Targeted Hybrid Capture for Accurate TCR Repertoire Profiling
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Solution Overview
Problem
Current methods for profiling T cell receptor (TCR) repertoires are indirect, prone to amplification biases, and lack error-correcting strategies, leading to overestimates of TCR diversity and distorted views of T cell populations, particularly in oncology where the efficacy of immune checkpoint inhibitors relies on pre-existing populations of inactive but potentially responsive tumor-specific killer T cells.
Innovation Solution
A method involving targeted hybrid capture technology with sequential hybridization steps using tagged probes to retrieve and quantify TCR repertoires, providing an accurate census of T cells by capturing all possible gene segments, including rearranged TCR genes, and eliminating false clones through molecular identifiers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional PCR-based methods are used to amplify TCR genes, then the process is simple and fast, but amplification biases occur leading to overestimates of TCR diversity
Solution Approach 1:
The patent extracts and removes amplification steps from the TCR repertoire profiling process. Instead of using PCR-based methods that introduce biases, the invention uses a direct hybridization capture approach where probes selectively bind to TCR gene sequences in genomic DNA, eliminating the amplification bias that leads to overestimates of TCR diversity.
Solution Approach 2:
The patent replaces the mechanical/chemical amplification system (PCR) with a hybridization-based capture system. Using probes that hybridize to TCR gene sequences, the method directly enriches and quantifies TCR repertoires without the need for amplification, thereby maintaining accuracy while preserving throughput.
2Ease of operation
If indirect methods are used to profile T cell repertoires, then the process is easier to implement, but false positive clones are generated leading to distorted views of T cell populations
Solution Approach 1:
The patent segments the TCR gene structure into distinct regions (V, D, J segments) and uses probes targeted at specific segments to identify rearranged genes. This segmentation allows for precise identification of true T cell clones while filtering out false positives, as only properly rearranged genes with the expected segment configuration will bind the probes.
Solution Approach 2:
The patent introduces hybridization probes as intermediaries between the genomic DNA and the detection system. These probes serve as mediators that specifically recognize and bind to rearranged TCR gene sequences, enabling accurate identification of true positives while eliminating false positives generated by indirect methods.
3Device complexity
If error-correcting strategies are not implemented, then the workflow is simpler, but sequencing errors lead to overestimates of TCR diversity
Solution Approach 1:
The patent performs preliminary action by using hybridization probes to pre-select and enrich for true TCR gene sequences before sequencing. This preliminary capture step filters out sequencing errors and false positives, as only sequences that successfully hybridize to the probes will be sequenced, thereby improving accuracy without requiring complex post-seq error correction.
4Measurement precision
If targeted hybrid capture with sequential hybridization is used, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The patent merges multiple functions into the hybridization capture process: probe design incorporates both capture and quantification capabilities, and the sequential hybridization steps integrate enrichment with specificity filtering. This merging reduces the need for separate validation steps, thereby managing complexity while maintaining high precision in TCR repertoire quantification.
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
This approach enables reproducible and specific profiling of adaptive immune response genes, providing a direct insight into T cell activities and monitoring the efficacy of immunotherapies by accurately counting individual T cell clones and distinguishing between true and false positive clones.
Implementation Method 1
capturing a rearranged adaptive immune response gene from the isolated genomic DNA by sequential hybridization; hybridizing the genomic DNA with a first set of probes specific to a first portion of the rearranged adaptive immune response gene to generate a hybridized sequence
Data Source
AI summary
The present disclosure relates generally to methods for targeted hybrid capture of rearranged T cell receptors. More particularly, some embodiments relate to a method for direct and quantitative, error-corrected counting of genomic sequences for determining immune response gene repertoires.