US2003124169A1PendingUtilityA1
Osteogenic devices
Priority: Apr 8, 1988Filed: Sep 19, 2001Published: Jul 3, 2003
Est. expiryApr 8, 2008(expired)· nominal 20-yr term from priority
A61P 43/00A61K 38/00A61F 2310/00365A61P 1/02C07K 14/51A61L 27/3604A61L 2430/02A61L 27/227A61L 27/365A61L 27/56A61C 8/0006A61L 27/3608A61L 27/24A61L 27/34A61K 9/0024A61L 27/3654
52
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Claims
Abstract
Disclosed are 1) osteogenic devices comprising a matrix containing osteogenic protein and methods of inducing endochondral bone growth in mammals using the devices; 2) amino acid sequence data, amino acid composition, solubility properties, structural features, homologies and various other data characterizing osteogenic proteins, 3) methods of producing osteogenic proteins using recombinant DNA technology, and 4) osteogenically and chondrogenically active synthetic protein constructs.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . An osteogenic device for implantation in a mammal, said device comprising:
a biocompatible, in vivo biodegradable matrix defining pores of a dimension sufficient to permit influx, proliferation and differentiation of migratory progenitor cells from the body of said mammal; and a protein, produced by expression of recombinant DNA in a host cell, comprising one or more polypeptide chains, each of which has an amino acid sequence sufficiently duplicative of the sequence of COP-5 or COP-7 such that said protein is capable of inducing endochondral bone formation in association with said matrix when implanted in a mammal.
2 . A device for implantation in a mammal, said device comprising:
a biocompatible, in vivo biodegradable matrix defining pores of a dimension sufficient to permit influx, proliferation and differentiation of migratory progenitor cells from the body of said mammal; and a protein, produced by expression of recombinant DNA in a host cell, comprising one or more polypeptide chains, each of which has less than about 200 amino acids, in a sequence sufficiently duplicative of the sequence of COP-5 or COP-7 such that said protein is capable of inducing cartilage formation in association with said matrix when implanted in a mammal.
3 . The device of claim 1 or 2 wherein the sequence comprises:
10 20 30 40 50
CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX
60 70 80 90 100
QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCX
CX
wherein each X independently represents an amino acid.
4 . The device of claim 1 or 2 wherein the sequence comprises:
10 20 30 40 50
LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX
60 70 80 90 100
QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCX
CX
wherein each X independently represents an amino acid.
5 . The device of claim 1 or 2 wherein the sequence comprises:
10 20 30 40 50
CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV
RRRS K S S L QE VIS E FD Y E A AY MPESMKAS VI
KE F E K I DN L N S Q ITK F P TL
Q A S K
60 70 80 90 100
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCG
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H
RF T S K DPV V Y N S H RN RS
N S K P E
CR
H
wherein, in each position where more than one amino acid is shown, any one of the amino acids shown may be in that position.
6 . The device of claim 1 or 2 wherein the sequence comprises:
10 20 30 40 50
LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV
K S S L QE VIS E FD Y E A AY MPESMKAS VI
F E K I DN L N S Q ITK F P TL
A S K
60 70 80 90 100
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCG
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H
RF T S K DPV V Y N S H RN RS
N S K P E
CR
H
wherein, in each position where more than one amino acid is shown, any one of the amino acids shown may be in that position.
7 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
Vg1
CKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE
50 60 70
ILNGSN--H-AILQTLVHSIEPED-IPLPCCVPTKMSP
80 90 100
ISMLFYDNNDNVVLRHYENMAVDECGCR
8 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
DPP
CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD
50 60 70
HFNSTN--H-AVVQTLVNNNNPGK-VPKACCVPTQLDS
80 90 100
VAMLYLNDQSTVVLKNYQEMTVVGCGCR
9 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
OP1
LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS
50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA
80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
10 . The device of claim 1 or 2 wherein the sequence comprises:
−5
HQRQA
1 10 20 30 40
OP1
CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS
50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA
80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
11 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
CBMP-2a
CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD
50 60 70
HLNSTN--H-AIVQTLVNSVNS-K-IPKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQDMVVEGCGCR
12 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
CBMP-2b
CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD
50 60 70
HLNSTN--H-AIVQTLVNSVNS-S-IPKACCVPTELSA
80 90 100
ISMLYLDEYDKVVLKNYQEMVVEGCGCR
13 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
CBMP-3
CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK
50 60 70
SLKPSN--H-ATIQSIVRAVGVVPGIPEPCCVPEKMSS
80 90 100
LSILFFDENKNVVLKVYPNMTVESCACR
14 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
COP1
LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFPSAD
50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA
80 90 100
ISMLYLDENSTVVLKNYQEMTVVGCGCR
15 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
COP3
LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD
50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
16 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
COP4
LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD
50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
17 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
COP5
LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD
50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
18 . The device of claim 1 or 2 wherein the sequence comprises:
1 10 20 30 40
COP7
LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD
50 60 70
HLNSTN--H-AVVQTLVNSVNSKI-PKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
19 . The device of claim 1 or 2 wherein the sequence comprises:
10
PKHHSQRARKKNKN
1 10 20 30 40
COP16
CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGECPFPLAD
50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
20 . The device of claim 1 or 2 wherein the osteogenics protein comprises a pair of separate polypeptide chains.
