Methods for identifying selective binding pairs

Patent metadata

Field	Value
Publication number	US-10508273-B2
Application number	US-201916392625-A
Country	US
Kind code	B2
Filing date	Apr 24, 2019
Priority date	Apr 24, 2018
Publication date	Dec 17, 2019
Grant date	Dec 17, 2019

Abstract

The present disclosure methods for identifying binding partners using cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of peptides that bind to targets of interest. The engineered peptides are preferably expressed in the cells under conditions that provide both secretion and display of the engineered peptides on the cell surfaces, thus providing access of the engineered peptides to identify potential binding pairs. The cell libraries cab be engineered using an automated editing system that provides for one or more targeted edits per cell.

First claim

We claim:

1. A method of producing a cell library expressing engineered peptides for identification of peptides that bind to a target of interest, 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 engineered peptides each comprising an N-terminus or C-terminus cell surface display conferring tag;
incubating the processed cells to facilitate nucleic acid editing in the cells, wherein the editing provides nucleic acids that encode engineered peptides each comprising an N-terminus or C-terminus cell surface display conferring tag in the cells; and
allowing the cells to express and display the engineered peptides on the surface of the cells.

2. The method of claim 1 , wherein the engineered peptides are putative GPCR binding peptides.

3. The method of claim 1 , wherein the engineered peptides comprise putative ion channel binding peptides.

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

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

6. A method of producing a cell library expressing engineered peptides 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 peptides each comprising an N-terminus or C-terminus cell surface display conferring tag;
incubating the processed cells to facilitate nucleic acid editing in the cells, wherein the editing provides nucleic acids that encode engineered peptides each comprising an N-terminus or C-terminus cell surface display conferring tag in the cells; and
allowing the cells to express and display the engineered peptides on the surface of the cells.

7. The method of claim 6 , wherein the engineered peptides are putative GPCR binding peptides.

8. The method of claim 6 , wherein the engineered peptides comprise putative ion channel binding peptides.

9. The method of claim 6 , wherein the cell surface display conferring tag is an HLA-A*02:01 heavy chain tag or an AGA2P tag.

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

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

12. The method of claim 6 , wherein one or more of the engineered peptides are binding regions of a target of interest.

13. The method of claim 12 , wherein one or more of the engineered peptides are binding regions of a GPCR target.

14. The method of claim 12 , wherein one or more of the engineered peptides are binding regions of an ion channel target.

15. A multiplexed method for identifying peptides that selectively bind one or more targets of interest, 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 comprises the steps of:
introducing nucleic acids that encode engineered peptides wherein each engineered peptide comprises an N-terminus or a C-terminus cell surface display conferring tag and a nuclease to a population of cells;
incubating the cells to facilitate nucleic acid editing in the cells; and
allowing the edited cells to express and display the engineered peptides on the surface of the edited cells via the N-terminus or a C-terminus cell surface display conferring tag;
screening the edited cells displaying the engineered peptides against one or more targets of interest; and
identifying the edited cells expressing engineered peptides that selectively bind to one or more targets of interest.

16. The method of claim 15 , further comprising isolating the nucleic acids encoding the engineered peptides that selectively bind to one or more targets of interest 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. The method of claim 15 , wherein the cells are identified and/or isolated using a barcode associated with the engineered peptides that selectively bind to one or more targets of interest from the cells.

Assignees

• Inscripta Inc

Inventors

• Church Deanna
• Federowicz Stephen
• Graige Michael

Classifications

• C07K14/723

  G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/1037 Primary

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