Review

BTK inhibitors: past, present, and future

Allison Cool1

, Tiffany Nong1

, Skye Montoya1,2, and Justin Taylor 1,*

Bruton’s tyrosine kinase (BTK) inhibitors have revolutionized the treatment landscape for B cell lymphomas such as chronic lymphocytic leukemia (CLL). The

first-in-class BTK inhibitor ibrutinib has recently been succeeded by covalent

BTK inhibitors that are safer but still face challenges of resistance mutations.

The noncovalent BTK inhibitor pirtobrutinib was recently approved for relapsed

and refractory CLL, and whether noncovalent BTK inhibitors will supplant covalent BTK inhibitors as upfront treatment options either alone or in combination

will be determined. Meanwhile, newer BTK inhibitors and BTK degraders are

vying for their place in the potential future landscape of B cell cancers as well as

autoimmune diseases. This review will cover the latest progress in BTK inhibitor

development and where the field is moving in light of these recent discoveries.

BTK is an attractive target prone to resistance

B cell receptor (BCR) signaling is an important signaling pathway that, under normal conditions,

plays a critical role in adaptive immunity [1]. When an antigen binds to the BCR, a signaling cascade is initiated, eventually leading to the proliferation (see Glossary) and differentiation of B

lymphocytes (Figure 1). Some of the key molecules involved in this pathway include the BCR itself,

Lck/Yes-related novel protein tyrosine kinase (LYN), spleen tyrosine kinase (SYK), BTK, and

phosphoinositide 3-kinase (PI3K) [2,3]. Both LYN and SYK play a role in phosphorylating BTK,

which activates it and allows activation of further downstream substrates such as phospholipase

C-γ2 (PLCγ2) [1]. BCR signaling is dysregulated in several B cell lymphomas such as CLL, mantle

cell lymphoma (MCL), Waldenström's macroglobulinemia (WM), and in autoimmune diseases

(Box 1) [4,5].

BTK is expressed in B cells and other hematopoietic cells including macrophages, mast cells, and

platelets, but not in T cells [6]. Due to the upstream and central location of BTK in the BCR signaling pathway, combined with its limited expression in other cell types, BTK is a good drug target

for inhibition. This is evident in how BTK inhibitors have become the standard of care for various B

cell malignancies (Figure 2) [7]. However, despite success in inhibiting BTK, there have been and

still are challenges that persist. These challenges include resistance mutations, resistance

through activation of alternate pathways, and adverse effects (Table 1) [2,8]. In this review of

the past, present, and future of BTK inhibitors, we discuss covalent BTK inhibitors, noncovalent

BTK inhibitors, and the basic, translational, and clinical studies that have been performed or are

underway and have informed our understanding of BTK as a target. The recent discovery of kinase impaired BTK resistance mutations have suggested that these mutations can confer resistance across classes of approved BTK inhibitors, so it is imperative to consider these liabilities

when designing future BTK inhibitor trials. We also attempt to illuminate the roadmap forward,

which includes alternative BTK inhibitors, BTK degraders, and combination therapies.

Covalent BTK inhibitors: powerful drugs in flux

Covalent BTK inhibitors form an irreversible covalent bond with the cysteine 481 (C481) residue of

BTK within its ATP binding pocket [9–11]. BTK inhibition leads to impaired activation of major

Highlights

One of the most successful and highly

developed drug targets in cancer is

Bruton’s tyrosine kinase (BTK). Remarkable basic and translational studies

have led to the clinical approval of several

generations of small-molecule BTK

inhibitors.

Recent discovery of kinase-deficient

BTK inhibitor resistance mutations

sheds light on still undiscovered roles of

BTK in B cell receptor signaling.

Noncovalent BTK inhibitors represent

potential future frontline treatment options in chronic lymphocytic leukemia

(CLL).

New dual-binding BTK inhibitors and

BTK degraders represent the future of

BTK targeting.

Successful development of BTK inhibitors in cancer has led to new applications

in other conditions such as autoimmune

disease.

1

Sylvester Comprehensive Cancer

Center at the University of Miami Miller

School of Medicine, Miami, FL, USA

2

Current affiliation: Yale, New Haven,

CT, USA

*Correspondence:

jxt1091@miami.edu (J. Taylor).

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© 2024 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

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TIPS 2142 No. of Pages 17

downstream signaling pathways such as PLCγ2, preventing proliferation and promoting apoptosis of malignant B cells [12]. Approved therapies in this class include ibrutinib, acalabrutinib, and

zanubrutinib [13]. However, long-term use of covalent BTK inhibitors can lead to development of

acquired resistance through mutations in the binding site of BTK (C481) or through activating mutations in the downstream substrate of BTK, PLCγ2 [14–16]. Preclinical profiling revealed little to

B-Cell Receptor Signaling

Antigeninduced

cross-linking

BCR

Activated

kinases

Lyn, SYK

P

P

P

P

P

P

P

P

PI3K P

P

P

NF-κB

PLCγ2

BTK Ibrutinib

cell proliferation

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Figure 1. B cell receptor (BCR) signaling. BCR signaling is an important signaling pathway for the survival of

B cells. When an antigen binds to the BCR, a signaling cascade is initiated, leading to the proliferation and differentiation

of B lymphocytes. Key molecules involved in this pathway include the BCR itself, the Src-family kinase LYN (LYN), spleen

tyrosine kinase (SYK), Bruton’s tyrosine kinase (BTK), and phosphoinositide 3-kinase (PI3K). Both LYN and SYK

phosphorylate BTK, which activates it and allows the activation of substrates further downstream such as phospholipase

C-γ2 (PLCγ2). BTK inhibitors inhibit BCR signaling by inhibiting BTK, which reduces the activity of nuclear factor κβ (NF-κB)

downstream in the signaling pathway. NF-κB contributes to the proliferation of B cell lymphomas when activated; therefore,

decreasing NF-κB leads to decreased proliferation. Created with BioRender.com.

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Glossary

Atrial fibrillation: an irregular heartbeat

that can lead to cardiac complications

such as stroke and heart failure.

Differentiation: the process by which

immature cells become mature cells with

a specific function.

