Paired TCR Alpha Beta Chain Isolation via Single-Cell Sorting

The patent segments the TCR isolation process into distinct stages: (1) enriching for antigen-specific T cells using tetramer staining, (2) sorting individual T cells by FACS, (3) performing targeted PCR amplification of TCR genes from sorted cells, and (4) sequencing. This segmentation allows parallel processing of multiple T cells simultaneously, dramatically reducing the time required compared to traditional bulk sequencing approaches while maintaining high identification accuracy through selective enrichment of relevant T cell clones.

The patent performs preliminary enrichment of antigen-specific T cells using tetramer-stained magnetic sorting before the actual TCR sequencing. This preliminary action concentrates the rare antigen-specific T cells (often less than 0.1% of total T cells) into a small population, allowing subsequent focused analysis of only the relevant cells. This preliminary enrichment step significantly reduces the time and computational resources needed for TCR identification while ensuring high precision by eliminating irrelevant T cell sequences from the analysis.

The patent merges the alpha and beta chain sequencing processes by performing PCR amplification and sequencing on the same sorted single T cell sample. Since each sorted cell contains both alpha and beta chain genes, the method naturally produces paired sequences from the same cellular source. This merging approach eliminates the complexity of manually pairing chains from different cells, as the physical separation of single cells during sorting ensures that each alpha-beta pair originates from the same antigen-specific T cell clone.

The patent uses single-cell sorting as an intermediary step that physically separates individual T cells before analysis. This intermediary action creates discrete, isolated samples where each cell's alpha and beta chains can be independently amplified and sequenced with unique barcodes or identifiers. The sorting intermediary thus facilitates accurate pairing by providing a clear physical and genetic link between alpha and beta chains from the same cell, avoiding the confusion that would arise from analyzing bulk mixed populations.

The patent applies local quality by creating heterogeneous samples through single-cell sorting, where each sorted cell represents a unique local population with specific TCR characteristics. By analyzing multiple such localized single-cell samples rather than one bulk population, the method captures TCR diversity across different antigen-specific clones while maintaining the ability to identify correct pairs within each local sample. The combination of multiple localized analyses provides both diversity coverage and identification precision.

The patent implements feedback through iterative enrichment and sorting steps. Initial tetramer staining identifies antigen-specific T cells, which are sorted and sequenced. The sequencing results provide feedback on which TCR sequences are present, allowing refinement of the enrichment strategy. This feedback loop enables the method to capture diverse antigen-specific TCRs while systematically identifying correct pairs through verification of co-expression in the same sorted cell, thereby resolving the contradiction between diversity capture and precise identification.