Protein binding domains stabilizing functional conformational states of GPCRs and uses thereof

• US 9,453,065 B2
• Filed: 07/18/2011
• Issued: 09/27/2016
• Est. Priority Date: 07/16/2010
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.), VIB VZW, Vrije Universiteit Brussel
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.), VIB VZW, Vrije Universiteit Brussel
• Inventors: Steyaert, Jan, Pardon, Els, Rasmussen, Soren, Fung, Juan, Kobilka, Brian, Laeremans, Toon
• Primary Examiner(s): Li, Ruixiang
• Application Number:
  US13/810,652
• Publication Number:
  US 20130183287A1
• Time in Patent Office: 1,898 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 2317/22 from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2317/33 Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/569 Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/75 Agonist effect on antigen
• CPC Class Codes: G01N 23/20 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6857 Antibody fragments
• CPC Class Codes: G01N 33/6872 : Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

The disclosure relates to the field of GPCR structure biology and signaling. In particular, it relates to protein binding domains directed against or capable of specifically binding to a functional conformational state of a G-protein-coupled receptor (GPCR). More specifically, it provides protein binding domains that are capable of increasing the stability of a functional conformational state of a GPCR, in particular, increasing the stability of a GPCR in its active conformational state. The protein binding domains hereof can be used as a tool for the structural and functional characterization of G-protein-coupled receptors bound to various natural and synthetic ligands, as well as for screening and drug discovery efforts targeting GPCRs. Moreover, also encompassed are the diagnostic, prognostic and therapeutic usefulness of these protein binding domains for GPCR-related diseases.

Claims

1. A complex comprising:

a nanobody able to specifically bind to and stabilize an active conformational state of a beta 2 adrenergic receptor (β

₂AR), wherein the nanobody is able to enhance the affinity of the β

₂AR for an agonist, and

β

₂AR in an active conformational state.

2. The complex according to claim 1 further comprising:

a receptor ligand.

3. The complex of claim 1, wherein said receptor ligand is selected from the group consisting of a small molecule, a protein, a peptide, a protein scaffold, a nucleic acid, an ion, a carbohydrate, an antibody, and any suitable fragment thereof.

4. The complex of claim 1, wherein said complex is in a solubilized form or immobilized to a solid support.

5. The complex of claim 1, wherein said complex is crystalline.

7. The complex of claim 1, wherein the nanobody is able to specifically bind to an agonist-bound β

₂AR.

8. The complex of claim 1, wherein the nanobody is able to increase the thermostability of an active conformational state of a β

₂AR upon binding.

9. The complex of claim 1, wherein the β

₂AR is a mammalian protein, a plant protein, a microbial protein, a viral protein, or an insect protein.

10. The complex of claim 1, wherein the β

₂AR is a human protein.

11. The complex of claim 1, wherein the nanobody mimics the binding of a G protein to the active conformational state of the β

₂AR.

12. The complex of claim 1, wherein the nanobody shows G protein-like behavior.

13. The complex of claim 1, wherein the nanobody is bound to the G protein binding site of the β

₂AR.

14. The complex of claim 1, wherein the β

₂AR, is in a conformation where the cytoplasmic end of transmembrane segment 6 (TM6) is moved outward and away from the core of the β

₂AR as compared to the β

₂AR when not bound to the nanobody.

15. The complex of claim 1, wherein the complex is in a cellular composition.

16. The complex of claim 1, wherein the nanobody binds to the β

₂AR in a pocket in the β

₂AR formed by amino acids from transmembrane segments 3, 5, 6, and 7.

17. The complex of claim 16, wherein the third complementary determining region of the nanobody is bound to the pocket.

6. A crystalline form of a complex comprising:

(i) a nanobody able to specifically bind to and stabilize an active conformational state of a beta 2 adrenergic receptor (β

₂AR), wherein the nanobody is able to enhance the affinity of the β

₂AR for an agonist,

(ii) a β

₂AR in an active conformational state, and

(iii) a ligand.

18. A complex comprising:

a nanobody able to specifically bind to and stabilize an active conformational state of a beta 2 adrenergic receptor (β

₂AR), wherein the nanobody is able to enhance the affinity of the β

₂AR for an agonist, and

a β

₂AR in an active conformational state, and

wherein the nanobody is bound in the G protein binding site of the β

₂AR;

wherein the β

₂AR is in a conformation where the cytoplasmic end of transmembrane segment 6 (TM6) is moved outward and away from the core of the β

₂AR as compared to the β

₂AR when not bound to the nanobody; and

wherein the β

₂AR has enhanced affinity for an agonist as compared to the β

₂AR when not bound to the nanobody.