Journal of Pharmaceutical Sciences 111 (2022) 2630−2638

Contents lists available at ScienceDirect

Journal of Pharmaceutical Sciences

jo urn a l h om ep ag e : w ww.jp harms ci.o rg

Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Preclinical Characterization of ASP2713, a Novel Igb and Fcg RIIB

Cross-Linking Antibody, for Prediction of Human
Pharmacokinetics and Clinically Effective Dose
Kentaro Konishia,1,*, Koji Nakamurab,1, Yuichi Hanadac,1, Yukihiro Kitanagab,
Satoshi Kubob, Fumitaka Kinugasaa, Daisuke Yamajukua, Masashi Maedaa,
Nobuchika Yamamotoa, Tsuyoshi Minematsuc, Masato Ohbuchia, Yuya Kondod,
Takayuki Sumidad
a
Applied Research & Operations, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan
b
Discovery Accelerator, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan
c
Immuno-Oncology, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan
d
Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan

A R T I C L E I N F O A B S T R A C T

Article history: Previously, we reported the fundamental pharmacological characteristics of a novel Igb and Fc gamma recep-
Received 10 February 2022 tor IIB cross-linking antibody, ASP2713, as a new treatment option for systemic lupus erythematosus. The
Revised 7 June 2022 aims of the present study were to investigate ASP2713’s characteristics with regard to pharmacological
Accepted 7 June 2022
effect, pharmacokinetics (PK), and receptor occupancy, and to predict its human PK and clinically effective
Available online 11 June 2022
dose. The relationship between the concentration and receptor occupancy of ASP2713 for Igb of B cell recep-
tors was examined using whole blood B cells. Calculated EC50 values in cynomolgus monkeys and healthy
Keywords:
volunteers were 0.35 and 0.058 mg/mL, respectively. Dose-dependent inhibition of anti-tetanus toxoid (TTx)
Antibody drug
Pharmacokinetics
antibody production, PK, and receptor occupancy of ASP2713 in TTx-sensitized cynomolgus monkeys sug-
Pharmacodynamics gested a minimally effective dose of 1 mg/kg by single intravenous (IV) administration. Scaling-up of monkey
Pharmacokinetic/pharmacodynamic (PK/PD) PK parameters to humans by allometric scaling predicted a clinically effective dose of 0.4 mg/kg IV adminis-
Correlation tration at 4-week intervals to maintain a trough concentration in humans which achieved the same receptor
Mechanistic modeling occupancy expected at the effective dose in monkeys. This study aids in understanding the characteristics of
Interspecies (dose) scaling ASP2713 and can be used as a basis for clinical dose setting.
Immunotherapy © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.
Receptor

Introduction

Abbreviations: APC, Allophycocyanin; AUC, Area under the curve; AUCtau, Area under
the concentration-time curve for a dosing interval; BCR, B cell receptor; CL, Linear com-
ponent of elimination clearance from the central compartment; Cmax, Maximum con-
centration; Ctrough, Concentration at trough; EC20, Concentration when 20% of
maximum efficacy is achieved; EC50, Concentration when 50% of maximum efficacy is
achieved; EC80, Concentration when 80% of maximum efficacy is achieved; Fcg R, Fc
Gamma receptor; IV, Intravenous; Km, Michaelis-Menten constant; MFI, Median fluo-
rescent intensity; PE, Phycoerythrin; PK, Pharmacokinetics; Q, Inter-compartment
clearance; SAV, Streptavidin; SLE, Systemic lupus erythematosus; TTx, Tetanus toxoid;
Vmax, Maximum non-linear component of elimination clearance velocity; V1, Distribu-
tion volume of the central compartment; V2, Distribution volume of the peripheral
compartment.
* Corresponding author at: Non-Clinical Biomedical Science, Applied Research &
Operations, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki, 305-8585,
Japan.
E-mail address: kentaro.konishi@astellas.com (K. Konishi).
1
These authors have contributed equally to this work.
Systemic lupus erythematosus (SLE) is an autoimmune disease
that causes damage to various organs.1-3 Symptoms typically appear
between the ages of 15 and 45, and about 90% of patients are
women.1 Overall prevalence in USA ranges from 42 to 300 per
100,000, and ethnic differences in SLE prevalence are known.1
Although the specific cause of SLE is still unclear, onset is associated
with multiple factors, including immune dysregulation, genetic fac-
tors, epigenetic factors, environmental factors, and sex hormones.2,4,5
In clinical practice, although a broad range of medications are used,
including glucocorticoids, immunosuppressive agents, antimalarial
agents, and nonsteroidal anti-inflammatory drugs, B cell-targeting
biologics such as rituximab and belimumab have not established a
mainstay position in the treatment of SLE because of the potential
issues described in the introduction of our previous report.6

