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 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 and comprises V regions from a plurality of V genes, and wherein the plurality of TCRs are exogenous to the plurality of cells;
• (b) contacting the plurality of cells with one or more target antigens, wherein a subset of the plurality of cells expressing the plurality of target-reactive TCRs bind to the one or more target antigens;
• and
• (c) identifying at least two cells of the subset of the plurality of cells, which at least two cells 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.

A method of identifying a target-reactive T-cell receptor (TCR), comprising:

• (a) providing a plurality of T cells expressing a plurality of TCRs, wherein each T cell of the plurality of T cells expresses a cognate pair of a TCR of the plurality of TCRs;
• (b) partitioning the plurality of T cells into a plurality of compartments, wherein each compartment comprises an individual T cell of the plurality of T cells;
• (c) within each compartment, linking a first polynucleotide encoding a first TCR chain and a second polynucleotide encoding a second TCR chain of the cognate pair of the TCR of the individual T cell, thereby generating a plurality of fused polynucleotides, wherein (i) the first polynucleotide and the second polynucleotide are transcribed or amplified products of endogenous nucleic acids of the individual T cell or (ii) the first polynucleotide and the second polynucleotide are not chemically synthesized using phosphoramidite;
• (d) delivering the plurality of fused polynucleotides into a plurality of cells, wherein each cell of the plurality of cells comprises at least one fused polynucleotide of the plurality of fused polynucleotides;
• (e) expressing the plurality of fused polynucleotides from the plurality of vectors in the plurality of cells, wherein a subset of the plurality of cells expresses a plurality of target-reactive TCRs;
• (f) contacting the plurality of cells with one or more target antigens, wherein the subset of the plurality of cells expressing the plurality of target-reactive TCRs binds to the one or more target antigens;
• and
• (g) identifying a target-reactive TCR of the plurality of target-reactive TCRs of the subset of the plurality of cells.

A method for identifying a target-reactive T-cell receptor (TCR), comprising:

• (a) contacting a plurality of T cells from a first sample with a second sample comprising tumor cells from a subject or a third sample, wherein the third sample is derived from the second sample, and wherein the third sample comprises:
• (i) a target antigen from the tumor cells of the second sample or a nucleic acid encoding the target antigen, and an MHC,
• (ii) a cell presenting the target antigen in an MHC, or
• (iii) a cell comprising an MHC and a protein product encoded by the nucleic acid, and
• wherein a subset of the plurality of T cells binds to the target antigen in complex with the MHC;
• (b) isolating the subset of the plurality of T cells or a portion thereof of the first sample;
• (c) partitioning the subset of the plurality of T cells or a portion thereof into a plurality of compartments, wherein each compartment comprises an individual T cell of the subset of the plurality of T cells or a portion thereof;
• and
• (d) within each compartment, identifying a first polynucleotide encoding a first TCR chain and a second polynucleotide encoding a second TCR chain of a cognate pair of TCR of the individual T cell, thereby generating one or more paired polynucleotides.