Method of identifying human compatible T cell receptors specific for an antigenic target
Methods are provided for identifying T cell receptors that specifically bind a particular antigenic target and can be used as therapeutics against disease.
1. A high-throughput method for identifying an antigen-specific T cell receptor for therapeutic use, the method comprising:
dividing one or more samples comprising T cells into a first subset and a second subset;
performing immunosequencing of rearranged nucleic acid molecules extracted from the first subset of cells to obtain a first plurality of unique sequence reads;
determining based on the first plurality of unique sequence reads a relative abundance of each unique T cell receptor (TCR) sequence out of a total number of T cells in the first subset;
enriching the second subset of cells with multimer molecules comprising an HLA- presented antigen to identify a population of antigen-specific T cells that bind the multimer molecules;
performing immunosequencing of rearranged nucleic acid molecules extracted from said sorted population of antigen-specific T cells to obtain a second plurality of unique sequence reads;
determining based on the second plurality of unique sequence reads a relative abundance of each unique TCR sequence out of a total number of T cells in the second subset;
determining a relative change in abundance of each antigen-specific TCR sequence based on the relative abundances of the antigen-specific TCR sequence in the first subset and the second subset; and
identifying an antigen-specific TCR sequence based on its relative change in abundance as a TCR sequence for therapeutic use for said HLA-presented antigen.
2. The method of claim 1 , wherein identifying an antigen-specific TCR sequence based on its relative change in abundance as a TCR sequence for therapeutic use for said HLA-presented antigen comprises ranking each of the antigen-specific TCR sequences based on its binding affinity for said HLA-presented antigen.
3. The method of claim 1 , wherein identifying an antigen-specific TCR sequence based on its relative change in abundance as a TCR sequence for therapeutic use for said HLA-presented antigen comprises identifying the top antigen-specific TCR sequences having the greatest relative change in abundance as therapeutics for said HLA-presented antigen.
4. The method of claim 3 , wherein the top antigen-specific TCR sequences comprise the top 100 ranked TCRs in the sample.
5. The method of claim 3 , wherein the top antigen-specific TCR sequences comprise the top 50 ranked TCRs in the sample.
6. The method of claim 3 , wherein the top antigen-specific TCR sequences comprise the top 10 ranked TCRs in the sample.
7. The method of claim 1 , further comprising pairing the antigen-specific TCR sequence with a second TCR sequence that forms its cognate pair in the T cell.
8. The method of claim 1 , further comprising identifying, is based on its determined relative change in abundance, a second TCR sequence that pairs with the antigen-specific TCR sequence to form an antigen-specific TCR cognate pair.
9. The method of claim 1 , wherein the enriching is performed by flow cytometry.
10. The method of claim 9 , wherein the enriching comprises binding the cells to a first concentration of the multimer molecules comprising the HLA- presented antigen and a diluted concentration of the multimer molecules comprising the HLA- presented antigen and identifying a population of antigen-specific T cells that bind the multimer molecules with a high affinity by identifying cells enriched using both the first concentration of the multimer molecules and the diluted concentration of the multimer molecules.
11. The method of claim 9 , wherein the enriching identifies a population of antigen-specific T cells that bind the multimer in a CD8-independent manner.
12. The method of claim 11 , wherein the HLA-presented antigen comprises HLA-A2 protein comprising the substitutions D227K and T228A that interfere with CD8 binding.
13. The method of claim 1 , wherein the one or more samples are blood samples.
14. The method of claim 1 , wherein the one or more samples are tissue samples.
15. The method of claim 1 , wherein the TCR sequence is a TCRB, TCRA, TCRG, or TCRD sequence.
16. The method of claim 1 , wherein the first subset of cell comprises at least 1 million T cells.