Antibody Sequence Mutation for Affinity and Stability
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Solution Overview
Problem
There is a need for methods to improve the properties of amino acid sequences used as single antigen-binding domains, such as affinity, specificity, stability, and reduced immunogenicity, which are not adequately addressed by conventional antibody fragments or domain antibodies.
Innovation Solution
A method involving PCR assembly of oligonucleotides with specific mutations to generate libraries of amino acid sequences that can be screened for desired properties, including affinity maturation, deimmunization, and stability, using techniques like degenerate codons and framework/complementarity determining region modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional antibody fragments or domain antibodies are used, then the structure is simpler and easier to produce, but the affinity, specificity, and stability are insufficient
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid positions in the CDR and FR regions of antibody sequences. This involves changing physical-chemical parameters of the protein structure through targeted amino acid substitutions, deletions, or insertions to optimize binding affinity and stability while maintaining structural integrity
Solution Approach 2:
The patent employs preliminary action through in silico screening and prediction methods before experimental validation. Computational algorithms predict which mutations will improve desired properties, allowing the selection of optimal candidate sequences before synthesis and testing, thereby reducing the complexity of the experimental process
2Reliability
If amino acid sequences are mutated to improve affinity and specificity, then binding properties are enhanced, but immunogenicity may increase
Solution Approach 1:
The patent implements feedback mechanisms by iteratively screening mutant libraries and using the results to guide subsequent rounds of mutation. Experimental binding data and immunogenicity assessments feed back into the design process, allowing optimization of sequences to simultaneously improve affinity while minimizing immunogenicity through selective pressure against highly immunogenic variants
3Reliability
If extensive mutation screening is performed to improve sequence properties, then affinity and stability are enhanced, but the time and resources required increase
Solution Approach 1:
The patent applies partial action by focusing mutations on specific critical regions (CDR and selected FR positions) rather than exhaustive screening of the entire sequence. This targeted approach concentrates resources on the most impactful positions, achieving significant stability improvements with reduced screening burden compared to comprehensive random mutagenesis
Solution Approach 2:
The patent uses preliminary computational filtering to predict stable mutants before experimental testing. In silico stability predictions pre-screen large numbers of potential mutants, allowing only the most promising candidates to proceed to wet-lab validation, thereby dramatically reducing the time and resources required for extensive mutation screening
Data Source
AI summary
The present invention relates to methods and techniques for providing improved amino acid sequences that can be used as single antigen-binding domains. In particular, the invention relates to methods and techniques for providing improved amino acid sequences that can be used as single antigen-binding domains that comprise or essentially consist of at least one immunoglobulin sequence. More in particular, the amino acid sequences provided herein may comprise or essentially consist of at least one variable domain sequence or a suitable fragment thereof such as at least one light chain variable domain sequence (e.g. a VL-sequence) or a suitable fragment thereof or at least one heavy chain variable domain sequence (e.g. a VH-sequence or VHH sequence) or a suitable fragment thereof.