Native-Conformation CB2 Antibodies for Flow Cytometry Assays
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Solution Overview
Problem
Existing monoclonal antibodies raised against synthetic peptides derived from the predicted sequence of the target protein often fail to recognize the native cannabinoid receptor 2 (CB2) protein due to alterations in protein confirmation during purification, necessitating a method to generate antibodies that specifically recognize CB2 in its native conformation for accurate detection and monitoring of CB2 agonist-induced internalization.
Innovation Solution
Development of antibodies with specific antigen binding moieties that target human CB2, utilizing antigens expressed on the surface of stably transfected mammalian cells for immunization and screening, bypassing the need for purified recombinant proteins, and employing flow cytometry for detecting CB2 expression changes in response to CB2 agonists.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If monoclonal antibodies are raised against synthetic peptides derived from the predicted sequence of the target protein, then the antibody generation process is simplified, but the antibodies fail to recognize the native protein due to alterations in protein conformation during purification
Solution Approach 1:
The patent uses stably transfected mammalian cells expressing CB2 on their surface as a biological copy or surrogate for the native protein, replacing the need for purified recombinant protein. These cell surfaces present the CB2 receptor in its native conformation, allowing antibodies to be generated and selected against the authentic native structure rather than against purified protein that may have lost its native conformation.
Solution Approach 2:
The patent introduces stably transfected mammalian cells as an intermediary system between the antibody generation process and the native CB2 protein. These cells serve as a platform that presents the native CB2 receptor surface, enabling antibody selection without requiring direct manipulation or purification of the native protein, thus preserving its conformational integrity.
2Quantity of substance
If purified recombinant proteins are used for antibody generation, then the protein can be obtained in sufficient yield and purity, but the proteins lose their native structure during purification processes
Solution Approach 1:
Instead of purifying the native protein and risking its structural degradation, the patent uses stably transfected mammalian cells as a biological copy that naturally presents the CB2 receptor in its correct native conformation. This approach obtains sufficient protein for antibody generation without subjecting it to harsh purification processes that would denature it.
Solution Approach 2:
The patent extracts or isolates the surface-expressed CB2 receptor from the stably transfected mammalian cells for antibody generation, rather than purifying recombinant CB2 protein. This extraction from the cell surface maintains the receptor's native structure while providing sufficient material for successful antibody production.
3Ease of operation
If standard hybridoma-screening protocols use recombinant proteins immobilized on solid supports, then the screening process is standardized, but the protein conformation is significantly altered
Solution Approach 1:
The patent replaces immobilized recombinant proteins on solid supports with stably transfected mammalian cells as the antigen presentation platform. These living cells naturally present CB2 in its native conformation, providing a biological copy that maintains proper protein structure while enabling standardized screening through flow cytometry.
Solution Approach 2:
The patent introduces stably transfected mammalian cells as an intermediary that bridges the gap between standardized screening protocols and native protein conformation. These cells can be processed using standard flow cytometry techniques while preserving the native CB2 structure, thus resolving the contradiction between protocol standardization and protein structural integrity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The antibodies provide sensitive and specific assays to monitor CB2 agonist-induced internalization, enabling effective drug effect monitoring in vitro and clinical settings, supporting the linkage of CB2 target engagement with downstream pharmacodynamic effects.
Implementation Method 1
an antibody which specifically binds to human cannabinoid receptor subtype 2 (CB2)
Implementation Method 2
a flow cytometry assay to detect changes in cell surface CB2 expression in response to treatment with a CB2 agonist
Implementation Method 3
CB2 agonist induced CB2 internalization as a target engagement biomarker
Data Source
AI summary
The present invention relates to novel antibodies which bind to human CB2. Further, the invention relates to a method of detecting changes in cell surface CB2 expression by flow cytometry in response to the treatment with a CB2 agonist. The method allows to assess and monitor target engagement of CB2 agonists thus supporting pre-clinical and clinical development of CB2 agonists.