7%, respectively) [3, 7]. A study and 15 of cycle 1, and then 1000 mg on day 1 of each of patients with relapsed/refractory non-Hodgkin lymphoma subsequent 28-day cycle for a total of 6 cycles). In the who were treated with obinutuzumab resulted in a similar rate chlorambucil-obinutuzumab arm, patients received 0.5- of IRRs (any grade: 69%; grade ≥ 3: 11%) [8]. It has been mg/kg chlorambucil on days 1 and 15 of each 28-day hypothesized that the structure of obinutuzumab (a type II, cycle for 6 cycles with obinutuzumab, as above (total of glycoengineered, humanized mAb) induces greater cellular 6 cycles). activation, thereby eliciting the increase in IRRs seen in these All patients received the same, standard prophylactic med- studies [9]. ications for obinutuzumab-related IRRs that included intrave- In the phase 3 iLLUMINATE (PCYC-1130) study that nous corticosteroids, oral analgesics/antipyretics, and an anti- c o m p ar e d f i r s t - l i n e i b r u t i n i b - o b i n u t u z u m a b a n d histamine. Based on the hypothesis that ibrutinib might reduce chlorambucil-obinutuzumab in patients with previously un- the incidence of IRRs, the oral study drug (ibrutinib or treated CLL, ibrutinib-obinutuzumab demonstrated superior chlorambucil) was administered approximately 30 to PFS including in patients with one or more high-risk genomic 120 min before the first obinutuzumab infusion. features (del[17p]/TP53 mutation, del[11q], and/or unmutated Plasma samples were collected at 4 time points on day IGHV) [1]. Of note, the rate of IRRs in iLLUMINATE was 1: before the ibrutinib/chlorambucil dose (Online lower in patients treated with ibrutinib-obinutuzumab Resource Table S1), immediately prior to infusion of Ann Hematol (2021) 100:1733–1742 1735 obinutuzumab, and 2 and 4 h into the obinutuzumab in- Results fusion. Baseline was defined as immediately before obinutuzumab infusion rather than prior to ibrutinib/ Analysis population and patient characteristics chlorambucil treatment, as there were no meaningful dif- ferences between the two time points (Online Resource Of 228 treated patients, 95 of 113 patients (84%) in the Table S2). Cytokines and chemokines that were evaluated ibrutinib-obinutuzumab arm and 88 of 115 patients (77%) included interferon-γ (IFNγ), interleukin (IL)-6, IL-8/ in the chlorambucil-obinutuzumab arm had cytokine/ CXCL8, IL-10, IL-18, monocyte chemoattractant chemokine results and were included in the cytokine/ protein-1 (MCP-1)/CCL2, macrophage inflammatory pro- chemokine analysis population. In the ibrutinib- tein (MIP)-1α/CCL3, MIP-1β/CCL4, and tumor necrosis obinutuzumab arm (n = 95), 15 patients (16%) had any factor-α (TNFα). A multi-analyte immunoassay panel grade IRRs on day 1, and 80 patients (84%) did not ex- (Myriad RBM, Austin, Texas, USA) was used to analyze perience IRRs. In the chlorambucil-obinutuzumab arm (n the level of plasma cytokines and chemokines. = 88), 45 patients (51%) had any grade IRRs on day 1, and 43 patients (49%) did not experience IRRs. Baseline characteristics were generally similar between Statistical analysis treatment arms in the analysis population (Table 1). Numerically lower platelet counts were observed in the Changes from baseline to post-obinutuzumab infusion peak ibrutinib-obinutuzumab arm compared with the cytokine/chemokine levels were compared between arms, as chlorambucil-obinutuzumab arm (median, 150 × 109/L versus well as between patients with and without IRRs, using 162 × 109/L, P = 0.0413), which was not a clinically relevant Wilcoxon rank sum test. A 2-sided P value of < 0.05 was difference. Baseline MCP-1 levels were also significantly considered significant with no adjustments for multiplicity. lower in the ibrutinib-obinutuzumab arm compared with the Analysis of covariance (ANCOVA) was performed to chlorambucil-obinutuzumab arm (median, 127 pg/mL versus evaluate each of the baseline factors associated with increase 191.5 pg/mL, P = 0.0089), though both of these values fall in post-obinutuzumab cytokine levels (log-adjusted) with within a normal range [10, 11]. Additionally, since a similar baseline of the cytokine level as a covariate in the model: arm difference was observed prior to administration of any sex (male versus female), bulky disease (lymph