Targeting Bruton tyrosine kinase using non-covalent inhibitors in B cell malignancies

Despite the clinical success ibrutinib has achieved, it should be noted that CLL progression or Richter transformation (RT) occurs in a subset of CLL patients administrated with ibrutinib [24]. Most of the patients who experience CLL relapse have BTK C481 mutations and less commonly PLCG2 mutations [24]. The BTK C481 mutations, most of which are BTK C481S, involve the cysteine where ibrutinib binding occurs, rendering ibrutinib unable to inhibit BTK and downstream pathways. BTK C481 mutations are also detected in approximately 30% of patients who underwent RT on ibrutinib, suggesting BTK C481 mutations could be involved in mediating RT on ibrutinib [25]. Ibrutinib resistance has also been observed in WM patients with active ibrutinib therapy. BTK C481 mutations are commonly observed in WM patients who experienced progression, suggesting BTK C481 mutations are also responsible for ibrutinib resistance in WM [26]. Both primary resistance (10.2–35%) and acquired resistance (17.5–54%) to ibrutinib are common in ibrutinib-treated patients with MCL [27]. Acquired BTK C481S mutation is detected in a small proportion of MCL patients who relapsed after ibrutinib therapy [28].

Disease progression due to drug resistance has also been reported in patients treated with other covalent inhibitors. According to the study by Woyach et al., with a median follow-up of 47.5 months, CLL relapse occurred in 17 of 105 CLL patients on acalabrutinib therapy [29]. BTK C481 mutations were identified in 11 of these 16 patients, indicating that the mechanism for acalabrutinib resistance is similar to that for ibrutinib resistance [29]. Thirty-one percent of R/R MCL patients on acalabrutinib had disease progression with a median follow-up of only 15.2 months. The mechanisms responsible for acalabrutinib resistance in MCL have not been characterized yet [30]. The acquired resistance also occurs in CLL patients treated with zanubrutinib, although at a relatively low rate [22]. CLL patients progressing on zanubrutinib had concurrent BTK Leu528Trp and BTK C481 mutations [31]. The co-occurrence of BTK Leu528Trp and BTK C481 mutations suggests these mutations may cooperate in mediating resistance to zanubrutinib.

In addition to ibrutinib resistance, ibrutinib intolerance is also a concern in patients treated with ibrutinib, frequently leading to ibrutinib discontinuation [32, 33]. Some kinases also harbor a modifiable cysteine residue that is homologous to C481 in BTK; therefore, these kinases could also be inhibited by ibrutinib [34]. A variety of receptor tyrosine kinases and non-receptor tyrosine kinases, including EGFR, ITK, TEC, ERBB4, BMK, JAK3, and HER2 [35], are covalently inhibited by ibrutinib. The inhibition of these non-BTK kinases accounts for part of the AEs related to ibrutinib. The common AEs in patients treated with ibrutinib include diarrhea, bleeding, atrial fibrillation, infection, and others, and serious AEs may cause discontinuation. According to a pooled analysis, diarrhea occurred in approximately half of the CLL patients treated with ibrutinib and 5% of patients had grade 3 diarrhea [36]. Ibrutinib-related diarrhea could be attributed to the inhibition of EGFR [37]; however, the exact mechanism remains to be determined. Bleeding/bruising events occurred in 55% of CLL patients on ibrutinib therapy, and 7.6% of patients experienced major hemorrhage events [36]. The inhibition of BTK and other Tec family kinases impairs glycoprotein VI signalings, suppressing platelet aggregation, and thereby contributing to bleeding events [38‐40]. Approximately 11% of CLL patients developed atrial fibrillation during ibrutinib monotherapy, and 5% of patients developed grade 3 atrial fibrillation [36]. It should be noted that the frequency of atrial fibrillation is various in different studies, with younger patients having a lower rate of atrial fibrillation (7.4% in the E1912 study) [15]. Ibrutinib inhibits BTK and TEC in cardiac tissue, resulting in the downregulation of the cardiac PI3K/AKT pathway. It has been reported that reduced PI3K/AKT activity increased the susceptibility to atrial fibrillation [41]. Therefore, ibrutinib may lead to atrial fibrillation by inhibiting the PI3K/AKT pathway in cardiac tissue [42]. Infection was prevalent (83%) in CLL patients on ibrutinib therapy, and grade 3 or 4 infection occurred in 29% of patients [36]. The significantly increased risk of infection could be caused by immune impairment, which is attributed to the inhibition of ITK in T cells and BTK in macrophages [43]. Other common AEs include arthralgia, fatigue, hypertension, and rash. Consistently, the second-generation covalent BTK inhibitors, including acalabrutinib, zanubrutinib, and orelabrutinib, bind to C481 residue. Current studies have shown that the second-generation covalent BTK inhibitors have higher specificity and fewer off-target toxicities, while still cause adverse events. For instance, headache (43%) and diarrhea (39%) were very common in acalabrutinib-treated relapsed CLL patients [44]. Further, the follow-up of patients treated with these second-generation covalent BTK inhibitors is relatively short compared to those treated with ibrutinib, so that longer follow-up is warranted to observe potential adverse events.

