Abstract
To date, more than 10 antibody-drug conjugates (ADCs) have been approved by the Food and Drug Administration (FDA) and contribute to the medical community. Interestingly, all of them are manufactured using chemical conjugation techniques [1]. This demonstrates that chemical conjugation is the primary approach to ADC manufacturing. Compared to other conjugation techniques (e.g., genetic engineering or enzymatic), chemical conjugation has the advantage of a relatively simple chemistry, manufacturing, and control (CMC) for ADC, prompting pharmaceutical companies to choose this technique for ADC projects [2].
To better understand this advantage, this article provides an overview of established full chemical conjugation methods that have already been applied in the scale-up phase for ADC production.
Stochastic conjugation
The most popular approach for fabricating ADCs is chemical stochastic conjugation. This methodology utilizes random modification, with either native lysine on the antibody surface, or cysteine reduced from the interchain disulfide bonds of the antibody. Native lysine conjugation is the traditional approach for linking drug-linkers to antibodies.
Activated ester groups (e.g., N-hydroxysuccinimide ) react with a primary amine on the side chains of the exposed lysine residues to form stable covalent bonds. This technique has been used to produce four U.S. Food and Drug Administration (FDA-) approved ADCs (gemtuzumab ozogamicin, trastuzumab emtansine, inotuzumab ozogamicin, and mirvetuximab soravtansine).
Contrastingly, native cysteine conjugation is known as a “semi-random” conjugation technology, which utilizes cysteine generated from partially reduced interchain disulfide bonds by treatment with a controlled equivalent of a reductant such as tris(2-carboxyethyl)phosphine. The conjugation strategy of this controlled reduction, followed by thiol-maleimide coupling, was applied to the manufacture of five commercially available ADCs (brentuximab vedotin, polatuzumab vedotin , enfortumab vedotin, belantamab mafodotin, and tisotumab vedotin). This cysteine-based approach can be considered a more favorable method than the lysine-based one due to the simplified CMC, which allows “some” control over the drug-antibody ratio (DAR) and ADC heterogeneity [2].
To minimize problems due to the heterogeneity of ADCs, several site-specific conjugation technologies have been developed. In this review, three promising chemical conjugation technologies for producing site-specific ADCs are discussed.
Site-specific ADCs
In 2019, site-specific ADCs first entered the clinical arena with the FDA’s approval of trastuzumab deruxtecan. While manufacturing this ADC, Daiichi-Sankyo overcame the challenges associated with ADCs with a DAR of 8, which were previously considered too hydrophobic to be practical. They succeeded in creating an ADC that is stable even at a DAR of 8 by using a unique payload and linker combination [3].
The conjugation technique used mimics conventional cysteine-based ADCs in that it utilizes the reduction of interchain disulfide bonds (although all disulfide bonds are cleaved). However, stochastic ADCs contain positional isomers, which make quality control, reproducibility, and analysis difficult. In contrast, ADCs with a DAR of 8 have the most cysteine groups bound to the payload, minimizing their heterogeneity. For the Daiichi-Sankyo payload, an exatecan analog was chosen to generate hydrophilic carboxylic acid in circulating blood. This payload was covalently attached to the antibody via an enzymatically cleavable linker: a tetrapeptide (Gly Gly Phe-Gly). This tetrapeptide linker enables to provide promising properties possessing a high contrast between plasma stability and sensitivity for the specific enzymes present in cancer cells. The combination of these factors enhances the stability of high-DAR ADC. This commercial success was followed by the approval of sacituzumab govitecan-hziy [1].
In 2014, the PolyTherics research group (currently Abzena) reported a unique chemical conjugation approach, called ThioBridge® [4]. After the reduction of interchain cysteines, this bridging technique provides stable covalent bonds and ADCs with a DAR of 4, in a selective manner. Using this method, the total process yield exceeds 70%. Furthermore, they established an appropriate separation strategy using preparative HPLC to obtain ADCs with a homogeneous DAR of increased purity.
Affinity labeling, an approach using affinity compounds, is a well-established tool in the field of chemical biology. In 2019, the Ajinomoto group applied this approach to produce site-specific ADCs by modifying the Fc region of antibodies using specific affinity reagents [5]. This technology, called AJICAP®, was rapidly applied to the production of good laboratory practice (GLP) materials for relevant preclinical studies [6].
These three chemical site-specific conjugation methods are well established. Their remarkable features are summarized in the table below (Table 1.0).
The DAR of 8 approach has a significant advantage in track-recorded technology, which is used for approved ADCs. However, the capability of this technology seems to be limited, especially in regards to target DAR and drug-linker compatibility.
For example, MC-VC-PAB-MMAE, the main drug-linker in clinical ADCs, cannot be applied to this high DAR method without redesigning the drug linker. The hydrophobicity of MC-VC-PAB-MMAE leads to a crucial amount of aggregation, which lowers its pharmacokinetics and safety profiles. Furthermore, the lack of disulfide bonds in the hinge region due to this conjugation chemistry may result in a potentially risky alteration of the physical properties of the resulting ADCs as compared to naked antibodies. Thus, sacituzumab govitecan-hziy (Trodelvy®; Gilead Oncology, approved in 2020) has significantly less antibody-dependent cell cytotoxicity activity than its naked antibody. The fact that these two ADCs were approved by the FDA suggests that this physical alteration due to hinge disulfides is not an intrinsic problem; however, it may limit the combination of antibodies and drug linkers to be applied to high-DAR ADCs.
