VH Domain Mutations and C-Terminal Extension for Low Antibody Binding
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Solution Overview
Problem
Existing immunoglobulin variable domains with exposed C-terminal regions are prone to binding with pre-existing antibodies found in the blood or serum of human subjects, particularly those with severe autoimmune disorders like systemic lupus erythematosus, leading to immune activation.
Innovation Solution
Introduce mutations at position 112 of the heavy-chain variable domain, specifically changing serine to lysine or glutamine, optionally combined with L11V and V89L mutations, to reduce binding of pre-existing antibodies, and include a C-terminal extension such as alanine to further enhance solubility.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If the heavy-chain variable domain has an exposed C-terminal region, then it maintains structural simplicity and ease of production, but it binds with pre-existing antibodies in human blood or serum, causing immune activation
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at position 112 (S112K or S112Q) and optionally at positions 11 (L11V) and 89 (V89L) in the heavy-chain variable domain. These mutations alter the chemical properties of the C-terminal region to reduce hydrophobicity and prevent binding with pre-existing antibodies, while maintaining the exposed C-terminal structure for ease of production and structural simplicity.
2Object-affected harmful factors
If mutations are introduced at position 112 to reduce binding of pre-existing antibodies, then immune activation is reduced, but the structural simplicity of the variable domain is compromised
Solution Approach 1:
The patent applies local quality by making targeted mutations only at specific positions (112, and optionally 11 and 89) in the heavy-chain variable domain, rather than altering the entire structure. This localized approach reduces binding with pre-existing antibodies while preserving the overall structural simplicity and exposed C-terminal region of the variable domain.
3Stability of the object's composition
If C-terminal extensions are added to enhance solubility, then protein stability is improved, but the simplicity of the variable domain structure is reduced
Solution Approach 1:
The patent applies segmentation by adding C-terminal extensions (such as VTVKS or VTVQS with additional amino acids) as separate segments appended to the heavy-chain variable domain. This modular approach enhances solubility and protein stability while maintaining the simplicity of the core variable domain structure.
Data Source
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Figure 2
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AI summary
VH domain, in which: (i) the amino acid residue at position 112 is one of K or Q; and/or (ii) the amino acid residue at position 89 is T; and/or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).