Journal of Pharmaceutical Sciences 111 (2022) 2630−2638

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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 Systemic lupus erythematosus (SLE) is an autoimmune disease
the concentration-time curve for a dosing interval; BCR, B cell receptor; CL, Linear com- that causes damage to various organs.1-3 Symptoms typically appear
ponent of elimination clearance from the central compartment; Cmax, Maximum con-
centration; Ctrough, Concentration at trough; EC20, Concentration when 20% of
between the ages of 15 and 45, and about 90% of patients are
maximum efficacy is achieved; EC50, Concentration when 50% of maximum efficacy is women.1 Overall prevalence in USA ranges from 42 to 300 per
achieved; EC80, Concentration when 80% of maximum efficacy is achieved; Fcg R, Fc 100,000, and ethnic differences in SLE prevalence are known.1
Gamma receptor; IV, Intravenous; Km, Michaelis-Menten constant; MFI, Median fluo- Although the specific cause of SLE is still unclear, onset is associated
rescent intensity; PE, Phycoerythrin; PK, Pharmacokinetics; Q, Inter-compartment
with multiple factors, including immune dysregulation, genetic fac-
clearance; SAV, Streptavidin; SLE, Systemic lupus erythematosus; TTx, Tetanus toxoid;
Vmax, Maximum non-linear component of elimination clearance velocity; V1, Distribu- tors, epigenetic factors, environmental factors, and sex hormones.2,4,5
tion volume of the central compartment; V2, Distribution volume of the peripheral In clinical practice, although a broad range of medications are used,
compartment. including glucocorticoids, immunosuppressive agents, antimalarial
* Corresponding author at: Non-Clinical Biomedical Science, Applied Research & agents, and nonsteroidal anti-inflammatory drugs, B cell-targeting
Operations, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki, 305-8585,
Japan.
biologics such as rituximab and belimumab have not established a
E-mail address: [email protected] (K. Konishi). mainstay position in the treatment of SLE because of the potential
1
These authors have contributed equally to this work. 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 2631

To provide a new treatment option for SLE and to overcome occupancy of ASP2713 for Igb of BCR was analyzed by BD FACSArray
the potential issues of rituximab and belimumab, we previously (BD Biosciences, NJ, USA) for samples from healthy cynomolgus mon-
reported the fundamental pharmacological characteristics of keys and by BD FACSVerse (BD Biosciences) for samples from healthy
ASP2713, a novel Igb and Fc gamma receptor (Fcg R) IIB cross- volunteers. B cells were defined as CD20+ cells and the free form
linking antibody.6 ASP2713 is an anti-Igb antibody with insertion of Igb of BCR was stained with the biotin-labeled ASP2713 F(ab’)2
of S239D, H268D, and L328W substitutions into the Fc domain to fragment.
increase affinity for Fcg RIIB and to crosslink Igb with Fcg RIIB.
