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ArticleAJICAP Second Generation: Improved Chemical Site-Specific
Conjugation Technology for Antibody−Drug Conjugate Production
Tomohiro Fujii,# Yutaka Matsuda,* Takuya Seki, Natsuki Shikida, Yusuke Iwai, Yuri Ooba,
Kazutoshi Takahashi, Muneki Isokawa, Sayaka Kawaguchi, Noriko Hatada, Tomohiro Watanabe,
Rika Takasugi, Akira Nakayama, Kazutaka Shimbo, Brian A. Mendelsohn, Tatsuya Okuzumi,
and Kei Yamada*,#Downloaded via 183.177.131.183 on March 31, 2023 at 06:32:08 (UTC).
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sı Supporting Information*

ABSTRACT: The site-directed chemical conjugation of antibodies remains an area of great interest and active efforts within the
antibody−drug conjugate (ADC) community. We previously reported a unique site modification using a class of immunoglobulin-G(IgG) Fc-affinity reagents to establish a versatile, streamlined, and site-selective conjugation of native antibodies to enhance thetherapeutic index of the resultant ADCs. This methodology, termed “AJICAP”, successfully modified Lys248 of native antibodies to
produce site-specific ADC with a wider therapeutic index than the Food and Drug Administration-approved ADC, Kadcyla.
However, the long reaction sequences, including the reduction−oxidation (redox) treatment, increased the aggregation level. In thismanuscript, we aimed to present an updated Fc-affinity-mediated site-specific conjugation technology named “AJICAP second
generation” without redox treatment utilizing a “one-pot” antibody modification reaction. The stability of Fc affinity reagents was
improved owing to structural optimization, enabling the production of various ADCs without aggregation. In addition to Lys248
conjugation, Lys288 conjugated ADCs with homogeneous drug-to-antibody ratio of 2 were produced using different Fc affinity
peptide reagent possessing a proper spacer linkage. These two conjugation technologies were used to produce over 20 ADCs fromseveral combinations of antibodies and drug linkers. The in vivo profile of Lys248 and Lys288 conjugated ADCs was also compared.
Furthermore, nontraditional ADC production, such as antibody−protein conjugates and antibody−oligonucleotide conjugates, wereachieved. These results strongly indicate that this Fc affinity conjugation approach is a promising strategy for manufacturing sitespecific antibody conjugates without antibody engineering.

■

INTRODUCTION
In the past decade, chemical conjugation to produce sitespecific antibody−drug conjugates (ADCs) has received
considerable attention in the oncology field.1,2 The firstexample of site-specific ADC approved by the Food and Drug
Administration was relished by Daiichi-Sankyo’s uniquechemical conjugation approach using a high drug-to-antibody
ratio (DAR) technology consisting of reduction of allinterchain disulfide bonds, followed by thiol-maleimide
coupling with their original drug linker (deruxutecan).3,4
Since this technique cleaves all interchain disulfide bonds, it ischaracterized by its ability to obtain near-homogeneous ADCs.
© XXXX The Authors. Published byAmerican Chemical Society

This elegant solution, even though the resultant ADCs containa few lower DAR species,5 enables the production of nearlyhomogeneous ADC with DAR = 8 without aggregation andloss of antibody properties, such as antigen binding.3,4
However, this technology is limited by compatible drug
Received: January 30, 2023
Revised: February 27, 2023

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ArticleFigure 1. Comparison of AJICAP first and second technology: (a) AJICAP first-generation technology, including redox treatment (steps 2 and 3).
(b) AJICAP second generation, including one-pot peptide or linker cleavage reaction. (c) Comparison of AJICAP first and second generation
reagents. (d) The structures of AJICAP reagents for Lys248 and Lys288 conjugation.

linkers. For example, MC-VC-MMAE, a commonly used ADC
drug linker in the market,6 cannot be applied to this high DAR
technology because hydrophobicity causes aggregation, lowering the physical and biological profile of the ADCs.7
Moreover, a high DAR ADC may not be an ideal molecularformat for every ADC. Another ADC manufactured by DaiichiSankyo’s conjugation technology using deruxutecan in theclinical stage has a DAR lower than 8, supporting that theappropriate DAR must be adjusted based on the pharmacologyof the target and/or the toxicity profiles of ADCs.8 The Seattle
Genetics (now Seagen) group published an in vivo
pharmacokinetics (PK) and efficacy comparison study between
chromatographically purified DAR = 2, DAR = 4, and DAR = 8
ADCs, suggesting that the lower DAR, especially DAR = 2, had
an ideal biological profile.9 Several developments have beenreported that improve the PK profile by reducing thehydrophobicity of high DAR ADCs;2,7 however, the DAR =2 ADC remains a promising ADC format because of its simple
structure that does not require hydrophilic linkers.
Owing to the limitations in high DAR technology and thepotential requirement of DAR = 2 production, the Ajinomotogroup commenced the development of site-specific conjugation technology utilizing Fc-affinity peptide reagents in 2019.10
The proof-of-concept study revealed that this affinity-guided
approach enabled the modification of a specific lysine in the Fc
region of various antibodies, including immunoglobulin-G(IgG)1, IgG2, and IgG4, to produce homogeneous DAR = 2
ADCs. This site occupancy was proved via several analyses,
including trypsin-digested peptide mapping, which revealed
that the conjugation site of the resultant ADCs is solely
Lys248.11 The Fc region is a constant domain of every
antibody;12 therefore, this Fc-affinity conjugation technology,
termed AJICAP, enables the application (theoretically) of all
antibodies without complicated reaction optimization. Furthermore, biological evaluation of site-specific AJICAP-ADC
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ArticleTable 1. Site-Specific Peptide Conjugation

