U.S. patent application number 10/753916 was filed with the patent office on 2004-07-15 for compositions and therapeutic methods using morphogenic proteins, hormones and hormone receptors.
This patent application is currently assigned to Stryker Corporation. Invention is credited to Lee, John C., Yeh, Lee-Chuan C..
Application Number | 20040138128 10/753916 |
Document ID | / |
Family ID | 31497979 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040138128 |
Kind Code |
A1 |
Lee, John C. ; et
al. |
July 15, 2004 |
Compositions and therapeutic methods using morphogenic proteins,
hormones and hormone receptors
Abstract
This invention features devices and methods for inducing tissue
formation in a mammal, involving the use of a morphogenic protein,
a hormone and a soluble receptor of the hormone. The hormone and
receptor thereof are used to enhance the tissue inductive activity
of the morphogenic protein.
Inventors: |
Lee, John C.; (San Antonio,
TX) ; Yeh, Lee-Chuan C.; (San Antonio, TX) |
Correspondence
Address: |
FISH & NEAVE
1251 AVENUE OF THE AMERICAS
50TH FLOOR
NEW YORK
NY
10020-1105
US
|
Assignee: |
Stryker Corporation
|
Family ID: |
31497979 |
Appl. No.: |
10/753916 |
Filed: |
January 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10753916 |
Jan 7, 2004 |
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09672224 |
Sep 27, 2000 |
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6696410 |
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60156261 |
Sep 27, 1999 |
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Current U.S.
Class: |
424/85.2 ;
514/16.7; 514/8.8 |
Current CPC
Class: |
A61K 38/1793 20130101;
A61K 38/1875 20130101; A61K 38/1875 20130101; A61K 38/1793
20130101; A61K 38/204 20130101; A61K 38/204 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/24; A61K
038/17 |
Claims
What is claimed is:
1. A method for improving the tissue inductive capability of a
morphogenic protein at a target locus in a mammal, the method
comprising administering to the target locus the morphogenic
protein and a first effective amount of a hormone and a second
effective amount of a soluble receptor of the hormone, wherein the
morphogenic protein is capable of inducing tissue formation when
accessible to a progenitor cell in the mammal, and the hormone and
the receptor in combination enhance that capability.
2. The method of claim 1, wherein the morphogenic protein comprises
a pair of subunits disulfide-bonded to produce a dimeric species,
wherein at least one of the subunits comprises a polypeptide
belonging to the BMP protein family.
3. The method of claim 1, wherein the morphogenic protein is an
osteogenic protein.
4. The method of claim 3, wherein the osteogenic protein is capable
of inducing the progenitor cell to form endochondral or
intramembranous bone.
5. The method of claim 3, wherein the osteogenic protein is capable
of inducing the progenitor cell to form cartilage.
6. The method of claim 1, wherein the morphogenic protein is
capable of inducing the progenitor cell to form tissue
tendon/ligament-like or neural-like tissue.
7. The method of claim 1, wherein the morphogenic protein comprises
an amino acid sequence sufficiently duplicative of the amino acid
sequence of BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 (OP-1), BMP-8,
BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, COP-5, or COP-7.
8. The method of claim 7, wherein the morphogenic protein comprises
a polypeptide selected from the group consisting of OP-1, BMP-2,
BMP-4 and BMP-6.
9. The method of claim 8, wherein the morphogenic protein is
OP-1.
10. The method of claim 2, wherein the dimer is a homo- or
heterodimer comprising a BMP-2 or BMP-7 (OP-1) subunit.
11. The method of claim 1, wherein the hormone is interleukin 6
(IL-6).
12. The method of claim 7, wherein the hormone is IL-6.
13. The method of claim 9, wherein the hormone is IL-6.
14. The method of claim 1, wherein the morphogenic protein, the
hormone, and the hormone receptor are administered simultaneously
to the target locus.
15. The method of claim 1, wherein the morphogenic protein, the
hormone, and the hormone receptor are administered separately to
the target locus.
16. The method of claim 1, wherein the hormone and the hormone
receptor are administered simultaneously to the target locus.
17. The method of claim 1, wherein the target locus is a jaw bone
defect.
18. The method of claim 1, wherein the target locus is a bone
defect selected from the group consisting of a fracture, a
non-union fracture, a critical size defect, a non-critical size
defect, an osteochondral defect, a fusion and a bony void.
19. The method of claim 1, wherein the target locus has a tissue
degenerative condition.
20. The method of claim 1, wherein the target locus is a cartilage
or soft tissue defect.
21. The method of claim 1, wherein the target locus is a neural
tissue defect.
22. The method of claim 1, wherein the morphogenic protein is
administered in a matrix-comprising carrier.
23. The method of claim 22, wherein the carrier comprises allogenic
bone.
24. The method of claim 1, wherein the morphogenic protein is
administered via a nucleic acid, the nucleic acid comprising a
sequence encoding the morphogenic protein and capable of expressing
the morphogenic protein in the progenitor cell.
25. A pharmaceutical composition for inducing tissue formation in a
mammal, comprising a morphogenic protein, a hormone and a soluble
receptor of the hormone, wherein the morphogenic protein is capable
of inducing tissue formation when accessible to a progenitor cell
in the mammal, and the hormone and the receptor in combination
enhance that capability.
26. The pharmaceutical composition of claim 25, further comprising
an implantable, biocompatible carrier.
27. The pharmaceutical composition of claim 26, wherein the carrier
comprises demineralized, protein-extracted, particulate, allogenic
bone.
28. The pharmaceutical composition of claim 26, wherein the carrier
comprises mineral-free, delipidated Type I insoluble bone collagen
particles, substantially depleted in noncollagenous protein.
29. The pharmaceutical composition of claim 25, wherein the
morphogenic protein comprises an amino acid sequence sufficiently
duplicative of the amino acid sequence of BMP-2, BMP-3, BMP-4,
BMP-5, BMP-6, BMP-7 (OP-1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12,
BMP-13, COP-5, or COP-7.
30. The pharmaceutical composition of claim 29, wherein the
morphogenic protein is human OP-1.
31. The pharmaceutical composition of claim 25, wherein the hormone
is IL-6.
32. A kit for inducing tissue formation in a mammal, comprising a
first receptacle containing a morphogenic protein, a second
receptacle containing a hormone, and a third receptacle containing
a soluble receptor of the hormone, wherein the morphogenic protein
is capable of inducing tissue formation when accessible to a
progenitor cell in the mammal, and the hormone and the receptor in
combination enhance that capability.
33. The kit of claim 32, wherein the second and the third
receptacles are the same.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] Under 35 U.S.C. .sctn. 119(e)(1), this application claims
the benefit of prior U.S. provisional application serial No.
60/156,261, filed Sep. 27, 1999.
BACKGROUND OF THE INVENTION
[0002] The Transforming Growth Factor-Beta ("TGF-.beta.")
superfamily represents a large number of evolutionarily conserved
morphogenic proteins with diverse activities in growth,
differentiation, tissue morphogenesis and repair. This superfamily
includes osteogenic proteins ("OPs") and bone morphogenic proteins
("BMPs"). OPs and BMPs share a highly conserved, bioactive
cysteine-rich domain near their C-termini and have a propensity to
form homo- and hetero-dimers.
[0003] Many morphogenic proteins belonging to the BMP family have
been described. Some were isolated using purification techniques on
the basis of osteogenic activity. Others were identified and cloned
by virtue of DNA sequence homologies within conserved regions that
are common to the BMP family. These homologs are referred to as
consecutively numbered BMPs whether or not they have demonstrable
osteogenic activity. While several of the earliest members of the
BMP family were identified by virtue of their ability to induce new
cartilage and bone, a number of other BMPs have different or
additional tissue-inductive capabilities. For example, BMP-12 and
BMP-13 (identified by DNA sequence homology) reportedly induce
tendon/ligament-like tissue formation in vivo (WO 95/16035).
Several BMPs, including some of those originally isolated on the
basis of their osteogenic activity, can induce neuron proliferation
and promote axon regeneration (WO 95/05846; Liem et al., Cell, 82,
pp. 969-79 (1995)). Thus, it appears that BMPs may have a variety
of potential tissue-inductive capabilities whose final expression
depends on a complex set of developmental and environmental
cues.
[0004] Many of the mammalian BMPs have been recombinantly expressed
as active bomo- or heterodimers in a variety of host systems,
making therapeutic treatments using morphogenic proteins feasible.
Implantable osteogenic devices comprising mammalian osteogenic
protein for promoting bone healing and regeneration have been
described (see, e.g., Oppermann et al., U.S. Pat. No. 5,354,557).
Some osteogenic devices contain porous, biocompatible matrices that
allow the diffusion of osteogenic proteins into the implantation
site as well as the influx and efflux of progenitor cells.
Osteogenic protein-coated prosthetic devices that enhance the bond
strength between the prosthesis and existing bone have also been
described (Rueger et al., U.S. Pat. No. 5,344,654).
SUMMARY OF THE INVENTION
[0005] This invention is based on the discovery that the
tissue-inductive activity of a morphogenic protein can be enhanced
by a hormone in the presence of a soluble receptor of the
hormone.
[0006] Accordingly, this invention features a method for improving
the tissue inductive capability of a morphogenic protein at a
target locus in a mammal. In this method, the morphogenic protein
and a first effective amount of a hormone and a second effective
amount of a soluble receptor of the hormone are administered to the
target locus, wherein the morphogenic protein is capable of
inducing tissue formation when accessible to a progenitor cell in
the mammal, and the hormone and the receptor in combination enhance
that capability. The morphogenic protein, hormone and hormone
receptor can be administered simultaneously to the target locus.
Alternatively, the three components are administered separately, in
any order: for instance, the morphogenic protein can be
administered first, and then the hormone and hormone receptor are
administered together; or the morphogenic protein and the hormone
are administered together first, and then the hormone receptor is
administered. In one embodiment, the morphogenic protein is
administered via a nucleic acid (e.g., a plasmid, a viral vector,
or naked DNA) that comprises a sequence encoding the morphogenic
protein and is capable of expressing the morphogenic protein in the
appropriate progenitor cells of a patient.