https://doi.org/10.1016/j.xphs.2022.06.006
0022-3549/© 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.
 K. Konishi et al. / Journal of Pharmaceutical Sciences 111 (2022) 2630−2638
To provide a new treatment option for SLE and to overcome
the potential issues of rituximab and belimumab, we previously
reported the fundamental pharmacological characteristics of
ASP2713, a novel Igb and Fc gamma receptor (Fcg R) IIB cross-
linking antibody.6 ASP2713 is an anti-Igb antibody with insertion
of S239D, H268D, and L328W substitutions into the Fc domain to
increase affinity for Fcg RIIB and to crosslink Igb with Fcg RIIB.
Cross-linking B cell receptor (BCR) and Fcg RIIB by antigen-IgG
immune complexes is considered to counteract excessive BCR-
mediated activation signals via induction of phosphorylation of
the immunoreceptor tyrosine-based inhibitory motif domain of
Fcg RIIB, followed by suppression of autoreactive B cell survival
and activation. The experimental results in our previous report
showed that ASP2713 has the potential to suppresses BCR signal-
dependent B cell activation by cross-linking Igb of BCR and
Fcg RIIB without exhibiting B cell-depleting properties. These
characteristics suggest that ASP2713 may be an attractive candi-
date for the treatment of SLE. To date, however, species differen-
ces in the relationship between concentration and the receptor
occupancy of ASP2713 have not been evaluated, and its in vivo
pharmacological effect has not been investigated.
Here, we investigated the compound’s characteristics with
regard to pharmacological effect, pharmacokinetics (PK), and
receptor occupancy of ASP2713. We then applied the acquired
data and analysis results to a mathematical model to predict
human PK and the clinically effective dose of this agent. It is
known that there are linear and non-linear elimination phases in
the concentration-time profile of antibody due to target-mediated
drug disposition. Therefore, in the present report, monkey PK
was analyzed by a two-compartment model with linear and non-
linear elimination described by the Michaelis-Menten equation
and scaled up to human PK by allometric scaling. Translation of
drug candidates from preclinical to clinical use clearly requires
the prediction of clinical dosage and regimen. This prediction not
only supports the developmental feasibility of the dosage and
regimen, but also provides a justification for the design of clinical
studies.
Materials and Methods
Human Samples and Ethics
Human whole blood samples were obtained from healthy adults
aged over 20 years who understood the purpose of this research and
participated voluntarily. The study was approved by Astellas
Research Ethics Committee. In addition, human whole blood samples
were obtained from patients under treatment for SLE at the Univer-
sity of Tsukuba. The study was approved by the Human Ethics Review
Committees of the Tsukuba University Hospital and Astellas Research
Ethics Committee.
Animal Welfare
The study was approved by the Institutional Animal Care and Use
Committee of Astellas Pharma Inc. and Shin Nippon Biomedical Labo-
ratories, Ltd., and was performed in accordance with the animal wel-
fare bylaws of Shin Nippon Biomedical Laboratories, Ltd., Drug Safety
Research Laboratories (Kagoshima, Japan). Both are accredited by
AAALAC International.
In Vitro Receptor Occupancy Assay
Whole blood samples treated with heparin from 3 healthy cyno-
molgus monkeys (Shin Nippon Biomedical Laboratories, Ltd.) and 3
healthy volunteers were used. The assay to determine the receptor
2631
occupancy of ASP2713 for Igb of BCR was analyzed by BD FACSArray
(BD Biosciences, NJ, USA) for samples from healthy cynomolgus mon-
keys and by BD FACSVerse (BD Biosciences) for samples from healthy
volunteers. B cells were defined as CD20+ cells and the free form
of Igb of BCR was stained with the biotin-labeled ASP2713 F(ab’)2
fragment.
Processing of Whole Blood Samples from Healthy Cynomolgus Monkeys
Fifty microliters of ASP2713 dilutions were added to 50 mL
of whole blood (final ASP2713 concentration: 0, 0.001, 0.01, 0.1,
1, 10, 100, and 1000 mg/mL) and the mixtures were incubated
for 3 hours at 37°C and 5% CO2. After 30 minutes incubation on
ice, 50 mL of biotin-labeled ASP2713 F(ab’)2 fragment solution
(10 mg/mL) was added into the blood and incubated for 30
minutes on ice. The cells were then centrifuged for 3 minutes
at 380 £ g, and supernatant was removed. The cell precipitate
was resuspended in 1.2 mL of FACS lysing buffer (BD Bioscien-
ces) and incubated for 10 minutes at room temperature. Cells
were washed with staining buffer (BD Biosciences) and sus-
pended in 50 mL of secondary antibody cocktail containing 5-
fold diluted allophycocyanin (APC) mouse anti-human CD20 and
1000-fold diluted streptavidin (SAV)-phycoerythrin (PE) anti-
bodies (BD Biosciences). The cell suspension was incubated for
30 minutes on ice, washed with staining buffer and analyzed on
a FACSArray.
Processing of Whole Blood Samples from Healthy Volunteers
Sixty microliters of ASP2713 dilutions were added to 60 mL of
whole blood (final ASP2713 concentration: 0, 0.0001, 0.001, 0.01,
0.03, 0.1, 1, and 10 mg/mL) and the mixtures were incubated for
3 hours at 37°C and 5% CO2. After incubation, 110 mL of each mix-
ture was obtained and placed on ice. Fifty-five microliters of biotin-
labeled ASP2713 F(ab’)2 fragment solution (1 mg/mL) or stain buffer
was added and incubated for 30 minutes on ice. Cells were then
centrifuged for 3 minutes at 400 £ g, and the supernatant was
removed. The cell precipitate was resuspended with 1.2 mL of FACS
lysing buffer and incubated for 12 minutes at room temperature
under light-resistant conditions. After incubation, cells were
washed twice with staining buffer and suspended with 90 mL of
antibody cocktail containing 5-fold diluted APC mouse anti-
human CD20 and 1000-fold diluted SAV-PE antibodies. The cell
suspension was incubated for 30 minutes on ice under light-resis-
tant conditions, washed twice, resuspended with staining buffer
and analyzed on a FACSVerse.
Calculation of Receptor Occupancy
Using the flow cytometry standard file obtained from each recep-
tor occupancy assay, Median Fluorescent Intensity (MFI) of PE on B
cells was calculated using FlowJoTM version 7.6 (Tree Star Inc., OR,
USA). Using the obtained MFI, receptor occupancy (%) was calculated
by the following equation.
Receptor occupancy of ASP 2713 for Ig bð%Þ
¼ ð1  ½MFI with ASP 2713  MFI background =½MFI without ASP 2713  MFI background Þ
 100
Calculation of EC20, EC50, and EC80 Values
Receptor occupancy (%) of ASP2713 for Igb of BCR was plotted
against the Log 10-transformed ASP2713 concentration and fitted
using nonlinear regression with the following equations by GraphPad
2632 K. Konishi et al. / Journal of Pharmaceutical Sciences 111 (2022) 2630−2638
Prism 7 (GraphPad Software, CA, USA) to calculate EC20, EC50, and
EC80 values.
Top  Bottom
Y ¼ Bottom þ
 five-parameter logistic regression model.
1 þ 10ððLogEC50  XÞ  HillSlopeÞ
Log 100F F
LogEC50 ¼ LogECF 
HillSlope
where X represents ASP2713 concentration (mg/mL), and Y repre-
sents receptor occupancy (%). Bottom and top were set to be 0 and
100, and F was 20 or 80, respectively. EC20, EC50, and EC80 represent
ASP2713 concentration when 20%, 50%, and 80% of the maximum
efficacy are achieved, respectively.
In Vivo Study in Cynomolgus Monkeys
In SLE patients, it was reported that B cells exhibit aberrant signal
transduction events and increased antigen-receptor-mediated phos-
phorylation.7 To mimic antibody production caused by B cell activa-
tion, cynomolgus monkeys were administrated tetanus toxoid (TTx)
and used to investigate the pharmacological effect of ASP2713. Male
cynomolgus monkeys aged 3 to 5 years (2.75 to 4.62 kg at initiation
of acclimation) were immunized with TTx antigen (Denka Seiken,
Tokyo, Japan) on Day 0 as described in a previous report.8 ASP2713
was administered once intravenously on Day 7 at the low dose levels
of 0.01, 0.03, 0.1, 0.3, and 1 mg/kg at a dosing rate of 2 mL/min and
volume of 1 mL/kg to 4 male cynomolgus monkeys at each dose and
at the high dose levels of 1, 3, and 10 mg/kg at a dosing rate of
2 mL/min and volume of 1 mL/kg to 4 or 5 male cynomolgus monkeys
at each dose. Anti-TTx IgG titer in serum was measured on Days 7
(before ASP2713 dosing), 8, 10, 14, 17, 21, 28, and 35 as described in
our previous report.6 The area under the curve (AUC) of anti-TTx IgG
titer was calculated from Day 7 to Day 35. When considering the effi-
cacy of ASP2713 in TTx-sensitized cynomolgus monkeys, we used the
index to determine whether the AUC increase in anti-TTx antibody
due to TTx administration to cynomolgus monkeys could be signifi-
cantly suppressed. Statistical comparison with the control group was
done using Dunnett’s multiple comparison test. Sampling points for
determination of ASP2713 concentration and anti-ASP2713 antibody
in serum were 1 hour, and 1, 3, 7, 10, 14, 21, and 28 days after dosing.
At the high dose levels only, competitive binding of biotinylated
ASP2713 F(ab’)2 in blood was measured on Days 7 (1 hour after
ASP2713 doing), 8, 10, 14, 17, 21, 28, and 35. In addition, ASP2713
was administered once intravenously at dose levels of 1 and
10 mg/kg at a dosing rate of 2 mL/min and volume of 1 mL/kg to 6
male healthy cynomolgus monkeys aged 3 to 4 years (2.86 to 3.98 kg
at initiation of acclimation) to determine ASP2713 concentration in
serum. Sampling points for determination of ASP2713 concentration
and anti-ASP2713 antibody in serum were 1 hour, and 1, 7, 14, 21,
28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 days after dosing.
PK Measurements
ASP2713 concentration was determined using the Gyrolab xP
workstation (Gyros Protein Technologies AB, Uppsala, Sweden). In
brief, biotin-labeled human Igb (Astellas Pharma Inc. Japan) and
Alexa-labeled anti-human IgG (Astellas Pharma Inc.) were used as
capture reagent and detection regent, respectively, to determine
ASP2713 in TTx-sensitized cynomolgus monkeys. The concentration
versus fluorescence relationship was determined through regression
according to a four-parameter logistic regression model. For determi-
nation of ASP2713 in healthy cynomolgus monkeys, biotin-labeled
primary anti-idiotype antibody (Shin Nippon Biomedical Laborato-
ries, Ltd.) and Alexa-labeled secondary anti-idiotype antibody (Shin
Nippon Biomedical Laboratories, Ltd.) were used as capture reagent
and detection regent, respectively. The concentration versus fluores-
cence relationship was determined through regression according to a
Anti-ASP2713 antibody was detected using a bridging assay. In
brief, diluted serum samples were mixed with biotin-labeled
ASP2713 (Astellas Pharma Inc.) and ruthenium-labeled ASP2713
(Astellas Pharma Inc.) for determination of anti-ASP2713 antibody
in TTx-sensitized cynomolgus monkeys. A signal ratio of post- to
pre-dose greater than 1.3 was defined as anti-ASP2713 antibody
positive. For determination of anti-ASP2713 antibody in healthy
cynomolgus monkeys, biotin-labeled ASP2713 (Shin Nippon Bio-
medical Laboratories, Ltd.) and ruthenium-labeled ASP2713 (Shin
Nippon Biomedical Laboratories, Ltd.) were mixed with diluted
serum samples. The mixtures were added to casein-coated wells.
The wells were shaken and washed several times. Diluted Read
buffer (Meso Scale Discovery Inc., MD, USA) was added and lumines-
cence intensity was measured using a SECTOR Imager (Meso Scale
Discovery Inc.). Using inhouse-validated criteria, anti-ASP2713 anti-
body was defined as positive or negative.
Competitive Binding Assay
Fifty microliters of biotinylated ASP2713 F(ab’)2 solution (20 mg/
mL) was added to 50 mL of blood sample drawn from the femoral
vein of cynomolgus monkeys and treated with heparin. For the sam-
ple before dosing only, 50 mL of cell staining buffer (BioLegend, CA,
USA) was added to the 50 mL blood sample as a background sample.
The sample was incubated for 30 minutes on ice under light-resistant
conditions and centrifuged for 3 minutes at 450 £ g. The supernatant
was removed and 1 mL of Pharm Lyse (BD Biosciences) was added.
The sample was incubated for 10 minutes at room temperature under
light-resistant conditions and centrifuged for 5 minutes at 200 £ g.
The supernatant was removed, 1 mL of cell staining buffer was added
and the mixture was centrifuged for 3 minutes at 750 £ g. Thereafter
the supernatant was removed, 50 mL of antibody mixture (1000-fold
diluted PE streptavidin solution [BD Biosciences], and 10-fold diluted
APC-CD20, [BioLegend]) was added, and the sample was incubated
for 30 minutes on ice under light-resistant conditions. After adding
cell staining buffer several times, centrifuging for 3 minutes at
750 £ g and removing the supernatant, the samples were analyzed
by FACSCalibur (BD Biosciences) and CellQuest Pro version 6.0 (BD
Biosciences). Receptor occupancy (%) was calculated from the geo-
metric mean of MFI of PE in CD20+ cells using the following equa-
tions.
!
MFIFðab0 Þ2þ
Receptor occupancy ð%Þ ¼ 1   100
MFIFðab0 Þ2
MFIFðab0 Þ2þ ¼ ðMFI value of after dosingÞ
 ðMFI value of background sampleÞ
MFIFðab0 Þ2 ¼ ðMean MFI value of before dosingÞ
 ðMFI value of background sampleÞ
PK Model
PK parameters in cynomolgus monkeys were estimated by fitting
a two-compartment model with linear and non-linear elimination
described by the Michaelis-Menten equation to the serum concentra-
tion in TTx-sensitized and healthy cynomolgus monkeys using NON-
MEM 7.3.0 (ICON Development Solutions, MD, USA) with a first order
 K. Konishi et al. / Journal of Pharmaceutical Sciences 111 (2022) 2630−2638 2633

