Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans

Article abstract of DOI:10.1124/jpet.113.203489

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2- (4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl) acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro and in the collagen-induced arthritis model of autoimmune disease. Recently, CC-292 has entered human clinical trials with a trial design that has provided rapid insight into safety, pharmacokinetics, and pharmacodynamics. This first-in-human healthy volunteer trial has demonstrated that a single oral dose of 2 mg/kg CC-292 consistently engaged all circulating Btk protein and provides the basis for rational dose selection in future clinical trials. This targeted covalent drug design approach has enabled the discovery and early clinical development of CC-292 and has provided support for Btk as a valuable drug target for B-cell mediated disorders. Copyright

Full text of DOI:10.1124/jpet.113.203489

Supplemental Material can be found at:
http://jpet.aspetjournals.org/content/suppl/2013/05/24/jpet.113.203489.DC1.html
1521-0103/346/2/219–228$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
http://dx.doi.org/10.1124/jpet.113.203489
J Pharmacol Exp Ther 346:219–228, August 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Inhibition of Btk with CC-292 Provides Early Pharmacodynamic
Assessment of Activity in Mice and Humanss
Erica K. Evans, Richland Tester, Sharon Aslanian, Russell Karp, Michael Sheets,
Matthew T. Labenski, Steven R. Witowski, Heather Lounsbury, Prasoon Chaturvedi,
Hormoz Mazdiyasni, Zhendong Zhu, Mariana Nacht, Martin I. Freed, Russell C. Petter,
Alex Dubrovskiy, Juswinder Singh, and William F. Westlin
Celgene Avilomics Research, Bedford, Massachusetts
Received January 23, 2013; accepted May 13, 2013
ABSTRACT
Targeted therapies that suppress B cell receptor (BCR) signaling demonstrate the quantity of Btk bound by CC-292 correlates
have emerged as promising agents in autoimmune disease and with the efficacy of CC-292 in vitro and in the collagen-induced
B cell malignancies. Bruton’s tyrosine kinase (Btk) plays a crucial arthritis model of autoimmune disease. Recently, CC-292 has
role in B cell development and activation through the BCR entered human clinical trials with a trial design that has provided
signaling pathway and represents a new target for diseases rapid insight into safety, pharmacokinetics, and pharmacody-
characterized by inappropriate B cell activity. N-(3-(5-fluoro-2- namics. This first-in-human healthy volunteer trial has demon-
(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phe- strated that a single oral dose of 2 mg/kg CC-292 consistently
nyl)acrylamide (CC-292) is a highly selective, covalent Btk engaged all circulating Btk protein and provides the basis for
inhibitor and a sensitive and quantitative assay that measures rational dose selection in future clinical trials. This targeted
CC-292-Btk engagement has been developed. This translational covalent drug design approach has enabled the discovery and
pharmacodynamic assay has accompanied CC-292 through early clinical development of CC-292 and has provided support
each step of drug discovery and development. These studies for Btk as a valuable drug target for B-cell mediated disorders.
2001). Once activated, Btk forms a signaling complex with
Introduction
proteins such as BLNK, Lyn, and Syk and phosphorylates
Bruton’s tyrosine kinase (Btk) is a kinase expressed exclusively
phospholipase C (PLC)g2 (Baba et al., 2001; Tsukada et al.,
in B cells and myeloid cells and has a well characterized, vital
2001). This leads to downstream release of intracellular Ca21
role in B cells highlighted by the human primary immune
stores and propagation of the BCR signaling pathway through
deficiency disease, X-linked agammaglobulinemia (XLA),
which results from mutation in the Btk gene (Smith et al.,
extracellular signal-regulated kinase and nuclear factor-kB
signaling, ultimately resulting in transcriptional changes to
1998). As a result of incomplete B cell differentiation, XLA
foster B cell survival, proliferation, and/or differentiation
patients have a near complete absence of mature B cells in the
(Baba et al., 2001; Maas and Hendriks, 2001; Mohamed et al.,
peripheral blood (Campana et al., 1990) and cannot produce
2009).
immunoglobulins (Conley, 1985; Nonoyama et al., 1998). The
While BCR signaling is essential in the normal develop-
human XLA phenotype is recapitulated, although less
ment and function of B cells, several pathologies have been
severely, in Btk knock-out mice (Khan et al., 1995) and in
attributed to dysregulated BCR activity. These include diseases
xid mice, which have a naturally occurring Btk mutation
(Rawlings et al., 1993).
of autoreactivity, such as that observed in lupus, multiple
sclerosis, and rheumatoid arthritis, in which B cells inappro-
Specifically, Btk plays an essential role in the B cell
priately break self-tolerance to produce antibodies contributing
receptor (BCR) signaling pathway. Antigen binding to the
to autoimmune disease (Edwards and Cambridge, 2005, 2006;
BCR results in B cell receptor oligomerization, Syk and Lyn
Teng et al., 2007). BCR signaling also contributes to several B cell
kinase activation (Gauld et al., 2002), followed by Btk kinase
malignancies, such as chronic lymphocytic leukemia (CLL) (Chen
activation (Park et al., 1996; Rawlings et al., 1996; Baba et al.,
et al., 2005; Hoellenriegel et al., 2011; Stevenson et al., 2011),
mantle cell lymphoma, and subsets of diffuse large B cell
leukemia (Chen et al., 2008; Lenz et al., 2008; Baran-Marszak
et al., 2010; Davis et al., 2010; Suljagic et al., 2010; Pighi et al.,
dx.doi.org/10.1124/jpet.113.203489.
s
This article has supplemental material available at jpet.aspetjournals.org.
