Front. Biosci. (Landmark Ed) 2022; 27(8): 234
https://doi.org/10.31083/j.fbl2708234

Original Research

Biological Evaluation of Maytansinoid-Based Site-Specific
Antibody-Drug Conjugate Produced by Fully Chemical Conjugation
Approach: AJICAP®
Takuya Seki1 , Kei Yamada1 , Yuri Ooba1 , Tomohiro Fujii1 , Takahiro Narita1 ,Akira Nakayama1 , Yoshiro Kitahara1 , Brian A. Mendelsohn2,§ , Yutaka Matsuda1,2, *,Tatsuya Okuzumi1, *1 Ajinomoto Co., Inc., 1-1, Suzuki-Cho, Kawasaki-Ku, Kawasaki-Shi, 210-8681 Kanagawa, Japan
2 Ajinomoto Bio-Pharma Services, San Diego, CA 92121, USA*Correspondence: Yutaka.Matsuda@US.AjiBio-Pharma.com (Yutaka Matsuda); tatsuya.okuzumi.e2m@asv.ajinomoto.com (Tatsuya Okuzumi)§ Present addresses: Exelixis Inc., Alameda, CA 94502, USA

Academic Editor: Graham Pawelec
Submitted: 31 May 2022 Revised: 4 July 2022

Accepted: 21 July 2022

Published: 5 August 2022

Abstract
Background: Trastuzumab-emtansine (T-DM1, commercial name: Kadcyla) is well-known antibody-drug conjugate (ADC) and was
first approved for human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. This molecular format consistingof trastuzumab and maytansinoid payload (emtansine) is very simple, however, T-DM1 has wide heterogeneity due to non-specific conjugation, lowering its therapeutic index (TI). Methods: To overcome this issue during the chemical modification of the random conjugation
approach to generate T-DM1, we developed a novel chemical conjugation technology termed “AJICAP®” for modification of antibodies
in site-specific manner by IgG Fc-affinity peptide based reagents. Results: In this study, we compared site-specific maytansinoidbased ADCs synthesized by AJICAP and T-DM1 in rat safety studies. The results indicated an increase in the maximum tolerated dose,demonstrating an expansion of the AJICAP-ADC therapeutic index compared with that of commercially available T-DM1. Gram scale
preparation of this AJICAP-ADC and the initial stability study are also described. Conclusions: Trastuzumab-AJICAP-maytansinoid
produced by this unique chemical conjugation methodology showed higher stability and tolerability than commercially available T-DM1.
Keywords: antibody drug conjugate; maytansinoid; toxicology study; site-specific conjugation; AJICAP

1. Introduction
Trastuzumab-emtansine (T-DM1, commercial name:
Kadcyla) is a maytansinoid-based antibody-drug conjugate(ADC), which was first approved for patients with human epidermal growth factor receptor 2 (HER2)-positive
metastatic breast cancer [1]. Since its success in 2013, morethan 10 ADCs been introduced into the market [2,3].
ADCs have becoming an innovative cancer medicinesthat consist of monoclonal antibodies (mAbs) and cytotoxic payloads via chemical linkage. This molecular concept is simple; however, most ADCs currently in the market have wide heterogeneity owing to non-specific conjugation. These nonspecific conjugations are classified into two
main categories: using reduced interchain native cysteine
and using native lysine. The “interchain-break” cysteinebased methodology is the major manufacturing approach to
produce “semi-random” ADC including mainly eight drugto-antibody ratio (DAR) species (DAR = 0, 2, 4, 6, 8)[4]. This conjugation provides six ADCs (Adcetris, Polivy, Padcev, Blenrep, Zynlonta and Tivdak) approved by
U.S. Food and Drug Administration (FDA). Native lysine
conjugation is a traditional conjugation technology using
activated ester groups (such as NHS) [5]. These high reac-

