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ArticleChemical Site-Speciﬁc Conjugation Platform to Improve thePharmacokinetics and Therapeutic Index of Antibody−Drug
Conjugates
Yutaka Matsuda,§ Takuya Seki,§ Kei Yamada, Yuri Ooba, Kazutoshi Takahashi, Tomohiro Fujii,
Sayaka Kawaguchi, Takahiro Narita, Akira Nakayama, Yoshiro Kitahara, Brian A. Mendelsohn,*
and Tatsuya Okuzumi*
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ABSTRACT: To overcome a lack of selectivity during the chemical
modiﬁcation of native non-engineered antibodies, we have developed atechnology platform termed “AJICAP” for the site-speciﬁc chemical conjugationof antibodies through the use of a class of IgG Fc-aﬃnity reagents. To date, alimited number of antibody−drug conjugates (ADCs) have been synthesized via
this approach, and no toxicological study was reported. Herein, we describe thecompatibility and robustness of AJICAP technology, which enabled the synthesisof a wide variety of ADCs. A stability assessment of a thiol-modiﬁed antibody
synthesized by AJICAP technology indicated no appreciable increase inaggregation or decomposition upon prolonged storage, indicating that theunexpectedly stable thiol intermediate has a great potential intermediate for
payload or linker screening or large-scale manufacturing. Payload conjugation
with this stable thiol intermediate generated several AJICAP-ADCs. In vivo
xenograft studies indicated that the AJICAP-ADCs displayed signiﬁcant tumor
inhibition comparable to benchmark ADC Kadcyla. Furthermore, a rat pharmacokinetic analysis and toxicology study indicated anincrease in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index, compared withstochastic conjugation technology. This is the ﬁrst report of the therapeutic index estimation of site-speciﬁc ADCs produced byutilizing Fc aﬃnity reagent conjugation. The described site-speciﬁc conjugation technology is a powerful platform to enable nextgeneration ADCs through reduced heterogeneity and enhanced therapeutic index.
KEYWORDS: antibody−drug conjugate, ADC, site-speciﬁc conjugation, therapeutic index, chemical conjugation, AJICAP■INTRODUCTION
More than a century ago, the German scientist Paul Ehrlich
proposed the idea of a “magic bullet” drug that targets invading
cells.1 Over the last two decades, antibody−drug conjugates(ADCs) have become a potential embodiment of Ehrlich’s
notion. ADCs are promising cancer therapeutics consisting ofrecombinant monoclonal antibodies.2−4 Although this concept
may seem simple, there is a critical issue with the synthesis of
traditional ADCs. Non-speciﬁc drug conjugation on naturally
available amino acid residues, such as lysine and reduced
interchain cysteine residues, is used to construct the chemicallinks between the antibodies and payloads. This stochastic
conjugation results in a heterogeneous distribution of cytotoxic
drugs over multiple sites on the ADCs and may lead to aclinically insuﬃcient and narrow therapeutic index (TI).5
With the goal of overcoming the potentially negative impact
of non-speciﬁc conjugation on the clinical use and eﬃcacy ofADCs, some eﬀective site-speciﬁc conjugation methods havebeen developed.6 A common strategy is the engineering of a
© 2021 The Authors. Published byAmerican Chemical Society

speciﬁc amino acid (or peptide moiety) into antibodies
followed by selective modiﬁcation using bioorthogonalchemistry for conjugation with payloads of interest. However,this protein engineering process can be costly and time
consuming due to the antibody optimization required.7
Some enzymatic approaches have also been developed tocreate ADCs from antibodies and have been used to introduce
biologically orthogonal functional groups at the appropriatesite(s) to antibodies both with and without the need forgenetic modiﬁcation or the introduction of sequence tags in
the antibody.8 For example, azido-labeled antibodies have beenobtained by enzymatically modifying the glycan present at
Received: June 11, 2021
Revised: September 10, 2021
Accepted: September 10, 2021
Published: September 28, 2021

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Articleminimum eﬀective dose (MED). A safety study in rats was also
conducted to determine the maximum tolerated dose (MTD),
resulting in the calculation of the TI of the AJICAP-ADCs,
which was then compared to that of a traditional partial
reduction cysteine-based ADC. These biological studies
indicated that AJICAP-ADCs may aﬀord an enhancement of
TI over ADCs constructed via traditional cysteine conjugation
technology. We conducted a pharmacokinetic (PK) experiment in rats with an AJICAP-conjugated ADC to compare theoverall clearance and detected deconjugation to that of astochastically conjugated ADC. The results of this PK study
lend credence to the notion that, ceteris paribus, ADCs
conjugated via this site-speciﬁc conjugation may aﬀord anenhanced TI. These overall results reported herein havedemonstrated that this chemical site-speciﬁc conjugation
technology is a powerful platform to enable next-generation
ADCs through the reduction of heterogeneity and enhancement of the TI.