21 . Osteogenic protein, produced by expression of recombinant DNA in a host cell, capable of inducing endochondral bone formation in association with a matrix when implanted in a mammal.
22 . A protein, produced by expression of recombinant DNA in a host cell, comprising one or more polypeptide chains less than about 200 amino acids long in a sequence sufficiently duplicative of the sequence of COP-5 or COP-7 such that said protein is capable of inducing cartilage formation in association with a matrix when implanted in a mammal.
23 . The osteogenic protein of claim 21 having an apparent molecular weight of about 30 kD when oxidized as determined by comparison to molecular weight standards in SDS-polyacrylamide gel.
24 . The osteogenic protein of claim 23 further characterized by being glycosylated.
25 . The osteogenic protein of claim 21 having an apparent molecular weight of about 27 kD as determined by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.
26 . The protein of claim 22 or 25 further characterized by being unglycosylated.
27 . The protein of claim 21 or 22 comprising a pair of separate polypeptide chains.
28 . The protein of claim 21 or 22 comprising the amino acid sequences:
10 20 30 40
CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAX
50 60 70 80 90
XQXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVX
100
XCXCX
wherein each X independently represents an amino acid.
29 . The protein of claim 21 or 22 comprising the amino acid sequences:
10 20 30 40 50
LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX
60 70 80 90 100
QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCX
CX
wherein each X independently represents an amino acid.
30 . The protein of claim 21 or 22 comprising the amino acid sequences:
10 20 30 40 50
CKPHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV
RRRS K S S L QE VIS E FD Y E A AY MPESMKAS VI
KE F E K I DN L N S Q ITK F P TL
Q A S K
60 70 80 90 100
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCG
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H
RF T S K DPV V Y N S H RN RS
N S K P E
CR
H
wherein, in each position where more than one amino acid is shown, any one of the amino acids shown may be in that position.
31 . The protein of claim 21 or 22 comprising the amino acid sequences:
10 20 30 40 50
LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV
K S S L QE VIS E FD Y E A AY MPESMKAS VI
F E K I DN L N S Q ITK F P TL
A S K
60 70 80 90 100
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCG
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H
RF T S K DPV V Y N S H RN RS
N S K P E
CR
H
wherein, in each position where more than one amino acid is shown, any one of the amino acids shown may be in that position.
32 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
Vg1
CKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE
50 60 70
ILNGSN--H-AILQTLVHSIEPED-IPLPCCVPTKMSP
80 90 100
ISMLFYDNNDNVVLRHYENMAVDECGCR
33 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
DPP
CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD
50 60 70
HFNSTN--H-AVVQTLVNNNNPGK-VPKACCVPTQLDS
80 90 100
VAMLYLNDQSTVVLKNYQEMTVVGCGCR
34 . The protein of claim 21 or 22 comprising the amino acid sequence:
1 10 20 30 40
OP1
LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS
50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA
80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
35 . The protein of claim 21 or 22 comprising the amino acid sequences:
−5
HQRQA
1 10 20 30 40
OP1
CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS
50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA
80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
36 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
CMP-2a
CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD
50 60 70
HLNSTN--H-AIVQTLVNSVNS-K-IPKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQDMVVEGCGCR
37 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
CBMP-2b
CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD
50 60 70
HLNSTN--H-AIVQTLVNSVNS-S-IPKACCVPTELSA
80 90 100
ISMLYLDEYDKVVLKNYQEMVVEGCGCR
38 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
CBMP-3
CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK
50 60 70
SLKPSN--H-ATIQSIVRAVGVVPGIPEPCCVPEKMSS
80 90 100
LSILFFDENKNVVLKVYPNMTVESCACR
39 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
COP1
LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFPSAD
50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA
80 90 100
ISMLYLDENSTVVLKNYQEMTVVGCGCR
40 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
COP3
LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD
50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
41 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
COP4
LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD
50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
42 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
COP5
LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD
50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
43 . The protein of claim 21 or 22 comprising the amino acid sequences:
1 10 20 30 40
COP7
LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD
50 60 70
HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
44 . The protein of claim 21 or 22 comprising the amino acid sequences:
−10
PKHHSSRARKKNKN
1 10 20 30 40
COP16
CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGECPFPLAD
50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
45 . The protein of claim 21 or 22 comprising the product of expression of a DNA in a procaryotic cell.
46 . A DNA sequence encoding an amino acid sequence sufficiently duplicative of that of the sequence encoded by the gene of Figure 1 A_ such that said encoded sequence induces bone or cartilage formation when implanted in a mammal in association with a matrix.
47 . The DNA of claim 46 encoding the same amino acid sequence as the gene set forth in FIG. 1A.
48 . The DNA sequence of claim 46 encoding:
1 10 20 30 40
OP1
LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS
50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA
80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
49 . The DNA sequence of claim 46 encoding:
−5
HQRQA
1 10 20 30 40
OP1
CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS
50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA
80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
50 . A cell line engineered to express the protein of claim 21 or 22 .
