1521-0103/355/2/199–205$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.115.224899
J Pharmacol Exp Ther 355:199–205, November 2015
Temporal Pharmacokinetic/Pharmacodynamic Interaction
between Human CD3« Antigen–Targeted Monoclonal Antibody
Otelixizumab and CD3« Binding and Expression in Human
Peripheral Blood Mononuclear Cell Static Culture s
Kevin R. Page, Enrica Mezzalana, Alexander J. MacDonald, Stefano Zamuner,
Giuseppe De Nicolao, and Andre van Maurik
GlaxoSmithKline, Stevenage, United Kingdom (K.R.P., A.J.M., S.Z., A.vM.); University of Pavia, Pavia PV, Italy (E.M., G.D.N.)
Received April 22, 2015; accepted August 17, 2015
Introduction
Monoclonal antibodies (mAb) directed against human lymphocyte antigen CD3« have been investigated clinically in
a number of autoimmune diseases such as type 1 diabetes
(Herold et al., 2002; Keymeulen et al., 2005, Hagopian et al.,
2013), ulcerative colitis (Sandborn et al., 2010), multiple
sclerosis (Weinshenker et al., 1991), and Crohn’s disease
(van der Woude et al., 2010). One such anti-CD3 mAb,
muromonab-CD3 (tradename Orthoclone OKT3), is approved
for the treatment of steroid-resistant acute rejection of
allogeneic renal, heart, and liver transplants. Despite encouraging results from clinical investigations in autoimmune
diseases, especially in new-onset type 1 diabetes, there has
been little progress in understanding the relationship between anti-CD3 mAb target engagement and its putative
mechanisms of action. Owing to this lack of pharmacological understanding, dosage regimens appear to have been
dx.doi.org/10.1124/jpet.115.224899.
s This article has supplemental material available at jpet.aspetjournals.org.
a shorter apparent half-life at low concentration. Correspondingly,
a rapid, otelixizumab concentration–, and time-dependent reduction in CD3/TCR expression was observed. These combined
observations were consistent with the phenomenon known as
target-mediated drug disposition (TMDD). A mechanistic, mathematical pharmacokinetic/pharmacodynamic (PK/PD) model was
then used to characterize the free otelixizumab-CD3 expressiontime relationship. CD3/TCR modulation induced by otelixizumab
was found to be relatively fast compared with the re-expression
rate of CD3/TCR complexes following otelixizumab removal from
supernatants. In summary, the CD3/TCR receptor has been shown
to have a major role in determining otelixizumab disposition. A
mechanistic PK/PD model successfully captured the PK and PD in
vitro data, confirming TMDD by otelixizumab.
determined largely empirically, or when animal disease or in
vitro models were used for extrapolation, the ability to adjust
for animal-human or in vitro–in vivo differences was limited.
The following pharmacology has been observed clinically with
anti-CD3 mAbs: 1) Binding leads to apparent partial T-cell
activation, resulting in release of proinflammatory cytokines,
and a temporary disruption of normal T-cell trafficking
(Chatenoud et al., 1982; Waldron Smith et al., 1997; Smith
et al., 1998; -Lynch et al., 2012) and 2) internalization of the
resultant mAb/CD3/T-cell receptor (TCR) complex causes loss
of the complex from the T-cell surface membrane and degradation and elimination of the antibody (Reinherz et al., 1982;
Press et al., 1988; Liu et al., 2000; Monjas et al., 2004),
a phenomenon known as target-mediated drug disposition
(TMDD) (Levy, 1994). Whereas some quantification of these
clinical observations has been possible (Wiczling et al., 2010),
it has been difficult separating and quantifying these individual pharmacological components at a mechanistic level owing
to the close temporal relationship between them.
In an attempt to overcome the limitations of studying antiCD3 mAb pharmacology in vivo, a static in vitro culture
ABBREVIATIONS: ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; KD, binding affinity; mAb, monoclonal
antibody; MESF, mean equivalent surface fluorescence; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PD,
pharmacodynamic; PerCP-Cy5.5, peridinin-chlorophyll-cyanine 5.5; PK, pharmacokinetic; SSC, side scatter; TCR, T-cell receptor; TMDD, targetmediated drug disposition.
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ABSTRACT
Otelixizumab is a monoclonal antibody (mAb) directed to human
CD3«, a protein forming part of the CD3/T-cell receptor (TCR)
complex on T lymphocytes. This study investigated the temporal
interaction between varying concentrations of otelixizumab, binding to human CD3 antigen, and expression of CD3/TCR complexes
on lymphocytes in vitro, free from the confounding influence of
changing lymphocyte frequencies observed in vivo. A static in vitro
culture system was established in which primary human peripheral
blood mononuclear cells (PBMCs) were incubated over an
extended time course with titrated concentrations of otelixizumab.
