Single-Cell TCR Characterization via Fluorescent Barcoding
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Solution Overview
Problem
Current methods for identifying and characterizing T cell receptors (TCRs) and TCR-like antigen-binding molecules (ABMs) are cumbersome, costly, and lack sensitivity and multiplexing capabilities, making it difficult to rapidly and reliably discover and characterize immunotherapeutic molecules that recognize specific antigens.
Innovation Solution
The development of methods and systems that involve partitioning a reaction mixture containing immune cells and MHC molecule complexes, using fluorescent molecules and nucleic acid barcode molecules to characterize ABMs such as TCRs or TCR-like antibodies, allowing for the identification of molecules that bind to target antigens with high confidence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high-throughput sequencing technology and bioinformatics are used for identification of TCRs, then the quantity of TCR sequences that can be identified increases, but the methods become cumbersome and costly
Solution Approach 1:
The method segments the identification process into distinct functional components: (1) immune cells are partitioned into discrete reactions with individual MHC complexes, (2) each partition receives unique barcode molecules for tracking, (3) binding events are detected through fluorescent signal changes, and (4) barcode sequencing identifies the TCR sequences. This segmentation transforms a cumbersome multi-step process into a streamlined workflow where each component performs a specific function, reducing overall method complexity while maintaining high throughput.
2Measurement precision
If conventional methods are used for characterizing TCR binding, then the process is simple, but the sensitivity and confidence of binding characterization are insufficient
Solution Approach 1:
The method introduces several intermediary elements that enhance measurement precision: (1) MHC molecule complexes serve as intermediaries that present target antigens in a standardized format, enabling consistent binding assessment, (2) Fluorescent molecules act as signal amplifiers that convert binding events into detectable optical signals, dramatically improving sensitivity, and (3) Barcode molecules serve as information carriers that link binding events to specific TCR sequences. These intermediaries transform weak or ambiguous binding signals into high-confidence, quantifiable measurements without requiring excessive time.
3Adaptability or versatility
If multiple TCR sequences need to be characterized simultaneously, then the multiplexing capability increases, but the cost and complexity of reagents increase
Solution Approach 1:
The method employs universal reagents that can serve multiple functions across different TCR characterization experiments: (1) MHC molecule complexes are designed to bind multiple different antigen peptides while maintaining consistent structural features, allowing the same MHC reagent to be used across various antigen-specific TCR identification assays, (2) Barcode molecules use a standardized sequence format that can be universally decoded and analyzed through common bioinformatics pipelines, and (3) Fluorescent detection systems use standard optical filters and wavelengths that work across multiple experiment types. This universality reduces per-experiment reagent costs while enabling high-level multiplexing capabilities.
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
These methods enable efficient and accurate characterization of ABMs, facilitating the development of new immunotherapies for cancer and infectious diseases by identifying molecules with specific binding affinities to target antigens.
Implementation Method 1
The target MHC molecule complex is coupled to a first fluorescent molecule. The non-target MHC molecule complex is coupled to the first fluorescent molecule and a second fluorescent molecule; the first fluorescent molecule is capable of emitting a first detectable signal and the second fluorescent molecule is capable of emitting a second detectable signal
Implementation Method 2
the first and the second fluorescent molecules, on the non-target MHC molecule complex, are capable of undergoing fluorescence resonance energy transfer (FRET), where the first fluorescent molecule is a donor and the second fluorescent molecule is an acceptor in the energy transfer
Implementation Method 3
The emergence of high-throughput sequencing technology and bioinformatics has provided opportunities for identification of these TCR and TCR-like ABMs
Data Source
AI summary
The present disclosure relates generally to compositions, methods, and systems for the characterization of T cell receptors and other antigen-binding molecules having T cell-like receptors, e.g., T cell receptor-like antibodies, using single-cell immune profiling methodologies. The compositions, methods and systems described herein permit rapid, high-throughput identification and characterization of T cell, and/or T cell-like, receptors having desired properties.