Gadolinium-enhancing lesions:

areas of active inflammatory lesions in

multiple sclerosis (MS), used as a

reference to assess the progression of

MS.

Pharmacodynamics: the effects of

drugs on the body, including their

mechanism of action and interactions

with cellular components.

Pharmacokinetics: the processes by

which a drug is absorbed, distributed,

metabolized, and eliminated by the

body.

Proliferation: an increase in cell

numbers as a result of growth and cell

division.

Salvage therapy: a treatment or

therapy used after the failure of an initial

first-line therapy.

Steric hindrance: a spatial

arrangement of atoms that causes bulky

areas, preventing proper binding.

Synergy: the interaction of two or more

agents to create a combined effect

greater than the sum of their individual

effects.

Box 1. Applications of BTK inhibitors in autoimmune diseases

In addition to hematologic malignancies, the potential of BTK inhibitors is being examined for treatment against various

autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune hemolytic anemia (AIHA), and multiple sclerosis (MS) [84–86]. Preclinical studies have shown in murine models that BTK inhibitors are

effective in treating both SLE and RA [87–89]. However, these preclinical data have not been clearly replicated in clinical

testing [88,89]. Various BTK inhibitors (fenebrutinib, spebrutinib, tirabrutinib, and evobrutinib) have been used in clinical

testing for RA, but despite the high safety profile, they have had limited success as a monotherapy [88]. However,

acalabrutinib and ibrutinib have been tested in murine models against AIHA, and acalabrutinib was found to significantly

reduce autoantibodies [90]. Additionally, in a retrospective study of ten patients with AIHA treated with ibrutinib, nine

achieved complete remission [91]. There are ongoing Phase 2 clinical trials testing the safety and efficacy of ibrutinib

(NCT04398459xxiii) and acalabrutinib (NCT04657094xxiv) for treating AIHA. Moreover, various studies have shown a

positive response to BTK inhibitors for treating MS [92]. A Phase 2b controlled trial was conducted, in which relapsing

MS patients were treated with an irreversible BTK inhibitor, tolebrutinib, for a period of 12 weeks [93]. At the endpoint of

this study, treatment with tolebrutinib resulted in a dose-dependent decrease in new gadolinium-enhancing lesions,

with only one serious adverse event out of 126 patients [93]. This study has led to several Phase 3 studies that are currently

ongoing to further assess the efficacy and safety of tolebrutinib in MS [92]. These studies include GEMINI 1

(NCT04410978xxv), GEMINI 2 (NCT04410991xxvi), PERSEUS (NCT04458051xxvii), and HERCULES (NCT04411641xxviii).

2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024

BTK inhibitors: timeline of FDA approval Covalent BTK inhibitors

Acalabrutinib

Ibrutinib

Zanubrutinib

Tirabrutinib

Orelabrutinib

Pirtobrutinib

Nemtabrutinib

Vecabrutinib

Not FDA Approved

Noncovalent BTK inhibitors

FDA approved

FDA approved

FDA approved

Not FDA Approved

Japan approval in 2020

China approval in 2020

In clinical trials, not yet FDA approved

In clinical trials, not yet FDA approved

FDA

approved

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Figure 2. Bruton’s tyrosine kinase (BTK) inhibitors: timeline of FDA approval. In the landscape of precision

medicine, BTK inhibitors have progressed the treatment of various diseases, notably hematologic malignancies such as

chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Visualizing the journey of FDA approvals for these

inhibitors uncovers a narrative of relentless innovation and transformative therapies. It begins with the discovery of ibrutinib,

the first-in-class BTK inhibitor. Its groundbreaking efficacy propelled it through clinical trials, leading to FDA approval in

2013. This milestone created a new era in targeted therapy. Following ibrutinib's success, acalabrutinib emerged as the next

BTK inhibitor, designed to enhance selectivity and mitigate off-target effects. The FDA granted accelerated approval for

acalabrutinib in 2017, in light of the promising results in relapsed or refractory MCL. To continue the momentum of

developing more tolerable BTK inhibitors, zanubrutinib was approved by the FDA in 2019. Noncovalent BTK inhibitors are

a newer class that can bind reversibly, with the first and only FDA-approved drug in this class being pirtobrutinib. The

additional therapies mentioned continue to be in the early stages of clinical trials. Vecabrutinib and nemtabrutinib were

discovered as early as 2017, with preclinical studies beginning at that time. The journey of FDA approval of BTK inhibitors

embodies the collaborative efforts of researchers, clinicians, and patients, driving innovation forward in the pursuit of

improved outcomes and minimizing adverse effects. The figure was made using Canva (https://www.canva.com).

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Table 1. BTK inhibitors and their notable features BTK inhibitor Type Approved indicationsa Common adverse effects Resistance mechanisms Novel inhibition/features Overall response rate NCT number Refs

Ibrutinib Covalent CLL, WM, cGVHD Atrial fibrillation (16%), hypertension (23.2%), and bleeding (51.3%) C481S mutation (acquired resistance mutations); downstream mutations in PLCγ2 A first-in-class BTK inhibitor that revolutionized treatment of various hematologic malignancies 91% (R/R CLL) NCT01578707xxx [23] 92% (TN CLL) NCT01722487iii [24] 93% (WM) NCT03053440vii [38]

Acalabrutinib Covalent CLL, MCL Headache (34.6%), diarrhea (34.6%), and cough (28.9%) C481S mutation (acquired resistance mutations); downstream mutations in PLCγ2 Increased selectivity, decreased off-target effects, and improved safety profile 81% (R/R CLL) NCT02477696ii [26] 89.9% (TN CLL) NCT02475681vi [36] 81% (MCL) NCT02213926xxxi [96]

Zanubrutinib Covalent CLL, MCL, WM, MZL, FL Atrial fibrillation (2%), hypertension (11.0%), neutropenia (33.7%), and upper respiratory tract infections (24%) C481S and L528W mutation (acquired resistance mutations); downstream mutations in PLCγ2 Higher selectivity than ibrutinib and acalabrutinib, decreased off-target effects, and improved safety profile 78.3% (R/R CLL) NCT03734016xxxii [39] 94.5% (TN CLL) NCT03336333xxxiii [97] 83.7% (MCL) NCT03206970xxxiv [98] 94% (WM) NCT03053440vii [38]

