Binding domains directed against GPCR:G protein complexes and uses derived thereof

Patent metadata

Field	Value
Publication number	US-10626154-B2
Application number	US-201715583540-A
Country	US
Kind code	B2
Filing date	May 1, 2017
Priority date	Jun 21, 2011
Publication date	Apr 21, 2020
Grant date	Apr 21, 2020

Abstract

The present disclosure relates to the field of G protein coupled receptor (GPCR) structural biology and signaling. In particular, the present disclosure relates to binding domains directed against and/or specifically binding to GPCR:G protein complexes. Also provided are nucleic acid sequences encoding such binding domains and cells expressing or capable of expressing such binding domains. The binding domains of the present disclosure can be used as universal tools for the structural and functional characterization of G-protein coupled receptors in complex with downstream heterotrimeric G proteins and bound to various natural or synthetic ligands, for investigating the dynamic features of G protein activation, as well as for screening and drug discovery efforts that make use of GPCR:G protein complexes.

First claim

What is claimed is:

1. A method of generating a library of binding domains, the method comprising:
immunizing an animal with a GPCR/G-protein complex;
allowing the animal to produce antibodies to an intracellular portion of the GPCR/G-protein complex;
isolating lymphocytes from the animal;
generating a library of binding domains;
screening the library of binding domains for binding domains that specifically bind to an intracellular portion of the GPCR/G-protein complex; and
isolating the binding domains that bind to the intracellular portion of the GPCR/G-protein complex.

2. The method according to claim 1 , wherein the animal is a camelid.

3. The method according to claim 1 , wherein the binding domains are V H H domains.

4. The method according to claim 1 , wherein the GPCR/G-protein complex further comprises a ligand of the GPCR.

5. The method according to claim 1 , wherein the animal is immunized with phospholipid vesicles comprising the GPCR/G-protein complex.

6. The method according to claim 5 , wherein at least one GPCR/G-protein complex is oriented with the intracellular loops of the GPCR outside the vesicle.

7. The method according to claim 1 , wherein the GPCR/G-protein complex is cross-linked.

8. The method according to claim 1 , wherein the lymphocytes are peripheral blood lymphocytes.

9. The method according to claim 1 , wherein generating the library comprises cloning into a vector cDNA sequences encoding variable domains of antibodies, which cDNA sequences are obtained from the lymphocytes isolated from the animal.

10. The method according to claim 9 , wherein said vector is a phage display vector.

11. A library of binding domains produced by the method according to claim 1 .

12. The method according to claim 1 , further comprising:
screening the isolated binding domains for binding domains that prevent the GPCR/G-protein complex from dissociation by GTPγS.

13. The method according to claim 1 , further comprising:
screening the isolated binding domains for binding domains that specifically bind to the G protein of the GPCR/G-protein complex.

14. The method according to claim 13 , further comprising:
screening the isolated binding domains for binding domains that specifically bind a conformational epitope of the G protein at the interface between the alpha and the beta subunit of said G protein.

15. The method according to claim 1 , wherein the GPCR/G-protein complex used in the screening step is different from the GPCR/G-protein complex used in the immunization step.

Assignees

• Vib Vzw
• Univ Brussel Vrije
• Univ Leland Stanford Junior
• Univ Michigan Regents

Inventors

• Steyaert Jan
• Pardon Els
• Laeremans Toon
• Kobilka Brian
• Rasmussen Soren G F
• Granier Sebastien
• Sunahara Roger K

Classifications

• C07K2317/22

  from camelids, e.g. camel, llama or dromedary · CPC title

• C07K14/70571 Primary

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

• G01N2500/02

  Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor) · CPC title

• G01N33/74

  involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors} · CPC title

• C07K14/435

  from animals; from humans · CPC title