Cross-linking B cell receptor (BCR) and Fcg RIIB by antigen-IgG Processing of Whole Blood Samples from Healthy Cynomolgus Monkeys
immune complexes is considered to counteract excessive BCR-
mediated activation signals via induction of phosphorylation of Fifty microliters of ASP2713 dilutions were added to 50 mL
the immunoreceptor tyrosine-based inhibitory motif domain of of whole blood (final ASP2713 concentration: 0, 0.001, 0.01, 0.1,
Fcg RIIB, followed by suppression of autoreactive B cell survival 1, 10, 100, and 1000 mg/mL) and the mixtures were incubated
and activation. The experimental results in our previous report for 3 hours at 37°C and 5% CO2. After 30 minutes incubation on
showed that ASP2713 has the potential to suppresses BCR signal- ice, 50 mL of biotin-labeled ASP2713 F(ab’)2 fragment solution
dependent B cell activation by cross-linking Igb of BCR and (10 mg/mL) was added into the blood and incubated for 30
Fcg RIIB without exhibiting B cell-depleting properties. These minutes on ice. The cells were then centrifuged for 3 minutes
characteristics suggest that ASP2713 may be an attractive candi- at 380 £ g, and supernatant was removed. The cell precipitate
date for the treatment of SLE. To date, however, species differen- was resuspended in 1.2 mL of FACS lysing buffer (BD Bioscien-
ces in the relationship between concentration and the receptor ces) and incubated for 10 minutes at room temperature. Cells
occupancy of ASP2713 have not been evaluated, and its in vivo were washed with staining buffer (BD Biosciences) and sus-
pharmacological effect has not been investigated. pended in 50 mL of secondary antibody cocktail containing 5-
Here, we investigated the compound’s characteristics with fold diluted allophycocyanin (APC) mouse anti-human CD20 and
regard to pharmacological effect, pharmacokinetics (PK), and 1000-fold diluted streptavidin (SAV)-phycoerythrin (PE) anti-
receptor occupancy of ASP2713. We then applied the acquired bodies (BD Biosciences). The cell suspension was incubated for
data and analysis results to a mathematical model to predict 30 minutes on ice, washed with staining buffer and analyzed on
human PK and the clinically effective dose of this agent. It is a FACSArray.
known that there are linear and non-linear elimination phases in
the concentration-time profile of antibody due to target-mediated Processing of Whole Blood Samples from Healthy Volunteers
drug disposition. Therefore, in the present report, monkey PK
was analyzed by a two-compartment model with linear and non- Sixty microliters of ASP2713 dilutions were added to 60 mL of
linear elimination described by the Michaelis-Menten equation whole blood (final ASP2713 concentration: 0, 0.0001, 0.001, 0.01,
and scaled up to human PK by allometric scaling. Translation of 0.03, 0.1, 1, and 10 mg/mL) and the mixtures were incubated for
drug candidates from preclinical to clinical use clearly requires 3 hours at 37°C and 5% CO2. After incubation, 110 mL of each mix-
the prediction of clinical dosage and regimen. This prediction not ture was obtained and placed on ice. Fifty-five microliters of biotin-
only supports the developmental feasibility of the dosage and labeled ASP2713 F(ab’)2 fragment solution (1 mg/mL) or stain buffer
regimen, but also provides a justification for the design of clinical was added and incubated for 30 minutes on ice. Cells were then
studies. centrifuged for 3 minutes at 400 £ g, and the supernatant was
removed. The cell precipitate was resuspended with 1.2 mL of FACS
Materials and Methods lysing buffer and incubated for 12 minutes at room temperature
under light-resistant conditions. After incubation, cells were
Human Samples and Ethics washed twice with staining buffer and suspended with 90 mL of
antibody cocktail containing 5-fold diluted APC mouse anti-
Human whole blood samples were obtained from healthy adults human CD20 and 1000-fold diluted SAV-PE antibodies. The cell
aged over 20 years who understood the purpose of this research and suspension was incubated for 30 minutes on ice under light-resis-
participated voluntarily. The study was approved by Astellas tant conditions, washed twice, resuspended with staining buffer
Research Ethics Committee. In addition, human whole blood samples and analyzed on a FACSVerse.
were obtained from patients under treatment for SLE at the Univer-
sity of Tsukuba. The study was approved by the Human Ethics Review
Calculation of Receptor Occupancy
Committees of the Tsukuba University Hospital and Astellas Research
Ethics Committee.
Using the flow cytometry standard file obtained from each recep-
tor occupancy assay, Median Fluorescent Intensity (MFI) of PE on B
Animal Welfare
cells was calculated using FlowJoTM version 7.6 (Tree Star Inc., OR,
USA). Using the obtained MFI, receptor occupancy (%) was calculated
The study was approved by the Institutional Animal Care and Use
by the following equation.
Committee of Astellas Pharma Inc. and Shin Nippon Biomedical Labo-
ratories, Ltd., and was performed in accordance with the animal wel- Receptor occupancy of ASP 2713 for Ig bð%Þ
¼ ð1  ½MFI with ASP 2713  MFI background =½MFI without ASP 2713  MFI background Þ
fare bylaws of Shin Nippon Biomedical Laboratories, Ltd., Drug Safety
 100
Research Laboratories (Kagoshima, Japan). Both are accredited by
AAALAC International.

