Analysis of Six Classic Point-Coupling Technologies for ADC

ADC drug conjugation refers to the method of linking various components of ADC, which determines the drug-antibody conjugation ratio (DAR) and homogeneity, affecting the drug’s activity, tolerance, and stability. It is generally divided into random coupling and site-specific coupling.

Non-Site-Specific Coupling Common methods include coupling at Lys residues and Cys residues.

Site-Specific Coupling, which includes the introduction of reactive cysteine, disulfide bridge re-bridging, non-natural amino acid technology, enzyme-catalyzed technology, glycosylation coupling technology, and proximity-induced antibody coupling (pClick) technology.

Site-specific coupling is easier to control in terms of its DAR value, and the ADC drugs produced have good homogeneity, making it a trend in the development of coupling methods.

In this article, we will introduce the advantages and disadvantages of the six major site-specific coupling technologies, and provide in-depth analysis using examples from relevant companies.

Introduction of Reactive Cysteine

The introduction of reactive cysteine, also known as Thiomab technology, was first developed by Genentech.

In simple terms, it involves inserting cysteine residues at specific points in the antibody through genetic engineering, and then coupling the hydroxyl group on the cysteine with the toxin to form a site-specific antibody-drug conjugate.

Analysis of Six Classic Point-Coupling Technologies for ADC

The Thiomab technology, engineered to introduce cysteine, does not interfere with the folding and assembly of immunoglobulins, nor does it change the binding mode of antibodies to antigens. The resulting ADC drugs retain their in vivo anti-tumor activity, improve tolerance, and reduce systemic toxicity.

This technology has advantages such as high homogeneity, good reactivity, and stability, but the drawback is that it requires genetic modification, typically resulting in a DAR of 2.

Currently, Roche’s ADC product targeting CD79b, Iladatuzumab vedotin (DCDS0780A), employs Thiomab technology.

Based on this, Seattle Genetics and Spirogen developed a similar technology called MAIA, which introduces a serine-cysteine mutation at position 239 in the key region of mAb for the bioconjugation of its PBD dimer.

Disulfide Bridge Re-Bridging

Disulfide bridge re-bridging, also known as Disulfide re-bridging, utilizes cysteine-selective crosslinking reagents (such as TECP or DTT) to reduce four inter-chain disulfide bonds in IgG1 antibodies. Subsequently, the small molecule payload is installed or further modifications of the antibody are completed while reconnecting the polypeptide chains using bifunctional reagents.

By covalently reconnecting cysteine residues, this method maintains the stabilizing effect of disulfide bonds while achieving controlled coupling where each disulfide bond links to one effective payload.

This coupling method has high homogeneity, does not affect the spatial structure of the antibody, and has a universal amino acid sequence with glycosylation modifications. However, it is prone to intra-chain erroneous bridging, typically resulting in a DAR of 4.

Lgenica Biotherapeutics’ SNAP platform and Abzena’s Thiobridge platform utilize disulfide bond reduction modification technology.

Non-Natural Amino Acid Technology

Non-natural amino acid technology, as the name suggests, artificially adds non-natural amino acids to the original sequence of the antibody, allowing specific sites to be presented on the antibody surface for convenient coupling, thus achieving site-specific, uniform DAR values for ADC.

The uniqueness of this coupling technology lies in its ability to randomly mutate non-natural amino acids, and to obtain ADCs with arbitrary DAR values. However, it requires genetic modification, has low antibody expression levels, and the non-natural amino acid sequence can cause immunogenicity and hydrophobicity-induced aggregation.

Ambrx’s ARX788 is the first antibody-drug conjugate developed using non-natural amino acids, and it is currently seeking market approval.

The non-natural amino acid chosen for ARX788 is acetylphenylalanine (pAF), where the ketone group on pAF can form an oxime bond with the hydroxyl group on the effective payload AS269, resulting in site-specific conjugation and producing homogeneous ADC.

Another ADC that also employs non-natural amino acid coupling technology is STRO-002, which is a third-generation ADC developed by Sutro targeting FRα and has global intellectual property rights.

The antibody portion of this drug’s synthetic process utilizes cell-free protein synthesis technology (XpressCF), which allows for the insertion of non-natural amino acids at specific sites in the protein, enabling the site-specific conjugation of ADC through click chemistry reactions with cathepsin B cleavable linkers.