68.2% (MZL) NCT03846427xxxv [99]

Tirabrutinib Covalent PCNSL- approved in Japan onlyb Rash (36.4%), neutropenia (27.3%), and leukopenia (25%) C481S mutation (acquired resistance mutations); downstream mutations in PLCγ2 Increased selectivity and decreased off-target effects 63.6% (PCNSL) jRCT2080223590xxxvi [42]

Orelabrutinib Covalent CLL, MCL- (approved in China onlyc) Thrombocytopenia (34%), upper respiratory tract infection (27.4%), and neutropenia (24.5%) C481S mutation (acquired resistance mutations); downstream mutations in PLCγ2 Similar selectivity to zanubrutinib, decreased off-target effects, and higher synergy with R-CHOP than ibrutinib 92.5% (CLL) NCT03493217xxxvii [100] 81.1% (MCL) NCT03494179ix [44]

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Pirtobrutinib Noncovalent CLL, MCL Infection (71%), bleeding (42.6%), and neutropenia (32.5%) Novel acquired BTK resistance mutations, kinase-proficient and -impaired mutations, and mutations leading to downstream activation of BCR signaling Overcomes resistance from C481X mutations, and only FDA-approved noncovalent BTK inhibitor 63% (CLL) NCT03740529x [51] 52% (MCL) NCT03740529x [51]

Nemtabrutinib Noncovalent N/A Hypertension (32%), weight gain (21%), cough (36%), fatigue (34%), and back pain (34%) Novel acquired BTK resistance mutations Overcomes resistance from C481X mutations, and less selective, so it can maintain inhibition of downstream BCR signaling when PLCγ2

has activating mutations

56% (CLL) NCT03162536xiv [60]

Vecabrutinib Noncovalent N/A Anemia (10%) and fatigue (10%) Novel acquired BTK resistance mutations Inhibits phosphorylation of BTK and PLCγ2, and promotes BCL-2 dependency N/A NCT03037645xv [64]

LP-168 Dual covalent and noncovalent N/A Neutropenia (29.4%), decreased platelets (26.5%), and anemia (23.5%) N/A Overcomes resistance to covalent inhibitors through noncovalent binding capabilities 77.4% (R/R MCL), 72.7% (MZL), 70.0% (non-GCB DLBCL) NCT04993690xxxviii NCT04775745xxxix [78]

NX-2127 PROTAC degrader N/A N/A N/A Overcomes resistance to both kinase-proficient and kinase-impaired mutations N/A NCT04830137xxix [57]

aFDA: orphan drug status

bFDA: breakthrough therapy designation for MCL only

cAbbreviations: cGVHD, chronic graft-versus-host disease; FL, follicular lymphoma; PCNSL, primary central nervous system lymphoma; non-GCB DLBCL, non-germinal center B-cell-like diffuse large B cell

lymphoma; N/A, not associated; R/R, relapsed or refractory; TN, treatment-naïve.

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no activity of covalent inhibitors against C481S mutant cells, posing a potential challenge to treating

patients with acquired resistance to these drugs [13]. Additional BTK inhibitors were developed to

address the side effects of ibrutinib inhibitors; and indeed, the improved selectivity of these new

covalent BTK inhibitors limits off-target toxicities of ibrutinib [13,15,17]. This presents an advantage

in the tolerability of newer covalent BTK inhibitors for treatment-naïve and relapsed or refractory

disease. The standard frontline treatment for diseases such as CLL has now shifted to the use of

covalent BTK inhibitors or the BCL-2 inhibitor venetoclax, in combination with the CD20 monoclonal antibody obinutuzumab [18]. Before getting to the development of newer covalent BTK inhibitors, we review the science behind the development of ibrutinib, the first-in-class BTK inhibitor.

Ibrutinib: the first-in-class BTK inhibitor

Ibrutinib was the inaugural BTK inhibitor approved by the FDA in 2013 (Figure 2) [9]. It revolutionized

the treatment landscape for hematologic malignancies such as MCL, marginal zone lymphoma

(MZL), WM, and, most importantly, CLL compared with traditional chemoimmunotherapy

[14,19]. However, as of 2023, its approval for the indications of MCL and MZL has been withdrawn

[20]. Leading up to its approval, multiple clinical trials proved that ibrutinib improved treatment outcomes. In a Phase 1b/2 study (NCT01105247i

) including 85 patients with relapsed or refractory

CLL, the overall response rate was 71% [95% confidence interval (CI 60–80)] [21]. Similarly, a

Phase 2 trial including 111 patients with relapsed or refractory MCL showed an overall response

rate of 67.6% (95% CI 58.9–76.3) [22]. The treatment of CLL has evolved from alkylating agents

to chemoimmunotherapy, with continued exploration to find an effective salvage regimen for relapsed or refractory CLL remaining a challenge even with agents such as ofatumumab [23]. Not

only did ibrutinib prove to be more effective than ofatumumab as a salvage therapy, but it was

found to be the most effective as a first-line therapy in CLL patients compared with the standard

of care at the time, chlorambucil [24]. In the Phase 3 RESONATE trial (NCT02477696ii), the overall

survival rate and overall response rate, respectively, were higher with the use of ibrutinib (90% and

42.6%) than with ofatumumab (81% and 4.1%) [23]. Furthermore, the Phase 3 RESONATE-2 trial

(NCT01722487iii) investigating ibrutinib as a first-line agent compared with chlorambucil demonstrated an 84% lower relative risk of death or progression, and the overall response rate was higher

in patients treated with ibrutinib (86%) than with chlorambucil (35%) [24]. However, with extended

observation in patients with CLL treated with ibrutinib, the RESONATE trial indicated 28% discontinuation due to adverse effects such as atrial fibrillation (16%), hypertension (23.2%), and bleeding (51.3%) [25]. These cardiac side effects are thought to be due to off-target inhibition of the

epidermal growth factor receptor (EGFR), Src, and Tec family kinases [26].

