Glycan Oxidation-based Antibody-Drug Conjugate (ADC) Development
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.
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.
We begin with a detailed discussion to understand your specific needs, including antibody and payload characteristics, therapeutic targets, and project timelines. Our experts provide a comprehensive feasibility assessment and a customized plan.
Your antibody is treated to create a uniform population of reactive glycan sites. This involves both enzymatic remodeling and the controlled periodate oxidation to generate the aldehyde groups required for conjugation.
The activated antibody is then conjugated with your drug-linker of choice. Our team optimizes reaction conditions to maximize conjugation efficiency and ensure the formation of a homogeneous product with a consistent DAR.
The resulting ADC is meticulously purified to isolate the homogeneous product and remove unreacted components. We then perform a full suite of characterization assays, including mass spectrometry and HPLC, to confirm the precise DAR, ensure product purity, and verify the integrity of the antibody.
We can support your preclinical development with a range of in vitro and in vivo studies, including assays for target-binding affinity, cytotoxicity, and in vivo efficacy in relevant disease models.
DOI.: 10.3390/ijms24065134
Journal: International Journal of Molecular Sciences
IF: 4.9
Published: 2023
Results: This work developed a site-specific ADC platform leveraging periodate oxidation of native IgG glycans to generate aldehyde handles for chemoselective conjugation. The core methodology involves oxidizing Fc glycans with sodium periodate, followed by oxime ligation with aminooxy-functionalized linkers containing cytotoxic payloads (doxorubicin or MMAE) and stoichiometry-tracking cyanine dyes. A key innovation is the integration of dye-mediated DAR control, where hydrophilic cyanine dyes (sCy3/sCy5) enable precise spectrophotometric quantification of drug-to-antibody ratios at each conjugation step. The approach was validated using an anti-PRAME antibody, yielding homogeneous ADCs with DARs of 2–3. While the doxorubicin conjugate showed limited efficacy, the MMAE-based ADC exhibited potent, target-specific cytotoxicity against PRAME⁺ cancer cells (IC50 ≈ 47 nM) with no off-target effects in PRAME models. This represents the first reported PRAME-targeting ADC and demonstrates the utility of glycan oxidation/oxime ligation for constructing functionally optimized ADCs.
Glycan oxidation-based ADC development leverages controlled periodate cleavage of antibody glycans to enable site-specific conjugation—optimizing therapeutic homogeneity while preserving antigen-binding integrity. To extend this precision to quality validation across diverse pharmaceuticals, we offer Pharmaceutical Analysis Services that ensure purity, stability, and compliance through:
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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