CheBc-Domain Protein Scaffold With Randomized Binding Loops
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Solution Overview
Problem
Existing antibody fragments are unstable and prone to aggregation, limiting their use in therapeutic and diagnostic applications, while non-immunoglobulin protein scaffolds face challenges in structural diversity and epitope recognition.
Innovation Solution
A recombinant, non-naturally occurring protein scaffold based on the CheBc domain, comprising alpha helices, beta strands, and a 310 helix with randomized loop regions, designed to mimic antibody variable regions for diverse binding capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If antibody fragments are used to reduce size and improve tissue penetration, then tissue penetration is improved, but stability and aggregation resistance deteriorate
Solution Approach 1:
The antibody is segmented into framework regions and loop regions, with only the essential framework structure retained while loop regions are randomized to create stable yet diverse binding molecules. This segmentation allows the molecule to maintain stability from the framework while achieving size reduction and binding diversity through loop variation.
Solution Approach 2:
The framework regions undergo parameter changes through randomization of specific amino acid positions, creating a library of variants with different stability and binding properties. This allows selection of optimal variants that balance stability, size, and binding affinity for therapeutic applications.
2Reliability
If non-immunoglobulin protein scaffolds are engineered to overcome antibody limitations, then stability is improved, but structural diversity and epitope recognition capability deteriorate
Solution Approach 1:
The scaffold incorporates dynamic loop regions that can adopt multiple conformations, allowing the same framework structure to recognize diverse epitopes. The loop regions are engineered to be flexible and adaptable, enabling the stable scaffold to maintain high structural stability while achieving versatility in target recognition through conformational diversity.
Solution Approach 2:
The framework structure is designed as a universal scaffold that can support multiple different loop configurations, each capable of binding different targets. This multi-functional design allows a single stable framework to serve multiple binding specificities, overcoming the limitation of structural diversity while maintaining stability.
3Reliability
If full length antibodies are used to achieve high specificity, then binding specificity is improved, but production complexity and cost deteriorate
Solution Approach 1:
The essential binding function is extracted from the complex full-length antibody structure, retaining only the framework regions and CDR loops necessary for antigen recognition. This extraction eliminates the need for complex mammalian cell production while preserving binding specificity, allowing production in simpler bacterial systems.
Solution Approach 2:
The scaffold uses a simplified, truncated antibody structure that can be produced more cheaply and quickly in bacterial expression systems. While smaller and potentially shorter-lived than full antibodies, these molecules provide sufficient binding specificity for many applications at lower production cost and complexity.
Data Source
AI summary
The invention provides a protein scaffold and methods of preparing, screening, engineering and using the protein scaffold.