In Vitro Receptor Occupancy Assay Calculation of EC20, EC50, and EC80 Values

Whole blood samples treated with heparin from 3 healthy cyno- Receptor occupancy (%) of ASP2713 for Igb of BCR was plotted
molgus monkeys (Shin Nippon Biomedical Laboratories, Ltd.) and 3 against the Log 10-transformed ASP2713 concentration and fitted
healthy volunteers were used. The assay to determine the receptor 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 Nippon Biomedical Laboratories, Ltd.) were used as capture reagent
EC80 values. and detection regent, respectively. The concentration versus fluores-
Top  Bottom cence relationship was determined through regression according to a
Y ¼ Bottom þ
 five-parameter logistic regression model.
1 þ 10ððLogEC50  XÞ  HillSlopeÞ
Anti-ASP2713 antibody was detected using a bridging assay. In
brief, diluted serum samples were mixed with biotin-labeled
Log 100F F ASP2713 (Astellas Pharma Inc.) and ruthenium-labeled ASP2713
LogEC50 ¼ LogECF 
HillSlope (Astellas Pharma Inc.) for determination of anti-ASP2713 antibody
in TTx-sensitized cynomolgus monkeys. A signal ratio of post- to
where X represents ASP2713 concentration (mg/mL), and Y repre-
pre-dose greater than 1.3 was defined as anti-ASP2713 antibody
sents receptor occupancy (%). Bottom and top were set to be 0 and
positive. For determination of anti-ASP2713 antibody in healthy
100, and F was 20 or 80, respectively. EC20, EC50, and EC80 represent
cynomolgus monkeys, biotin-labeled ASP2713 (Shin Nippon Bio-
ASP2713 concentration when 20%, 50%, and 80% of the maximum
medical Laboratories, Ltd.) and ruthenium-labeled ASP2713 (Shin
efficacy are achieved, respectively.
Nippon Biomedical Laboratories, Ltd.) were mixed with diluted
serum samples. The mixtures were added to casein-coated wells.
In Vivo Study in Cynomolgus Monkeys The wells were shaken and washed several times. Diluted Read
buffer (Meso Scale Discovery Inc., MD, USA) was added and lumines-
In SLE patients, it was reported that B cells exhibit aberrant signal cence intensity was measured using a SECTOR Imager (Meso Scale
transduction events and increased antigen-receptor-mediated phos- Discovery Inc.). Using inhouse-validated criteria, anti-ASP2713 anti-
phorylation.7 To mimic antibody production caused by B cell activa- body was defined as positive or negative.
tion, cynomolgus monkeys were administrated tetanus toxoid (TTx)
and used to investigate the pharmacological effect of ASP2713. Male Competitive Binding Assay
cynomolgus monkeys aged 3 to 5 years (2.75 to 4.62 kg at initiation
of acclimation) were immunized with TTx antigen (Denka Seiken, Fifty microliters of biotinylated ASP2713 F(ab’)2 solution (20 mg/
Tokyo, Japan) on Day 0 as described in a previous report.8 ASP2713 mL) was added to 50 mL of blood sample drawn from the femoral
was administered once intravenously on Day 7 at the low dose levels vein of cynomolgus monkeys and treated with heparin. For the sam-
of 0.01, 0.03, 0.1, 0.3, and 1 mg/kg at a dosing rate of 2 mL/min and ple before dosing only, 50 mL of cell staining buffer (BioLegend, CA,
volume of 1 mL/kg to 4 male cynomolgus monkeys at each dose and USA) was added to the 50 mL blood sample as a background sample.
at the high dose levels of 1, 3, and 10 mg/kg at a dosing rate of The sample was incubated for 30 minutes on ice under light-resistant
2 mL/min and volume of 1 mL/kg to 4 or 5 male cynomolgus monkeys conditions and centrifuged for 3 minutes at 450 £ g. The supernatant
at each dose. Anti-TTx IgG titer in serum was measured on Days 7 was removed and 1 mL of Pharm Lyse (BD Biosciences) was added.