Peptide antibody ratio (PAR)a
Entry

Antibody

Conjugation site

Conjugation method

AJICAP reagent

0

1

2

3

Averageb PAR

1
2
3
4
5
6

Trastuzumab
Trastuzumab
Trastuzumab
Trastuzumab
Rituximab
Rituximab

K248
K248
K288
K288
K248
K288

First Gen.
Second Gen.
First Gen.
Second Gen.
Second Gen.
Second Gen.

1a
1b
6a
6b
1b
6b

23%
-

5%
1%
50%
2%
10%
-

95%
98%
26%
95%
90%
97%

1%
1%
3%
3%

1.9
2.0
1.1
2.0
2.0
2.0

a

Calculated by Q-TOF-MS. bAnalyzed by HIC-HPLC.

consisting of cytotoxic monomethyl auristatin E (MMAE)
indicated that AJICAP first-generation technology enhanced
therapeutic index compared with stochastic cysteine-based
ADCs.13 This improvement in the in vivo profile was also
observed with different payload (maytansinoid) cases. Sitespecific AJICAP-ADC, consisting of maytansinoid, demonstrated higher in vivo efficacy and tolerability than Kadcyla,14 aclinical ADC approved by the Food and Drug Administration.
These results indicate that AJICAP technology has great
potential for producing next-generation ADCs.
Although AJICAP first-generation technology is a promising
approach for manufacturing site-specific ADCs, several
challenges remain (Figure 1a). This previous approach requirestris(2-carboxyethyl)-phosphine hydrochloride (TCEP) reduction to cleave the linkage between antibodies and AJICAP
peptide reagent to install thiol groups on specific lysine.
However, this reduction also cleaves the disulfide bonds ofinterchain cysteines in antibodies. Therefore, the disulfides
must be reconstructed following the reoxidation step using
dehydroascorbic acid. This redox treatment is also used in
THIOMAB conjugation technology;15 however, it can lead todisulfide bond scrambling.2 A more critical concern in thisapproach is the risk of aggregation. Small amounts ofaggregates (5−10%) were found in ADCs produced using
the AJICAP first-generation technology.13 Additionally, a morestreamlined manufacturing sequence consisting of fewer
reaction steps is preferred from the chemical manufacturingcontrol perspective.
Our research group performed optimization studies tostreamline the conjugation reaction sequence to overcome
these issues (Figure 1b). This improved technology, termed“AJICAP second generation,” enables the production of sitespecific ADC while preventing aggregation due to selective
cleavage reaction utilizing thioester chemistry. The stability of
the AJICAP peptide reagent in buffer solution was improved,
resulting in higher selectivity to target lysine and improved
DAR value of ADCs. This thioester-based strategy provides
two different AJICAP peptide reagents to access two different
conjugation sites (Lys248 and Lys288). In vivo biologicalstudies of Lys248- and Lys288-based ADCs revealed
comparable efficacy and tolerability. Furthermore, theapplication of conjugation chemistry to nontraditional ADC
production was investigated.

■

RESULTS AND DISCUSSION

AJICAP Reagent Design and Antibody Modification.
There are two major challenges to improving AJICAP peptide
reagents (Figure 1c): (1) replacing the unstable N-succinimide(NHS) group with an amine group of lysine in the antibody
and (2) replacing disulfide bonds in the AJICAP reagent
requiring redox treatment in the ADC synthesis scheme.
Considering the shelf life and reactivity balance, thethiophenyl ester group was selected as a reactive group for
antibody modification.16 The thiophenol group has higher
stability and slightly lower reactivity than the NHS group.
However, we expected that the proximity effect of the Fc
affinity peptide would enhance reactivity for antibody
modification. Additionally, the longer shelf life of thiophenyl
ester in the reaction buffer solution compared to NHS ester
improved the modification yield and site-specificity. An
alkylthioester linkage was selected to replace the disulfide
bond in the AJICAP first-generation reagent. Alkylthioesters
are well-known protecting groups of thiols. N-Succinimidyl Sacetylthioacetate is a widely used alkylthioester reagent forprotein functionalization.17 Modification of lysine of proteinusing N-succinimidyl S-acetylthioacetate followed by specificdeprotection using hydroxylamine can install sulfhydryl groups
on the lysine residue. An optimized AJICAP reagent (1b) was
produced from these thioesters (alkyl and phenyl) (Figure 1d;
Supporting Information (SI), Figure S1−S3). Furthermore,another AJICAP peptide reagent (6b) possessing different
affinity peptide sequence was designed to modify the Lys288 ofantibodies (SI, Figure S4 and S5). In previous studies, themodification yield of Lys288 was lower than that of Lys248;
therefore, we installed a rigid architecture18 to allow thereactive thiophenyl ester group of the AJICAP reagent to beclose to Lys288 in buffer conditions.
Trastuzumab and rituximab were used to demonstrate
thiophenyl ester chemistry (Table 1).
The conversion yield from antibodies to conjugates wascalculated by measuring the peptide-to-antibody ratio (PAR)using quadrupole time-of-flight mass spectrometry (Q-TOF

