Title: Osteogenic devices
United States Patent: 6,297,213
Inventors: Oppermann; Hermann (Medway, MA); Kuberasampath; Thangavel (Medway, MA); Rueger; David C. (West Roxbury, MA); Ozkaynak; Engin (Milford, MA)Assignee: Stryker Corporation (Kalamazoo, MI)
Appl. No.: 074299Filed: May 7, 1998
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.
SUMMARY OF THE INVENTION
This invention involves osteogenic devices which, when
implanted in a mammalian body, can induce at the locus of the implant the
full developmental cascade of endochondral bone formation and bone marrow
differentiation. Suitably modified as disclosed herein, the devices also
may be used to induce cartilage formation. The devices comprise a carrier
material, referred to herein as a matrix, having the characteristics
disclosed below, containing dispersed osteogenic protein either in its
native form or in the form of a biosynthetic construct.
A key to these developments was the elucidation of amino acid sequence and
structure data of native osteogenic protein. A protocol was developed
which results in retrieval of active, substantially pure osteogenic
protein from mammalian bone. Investigation of the properties and structure
of the native form osteogenic protein then permitted the inventors to
develop a rational design for non-native forms, i.e., forms never before
known in nature, capable of inducing bone formation. As far as applicants
are aware, the constructs disclosed herein constitute the first instance
of the design of a functional, active protein without preexisting
knowledge of the active region of a native form nucleotide or amino acid
sequence.
A series of consensus DNA sequences were designed with the goal of
producing an active osteogenic protein. The sequences were based on
partial amino acid sequence data obtained from the natural source product
and on observed homologies with unrelated genes reported in the
literature, or the sequences they encode, having a presumed or
demonstrated developmental function. Several of the biosynthetic consensus
sequences have been expressed as fusion proteins in procaryotes, purified,
cleaved, refolded, combined with a matrix, implanted in an established
animal model, and shown to have endochondral bone-inducing activity. The
currently preferred active totally biosynthetic proteins comprise two
synthetic sequences designated COPS and COP7. The amino acid sequences of
these proteins are set forth below.
1 10 20 30 40
COP5 LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD
50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
1 10 20 30 40
COP7 LYVDFS-DVGWNDWIVAPPGYAHFYCHGECPFPLAD
50 60 70
HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
In these sequences and all other amino acid sequences disclosed herein, the dashes (-) are used as fillers only to line up comparable sequences in related proteins, and have no other function. Thus, amino acids 45-50 of COP7, for example, are NHAVV. Also, the numbering of amino acids is selected solely for purposes of facilitating comparisons between sequences. Thus, for example, the DF residues numbered at 9 and 10 of COP5 and COP7 may comprise residues, e.g., 35 and 36, of an osteogenic protein embodying invention.
Thus, in one aspect, the invention comprises a protein comprising an amino acid sequence sufficiently duplicative of the sequence of COP5 or COP7 such that it is capable of inducing endochondral bone formation when properly folded and implanted in a mammal in association with a matrix. Some of these sequences induce cartilage, but not bone. Also, the bone forming materials may be used to produce cartilage if implanted in an avascular locus, or if an inhibitor to full bone development is implanted together with the active protein. Thus, in another aspect, the invention comprises a protein less than about 200 amino acids long in a sequence sufficiently duplicative of the sequence of COP5 or COP7 such that it is capable at least of cartilage formation when properly folded and implanted in a mammal in association with a matrix.
In one preferred aspect, these proteins comprise species of the generic amino acid sequences:
1 10 20 30 40 50
LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAAK
60 70 80 90 100
QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXVXXLXXYXXMXVXXCXCX
or
1 10 20 30 40 50
CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX
60 70 80 90 100
QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX
where the letters indicate the amino acid residues of standard single letter code, and the Xs represent amino acid residues. Preferred amino acid sequences within the foregoing generic sequences are:
1 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
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGCR
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H
RF T S K DPV V Y N S H RN RS
N S K P E
and
1 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
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGCR
SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE T DA H H
RF T S K DPV V Y N S H RN RS
N S K P E
wherein each of the amino acids arranged vertically at each position in the sequence may be used alternatively in various combinations. Note that these generic sequences have 6 and preferably 7 cysteine residues where inter- or intramolecular disulfide bonds can form, and contain other critical amino acids which influence the tertiary structure of the proteins. These generic structural features are found in previously published sequences, none of which have been described as capable of osteogenic activity, and most of which never have been linked with such activity.