Asn297 of the antibody Fc region to which appropriate linkers
can be covalently attached via dipolar cycloaddition reactions.9
However, there are two barriers that must be overcome todevelop ADCs employing enzymatic conjugation methods:
ﬁrst, the establishment of large-scale enzyme production
required under strict regulatory controls such as good
manufacturing practice (GMP), and second, the potentially
diﬃcult optimization of the removal of the enzyme from theconjugation reaction matrix. The industry is breaking throughthese barriers as enzyme-produced ADCs progress throughclinical development. For example, the transglutaminase
enzyme can be used to conjugate antibodies to drug-linkers,
and a description of the large-scale enzyme manufacturingfrom E. coli was reported.10
Chemical site-speciﬁc conjugation methods have emerged as
an alternate method that requires neither antibody modiﬁcations through protein engineering nor complicated processdevelopment.11 Daiichi Sankyo and AstraZeneca havelaunched the ﬁrst site-speciﬁc ADCs onto the commercial
market, which are conjugated by saturation of the reduced
interchain cysteine residues using a highly loaded drug
methodology.12 While this approach using fully reduced
antibodies is a solution to obtain ADCs with a nearlyhomogeneous drug antibody ratio (DAR), it may be of limited
utility due to the requirements of their speciﬁc drug-linkers.
Many drug-linkers applied in commercial and clinical ADCs
are highly hydrophobic (and potent) and therefore may not besuitable for use in a high DAR ADC format.13
There are reports in the literature of alternative conjugation
approaches employing aﬃnity labeling, which leverage aproximity eﬀect between an aﬃnity compound and theantibody, usually involving ultraviolet light to initiate a
covalent chemical reaction. This strategy has been applied
for site-speciﬁc antibody modiﬁcations; however, there are noreports in the chemical literature describing this method for alate-stage pre-clinical or clinical stage.11
From these limitations of site-speciﬁc chemical conjugation,
we conceived a unique strategy to use an Fc-aﬃnity peptide forthe site-speciﬁc functionalization of the biorthogonal group
into antibodies. This platform, named “AJICAP”, has beenpreviously described to enable the highly eﬃcient and versatile
conjugation of IgG1, IgG2, and IgG4 antibodies.14 The sitespeciﬁcity of the resulting ADC was conﬁrmed through peptide
mapping analyses, which conﬁrmed the desired conjugationsite at Lys248 located in the antibody Fc region.15 The
scalability of AJICAP technology was conﬁrmed by gram-scale
ADC synthesis based on a GMP strategy, demonstrating that
this technology is suitable for scale-up ADC manufacturing.16,17 However, limited ADCs have been synthesized by thisconjugation technology, and neither detailed analyses of theintermediate stability nor toxicology studies using animals havebeen reported.
Herein, we report a full investigation into the capability,
robustness, and biological activity of AJICAP technology. A
long-term stability assessment of site-speciﬁc thiol compounds,
the precursor for AJICAP-ADCs, indicated that the thiol
intermediate produced by AJICAP technology is stable formore than 1 year of storage and retained reactivity useful forthe synthesis of a variety of site-speciﬁc ADCs during this time.
To evaluate the drug-linker compatibility, over multiple
AJICAP-ADC syntheses were performed. In vitro potency
and in vivo xenograft eﬃcacy in the HER2 + NCI-N87 model
were evaluated for each AJICAP-ADC to determine the■

MATERIALS AND METHODS
Materials. Human IgG1 trastuzumab (Herceptin) andrituximab (Rituxan) were purchased from Roche Pharmaceutical Company (Switzerland). Human IgG1 inﬂiximab(Humira) was purchased from Sigma-Aldrich (USA). MCCmaytansinoid (Catalog No: TCRS-1262), MC-MMAF (CAS#:
863971-19-1), and MC-VA-PBD (CAS#: 1342820-51-2) werepurchased from Abzena (USA). MC-VC-MMAE (CAS#:
646502-53-6) was purchased from NJ Biopharmaceuticals
LLC (USA). The peptide reagent (1) was prepared aspreviously reported.14 All other chemical reagents wereacquired from Sigma-Aldrich (USA). All cell lines (HCC1954, NCI-N87, PC-3, and SU-DUL-4 cancer cell lines) werepurchased from ATCC (USA).
Instruments and Analytical Methods. ADC concentration and recovery were checked by the Slope Spectroscopy
method with a Solo-VPE system.14
Q-TOF MS analysis was performed as previously reported.14
SEC-HPLC analysis was performed as previously reported.17
ADC Synthesis. Site-speciﬁc ADCs were synthesizedaccording to our previous procedure.14
Stability Assessment. Three sets of standard solutions of
trastuzumab-thiol (3A) were prepared in storage buﬀer (50mM PBS, 10 mM EDTA, pH 7.4, 5 mg/mL). All the samples
were stored at −80 °C for a period of 365 days. Analysis of theaggregation was conducted by SEC.
Molecular Modeling. The model structure of the FcAJICAP peptide was generated using the Protein Preparation
module facilitated by Maestro Suite.18,19 Based on the crystal
structure of Fc-peptide (PDB: 6IQG),18 Arg was mutated to
Lys and then energy minimized.
In Vitro Cell-Based Assay. Trastuzumab-derived ADCs
(4A-4D) and trastuzumab were analyzed using the PC3, HCC1954, and NCI-N87 cancer cell lines. Rituximab-derived ADCs
(5A-5D) and trastuzumab were analyzed using the SU-DUL-4
and NCI-N87 cancer cell lines. Cells were seeded in clear,tissue culture-treated 96-well plates at approximately 2500−
5000 cells per well in 100 μL of growth media (RPMI-1640 +10% heat-inactivated fetal bovine serum) and incubated for 24
h in a humidiﬁed incubator, at 37 °C with 5% CO2, to allow
the cells to attach. Stock solutions of ADCs in 20 mM histidine
buﬀer were diluted with growth media (RPMI-1640 + 10%
heat-inactivated fetal bovine serum). The next day, 100 μL of
ADCs at varying concentrations was added to each well in4059