nodes < study drug (Online Resource Table S1), this indicates that 5 cm versus ≥ 5 cm), Rai stage (stage III/IV versus others), the difference was due to the chance of randomization and splenomegaly (yes/no), age (continuous variable), and log- was not related to any study treatment. Patients in both arms transformed hematologic parameters (hemoglobin, platelet had median baseline levels of IFNγ and IL-6 that were below counts, absolute neutrophil count, and absolute lymphocyte the detectable threshold of the assay (Table 1), which preclud- count, all as continuous variables). ed their inclusion in the analysis as a predictor for the devel- Multivariable logistic regression was performed to opment of IRRs. identify predictors of IRRs. Explanatory variables were first selected based on a univariate logistic regression Analysis of change in cytokine levels by treatment model at a P < 0.2 level among the following factors: arm and IRR status treatment arm, baseline clinical factors (stated above), and log-transformed baseline cytokine levels (IFNγ, IL- Overall, increases over baseline levels were observed post- 6, IL-8, IL-10, IL-18, TNFα, MCP-1, MIP-1α, and MIP- obinutuzumab infusion across all cytokines and chemokines 1β). Correlation between selected factors was also exam- analyzed regardless of treatment arm or IRR occurrence. ined. Independent variables (absolute value of Pearson Median change from baseline levels was significantly lower correlation coefficient < 0.2) were included in the multi- (P < 0.01) in patients treated with ibrutinib-obinutuzumab variable model. If any pair of the variables correlated, compared with chlorambucil-obinutuzumab for IFNγ, IL-6, only the variable with the smaller P value in the univar- IL-18, IL-8, IL-10, TNFα, MCP-1, and MIP-1α (Fig. 1). iate model was entered into the multivariable model. MIP-1β was the only chemokine for which the change from baseline levels was not significantly different between treat- ment arms (P = 0.7747). Data sharing statement Across both treatment arms, increases in median changes from baseline levels were significantly greater (P < 0.001) for Requests for access to individual participant data from clinical patients with IRRs (n = 60) compared with patients without studies conducted by Pharmacyclics LLC, an AbbVie IRRs (n = 123) for all cytokines and chemokines analyzed Company, can be submitted through the Yale Open Data except for MIP-1β (P = 0.6290) (Fig. 2, Online Resource Access (YODA) Project site at http://yoda.yale.edu. Fig. S1). 1736 Ann Hematol (2021) 100:1733–1742 Table 1 Patient demographics and baseline characteristics in the Characteristic Ibrutinib- Chlorambucil- cytokine analysis population obinutuzumab obinutuzumab n = 95a n = 88b Median age, years (range) 70 (47–87) 72 (40–86) Male, n (%) 60 (63) 63 (72) Rai stage III/IV, n (%) 49 (52) 45 (51) Bulky disease ≥ 5 cm, n (%) 25 (26) 33 (38) Splenomegaly, n (%) 80 (84) 74 (84) Median hemoglobin, g/dL (range) 115 (58–163) 112 (72–153) Median platelet counts, × 109/L (range) 150 (48–359) 162 (48–463) Median absolute neutrophil count, × 109/L (range) 6 (0–362) 5 (0–61) Median absolute lymphocyte count, × 109/L (range) 54 (1–350) 48 (1–386) Median baseline cytokine levels, pg/mL (range) IFNγ < 6.8 (NA) < 6.8 (NA) IL-6 < 3.4 (NA–957) < 3.4 (NA–252) IL-8 6.6 (6.0–750) 9.0 (6.0–306) IL-10 9.0 (5.2–1090) 11.0 (5.2–155) IL-18 416.0 (69–5540) 401.0 (117–3010) MCP-1 127.0 (110–1920) 191.5 (110–3090) MIP-1α 34.0 (25–4070) 30.0 (25–741) MIP-1β 384.0 (45–44400) 446.0 (45–5920) TNFα 26.0 (18–887) 24.5 (18–335) a Enrolled in iLLUMINATE, n = 113 b Enrolled in iLLUMINATE, n = 116 IFN, interferon; IL, interleukin; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory pro- tein; NA, not available (below the lower limit of detection); TNF, tumor necrosis factor Analysis of cytokine/chemokine patterns in patients within obinutuzumab levels of IL-18, TNFα, MCP-1, and MIP-1α each treatment arm showed that greater increases from base- (P < 0.04) (Fig. 3) than patients without IRRs. In the line levels in IL-6 and IL-8 post-obinutuzumab were signifi- chlorambucil-obinutuzumab arm, patients with IRRs also cantly associated with IRRs in both ibrutinib-obinutuzumab had greater increases than patients without IRRs in post- (P = 0.0012 and P = 0.0126, respectively) and chlorambucil- obinutuzumab levels of IFNγ and IL-10 (P < 0.03) (Fig. 3). obinutuzumab treated patients (P = 0.0097 and P = 0.0234, ANCOVA was performed to evaluate potential baseline respectively). In the ibrutinib-obinutuzumab arm, patients characteristics associated with the increase in absolute values with IRRs also had significantly greater increases in post- from baseline of cytokine and chemokine levels observed Fig. 1 Median changes in cytokine and chemokine levels from baseline cytokines and chemokines examined by treatment arm. IFN, interferon; to post-obinutuzumab peak by treatment arm. Median changes over IL, interleukin; MCP, monocyte chemoattractant protein; MIP, baseline (i.e., immediately before obinutuzumab) are shown for all macrophage inflammatory protein; TNF, tumor necrosis factor Ann Hematol (2021) 100:1733–1742 1737 Fig. 2 Median changes in cytokine and chemokine levels from baseline their IRR status. IFN, interferon; IL, interleukin; IRR, infusion-related to post-obinutuzumab peak by occurrence of IRR on day 1. Median reaction; MCP, monocyte chemoattractant protein; MIP, macrophage changes over baseline (i.e., immediately before obinutuzumab) are inflammatory protein; TNF, tumor necrosis factor shown for all cytokines and chemokines examined by all patients and post-obinutuzumab infusion in each treatment arm. In the splenomegaly (IFNγ, IL-18, IL-6, IL-8, MIP-1α, TNFα), ibrutinib-obinutuzumab arm, factors associated with an in- lower hemoglobin levels (IL-10, IL-8), lower platelet count crease in cytokine and chemokine levels included splenomeg- (IL-18, MCP-1, MIP-1β), and higher absolute lymphocyte aly (IL-6, IL-8, MCP-1, MIP-1α, TNFα), lower hemoglobin count (IFNγ, IL-10, IL-6, IL-8, MCP-1, MIP-1α, TNFα) levels (IL-8), higher absolute neutrophil count (IL-8, MCP-1, (Online Resource Table S3). MIP-1α, TNFα), and higher absolute lymphocyte count In patients with IRRs and comparing between treatment (IFNγ, IL-6, IL-8, MCP-1, MIP-1α, TNFα). In the arms, patients treated with ibrutinib-obinutuzumab had lower chlorambucil-obinutuzumab arm, factors associated with an increases over baseline in IL-6, IL-8, IL-10, and MCP-1 com- increase in cytokine and chemokine levels included Rai stage pared with patients treated with chlorambucil-obinutuzumab III/IV (IL-8, IL-18), bulky disease (≥ 5 cm: IL-10, TNFα), (P < 0.04) (Fig. 4). a b Fig. 3 Median changes in cytokine and chemokine levels from baseline ibrutinib-obinutuzumab; b chlorambucil-obinutuzumab) and IRR to post-obinutuzumab peak by occurrence of IRR on day 1. Median occurrence. IFN, interferon; IL, interleukin; IRR, infusion-related changes over baseline (i.e., immediately before obinutuzumab) are reaction; MCP, monocyte chemoattractant protein; MIP, macrophage shown for all cytokines and chemokines examined by treatment arm (a inflammatory protein; TNF, tumor necrosis factor 1738 Ann Hematol (2021) 100:1733–1742 Fig. 4 Median changes in cytokine and chemokine levels from baseline for only patients who experienced an IRR. IFN, interferon; IL, to post-obinutuzumab peak by treatment arm in patients with IRRs on day interleukin; MCP, monocyte chemoattractant protein; MIP, macrophage 1. Median changes over baseline (i.e., immediately before obinutuzumab) inflammatory protein; TNF, tumor necrosis factor are shown for all cytokines and chemokines examined by treatment arm Multivariable analysis of baseline predictors of IRRs of ibrutinib-obinutuzumab in first-line CLL, Velez Lujan et al. reported that only 19% of treated patients developed IRR Among all baseline factors, five were selected as explanatory symptoms (grades 1–2: 15%; grade 3: 3%) [13], which com- variables for the multivariable logistic model: treatment arm, pares favorably with historical rates of IRRs with sex, absolute lymphocyte count, IL-18, and MCP-1. IFNγ and obinutuzumab as single-agent or in other (non-ibrutinib) com- IL-6 had undetectable levels at baseline and could not be in- bination regimens (all grades: 65–90%; grade ≥ 3: 2.5–20%) cluded in the evaluation of IRR predictors. Two baseline fac- [3, 14, 15]. Ibrutinib has also been shown to reduce the sever- tors had a significance of P < 0.2 in the univariate model but ity of cytokine release