Understanding Antibody-Drug Conjugates (ADC)

Understanding Antibody-Drug Conjugates (ADC)

Introduction

Antibody-drug conjugates (ADCs) typically consist of: highly specific and high-affinity antibodies, stable linkers, and potent small-molecule cytotoxic drugs1. The key to the development of ADCs lies in the selection of targets, antibodies, cytotoxic payloads, linkers, and the conjugation method. Among these, the conjugation method of ADCs directly determines the drug-to-antibody ratio (DAR), the distribution of conjugation sites, and the stability of the conjugation, among other properties. This is also the most challenging barrier in the technological development of ADCs. This article will focus on the conjugation methods of ADCs.

Previous Articles:

ADC Series – Target Section

ADC Series – Antibody Section

ADC Series – Linker Section

ADC Series – Payload Section

Understanding Antibody-Drug Conjugates (ADC)

Figure 1 Schematic of ADC Structure

Classification of ADC Conjugation Methods

The first type is non-specific conjugation technology (random conjugation technology) : lysine-based conjugation technology and cysteine-based conjugation technology;The second type is specific conjugation technology: introducing reactive cysteine conjugation (Thio-mab) technology, interchain disulfide bond modification, unnatural amino acid conjugation technology, enzyme modification conjugation, etc..2-3

Non-Specific Conjugation Technology

In non-specific conjugation technology, the earliest used is lysine residue conjugation (Figure 2A). Lysine residues have a high natural abundance (7.2%) and surface accessibility, and immunoglobulin G1 (IgG1) contains about 90 lysine residues, of which more than 30 can be chemically modified. Therefore, this conjugation method is rapid and convenient, but its selectivity is relatively poor, and uniformity is insufficient, usually resulting in multiple ADCs with variable DAR and conjugation sites, affecting the PK/PD of ADCs. Currently, Kadcyla, Mylotarg, and Besponsa on the market use lysine residue conjugation.
Cysteine-based reactions provide another conjugation method: conjugation with cysteine residues (Figure 2A), which means that after reducing the disulfide bonds, they are converted into cysteine residues for conjugation reactions. Typically, IgG1 antibodies have interchain disulfide bonds and intrachain disulfide bonds. The interchain disulfide bonds are exposed on the outside of the antibody and can be easily reduced to expose free cysteine residues, providing available sites for the conjugation of the payload with the antibody. Due to the limited number of binding sites and the unique reactivity of thiol groups, using cysteine as a conjugation site helps reduce the heterogeneity of ADCs. After reduction, up to 8 cysteine residue sites can be exposed, generating drugs with DARs of 2, 4, 6, and 8. Cysteine conjugation can significantly reduce the heterogeneity of ADCs and achieve higher uniformity than ADCs based on lysine conjugation. Market products like Polivy, Padcev, and Adcetris use this method for conjugation.2-5

Understanding Antibody-Drug Conjugates (ADC)

Figure 2 A Lysine Residue Conjugation and Its DAR Distribution; B Cysteine Residue Conjugation and Its DAR Distribution

Specific Conjugation Technology

The first is the introduction of reactive cysteine technology.The ThioMab technology developed by Genentech is a representative of this technology (Figure 3). It is achieved by genetic engineering, inserting cysteine residues at different positions of the heavy chain (HC) or light chain (LC) of the antibody for drug conjugation. The percentage of ADCs generated with a DAR of 2 can be as high as 92.1%. Additionally, the ThioMab technology does not affect the folding and assembly of immunoglobulins or the binding of antibodies to antigens. One major drawback of ThioMab technology is that the introduction of thiol groups may lead to the formation of incorrect disulfide bonds between the two fabs in the antibody, which remains an issue to be resolved.

Understanding Antibody-Drug Conjugates (ADC)

Figure 3 THIOMAB Technology Process

The second is disulfide bond modification technology (Figure 4).Disulfide bond re-bridging has also attracted attention, although its conjugation efficiency is lower and there are intrachain cross-bridges. Similar to traditional cysteine conjugation, the conjugation sites are also obtained by reducing interchain disulfide bonds. Unlike random conjugation, disulfide re-bridging involves reactions with cysteine-selective crosslinking reagents, such as next-generation maleimides (NGMs), pyridazinediones (PDs), etc.

Understanding Antibody-Drug Conjugates (ADC)

Figure 4 Re-bridging of Interchain Disulfide Bonds

The third is unnatural amino acid conjugation technology (Figure 5).By artificially incorporating unnatural amino acids into the original sequence of the antibody, specific sites that are convenient for conjugation can be presented on the antibody surface, thus obtaining site-specific and uniform DAR ADCs. Unnatural amino acid conjugation allows for arbitrary mutations of unnatural amino acids and the generation of ADCs with arbitrary DAR values. ADCs generated through the introduction of unnatural amino acid technology exhibit longer circulation half-lives, better efficacy, and safety.

Understanding Antibody-Drug Conjugates (ADC)

Figure 5 Process of Unnatural Amino Acid Conjugation

The fourth is enzyme modification (Figure 6).Through genetic engineering, specific amino acid sequences are artificially induced to be expressed in the antibody, which can be recognized by specific enzymes. Subsequently, the specific amino acid residues are modified by enzymes, achieving site-specific binding. Currently, commonly used enzymes include formylglycine-generating enzyme (FGE) and transglutaminase (TG).

Understanding Antibody-Drug Conjugates (ADC)

Figure 6 Microbial Transglutaminase (MTGase) Method

In addition to the above common four specific conjugation technologies, there are also glycosylation technology, pClick technology, etc.2-6

Conclusion

Compared with random conjugation technology, specific conjugation technology shows more promising preclinical activity and higher therapeutic indices, improving safety, PK, and efficacy. Therefore, in the future, with the continuous development and innovation of conjugation technologies, these new technologies will play an important role in the R&D and application of ADC drugs.

Understanding Antibody-Drug Conjugates (ADC)

Table 1 Characteristics of Common Conjugation Methods for ADCs

References

1. Chinese Anti-Cancer Association Clinical Research Professional Committee of Tumor Drugs, National Expert Committee for Clinical Application Monitoring of Anti-Cancer Drugs, National Tumor Quality Control Center Breast Cancer Expert Committee, et al. Expert Consensus on the Clinical Application of Antibody-Drug Conjugates for Malignant Tumors in China (2023 Edition) [J]. Chinese Journal of Oncology, 2023, 45(9):741-762.

2. Li M, Zhao X, Yu C, et al. Antibody-Drug Conjugate Overview: a State-of-the-art Manufacturing Process and Control Strategy. Pharm Res. 2024 Mar;41(3):419-440.

3. Tsuchikama K, An Z. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell. 2018 Jan;9(1):33-46.

4. Metrangolo V, Engelholm LH. Antibody-Drug Conjugates: The Dynamic Evolution from Conventional to Next-Generation Constructs. Cancers (Basel). 2024 Jan 20;16(2):447.

5. Fu Z, Li S, Han S, et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Signal Transduct Target Ther. 2022 Mar 22;7(1):93.

6. Kostova V, Désos P, Starck JB, et al. The Chemistry Behind ADCs. Pharmaceuticals (Basel). 2021 May 7;14(5):442.

* This article is for the purpose of providing scientific information to medical professionals and does not represent the views of this platform.

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Understanding Antibody-Drug Conjugates (ADC)
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Understanding Antibody-Drug Conjugates (ADC)

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