tive compounds enable to react with exposed lysine residue
to form stable covalent bond between antibodies and druglinkers. This methodology produced three FDA-approved
ADCs (Mylotarg, Kadcyla and Besponsa). T-DM1 (commercially name: Kadcyla) is produced by natural conjugation with available lysine residues, thereby resulting ina heterogeneous distribution of high potent payload-linker
over multiple ADC sites; this process could lead to a clinically insufficient and narrow therapeutic index (TI) [6].
In addition to limited in vivo profile, the heterogeneity of T-DM1 could be problematic from a chemical manufacturing and control point-of-view. Some HPLC analyses,
which are the most common approaches for determining theDAR, fail to provide clear chromatographic results [7,8].
To overcome ADC heterogeneity, several site-specific conjugation methods to produce more homogeneous ADCs
have been developed [9]. Thus, our research group conceived a proprietary technology utilizing an Fc-affinity peptide, which can install a bi-orthogonal group onto antibodies
in a site-selective manner, thereby enabling the site-specific
ADCs syntheses (Fig. 1) [10,11].
This platform, termed “AJICAP”, has been advantageous in several studies. First, its proof of concept was re-

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Fig. 1. Summary of the conjugation chemistry for ADC production. (A) Upper: Synthesis of T-DM1 by traditional native lysine conjugation; Lower: AJICAP site-specific conjugation to synthesize trastuzumab-AJICAP-maytansinoid. T-DMI, Trastuzumab-emtansine;
ADC, antibody-drug conjugate. (B) Reaction scheme of AJICAP technology.

ported in 2019, showing highly efficient and versatile conjugation applied to several mAb subtypes such as IgG1,
IgG2, and IgG4 [10]. Peptide mapping analysis of the resulting homogeneous ADC confirmed its conjugation siteoccupancy at Lys 248 in the antibody Fc region [12]. The
larger scale ADC preparation using a glass reactor vial
based on the good manufacturing practice strategy indicatedthe high productivity and robustness of the AJICAP technology [13]. In addition, site-specific AJICAP-ADCs havebeen used as a demonstration tool to develop novel analytical approaches [14].
In contrast to the numerous synthetic/analytical reports on AJICAP-ADCs, there have been limited studies
on their biological profile. Furthermore, no studies have
performed a direct comparison between AJICAP-ADC andcommercially available ADCs.
In order to provide a TI comparison between AJICAPADCs and traditional ADCs in the market, we decided
to demonstrate the rat toxicology study of trastuzumabAJICAP-maytansinoid (DAR = 2) as compared to commercially available T-DM1 (DAR = 3.4). The combination of this safety study and a previously reported efficacy
2

study enables comparison of the TI, thereby resulting ina wide enhancement of TI by AJICAP technology [15].
To supply a sufficient quantity of trastuzumab-AJICAPmaytansinoid, gram-scale ADC preparation was also performed, thus, supporting the robustness of the previously
established conjugation process [13]. To adapt the AJICAP method of conjugation, a cysteine-reactive maytansinoid (MCC-maytansinoid, Fig. 1) was synthesized as previously reported [16]. The stability of trastuzumab-AJICAPmaytansinoid was assessed to determine its appropriate
storage conditions [17,18]. Overall, the results describedherein support the previously reported advantages of AJICAP conjugation platform enabling site-specific ADC production.

2. Materials and Methods
2.1 Materials
Human IgG1 trastuzumab (Herceptin®) was purchased from Roche Pharmaceutical Company (Basel
Switzerland). MCC-maytansinoid (catalog no: TCRS1262) was purchased from Abzena (San Diego, CA, USA).

