Automated instrumentation for production of T-cell receptor peptide libraries

Patent metadata

Field	Value
Publication number	US-10557216-B2
Application number	US-201916392605-A
Country	US
Kind code	B2
Filing date	Apr 23, 2019
Priority date	Apr 24, 2018
Publication date	Feb 11, 2020
Grant date	Feb 11, 2020

Abstract

The present disclosure provides instrumentation and automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of antigens that bind to T-cell receptors. The engineered peptides may be putative antigens or binding regions of the T-cell receptors.

First claim

We claim:

1. An automated method of creating a cell library expressing putative TCR binding antigens using instrumentation, the method comprising:
providing a population of cells;
processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nucleic acid-directed nuclease to create cells comprising nucleic acids that encode putative TCR binding antigens configured to be displayed on a surface of the cells;
incubating the processed cells to facilitate nucleic acid editing in the cells, wherein the editing provides nucleic acids that encode the putative TCR binding antigens in the cells; and
allowing the cells to express and display the putative TCR binding antigens on the surface of the cells.

2. The method of claim 1 , wherein the population of cells comprises mammalian cells.

3. The method of claim 1 , wherein the putative TCR binding antigens comprise predicted TCR binding regions.

4. The method of claim 1 , wherein the population of cells are yeast cells.

5. The method of claim 1 , wherein the nuclease is an RNA-directed nuclease.

6. An automated method of creating a cell library expressing engineered putative T-cell receptor (TCR) antigens on the surface of the cells, the method comprising:
providing a population of cells;
processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nuclease to create cells comprising nucleic acids that encode engineered putative TCR antigens configured to be displayed on a surface of the cells;
incubating the processed cells to facilitate nucleic acid editing in the cells, wherein the editing provides nucleic acids that encode the engineered putative TCR antigens in the cells; and
allowing the cells to express and display the engineered putative TCR antigens on the surface of the cells.

7. The method of claim 6 , wherein the population of cells are yeast cells.

8. The method of claim 6 , wherein the population of cells are mammalian cells.

9. The method of claim 6 , wherein the cells display the engineered putative TCR antigens are displayed on the cells as part of a ligand.

10. The method of claim 6 , wherein the cells co-express the engineered putative TCR antigens and MHC molecules.

11. The method of claim 6 , wherein the nuclease is a nucleic acid-directed nuclease.

12. The method of claim 6 , wherein the nuclease is an RNA-directed nuclease.

13. The method of claim 6 , wherein one or more of the engineered putative TCR binding antigens are putative antigens of one or more orphan TCRs.

14. The method of claim 6 , wherein one or more of the engineered putative TCR binding antigens are known antigens of one or more TCRs.

15. An automated, multiplexed method for identifying peptides that selectively bind one or more T-cell receptors (TCRs), the method comprising:
providing a population of cells;
processing the population of cells using an automated system for multiplexed nuclease-directed genome editing, wherein the system performs the steps of:
introducing nucleic acids that encode one or more TCRs and a nuclease to a population of cells;
incubating the cells to facilitate nucleic acid editing in the cells;
allowing the edited cells to express and display the one or more TCRs on the surface of the edited cells;
screening the edited cells displaying the one or more TCRs against one or more peptides; and
identifying the peptides that selectively bind to the one or more TCRs.

16. The method of claim 15 , further comprising isolating nucleic acids encoding the peptides that selectively bind to one or more TCRs from the cells.

17. The method of claim 15 , wherein the nuclease is a nucleic acid-directed nuclease.

18. The method of claim 15 , wherein the nuclease is an RNA-directed nuclease.

19. A cell library produced using the method of claim 15 .

Assignees

• Inscripta Inc

Inventors

• Federowicz Stephen
• Church Deanna
• Graige Michael

Classifications

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C40B20/04

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title

• C07K2317/92

  Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value · CPC title

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C12N15/81

  for yeasts · CPC title