Complexes comprising transmembrane receptors and antibodies that bind the receptors in a conformationally selective manner

Patent metadata

Field	Value
Publication number	US-10520516-B2
Application number	US-201715844319-A
Country	US
Kind code	B2
Filing date	Dec 15, 2017
Priority date	Feb 5, 2013
Publication date	Dec 31, 2019
Grant date	Dec 31, 2019

Abstract

Provided herein are several methods for selecting agents that bind to transmembrane receptors in a conformationally-selective way. In some embodiments, the method may comprise producing: a transmembrane receptor in an active conformation; and said transmembrane receptor in an inactive conformation and using cell sorting to select, from a population of cells comprising a library of cell surface-tethered extracellular capture agents, cells that are specifically bound to either the transmembrane receptor in its active conformation or the transmembrane receptor in its inactive conformation, but not both. In other embodiments, the method may comprise: contacting a GPCR with a population of cells that comprise a library of surface-tethered extracellular proteins; labeling the cell population with a conformationally-specific binding agent, e.g., a G-protein or mimetic thereof; and using cell sorting to select from the cell population cells that bind to the agent.

First claim

What is claimed is:

1. A composition comprising:
a complex comprising a transmembrane receptor and a modulator of said transmembrane receptor, wherein said transmembrane receptor is maintained in an active or inactive conformation by the modulator; and
a cell expressing a surface-tethered extracellular antibody, wherein the surface-tethered extracellular antibody binds to the transmembrane receptor in its active or inactive conformation and does not bind to both the active and inactive conformation of said transmembrane receptor.

2. The composition of claim 1 , wherein said transmembrane receptor is maintained in the active conformation by the modulator.

3. The composition of claim 1 , wherein said transmembrane receptor is maintained in the inactive conformation by the modulator.

4. The composition of claim 1 , wherein said transmembrane receptor is further labeled with a first fluorescent label.

5. The composition of claim 4 , wherein said transmembrane receptor is further labeled with a second fluorescent label that is distinguishable from the first fluorescent label.

6. The composition of claim 1 , wherein said transmembrane receptor in the active confirmation and said transmembrane receptor in the inactive confirmation are distinguishably tagged using different epitope tags.

7. The composition of claim 1 , wherein said transmembrane receptor is a G protein-coupled receptor (GPCR), an ion channel, a progestin and adipoQ receptor (PAQR) family receptor, or carrier transporter.

8. The composition of claim 1 , wherein said transmembrane receptor is a GPCR.

9. The composition of claim 1 , wherein the cell is a yeast cell.

10. The composition of claim 1 , wherein said antibody is a single chain antibody.

11. The composition of claim 1 , wherein said antibody is encoded by cDNA obtained from an animal that has been immunized with said transmembrane receptor in its active or inactive conformation.

12. The composition of claim 1 , wherein said antibody is a camelid antibody.

13. The composition of claim 1 , wherein said antibody is a variant of a capture agent that is known to bind said transmembrane receptor in its active or inactive conformation and not bind to both the active and inactive conformation of said transmembrane receptor.

14. The composition of claim 1 , wherein said antibody is a nanobody.

15. The composition of claim 1 , wherein the modulator is a G-protein or an antibody.

16. The composition of claim 15 , wherein the G-protein is labeled with a fluorophore or using a labeled antibody.

17. The composition of claim 16 , wherein said labeled G-protein is a heterotrimer G-protein comprising G s , G i , G q , G 12 , or G t .

18. The composition of claim 15 , wherein said modulator is an antibody.

19. The composition of claim 1 , wherein the composition comprises 0.005% to 0.15% (w/v) of a detergent.

20. The composition of claim 1 , wherein the composition comprises 0.05% to 0.02% (w/v) of cholesterol or an analog thereof.

21. The composition of claim 20 , wherein the cholesterol analog is cholesterol hemisuccinate.

Assignees

• Univ Leland Stanford Junior

Inventors

• Ring Aaron Michael
• Manglik Aashish
• Kruse Andrew
• Kobilka Brian K

Classifications

• G01N33/566

  using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} · CPC title

• G01N2333/726

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

• C07K14/70571

  for neuromediators, e.g. serotonin receptor, dopamine receptor · CPC title

• G01N33/6842

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title

• G01N2570/00

  Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes · CPC title