PNGase F and GlycoBuffer 2 were purchased from New
England Biolabs (Ipswich, Massachusetts, UK). All otherchemical reagents were purchased from Sigma-Aldrich (St.
Louis, MO, USA).
2.2 ADC Concentration
The SoloVPE spectroscopy system (C Technologies,
Inc. Bridgewater, NJ, USA) provided ADC concentration.
2.3 Synthesis of Thiol-Modified Trastuzumab
Site-specific thiol intermediate was produced by a previously established procedure [13].
2.4 Synthesis of AJICAP-ADC: Payload Conjugation
To a solution of site-specific thiol intermediate (6.8mg/mL, 1.63 g) in a PBSE buffer (50 mM phosphatebuffered saline (PBS), 10 mM ethylenediaminetetraacetic
acid (EDTA), pH 7.4), dimethylformamide (DMF) (15 mL)
and a 10-mM DMF solution of MCC-maytansinoid (10 eq.,11.7 mL) were added to form a mixture, which was thenincubated at 20 °C. After 2 h, a small amount of the reaction mixture (0.5 mL) was sampled for the in-processcontrol (IPC) analysis. Subsequently, the reaction mixture
was quenched with an excess amount of a 50-mM aqueous solution of N-acetyl cysteine (NAC) and then incubated at 25 °C for 15 min. This reaction mixture was purified with a tangential flow filtration (TFF) system usinga Sartocon Slice 200 Eco Hydrosart membrane (30 kDa;
Sartorius) and diafiltration (DF) buffer as the conjugationbuffer at an antibody concentration of 20 mg/mL. Next,
buffer exchange of this solution was performed using a
TFF system with a Sartocon Slice 200 Eco Hydrosart membrane (30 kDa; Sartorius) and DF buffer as the formulationbuffer (20 mM histidine containing 5% trehalose, pH 5.2)
at an antibody concentration of 5.7 mg/mL. TrastuzumabAJICAP®-maytansinoid 4 (1.67 g, 98% yield) was then afforded in the formulation buffer.
2.5 Deglycosylated Pre-Treatment for Q-TOF Analysis
Trastuzumab-AJICAP-maytansinoid (50 µL; 1
mg/mL in 50 mM Tris-HCl buffer, pH 7.6), PNGase F (2µL; 500,000 units/mL), 10 µL of GlycoBuffer 2 (10×),and 48 µL of water were incubated at 37 °C for 16 h. Then
5% v/v aqueous solution of trifluoroacetic acid as quencher
was added to this reaction mixture and incubated at 37 °C
for 30 min. The buffer exchange and sample concentration
were carried out using 25 mM ammonium bicarbonate
buffer with centrifugal concentrator (Vivaspin 500, 10,000
MWCO, Polyethersulfone (PES)).
2.6 Q-TOF MS Analysis
Q-TOF MS analysis was performed using a PLRPS
1000 Å 8 µm column (Agilent) connected to an Agilent
1260 HPLC system, as well as with an Agilent 6550 Q-TOF
system containing a binary gradient pump, temperature-controlled column compartment, autosampler, and diodearray detector. The system ran at 0.3 mL/min at 80 °C using
mobile phase A (MPA: 0.1% formic acid (FA), 0.01% TFA,
2% ACN, and 98% water) and mobile phase B (MPB: 0.1%
FA, 0.01% TFA, and 99.9% acetonitrile). The absorbancewas estimated at 280 nm using a reference wavelength of
450 nm. Intact or deglycosylated ADC (1 mg/mL, 5 µL)
was injected into the system and eluted over a 15-min run
consisting of a 2-min isocratic hold at 20% MPB, a 15-minlinear gradient from 20% to 95% MPB, a 2-min wash using
95% MPB, and a 3-min re-equilibration at 20% MPB. Dataacquisition was performed using MassHunter BioConfirm
software (Agilent) to analyze the intact MS spectra for intact deconvolution, a mass range of 100,000–180,000 anda limited m/z range of 1000–5000 were set for standardization. The DAR was determined using DAR Calculator
software (Agilent).
2.7 Extracted Ion Chromatogram (EIC) Analysis
The EIC analysis was performed using Q-TOF MS
system with m/z ratio of 1103.4 (M+).
2.8 Pre-Treatment for RP-HPLC Analysis
The reductive pre-treatment was performed as previously reported [19].
2.9 RP-HPLC Analysis for Reduced ADCs
RP-HPLC analysis was performed according to a previously reported study [19].
2.10 Hydrophobic Interaction Chromatography(HIC)-HPLC Analysis
HIC HPLC analysis was performed according to a previously reported study [20].
2.11 Stability Assessment
Three sets of standard solutions of trastuzumabAJICAP-maytansinoid (4) were prepared in a formulationbuffer. All the samples were stored at various temperatures(–80, –20, 4, 25, and 37 °C) for 4 weeks. Aggregation wasthen analyzed using size-exclusion chromatography (SEC)HPLC.
2.12 SEC Analysis of ADCs
Aggregation analysis using SEC was performed aspreviously reported [20].
2.13 Rat Safety Study
2.13.1 Animal Experiments
Sixty 8-week-old female Sprague-Dawley rats
(Charles River Japan, Tokyo, Japan) were provided ad
libitum access to a standard diet (Oriental Yeast, Tokyo,
Japan) and water. Following the acclimatization period(1 week), the animals were stratified by body weight andrandomly assigned to the following groups: (1) control
3