(before ASP2713 dosing), 8, 10, 14, 17, 21, 28, and 35 as described in The sample was incubated for 10 minutes at room temperature under
our previous report.6 The area under the curve (AUC) of anti-TTx IgG light-resistant conditions and centrifuged for 5 minutes at 200 £ g.
titer was calculated from Day 7 to Day 35. When considering the effi- The supernatant was removed, 1 mL of cell staining buffer was added
cacy of ASP2713 in TTx-sensitized cynomolgus monkeys, we used the and the mixture was centrifuged for 3 minutes at 750 £ g. Thereafter
index to determine whether the AUC increase in anti-TTx antibody the supernatant was removed, 50 mL of antibody mixture (1000-fold
due to TTx administration to cynomolgus monkeys could be signifi- diluted PE streptavidin solution [BD Biosciences], and 10-fold diluted
cantly suppressed. Statistical comparison with the control group was APC-CD20, [BioLegend]) was added, and the sample was incubated
done using Dunnett’s multiple comparison test. Sampling points for for 30 minutes on ice under light-resistant conditions. After adding
determination of ASP2713 concentration and anti-ASP2713 antibody cell staining buffer several times, centrifuging for 3 minutes at
in serum were 1 hour, and 1, 3, 7, 10, 14, 21, and 28 days after dosing. 750 £ g and removing the supernatant, the samples were analyzed
At the high dose levels only, competitive binding of biotinylated by FACSCalibur (BD Biosciences) and CellQuest Pro version 6.0 (BD
ASP2713 F(ab’)2 in blood was measured on Days 7 (1 hour after Biosciences). Receptor occupancy (%) was calculated from the geo-
ASP2713 doing), 8, 10, 14, 17, 21, 28, and 35. In addition, ASP2713 metric mean of MFI of PE in CD20+ cells using the following equa-
was administered once intravenously at dose levels of 1 and tions.
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 MFIFðab0 Þ2þ
Receptor occupancy ð%Þ ¼ 1   100
at initiation of acclimation) to determine ASP2713 concentration in MFIFðab0 Þ2
serum. Sampling points for determination of ASP2713 concentration
and anti-ASP2713 antibody in serum were 1 hour, and 1, 7, 14, 21,
MFIFðab0 Þ2þ ¼ ðMFI value of after dosingÞ
28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 days after dosing.
 ðMFI value of background sampleÞ
PK Measurements
MFIFðab0 Þ2 ¼ ðMean MFI value of before dosingÞ
ASP2713 concentration was determined using the Gyrolab xP
workstation (Gyros Protein Technologies AB, Uppsala, Sweden). In  ðMFI value of background sampleÞ
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 PK Model
ASP2713 in TTx-sensitized cynomolgus monkeys. The concentration
versus fluorescence relationship was determined through regression PK parameters in cynomolgus monkeys were estimated by fitting
according to a four-parameter logistic regression model. For determi- a two-compartment model with linear and non-linear elimination
nation of ASP2713 in healthy cynomolgus monkeys, biotin-labeled described by the Michaelis-Menten equation to the serum concentra-
primary anti-idiotype antibody (Shin Nippon Biomedical Laborato- tion in TTx-sensitized and healthy cynomolgus monkeys using NON-
ries, Ltd.) and Alexa-labeled secondary anti-idiotype antibody (Shin 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 from Day 7 to Day 35 values for anti-TTx IgG titer at all doses tended
serum tended to be rapid at lower doses, indicating nonlinear PK at to be lower than those in the control group. The mean AUC from Day
the dose range from 0.01 to 1 mg/kg. On the other hand, in TTx-sensi- 7 to Day 35 value reached nearly plateau at 1 mg/kg and above
tized cynomolgus monkeys at the high dose levels of 1, 3, and (Fig. 2e). Considering the results at both low and high dose levels, we
10 mg/kg, mean anti-TTx IgG titer was gradually increased from Day concluded that 1 mg/kg was the minimum effective dose in TTx-sen-
10 or Day 14 to Day 35 (Fig. 2d). No statistically significant change in sitized cynomolgus monkeys. In Fig. 2f, a dose-dependent concentra-
