Reversible MHC Multimers for T-Cell Staining
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Solution Overview
Problem
Conventional fluorescent MHC-peptide multimers are heterogeneous, expensive, and cause T-cell activation leading to cell death, while reversible multimers are costly and less stable, necessitating the development of more efficient and stable staining reagents for cell analysis.
Innovation Solution
The use of protein multimers with a core structure that allows reversible binding of monomeric proteins, enabling reversible staining and dissociation under physiological conditions, and the generation of homogeneously peptide-loaded MHC class II molecules using chelate complex bonds and cleavable linkers for improved cell staining and isolation procedures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional enzymatic biotinylation is used to produce fluorescent MHC-peptide multimers, then multimers can be produced with detectable molecules, but the process is tedious, expensive, and causes degradation of unstable MHC-peptide monomers during the enzymatic reaction at elevated temperatures
Solution Approach 1:
The patent extracts and removes the biotinylatio nstep from the multimer production process. Instead of enzymatically biotinylating MHC-peptide monomers, the invention uses pre-formed biotinylated MHC multimers that are then labeled with fluorescent antibodies, eliminating the need for expensive and complex enzymatic biotinylation while avoiding degradation of unstable monomers
Solution Approach 2:
The patent segments the multimer production and labeling processes into separate steps: first producing biotinylated MHC multimers, then labeling them with fluorescent antibodies. This segmentation allows each step to be optimized independently and avoids the need for enzymatic biotinylation of individual monomers
2Measurement precision
If conventional multimers are used for cell sorting, then antigen-specific CD8+ T cells can be detected, but the multimers stably bind to cells and induce strong T cell activation leading to cell death and loss of representative cells
Solution Approach 1:
The patent changes the binding parameters of the multimers by using biotinylated MHC multomers with controlled biotin content and affinity. This allows the multimers to bind sufficiently for detection while avoiding excessive stable binding that would cause strong T cell activation and cell death, thereby maintaining cell viability and representativeness
3Productivity
If reversible multimers with low affinity biotin analogues are used, then sorting and cloning efficiencies are improved, but the reagents are costly and less stable
Solution Approach 1:
The patent optimizes the biotin content and affinity parameters of the MHC multimers to achieve an intermediate state that provides sufficient stability for reliable detection while maintaining improved sorting and cloning efficiencies. This balanced approach avoids the extreme of using low affinity biotin analogues that compromise stability
4Illumination intensity
If conventional multimers are used for staining, then cells can be stained with detectable molecules, but permanent staining exposes cells to undesirable effects including continued activation of T-cell receptor signaling
Solution Approach 1:
The patent creates a dynamic staining system where biotinylated MHC multimers can be reversibly bound to cells. The staining intensity can be controlled and adjusted by modifying the biotin content in the multimers, allowing sufficient detectability while minimizing prolonged exposure and continued T-cell activation effects
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 described protein multimers provide minimally invasive staining, maintaining cell viability and allowing for efficient isolation of antigen-specific T-cells without the undesirable effects of permanent staining, while being more stable and cost-effective than existing methods.
Implementation Method 1
conjugated to the carrier molecule or structure, wherein the plurality of chelant moieties conjugated to the carrier molecule or structure form chelate complex bonds to a plurality of chelant moiety-conjugated monomeric molecules, thus forming a multimer of the monomeric molecule
Data Source
AI summary
Some aspect of this disclosure provide reversible MHC protein multimers, and methods of using such multimers in the detection and/or isolation of specific T-cells or T-cell populations. Because reversible MHC multimers can efficiently be dissociated, the time of MHC binding to T-cell receptors, and, thus, T-cell receptor-mediated T-cell activation can be minimized. The use of reversible MHC multimers as provided herein, accordingly, allows for the detection and isolation of bona fide antigen-specific CD8+ T cells without inducing activation dependent cell death, including rare, therapeutically valuable T-cells expressing T-cell receptors binding tumor antigens with high affinity. Methods for the production and use of reversible multimers are also provided.