At each time point, free, bound, and total CD3/TCR expression on
both CD41 and CD81 T cells and the amount of free otelixizumab
antibody in the supernatant were measured. The pharmacokinetics
of free otelixizumab in the culture supernatants was saturable, with
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Materials and Methods
Experimental Design. PBMCs isolated from healthy volunteer
bloods were cultured in the presence of a titration of otelixizumab over
an extended time course (Fig. 1). Flow cytometry was used to measure
free and bound CD3« and TCR at the indicated time points.
Simultaneously, an enzyme-linked immunosorbent assay (ELISA)
was used to measure the amount of free antibody in the supernatant of
these cultures. In addition, some cells were washed to remove
exogenous antibody after 2 days and measurement of CD3/TCR reexpression examined at the indicated time points postwash.
Antibodies. Otelixizumab (also known as ChAglyCD3) is an
aglycosylated nonmitogenic recombinant antibody (human g1) directed against CD3« chain, a protein forming part of the CD3/T-cell
receptor complex (TCR) on T lymphocytes (Routledge et al., 1991; Bolt
et al., 1993). Otelixizumab reference standard at 12.1 mg/ml was used
for all experiments.
Isolation of PBMC. Healthy volunteer blood were obtained from
the on-site blood donation unit and appropriate informed consent was
obtained from all donors. Blood was withdrawn into bags containing
1 IU/ml sodium heparin (MP Biomedicals, Santa Ana, CA) as anticoagulant. Heparinized blood was kept at room temperature and processed
within 2 hours of withdrawal. PBMC separation was performed by
density gradient centrifugation using 50-ml Leucosep tubes (Greiner
Bio-One, Kremsmünster, Upper Austria) containing Ficoll-Paque.
Blood (25 ml per tube) was centrifuged for 20 minutes at 800g with
the brake off. PBMCs recovered from the interface were washed twice
with Dulbecco’s phosphate-buffered saline (PBS; Gibco/Thermo
Fisher Scientific, Sunnyvale, CA) and finally resuspended in complete
medium for counting using the guava easyCyte flow cytometer (EMD
Millipore, Billerica, MA). Complete medium comprised RPMI 1640
(Gibco) 1 1 mM glutamine (Gibco), 100 IU/ml penicillin/100 mg/ml
Fig. 1. Experimental design. Schematic of cell culture system.
streptomycin (Gibco) and 5% heat-inactivated human AB serum
(Gemini Bio-Products, West Sacramento, CA).
Time Course of PBMC Treatment with Otelixizumab. Freshly
isolated PBMC from two healthy donors (1 106/ml) were cultured in
24-well plates (Costar/Corning, Corning, NY) in RPMI 1 5% AB serum
with titrated concentrations of otelixizumab (0, 1, 3, 10, 30, 100, 300, 1000,
3000, and 10,000 ng/ml), 16 wells for each concentration. At time points
0.25, 0.5, 1, 2, 4, 8, 16 hours and 1, 2, 3, 4, 6, 8, 10, 12, and 14 days the
cell suspension was removed from one well for each donor transferred
to fluorescence-activated cell sorting (FACS) tubes and centrifuged
5 minutes at 1350 rpm. The supernatant was removed and stored for
subsequent ELISA at –20°C and the cells were resuspended in 4 ml FACS
buffer and split between two tubes for FACS staining. For CD3/TCR reexpression PBMC from each donor were cultured at 1 106/ml (10 ml)
in RPMI 1 5% AB serum with the same titrated concentrations of
otelixizumab. After 48 hours cells were removed from wells and transferred to 50-ml tubes and centrifuged at 1350 rpm for 5 minutes. Cells
were resuspended in 50 ml PBS and centrifuged again. After resuspension in fresh RPMI 1 5% AB serum, counting cells were adjusted to 106/ml
and 1 ml/well was added to 24-well plates, eight replicate wells for each
population. One well of each replicate was harvested and the cells stained
for FACS as for other cells at 8 hours, and 1, 2, 4, 6, 8, 10, and 12 days.
Quantification of CD3/TCR Modulation by Flow Cytometry.
At the indicated time points the cells were removed, split between two
tubes, and washed twice with FACS buffer (PBS 1 1% FCS 1 sodium
azide 0.1%). After blocking of Fc receptors for 5 minutes with TruStain
FcX (BioLegend, Sacramento, CA) cells were surface-stained with
the following murine antibodies: Tube A: CD4-PerCP-Cy5.5 (clone
RPA-T4), CD8-APC (clone RPA-T8), CD3-FITC (clone SK7), and antiHuman IgG Fc-PE (clone HP6017); and Tube B: CD4-PerCP-Cy5.5
(clone RPA-T4), CD8-APC (clone RPA-T8), and TCRab-PE (clone
IP26) (all BioLegend) for 30 minutes at room temperature protected
from light. After washing twice with 3 ml FACS buffer the cells were
resuspended in 300 ml Cytofix (BD Biosciences, San Jose, CA).
Lymphocytes were gated on forward scatter versus side scatter and
5000 CD4 events were acquired. The mean fluorescence intensity data
for phycoerythrin and fluorescein isothiocyanate fluorescence for
gated CD4 and CD8 cells were converted to mean equivalent surface
fluorescence (MESF) values using Quantum Simply Cellular beads
(Bangs Laboratories, Fishers, IN) per the manufacturer’s instructions.