conditional estimation method. Residual variabilities were assumed receptor occupancy for Igb of BCR on whole blood B cells between cyn-
to be in accordance with the proportional model. The results of fitting omolgus monkeys and humans; and Vmax in humans was assumed to
were confirmed by goodness-of-fit plots using R version 3.6.3.9 The be the same as Vmax in cynomolgus monkeys. Microsoft Excel 2016
equations used for model fitting are described as follows. To analyze (Microsoft Corporation, WA, USA) was used for calculations.
under the condition in which anti-ASP2713 antibody does not
appear, serum concentrations after those time points at which anti- Prediction of Clinical Efficacious Dose
ASP2713 antibody was positive were excluded from the analysis.
0 1
The trough concentration required for clinical use was predicted
@
Vmax A from the results in TTx-sensitized cynomolgus monkeys, assuming
that the same receptor occupancy of ASP2713 for Igb of BCR in
X1
dX1 CL Km þ V1 Q
¼   X1   X1   X1
dt V1 V1 V1 humans at trough is required as that in cynomolgus monkeys to
Q achieve an equivalent pharmacological effect. The clinically effective
þ  X2 ð1Þ dose was predicted from simulated human PK profiles for keeping
V2
the required trough concentration. Microsoft Excel 2016 and R ver-
dX2 Q Q sion 3.6.3 was used to calculate the required trough concentration,
¼  X1   X2 ð2Þ
dt V1 V2 simulate human PK profiles, and produce the related illustration.