syndrome (CRS) in recent studies of were excluded due to correlation: baseline bulky disease cor- patients with relapsed/refractory CLL receiving chimeric an- related with baseline absolute lymphocyte count (Pearson cor- tigen receptor-modified T-cell (CAR-T) therapy [16, 17]. relation coefficient 0.200) and baseline IL-10 correlated with Taken together, these studies indicate a role for ibrutinib in both baseline IL-18 (Pearson coefficient 0.592) and MCP-1 modulating the immune response to certain therapies. (Pearson coefficient 0.284). All other baseline factors were While the mechanism of IRRs is not fully understood, excluded due to P > 0.2 in the univariate analysis. In the final the development of IRRs has been associated with the multivariable model, treatment arm, baseline absolute lym- release of proinflammatory cytokines. In the case of the phocyte count, and baseline IL-18 were independently asso- anti-CD20 mAbs rituximab and obinutuzumab, these pro- ciated with the likelihood of IRRs (P < 0.05). Within each inflammatory cytokines include TNFα, IL-6, and IL-8 arm, the multivariable model demonstrated that baseline IL- [18, 19]. Freeman et al. used multivariable logistic regres- 18 was associated with the likelihood of IRRs in the ibrutinib- sion models to determine baseline factors associated with obinutuzumab arm, and baseline absolute lymphocyte count IRR risk in the CLL11 study and determined that CD20 was associated with the likelihood of IRRs in the expression level was a main predictor of IRRs post- chlorambucil-obinutuzumab arm. obinutuzumab or rituximab infusion [9]. Additionally, an absolute lymphocyte count of ≥ 50 × 10 9/L has been shown to be a risk factor for IRRs, particularly for severe Discussion IRRs [18]. In a pre-clinical model of CAR-T therapy, the addition In iLLUMINATE, the frequency and severity of IRRs were of ibrutinib significantly reduced serum cytokines associ- reduced in patients treated with ibrutinib-obinutuzumab com- ated with CRS including IFNγ, IL-6, TNFα, IL-2, and pared with those treated with chlorambucil-obinutuzumab [1]. GM-CSF [20]. In patients with CLL, treatment with This decrease in IRRs with ibrutinib-based treatment is con- ibrutinib has been shown to decrease regulatory T cells sistent with results from the phase 3 iNNOVATE study, and modulate the cytokine and chemokine milieu in vivo which evaluated ibrutinib combined with rituximab versus through the decrease of several cytokines (including IL- rituximab alone in patients with Waldenström’s macroglobu- 10, IFNγ, IL-6, and TNFα) and chemokines (including linemia, and showed that any grade and grade ≥ 3 IRRs oc- IL-8, MCP-1, MIP-1α, and MIP-1β) [21, 22]. We hy- curred less frequently with ibrutinib-rituximab versus pothesized that pretreatment with ibrutinib in patients re- placebo-rituximab (any grade: 43% versus 59%; grade ≥ 3: ceiving obinutuzumab infusion led to decreased IRRs be- 1% versus 12%, respectively) [12]. In a recent phase 1/2 study cause of the potential modulatory effect of ibrutinib on the Ann Hematol (2021) 100:1733–1742 1739 release of proinflammatory cytokines. In iLLUMINATE, cytokines and chemokines, except MIP-1β. The pattern serial measurements of plasma-based inflammatory of cytokines released during IRRs appeared to differ by markers were prospectively performed for various cyto- treatment regimen, where significantly lower increases in kines and chemokines known to be associated with IRRs post-obinutuzumab cytokine levels were observed with and anti-CD20 therapy (Online Resource Table S4). ibrutinib-obinutuzumab versus chlorambucil- In our analysis, significant increases in post-obinutuzumab obinutuzumab for IL-6, IL-8, IL-10, and MCP-1. levels of IL-6 and IL-8 were observed across both treatment Increases in IL-6 and IL-8 levels have been shown to be arms in patients with IRRs versus patients who did not expe- related to IRRs induced by obinutuzumab [19]. IL-10 is rience IRRs. This finding is consistent with prior reports of an anti-inflammatory cytokine that functions to suppress obinutuzumab-induced IRRs associated with IL-6 and IL-8 and dampen immune responses. Increases in IL-10 were [9]. While the post-infusion increase in IL-6 and IL-8 levels observed in a patient with diffuse large