Stable Autonomous Antibody VH Domain
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Solution Overview
Problem
Current methods for stabilizing VH domains are hindered by poor stability due to the loss of light-chain interactions, and there is a lack of success in generating disulfide-free VH domains suitable for intracellular applications.
Innovation Solution
Development of single domain antibodies with specific modifications such as H35D, A78V, S93V, S93G, and W103R, and optionally C22S, A24I, A24L, or C92T, based on the 4D5 antibody scaffold, which allows for autonomous folding and the absence of intramolecular disulfide bridges, enhancing stability and solubility for intracellular targeting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If VH domains are used as single domain antibodies, then tumor penetration and clearance are enhanced, but stability deteriorates due to loss of light-chain interactions
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (H35D, A78V, S93V, S93G, W103R) in the VH domain framework regions. These mutations alter the structural parameters of the VH domain to compensate for the loss of light-chain interactions, thereby enhancing thermal stability while preserving the small size and fast clearance characteristics of single domain antibodies
Solution Approach 2:
The patent creates a composite structure by combining multiple stabilizing mutations within the VH domain framework. This composite approach integrates several amino acid changes that work synergistically to provide enhanced stability, analogous to creating a composite material with improved properties by combining multiple components
2Ease of operation
If conventional VH domains are used, then autonomous folding is achieved, but disulfide bridges are present which limit intracellular applications
Solution Approach 1:
The patent applies the extraction principle by removing the disulfide bridge-forming cysteine residues (C22, C92) from the VH domain sequence. This elimination of the disulfide bonding capability allows the antibody to function in the reducing intracellular environment while preserving autonomous folding through the stabilized framework structure
Solution Approach 2:
The patent changes the chemical parameter of the VH domain by substituting cysteine residues with serine or threonine (C22S, C92T mutations). This chemical modification eliminates disulfide bridge formation while maintaining the overall structural integrity and folding autonomy of the domain, enabling intracellular application
3Stability of the object's composition
If VH domains are stabilized through conventional methods, then some stability is achieved, but production costs and time remain high due to required post-translational modifications
Solution Approach 1:
The patent applies self-service by designing VH domains that achieve high thermal stability and proper folding through intrinsic amino acid sequence modifications alone, without requiring external post-translational modifications. The stabilized framework structure autonomously provides the necessary stability, enabling direct expression in bacterial systems
Solution Approach 2:
The patent extracts the requirement for post-translational modifications by incorporating all necessary stabilizing features directly into the amino acid sequence during gene synthesis. This eliminates the need for subsequent chemical or enzymatic modification steps, thereby reducing production time and cost
Data Source
Figure 1A~1C
Figure 2A~3
Figure 4~5B
AI summary
The present invention relates to single domain antibodies as defined herein. Further encompassed are the multi-modular antibody molecules and antibody conjugates comprising the single domain antibodies, as well as methods for producing them and methods for diagnosing or treating disorders or diseases using them. The invention in particular provides a library of the single domain antibodies or multi-modular antibody molecules and a method of selecting an antibody molecule that binds an antigen.