[0007] The morphogenic protein may comprise a pair of subunits
disulfide-bonded to produce a dimeric species, wherein at least one
of the subunits comprises a polypeptide belonging to the BMP
protein family. For instance, the morphogenic protein may comprise
an amino acid sequence sufficiently duplicative of the amino acid
sequence of a reference BMP such as BMP-2, BMP-3, BMP-4, BMP-5,
BMP-6, BMP-7 (OP-1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13,
COP-5, or COP-7, such that it has morphogenic activity similar to
that of the reference BMP. In one preferred embodiment, the
morphogenic protein is a homo- or heterodimer comprising a BMP-2 or
BMP-7 (OP-1) subunit.
[0008] The morphogenic protein is capable of inducing tissue
formation. For instance, it may be capable of inducing the
progenitor cell to form tissue tendon/ligament-like or neural-like
tissue; or it may be an osteogenic protein that is capable of
inducing the progenitor cell to form endochondral or
intramembranous bone, or cartilage. The method of this invention
thus can be used to induce tissue regeneration or repair in a
variety of tissue defects such as bone, cartilage, soft tissue and
neural tissue defects.
[0009] Hormones useful in this invention include but are not
limited to cytokines (e.g., interleukins 1 through 18), growth
factors (e.g., fibroblast growth factor, vascular endothelial
growth factor, platelet-derived growth factor, TGF-.beta., or
prostaglandin) or morphogenic proteins.
[0010] The invention also features pharmaceutical compositions and
kits comprising a hormone and a soluble receptor thereof for
improving the tissue inductive activity of a morphogenic protein.
This invention also provides implantable morphogenic devices for
inducing tissue formation in allogeneic and xenogeneic implants.
Such devices comprise a morphogenic protein, a hormone and a
soluble receptor thereof disposed within a carrier. Methods for
inducing local tissue formation from a progenitor cell in a mammal
using those compositions and devices are also provided. A method
for accelerating allograft repair in a mammal using those
morphogenic devices is provided. This invention also provides a
prosthetic device comprising a prosthesis coated with a morphogenic
protein, a hormone and a soluble receptor thereof, and a method for
promoting in vivo integration of an implantable prosthetic device
to enhance the bond strength between the prosthesis and the
existing target tissue at the joining site. Methods for treating
tissue degenerative conditions in a mammal using the pharmaceutical
compositions are also provided.
[0011] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Exemplary methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention. All publications and other references mentioned herein
are incorporated by reference in their entirety. In case of
conflict, the present specification, including definitions, will
control. The materials, methods, and examples are illustrative only
and not intended to be limiting.
[0012] Other features and advantages of the invention will be
apparent from the following drawings, detailed description, and the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a bar graph showing that a combination of
interleukin 6 ("IL-6") and soluble IL-6 receptor ("sIL-6R")
significantly increases the ability of OP-1 to induce alkaline
phosphatase ("AP") activity in fetal rat calvaria ("FRC") cells.
"OP" stands for "OP-1"; "IL-6R" refers to "sIL-6R." The
parenthesized numbers indicate the protein concentrations (ng/ml)
used in the assay; in the case of IL-6/sIL-6R combinations, the two
numbers separated by a backslash in the parenthesis indicate the
protein concentrations of IL-6 and sIL-6R, respectively.
[0014] FIG. 2 is a photograph showing results of a mineralized bone
nodule formation assay using OP-1 and IL-6. Dark spots inside the
wells represent mineralized bone nodules.
[0015] FIG. 3 is a photograph showing results of a mineralized bone
nodule formation assay using OP-1, IL-6 and sIL-6R. Dark spots
inside the wells represent mineralized bone nodules.
[0016] FIG. 4 is a bar graph showing the mRNA levels of BMPR-IA,
BMPR-IB, ActR-I, and BMPR-II in various test groups. "sR" stands
for sIL-6R. Values in the graph represent the means.+-.SE of twelve
Northern blots with RNA isolated from two different FRC cell
preparations.
[0017] FIG. 5 is a bar graph showing that the AP activity in FRC
cells transfected with the OP-1-encoding pW24 plasmid is enhanced
by exogenous sIL-6R alone or a combination of IL-6 and sIL-6R
("IL-6/R"). "IL6R" stands for sIL-6R. Values in the graph represent
the mean.+-.SE of five independent determinations with three
different FRC cell preparations and two different DNA preparations.
"IL-6/R (X/Y)" refers to X ng/ml IL-6 and Y ng/ml sIL-6R.
DETAILED DESCRIPTION OF THE INVENTION
[0018] In order that the invention herein described may be fully
understood, the following detailed description is set forth.
[0019] The term "biocompatible" refers to a material that does not
elicit detrimental effects associated with the body's various
protective systems, such as cell and humoral-associated immune
responses, e.g., inflammatory responses and foreign body fibrotic
responses. This term also implies that no specific undesirable
effects, cytotoxic or systemic, are caused by the material when it
is implanted into the patient.
[0020] The term "BMP" refers to a protein belonging to the BMP
family of the TGF-.beta. superfamily of proteins defined on the
basis of DNA and amino acid sequence homology. According to this
invention, a protein belongs to the BMP family when it has at least
50% (e.g., at least 70% or even 85%) amino acid sequence homology
with a known BMP family member within the conserved C-terminal
cysteine-rich domain that characterizes the BMP family. Members of
the BMP family may have less than 50% DNA or amino acid sequence
homology overall.
[0021] The term "morphogenic protein" refers to a protein having
morphogenic activity. For instance, this protein is capable of
inducing progenitor cells to proliferate and/or to initiate
differentiation pathways that lead to the formation of cartilage,
bone, tendon, ligament, neural or other types of tissue, depending
on local environmental cues. Thus, morphogenic proteins useful in
this invention may behave differently in different surroundings. A
morphogenic protein of this invention may comprise at least one
polypeptide belonging to the BMP family.
[0022] The term "osteogenic protein" refers to a morphogenic
protein that is capable of inducing a progenitor cell to form
cartilage and/or bone. The bone may be intramembranous bone or
endochondral bone. Most osteogenic proteins are members of the BMP
family and are thus also BMPs. However, the converse may not be
true. According to this invention, a BMP identified by sequence
homology must have demonstrable osteogenic or chondrogenic activity
in a functional bioassay to be an osteogenic protein.
[0023] The terms "morphogenic activity," "inducing activity" and
"tissue inductive activity" alternatively refer to the ability of
an agent to stimulate a target cell to undergo one or more cell
divisions (proliferation) that may optionally lead to cell
differentiation. Such target cells are referred to generically
herein as progenitor cells. Cell proliferation is typically
characterized by changes in cell cycle regulation and may be
detected by a number of means which include measuring DNA synthetic
or cellular growth rates. Early stages of cell differentiation are
typically characterized by changes in gene expression patterns
relative to those of the progenitor cell; such changes may be
indicative of a commitment towards a particular cell fate or cell
type. Later stages of cell differentiation may be characterized by
changes in gene expression patterns, cell physiology and
morphology. Any reproducible change in gene expression, cell
physiology or morphology may be used to assess the initiation and
extent of cell differentiation induced by a morphogenic
protein.
[0024] The term "synergistic interaction" refers to an interaction
in which the combined effect of two or more agents is greater than
the algebraic sum of their individual effects.
[0025] The term "hormone/receptor pair" refers to a combination of
a hormone and a soluble receptor thereof. The hormone (e.g., a
cytokine, a growth factor, or a morphogenic protein) can be of any
mammalian origin (e.g., human, bovine, or murine). A soluble
receptor of a hormone is a compound that binds specifically to the
hormone, and can, for example, be a polypeptide containing only the
hormone-binding domain (e.g., an extracellular domain) of a native
cellular receptor of the hormone, an antibody specific for the
hormone, or a chemical compound that specifically interacts with
the hormone. A soluble receptor can also be a compound (e.g., a
protein) containing a domain that specifically binds to the hormone
and another domain that specifically binds to the native cellular
receptor of the hormone such that the soluble receptor facilitates
the binding of the hormone to its native cellular receptor; an
example of such a soluble receptor is the IGF-binding protein.
[0026] Morphogenic Proteins
[0027] The morphogenic proteins of this invention are capable of
stimulating a progenitor cell to undergo cell division and/or
differentiation. They may belong to the TGF-13 protein superfamily,
and include, but are not limited to, OP-1, OP-2, OP-3, BMP-2,
BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10; BMP-11, BMP-12,
BMP-13, BMP-14, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7,
GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, DPP, Vg-1, Vgr-1, 60A
protein, NODAL, UNIVIN, SCREW, ADMP, NEURAL, and TGF-.beta..
[0028] One of the preferred morphogenic proteins is OP-1.
Nucleotide and amino acid sequences for hOP-1 are provided in SEQ
ID NOs:1 and 2, respectively. For ease of description, hOP-1 is
recited as a representative morphogenic protein. It will be
appreciated by the ordinarily skilled artisan that OP-1 is merely
representative of a family of morphogens.
[0029] Other useful morphogenic proteins also include polypeptides
having at least 50% (e.g., at least 70% or even 85%) sequence
homology with a known morphogenic protein, particularly with a
known BMP within the conserved C-terminal cysteine-rich domain that
characterizes the BMP protein family. These morphogenic proteins
include biologically active variants of any known morphogenic
protein, including variants containing conservative amino acid
changes. For instance, useful morphogenic proteins include those
containing sequences that share at least 70% amino acid sequence
homology with the C-terminal seven-cysteine domain of hOP-1, which
domain corresponds to the C-terminal 102-106 amino acid residues of
SEQ ID NO:2. The C-terminal 102 amino acid residues corresponds to
residues 330-431 of SEQ ID NO:2. In one embodiment of this
invention, the morphogenic protein consists of a pair of subunits
disulfide-bonded to produce a dimer, wherein at least one of the
subunits comprises a recombinant polypeptide belonging to the BMP
family.
[0030] As used herein, "amino acid sequence homology" is understood
to include both amino acid sequence identity and similarity.
Homologous sequences share identical and/or similar amino acid
residues, where similar residues are conservative substitutions
for, or "allowed point mutations" of, corresponding amino acid
residues in an aligned reference sequence. Thus, a candidate
polypeptide sequence that shares 70% amino acid homology with a
reference sequence is one in which any 70% of the aligned residues
are either identical to, or are conservative substitutions of, the
corresponding residues in a reference sequence. Certain
particularly preferred morphogenic polypeptides share at least 60%
(e.g., at least 65%) amino acid sequence identity with the
C-terminal seven-cysteine domain of human OP-1.