where X1 and X2 represent the amount of ASP2713 in the central

compartment and peripheral compartment, respectively. CL, V1, Results
Vmax, Km, Q, and V2 represent the linear component of elimination
clearance of ASP2713 from the central compartment, distribution In Vitro Receptor Occupancy of ASP2713 for Igb of BCR on Whole Blood B
volume of the central compartment, maximum non-linear compo- Cells in Monkeys and Humans
nent of elimination clearance velocity, Michaelis-Menten constant,
inter-compartment clearance, and distribution volume of the periph- ASP2713 bound to Igb of BCR on whole blood B cells in both
eral compartment, respectively. healthy cynomolgus monkeys (Fig. 1a) and healthy volunteers
(Fig. 1b) in a concentration-dependent manner. In the concentration-
response analysis, EC20, EC50, and EC80 values of ASP2713 were 0.068,
Allometric Scaling 0.35, and 1.8 mg/mL in healthy cynomolgus monkeys and 0.018,
0.058, and 0.19 mg/mL in healthy volunteers, respectively (Table 1).
To predict the human PK profile of ASP2713, PK parameters in Although ASP2713 is an Igb and Fcg RIIB cross-linking antibody, we
humans (CL, V1, Vmax, Km, Q, and V2) were extrapolated from esti- focused on in vitro receptor occupancy only for Igb of BCR from the
mated PK parameters in cynomolgus monkeys by an allometric scal- point of view of the pharmacological analysis, because the binding
ing method described as follows. affinity of ASP2713 for Igb is substantially higher than that for
  Fcg RIIB.6
BWhuman b
CLhuman ¼ CLmonkey 
BWmonkey
Pharmacological Effect, PK, and Receptor Occupancy in Cynomolgus
 b0 Monkeys
BWhuman
Vhuman ¼ Vmonkey 
BWmonkey
In TTx-sensitized cynomolgus monkeys at the low dose levels of
EC50;human 0.01, 0.03, 0.1, 0.3, and 1 mg/kg, mean anti-TTx IgG titer was gradu-
Km;human ¼ Km;monkey  ally increased from Day 10 to Day 35 (Fig. 2a). Mean AUC from Day 7
EC50;monkey
to Day 35 for anti-TTx IgG titer decreased with an increase in dose
where b and b’ are 0.75 and 1, respectively, corresponding to the scaling level of ASP2713 on single intravenous (IV) administration, and a sta-
exponent for the clearance (CL and Q) and distribution volume10; tistically significant decrease in mean AUC from Day 7 to Day 35 for
BWmonkey and BWhuman are 5 kg and 70 kg, respectively11; Km,human anti-TTx IgG titer was noted at 1 mg/kg compared with the control
was calculated from Km,monkey corrected by EC50 differences in ASP2713 group (Fig. 2b). In Fig. 2c, a dose-dependent concentration-time

