review
`
`Breaking good: the inexorable rise of BTK inhibitors in the
`treatment of chronic lymphocytic leukaemia
`
`Claire V. Hutchinson1,2 and Martin J. S. Dyer1,2,3
`
`1Department of of Cancer Studies and Molecular Medicine, University of Leicester, 2The Ernest and Helen Scott Haematological
`Research Institute, University of Leicester, and 3Department of Biochemistry, University of Leicester, Leicester, UK
`
`Summary
`
`lineages and
`Although expressed in several haematological
`involved in multiple different signalling pathways, Bruton
`tyrosine kinase (BTK) plays an indispensible role in B cells in
`signalling from the B cell receptor (BCR) for antigen. Many
`B cell malignancies remain dependent on constitutive BCR
`signalling, making BTK a functional therapeutic target. Sev-
`eral BTK inhibitors (BTKi) with different kinomes and
`modes of action are being assessed clinically. This review
`documents the efficacy and toxicity of BTKi in chronic lym-
`phocytic leukaemia (CLL). Clinically, the furthest in develop-
`ment is ibrutinib (trade name, Imbruvica), an irreversible
`BTKi, which has shown spectacular preliminary efficacy, with
`rapid reductions in lymph nodes accompanied by peripheral
`blood lymphocytosis. The lymphocytosis resolves slowly and
`most patients do not enter a complete remission. Neverthe-
`less, it is possible to maintain many CLL patients, even those
`with adverse cytogenetic features, on drug for many months
`with minimal
`toxicities,
`thus potentially transforming the
`therapeutic paradigms for CLL. The efficacy, lack of toxicity
`and oral administration of BTKi will ensure their adoption
`in a wide range of B cell malignancies. An outstanding chal-
`lenge is to incorporate BTKi with other precision medicines
`in a mechanism-based manner in order to dispense with
`conventional chemotherapy.
`
`Keywords: chronic lymphocytic leukaemia, Bruton tyrosine
`kinase, ibrutinib, B cell receptor signalling.
`
`If the multi-billion dollar industry that has developed since
`the ‘war on cancer’ was announced by Richard Nixon in
`1971 (Sporn, 1997) is eventually to succeed in ‘eliminating
`the suffering and death from cancer’ (von Eschenbach,
`2006), then it must provide tools that allow either eradica-
`tion or long-term control of disease with minimal toxicity.
`In terms of the malignancies of mature B cells, significant
`
`Correspondence: Professor Martin J. S. Dyer, Room 3/57, Henry
`
`Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK.
`
`E-mail: mjsd1@le.ac.uk
`
`progress has been made and there is now a plethora of
`‘precision medicines’
`including both engineered antibodies
`and targeted small molecules that are having great therapeu-
`tic impact even in early phase clinical trials (Dyer et al,
`2013).
`One class of small molecule inhibitor currently arousing
`intense interest comprises compounds targeting Bruton tyro-
`sine kinase (BTK). Importantly, BTK is not a genetically-
`defined therapeutic target; there are no mutations in the
`BTK gene or gene fusions that result in constitutive activity.
`Rather, BTK is defined functionally as a therapeutic target,
`playing a key role in several pathways that maintain B cell
`survival. The efficacy of BTK inhibitors (BTKi) in several
`forms of mature B cell malignancy has been spectacular,
`resulting in the first-in-class BTKi (Ibrutinib; Pharmacyclics,
`Sunnyvale, CA, USA) being awarded ‘Breakthrough Drug’
`status by the US Food and Drug Administration (FDA) for
`mantle cell lymphoma (MCL), CLL and Waldenstrom mac-
`roglobulinaemia (Dolgin, 2013); breakthrough drug status is
`given if preliminary clinical evidence indicates the drug may
`offer a substantial improvement over available therapies for
`patients with serious or life-threatening diseases. A product
`licence for ibrutinib, for the treatment of relapsed MCL, was
`awarded by the US FDA on 13 November 2013 and for
`relapsed CLL on 11 February 2014; at the time of writing,
`similar licences are anticipated imminently in Europe.