[0031] As used herein, "conservative substitutions" are residues
that are physically or functionally similar to the corresponding
reference residues. That is, a conservative substitution and its
reference residue have similar size, shape, electric charge,
chemical properties including the ability to form covalent or
hydrogen bonds, or the like. Preferred conservative substitutions
are those fulfilling the criteria defined for an accepted point
mutation in Dayhoff et al., Atlas of Protein Sequence and Structure
5:345-352 (1978 & Supp.). Examples of conservative
substitutions are substitutions within the following groups: (a)
valine, glycine; (b) glycine, alanine; (c) valine, isoleucine,
leucine; (d) aspartic acid, glutamic acid; (e) asparagine,
glutamine; (f) serine, threonine; (g) lysine, arginine, methionine;
and (h) phenylalanine, tyrosine. The term "conservative variant" or
"conservative variation" also includes the use of a substituting
amino acid residue in place of an amino acid residue in a given
parent amino acid sequence, where antibodies specific for the
parent sequence are also specific for, i.e., "cross-react" or
"immuno-react" with, the resulting substituted polypeptide
sequence.
[0032] Amino acid sequence homology can be determined by methods
well known in the art. For instance, to determine the percent
homology of a candidate amino acid sequence to the sequence of the
seven-cysteine domain, the two sequences are first aligned. The
alignment can be made with, e.g., the dynamic programming algorithm
described in Needleman et al., J. Mol. Biol. 48:443 (1970), and the
Align Program, a commercial software package produced by DNAstar,
Inc. The teachings by both sources are incorporated by reference
herein. An initial alignment can be refined by comparison to a
multi-sequence alignment of a family of related proteins. Once the
alignment is made and refined, a percent homology score is
calculated. The aligned amino acid residues of the two sequences
are compared sequentially for their similarity to each other.
Similarity factors include similar size, shape and electrical
charge. One particularly preferred method of determining amino acid
similarities is the PAM250 matrix described in Dayhoff et al.,
supra. A similarity score is first calculated as the sum of the
aligned pairwise amino acid similarity scores. Insertions and
deletions are ignored for the purposes of percent homology and
identity. Accordingly, gap penalties are not used in this
calculation. The raw score is then normalized by dividing it by the
geometric mean of the scores of the candidate sequence and the
seven-cysteine domain. The geometric mean is the square root of the
product of these scores. The normalized raw score is the percent
homology.
[0033] Morphogenic proteins useful herein include any known
naturally occurring native proteins, including allelic,
phylogenetic counterparts and other variants thereof. These
variants include forms having varying glycosylation patterns,
varying N-termini, and active truncated or mutated forms of a
native protein. Useful morphogenic proteins also include those that
are biosynthetically produced (e.g., "muteins" or "mutant
proteins") and those that are new, morphogenically active members
of the general morphogenic family of proteins. Particularly useful
sequences include those comprising the C-terminal 96 to 102 amino
acid residues of: DPP (from Drosophila), Vg-1 (from Xenopus), Vgr-1
(from mouse), the OP1 and OP2 proteins (U.S. Pat. No. 5,011,691),
as well as the proteins referred to as BMP-2, BMP-3, BMP-4 (WO
88/00205, U.S. Pat. No. 5,013,649 and WO 91/18098), BMP-5 and BMP-6
(WO 90/11366), BMP-8 and BMP-9. Other proteins useful in the
practice of the invention include active forms of OP1, OP2, OP3,
BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-11,
BMP-12, BMP-13, BMP-14, BMP-15, DPP, Vg-1, Vgr-1, 60A protein,
GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, and GDF-10,
GDF-11, GDF-12, GDF-13, UNIVIN, NODAL, SCREW, ADMP, NEURAL, and
TGF-.beta..
[0034] Osteogenic proteins useful as morphogenic proteins of this
invention include those containing sequences that share greater
than 60% identity with the seven-cysteine domain. In other
embodiments, useful osteogenic proteins are defined as
osteogenically active proteins having any one of the generic
sequences defined herein, including OPX (SEQ ID NO:3) and Generic
Sequences 7 (SEQ ID NO:4), 8 (SEQ ID NO:5), 9 (SEQ ID NO:6) and 10
(SEQ ID NO:7).
[0035] Generic Sequence 7 (SEQ ID NO:4) and Generic Sequence 8 (SEQ
ID NO:5), disclosed below, accommodate the homologies shared among
preferred protein family members identified to date, including
OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, 60A, DPP,
Vg-1, Vgr-1, and GDF-1. The amino acid sequences for these proteins
are described herein and/or in the art. The generic sequences
include the identical amino acid residues shared by these sequences
in the C-terminal six- or seven-cysteine skeletal domains
(represented by Generic Sequences 7 and 8, respectively), as well
as alternative residues for the variable positions within the
sequences. The generic sequences provide an appropriate cysteine
skeleton where inter- or intra-molecular disulfide bonds can form.
Those sequences contain certain specified amino acids that may
influence the tertiary structure of the folded proteins. In
addition, the generic sequences allow for an additional cysteine at
position 36 (Generic Sequence 7) or position 41 (Generic Sequence
8), thereby encompassing the biologically active sequences of OP-2
and OP-3.
1 GENERIC SEQUENCE 7 Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa
Xaa Xaa Xaa Xaa Xaa (SEQ ID NO:4) 1 5 10 15 Pro Xaa Xaa Xaa Xaa Ala
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa 20 25 30 Xaa Xaa
Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35
40 45 50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa
Xaa Xaa Xaa Xaa 55 60 65 70 Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Val Xaa Leu Xaa Xaa Xaa Xaa Xaa 75 80 85 Met Xaa Val Xaa Xaa Cys
Xaa Cys Xaa 90 95
[0036] wherein each Xaa is independently defined as follows ("Res."
means "residue"): Xaa at res.2=(Tyr or Lys); Xaa at res.3=(Val or
Ile); Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser,
Lys or Ala); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu, Val or
Ile); Xaa at res.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at
res.12=(Asp, Arg, Asn or Glu); Xaa at res. 13=(Trp or Ser); Xaa at
res.14=(Ile or Val); Xaa at res.15.=(Ile or Val); Xaa at res. 16
(Ala or Ser); Xaa at res. 18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa
at res. 19=(Gly or Ser); Xaa at res.20 (Tyr or Phe); Xaa at
res.21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at
res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp, Gln,
Ala or Ser); Xaa at res.28=(Glu, Lys, Asp, Gln or Ala); Xaa at
res.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res.31=(Phe, Leu or
Tyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp,
Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys);
Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe,
Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala,
Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser); Xaa at
res.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu, Met or Ile); Xaa
at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at
res.48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at
res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val, Gly or Leu); Xaa at
res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or
Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa at
res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa at
res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at
res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala); Xaa at
res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at
res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa
at res.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser);
Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or
Pro); Xaa at res.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or
Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn
or Leu); Xaa at res.77=(Asp, Glu, Asn, Arg or Ser); Xaa at
res.78=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser, Asn, Asp,
Glu or Lys); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile,
Val or Asn); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn,
Gln, His, Arg or Val); Xaa at res.86=(Tyr, Glu or His); Xaa at
res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu, Trp or
Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,
Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser);
Xaa at res.95=(Gly or Ala); and Xaa at res.97=(His or Arg).
[0037] Generic Sequence 8 (SEQ ID NO:5) includes all of Generic
Sequence 7 and in addition includes the following five amino acid
at its N-terminus: Cys Xaa Xaa Xaa Xaa (SEQ ID NO:8), wherein Xaa
at res.2=(Lys, Arg, Ala or Gln); Xaa at res.3=(Lys, Arg or Met);
Xaa at res.4=(His, Arg or Gln); and Xaa at res.5=(Glu, Ser, His,
Gly, Arg, Pro, Thr, or Tyr). Accordingly, beginning with residue 7,
each "Xaa" in Generic Sequence 8 is a specified amino acid as
defined as for Generic Sequence 7, with the distinction that each
residue number described for Generic Sequence 7 is shifted by five
in Generic Sequence 8. For example, "Xaa at res.2=(Tyr or Lys)" in
Generic Sequence 7 corresponds to Xaa at res.7 in Generic Sequence
8.
[0038] Generic Sequences 9 (SEQ ID NO:6) and 10 (SEQ ID NO:7) are
composite amino acid sequences of the following proteins: human
OP-1 ("hOP-1"), hOP-2, hOP-3, hBMP-2, hBMP-3, hBMP-4, hBMP-5,
hBMP-6, hBMP-9, hBMP10, hBMP-11, Drosophila 60A, Xenopus Vg-1, sea
urchin UNIVIN, hCDMP-1 (mouse GDF-5 or "mGDF-5"), hCDMP-2 (mGDF-6,
hBMP-13), hCDMP-3 (mGDF-7, hBMP-12), mGDF-3, hGDF-1, mGDF-1,
chicken DORSALIN, DPP, Drosophila SCREW, mouse NODAL, mGDF-8,
hGDF-8, mGDF-9, mGDF-10, hGDF-11, mGDF-11, hBMP-15, and rat BMP3b.
Like Generic Sequence 7, Generic Sequence 9 accommodates the
C-terminal six-cysteine skeleton and, like Generic Sequence 8,
Generic Sequence 10 accommodates the C-terminal seven-cysteine
skeleton.
