Pre-Humanized VNAR Libraries for High-Throughput Affinity Screening
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Solution Overview
Problem
Existing methods for producing antigen-specific binding molecules, such as those described in WO 2014/173959, rely on naturally occurring VNAR sequences that require subsequent humanization and re-testing, which can diminish the relevance of testing data and are not efficient for high-throughput screening.
Innovation Solution
A method for producing humanized antigen-specific binding molecules with a peptide domain structure FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4, involving DNA amplification, ligation, cloning into a display vector, and transformation to create a library, allowing for high-throughput screening and efficient identification of high-affinity binders.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If naturally occurring VNAR sequences are used, then diversity and affinity are improved, but subsequent humanization and re-testing are required which diminishes data relevance and reduces efficiency
Solution Approach 1:
The framework regions of the VNAR sequences are pre-humanized before antigen binding testing, so that the sequences are already in their final therapeutic form. This eliminates the need for subsequent humanization and re-testing, making the testing data directly relevant and saving time.
Solution Approach 2:
Human framework region sequences are copied and integrated into the VNAR structure, creating hybrid molecules that retain the binding capabilities of natural VNARs while having human-compatible framework regions. This allows direct testing without further modification.
2Adaptability or versatility
If framework regions are humanized, then therapeutic potential is improved, but structural perturbations may occur
Solution Approach 1:
Only the framework regions are humanized while the CDR regions retain their natural sequences. This localized modification approach maintains the binding specificity determined by CDRs while improving therapeutic compatibility through humanized frameworks, minimizing structural perturbations.
Solution Approach 2:
The amino acid sequences of framework regions are modified to match human germline sequences, changing the biochemical parameters (charge, hydrophobicity, glycosylation patterns) to be more compatible with human physiology while maintaining overall fold stability through conservation of key structural residues.
3Adaptability or versatility
If diversity is maximized in CDR regions, then binding specificity is improved, but structural perturbations increase
Solution Approach 1:
Diversity is concentrated in the CDR regions which are responsible for antigen binding, while the framework regions are kept stable and humanized. This localized diversity strategy maximizes binding specificity without compromising overall structural integrity, as the stable frameworks provide a rigid scaffold for the variable CDR loops.
Data Source
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AI summary
The present invention relates to a synthetic library of humanised antigen specific binding molecules derived from a member of a species in the Elasmobranchii subclass, processes for the production thereof and specific antigen specific binding molecules isolated from said library. The present invention also relates to the multi-domain specific binding molecules comprising humanised Ig-like Novel Antigen Receptor variable domains (VNARs). Specific binding domains that bind to Tumour Necrosis Factor alpha (TNFα) are also provided.