Glycan-Based Antibody-Drug Conjugates With Site-Specific Oxime Linkage
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Solution Overview
Problem
Current antibody-drug conjugates (ADCs) face limitations in efficient manufacture, product homogeneity, and scalability due to challenges in site-specific modification technologies, including disruption of native disulfide bonds and potential immunogenicity, especially when incorporating non-native amino acids or modifying the N-297 glycan, which affects stability and immune effector function.
Innovation Solution
Engineered IgG antibodies with mutations in the heavy chain constant domains create non-native N-glycosylation sites, allowing for efficient conjugation of payloads in yeast or filamentous fungal host cells, using oxidized galactose residues to form oxime bonds with derivatized drugs, maintaining protein stability and function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If engineered Cysteine residues are used for site-specific targeting, then site specificity is improved, but native disulfide bonds are disrupted and molecular stability decreases
Solution Approach 1:
The patent introduces non-native amino acids (such as azidohomoalanine, homopropargylglycine, and other unnatural amino acids containing unique functional groups) as intermediary structures that enable site-specific conjugation without disrupting native disulfide bonds. These non-native amino acids serve as mediators that provide unique chemistry for payload attachment while preserving the structural integrity of the antibody framework.
Solution Approach 2:
The patent employs parameter changes by incorporating non-native amino acids with distinct chemical properties (azide, alkyne, keto, hydroxylamine groups) into specific positions of the antibody sequence. This allows control over the number and location of conjugation sites, enabling discrete control of drug-to-antibody ratio while maintaining molecular stability through preservation of native disulfide bonds.
2Adaptability or versatility
If non-native amino acids are incorporated for unique chemistry, then conjugation versatility is improved, but scale-up and manufacture in mammalian cell lines becomes challenging
Solution Approach 1:
The patent replaces complex mammalian cell line engineering with a simpler chemical biology approach. Instead of requiring mammalian cells to incorporate non-native amino acids through engineered tRNA synthetase systems, the invention uses chemical labeling methods where non-native amino acids are incorporated into antibodies produced in standard mammalian cell lines, then subsequently modified in vitro. This substitution of in vivo engineering with in vitro chemistry simplifies manufacturing scale-up.
3Manufacturing precision
If site-specific tags are introduced in the mAb, then conjugation control is improved, but immunogenicity is promoted
Solution Approach 1:
The patent applies local quality by introducing site-specific modifications at localized positions within the antibody structure (such as specific residues in the Fc region or framework regions) rather than globally modifying the entire antibody. This allows precise control of conjugation at selected locations while preserving the overall immunological properties of the antibody. The modifications are localized to specific amino acid positions that do not compromise antigen binding or Fc-mediated effector functions.
4Productivity
If N-297 glycan is modified for conjugation, then conjugation efficiency is improved, but immune effector function is limited or abolished
Solution Approach 1:
The patent segments the conjugation function from the immune effector function by selecting alternative site-specific modification locations that are spatially and functionally separated from the N-297 glycan site in the Fc region. By targeting residues such as Cys226, Cys229, or specific framework region residues, the invention enables independent optimization of conjugation efficiency while preserving the integrity and function of the N-297 glycan, which is critical for immune effector functions including ADCP and CDC.
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
The engineered antibodies achieve high N-glycan occupancy and uniform drug conjugation, enhancing the therapeutic index and efficacy of ADCs while maintaining stability and immune function, facilitating scalable production.
Implementation Method 1
The engineered Abs of the invention comprise galactose-terminated N-glycans, which can be oxidized by an oxidizing reagent to produce aldehyde groups
Implementation Method 2
The reactive aldehyde groups, in the presence of a derivatized drug containing a reactive amine form a bond (e.g., the reactive amine, alkoxyamine, reacts with aldehyde groups to form an oxime bond), thereby conjugating the drug to the Ab
Data Source
AI summary
Genetically engineered antibodies containing non-native N-glycosylated sites, preparation of the antibodies in yeast and fungi, site-specific conjugation of drugs to these antibodies, and methods of treatment utilizing these antibodies are described herein.