# US 7,612,169 B2 · Osteoprotegerin variant proteins

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- Number: US 7,612,169 B2
- Document ID: US7612169B2
- Status: Granted
- Kind: B2
- Application number: 11/303,169
- Filed: 2005-12-13
- Published: 2006-08-24
- Granted: 2009-11-03
- Earliest claimed: 2004-12-13
- Assignees: EvoGenix, Ltd.
- Inventors: Anthony Roberts; George Kopsidas; Gregory Coia; Merilyn Sleigh; Vincent Batori
- Web page: https://patentgrep.com/patent/US7612169B2

## CPC

- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 19/00: Drugs for skeletal disorders
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 19/10: for osteoporosis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention relates to novel osteoprotegerin variant proteins (OVPs) that demonstrate reduced binding affinity for their ligand TRAIL when compared to wild-type osteoprotegerin. Nucleic acids which encode these OVPs are also provided. Recombinant vectors and host cells expressing these OVPs are also encompassed as are methods of producing recombinant OVPs. The present invention also relates to compositions comprising these OVPs, and to methods of treating bone diseases characterised by increased bone turnover and/or loss. The OVPs of the invention are useful for preventing bone resorption and may be used to treat any condition resulting in abnormal bone turnover or bone loss such as osteoporosis, hypercalcemia, Paget's disease of bone, multiple myeloma, bone cancer and bone loss due to rheumatoid arthritis or osteomyelitis, and the like.

## Claims

### Claim 1

An osteoprotegerin (OPG) variant protein and conservative substitutions thereof, the variant protein consisting of one to three modifications, one of which is a modification at residue 115 when compared to wild-type OPG, wherein the OPG variant protein or conservative substitution thereof retains binding affinity for receptor activator of NFκB ligand (RANKL) but exhibits reduced binding affinity for TNF-related apoptosis inducing ligand (TRAIL) when compared to wild-type OPG.

### Claim 2 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein Ile at position 115 is substituted with Thr, Met, Val, Asp, Gly, Ser or Arg.

### Claim 3 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein one of the modifications occurs at residue 122.

### Claim 4 (depends on 3)

An OPG variant protein as claimed in claim 3 wherein Arg at position 122 is substituted with Gly, Gln, Ser, Asn or Glu.

### Claim 5 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein one of the modifications occurs at residue 128.

### Claim 6 (depends on 5)

An OPG variant protein as claimed in claim 5 wherein Phe at position 128 is substituted with Val, Ala, Leu, Ile or Ser.

### Claim 7 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein one of the modifications occurs at residue 130.

### Claim 8 (depends on 7)

An OPG variant protein as claimed in claim 7 wherein Val at position 130 is substituted with Glu or Ala.

### Claim 9 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein at least one of the modifications occurs in the region encompassed by residues 122-130.

### Claim 10 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein two of the one to three modifications are selected from the group consisting of:
(i) R122N and I115M;
(ii) F128S and I115M;
(iii) F128I and I115M; and
(iv) F128L and I115M.

### Claim 11 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein the modifications occur at any one or more of residues 31, 40, 51, 100, 155, 167 or 168.

### Claim 12 (depends on 1)

An OPG variant protein as claimed in claim 1, wherein the modifications occur at any one or more of residues Gln21, Glu22, Thr23, Phe24, Pro25, Pro26, Lys27, Tyr28, Leu29, His30, Tyr31, Asp32, Glu33, Glu34, Thr35, Ser36, His37, Gln38, Leu40, Asp42, Lys43, Pro45, Pro46, Thr48, Lys51, Gln52, His53, Cys54, Thr55, Ala56, Lys57, Trp58, Lys59, Thr60, Val61, Ala63, Pro64, Pro66, Asp67, His68, Tyr69, Asp72, Ser73, Trp74, Thr76, Ser77, Asp78, Glu79, Leu81, Tyr82, Ser84, Pro85, Val86, Lys88, Glu89, Leu90, Tyr92, Val93, Lys94, Gln95, Glu96, Asn98, Arg99, Thr100, His101, Val131, Gln132, Ala133, Gly134, Thr135, Pro136, Glu137, Arg138, Val141, Lys143, Arg144, Cys145, Pro146, Asp147, Gly148, Phe149, Phe150, Ser151, Asn152, Glu153, Thr154, Ser155, Ser156, Lys157, Ala158, Pro159, Cys160, Arg161, Lys162, His163, Thr164, Asn165, Cys166, Ser167, Val168, Phe169, Gly170, Leu171, Leu172, Leu173, Thr174, Gln175, Lys176, Gly177, Asn178, Ala179, Thr180, His181, Asp182, Asn183, Ile184, Cys185, Ser186, Gly187, Asn188, Ser189, Glu190, Ser191, Thr192, Gln193, Lys194, Cys195, Gly196, Ile197, Asp198, Val199, Thr200 or Leu201.