Fig. 2. Synthesis and analysis of trastuzumab-AJICAP-maytansinoid. (A) Synthetic scheme of the gram-scale ADC preparation. (B)Analytical summary of resulting ADC. (C) Aggregation comparison of T-DM1 (literature data) and trastuzumab-AJICAP-maytansinoid(tested data) at different temperatures after 4 weeks of storage. T-DMI, Trastuzumab-emtansine.

group, treated with the vehicle (histidine buffer); (2) two
T-DM1 groups, treated with 20 mg/kg and 60 mg/kg;and (3) three trastuzumab-AJICAP-maytansinoid (4)groups, treated with 20 mg/kg, 60 mg/kg, and 120 mg/kg.
Each group consisted of five animals for blood chemistry
test as well as five animals for clinical signs and bodyweight measurements. All rats were administered once
through intravenous injection via the caudal vein using abutterfly needle, a 30-mL polypropylene syringe, and asyringe pump (Pump 11 Elite, Harvard Apparatus). The
experimental procedures were approved by the institutional
ethics committee.
2.13.2 Clinical Observations
The animals were observed once daily for any clinicalsigns. Individual body weights were measured on Days 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 19, 20, and21; the first day of administration was defined as Day 0.
2.13.3 Blood Chemistry Test
Blood chemistry parameters were evaluated 2 d after administration. The animals were fasted overnight andanesthetized with isoflurane prior to collecting blood fromthe caudal vena cava. Heparin-anticoagulated plasma samples were evaluated using an automated clinical chemistry analyzer (TBA-120FR; Toshiba Medical Systems,
Inc., Tokyo, Japan) to determine aspartate (AST) and alanine aminotransferase (ALT) levels. EDTA-anticoagulated
blood samples were evaluated using an automated hematology analyzer (ADVIA2120, Siemens Healthineers AG.,
Munich, Germany) to determine platelet (PLT) counts.

