VH Domain Disulfide Bond Engineering for Stable Single-Domain Antibodies
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Solution Overview
Problem
Single-domain antibodies derived from human IgGs suffer from low stability and solubility, leading to aggregation and reduced therapeutic efficacy, while introduction of non-canonical disulfide bonds can enhance stability but may affect affinity and specificity.
Innovation Solution
Introduce non-canonical cysteines at specific positions (52a and 71 or 33 and 52) in VH domains to form disulfide bonds, enhancing stability and solubility, and incorporate these domains into engineered VH libraries with high sequence identity to human frameworks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If non-canonical disulfide bonds are introduced into VH domains to improve stability, then conformational stability increases, but affinity and specificity may be affected
Solution Approach 1:
The patent systematically varies parameters including disulfide bond positions (C23-C104 canonical bond combined with non-canonical bonds at positions 33-52, 52a-71, or 78-92), amino acid substitutions at framework positions, and CDR region sequences to optimize both stability and binding properties. This parameter optimization allows achieving enhanced conformational stability while preserving or maintaining affinity and specificity for target antigens
Solution Approach 2:
The patent introduces disulfide bonds at specific localized positions within the VH domain structure (framework regions FR1-FR4) rather than throughout the entire molecule. The canonical disulfide bond at C23-C104 is supplemented with non-canonical bonds at strategically selected positions to stabilize specific structural elements while leaving the CDR regions relatively undisturbed, thereby maintaining binding functionality
2Object-affected harmful factors
If single-domain antibodies are derived from human IgGs to reduce immunogenicity, then immunogenicity decreases, but stability and solubility are reduced leading to aggregation
Solution Approach 1:
The patent creates composite VH domain structures that combine human framework regions (for low immunogenicity) with stabilizing elements including non-canonical disulfide bonds and specific amino acid substitutions. This composite approach integrates the advantages of human-derived sequences (reduced immunogenicity) with engineered stability features (disulfide bonds at positions 33-52, 52a-71, or 78-92) to achieve both low immunogenicity and high stability
3Stability of the object's composition
If disulfide bonds are introduced at inappropriate positions to enhance stability, then conformational stability may improve, but unfavorable effects occur on surrounding amino acids and existing interactions
Solution Approach 1:
The patent uses the canonical disulfide bond at C23-C104 as a stable structural intermediary that anchors the framework structure. Non-canonical disulfide bonds are then introduced at carefully selected positions (33-52, 52a-71, or 78-92) that act as additional stabilizing intermediaries without interfering with the canonical bond's function. This intermediary approach allows multiple disulfide bonds to work cooperatively while minimizing adverse effects on surrounding residues
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 VH domains exhibit improved stability and solubility, reducing aggregation and maintaining target-binding affinity, making them suitable for therapeutic applications.
Implementation Method 1
Introduce non-canonical cysteines at specific positions (52a and 71 or 33 and 52) in VH domains to form disulfide bonds, enhancing stability and solubility
Data Source
AI summary
The invention relates autonomous VH domains (aVH) with cysteines in positions 52a and 71 or in positions 33 and 52 in order to stabilize the autonomous VH domains. Said cysteines are capable of forming a disulfide bond and/or form a disulfide bond under suitable conditions. The invention further relates to aVH libraries.