2 GENERIC SEQUENCE 9 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa (SEQ ID NO:6) 1 5 10 15 Pro Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 50
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa
55 60 65 Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Cys Xaa 85 90 95
[0039] wherein each Xaa is independently defined as follows: Xaa at
res.1=(Phe, Leu or Glu); Xaa at res.2=(Tyr, Phe, His, Arg, Thr,
Lys, Gln, Val or Glu); Xaa at res.3=(Val, Ile, Leu or Asp); Xaa at
res.4=(Ser, Asp, Glu, Asn or Phe); Xaa at res.5=(Phe or Glu); Xaa
at res.6=(Arg, Gln, Lys, Ser, Glu, Ala or Asn); Xaa at res.7=(Asp,
Glu, Leu, Ala or Gln); Xaa at res.8=(Leu, Val, Met, Ile or Phe);
Xaa at res.9=(Gly, His or Lys); Xaa at res.10=(Trp or Met); Xaa at
res. 11=(Gln, Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at
res.12=(Asp, Asn, Ser, Lys, Arg, Glu or His); Xaa at res.13=(Trp or
Ser); Xaa at res.14=(Ile or Val); Xaa at res. 15=(Ile or Val); Xaa
at res. 16=(Ala, Ser, Tyr or Trp); Xaa at res. 18=(Glu, Lys, Gln,
Met, Pro, Leu, Arg, His or Lys); Xaa at res. 19=(Gly, Glu, Asp,
Lys, Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at
res.21=(Ala, Ser, Gly, Met, Gln, His, Glu, Asp, Leu, Asn, Lys or
Thr); Xaa at res.22=(Ala or Pro); Xaa at res.23=(Tyr, Phe, Asn, Ala
or Arg); Xaa at res.24=(Tyr, His, Glu, Phe or Arg); Xaa at
res.26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln or Gly); Xaa at
res.28.=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gln); Xaa at
res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Phe, Gln or Leu); Xaa at
res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res.32=(Pro, Ser,
Ala or Val); Xaa at res.33=(Leu, Met, Glu, Phe or Val); Xaa at
res.34=(Asn, Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at
res.35=(Ser, Ala, Glu, Asp, Thr, Leu, Lys, Gln or His); Xaa at
res.36=(Tyr, His, Cys, Ile, Arg, Asp, Asn, Lys, Ser, Glu or Gly);
Xaa at res.37=(Met, Leu, Phe, Val, Gly or Tyr); Xaa at res.38=(Asn,
Glu, Thr, Pro, Lys, His, Gly, Met, Val or Arg); Xaa at res.39=(Ala,
Ser, Gly, Pro or Phe); Xaa at res.40=(Thr, Ser, Leu, Pro, His or
Met); Xaa at res.41=(Asn, Lys, Val, Thr or Gln); Xaa at
res.42=(His, Tyr or Lys); Xaa at res.43=(Ala, Thr, Leu or Tyr); Xaa
at res.44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaa at
res.45=(Val, Leu, Met, Ile or His); Xaa at res.46=(Gln, Arg or
Thr); Xaa at res.47=(Thr, Ser, Ala, Asn or His); Xaa at
res.48=(Leu, Asn or Ile); Xaa at res.49=(Val, Met, Leu, Pro or
Ile); Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gln); Xaa at
res.51=(Phe, Leu, Ser, Asn, Met, Ala, Arg, Glu, Gly or Gln); Xaa at
res.52=(Ile, Met, Leu, Val, Lys, Gln, Ala or Tyr); Xaa at
res.53=(Asn, Phe, Lys, Glu, Asp, Ala, Gin, Gly, Leu or Val); Xaa at
res.54=(Pro, Asn, Ser, Val or Asp); Xaa at res.55=(Glu, Asp, Asn,
Lys, Arg, Ser, Gly, Thr, Gln, Pro or His); Xaa at res.56=(Thr, His,
Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaa at res.57=(Val, Ile,
Thr, Ala, Leu or Ser); Xaa at res.58=(Pro, Gly, Ser, Asp or Ala);
Xaa at res.59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg or Gly); Xaa at
res.60=(Pro, Ala, Val, Thr or Ser); Xaa at res.61=(Cys, Val or
Ser); Xaa at res.63=(Ala, Val or Thr); Xaa at res.65=(Thr, Ala,
Glu, Val, Gly, Asp or Tyr); Xaa at res.66=(Gln, Lys, Glu, Arg or
Val); Xaa at res.67=(Leu, Met, Thr or Tyr); Xaa at res.68=(Asn,
Ser, Gly, Thr, Asp, Glu, Lys or Val); Xaa at res.69=(Ala, Pro, Gly
or. Ser); Xaa at res.70=(Ile, Thr, Leu or Val); Xaa at res.71=(Ser,
Pro, Ala, Thr, Asn or Gly); Xaa at res.72=(Val, Ile, Leu or Met);
Xaa at res.74=(Tyr, Phe, Arg, Thr, Tyr or Met); Xaa at res.75=(Phe,
Tyr, His, Leu, Ile, Lys, Gln or Val); Xaa at res.76=(Asp, Leu, Asn
or Glu); Xaa at res.77=(Asp, Ser, Arg, Asn, Glu., Ala, Lys, Gly or
Pro); Xaa at res.78=(Ser, Asn, Asp, Tyr, Ala, Gly, Gln, Met, Glu,
Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln
or Arg); Xaa at res.80=(Asn, Lys, Thr, Pro, Val, Ile, Arg, Ser or
Gln); Xaa at res.81=(Val, Ile, Thr or Ala); Xaa at res.82=(Ile,
Asn, Val, Leu, Tyr, Asp or Ala); Xaa at res.83=(Leu, Tyr, Lys or
Ile); Xaa at res.84=(Lys, Arg, Asn, Tyr, Phe, Thr, Glu or Gly); Xaa
at res.85=(Lys, Arg, His, Gln, Asn, Glu or Val); Xaa at
res.86=(Tyr, His, Glu or Ile); Xaa at res.87=(Arg, Glu, Gln, Pro or
Lys); Xaa at res.88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa at
res.89=(Met or Ala); Xaa at res.90=(Val, Ile, Ala, Thr, Ser or
Lys); Xaa at res.91=(Val or Ala); Xaa at res.92=(Arg, Lys, Gln,
Asp, Glu, Val, Ala, Ser or Thr); Xaa at res.93=(Ala, Ser, Glu, Gly,
Arg or Thr); Xaa at res.95=(Gly, Ala or Thr); and Xaa at
res.97=(His, Arg, Gly, Leu or Ser). Further, after res.53 in rat
BMP3b and mGDF-10 there is an Ile; after res.54 in GDF-I there is a
Thr; after res.54 in BMP3 there is a Val; after res.78 in BMP-8 and
DORSALIN there is a Gly; after res.37 in hGDF-1 there are Pro, Gly,
Gly, and Pro.
[0040] Generic Sequence 10 (SEQ ID NO:7) includes all of Generic
Sequence 9 and in addition includes the following five amino acid
residues at its N-terminus: Cys Xaa Xaa Xaa Xaa (SEQ ID NO:9),
wherein Xaa at res.2=(Lys, Arg, Gin, Ser, His, Glu, Ala, or Cys);
Xaa at res.3=(Lys, Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at
res.4=(His, Gin, Arg, Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at
res.5=(Gin, Thr, His, Arg, Pro, Ser, Ala, Gin, Asn, Tyr, Lys, Asp,
or Leu). Accordingly, beginning at res.6, each "Xaa" in Generic
Sequence 10 is a specified amino acid defined as for Generic
Sequence 9, with the distinction that each residue number described
for Generic Sequence 9 is shifted by five in. Generic Sequence 10.
For example, "Xaa at res.1=(Phe, Leu or Glu)" in Generic Sequence 9
corresponds to Xaa at res.6 in Generic Sequence 10.
[0041] As noted above, certain preferred bone morphogenic proteins
useful in this invention have greater than 60%, preferably greater
than 65%, identity with the C-terminal seven-cysteine domain of
hOP-1. These particularly preferred sequences include allelic and
phylogenetic variants of the OP-1 and OP-2 proteins, including the
Drosophila 60A protein. Accordingly, in certain particularly
preferred embodiments, useful proteins include active proteins
comprising dimers having the generic amino acid sequence "OPX" (SEQ
ID NO:3), which defines the seven-cysteine skeleton and
accommodates the homologies between several identified variants of
OP-1 and OP-2. Each Xaa in OPX is independently selected from the
residues occurring at the corresponding position in the C-terminal
sequence of mouse or human OP-1 or OP-2.
3 OPX Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp
Xaa Asp Trp (SEQ ID NO:3) 1 5 10 15 Xaa Ile Ala Pro Xaa Gly Tyr Xaa
Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe 20 25 30 35 Pro Leu Xaa Ser
Xaa Met Asn Ala Thr Asn His Ala Ile Xaa Gln Xaa Leu Val 40 45 50 55
His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa
Leu 60 65 70 Xaa Ala Xaa Ser Val Leu Tyr Xaa Asp Xaa Ser Xaa Asn
Val Ile Leu Xaa Lys 75 80 85 90 Xaa Arg Asn Met Val Val Xaa Ala Cys
Gly Cys His 95 100
[0042] wherein Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or
Arg); Xaa at res.11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa
at res. 19=(Ile or Val); Xaa at res.23=(Glu or Gln); Xaa at
res.26=(Ala or Ser); Xaa at res.35=(Ala or Ser); Xaa at res.39=(Asn
or Asp); Xaa at res.41=(Tyr or Cys); Xaa at res.50=(Val or Leu);
Xaa at res.52=(Ser or Thr); Xaa at res.56=(Phe or Leu); Xaa at
res.57=(Ile or Met); Xaa at res.58=(Asn or Lys); Xaa at
res.60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at
res.65=(Pro or Ala); Xaa at res.71=(Gln or Lys); Xaa at res.73=(Asn
or Ser); Xaa at res.75 (Ile or Thr); Xaa at res.80=(Phe or Tyr);
Xaa at res.82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at
res.89=(Lys or Arg); Xaa at res.91=(Tyr or His); and Xaa at
res.97=(Arg or Lys).
[0043] In another embodiment, the morphogenic proteins comprise
species of the generic amino acid sequence
4 1 10 20 30 40 50
CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX (SEQ ID NO:10)
60 70 80 90 100
QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX
[0044] or residues 6-102 of SEQ ID NO:10, where the letters
indicate the amino acid residues of standard single letter code,
and the Xs represent any amino acid residues. Cysteine residues are
highlighted.
[0045] Preferred amino acid sequences within the foregoing generic
sequence (SEQ ID NO:10) are:
5 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
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGC- R SI HAI SEQV
EP 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
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCG- CR SI HAI SEQV
EP 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
[0046] wherein each of the amino acids arranged vertically at each
position in the sequence may be used alternatively in various
combinations (SEQ ID NO:10). These generic sequences have 6 or 7
cysteine residues where inter- or intra-molecular disulfide bonds
can form. These sequences also contain other critical amino acids
that influence the tertiary structure of the proteins.