4

3. Results and Discussions
3.1 Gram-Scale Preparation and Analysis of
Trastuzumab-AJICAP-Maytansinoid
Using a well-established previously described approach [13], a site-specific thiol-modified mAb, the intermediate ADC precursor, was prepared from trastuzumab,
which is the same antibody as that in T-DM1 upon completion of a gram-scale synthesis (Fig. 2). The final stepin ADC preparation is the conjugation of this thiol intermediate with MCC-maytansinoid (Fig. 2A). This reaction completion was detected by IPC analysis using HIC
HPLC, as per a previous good laboratory practice (GLP)
material preparation [13]. Purification to remove residual drug linkers was performed in a straightforward manner using tangential flow filtration (TFF), which is a commonly used technique that can be applied to kilogramscale ADC manufacturing [21,22]. Compared to MC-VCMMAE, which was previously used for preparing AJICAPADC, TFF could clear MCC-maytansinoid from the ADC
composition. This can be attributed to the hydrophobicity difference of each drug linker. MC-VC-MMAE is relatively more hydrophobic than MCC-maytansinoid [23],thus, requiring careful monitoring of its clearance level.
The removal rate of highly potent MCC-maytansinoid druglinker was calculated by the extracted ion chromatogram(EIC) analysis, which showed that the rate was supposed tobe near the detection limit in this analysis (data not shown).
Three different analytical methods were used to determine the DAR of trastuzumab-AJICAP-maytansinoid(Fig. 2B). Q-TOF MS (Intact ADC: Supplementary Fig. 1,
deglycosylated ADC: Supplementary Fig. 2), RP-HPLC(Supplementary Fig. 3) and HIC-HPLC (Supplementary
Fig. 4) analyses revealed that this ADC has a highDAR value (1.8–1.9). The DAR results were slightly dif-Fig. 3. Rat acute toxicity study to determine the MTD of ADCs. (A) Body weight change (N = 5 female SD rats). (B) Liver weightin rat serum at 2 days after administration (N = 5 female SD rats. (C) AST in rat serum at 2 d after administration (N = 5 female SD rats).
(D) ALT levels in rat serum at 2 d after administration (N = 5 female SD rats). MTD, maximum tolerated dose; ADCs, antibody-drug
conjugates; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Significantly different from Histidine Buffer: * p < 0.05,*** p < 0.001. DT, Dunnett test (two-side); ST, Steel test (two-side).

ferent among the three methods; however, this observation was also reported in several previous studies [24–26]. Aggregation percentage was within acceptance level(Supplementary Fig. 5).
Next, a thermal stability study of trastuzumabAJICAP-maytansinoid was examined to estimate the suitable formulation and storage conditions towards forthcoming manufacturing processes (Fig. 2C). Several previous
studies, including ours, had reported stability assessments
results of T-DM1 in peer-reviewed literature; and we followed the same procedures for one of the processes applied
to trastuzumab-AJICAP-maytansinoid [13,16–18].
T-DM1 was found to be relatively stable at lower
temperatures (<4 °C) [11,15]. At room temperature or
higher, the aggregation percentage increased significantly;
however, in the current study, trastuzumab-AJICAPmaytansinoid showed more resistance to high temperaturethan T-DM1. As described above, T-DM1 has a heterogeneous mixture of possibly more than 80 species [27], someof which may be unstable and prone to aggregation. In contrast, AJICAP-ADC is a low heterogeneous ADC; its conjugation position is known to be stable under thermal/blood
circulation. This high stability of trastuzumab-AJICAPmaytansinoid reflects the stable linkage/conjugation site
produced by the AJICAP technology.
3.2 Toxicology Study in Rats
A rat acute toxicity study was performed for the
MTD determination of the ADCs (Fig. 3). Mortality
or severe toxicity was observed with T-DM1 at a doseof 40 mg/kg; however, no toxicity was observed with
AJICAP-ADC even at a dose of 120 mg/kg (Fig. 3A). Decreases in body weight or PLT levels and increased ASTlevels were not observed at doses of up to 120 mg/kg
5

Table 1. Therapeutic index comparison of T-DM1 and trastuzumab-AJICAP-maytansinoid.
ADC
T-DM1 (Kadcyla)
Trastuzumab-AJICAP-maytansinoid

Minimum effective dose (MED)

