Single Chain VH Antibody Design for Stable HCAb Production
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Solution Overview
Problem
There is a need for efficient and cost-effective methods to produce heavy chain only antibodies (HCAbs) for diagnostic and therapeutic use, particularly requiring small, rapidly breeding animals capable of producing antigen-specific HCAbs for large-scale production.
Innovation Solution
Transgenic non-human animals are developed to produce single chain VH antibodies (scVHAb) comprising an antigen-binding part with a VH domain and immunoglobulin constant domains, including a CL/CH1 dimer formed by beta-sheet contact, stabilized by interdomain disulfide bonds, and linked by peptidic linkers, allowing for efficient production of HCAbs in both transgenic animals and in vitro cell culture.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional H2L2 antibody structures are used, then antigen binding capability is achieved, but production efficiency and cost-effectiveness are reduced
Solution Approach 1:
The invention extracts and utilizes only the essential components for antigen binding: the VH domain paired with CL and CH1 domains. By removing the light chain variable domain (VL) and light chain constant domain (CLight), the patent creates a simplified HCAb structure that retains antigen binding capability while improving production efficiency and reducing structural complexity.
Solution Approach 2:
The antibody structure is segmented into functional modules: the VH domain for antigen recognition, paired with CL and CH1 constant domains for structural stability. This segmentation allows independent optimization of each domain and simplifies the overall production process compared to conventional H2L2 structures.
2Productivity
If HCAbs are produced in transgenic animals, then large-scale production is enabled, but animal breeding time and facility requirements increase
Solution Approach 1:
Instead of breeding entire transgenic animals, the invention copies the essential antibody-producing capability by expressing HCAbs in cell culture systems using transfected plasmids. This approach replicates the antibody production function without requiring animal breeding, significantly reducing time and facility requirements while maintaining large-scale production capability.
3Stability of the object's composition
If scVHAb constructs with CL/CH1 dimers are used, then antibody stability is enhanced, but construction complexity increases
Solution Approach 1:
The invention merges the CL and CH1 domains into a single polypeptide chain connected by a flexible linker, forming a CL/CH1 dimer structure. This merging approach enhances antibody stability through improved structural organization and reduced immunogenicity, while the modular design actually simplifies construction compared to assembling multiple separate chains.
Solution Approach 2:
A flexible linker sequence acts as an intermediary between the CL and CH1 domains, allowing proper folding and stable dimer formation. This intermediary element facilitates the association of domains while maintaining structural flexibility, enhancing overall antibody stability without requiring complex construction protocols.
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 enables the easy production of HCAbs with enhanced stability and accessibility to previously inaccessible epitopes, facilitating their use in diagnostics and therapeutics while minimizing immunogenicity issues in human and animal subjects.
Implementation Method 1
CL/CH1 dimer formed by beta-sheet contact
Implementation Method 2
stabilized by interdomain disulfide bonds
Data Source
AI summary
The present invention provides cells, transgenic animals, including transgenic mammals and particularly rodents comprising engineered immunoglobulin (Ig) alleles. Such engineered alleles, wherein an Ig light chain CL exon [Cκ or Cλ (Cλ1, Cλ2 or Cλ3)] is incorporated into the Ig heavy chain locus, are capable of producing heavy chain-only antibodies as a single chain VH antibody (scVHAb) or heavy chain antibody (HCAb) comprising two extended scVHAbs. The scVHAb comprises an antigen-binding part consisting of a VH domain and the immunoglobulin constant domains CL, which is either Cκ or Cλ, and CH1, in the order from N-terminus to C-terminus: VH-L1-CL-L2-CH1, wherein L1 and L2 are each, independently, peptidic linkers; and wherein CL is paired with CH1 through beta-sheet contact thereby obtaining a CL/CH1 dimer.