Otelixizumab ELISA. ELISA plates (96-well; Greiner Bio-One)
were coated with capture antibody (anti-otelixizumab mAb clone
T54.1C9.A9) at 5 mg/ml in 50 mM bicarbonate buffer overnight at 4°C,
washed 5 with PBS 1 0.05%Tween 20, and blocked with Superblock
(Thermo Scientific). Assay samples and otelixizumab standards (prepared in culture supernatant) were added to the prepared plates for 2
hours. After washing, 1 mg/ml detection antibody (biotinylated antiotelixizumab mAb clone 6H12.B12.A3.G10) was added for 1 hour
followed by addition of a streptavidin–horseradish peroxidase conjugate
(Invitrogen, Carlsbad, CA) at 1/10,000 dilution for 1 hour. A chromogenic substrate tetramethylbenzidine (Sigma-Aldrich, St. Louis, MO)
was used and color allowed to develop for 10 minutes before reaction
was stopped with 0.5 M sulfuric acid. A Molecular Devices SpectroMax
plate reader provided readings at 450 nm, and otelixizumab concentrations were calculated from a four-parameter fit standard curve using
analytical software (Softmax Pro; Molecular Devices, Sunnyvale, CA).
The lower limit of quantification for this assay was 2.5 ng/ml.
In Vitro PK/PD Model. A TMDD model (Mager and Jusko, 2001)
accounting for otelixizumab binding on both CD41 and CD81 lymphocytes was proposed to describe in vitro experimental data, including the extension of two binding sites (Gibiansky and Gibiansky,
2010). The proposed model is illustrated in Fig. 2.
Otelixizumab (here denoted by Cp) is administered in the central
compartment, where it can bind to free CD3/TCR receptor complex on
both CD41 and CD81 lymphocytes (FR4 and FR8, respectively) to
form drug-receptor complexes (DR4 and DR8, respectively).
The model is described through the following differential equations
expressed as molar concentrations:
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system was established to investigate the time course of the
interaction between anti-CD3 mAb, CD3, and CD3/TCR
complex, free from the confounding influence of changes in
number of circulating T lymphocytes, observed as transient
peripheral lymphopenia in vivo. A mechanistic nonlinear
mixed effect pharmacokinetic/pharmacodynamic (PK/PD)
model was then used to analyze and describe the complex
pharmacological interactions in this static in vitro system. The
in vitro pharmacokinetics was investigated in which primary
human peripheral blood mononuclear cells (PBMCs) were
used; CD31 T lymphocytes in PBMCs is typically in the range
of 50–70%. PBMCs were cultured over an extended time
course with titrated concentrations of otelixizumab. At each
time point, free, bound, and total CD3/TCR expression on both
CD41 and CD81 T cells and the amount of free antibody in
the supernatant were measured so that PK could be related to
PD effects. To investigate the kinetics of CD3/TCR reexpression, cells were washed on day 2 to remove exogenous
otelixizumab and thereby allow the rate of CD3/TCR complex
re-expression to be monitored.
PK/PD Model of Anti-CD3« mAb Activity In Vitro
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Fig. 2. Mechanism-based PK/PD model for otelixizumab disposition and effect on CD3/TCR expression. Otelixizumab (here denoted by Cp) is
administered in the central compartment, where it can bind to free receptors, CD3/TCR, on both CD4+ and CD8+ lymphocytes (FR4 and FR8,
respectively) to form drug-receptor complexes (DR4 and DR8, respectively). The binding processes are governed by the second-order rate constants kon4
and kon8. Free receptors on both CD4+ and CD8+ lymphocytes are synthesized at a zero-order rate (ksyn4, ksyn8) and degraded at a first-order rate
(kdeg4, kdeg8). Complexes, DR4 and DR8, may either dissociate (koff4, koff8) or be internalized and degraded, (kint4, kint8).
dCp=dt 5 InðtÞ=V-kon4 × Cp × FR4 1 koff 4 × DR4-kon8 × Cp× FR8
1 koff 8 × DR8
(1)
(2)
dDR4=dt 5 1 kon4 × Cp×FR4 koff 4 × DR4 kint4 × DR4
(3)
dFR8=dt 5 -kon8 × Cp × FR8 1 koff 8 × DR8 1 ksyn8 kdeg8 × FR8
(4)
dDR8=dt 5 1 kon8 × Cp × FR8 1 koff 8 × DR8 kint8 × DR8
(5)
with initial conditions given by Cp(0) 5 0, FR4(0) 5 ksyn4/kdeg4 5
BAS4, DR4(0) 5 0, FR8(0) 5 ksyn8/kdeg8 5 BAS8, DR8(0) 5 0.
Assuming equal otelixizumab binding to CD41 and CD81 lymphocytes, the model can be simplified by imposing equal association
and dissociation rate constants (i.e., kon4 5 kon8 5 kon and koff4 5
koff8 5 koff).
