Hinge-Deleted Fc Polypeptides With CH3-Interface Disulfides
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Solution Overview
Problem
The hinge region in Fc-containing polypeptides can interfere with folding and lead to aggregation due to internal disulfide bonds or free cysteine residues, causing disassociation during manufacturing or in vivo, which affects the stability of Fc dimers.
Innovation Solution
Introduce a disulfide bond at the CH3 domain interface by replacing specific amino acids with sulfhydryl-containing residues like cysteine, stabilizing the interaction between Fc chains without the hinge region, using engineered cysteine residues to form covalent links.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-generated harmful factors
If the hinge region is deleted to remove interference with folding, then aggregation is reduced, but the covalent link between Fc chains is eliminated leading to disassociation
Solution Approach 1:
The hinge region is extracted/deleted from the Fc structure to eliminate the harmful disulfide bonds and free cysteine residues that cause aggregation and instability. This removes the source of harmful factors while the patent compensates by introducing a new stabilizing disulfide bond at the CH3 interface.
Solution Approach 2:
The patent changes the structural parameter by introducing a disulfide bond at the CH3 domain interface (residues 351-351 or 394-394) to compensate for the removal of the hinge region. This parameter change restores covalent linkage and stabilizes the dimer structure without the harmful effects of the hinge region disulfides.
2Reliability
If hinge disulfide bonds are present, then covalent linkage between Fc chains is maintained, but interference with folding and aggregation occurs
Solution Approach 1:
The harmful hinge region disulfide bonds are extracted and removed from the Fc structure. This eliminates the interference with folding and aggregation-prone free cysteine residues while maintaining dimer stability through an alternative disulfide bond placement.
Solution Approach 2:
The disulfide bond is relocated from the hinge region to the CH3 domain interface. This parameter change in bond location eliminates the harmful effects of hinge disulfides (aggregation and folding interference) while preserving the essential covalent linkage function for maintaining Fc dimer stability.
3Adaptability or versatility
If free cysteine residues are present in the hinge region, then flexibility is maintained, but disassociation during manufacturing and in vivo occurs
Solution Approach 1:
The hinge region containing free cysteine residues is extracted and deleted. This removes the source of disassociation problems during manufacturing and in vivo, while the patent compensates by introducing a disulfide bond at the CH3 interface to maintain structural integrity.
Solution Approach 2:
The patent changes the chemical parameter by introducing a disulfide bond at the CH3 domain interface to compensate for the loss of flexibility from hinge deletion. This new disulfide bond provides covalent linkage without the problematic free cysteine residues that caused disassociation.
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 stabilization of Fc-containing molecules is achieved, maintaining the integrity of the dimer structure both in vitro and in vivo, enhancing thermal stability and preventing disassociation.
Implementation Method 1
Introduce a disulfide bond at the CH3 domain interface by replacing specific amino acids with sulfhydryl-containing residues like cysteine, stabilizing the interaction between Fc chains without the hinge region
Data Source
AI summary
This disclosure provides polypeptides comprising an antibody Fc region having a deletion of one or more cysteine residues in the hinge region and substitution with a sulfhydryl-containing residue of one or more CH3-inteface amino acids. Also, provided are Fc-fusion proteins and antibodies containing said polypeptides, nucleic acids and vectors encoding said polypeptides, along with host cells and methods for making said polypeptides.