[0047] In still another embodiment, useful morphogenic proteins
comprise an amino acid sequence encoded by a nucleic acid that
hybridizes, under low, medium or high stringency hybridization
conditions, to DNA or RNA encoding reference morphogenic protein
coding sequences. Exemplary reference sequences include the
C-terminal sequences defining the conserved seven-cysteine domains
of OP-1, OP-2, BMP-2, BMP-4, BMP-5, BMP-6, 60A, GDF-3, GDF-5,
GDF-6, GDF-7, and the like. High stringent hybridization conditions
are herein defined as hybridization in 40% formamide, 5.times.SSPE,
5.times. Denhardt's Solution, and 0.1% SDS at 37.degree. C.
overnight, and washing in 0.1.times. SSPE, 0.1% SDS at 50.degree.
C. Standard stringency conditions are well characterized in
commercially available, standard molecular cloning texts. See, for
example, Molecular Cloning, A Laboratory Manual, 2nd Ed., ed. by
Sambrook et al. (Cold Spring Harbor Laboratory Press 1989); DNA
Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide
Synthesis (M. J. Gait ed., 1984); Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); and B. Perbal, A Practical
Guide To Molecular Cloning (1984).
[0048] Suitable in vitro, ex vivo and in vivo bioassays known in
the art, including those described herein, may be used to ascertain
whether a new BMP-related gene product has a morphogenic activity.
Expression and localization studies defining where and when the
gene is expressed may also be used to identify potential
morphogenic activities. Nucleic acid and protein localization
procedures are well known to those of skill in the art (see, e.g.,
Ausubel et al., eds. Current Protocols in Molecular Cloning, Greene
Publishing and Wiley Interscience, New York, 1989).
[0049] Many of the identified BMPs are osteogenic and can induce
bone and cartilage formation when implanted into mammals. Some BMPs
identified based on sequence homology to known osteogenic proteins
possess other morphogenic activities and a combination of a hormone
and a soluble receptor thereof may be used to enhance those
activities. For example, BMP-12 and BMP-13 reportedly induce
ectopic formation of tendon/ligament-like tissue when implanted
into mammals (Celeste et al., WO 95/16035). Using this bioassay, a
skilled practitioner can readily identify one or more combinations
of hormones and soluble receptors thereof that can stimulate the
ability of the BMP to induce tendon/ligament-like tissue
formation.
[0050] Certain BMPs which are known to be osteogenic can also
induce neuronal cell differentiation. Embryonic mouse cells treated
with BMP-2 or OP-1 differentiate into astrocyte-like (glial) cells,
and peripheral nerve regeneration using BMP-2 has been reported
(Wang et al., WO 95/05846). In addition, BMP-4, BMP-5 and OP-1 are
expressed in epidermal ectoderm flanking the neural plate. Ectopic
recombinant BMP-4 and OP-1 proteins can induce neural plate cells
to initiate dorsal neural cell fate differentiation (Liem et al.,
Cell, 82, pp. 969-79 (1995)). At the spinal cord level, OP-1 and
other BMPs can induce neural crest cell differentiation. It is
suggested that OP-1 and these BMPs can induce many or all dorsal
neural cell types, including roof plate cells, neural crest cells,
and commissural neurons, depending on localized positional
cues.
[0051] That several osteogenic proteins originally derived from
bone matrix are involved in neural development suggests that these
and other members of the BMP family have additional tissue
inductive properties that are not yet disclosed. It is envisioned
that the hormone/receptor combinations set forth in this invention
can be used to enhance new or known tissue inductive properties of
various known morphogenic proteins. It is also envisioned that the
invention described herein will be useful for stimulating tissue
inductive activities of new morphogenic proteins as they are
identified in the future.
[0052] Production of Morphogenic Proteins
[0053] The morphogenic proteins of this invention can be derived
from a variety of sources. For instance, they may be isolated from
natural sources, recombinantly produced, or chemically
synthesized.
[0054] A. Naturally Derived Morphogenic Proteins
[0055] The morphogenic proteins of the invention can be purified
from tissue sources, e.g., mammalian tissue sources, using well
known techniques. See, e.g., Oppermann et al., U.S. Pat. Nos.
5,324,819 and 5,354,557. If a purification protocol is unpublished,
as for a newly identified morphogenic protein, conventional protein
purification techniques (e.g., immunoaffinity) may be performed in
combination with morphogenic activity assays. Such assays allow the
trace of the morphogenic activity through a series of purification
steps.
[0056] B. Recombinantly Expressed Morphogenic Proteins
[0057] In another embodiment of this invention, the morphogenic
protein is produced by expressing an appropriate recombinant DNA
molecule in a host cell. The DNA and amino acid sequences of many
BMPs and OPs have been reported, and methods for their recombinant
production are published and otherwise known to those of skill in
the art. For a general discussion of cloning and recombinant DNA
technology, see Ausubel et al., supra; see also Watson et al.,
Recombinant DNA, 2d ed. 1992 (W. H. Freeman and Co, New York).
[0058] The DNA sequences encoding bovine and human BMP-2 (formerly
BMP2A) and BMP-4 (formerly BMP-2B), and processes for recombinantly
producing the corresponding proteins are described in U.S. Pat.
Nos. 5,011,691, 5,013,649, 5,166,058 and 5,168,050. The DNA and
amino acid sequences of human and bovine BMP-5 and BMP-6, and
methods for their recombinant production, are disclosed in U.S.
Pat. No. 5,106,748, and 5,187,076, respectively; see also U.S. Pat.
Nos. 5,011,691 and 5,344,654. Methods for OP-1 recombinant
expression are disclosed in Oppermann et al., U.S. Pat. Nos.
5,011,691 and 5,258,494. For an alignment of BMP-2, BMP-4, BMP-5,
BMP-6 and OP-1 (BMP-7) amino acid sequences, see WO 95/16034. DNA
sequences encoding BMP-8 are disclosed in WO 91/18098, and DNA
sequences encoding BMP-9 in WO 93/00432. DNA and deduced amino acid
sequences encoding BMP-10 and BMP-11 are disclosed in WO 94/26893,
and WO 94/26892, respectively. DNA and deduced amino acid sequences
for BMP-12 and BMP-13 are disclosed in WO 95/16035. The above
patent disclosures, which describe DNA and amino acid sequences,
and methods for producing the BMPs and OPs encoded by those
sequences, are incorporated herein by reference.
[0059] To clone genes that encode new BMPs, OPs and other
morphogenic proteins identified in extracts by bioassay, methods
entailing "reverse genetics" may be employed. Such methods start
with a protein of known or unknown function to obtain the gene that
encodes that protein. Standard protein purification techniques may
be used as an initial step. If enough protein can be purified to
obtain a partial amino acid sequence, a degenerate DNA probe
capable of hybridizing to the DNA sequence that encodes that
partial amino acid sequence may be designed, synthesized and used
as a probe to isolate full-length clones that encode that or a
related morphogenic protein.
[0060] Alternatively, a partially-purified extract containing the
morphogenic protein may be used to raise antibodies directed
against that protein. Morphogenic protein-specific antibodies may
then be used as a probe to screen expression libraries made from
cDNAs (see, e.g., Broome and Gilbert, Proc. Natl. Acad. Sci.
U.S.A., 75, pp. 2746-49 (1978); Young and Davis, Proc. Natl. Acad.
Sci. U.S.A., 80, pp. 31-35 (1983)).
[0061] For cloning and expressing new BMPs, OPs and other
morphogenic proteins identified based on DNA sequence homology, the
homologous sequences may be cloned and sequenced using standard
recombinant DNA techniques. With the DNA sequence available, a DNA
fragment encoding the morphogenic protein may be inserted into an
expression vector selected to work in conjunction with a desired
host expression system. The DNA fragment is cloned into the vector
such that its transcription is controlled by a heterologous
promoter in the vector, preferably a promoter which may be
optionally regulated.
[0062] Some host-vector systems appropriate for the recombinant
expression of BMPs and OPs are disclosed in the references cited
above. Useful host cells include but are not limited to bacteria
such as E. coli, yeasts such as Saccharomyces and Picia, insects
cells and other primary, transformed or immortalized eukaryotic
cultured cells. Preferred eukaryotic host cells include CHO, COS
and BSC cells (see below).
[0063] An appropriate vector is selected according to the host
system selected. Useful vectors include but are not limited to
plasmids, cosmids, bacteriophage, insect and animal viral vectors,
including those derived from retroviruses and other single and
double-stranded DNA viruses.
[0064] In one embodiment of this invention, the morphogenic protein
may be derived from a recombinant DNA molecule expressed in a
prokaryotic host. Using recombinant DNA techniques, various fusion
genes have been constructed to induce recombinant expression of
naturally sourced osteogenic sequences in E. coli (see, e.g.,
Oppermann et al., U.S. Pat. No. 5,354,557, incorporated herein by
reference). Using analogous procedures, DNAs comprising truncated
forms of naturally sourced morphogenic sequences may be prepared as
fusion constructs linked by a sequence coding for the acid labile
cleavage site (Asp-Pro) to a leader sequence (such as the "MLE
leader") suitable for promoting expression in E. coli.
[0065] In another embodiment of this invention, the morphogenic
protein is expressed using a mammalian host-vector system (e.g.,
transgenic production or tissue culture production). A morphogenic
protein so expressed may resemble more closely the naturally
occurring protein. While the glycosylation pattern of the
recombinant protein may sometimes differ from that of the natural
protein, such differences are often not essential for biological
activity of the recombinant protein. Techniques for transfection,
expression and purification of recombinant proteins are well known
in the art. See, e.g., Ausubel et al., supra, and Bendig, Genetic
Engineering, 7, pp. 91-127 (1988).
[0066] Mammalian DNA vectors should include appropriate sequences
to promote expression of the gene of interest. Such sequences
include transcription initiation, termination and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; mRNA-stabilizing sequences;
translation-enhancing sequences (e.g., Kozak consensus sequence);
protein-stabilizing sequences; and when desired, sequences that
enhance protein secretion.
[0067] Restriction maps and sources of various exemplary expression
vectors designed for OP-1 expression in mammalian cells have been
described in U.S. Pat. No. 5,354,557. Each of these vectors employs
a full-length hOP-1 cDNA sequence inserted into the pUC-18 vector.
It will be appreciated by those of skill in the art that DNA
sequences encoding truncated forms of morphogenic proteins may also
be used, provided that the expression vector or host cell provides
the sequences necessary to direct processing and secretion of the
expressed protein.
