Trogocytosis mediated epitope discovery methods
Disclosed herein are trogocytosis based TCR ligand discovery platforms and methods of using the same, trogocytosis based TCR discovery platforms and methods of using the same, and trogocytosis based ligand discovery platforms and methods of using the same. Also disclosed herein are isolated cells, nucleotides, sequences, and sequence databases produced using trogocytosis based discovery platforms.
1. A method of antigen or antigen-specific TCR identification, comprising:
a) providing one or more T cell receptor (TCR)-expressing cell populations comprising a first immortalized cell line engineered to express one or more exogenous TCRs;
b) providing one or more target cell populations comprising a second immortalized cell line, wherein target cells of the one or more target cell populations are engineered to present one or more peptides that are 7-35 amino acids in length on one or more major histocompatibility complex (MHC) alleles;
c) contacting the one or more TCR-expressing cell populations with the one or more target cell populations under conditions sufficient to allow at least one TCR-expressing cell of the one or more TCR-expressing cell populations to specifically interact with at least one target cell of the one or more target cell populations,
whereby one or more membrane components are transferred between the at least one TCR-expressing cell and the at least one target cell;
d) isolating or having isolated
i) the at least one target cell, at least based on detecting in the at least one target cell the one or more membrane components transferred from the at least one TCR-expressing cell following the contacting step (c), and/or
ii) the at least one TCR-expressing cell, at least based on detecting in the at least one TCR-expressing cell
(1) the one or more membrane components transferred from the at least one target cell following the contacting step (c), and/or
(2) the loss of the one or more membrane components transferred from the at least one TCR-expressing antigen cell following the contacting step (c); and
e) determining or having determined
i) a sequence of the one or more peptides associated with the isolated at least one target cell,
ii) at least a portion of a TCR sequence of a TCR peptide associated with the isolated at least one TCR-expressing cell, or
iii) a sequence of one or more peptides associated with the isolated at least one target cell and at least a portion of a TCR sequence of a TCR peptide associated with the isolated at least one TCR-expressing cell.
2. The method of claim 1 , wherein at least one of the one or more antigen-specific T cell populations is isolated from a subject known or suspected to have cancer.
3. The method of claim 1 , wherein the one or more exogenous TCRs are identified or have been identified in a tumor sample.
4. The method of claim 1 , wherein at least one of the one or more MHC alleles comprises an exogenous MHC allele.
5. The method of claim 1 , wherein at least one of the one or more MHC alleles comprises a single chain trimer (SCT).
6. The method of claim 1 , wherein the one or more peptides comprises a neoepitope, and wherein the method comprises analyzing tumor, viral, or bacterial sequencing data from a subject to identify one or more somatic mutations, to select the neoepitope.
7. The method of claim 1 , wherein
the one or more membrane components transferred from the at least one TCR-expressing cell comprises a TCR-expressing cell membrane component selected from the group consisting of: a TCR; a TCR co-receptor, a CD3 TCR co-receptor, a CD4 TCR co-receptor, and a CD8 TCR co-receptor; and/or
the one or more membrane components transferred from the at least one target cell comprises a target cell membrane component comprising an MHC allele.
8. The method of claim 1 , wherein at least one of the one or more membrane components comprises a labeled membrane component.
9. A method of antigen identification, comprising:
a) providing one or more T cell receptor (TCR)-expressing cell populations comprising a first immortalized cell line engineered to express one or more exogenous TCRs;
b) providing one or more target cell populations comprising a second immortalized cell line, wherein target cells of the one or more target cell populations are engineered to present one or more peptides that are 7-35 amino acids in length on one or more major histocompatibility complex (MHC) alleles;
c) contacting the one or more TCR-expressing cell populations with the one or more target cell populations under conditions sufficient to cause an antigen-specific interaction between at least one TCR-expressing cell of the one or more TCR cell populations and at least one target cell of the one or more target cell populations,
whereby one or more membrane components are transferred between the at least one TCR-expressing cell and the at least one target cell;
d) isolating or having isolated the at least one target cell, at least based on detecting in the at least one target cell the one or more membrane components transferred from the at least one TCR-expressing cell following the contacting step (c); and
e) determining or having determined a sequence of the one or more peptides associated with the isolated at least one target cell.