`This review addresses the rationale for targeting BTK in
`CLL, the nature and mode of action of BTKi and the data
`emerging from clinical trials in CLL, alone and in combina-
`tion with other agents. Despite their huge promise and huge
`enthusiasm for their use from both patients and clinicians
`alike, there remain several barriers to the incorporation of
`these novel agents into CLL therapy.
`
`An overview of BTK structure and functions
`
`BTK takes its name from Colonel Ogden C. Bruton (1908–
`2003), an American Army paediatrician based at the Walter
`Reed Army Hospital
`in Washington DC, who,
`in 1952,
`described a condition now referred to as X-linked agamma-
`globulinaemia,
`following the study of an 8-year-old boy,
`Joseph S. Holtoner, Jr., who had recurrent pneumonia and
`
`First published online 18 April 2014
`doi: 10.1111/bjh.12895
`
`ª 2014 John Wiley & Sons Ltd
`British Journal of Haematology, 2014, 166, 12–22
`
` 13652141, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.12895, Wiley Online Library on [29/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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`absence of gamma globulin in his serum (Bruton, 1952). Dr
`Bruton and his discovery were featured in ‘TIME’ magazine
`18 May 1953. It was noted that all patients were boys; disease
`identification followed the development of successful antibi-
`otic therapy for recurrent bacterial
`infections. Forty years
`later,
`following extensive mapping experiments,
`the gene
`responsible for this condition (BTK), was cloned and charac-
`terized (Tsukada et al, 1993; Vetrie et al, 1993). Mapping to
`chromosome Xq21.3-q22, BTK encodes a kinase of the TEC
`subfamily (Yu & Smith, 2011; and other articles in that issue).
`Collectively, these kinases (BTK, ITK, BMX, TXK and TEC)
`are characterized by five structural domains including PH
`(pleckstrin homology) mediating phosphatidylinositol (3,4,5)-
`trisphosphate (PIP3) binding, TEC homology (BH), SH3 (Src
`homology 3), SH2 (Src homology 2) and kinase domains
`(Fig 1). The crystal structures of the major BTK domains
`have been solved (Mao et al, 2001; Marcotte et al, 2010).
`These structures have been of fundamental
`importance in
`terms of the design of targeted inhibitors, especially the irre-
`versible inhibitors, such as ibrutinib, AVL-292/CC-292 and
`ONO-4059 (Kuglstatter et al, 2011). BTK is localized pre-
`dominantly in the cytoplasm (but interestingly, also in the
`nucleus) with translocation to the plasma membrane via the
`PH domain following PIP3 binding,
`for phosphorylation
`and activation. Within BTK there are two major tyrosine
`
`Review
`
`phosphorylation sites. Phosphorylation of tyrosine (Tyr)551
`within the kinase domain regulates the transition between
`active (open) and inactive (closed) states. Phosphorylation of
`Tyr551 occurs on localization to the membrane by SRC fam-
`ily kinases (LYN/FYN/BLK) or SYK kinase. Conformational
`change then enables auto-phosphorylation at Tyr223. Once
`activated, BTK can then phosphorylate phospholipase Cc2,
`leading to calcium mobilization (King & Freedman, 2009)
`and subsequent activation of downstream signalling pathways,
`cytoskeletal
`rearrangements and transcriptional pathways
`(including NF-jB, NFAT and ARID3A) involved in prolifera-
`tion, cell survival and migration (Fig 1). Over 600 unique
`inherited BTK mutations have been described that lead to
`profound defects in B cell development (failure of develop-
`ment of pro to pre B cells) and immunoglobulin production
`(Valiaho
`et al,
`2006;
`http://structure.bmc.lu.se/idbase/
`BTKbase/). BTK mutations fall within all domains with the
`exception of the SH3 domain and many affect crucial residues
`involved in either ATP binding or in the catalytic site; others
`affect substrate recognition. Affected boys have normal levels
`of pre-B cells in their bone marrow but virtually no circulat-
`ing mature B lymphocytes, with a resultant lack of immuno-
`globulins of all classes and recurrent bacterial infections in
`the first few years of life. Interestingly, mice with identical Btk
`mutations have a much milder phenotype,
`indicating a
`
`pPLCγ2
`
`RAS
`
`PKCβ
`
`pERK
`
`IKK
`
`B-cell activation /
`Proliferation
`
`CD79B
`
`CD79A
`
`CD19
`
`PI3K
`
`PIP3
`
`pSYK
`
`pLYN
`
`BTK
`
`pTyr223
`
`Cys481
`
`pTyr551
`
`1
`
`PH
`
`BH PPR SH3
`
`SH2
`
`TK
`
`659
`
`Fig 1. BTK and its involvement in BCR signalling. Bruton tyrosine kinase (BTK) comprises several domains including: (i) pleckstrin homology
`(PH) domain. Mediates binding to inositol polyphosphate and phosphoinositides, leading to targeting of BTK to the plasma membrane. Plasma
`membrane localization is a critical step in the activation of BTK. (ii) BTK homology (BH) domain. Contains a highly conserved zinc finger motif
`that mediates Zn2+ ion binding necessary for protein stability. (iii) Polyproline (PPR) domain. (iv) Src homology (SH3 and SH2) domains. The
`SH2 domain of BTK binds to the B cell adapter protein BLNK, which is required for full BTK activation. (v) Tyrosine kinase (TK) domain.