mean AUC from Day 7 to Day 35 for anti-TTx IgG titer was noted at tion-time profile is observed, indicating linear PK at the dose range
any dose compared with the control group. However, mean AUC 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- Prediction of Clinically Effective Dose
pancy at all doses plateaued at a high level from Day 7 to Day 14
(Fig. 2g). Thereafter, high mean receptor occupancy was maintained To avoid underestimating the clinically effective dose, we use the
at 10 mg/kg until Day 35, but not at 1 and 3 mg/kg. The number of maximum individual serum concentration at trough after single IV
animals in which individual values of receptor occupancy decreased administration of ASP2713 at 1 mg/kg in TTx-sensitized cynomolgus
was 4 of 5 and 2 of 4 animals at 1 and 3 mg/kg, respectively. In monkeys (2.2 mg/mL) rather than the average concentration (refer to
healthy cynomolgus monkeys, a dose-dependent concentration-time Fig. 2c and f). From the results of the concentration-response curve of
profile was observed, indicating linear PK at a dose range from 1 to ASP2713 for Igb of BCR on whole blood B cells in cynomolgus mon-
10 mg/kg (Fig. 2h). 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
Analysis of PK data and Extrapolation to Humans receptor occupancy. Thereafter, from the concentration-response
curve of ASP2713 for Igb of BCR on whole blood B cells in healthy vol-
Estimated PK parameters (CL, V1, Vmax, Km, Q, and V2) in monkeys unteers, the trough concentration required to obtain 82.4% as recep-
obtained by fitting a two-compartment model with non-linear elimi- tor occupancy was calculated to be 0.21 mg/mL (refer to Fig. 1b),
nation described by the Michaelis-Menten equation to the serum which was defined as the required trough concentration. The clini-
concentration in TTx-sensitized and healthy cynomolgus monkeys cally effective dose to exceed the required trough concentration (0.21
(refer to Fig. 2c, f, and h) are summarized in Table 2. The goodness- mg/mL) at Day 28 was predicted to be 0.4 mg/kg IV administration at
of-fit plots indicated acceptable fitness (Supplementary Fig. 1). Using 4-week intervals, based on the simulated human PK profile Fig. 3)
the allometric scaling method, PK parameters in humans were and using Eqs. (1) and ((2) with the predicted human PK parameters
extrapolated from the estimated PK parameters in cynomolgus mon- (Table 2). Predicted concentration at trough (Ctrough), maximum con-
keys and are summarized in Table 2. 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 2637

Figure 4. Overall procedure used to predict clinically effective dose.

therefore suggest that the species difference in KD values is (Table 2). This concentration is reasonable given that the results for
related to that of EC50. the in vitro receptor occupancy of ASP2713 for Igb of BCR on whole
The EC20, EC50, and EC80 values of ASP2713 receptor occupancy for blood B cells in cynomolgus monkeys was 50% at 0.35 mg/mL (Table 1),
Igb of BCR on whole blood B cells in 5 SLE patients was measured to which is almost the same as the Km value. We consider that the non-
be 0.019, 0.076, and 0.30 mg/mL, respectively, by the same analysis linear PK was caused by the decrease in in vivo receptor occupancy of
as that used in the in vitro receptor occupancy assay for healthy vol- ASP2713 for Igb of BCR on whole blood B cells in cynomolgus mon-
unteers described in the Materials and Methods section (data not keys. Since the same kind of non-linearity may be observed in humans,
shown). These values in SLE patients are similar to those in healthy it is reasonable to calculate the Km value in humans from that in cyno-
volunteers (Table 1), indicating no large differences in the binding molgus monkeys corrected by the species difference of EC50 values of
affinity and serum concentration of ASP2713 in occupying Igb of BCR ASP2713 receptor occupancy for Igb of BCR on whole blood B cells in
on whole blood B cells between healthy volunteers and SLE patients. cynomolgus monkeys and healthy humans.