ABBREVIATIONS: BCR, B cell receptor; BSA, bovine serum albumin; Btk, Bruton’s tyrosine kinase; CC-292, N-(3-(5-fluoro-2-(4-(2-
methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide; CIA, collagen-induced arthritis; CLL, chronic lymphocytic leukemia; CNX-500,
N1-(3-(3-(4-(3-acrylamidophenylamino)-5-methylpyrimidin-2-ylamino)phenoxy)propyl)-N5-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-
d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide; CST, Cell Signaling Technology; ELISA, enzyme-linked immunosorbet assay;
PBS, phosphate-buffered saline; PD, pharmacodynamic; PK, pharmacokinetic; PLC, phospholipase C; XLA, X-linked agammaglobulinemia.
219

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Evans et al.
incubated in the presence of compound for 1 hour at 37°C. Following
incubation, cells were centrifuged and resuspended in 100 ml of
serum-free RPMI and BCR was stimulated with addition of 5 mg/ml
a-human IgM. Samples were centrifuged, washed in phosphate-
buffered saline (PBS), and lysed in 100 ml of Cell Extraction Buffer
[cat. no. FNN0011; Life Technologies (Invitrogen), Carlsbad, CA] plus
1:10 (v/v) PhosSTOP Phosphatase Inhibitor (Roche, Basel, Switzerland)
and 1:10 (v/v) Complete Protease Inhibitor (cat. no. 11836145001;
Roche). Antibodies used for immunoblot analysis include P-PLCg2 [cat.
no. 3872; Cell Signaling Technology, Beverly, MA (CST)], PLCg2 (3871;
CST), Syk (2712; CST), P-Syk (2710; CST), Btk (cat. no. 611116; BD
Biosciences, Franklin Lakes, NJ), P-Btk (cat. no. 2207-1; Epitomics,
Berlingame, CA), and Tubulin (cat. no. T6199; Sigma-Aldrich, St. Louis,
2011). However, until recently, therapies that target the B cell
have resulted in depletion of the B cell repertoire, while
therapeutic strategies that reduce BCR activity are rela-
tively new for treatment of these diseases.
Promising recent clinical data generated by inhibition of
distinct BCR signaling components, including Syk, phospha-
tidylinositide 3-kinase d, and Btk with fostamatinib, idelalisib
(also known as GS-1101 or CAL-101), and ibrutinib (PCI-
32765), respectively, have provided great excitement for this
approach. Inhibition of Syk with fostamatinib has demon-
strated efficacy in human clinical trials in rheumatoid
arthritis as well as in B cell malignancies dependent on
BCR signaling such as CLL (Braselmann et al., 2006; Chen MO). Membranes were scanned on a Li-Cor Odyssey scanner using
infrared fluorescence detection (Li-Cor Biosciences, Lincoln, NB).
B-Lymphocyte Proliferation (3H-Thymidine Incorporation).
A suspension of resting purified naïve human B cells isolated by
negative selection (MACS reagent 130-091-150) in RPMI was pre-
pared at 0.4–0.5 Â 106 cells/ml. Cells were mixed together with a-human
IgM (final concentration of 5 mg/ml in each well) and vehicle (dimethyl
sulfoxide) or CC-292 (final concentrations of 0.01, 0.1, 1.0, 10.0, 100.0, or
1000 nM per well) and seeded in a 96-well plate. Cells were incubated
for 56 hours in a humidified incubator maintained at 37°C and 5%
CO2. 3H-Thymidine was added (final concentration of 1 mCi in each well)
and cells were incubated overnight, harvested, and measured for 3H
incorporation. Experiments were performed in triplicate.
et al., 2008; Podolanczuk et al., 2009; Friedberg et al., 2010;
Genovese et al., 2011). Similarly, inhibition of phosphatidy-
linositide 3-kinase d with GS-1101 has also shown promising
results in CLL (Herman et al., 2010; Hoellenriegel et al., 2011;
Lannutti et al., 2011). Btk, downstream of Syk in the BCR
signaling pathway, also represents an attractive drug target
in diseases characterized by aberrant B cell activity. More-
over, owing to its highly restricted expression pattern in
B cells and myeloid cells, Btk provides an opportunity for
selective therapeutic targeting. Preclinically, small molecule
inhibition of Btk with CGI1746 and ibrutinib demonstrated
therapeutic activity in several models of autoimmune disease
(Honigberg et al., 2010; Chang et al., 2011; Di Paolo et al.,
2011). Ibrutinib has shown promising results in early clinical
Btk Target Site Occupancy Enzyme-Linked Immunosor-
bent Assay. An enzyme-linked immunosorbent assay (ELISA)
method for the detection of free uninhibited Btk in mouse, rat, dog,
monkey, and human lysates was developed at Celgene Avilomics
Research, and a validation of this method in human B cell lysate
was performed by a federal Certified Laboratories Improvement
Amendments-certified laboratory (Cambridge Biomedical Laborato-
ries, Boston, MA) The parameters that were assessed included: accuracy,
linearity, dilution, precision (intra- and interassay), stability, refer-
ence range, freeze-thaw cycles, reportable range, specificity, sensitiv-
ity, and carryover. All specifications for linearity, precision (intra- and
interassay), accuracy, and carryover defined in the validation protocol
were met. Samples were stable at –80°C for 5 weeks and the re-
portable range of the Btk ELISA was 12–12,800 pg of free Btk. Cell
lysates or spleen homogenates were incubated with N1-(3-(3-(4-(3-
acrylamidophenylamino)-5-methylpyrimidin-2-ylamino)phenoxy)propyl)-
N5-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-
4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide (CNX-500) (Celgene
Avilomics Research; final concentration 1 mM) in a PBS, 0.05% Tween-
20, 1% bovine serum albumin (BSA) solution for 1 hour at room
temperature. Standards and samples were transferred to a streptavi-
din-coated 96-well ELISA plate and mixed while shaking for 1 hour at
room temperature. The a-Btk antibody (BD 611116, 1:1000 dilution in
PBS 1 0.05% Tween-20 1 0.5% BSA) was then incubated for 1 hour at
room temperature. After wash, goat anti-mouse horseradish peroxi-
dase (1:5000 dilution in PBS 1 0.05% Tween-20 1 0.5% BSA) was
added and incubated for 1 hour at room temperature. The ELISA was
developed with addition of tetramethyl benzidine followed by Stop
Solution (CST) and read at optical density 450 nm. The standard
curve (11.7–3000 pg/ml) was generated with human full-length
recombinant Btk protein and plotted using a 4-parameter curve fit in
Gen5 software. Uninhibited Btk detected from samples was normal-
ized to mg total protein as determined by BCA protein analysis (cat.
