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Institute Reference: INV-21035
Background
Antibodies are a predominant class of macromolecules with wide-spread use throughout the scientific community including laboratory assays, imaging techniques, and medical treatments. Antibodies can be costly and challenging to properly manufacture or engineer whilst maintaining initial structural integrity and function of the antibody itself. Non-specific chemical methods – such as acylation of amines (e.g., lysine) or alkylation of thiols (e.g., cysteine) – are widely used to construct protein conjugates for a myriad of applications. These techniques have several shortcomings. First, the lack of site-specificity results in heterogenous products, as well as a synthetic route that is irreproducible and prone to side reactions. Second, modifications at or near the functional domains of proteins may significantly alter and/or reduce their activity and specificity.
Conversely, site-specific methods produce homogenous, reproducible, and well-defined products that are likely to achieve their desired function. A versatile and chemoenzymatic tool to modify native proteins site-specifically is transglutaminase (TGase). Northeastern researchers have developed several methodologies overcoming current limitations in antibody engineering and demonstrate their usefulness in analysis, imaging, and photoactivation.
Technical
This invention centers on a new method for modification of antibodies and their related fragments via a chemoenzymatic process. The researchers have developed a method to trim native or engineered glycans on antibodies – namely glycan remodeling – to render antibody glutamine residues accessible for conjugation by TGase. This newly developed process maintains the antibody’s core glycan without generating charged intermediary side residues as seen with current methods. This initial remodeling can be followed up to conjugate native or engineered glutamine residues with an amine containing reagents or other suitable substitutions (i.e., fluorophores, reactive side chains enabling click chemistry, PEGs, modified Fc regions reducing ADCC, photocleavable conjugates) via a transamidation reaction mediated by TGase. This allows for a facile process of site-specific antibody engineering resulting in higher binding efficiency antibody-conjugates. The final engineered product is homogenous with a single glycoform and more likely to have reduced immunogenicity than previous PNGaseF hydrolysis methods. These specific alterations can not only change an antibody’s biological form and function, but abolishment of effector function can result in better imaging agents by decreasing non-specific binding.
Benefits
• Design of the molecular entity with single, dual, or multiple sites of modifications on macromolecules: i.e., glycan and glutamine residues.
• No generation of charge variants (i.e., aspartic acid) compared to previous and concomitant conversion of the asparaginyl amide to aspartyl carboxylic acid.
• Production of homogenous glycoengineering of macromolecules, e.g., modulation of effector functions such as antibody-dependent cellular cytotoxicity (ADCC), imaging, radioimmunoconjugates, PEGylation, photocleavable side conjugates, glycoengineering.
• Introduction of isotopes eliminates need for metal isotopic labeling or recombinant antibody production.
Applications
• Antibody drug conjugates
• Radioimmunoconjugates
• PEGylated drugs
• Bioconjugation
• Medical applications such as protein drugs
• Preparation of imaging and research reagents
• Engineering of synthetic biology approaches for cell therapies and biologics