10. The method of claim 9 , wherein the one or more membrane components transferred from the at least one TCR-expressing cell comprises a TCR-expressing cell membrane component selected from the group consisting of: a TCR; a TCR co-receptor, a CD3 TCR co-receptor, a CD4 TCR co-receptor, and a CD8 TCR co-receptor.
11. The method of claim 1 , wherein the first immortalized cell line comprises an immortalized T cell line.
12. The method of claim 1 , wherein the first immortalized cell line and the second immortalized cell line are each selected from: an immortalized T cell line, K562 cells, HEK293 cells, 3T3 cells, Chinese Hamster Ovary (CHO) cells, or HeLa cells.
13. The method of claim 1 , wherein the first immortalized cell line and the second immortalized cell line are each selected from: Jurkat cells, K562 cells, HEK293 cells, 3T3 cells, Chinese Hamster Ovary (CHO) cells, or HeLa cells.
14. The method of claim 1 , wherein the first immortalized cell line and the second immortalized cell line are different immortalized cell lines.
15. The method of claim 9 , wherein the first immortalized cell line comprises an immortalized T cell line.
16. The method of claim 9 , wherein the first immortalized cell line and the second immortalized cell line are each selected from: an immortalized T cell line, K562 cells, HEK293 cells, 3T3 cells, Chinese Hamster Ovary (CHO) cells, or Hela cells.
17. The method of claim 9 , wherein the first immortalized cell line and the second immortalized cell line are each selected from: Jurkat cells, K562 cells, HEK293 cells, 3T3 cells, Chinese Hamster Ovary (CHO) cells, or Hela cells.
18. The method of claim 9 , wherein the first immortalized cell line and the second immortalized cell line are different immortalized cell lines.
19. The method of claim 1 , wherein contacting at (c) comprises co-incubating the one or more TCR-expressing cell populations with the one or more target cell populations at a ratio of TCR-expressing cells to target cells of about 1:1 to about 5:1.
20. The method of claim 1 , wherein the ratio of (1) the number of target cells presenting on one or more MHC alleles a peptide that is specifically bound by a TCR expressed by a TCR-expressing cell of the one or more TCR-expressing cell populations, to (2) the number of target cells not presenting on one or more MHC alleles the specifically bound peptide, is about 1:1 to about 1:10,000.
21. A method of antigen or antigen-specific TCR identification, comprising:
a) providing one or more T cell receptor (TCR)-expressing cell populations comprising a first immortalized cell line selected from an immortalized T cell line, K562 cells, HEK293 cells, 3T3 cells, Chinese Hamster Ovary (CHO) cells, or HeLa cells, wherein the first immortalized cell line is engineered to express one or more exogenous TCRs;
b) providing one or more target cell populations comprising a second immortalized cell line selected from an immortalized T cell line, K562 cells, HEK293 cells, 3T3 cells, CHO cells, or HeLa cells, wherein the one or more target cell populations are engineered to present one or more peptides that are 7-35 amino acids in length on one or more major histocompatibility complex (MHC) alleles;
c) co-incubating the one or more TCR-expressing cell populations with the one or more target cell populations under conditions sufficient to induce trogocytosis, whereby one or more membrane components are transferred between at least one TCR-expressing cell of the one or more TCR-expressing cell populations and at least one target cell of the one or more target cell populations;
d) isolating or having isolated
i) the at least one target cell, at least based on detecting in the at least one target cell the one or more membrane components transferred from the at least one TCR-expressing cell following the co-incubating in (c), and/or
ii) the at least one TCR-expressing cell, at least based on detecting in the TCR-expressing cell
(1) the one or more membrane components transferred from the at least one target cell following the co-incubating in (c), and/or
(2) the loss of the one or more membrane components transferred from the at least one TCR-expressing cell following the co-incubating in (c); and
e) determining or having determined
i) a sequence of the one or more peptides associated with the isolated at least one target cell,
ii) at least a portion of a TCR sequence of a TCR peptide associated with the isolated at least one TCR-expressing cell, or
iii) both e-i) and e-ii).