Otelixizumab molar concentration was converted into the observed
concentration (ng/ml) using its molecular weight (150 kDa) and
appropriate scaling (apparent volume of distribution V). Receptor
dynamic measurements were expressed in MESF units assumed to be
proportional to receptors molar concentrations. Otelixizumab bound
to CD3 blocked the binding of all available anti-TCR and anti-CD3
detection antibodies that were tested. As a consequence, free and total
receptors could be detected only where otelixizumab was not bound to
CD3. This implied that total receptor measurements (MTR4 and
MTR8) were proportional to free receptors molar concentration (and
not to the sum of free and bound receptor concentrations):
MFR4 5 g FR × FR4 MFR8 5 g FR × FR8
(6)
MDR4 5 g DR × DR4 MDR8 5 g DR × DR8
(7)
MTR4 5 g TR × FR4 MTR8 5 gTR × FR8
(8)
where g FR, gDR, and g TR are conversion factors between variables and
the measured quantities MFR4, MFR8, MDR4, MDR8, MTR4, and
MTR8.
The PK/PD model was fitted to the individual PK and PD (free,
engaged, and total CD3/TCR receptors) data simultaneously using
nonlinear mixed-effect statistical analysis. Model development included differences for the two donors in baseline values for CD41 and
CD81 T lymphocytes and different otelixizumab affinities for binding,
respectively, on CD41 and CD81 T lymphocytes. Additive, proportional, and combined residual error models were tested for each
variable. The data fitting software used was NONMEM version 7.2
(Beal et al., 2009) embedded in a wrapper program Perl-Speaks
Nonmem (PsN version 3.6.2) (Lindbom et al., 2004) in a Windows 7
environment. The first-order conditional estimation with interaction
Results
Pharmacokinetics of Free Otelixizumab. To describe
pharmacological interactions in a static in vitro culture
system, the pharmacokinetics of otelixizumab was investigated. Free otelixizumab concentration time-course profiles
are shown in Fig. 3 from two donors at different initial drug
concentrations over a 48-hour incubation period. At initial
drug concentrations higher than 100 ng/ml, otelixizumab
concentration levels were relatively static over 48 hours. In
contrast, there was an apparent initial concentrationdependent and time-dependent reduction in free otelixizumab
concentrations observed with both donors at initial concentrations lower than 100 ng/ml. At the lowest starting
concentrations (3 and 1 ng/ml), free otelixizumab concentrations were below the limit of quantification for both
donors. Some values were obtained by extrapolation of the
standard curve at early time points, but antibody was
undetectable at later time points. Collectively, these observations suggest target-mediated elimination of otelixizumab
in this system.
Pharmacodynamics of Free/Bound CD3 and Total
CD3 Expression. Next, the in vitro pharmacodynamic time
courses of free and bound CD3« together with total CD3«
expression on CD41 and CD81 T lymphocytes in the PBMC
culture following incubation with otelixizumab were determined. CD41 T lymphocytes pharmacodynamic time-course
profiles selected initial concentrations for donor 1 are shown in
Fig. 4. Free CD3« and total CD3« expression followed similar
time courses. There was a rapid decline in both free CD3« and
total CD3« expression relative to preincubation levels. In both
cases, the rate and extent of reduction appeared to increase
with higher initial concentrations, the nadirs occurring as
early as 0.25 hours following the start of incubation and being
maintained for the whole 48-hour incubation period at initial
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dFR4=dt 5 -kon4 × Cp × FR4 1 koff 4 × DR4 1 ksyn4 kdeg4 × FR4
(FOCEI) method was used throughout the analysis, and LSODA
algorithm was used to integrate differential equations (ADVAN13
subroutine with TOL 5 6). A nonparametric bootstrap method (n 5
1000) was used to study the uncertainty of all TMDD model parameter
estimates. From the bootstrap empirical posterior distribution, relative standard errors (RSE) were obtained for the parameters.
The criteria for model selection included: 1) improved fitting of the
diagnostic scatter plots (observations versus predictions, weighted
residual versus predictions or time, individual weighted residual
versus predictions or time), 2) convergence of the minimization, 3)
residual standard errors on all the estimated parameters, 4) significant decrease in the objective function value (OFV).
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concentrations $1000 ng/ml. Similar results were obtained
with both CD41 and CD81 cells and results from both donors
were comparable (data not shown).
Following washout of otelixizumab after 48 hours, there was
a gradual but steady increase in free CD3« and re-expression
of total CD3«. Based on visual inspection of the data, the rate
of re-expression of total CD3« appeared to be similar for all
initial concentrations and independent of the degree of reduction of CD3« expression induced by incubation with
otelixizumab. For low initial concentrations (,3 ng/ml) expression levels returned to preincubation levels by approximately day 6 following wash out of otelixizumab. In contrast,
at the highest initial concentration (10,000 ng/ml) expression
had recovered to only around 80% of preincubation levels by
the end of the experiment (day 14). Again, results were
consistent for both CD41 and CD81 cells and comparable
between donors (data not shown).