The effectiveness of ibrutinib in treating primary or secondary central nervous system conditions

relies on its ability to be distributed across the blood–brain barrier [27]. Recurrence rates are extremely high, with no established salvage therapy for primary central nervous system lymphoma

(PCNSL), for which ibrutinib shows promise [28]. Ibrutinib impairs BCR signaling, reducing the

activity of nuclear factor κβ (NF-κB) downstream in the signaling pathway, which contributes to

the proliferation of B cell lymphomas when activated [29].

In the Phase 3 PHOENIX (NCT01855750iv) trial, when added to the R-CHOP regimen, the standard frontline diffuse large B cell lymphoma (DLBCL) treatment, ibrutinib, improved outcomes in

younger patients (<60 years) only, possibly due to better tolerability of adverse effects [30]. In

these patients (<60), the overall survival was improved in ibrutinib plus R-CHOP versus a placebo

plus R-CHOP: 93.2% and 80.9%, respectively. Serious adverse effects were higher in the

ibrutinib plus R-CHOP arm (53.1%) than in the R-CHOP plus placebo arm (34.0%) [30]. Older

adults (≥60 years) experienced increased toxicity, leading to compromised administration of RCHOP [31]. This highlights that ibrutinib remains a very efficacious drug; however, its toxicity

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remains a major challenge. This led to the development of more covalent BTK inhibitors, with

acalabrutinib being the next to gain approval.

Acalabrutinib: making strides with BTK inhibitors

The development of covalent BTK inhibitors after ibrutinib aimed to minimize the off-target

adverse effects while preserving the on-target potency against BTK. Acalabrutinib demonstrated

efficacy in treating relapsed or refractory CLL patients, including patients who are intolerant of

ibrutinib [32]. However, this agent was safe and effective in treatment-naïve CLL patients as

well, according to a randomized Phase 2 study (NCT02337829v

) investigating the use of

acalabrutinib in relapsed or refractory and treatment-naïve patients [33]. This study showed estimated progression-free survival (PFS) rates at 24 months of 100% in treatment-naïve patients

and 84.3% in relapsed or refractory CLL patients [33]. Acalabrutinib has an improved safety profile compared with ibrutinib due to minimal inhibition of the off-target effects of ibrutinib mentioned

previously [26,34]. Designed as a noninferiority study, a head-to-head comparison in the Phase 3

ELEVATE-RR trial (NCT02477696ii) found that acalabrutinib had noninferior PFS to ibrutinib, with

a median of 38.4 months in both arms [25]. Of the 533 patients randomly assigned, 14.7% of the

individuals discontinued acalabrutinib due to toxicity, compared with 21.3% discontinuation in

the case of ibrutinib, demonstrating acalabrutinib's superior tolerability [25]. Despite a reduction

in adverse cardiac effects, a smaller percentage of CLL patients still experienced atrial fibrillation

during treatment with acalabrutinib (6.2%) versus ibrutinib (14.9%) [25]. Within that trial, the most

common side effects of acalabrutinib included headache (34.6%), diarrhea (34.6%), and

coughing (28.9%) [25]. Similarly, a pooled safety profile analysis of acalabrutinib in seven trials

(n = 610) identified the most common treatment-related adverse events to be headache

(29.2%) and diarrhea (16.6%) [35]. The development of optimal combination therapies is a

major current focus in CLL. One that has shown promise in the ELEVATE-TN clinical trial

(NCT02475681vi) was the combination of acalabrutinib plus obinutuzumab, with a 24-month

PFS of 93% versus 87% with acalabrutinib monotherapy [34,36]. Although this drug is welltolerated, resistance mechanisms still pose an issue, with patients discontinuing the drug typically

due to progressive disease rather than toxicity [26,37]. Due to perceived unmet needs still in the

field of BTK inhibitors, zanubrutinib was developed and proved to be potent and selective.

Zanubrutinib: the next step in BTK inhibition – reducing toxicities and enhancing efficacy

Similar to the rationale behind the development of acalabrutinib, the development of zanubrutinib

was intended to reduce toxicities in patients compared with ibrutinib. It was FDA-approved in

2021 for the treatment of WM, reflecting this objective [14]. Leading up to its approval was the

ASPEN study (NCT03053440vii), a Phase 3 trial comparing zanubrutinib with ibrutinib in WM patients [38]. Zanubrutinib had fewer rates of toxicities than were present with ibrutinib, such as atrial

fibrillation (2% for zanubrutinib versus 15% for ibrutinib) and hypertension (11% versus 16%)

[14,38]. There were higher rates of neutropenia with zanubrutinib than with ibrutinib, 33.7%

and 19.4%; however, their rates of upper respiratory tract infections were similar: 24% and

29%, respectively [38]. Before its FDA approval for use in CLL, the ALPINE study, a head-tohead superiority trial with 652 patients found that zanubrutinib was superior to ibrutinib in terms

of the overall response in CLL patients as determined by an independent review committee

(86.2% and 75.7%, respectively). The rates of atrial fibrillation in CLL patients were lower with

zanubrutinib (5.2%) than with ibrutinib (13.3%); however, the rates of hypertension were similar

(23.5% and 22.8%, respectively) [39]. This exhibits a difference in the profile of adverse effects

compared with the WM patients in the ASPEN study mentioned earlier, where rates of both atrial

fibrillation and hypertension were lower in the zanubrutinib group [38,39]. Resistance remains a

challenge in zanubrutinib, similar to the other covalent BTK inhibitors. In addition to mutations

at the C481 site, resistance to zanubrutinib may be due to acquired mutations at other sites

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such as L528W [40]. Other BTK inhibitors have been developed but are currently aiming to target

other types of lymphoma besides CLL, such as PCNSL and DLBCL.