B-cell lymphoma among patients with IRRs was common to the 2 arms, the who experienced a fatal IRR after treatment with rituxi- patterns of other cytokine and chemokine levels released mab [23]. MCP-1 is a chemokine that acts as a main post-obinutuzumab differed. chemoattractant for monocytes and macrophages, and el- Within the ibrutinib-obinutuzumab arm, patients with evated levels of MCP-1 were associated with severe cy- IRRs (versus those without IRRs) had significantly higher tokine release syndrome in patients with B-cell malignan- levels of IL-6, IL-8, TNFα, IL-18, MCP-1, and MIP-1α, cies treated with chimeric antigen receptor T-cell therapy whereas in the chlorambucil-obinutuzumab arm, patients [24]. with IRRs had significantly higher levels of IFNγ, IL-6, The mechanism by which ibrutinib treatment decreases IL-8, and IL-10 compared with those without IRRs. In a or inhibits circulating cytokine and chemokine levels that multivariable analysis of baseline factors associated with contribute to IRRs remains unknown. An in vitro study IRRs during ibrutinib-obinutuzumab treatment, only showed that rituximab mediated the CD20 trogocytosis of higher baseline levels of IL-18 predicted the risk of de- B cells that led to the rapid production and release of IL-6 veloping IRRs. For patients in the chlorambucil- [25]. Ibrutinib has been shown to potently inhibit obinutuzumab arm, elevated baseline absolute lymphocyte trogocytosis mediated by anti-CD20 mAbs in CLL cells counts and MCP-1 were factors independently predictive in vitro [26]. This inhibition of trogocytosis by ibrutinib of IRR risk. Results from the multivariable analysis are could potentially explain the attenuated release of IL-6 limited given that some cytokines (particularly IFNγ and observed in the current analysis; however, further studies IL-6) had baseline levels that were below the detectable are needed to investigate the exact mechanism. Although threshold of the assay, preventing their inclusion in the several pre-clinical studies have shown that treatment with multivariable analysis assessing for predictive variables ibrutinib may antagonize the mechanism of anti-CD20 for IRRs. In addition, the small number of patients who antibodies (particularly as it relates to cell-mediated effec- experienced IRRs within the ibrutinib-obinutuzumab arm tor mechanisms such as antibody-dependent cellular cyto- (n = 15) may also limit interpretations of the current toxicity) [27–29], these observations have not translated analysis. to the clinical setting [14, 30–32]. In the recent phase 3 These findings differ from a recent report of cytokine anal- Alliance (A041202) study that evaluated ibrutinib-based ysis from a phase 1/2 study in 32 patients treated with first-line treatment (single-agent ibrutinib or ibrutinib-rituximab ibrutinib-obinutuzumab [13]. In that study, significant in- combination) versus bendamustine-rituximab in first-line creases from baseline in IFNγ, MIP-1α, and TNFα post- CLL, slightly higher responses were observed for obinutuzumab infusion were observed in patients who devel- ibrutinib-rituximab than for single-agent ibrutinib (com- oped IRRs. In univariate analysis, baseline levels of IFNγ, IL- plete response [CR] rate: 12% and 7%; undetectable min- 6, and MIP-1α were found to be significantly higher in pa- imal residual disease [MRD] 4% and 1%). There was no tients who developed IRRs. Differences between findings difference, however, in PFS between the two arms (88% from our current analysis and the published phase 1/2 report versus 87% at 2 years), although it should be noted the with ibrutinib-obinutuzumab may be partially due to patient study was not designed to compare between the two population differences in burden of disease, panels of cyto- ibrutinib-based treatment arms [31]. kines and chemokines analyzed (iLLUMINATE included The current analysis demonstrates that ibrutinib sup- MCP-1), methods of analysis, and sample size (the larger presses obinutuzumab-induced increases in multiple cyto- number of patients in iLLUMINATE allowed multivariable kines and chemokines related to IRRs and suggests that analysis). the reduction in IRR events in ibrutinib-treated patients is Our analysis shows that patients treated with ibrutinib the result of a