no. 23225; Pierce, Rockford, IL).
development for the treatment of
B cell malignancies
(Harrison, 2012; Advani et al., 2013) and is currently in phase
III trials in CLL, providing evidence that Btk represents
a viable and efficacious therapeutic target.
We describe our work on N-(3-(5-fluoro-2-(4-(2-methoxyeth-
oxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide
(CC-292), which is a potent, highly selective, covalent in-
hibitor of Btk that inhibits BCR signaling and has efficacy in
a rheumatoid arthritis disease model. We also describe
a pharmacodynamic (PD) assay that has been implemented
throughout all stages of preclinical development to measure
activity of CC-292 and correlate Btk inhibition with functional
outcome both in vitro and in vivo. Finally, we report the
substantial oral exposure to CC-292 in humans and use this
PD assay to unequivocally and quantitatively demonstrate
complete Btk engagement in a first-in-human setting. This
work represents the first report of a selective Btk inhibitor
appropriate for use in a human clinical setting of autoimmune
disease and uses a powerful translational approach to confirm
on-target activity in human B cells.
Materials and Methods
B-Lymphocyte Isolation for In Vitro Signaling, Prolifera-
tion, and Activation. Human naïve, primary B cells (CD191, IgD1)
were isolated from anticoagulated whole blood by density centrifuga-
tion through Histopaque-1077 and peripheral blood mononuclear cell
(PBMC) isolation. PBMCs were subject to red blood cell lysis using
Red Blood Cell Lysis Buffer (Boston BioProducts, Ashland, MA) followed
by incubation with MACS reagent (130-091-150) and negative selection
over a MACS column to obtain naïve primary B cells with .85%
purity.
Spleen Homogenization. Spleens were harvested from mice,
frozen immediately in liquid nitrogen, and stored at –80°C. To
generate spleen lysates, each spleen was sliced in half and lysed using
a Precellys 24 Bead Homogenizer in 500 ml of Bio-Rad Bio-Plex Lysis
Buffer plus protease inhibitors (Bio-Rad, Hercules, CA). Supernatant
Immunoblot Analysis. Cells were incubated in serum-free RPMI
media for 1–1.5 hours. Isolated human B cells were incubated with
CC-292 at a final concentration of 0.001, 0.01, 0.1 and 1 mM. Ramos was transferred to a fresh microfuge tube and stored frozen at –80°C
cells were incubated with 0.1 nM–3 mM CC-292. Cells were then until analysis.

Btk Inhibition with the Covalent Inhibitor CC-292
221
Collagen-Induced Arthritis Model. Experiments were carried
out at Bolder Biopath, Boulder, CO. All experiments were carried out
in compliance with regulations of the Institutional Animal Care and
Use Committee and were conducted in accordance with principles and
procedures dictated by the highest standards of humane animal care.
Dba1 mice were injected at the base of the tail with 150 ml of Freund’s
Complete Adjuvant (Sigma-Aldrich) containing bovine type II
collagen (Elastin Products, Owensville, MO) (2 mg/ml) on day 0 and
again on day 21. On study days 25–27, onset of arthritis occurred, and
mice were randomized into treatment groups (10 per treatment
group, four per group for normal). Randomization into each group was
done after swelling was obviously established in at least one paw, and
attempts were made to assure approximately equal mean scores
across the groups at the time of enrollment. Treatment was initiated
after enrollment. Treatment continued daily (QD at 24-hour inter-
vals) through arthritis day 14. Clinical scores were assessed for each
of the paws on study arthritis days 1–15 using the following scoring
system: 0 5 normal, 1 5 one hind or fore paw joint affected or minimal
diffuse erythema and swelling, 2 5 two hind or fore paw joints affected
or mild diffuse erythema and swelling, 3 5 3 hind or fore paw joints
affected or moderate diffuse erythema and swelling, 4 5 marked
diffuse erythema and swelling or 4 digit joints affected, 5 5 severe
diffuse erythema and severe swelling entire paw, unable to flex digits.
Spleens and plasma were harvested 2 or 24 hours after the last dose of
CC-292 on arthritis day 14 and paws were removed and fixed in
formalin for histopathological analysis.
potent, selective inhibitor of Btk. CC-292 was rationally
designed to possess high affinity for the ATP binding pocket
and to form a specific covalent bond with cysteine 481 in Btk,
a poorly conserved amino acid among kinases. In biochemical
assays, CC-292 is a potent inhibitor of Btk kinase activity
(IC50apparent , 0.5 nM, kinact/KI 5 7.69 Â 104 M–1s21) and is
highly selective (Supplemental Tables 1 and 2; see Supple-
mental Methods for more information). Because biochemical
kinase assays may overestimate the potency of small molecule
kinase inhibitors due to high ATP concentrations found in the
cellular environment, cell activity for several of these closely
related kinase family members was assessed. CC-292 dem-
onstrated a high degree of selectivity against kinases with
a cysteine in a homologous position as Cys481 in Btk (epidermal
growth factor receptor, Itk, Janus kinase 3; Supplemental
Tables 3 and 4). Full details of the in vitro properties of
CC-292 as well as confirmation of the covalent mechanism of
action by mass spectrometry are shown in Supplemental Figs.