`Unphosphorylated BTK is catalytically inactive. As shown in the figure, full activation of BTK requires both phosphorylation and binding of
`PIP3. Following BCR engagement and activation, and following PIP3 binding, BTK translocates to the plasma membrane, where it is phosphory-
`lated at Tyr551 by LYN and SYK, leading to autophosphorylation of Tyr223. Once bound to BLNK, BTK phosphorylates PLCG2 at several sites,
`leading to calcium mobilization and activation of the protein kinase C (PKC) family members and other effectors including RAS and lead to
`NFjB activation and phosphorylation of ERK. The BTK inhibitors ibrutinib, CC-292 and ONO-4059 all bind irreversibly to Cys481 within the
`ATP binding site of the BTK kinase domain, thus preventing BTK activation. In contrast, the highly specific BTKi, CGI-1746, binds to the ATP
`binding pocket of BTK and stabilizes the non-phosphorylated form of the protein by internalizing Tyr551. Apart from its role in BCR signalling,
`BTK is also implicated in cytokine and Toll receptor signalling pathways (not shown).
`
`ª 2014 John Wiley & Sons Ltd
`British Journal of Haematology, 2014, 166, 12–22
`
`13
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`Review
`
`species-specific functional redundancy; combined inactivation
`of both BTK and TEC is necessary to produce a similar phe-
`notype in mice.
`Although recent focus has been on the role of BTK in
`terms of modulating signalling from the BCR as outlined
`below, BTK is, in fact, activated in response to a very broad
`range of growth/differentiation stimuli in haemopoietic tis-
`sues, including growth factor receptors, cytokine receptors,
`G-protein coupled receptors and integrins (Qiu & Kung,
`2000). BTK is implicated in the control of several pathways
`of central importance to mature B cells. Moreover, BTK is
`expressed in all bone marrow-derived cell lineages, with the
`exception of T lymphocytes and plasma cells (de Weers et al,
`1993; Genevier et al, 1994; Smith et al, 1994). In terms of
`these other lineages the functions of BTK are relatively unex-
`plored, but recent data indicate key functions in myeloid
`cells (Honda et al, 2012). Recent exciting in vitro data indi-
`cate that BTK may be a therapeutic target in acute myeloid
`leukaemias (Rushworth et al, 2014) as well as B cell malig-
`nancies. In macrophages, BTK functions downstream of sev-
`eral toll-like receptors and regulates apoptotic cell uptake
`(Byrne et al, 2013). These and other observations may also
`have important clinical implications in terms of design of
`BTKi combination studies,
`for example, with therapeutic
`monoclonal antibodies dependent on macrophages for their
`clinical efficacy. BTKi may also interfere with platelet activa-
`tion (Byrd et al, 2013a; Hsu et al, 2013; Rushworth et al,
`2013). Rushworth et al
`(2013)
`showed that
`ibrutinib
`impaired the platelet activation in response to collagen and
`ADP; however, the clinical significance of these observations
`is not clear (Farooqui et al, 2012). Finally, BTK is highly
`homologous to the interleukin-2 inducible T cell kinase
`(ITK) expressed in T cells, a homology that may be of thera-
`peutic relevance (Dubovsky et al, 2013). Inherited mutations
`in ITK predispose to viral infections, including Epstein–Barr
`virus (EBV) infection as well as EBV-driven lymphoprolifera-
`tive conditions (Huck et al, 2009; Stepensky et al, 2011).