Figure 1. Concentration-response curve of ASP2713 receptor occupancy for Igb of BCR on whole blood B cells. The percent receptor occupancy value was calculated using Median
Fluorescent Intensity on B cells from each assay in healthy cynomolgus monkeys (a) and healthy volunteers (b). Data represent the mean § SE (n=3).
2634 K. Konishi et al. / Journal of Pharmaceutical Sciences 111 (2022) 2630−2638

Table 1
EC20, EC50, and EC80 Values of ASP2713 for Receptor Occupancy for Igb of BCR on Whole Blood B Cells in Cynomolgus Monkeys and Healthy Volunteers

Species EC20 (mg/mL) EC50 (mg/mL) EC80 (mg/mL)

Cynomolgus monkeys 0.068 (0.045 − 0.10) 0.35 (0.28 − 0.45) 1.8 (1.3 − 2.8)
Healthy volunteers 0.018 (0.014 − 0.021) 0.058 (0.051 − 0.065) 0.19 (0.15 − 0.24)
EC20, EC50, and EC80 were calculated using a fitted curve which incorporated all data of receptor occupancies of ASP2713 for Igb from 3 cynomolgus monkeys and 3 healthy volun-
teers. Hill slope in cynomolgus monkeys and humans are 0.84 and 1.171, respectively. Values in parentheses represent 95% confidence intervals.
EC20: ASP2713 concentration when 20% of maximum efficacy is achieved, EC50: ASP2713 concentration when 50% of maximum efficacy is achieved, EC80: ASP2713 concentration
when 80% of maximum efficacy is achieved.

profile is observed, and the decrease in ASP2713 concentration in
serum tended to be rapid at lower doses, indicating nonlinear PK at
the dose range from 0.01 to 1 mg/kg. On the other hand, in TTx-sensi-
tized cynomolgus monkeys at the high dose levels of 1, 3, and
10 mg/kg, mean anti-TTx IgG titer was gradually increased from Day
10 or Day 14 to Day 35 (Fig. 2d). No statistically significant change in
mean AUC from Day 7 to Day 35 for anti-TTx IgG titer was noted at
any dose compared with the control group. However, mean AUC
from Day 7 to Day 35 values for anti-TTx IgG titer at all doses tended
to be lower than those in the control group. The mean AUC from Day
7 to Day 35 value reached nearly plateau at 1 mg/kg and above
(Fig. 2e). Considering the results at both low and high dose levels, we
concluded that 1 mg/kg was the minimum effective dose in TTx-sen-
sitized cynomolgus monkeys. In Fig. 2f, a dose-dependent concentra-
tion-time profile is observed, indicating linear PK at the dose range
from 1 to 10 mg/kg. On analysis of competitive binding of

Figure 2. Inhibition of anti-tetanus toxoid (TTx) antibody production, PK, and receptor occupancy of ASP2713 in TTx-sensitized and healthy cynomolgus monkeys. (a) Anti-TTx IgG
titer-time profiles in TTx-sensitized cynomolgus monkeys at 0 (open circle, n=4), 0.01 (open triangle, n=4), 0.03 (open square, n=4), 0.1 (closed circle, n=4), 0.3 (closed triangle, n=4),
and 1 (closed square, n=4) mg/kg. The X-axis represents the time (day) after TTx dosing. Day 7 is the timing of ASP2713 dosing. The Y-axis represents the logarithm of anti-TTx IgG
titer (log10(units/mL)). Each plot represents mean § SE. (b) Value of the area under the curve (AUC) from day 7 to day 35 described in Figure 2A at each dose. The Y-axis represents
the logarithm of anti-TTx IgG titer multiplied by time ((log10(units/mL)) £ day). Data represent the mean § SE. *: Significantly different (P < 0.05) from the control group by Dun-
nett's multiple comparison test (c) Individual serum concentration-time profiles in TTx-sensitized cynomolgus monkeys at 0.01 (open triangle), 0.03 (open square), 0.1 (closed cir-
cle), 0.3 (closed triangle), and 1 (closed square) mg/kg. The X-axis represents the time (day) after ASP2713 dosing. The lower limit of quantification was 0.001 mg/mL, and serum
concentrations at the time points at which anti-ASP2713 antibody was positive were excluded. (d) Anti-TTx IgG titer-time profiles in TTx-sensitized cynomolgus monkeys at 0
(open circle, n=5), 1 (open triangle, n=5), 3 (open square, n=4), and 10 (closed circle, n=5) mg/kg. The X-axis represents the time (day) after TTx dosing. Day 7 is the timing of
ASP2713 doing. Y-axis represents the logarithm of anti-TTx IgG titer (log10(units/mL)). Each plot represents mean § SE. (e) Value of AUC from day 7 to day 35 described in Figure 2D
at each dose. The Y-axis represents the logarithm of anti-TTx IgG titer multiplied by time ((log10(units/mL)) £ day). Data represents mean § SE. (f) Individual serum concentration-
time profiles in TTx-sensitized cynomolgus monkeys at 1 (open triangle), 3 (open square), and 10 (closed circle) mg/kg. The X-axis represents the time (day) after ASP2713 dosing.
The lower limit of quantification was 0.001 mg/mL, and serum concentrations at time points at which anti-ASP2713 antibody was positive were excluded. (g) Receptor occupancy-
time profiles in TTx-sensitized cynomolgus monkeys at 0 (open circle), 1 (open triangle), 3 (open square), and 10 (closed circle) mg/kg. The X-axis represents the time (day) after
TTx dosing. Day 7 is the time of ASP2713 doing. The Y-axis represents receptor occupancy (%). Each plot represents mean § SE. (h) Individual serum concentration-time profiles in
healthy cynomolgus monkeys at 1 (open triangle, n=6) and 10 (open square, n=6) mg/kg. The X-axis represents the time (day) after ASP2713 dosing. The lower limit of quantification
was 0.1 mg/mL, and serum concentrations at time points at which anti-ASP2713 antibody was positive were excluded.
 K. Konishi et al. / Journal of Pharmaceutical Sciences 111 (2022) 2630−2638 2635

