Micropipette Adhesion Assay for Single TCR Affinity
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Solution Overview
Problem
Current methods for measuring T cell receptor (TCR) affinity and sequence are laborious and not feasible for analyzing large or polyclonal TCR repertoires, particularly from primary human samples, as they require high frequencies or counts of antigen-specific CTLs, which are rarely obtainable, and cannot easily link biophysical binding parameters to TCR sequences.
Innovation Solution
A method involving micropipette adhesion assays to measure two-dimensional TCR affinity and paired TCRα/TCRβ sequencing, using pMHC-tagged streptamers for antigen-specific T cell isolation and subsequent sequencing, allowing for the analysis of TCR affinity and sequence from a small number of T cells, including less than 200 CD8+ CTLs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current gold standard methods (SPR) are used to measure TCR affinity, then measurement precision is improved, but device complexity and ease of operation deteriorate due to laborious procedures and requirement for recombinant soluble TCR protein expression
Solution Approach 1:
The patent replaces the mechanical/biochemical SPR measurement system with a micropipette adhesion assay that uses simple mechanical manipulation of cells and micropipettes to measure TCR-pMHC binding forces, eliminating the need for complex SPR instrumentation and recombinant protein expression systems
Solution Approach 2:
The method uses primary T cells themselves to perform the measurement function, where antigen-specific CTLs naturally bind to pMHC-coated micropipettes, eliminating the need for external recombinant TCR protein expression and complex assay setup
2Measurement precision
If current methods are used to characterize TCR-pMHC kinetics, then measurement precision is improved, but productivity deteriorates due to inability to analyze large or polyclonal TCR repertoires
Solution Approach 1:
The patent segments the TCR repertoire analysis into individual cell measurements using micropipette adhesion assays on single antigen-specific CTLs, allowing parallel analysis of multiple cells and TCR variants without requiring bulk population measurements
Solution Approach 2:
The method changes the measurement parameter from bulk population kinetics to single-cell adhesion forces, enabling analysis of rare antigen-specific T cells (frequency of one in 10^4-10^7) that cannot be detected by conventional kinetic methods requiring high cell counts
3Measurement precision
If current methods are used to measure TCR affinity, then measurement precision is improved, but loss of information occurs due to inability to link biophysical binding parameters to TCR sequence
Solution Approach 1:
The patent merges the affinity measurement process with TCR sequencing by performing micropipette adhesion assays on the same single T cells that are subsequently subjected to paired TCRα/TCRβ sequencing, thereby linking biophysical binding parameters directly to TCR sequence information
Solution Approach 2:
The method uses antigen-specific CTLs as intermediaries that naturally express the TCR of interest, allowing simultaneous measurement of functional affinity and sequencing of the TCR without requiring recombinant protein expression systems that separate function from sequence
4Measurement precision
If current methods are used to analyze TCR repertoire, then measurement precision is improved, but quantity of substance required increases due to need for high frequency or high count of antigen-specific CTLs
Solution Approach 1:
The patent replaces bulk biochemical assays requiring large cell numbers with single-cell micropipette adhesion measurements, enabling precise affinity determination from individual antigen-specific CTLs regardless of their frequency in the total T cell population
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
Enables the efficient measurement of TCR affinity and sequence from a limited number of T cells, correlating with CTL lysis capacity and providing insights into immune responses, suitable for adoptive immunotherapies and monitoring T cell responses in cancer and viral infections.
Implementation Method 1
performing a micropipette adhesion assay on said antigen-specific T cells, thereby measuring the two-dimensional (2D) TCR affinity to a peptide-major histocompatibility complex (pMHC)
Implementation Method 2
sequencing the TCR by paired TCRα/TCRβ sequencing, thereby obtaining the sequence of said TCR
Data Source
AI summary
Provided herein are methods for determining the T cell receptor affinity and sequence of antigen-specific T cells using a micropipette adhesion assay and single cell paired TCRα/TCRβ sequencing. Further provided are methods for the treatment of viral infections or cancers by adoptive transfer of high affinity functional T cells.