`Collectively, these data do not appear to augur well for
`targeting BTK therapeutically. The expression of BTK in
`multiple haemopoietic lineages, the involvement of BTK in
`multiple different pathways, and the complete block of nor-
`mal B cell development in individuals with constitutional
`BTK mutations suggest that BTK inhibition might be cata-
`strophic! However, all of the above appear to be trumped by
`functional data concerning the central role of BTK activity in
`the maintenance of several forms of mature B cell malig-
`nancy.
`
`Functions of BTK in malignant B cells; BTK as a
`potential therapeutic target
`
`In mouse models, BCR signalling is vital for the survival of
`normal mature B cells (Kraus et al, 2004): interestingly, in
`mice this defect is rescued by phosphatidylinositide 3-kinase
`(PI3K) rather than BTK signalling (Srinivasan et al, 2009). In
`
`14
`
`human B cell malignancies, maintained BCR signalling has
`been shown to play an essential role in the survival of the
`activated B cell subtype of diffuse large B cell
`lymphoma
`(DLBCL) and in CLL (please see two recent reviews, Burger
`& Chiorazzi, 2013; Young & Staudt, 2013). In activated B cell
`(ABC)-DLBCL, constitutive BCR signalling is largely driven
`by somatic mutations in key domains of signalling molecules
`including CD79A/B and CARD11 (Davis et al, 2010). In CLL
`however these mutations are not present, but a fascinating
`recent finding indicates that the configuration of the BCR
`itself may drive antigen-independent cell-autonomous signal-
`ling (Duhren-von Minden et al, 2012). Inhibition of BTK
`may therefore be effective therapy in all cases of CLL rather
`than just with maintained signalling capacity following BCR
`cross-linking in vitro (Apollonio et al, 2013).
`However,
`the above scenario is complicated by the
`involvement of BTK in other signalling pathways controlling
`B cell migration and motility. Mature lymphocytes are not
`static; they are constantly moving between blood and tissues
`(Gowans & Knight, 1964). For normal lymphocytes this pro-
`cess enables immune surveillance; for malignant lymphocytes
`it offers a protective niche in which to receive survival and
`proliferation signals from the microenvironmental and solu-
`ble factors within the lymph node. Lymphocyte movement
`is not random and depends on a complex process of signal-
`ling pathways involving adhesion molecules and chemokines,
`which ultimately control the cytoskeleton. Two such path-
`ways of particular importance are the chemokine receptor-
`ligand pathways CXCR4-CXCL12 and CXCR5-CXCL13.
`BTK is known to be important for B cell migration and
`homing (Ortolano et al, 2006; de Gorter et al, 2007), and is
`activated upon chemokine binding to CXCR4 and CXCR5
`through direct interaction with the chemokine receptor G
`protein subunits (Jiang et al, 1998; Lowry & Huang, 2002).
`Thus, BTKi may have a significant effect on lymphocyte
`homing and recirculation; this has been observed clinically
`in CLL patients receiving BTKi, as discussed below. The rel-
`ative importance of BTKi-mediated blockade of the various
`different pathways will presumably vary from patient
`to
`patient, and moreover in CLL cells at different anatomical
`locations.