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ArticleFigure 1. Long-term stability assessment of the AJICAP-thiol intermediate. (a) Overview of the synthesis of the thiol intermediate. (b) Interaction
between human IgG1 Fc and the peptide reagent (PDB: 6IQG). (c) Molecular modeling of the thiol intermediate. (d) Analytical comparison
before and after storage for 365 days.

consisted of four animals for pharmacokinetics analysis. All rats
were administered once via the caudal vein with a butterﬂyneedle for intravenous injection, a 30 mL polypropylene
syringe, and a syringe pump (Pump 11 Elite, Harvard
Apparatus). The experimental procedure was approved by
the institutional ethics committee.
Clinical Observations. The animals were observed once
daily for clinical signs. Individual body weights were measured
on days 0, 7, 14, and 21, with the ﬁrst day of administration
deﬁned as day 0.
Pharmacokinetics Analysis. The blood samples werecollected on eight time points (immediately after administration, 6 h after administration, and 1, 2, 3, 7, 14, and 21 hafter administration) via the caudal vein. The concentrations of
ADC or total antibody were measured by a double sandwich
ELISA method.
Rat Safety Study. Animal Experiments. Sixty 8-week-old
female Sprague−Dawley rats (Charles River Japan, Tokyo,
Japan) were provided with a standard diet (Oriental Yeast,
Tokyo, Japan) and water ad libitum. Following theacclimatization period (1 week), the animals were stratiﬁed
by body weight and randomly assigned as the control groupthat were treated with the vehicle (histidine buﬀer), the three
trastuzumab-stochastic-MMAE(6) groups that were treatedwith 40, 80, and 120 mg/kg, and the two trastuzumabAJICAP-MMAE(4B) group that were treated with 10 and 40
mg/kg. Each group consisted of ﬁve animals for the blood
chemistry test and ﬁve animals for clinical signs and bodyweight measurement. All rats were administered once via thecaudal vein with a butterﬂy needle for intravenous injection, a
30 mL polypropylene syringe, and a syringe pump (Pump 11
Elite, Harvard Apparatus). The experimental procedure wasapproved by the institutional ethics committee.
Clinical Observations. The animals were observed once
daily for clinical signs. Individual body weights were measured
on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 19, and20, with the ﬁrst day of administration deﬁned as day 0.
Blood Chemistry Test. Blood chemistry parameters were
evaluated on 2 days after administration. The animals were
fasted overnight and then anesthetized with isoﬂurane before
blood was collected from the caudal vena cava. Heparin-

triplicate. The plates were incubated in a humidiﬁed tissue
culture incubator, with 5% CO2 at 37 °C for 5−8 days after the
addition of ADCs, to measure cytotoxicity. After 5−8 days, 20
μL of premix WST-1 reagent (TaKaRa Bio) was added to eachwell. Plates were then incubated at 37 °C for 1−6 h. After the
incubation period, the cell viability was measured using a plate
reader (PerkinElmer) at 450 nm. The IC50 values were
calculated from the cell viability data using Graph-Pad Prism
Software. Data for all ADCs tested in this assay were graphedas percent survival compared with untreated control wells.
In Vivo Xenograft Study. Cells. NCI-N87 cells (Cat#CRL-5822) were purchased through ATCC. The cells were
cultured in RPMI supplemented with 10% FBS and 1% P/S ina humidiﬁed incubator at 37 °C and 5% CO2.
Animals. NOD.CB17 homozygous mice were procured
through Envigo. Mice were fed a Teklad irradiated (sterilized)mouse diet and bedded with Teklad irradiated (sterilized)
corncob bedding from Envigo (Indianapolis, IN). Mice werehoused in Optimice carousel sterile quarters with ﬁltered air
supply in disposable cages from Animal Care Systems, Inc.
(Centennial, CO).
Implantation. On the day of implantation, the cells weretrypsinized and allowed to detach from ﬂasks. Trypsin wasthen neutralized with complete media, and the cells were spunat 400g. Media were aspirated, and the cells were resuspended
in 50:50 (Cultrex: RPMI) at a concentration of 5 × 107 cells/
mL. A volume of 100 μL was injected into the right hind ﬂank
of each animal (a total of 5 × 106 cells).
Study Arms and Treatments for NCI-N87. The tumor
volumes were monitored, and on the ﬁrst day (when mean
tumor volume reached ∼120 mm3), mice were stratiﬁed andplaced into 7 or 5 treatment groups of (10) mice as outlined inTables S1 and S2.
Rat PK Study. Animal Experiments. Eight 8-week-old
female Sprague−Dawley rats (Charles River Japan, Tokyo,
Japan) were provided with a standard diet (Oriental Yeast,
Tokyo, Japan) and water ad libitum. Following theacclimatization period (1 week), the animals were stratiﬁed
by body weight and randomly assigned as the AJICAP-ADC
groups that were treated with 5 mg/kg and the traditional
ADC group that were treated with 5 mg/kg. Each group4060

