GREMLIN-1 CRYSTAL STRUCTURE AND INHIBITORY ANTIBODY

• US 20190330323A1
• Filed: 12/19/2017
• Published: 10/31/2019
• Est. Priority Date: 12/19/2016
• Status: Active Grant

• Current Assignee: UCB Biopharma SRL (UCB SA)
• Original Assignee: Ucb Biopharma Sprl (UCB SA)
• Inventors: DEDI, NEESHA, TWOMEY, BREDA, WRIGHT, MICHAEL JOHN, DAVIES, GARETH, MCMILLAN, DAVID JAMES
• Granted Patent: US 10,947,304 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/12 Antihypertensives
• CPC Class Codes: C07B 2200/13 Crystalline forms, e.g. polymorphs
• CPC Class Codes: C07K 14/51 Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

This invention relates to crystals of the human Gremlin-1 protein, and the human Gremlin-1 protein in complex with an inhibitory antibody. The invention also relates to the structure of human Gremlin-1 (on its own, or in complex with the antibody) and uses of these structures in screening for agents which modulate Gremlin-1 activity. The invention further provides antibodies which bind an allosteric inhibitory site on Gremlin-1, together with pharmaceutical compositions and medical uses of such antibodies and agents identified by the screening methods.

Claims

1-54. -54. (canceled)

55. An antibody which binds to an epitope on Gremlin-1 comprising at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, wherein the residue numbering is according to SEQ ID NO:

1.

56. The antibody according to claim 55, which binds an epitope comprising all of Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175.

57. The antibody according to claim 55, wherein Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175 are located on the same Gremlin-1 monomer and Ile131 is located on the second Gremlin-1 monomer.

58. The antibody according to claim 55, wherein an antibody binds a Gremlin-1 residue if the antibody paratope is within 4 Å

of the Gremlin-1 residue as determined by x-ray crystallography.

59. The antibody according to claim 55, which restores signal in the Hek-Id1 reporter gene assay.

60. An anti-Gremlin-1 antibody which comprises:

a) a heavy chain complementarity determining region (HCDR) sequences contained within a heavy chain variable region (HCVR) of SEQ ID NO;

10 or 12 and/or light chain complementarity determining region (LCDR) sequences contained within a light chain variable region (LCVR) of SEQ ID NO;

11 or 13;

b) at least one HCDR sequence selected from SEQ ID NOs;

3, 4, 5 and 6 and/or at least one LCDR sequence selected from SEQ ID NOs;

7, 8 and 9;

c) a HCDR3 sequence of SEQ ID NO;

6;

d) a HCDR1/HCDR2/HCDR3 sequence combination selected from SEQ ID NOs;

4/5/6 or from SEQ ID NOs;

3/5/6 and/or a LCDR1/LCDR2/LCDR3 sequence combination selected from SEQ ID NOs;

7/8/9;

e) a HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequence combination of SEQ ID NOs;

4/5/6/7/8/9 or SEQ ID NOs;

3/5/6/7/8/9;

f) a heavy chain variable region (HCVR) sequence of SEQ ID NO;

10 or 12 and/or a light chain variable region (LCVR) sequence of SEQ ID NO;

11 or 13, or sequences which are at least 95% identical thereto;

g) a HCVR and LCVR sequence pair of SEQ ID NOs;

10/11 or 12/13 or sequences which are at least 95% identical thereto;

h) HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consisting of SEQ ID NOs;

4/5/6/7/8/9 or SEQ ID NOs;

3/5/6/7/8/9 and the remainder of the HCVR and LCVR have at least 95% identity to SEQ ID NOs;

10, 11, 12 and/or 13, respectively;

i) a heavy chain of SEQ ID NO;

14, 16, 18, 22, 28, 30, 32 or 34 and/or a light chain of SEQ ID NO;

15, 17, 19, 23, 29, 31, 33 or 35, or sequences which are at least 95% identical thereto;

j) a heavy and light chain pair of SEQ ID NOs;

14/15, 16/17, 18/19, 22/23, 28/29 or 30/31, 32/33, 34/35, or sequences which are at least 95% identical thereto;

k) HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consisting of SEQ ID NOs;

4/5/6/7/8/9 or SEQ ID NOs;

3/5/6/7/8/9 and the remainder of the heavy and light chains having at least 95% identity to SEQ ID NOs;

14, 15, 16 and/or 17, respectively;

l) an antibody that competes for binding to Gremlin-1 with an antibody as defined in any one of a)-k);

or

m) an antibody which binds the same epitope on Gremlin-1 as an antibody defined in any one a)-k).