2–4 and Supplemental Tables 1–3. To demonstrate specific
inhibition of Btk in cells, CC-292 was evaluated in Ramos
cells, which express an intact BCR signaling pathway that is
activated robustly by addition of anti-IgM. CC-292 potently
inhibited Btk autophosphorylation on Tyr223 (EC50 5 8 nM;
Fig. 2A; Supplemental Fig. 5), phosphorylation of the Btk
substrate, PLCg2, as well as activation of the downstream
kinase extracellular signal-regulated kinase, all previously
shown to be sensitive to Btk inhibition (Honigberg et al., 2010;
Di Paolo et al., 2011). It is noteworthy that while CC-292
inhibited autophosphorylation of Btk, it had no effect on the
phosphorylation of Btk on Tyr551, a site phosphorylated by
Lyn and Syk and required for Btk activation (Afar et al.,
1996). These data demonstrate CC-292 is selective for Btk and
does not inhibit the Src-family kinases upstream of Btk in the
BCR signaling pathway (Fig. 2A).
Consistent with its covalent mechanism of action, CC-292
provided prolonged inhibition of kinase activity hours after
the drug was removed from cells. In contrast to reversible
inhibition with the potent Btk inhibitor dasatinib (Hantschel
et al., 2007), for which kinase activity had almost completely
returned 6 hours after drug removal, recovery of Btk activity
following a 1-hour exposure to CC-292 continued to be suppressed
∼8 hours in drug-free media (Fig. 2B). This prolonged period
of Btk inhibition correlated well with Btk protein turnover
assayed in the presence of the protein synthesis inhibitor
cyclohexamide. These experiments indicated that existing
cellular Btk was degraded slowly (36% reduction of protein in
8 hours and 63% reduction at 17 hours) (Supplemental Fig. 6).
Since Btk exposed to CC-292 is irreversibly bound and inhibited,
the return of Btk-dependent signaling relies on the appearance
of new Btk protein as a result of protein synthesis in a CC-292–free
environment.
Clinical Study. A double-blind, placebo-controlled, ascending
single-dose, randomized study in normal healthy human volunteers
was conducted at a single clinical research unit in accordance with
Declaration of Helsinki principles. Informed consent statements were
obtained from all subjects prior to inclusion in the study. Subjects
were admitted to the unit 1 day before dosing and discharged 96 hours
after dosing. Six subjects were administered a single oral dose of
2 mg/kg CC-292, monitored for safety and evaluated for drug action by
pharmacokinetic (PK) and PD analysis.
Isolation of Enriched
B Lymphocyte Population from
Human Healthy Volunteers. Human whole blood (21 ml) was
collected from each subject at each time point into BD Vacutainer
CPT Cell Preparation Tubes containing sodium heparin. RosetteSep
Human B Cell Enrichment Cocktail (cat. no. 15024; StemCell Technol-
ogies, Vancouver, BC, Canada) was added to each CPT tube and
centrifuged for 25 minutes at 1800g at room temperature. Isolated
cells were harvested into a clean 50-ml conical tube that was pooled by
subject. Each enriched B cell suspension was centrifuged at 400g for
15 minutes at room temperature. Cell suspensions were diluted in
1 ml of red blood cell lysis buffer for 3 minutes at room temperature.
Cell pellets were lysed with 150 ml Bio-Plex lysis buffer (cat. no.
171-304012; Bio-Rad). The lysates were stored frozen at #–70°C until
Btk target site occupancy analysis by ELISA.
Results
CC-292: A Potent, Highly Selective Btk Inhibitor. We
have identified CC-292 (Fig. 1; Supplemental Fig. 1) as a
Quantitative Analysis of Btk Occupancy. The covalent
mechanism of action of CC-292 has enabled design of a
companion PD assay that directly quantifies covalent bonding
to Btk protein after drug exposure. A probe (CNX-500) was
developed consisting of a covalent Btk inhibitor chemically
linked to biotin (Fig. 3A; Supplemental Fig. 7). This molecule
retains inhibitory activity against Btk (IC50app 5 0.5 nM)
as well as the ability to form a covalent bond with Btk
(Supplemental Fig. 8) and has demonstrated selectivity
against the structurally related kinase epidermal growth
factor receptor (IC50app . 25 nM), and upstream Src-family
Fig. 1. Chemical structure of CC-292 (N-(3-(5-fluoro-2-(4-(2-methoxye-
thoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide).

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Evans et al.
Fig. 2. CC-292 demonstrates concentration-dependent silencing of Btk activity and prolonged duration of action after a-IgM stimulation of the B cell
receptor in Ramos cells. (A) Ramos cells were treated with increasing concentrations of CC-292 (0.3–3000 nM) and then stimulated with 5 mg/ml of the
BCR ligand a-IgM. Btk autophosphorylation as well as Btk substrate phosphorylation (P-Y1217-PLCg2) and downstream activation of extracellular
signal-regulated kinase (Erk) were assayed by immunoblot. Quantitation of immunoblot demonstrated that CC-292 inhibits Btk autophosphorylation
with EC50 = 8 nM (n = 4) (Supplemental Fig. 4). (B) Ramos cells were treated with compound for 1 hour. Cells were then resuspended in compound-free
media and stimulated with 5 mg/ml a-IgM at 0, 4, 6, or 8 hours after compound removal. Btk substrate phosphorylation was measured by immunoblot.