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ArticleFigure 2. Capability of AJICAP technology. (a) Syntheses of four diﬀerent ADCs derived from trastuzumab. (b) Syntheses of four diﬀerent ADCs
derived from rituximab. (c) Compatibility with various antibodies and toxic/non-toxic payloads. (d) Summary of DAR analysis and aggregation
after conjugation with various payloads.

described in Figure S1 in the Supporting Information) werereacted with 3A to produce two ADCs, and the respective
DARs were determined by quadrupole time-of-ﬂight mass
spectrometry (Q-TOF MS). Next, an investigation of the eﬀectof long-term storage on 3A was conducted. Compound 3A wasstored as a frozen matrix in a −80 °C freezer for 1 year, andthen the same material lots, before and after 365 days ofstorage, were evaluated by size exclusion chromatography(SEC) to determine the aggregation status. Conjugationeﬃcacy of the “1-year-old” thiol-mAb (3B) was also conﬁrmed
by reaction to the drug-linkers to produce ADCs. Surprisingly,
there was no appreciable diﬀerence between the antibodies
before and after 1 year of storage in both investigations(Figures S3 and S4). An in silico molecular modeling study(Figure 1c and Figure S2) revealed that the thiol group in 3A
was moderately exposed.19
Capability of AJICAP Technology. Next, the breadth ofcompatibility of AJICAP technology was conﬁrmed through 10
diﬀerent ADC syntheses (Figure 2 and Figures S5−S14)
including a pyrrolobenzodiazepine dimer (PBD) payload. In
addition to previously reported ADC syntheses [trastuzumabAJICAP-maytansinoid (4A) and trastuzumab-AJICAP MMAE(4B)],14,21 MC-MMAF (for trastuzumab-AJICAP-MMAF(4C)) and MC-VA-PBD (for trastuzumab-AJICAP-PBD(4D)) (the chemical structures of these payloads are describedin Figure S1) were reacted with the trastuzumab-thiol (3A).
Conjugation with a hydrophobic PBD drug-linker required theuse of propylene glycol as a co-solvent as Jeﬀery et al. havereported.22 Rituximab, an antibody reported to be less stable
than trastuzumab,23 was labeled using our standard platformconjugation conditions to successfully produce ADCs 5A−5D
obviating protocol optimizations or modiﬁcations. To date,dozens of diﬀerent antibodies among IgG1, IgG2, and IgG4
isotypes have demonstrated compatibility with this site-speciﬁc
conjugation protocol, including previous published reports:

anticoagulated plasma samples were evaluated with anautomated clinical chemistry analyzer (TBA-120FR, Toshiba
Medical Systems, Inc., Tokyo, Japan) to determine aspartate
aminotransferase (AST). EDTA-anticoagulated blood samples
were evaluated with an automated hematology analyzer(ADVIA2120, Siemens Healthineers AG., Munich, Germany)
to determine the platelet count (PLT).

■RESULTS
Stability Assessment of the AJICAP-Thiol Intermediate. Crucial to the establishment of this conjugation
technology’s breadth and robustness, the long-term stabilityof the ADC intermediate precursor containing site-speciﬁcfree-sulfhydryls was evaluated (Figure 1). As previously
reported,14 the peptide reagent (1) was reacted withtrastuzumab, a commonly used antibody for conjugation
studies, to aﬀord trastuzumab-peptide conjugates (2). Ourgroup has already reported the completion of a gram-scale
synthesis utilizing this well-established approach.17 The
proximity eﬀect of the peptide reagent assists this conjugation
to proceed in a site-speciﬁc manner due to the high aﬃnity ofthe peptide toward the Fc region (Figure 1b).18 Removal of
the peptide portion of (1) via tris-(2-carboxyethyl)-phosphinehydrochloride (TCEP)-mediated reduction and concomitantantibody interchain disulﬁde bond cleavage followed byoxidation using dehydroascorbic acid (DHAA) under mild
conditions to reform the reduced interchain disulﬁde bridges
provided the site-speciﬁcally thiol-modiﬁed trastuzumab (3A).
All puriﬁcation steps to remove impurities, including peptiderelated fragments, were carried out straightforwardly utilizing
tangential ﬂow ﬁltration, a well-known technique that enables
facile scale-up and future translation to kilogram-scale ADC
preparations in a manufacturing facility.20 To examine thereactivity of the installed thiol groups, drug-linkers MCCmaytansinoid and MC-VC-MMAE (their chemical structures
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ArticleFigure 3. DAR analysis and cell-based assays. (a) Summary of the in vitro cytotoxic activities of trastuzumab-based AJICAP-ADCs and unlabeledtrastuzumab (as control). (b) Summary of the in vitro cytotoxic activities of rituximab-based AJICAP-ADCs and unlabeled rituximab (as control).

