Single-Chain Immunoglobulin Assembly Without Heavy-Light Stoichiometry
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Solution Overview
Problem
Existing methods for producing recombinant immunoglobulins face challenges due to the stoichiometric expression requirement of heavy and light chains and high endogenous Ig expression in B cells, leading to unpredictable hybrid molecule formation and low efficiency.
Innovation Solution
A recombinant nucleic acid molecule structure comprising VH-PL1-VL-CL-PL2 or VH-PL1-VL-CL-PL2-CH, with identical or different peptide linkers, allows for single-chain expression of immunoglobulins, overcoming stoichiometry issues and enabling high expression of recombinant antibodies by integrating into the immunoglobulin heavy locus using viral vectors like AAV6.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If simple addition of donor Hd and Ld chains is performed in recipient cells expressing endogenous Ig, then B-cell specificity re-engineering is attempted, but unpredictable assembly of hybrid molecules occurs due to stoichiometric requirements and high endogenous Ig expression
Solution Approach 1:
The patent merges the heavy chain variable region (VH) and light chain variable region (VL) into a single polypeptide chain connected by a peptide linker, creating a single-chain Fv region. This merging eliminates the need for separate heavy and light chain expression and assembly, thereby preventing the formation of unpredictable hybrid molecules while enabling precise control over antibody specificity through single-gene transduction.
2Reliability
If stoichiometric expression of H and L chains is required for correct Ig production, then proper antibody assembly is achieved, but complexity of simultaneous expression of two recombinant proteins increases
Solution Approach 1:
The patent combines the coding sequences for heavy chain variable region and light chain variable region into a single open reading frame separated by a peptide linker sequence. This merging reduces the expression system from requiring two separate recombinant proteins to requiring only one recombinant polypeptide, thereby simplifying the expression system while ensuring stoichiometric correctness of the antibody structure.
Solution Approach 2:
The patent segments the antibody variable region into VH and VL domains connected by a flexible peptide linker, allowing each domain to fold independently while maintaining the correct spatial relationship. This segmentation enables proper folding and assembly within a single-chain structure, achieving reliable antibody production without the complexity of co-expressing multiple proteins.
3Reliability
If high amounts of endogenous Ig are expressed in B cells, then normal B-cell function is maintained, but interference with donor chain expression and unpredictable hybrid molecule formation occurs
Solution Approach 1:
The patent merges VH and VL into a single-chain Fv region that can be expressed as a unified polypeptide. This merged structure is recognized by the cell as a single functional unit rather than separate chains, allowing it to compete effectively with endogenous Ig for assembly partners while preventing the formation of hybrid molecules through its unified structure.
Solution Approach 2:
The peptide linker acts as an intermediary between the VH and VL domains, providing a flexible connection that allows proper spatial arrangement and folding. This intermediary structure facilitates the correct assembly of the single-chain Fv region even in the presence of high endogenous Ig expression, preventing harmful hybrid molecule formation while maintaining B-cell function.
Data Source
AI summary
The present invention relates to an antibody comprising an immunoglobulin heavy chain variable region (VH), an immunoglobulin light chain variable region (VL), an immunoglobulin light chain constant region (CL), an immunoglobulin heavy chain constant region (CH), and two sequences encoding peptide linkers (PL1 and PL2), wherein VH is fused to VL through PL1 and CL is fused to CH through PL2, and its uses for therapeutic or diagnostic purposes. The invention further relates to a recombinant nucleic acid molecule encoding said antibody, and an expression cassette, vector, viral particle, host cell, transgenic organism or pharmaceutical composition comprising said recombinant nucleic acid molecule.