Btk remains inhibited up to 8 hours after treatment with the covalent modifier CC-292, whereas Btk activity returns quickly after treatment with the
reversible inhibitor, dasatinib. Representative immunoblot of n = 3 experiments.
kinases including Syk (IC50app . 1000 nM) and Lyn (IC50app
.
B cells ex vivo. In naïve human B cells, the kinase activity of
3500 nM). Moreover, the specificity of the Btk target Btk was inhibited 42% at 10 nM, a concentration that
occupancy ELISA derives from the use of a detection mono- produced 37% Btk occupancy (Fig. 4A). It is noteworthy that
clonal antibody that selectively recognizes Btk immobilized kinase inhibition and occupancy also reflected efficacy in
on the streptavidin substrate by the covalent probe and, B cell functional assays such as B cell proliferation (EC50
5
therefore, this assay measures only Btk bound to the covalent 3 nM; Fig. 4B) and activation as determined by inhibition of
probe. By building a standard curve with known amounts of upregulation of the activation marker, CD69, in response to
recombinant Btk protein bound to CNX-500, the amount of stimulation by anti-IgM (Supplemental Table 5). These data
Btk in any sample can be precisely quantitated. Used in demonstrate a strong quantitative relationship among CC-292
a competition assay, this probe detected free, uninhibited Btk concentration, extent of Btk enzyme inhibition, and level of
and was excluded from interaction with Btk previously Btk occupancy. Therefore, measurement of Btk occupancy
bonded by CC-292 (Fig. 3B). Results from this analysis can can serve as a robust surrogate measurement of Btk kinase
be reported in absolute values, such as picograms of free Btk inhibition that correlates with inhibition of BCR signaling
per microgram of total protein or in relative terms by and its functional consequences.
normalization to control samples not exposed to inhibitor. In
As described above, once covalently bound by CC-292, an
Ramos cells exposed to a range of CC-292 concentrations, the individual Btk protein is permanently silenced. Therefore, the
amount of Btk captured by the probe was compared with return of activity must depend on new Btk protein synthesis.
untreated samples and the extent of Btk bonded was Determination of Btk protein resynthesis rates in mice in vivo
demonstrated to be proportional to CC-292 drug concentra- was enabled by maximally inhibiting Btk with a single dose of
tion (Supplemental Fig. 9). It is noteworthy that the degree of CC-292 and then monitoring the return of Btk in spleen
Btk covalently bonded by CC-292, herein referred to as Btk lysates over time with the covalent probe. Mouse spleens were
occupancy, correlated with inhibition of Btk kinase activity. collected at several time points after a single oral dose of
Extensive analysis has revealed that the EC50 of Btk 50 mg/kg CC-292, a dose level projected to achieve complete
occupancy from a CC-292 dose-response in Ramos cells Btk engagement, and assayed with the covalent probe to track
(EC50 5 6 nM) correlated directly with the cellular EC50 of emergence of new Btk protein. New Btk protein was detected
Btk kinase inhibition with CC-292 (EC50 5 8 nM) (Supple- at low levels 8 hours after compound administration, and
mental Figs. 5 and 9). Furthermore, the concentration at achieved 43% of predose Btk protein levels at 24 hours and
which CC-292 inhibited 90% of Btk activity in Ramos cells 71% of predose levels 48 hours after drug administration
was 35 nM while the concentration of CC-292 required for (Fig. 5). It is noteworthy that PK analysis of mouse plasma
90% occupancy of Btk was 39 nM, supporting a direct from this experiment indicated circulating CC-292 was absent
stoichiometric correlation between target occupancy and in five of six animals by the 8-hour time point (unpublished
inhibition of Btk activity. This correlative relationship was data). Presently, the potential contribution of active metabo-
also demonstrated in freshly isolated human primary naïve lites of CC-292 cannot be excluded. These data provide precise

Btk Inhibition with the Covalent Inhibitor CC-292
223
Fig. 3. Covalent probe CNX-500 allows direct assessment of Btk occupancy in vitro and in vivo. (A) Covalent probe CNX-500. (B) Covalent probe CNX-
500 detects free, uninhibited Btk in lysates derived from tissue culture, animal tissues, or clinical samples. Samples treated with CC-292 are lysed and
then incubated with 1 mM CNX-500. Uninhibited Btk in the lysate is captured by CNX-500 and quantitated by streptavidin (SA)-coated ELISA plate.
Normalization to untreated control sample allows determination of the percentage of Btk occupancy.
determination of the extent and duration of covalent in- dose were assayed for Btk occupancy. Occupancy in spleen
hibition of Btk protein in mice.