Characterization of the PK/PD–Time Relationship.
Mechanistic, mathematical, TMDD PK/PD models were fitted
simultaneously to the PK and PD (free, engaged, and total
CD3/TCR receptors) data. In terms of the model iteration
and adequacy, the most robust best fit model included
different baseline levels for CD3/TCR on CD41 and CD81
T lymphocytes (BAS4 and BAS8 respectively) for the two
donors. The final model did not include any between-subject
variability on model parameters, so that the resulting analysis
can be considered as a naïve-pooling approach. The binding
affinities for otelixizumab for CD3« on CD41 or CD81 lymphocytes were found to be similar and the model was
simplified by assuming equal affinity for both lymphocyte
subsets (i.e., kon4 5 kon8 5 kon and koff4 5 koff8 5 koff).
The final model included combined residual error model for
total CD3/TCR receptor measurements and free otelixizumab
concentration, and an additive residual error model was adopted
for free and engaged CD3/TCR receptors. All parameters were
estimated with reasonable precision (,40%). Goodness-of fit
diagnostic plots, including comparisons of observed and
predicted values, as well as residual analysis, suggested that
although the model tended to underpredict the lowest free
and total CD3/TCR observations, it provided a reasonable
approximation for the data (Supplemental Data). The proposed
model captured the PK/PD time-course profiles reasonably well
for both donors and all initial concentrations of otelixizumab
(Figs. 3 and 4).
The parameter estimates for the model are reported in
Table 1 together with relative standard errors (RSE) based on
1000 bootstrapped datasets. The binding constants were
estimated as kon 5 51.5/nM per day and koff 5 4.64 per day,
suggesting high affinity of otelixizumab to human CD3«.
Correspondingly, the derived equilibrium dissociation constant, KD (koff/kon) was 90 pM.
Estimated internalization rates (kint4 5 1.26/day and kint8 5
1.29/day) were five times higher than degradation rates (kdeg4 5
0.273/day and kdeg8 5 0.275/day).
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Fig. 3. In vitro pharmacokinetics of otelixizumab. Otelixizumab concentration versus time profiles after incubation with PBMCs from the two donors.
Different symbols are used for different initial concentrations. Solid lines represent predictions from final TMDD model. Dashed horizontal lines indicate
the lower limit of quantification (LLOQ). Values below the LLOQ occur in the lowest curves for both subjects.
PK/PD Model of Anti-CD3« mAb Activity In Vitro
203
Discussion
We established a static in vitro culture system to investigate
and characterize otelixizumab pharmacology using human
cells. We were able to study the dose range and time course
with sufficient resolution to observe and quantify the concentration and time-dependent PK behavior consistent with
target-mediated disposition of the antibody. A mechanistic,
mathematical model (TMDD model) used to describe this
phenomena was fitted to the PK/PD–time data. The model
adequately described all the data and was sufficient to explain
the nonlinear disposition of otelixizumab in this culture
system. The rate and degree of change of free, bound, and
total CD3« expression was found to be determined by the
concentration of otelixizumab. A moderate increase was
observed in total CD3/TCR receptor profiles at the end of the
experiment. Although data described in this paper does not
allow any conclusion regarding the cause of this modest
increase, it is possible that a small degree of cell death
(approximately 10% was observed during the 14-day culture)
is a contributing factor. This was, however, not considered
a relevant feature to implement in the final model as it was
judged to be relatively minor compared with the degree of
CD3/TCR decreases achieved rapidly following otelixizumab
engagement. Although the use of a constant synthesis rate for
CD3/TCR receptors does not account for possible changes in
cell culture compositions attributable to cell death, the proposed model captured the concentration-dependent kinetic
profiles for free, bound, and total CD3« reasonably well. At
saturating concentrations, the engagement of target CD3«
molecules was found to be rapid, leading to full occupancy of
CD3« within minutes. At lower concentrations, binding of
antibody gradually achieved equilibrium. The estimated
parameters appeared reasonable and consistent with an
antibody of this type. According to the model, the affinity
constant for otelixizumab binding to CD3« is KD 5 90 pM.
This finding is in line with observations made in a previous
study (Wiczling et al., 2010), in which the estimated concentration of otelixizumab producing 50% reduction in free CD3/
TCR sites was in the range of 14–16 ng/ml (5 93.8–107.2 pM).
However, Wiczling et al. estimated a nonlinear PK model
(Michaelis-Menten elimination) with a Michaelis-Menten
constant (Km 5 0.968 mg/ml) that is not in agreement with
our in vitro results. In fact, a Michaelis-Menten PK model can
be considered still an approximation of a full TMDD model in
which the nonlinear elimination Km is related to the binding
and internalization process (Km 5 (koff 1 kint)/kon) (Ma,
2012). In our specific case, since the estimated dissociation
constant is faster than the internalization rate, the net
result of the binding-internalization process is driven by the
affinity estimate (Km ∼ KD). Wiczling et al. suggested that
binding to CD3/TCR complexes did not affect otelixizumab
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Fig. 4. In vitro pharmacodynamics of otelixizumab. Free (A), engaged (B), and total (C) CD3/TCR receptor kinetic profiles on CD4+ cells from Donor 1
after incubation with otelixizumab for selected different initial concentrations. Open circles represent the available in vitro data and lines are
predictions from the final TMDD model.