Figure 2. Continued.

biotinylated ASP2713 F(ab’)2, the mean percentages of receptor occu-
pancy at all doses plateaued at a high level from Day 7 to Day 14
(Fig. 2g). Thereafter, high mean receptor occupancy was maintained
at 10 mg/kg until Day 35, but not at 1 and 3 mg/kg. The number of
animals in which individual values of receptor occupancy decreased
was 4 of 5 and 2 of 4 animals at 1 and 3 mg/kg, respectively. In
healthy cynomolgus monkeys, a dose-dependent concentration-time
profile was observed, indicating linear PK at a dose range from 1 to
10 mg/kg (Fig. 2h).
Analysis of PK data and Extrapolation to Humans
Estimated PK parameters (CL, V1, Vmax, Km, Q, and V2) in monkeys
obtained by fitting a two-compartment model with non-linear elimi-
nation described by the Michaelis-Menten equation to the serum
concentration in TTx-sensitized and healthy cynomolgus monkeys
(refer to Fig. 2c, f, and h) are summarized in Table 2. The goodness-
of-fit plots indicated acceptable fitness (Supplementary Fig. 1). Using
the allometric scaling method, PK parameters in humans were
extrapolated from the estimated PK parameters in cynomolgus mon-
keys and are summarized in Table 2.
Prediction of Clinically Effective Dose
To avoid underestimating the clinically effective dose, we use the
maximum individual serum concentration at trough after single IV
administration of ASP2713 at 1 mg/kg in TTx-sensitized cynomolgus
monkeys (2.2 mg/mL) rather than the average concentration (refer to
Fig. 2c and f). From the results of the concentration-response curve of
ASP2713 for Igb of BCR on whole blood B cells in cynomolgus mon-
keys, target receptor occupancy at 2.2 mg/mL was calculated to be
82.4% (refer to Fig. 1a), and this was accordingly defined as the target
receptor occupancy. Thereafter, from the concentration-response
curve of ASP2713 for Igb of BCR on whole blood B cells in healthy vol-
unteers, the trough concentration required to obtain 82.4% as recep-
tor occupancy was calculated to be 0.21 mg/mL (refer to Fig. 1b),
which was defined as the required trough concentration. The clini-
cally effective dose to exceed the required trough concentration (0.21
mg/mL) at Day 28 was predicted to be 0.4 mg/kg IV administration at
4-week intervals, based on the simulated human PK profile Fig. 3)
and using Eqs. (1) and ((2) with the predicted human PK parameters
(Table 2). Predicted concentration at trough (Ctrough), maximum con-
centration (Cmax), and AUC for a dosing interval (AUCtau) on multiple

Table 2
PK Parameters Estimated in Monkeys and those Extrapolated to Humans

Species CL (L/day/kg) V1(L/kg) Vmax (mg/day/kg) Km (mg/mL) Q (L/day/kg) V2(L/kg)

Monkeys 0.0030 (0.00058) 0.039 (0.0013) 0.0096 (0.0082) 0.85 (0.98) 0.012 (0.0028) 0.029 (0.0023)
Humans 0.0016 0.039 0.0096 0.14 0.0061 0.029
Values in parentheses represent standard error.
CL: Linear component of elimination clearance of ASP2713 from the central compartment, Km: Michaelis-Menten constant, Q: Inter-compartment clearance, Vmax: Maximum reac-
tion velocity, V1: Distribution volume of central compartment, V2: Distribution volume of peripheral compartment.
2636 K. Konishi et al. / Journal of Pharmaceutical Sciences 111 (2022) 2630−2638

