High throughput cloning of paired bipartite immunoreceptor polynucleotides and applications thereof

A method of identifying a plurality of target-reactive T-cell receptors (TCRs), comprising:

• (a) providing a mixture comprising a plurality of cells expressing a plurality of TCRs, each cell of the plurality of cells expressing a TCR of the plurality of TCRs, wherein the plurality of TCRs comprises at least 50 different cognate pairs from a sample from a subject and comprises V regions from a plurality of V genes, wherein each cognate pair of the at least 50 different cognate pairs comprises a TCR alpha (TCRα) chain paired with a TCR beta (TCRβ) chain, and wherein the plurality of TCRs are exogenous to the plurality of cells;
• (b) contacting the mixture comprising the plurality of cells from (a) with one or more target peptide antigens in complex with a major histocompatibility complex (MHC) molecule, wherein a subset of the plurality of cells expressing the plurality of target-reactive TCRs bind to the one or more target peptide antigens in complex with the MHC molecule;
• and
• (c) identifying at least two cells of the subset of the plurality of cells from (b) that express at least two target-reactive TCRs of the plurality of target-reactive TCRs, thereby identifying the at least two target-reactive TCRs of the plurality of target-reactive TCRs.