Fc-Fused BChE Fusion Proteins for High-Yield Expression

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for producing recombinant butyrylcholinesterase (BChE) and its mutants face challenges such as low yield and short biological half-life, limiting their clinical application for treating organophosphorus nerve agent toxicity and cocaine abuse, due to improper post-translational modification and immunogenicity issues.

Innovation Solution

Development of fusion proteins comprising BChE polypeptides with an Fc polypeptide joined to the N-terminal or C-terminal end, using linkers to enhance protein production yield and biological half-life, specifically through the use of Fc(M3)-linker-CocH3 entities in CHO cells, which improve catalytic activity and expression levels while maintaining a long biological half-life.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If BChE is expressed in bacteria, then production cost is reduced, but the protein cannot fold appropriately to become an active enzyme

Engineering Contradiction:

Improveproduction costVSAvoidprotein folding and activity

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The patent uses a fusion protein strategy where BChE is fused to a carrier protein (such as albumin or immunoglobulin) that can be properly expressed in bacterial systems. This intermediary fusion protein acts as a mediator that allows the BChE portion to achieve proper folding and activity while benefiting from the ease of bacterial expression. The carrier protein provides a favorable structural context that enables correct folding of the BChE domain.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If BChE is produced in transgenic plants and animals, then production efficiency is improved, but the proteins have significantly shorter biological half-lives

Engineering Contradiction:

Improveproduction efficiencyVSAvoidbiological half-life

Core Design Contradiction:

ProductivityVSDuration of action of stationary object

Solution Approach 1:

The patent combines BChE with a carrier protein that has a long biological half-life (such as human serum albumin or IgG Fc region). This merging of BChE with the long-lived carrier protein creates a fusion protein that inherits the extended circulation time of the carrier while retaining BChE's enzymatic activity. The carrier protein protects the BChE from rapid clearance by the reticuloendothelial system.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The fusion protein represents a composite structure combining BChE with a carrier protein that has complementary properties. The carrier protein component provides long circulation half-life and resistance to proteolysis, while the BChE component provides the desired enzymatic function. This composite approach allows the final product to exhibit both high production efficiency and extended biological half-life.

Inventive Principle:

Principle #40Composite materials

3Manufacturing precision

If BChE is produced in CHO cells, then post-translational modification is improved, but expression yield remains low

Engineering Contradiction:

Improvepost-translational modificationVSAvoidexpression yield

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent merges BChE with a carrier protein to create a fusion protein that can be expressed at high levels in CHO cells. The fusion construct leverages the strong expression capabilities of CHO cells for the carrier protein portion while ensuring proper post-translational modification of the BChE domain. This merging strategy overcomes the low expression yield limitation by using the carrier protein as an expression vehicle.

Inventive Principle:

Principle #5Merging (Combining)

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 Fc-fused BChE proteins demonstrate significantly improved yield and biological half-life, up to 21-fold and 6.6-fold longer than previous methods, respectively, while maintaining catalytic activity against cocaine, making them suitable for therapeutic applications.

Implementation Method 1

The short biological half-life is mainly explained by possibly incomplete post-translational modification causing the BChE or CocH to be taken up by asialo receptors in the liver

Methodology Applied

Scientific EffectFcRn-mediated recycling:

Implementation Method 2

BChE is a major metabolic enzyme that catalyzes the hydrolysis of cocaine to produce biologically inactive metabolites

Methodology Applied

Scientific EffectCatalysis: Catalysis

Implementation Method 3

BChE can intercept and destroy the organophosphorus (OP) nerve poisons before they reach their target—acetylcholinesterase (AChE)

Methodology Applied

Scientific EffectEnzymatic degradation: Enzyme

Data Source

PatentUS11473070B2Increased polypeptide production yields of butyrylcholinesterase polypeptides for therapeutic use

Publication Date: 2022.10.18 UNIVERSITY OF KENTUCKY RESEARCH FOUNDATION

AI summary

The presently-disclosed subject matter describes fusion proteins comprising butyrylcholinesterase (BChE) having an improved production yield and biological half-life and nucleotides encoding the same.