### Claim 13 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein the one to three modifications are selected from I115V; I115L; I115M; I115W; I155A; I115G; I115N; I115S; I115R; I115K; I115Y; I115D; I115E; N102D; I115M and L40S; I115M and N139D; I115T and K51R; I115M and R122G; I115M and R122N; I115M and R122Q; I115M and R122S; I115M and R122D; I115M and R122E; I115M and F128L; I115M and F128A; I115M and F128 S; I115M and F128T; I115M and F128I; I115T and K51R and R111H; or I115T and K51R and S167G.

### Claim 14 (depends on 1)

An OPG variant protein as claimed in claim 1 which exhibits a binding affinity for RANKL that is at least 80% that of wild type OPG.

### Claim 15 (depends on 1)

An OPG variant protein as claimed in claim 1 which has an EC50 (nM) for RANKL of less than 1 nM under ELISA assay conditions wherein the OPG variant protein and wild type OPG are each tested as a fusion with a human Fc domain.

### Claim 16 (depends on 1)

An OPG variant protein as claimed in claim 1 which exhibits a binding affinity for TRAIL that is less than 20% that of wild type OPG.

### Claim 17 (depends on 1)

An OPG variant protein as claimed in claim 1 which has an EC50 (nM) for TRAIL of greater than 10 nM under ELISA assay conditions wherein the OPG variant protein and wild type OPG are each tested as a fusion with a human Fc domain.

### Claim 18 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein the OPG variant protein is conjugated to a polypeptide.

### Claim 19 (depends on 18)

An OPG variant protein as claimed in claim 18 wherein the polypeptide is an immunoglobulin constant domain.

### Claim 20 (depends on 1)

A dimer or multimer comprising at least two OPG variant proteins as claimed in claim 1.

### Claim 21 (depends on 1)

An isolated polynucleotide encoding an OPG variant protein as claimed in claim 1.

### Claim 22 (depends on 1)

A composition for inhibiting or reducing binding of RANK to RANKL, the composition comprising an OPG variant protein as claimed in claim 1, and one or more acceptable carriers.

### Claim 23 (depends on 1)

An OPG variant protein as claimed in claim 1 which exhibits a binding affinity for RANKL that is at least 50% that of wild type OPG.

### Claim 24 (depends on 1)

An OPG variant protein as claimed in claim 1 which exhibits a binding 8affinity for RANKL that is at least 60% that of wild type OPG.

### Claim 25 (depends on 1)

An OPG variant protein as claimed in claim 1 which exhibits a binding affinity for RANKL that is at least 70% that of wild type OPG.

### Claim 26 (depends on 1)

An OPG variant protein as claimed in claim 1 which exhibits a binding affinity for TRAIL that is less than 50% that of wild type OPG.

### Claim 27 (depends on 1)

An OPG variant protein as claimed in claim 1 which exhibits a binding affinity for TRAIL that is less than 40% that of wild type OPG.

### Claim 28 (depends on 1)

An OPG variant protein as claimed in claim 1 which exhibits a binding affinity for TRAIL that is less than 30% that of wild type OPG.

### Claim 29 (depends on 1)

An OPG variant protein as claimed in claim 1 wherein the protein is a modified fragment of OPG comprising amino acids 22-194.

### Claim 30

An osteoprotegerin (OPG) variant protein and conservative substitutions thereof, the variant protein consisting of a modification at residue 115 and one or more additional modifications when compared to wild-type OPG, and wherein the one or more additional modifications occur at one or more residues selected from the group consisting of: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 43, 45, 46, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 100, 101, 102, 111, 122, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 141, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 and 201; and wherein the OPG variant protein or conservative substitution thereof retains binding affinity for receptor activator of NFκB ligand (RANKL) but exhibits reduced binding affinity for TNF-related apoptosis inducing ligand (TRAIL) when compared to wild-type OPG.

