Overview

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.

Introduction of ADC

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.

Key Technologies

• Glycoengineering for Homogeneous Antibodies

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.

• Chemoenzymatic Glycan Remodeling

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:

• Glycan trimming: Endogenous, heterogeneous N-glycans are systematically truncated to a solitary N-acetylglucosamine (GlcNAc) residue at the conserved N297 glycosylation site, yielding a standardized attachment locus for subsequent functionalization.
• Enzymatic labeling: Utilizing highly specific enzymes such as galactosyltransferases in concert with purified sugar-nucleotide donors, we introduce synthetic carbohydrate analogs bearing bioorthogonal handles; a representative example involves the site-specific incorporation of an azide-functionalized sugar (e.g., GalNAz) onto the homogenized glycan core.

• Bioorthogonal Chemistry for Site-Specific Conjugation

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.

Revolutionizing ADC Development with Advanced Glycoengineering

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.

Workflow

• Project Consultation & Design

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.

• Enzymatic Glycan Remodeling

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.

• Site-Specific Glycan Labeling

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.

• Bioorthogonal Drug Conjugation

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.

• Purification & Quality Control

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.

Publication Data

Advantages

• ADCs produced via our glycoengineering platform demonstrate improved in vivo properties.
• We possess the unique capability to produce our own high-purity sugar nucleotides, the essential building blocks for glycan remodeling.
• We utilize various enzymatic and bioorthogonal chemistry methods, and our process can be applied to antibodies produced in different host systems, including CHO, HEK, and SF9 cells. This flexibility allows us to tailor our services to your specific project needs.

Application

• ADCs are being explored for autoimmune diseases, representing a significant expansion beyond oncology.
• ADCs are primarily used to research various blood cancers (like lymphomas and leukemias) and solid tumors (such as breast, lung, and ovarian cancers).
• A cutting-edge development involves bispecific ADCs that can target two different antigens. This aims to enhance selectivity, overcome drug resistance, increase internalization into cancer cells, and improve the overall therapeutic profile.

Frequently Asked Questions

Associated Services

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.