Single B-Cell IgG Cloning for Homogeneous Epitope-Specific Antibodies
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Solution Overview
Problem
Existing methods for producing antigen-binding proteins from a single B-lymphocyte often result in heterogeneous populations due to varying V H DJ H and V L J L recombination patterns, leading to proteins with non-specific epitope binding, avidity, and affinity.
Innovation Solution
A method involving cloning immunoglobulin heavy and light chains from a single B-cell, expressing them in host cells, screening for specificity and affinity, and isolating the desired antigen-binding proteins in eukaryotic cells to produce homogeneous antibodies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional B-cell derived monoclonal antibody discovery techniques are used, then antibody production from single B-cell is achieved, but heterogeneous antibody populations result due to multiple V H DJ H and V L J L recombination patterns
Solution Approach 1:
The patent extracts and isolates only the functional immunoglobulin genes (VH, VL, CH, CL regions) from the B-cell genome, separating them from non-functional recombination patterns. By specifically targeting and cloning only the productive antibody genes that bind to the antigen of interest, the method eliminates heterogeneous populations while maintaining high antibody production.
Solution Approach 2:
The patent applies local quality by focusing on specific regions of the immunoglobulin genes (VH, VL, CH, CL) that are responsible for antigen binding and antibody function. By selectively amplifying and expressing only these functional regions with desired specificity and affinity characteristics, the method ensures homogeneous antibody production while maintaining the necessary functional properties.
2Adaptability or versatility
If multiple V H DJ H and V L J L recombination patterns are present in single B-lymphocyte, then diverse immunoglobulin classes are produced, but epitope specificity, avidity, and affinity cannot be ensured
Solution Approach 1:
The patent performs preliminary screening and selection of B-cells that specifically bind to the target antigen before cloning and expression. By pre-selecting B-cells with desired epitope specificity through antigen binding assays, and then isolating and expressing only their functional immunoglobulin genes, the method ensures high epitope binding specificity while maintaining the ability to produce diverse immunoglobulin classes from different B-cell clones.
3Productivity
If functional immunoglobulin genes are cloned and expressed from single B-cell, then antigen-binding protein is produced, but heterogeneous populations result with non-specific epitope binding
Solution Approach 1:
The patent implements feedback control through multiple screening steps where produced antibodies are tested for epitope binding specificity and affinity. By iteratively selecting B-cells that produce antibodies with desired binding characteristics and re-expressing their immunoglobulin genes, the method ensures high reliability of epitope binding while maintaining high productivity through scalable expression systems.
Data Source
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AI summary
The present disclosure provides a method of producing an antigen-binding protein (e.g., antigen-binding region, immunoglobulin, or antigen-binding fragment thereof), including cloning from a single B-cell at least one immunoglobulin heavy chain or an antigen-binding fragment thereof and at least one immunoglobulin light chain or an antigen-binding fragment thereof; producing at least one antigen-binding protein expressing host cell that expresses one or more of a single antigen-binding protein comprising one of the at least one immunoglobulin heavy chain or antigen-binding fragment thereof and one of the at least one immunoglobulin light chain or antigen-binding fragment thereof; screening/examining each of the at least one antigen-binding protein expressing host cell for the specificity, avidity, and/or affinity of the antigen-binding protein for a protein or peptide epitope of interest to find or select at least one antigen-binding protein; and expressing the at least one antigen-binding protein of interest in a eukaryotic cell.