### Claim 31 (depends on 30)

The OPG variant protein of claim 30, wherein Ile at position 115 is substituted with Thr, Met, Val, Asp, Gly, Ser or Arg.

### Claim 32 (depends on 30)

The OPG variant protein of claim 30, wherein one of the additional modifications occurs at residue 122.

### Claim 33 (depends on 32)

The OPG variant protein of claim 32, wherein Arg at position 122 is substituted with Gly, Gln, Ser, Asn or Glu.

### Claim 34 (depends on 32)

The OPG variant protein of claim 32, wherein one of the additional modifications occurs at residue 128.

### Claim 35 (depends on 34)

The OPG variant protein of claim 34, wherein Phe at position 128 is substituted with Val, Ala, Leu, Ile or Ser.

### Claim 36 (depends on 30)

The OPG variant protein of claim 30, wherein one of the additional modifications occurs at residue 130.

### Claim 37 (depends on 36)

The OPG variant protein of claim 36 wherein Val at position 130 is substituted with Glu or Ala.

### Claim 38 (depends on 30)

The OPG variant protein as claimed of claim 30, wherein at least one of the additional modifications occurs in the region encompassed by residues 122-130.

### Claim 39 (depends on 30)

The OPG variant protein of claim 30, wherein Ile at position 115 is substituted with Met and the one or more additional modifications are selected from the group consisting of:
(i) R122N;
(ii) F128S;
(iii) F128I; and
(iv) F128L.

### Claim 40 (depends on 30)

The OPG variant protein of claim 30, which exhibits a binding affinity for RANKL that is at least 80% that of wild type OPG.

### Claim 41 (depends on 30)

The OPG variant protein of claim 30, which has an EC50 (nM) for RANKL of less than 1 nM under ELISA assay conditions wherein the OPG variant protein and wild type OPG are each tested as a fusion with a human Fc domain.

### Claim 42 (depends on 30)

The OPG variant protein of claim 30, which exhibits a binding affinity for TRAIL that is less than 20% that of wild type OPG.

### Claim 43 (depends on 30)

The OPG variant protein of claim 30, which has an EC50(nM) for TRAIL of greater than 10 nM under ELISA assay conditions wherein the OPG variant protein and wild type OPG are each tested as a fusion with a human Fc domain.

### Claim 44 (depends on 30)

An OPG variant protein as claimed in claim 30, wherein the OPG variant protein is conjugated to a polypeptide.

### Claim 45 (depends on 44)

An OPG variant protein as claimed in claim 44, wherein the polypeptide is an immunoglobulin constant domain.

### Claim 46 (depends on 30)

A dimer or multimer comprising at least two OPG variant proteins as claimed in claim 30.

### Claim 47 (depends on 30)

An isolated polynucleotide encoding an OPG variant protein as claimed in claim 30.

### Claim 48 (depends on 30)

A composition for inhibiting or reducing binding of RANK to RANKL, the composition comprising an OPG variant protein as claimed in claim 30, and done or more acceptable carriers.

### Claim 49 (depends on 30)

An OPG variant protein as claimed in claim 30 which exhibits a binding affinity for RANKL that is at least 50% that of wild type OPG.

### Claim 50 (depends on 30)

An OPG variant protein as claimed in claim 30 which exhibits a binding affinity for RANKI that is at least 60% that of wild type OPG.

### Claim 51 (depends on 30)

An OPG variant protein as claimed in claim 30 which exhibits a binding affinity for RANKL that is at least 70% that of wild type OPG.

### Claim 52 (depends on 30)

An OPG variant protein as claimed in claim 30 which exhibits a binding affinity for TRAIL that is less than 50% that of wild type OPG.

### Claim 53 (depends on 30)

An OPG variant protein as claimed in claim 30 which exhibits a binding affinity for TRAIL that is less than 40% that of wild type OPG.

### Claim 54 (depends on 30)

An OPG variant protein as claimed in claim 30 which exhibits a binding affinity for TRAIL that is less than 30% that of wild type OPG.

### Claim 55 (depends on 30)

An OPG variant protein as claimed in claim 30 wherein the protein is a modified fragment of OPG comprising amino acids 22-194.

### Claim 56

An osteoprotegerin (OPG) variant protein and conservative substitutions thereof, the variant protein consisting of a modification at residue 115 and one or more additional modifications when compared to wild-type OPG, wherein the one or more additional modifications occur at residues selected from the group consisting of: 107-154; and wherein the OPG variant protein or conservative substitution thereof retains binding affinity for receptor activator of NFκB ligand (RANKL) but exhibits reduced binding affinity for TNF-related apoptosis inducing ligand (TRAIL) when compared to wild-type OPG.