[0068] Useful promoters include, but are not limited to, the SV40
early and late promoters, the adenovirus-major late promoter, the
mouse metallothionein-I ("mMT") promoter, the Rous sarcoma virus
("RSV") long terminal repeat ("LTR"), the mouse mammary tumor virus
("MMTV") LTR, and the human cytomegalovirus ("CMV") major
intermediate-early promoter. For instance, a combination of the CMV
or MMTV promoter with an enhancer sequence from the RSV LTR has
been found to be particularly useful in expressing human osteogenic
proteins.
[0069] Preferred DNA vectors also include a marker gene (e.g.,
neomycin or DHFR) and means for amplifying the copy number of the
gene of interest. DNA vectors may also comprise stabilizing
sequences (e.g., ori- or ARS-like sequences and telomere-like
sequences), or may alternatively be designed to favor directed or
non-directed integration into the host cell genome.
[0070] One method of gene amplification in mammalian cell systems
is the use of the selectable dihydrofolate reductase (DHFR) gene in
a dhfr.sup.- cell line. Generally, the DHFR gene is provided on the
vector carrying the gene of interest, and addition of increasing
concentrations of the cytotoxic drug methotrexate (MTX) leads to
amplification of the DHFR gene copy number, as well as that of the
gene physically associated with it. DHFR as a selectable,
amplifiable marker gene in transfected Chinese hamster ovary (CHO)
cell lines is particularly well characterized in the art. Other
useful amplifiable marker genes include the adenosine deaminase
(ADA) and glutamine synthetase (GS) genes.
[0071] Gene amplification can be further enhanced by modifying
marker gene expression regulatory sequences (e.g., enhancer,
promoter, and transcription or translation initiation sequences) to
reduce the levels of marker protein produced. Lowering the level of
DHFR transcription increases the DHFR gene copy number (and the
physically-associated gene) to enable the transfected cell to adapt
to growth in even low levels of methotrexate (e.g., 0.1 .mu.M MTX).
Preferred expression vectors such as pH754 and pH752 (Oppermann et
al., U.S. Pat. No. 5,354,557, FIGS. 19C and D) have been
manipulated, using standard recombinant DNA technology, to create a
weak DHFR promoter. As will be appreciated by those skilled in the
art, other useful weak promoters, different from those disclosed
herein, can be constructed using standard methods. Other regulatory
sequences also can be modified to achieve the same effect.
[0072] Another gene amplification scheme relies on the temperature
sensitivity (ts) of BSC40-tsA58 cells transfected with an SV40
vector. Temperature reduction to 33.degree. C. stabilizes the
temperature-sensitive SV40 T antigen, which leads to the excision
and amplification of the integrated transfected vector DNA, thereby
amplifying the physically-associated gene of interest.
[0073] The choice of cells/cell lines depends on the needs of the
skilled practitioner. Monkey kidney cells (COS) provide high levels
of transient gene expression and are thus useful for rapidly
testing vector construction and the expression of cloned genes. COS
cells expressing the gene of interest can be established by
transfecting the cells with, e.g., an SV40 vector carrying the
gene. Stably transfected cell lines, on the other hand, can be used
for long term production of morphogenic proteins. By way of
example, both CHO cells and BSC40-tsA58 cells can be used as host
cells. Recombinant OP-1 has been expressed in three different cell
expression systems: COS cells for rapidly screening the
functionality of the various expression constructs, CHO cells for
the establishment of stable cell lines, and BSC40-tsA58 cells as an
alternative means of producing recombinant OP-1 protein.
[0074] Several bone-derived osteogenic proteins (OPs) and BMPs are
found as homo- and heterodimers comprising interchain disulfide
bonds in their active forms. For instance, BMP-2, BMP-4, BMP-6 and
BMP-7 (OP-1)--originally isolated from bone--are bioactive as
either homodimers or heterodimers. The ability of OPs and BMPs to
form heterodimers may confer additional or altered morphogenic
activities on morphogenic proteins. Heterodimers may exhibit
qualitatively or quantitatively different binding affinities than
homodimers for OP and BMP receptors. Altered binding affinities may
in turn result in differential activation of receptors that mediate
different signalling pathways, ultimately leading to different
biological activities. Altered binding affinities can also be
manifested in a tissue or cell type-specific manner, thereby
inducing only particular progenitor cell types to undergo
proliferation and/or differentiation.
[0075] The dimeric proteins can be isolated from the culture media
and/or refolded and dimerized in vitro to form biologically active
compositions. Heterodimers can be formed in vitro by combining
separate, distinct polypeptide chains. Alternatively, heterodimers
can be formed in a single cell by co-expressing nucleic acids
encoding separate, distinct polypeptide chains. See, e.g., WO
93/09229 and U.S. Pat. No. 5,411,941, for exemplary protocols for
heterodimer protein production.
[0076] C. In Vivo Expression of Morphogenic Proteins
[0077] The morphogenic protein of the invention can also be
produced in vivo in a patient. To achieve this, an expression
vector comprising a promoter operatively linked to a coding
sequence of the morphogenic protein may be introduced into
progenitor cells in the patient. Alternatively, one can isolate the
appropriate progenitor cells from the patient, transfect or
transduce the cells with the expression vector, and re-introduce
the treated cells to the patient at a desired locus.
[0078] (1) Vectors
[0079] A nucleic acid construct according to this invention is
derived from a non-replicating linear or circular DNA or RNA
vector, or from an autonomously replicating plasmid or viral
vector. Alternatively, the construct is integrated into the host
genome. Any vector that can transfect or transduce the desired
progenitor cell may be used. Preferred vectors are viral vectors,
including those derived from replication-defective retroviruses
(see, e.g., WO89/07136; Rosenberg et al., N. Eng. J. Med. 323(9):
570-578 (1990)), adenovirus (see, e.g., Morsey et al., J. Cell.
Biochem., Supp. 17E (1993)), adeno-associated virus (Kotin et al.,
Proc. Natl. Acad. Sci. USA 87:2211-2215 (1990)),
replication-defective herpes simplex viruses (HSV; Lu et al.,
Abstract, page 66, Abstracts of the Meeting on Gene Therapy, Sep.
22-26, 1992, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.), vaccinia virus (Mukherjee et al., Cancer Gene Ther. 7:663-70
(2000)), and any modified versions of these vectors. Methods for
constructing expression vectors are well known in the art. See,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, 2nd Edition, Cold Spring Harbor, N.Y.,
1989).
[0080] (2) Expression Control Sequences
[0081] In these vectors, expression control sequences are operably
linked to the nucleic acid sequence encoding the morphogenic
protein useful in this invention. For eukaryotic cells, expression
control sequences may include a promoter, an enhancer, such as one
derived from an immunoglobulin gene, SV40, cytomegalovirus, etc.,
and a polyadenylation sequence. A nucleic acid construct of this
invention may also contain an internal ribosome entry site
("IRES"), and an intron that may be desirably located between the
promoter/enhancer sequence and the morphogenic protein-coding
sequence. Selection of these and other common vector elements are
conventional. See, e.g., Sambrook et al, supra; Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, (1989); and references cited therein.
[0082] In one embodiment of the present invention, high-level
constitutive expression is desired. Exemplary promoters for this
purpose include, without limitation, the retroviral Rous sarcoma
virus (RSV) LTR promoter/enhancer, the cytomegalovirus (CMV)
immediate early promoter/enhancer (see, e.g., Boshart et al, Cell
41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase
promoter, the cytoplasmic .beta.-actin promoter, the
phosphoglycerol kinase (PGK) promoter. Useful promoters for BMP
expression in osteoblasts also include the Type I collagen gene
promoter and the CBFA gene promoter. Useful promoters for BMP
expression in chondrocytes and chondroblasts include the Type II
collagen gene promoter and the Type X collagen gene promoter. In
another embodiment, the native transcription-regulatory elements of
the desired morphogenic protein can be used.
[0083] Using the guidance provided by this application, one of
skill in the art may make a selection among the above expression
control sequences and modified versions thereof without departing
from the scope of this invention.
[0084] (3) Administration of Nucleic Acid Constructs
[0085] The nucleic acid constructs of this invention may be
formulated as a pharmaceutical composition for use in any form of
transient and/or stable gene transfer in vivo and in vitro. The
composition comprises at least the nucleic acid construct and a
pharmaceutically acceptable carrier such as saline. Other aqueous
and non-aqueous sterile suspensions known to be pharmaceutically
acceptable carriers and well known to those of skill in the art may
be employed also. The construct may be used for in vivo and ex vivo
gene therapy, in vitro protein production and diagnostic
assays.
[0086] The nucleic acid construct can be introduced into target
cells as naked DNA, or by, e.g., liposome fusion (see, e.g., Nabel
et al., Science 249:1285-8 (1990); Ledley, J Pediatrics 110:1-8 and
167-74 (1987); Nicolau et al., Proc Natl Acad Sci USA 80:1068-72
(1983)), erythrocyte ghosts, or microsphere methods.
(microparticles; see, e.g., U.S. Pat. No. 4,789,734; U.S. Pat. No.
4,925,673; U.S. Pat. No. 3,625,214; Gregoriadis, Drug Carriers in
Biology and Medicine, pp. 287-341, Academic Press, 1979).
[0087] If the nucleic acid construct is viral-based, it can also be
packaged as a virion which then is used to transduce a cell (e.g.,
an autologous T cell isolated from a patient) in vitro. The
infected cell is then introduced into the patient. Alternatively,
the recombinant virus may be administered to a patient directly,
e.g., locally at the tissue defect site; or intravenously,
intraperitoneally, intranasally, intramuscularly, subcutaneously,
and/or intradermally, as determined by one skilled in the gene
therapy art. A slow-release device, such as an implantable pump,
may be used to facilitate delivery of the recombinant virus to a
cell. Where the virus is administered to a subject, the specific
cells to be infected may be targeted by controlling the method of
delivery. The treatments of the invention may be repeated as
needed, as determined by one skilled in the art.
[0088] Dosages of the nucleic acid construct of this invention in
gene therapy will depend primarily on factors such as the condition
being treated. The dosage may also vary depending upon the age,
weight and health of the patient. For example, an effective human
dosage of a BMP-coding virus is generally in the range of from
about 0.5 ml to 50 ml of saline solution containing the virus at
concentrations of about 1.times.10.sup.7, 1.times.10.sup.8,
1.times.10.sup.9, 1.times.10.sup.10, 1.times.10.sup.11,
1.times.10.sup.12, 1.times.10.sup.13, 1.times.10.sup.14,
1.times.10.sup.15, or 1.times.10.sup.16 viral particles per dose
administered. The dosage will be adjusted to balance the corrective
benefits against any adverse side effects. The levels of expression
of BMP may be monitored to determine the type and frequency of
dosage administration.