`
`Pharmacological development of BTKi
`
`As is so often the case in drug development, there are several
`BTKi currently in clinical trial or in late preclinical develop-
`ment. Some of the BTKi being assessed are listed in Table I;
`Ibrutinib (Pharmacyclics) is furthest along in terms of clini-
`cal development but there are a currently a number of others
`undergoing clinical trials including AVL-292/CC-292 (Cel-
`gene, Summit, NJ, USA) and ONO-4059 (ONO Pharmaceu-
`ticals, Osaka, Japan). Whilst this plethora of new molecules
`is of considerable scientific and clinical interest and reflects
`the activities of unfettered market forces, one must question
`the logic of commercially developing so many molecules
`
`ª 2014 John Wiley & Sons Ltd
`British Journal of Haematology, 2014, 166, 12–22
`
` 13652141, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.12895, Wiley Online Library on [29/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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`Table I. BTKi currently in clinical trial or preclinical development.
`
`Molecule
`
`Company
`
`Irreversible
`inhibitor?
`
`Ibrutinib
`
`Pharmacyclics/Jansen Yes
`
`AVL-292/CC-292 Avila/Celgene
`
`Yes
`
`BTK
`BTK IC50
`specific?
`0 5 nmol/l No – broad
`kinome
`<0 5 nmol/l Yes
`
`ONO-4059
`
`Ono Pharma
`
`Yes
`
`2 2 nmol/l Yes
`
`HM71224
`
`Hanmi Pharma
`
`GDC-0834
`
`CGI-1746
`
`CGI Pharma
`Genentech
`CGI Pharma
`
`NK
`
`No
`
`No
`
`NK
`Yes
`5 9 nmol/l NK
`1 9 nmol/l Yes
`
`Review
`
`Clinical
`development
`
`Clinical trials
`
`References
`
`Multiple Phase
`II and Phase III
`clinical trials
`Phase II clinical trial NCT01766583
`With Lenalidomide
`in ABC-DLBCL
`NCT01659255
`Dose escalation
`in B cell
`malignancies
`NCT01765478
`
`Phase I clinical trial
`
`Phase I in normal
`individuals
`Phase I normal
`individuals
`Preclinical
`
`See text
`
`See text
`
`Salles et al
`(2013) and
`Rule et al
`(2013)
`NK
`
`Liu et al
`(2011)
`Di Paolo
`et al (2011)
`
`BTK, Bruton tyrosine kinase; BTKi, Bruton tyrosine kinase inhibitor; IC50, 50% inhibitory concentration; ABC-DLBCL, activated B cell-diffuse
`large B cell; NK, not known.
`
`against the same target. Which might be best clinically is
`quite unclear, and given the clinical success of ibrutinib may
`well be impossible to assess. The chemical structures of the
`three irreversible BTKi are very similar and yet the kinomes
`of each molecule are very different.
`The development of ibrutinib is of interest for at least two
`reasons. Firstly, and most
`importantly,
`ibrutinib is an
`irreversible BTKi that binds covalently to BTK (Pan et al,
`2007). A series of highly selective and irreversible BTKi were
`developed at Celera using a bioinformatics approach using
`the crystal structures of BTK. These molecules inactivate BTK
`by covalent binding to Cys481 near the ATP binding domain
`of BTK and thus inhibited phosphorylation at Tyr223. The
`lead molecule (PCI-32765) was tested in in vivo models of
`both rheumatoid arthritis and lymphoma (using a spontane-
`ously occurring canine lymphoma model) and showed con-
`siderable activity (Honigberg et al, 2010). Historically, the
`pharmaceutical industry has been wary of irreversible kinase
`inhibitors because of concerns over possible toxicity, particu-
`larly so in kinases with long protein half-lives. (Interestingly,
`BTK is a long-lived protein with a half-life in B cells of over
`8 h). However, there are now an increasing number of irre-
`versible kinase inhibitors entering the market, including afati-
`nib an irreversible inhibitor of EGFR (Sanderson, 2013). The
`second interesting aspect about ibrutinib is that its kinome is
`very broad and includes several other kinases with pivotal
`roles in normal and malignant B cell signalling,
`including
`numerous SRC kinase family members including LYN (Pan
`et al, 2007). Not unsurprisingly, given the close homology of
`the two proteins, ibrutinib irreversibly inactivates ITK as well
`as BTK (Dubovsky et al, 2013). One of the implications of
`this finding might be a role for ibrutinib in T cell malignan-
`cies dependent on ITK signalling as well as those with ITK
`
`ª 2014 John Wiley & Sons Ltd
`British Journal of Haematology, 2014, 166, 12–22
`
`fusions (Streubel et al, 2006; Pechloff et al, 2010). Less
`positively, it is difficult to reconcile the lack of observed tox-
`icity seen in patients taking ibrutinib for many months with
`this broad kinome, especially perhaps in patients immuno-
`compromised by their disease and prior therapies.