Cysteine rebridging
Cysteine rebridging is an excellent method to form stable covalent bonds while overcoming the issues caused by the retro-Michael reaction. This known side reaction detaches linker payloads in the blood circulation, causing undesired toxicity. This feature is a great advantage and makes this technique superior to other chemical conjugation technologies. However, one possible disadvantage of cysteine rebridging is that some undesired “mis-bridged” ADCs are generated in conjugation reactions, resulting in reaction-related heterogeneity. This potential issue can likely be addressed with the use of novel bisalkylating reagents.
The Fc-affinity compound approach is promising and can potentially be applied to a variety of antibodies and drug-linkers without reaction optimizations. Furthermore, this conjugation method, which provides site-specific ADCs with a DAR of 2, does not require hydrophilic linkers or bisalkylating reagents. However, one possible disadvantage of this conjugation technology is the need for a relatively long synthetic sequence. Research groups at Ajinomoto have indicated that optimizations to streamline the sequence were recently undertaken, and this issue may be resolved.
Conclusion
In conclusion, all three conjugation approaches can be recommended to drug developers to use their ADC pipeline project, depending on specific objectives (e.g. biological properties, target DAR, etc). I am confident that further chemical conjugation technologies including these three technologies described herein will further advance to provide the next generation of ADCs.
Highlights of prescribing information
Gemtuzumab ozogamicin (Mylotarg™; Pfizer) [Prescribing information]
Trastuzumab emtansine (Kadcyla®; Genentech/Roche) [Prescribing Information]
Inotuzumab ozogamicin (Besponsa®; Pfizer)[Prescribing Information]
Mirvetuximab soravtansine (Elahere®;ImmunoGen) [Prescribing Information]
Brentuximab vedotin (Adcetris®; Seagen)[Prescribing Information]
Polatuzumab vedotin (Polivy®; Genentech/Roche)[Prescribing Information]
Enfortumab vedotin (Padcev®; Astellas/Seagen)[Prescribing Information]
Belantamab mafodotin (Blenrep®; GSK) [Prescribing Information]
Tisotumab vedotin (Tivdak®; Seagen/Genmap) [Prescribing Information]
Sacituzumab govitecan (Trodelvy®; Gilead Oncology)[Prescribing Information]
Trastuzumab deruxtecan (Enhertu®; Daiichi Sankyo/AstraZeneca) [Prescribing Information]
References
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[2] Matsuda Y, Mendelsohn BA. Recent Advances in Drug-Antibody Ratio Determination of Antibody-Drug Conjugates. Chem Pharm Bull (Tokyo). 2021;69(10):976-983. doi: 10.1248/cpb.c21-00258. PMID: 34602579.
[3] Nakada T, Sugihara K, Jikoh T, Abe Y, Agatsuma T. The Latest Research and Development into the Antibody-Drug Conjugate, [fam-] Trastuzumab Deruxtecan (DS-8201a), for HER2 Cancer Therapy. Chem Pharm Bull (Tokyo). 2019;67(3):173-185. doi: 10.1248/cpb.c18-00744. PMID: 30827997.
[4] Badescu G, Bryant P, Bird M, Henseleit K, Swierkosz J, Parekh V, Tommasi R, Pawlisz E, Jurlewicz K, Farys M, Camper N, Sheng X, Fisher M, Grygorash R, Kyle A, Abhilash A, Frigerio M, Edwards J, Godwin A. Bridging disulfides for stable and defined antibody drug conjugates. Bioconjug Chem. 2014 Jun 18;25(6):1124-36. doi: 10.1021/bc500148x. Epub 2014 May 23. PMID: 24791606.
[5] Yamada K, Shikida N, Shimbo K, Ito Y, Khedri Z, Matsuda Y, Mendelsohn BA. AJICAP: Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies. Angew Chem Int Ed Engl. 2019 Apr 16;58(17):5592-5597. doi: 10.1002/anie.201814215. Epub 2019 Mar 29. PMID: 30854738.
[6] Matsuda Y, Seki T, Yamada K, Ooba Y, Takahashi K, Fujii T, Kawaguchi S, Narita T, Nakayama A, Kitahara Y, Mendelsohn BA, Okuzumi T. Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody-Drug Conjugates. Mol Pharm. 2021 Nov 1;18(11):4058-4066. doi: 10.1021/acs.molpharmaceut.1c00473. Epub 2021 Sep 27. PMID: 34579528.
Authors:
Corresponding Author: Yutaka Matsuda, Ph.D. E-mail: Yutaka Matsuda, Ph.D.
Key terms: ADC, antibody-drug conjugate, chemical conjugation, cysteine rebridging, Fc affinity peptide conjugation
Published In: ADC Review| Journal of Antibody-drug Conjugates
DOI: https://doi.org/10.14229/jadc.2023.01.05.002.
How to cite:
Matsuda Y.1 Recent advances in antibody-drug conjugates produced using chemical conjugation technology – J. ADC. January 5, 2023. DOI: 10.14229/jadc.2023.01.05.002.
1Ajinomoto Bio-Pharma Services, San Diego, CA 92121
Last Editorial Review: December 26, 2022
Article History:
- Original Manuscript Received December 15, 2022
- Review results received December 20, 2022
- Manuscript accepted for publication December 27, 2023
Featured image: Antibody. Photo courtesy: © 2016 – 2023 – Fotolia/Adobe. Used with permission.
