Protease-Cleavable Peptide Linkers for Oral Polypeptide Release
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Solution Overview
Problem
Existing polypeptide constructs with stable linkers face issues such as misfolding, low yield, impaired bioactivity, and steric hindrance, particularly during in vivo delivery and recombinant production, necessitating a linker that allows controlled release of polypeptides in specific intestinal regions for oral administration.
Innovation Solution
Development of labile peptide linkers that are resistant to intestinal proteases and engineered for controlled cleavage, allowing targeted release of polypeptides in the intestinal tract, with options for shielded and non-shielded labile sites to manage cleavage resistance and lability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If stable linkers are used to covalently join polypeptides, then plasma half-life is prolonged and resistance to protease cleavage is improved, but steric hindrance between polypeptides increases and bioactivity decreases
Solution Approach 1:
The stable linker is segmented into two functional parts: a stable core region that provides protease resistance and plasma half-life extension, and a labile peptide linker region that can be cleaved by specific proteases to release the polypeptides. This segmentation allows the construct to maintain stability during circulation while enabling controlled release at the target site to reduce steric hindrance and restore bioactivity.
Solution Approach 2:
The linker transitions from a static stable state during circulation to a dynamic cleavable state at the target site. The labile peptide linker is designed to be stable under physiological conditions but becomes susceptible to proteolytic cleavage in the presence of specific intestinal proteases, allowing the system to adapt its properties based on the biological environment.
2Duration of action of stationary object
If stable linkers are used to join polypeptides, then plasma half-life is prolonged, but release of free polypeptides in vivo cannot occur
Solution Approach 1:
A protease-cleavable peptide sequence is introduced as an intermediary element within the linker region. This intermediary is flanked by shielding residues that protect it from cleavage during circulation, but becomes accessible to proteases at the target site, enabling controlled release of the polypeptides while maintaining prolonged plasma half-life during the circulation phase.
Solution Approach 2:
The linker is designed with non-uniform properties: the core region maintains stability for prolonged circulation, while specific local regions contain protease recognition sites that are shielded during circulation but become accessible at the target site. This local differentiation allows simultaneous achievement of prolonged half-life and controlled release capability.
3Ease of manufacture
If direct fusion of polypeptides without a linker is performed, then production simplicity is improved, but misfolding and impaired bioactivity occur
Solution Approach 1:
A short, simple peptide linker sequence is used as a disposable element between the polypeptides. This linker is genetically encoded and easily incorporated during recombinant production, providing necessary spacing for proper folding while being simple enough to maintain ease of manufacture. The linker performs its structural function during production and can be cleaved off if needed.
4Reliability
If stable linkers are used, then resistance to cleavage by host organism proteases is improved, but decreased bioactivity and altered biodistribution occur
Solution Approach 1:
The linker is segmented into a stable core region that provides protease resistance during circulation and a labile peptide region that can be cleaved to release the active polypeptides at the target site. This segmentation allows the construct to maintain stability during delivery while enabling restoration of full bioactivity upon cleavage, resolving the contradiction between stability and activity.
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
Enhances suitability for oral administration, local delivery, targeted cleavage, improved bioactivity, and stability against protease degradation, facilitating treatment of gastrointestinal diseases and infections, and increased yield and bioactivity of polypeptides.
Implementation Method 1
the labile peptide linker is labile to one or more proteases present in the intestinal tract
Data Source
AI summary
There is provided inter alia a construct suitable for oral administration comprising a first polypeptide and a second polypeptide connected by a labile peptide linker, wherein the labile peptide linker is labile to one or more proteases present in the intestinal tract and wherein the first and second polypeptides are substantially resistant to said one or more proteases.