Antibody Generation Against T Cell Receptors Using Exogenous Expression
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Solution Overview
Problem
Generating and screening antibodies against cell surface proteins, particularly T cell receptors (TCRs), is challenging due to the need for the proteins to fold correctly in their natural cellular environment, making it difficult to produce effective antibodies.
Innovation Solution
A method involving the use of non-human cells that express exogenous forms of TCR proteins, followed by immunization, hybridoma generation, and screening with human cells expressing specific TCR forms to identify binding antibodies, utilizing a mixture of cells with and without functional TCRs and selection markers for accurate binding identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If cell surface proteins are used in their natural cellular environment to ensure correct folding, then protein structure integrity is improved, but accessibility for antibody generation and screening deteriorates
Solution Approach 1:
The patent extracts the cell surface protein from its natural cellular environment by using cells that overexpress the protein of interest. This allows the protein to be presented on the cell surface in high quantities while maintaining its native folded structure, making it accessible for antibody generation without requiring extraction from the natural membrane context
Solution Approach 2:
The patent uses a non-human cell line as an intermediary system that expresses the human cell surface protein. This intermediary cell line allows the protein to fold correctly (maintaining structure integrity) while providing abundant surface expression for easy access during immunization and screening processes
2Measurement precision
If antibodies are screened against cells expressing the surface protein, then binding specificity is improved, but differentiation between specific and non-specific binding deteriorates
Solution Approach 1:
The patent introduces a local quality difference between test cells and control cells by using cells with different surface protein expression levels or characteristics. This allows specific binding to be distinguished from non-specific binding by comparing signals from cells with the target protein versus cells without it, creating a localized differentiation strategy
Solution Approach 2:
The patent employs asymmetric cell populations in the screening process - one population expressing the surface protein of interest and another serving as a negative control without the protein. This asymmetric setup creates a clear contrast that enables differentiation between specific and non-specific antibody binding
3Ease of manufacture
If non-human cells expressing exogenous human protein segments are used for immunization, then antibody generation feasibility is improved, but cross-species compatibility challenges worsen
Solution Approach 1:
The patent inverts the typical approach by using non-human cells to express human protein segments, rather than using human cells. This inversion allows the use of robust non-human cell lines for protein expression while still generating antibodies against human targets, effectively reversing the conventional cell source paradigm
Solution Approach 2:
The patent changes the species parameter of the expression system from human to non-human while maintaining the human identity of the target protein. This parameter change enables the use of well-established non-human cell culture systems while still producing antibodies against human cell surface proteins
Data Source
AI summary
The invention relates to a method for generating an antibody binding to a cell surface protein of interest, the method comprising the following steps:(a) providing a non-human cell which does not express the endogenous form of the cell surface protein of interest but expresses an exogenous form of the cell surface protein of interest comprising at least one human segment;(b) immunization of a non-human animal with the cell line provided in step (a);(c) generation of hybridomas from the immunized non-human animal of step (b);(d) screening for an antibody that binds to the cell surface protein of interest by contacting the antibodies secreted by the hybridomas of step (c) with human cells which do not express the endogenous form of the cell surface protein of interest but express an exogenous form of the cell surface protein of interest comprising at least one human segment.