Fibronectin Scaffold Proteins With C-Terminal Stabilizing Moieties
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Solution Overview
Problem
Wild-type 10Fn3 proteins with target-binding mutations often suffer from reduced stability and solubility, posing a challenge in maintaining their therapeutic efficacy.
Innovation Solution
Stabilized fibronectin-based scaffold proteins, such as 10Fn3 molecules, are modified at their C-terminus with a PmXn moiety, where P is proline and X is any amino acid, to enhance thermostability and solubility.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mutations are introduced into wild-type 10Fn3 sequence to enable target binding, then binding affinity is improved, but protein stability is reduced
Solution Approach 1:
The patent applies parameter changes by modifying the C-terminal region of the 10Fn3 protein through specific amino acid substitutions (such as E94Q, E94N, E94K, E94R, E94D, E94E, E94G, E94H, E94L, E94M, E94P, E94S, E94T, E94V, E94W, E94Y) to enhance thermostability while preserving target binding capability. These parameter changes in the C-terminal region allow the protein to maintain both high binding affinity and improved thermal stability.
2Reliability
If mutations are introduced into wild-type 10Fn3 sequence to enable target binding, then binding affinity is improved, but solubility is reduced
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions in the C-terminal region of 10Fn3 that simultaneously improve both target binding affinity and solubility. The modifications at positions 94 and surrounding residues create a C-terminal moiety that enhances protein solubility while maintaining or improving target binding characteristics.
3Stability of the object's composition
If wild-type 10Fn3 sequence is used, then protein stability is maintained, but target binding capability is lost
Solution Approach 1:
The patent applies local quality by making targeted modifications specifically in the C-terminal region of the 10Fn3 protein while leaving the core structural domains intact. This localized approach allows the protein to retain its inherent stability from the wild-type structure while gaining new target binding capabilities through specific C-terminal modifications.
Solution Approach 2:
The patent applies parameter changes by systematically testing various amino acid substitutions at position 94 and surrounding C-terminal residues to optimize both stability and binding affinity. These parameter changes enable the protein to achieve an optimal balance between maintaining wild-type stability and acquiring target binding functionality.
Data Source
AI summary
Provided herein are proteins comprising a fibronectin based scaffold (FBS) domain, e.g., 10Fn3 molecules, that bind specifically to a target, and wherein the FBS domain is linked at its C-terminus to a region consisting of PmXn, wherein P is proline, X is any amino acid and wherein n is 0 or an integer that is at least 1 and m is an integer that is at least 1, and wherein the PmXn moiety provides an enhanced property to the FBS domain, e.g., enhanced stability, relative to the protein that is not linked to the PmXn moiety.