[0089] D. Synthetic Non-native Morphogenic Proteins
[0090] In another embodiment of this invention, a morphogenic
protein may be prepared synthetically. Morphogenic proteins
prepared synthetically may be native, or may be non-native
proteins, i.e., those not otherwise found in nature.
[0091] Non-native morphogenic proteins can be made by mutating
native morphogenic proteins. Methods for making mutations that
favor refolding and/or assembling subunits into forms that exhibit
greater morphogenic activity have been described. See, e.g., U.S.
Pat. No. 5,399,677.
[0092] Non-native morphogenic proteins can also be synthesized
using a series of consensus sequences (U.S. Pat. No. 5,324,819).
These consensus sequences were designed based on partial amino acid
sequence data obtained from native osteogenic products and on their
homologies with other proteins reportedly having a presumed or
demonstrated developmental function. Several biosynthetic consensus
sequences (called consensus osteogenic proteins or "COPs") have
been expressed as fusion proteins in prokaryotes. Purified fusion
proteins may be cleaved, refolded, combined with a hormone and a
soluble receptor thereof, implanted in an established animal model
and examined for their bone- and/or cartilage-inducing activity.
Certain preferred synthetic osteogenic proteins comprise one or
both of two synthetic amino acid sequences designated COP5 (SEQ ID
NO:11) and COP7 (SEQ ID NO: 12).
[0093] The amino acid sequences of COP5 and COP7 are shown below,
as set forth in Oppermann et al., U.S. Pat. Nos. 5,011,691 and
5,324,819, which are incorporated herein by reference:
6 COP5 LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD COP7
LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD COP5
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA COP7
HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA COP5
ISMLYLDENEKVVLKYNQEMVVEGCGCR (SEQ ID NO:11) COP7
ISMLYLDENEKVVLKYNQEMVVEGCGCR (SEQ ID NO:12)
[0094] In these amino acid sequences, the dashes (-) are used as
fillers only to line up comparable sequences in related proteins.
Differences between the aligned amino acid sequences are
highlighted.
[0095] In one embodiment of this invention, the morphogenic protein
is a synthetic osteogenic protein comprising a partial or complete
sequence of a generic sequence described above (SEQ ID NO:4, 5, 6,
7, or 10) such that it is capable of inducing tissue formation when
properly folded and implanted in a mammal. For instance, the
synthetic protein can induce bone formation from osteoblasts when
implanted in a favorable environment; or it can promote cartilage
formation when implanted in an avascular locus or when
co-administered with an inhibitor of full bone development.
[0096] In another embodiment, the synthetic morphogenic protein of
this invention comprises a sequence sufficiently duplicative of a
partial or complete sequence of a COP, e.g., COP5 (SEQ ID NO:11) or
COP7 (SEQ ID NO:12). Biosynthetic COP sequences are believed to
dimerize during refolding and appear not to be active when reduced.
Both homodimeric and heterodimeric COPs are contemplated in this
invention. In certain embodiments, this synthetic protein is less
than about 200 amino acids long.
[0097] These and other synthetic non-native osteogenic proteins may
be used in concert with a hormone/receptor pair and tested using in
vitro, ex vivo or in vivo bioassays for progenitor cell induction
and tissue regeneration. The proteins in conjunction with the
hormone/receptor pairs of this invention are envisioned to be
useful for the repair and regeneration of bone, cartilage, tendon,
ligament, neural and potentially other types of tissue.
[0098] Homologous Proteins Having Morphogenic Activity
[0099] The morphogenic proteins useful in this invention may be
produced by recombinant expression of DNA sequences isolated based
on homology with the osteogenic COP consensus sequences described
above. Synthetic COP DNA sequences may be used as probes to
retrieve related DNA sequences from a variety of species (see,
e.g., Oppermann et al., U.S. Pat. Nos. 5,011,591 and 5,258,494,
which are incorporated herein by reference).
[0100] Morphogenic proteins encoded by a gene that hybridizes with
a COP sequence probe are assembled into two subunits
disulfide-bonded to produce a heterodimer or homodimer capable of
inducing tissue formation when implanted into a mammal. Recombinant
BMP-2 and BMP-4 have been shown to have cross-species osteogenic
activity as homodimers and as heterodimers assembled with OP-1
subunits. Morphogenic protein-encoding genes that hybridize to
synthetic COP sequence probes include genes encoding Vg1, inhibin,
DPP, OP-1, BMP-2 and BMP-4. Vg1 is a known Xenopus laevis
morphogenic protein involved in early embryonic patterning. Inhibin
is another developmental gene that is a member of the BMP family of
proteins from Xenopus laevis. DPP is an amino acid sequence encoded
by a Drosophila gene responsible for development of the
dorso-ventral pattern. OP-1, BMP-2 and BMP-4 are osteogenic
proteins that can induce cartilage, bone and neural tissue
formation.
[0101] In another embodiment of this invention, a morphogenic
protein may comprise a polypeptide encoded by a nucleic acid that
hybridizes under stringent conditions to an "OPS" nucleic acid
probe (Oppermann et al., U.S. Pat. No. 5,354,557). "OPS"--standing
for OP-1 "short"--refers to the portion of the human OP-1 protein
defining the conserved 6 cysteine skeleton in the C-terminal active
region (97 amino acids; SEQ ID NO:2, residues 335-431).
[0102] One example of a stringent hybridization condition is
hybridization in 4.times. SSC at 65.degree. C. (or 10.degree. C.
higher than the calculated melting temperature for a hybrid between
the probe and a nucleic acid sequence containing no mis-matched
base pairs), followed by washing in 0.1.times. SSC at the
hybridization temperature. Another stringent hybridization
condition is hybridization in 50% formamide, 4.times. SSC at
42.degree. C.
[0103] Thus, in view of this disclosure, the skilled practitioner
can readily design and synthesize genes, or isolate genes from cDNA
or genomic libraries that encode amino acid sequences having
morphogenic activity. These genes can be expressed in prokaryotic
or eukaryotic host cells to produce large quantities of active
osteogenic or otherwise morphogenic proteins. The recombinant
proteins may be in native, truncated, mutant, fusion, or other
active forms capable of inducing formation of bone, cartilage, or
other types of tissue, as demonstrated by in vitro and ex vivo
bioassays and in vivo implantation in mammals, including
humans.
[0104] Hormones and Receptors Thereof
[0105] A hormone/receptor pair of this invention is capable of
stimulating the ability of a morphogenic protein to induce tissue
formation from a progenitor cell. In a method of this invention,
the tissue inductive activity of a morphogenic protein in a mammal
is improved by co-administering effective amounts of a hormone and
a soluble receptor thereof. Alternatively, the morphogenic protein
and the hormone/receptor pair are administered sequentially. It has
been found that the synergism between a morphogenic protein and a
hormone/receptor pair is preserved even if the morphogenic protein
is administered 4 to 8 hours before the hormone/receptor pair. The
morphogenic protein, the hormone, and the hormone receptor can also
be administered separately.
[0106] One or more hormone/receptor pairs can be selected for use
in concert with one or more morphogenic proteins according to the
desired tissue type to be induced and the site at which the
treatment will be administered. The particular choice of a
morphogenic protein(s)/hormone(s)/receptor(s) combination and the
relative concentrations at which they are combined may be varied
systematically to optimize the tissue type induced at a selected
treatment site using the procedures described herein.
[0107] Hormones useful in this invention include, but are not
limited to, interleukins. 1 through 8, fibroblast growth factor,
vascular endothelial growth factor, platelet-derived growth factor,
TGF-.beta., and prostaglandins (e.g., E1 and E2). It may be
preferred that the target cell has a cell-surface receptor for the
hormone. The hormones can also be morphogenic proteins such as
GDFs; as a result, the composition of this invention will contain
two morphogenic proteins and a soluble receptor of one of these
proteins.
[0108] One preferred hormone/receptor pair of this invention is
IL-6/sIL-6R. IL-6 is a member of a subfamily of multifunctional
hormones. It appears to play a role in both bone formation and bone
resorption by affecting mitogenesis of target cells and regulating
the synthesis of other local factors. Clinical studies show that
IL-6 is involved in a variety of diseases, such as fibrous
dysplasia, osteopenia, osteoporosis and Paget's disease.
Recombinant full length human IL-6 (26 kD) expressed from E. coli
can be obtained from Sigma (St. Louis, Mo.) and Promega (Madison,
Wis.). Recombinant sIL-6R produced from baculovirus and containing
the entire extracellular domain (residues 1-339; 38 kD) of human
IL-6R can be obtained from Sigma and R&D Systems (Minneapolis,
Minn.). See also Examples 1 and 2, infra. Active allelic, species
or other variants of these IL-6 and sIL-6 products can also be
used.
[0109] The hormone or the hormone receptor of this invention can be
associated with an agent that is capable of increasing the
hormone's or receptor's bio-activity, e.g., synthesis, half-life,
bio-availability, and reactivity with other bio-molecules such as
binding proteins and receptors. These agents may contain carrier
molecules such as proteins and lipids.
[0110] The hormone and hormone receptor are present in amounts
capable of synergistically stimulating the tissue inductive
activity of the morphogenic protein in a mammal. The relative
concentrations of morphogenic protein, hormone and hormone receptor
that optimally induce tissue formation may be determined
empirically by the skilled practitioner using the procedures
described herein.
[0111] Progenitor Cells
[0112] The progenitor cell that is induced to proliferate and/or
differentiate by the morphogenic protein of this invention is
preferably a mammalian cell. Examples of useful progenitor cells
are mammalian chondroblasts, osteoblasts and neuroblasts, all
earlier developmental precursors thereof, and all cells that
develop therefrom (e.g., prechondroblasts and chondrocytes). The
progenitor cell may be induced to form one or more tissue types
such as endochondral or intramembranous bone, cartilage,
tendon/ligament-like tissue, neural tissue and kidney tissue. The
specific morphogenic activity exhibited by a morphogenic protein
will depend in part on the type of the progenitor cell as well as
the treatment site. These variables may be tested empirically.