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TABLE 1
Parameter estimates from the TMDD model
Parameter (Units)
kon (/nM/day)
koff (/day)
kint4 (/day)
kint8 (/day)
Donor 1 BAS4
Donor 1 BAS8
Donor 2 BAS4
Donor 2 BAS8
kdeg4 (/day)
(pM)
(pM)
(pM)
(pM)
kdeg8 (/day)
sAD (FR) (103 MESF)
sAD (DR) (103 MESF)
sAD (TR) (103 MESF)
sPR (TR)(-)
pharmacokinetics, and they considered the short terminal
half-life (t1/2 of approximately 0.5 days) to be related to the
absence of FcRn recycling. The data presented here suggest
that the process of CD3/TCR internalization is stimulated as
a consequence of antibody binding, with an estimated internalization rate of drug-receptor complexes significantly
faster than the free receptor degradation (kint 5 1.26–1.29
day21 . kdeg 5 0.27–0.28 day21), resulting in a mean
residence time of the free receptor of approximately five times
higher than the one of the drug-receptor complexes. This
phenomenon is a common mechanism when natural ligand
binds to its corresponding receptor and has been described for
antigen binding to the T-cell receptor (Huang et al., 1999), and
the B-cell receptor (Caballero et al., 2006), and for ligand
binding to the granulocyte-macrophage–colony-stimulating
factor receptor (Vainshtein et al., 2015). Following removal
of antibody from the culture, the rate of re-expression of CD3«
was observed to be slower than otelixizumab-induced downregulation. Model estimates for internalization and degradation rate constants for CD3/TCR receptors reflect these
findings. At saturating concentrations, complete modulation
was achieved within about 30 minutes, whereas full CD3/TCR
re-expression to pretreatment levels required about 8 days.
These findings are in agreement with data obtained from
mouse models using surrogate anti-CD3 antibody fragments
(Mehta et al., 2010). These observations are supportive of the
notion that CD3« reappearance requires new synthesis and
assembly of complexes, known to be a slow process that takes
days, as opposed to re-expression of transiently internalized
existing complexes, which has been described as a relatively
rapid process (minutes to hour) (Menné et al., 2002). It should
be noted that the exact fate of otelixizumab was not investigated in this study. Several mechanisms are possible,
with some studies demonstrating endocytosis of antibody
receptor complexes (Liu et al., 2000; Monjas et al., 2004;
Kuhn et al., 2011), whereas others suggest shedding of the
Value
RSE [%]
51.47
4.64
1.26
9.60
28.88
10.48
1.29
8.22
42.36
34.47
39.22
33.32
0.27
22.99
23.06
22.79
22.69
7.68
0.28
7.83
1.12
4853.40
5885.90
760.88
0.924
0.207
3.20
16.74
16.61
16.85
38.56
11.28
12
20.4
2.11
0.14
5.65
9.39
33.27
18.48
complex from the cell surface (Reinherz et al., 1982; Press
et al., 1988).
In contrast, despite considerable investigation, the degree,
duration of action of otelixizumab binding to CD3«, CD3
expression, and its relationship to unbound antibody has only
been partially elucidated from clinical data alone. For practical reasons, it has been difficult to study a sufficiently wide
dose range with an adequate blood sampling schedule following treatment of otelixizumab in patients to determine
whether the dynamics of antibody binding can be separated
from the dynamics of the observed transient lymphopenia.
Thus, attempts to quantify these relationships clinically have
been largely empirical and simple (Wiczling et al., 2010).
Whereas the published models describe the data adequately,
and may be useful for limited interpolation or extrapolation,
they lack the mechanistic detail to provide insight about
the underlying pharmacological behavior of otelixizumab.
Understanding the in vivo interaction of otelixizumab to the
CD3 target is challenging since observed data may not be
sufficient to estimate relevant system parameters. In this
case, the in vitro model can provide relevant information for
the receptor synthesis (ksyn), degradation/internalization
rate (kdeg and kint), and binding affinity (KD) that can help
to better inform any in vivo model in which other phenomena
(mAb distribution, disposition, and systemic clearance
attributable to nonspecific target binding, together with target
expression, lymphocytes trafficking, etc.) would increase the
complexity of the model structure, including identifiability issues
for relevant system parameters. In general, the development of
mechanistic models from in vitro experiments offers a valuable
approach to addressing the complexity of in vivo systems, for
example, by using the in vitro estimates as priors for the in vivo
models.
