Second generation <scp>BTK</scp> inhibitors impair the anti‐fungal response of macrophages and neutrophils
Authors: Ana Colado, José L. Marín Franco, Esteban E. Elías, Mikele Amondarain, Maricef Vergara Rubio, Valeria Sarapura Martínez, Gregorio Cordini, Federico Fuentes, Luciana Balboa, Horacio Fernandez Grecco, Miguel Pavlovsky, Fernando Bezares, Pablo Morande, Mirta Giordano, Romina Gamberale, Mercedes Borge
Journal: American Journal of Hematology (2020)

Bruton's tyrosine kinase inhibitors (BTKi) have changed the treatment paradigm for chronic lymphocytic leukemia (CLL) and other B-cell malignancies. Ibrutinib is a first in-class BTKi approved for CLL treatment, and although it shows impressive clinical efficacy in different risk groups, most patients do not achieve complete responses and require continuous administration. Infection rates during ibrutinib treatment are similar or lower compared to other treatments, but they are still one of the most frequent adverse events leading to drug-discontinuation, especially in relapsed/refractory patients.1 The emergence of invasive fungal infections in some treated patients, including cases of invasive aspergillosis with central nervous system involvement, suggests that ibrutinib may disturb immunological mechanisms implicated in fungal defense.2 In line with these clinical observations, substantial evidence showed that ibrutinib affects functions on macrophages and neutrophils,3-6 both critical players for antimicrobial immune response. Aside from BTK, ibrutinib also targets other kinases such as ITK, TEC and EGFR, which may account for some of its adverse effects. Second generation BTKi, endowed with higher selectivity, are being tested in CLL patients. Acalabrutinib (ACP-196) is the first second-generation BTKi approved for medical use, currently for patients with mantle cell lymphoma and CLL. Probably due to its greater selectivity, compared to ibrutinib, acalabrutinib has fewer effects on platelet functionality and does not inhibit some of the effector mechanisms of anti-CD20 antibodies. Spebrutinib (CC-292) is another second generation BTKi shown to be well-tolerated in relapsed/refractory CLL patients, although its clinical activity was inferior to that of ibrutinib. The aim of this study was to evaluate the effect of the second generation BTKi, acalabrutinib and spebrutinib, on macrophage and neutrophil functions relevant for anti-fungal immune response, and on cell-mediated mechanisms of action of anti-CD20 antibodies. Peripheral blood samples from healthy donors and CLL patients were obtained with their informed consent in accordance with the Declaration of Helsinki. Experiments were carried out with healthy donors' cells and confirmed with those from CLL patients. Results with CLL samples are shown in Figures S11 to S14. BTKi were used at clinically achievable concentrations (0.3 and 1 μM) which did not affect the viability of macrophages and neutrophils in vitro (Figure S1). Detailed experimental procedures are depicted in the online Supporting Information, Appendix S1. We found that, as reported for ibrutinib,3, 5 acalabrutinib, but not spebrutinib, impaired M1 polarization in macrophages, which is a profile associated with an efficient anti-microbial immune response. This was shown by an increase in the expression of the M2-associated markers CD14, CD16, CD206 and CD163 and on IL-10 secretion, and also, by a decrease in the M1-associated markers CD86 and HLA-DR and on TNF-α secretion (Figure 1A and Figure S2). When we evaluated the response of macrophages to fungal stimulation we found that acalabrutinib, as reported for ibrutinib,4 significantly decreased TNF-α secretion in response to heat inactivated Aspergillus fumigatus conidia or Candida albicans yeast (Figure 1B,C and Figure S3A,B). In line with this, BTKi impaired TNF-α secretion in response to zymosan, a ligand of TLR2 and Dectin-1, which are receptors involved in macrophage-recognition of fungi (Figure S3C,D). On the other hand, spebrutinib showed a lower inhibitory effect on macrophages, and only impaired TNF-α secretion in response to A. fumigatus when used at 1 μM (Figure 1B), but not at 0.3 μM (Figure S3A). In contrast to TNF-α secretion, we found that none of the BTKi affected the phagocytosis of A. fumigatus, C. albicans or zymosan by macrophages (Figure 1D and Figure S4). When we evaluated the effect of second generation BTKi on neutrophil response to fungi, we found that BTKi inhibited classical markers of neutrophil activation, as CD11b upregulation, CD62L downregulation and cell enlargement, in response to heat inactivated C. albicans yeast (Figure 1E and Figure S5A, and S6A) or zymosan stimulation (Figures S5B and S6B). On the other hand, cell enlargement was the only parameter affected by BTKi when neutrophils were stimulated by A. fumigatus conidia (Figure 1F and Figures S5C and S6C). Interestingly, in contrast to what we found in macrophages, BTKi decreased neutrophil capacity to uptake C. albicans, A. fumigatus and zymosan (Figure 1G,H and Figures S7 and S8). Moreover, oxidative burst in response to fungal stimulation was impaired by the three BTKi (Figure 1I,J and Figure S9). Then, we evaluated the effect of second generation BTKi on neutrophil-mediated inhibition of A. fumigatus conidia germination, which