Figure 4. In vivo xenograft assay to determine the MED of AJICAP-ADC. (a) Anti-tumor activity of anti-HER2 AJICAP-ADCs (DAR = 2) and TDM1 (DAR = 3.4) in the NCI-N87 xenograft tumor model. (b) ADC dose comparison plots. (c) Anti-tumor activity of anti-HER2 AJICAP-ADC(4B, DAR = 1.6) and stochastic cysteine-based ADC (6, DAR = 4.0) in the NCI-N87 xenograft tumor model. (d) Synthesis of stochastic cysteinebased ADC (6).

trastuzumab (IgG1), rituximab (IgG1), inﬂiximab (IgG1);
adalimumab (IgG1);14 denoxumab (IgG2);14 dupilumab(IgG4).14 A ﬂuorophore payload (Alexa Fluor) was also tested
for conjugation eﬃciency with AJICAP-thiol intermediates(Figures S3−S23). DAR analyses of the resulting ADCs wereconducted by Q-TOF MS (Figure 2d). Some variability in the
DAR values between each linker-payload was observed (range
1.5−1.9), attributed to diﬀerences in the eﬃciency of
conjugation for each drug-linker unit under unoptimized
conditions; however, no signiﬁcant diﬀerences in conjugationeﬃciency were observed between trastuzumab and rituximab
employing a platform conjugation method. Aggregation levels
measured after conjugation of the diﬀerent linker-payloads
were conducted by an SEC method that indicated that

rituximab-based ADCs contained slightly more aggregates than
trastuzumab-based ADCs.
In Vitro Cell-Based Assays. In vitro cell-based assays of
the ADCs were also conducted (Figure 3). To compare thecytotoxic activities of four diﬀerent AJICAP-ADCs derived
from trastuzumab, cell-based activity assays using HER2
antigen-overexpressing cell lines (NCI-N87: gastric cancer
cell and HCC1954: breast cancer cell) and a low HER2
antigen-expressing cell line (PC-3: human prostate cancer cell)were performed (Figure 3a). For rituximab-based ADCs, botha CD20 antigen-overexpressing cell line (SU-DHL-4) and an
antigen-negative expressing cell line (NCI-N87) were
evaluated. Both trastuzumab-based and rituximab-based
ADCs showed appreciable anti-tumor activity against the4062

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Articlevia the liver.27 On the other hand, due to reduced drug
decorating the antibody, lower DAR ADCs may retain
properties more similar to its parent antibody resulting in an
enhanced safety proﬁle; however, these low DAR stochastic
ADC mixtures often contain signiﬁcant DAR = 0 (naked
antibody), which may compete for limited tumor-associatedantigen and therefore result in lower in vivo eﬃcacy. Sitespeciﬁc ADCs created with AJICAP technology (and othersite-speciﬁc methods) have a narrow degree of heterogeneity
compared to stochastic DAR4 ADCs and can be more easily
optimized for a desired biological outcome.
Toxicology Study in Rats. To determine the MTD of the
ADCs, a rat acute toxicity study was conducted (Figure 5).
Mortality or severe toxicity could be observed at doses of 120
mg/kg of trastuzumab-AJICAP-MMAE (4B) and 40 mg/kg of
trastuzumab-stochastic-MMAE (6). Decreases in body weightor platelet (PLT) levels, and importantly increases in theserum aspartate aminotransferase (AST) levels, were notobserved at doses of up to 80 mg/kg of trastuzumab-AJICAPMMAE (4B) and 10 mg/kg of trastuzumab-stochastic-MMAE(6). Thus, the MTD values of trastuzumab-AJICAP-MMAE(4B) and trastuzumab-stochastic-MMAE (6) were estimatedto be at least 80 and 10 mg/kg, respectively.
Plasma Pharmacokinetics in Rats. To provide arationale for the observed expansion of the TI for our sitespeciﬁc-ADCs versus traditional stochastic ADCs, an in vivo rat
pharmacokinetics study was conducted (Figure 6). For thestochastic cysteine-based ADC, signiﬁcant detachment of the
payload was detected after several days (Figure 6a). In
contrast, signiﬁcantly less payload detachment was observed
over the course of the study by comparing the curves of total
mAb and the total ADC concentrations of the AJICAP-ADC(Figure 6b). Furthermore, over 21 days of the study, the totalmAb clearance of the AJICAP-ADC did not change
signiﬁcantly.