### Claim 57 (depends on 56)

The OPG variant protein of claim 56, wherein lie at position 115 is substituted with Thr, Met, Val, Asp, Gly, Ser or Arg.

### Claim 58 (depends on 56)

The OPG variant protein of claim 56 wherein one of the additional modifications occurs at residue 122.

### Claim 59 (depends on 58)

The OPG variant protein of claim 58, wherein Arg at position 122 is substituted with Gly, Gln, Ser, Asn or Glu.

### Claim 60 (depends on 56)

The OPG variant protein of claim 56, wherein one of the additional modifications occurs at residue 128.

### Claim 61 (depends on 60)

The OPG variant protein of claim 60, wherein Phe at position 128 is substituted with Val, Ala, Leu, Ile or Ser.

### Claim 62 (depends on 56)

The OPG variant protein of claim 56, wherein one of the additional modifications occurs at residue 130.

### Claim 63 (depends on 62)

The OPG variant protein of claim 62, wherein Val at position 130 is substituted with Glu or Ala.

### Claim 64 (depends on 56)

The OPG variant protein as claimed of claim 56, wherein at least one of the additional modifications occurs in the region encompassed by residues 122-130.

### Claim 65 (depends on 56)

The OPG variant protein of claim 56, wherein lie at position 115 is substituted with Met and the one or more additional modifications are selected from the group consisting of:
(i) R122N;
(ii) F128S;
(iii) F128I; and
(iv) F128L.

### Claim 66 (depends on 56)

The OPG variant protein of claim 56 which exhibits a binding affinity for RANKL that is at least 80% that of wild type OPG.

### Claim 67 (depends on 56)

The OPG variant protein of claim 56 which has an EC50 (nM) for RANKL of less than 1 nM under ELISA assay conditions wherein the OPG variant protein and wild type OPG are each tested as a fusion with a human Fc domain.

### Claim 68 (depends on 56)

The OPG variant protein of claim 56, which exhibits a binding affinity for TRAIL that is less than 20% that of wild type OPG.

### Claim 69 (depends on 56)

The OPG variant protein of claim 56 which has an EC50 (nM) for TRAIL of greater than 10 nM under ELISA assay conditions wherein the OPG variant protein and wild type OPG are each tested as a fusion with a human Fc domain.

### Claim 70 (depends on 56)

An OPG variant protein as claimed in claim 56 wherein the OPG variant protein is conjugated to a polypeptide.

### Claim 71 (depends on 70)

An OPG variant protein as claimed in claim 70, wherein the polypeptide is an immunoglobulin constant domain.

### Claim 72 (depends on 56)

A dimer or multimer comprising at least two OPG variant proteins as claimed in claim 56.

### Claim 73 (depends on 56)

An isolated polynucleotide encoding an OPG variant protein as claimed in claim 56.

### Claim 74 (depends on 56)

A composition for inhibiting or reducing binding of RANK to RANKL, the composition comprising an OPG variant protein as claimed in claim 56, and one or more acceptable carriers.

### Claim 75 (depends on 56)

An OPG variant protein as claimed in claim 56 which exhibits a binding affinity for RANKL that is at least 50% that of wild type OPG.

### Claim 76 (depends on 56)

An OPG variant protein as claimed in claim 56 which exhibits a binding affinity for RANKL that is at least 60% that of wild type OPG.

### Claim 77 (depends on 56)

An OPG variant protein as claimed in claim 56 which exhibits a binding affinity for RANKL that is at least 70% that of wild type OPG.

### Claim 78 (depends on 56)

An OPG variant protein as claimed in claim 56 which exhibits a binding affinity for TRAIL that is less than 50% that of wild type OPG.

### Claim 79 (depends on 56)

An OPG variant protein as claimed in claim 56 which exhibits a binding affinity for TRAIL that is less than 40% that of wild type OPG.

### Claim 80 (depends on 56)

An OPG variant protein as claimed in claim 56 which exhibits a binding affinity for TRAIL that is less than 30% that of wild type OPG.

### Claim 81 (depends on 56)

An OPG variant protein as claimed in claim 56 wherein the protein is a modified fragment of OPG comprising amino acids 22-194.