Particular useful sequences include:
1 10 20 30 40
Vg1 CKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE
50 60 70
ILNGSN--H-AILQTLVHSIEPED-IPLPCCVPTKMSP
80 90 100
ISMLFYDNNDNVVLRHYENMAVDECGCR
1 10 20 30 40
DPP CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD
50 60 70
HFNSTN--H-AVVQTLVNNNNPGK-VPKACCVPTQLDS
80 90 100
VAMLYLNDQSTVVLKNYQEMTVVGCGCR
-5
HQRQA
1 10 20 30 40
OP 1 CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS
50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA
80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
1 10 20 30 40
CBMP-2a CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD
50 60 70
HLNSTN--H-AIVQTLVNSVNS-K-IPKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQDMVVEGCGCR
1 10 20 30 40
CBMP-2b CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD
50 60 70
HLNSTN--H-AIVQTLVNSVNS-S-IPKACCVPTELSA
80 90 100
ISMLYLDEYDKVVLKNYQEMVVEGCGCR
1 10 20 30 40
CBMP-3 CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK
50 60 70
SLKPSN--H-ATIQSIVRAVGVVPGIPEPCCVPEKMSS
80 90 100
LSILFFDENKNVVLKVYPNMTVESCACR
1 10 20 30 40
COP 1 LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFPSAD
50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA
80 90 100
ISMLYLDENSTVVLKNYQEMTVVGCGCR
1 10 20 30 40
COP3 LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD
50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
1 10 20 30 40
COP4 LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD
50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
-10
PKHHSQRARKKNKN
1 10 20 30 40
COP16 CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGECPFPLAD
50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA
80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
Vg1 is a known Xenopus sequence heretofore not associated with bone formation. DPP is an amino acid sequence encoded by a drosophila gene responsible for development of the dorsoventral pattern. OP1 is a region of a natural sequence encoded by exons of a genomic DNA sequence retrieved by applicants. The CBMPs are amino acid sequences comprising subparts of mammalian proteins encoded by genomic DNAs and cDNAs retrieved by applicants. The COPs are biosynthetic protein sequences expressed by novel consensus gene constructs, designed using the criteria set forth herein, and not yet found in nature.
These proteins are believed to dimerize during refolding. They appear not to be active when reduced. Various combinations of species of the proteins, i.e., heterodimers, have activity, as do homodimers. As far as applicants are aware, the COP5 and COP7 constructs constitute the first instances of the design of a bioactive protein without preexisting knowledge of the active region of a native form nucleotide or amino acid sequence.
The invention also provides native forms of osteogenic protein, extracted from bone or produced using recombinant DNA techniques. The substantially pure osteogenic protein may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native protein, no matter how derived. The osteogenic protein in its native form is glycosylated and has an apparent molecular weight of about 30 kD as determined by SDS-PAGE. When reduced, the 30 kD protein gives rise to two glycosylated polypeptide chains having apparent molecular weights of about 16 kD and 18 kD. In the reduced state, the 30 kD protein has no detectable osteogenic activity. The deglycosylated protein, which has osteogenic activity, has an apparent molecular weight of about 27 kD. When reduced, the 27 kD protein gives rise to the two deglycosylated polypeptides have molecular weights of about 14 kD to 16 kD.
Analysis of digestion fragments indicate that the native 30 kD osteogenic protein contains the following amino acid sequences (question marks indicate undetermined residues):
(1) S-F-D-A-Y-Y-C-S-G-A-C-Q-F-P-M-P-K;
(2) S-L-K-P-S-N-Y-A-T-I-Q-S-I-V;
(3) A-C-C-V-P-T-E-L-S-A-I-S-M-L-Y-L-D-E-N-E-K;
(4) M-S-S-L-S-I-L-F-F-D-E-N-K;
(5) S-Q-E-L-Y-V-D-F-Q-R;
(6) F-L-H-C-Q-F-S-E-R-N-S;
(7) T-V-G-Q-L-N-E-Q-S-S-E-P-N-I-Y;
(8) L-Y-D-P-M-V-V;
(9) V-G-V-V-P-G-I-P-E-P-C-C-V-P-E;
(10) V-D-F-A-D-I-G;
(11) V-P-K-P-C-C-A-P-T;
(12) I-N-I-A-N-Y-L;
(13) D-N-H-V-L-T-M-F-P-I-A-I-N;
(14) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-?-P;
(15) D-I-G-?-S-E-W-I-I-?-P;
(16) S-I-V-R-A-V-G-V-P-G-I-P-E-P-?-?-V;
(17) D-?-I-V-A-P-P-Q-Y-H-A-F-Y;
(18) D-E-N-K-N-V-V-L-K-V-Y-P-N-M-T-V-E;
(19) S-Q-T-L-Q-F-D-E-Q-T-L-K-?-A-R-?-K-Q;
(20) D-E-Q-T-L-K-X-A-R-R-K-Q-W-I-E-P-R-N-?-A-R-R-Y-L;
(21) A-R-R-K-Q-W-I-E-P-R-N-?-A-?-R-Y-?-?-V-D; and
(22) R-?-Q-W-I-E-P-?-N-?-A-?-?-Y-L-K-V-D-?-A-?-?-G.