Tirabrutinib: navigating efficacy and tolerance in PCNSL

Tirabrutinib is an irreversible BTK inhibitor that binds covalently to Cys481 [41]. In a preclinical

study, tirabrutinib inhibited BTK at a rate that was similar to that of acalabrutinib but greater

than tenfold less than that of ibrutinib [41]. It is currently only approved in Japan for the treatment

of PCNSL [12]. However, enrollment has just begun for the PROSPECT study (NCT04947319viii),

a Phase 2 trial evaluating tirabrutinib in US patients with relapsed or refractory PCNSL. A 3-year

follow-up of a Phase 1/2 trial in Japan evaluating tirabrutinib in 44 patients with PCNSL reported

the overall response rate to be 63.6% (95% CI: 47.8–77.6). The most common adverse effects

reported were rash (36.4%), neutropenia (27.3%), and leukopenia (25.0%) [42]. Early-phase clinical trials have shown that tirabrutinib also has activity against relapsed or refractory CLL and WM

[12]. Due to the lack of salvage therapies for PCNSL, tirabrutinib may be a safer alternative for patients who are intolerant of the toxicities associated with ibrutinib; however, it is not approved in

the USA. Another BTK inhibitor approved in other countries but not the USA is orelabrutinib.

Orelabrutinib: unveiling the potential of BTK inhibitors

Orelabrutinib is currently only approved in China for MCL and CLL, although further studies are

currently being carried out to investigate its therapeutic potential [28]. Orelabrutinib has comparable kinase selectivity with zanubrutinib, which demonstrated higher selectivity than acalabrutinib

and ibrutinib [43]. A Phase 1/2 trial conducted in China (NCT03494179ix) included 106 patients

with relapsed or refractory MCL treated with orelabrutinib. They only assessed the treatment outcomes in 86 out of 106 patients treated with 150 mg once daily versus 20 out of 106 patients

treated with 100 mg twice daily, due to a superior overall response of 81.1% (95% CI: 72.4–

88.1) [44]. The 150 mg once-daily dose will be the preferred Phase 2 dose and was also predicted to be superior in pharmacokinetic and pharmacodynamic studies [44]. They reported

the most common adverse events to be thrombocytopenia (34.0%), upper respiratory tract infection (27.4%), and neutropenia (24.5%) [44]. In vitro studies have shown that orelabrutinib demonstrated superior synergy when added to R-CHOP therapy in DLBCL patients compared with

ibrutinib, which antagonizes the efficacy of rituximab, an element of R-CHOP therapy [45]. A

Phase 3 randomized, double-blind, multicenter trial is currently ongoing to evaluate the efficacy

and safety of combining orelabrutinib plus R-CHOP versus R-CHOP alone [46].

Although approved covalent BTK inhibitors have had a strong clinical impact, their toxicities and

ability to develop resistance can limit their long-term efficacy, and this has spurred the development of noncovalent BTK inhibitors [13].

Noncovalent BTK inhibitors can overcome BTK C481 resistance mutations

Noncovalent BTK inhibitors function without binding to Cys481 and blocking the activity of both

wild-type (WT) and Cys481-mutant forms of BTK [15,47,48]. The development of this new class

created an alternative for patients with progressive disease due to C481 mutations after treatment with covalent BTK inhibitors [47]. The only noncovalent BTK inhibitor approved thus far,

pirtobrutinib, interacts with water molecules and BTK residues near ATP binding sites through extensive hydrogen bonding [13]. While noncovalent BTK inhibitors can effectively treat ibrutinibresistant C481-mutant BTK, the development of resistance downstream in the signaling pathway

still remains a challenge, particularly resistance through activating mutations involving PLCγ2

[15,49]. Furthermore, novel acquired BTK mutations were revealed by sequencing of patients

progressing on noncovalent BTK inhibitors (Figure 3). The most mature data for noncovalent

BTK inhibitors are for pirtobrutinib.

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Pirtobrutinib: a first-in-class reversible BTK inhibitor

Pirtobrutinib is the first and only noncovalent, reversible BTK inhibitor that is currently FDAapproved. It is highly selective for the inhibition of C481S-mutant cells, among other C481X mutations [12,47]. Pirtobrutinib also exhibited activity to resistant B cell malignancies even without

C481 mutations [13,15,50]. This mechanism marks an important development for BTK inhibitors,

as the C481 mutation is the predominant resistance mechanism of covalent BTK inhibitors in CLL

[13,15]. In the BRUIN Phase 1/2 trial (NCT03740529x

), 317 patients with CLL were treated with

pirtobrutinib as monotherapy after disease progression on a covalent BTK inhibitor. This showed

promise as the first-in-class reversible BTK inhibitor with an overall response rate of 73.3% (95%

T474I

C481S

L528W

PH TH SH3 SH2 Kinase

T316A

T474I

PH SH3 SH2 Kinase TH

L528W

PH SH3 SH2 Kinase

V416L

L528W A428D

M437R

PH SH3 SH2 Kinase

T474I

Ibrutinib

Acalabrutinib

Zanubrutinib

Pirtobrutinib

Kinase Impaired Resistance

Locations of Mutations at the

BTK Protein Domain

C481F/R/Y

C481S

C481F/R/Y

C481S

C481F/R/Y

TH

TH

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Figure 3. Locations of mutations at the Bruton’s tyrosine kinase (BTK) protein domain. BTK inhibitors (ibrutinib,

acalabrutinib, zanubrutinib, and pirtobrutinib) have revolutionized the treatment of various B cell lymphomas; however,

resistance mutations are a major challenge affecting their success. Most of these mutations arise within the kinase domain

of BTK, which affects the binding pocket of the BTK inhibitor. However, in ibrutinib, the T316A mutation occurs in the SH2

domain, which affects the binding of the BTK inhibitor through steric hindrance. In addition to their location, BTK

mutations can be classified as being kinase-proficient or kinase-impaired. Kinase-proficient mutations promote B cell

receptor signaling by maintaining BTK’s kinase activity, whereas kinase-impaired mutations have disabled kinase activity

of BTK but still maintain B cell receptor (BCR) signaling by activating alternative kinases downstream. Created with

BioRender.com.

