Nanobody Humanization With CDR-Aware Human VH Frameworks
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Solution Overview
Problem
Current methods for humanizing nanobodies lack systematic and structural investigations, leading to potential anti-drug antibody responses and toxicity due to non-human antibodies, undermining structural compatibility and antigen recognition.
Innovation Solution
A method involving sequence alignment, intramolecular interaction analysis, solvent accessibility analysis, and residue substitution to minimize immunogenicity while maintaining structural integrity and binding ability, using Llamanade software for automated humanization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If CDR grafting to a universal Nb framework is used, then the humanization process is simplified, but structural compatibility with many CDRs is undermined
Solution Approach 1:
The patent applies local quality by selecting different human VH framework sequences tailored to match specific Nb CDR characteristics. Instead of using a single universal framework, the method analyzes individual Nb sequence features and pairs them with complementary human VH frameworks that ensure optimal structural compatibility and folding for each specific CDR configuration.
2Stability of the object's composition
If solvent-exposed framework residues are resurfaced without changing buried residues, then structural integrity is maintained, but antigen engagement is compromised
Solution Approach 1:
The patent segments the framework residues into distinct categories based on their functional roles: buried residues critical for structural integrity, solvent-exposed residues involved in antigen engagement, and intermediate residues. This segmentation enables selective modification of only those solvent-exposed residues that do not participate in antigen binding, while preserving both structural integrity and antigen engagement capabilities.
Solution Approach 2:
The method employs parameter changes by systematically evaluating each solvent-exposed framework residue's contribution to antigen engagement through structural analysis and computational modeling. Residues are selectively substituted based on quantified parameters such as solvent accessibility, distance to antigen contact points, and structural flexibility, allowing precise optimization of immunogenicity without compromising function.
3Object-affected harmful factors
If extensive framework residues are substituted to reduce immunogenicity, then humanization is improved, but structural stability and binding ability are reduced
Solution Approach 1:
The patent applies partial action by performing selective substitution only on the specific subset of framework residues that are both solvent-exposed and non-critical for structural stability or antigen binding. This targeted approach substitutes enough residues to significantly reduce immunogenicity while maintaining the minimal necessary framework diversity to preserve structural integrity and binding capability.
Data Source
AI summary
Disclosed are methods for humanizing antibodies and nanobodies and systems for performing the same.