The substantially pure (i.e., free of contaminating proteins having no osteoinductive activity) osteogenic proteins and the synthetics are useful in clinical applications in conjunction with a suitable delivery or support system (matrix). The matrix is made up of particles or porous materials. The pores must be of a dimension to permit progenitor cell migration and subsequent differentiation and proliferation. The particle size should be within the range of 70-850 mm, preferably 70-420 mm. It may be fabricated by close packing particulate material into a shape spanning the bone defect, or by otherwise structuring as desired a material that is biocompatible (non-inflammatory) and, biodegradable in vivo to serve as a "temporary scaffold" and substratum for recruitment of migratory progenitor cells, and as a base for their subsequent anchoring and proliferation. Currently preferred carriers include particulate, demineralized, guanidine extracted, species-specific (allogenic) bone, and particulate, deglycosglated, protein extracted, demineralized, xenogenic bone. Optionally, such xenogenic bone powder matrices also may be treated with proteases such as trypsin. Other useful matrix materials comprise collagen, homopolymers and copolymers of glycolic acid and lactic acid, hydroxyapatite, tricalcium phosphate and other calcium phosphates.
The availability of the protein in substantially pure form, and knowledge of its amino acid sequence and other structural features, enable the identification, cloning, and expression of native genes which encode osteogenic proteins. When properly modified after translation, incorporated in a suitable matrix, and implanted as disclosed herein, these proteins are operative to induce formation of cartilage and endochondral bone.
The consensus DNA sequences are also useful as probes for extracting genes encoding osteogenic protein from genomic and cDNA libraries. One of the consensus sequences has been used to isolate a heretofore unidentified genomic DNA sequence, portions of which when ligated encode a protein having a region capable of inducing endochondral bone formation. This protein, designated OP1, has an active region having the sequence set forth below.
1 10 20 30 40
OP1 LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS
50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA
80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
A longer active sequence is:
-5
HQRQA
1 10 20 30 40
OP1 CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS
50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA
80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
The probes have also retrieved the DNA sequences identified in PCT/087/01537, referenced above, designated therein as BMPII(b) and BMPIII. The inventors herein have discovered that certain subparts of these genomic DNAs, and BMPIIa, from the same publication, when properly assembled, encode proteins (CBMPIIa, CBMPIIb, and CBMPIII) which have true osteogenic activity, i.e., induce the full cascade of events when properly implanted in a mammal leading to endochondral bone formation.
Thus, in view of this disclosure, skilled genetic engineers can design and synthesize genes or isolate genes from cDNA or genomic libraries which encode appropriate amino acid sequences, and then can express them in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active proteins in native forms, truncated analogs, muteins, fusion proteins, and other constructs capable of inducing bone formation in mammals including humans.
The osteogenic proteins and implantable osteogenic devices enabled and disclosed herein will permit the physician to obtain optimal predictable bone formation to correct, for example, acquired and congenital craniofacial and other skeletal or dental anomalies (Glowacki et al. (1981) Lancet 1:959-963). The devices may be used to induce local endochondral bone formation in non-union fractures as demonstrated in animal tests, and in other clinical applications including periodontal applications where bone formation is required. The other potential clinical application is in cartilage repair, for example, in the treatment of osteoarthritis.
Claim 1 of 24 Claims
What is claimed is:
1. A device for implantation in a mammal, comprising:
a biocompatible particulate matrix, the matrix comprising collagen and at
least one material selected from the group consisting of polymers
comprising lactic acid monomer units, polymers comprising glycolic acid
monomer units, bone, hydroxyapatite, calcium phosphate, muscle, and
tissue; and
a substantially pure osteogenic or chondrogenic protein disposed in or on
said matrix, wherein said protein induces the influx, proliferation, and
differentiation of migratory precursor cells from the body of said mammal
into or on said matrix.
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