Tandem Protease-Cleavable Polypeptides for Specific Enzyme Activation
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Solution Overview
Problem
There is a need to identify new substrates for proteases that can be used in therapeutic, diagnostic, and prophylactic indications, particularly for matrix metalloproteases (MMPs), serine proteases (SPs), and cysteine proteases (CPs), as existing substrates may not effectively target these enzymes for specific applications.
Innovation Solution
Development of polypeptides with tandem substrates comprising a first cleavable moiety (CM1) for MMPs and a second cleavable moiety (CM2) for SPs or CPs, including specific amino acid sequences such as AHGL or PRQV, which are designed to be cleaved by these proteases, allowing for targeted activation and binding to targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing substrates are used for proteases, then the substrate structure is simple and well-understood, but the substrate may not effectively target specific proteases (MMPs, SPs, CPs) for therapeutic applications
Solution Approach 1:
The substrate is divided into multiple distinct cleavable moieties (CM1, CM2, CM3) that can be independently recognized by different protease families. Each moiety contains specific amino acid sequences tailored to bind particular proteases, allowing the substrate to target multiple proteases simultaneously or sequentially with high specificity.
Solution Approach 2:
The invention creates a composite substrate structure that integrates multiple cleavable moieties with different protease specificities into a single polypeptide chain. This composite design enables the substrate to function as a multi-specific targeting agent, combining the properties of multiple individual substrates for MMPs, SPs, and CPs.
2Reliability
If a single cleavable moiety is used, then the substrate structure is simple, but the activation specificity and control are limited
Solution Approach 1:
The substrate incorporates multiple cleavable moieties (CM1, CM2, CM3) that can be independently cleaved by different protease families. This segmentation allows for staged or sequential activation where different portions of the substrate can be processed by different enzymes, providing fine-tuned control over activation specificity.
Solution Approach 2:
Each cleavable moiety is designed with specific amino acid sequence parameters optimized for recognition by particular proteases. By varying the sequence parameters of each CM, the substrate can be tuned to respond to different protease activities, enabling precise control over activation conditions and specificity.
3Adaptability or versatility
If multiple protease-specific cleavable moieties are incorporated, then the substrate can effectively target multiple proteases, but the substrate complexity and design difficulty increase
Solution Approach 1:
The substrate is segmented into distinct cleavable moieties (CM1 for MMPs, CM2 for SPs, CM3 for CPs) that can be independently designed and optimized for specific protease families. This modular segmentation allows researchers to select and combine appropriate sequences for desired protease targeting without redesigning the entire substrate.
Solution Approach 2:
The multi-cleavable moiety substrate design provides universal applicability across different protease families and therapeutic contexts. The same substrate architecture can be adapted to target MMPs, SPs, and CPs by simply changing the specific sequence of the cleavable moieties, making it a versatile platform for various therapeutic applications.
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 tandem substrates demonstrate high cleavability and stability, enabling effective activation and binding to targets, particularly in therapeutic applications like cancer treatment, with efficacy comparable to unmasked antibodies.
Implementation Method 1
Proteases are enzymes that degrade proteins by cleaving the peptide bonds between amino acid residues. Some proteases are known to break specific peptide bonds based on the presence of a particular amino acid sequence within a protein.
Data Source
AI summary
The invention relates generally to cleavable polypeptides, method of producing a cleavable moiety (CM) containing polypeptide by culturing a cell under conditions that lead to expression of the polypeptide, methods of manufacturing a cleavable moiety (CM) containing polypeptide by culturing a cell comprising a nucleic acid construct that encodes the cleavable polypeptide to express the cleavable polypeptide, and recovering the cleavable polypeptide, nucleic acids encoding the cleavable polypeptides, and vectors including the nucleic acids encoding the cleavable polypeptides.