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ArticleTable 2. Stepwise and One-Pot Conversion from Native Mabs to Antibody-Thiol (4)Entry

Antibody

Subtype

Conjugation site

AJICAP reagent

Average PARa of 2b

Aggregation of 4

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

Trastuzumab
Trastuzumab
Rituximab
Rituximab
Infliximab
Infliximab
Cetuximab
Cetuximab
Denosumab
Denosumab
Pembrolizumab
Pembrolizumab
Fc-Protein
Fc-Protein
Polyclonal antibody
Polyclonal antibody

IgG1
IgG1
IgG1
IgG1
IgG1
IgG1
IgG1
IgG1
IgG2
IgG2
IgG4
IgG4
-

K248
K288
K248
K288
K248
K288
K248
K288
K248
K288
K248
K288
K248
K288
K248
K288

1b
6b
1b
6b
1b
6b
1b
6b
1b
6b
1b
6b
1b
6b
1b
6b

2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0b
2.0b
2.0
2.0

<1%
1.0%
<1%
1.0%
<1%
<1%
<1%
1.7%
<1%
<1%
<1%
<1%
1.0%
1.0%
2.6%
3.3%

a

Analyzed by HIC-HPLC. bAnalyzed by Q-TOF. cAnalyzed by SEC-HPLC.

MS) (SI, Figures S6−S10)13 and hydrophobic interaction
chromatography (HIC)-HPLC (SI, Figures S11−S21).19
In the previous Lys248 conjugation, AJICAP reagent (1a)provided higher PAR (entry 1);10 however, AJICAP reagent(1b), consisting of a thiophenyl group, achieved further
reactivity optimization to meet the targeted PAR and improved
PAR = 2 selectivity (entry 2). In the case of Lys288
conjugation, further optimization studies were needed. In
previous studies, the first generation AJICAP reagent (6a)provided insufficient PAR (entry 310). To understand thisphenomenon, we performed a structural analysis of thecomplex of the base peptide (termed Z34C) of reagents 6aand 6b, with the Fc protein;20,21 the predicted distance fromthe Z34C peptide to Lys288 of the antibody was farther thanthe predicted distance from FcIII-like peptide22 to Lys248 (SI,
Figure S22). These results indicate that the AJICAP reagent
for Lys288 modification requires a longer length linker thanthe AJICAP reagent for Lys248 modification (SI, Figures S5,
S8, and S14).23 Therefore, we tuned the linker lengths of thepeptide reagents. Several groups have reported unique affinityguided protein modification technologies; in some cases, rigid
architectures, such as piperazine18 and proline24 in the linker
portion, are powerful tools for reaching the reactive group to
target amino acids with improved conversion. These reports
prompted us to screen linkers to improve the modification

efficiency of Lys288, and the aromatic ring was found to be asuitable linkage. The buffer pH in peptide conjugation step
depended on the binding affinity of the peptide moiety of the
AJICAP reagents; in the case of Lys 248 conjugation, FcIII-like
peptide showed the highest binding affinity in acidic buffer(around pH 5.5). On the other hand, Z34C, which was usedfor Lys 288 conjugation, showed favorable affinity strength inslightly alkaline conditions (around pH 8.0, data not shown).
In both reagents (1b and 6b), the following linker cleavage
using hydroxylamine was completed (SI, Figures S15, S16, S20,
and S21), indicating that the thiophenyl group reacted withlysine in the antibody, while no side reaction via alkylthioester
occurred during peptide conjugation.
The Capability of AJICAP Technology. The compatibility of this conjugation chemistry using several antibodies wasevaluated via HIC-HPLC analysis (SI, Figures S23−S44). In
the traditional cysteine-conjugation case, the antibody
characteristics affected the reactivity and DAR of the resultant
ADCs;25 therefore, a conjugation examination using several
ADCs with different isoelectric points (PIs)26 was conducted
to understand the reaction tolerability of AJICAP secondgeneration technology. In addition to trastuzumab (PI = 8.8,
entries 1 and 2) and rituximab (PI = 9.1, entries 3 and 4),
infliximab (PI = 7.6, entries 5 and 6), cetuximab (PI = 8.7,
entries 7 and 8), denosumab (PI = 8.9, IgG2, entries 9 and 10),Dhttps://doi.org/10.1021/acs.bioconjchem.3c00040
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ArticleTable 3. ADC Syntheses

Entry

mAb

Drug-linker

Conj. site

DAR by Q-TOF

DAR by HIC

Aggregation

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Trastuzumab
Trastuzumab
Trastuzumab
Trastuzumab
Trastuzumab
Trastuzumab
Trastuzumab
Trastuzumab
Trastuzumab
Trastuzumab
Rituximab
Rituximab
Rituximab
Rituximab
Rituximab
Rituximab
Rituximab
Rituximab
Rituximab
Rituximab