target antigen-overexpressing cells. In contrast, no activity
against the low target antigen-expressing cells was observed.
In Vivo Xenograft Assay to Determine the MED of
AJICAP-ADC. To determine the MED of AJICAP-ADCs,
mouse xenograft studies were evaluated (Figure 4).24
Trastuzumab-derived AJICAP-ADCs and Kadcyla (T-DM1),
a commercially available lysine-based stochastic ADC, were
evaluated in the HER2-positive NCI-N87 gastric cancer
xenograft model. The AJICAP-ADCs at doses of 5 mg/kg
displayed signiﬁcant tumor regressions comparable to Kadcyla(Figure 4a,b).
In addition to the lysine-based stochastic ADC, an AJICAPADC was also compared to a cysteine-based stochastic ADC
modeled after a well-established established structural format
in the ADC ﬁeld. This stochastic ADC was synthesized fromtrastuzumab by TCEP-mediated partial reduction of theinterchain disulﬁde bonds followed by conjugation with MCVC-MMAE to a DAR of 4 in the same manner as commerciallyapproved ADCs Adcetris, Polivy, and Padcev (Figure 4d).25,26
The stochastic MMAE-ADC 6 was compared against thesimilar AJICAP-MMAE-ADC 4B in eﬃcacy (Figure 4c), safety(Figure 5), and PK (Figure 6) studies. The optimal DAR for■DISCUSSION
A stable site-speciﬁcally labeled thiol intermediate is useful forat least two purposes. In terms of ADC manufacturing, a stableintermediate can be considered a “hold point” in the
conjugation process, enabling the possibility of minimizing a
manufacturing operation workload. In addition to thismanufacturing advantage, a stable thiol-modiﬁed antibody isa convenient intermediate in a discovery setting in that it may
be convergently converted to a variety of desired ADCs forearly-stage ADC screening (Figure 2c). These two desirable
attributes prompted us to perform a stability assessment of an
AJICAP-thiol antibody intermediate. In a previous literaturereport, a site-speciﬁc thiol intermediate was either immediately
used for payload conjugation once generated or was stored at−80 °C for later conjugation.5,29 There are no reports
speciﬁcally describing the eﬀects of long-term storage on asite-speciﬁc sulfhydryl-labeled antibody. Furthermore, unconjugated thiol groups present in the Fc region may trigger
undesired disulﬁde scrambling or cross-linking.29 Recently,
Tumey and co-workers synthesized a site-speciﬁc thiol
modiﬁed antibody via a novel enzymatic strategy; however,
the application of a carefully controlled re-oxidation step was
required to avoid undesired S−S bond formation by spatiallyadjacent thiol groups.29 This remarkable previous result
prompted our group to conduct stability assessments and in
silico molecular modeling analyses of installed thiol groups on
Lys248 introduced by our Fc-aﬃnity reagent. No observations

Figure 5. Rat acute toxicity study to determine the MTD of ADCs.
(a) Body weight change. (N = 5 female SD rats). (b) Aspartate
aminotransferase (AST) in rat serum at 2 days after administration.
(N = 5 female SD rats). (c) Platelet (PLT) levels in rat serum at 2days after administration. (N = 5 female SD rats).

an ADC depends on a large number of complex variables,
including the in-part expression level of the target antigen,hydrophobicity of linker-payload, and payload class.27,28
Stochastic ADCs produced by cysteine conjugation are a
mixture of diﬀerent compounds with varying DARs (DAR = 0,2, 4, 6, and 8 are typically the major species, and there areseveral forms of these ADCs with regard to payload placementon the antibody). Higher DAR ADCs may show greater
potency in in vitro and in vivo eﬃcacy models; however, they
may suﬀer greater oﬀ-target toxicities due to faster clearance
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ArticleFigure 6. Plasma concentration of total mAb and total ADC in rats measured by ELISA. (a) Trastuzumab-stochastic-MMAE (6). (b)Trastuzumab-AJICAP-MMAE (4B).