Preclinical investigation of these phenomena in vivo is
limited by the almost complete lack of crossreactivity of
otelixizumab with animal CD3 and lack of suitable animal
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
V (ml)
g FR (103 MESF/nM)
g DR (103 MESF/nM)
g TR (103 MESF/nM)
sAD (Cp)(ng/ml)
sPR (Cp) (-)
Description
Second-order rate constant for otelixizumab-CD3/TCR association
First-order rate constant for otelixizumab-CD3/TCR dissociation
First-order rate constant for internalization of otelixizumab-CD3/TCR
complexes on CD4+ cells
First-order rate constant for internalization of otelixizumab-CD3/TCR
complexes on CD8+ cells
CD3/TCR receptor complex baseline on CD4+ cells for Donor 1
CD3/TCR receptor complex baseline on CD8+ cells for Donor 1
CD3/TCR receptor complex baseline on CD4+ cells for Donor 2
CD3/TCR receptor complex baseline on CD8+ cells for Donor 2
First-order rate constant for degradation of CD3/TCR receptor
complex on CD4+ cells
First-order rate constant for degradation of CD3/TCR receptor
complex on CD8+ cells
Apparent volume of Distribution
Conversion factor from FR to MFR
Conversion factor from DR to MDR
Conversion factor from FR to MTR
Additive residual error model for free Otelixizumab concentrations
Proportional residual error model for free Otelixizumab
concentrations
Additive residual error model for free CD3/TCR receptors
Additive residual error model for engaged CD3/TCR receptors
Additive residual error model for total CD3/TCR receptors
Proportional residual error model for total CD3/TCR receptors
PK/PD Model of Anti-CD3« mAb Activity In Vitro
surrogate antibody. In conclusion, we have demonstrated using
an in vitro cell culture system and a mechanistic model–based
approach to data analysis, that we can successfully study and
quantify the complex antibody-receptor complex PK/PD interactions that were hypothesized for otelixizumab. This will
probably be applicable to other membrane-bound targets,
particularly for immune cell targets in which antibodies or
other protein therapeutics may cause altered cell trafficking.
The parameters estimated from such analysis may allow
investigators to compare and contrast other data, and the
models can be used to design dosage regimens and optimize
sample schedules for future clinical trials.
Authorship Contributions
References
Beal S, Sheiner LB, Boeckmann A, and Bauer RJ (2009) Nonmem User’s Guides
(1989-2009), Icon Development Solutions, Ellicott City, MD.
Bolt S, Routledge E, Lloyd I, Chatenoud L, Pope H, Gorman SD, Clark M,
and Waldmann H (1993) The generation of a humanized, non-mitogenic CD3
monoclonal antibody which retains in vitro immunosuppressive properties. Eur J
Immunol 23:403–411.
Caballero A, Katkere B, Wen X-Y, Drake L, Nashar TO, and Drake JR (2006)
Functional and structural requirements for the internalization of distinct BCRligand complexes. Eur J Immunol 36:3131–3145.
Chatenoud L, Baudrihaye MF, Kreis H, Goldstein G, Schindler J, and Bach JF (1982)
Human in vivo antigenic modulation induced by the anti-T cell OKT3 monoclonal
antibody. Eur J Immunol 12:979–982.
Gibiansky L and Gibiansky E (2010) Target-mediated drug disposition model for drugs
that bind to more than one target. J Pharmacokinet Pharmacodyn 37:323–346.
Hagopian W, Ferry RJ, Jr, Sherry N, Carlin D, Bonvini E, Johnson S, Stein KE,
Koenig S, Daifotis AG, and Herold KC, et al. Protégé Trial Investigators (2013)
Teplizumab preserves C-peptide in recent-onset type 1 diabetes: two-year results
from the randomized, placebo-controlled Protégé trial. Diabetes 62:3901–3908.
Herold KC, Hagopian W, Auger JA, Poumian-Ruiz E, Taylor L, Donaldson D,
Gitelman SE, Harlan DM, Xu D, and Zivin RA, et al. (2002) Anti-CD3 monoclonal
antibody in new-onset type 1 diabetes mellitus. N Engl J Med 346:1692–1698.
Huang J-F, Yang Y, Sepulveda H, Shi W, Hwang I, Peterson PA, Jackson MR, Sprent
J, and Cai Z (1999) TCR-Mediated internalization of peptide-MHC complexes acquired by T cells. Science 286:952–954.
Keymeulen B, Vandemeulebroucke E, Ziegler AG, Mathieu C, Kaufman L, Hale G,
Gorus F, Goldman M, Walter M, and Candon S, et al. (2005) Insulin needs after
CD3-antibody therapy in new-onset type 1 diabetes. N Engl J Med 352:2598–2608.
Kuhn C, You S, Valette F, Hale G, van Endert P, Bach JF, Waldmann H, Chatenoud
L (2011) Human CD3 transgenic mice: preclinical testing of antibodies promoting
immune tolerance. Sci Transl Med 3:68ra10.
Levy G (1994) Pharmacologic target-mediated drug disposition. Clin Pharmacol Ther
56:248–252.
