Glycoengineering-based Antibody-Drug Conjugate (ADC) Development
ADCs are prepared by nonspecific modification of antibody lysine or cysteine residues by drug linker reagents, which results in a highly heterogeneous mixture. With the development of the fields of bioorthogonal chemistry and protein engineering, there is a growing interest in site-specific conjugation. CD BioGlyco provides ADC development services through glycosylation site-specific conjugation strategy for our clients, such as glycoengineering-based ADC development.
ADCs have both the targeting ability of antibody macromolecules and the anti-tumor activity of small molecule toxins and use the characteristics of antibody binding to target antigens to carry small molecule toxins into tumor cells to play an anti-tumor effect. The structure of ADCs is generally composed of three parts: 1) Antibodies, which are generally specific monoclonal antibodies designed for target antigens on the surface of tumor cells; 2) Highly active cytotoxins; 3) Linkers, which make toxins and antibodies stable connection by covalent bond. Due to the existence of many functional groups on the antibody molecule, the coupling reaction may be carried out at multiple coupling sites. This leads to the fact that ADCs are highly heterogeneous mixtures of conjugates with different drug-antibody ratios (DAR) and random conjugation sites, which affect the safety and efficacy of the drugs. In recent years, researchers have used unnatural amino acid mutation or used steric hindrance to block other conjugation sites, so that the linker-toxin is site-specific conjugated with specific amino acids of antibodies, which greatly reduces the heterogeneity of ADCs products.
There is a conserved glycosylation site N297 in IgG-type antibodies, which is located in the CH2 domain and is far from the functional domain of the antibody that binds the antigen. Therefore, the use of this site for coupling will not affect the binding function of the antibody. The glycans of the side chains are targeted for site-specific conjugation. This coupling is mainly by introducing a new reactive group on the sugar group and then using related reagents for site-specific coupling.
Antibody glycosylation constitutes an indispensable post-translational modification that profoundly sculpts immunological behavior, circulatory persistence, and clinical efficacy. Our glycoengineering methodology facilitates the manufacture of glycan-defined antibodies featuring exacting site-specific modifications, thereby ensuring batch-wide monosaccharide uniformity and functional fidelity.
Our site-directed bioconjugation methodology employs chemoenzymatic glycan remodeling—an in vitro process enabling meticulous incorporation of functional moieties into the antibody's glycosylation architecture. This strategy comprises two pivotal stages:
Employing the antibody furnished with a uniquely reactive and site-localized handle, we subsequently engage bioorthogonal ligation techniques—exemplified by click chemistry—for covalent payload installation. This classification of reactions encompasses rapid, high-yielding, and biocompatible coupling events, permitting molecular conjugation with exceptional selectivity absent off-target interactions. The cycloaddition between an alkyne-functionalized cytotoxic agent and the azide-modified glycan proceeds with remarkable efficiency, furnishing a homogeneous ADC characterized by a uniform drug-to-antibody ratio (DAR). Such precision stands in stark opposition to conventional stochastic conjugation techniques, which generate heterogeneous adduct populations, thereby affording a more consistent and therapeutically predictable agent.
In addition to the engineering of antibody glycans using in vitro chemical and enzymatic modification strategies, ADCs are also developed by introducing saccharide analogs containing bioorthogonal reactive groups through metabolic engineering. Introduction of 6-thiofucose into the expression medium, which replaces fucose in antibody glycans with 60-70% efficiency, yields antibodies with thiol-functionalized glycans. Antibodies to the thiol-functionalized glycans are conjugated to maleimide-linked drugs to produce ADCs. CD BioGlyco provides ADC development services using this strategy for our clients worldwide.
Case: Metabolic incorporation of 6-thiofucose followed by conjugation with maleimide to produce ADC.
We begin with an in-depth discussion to understand your specific goals, including the antibody and drug payload you wish to use. Our team of experts will then design a tailored glycoengineering and conjugation strategy, selecting the optimal enzymes and reagents for your project.
The starting antibody is enzymatically treated to precisely trim the native N-glycans down to a single GlcNAc residue at the Asn-297 site. This critical step creates a uniform foundation for subsequent site-specific modification.
Next, a highly specific enzyme (e.g., galactosyltransferase) is used to attach a bioorthogonal sugar, such as an azido-derivatized nucleotide sugar, to the exposed GlcNAc residue. Our in-house capabilities ensure a reliable supply of these high-purity sugar nucleotides, which are essential for this step.
The drug payload, which has been functionalized with a complementary bioorthogonal group, is then reacted with the glycan-labeled antibody via highly efficient click chemistry. This process ensures that the drug is attached exclusively at the glycan sites, yielding a homogeneous ADC with a consistent DAR.
The final ADC is purified to remove any unreacted payload, excess reagents, and byproducts. A rigorous quality control (QC) process, including advanced analytical techniques such as mass spectrometry (MS) and high-performance liquid chromatography (HPLC), confirms the identity, purity, and DAR of the final product.
DOI.: 10.1016/j.jbc.2024.108005
Journal: Journal of Biological Chemistry
IF: 3.9
Published: 2024
Results: This study utilizes glycan remodeling enzymes like endoglycosidases to trim complex glycans, exposing core GlcNAc residues for chemoenzymatic functionalization. Alternatively, periodate oxidation generates aldehyde handles on sialic acid or galactose residues for oxime ligation with aminooxy-bearing linkers. Key advantages include preservation of antigen-binding affinity, avoidance of antibody engineering, and generation of homogeneous ADCs with defined drug-to-antibody ratios (DAR ≈ 2–4). The methodology allows precise attachment of cytotoxic payloads (e.g., MMAE, doxorubicin) via bioorthogonal chemistry while maintaining Fc-mediated effector functions. Recent advances demonstrate improved pharmacokinetics and potent in vitro/in vivo efficacy against target-expressing tumors, positioning glycoengineering as a robust platform for next-generation ADC therapeutics.
While the strategic application of glycoengineering empowers the development of ADCs with enhanced homogeneity and therapeutic efficacy, unlocking the full potential of this technology necessitates a profound understanding of the glycosylation profiles themselves. This imperative extends beyond ADCs to encompass the entire spectrum of biopharmaceuticals, mandating comprehensive Characterization of Glycosylation in Drugs. This critical analysis verifies glycan attributes across diverse modalities, including:
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 are interested in our glycoengineering-based ADC development services, please feel free to contact us for more information.
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