Conditionally Active Proteins via Temperature-Shifted Mutagenesis
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Solution Overview
Problem
Current methods struggle to engineer proteins that are reversibly or irreversibly inactivated at wild-type conditions while maintaining or exceeding activity at non-normal conditions, particularly for therapeutic proteins like enzymes and antibodies, which is crucial for avoiding harmful side effects and achieving targeted therapeutic effects.
Innovation Solution
A method involving the selection and evolution of wild-type proteins using DNA mutagenesis techniques to create conditionally active biologic proteins that are virtually inactive at normal physiological conditions but active at aberrant conditions, such as lower temperatures or specific microenvironments, allowing for controlled therapeutic activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a protein is engineered to be active at non-normal conditions (e.g., lower temperatures), then therapeutic activity at targeted conditions is improved, but activity at wild-type normal physiological conditions increases causing harmful side effects
Solution Approach 1:
The patent applies parameter changes by engineering proteins with altered temperature sensitivity through mutagenesis. The evolved proteins exhibit shifted temperature-activity profiles, being virtually inactive at wild-type physiological temperatures (37°C) but active at lower temperatures (e.g., 20-30°C), thereby resolving the contradiction between targeted therapeutic activity and harmful side effects
Solution Approach 2:
The patent inverts the conventional approach by designing proteins that are inactive at normal physiological conditions and active only under aberrant conditions. This inversion of the typical activity profile allows therapeutic proteins to remain dormant during normal body function and activate only when needed, such as in inflamed tissues with lower temperatures
2Object-generated harmful factors
If a protein is engineered to be virtually inactive at wild-type conditions, then harmful side effects are reduced, but therapeutic activity at normal conditions is lost
Solution Approach 1:
The patent changes the temperature parameter at which the protein becomes active. Through evolutionary engineering, the proteins are designed to have their activity window shifted to lower temperatures, maintaining therapeutic reliability at targeted sites while remaining inactive at normal physiological temperatures to avoid side effects
Solution Approach 2:
The patent applies local quality by creating proteins with spatially selective activity. The conditionally active proteins are inactive in normal physiological environments (most of the body) but become active in specific localized areas with aberrant conditions such as inflamed or tumor tissues, providing targeted therapy without systemic side effects
3Object-generated harmful factors
If destabilizing mutations are introduced to inactivate protein at wild-type conditions, then side effects are reduced, but overall protein stability and activity are compromised
Solution Approach 1:
The patent changes the stability parameter of the protein by introducing mutations that destabilize the protein structure at wild-type temperatures. However, these same mutations confer stability and activity at lower temperatures, effectively trading temperature-independent stability for temperature-dependent stability that enables conditional activation
Solution Approach 2:
The patent introduces dynamic behavior into the protein by making its stability and activity state dependent on temperature. The proteins dynamically transition between inactive (at high temperature) and active (at low temperature) states, allowing them to adapt their stability characteristics to the physiological conditions they encounter
Data Source
AI summary
Methods of generating conditionally active biologic proteins, in particular therapeutic or diagnostic proteins, which are more active at an aberrant condition than at a normal physiological condition. The methods include discovery methods using libraries of proteins and assays employing physiological concentrations of components of bodily fluids. The conditionally active biologic proteins may be further evolved, conjugated to other molecules, masked, reduced in activity by attaching a cleavable moiety. Criteria for selecting starting proteins for the discovery methods, as well as formats of the proteins are also disclosed.