Lindbom L, Ribbing J, and Jonsson EN (2004) Perl-speaks-NONMEM (PsN)–a Perl
module for NONMEM related programming. Comput Methods Programs Biomed
75:85–94.
Liu H, Rhodes M, Wiest DL, and Vignali DA (2000) On the dynamics of TCR:CD3
complex cell surface expression and downmodulation. Immunity 13:665–675.
Ma P (2012) Theoretical considerations of target-mediated drug disposition models:
simplifications and approximations. Pharm Res 29:866–882.
Mager DE and Jusko WJ (2001) General pharmacokinetic model for drugs exhibiting
target-mediated drug disposition. J Pharmacokinet Pharmacodyn 28:507–532.
Mehta DS, Christmas RA, Waldmann H, and Rosenzweig M (2010) Partial and
transient modulation of the CD3-T-cell receptor complex, elicited by low-dose
regimens of monoclonal anti-CD3, is sufficient to induce disease remission in nonobese diabetic mice. Immunology 130:103–113.
Menné C, Møller Sørensen T, Siersma V, von Essen M, Ødum N, and Geisler C (2002)
Endo- and exocytic rate constants for spontaneous and protein kinase C-activated
T cell receptor cycling. Eur J Immunol 32:616–626.
Monjas A, Alcover A, and Alarcón B (2004) Engaged and bystander T cell receptors
are down-modulated by different endocytotic pathways. J Biol Chem 279:
55376–55384.
Press OW, Hansen JA, Farr A, and Martin PJ (1988) Endocytosis and degradation
of murine anti-human CD3 monoclonal antibodies by normal and malignant
T-lymphocytes. Cancer Res 48:2249–2257.
Reinherz EL, Meuer S, Fitzgerald KA, Hussey RE, Levine H, and Schlossman SF
(1982) Antigen recognition by human T lymphocytes is linked to surface expression
of the T3 molecular complex. Cell 30:735–743.
Routledge EG, Lloyd I, Gorman SD, Clark M, and Waldmann H (1991) A humanized
monovalent CD3 antibody which can activate homologous complement. Eur J
Immunol 21:2717–2725.
Sandborn WJ, Colombel JF, Frankel M, Hommes D, Lowder JN, Mayer L, Plevy S,
Stokkers P, Travis S, and Van Assche G, et al. (2010) Anti-CD3 antibody
visilizumab is not effective in patients with intravenous corticosteroid-refractory
ulcerative colitis. Gut 59:1485–1492.
Smith JA, Tso JY, Clark MR, Cole MS, and Bluestone JA (1997) Nonmitogenic antiCD3 monoclonal antibodies deliver a partial T cell receptor signal and induce
clonal anergy. J Exp Med 185:1413–1422.
Smith JA, Tang Q, and Bluestone JA (1998) Partial TCR signals delivered by FcRnonbinding anti-CD3 monoclonal antibodies differentially regulate individual Th
subsets. J Immunol 160:4841–4849.
Vainshtein I, Roskos LK, Cheng J, Sleeman MA, Wang B, and Liang M (2015)
Quantitative measurement of the target-mediated internalization kinetics of biopharmaceuticals. Pharm Res 32:286–299.
van der Woude CJ, Stokkers P, van Bodegraven AA, Van Assche G, Hebzda Z,
Paradowski L, D’Haens G, Ghosh S, Feagan B, and Rutgeerts P, et al.; Initiative on
Crohn’s and Colitis, The Netherlands (2010) Phase I, double-blind, randomized,
placebo-controlled, dose-escalation study of NI-0401 (a fully human anti-CD3
monoclonal antibody) in patients with moderate to severe active Crohn’s disease.
Inflamm Bowel Dis 16:1708–1716.
Waldron-Lynch F, Henegariu O, Deng S, Preston-Hurlburt P, Tooley J, Flavell R,
and Herold KC (2012) Teplizumab induces human gut-tropic regulatory cells in
humanized mice and patients. Sci Transl Med 4:118ra12.
Weinshenker BG, Bass B, Karlik S, Ebers GC, and Rice GP (1991) An open trial of
OKT3 in patients with multiple sclerosis. Neurology 41:1047–1052.
Wiczling P, Rosenzweig M, Vaickus L, and Jusko WJ (2010) Pharmacokinetics
and pharmacodynamics of a chimeric/humanized anti-CD3 monoclonal antibody,
otelixizumab (TRX4), in subjects with psoriasis and with type 1 diabetes mellitus. J
Clin Pharmacol 50:494–506.
Address correspondence to: Dr. Kevin Page, Experimental Medicine Unit,
ImmunoInflammation TA, Medicines Research Centre, Gunnelswood Road,
Stevenage, Hertfordshire, UK SG1 2NY. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Participated in research design: van Maurik, MacDonald, Page.
Conducted experiments: Page.
Performed data analysis: Mezzalana, De Nicolao.
Wrote or contributed to the writing of the manuscript: van Maurik,
MacDonald, Page, Mezzalana, Zamuner, De Nicolao.
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