AJICAP Second-generation: Realizing Versatility and Robustness through Fc-Affinity Guided Chemical Conjugation Technology

Over the past decade, site-specific chemical conjugation has emerged as a promising strategy to produce antibody-drug conjugates (ADCs) in the field of oncology [1]. Daiichi-Sankyo’s chemical conjugation method, which uses high drug-to-antibody ratio (DAR) technology, was approved by the U.S. Food and Drug Administration as the first site-specific ADC (fam-trastuzumab-deruxtecan-nxki; Enhertu® | Daiichi Sankyo and AstraZeneca).

However, owing to limitations in high-DAR technology and the potential need for DAR=2 production, the Ajinomoto group has been developing a chemical site-specific conjugation technology using Fc-affinity peptide reagents since 2019 [2]. This approach modifies a specific lysine in the Fc region of various antibodies such as IgG1, IgG2, and IgG4 to create homogeneous DAR=2 ADCs, as demonstrated in a proof-of-concept study.

The resulting ADCs were confirmed to be solely conjugated to Lys248 using various analyses, including trypsin-digested peptide mapping. As the Fc region of any antibody is constant, the Fc-affinity conjugation technology, AJICAP first-generation technology, has potential to be used with all antibodies without complex reaction optimization. The therapeutic index of the site-specific AJICAP-ADC made with cytotoxic monomethyl auristatin E (MMAE) was found to be superior to that of stochastic cysteine-based ADCs, and the site-specific AJICAP-ADC made with maytansinoid demonstrated higher in vivo efficacy and tolerability than Kadcyla, an FDA-approved clinical ADC [3][4]. These findings indicate that AJICAP technology has immense potential as next-generation ADCs.

However, there are still challenges associated with AJICAP first-generation technology. For example, this approach requires TCEP reduction to install thiol groups on a specific lysine residue, which cleaves the disulfide bonds of interchain cysteines in antibodies. In addition, reoxidation using dehydroascorbic acid following reduction can cause disulfide bond scrambling. Aggregation is also a significant concern with this approach, as small amounts of aggregates (5–10%) were found in ADCs produced using AJICAP first-generation technology. A streamlined manufacturing sequence with fewer reaction steps is desirable from the perspective of chemical manufacturing control.

Figure 1.0: 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.

In recent optimization studies, the Ajinomoto group tackled the challenges in their conjugation reaction sequences, as illustrated in Figure 1 [5]. These efforts have yielded an improved technology known as “AJICAP second-generation,” which enables the production of site-specific ADCs without aggregation problems by utilizing selective cleavage reactions via thioester chemistry. By enhancing the stability of the AJICAP peptide reagent in a buffer solution, this new approach achieved greater selectivity in targeting lysine and higher DAR values for ADCs.

Furthermore, the thioester-based method enabled two different AJICAP peptide reagents to access two distinct conjugation sites (Lys248 and Lys288), and in vivo studies indicated comparable efficacy and tolerability. The potential of this conjugation chemistry for non-traditional ADC production was also explored (Figure 2).

Figure 2.0: Application to other antibody conjugates.

The encouraging outcomes of site-specific ADCs generated using AJICAP second-generation technology highlight the need to produce large-scale ADCs to facilitate further biological investigations, including non-human primate safety studies.

Therefore, it is crucial to develop a scalable process for large-scale ADC production to implement an affinity peptide strategy (Figure 3). To this end, the Ajinomoto group conducted additional studies to demonstrate the scalability and robustness of the AJICAP second-generation technology [6]. The parameters of the peptide conjugation step, unique to the AJICAP strategy, were examined and found to operate effectively under a broad range of conditions. Synthetic intermediates, such as antibody-affinity peptide conjugates (2) and antibody-thiol (3), were determined to be sufficiently stable as holding point compounds during the manufacturing process.

Gram-scale synthesis of Lys248- and Lys288-conjugated ADCs was successfully accomplished using two different Fc-affinity peptide reagents and a scaled-down manufacturing approach involving tangential flow filtration (TFF). TFF purification efficiently reduced residual peptide reagents from Lys248 and Lys288 modifications to acceptable levels. The overall manufacturing yield was more than 80%, leading to the production of 13.2 g of trastuzumab-Lys248-MMAE and 1.26 g of trastuzumab-Lys288-MMAE with a high DAR. These findings indicate that the second-generation AJICAP strategy is practical and robust for large-scale ADC production.

