CDR Histidine Substitution in Polypeptides for Intestinal Stability
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Solution Overview
Problem
Therapeutic polypeptides, such as immunoglobulin chain variable domains, are unstable in the intestinal tract, limiting their oral administration and affecting their stability and binding affinity, particularly in the presence of proteases and inflammatory conditions.
Innovation Solution
Substituting lysine and arginine residues in the complementarity determining regions (CDRs) of immunoglobulin chain variable domains with histidine residues to enhance intestinal stability while maintaining binding affinity, suitable for oral administration and resistance to proteases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If amino acid substitutions are made in CDR regions to enhance intestinal stability, then protease resistance is improved, but binding affinity to target antigen deteriorates
Solution Approach 1:
The patent applies local quality by making specific amino acid substitutions only in non-CDR regions (framework regions) of the immunoglobulin variable domain, while leaving the CDR regions unchanged. This localized approach allows the polypeptide to gain intestinal stability from the substitutions in framework regions without compromising the binding affinity determined by the intact CDR regions.
Solution Approach 2:
The patent segments the immunoglobulin variable domain into functional regions (CDRs for binding and framework regions for structural stability) and applies modifications only to the framework regions. This segmentation allows independent optimization of binding affinity (maintained in CDRs) and intestinal stability (improved in framework regions through substitutions).
2Reliability
If additional cysteine bridges are introduced to increase proteolytic stability, then intestinal stability is improved, but recombinant production levels deteriorate
Solution Approach 1:
The patent changes the amino acid sequence parameters by substituting specific residues in framework regions with amino acids that enhance protease resistance (such as replacing protease cleavage sites). This parameter change approach improves proteolytic stability without introducing additional cysteine bridges that would complicate the protein structure and reduce recombinant production efficiency.
3Ease of operation
If conventional small molecule oral dosage forms are used for polypeptide delivery, then oral administration is enabled, but intestinal stability deteriorates
Solution Approach 1:
The patent extracts the polypeptide from the conventional small molecule dosage form context and applies direct amino acid sequence modifications to the polypeptide itself. This extraction from the formulation approach and implementation of molecular-level modifications enables the polypeptide to inherently resist intestinal proteases while maintaining oral administrability.
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3B
AI summary
There is provided inter alia a polypeptide comprising an immunoglobulin chain variable domain comprising three complementarity determining regions (CDR1 -CDR3) and four framework regions, wherein: (a) at least one lysine residue in CDR1, CDR2 and/or CDR3 has been substituted with at least one histidine residue, and/or (b) at least one arginine residue in CDR1, CDR2 and/or CDR3 has been substituted with at least one histidine residue; wherein the polypeptide has increased intestinal stability relative to a corresponding polypeptide not having said histidine substitutions.