MCC-Maytansinoid
MC-VC-PAB-MMAE
MC-MMAF
Tesirine
MC-GGFG-Dxd
MCC-Maytansinoid
MC-VC-PAB-MMAE
MC-MMAF
Tesirine
MC-GGFG-Dxd
MCC-Maytansinoid
MC-VC-PAB-MMAE
MC-MMAF
Tesirine
MC-GGFG-Dxd
MCC-Maytansinoid
MC-VC-PAB-MMAE
MC-MMAF
Tesirine
MC-GGFG-Dxd

K248
K248
K248
K248
K248
K288
K288
K288
K288
K288
K248
K248
K248
K248
K248
K288
K288
K288
K288
K288

1.9
1.9
1.8
1.9
2.0
1.8
1.7
1.9
2.0
2.0
1.9
1.8
1.8
1.8
1.9
1.9
1.7
1.9
1.9
2.0

1.9
1.9
1.9
1.9
1.9
1.9
1.8
1.9
1.9
1.8
1.9
1.9
1.8
1.8
1.8
1.8
1.8
1.9
1.9
1.8

1.8%
1.9%
1.8%
2.0%
1.9%
2.5%
2.0%
2.5%
2.6%
2.5%
2.0%
1.8%
2.2%
3.8%
2.0%
3.3%
3.0%
3.2%
3.2%
2.0%

and pembrolizumab (PI = 8.7, IgG4, entries 11 and 12) were
evaluated. In all cases, both AJICAP reagents (1b and 6b)reacted sufficiently with these mAbs to produce conjugateswith PAR = 2.0, indicating that this conjugation does notdepend on the PI difference or IgG subtypes. These AJICAP
reagents also modified the Fc protein (entries 13 and 14,
confirmed using Q-TOF analysis (SI, Figures S45 and S46)),
demonstrating the potential use of this Fc-modification
technology for Fc-fusion protein production. Fc-Fusion is acommonly used technique for prolonging the in vivo half-life ofsmall-and medium-sized molecules.27 Furthermore, this powerful conjugation technology enabled the modification ofpolyclonal antibodies (whole IgGs purified from humanserum) (entries 15 and 16).28 Sequential linker cleavage
using excess equivalents of hydroxylamine enables the releaseof an affinity peptide portion, providing site-specific thiolincorporating antibodies (4). In all reaction cases, aggregation
percentages analyzed using size exclusion chromatography(SEC) were retained postconjugation or linker cleavage (SI,
Figures S47−S62).
In addition, we succeeded in omitting the purification stepafter AJICAP peptide conjugation. As described in Table 2, theone-pot reaction of peptide conjugation followed by linker
cleavage modified all antibodies, with the same efficiency andaggregation profile as the stepwise conversion. This streamlined process is applicable to ADC manufacturing.
ADC Syntheses. Over 20 ADCs were synthesized todemonstrate the compatibility of antibody-thiols (4) produced
by AJICAP second-generation technology (Table 3).
There are various drug linkers in the current ADC field, eachwith unique hydrophobicity and reactivity.29 Therefore, we
evaluated five drug linkers (MCC-maytansinoid, MC-VC-PAB-

MMAE, MC-MMAF, Tesirine, and MC-GGFG-Dxd; Figure
S63 in SI presents their structures). All ADCs converted fromtrastuzumab-Lys248-thiol, trastuzumab-Lys288-thiol, rituximab-Lys248-thiol, and rituximab-Lys288-thiol had higher
DAR values than previously reported (AJICAP first generation
production).10 DAR was determined using two different
analytical methods (Q-TOF MS and HIC-HPLC) (SI, Figures
S64−S83 and S84−S103). The average DAR values derived
from two different analyses were indistinguishable. The
measured differences are attributed to method sensitivity,
accuracy, or linearity.30,31 The aggregation percentage of all
ADCs was less than 3.0% (SI, Figures S104−S123), whichwere not significantly different from the naked antibodies(trastuzumab and rituximab). These results indicated that thisimproved technology could be a more practical approach forvarious ADCs production than previous AJICAP technology.32
Conjugation Site Determination. The conjugation siteof these trastuzumab-thiols (4a derived from 1b and 4b
derived from 6b) was determined using peptide mapping
analysis (Figure 2). In our previous peptide mapping analysis,
the sequence coverage was approximately 80% because of asingle enzyme (trypsin) digestion.10 To increase this coverage,
double enzymatic digestion was performed using trypsin andLys-C.33 In addition, we optimized HPLC condition including
a long gradient and newly constructed HPLC system toincrease peak resolution (see Experimental Section). Consequently, the sequence coverage was improved to more than
95%. The thiol adduct was compared to the results, and targetspecific modification of AJICAP-antibody-thiols (Lys248 andLys288) was indicated (Figure 3). Among detected lysines, thetarget site (Lys248 and Lys288, respectively) was specificallysubject to 3-(2-amino-2-oxo-ethyl) sulfanylpropionate modE

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ArticleFigure 2. Summary of lysine residue search by BioPharma Finder: (a) analysis of trastuzumab-Lys248-thiol (4a); (b) analysis of trastuzumabLys288-thiol (4b). Each result shows the high specificity of the AJICAP reaction.