reduction in the release of inflammatory versus chlorambucil prior to obinutuzumab infusion had cytokines and chemokines. These observations may have significantly lower increases from baseline for all implications for immunotherapeutic approaches where the 1740 Ann Hematol (2021) 100:1733–1742 tuning of the inflammatory reaction may modify the type Janssen-Cilag, Gilead, and Roche; travel, accommodations, or expenses: AbbVie, Gilead, Janssen-Cilag, and Roche. DG: honoraria and and degree of cytokine release patterns (activated or consulting/advisory role: Janssen-Cilag. JGG: honoraria: AbbVie, dampened) and their role in the treatment effect or safety Bristol-Myers Squibb, AstraZeneca, Janssen, and Roche; consultancy/ profile of immunotherapies. advisory role: AbbVie, Janssen, Gilead; research funding: AstraZeneca, Celgene, and Janssen. IWF: consultancy/advisory role: AbbVie, AstraZeneca, BeiGene, Genentech, Gilead, Great Point Partners, Iksuda Therapeutics, Janssen, Juno Therapeutics, Kite Pharma, MorphoSys, Supplementary Information The online version contains supplementary Nurix Therapeutics, Pharmacyclics LLC, an AbbVie Company, Roche, material available at https://doi.org/10.1007/s00277-021-04536-6. Seattle Genetics, Takeda, TG Therapeutics, Unum Therapeutics, Verastem, and Yingli Pharmaceuticals; research funding: AbbVie, Acknowledgements The authors thank the patients who participated in Acerta Pharma, Agios, ArQule, AstraZeneca, BeiGene, Calithera, this study and their supportive families, as well as the investigators, sub- Celgene, Constellation, Curis, F. Hoffman-la Roche Ltd, Forma, Forty investigators, and coordinators at each of the study sites. This study was Seven, Genentech, Gilead, IGM Biosciences, Incyte, Infinity, Janssen, sponsored by Pharmacyclics LLC, an AbbVie Company. Editorial sup- Juno, Karyopharm, Kite, Loxo, Merck, MorphoSys AG, Novartis, port was provided by Emily Chastain, PhD, an employee of Pfizer, Pharmacyclics LLC, an AbbVie Company, Portola, Rhizen Pharmacyclics LLC, an AbbVie Company. Pharmaceuticals, Roche, Seattle Genetics, Takeda, Teva, TG Therapeutics, Trillium, Triphase Research & Development Corp, Author contribution Data were collected by investigators and their re- Unum, and Verastem. ZW: employment: currently with Ambry search teams. The sponsor confirmed the accuracy of the data and com- Genetics; previously with Pharmacyclics LLC, an AbbVie Company; piled them for analysis. All authors had full access to the data and stock or other ownership: AbbVie, AnaptysBio, Bristol-Myers Squibb, interpreted the data. All authors contributed to revisions and final approv- and Luxvitae; travel, accommodations, or other expenses: Ambry al of the manuscript and made the decision to submit the manuscript for Genetics. LWKC: employment: Pharmacyclics LLC, an AbbVie publication. Company; stock or other ownership: AbbVie and Eli Lilly; patents, roy- alties, or other intellectual property: Pharmacyclics LLC, an AbbVie Company. ANN: employment: currently with SparcBio; previously with Funding Open access funding provided by Paracelsus Medical Celgene and Pharmacyclics LLC, an AbbVie Company; stock or other University. The study was sponsored by Pharmacyclics LLC, an ownership: AbbVie, Celgene, and SparcBio; patents, royalties, or other AbbVie Company, and was designed by the sponsor in collaboration with intellectual property: Celgene; travel, accommodations, or other ex- investigators of the Steering Committee. Janssen Research and penses: Celgene. CZ and LS: employment: Pharmacyclics LLC, an Development provided funding for the study and was permitted courtesy AbbVie Company; stock or other ownership: AbbVie. FD: consulting/ review of the manuscript. advisory role: AbbVie AstraZeneca, Amgen, and Roche; research funding: AbbVie, Janssen, Pharmacyclics LLC, an AbbVie Company, Data availability Requests for access to individual participant data from and AstraZeneca; speakers bureau: AbbVie, Amgen, and Janssen; travel, clinical studies conducted by Pharmacyclics LLC, an AbbVie Company, accommodations, or other expenses: AbbVie, Amgen, Pfizer, and can be submitted through the Yale Open Data Access (YODA) Project Janssen. site at http://yoda.yale.edu. 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