Modified Fn3 Domain Proteins for Reduced Aggregation
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Solution Overview
Problem
Protein aggregation is a challenge for fibronectin-based scaffolds, particularly in high concentration formulations, leading to issues like unintended receptor agonism and potential immune responses, which affects drug safety and manufacturing processes.
Innovation Solution
Modified fibronectin type III (Fn3) domains with enhanced solubility are developed by mutating specific amino acids, such as residue 58, and optimizing loop and scaffold regions to reduce aggregation propensity while maintaining binding affinity to targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If high protein concentration formulations are used for fibronectin-based scaffolds, then drug delivery efficiency is improved, but protein aggregation occurs leading to unintended receptor agonism and immune responses
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (such as position 58, 64, or 89) in the fibronectin type III domain sequence to alter the protein's physicochemical properties. These mutations change the local charge distribution and hydrophilicity, thereby reducing aggregation propensity while maintaining solubility at high concentrations required for effective drug delivery.
2Stability of the object's composition
If amino acid mutations are introduced to enhance solubility, then protein aggregation is reduced, but structural stability and binding affinity may be compromised
Solution Approach 1:
The patent applies local quality by introducing mutations only at specific, strategically chosen positions (such as residues 58, 64, or 89) within the Fn3 domain rather than throughout the entire protein sequence. These localized mutations are designed to specifically address solubility and aggregation issues in critical regions while preserving the overall fold and binding interface integrity, thus maintaining binding affinity to target receptors.
3Adaptability or versatility
If fibronectin-based scaffolds are used as protein therapeutics, then targeted binding capability is achieved, but manufacturing challenges arise due to aggregation and quality control issues
Solution Approach 1:
The patent applies preliminary action by pre-engineering solubility-enhancing mutations into the fibronectin type III domain sequence during the protein design and development phase. This proactive modification ensures that the protein inherently possesses reduced aggregation propensity before entering manufacturing processes, thereby simplifying downstream purification, formulation, and quality control steps while maintaining the targeted binding capability essential for therapeutic efficacy.
Data Source
AI summary
Provided herein are polypeptides comprising a modified fibronectin type III (Fn3) domain, wherein the amino acid corresponding to residue 58 of SEQ ID NO: 1 is mutated, and wherein the solubility is enhanced relative to the solubility of a Fn3 domain in which the amino acid corresponding to residue 58 of SEQ ID NO: 1 is not mutated. Also provided are libraries comprising a plurality of the polypeptides and a method for identifying a polypeptide that binds to a target.