Figure 3. In vivo efficacy comparison between Lys248-ADC and Lys288-ADC: (a) Antitumor activity of anti-HER2 trastuzumab-Lys248-MMAE(5a) in the NCI-N87 xenograft tumor model. (b) Antitumor activity of anti-HER2 trastuzumab-Lys288-MMAE (5b) in the NCI-N87 xenograft
tumor model.

ification. In fact, for the AJICAP-Lys248-thiol sample, we
detected a peptide fragment including two candidate residues
Lys246 and Lys248 (THTCPPCPAPELLGGPSVFLFPPK246PK248DTLMISR) for modification. In this analysis, Lys-C andtrypsin for digestion were used; therefore lysines and arginines
excepting followed by proline are recognized and cleaved.
Lys246 is located just behind the proline and is inhibited fromenzymatic cleavage, while Lys248 should be cleaved by the
enzyme. From these considerations, we concluded Lys248 is
specifically modified. Similarly for AJICAP-Lys288-thiol, we
identified FNWYVDGVEVHNAK288TK290PR, having two
lysines Lys288 and Lys290. We concluded that AJICAP-

Lys288-thiol achieved specific modification at Lys288 for thesame reason. Detailed extracted ion chromatogram (XIC) andMS/MS results supporting the site-specificity of two antibodythiols (Lys248 and Lys288) are shown in SI, Figures S124 and
S125.
In Vivo Biological Evaluation of AJICAP-ADCs. In
previous studies, our research group revealed enhanced
therapeutic index using AJICAP conjugation technology thanstochastically conjugated ADCs.13,14 Following these evaluations, we examined the biological evaluation of AJICAP-ADCs
produced using second-generation methods (Figure 3). In
addition, Lys288-conjugated ADC was evaluated to compare
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ArticleFigure 4. PK profile comparison between Lys248-ADC and Lys288-ADC: (a) Plasma concentration of total mAb (black line) and total ADC (blueline) of anti-HER2 trastuzumab-Lys248-MMAE (5a) in rats measured using ELISA. (b) Plasma concentration of mAb (black line) and total ADC(blue line) of trastuzumab-Lys288-MMAE (5b) in rats measured using ELISA.

Table 4. Binding Kinetics against Human FcRn
Entry
1
2
3

AnalyteTrastuzumab
Trastuzumab-K248-MMAE (5a)Trastuzumab-K288-MMAE (5b)Conjugation site

Kd (M)−95.65 × 10
5.60 × 10−9
7.22 × 10−9

K248
K288

Kon (1/Ms)

Kdis (1/s)

5

3.77 × 10−3
8.48 × 10−4
3.83 × 10−3

6.67 × 10
1.52 × 105
5.30 × 105

Figure 5. Synthesis of novel format antibody conjugates. The production of an antibody-SiRNA conjugate (8) (upper). The production of an
antibody−protein conjugate (12) (lower).

the in vivo efficacy of these two conjugation sites.
Trastuzumab-Lys248-MMAE (5a) and trastuzumab-Lys288MMAE (5b) were selected in these biological studies.
First, two different doses of trastuzumab-derived AJICAPADC (5a) (conjugation site: Lys248, payload: MMAE) were
evaluated in a HER2-positive NCI-N87 gastric cancer
xenograft model (Figure 4a). The Lys248 conjugated ADC(5a) at a dose of 5 mg/kg displayed significant tumor
regression, comparable to previous ADC produced using the
AJICAP first-generation technology.13 The other AJICAPADC (5b) (conjugation site: Lys288, payload: MMAE) also
displayed significant efficacy at a 5 mg/kg dosage (Figure 4b).
These results indicate that AJICAP conjugation technology can
produce ADCs with comparable efficacy that are independentof conjugation site and generation. In previous studies,34 theminimum effective dose of stochastic DAR = 4 trastuzumabMMAE ADC produced via native cysteine conjugation was 2.5
mg/kg. Despite the DAR number difference (stochastic ADC= 4, AJICAP-ADC = 2), the in vivo efficacy of AJICAP-ADCs
was comparable to that of stochastic ADCs.
AJICAP conjugation technology modified the antibody’s Fc
region; therefore, the resultant ADC was at risk of losing

binding affinity to FcRn because of steric interference by the
payload linker conjugated close to the FcRn binding site.22
However, in 2021, our research group discovered that AJICAPADC (conjugation site: Lys248, payload: MMAE) retained
FcRn binding affinity postconjugation.35 These findings arereasonable considering the plasma stability of AJICAP-ADCs
measured in a rat PK study.13 In this study, the FcRn binding
affinity and PK profile of two different AJICAP-ADCs (5a and5b) (conjugation site: Lys248 or Lys288, payload: MMAE)
produced using second-generation technology were confirmed.
Biolayer interferometry analysis36 of these ADCs, as in aprevious study35 revealed that both ADCs had comparable
binding strength for immobilized FcRn with naked trastuzumab (Table 4). These affinity results of two different ADCs
provided via biolayer interferometry can be rationalized fromtheir molecular structures (SI, figure S126). Lys248 andLys288 are acceptable sites for FcRn binding that avoids sterichindrance due to conjugated payload linkers. Rat PK studies
supported this FcRn binding result (Figures 4). Total antibody
levels from two AJICAP-conjugated ADCs (5a and 5b)(conjugation site: Lys248 or Lys288, payload: MMAE)
indicated a half-life similar to that of an unconjugatedG

