Antibody Site-Specific Conjugation via Enzymatic Deglycosylation
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Solution Overview
Problem
Current methods for site-specific conjugation of antibodies with chelating agents result in heterogeneous products with variable pharmacokinetic properties, lacking a defined Chelator-to-antibody ratio (CAR), which affects their therapeutic efficacy and tumor uptake.
Innovation Solution
A method involving enzymatic deglycosylation of antibodies followed by coupling with a Linker-Chelator compound using transglutaminase, ensuring site-specific attachment at Glutamine 295, allowing for a controlled and defined CAR between 0 and 2, without the need for genetic engineering.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If non-site specific chemical conjugation is used to attach chelators to antibodies, then the conjugation process is simple and fast, but the resulting product is highly heterogeneous with undefined Chelator-to-antibody ratio (CAR) and variable pharmacokinetic properties
Solution Approach 1:
The antibody is pre-modified by removing the N297 glycan moiety with PNGase F before conjugation. This preliminary action creates a unique exposed glutamine residue at position 295 that serves as the sole conjugation site, ensuring site-specific attachment of chelators with defined CAR while maintaining efficient reaction conditions
2Ease of manufacture
If site-specific enzymatic conjugation with MTG is used without removing the N297 glycan, then the process avoids additional steps, but quantitative conjugation to Q295 is not possible due to glycosylation blocking the site
Solution Approach 1:
The N297 glycan moiety is selectively removed using PNGase F enzyme treatment. This extraction of the blocking glycan structure exposes the underlying Q295 residue, enabling MTG to catalyze quantitative conjugation at this specific site while maintaining overall process efficiency and simplicity
3Manufacturing precision
If genetic engineering is performed to create acyl glutamine-containing amino donor sequences, then site-specific conjugation can be achieved, but the process becomes cumbersome requiring engineering, growing and purifying each antibody
Solution Approach 1:
The antibody's own N297 glycan structure is exploited as a removable blocking element that, when removed by PNGase F, automatically exposes the Q295 conjugation site. This self-service approach eliminates the need for external genetic engineering, allowing any antibody to undergo site-specific conjugation through simple enzymatic treatment followed by MTG-catalyzed reaction
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach produces homogeneous bioconjugated antibodies with a defined CAR, enhancing tumor uptake and reducing non-targeted tissue uptake, thereby improving pharmacokinetics and therapeutic indexes.
Implementation Method 1
the enzymatic deglycosylation of said antibody
Implementation Method 2
The MTG catalyzes under physiological conditions a transamidation reaction between a 'reactive' glutamine of a protein or peptide and a 'reactive' lysine residue of a protein or peptide
Data Source
AI summary
A process is for preparing a site-specific bioconjugated antibody of a formula (I): Ab-(Linker-Chelator)n (I). The Linker is an oligopeptide with an N-terminal end. The Chelator is a metal chelating agent. n is a Chelator-to antibody ratio (CAR), wherein 0<n≤2. The process includes enzymatic deglycosylation of the antibody; coupling of the obtained deglycosylated antibody with a compound of a formula (A): Linker-Chelator (A) in the presence of a transglutaminase. The Linker is bound to the Ab at its N-terminal end, and comprising a sequence chosen among (*G-G-G), (*K-G-G) and (*A-K-A), where * denotes the N-terminal end of the Linker which is covalently bound to the Ab.