lysates tracked closely with inhibition of the clinical signs of
Relationship of Btk Occupancy and Efficacy of disease: 34% occupancy at 3 mg/kg at 2 hours correlated
CC-292 in the Collagen-Induced Arthritis Model of with 50% inhibition of disease, Btk occupancy of 84% was
Arthritis. The collagen-induced arthritis (CIA) model has detected 2 hours after dosing with 10 (85% inhibition of
been shown previously to respond to both B cell modulating disease) or 30 mg/kg (97% occupancy, 95% inhibition of
therapies as well as direct Btk inhibition (Pine et al., 2007; disease) of CC-292. Consistent with Btk resynthesis experi-
Honigberg et al., 2010; Chang et al., 2011; Di Paolo et al., ments described earlier, only 19% Btk occupancy remained 24
2011; Liu et al., 2011b). Oral efficacy of CC-292 in an hours after the 3-mg dose, whereas sustained occupancy of
established CIA model in mice was measured. Dose- .40% at 24 hours was achieved with dose levels of 10 and 30
dependent inhibition of the clinical signs of inflammatory mg/kg. This analysis demonstrated that once-a-day dosing at
disease was observed during the in-life portion of the model, the higher doses resulted in continuous CC-292–Btk engage-
including reduction in joint and paw swelling and visible ment at levels greater than 40% and that this was sufficient
redness of the affected paws. Reduction of clinical signs of for .85% inhibition of disease with therapeutic dosing of CC-
disease was measured at 95, 85, and 50% for 30, 10, and 3 mg/kg, 292 (Fig. 6B). Morphologic and histopathologic analysis of six
respectively (Fig. 6A). Moreover, all three dose levels of CC-292 affected joints (four paws, two knees) demonstrated a dose-
prevented the loss in body weight typically associated with dependent protection from joint damage, including pannus
severity of disease observed in this model (Supplemental formation, cartilage degradation, and bone erosion. The
Fig. 10). It is noteworthy that CC-292 also demonstrated disease-modifying activity of CC-292 correlated with both
significant effects on the generation of inflammatory chemo- Btk occupancy and the pronounced inhibition of the clinical
kines and cytokines in this model, including KC (mouse inflammation characteristic of arthritis in this model (Fig.
equivalent of interleukin-8), interleukin-6, and tumor necrosis 6C). This correlation between Btk occupancy and inhibition
factor a (Supplemental Table 6). The precise mechanism of disease strongly suggests that selective inhibition of Btk
for this protective effect is currently under investigation but provided the protective effect of CC-292 activity in this
suggests direct or indirect modulation of effector cell function collagen-induced arthritis model.
and may be independent of the role of Btk in B cells. To
Human Clinical PK-PD Relationship with CC-292.
demonstrate the relationship between inhibition of inflam- CC-292 demonstrated covalent bonding, prolonged, selective
matory activity and direct engagement of CC-292 with Btk, inhibition of Btk in vitro, and efficacy in preclinical models in
spleens collected either 2 or 24 hours after the last CC-292 vivo. In addition, there was a strong correlation between the

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Evans et al.
Fig. 4. Btk occupancy with CC-292 in human primary B
cells correlates with inhibition of Btk signaling and in-
hibition of B cell proliferation. (A) Human naïve B cells were
isolated by negative selection, treated with the indicated
concentrations of CC-292 for 1 hour, stimulated with 5 mg/ml
a-IgM for 10 minutes, and lysed. Lysates were split in two for
immunoblot and occupancy analyses. Representative immu-
noblot from three independent experiments shown and
occupancy from three experiments plotted as mean 6 S.D.
(B) Human naïve B cells (CD19+, IgD+) were purified from
whole blood by negative selection with MACS purification.
Purified naïve B cells were stimulated with 5 mg/ml a-IgM for
72 hours. 3H-Thymidine was added to the media for the final
16 hours and 3H incorporation measured. Experiments were
done in triplicate. The mean 6 S.D. were plotted and the
EC50 calculated from n = 3 separate experiments. CC-292
inhibits B cell proliferation EC50 = 3 nM.
concentration of CC-292 required for Btk occupancy, paired traditional pharmacokinetic analysis of plasma
inhibition of BCR signaling, and consequent functions drug levels with Btk occupancy analysis in a B cell–enriched
such as B cell proliferation. As part of a larger clinical fraction from freshly isolated human blood to determine the
study with CC-292 in healthy adult human volunteers, we PK-PD relationship of CC-292 following single oral admin-
istration in humans. After initial dose escalation, 2 mg/kg
CC-292 was found to be optimal for analysis of this PK-PD
relationship. Six healthy adult subjects were adminis-
tered a single oral dose of CC-292 (2.0 mg/kg) and
sequential blood samples were isolated over time to
determine the relationship between the plasma concen-
tration of CC-292 and Btk occupancy in an enriched B cell
population.
There was rapid absorption of 2 mg/kg CC-292 in all
subjects, with peak plasma concentrations achieved within
30–120 minutes after dose administration and a mean
measured maximum plasma concentration of 542 ng/ml
(Cmax) was attained. Plasma concentrations declined to near
or below the lower limit of detection (0.1 ng/ml) within 24
hours post-dose with a median terminal elimination half-life
of 1.9 hours. Plasma concentrations of CC-292 at 48 hours
post-dose were below the lower limit of quantification in all
subjects (Fig. 7).
Analysis of Btk occupancy was determined at each time
point using the covalent probe ELISA. The absolute value of
free Btk in lysates of enriched B cells isolated before CC-292
administration averaged 465 6 67 pg free Btk/mg total protein
(mean 6 S.E.M.) for the six subjects administered CC-292 at
a dose of 2.0 mg/kg. Within 4 hours after dose administration,
five of six subjects had greater than 98% Btk occupancy with
Fig. 5. Btk occupancy and Btk protein resynthesis can be detected in mice in
vivo. Mice were treated orally once with 50 mg/kg CC-292 to inhibit all Btk
protein. At 4, 8, 16, 24, 48, 72, and 96 hours after treatment, spleens and
serum were harvested (n = 6 mice/time point). Spleens were homogenized
and assayed on the covalent probe ELISA for Btk target site occupancy and
compound concentration in serum was measured. Btk protein recovered to
50% predose levels 24–48 hours after CC-292 administration. Pharmacoki-
netic analysis of compound concentration in plasma at each time point
demonstrated compound was undetectable in five of six mice by the 8-hour
time point. Plotting the mean plasma level of CC-292 in mice versus the mean
percentage Btk occupancy (both 6 S.E.M.) demonstrates CC-292 action on Btk
protein persists for .12 hours after circulating compound has disappeared.

