Glycosylation Site-specific Antibody-Drug Conjugate (ADC) Development Platform
CD BioGlyco has been focusing on glycosylation site-specific ADC development for many years. We provide three glycosylation site-specific conjugation strategies to bind payloads: 1) Glycan oxidation; 2) Glycan enzymatic modification; 3) Glycoengineering.
Due to the limited selectivity, systemic toxicity, and drug resistance of traditional small-molecule anticancer drugs against cancer cells, emerging targeted ADCs have received extensive attention from researchers. ADC is a humanized or human monoclonal antibody conjugated to a biologically active cytotoxic small molecule (payload) via a chemical linker. It targets cancer cells more effectively, thereby increasing the efficacy of the drug and reducing systemic toxicity. ADC is composed of three parts: antibody, cytotoxic drug, and the linker between them. During the development of ADCs, antibodies are the key to target specificity, and the payload drug is attached to different sites on the antibody through linker chemistry. Commonly used coupling methods include cysteine conjugation, glycoconjugation, unnatural or noncanonical amino acid incorporation, and peptide tags.
After the ADC enters the bloodstream, the antibody component of the ADC recognizes and binds to cell surface antigens that are highly expressed in target cells to form an ADC-antigen complex, resulting in the endocytosis of the complex. The internalized complex is processed within the lysosome and releases the cytotoxic payload inside the cell. The released payload binds to its targets such as deoxyribonucleic acid (DNA) strands or microtubules, or exerts topoisomerase or ribonucleic acid (RNA) polymerase inhibition, ultimately leading to cell death. Cytotoxic chemical agents that typically have high potency against cancer cells but have low off-target cytotoxicity are used as payloads.
Chemically oxidizes native antibody glycans to generate aldehyde groups for site-specific conjugation with payloads.
Uses enzymatic strategies to modify glycans, including:
Employs metabolic engineering to incorporate non-natural sugars (e.g., 6-thiofucose) into antibodies, followed by targeted conjugation (e.g., with maleimide linkers).
Immunoglobulin G (IgG) molecules have a conserved glycosylation site at each N297 residue in the CH2 domain, and this site is sufficiently distant from the variable region that regulates antigen interactions. Therefore, binding to linked glycans is unlikely to impair antigen-binding affinity and may serve as an excellent surrogate for site-specific ADC generation. At CD BioGlyco, we offer three glycosylation site-specific conjugation strategies to bind payloads: 1) Glycan oxidation; 2) Glycan enzymatic modification; 3) Glycoengineering.
The process begins with the enzymatic remodeling of the client's monoclonal antibody. This step involves transferring galactose and sialic acid residues onto the native glycans, preparing the antibody for subsequent chemical modification. We ensure this process is meticulously controlled for consistency and high yield.
The enzymatically modified antibody is then subjected to a mild oxidation using sodium periodate. This step is carefully optimized to selectively generate reactive aldehyde groups on the terminal sialic acids while minimizing any off-target oxidation that could compromise the antibody's integrity and function.
The aldehyde-functionalized antibody is then reacted with a cytotoxic drug payload that has been synthesized with a corresponding aminooxy group. This reaction forms a highly stable oxime bond, creating an ADC with a precise and predictable DAR.
The final ADC product is purified to remove unconjugated drug-linker and other impurities. We perform extensive characterization using advanced analytical techniques such as hydrophobic interaction chromatography (HIC) and LC-MS to confirm homogeneity and the exact DAR, ensuring a final product of the highest quality.
DOI.: 10.3390/antib12040071
Journal: Antibodies
IF: 2.7
Published: 2023
Results: This research systematically evaluated two chemoenzymatic approaches for generating site-specific ADCs via Fc-glycan remodeling. The authors optimized scalable synthesis of azido-glycan oxazolines (disaccharide and sialylated complex-type) and applied both one-pot (EndoS2-catalyzed transglycosylation with azido-disaccharide) and two-step (deglycosylation followed by glycosynthase-mediated transfer of azido-SCT glycan) strategies to trastuzumab. Resulting azide-functionalized antibodies were conjugated to MMAE (cleavable linker) or DM1 (non-cleavable linker) payloads via SPAAC click chemistry. All ADCs exhibited high homogeneity (DAR 4), <2% aggregation, exceptional serum stability, and potent in vitro cytotoxicity against BT-474 cells. In vivo xenograft studies demonstrated dose-dependent tumor eradication (e.g., complete regression at 10–15 mg/kg), comparable efficacy to FDA-approved T-DM1, and no significant weight loss, confirming therapeutic potential. The study validates both glycoengineering routes as robust platforms for next-generation ADC development.
The primary application for ADCs, our platform is optimized for the development of potent and selective anti-cancer agents.
Targeted delivery of immunomodulatory or immunosuppressive agents can provide a more effective and less toxic treatment for autoimmune disorders.
ADCs can be engineered to target and deliver antimicrobial agents to specific infected cells.
The platform can be used to site-specifically label antibodies for diagnostic imaging or to deliver genetic material to target cells.
Glycosylation site-specific ADC development platforms empower precision engineering of ADCs with optimized therapeutic indices—leveraging conserved glycosylation sites for homogeneous payload attachment. To validate the safety, purity, and efficacy of such advanced therapeutics and other critical pharmaceuticals, we provide Pharmaceutical Analysis Services that rigorously characterize drug composition, stability, and impurities:
CD BioGlyco is dedicated to offering glycosylation site-specific ADC development services to help our global clients in this field. If you have a related ADC development project in hand, please feel free to contact us for more information.
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