Methods of using an antibody to stabilize and analyze a BRIL fusion protein

Patent metadata

Field	Value
Publication number	US-11091545-B2
Application number	US-201816630181-A
Country	US
Kind code	B2
Filing date	Jul 12, 2018
Priority date	Jul 13, 2017
Publication date	Aug 17, 2021
Grant date	Aug 17, 2021

Abstract

An object of the present invention is to provide a method of enabling efficient structural analysis of a target protein that has been impossible or difficult to analyze so far, by stabilizing the target protein. The present invention provides an anti-BRIL antibody which specifically binds to BRIL or a BRIL fusion protein and an antigen-binding fragment thereof, a nucleic acid encoding the anti-BRIL antibody and the antigen-binding fragment thereof, a vector containing the nucleic acid, an antibody producing cell containing the vector, a method of producing the antibody, and a method of using the antibody in a protein structural analysis.

First claim

The invention claimed is:

1. A method of stabilizing a BRIL fusion protein in which BRIL is fused to a target protein, comprising:
reacting an antibody which binds to BRIL or an antigen-binding fragment thereof with the BRIL fusion protein, and
stabilizing the BRIL fusion protein by binding the antibody which binds to BRIL or the antigen-binding fragment thereof to BRIL of the BRIL fusion protein,
wherein BRIL is a heat resistant apocytochrome b562 modified protein,
wherein the BRIL fusion protein is a protein in which two adjacent helix structures of the target protein are fused by BRIL,
wherein the target protein is a membrane-expressed protein selected from the group consisting of a G protein-coupled receptor (GPCR), an ion channel, and a transporter, and
wherein the antibody which binds to BRIL or the antigen-binding fragment thereof is any one selected from the following (1) to (3):
(1) an antibody which binds to at least any one of a third helix structure and a fourth helix structure of BRIL or an antigen-binding fragment thereof,
(2) an antibody which binds to at least a portion of the third helix structure to the fourth helix structure of BRIL or an antigen-binding fragment thereof, and
(3) an antibody which binds to at least one amino acid residue selected from amino acid residues of 67 th Ile (I), 71 st Gln (Q), 74 th Asp (D), 77 th Lys (K), 78 th Leu (L), 83 rd Lys (K), 85 th Lys (K), 86 th Glu (E), 88 th Gln (Q), 89 th Ala (A), 90 th Ala (A), 92 nd Glu (E), 93 rd Gln (Q), 96 th Thr (T), 97 th Thr (T), 99 th Asn (N), and 100 th Ala (A) from an N-terminus of BRIL or an antigen-binding fragment thereof.

2. The method according to claim 1 ,
wherein the antibody which binds to BRIL or the antigen-binding fragment thereof is any one selected from the following (i) to (iii):
(i) an antibody which binds by competing with an antibody in which complementarity determining regions (CDRs) 1 to 3 of a heavy chain variable region (VH) of the antibody comprise amino acid sequences represented by SEQ ID NOs: 1 to 3, respectively, and in which CDRs 1 to 3 of a light chain variable region (VL) of the antibody comprise amino acid sequences represented by SEQ ID NOs: 4 to 6, respectively, or an antigen-binding fragment thereof,
(ii) an antibody which binds to an epitope to which an antibody in which CDRs 1 to 3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs: 1 to 3, respectively, and in which CDRs 1 to 3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs: 4 to 6, respectively, binds or an antigen-binding fragment thereof, and
(iii) an antibody which binds to the same epitope as an epitope to which an antibody in which CDRs 1 to 3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs: 1 to 3, respectively, and in which CDRs 1 to 3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs: 4 to 6, respectively, binds or an antigen-binding fragment thereof.

3. The method according to claim 1 ,
wherein the antibody which binds to BRIL or the antigen-binding fragment thereof comprises VH CDRs 1 to 3 represented by SEQ ID NOs: 1 to 3, respectively, and VL CDRs 1 to 3 represented by SEQ ID NOs: 4 to 6, respectively.

4. A method of analyzing a three-dimensional structure of a BRIL fusion protein in which BRIL is fused to a target protein, comprising:
reacting an antibody which binds to BRIL or an antigen-binding fragment thereof with the BRIL fusion protein, and
performing structural analysis on the three-dimensional structure of the BRIL fusion protein,
wherein BRIL is a heat resistant apocytochrome b562 modified protein,
wherein the BRIL fusion protein is a protein in which two adjacent helix structures of the target protein are fused by BRIL,
wherein the target protein is a membrane-expressed protein selected from the group consisting of a G protein-coupled receptor (GPCR), an ion channel, and a transporter, and
wherein the antibody which binds to BRIL or the antigen-binding fragment thereof is any one selected from the following (1) to (3):
(1) an antibody which binds to at least any one of a third helix structure and a fourth helix structure of BRIL or an antigen-binding fragment thereof,
(2) an antibody which binds to at least a portion of the third helix structure to the fourth helix structure of BRIL or an antigen-binding fragment thereof, and
(3) an antibody which binds to at least one amino acid residue selected from amino acid residues of 67 th Ile (I), 71 st Gln (Q), 74 th Asp (D), 77 th Lys (K), 78 th Leu (L), 83 rd Lys (K), 85 th Lys (K), 86 th Glu (E), 88 th Gln (Q), 89 th Ala (A), 90 th Ala (A), 92 nd Glu (E), 93 rd Gln (Q), 96 th Thr (T), 97 th Thr (T), 99 th Asn (N), and 100 th Ala (A) from an N-terminus of BRIL or an antigen-binding fragment thereof.

5. The method according to claim 4 , wherein the structural analysis is performed using an electron microscope.

6. The method according to claim 4 , wherein the structural analysis is X-ray crystallography.

7. The method according to claim 4 , wherein the structural analysis is performed using a cryo electron microscope.

Assignees

• Kyowa Kirin Co Ltd

Inventors

• Miyagi Hikaru
• Suzuki Michihiko
• Saito Junichi
• Asada Hidetsugu
• Iwata So

Classifications

• C07K2317/32

  specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• C07K14/723

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

• C07K2319/32

  fusions with soluble part of a cell surface receptor, "decoy receptors" · CPC title