Single-Cell Antibody Screening Preserves Heavy-Light Chain Pairing
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Solution Overview
Problem
Current methods for isolating antibodies with specific binding specificity are inefficient and prone to cross-reactivity, as they often disrupt the original pairing of immunoglobulin heavy and light chain variable region encoding sequences, leading to low affinity and stability issues in antibody production.
Innovation Solution
A methodology involving the use of microwells to confine and analyze individual cells, where antibodies are produced and then contacted with antigens, allowing for the simultaneous screening and sequencing of DNA sequences encoding the antibodies, enabling the identification and isolation of specific antibody-producing cells and their corresponding nucleotide sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If combinatorial libraries are generated by separate isolation of variable region encoding sequences, then large repertoires of antibody clones can be generated, but the original pairing of heavy and light chain sequences is lost leading to low affinity and cross-reactivity
Solution Approach 1:
The invention separates the analysis into single-cell units, where each cell is individually processed to maintain the integrity of heavy and light chain pairings. This segmentation approach allows large-scale screening while preserving the original cognate relationships between antibody chains that would otherwise be lost in bulk combinatorial library methods.
Solution Approach 2:
The patent introduces an intermediary tagging system where oligonucleotide tags are attached to both heavy and light chain variable region sequences within the same cell. These tags serve as identifiers that allow the original pairings to be tracked and reconstructed, acting as a mediator that preserves information through the sequencing and analysis process.
2Measurement precision
If hybridoma technology is used to isolate specific antibody clones, then functional screening can be performed, but the process takes 5-15 months and requires stable cell line generation
Solution Approach 1:
The invention performs preliminary functional screening at the single-cell level before committing to long-term cell line generation. By screening intact cells for desired binding properties first, the method identifies promising candidates early, avoiding the time-consuming process of generating and screening hybridoma clones that would ultimately fail functional assays.
Solution Approach 2:
The patent creates a digital copy of the antibody repertoire through sequencing, allowing virtual screening and analysis of antibody sequences and their predicted properties. This computational approach enables rapid evaluation of large numbers of clones without the time investment required for physical hybridoma generation and functional testing.
3Measurement precision
If conventional antibody screening methods are used, then specific binding can be identified, but cross-reactivity against self-antigens increases due to lack of in vivo negative selection
Solution Approach 1:
The invention implements feedback by sequencing and analyzing the actual antibody sequences produced by screened cells, then using this information to evaluate binding specificity and predict cross-reactivity. This feedback loop allows for the identification and exclusion of clones with potential cross-reactivity issues before they are advanced to later development stages.
Solution Approach 2:
The patent replaces the biological in vivo negative selection mechanism with an in silico computational approach. By using bioinformatics tools to analyze sequence features and predict binding specificity, the method substitutes the natural immune system's filtering mechanism with computational prediction and screening methods.
4Productivity
If large numbers of cells are screened simultaneously, then productivity increases, but maintaining single-cell resolution for sequence correlation becomes difficult
Solution Approach 1:
The invention applies universal tagging strategies where oligonucleotide tags with standardized structures are used across all cells in the screen. This universal approach allows for high-throughput processing while maintaining the ability to correlate sequences to their source cells through the consistent tagging system, enabling both high productivity and information preservation.
Data Source
AI summary
The invention is a methodology which makes it possible to select from a very large number of cells, a single cell or cells of interest and obtain specific information from those cells in a rapid and efficient manner. As an example of the methodology, a large number of antibody producing cells such as plasma cells are separated so that these individual antibody producing plasma cells are placed in individual wells. The cells are allowed to produce antibodies and the antibodies in the wells are then contacted with a protein bound to a solid surface such as a well top. The protein universally and specifically binds antibodies in the wells. The surface or well tray top includes addresses configured such that each address is specifically related to one of the individual wells containing a cell producing antibodies.