Vecabrutinib is a selective, reversible, non-covalent BTK inhibitor with nanomolar potency. In vitro studies have demonstrated vecabrutinib shows activity against both wild-type and C481S-mutated BTK. Vecabrutinib decreases surface expression of B cell activation markers and viability of primary cells from CLL patients, resulting in a phenotypic alteration that is comparable to ibrutinib [45]. Vecabrutinib also inhibits ITK but not EGFR; therefore, it may have less EGFR-mediated toxicities including rash and diarrhea [46].

A phase I study of vecabrutinib that enrolled 32 healthy participants was completed [47]. All AEs were grade 1, including headache (n = 5) and nausea, constipation, bronchitis, fatigue, orthostatic hypotension, and supraventricular tachycardia (n = 1 each), except for 1 subject who received 300 mg vecabrutinib experiencing grade 2 headache and fatigue [47]. The occupation produced by vecabrutinib and duration of BTK inhibition is encouraging [47]. A phase Ib/II dose-escalation and cohort-expansion study is ongoing in patients with relapsed/refractory advanced B cell malignancies who progressed on covalent BTK inhibitor therapy. According to the reported data, 27 patients (CLL, n = 21; MCL, n = 2; WM, n = 3; MZL = 1) have been treated with doses ranging from 25 to 300 mg, twice daily [48]. The maximum tolerated dose (MTD) has not been reached. Data regarding safety are available for 24 patients; the most frequent AEs included anemia (37.5%), neutropenia (25%), night sweats (25%), and headache (25%) [48]. Grade 3 drug-related AEs included alanine aminotransferase (ALT) elevation, neutropenia and worsening anemia (all in 1 patient), and leukocytosis (2 patients). Regarding efficacy, no response was observed, and 4 CLL patients, including three with BTK C481S mutation, showed stable disease. Preliminary results revealed that vecabrutinib in dose levels from 25 to 200 mg twice daily was safe in patients with B cell malignancies. The 300 mg twice daily dose level was being evaluated when the study was presented at the 2019 American Society of Hematology (ASH) meeting [48]. Vecabrutinib shows manageable safety profiles, while its efficacy in patients with B cell malignancies remains to be explored.

Fenebrutinib is a highly selective, reversible, non-covalent BTK inhibitor that does not bind to the C481 residue for its action and does not inhibit EGFR or ITK [49, 50]. Fenebrutinib potently inhibits BCR signaling through BTK inhibition. In vitro studies showed that fenebrutinib decreased the activation of BTK and its downstream targets upon stimulation with αIgM and reduced viability, NF-κB gene transcription, activation, and migration in CLL cells [49]. Fenebrutinib inhibits C481S BTK mutant that mediates ibrutinib resistance and is toxic to CLL cells with BTK C481S mutation. Unlike ibrutinib, which antagonizes rituximab-mediated NK cell–mediated cytotoxicity through ITK inhibition, fenebrutinib does not inhibit ITK and preserves NK cell-mediated cytotoxicity that is dependent on anti-CD20 antibodies [51]. Thus, exploration of fenebrutinib as monotherapy and in combination with anti-CD20 antibodies is promising, especially in patients with acquired resistance to ibrutinib [49, 52].

Fenebrutinib was well-tolerated with favorable safety, selectivity, and pharmacokinetic/pharmacodynamic (PK/PD) profiles in healthy volunteers [53]. A phase I study has evaluated fenebrutinib in 24 patients with relapsed/refractory B cell malignancies (CLL, n = 14; follicular lymphoma [FL], n = 4; diffuse large B cell lymphoma [DLBCL], n = 3; MCL, n = 2; prolymphocytic leukemia plus WM, n = 1) [54]. The enrolled patients were treated at 100, 200, or 400 mg once daily, orally. There was no dose-limiting toxicity. This phase I trial of fenebrutinib was prematurely terminated during dose escalation and the MTD was not reached. The most common AEs included fatigue (37%), nausea (33%), diarrhea (29%), thrombocytopenia (25%), and headache (20%). Eight of 24 patients had a response to fenebrutinib. Of these 8 patients who responded, an MCL patient achieved complete response (CR) and 7 CLL patients achieved partial response (PR) or PR with lymphocytosis (PR-L), including 1 of 5 heavily pretreated patients with BTK C481S mutant CLL [54]. Two additional patients with BTK C481S mutation showed a decrease in the size of target tumors (− 23% and − 44%). The median duration of response in all responding patients and patients with CLL is 3.8 months and 2.5 months, respectively. As the MTD that may provide greater BTK inhibition was not reached, the short duration of response should be interpreted cautiously. Despite this, this study provides evidence of clinical activities of reversible non-covalent BTK inhibitors in B cell malignancies [54].

In addition to the several non-covalent BTK inhibitors that have shown promising efficacy in clinical trials, there are also some other non-covalent BTK inhibitors with significant antitumor effects in preclinical studies (Table 4). For instance, XMU-MP-3, a non-covalent inhibitor with potent BTK inhibitory activity, inhibited B cell lymphoma cells with or without BTK C481S mutation in vitro and in vivo [65], suggesting it could be effective in treating B cell lymphomas including those resistant to ibrutinib. Several other non-covalent BTK inhibitors including CB1763, GNE-431, and CGI-1746 have shown potent inhibitory effects on both wildtype and C481S mutant BTK [66, 67]. Further clinical trials are warranted to investigate the safety and efficacy of these novel agents.