of cross-linking or disulﬁde-bridge formation were noted postintroduction of the thiol groups at Lys248 under long-term
storage conditions (Figure 1d) and multiple several freeze/
thaw cycles (data not reported). This discrepancy with Tumeyand co-workers’ investigation29 may be attributed to thediﬀerence in the nature of the modiﬁed sites. The thiol groups
installed on Lys248 are located in a hydrophobic pocket of the
Fc region (Figure 1c) and are moderately solvent exposed withthe sulfhydryl groups of the AJICAP-thiol potentially stericallyinhibited from reacting with other antibody thiol groups.
Furthermore, the distance between two newly formed thiolgroups was relatively far (over 70 Å apart, Figure S2), which
was considered to avoid cross-linking to form an intramolecular disulﬁde bond.31 These moderate reactivity andappropriate location of a thiol group could lead to keep
aggregation percentage via intermolecular disulﬁde formation
within the acceptance level and to avoid undesired crosslinking via intramolecular disulﬁde formation. This hypothesis
was supported by in silico molecular modeling studies (FigureS2). In 2018, Kozak and co-workers reported cysteine scanning
via antibody engineering to identify optimal conjugation sites
balancing aggregation propensity, payload conjugation eﬃciency, and plasma stability.30 In Kozak and co-workers’ report,
the installation of cysteine amino acids in the vicinity of theProtein A binding site resulted in either lower DARs after
conjugation or poor reoxidation-step outcomes. They used
immobilized antibody bound to Protein A beads for thiscysteine scanning study, and the beads may have interferedwith the conjugation process by steric hindrance for thosecysteines introduced in the vicinity of the Protein A binding
region. In our case, the Fc-aﬃnity reagent binds to theantibody similarly to Protein A and results in the introductionof a sulfhydryl group on the side-chain of a nearby lysine
residue. This newly installed moiety at Lys248 does notinterfere with the Protein A binding site due to its introduction
while the Fc-aﬃnity reagent is still bound to the antibody (theaﬃnity reagent is cleaved and removed after the covalent
modiﬁcation of Lys248), and antibodies conjugated in thismanner retain binding to Protein A. Additionally, thiols
installed at Lys248 in this manner displayed desirable reactivity(i.e., reoxidation chemistry, conjugation reactivity, andstability).
The conjugation investigation described in Figure 2
indicates compatibility of the installed AJICAP-thiol with
commonly employed ADC drug-linkers. During the reaction
with a relatively hydrophobic payload such as a PBD, the
AJICAP-thiol reacted with the drug-linker as desired. These
results indicated that AJICAP-thiol intermediates havepotential application to create a wide variety of antibody-

linked compounds. In addition to wide drug-linker compatibility, multiple antibodies, including IgG1, IgG2, and IgG4
subtypes, can be used for AJICAP conjugation as the Fcaﬃnity reagent binds similarly across these subtypes. The Fcaﬃnity reagent binds to an antibody Fc in a similar manner as
Protein A, and the binding site of this reagent to antibodies
overlaps with the binding site of the domain B of Protein A(Figure 1b). These results indicate that antibodies that can bebound to or puriﬁed with Protein A should also possess
potential reactivity for site-speciﬁc conjugation mediated by
the AJICAP Fc-aﬃnity reagent.
By applying a general, unoptimized method for conjugationand puriﬁcation for each ADC, we measured the resulting DAR
and aggregation (Figure 2d). Notably, the aggregation present
in the AJICAP-ADCs using the platform conjugation method14
resulted in ADCs containing 90−95% monomer. Our researchgroup has completed additional optimization studies to further
streamline the conjugation reaction sequence as well as
improve the characteristics of the resulting bioconjugates(these advancements will be discussed in future reports).
Cell-based assays (Figure 3) of the AJICAP-ADCs illustrated
antigen-mediated target selectivity and in vitro cytotoxic
activity reﬂective of the payload potency. Our previous
published work14 has demonstrated the retained aﬃnity of
AJICAP-ADCs based on trastuzumab toward HER2 measured
by surface plasmon resonance (SPR), and the presently
described in vitro assay results further supported that result.
CD20-reactive rituximab-based ADCs also showed expected
potency and target selectivity. These results indicated that
ADCs produced by AJICAP technology have multi-antibody
compatibility and the potential for use in ADC development
regardless of the antibody target or isotype so long as theantibody is reactive to the Fc-aﬃnity reagent and possesses alysine residue at position 248.
In vivo, the HER2+ NCI-N87 xenograft model (Figure 4a−c) was utilized to demonstrate that the eﬃcacy of trastuzumabAJICAP-MMAE was comparable to stochastic cysteine-based
ADCs. The MED of the stochastic cysteine-based ADC wasdetermined to be 2.5 mg/kg (Figure S26). TrastuzumabAJICAP-MMAE (DAR = 2) at 5 mg/kg showed tumor
inhibition comparable to 2.5 mg/kg stochastic trastuzumabMMAE (DAR = 4) (Figure 4c). An exploratory rat safety
toxicology study showed that trastuzumab-AJICAP-MMAE(4B) was well tolerated compared with trastuzumabstochastic-MMAE (6). Given the limited dosing of ourstudy, the MTD of trastuzumab-AJICAP-MMAE (4B) was
estimated to be at least 80 mg/kg, while the MTD of
trastuzumab-stochastic-MMAE (6) was estimated to be at least
10 mg/kg. Thus, the TI of trastuzumab-AJICAP-MMAE (4B)4064

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Mol. Pharmaceutics 2021, 18, 4058−4066

