BoNT/B4 Receptor Binding Domain Mutations for Human SytII Affinity
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Solution Overview
Problem
Botulinum neurotoxins, particularly serotype B, face challenges in binding to human SytII receptors, leading to reduced efficacy and increased adverse effects due to neutralizing antibodies and toxin diffusion, necessitating improved receptor binding affinity to enhance therapeutic efficacy and specificity.
Innovation Solution
Site-directed mutations in the receptor-binding domain of Clostridium botulinum serotype B, such as V1113K, S1117P, S1196A, and I1197P, are introduced to enhance the binding of BoNT/B4 to human SytII, increasing its affinity and specificity for human neurons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild type BoNT/B is used, then the toxin can bind to rodent SytII receptors, but it does not bind efficiently to human SytII receptors, resulting in reduced efficacy
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (V1113K, S1117P, S1196A, I1197P) at defined positions in the heavy chain sequence to alter the binding properties of the toxin. These sequence parameter modifications enable the toxin to recognize and bind to human SytII receptors with high affinity, resolving the incompatibility between wild-type BoNT/B and human neuronal receptors.
Solution Approach 2:
The patent applies local quality by making targeted modifications only in the receptor binding domain (heavy chain) while maintaining the rest of the toxin structure unchanged. The specific amino acid changes are localized to residues involved in SytII interaction, allowing the toxin to adapt its binding specificity to human neurons without affecting other functional domains.
2Reliability
If higher toxin doses are administered to achieve sufficient binding, then therapeutic efficacy improves, but adverse effects and neutralizing antibody generation increase
Solution Approach 1:
By changing the sequence parameters of the heavy chain to enhance binding affinity to human SytII, the patent enables therapeutic efficacy at lower toxin doses. The modified toxin achieves sufficient receptor binding and neuronal inhibition with reduced dosing, thereby minimizing adverse effects and reducing the likelihood of neutralizing antibody generation.
Solution Approach 2:
The patent converts the originally harmful effect of low binding affinity (which caused the need for high doses and subsequent adverse effects) into a benefit by introducing specific mutations that create high-affinity binding to human receptors. This transforms the toxin into a more precise and safer therapeutic agent.
3Reliability
If higher toxin doses are used to ensure sufficient neuronal inhibition, then therapeutic effect is achieved, but toxin diffusion to other regions increases causing adverse effects
Solution Approach 1:
The patent changes the binding parameters of the toxin to achieve high-affinity interaction with human SytII receptors. This enhanced specificity ensures that the toxin is efficiently captured by target neurons at lower doses, preventing diffusion to non-target regions and reducing adverse effects while maintaining therapeutic efficacy.
Solution Approach 2:
The patent applies local quality by concentrating the toxin's binding activity specifically at the receptor interaction interface through localized amino acid substitutions. This creates a highly specific interaction that prevents off-target binding and diffusion, while maintaining strong neuronal inhibition at the intended target sites.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified receptor-binding domain significantly enhances the ability of BoNT polypeptides to inhibit neuronal activity, reducing the need for higher toxin doses and minimizing unwanted immune responses, thereby improving therapeutic outcomes and safety.
Implementation Method 1
The modified receptor binding domain of Clostridium botulinum serotype B, strain 4 (B4-Hc) binds human SytII
Implementation Method 2
These toxins act by blocking neurotransmitter release from neurons, thus paralyzing animals and humans
Data Source
AI summary
Disclosed herein are botulinum neurotoxin (BoNT) polypeptides with a modified BoNT/B4 receptor binding domain (B4-HC) having amino acid mutations that modify the binding of the BoNT to the human SytII receptor. Specific mutations and combinations of mutations are disclosed. Isolated modified HCs, polypeptides comprising such modified HCs, chimeric molecules, pharmaceutical compositions, and methods of making and using the same are also disclosed. Methods of identifying additional such modified receptor binding domains, are further disclosed.