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Trends in Pharmacological Sciences, Month 2024, Vol. xx, No. xx 9

CI: 67.3–78.7) [51]. There was discontinuation in only 2.6% of patients due to treatment-related

adverse effects, a stark difference from ibrutinib. The most common adverse events were infection (71.0%), bleeding (42.6%), and neutropenia (32.5%) [51]. Later cohorts were enrolled in the

BRUIN study, where the authors reported the overall response rate for the combination of

pirtobrutinib plus venetoclax (93.3%, n = 15) and pirtobrutinib, venetoclax, and rituximab

(100%, n = 10) [52]. The potential for the combination of pirtobrutinib plus venetoclax and rituximab has progressed to investigation in a Phase 3 head-to-head trial, BRUIN CLL-322

(NCT04965493xi), where it is being compared with venetoclax and rituximab alone. Another

Phase 3 study, BRUIN MCL-321 (NCT04662255xii), is underway where pirtobrutinib is being

compared with three other BTK inhibitors in BTK-naïve MCL patients with one prior therapy

[53,54]. A new Phase 2 study has recently started investigating the combination of pirtobrutinib

and obinutuzumab as a first-line therapy in treatment-naïve CLL patients (NCT06333262xiii). If

this shows promising treatment outcomes, it will warrant a head-to-head trial with the current

first-line therapy, potentially leading to a new standard of care amongst this patient population.

Challenges present in the use of pirtobrutinib are due to the many non-C481 BTK mutations identified in genomic analyses of relapsed CLL patients [15]. These off-target mutations allowed BCR

signaling through activation of the AKT signaling pathway, demonstrating its autonomy from BTK

[15,47]. Kinase-proficient mutations promote BCR signaling by maintaining the kinase activity of

BTK, whereas kinase-impaired mutations disabled the kinase activity of BTK but still maintain

BCR signaling by activating alternative kinases downstream. Additional kinase domain mutations

were identified in noncovalent BTK inhibitors such as pirtobrutinib, with L528W being noteworthy

[55,56]. This resistance mechanism renders BTK kinase-impaired and activates BCR signaling

independent of BTK [57]. It remains to be seen if other noncovalent BTK inhibitors such as

nemtabrutinib will be susceptible to these novel resistance mechanisms in clinical use, but

preclinical data suggest that they may.

Nemtabrutinib: navigating resilience for improved therapeutic outcomes

Nemtabrutinib has a similar mechanism as pirtobrutinib, both with the ability to reversibly inhibit

WT BTK and C481 ibrutinib-resistant BTK [12]. It maintains its inhibition through formation of hydrogen bonds with the E475 and Y476 residues of BTK [48,58]. In contrast to other noncovalent

BTK inhibitors, preclinical studies have shown that it can maintain the inhibition of downstream

BCR signaling, even in patients with activating mutations in PLCγ2 [59]. The development of

nemtabrutinib aimed to target additional kinases, such as Tec and Src, stemming from the hypothesis that its response would be more robust and resilient for improved therapeutic outcomes

in relapsed or refractory patients [59,60]. However, additional kinase inhibition can potentially lead

to adverse effects, as seen with the high discontinuation rates for ibrutinib, mainly due to cardiac

effects. In a recent Phase 1 trial, MK-1026 (NCT03162536xiv), the use of nemtabrutinib in patients

with B cell malignancies showed manageable safety overall, with most adverse effects being at

Grade 1 and 2, although 87% had at least one adverse event at Grade 3 or higher [60]. The

most common toxicities included hypertension (32%), weight gain (21%), cough (36%), fatigue

(34%), and back pain (34%) [60]. There are currently very limited efficacious therapies for relapsed

or refractory disease, with one being venetoclax, a B cell lymphoma 2 (BCL-2) inhibitor [60].

Preclinical studies have shown the potential of a combination therapy of nemtabrutinib and

venetoclax [61].

Vecabrutinib: setbacks and pioneering pathways for combination therapy

Preclinical characterization of vecabrutinib highlighted its ability to inhibit WT and mutant BTK

cells by inhibiting the phosphorylation of BTK and its downstream target, PLCγ2 [62]. In vitro

studies on relapsed DLBCL showed sensitivity to vecabrutinib but resistance to ibrutinib [63]. A

Trends in Pharmacological Sciences

10 Trends in Pharmacological Sciences, Month 2024, Vol. xx, No. xx

Phase 1b study (NCT03037645xv) interrogated the clinical responses to vecabrutinib but failed to

meet its endpoint. As a result, it did not move on to Phase 2 studies, despite being well tolerated

in patients [62,64]. Vecabrutinib was tolerated well, and the investigators primarily found anemia

(10%) and fatigue (10%) to be the most prevalent treatment-related adverse events observed

[64]. According to the National Cancer Institutexvi, no clinical trials are investigating vecabrutinib

at the time of writing. Although vecabrutinib has not shown promise as a monotherapy in CLL,

its potential as part of combination therapy with the BCL-2 inhibitor venetoclax has been investigated in preclinical studies [65]. A novel attribute that has been identified in vecabrutinib is its ability to promote BCL-2 dependency, unlike ibrutinib, making it an option for combination therapies,

similar to venetoclax [66]. This synergy with venetoclax could be a way for noncovalent BTK inhibitors to overcome the potential mechanisms of resistance that might limit their utility as upfront

monotherapy treatments.

Future directions in targeting BTK to circumvent resistance

A new direction in targeting BTK involves the use of combination therapies of BTK inhibitors

with BCL-2 inhibitors to help overcome resistance and create better treatment options for

patients. BCL-2 can become upregulated as part of a prosurvival pathway in B cell lymphoma

patients, leading to resistance to BTK inhibitors; therefore, treatment with a BCL-2 inhibitor

has the potential to resensitize patients to BTK inhibition [67,68]. The synergistic effects

between BTK inhibitors and BCL-2 inhibitors were well supported by both in vitro and in vivo

studies [69,70].

Ibrutinib and venetoclax: new strides in overcoming resistance

Ibrutinib and venetoclax both exist as monotherapies for various B cell malignancies, however,

they each have independent limitations, including resistance, adverse effects, and dose-limiting

toxicities [19,21,71]. Various clinical trials have aimed to see if the combined effects of ibrutinib

with venetoclax offer a more therapeutic treatment option for patients with B cell lymphoma.