61. The antibody according to claim 60, wherein said antibody is a chimeric, human or humanised antibody.

62. The antibody according to claim 60, wherein said antibody is a Fab, modified Fab, Fab′

, modified Fab′

, F(ab′

)₂, Fv, single domain antibody or an scFv.

63. An isolated polynucleotide encoding an antibody as defined in claim 60.

64. An expression vector comprising the polynucleotide of claim 63.

65. A host cell comprising the vector as defined in claim 64.

66. A method of producing an antibody comprising culturing the host cell of claim 65 under conditions permitting production of the antibody, and recovering the antibody produced.

67. A pharmaceutical composition comprising an antibody as defined in claim 60 and a pharmaceutically acceptable adjuvant and/or carrier.

68. A method of treating renal fibrosis, diabetic nephropathy, idiopathic pulmonary fibrosis, pulmonary arterial hypertension, angiogenesis and/or cancer, comprising administering a therapeutically effective amount of an antibody according to claim 60 or a pharmaceutical composition thereof to a patient in need thereof.

69. A crystal of Gremlin-1 or a crystal of human Gremlin-1 in complex with an antibody.

70. The crystal of claim 69, wherein the Gremlin-1 is human Gremlin-1.

71. The crystal of claim 70, consisting of a C2 space group with unit cell dimensions of a=84.55 Å

, b=107.22 Å and

c=77.09 Å

for human Gremlin-1.

72. The crystal of claim 69, wherein the antibody in complex with human Gremlin 1 comprises a heavy chain of SEQ ID NO:

18 and a light chain of SEQ ID NO;

19.

73. A machine readable data storage medium which comprises data storage material encoded with machine readable data defined by the structure coordinates of Gremlin-1 in Table 1 or coordinates defining homologues of the structure.

74. A method of screening for modulatory agents of Gremlin-1 activity, comprising the steps of:

(a) identifying a ligand binding site from the structural coordinates in Table 1;

(b) identifying candidate agents which interact with at least part of the ligand binding site; and

(c) obtaining or synthesising said agent.

75. The method of claim 74, wherein steps (a) and (b) are performed in silico.

76. The method of claim 74, wherein the agent is an inhibitor of Gremlin-1 activity.

77. The method of claim 74, wherein the method comprises identifying candidate agents which interact with at least one of the following residues of Gremlin-1:

Trp93;

Phe 117;

Tyr119;

Phe125;

Tyr126 and/or

Phe138,

wherein the residue numbering is based on SEQ ID NO;

1.

78. The method of claim 77, wherein the agent interacts with all the following residues of Gremlin-1:

Trp93, Phe117, Tyr119, Phe125, Tyr126 and Phe138.

79. The method of claim 77, wherein a candidate agent interacts with Gremlin-1 if the agent is within 6 Å

of a residue as determined by x-ray crystallography.

80. The method of claim 74, wherein the ligand binding site is an allosteric site.

81. The method of claim 80, wherein the method comprises identifying candidate agents which interact with at least one of the following residues of Gremlin-1:

Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, wherein the residue numbering is based on SEQ ID NO;

1.

82. The method of claim 81, wherein the agent interacts with all the following residues of Gremlin-1:

Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175.

83. The method of claim 81, wherein Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and G1n175 are located on the same Gremlin-1 monomer and Ile131 is located on the second Gremlin-1 monomer.

84. The method of claim 80, wherein a candidate agent interacts with Gremlin-1 if the agent is within 4 Å

of a residue as determined by x-ray crystallography.

85. The method of claim 76, wherein the agent is a small molecule or antibody.

86. A Gremlin-1 modulating agent identified by the method of claim 74.