is a central mechanism exerted by neutrophils to prevent fungal invasion. To that aim, neutrophils were co-cultured with viable conidia for 6 hours, in the presence or absence of the BTKi, and then conidia germination was observed by microcopy and quantified as described in supporting information. Interestingly, we found that the presence of second generation BTKi decreased the capacity of neutrophils to control A. fumigatus conidia germination (Figure 1K,L), as it was recently reported with ibrutinib.6 It is well-established that BTKi interfere with Fcγ receptor-dependent functions which in CLL patients are critical not only in host defense but also for effective therapy with anti-CD20 mAbs. Therefore we compared the effects of the three BTKi on antibody-dependent phagocytosis (ADCP) mediated by macrophages and antibody dependent cytotoxicity (ADCC) mediated by NK-cells. We found that spebrutinib, as reported for acalabrutinib,7 did not affect macrophage ADCP, but impaired NK-cell degranulation and ADCC at comparable levels that ibrutinib (Figure S10). Although spebrutinib is a more selective BTKi than ibrutinib, they both have comparable affinity for ITK, which may explain its effect on NK-cells. Meanwhile, the fact that none of the second generation BTKi affects macrophage-ADCP supports the notion that ibrutinib-mediated inhibition on the anti-CD20 mechanism of actions are BTK-independent, and will be overcome with next-generation BTKi. Because infections are a leading cause of morbidity and mortality in patients with CLL, it is critical to increase our knowledge on how novel agents affect immune cells in order to prevent or manage infectious complications. Here we found that as reported for ibrutinib, second generation BTKi showed a strong inhibitory effect on macrophage and neutrophil anti-fungal response. First, we found that second generation BTKi impaired macrophage polarization and TNF-α secretion in response to fungal stimulation. Pro-inflammatory cytokines secreted by macrophages strengthen the immune response to fungi, essentially by promoting local inflammation which in turns induces cell-recruitment to tissue. We also found that second generation BTKi dampened key functions in neutrophils that promote fungal control as phagocytosis, oxidative burst and A. fumigatus conidia germination. Indeed, defects in phagocyte number or function as found in patients with chronic granulomatous disease (CGD) are associated with an increased risk of fungal infections. The effect on macrophages seems to be different between the two second generation BTKi evaluated here, being more pronounced with acalabrutinib than with spebrutinib. Although in terms of potency, spebrutinib was found inferior to acalabrutinib when their inhibitory capacity was compared in a cellular assay,8 the concentrations of spebrutinib used in our work are in the micromolar range, and therefore high enough to inhibit BTK in a cellular assay according to that report. This suggests that ibrutinib and acalabrutinib might impair cytokine secretion and macrophage polarization by inhibiting an off-target kinase for which spebrutinib has no affinity or a pronounced lower potency. On the other hand, the three BTKi have equal effect on neutrophil fungal-response, thus it is likely that the effect of BTKi on neutrophils is an on-target effect and therefore will be present even with inhibitors with the highest selectivity for BTK. Regarding infections in patients treated with second generation BTKi, a pooled analysis of safety data from different clinical trials with 610 patients treated with acalabrutinib monotherapy showed that grade ≥ 3 infections occurred in 16.2% patients, with pneumonia being the most frequent.9 Opportunistic infections including Pneumocystis jirovecii or cryptococcal pneumonia and aspergillosis were also reported in four patients of that same study. Our results suggest that acalabrutinib may be compromising the innate immune response in treated patients. Combination of BCR-associated kinase inhibitors with anti-CD20 antibodies has been an attractive and extensively investigated treatment strategy in CLL. Here we found that macrophage ADCP, which is proposed as a central mechanism of action of anti-CD20 mAbs in vivo, was not affected by spebrutinib, suggesting that this BTKi can be considered as a candidate for combinatory treatment with anti-CD20 mAbs. However, given its lower clinical efficacy compared to other BTKi, it is not clear whether the development of spebrutinib in CLL will continue. In conclusion our results show that BTKi have inhibitory effects on innate immune cells. Second-generation BTKi, particularly acalabrutinib, showed detrimental effects on macrophages and neutrophils, comparable to those of ibrutinib, which may impair the anti-microbial innate-immune response in vivo and should be taken into account especially in the presence of other factors that weaken the immune system. On the other hand, second generation BTKi show fewer effects on anti-CD20 mAbs-activity and should be considered as better partners for combinatory treatment. The authors would like to thank to María Tejeda, Romina Mariel Pagano, Federico Remes Lenicov and Agustina Mena for their technical assistance. Appendix S1. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.