Soluble Antibody Variable Domains in Reducing Cytoplasm
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Solution Overview
Problem
Current methods for expressing antibodies in bacterial hosts face challenges such as low folding stability and propensity for aggregation due to diverged subunits, requiring oxidizing environments for proper folding, which limits cytoplasmic expression and yields in bacterial hosts like E. coli.
Innovation Solution
Development of polypeptides comprising antibody heavy and light chain variable regions with scaffold regions at least 90% identical to specific germline sequences (e.g., IGHV3-23 and IGLV1-40, IGLV3-1) that can form stable antigen-binding sites in reducing environments, allowing for soluble expression and high yields in both bacterial and mammalian cytoplasmic environments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If antibody variable domains are expressed in bacterial cytoplasm, then production cost is reduced and accessibility is improved, but folding stability is insufficient and aggregation occurs
Solution Approach 1:
The patent changes the amino acid sequence parameters of the antibody variable domains by introducing stabilizing mutations. Specifically, it modifies the framework regions of VH and VL domains to enhance their folding stability in reducing environments while maintaining antigen binding capability. This resolves the contradiction by altering the molecular parameters of the antibody domains themselves.
2Stability of the object's composition
If disulphide bonds are required for proper folding, then structural stability is achieved, but expression in reducing cytoplasmic environments is prevented
Solution Approach 1:
The patent inverts the conventional approach by designing antibody variable domains that do not require disulphide bonds for stability. Instead of relying on oxidizing environments and disulphide bond formation, the invention creates reduction-stable domains through specific amino acid sequence modifications in the framework regions, enabling cytoplasmic expression while maintaining structural integrity.
3Stability of the object's composition
If periplasmic expression is used to ensure proper folding, then folding stability is improved, but expression yield is reduced due to membrane export saturation
Solution Approach 1:
The patent creates a copy of the antibody variable domain structure that is stabilized through amino acid sequence modifications rather than relying on the periplasmic environment. The stabilized VH and VL domains can fold correctly in the cytoplasm without needing to be exported to the periplasm, thus avoiding membrane export saturation and increasing expression yield while maintaining folding stability.
4Adaptability or versatility
If antibody domains with high diversity are used to expand repertoire, then binding capability is improved, but interfacial fit and folding stability deteriorate
Solution Approach 1:
The patent applies local quality by differentiating the functional requirements of different antibody regions. The framework regions are modified to provide structural stability and proper folding, while the complementarity determining regions (CDRs) maintain high diversity for antigen binding. This localized optimization allows the interface between VH and VL to remain stable even as CDR sequences diversify to expand the antibody repertoire.
Data Source
AI summary
The invention relates generally to polypeptides, such as antibody molecules, that demonstrate high stability and solubility. In particular, the invention relates to polypeptides comprising paired VL and VH domains that demonstrate soluble expression and folding in a reducing or intracellular environment. The invention also relates to polynucleotides encoding such polypeptides, to libraries of such polypeptides or polynucleotides, and to methods of using such polypeptides in research, diagnostic and therapeutic applications.