ADCs are a class of drugs that use monoclonal antibodies to achieve targeted delivery of cytotoxic drugs to antigen-expressing cells. CD BioGlyco provides customers with ADC development services through site-specific conjugation based on glycan oxidation.
ADCs combine the targeting specificity of monoclonal antibodies with the potency of cytotoxic drugs to achieve targeted delivery of cytotoxic drugs to antigen-expressing cells, thereby enhancing therapeutic activity. ADCs generally consist of an antibody, a cytotoxic drug, and a linker between the two. During the development of ADC drugs, it is necessary to select monoclonal antibodies that recognize cell surface antigens and be endocytosed by cells. And cytotoxic molecules and coupling methods also need to be comprehensively considered. The traditional coupling method is to use drug linker reagents to non-specifically modify antibody lysine or cysteine residues, but this method has the problem of lack of specificity of coupling sites and the number of coupled small molecules. Therefore, the traditional coupling method is difficult to control the uniformity of the drug, and moreover, the efficacy, biosafety, and kinetic parametersof ADCs with different payloads are also different. The site-specific conjugation of ADC can effectively solve this problem.
There are various methods to achieve site-specific conjugation, such as cysteine conjugation, glycoconjugation, incorporation of unnatural amino acids, peptide tags, etc. Cysteine residues are a classic method of site-specific protein modification by engineering or reducing native disulfide bonds to generate cysteine residues, which are then selectively reacted with many electrophiles. Glyco-based site-specific conjugation is based on human IgG molecules having a conserved glycosylation site at each N297 residue in the CH2 domain, making the N-glycans of the side chain site-specific conjugated target. Unnatural amino acids are incorporated into the antibody sequence via amber stop codon-mediated transcription, enabling site-specific conjugation. Biotin ligases and transglutaminases, etc. provide site-specific attachment of small molecules to peptide substrates in various proteins.
| Category | Specific Method | Pros | Cons |
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| Cysteine conjugation | 1. Global reduction/alkylation 2. Cysteine to serine mutation 3. THIOMAB 4. N-Terminal cysteine conjugation |
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| Glycosylation site-specific conjugation | 1. Periodate oxidation of fucose or sialic acid 2. Enzymatic transfer of azidosugars 3. Metabolic incorporation of thiofucose |
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| Unnatural or noncanonical amino acid incorporation | 1. Amber codon suppression 2. Cell-free amber codon suppression 3. Selenocysteine incorporation |
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| Peptide tags | 1. Deglycosylation followed by transglutaminase treatment 2. Transglutaminase tag 3. Sortase tag 4. Aldehyde tag |
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Tab.1 Construction methods of site-specific ADCs. (Agarwal & Bertozzi, 2015)
Oxidative cleavage of the terminal cis-diol oligosaccharide results in an aldehyde group that is chemically reacted with some reagents containing aminooxy, hydrazine, or hydrazide functional groups. The reaction allows site-specific conjugation of the load to the antibody. Antibody glycans typically contain vicinal diols that are cleaved by oxidation to generate aldehyde groups. At CD BioGlyco, we oxidize the sugar group in the antibody's native glycan to an aldehyde group by the periodate oxidation method, and then the aldehyde group is combined with a small molecule hydrazide or aminooxy compound.
Case: The fucose in the antibody's native glycan is oxidized by sodium periodate (NaIO4) and coupled to hydrazone. Thereby the drug-linker molecule binds to the N-glycan of IgG.
Fig.1 Fucose-based site-specific conjugation. (Agarwal & Bertozzi, 2015)
CD BioGlyco is committed to providing ADC development services utilizing glycosylation site-specific conjugation technology to meet the research needs of our clients. If you have glycan oxidation-based ADC development services, please feel free to contact us for more information.
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