The use of EC50 values in healthy volunteers to predict the clinically From the results of the in vivo study in TTx-sensitized cyno-
effective dose therefore appears acceptable. molgus monkeys, it was confirmed that almost full receptor occu-
Concerning competitive binding of biotinylated ASP2713 F(ab’)2, pancy for Igb of BCR on whole blood B cells was observed at the
individual animals without anti-ASP2713 antibody at Day 35 showed minimum effective dose of 1 mg/kg in monkeys without anti-
90% or higher receptor occupancy at doses of 1, 3, and 10 mg/kg ASP2713 antibody, as described above. In humans, to maintain the
(detailed data not shown). At 1 mg/kg, mean receptor occupancy was target receptor occupancy for Igb of BCR on whole blood B cells,
65% at Day 35 (Fig. 2g); nevertheless, 4 of 5 individuals were shown to the required trough concentration in humans was calculated to be
be anti-ASP2713 antibody-positive. The only anti-ASP2713 antibody- 0.21 mg/mL from the target trough concentration of ASP2713 at
negative individual had a receptor occupancy of 96% at Day 35, indicat- the minimum effective dose of 1 mg/kg in TTx-sensitized cynomol-
ing almost full occupancy. Considering that the inhibitory effect of gus monkeys and the relationship between concentration and
ASP2713 on anti-TTx IgG titer peaks at 1 mg/kg (Fig. 2a, b, d, and e), the receptor occupancy of ASP2713 for Igb of BCR on whole blood B
result of competitive binding assay using biotinylated ASP2713 F(ab’)2 cells in cynomolgus monkeys and healthy volunteers. Considering
supports the idea that 1 mg/kg is not only the minimum effective dose that the inflection point of the predicted concentration-time pro-
but also the dose at which the maximum effect against anti-TTx IgG file due to target-mediated drug disposition appeared at around
titer inhibition is expected in TTx-sensitized cynomolgus monkeys. the trough in humans after multiple IV administration of ASP2713
Since the concentration-time profile that seemed to be derived at 0.4 mg/kg (Fig. 3), it can be expected that high receptor occu-
from target-mediated drug disposition was observed at the dose range pancy for Igb of BCR on whole blood B cells is maintained during
of 0.01 to 1 mg/kg (Fig. 2c), a two-compartment model with linear treatment with ASP2713, which will in turn enable ASP2713 to
and non-linear elimination described by the Michaelis-Menten equa- exert its pharmacological effect. This hypothesis from prediction
tion was selected in the present study. According to the PK analysis results using mathematical modeling and simulation of the rela-
results, Km value estimated from serum concentrations of ASP2713 in tionship among PK, receptor occupancy, and pharmacological
TTx-sensitized and healthy cynomolgus monkeys was 0.85 mg/mL 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 Supplementary Materials
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- Supplementary material associated with this article can be found
cant decrease in mean AUC from Day 7 to Day 35 for anti-TTx IgG titer in the online version at doi:10.1016/j.xphs.2022.06.006.
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-
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The authors thank all the members of ASP2713 research and devel- 567.
opment team at Astellas Pharma Inc. for their support. The authors 17. Xue J, Kong D, Yao Y, Yang L, Yao Q, Zhu Y, Ding Y, Yang F, Gong J, Shen L, Zhou T.
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would also like to thank to Yasuhisa Nagasaka (Astellas Pharma Inc.) EGFR/HER3 Bi-specific Monoclonal Antibody. J Pharm Sci. 2020;109(10):3172–
and Masayo Oishi (Astellas Pharma Inc.) for their useful advice. 3180.