Enzyme-Catalyzed Technology

Enzymes have high specificity and efficiency, and are now also applied in the development of ADCs.

Enzyme-catalyzed technology inserts specific amino acid markers that can be recognized by certain specialized enzymes into the antibody sequence, allowing toxins to selectively connect with the antibody.

This technology allows for site selection and customizable DAR values, but requires genetic modification to insert enzyme recognition sequences, and there is a risk of immunogenicity due to foreign amino acid sequences.

In China, Qide Pharmaceutical is one of the early innovative high-tech enterprises to develop ADC drugs using differential enzyme-catalyzed site-specific coupling technology.

Unlike traditional chemical coupling methods, Qide Pharmaceutical has uniquely developed two systems based on enzyme-catalyzed site-specific coupling technology iLDC and iGDC through years of accumulation in the field of synthetic biology, achieving the preparation of highly homogeneous ADC drugs through engineered transpeptidase and glycosyltransferase modifications.

The company’s GQ1001 is an ADC generated through the specific coupling of toxin DM1 with trastuzumab based on its unique enzyme-catalyzed coupling technology and patented open-loop linker technology.

Additionally, Shiyao Group’s SYS6002 employs its proprietary enzyme-catalyzed site-specific antibody coupling technology to direct effective mitotic inhibitor MMAE specifically to Nectin-4 expressing cancer cells, while the stability of its linker aids in delivering high concentrations of MMAE to tumors while reducing adverse systemic exposure and side effects.

Glycosylation Coupling Technology

Glycosylation coupling technology modifies different glycosyl groups in natural proteins using glycosidase, exposing N-acetylglucosamine, and then connects N-acetylgalactosamine modified by azide chemistry to the N-acetylglucosamine on the antibody using glycosyltransferase, ultimately achieving site-specific conjugated ADC through click chemistry reactions.

The advantage of this technology is that the drug linker is coupled with the glycan without changing the amino acid sequence, and they are connected far from the amino acid residues.

However, this method requires special reagents and enzymes needed for glycoengineering. For reagents that may introduce foreign factors during the modification process, the purification process should carefully consider the removal of foreign factors and conduct viral clearance studies in accordance with ICH Q5A guidelines.

Synaffix’s core platform GlycoConnect™ is based on glycosylation site-specific coupling technology.

GlycoConnect™ achieves efficient site-specific coupling by anchoring effective payloads to the N297 glycan of the antibody. By modifying each N-glycan with glycosidase and precise azide labeling, a unique anchor point is introduced for copper-free click reactions with effective payloads.

GlycoLink’s glycosylation coupling technology is called “DisacLink” technology, which uses only one enzyme, has a short reaction time, and high and stable coupling rates.

This technology not only greatly reduces the complexity of preparing site-specific ADC drugs, lowers the R&D costs of site-specific ADC drugs, but also accommodates various drug and linker structures, achieving structural diversity in the preparation of site-specific ADC compounds, providing rich structural diversity for candidate drug screening.

Proximity-Induced Antibody Coupling (pClick) Technology

The pClick technology introduces proximity-activated crosslinkers, allowing azide-modified peptides to spontaneously react with the nearest lysine residues on the antibody. The azide group provides a biorthogonal handle for click chemistry to modify effective payloads.

The pClick technology does not require any modifications to the antibody, but connects the toxin-linker to the antibody affinity protein, which then undergoes a proximity-induced reaction with the antibody to obtain ADC molecules with arbitrary DAR values.

Additionally, this technology can attach effective payloads site-specifically to natural antibodies under mild conditions, thereby minimizing interference with binding to antigen receptors or FcγRIII receptors (which are responsible for activating antibody-dependent cellular cytotoxicity).

The pClick technology provides a new option for ADC development, allowing for a more convenient and efficient way of site-specific coupling.

Reference link:

https://www.nature.com/articles/s41392-022-00947-7/tables/2

Analysis of Six Classic Point-Coupling Technologies for ADC

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Analysis of Six Classic Point-Coupling Technologies for ADC

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Analysis of Six Classic Point-Coupling Technologies for ADC

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