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Site-specific conjugation is an important aspect of antibody-drug conjugates (ADCs). Compared with non-specific conjugation, site-specific bioconjugation can produce more uniform, well-defined, and developable antibody conjugates. At BOC Sciences, we offer a range of site-specific conjugation services to meet the diverse needs of our customers in the ADC space. Our experienced team of scientists is well-versed in the latest conjugation technologies and can work with a variety of antibody formats, including monoclonal antibodies, antibody fragments, and engineered antibodies.
Although non-specific chemical couplings such as lysine amine acylation or cysteine thiol alkylation are widely used, they still have a series of shortcomings. For example, a lack of site specificity leads to product heterogeneity and process irreproducibility; potential modifications in complementarity-determining regions may reduce the binding affinity and specificity of the antibody. Site-specific conjugation can produce more uniform antibody conjugates, thereby ensuring product development and clinical application. Recent comparisons between site-specific and random modifications have shown that site-specific modifications can achieve the desired characteristics and functions, such as improving plasma stability, reducing variability in dose-dependent studies (especially at low concentrations), enhancing binding affinity, and increasing tumor uptake and absorption.
Fig. 1. Categories of site-specific ADCs (Biomedicines. 2017, 5(4): 64).
In recent years, many research groups have reported site-specific conjugation strategies for the preparation of homogeneous ADC drugs. Some studies use genetic engineering technology to introduce some specific reaction sites at a certain position of the antibody, and achieve site-specific coupling of antibodies and drugs through the selective reaction of drug-loaded groups with specific sites on the antibody. Among them, Thiomab technology may be the earliest strategy to realize site-specific conjugation of antibodies and drugs. It inserts two cysteine residues into the light and heavy chains of the antibody through genetic engineering technology. The sulfhydryl group of cysteine can be coupled with the microtubule-related inhibitor MMAE to obtain a homogeneous ADC drug with a DAR of 2.
With our state-of-the-art facilities, cutting-edge technology, and stringent quality control measures, BOC Sciences is committed to providing high-quality site-specific coupling services for ADC development. Whether you want to improve the stability, efficacy or safety of your ADC, we have the expertise and capabilities to help you achieve your development goals.
Site-specific conjugation of amino acids often requires the selection of several natural or engineered amino acids, such as cysteine and glutamine, as conjugation sites. Among them, unpaired cysteine-mediated conjugation was the first site-specific ADC to be described. Cysteine residues are engineered into different positions on the antibody heavy chain (HC) or light chain (LC) for conjugation. Because engineered cysteines are always capped by glutathione or other substances during expression, partial reduction of the antibody is required to remove the cap. The uncapped cysteine is then coupled to a cytotoxic-containing thiol-reactive linker using thiol-maleimide chemistry.
There are also researchers constructing ADC drugs by modifying different types of glycosyl-groups on natural antibodies. These glycosyl-groups on the antibody can be exposed to N-acetylglucosamine under the action of endoglycosidase, and then N-acetylgalactosamine modified by azide is linked to the N-acetylglucose of the antibody under the action of glycosyltransferase. Finally, azide-modified antibodies can achieve site-specific coupling of antibodies and drugs by clicking chemical reactions with alkynyl linker toxins.
In addition to modifying natural antibodies, non natural amino acids (such as acetylphenylalanine, azidylmethyl-L-phenylalanine with bioorthogonal functionality, and azidyllysine) can be integrated into antibodies by using enzymes that can specifically recognize non natural amino acids. These unnatural amino acids contain specific functional groups (such as ketone groups, azide groups, etc.) that can react specifically with other functional groups in the drug linker (such as hydroxylamine groups, alkynyl groups, etc.) to obtain ADC drugs with uniform DAR. In addition, enzymes have high specificity and efficiency. Some researchers recognize specific sequences or sites of antibodies through some enzymes (such as transglutaminase, formylglycine synthetase and sortase A, etc.), and then introduce some specific functional groups through the catalysis of enzymes to achieve site specificity.
Several site-specific conjugation methods are being developed by coupling cytotoxins to specific short peptide tags containing four to six amino acid residues. Such methods rely on the introduction of unique short peptide tags into antibodies for enzymatic modification in vivo or in vitro. They allow specific amino acids in the peptide tag to be functionalized and conjugated to drug linkers.
A pharmaceutical company is struggling to develop effective ADCs due to a lack of site-specific conjugation expertise. They turned to BOC Sciences for help, and our combination of site-specific services proved to be key to their success.
Utilizing our cutting-edge technology and state-of-the-art facilities, we are able to engineer cysteine residues into specific locations on antibodies for conjugation. Through precise manipulation and partial reduction, we successfully opened engineered cysteines and coupled them to cytotoxic-containing thiol-reactive linkers using thiol-maleimide chemistry. This has led to the creation of highly stable, effective and DAR consistent ADCs. Thanks to BOC Sciences' expertise and capabilities, the pharmaceutical company was able to achieve its development goals and successfully advance its ADC into clinical studies.
Site-specific ADC conjugation is a method used to attach cytotoxic drugs to specific sites on antibody molecules in antibody-drug conjugates (ADCs). This allows for more precise and controlled drug conjugation compared to non-specific conjugation methods, potentially improving efficacy and reducing side effects. Site-specific conjugation can be achieved by various techniques, such as using engineered antibodies with specific conjugation sites or utilizing chemical modifications to target specific amino acid residues on the antibody.
Cysteine-conjugated ADCs are a type of antibody-drug conjugate (ADC) in which the drug payload is linked to the antibody through a cysteine residue. Cysteine conjugation involves the use of linker molecules containing reactive groups that can specifically bind to cysteine residues on antibodies, allowing site-specific attachment of drug payloads. This approach could improve ADC stability and efficacy by reducing off-target binding and increasing the specificity of drug delivery to tumor cells.
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