Btk Inhibition with the Covalent Inhibitor CC-292
225
Fig. 7. Pharmacokinetics and pharmacodynamics are uncoupled with
covalent inhibitor CC-292 in healthy human volunteers. PK analysis of six
subjects dosed with CC-292 demonstrated rapid absorption and mean
peak plasma levels (Cmax) of 542 ng/ml CC-292. PD analysis of Btk target
occupancy in the same six subjects displayed maximal occupancy at 4
hours (average cohort occupancy .97%) with sustained occupancy
through 24 hours and recovery of Btk protein levels toward 50% 48–72
hours after CC-292 administration. Mean 6 S.E.M. for plasma level and
percentage Btk occupancy depicted.
plasma concentrations of CC-292 were low or approaching the
limit of quantification. Thus, as in the experiment in mice
described above, detection of free Btk over the ensuing 24–96-
hour period postadministration of CC-292 was a reflection of
the resynthesis rate of Btk by existing B cells plus the
addition of any new B cells circulating in the periphery. Free
Btk protein levels recovered toward 75% predose values
within 96 hours with a resynthesis half-life of 48–72 hours
and an average resynthesis rate of 3.0 pg Btk/mg protein
per hour. These data demonstrate an uncoupling of PD
from PK and, similar to data generated in preclinical
models, reveal that CC-292 action on Btk in human clinical
trials was sustained for several hours after circulating
drug levels declined to undetectable levels (Fig. 7). The
generation of metabolites was not evaluated in this clinical
study and, therefore, the potential contribution of active
metabolites of CC-292 cannot be excluded. These trans-
lational studies demonstrate the capability to precisely
determine the concentration of CC-292 required for complete
inhibition of Btk in human subjects to inform subsequent drug
development.
Fig. 6. CC-292 is efficacious in an established collagen-induced arthritis
model. (A) Male DBA/1 mice were injected with bovine type II collagen in
Freund’s complete adjuvant on day 0 and day 21. On study days 25–27, onset
of arthritis occurred, and mice were randomized into treatment groups (10/
treatment group, 4/group for normal). Randomization into each group was
done after swelling was obviously established in at least one paw. Treatment
was initiated after enrollment. Treatment continued daily (QD at 24-hour
intervals) through arthritis day 14. CC-292 demonstrated dose-dependent
inhibition of disease symptoms as measured by the daily clinical arthritis
score plotted over 14 days of treatment. CC-292 administered at either 10 or
30 mg/kg was similar to dexamethasone (Dex) control in inhibiting disease
symptoms. P , 0.05 for 10 and 30 mg/kg CC-292 (analysis of variance). In-life
study conducted at Bolder Biopath, plotted as mean 6 S.E.M. (B) Spleens
were collected 2 and 24 hours following the final administration of CC-292
(n = 6 mice per group). Lysates were made and run on the covalent probe
ELISA to determine Btk occupancy. CC-292 at 3 mg/kg occupied 34% of the
Btk at 2 hours; 10 and 30 mg/kg occupied 84 and 95% Btk at 2 hours,
respectively. Occupancy shown for each individual mouse with mean of each
group indicated by bar. (C) Histopathologic analysis of the six joints in
Discussion
Recent analysis suggests that attrition rates in clinical
development are highest in Phase 2 clinical trials, where lack
affected CIA mice demonstrated decreased cartilage and bone damage as of PD activity and/or efficacy leads to failure rates of
well as inflammation and pannus in CC-292–treated animals. CC-292’s
approximately a third of drug candidates (Kola and Landis,
2004). To address this, the development of novel strategies
enabling earlier insights into PD activity is an intense area of
translational research.
inhibition of histopathologic signs of disease was significant (P , 0.05) and
dose-dependent, such that 10 and 30 mg/kg had effects similar to that of the
positive control dexamethasone (inhibition 82% with dexamethasone, 87%
with 10 mg/kg CC-292, 96% with 30 mg/kg CC-292).
As a critical component of BCR signaling, Btk has emerged
the sixth subject achieving 84% occupancy (Supplemental as an important drug target for the treatment of B cell
Table 7). Complete or near-complete Btk occupancy was disorders. CC-292 is a novel, potent, selective covalent
sustained in all six subjects for between 8 and 24 hours post- inhibitor of Btk that has advanced rapidly into human clinical
administration of CC-292, and this occurred at a time when studies using an innovative translational medicine strategy to

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Evans et al.
assess PD activity at each step of drug discovery into early recover to 50% of baseline protein levels. This may reflect
clinical development. a more rapid resynthesis rate of Btk in mice. However, this
Covalent inhibition of Btk has allowed creation of an assay may also reflect the fact that in mice, Btk return was
to measure on-target activity, facilitating both preclinical measured in spleen lysates whereas Btk resynthesis in
research and clinical development by enabling a quantitative humans was measured in isolated peripheral B cells. We
understanding of the relationship among dose, exposure, cannot rule out the possibility that either different cellular
target engagement, functional consequence, and efficacy. The populations or B cell subsets resident in the spleen may
translational method described here quantitatively measures synthesize Btk protein more quickly resulting in an increased
CC-292–Btk engagement by direct assay with an ELISA that resynthesis rate in mice. In both mouse and human studies
sensitively detects the presence of drug-free Btk protein. In however, CC-292–Btk engagement persisted well after circu-
vitro, the concentrations of CC-292 required for inhibition of lating drug had disappeared. In this way, covalent inhibitors
Btk activity and Btk occupancy were virtually equivalent allow a departure from the confines of traditional drug design;
(EC50s 5 8 versus 6 nM, respectively) demonstrating a near a pharmacokinetic profile that includes a long circulating
stoichiometric relationship. In freshly isolated primary half-life to ensure 24-hour target coverage is not necessary.
