CH3 Domain Mutant Pair for Antibody Heterodimer Formation
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Solution Overview
Problem
Current methods for producing bispecific antibodies face challenges in achieving high yields and stability of heterodimeric heavy chain constant regions, leading to difficulties in purification and therapeutic efficacy due to issues like low expression, aggregation, and immunogenicity.
Innovation Solution
A method using yeast mating and surface display is employed to evaluate and enhance the formation yield of heterodimeric heavy chain constant regions, introducing specific mutations in the CH3 domain interaction surfaces to promote non-covalent bonding, such as hydrogen bonding and cation-π bonding, resulting in a heterodimeric heavy chain constant region library with improved stability and expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to produce heterodimeric heavy chain constant regions, then production cost is reduced, but heterodimer formation yield is low and aggregation occurs
Solution Approach 1:
The patent introduces specific amino acid mutations in the CH3 domain interaction surfaces to alter the chemical properties and bonding characteristics. These parameter changes in the molecular structure enable non-covalent bonding (hydrogen bonding and cation-π bonding) that significantly increases heterodimer formation yield to 90% or more while maintaining stability
Solution Approach 2:
The patent creates a composite interaction interface by combining multiple types of non-covalent bonds (hydrogen bonds and cation-π bonds) between the CH3 domains. This composite bonding mechanism provides both high formation yield and enhanced stability, resolving the contradiction between productivity and reliability
2Productivity
If mutation is introduced in CH3 domain to promote heterodimer formation, then heterodimer formation yield is improved, but expression level may be reduced
Solution Approach 1:
The patent applies local quality changes by introducing mutations only in the specific CH3 domain interaction surfaces rather than throughout the entire antibody structure. This localized modification promotes heterodimer formation while preserving the overall expression levels and functional properties of the antibody
3Ease of manufacture
If conventional purification methods are used, then purification cost is reduced, but purification efficiency is low due to aggregation
Solution Approach 1:
The patent applies preliminary anti-action by pre-preventing aggregation through the introduction of non-covalent bonding mutations in the CH3 domain. This preliminary structural modification eliminates the aggregation problem before purification begins, making the purification process more efficient and reducing substance loss
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 method achieves a heterodimer formation yield of 90% or more, maintaining the intrinsic functions of the wild-type antibody, including long serum half-life and efficient purification, while enhancing therapeutic potential.
Implementation Method 1
introducing specific mutations in the CH3 domain interaction surfaces to promote non-covalent bonding, such as hydrogen bonding and cation-π bonding
Implementation Method 2
introducing specific mutations in the CH3 domain interaction surfaces to promote non-covalent bonding, such as hydrogen bonding and cation-π bonding
Implementation Method 3
formation of a homodimer between two identical heavy chains is induced through non-covalent interaction between last domains of a constant region (Fc, crystallizable fragment) of an antibody
Data Source
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AI summary
The present disclosure relates to a method for evaluating and screening a mutant inducing the high-efficiency formation of heterodimers from a human antibody heavy chain constant region mutant pair combination library in order to increase the yield of formation of human antibody heterodimeric heavy chain constant regions, and to a heterodimeric heavy chain constant region (heterodimeric F) library thereby. In addition, the present disclosure relates to a CH3 domain mutant pair in which the formation of the heterodimeric heavy chain constant regions is preferred in the library, to a heterodimeric heavy chain constant region pair comprising the CH3 mutant pair, to a bispecific antibody, to a fusion protein, and a use thereof. The CH3 domain mutant pair according to the present disclosure forms heterodimeric heavy chain constant regions at a high yield of at least 80-90%, and also has excellent thermal stability and retains binding ability to the heavy chain constant region receptor (FcRn). The CH3 domain mutant pair, the bispecific antibody comprising the same, or the antibody constant region fusion protein of the present disclosure can be usefully applied to the treatment or prevention of diseases associated with target antigens or target proteins.