Modified IL-12p40 Variants Proteolytic Cleavage Resistance
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Solution Overview
Problem
The instability of IL-12 and IL-23 proteins due to proteolytic cleavage in the C-terminal region of the IL-12p40 subunit complicates their production and clinical use as therapeutic agents, leading to heterogeneity and reduced efficacy.
Innovation Solution
Engineering amino acid alterations in the D3 domain of the IL-12p40 subunit to remove the proteolytic site between Lys260 and Arg261, increasing stability and resistance to proteolytic cleavage, while avoiding the creation of immunogenic T-cell epitopes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If wild-type IL-12p40 subunit is used, then the protein can be produced, but it undergoes proteolytic cleavage in the C-terminal region leading to heterogeneity and reduced stability
Solution Approach 1:
The patent removes the proteolytic cleavage site (Lys260-Arg261) from the IL-12p40 subunit sequence, extracting the harmful element that causes instability. This results in a modified p40 subunit that resists proteolytic cleavage and maintains structural integrity, directly resolving the contradiction between protein stability and susceptibility to proteolytic degradation.
Solution Approach 2:
The patent changes the amino acid sequence parameters at positions 260-261 in the p40 subunit, altering the local chemical properties to eliminate the proteolytic cleavage site. This parameter change transforms the protein from a proteolytically unstable form to a stable variant, improving both stability and reliability.
2Stability of the object's composition
If amino acid alterations are made in the D3 domain to remove the proteolytic site, then stability improves, but there is a risk of creating immunogenic T-cell epitopes
Solution Approach 1:
The patent applies local quality by making targeted amino acid alterations only at the specific proteolytic cleavage site (Lys260-Arg261) in the D3 domain, while preserving the rest of the protein's native structure and function. This localized modification eliminates proteolytic susceptibility without creating widespread immunogenic epitopes, as the changes are confined to a specific region rather than altering the overall protein architecture.
3Productivity
If wild-type IL-12p40 is used, then the protein can be secreted in free form, but it exhibits heterogeneity in the purified product due to proteolytic cleavage
Solution Approach 1:
By removing the proteolytic cleavage site from the p40 subunit sequence, the patent eliminates the source of heterogeneity that arises during protein purification. The modified p40 subunit maintains its ability to be secreted in free form while producing a homogeneous purified product, as the proteolytic cleavage event that creates heterogeneity is prevented by the removed cleavage site.
Data Source
AI summary
Modified interleukin-12 (IL-12) p40 polypeptides are disclosed. The modified polypeptides have alterations in the IL-12p40 subunit to eliminate the protease site between positions Lys260 and Arg261. The modified IL-12p40 polypeptides according to the invention have improved stability compared to wild-type mature human IL-12p40 polypeptides.