51 . The protein of claim 21 having a half maximum bone forming activity of about 20-25 ng per 25 mg of implant.
52 . A biocompatible, in vivo biodegradable deglycosylated collagenous matrix defining pores of dimensions sufficient to permit influx, proliferation, and differentiation of migratory progenitor cells from the body of a mammal.
53 . The matrix of claim 52 comprising close-packed particulate matter having a particle size within the range of 70-850 mm.
54 . The matrix of claim 53 wherein said particulate matter has a particle size within the range of 70-420 mm.
55 . The matrix of claim 52 defining a shape to span a non-union fracture in said mammal.
56 . The matrix of claim 52 comprising demineralized, protein-extracted, deglycosylated, particulate xenogenic bone.
57 . The matrix of claim 52 comprising a material selected from the group consisting of hydroxyapatite, tricalcium phosphate, polymers comprising lactic acid monomer units, polymers comprising glycolic acid monomer units, demineralized, guanidine-extracted, deglycosylated xenogenic bone, and mixtures thereof.
58 . An osteogenic device for implantation in a mammal, said device comprising:
a biocompatible, in vivo biodegradable matrix defining pores of a dimension sufficient to permit influx, proliferation and differentiation of migratory progenitor cells from the body of said mammal; and substantially pure osteogenic protein capable of inducing endochondral bone formation in said mammal disposed in said matrix and accessible to said cells.
59 . The device of claim 1 , 2 , or 58 wherein said matrix comprises close-packed particulate matter having a particle size within the range of 70-850 mm.
60 . The device of claim 1 , 2 , or 58 wherein said particulate matter has a particle size within the range of 70-420 mm.
61 . The device of claim 1 , 2 , or 58 wherein said matrix comprises demineralized, protein-extracted, particulate, allogenic bone.
62 . The device of claim 1 , 2 , or 58 wherein said matrix comprises a material selected from the group consisting of collagen, hydroxyapatite, tricalcium phosphate, polymers comprising lactic acid monomer units, polymers comprising glycolic acid monomer units, demineralized, guanidine-eitracted allogenic bone, and mixtures thereof.
63 . The device of claim 1 , 2 , or 58 wherein said matrix is shaped to span a non-union fracture in said mammal.
64 . The device of claim 1 , 2 , or 58 disposed within the marrow cavity of allogenic bone.
65 . The device of claim 1 , 2 , or 58 wherein said matrix comprises demineralized, protein extracted, particulate, deglycosylated zenogeneic bone.
66 . The device of claim 65 wherein said matrix is treated with a protease.
67 . The device of claim 58 wherein said osteogenic,protein is unglycosylated.
68 . The device of claim 67 wherein said osteogenic protein has at apparent molecular weight of about 27 kD when oxidized as determined by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.
69 . The device of claim 58 wherein said osteogenic protein is glycosylated.
70 . The device of claim 69 wherein said osteogenic protein has an apparent molecular weight of about 30 kD when oxidized as determined by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.
71 . The device of claim 58 wherein said osteogenic protein comprises a pair of ppolypeptide chains.
72 . The device of claim 71 wherein one chain of said pair of polypeptide chains has an apparent molecular weight of about 14 kD and the other has an apparent molecular weight of about 16 kD, both as determined after reduction by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.
73 . The device of claim 71 wherein one chain of said pair of polypeptide chains has an apparent molecular weight of about 16 kD and the other has an apparent molecular weight of about 18 kD, both as determined after reduction by comparison to molecular weight standards in SDS-polyacrylamide gel electrophoresis.
74 . The device of claim 58 wherein said osteogenic protein has the approximate amino acid composition set forth below:
Amino acid
Rel. no.
Amino acid
Rel. no.
residue
res./molec.
residue
res./molec.
Aspartic acid/
22
Tyrosine
11
Asparagine
Valine
14
Glutamic acid/
24
Methionine
3
Glutamine
Cysteine
16
Serine
24
Isoleucine
15
Glycine
29
Leucine
15
Histidine
5
Proline
14
Arginine
13
Phenylalanine
7
Threonine
11
Tryptophan
ND
Alanine
18
Lysine
12
75 . The device of claim 58 wherein said osteogenic protein comprises the amino acid sequence:
VPKPCCAPT
76 . The device of claim 1 or 58 wherein the half maximum bone inducing activity of said protein is 0.8 to 1.0 ng per mg of said matrix.
77 . A method of inducing local cartilage or bone formation in a mammal comprising the step of implanting the device of claim 1 , 2 , or 58 in said mammal at a locus accessible to migratory progenitor cells of said mammal.
78 . A method of inducing endochondral bone formation in a mammal comprising the step of implanting the device of claim 1 or 58 in said mammal at a locus accessible to migratory progenitor cells of said mammal.
79 . A method of inducing endochondral bone formation in a non-union fracture in a mammal comprising the step of implanting in the fracture in said mammal the device of claim 63 .
80 . Antibodies reactive with an epitope of the protein of claim 21 or 22 .Join the waitlist — get patent alerts
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