Methods for high throughput receptor:ligand identification

Patent metadata

Field	Value
Publication number	US-10048271-B2
Application number	US-201314649176-A
Country	US
Kind code	B2
Filing date	Dec 5, 2013
Priority date	Dec 11, 2012
Publication date	Aug 14, 2018
Grant date	Aug 14, 2018

Abstract

Methods and systems for high-throughput identification of receptor:ligand interactions are provided.

First claim

What is claimed is:

1. A system comprising:
a) a first plurality of suspension-adapted mammalian or insect cells, each of which first plurality of cells expresses, on the cell surface, a first heterologous polypeptide fused to a first fluorescent polypeptide; and
b) a challenge component, wherein the challenge component comprises a second plurality of suspension-adapted mammalian or insect cells, each of which second plurality of cells expresses, on the cell surface, a second heterologous polypeptide fused to a second fluorescent polypeptide, wherein the second heterologous polypeptide is a ligand or a co-receptor for the first heterologous polypeptide, and wherein the second fluorescent polypeptide is distinguishable from the first fluorescent polypeptide.

2. The system of claim 1 , wherein the first plurality of cells is immobilized on a surface.

3. The system of claim 2 , wherein the first plurality of cells is immobilized on a surface in a spatially restricted manner.

4. The system of claim 1 , wherein the first plurality of cells is suspended in a liquid medium.

5. The system of claim 1 , wherein the first heterologous polypeptide is a polypeptide that mediates cell-cell interactions, an immunoglobulin superfamily polypeptide, a TNF superfamily polypeptide, a TNF receptor superfamily polypeptide, a G-protein coupled receptor, a growth factor receptor, a nectin, an interleukin receptor, an ion channel, a T-cell costimulatory receptor, a T-cell costimulatory receptor ligand, or a cytokine receptor.

6. The system of claim 1 , wherein the first heterologous polypeptide is a PD-L1 polypeptide, a PD1 polypeptide, a CTLA-4 polypeptide, a B7-1 polypeptide, a CD200R polypeptide, or a CD200 polypeptide.

7. The system of claim 1 , wherein the first heterologous polypeptide is encoded by an expression vector, wherein the expression vector comprises a unique predetermined 15-35 nucleotide sequence for the first heterologous polypeptide, the unique sequence capable of being primed by one or more universal primer(s).

8. The system of claim 1 , wherein each of which first plurality of cells expresses a different first heterologous polypeptide fused to a first fluorescent polypeptide, wherein the different first heterologous polypeptides differ from one another in amino acid sequence by from 1 to 25 amino acids.

9. The system of claim 1 , wherein the challenge component comprises a plurality of different second heterologous polypeptides, wherein the different second heterologous polypeptides differ from one another in amino acid sequence by from 1 to 25 amino acids.

10. The system of claim 1 , wherein the challenge component comprises a second plurality of suspension-adapted mammalian or insect cells, each of which second plurality of cells expresses a second heterologous polypeptide fused to a second fluorescent polypeptide, wherein the second heterologous polypeptide is a ligand or a co-receptor for the first heterologous polypeptide, and wherein the second fluorescent polypeptide is distinguishable from the first fluorescent polypeptide.

11. The system of claim 1 , wherein the first plurality of cells are mammalian cells.

12. The system of claim 1 , wherein the second plurality of cells are insect cells.

13. A method of identifying a polypeptide that binds to a target polypeptide, the method comprising:
a) contacting a first plurality of suspension-adapted cells, each of which first plurality of cells expresses, on the cell surface, a first heterologous polypeptide fused to a first fluorescent polypeptide, with a challenge component, wherein the challenge component comprises a second plurality of suspension-adapted cells, each of which second plurality of cells expresses, on the cell surface, a second heterologous polypeptide fused to a second fluorescent polypeptide, wherein the second heterologous polypeptide is a ligand or a co-receptor for the first heterologous polypeptide, and wherein the second fluorescent polypeptide is distinguishable from the first fluorescent polypeptide; and
b) determining whether the second heterologous polypeptide binds to the first heterologous polypeptide.

14. The method of claim 13 , wherein detection of both the first fluorescent polypeptide and the second fluorescent polypeptide on a cell in the first plurality of cells indicates that the second heterologous polypeptide expressed on the surface of the second cell binds to the first heterologous polypeptide.

15. The method of claim 13 , wherein the first plurality of cells and the second plurality of cells are suspended in a liquid medium, and wherein said determining is via fluorescence activated cell sorting.

16. The method of claim 13 , wherein the first plurality of cells are mammalian cells or insect cells.

17. The method of claim 13 , wherein the second plurality of cells are mammalian cells or insect cells.

Assignees

• Albert Einstein College Medicine Yeshiva Univ
• Albert Einstein College Medicine Inc

Inventors

• Almo Steven C
• Seidel Iii Ronald D
• Hillerich Brandan S
• Garrett-Thomson Sarah C
• Love James D

Classifications

• G01N33/6845 Primary

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• G01N33/54326

  Magnetic particles · CPC title

• G01N33/54366

  Apparatus specially adapted for solid-phase testing · CPC title

• G01N15/14 Primary

  Optical investigation techniques, e.g. flow cytometry · CPC title

• G01N2015/149

  Physics · mapped topic