Beta-sheet Polypeptide Libraries for Targeted Blood-Brain Barrier Delivery
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Solution Overview
Problem
Current techniques for engineering proteins to bind to specific targets often result in undesired properties, such as non-specific binding or lack of developability, due to the randomization of amino acid positions.
Innovation Solution
The development of beta-sheet libraries with limited liability approaches, where 10-60% of randomized positions exclude certain amino acids like Cys, Trp, Met, Arg, or Gly, to reduce the frequency of undesirable characteristics, allowing for the discovery of polypeptides with novel binding sites, including those targeting CD98 heavy chain (CD98hc) and transferrin receptor (TfR).
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If randomization of amino acid positions is performed to engineer novel binding sites, then binding diversity is improved, but non-specific binding and undesirable characteristics increase
Solution Approach 1:
The patent applies local quality by differentiating between randomized positions and fixed positions within the polypeptide sequence. Specifically, positions 422, 424, 426, 438, and 440 are randomized to generate binding diversity, while positions 380, 382, 384, 386, and 428 are fixed to maintain structural integrity and avoid non-specific binding. This localized randomization strategy enables the polypeptide to achieve both binding diversity and specificity simultaneously.
2Adaptability or versatility
If randomization of amino acid positions is performed to engineer novel binding sites, then binding diversity is improved, but developability deteriorates
Solution Approach 1:
The patent implements local quality by restricting randomization to specific positions (422, 424, 426, 438, 440) while maintaining fixed residues at other positions (380, 382, 384, 386, 428). This approach ensures that the polypeptide maintains proper folding, stability, and manufacturability while still achieving sufficient binding diversity through the randomized positions.
Solution Approach 2:
The patent applies parameter changes by controlling the diversity at each randomized position to include only 8 specific amino acids, excluding Cys, Trp, Met, Arg, and Gly. This parameter constraint prevents the formation of undesirable characteristics such as aggregation-prone sequences while maintaining adequate binding diversity for engineering novel binding sites.
3Adaptability or versatility
If beta-sheet structures are engineered into polypeptides, then novel binding sites are created, but structural complexity increases
Solution Approach 1:
The patent applies local quality by incorporating beta-sheet structures at specific regions of the polypeptide (positions 422-440) while maintaining a simpler overall framework. The beta-sheet forming randomized positions create novel binding sites through local structural complexity, while the fixed positions maintain global structural simplicity and facilitate easier manufacturing.
Data Source
AI summary
The present disclosure includes engineering methods and polypeptide libraries that are useful for introducing non-native binding sites into polypeptides. Also provided herein are polypeptides that bind to a CD98hc or transferrin receptor (TfR) protein, methods of generating such polypeptides, and methods of using the polypeptides to target a composition across the blood-brain barrier or to a CD98hc-expressing or TfR-expressing cell.