Disulfide-Stabilized Multivalent Antibody Format
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Solution Overview
Problem
The development of multivalent antibodies with two antigen binding sites has been hindered by stability and expression challenges, leading to increased costs and reduced immunogenicity, as well as undesirable pharmacokinetics, particularly in non-natural formats which often result in heterogeneous products requiring elaborate purification.
Innovation Solution
A stable multivalent antibody format is achieved by directly linking variable domains of the heavy and light chains with peptides and forming disulfide bonds between specific cysteine residues, such as VH44 and VL100, to enhance stability and prevent aggregation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If non-natural antibody formats are used to create multivalent antibodies with two antigen binding sites, then antigen-binding capability is improved, but stability and expression levels deteriorate
Solution Approach 1:
The patent introduces a disulfide bond at a specific position (VH44-Cys to VL100-Cys) within the variable domain interface to stabilize the non-natural antibody format. This parameter change (adding a covalent bond) maintains the multivalent antigen-binding capability while significantly improving structural stability and expression levels by preventing misfolding and aggregation of the engineered format.
2Adaptability or versatility
If non-natural antibody formats are used to create multivalent antibodies, then multivalency is achieved, but expression levels and cost-effectiveness deteriorate
Solution Approach 1:
By introducing a disulfide bond between VH44-Cys and VL100-Cys, the patent stabilizes the variable domain interface in the multivalent antibody format. This structural stabilization improves expression levels by preventing aggregation and misfolding during production, thereby enhancing cost-effectiveness while maintaining the desired multivalent functionality.
3Adaptability or versatility
If non-natural antibody formats are used, then multivalent binding is achieved, but immunogenicity increases
Solution Approach 1:
The disulfide bond introduction stabilizes the non-natural antibody format, reducing conformational heterogeneity and aggregation. This stabilization decreases immunogenicity by presenting a more consistent and native-like structure to the immune system, while preserving the multivalent binding capability through proper positioning of the variable domains.
4Manufacturing precision
If elaborate purification methods are used to isolate homogeneous products, then product purity is improved, but manufacturing complexity increases
Solution Approach 1:
The disulfide bond is introduced during the antibody design and expression phase, stabilizing the protein structure before purification is needed. This preliminary structural stabilization ensures that the antibody remains homogeneous during production, significantly reducing the complexity of purification steps required to achieve the desired product purity.
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
This approach results in improved stability and expression levels, reducing aggregation in liquid formulations and maintaining high antigen-binding affinity, thus overcoming previous challenges in commercial viability and immunogenicity.
Implementation Method 1
wherein: there is a disulfide bond between VH1 and VL1; and wherein the disulfide bond is between cysteine residues VH44 and VL100 numbered according to the Kabat numbering system
Data Source
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AI summary
The present disclosure provides a binding protein comprising: a polypeptide heavy chain comprising: VH1-(X1)n-VH2-CH (X2)y wherein VH1 is a first variable domain, VH2 is a second variable domain, CH is a constant domain, X1 represents an amino acid or peptide, X2 represents an Fc region, n is 0 or 1 and y is independently 1 or 2, and a polypeptide light chain comprising: VL1-(X1)n-VL2-C wherein VL1 is a first variable domain, VL2 is a second variable domain, C is a constant domain, X1 represents an amino acid or peptide and n is 0 or 1, wherein the heavy chain and light chain are aligned such that VH1 and VL1 form a first binding domain, and VH2 and VL2 form a second binding domain and wherein: there is a disulfide bond between VH1 and VL1, and/or there is a disulfide bond between VH2 and VL2, and use thereof in treatment.