T-CELL EPITOPE IDENTIFICATION

• US 20180052176A1
• Filed: 03/25/2015
• Published: 02/22/2018
• Est. Priority Date: 03/27/2014
• Status: Active Grant

• Current Assignee: Provincial Health Services Authority (Province of British Columbia)
• Original Assignee: British Columbia Cancer Agency
• Inventors: Holt, Robert, Sharma, Govinda
• Granted Patent: US 10,627,411 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6878 in eptitope analysis

Abstract

The present invention is a method for determining the identity of the epitopes recognized by T-cells. The method consists of expressing an encoded library of candidate epitope sequences in a recipient reporter cell capable of providing a detectable signal upon cytotoxic attack from a single cognate T-cell followed by contacting the reporter cells with T-cells of interest. The reporter cells with a single indicating cytotoxic attack from a T-cell are isolated and then analyzed by next-generation sequencing in order to identify the epitope sequences. Specifically disclosed is a method in which a library of candidate epitope-encoding nucleic acids are expressed in cells which feature a membrane-bound major histocompatibility complex (MHC) protein, said library produced by transfection of plasmids featuring both a nucleotide encoding the candidate epitope and a nucleotide encoding a FRET-based fluorescent protein cleaved by granzyme.

Claims

1. A method for determining epitopes recognized by cytotoxic lymphocytes, the method comprising the steps of:

expressing a library of candidate epitope-encoding nucleic acids in reporter cells capable of presenting expressed peptides of such candidate epitope-encoding nucleic acids in the context of a membrane-bound major histocompatibility complex (MHC) protein and generating a signal upon recognition thereof by a cytotoxic lymphocyte and activation of effector functions of the cytotoxic T-cell;

contacting the reporter cells containing the expressed library of candidate epitope-encoding nucleic acids with a sample comprising cytotoxic lymphocytes; and

analyzing the candidate epitope-encoding nucleic acids of reporter cells that generate a signal indicating recognition by a cytotoxic lymphocyte of the sample to identify sequences of the epitope-encoding nucleic acids.

2. The method of claim 1 wherein said sample is from an individual and said MHC proteins of said reporter cells are matched with MHC proteins expressed by the individual.

3. The method of claim 2 wherein said reporter cells comprise autologous cells of said individual genetically modified to express candidate epitopes of said library.

4. The method of claim 3 wherein said autologous cells are B cells of said individual.

5. The method of claim 4 wherein said autologous cells are stably transfected or transformed by a vector capable of expressing said candidate epitopes of said library.

6. The method of claim 3 further including a step of isolating said reporter cells generating a signal indicating recognition by said cytotoxic lymphocyte.

7. The method of claim 6 wherein said reporter cells are further genetically modified to express a FRET-based fluorescent protein signaling system that generates a fluorescent signal whenever enzymatically cleaved by an effector agent.

8. The method of claim 7 wherein said effector agent is a granzyme.

9. The method of claim 3 wherein said step of analyzing includes identifying sequences of the epitope-encoding nucleic acids by sequencing cell-free nucleic acid released by said reporter cells after recognition by said cytotoxic lymphocytes.

10. A method for determining epitopes recognized by cytotoxic lymphocytes, the method comprising the steps of:

(a) expressing a library of candidate epitope-encoding nucleic acids or an enriched library of candidate epitope-encoding nucleic acids in reporter cells capable of presenting expressed peptides of such candidate epitope-encoding nucleic acids in the context of a membrane-bound major histocompatibility complex (MHC) protein and generating a signal upon recognition thereof by a cytotoxic lympohcyte and activation of effector functions of the cytotoxic lymphocyte;

(b) contacting the reporter cells containing the expressed library of candidate epitope-encoding nucleic acids with a sample comprising cytotoxic lymphocytes;

(c) isolating reporter cells generating a signal indicating recognition by a cytotoxic lymphocyte;

(d) extracting candidate epitope-encoding nucleic acids from the isolated reporter cells and generating therefrom an enriched library of candidate epitope-encoding nucleic acids;

(e) repeating steps (a)-(d) with the enriched library of candidate epitope-encoding nucleic acids until a frequency of reporter cells generating the signal indicating recognition by a cytotoxic lymphocyte is greater than or equal to a predetermined value;

(f) analyzing the candidate epitope-encoding nucleic acids of reporter cells generating a signal to identify sequences of the epitope-encoding nucleic acids.

11. The method of claim 10 wherein said step of analyzing includes isolating said reporter cells generating said signal, extracting said epitope-encoding nucleic acids, and sequencing said eptitope-encoding nucleic acids.

12. The method of claim 10 wherein said signal indicating recognition by a cytotoxic lymphocyte is fluorescent signal and said step of isolating includes sorting said reporter cells by a fluorescently activated cell sorter (FACS).

13. A reporter cell for determining antigen-specific cytotoxic lymphocytes of an individual comprising:

an autologous cell of the individual having MHC molecules capable of forming an MHC-epitope complex on a cell surface;

an epitope displayed in an MHC-epitope complex on the cell surface, the epitope being expressed in the autologous cell by a transgene comprising a candidate epitope-encoding nucleic acid; and

a signal generation product capable of generating a signal in response to action by an effector agent of a cytotoxic lymphocyte, the signal generation product being expressed in the autologous cell by a transgene comprising nucleic acids encoding the signal generation product.

14. The reporter cell of claim 13 wherein said signal generation product is a FRET-based fluorescent protein signaling system.

15. A method for testing the presence and/or level of cellular immunity in an individual to a predetermined antigen or class of antigens comprising the steps of:

(a) providing reporter cells expressing a library of epitopes derived from the predetermined antigen or class of antigens, the reporter cells being MHC matched to the individual, the reported cells capable of generating a detectable signal in response to recognition by a cytotoxic T cell and activation of an effector response by cytotoxic T cell;

(b) combining the reporter cells with a sample of cytotoxic T cells from the individual;

(c) determining the presence and/or level of cellular immunity of the individual to the predetermined antigen or class of antigens by a number or frequency of reporter cells generating a signal.

16. The method of claim 15 wherein said effector response of said cytotoxic T cell includes formation of lytic granules and transport and exocytosis thereof adjacent to said reporter cell, thereby providing delivery to said reporter cell of effector agents.

17. The method of claim 16 wherein said signal of said reporter cell is generated by a FRET-based fluorescent protein signaling system and wherein the FRET-based fluorescent protein signaling system is activated to generate a fluorescent signal by cleavage of a peptide recognition site by a granzyme.

18. The method of claim 17 wherein said predetermined antigen or class of antigens comprise one or more allergens.

19. The method of claim 17 wherein said predetermined antigen or class of antigens are proteins from an infectious agent.

20. The method of claim 19 wherein said infectious agent is a virus, a bacteria, a fungus, or a protozoa.