Engineered AAT Polypeptides for Thermal Stability and Refolding
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Solution Overview
Problem
Current AAT therapies are limited by availability, cost, heterogeneity, instability, and misfolding, which hinder their use for treating inflammatory and respiratory conditions.
Innovation Solution
Development of recombinant alpha-1 antitrypsin (AAT) polypeptides with specific amino acid substitutions and modifications, such as G99A, Y144W, Y171A, L275F, S276K, T278E, T323E, I324V, D325N, K327E, and M358I, to enhance stability and functionality, allowing for improved manufacturing and therapeutic efficacy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If blood-derived AAT is used for therapy, then safety and tolerance are improved, but availability is limited and cost is very high
Solution Approach 1:
The patent creates recombinant copies of the AAT protein through genetic engineering. The wild-type AAT cDNA is cloned into expression vectors and produced in host cells (E. coli, CHO cells), generating multiple copies of the therapeutic protein without relying on blood donations. This copying approach maintains the safety profile while dramatically increasing availability.
Solution Approach 2:
The patent employs inexpensive bacterial expression systems (E. coli) to produce AAT protein. By using simple, well-established bacterial culture methods rather than complex blood fractionation processes, the cost of production is significantly reduced, making the therapy more accessible and affordable.
2Reliability
If blood-derived AAT is used for therapy, then safety and tolerance are improved, but manufacturing cost is very high
Solution Approach 1:
The patent creates recombinant copies of the AAT protein through genetic engineering. The wild-type AAT cDNA is cloned into expression vectors and produced in host cells (E. coli, CHO cells), generating multiple copies of the therapeutic protein without relying on blood donations. This copying approach maintains the safety profile while dramatically increasing availability.
Solution Approach 2:
The patent employs inexpensive bacterial expression systems (E. coli) to produce AAT protein. By using simple, well-established bacterial culture methods rather than complex blood fractionation processes, the cost of production is significantly reduced, making the therapy more accessible and affordable.
3Reliability
If blood-derived AAT is used for therapy, then therapeutic effect is achieved, but heterogeneity and instability increase
Solution Approach 1:
The patent applies local quality control by engineering specific features into the recombinant AAT protein. The wild-type AAT sequence is precisely replicated without the variability inherent in blood-derived products. Additionally, the patent introduces specific stabilizing mutations at key positions in the protein structure to enhance stability while maintaining the core therapeutic function.
Solution Approach 2:
The patent changes the stability parameters of AAT through site-directed mutagenesis. Specific amino acid substitutions are introduced to increase the melting temperature and reduce aggregation, transforming the protein's physical parameters while preserving its biological activity. This allows the protein to remain stable under various storage and administration conditions.
4Stability of the object's composition
If AAT is engineered for improved stability, then thermal stability and re-fold yield are enhanced, but manufacturing complexity increases
Solution Approach 1:
The patent changes the stability parameters of AAT through site-directed mutagenesis. Specific amino acid substitutions are introduced to increase the melting temperature and reduce aggregation, transforming the protein's physical parameters while preserving its biological activity. This allows the protein to remain stable under various storage and administration conditions.
Solution Approach 2:
The patent employs a refolding strategy where the protein is initially produced in a denatured or inclusion body state, then selectively refolded under controlled conditions. The stabilizing mutations ensure that during this refolding process, the protein adopts the correct native conformation with high efficiency, recovering functional protein from what would otherwise be misfolded aggregates.
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 AAT polypeptides demonstrate enhanced thermal stability, re-fold yield, and maintained inhibitory activity, facilitating their use in treating conditions like COPD, cystic fibrosis, and viral infections, with potential for aerosol delivery and reduced aggregation.
Implementation Method 1
The modified AAT polypeptides demonstrate enhanced thermal stability, re-fold yield
Data Source
AI summary
This disclosure provides an alpha-1 antitrypsin (AAT) polypeptide as defined herein. Also provided herein are pharmaceutical compositions comprising the polypeptide, nucleic acids encoding the polypeptide, vectors comprising the nucleic acid, cells, and methods of producing the AAT polypeptide and method of using the AAT polypeptide in therapy.