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Authors

was estimated to be qualitatively greater than trastuzumabstochastic-MMAE (6), taking both the respective MTDs and
MEDs into consideration.
The conjugation site (Lys248) is near to the antibody FcRnbinding region, and thus, potential interference with FcRnbinding has to be determined to fully characterize ADCs
conjugated via this site-speciﬁc methodology. A pharmacokinetic study in rats indicated that the AJICAP-ADC maintained
reasonable stability in circulation over a 3 week study (Figure6) compared to stochastic ADC 6. In our study, the measured
levels of total antibody from AJICAP-conjugated ADC 4B
indicated a half-life similar to that expected for an
unconjugated antibody (Figure 6b), and this ﬁnding was
supported by a recently published in vitro aﬃnity study using
biolayer interferometry (BLI) that indicates that conjugation at
Lys248 in this manner does not aﬀect FcRn interactions.32
This result and the trend of total ADC likely correspond to theincreased tolerability observed for the AJICAP-ADCs andsuggest that site-speciﬁc conjugation at Lys248 results in asuperior proﬁle with regard to both safety and in vivo stability
compared to stochastic-conjugated ADCs.
In conclusion, our research group has developed a chemical
conjugation technology to produce site-speciﬁc antibody−drugconjugates without the need for antibody engineering nor
enzyme application. This novel methodology termed AJICAP
utilizes an Fc-aﬃnity reagent and is compatible with a wide
range of antibody formats and can easily introduce two linkerpayloads per antibody using native IgGs. We succeeded increating a convenient set of thiol-labeled antibodies for use indiscovery research or as a hold point in manufacturing.
Furthermore, when compared against stochastically conjugated
ADCs in mouse eﬃcacy and rat PK and toxicology studies,
results from ADCs generated using this chemical site-speciﬁc
conjugation technique indicate a widening of the therapeuticindex, which may translate into clinically relevant beneﬁts asadditional pre-clinical study data becomes available. The
platform conjugation technology described herein enables
rapid, site-speciﬁc, and eﬃcient conjugation to a wide varietyof native antibodies with various linker-payloads to produce
next generation low-heterogeneity ADCs.

■

Yutaka Matsuda − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan; Ajinomoto Bio-Pharma
Services, San Diego, California 92121, United States;
orcid.org/0000-0003-2801-4706
Takuya Seki − Ajinomoto Co., Inc., Kawasaki-Shi, Kanagawa
210-8681, JapanKei Yamada − Ajinomoto Co., Inc., Kawasaki-Shi, Kanagawa
210-8681, Japan; orcid.org/0000-0002-7563-0176
Yuri Ooba − Ajinomoto Co., Inc., Kawasaki-Shi, Kanagawa
210-8681, Japan
Kazutoshi Takahashi − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan; orcid.org/0000-00033275-4109
Tomohiro Fujii − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Sayaka Kawaguchi − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Takahiro Narita − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Akira Nakayama − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Yoshiro Kitahara − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan

Complete contact information is available at:https://pubs.acs.org/10.1021/acs.molpharmaceut.1c00473
Author Contributions§

Y.M. and T.S. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
The authors wish to thank our colleagues from Ajinomoto Co.,
Inc. and Ajinomoto Bio-Pharma Services, Inc., as follows: Ms.
Noriko Hatada, Ms. Yumiko Suzuki, Dr. Shigeo Hirasawa, and
Mr. Ryusuke Hirama for technical assistance with AJICAP
conjugations; Ms. Natsuki Shkida, Ms. Reiko Yuji, and Dr.
Kazutaka Shimbo for critical opinion for antibody analysis; Ms.
Zhala Tawﬁq for technical assistance for ADC preparation; andDr. Akira Chiba for helpful discussions and suggestions inmanuscript preparation. The authors also wish to thank ourpartners as follows: Dr. Bret Stephens from Rincon Bioscience,
LLC for assistance with the xenograft assay; Dr. Noriko
Nakamura from Kamakura Techno-Science, Inc. for assistancewith cell-based assays.

ASSOCIATED CONTENT

sı Supporting Information*

The Supporting Information is available free of charge at
h t t p s : / / p u b s . a c s . o r g / d o i / 10 . 1 02 1 / a c s .m o l p h a rm a ceut.1c00473.

■

Details of the in vivo studies, ﬁgures supporting themolecular modeling, HPLC chromatograms, and QTOF MS analyses (PDF)■

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AUTHOR INFORMATION

Corresponding Authors

Brian A. Mendelsohn − Ajinomoto Bio-Pharma Services, San
Diego, California 92121, United States; Present
Address: Present address: Exelixis Inc., 1851 Harbor Bay
Pkwy, Alameda, California 94502, United States(B.A.M.); orcid.org/0000-0002-6339-2377;
Email: bmendelsohn@exelixis.com
Tatsuya Okuzumi − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan; orcid.org/0000-00015839-6958; Email: tatsuya_okuzumi@ajinomoto.com
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