Antibody Selection Using Triple Knockout Pro-B Cells
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Solution Overview
Problem
Current methods fail to produce antibodies that selectively bind to autonomously active B-cell receptors, leading to non-specific binding and significant side effects, as they cannot accurately target the modified receptor regions characteristic of tumor cells in conditions like chronic lymphocytic leukemia.
Innovation Solution
The development of a method using genetically modified cells with autonomously active B-cell receptors in a native and activated state to select antibodies that specifically bind to epitopes unique to these receptors, employing triple knockout pro-/pre-B cells to ensure correct folding and presentation of BCRs, allowing for the identification of highly specific monoclonal antibodies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional antibody production methods are used, then antibody production is simple and fast, but the antibodies bind non-specifically to receptors leading to side effects
Solution Approach 1:
The patent uses an intermediary selection system comprising cells expressing autonomously active B-cell receptors that serve as a bridge between the antibody candidate and the target. This intermediary system enables specific selection of antibodies that bind to the modified receptor regions characteristic of tumor cells, while excluding antibodies that bind non-specifically to healthy cell receptors.
Solution Approach 2:
The patent performs preliminary action by first establishing a selection system with cells expressing autonomously active B-cell receptors before conducting antibody selection. This preliminary setup creates a controlled environment that pre-defines the selection criteria, allowing subsequent antibody candidates to be screened against the specific target characteristics of tumor cell receptors.
2Reliability
If antibodies are selected to bind to B-cell receptors, then therapeutic effect is achieved, but side effects occur due to binding to healthy cells
Solution Approach 1:
The patent applies local quality by targeting specific modified regions (epitopes) on the B-cell receptor that are characteristic of autonomously active receptors in tumor cells. The selected antibodies exhibit different binding properties to these local modified regions compared to the unmodified regions on healthy cell receptors, enabling selective binding to tumor cells while sparing healthy cells.
Solution Approach 2:
The patent utilizes parameter changes in the receptor structure - specifically the modified amino acid sequences in the variable regions of the heavy and light chains that characterize autonomously active B-cell receptors. These parameter changes in the target structure enable the selection of antibodies with altered binding specificity that distinguish between tumor and healthy cells.
3Measurement precision
If genetically modified cells are used for selection, then antibody specificity is improved, but production complexity increases
Solution Approach 1:
The patent uses copying by creating cell lines that express copies of the autonomously active B-cell receptor constructs. These copied receptor expressions in the selection system cells replicate the target characteristics, enabling precise selection of antibodies without requiring direct access to primary tumor cells for each selection round.
Solution Approach 2:
The patent achieves universality by developing a multi-functional selection system using genetically modified cells that can serve multiple purposes: expressing the target autonomously active B-cell receptors, providing a platform for iterative antibody selection, and enabling characterization of antibody binding specificity. This universal system replaces multiple separate selection approaches.
Data Source
Figure 1A~1F
AI summary
The present invention relates to the production, identification, and selection of biological binding molecules, in particular antibodies or fragments thereof, that selectively bind to autonomously active B-cell receptors or B-cell receptor complexes. The method serves to select a biological binding molecule that specifically binds to an autonomously active or activated B-cell receptor as its target receptor, but not to an inactive or non-activated B-cell receptor, and is carried out within a cell-based system using immature B cells that are in the pro/pre stage and exhibit a triple knockout of the genes for RAG2 or Rag1, Lambda5, and SLP65.