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antibody. Total ADC analysis in rat PK studies indicated thatthese AJICAP-ADCs demonstrated sufficient stability in blood
circulation. In contrast, stochastic ADC produced via cysteine
conjugation technology showed insufficient stability in rats.13
Furthermore, we conducted an initial toxicology study of
AJICAP-ADC (5b) (conjugation site: Lys288, payload:
MMAE), which showed that this ADC did not negatively
impact body weight changes in rats at the 80 mg/kg dosage(single dose, SI, Figure S127). The other ADC (5a)(conjugation site: Lys248, payload: MMAE) has alreadybeen evaluated in a rat toxicology study, which indicatedthat the maximum tolerated dose was >80 mg/kg.13 In
previous studies, the estimated maximum tolerated dose valueof trastuzumab-stochastic MMAE was approximately 10 mg/kg(single dose, previously reported13). These results indicate thatthe two AJICAP-ADCs have significantly wider tolerability
than traditional stochastic ADCs. Further toxicological studies
to determine the maximum tolerated dose of Lys288
conjugated ADC are underway.
Based on these in vivo efficacy, PK, and initial safety studies,
we concluded that AJICAP second-generation technology
enables the production of site-specific ADCs with higher
therapeutic indexes than traditional stochastic ADCs.
In these in vivo data sets, no significant differences wereobserved in Lys248-ADCs and Lys288-ADCs. In the antibody
development, some point mutations in the Fc region of an
antibody to enhance FcRn binding were reported.37 Some of
their mutated antibodies might lose their affinity for the
AJICAP-Lys248 peptide reagent. The discovery of a novel
AJICAP-ADC conjugated to Lys288 described herein may
provide another alternative to using Fc region-mutatedantibodies for AJICAP technology.
Application to Novel Format Antibody Conjugates.
Recently, innovations have enlightened the oncology or
bioconjugation community and broadened the research anddevelopment area to include ADC alternative antibody−oligonucleotide conjugates38 and antibody−protein conjugates.39 Feasibility studies were performed using model
conjugates to meet the potential requirements for producing
these novel format antibody conjugates (Figure 5).
As a model SiRNA compound, we used a SiRNA compound
whose sequence targets peptidylprolyl isomerase B (PPIB,
cyclophilin B) reported by the Genentech group.40 Following
their report, model SiRNA possessing C6 amine linker wasconverted to a maleimide conjugate (7) by amidation. This
maleimide labeled SiRNA was conjugated with antibodyLys248-thiol (4a) to afford the antibody−SiRNA conjugate(8) by thiol-maleimide coupling (detailed information is in theExperimental Section in SI). The resulting conjugate (8) was
purified using preparative SEC-HPLC to remove excess
unreacted SiRNA from the antibody-conjugate composition.
SDS-PAGE analysis of this conjugate revealed a considerable
conversion yield (SI, Figure S128). Lysozyme was selected as
the model compound for protein conjugate production.
Random lysine conjugation using an azide-NHS reagent
produced the azide-incorporated lysozyme (11). AntibodyDBCO (10) transformed from antibody-Lys248-thiol (4a) andreacted with this azide-lysozyme (11) to form an antibody−protein conjugate (12). A Q-TOF MS analysis of (12)revealed that this conjugation proceeded smoothly (SI, Figures
S129−131). These results indicate that the AJICAP conjugation strategy can be applied to produce ADCs with small
cytotoxic molecules and ADC alternatives whose payloads are

medium-sized molecules. Further investigations, including
DAR determination, process development, and in vitro andin vivo evaluation, are currently ongoing.

Article■CONCLUSION
Our research group has completed proof-of-concept studies of
AJICAP second-generation and improved chemical conjugation technology using Fc-affinity reagents. This novel methodology is compatible with various antibody formats, including
antibody fragments and polyclonal antibodies. The linker
structural tuning enabled site-specific modification of native
IgGs at the novel conjugation site (Lys288) with a highconversion yield. This conjugation technology can easily
introduce two payload linkers per native antibody, producing
over 20 site-specific ADCs without aggregation during thereaction. In vivo biological studies, including mouse efficacy, rat
PK, and toxicology studies, of ADCs produced by this chemicalsite-specific conjugation technology indicate a widened
therapeutic index.
Furthermore, preliminary feasibility studies for application to
nontraditional ADCs, including antibody−oligonucleotide
conjugates and antibody−protein conjugates, were completed.
This technology enables rapid, versatile, streamlined, sitespecific conjugation to various native antibodies with several
linker payloads, including medium-sized molecules, to produce
next-generation antibody conjugates.