Maximum tolerated dose (MTD)<5 mg/kg>20 mg/kg<5 mg/kg>120 mg/kg

trastuzumab-AJICAP-maytansinoid and 20 mg/kg T-DM1.
Additionally, Liver weight did not change significantly at
a 120 mg/kg. Thus, the MTD of trastuzumab-AJICAPmaytansinoid and T-DM1 was estimated to be at least 120
and 20 mg/kg, respectively. Neutrophil, reticulocyte, lymphocyte and platelet count data supported these estimations, considering the mode of action of maytansinoids(Supplementary Fig. 6) [28,29]. An exploratory rat
safety toxicology study showed that the rats showed better tolerance to trastuzumab-AJICAP-maytansinoid than to
T-DM1. Given the limited dosing in our study, the MTDof trastuzumab-AJICAP-maytansinoid was estimated to beat least 120 mg/kg, whereas the MTD of T-DM1 was estimated to be at least 20 mg/kg. This initial safety analysis
indicated that the conjugation approach generated a stable
ADC, although further biological/analytical studies are currently ongoing, such as PK. In previous report [15], a rat PK
study showed that the trastuzumab-AJICAP-MMAE ADC
is stable in blood circulation compared to native cysteinebased stochastic ADC. The measured levels of total antibody from trastuzumab-AJICAP-MMAE indicated a halflife similar to that expected for parent trastuzumab. Furthermore, in vitro binding assay using biolayer interferometry
method concluded that trastuzumab-AJICAP-maytansinoid
does not affect FcRn interactions [30]. These results suggest that site-specific conjugated ADCs at Lys248 result ina superior profile with regard to both safety and in vivo stability compared to the approved ADC, T-DM1.
Furthermore, in a previous study, we reported thatthe minimum effective dose (MED) of both ADCs was 5
mg/kg based on the NCI-N87 xenograft study. Thus, the TIof trastuzumab-AJICAP-maytansinoid was estimated to bequalitatively greater than that of T-DM1, considering theirrespective MTDs and MEDs (Table 1).

therapeutic index expansion. Therefore, this platform conjugation technology has a strong potential to produce nextgeneration ADCs with high homogeneity and significant invivo stability.

4. Conclusions

Acknowledgment

In conclusion, we applied a full chemical conjugation technology, AJICAP, to produce a maytansinoid-basedsite-specific ADC. The scale-down manufacturing processusing TFF purification effectively removed the residual
drug linkers from the final ADC composition, thus, producing DAR of nearly 2.0 ADCs without including a significant level of aggregates. The resulting site-specific ADCs
showed sufficient long-term stability under different storage conditions.
Furthermore, the comparative toxicology study between trastuzumab-AJICAP-maytansinoid and commercially available T-DM1 indicated that ADCs produced bythis unique chemical conjugation methodology enables
6

Abbreviations
T-DMI, Trastuzumab-emtansine; ADCs, antibodydrug conjugates; HER2, human epidermal growth factor
receptor 2; TI, therapeutic index; DAR, drug-to-antibody
ratio; DMF, dimethylformamide; MPA, mobile phase A;
MPB, mobile phase B; FA, formic acid; AST, aspartate
aminotransferase; ALT, alanine aminotransferase; PLT,
platelet; TFF, tangential flow filtration; EIC, extracted
ion chromatogram; Q-TOF MS, Quadrupole time-of-flightmass spectrometry; MED, minimum effective dose; MTD,
maximum tolerated dose.

Author Contributions
TS—Animal study, Validation, Formal analysis, Data
curation and Writing—original draft preparation. KY—
Conceptualization and ADC preparation. YO—Animal
study, Formal analysis, Data curation. TF—Resource,
Stability assessment and ADC preparation. TN, AN andYK—Methodology and Validation. BAM—Supervision,
Writing—review and editing. YM—Conceptualization,
Resources, ADC preparation, Writing—original draft
preparation, review and editing. TO—Conceptualization,
Funding acquisition, Supervision, Writing—review andediting. All authors have read and agreed to the publishedversion of the manuscript.

Ethics Approval and Consent to Participate
Not applicable.

The authors wish to express the gratitude to colleagues
from Ajinomoto Co., Inc., as follows: Natsuki Shkida,
Reiko Yuji, Kazutoshi Takahashi, Kunio Nakata and Kazutaka Shimbo for useful opinions on ADC analysis; Akira
Chiba for support and encouragement during manuscript
preparation.

Funding
This research received no external funding.

Conflict of Interest
The authors declare no conflict of interest. YM andBAM are serving as the Guest editors of this journal. We

declare that YM and BAM had no involvement in the peer
review of this article and has no access to information regarding its peer review. Full responsibility for the editorial
process for this article was delegated to GP.

Supplementary Material
Supplementary material associated with this article
can be found, in the online version, at https://doi.org/10.
31083/j.fbl2708234.

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