The AIM study (NCT02471391xvii), a Phase 2 trial conducted in Australia, enrolled patients who

had either relapsed or refractory MCL, untreated MCL, or those who were not suitable for chemotherapy. The patients received 560 mg per day of orally administered ibrutinib for 4 weeks and

then, during Week 5, venetoclax was introduced at 50 mg per day and increased weekly up to

400 mg. At the primary end-point (Week 16), the complete response rate assessed without positron emission tomography (PET) was 42% in patients treated with the combination. The study

compared this with a historical cohort of patients treated with ibrutinib monotherapy, which

showed a complete response rate assessed without PET of only 9% [72]. The most common adverse events were diarrhea (83%), nausea and vomiting (71%), gastroesophageal reflux (38%),

fatigue (75%), and bleeding (54%). As a result of this study, it was determined that 50 mg per

day was too high a starting dose for venetoclax, and patients had less severe adverse effects

when starting at 20 mg per day [72]. A Phase 2 nonrandomized trial of ibrutinib and venetoclax

was conducted on CLL patients who had not been previously treated [73]. Patients were treated

with 420 mg per day of ibrutinib for the first three cycles (1 cycle = 28 days) and then venetoclax

was introduced, starting at 20 mg per day followed by a weekly ramp-up to 400 mg per day. Five

patients discontinued treatment during the monotherapy; the remaining 75 patients continued on

to combination treatment. At the conclusion of the study the estimated 3-year PFS was 93%, and

66% of the patients had undetectable measurable residual disease (U-MRD) remission in bone

marrow, which was greater than that achieved with monotherapy [73]. These results are similar

to what was found in CAPTIVATE, another Phase 2 trial (NCT02910583xviii) using ibrutinib and

venetoclax for CLL [73,74]. In the CAPTIVATE trial, the researchers found that after 12 cycles

of combination treatment, the U-MRD rate was 68% [73,74]. The CAPTIVATE trial found that

most of the adverse events from the combination treatment were at Grade 1 and 2 and occurred

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Trends in Pharmacological Sciences, Month 2024, Vol. xx, No. xx 11

early in the treatment. The most common Grade 3 and 4 adverse events were neutropenia (36%),

hypertension (10%), thrombocytopenia (5%), and diarrhea (5%) [74]. Another trial examining the

effects of ibrutinib and venetoclax was GLOW, a Phase 3 trial that, in combination with the

CAPTIVATE trial, led to European approval for the use of ibrutinib and venetoclax in the treatment

of CLL (NCT03462719xix) [75]. However, this combination has yet to be approved by the FDA.

Additionally, newer BTK inhibitors are being tested in clinical trials in combination with venetoclax,

such as zanubrutinib (NCT05168930xx) and pirtobrutinib (NCT05734495xxi) for treatment of

hematologic malignancies. Furthermore, zanubrutinib is being tested in CLL with a more potent

BCL-2 inhibitor, sonrotoclax (NCT06073821xxii).

Moving beyond traditional BTK inhibitors

In addition to combination therapies, a new agent of BTK inhibitors is under development

that aims to minimize side effects and reduce resistance. This novel inhibitor, LP-168, is a

dual inhibitor capable of binding covalently and noncovalently, depending on the presence of

mutations [76]. LP-168 binds covalently to WT BTK and noncovalently to mutated BTK residues [77]. A Phase 1 study examined the safety and efficacy of LP-168 [78]. Sixty-eight

patients with various B cell malignancies who had received at least one prior round of covalent

BTK inhibitors were enrolled. Patients were treated following a 3 + 3 dose escalation from

100 mg to 150 mg to 200 mg [78]. Additionally, the adverse effects were minimal, with the

majority being at Grade 1 or 2. Neutropenia (29.4%), decreased platelets (26.5%), and anemia

(23.5%) were among the most common adverse effects [78]. Another Phase 1 study found that

LP-168 was well tolerated in patients, and doses up to 300 mg did not reach the maximum

tolerated dose [76].

Moreover, another emerging treatment option for targeting BTK is the use of BTK degraders

(Box 2). A first-in-class BTK degrader, NX-2127, is currently undergoing a Phase 1 clinical trial

in relapsed or refractory B cell malignancies. An interim analysis of the patients enrolled with

CLL or small lymphocytic lymphoma (SLL) revealed that despite 10 out of the 23 patients having

BTK mutations, they still responded to NX-2127 [57]. Additionally, two other BTK degraders,

NX-5948 and BGB-16673, are undergoing clinical trials [79,80]. The initial findings of the NX5948 clinical trial showed that it has clinical efficacy and is tolerated well by patients, with the

most common adverse effects being purpura or contusion (57.1%), nausea (35.7%), and

thrombocytopenia (35.7%) [80]. The preliminary findings of the Phase 1 trial for BGB-16673

showed that out of the 18 patients with an evaluable response, 12 responded and the remaining had a partial response [79]. Despite the success of BTK degraders, it is important to recognize

the potential challenges that may arise over time. As with most drugs, extended use may result in

the development of resistant mutations. These mutations could potentially arise within BTK itself or

within cereblon (CRBN), the E3 ligase used for most of the current degraders under evaluation [81].

Concluding remarks and future perspectives

It has been over a decade since BTK inhibitors were first approved for treatment of B cell lymphomas and they have become blockbuster therapies. Advances in small-molecule BTK inhibitors

have led to more selective, more potent, and less toxic compounds. Despite the majority of

patients with CLL experiencing years of response to monotherapy BTK inhibitors, continuous

treatment is required and resistant mutations in BTK or PLCγ2 eventually develop. The covalent

inhibitors are uniquely susceptible to C481 mutations due to their dependence on binding to this

residue. Noncovalent BTK inhibitors overcome this limitation but are still susceptible to novel BTK

kinase domain mutations. BTK L528W, V416L, and A428D are classified as kinase-impaired

resistance mutations, while BTK C481S and T474I are kinase-proficient. BTK degraders

are still undergoing clinical trials, but early results have suggested they can overcome both kinaseTrends in Pharmacological Sciences

12 Trends in Pharmacological Sciences, Month 2024, Vol. xx, No. xx

Outstanding questions

What is the optimal sequencing of

covalent and noncovalent BTK inhibitors in CLL?

If we must select a covalent BTK inhibitor for frontline CLL treatment, is

acalabrutinib or zanubrutinib better?