Table 3 values between monkeys and humans was observed. The dose-
Predicted Ctrough, Cmax, and AUCtau in Humans After Multiple 0.4 mg/kg IV Administra- dependent inhibition of anti-TTx antibody production, PK, and recep-
tion of ASP2713 at 4-Week Intervals
tor occupancy of ASP2713 in TTx-sensitized and healthy cynomolgus
Ctrough Cmax AUCtau monkeys were evaluated, and we concluded that 1 mg/kg was the
(mg/mL) (mg/mL) (mg £ day/mL) minimum effective dose in TTx-sensitized cynomolgus monkeys.
First Dose 0.24 10.3a 77.3d These findings aid our understanding of the characteristics of
Second Dose 0.40 10.6b 86.0e ASP2713 and can be used as a basis for clinical dose setting.
Last Dose 0.48 10.7c 89.7f ASP2713 is an Igb and Fcg RIIB cross-linking antibody. In the pres-
Ctrough: Concentration at trough, Cmax: Maximum concentration, AUCtau: Area under ent study, however, the receptor occupancy of ASP2713 only for Igb
the concentration-time curve for a dosing interval. of BCR on whole blood B cells was measured and used to predict the
a
Cmax at first dose was calculated as dose divided by distribution volume in the cen- clinically effective dose. This process appears reasonable because the
tral compartment (C0).
b
Cmax at second dose was calculated as Ctrough at first dose plus C0.
results of our previous report indicated that the binding of ASP2713
c
Cmax at last dose was calculated as Ctrough at second dose plus C0. to Igb correlates with the inhibitory effect of B cell proliferation via
d
AUCday0-28. Fcg RIIB phosphorylation.6 In addition, the dissociation constant of
ASP2713 for human Igb was 3.0 £ 1010 mol/L, which was substan-
e
AUCday28-56.
f
AUCday56-84.
tially higher than that for human Fcg RIIB (6.2 £ 107 mol/L) mea-
sured using a surface plasmon resonance technique as described in a
IV administration of ASP2713 at 0.4 mg/kg at 4-week intervals are previous report.6 These observations suggested that ASP2713 binds
shown in Table 3. In addition, to compare the concentration-time to Igb of BCR initially, and then crosslinks to Fcg RIIB, resulting in an
profiles of ASP2713 at different doses, human PK profiles were simu- inhibitory effect on B cell proliferation. Because of these mechanisms,
lated at 0.1 and 1 mg/kg multiple IV administrations of ASP2713 at 4- the receptor occupancy of ASP2713 for Igb is considered a surrogate
week intervals and shown in Fig. 3. The overall procedure of clinical efficacy marker, and measurement of the receptor occupancy of
efficacious dose prediction is summarized in Fig. 4. ASP2713 for Igb is more important than that for Fcg RIIB from the
point of view of pharmacological analysis.
With regard to the EC50 values of ASP2713 receptor occupancy
Discussion for Igb of BCR on whole blood B cells in cynomolgus monkeys
and healthy volunteers, a 6-fold difference was observed (Table 1).
In the present study, we investigated ASP2713’s characteristics in This can be explained by the binding affinity of ASP2713 for
preclinical studies, and applied the acquired data and analysis results human and monkey Igb. The dissociation constant of ASP2713 for
in a mathematical model to predict the clinically effective dose of human Igb was measured to be 3.0 £ 1010 mol/L in a previous
0.4 mg/kg IV administration at 4-week intervals. From the results of report;6 and in line with the EC50 values, ASP2713 showed
in vitro receptor occupancy study, ASP2713 bound to Igb of BCR in a approximately 10-fold lower binding affinity to monkey Igb using
concentration-dependent manner, and a species difference in EC50 a surface plasmon resonance technique (data not shown). We