human B lymphocytes, there was a close correlation between Instead, a covalent inhibitor must achieve concentrations
the concentration of CC-292 required to inhibit Btk signaling sufficient to engage all available molecular target only for
and B cell proliferation, and to achieve Btk occupancy, a short period and then the resynthesis rate of the target itself
suggesting a quantitative relationship among inhibition of dictates the duration of drug action. As demonstrated here,
Btk kinase activity, target occupancy, and functional assays the level and length of drug action can be empirically
in vitro. This direct correlation supports the use of Btk occupancy determined by target occupancy measurements to rationally
as a surrogate marker for inhibition of Btk activity. It is adjust dosing.
noteworthy that this relationship between the inhibition
achieved and the extent of CC-292-target engagement mea- was assessed in this first-in-human trial. This represents
sured by the covalent probe was maintained in vivo. a marked acceleration of clinical PD evaluation, typically not
The action of CC-292 on Btk was confirmed and PD
CC-292 demonstrated therapeutic efficacy in a mouse CIA available until Phase 2 clinical testing. The rapid identification
model, with 85 and 95% inhibition of disease observed at of doses providing Btk target engagement provides an ad-
doses of 10 mg/kg per day and 30 mg/kg per day, respectively. vantage in the design of subsequent human clinical trials and
The reduced disease severity seen with CC-292 treatment supports Phase 2 dose selection to incorporate safety,
recapitulates xid mice, which harbor an inactivating mutation tolerability, and on-target activity. Future clinical trials in
in the Btk gene and have a reduced incidence and severity of patients will appropriately assess the relationship of complete
CIA disease induction (Mangla et al., 2004), as well as or partial Btk inhibition to therapeutic outcomes. However,
previous studies using pharmacological inhibition of Btk to direct quantification of Btk engagement in these trials will
reduce disease activity in autoimmune models (Honigberg reduce uncertainty about the dose required for target
et al., 2010; Chang et al., 2011; Di Paolo et al., 2011; Liu et al., inhibition and enable selection of the optimal pharmaco-
2011a). In addition to a full phenotypic response, once daily, logical dose and dosing schedule. By providing this informa-
oral dosing of 10 mg/kg CC-292 resulted in 84% Btk inhibition tion to clinicians early in clinical development, subtherapeutic
verified by Btk occupancy analysis assayed 2 hours after dose drug administration in initial patient cohorts may be avoided
administration. Btk occupancy measured 24 hours after a 10 and provide time efficiencies in clinical testing to more rapidly
mg/kg dose suggested drug-free Btk protein was resynthe- impact patient health. CC-292 has advanced to Phase 1b
sized to approximately 60% of predose levels. That sustained clinical testing in relapsed, refractory B cell malignancies
protection from the clinical signs of arthritis were provided at with initial patient cohorts dosed at levels identified in this
this dose suggests 100% Btk inhibition may not be required study. By refining the number of cohorts required for dose
throughout a 24-hour time period and that intermittent finding, it is anticipated that this trial will quickly provide
inhibition of Btk may be sufficient for modulation of autoim- recommended Phase 2 doses allowing rapid advancement
mune disease in a clinical setting (Liu et al., 2011a). To guide into Phase 2 testing. Furthermore, CC-292 is the only Btk
this determination, future studies with the translational Btk inhibitor to our knowledge with suitable safety profile to support
occupancy assay will enable establishment of the optimal dose, its advance into clinical trials in patients with autoimmune
dose frequency, and degree of Btk inhibition required for full disease, and human clinical development of this molecule is
disease modification in animal models. Given the inhibitory occurring in parallel for both oncology and autoimmune
activity on other members of the Tec family of kinases we indications.
cannot rule out the possibility of their inhibition playing a role
in the efficacy seen in vivo.
Given a long protein half-life, highly restricted expression
pattern, and the presence of a poorly conserved cysteine in the
As expected, CC-292 PK was dissociated from its PD in vivo, ATP binding pocket, Btk represents an excellent target for
a feature confirmed by analysis of Btk occupancy. CC-292 selective covalent inhibition. The long protein half-life of Btk,
remained active on Btk for a prolonged duration in vivo after shown previously to be .12 hours in human primary B cells
plasma drug levels had declined to undetectable levels. (Saffran et al., 1994), provides for prolonged duration of drug
Recovery from CC-292 treatment occurred slowly in both action that extends well beyond the time frame of systemic
mice and humans as new Btk protein was made. In mice, covalent drug exposure. The uncommon cysteine targeted by
where Btk occupancy was measured in spleen homogenates, CC-292 confers the opportunity for selective inhibition of Btk,
Btk protein was resynthesized to 50% of predose levels 24–48 as only 10 of the approximately 500 human kinases share
hours after a single dose. This differs from the resynthesis of placement of the cysteine in a homologous location within the
Btk protein seen in humans which required 48–72 hours to ATP binding pocket. However, it is noteworthy that, even in

Btk Inhibition with the Covalent Inhibitor CC-292
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Acknowledgments
The authors thank Robert Tjin for help in overseeing the cellular
selectivity experiments.
Authorship Contributions
Participated in research design: Evans, Aslanian, Sheets, Witow-
ski, Lounsbury, Chaturvedi, Nacht, Freed, Westlin.
Conducted experiments: Evans, Aslanian, Sheets, Karp, Labenski,
Witowski, Chaturvedi, Dubroviskiy.
Contributed new reagents or analytic tools: Tester, Mazdiyasni,
Zhu, Petter, Singh.
Performed data analysis: Evans, Aslanian, Sheets, Witowski,
Chaturvedi, Nacht, Freed, Dubroviskiy, Westlin.
Wrote or contributed to the writing of the manuscript: Evans, Singh,
Westlin.
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