`A variety of more specific BTKi have been developed
`and three of these are currently undergoing clinical trials in
`B cell malignancies. Some of these are shown in Table I.
`AVL-292/CC-292 and ONO-4059 are both irreversible
`inhibitors, which are closely related to ibrutinib structurally
`but are considerably more specific in terms of other kinases
`inhibited; both are being assessed in patients with B cell
`malignancies. ONO-4059 is in early phase I development
`but
`initial results presented at
`the American Society of
`Hematology (ASH) annual meeting in 2013 indicate compa-
`rable activity to ibrutinib in CLL with possibly less toxicity;
`significant efficacy was also observed in MCL (Salles et al,
`2013; Rule et al, 2013). Data with AVL-292/CC-292 in CLL
`is given below and with twice daily dosing suggests
`improved nodal responses (Harb et al, 2013). The combina-
`tion of AVL-292/CC-292 and lenalidomide in ABC-DLBCL
`is of
`interest, given that both molecules have significant
`activity against this disease subtype (Yang et al, 2012; Vose
`et al, 2013; NCT01766583). This combination is also being
`assessed in CLL (NCT01732861); it is a mechanistically and
`financially logical combination, given that both lenalidomide
`and CC-292 are now owned by Celgene. Another BTKi,
`CGI-1746 [CGI Pharmaceuticals (Branford, CT, USA) (now
`part of Gilead)]
`is highly BTK-specific and may have
`unique and potentially advantageous properties. CGI-1746
`binds to the ATP-binding pocket of BTK and stabilizes the
`non-phosphorylated form of
`the protein by internalizing
`the crucial residue Tyr551, which has to be phosphorylated
`
`15
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`Review
`
`for BTK activation. CGI-1746 therefore has exquisite sensi-
`tivity for BTK (Di Paolo et al, 2011). Whether this mole-
`cule is now being developed for clinical use is not clear.
`There are insufficient data to compare the BTKi even indi-
`rectly at the current time. Paradoxically, given the data from
`the clinical studies to date, the ‘dirtier’ inhibitor, ibrutinib
`might possibly have significant advantages.
`
`Clinical studies with BTKi
`
`Ibrutinib as single agent has been used both in elderly patients
`(≥65 years) with treatment-na€ıve CLL and those with relapsed
`and refractory disease. A phase Ib-II trial was recently reported
`for relapsed and refractory CLL (Byrd et al, 2013b). Eighty-five
`patients considered to be ‘high risk’ were treated with either
`420 mg (51 patients) or 840 mg (34 patients). The overall
`response rate (ORR) was 71% in both cohorts [two complete
`responses (CR) and 34 partial responses (PR)] with a further
`20% (420 mg cohort) and 15% (840 mg cohort) achieving a
`PR with lymphocytosis. The response was independent of the
`number of previous therapies or high-risk features, including
`17p 13 deletion. At 26 months, progression-free survival (PFS)
`was estimated at 75% and OS at 83%. A lymphocytosis was
`observed in 78% of patients. Ibrutinib was very well tolerated;
`however, the higher dose was associated with more adverse
`events leading to discontinuation of treatment (two patients
`on 420 mg and four patients on the 840 mg dose). Perhaps
`not surprizingly in this patient group, pneumonia was the
`most common grade 3 or higher adverse event, seen in 10
`patients, and infections were seen more commonly early in
`treatment. Interestingly, despite the low levels of immunoglob-
`ulins seen in patients with the genetic mutation of BTK,
`immunoglobulin levels remained relatively stable during treat-
`ment. Indeed, IgA and IgM serum levels increase on treatment,
`signifying recovery of humoral
`immunity (Farooqui et al,
`2013). The treatment-na€ıve arm of this trial in patients older
`than 65 years (n = 31) was updated at the International Work-
`shop
`on Chronic
`Lymphocytic
`Leukaemia
`in
`2013
`(iwCLL2013): with a median follow up of 22 1 months, the
`ORR was 71% (CR 13%) with a 96% PFS (O’Brien et al,
`2014).