Figure 3. Predicted serum concentration-time profiles in humans after multiple IV administration of ASP2713 at 4-week intervals. Dotted, dashed, and solid lines represent pre-
dicted serum concentration-time profiles at 0.1, 0.4, and 1 mg/kg, respectively. The gray line shows the required trough concentration (0.21 mg/mL).
 K. Konishi et al. / Journal of Pharmaceutical Sciences 111 (2022) 2630−2638
Figure 4. Overall procedure used to predict clinically effective dose.
therefore suggest that the species difference in KD values is
related to that of EC50.
The EC20, EC50, and EC80 values of ASP2713 receptor occupancy for
Igb of BCR on whole blood B cells in 5 SLE patients was measured to
be 0.019, 0.076, and 0.30 mg/mL, respectively, by the same analysis
as that used in the in vitro receptor occupancy assay for healthy vol-
unteers described in the Materials and Methods section (data not
shown). These values in SLE patients are similar to those in healthy
volunteers (Table 1), indicating no large differences in the binding
affinity and serum concentration of ASP2713 in occupying Igb of BCR
on whole blood B cells between healthy volunteers and SLE patients.
The use of EC50 values in healthy volunteers to predict the clinically
effective dose therefore appears acceptable.
Concerning competitive binding of biotinylated ASP2713 F(ab’)2,
individual animals without anti-ASP2713 antibody at Day 35 showed
90% or higher receptor occupancy at doses of 1, 3, and 10 mg/kg
(detailed data not shown). At 1 mg/kg, mean receptor occupancy was
65% at Day 35 (Fig. 2g); nevertheless, 4 of 5 individuals were shown to
be anti-ASP2713 antibody-positive. The only anti-ASP2713 antibody-
negative individual had a receptor occupancy of 96% at Day 35, indicat-
ing almost full occupancy. Considering that the inhibitory effect of
ASP2713 on anti-TTx IgG titer peaks at 1 mg/kg (Fig. 2a, b, d, and e), the
result of competitive binding assay using biotinylated ASP2713 F(ab’)2
supports the idea that 1 mg/kg is not only the minimum effective dose
but also the dose at which the maximum effect against anti-TTx IgG
titer inhibition is expected in TTx-sensitized cynomolgus monkeys.
Since the concentration-time profile that seemed to be derived
from target-mediated drug disposition was observed at the dose range
of 0.01 to 1 mg/kg (Fig. 2c), a two-compartment model with linear
and non-linear elimination described by the Michaelis-Menten equa-
tion was selected in the present study. According to the PK analysis
results, Km value estimated from serum concentrations of ASP2713 in
TTx-sensitized and healthy cynomolgus monkeys was 0.85 mg/mL
2637
(Table 2). This concentration is reasonable given that the results for
the in vitro receptor occupancy of ASP2713 for Igb of BCR on whole
blood B cells in cynomolgus monkeys was 50% at 0.35 mg/mL (Table 1),
which is almost the same as the Km value. We consider that the non-
linear PK was caused by the decrease in in vivo receptor occupancy of
ASP2713 for Igb of BCR on whole blood B cells in cynomolgus mon-
keys. Since the same kind of non-linearity may be observed in humans,
it is reasonable to calculate the Km value in humans from that in cyno-
molgus monkeys corrected by the species difference of EC50 values of
ASP2713 receptor occupancy for Igb of BCR on whole blood B cells in
cynomolgus monkeys and healthy humans.
From the results of the in vivo study in TTx-sensitized cyno-
molgus monkeys, it was confirmed that almost full receptor occu-
pancy for Igb of BCR on whole blood B cells was observed at the
minimum effective dose of 1 mg/kg in monkeys without anti-
ASP2713 antibody, as described above. In humans, to maintain the
target receptor occupancy for Igb of BCR on whole blood B cells,
the required trough concentration in humans was calculated to be
0.21 mg/mL from the target trough concentration of ASP2713 at
the minimum effective dose of 1 mg/kg in TTx-sensitized cynomol-
gus monkeys and the relationship between concentration and
receptor occupancy of ASP2713 for Igb of BCR on whole blood B
cells in cynomolgus monkeys and healthy volunteers. Considering
that the inflection point of the predicted concentration-time pro-
file due to target-mediated drug disposition appeared at around
the trough in humans after multiple IV administration of ASP2713
at 0.4 mg/kg (Fig. 3), it can be expected that high receptor occu-
pancy for Igb of BCR on whole blood B cells is maintained during
treatment with ASP2713, which will in turn enable ASP2713 to
exert its pharmacological effect. This hypothesis from prediction
results using mathematical modeling and simulation of the rela-
tionship among PK, receptor occupancy, and pharmacological
effect should be confirmed in clinical trials.
2638 K. Konishi et al. / Journal of Pharmaceutical Sciences 111 (2022) 2630−2638
There are two limitations in the present study. First, the number of
monkeys was limited and variability in anti-TTx IgG titer was present
in the in vivo study. As described in the Results, a statistically signifi-
cant decrease in mean AUC from Day 7 to Day 35 for anti-TTx IgG titer
was noted at 1 mg/kg compared with the control group in Fig. 2b;
however, no statistically significant change was noted at 1 mg/kg and
above compared with the control group in Fig. 2e. Although it is possi-
ble to resolve this discrepancy by optimizing the experimental design,
the tendency of a decrease in anti-TTx IgG titer was observed and the
mean AUC from Day 7 to Day 35 value reached near-plateau at
1 mg/kg and above. We therefore concluded that 1 mg/kg was the min-
imum effective dose in TTx-sensitized cynomolgus monkeys. Second,
regarding the allometric scaling exponent for Vmax, although Vmax is
the maximum non-linear component of elimination clearance velocity
that relates to total target concentration profile,12 no information is
available about a possible species difference between monkeys and
humans in the amount of Igb of BCR on whole blood B cells. It has
been reported that values from 0.75 to 1 were used for the allometric
scaling exponent of Vmax;13-17 however, to avoid underestimation of
the clinically effective dose, we assumed that Vmax in humans was the
same as that in cynomolgus monkeys in the present study.
Conclusion
We examined the relationship between the concentration and
receptor occupancy of ASP2713 for Igb of BCR using whole blood B
cells. The results showed that ASP2713 bound to Igb of BCR on whole
blood B cells in both cynomolgus monkeys and healthy volunteers in
a concentration-dependent manner. In addition, we evaluated the
dose-dependency of inhibition of anti-TTx antibody production, PK,
and receptor occupancy of ASP2713 in TTx-sensitized and healthy
cynomolgus monkeys and concluded that the minimum effective
dose was 1 mg/kg by single IV administration. By mathematical
modeling and simulation which integrated the acquired data and
analysis results, we predicted that the clinically effective dose
required to maintain a concentration in humans which achieved the
same receptor occupancy expected in monkeys at the effective dose
was 0.4 mg/kg by IV administration at 4-week intervals. This study
aids our understanding of the characteristics of ASP2713 and can be
used as a basis of clinical dose setting.
Declaration of Competing Interest
Kentaro Konishi, Koji Nakamura, Yuichi Hanada, Yukihiro Kita-
naga, Satoshi Kubo, Fumitaka Kinugasa, Daisuke Yamajuku, Masashi
Maeda, Nobuchika Yamamoto, Tsuyoshi Minematsu, and Masato
Ohbuchi are employees of Astellas Pharma Inc. This study was funded
by Astellas Pharma Inc. Medical writing/editing support was pro-
vided by Guy Harris of DMC Corp. and funded by Astellas Pharma Inc.
Acknowledgment
The authors thank all the members of ASP2713 research and devel-
opment team at Astellas Pharma Inc. for their support. The authors
would also like to thank to Yasuhisa Nagasaka (Astellas Pharma Inc.)
and Masayo Oishi (Astellas Pharma Inc.) for their useful advice.
Supplementary Materials
Supplementary material associated with this article can be found
in the online version at doi:10.1016/j.xphs.2022.06.006.
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