Figure 3.0: Scale-up status of AJICAP second-generation.

In summary, AJICAP second-generation technology represents a potential breakthrough method for addressing the challenges of site-specific antibody modification and fostering the emergence of next-generation ADCs.

In contrast to the first-generation approach, the second-generation iteration offers enhanced stability, greater specificity towards lysine targets, better DAR values for ADCs, and suitability for gram-scale synthesis of ADCs. Moreover, it features two distinct AJICAP peptide reagents that enable access to different conjugation sites, as confirmed by in vivo studies demonstrating equivalent effectiveness and tolerability.

The authors would like to express their gratitude to their former/present colleagues at Ajinomoto Co., Inc. and Ajinomoto Bio-Pharma Services, Inc. for their invaluable contributions. Specifically, we extend our thanks to Tomohiro Watanabe, Rika Takasugi, Noriko Hatada, Yumiko Suzuki, Ryusuke Hirama, and Kei Yamada for their technical assistance with AJICAP conjugations; Monica Leung, Veronica Robles, Zhala Tawfiq for her technical support with ADC preparation; and Natsuki Shikida, Muneki Isokawa, Kazutoshi Takahashi, Kenichiro Ito, and Kazutaka Shimbo for their contributions with ADC analysis; Takuya Seki, Yuri Ooba, Sayaka Kawaguchi, Yusuke Iwai, Ryo Yamada, and Akira Nakayama for their contributions with in vivo and in vitro studies. We also acknowledge the critical suggestions on protein purification from Jason T. Stofleth, John Patrick McCurley and Rocco Vendegna, and the valuable discussions and suggestions in manuscript preparation and ADC project from Tessa Ente Barnes, Akira Chiba, Hiroki Imai, Brian A. Mendelsohn, and Tatsuya Okuzumi.

Disclosure of Interest
The authors do not have any competing interests.

Highlights of Prescribing information
Fam-trastuzumab-deruxtecan-nxki (Enhertu® | Daiichi Sankyo and AstraZeneca) [Prescribing Information]

[1] Matsuda Y.  J. ADC. January 5, 2023. DOI: 10.14229/jadc.2023.01.05.002.
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[5] T. Fujii, Y. Matsuda, T. Seki, N. Shikida, Y. Iwai, Y. Ooba, K. Takahashi, M. Isokawa, S. Kawaguchi, N. Hatada, T. Watanabe, R. Takasugi, A. Nakayama, K. Shimbo, B. A. Mendelsohn, T. Okuzumi, K. Yamada, AJICAP Second Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody–Drug Conjugate Production. Bioconjugate. Chem. 2023; 34: 728.
[6] T. Watanabe, T. Fujii, J. T. Stofleth, R. Takasugi, K. Takahashi, Y. Matsuda, Scale-Up Synthesis of Site-Specific Antibody-Drug Conjugates Using AJICAP Second-Generation Technology. Org. Process Res. Dev. 2023, in press.

Authors: Tomohiro Fujii, Ph.D.1, and Yutaka Matsuda, Ph.D, Ph.D, MBA,2

Corresponding Author: Tomohiro Fujii, Ph.D

Key terms: ADC, antibody-drug conjugate, drug-to-antibody ratio, DAR, chemical conjugation, site-specific conjugation, Fc affinity peptide conjugation, AJICAP
Published In: ADC Review| Journal of Antibody-drug Conjugates

DOI: https://doi.org/10.14229/jadc.2023.07.28.003.

How to cite:

Tomohiro Fujii1, Yutaka Matsuda2
AJICAP Second-generation: Realizing Versatility and Robustness through Fc-Affinity Guided Chemical Conjugation Technology – J. ADC. July 28, 2023. DOI: 10.14229/jadc.2023.07.28.003.

1 Ajinomoto Co., Inc., 1-1, Suzuki-Cho, Kawasaki-Ku, Kawasaki-Shi, Kanagawa 210-8681, Japan.
2 Ajinomoto Bio-Pharma Services, 11040 Roselle Street, San Diego, CA 92121, United States

Last Editorial Review: July 12, 2023

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Article History:

  • Original Manuscript Received May 23, 2023
  • Review results received July 17, 2022
  • Manuscript accepted for publication  July 28, 2023