■

EXPERIMENTAL PROCEDURES
Materials. Human IgG1 trastuzumab (Herceptin) andrituximab (Rituxan) were purchased from the Roche
Pharmaceutical Company (Switzerland). Human IgG1 infliximab (Remicade) was purchased from Sigma-Aldrich (USA).
Human IgG1 cetuximab (Erbitux), Human IgG2 denosumab(Prolia), and human IgG4 pembrolizumab (Keytruda) werepurchased from Midwinter. Polyclonal antibody (human IgG
Whole molecule, cat#: 143−09501) was purchased from
Fujifilm Wako. Human IgG1 Fc Recombinant Protein (cat#:
A42561) was purchased from Thermo Fisher Scientific. MCCmaytansinoid (cat#: TCRS-1262) and MC-MMAF(CAS#:1228105−51−8) were purchased from Abzena(USA). MC-VC-MMAE (CAS#: 646502−53−6), MCPEG8-VA-PBD (CAS#: 1595275−62−9), and MC-GGFGDxd (CAS#: 1599440−13−7) were purchased from NJ
Biopharmaceuticals, LLC (USA). All other chemical reagentswere purchased from Sigma-Aldrich (USA).
Instruments and Analytical Methods. The ADC
concentration and recovery were measured using the Slope
Spectroscopy method with a Solo-VPE system.13
Q-TOF MS analysis was performed as previously reported.13
Hydrophobic interaction chromatography-HPLC analysis
was performed as previously reported.19
The SEC-HPLC analysis of antibody-thiols (4) was
performed using an AdvanceBio SEC column (200 Å, 4.6 ×
300 mm, 1.9 μm) as previously reported.28
SEC-HPLC analysis of ADCs (5) was performed using
Waters ACQUITY UPLC Protein BEH SEC column (200 Å,
4.6 × 300 mm, 1.7 μm) as previously reported.28
Biolayer interferometry assay to analyze FcRn binding was
performed as previously reported.36
Synthetic Protocols to Produce AJICAP Reagents.
Supporting Information contains detailed synthetic protocols
for producing AJICAP reagents.

H

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Bioconjugate Chemistrypubs.acs.org/bc

AJICAP Peptide Reagent Conjugation. Lys248 Conjugation Using AJICAP Reagent (1b). We added 2.5 equiv of
AJICAP reagent (1b) (20 mM in DMF) to each mAb (10 mg/
mL, 20 mM acetate buffer, pH 5.5), and the mixture wasincubated for 1 h at 20 °C. After 1 h, the reaction mixture waspurified using NAP-25 desalting columns and eluted with 20
mM acetate buffer (pH 5.5).
Lys288 Conjugation Using AJICAP Reagent (6b) or (6c).
Six equivalents of the AJICAP reagent (6b or 6c (structure inSI)) (20 mM in DMF) were added to each mAb (10 mg/mL,
20 mM borate buffer, pH 8.2), and the mixture was incubatedfor 1 h at 20 °C. After 1 h, the reaction mixture was purified
using NAP-25 desalting columns and eluted with 20 mM
acetate buffer (pH 5.5).
Lys248 Conjugation Using AJICAP Reagent (1b) Followed
by Linker Cleavage (One-Pot). We added 2.5 equiv of AJICAP
reagent (1b) (20 mM in DMF) to each mAb (10 mg/mL, 20
mM acetate buffer, pH 5.5), and the mixture was incubated for
1 h at 20 °C. After 1 h, excess NH2OH HCl was added, andthe mixture was stirred for an additional 1 h. This reaction
mixture was purified using a NAP-25 desalting column andeluted with 20 mM acetate buffer at pH 5.5.
Lys288 Conjugation Using AJICAP Reagent (6b) Followed
by Linker Cleavage (One-Pot). Six equivalents of AJICAP
reagent (6b) (20 mM in DMF) were added to each mAb (10mg/mL, 20 mM borate buffer, pH 8.2), and the mixture wasincubated for 1 h at 20 °C. After 1 h, excess NH2OH HCl and
1 M acetate buffer (pH 4.7) for adjusting the pH of thereaction mixture (less than pH 6.0) were added and stirred foran additional 1 h. Subsequently, the reaction mixture waspurified using a Centripure P50 desalting column and eluted
with 20 mM acetate buffer (pH 5.5).
ADC Synthesis. Payload conjugation with antibody-thiol(4) was achieved using a previously established procedure.13
The SI shows the payload linkers used in this study.
Peptide Mapping for Conjugation Site Determination. For peptide mapping, 20 μg of each sample(trastuzumab, trastuzumab-Lys248-thiol, and trastuzumabLys288-thiol) was diluted to 110 μL with 0.25 M Tris-HCl(pH 7.5)/0.75 M GnHCl buffer. Reductive alkylation wasachieved by adding dithiothreitol (DTT) and iodoacetamide(IAM) successively. The sample was then buffer exchanged to
100 mM Tris-HCl (pH 7.5) by Zeba Spin Desalting columns.
For enzymatic digestion, we added 3:1 mixture of trypsin andLys-C, then incubated at 37 °C for 18 h. Digestion was
quenched by adding formic acid and acetonitrile.
The resulting peptide mixture was analyzed on an Orbitrap
Fusion Tribrid (Thermo Fisher Scientific) interfaced with a
Vanquish Duo (Thermo Fisher Scientific). We used an
ACQUITY UPLC CSH C18 Column (1.7 μm, 2.1 × 150
mm, Cat #186005298, Waters) with a column temperature of
50 °C. The chromatographic method consisted of a 2 min holdat 2% solvent B (0.1% formic acid in acetonitrile) and a 55 minlinear gradient from 2 to 42% solvent B. After a linear rise to90% solvent B in 5 min, a wash step was performed with a 5
min hold at 90% solvent B. Subsequently, solvent B was
reduced to 2% in 1 min and maintained at 2% for 12 min. The
flow rate was set to 250 μL/min, and solvent A contained 0.1%
formic acid. Mass spectrometry analysis was conducted in datadependent acquisition mode with full scans (350−2,000 m/z)
acquired at a mass resolution of 120,000. Tandem mass spectra
were produced using the higher energy collision-induced
dissociation method.

