Discovery of Rare T Cell Receptors of Therapeutic Value by Simultaneous Detection of Sequence, Antigen Specificity and Cell Phenotype from Millions of Single Cells in Emulsion
Stephen J. Goldfless*, Alexandra Croft, Cameron S. Brandt, Eric W. Jeffery, Katherine L. Connor, Christopher R. Clouser, Yue Jiang, Timothy G. Johnstone, David Koppstein, Hieu Nguyen, Haley Peper, Dean Toy, James Sissons, Ronald J. Hause, Adrian W. Briggs, and Francois Vigneault
Juno Therapeutics, Seattle, WA
Recognition of tumor-specific antigens by T and B lymphocytes is a critical component of the immune response to cancer. Molecular characterization of T cell receptors (TCRs) and B cell receptors (antibodies) at the mRNA sequence level allows the engineering of targeted immunotherapies against cancer, including chimeric antigen receptor T cell (CAR-T) and TCR-engineered T cell therapies. We previously demonstrated the use of our emulsion-based AbPair technology for massively high-throughput capture of natively paired adaptive immune receptors.
Although mRNA sequence alone can enable the inference of target-specific receptors, interrogation of lymphocyte phenotype and functionality is key to identifying the best therapeutic leads likely to have high activity in a recombinant context. We present here the coupling of paired TCR sequencing with single-cell measurements of antigen binding, cell surface protein phenotype, and functional response to antigen recognition. Emulsion droplet barcoding of total single-cell RNA captures cell state, and DNA-tagged affinity reagents (antibodies for surface protein or peptide-MHC multimers) allow simultaneous quantitation of phenotype and TCR specificity by sequencing.
Analyzing millions of cells per experiment, our method recovers the natively paired TCR of target specific T cells even at clonal abundances below 0.1%. We eliminate the requirement for lengthy in vitro culture or cell sorting, both of which can greatly bias the immune repertoire and introduce false positive signal. Other applications of this technology include antibody discovery, tumor profiling, and characterization of T cell dynamics during CAR-T manufacturing.