`An unanticipated finding was that ibruitinib induced a
`prompt lymphocytosis in the peripheral blood. A similar
`effect was observed in patients receiving PI3 delta kinase
`inhibitors (reviewed in Macias-Perez & Flinn, 2013) as well
`as CXCR4 antagonists. In ibrutinib-treated CLL patients the
`lymphocytosis is usually seen by 7 d, peaking within 4 weeks
`and then slowly decreasing with time, which is generally
`slower than that seen with treatment in MCL. The lympho-
`cytosis is not restricted to subtypes of CLL but patients with
`unmutated immunoglobulin variable-region heavy chain
`(IGHV) gene segments had an earlier resolution of lympho-
`cytosis compared to patients with mutated IGHV rearrange-
`ments (median 6 4 vs. 14 8 months) (Byrd et al, 2013a). It is
`not yet clear whether the rate of fall in lymphocyte count
`
`16
`
`will perhaps become a prognostic factor as important as
`lymphocyte doubling time.
`
`What are the practicalities?
`
`Ibrutinib is given orally once a day. In CLL patients, the
`optimal dose, determined from the above trials and being
`taken forward in future trials, is 420 mg. Given the broad
`expression of BTK in haematological cells and the ibrutinib
`kinome, it is surprising that the drug is so well tolerated.
`Toxicities are very manageable on an out-patient basis with
`the most common treatment-related adverse event being
`mild to moderate diarrhoea, fatigue and nausea, with pneu-
`monia more commonly seen in CLL patients. Haematological
`grade 3 or higher neutropenia, thrombocytopenia and anae-
`mia are seen in 10–20% of patients with previously treated
`CLL and these improve with treatment. Bleeding is seen in a
`subset of patients and current trials exclude the concurrent
`use of warfarin but not other anticoagulant therapies.
`
`How will ibrutinib compare to other single agents?
`
`Two randomized, multicentre phase III trials are currently
`ongoing to address this question. RESONATE-2 is comparing
`chlorambucil against
`ibrutinib in a 1:1 randomization in
`treatment–na€ıve CLL/SLL patients older
`than 65 years
`(NCT01722487), whilst RESONATE compared the novel
`CD20 monoclonal antibody ofatumumab against ibrutinib in
`relapsed/refractory CLL/SLL patients (NCT01578707). The
`latter study was stopped early by the data monitoring com-
`mittee after an an interim analysis showed that patients on
`ibrutinib had a statistically significant improvement not only
`in PFS (the primary study end point), but also in OS (a sec-
`ondary end point), when compared with ofatumumab
`(http://www.pmlive.com/pharma_news/j_and_js_imbruvica_
`tops_arzerra_in_phase_iii_trial_531853 and please see also
`http://www.btktrials.com.)
`
`Will remission be achieved using single agent ibrutinib?
`
`The present indication is that there are very few complete
`responses with ibrutinib (2% in previously treated CLL, 13%
`in treatment na€ıve CLL) and progression is rapidly seen when
`treatment is interrupted. However, most patients continue to
`have an ongoing response with continued treatment and the
`role of combination therapy to improve responses, with the
`aim of attaining a minimal residual disease (MRD) negative
`response without significant toxicity is keenly debated.
`
`What about the chance of developing resistance to BTK
`inhibition?
`
`In CLL patients who had developed resistance to ibrutinib,
`comparative DNA sequence analysis of
`the genome was
`performed in samples at baseline and at disease progression.
`
`ª 2014 John Wiley & Sons Ltd
`British Journal of Haematology, 2014, 166, 12–22
`
` 13652141, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.12895, Wiley Online Library on [29/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1019, p. 5 of 11
`
`

`

`In five patients analysed, four patients developed a mutation
`resulting in the substitution of cysteine for serine at position
`481 with a further mutation seen in the major downstream
`BTK target, PLCc2. All five patients had high risk cytogenet-
`ics. It is therefore likely that mutations will develop with
`prolonged therapy, highlighting the necessity for the develop-
`ment of mechanism-based combinations.