The resulting MS/MS data were compared against the
trastuzumab sequence (Figure S22) using BioPharma Finder
3.2 (Thermo Fisher Scientific). Carbamidomethylation ofcysteine (+57.021 Da) was specified as a fixed modification,and oxidation of methionine (+15.995 Da) and 3-(2-amino-2oxo-ethyl) sulfanylpropionate of lysine (+145.019 Da) were
included as variable modifications.
Biological Evaluation Procedure. Detailed biologicalevaluation procedure can be found in the SI.

■

ArticleASSOCIATED CONTENT

sı Supporting Information*

The Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acs.bioconjchem.3c00040.
Details of the in vivo studies, figures supporting themolecular modeling, HPLC chromatograms, and QTOF
MS analyses (PDF)■

AUTHOR INFORMATION

Corresponding Authors

Kei Yamada − Ajinomoto Co., Inc., Kawasaki-Shi, Kanagawa
210-8681, Japan; Email: kei.yamada.wa9@
asv.ajinomoto.com
Yutaka Matsuda − Ajinomoto Bio-Pharma Services, San
Diego, California 92121, United States; orcid.org/00000003-2801-4706; Email: yutaka.matsuda@us.ajibiopharma.com
Authors

Tomohiro Fujii − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Takuya Seki − Ajinomoto Co., Inc., Kawasaki-Shi, Kanagawa
210-8681, Japan
Natsuki Shikida − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Yusuke Iwai − Ajinomoto Co., Inc., Kawasaki-Shi, Kanagawa
210-8681, Japan
Yuri Ooba − Ajinomoto Co., Inc., Kawasaki-Shi, Kanagawa
210-8681, Japan
Kazutoshi Takahashi − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Muneki Isokawa − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Sayaka Kawaguchi − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Noriko Hatada − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Tomohiro Watanabe − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Rika Takasugi − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Akira Nakayama − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Kazutaka Shimbo − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, Japan
Brian A. Mendelsohn − Ajinomoto Bio-Pharma Services, San
Diego, California 92121, United States; Present
Address: Exelixis Inc., 1851 Harbor Bay Pkwy, Alameda,
CA 94502, United States; orcid.org/0000-0002-63392377
Tatsuya Okuzumi − Ajinomoto Co., Inc., Kawasaki-Shi,
Kanagawa 210-8681, JapanI

https://doi.org/10.1021/acs.bioconjchem.3c00040
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistrypubs.acs.org/bc

Complete contact information is available at:https://pubs.acs.org/10.1021/acs.bioconjchem.3c00040

First Generation. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
2020, 1140, 121981.
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(23) Utilizing reagent 6c consisting of the same linker as 1b did not
improve the PAR value sufficiently. See Figure S5, S8, and S14.
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(27) Strohl, W. R. Fusion Proteins for Half-Life Extension of
Biologics as a Strategy to Make Biobetters. BioDrugs 2015, 29, 215−
239.
(28) From these conjugates, two polyclonal mAb derived ADCs
(conjugation site: Lys248 and Lys288, payload: MMAE) werecreated. See Supporting Information, Figures S38 and S39.
(29) Fujii, T.; Reiling, C.; Quinn, C.; Kliman, M.; Mendelsohn, B.
A.; Matsuda, Y. Physical Characteristics Comparison Between
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(32) Representative data of AJICAP-ADC produced by first
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(33) Hernandez-Alba, O.; Houel, S.; Hessmann, S.; Erb, S.; Rabuka,
D.; Huguet, R.; Josephs, J.; Beck, A.; Drake, P. M.; Cianférani, S. A
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Author Contributions#

T.F. and K.Y. contributed equally.

Notes

The authors declare the following competing financial
interest(s): This work was supported by Ajinomoto Co., Inc.

■ACKNOWLEDGMENTS
The authors wish to thank our colleagues from Ajinomoto Co.,
Inc. and Ajinomoto Bio-Pharma Services, Inc., as follows: Ms.
Yumiko Suzuki and Mr. Ryusuke Hirama for technicalassistance with AJICAP conjugations; Ms. Zhala Tawfiq fortechnical assistance with ADC preparation; Dr. Kenichiro Ito
for critical suggestions regarding antibody-SiRNA conjugates;
Dr. Akira Chiba and Mr. Hiroki Imai for helpful discussions
and suggestions in manuscript preparation.

■

ABBREVIATIONSADC, antibody−drug conjugate; IgG, immunoglobulin-G;
DAR, drug-to-antibody ratio; NHS, N-succinimide; PAR,
peptide-to-antibody ratio; PK, pharmacokinetic; Q-TOF MS,
quadrupole time-of-flight mass spectrometry; SEC, size
exclusion chromatography■

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