Do we already have the best-of-class

covalent BTK inhibitors or do we need

more?

Do kinase-deficient BTK resistance

mutations confer clinical resistance to

all BTK inhibitors?

What resistance mechanisms might

arise from a fixed-duration combination therapy with BTK inhibitors and

BCL-2 inhibitors?

Box 2. Targeting BTK through degradation

A novel approach to targeting BTK is through the use of proteolysis-targeting chimera (PROTAC) degraders to selectively

degrade the protein by utilizing the ubiquitin–proteasome system (UPS) [94]. PROTAC degraders structurally consist of

three components: the binding region of the protein of interest (POI) (the hook); the E3 ligase binding region (the harness),

which is commonly cereblon (CRBN); and a linker to connect the two regions (Figure I) [57]. When the hook and harness

bind their targets, they bring CRBN in proximity to the POI; this is followed by polyubiquitination of the POI by CRBN

[57,94]. The polyubiquitination of the POI targets the protein for degradation by the proteosome [94]. PROTACs bind to

their target transiently and then are available to bind and initiate another round of degradation [95]. BTK degraders offer

various benefits for overcoming the resistance mechanisms seen with BTK inhibitors. A clinical-grade BTK degrader,

NX-2127, has been evaluated both in clinical studies (NCT04830137xxix) and mechanistically in vivo and in vitro [57]. Fluorescence resonance Energy transfer (FRET)-based assays have shown that despite the lower binding affinity of NX-2127 in

the presence of various BTK mutations that have caused resistance in BTK inhibitors (C481S, L528W, T474I, M437R, and

V416L), the degrader still bound and degraded BTK [57]. Furthermore, since the POI was degraded rather than enzymatically inhibited, the degrader can overcome resistance due to both kinase-proficient and kinase-impaired mutations [57].

Ub

Ub

Ub

Ub

E3

E2

Ub

E3

E2

Ub

E3

E2

Ub

Ub

POI

binding

domain

BTK PROTAC

Linker

E3

ligase

binding

domain

(A)

(C) (B)

(D) BTK

Trends in Pharmacological Pharmacological Sciences Sciences

Figure I. Mechanism of action for proteolysis-targeting chimera (PROTAC) degraders. The mechanism of

action for Bruton’s tyrosine kinase (BTK) PROTAC degraders utilizes the ubiquitin (Ub)–proteasome system (UPS). (A)

The binding domain of the protein of interest (POI) binds to BTK; meanwhile, the E3 ligase-binding domain binds to the

E3 ubiquitin ligase cereblon (CRBN). This brings BTK into close proximity with the E3 ubiquitin ligase. (B) Once in

proximity, the E3 ubiquitin ligase polyubiquitinates BTK. The PROTAC is then released and is able to repeat the cycle.

(C) The BTK is polyubiquitinated, which marks it for degradation by the proteasome. (D) The proteasome then

recognizes the polyubiquitinated BTK and degrades the protein. Created with BioRender.com.

Trends in Pharmacological Sciences

Trends in Pharmacological Sciences, Month 2024, Vol. xx, No. xx 13

impaired and kinase-proficient acquired BTK resistance mutations, but it is unknown if they can target

the downstream PLCγ2-activating mutations. A fixed duration of combination therapy of BTK inhibitors with BCL-2 inhibition has shown promise as a time-limited therapy. So far, patients with CLL who

relapse after the combination therapy do not acquire BTK, PLCγ2, or BCL-2 resistance mutations.

Despite remarkable basic, translational, and clinical advances in understanding BTK as a target,

many questions remain unanswered (see Outstanding questions). In CLL, noncovalent BTK inhibitors are only approved after two lines of therapy, including a previous covalent BTK inhibitor; however, if clinical trials of frontline treatment with noncovalent BTK inhibitors lead to approval in this

space, optimal sequencing of covalent and noncovalent BTK inhibitors may be determined by the

side effect profiles of the individual BTK inhibitors. Otherwise, further clinical trials would be required

to clarify the optimal sequencing of BTK inhibitors. Even now, when selecting a covalent BTK inhibitor in frontline CLL, we do not have data to suggest whether acalabrutinib or zanubrutinib is better.

They are both better tolerated than ibrutinib in terms of the side effect profile, but there has not been

a head-to-head trial and the study designs of each drug versus ibrutinib are very different, preventing

cross-trial comparisons. Matching-adjusted indirect comparisons (MAIC) results have come back

with mixed results [82,83]. From the treating CLL physician’s standpoint, BTK inhibitors are good

options, and a discussion of the side effect profile and frequency of administration can help distinguish which one might be best suited to the individual patient. With other covalent BTK inhibitors

being studied, it must be asked whether we already have the best-of-class covalent BTK inhibitors

or if we need more. Alternatively, should the focus be on developing BTK-targeting therapies or

combinations that are less susceptible to resistance mechanisms? We do not know whether

kinase-impaired BTK resistance mutations confer clinical resistance to all approved BTK inhibitors.

Kinase-impaired BTK L528W resistance mutations have been reported to occur in patients treated

with ibrutinib, zanubrutinib, and pirtobrutinib. These mutations are susceptible to BTK degraders,

though the safety and efficacy of these are still under investigation. Combination treatment with

BTK inhibitors and BCL-2 inhibitors appears to be safe and effective, offering a fixed-duration

therapy that may be equally effective as sequential therapy, but long-term results are still pending

as the data from these trials mature. Randomized trials comparing the different options are underway. Currently, there are no reports of resistance mechanisms to combination therapy with BTK

inhibitors and BCL-2 inhibitors, but understanding what leads to relapses will be important for

improving the combination therapy and potentially developing long-lasting remission for patients.

Acknowledgments

J.T. is supported by the National Institute of General Medical Sciences (NIGMS)/NIH (R35GM151109), the Doris Duke Charitable

Foundation, the Edward P. Evans Foundation, and the NCI Cancer Center Support Grant to Sylvester Comprehensive Cancer

Center (P30CA240139).

Declaration of interests

The authors declare no competing interests.

Resources

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https://clinicaltrials.gov/study/NCT01105247

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