`
`What are the mechanisms of action of BTKi in vivo?
`
`Despite the clinical success of ibrutinib, there remain some
`fundamental questions
`concerning the mechanism(s) of
`action of this drug and other BTKi
`in vivo. Specifically,
`how much of the tumour cell death induced by BTK inhi-
`bition is due to a direct cytotoxic effect on the malignant B
`cells and how much is due to death by deprivation of pro-
`survival signalling? The relative importance of
`these two
`mechanisms may vary according to disease type. Firstly, it
`is clear that BTKi can have significant activity against some
`cases of chemotherapy-refractory non-germinal center B
`cell-like (GCB) DLBCL (deVos et al, 2013; Yoshizawa et al,
`2013). Given that these malignancies are rapidly prolifera-
`tive,
`this suggests that BTK inhibition directly results in
`DLBCL cell death without significant
`lymphocytosis. The
`precise mechanism by which this occurs remains unclear;
`only one ABC-DLBCL cell line (TMD8) is sensitive to BTKi
`in vitro (Kozaki et al, 2013). Similar remarks may apply to
`nodal MCL. However,
`this
`simple mechanism is con-
`founded in CLL where the malignant lymphocytes initially
`increase significantly and persist in the peripheral blood for
`many months during therapy and decline only slowly with
`time. These clinical data suggest ‘death by deprivation’. In
`contrast however,
`lymph node size diminishes rapidly in
`CLL, with the majority of effects seen in the first 2 months
`of therapy (Byrd et al, 2013b). The egress of CLL cells into
`the peripheral blood is not sufficient to account for the
`observed decreases in lymph node size (Garg et al, 2013;
`CV Hutchinson and MJS Dyer, unpublished observations).
`BTK inhibition clearly has significant activity across all sub-
`types of CLL including patients with poor risk disease indi-
`cating a TP53 independent mechanism of action. However,
`precise mechanisms of action of BTKi in vivo need to be
`determined in order to design rational combination thera-
`pies.
`
`What about combination therapy?
`
`In CLL, the combination with rituximab would seem logical
`and potentially synergistic;
`‘mobilize’ the cells with BTKi
`and then target them in the circulation with a monoclonal
`antibody. Burger et al (2012) reported on a single centre
`study of 40 patients with high risk CLL [del17p or TP53
`mutation (treated or untreated), PFS <36 months after
`frontline
`chemoimmunotherapy or
`relapsed CLL with
`del11q]. Patients received daily ibrutinib at 420 mg plus
`
`ª 2014 John Wiley & Sons Ltd
`British Journal of Haematology, 2014, 166, 12–22
`
`Review
`
`rituximab 325 mg/m2 weekly for
`then
`the first month,
`monthly for a total 6 months and then continued on daily
`ibrutinib. The results were recently updated at
`the ASH
`annual meeting in 2013 (Keating et al, 2013), at a median
`follow up of 14 months, 32 of 40 patients continued on
`therapy without disease progression (16 out of 20 with
`del17p or TP53 mutation). Of the 39 patients evaluable,
`there was an ORR of 95% (PR 87%, CR 8%). The regimen
`was generally well
`tolerated although infections were the
`most common complication (six cases of pneumonia, three
`cases of upper respiratory infections). Interestingly, the lym-
`phocytosis peaked earlier
`(within first month) and the
`duration was
`shorter
`(back
`to at
`least baseline
`at
`3 months). Whether this will equate to better responses is
`keenly anticipated. The combination of
`ibrutinib and of-
`atumumab, the fully humanized CD20 antibody, also has
`shown favourable responses in patients with relapsed/refrac-
`tory CLL/small lymphocytic lymphoma/prolymphocytic leu-
`kaemia, with 100% ORR (24/24 patients) within six cycles
`with minimal toxicity and rapid response rate (Jaglowski
`et al, 2012).
`Going one step further, will improved responses be seen
`with BTKi in combination with chemoimmunotherapy? The
`final result