TIGIT-TGF-β Fusion Protein Variants for Cleavage-Stable Binding
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Solution Overview
Problem
Current fusion proteins containing TGF-βRII extracellular region fragments are unstable due to cleavage and degradation issues, affecting their purity and quality uniformity, which poses challenges in drug safety and effectiveness.
Innovation Solution
A fusion protein is developed comprising an anti-TIGIT antibody and a variant TGF-βRII extracellular region fragment, where the first serine at the N-terminus is replaced with alanine, glycine, or threonine, and/or a specific amino acid sequence is deleted, enhancing stability and binding capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a fusion protein is constructed using TGF-βRII extracellular region fragment, then binding activity to TGF-β is achieved, but the protein is unstable due to cleavage and degradation
Solution Approach 1:
The patent extracts and removes the unstable N-terminal region (residues 1-20) containing the serine that is prone to cleavage and degradation. By taking out this problematic segment, the fusion protein maintains its binding activity while eliminating the source of instability, thus resolving the contradiction between binding activity and protein stability.
Solution Approach 2:
The patent changes the amino acid sequence parameters by deleting the N-terminal region and potentially introducing linkers or stabilizing mutations. This parameter change in the protein structure eliminates cleavage sites and improves folding stability, allowing the fusion protein to maintain its functional properties while gaining structural stability.
2Ease of manufacture
If the N-terminal serine is present in TGF-βRII extracellular region fragment, then the fragment can be expressed, but it undergoes easy cleavage and degradation
Solution Approach 1:
The patent removes the N-terminal serine and its surrounding region (residues 1-20) that are susceptible to cleavage and degradation. This extraction eliminates the harmful cleavage sites while preserving the essential binding function, thus improving reliability without significantly affecting expressibility.
Solution Approach 2:
The patent segments the TGF-βRII extracellular region into functional domains, retaining only the stable and functional portions (residues 21-...) while discarding the unstable N-terminal segment. This segmentation allows the protein to be expressed while resisting cleavage, as the remaining segment is structurally stable.
3Ease of manufacture
If fusion protein structure is simplified, then manufacturing is easier, but quality uniformity and purity are affected
Solution Approach 1:
The patent extracts and removes the problematic N-terminal region that causes cleavage and degradation, simplifying the overall protein structure. This extraction improves manufacturing simplicity while enhancing quality uniformity and purity by eliminating the source of heterogeneity and degradation products.
Solution Approach 2:
The patent changes the protein structure parameters by deleting the unstable N-terminal segment and optimizing the remaining sequence. This parameter change simplifies manufacturing while improving quality uniformity, as the modified structure is more resistant to degradation and more consistent in its functional properties.
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 fusion protein effectively binds to TIGIT and TGF-β, overcoming stability issues and demonstrating potential for anti-tumor drug applications by inhibiting immune checkpoints, thereby enhancing tumor treatment efficacy.
Implementation Method 1
the fusion protein can effectively bind to TIGIT and TGF-β at the same time
Data Source
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AI summary
The present disclosure pertains to the fields of biology and pharmaceutics and relates to a fusion protein comprising a TGF-βRII extracellular region fragment, a pharmaceutical composition thereof, and use thereof. Specifically, the fusion protein of the present disclosure comprises: a first protein functional region targeting an immune checkpoint and a second protein functional region with TGF-β binding activity, wherein the second protein functional region is a variant of a TGF-βRII extracellular region fragment, and the variant of the TGF-βRII extracellular region fragment is: the first serine at the N-terminus of the TGF-βRII extracellular region fragment is replaced with alanine, glycine, or threonine, and/or a fragment comprising the amino acid sequence set forth in SEQ ID NO: 59 is deleted from the TGF-βRII extracellular region fragment. The fusion protein of the present disclosure can simultaneously inhibit TIGIT and reduce TGF-β levels, demonstrating good potential for preparing anti-tumor drugs.