DNA-Barcoded pMHC Multimers for Single-Cell TCR Mapping
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Solution Overview
Problem
Current methods for identifying antigen-specific T cells are limited by the inability to assess cross-reactivity at the single-cell level and link peptides with TCR sequences, and the high cost of generating pMHC libraries prevents quick adaptation to pathogens or diseases.
Innovation Solution
The development of DNA-barcoded pMHC multimer libraries that allow for the simultaneous analysis of hundreds or thousands of peptides, linking peptide-encoding oligonucleotides to multimer backbones, enabling single-cell level assessment of T cell receptor sequences and antigen specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If fluorescent pMHC tetramers are used to identify antigen-binding T cells, then T cell detection is enabled, but the number of peptides that can be examined at a time is limited due to fluorescence spectral overlapping
Solution Approach 1:
The patent replaces the optical detection system (fluorescence) with a mass-based detection system (mass cytometry using isotopic labels). This substitution eliminates spectral overlapping issues inherent in fluorescence-based methods, allowing simultaneous examination of many more peptides without signal interference.
Solution Approach 2:
The patent changes the detection parameter from fluorescence intensity to mass-to-charge ratio. By using isotopically labeled metals with distinct mass signatures instead of fluorophores with overlapping emission spectra, the system can resolve and quantify multiple peptide-specific T cell populations simultaneously with higher dimensional separation.
2Quantity of substance
If isotope-labeled pMHC tetramers with mass cytometry are used to interrogate a larger number of peptides, then peptide diversity increases, but cross-reactivity assessment and TCR sequence linking are not demonstrated
Solution Approach 1:
The patent merges two previously separate analytical capabilities into a single integrated platform: (1) mass cytometry-based multi-peptide interrogation and (2) TCR sequence sequencing. By combining these methods, the system simultaneously achieves high-dimensional peptide screening and preserves TCR sequence information for cross-reactivity analysis.
Solution Approach 2:
The patent introduces a molecular intermediary (e.g., a linker or barcode) that physically connects the isotopically labeled pMHC tetramer to the TCR sequence. This intermediary allows the mass cytometry signal to be directly associated with the corresponding TCR sequence, enabling both peptide identification and cross-reactivity assessment in the same experiment.
3Productivity
If DNA-barcoded pMHC multimer technology is used for bulk analysis of antigen-binding T cell frequencies, then analysis throughput increases, but information on binding to individual T cells is lost
Solution Approach 1:
The patent segments the bulk population analysis into single-cell resolution measurements. By maintaining DNA barcode integrity at the single-cell level during sorting and sequencing, the system recovers individual T cell binding information that was previously lost in bulk analyses, enabling assessment of antigen specificity and cross-reactivity at the single-cell level.
4Quantity of substance
If each peptide is chemically synthesized for each pMHC species to scale up the peptide library, then library size increases, but cost and time requirements increase
Solution Approach 1:
The patent uses DNA barcodes as informational copies that represent each peptide sequence. Instead of chemically synthesizing each unique peptide, the system synthesizes a universal pMHC multimer platform and uses DNA barcode variation to represent different peptide specificities. This copying approach dramatically reduces the manufacturing complexity and cost of generating large peptide libraries.
Solution Approach 2:
The patent creates a universal pMHC multimer platform that can be used to interrogate multiple different peptides simultaneously. The DNA-barcoded system allows a single multimer construct to represent multiple peptide specificities through barcode variation, enabling one platform to serve multiple functions and eliminating the need for separate chemical synthesis for each peptide.
Data Source
AI summary
Provided herein are methods compositions and methods to generate pMHC libraries, and methods of using the pMHC libraries to determine the sequences of T cell receptors, and T cell developmental and activation status.