[0113] Morphogenic proteins are highly conserved throughout
evolution, and non-mammalian progenitor cells are likely to be
stimulated by same- or cross-species morphogenic proteins and
hormone/receptor combinations. It is thus envisioned that where
schemes are available for implanting xenogeneic cells into humans
without adverse immunological reactions, non-mammalian progenitor
cells stimulated by morphogenic protein and a hormone/receptor pair
according to the procedures set forth herein will be useful for
tissue regeneration and repair in humans.
[0114] Testing Morphogenic Activity
[0115] To identify a hormone/receptor pair capable of stimulating
the tissue inductive activity of a chosen morphogenic protein, an
appropriate assay is selected. Initially, in vitro assays can be
performed. A useful in vitro assay may monitor a nucleic acid or
protein marker whose expression is known to correlate with the
associated cell differentiation pathway. See, e.g., Examples 3 and
4 of U.S. Pat. No. 5,854,207, Lee et al.; and Examples 1 and 2,
infra.
[0116] Examples 1 and 2, infra, describe experiments using OP-1 to
identify and to optimize an effective concentration of IL-6 and
sIL-6R. OP-1 is known to have osteogenic and neurogenic activity.
Thus, to identify a hormone/receptor pair having synergistic
effects with OP-1, one can conduct an in vitro assay that examines
the expression of a molecular marker, e.g., an osteogenic- or a
neurogenic-associated marker, in appropriate progenitor cells.
[0117] One useful assay for testing potential hormone/receptor
pairs with OP-1 for osteogenic activity is the alkaline phosphatase
("AP") enzymatic assay. AP is an osteoblast differentiation marker
in primary osteoblastic fetal rat calvarial ("FRC") cells. The
OP-1stimulated AP activity results from increased steady-state AP
mRNA levels. Other useful protein markers for monitoring osteogenic
activity of a composition include, but are not limited to, type I
collagen, osteocalcin, osteopontin, bone sialoprotein and
PTH-dependent cAMP levels.
[0118] An AP assay is performed generally as follows. First, a
hormone/receptor pair is identified by picking various
concentrations and ratios of the hormone and hormone receptor and
testing them in the absence and presence of a morphogenic protein.
Second, the amounts of hormone and hormone receptor required to
achieve optimal, preferably synergistic, tissue induction in
concert with the morphogenic protein is determined by generating
dose response curves.
[0119] Optionally, additional hormone/receptor pairs that further
stimulate or otherwise alter the morphogenic activity induced by a
morphogenic protein and a first hormone/receptor pair may be
identified and a new multi-factor dose response curve generated.
See, e.g., Example 5 of U.S. Pat. No. 5,854,207.
[0120] Bone Induction Assays
[0121] The various morphogenic compositions and devices of this
invention can also be evaluated with ex vivo or in vivo bioassays.
A rat bioassay for bone induction may be used to monitor osteogenic
activity of osteogenic proteins in concert with one or more
hormone/receptor pairs. See, e.g., Sampath et al., Proc. Natl.
Acad. Sci. USA, 80, pp. 6591-95 (1983), and U.S. Pat. No.
5,854,207, Example 7. Rat bioassays are useful as the first step in
moving from in vitro studies to in vivo studies.
[0122] Large animal efficacy models for osteogenic device testing
are known in the art. Exemplary models are the feline femoral
model, the rabbit ulnar model, the dog ulnar model and the monkey
model. See, e.g., U.S. Pat. No. 5,354,557, and 5,854,207 (Examples
10 and 11 therein).
[0123] In general, about 500-1000 ng of active morphogenic protein
and about 10-200 ng of active hormone and active hormone receptor
are combined with 25 mg of a carrier matrix for rat bioassays. In
larger animals, typically about 0.8-Img of active morphogenic
protein per gram of carrier is combined with about 100 ng or more
of an active hormone and hormone receptor. The optimal ratios of
morphogenic protein to hormone and of hormone to hormone receptor
for a specific tissue type may be determined empirically by those
of skill in the art according to the procedures set forth herein.
Greater amounts may be used for large implants.
[0124] Tendon/Ligament-Like Tissue Formation Bioassay
[0125] Assays for monitoring tendon and ligament-like tissue
formation induced by morphogenic proteins are known in the art.
See, e.g., Celeste et al., WO 95/16035, hereby incorporated by
reference. Such assays can be used to identify hormone/receptor
pairs that stimulate tendon/ligament-like tissue formation by
BMP-12, BMP-13 or other morphogenic proteins in a particular
treatment site. The assays may also be used to optimize
concentrations and treatment schedules for therapeutic tissue
repair regiments.
[0126] These assays may be used to test various combinations of
morphogenic protein and hormone/receptor combinations, and to
produce an in vivo dose response curve for determining the
effective relative concentrations of morphogenic proteins and
hormones/receptors.
[0127] Neural Assays
[0128] The osteogenic proteins BMP-4 and BMP-7 (OP-1) can induce
ventral neural plate explants to undergo differentiation into
dorsal neural cell fates (Liem et al., Cell, 82, pp. 969-79
(1995)). Molecular markers of dorsal cell differentiation are
described in Liem et al. These markers include PAX3 and MSX, whose
expression delineates an early stage of neural plate cell
differentiation; DSL-1, a BMP-like molecule delineating
differentiation of dorsal neural plate cells at a stage after
neural tube closure; and SLUG protein, whose expression after
neural tube closure defines pre-migratory neural crest cells.
Expression of these dorsal markers can be induced in ventral neural
plate explants by ectopic BMP4 and OP-1.
[0129] A peripheral nerve regeneration assay using BMP-2 has been
described (Wang et al., WO 95/05846, hereby incorporated by
reference). The assay involves the implantation of neurogenic
devices in the vicinity of severed sciatic nerves in rats. This
procedure may be used to assess the ability of a putative
hormone/receptor pair to enhance the neuronal inductive activity of
homo- and heterodimers of morphogenic proteins having neurogenic
activity, such as BMP-2, BMP-4, BMP-6 and OP-1, or of any selected
neurogenic protein/hormone/hormone receptor combinations.
[0130] Pharmaceutical Compositions
[0131] The pharmaceutical compositions of this invention contain at
least one (e.g., at least 2, 3 or 5) morphogenic protein, and at
least one (e.g., at least 2 or 3) hormone/receptor pair. These
compositions are capable of inducing tissue formation when
administered, e.g., implanted, into a patient. The compositions
will be administered at an effective dose to induce the particular
type of tissue at the desired treatment site. Determination of a
preferred pharmaceutical formulation and a therapeutically
efficient dose regiment for a given application is well within the
skill of the art. Factors that need to be considered include, for
example, the administration mode, the condition and weight of the
patient, the extent of desired treatment and the tolerance of the
patient for the treatment.
[0132] Doses expected to be suitable starting points for optimizing
treatment regiments are based on the results of in vitro assays,
and ex vivo or in vivo assays. Based on the results of such assays,
a range of suitable morphogenic protein and hormone/receptor
concentration ratios can be selected to test at a treatment site in
animals and then in humans.
[0133] The pharmaceutical compositions of this invention may be in
a variety of forms. These include, for example, solid, semi-solid
and liquid forms such as tablets, pills, powders, liquids,
suspensions, suppositories, gels, pastes, and other injectable and
infusible solutions. The preferred form depends on the intended
mode of administration and therapeutic application. Modes of
administration may include oral, parenteral, subcutaneous,
intravenous, intralesional or topical administration. In most
cases, the pharmaceutical compositions of this invention will be
administered in the vicinity of or at the treatment site in need of
tissue regeneration or repair.
[0134] The pharmaceutical compositions of this invention may, for
example, be placed into sterile, isotonic formulations with or
without co-factors which stimulate uptake or stability. For
example, the compositions may contain a formulation buffer
comprising 5.0 mg/ml citric acid monohydrate, 2.7 mg/ml trisodium
citrate, 41 mg/ml mannitol, 1 mg/ml glycine and 1 mg/ml polysorbate
20. This solution can be lyophilized, stored under refrigeration
and reconstituted prior to administration with sterile
Water-For-Injection (USP).
[0135] The compositions may also include pharmaceutically
acceptable carriers well known in the art. See, for example,
Remington's Pharmaceutical Sciences, 16th Edition, 1980, Mac
Publishing Company. Such pharmaceutically acceptable carriers may
include other medicinal agents, carriers, genetic carriers,
adjuvants, and excipients such as human serum albumin or plasma
preparations. The compositions may be in the form of a unit dose
and will usually be administered as a dose regiment that depends on
the particular tissue treatment.
[0136] The pharmaceutical compositions of this invention may also
be administered in form of a morphogenic device using, for example,
microspheres, liposomes, other microparticulate delivery systems or
sustained release formulations placed in, near, or otherwise in
communication with affected tissues or the bloodstream bathing
those tissues.
[0137] Liposomes containing the polypeptide mixtures of this
invention can be prepared by well-known methods. See, e.g. DE
3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82, pp.
3688-92 (1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77,
pp. 4030-34 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545.
Ordinarily the liposomes are of the small (about 200-800 Angstroms)
unilamellar type in which the lipid content is greater than about
30 mol. % cholesterol. The proportion of cholesterol is selected to
control the optimal release rate of the polypeptides of
interest.
[0138] The polypeptide mixtures of this invention may also be
attached to liposomes containing other biologically active
molecules to modulate the rate and characteristics of tissue
induction. Such attachment may be accomplished by cross-linking
agents such as heterobifunctional cross-linking agents that have
been widely used to couple toxins or chemotherapeutic agents to
antibodies for targeted delivery. Conjugation to liposomes can also
be accomplished using the carbohydrate-directed cross-linking
reagent 4-(4-maleimidophenyl)butyric acid hydrazide. See, e.g.,
Duzgunes et al., J. Cell. Biochem. Abst., Suppl. 16E 77 (1992).
[0139] Morphogenic Devices
[0140] The pharmaceutical compositions of this invention can
additionally contain an implantable, biocompatible carrier. Such
compositions are also called morphogenic devices. The carrier
functions as a sustained release delivery system for the
therapeutic proteins and protects the proteins from non-specific
proteolysis. The carrier may be biodegradable in vivo. A sustained
release carrier may contain semipermeable polymer matrices in the
form of shaped articles, e.g., suppositories or capsules. Such a
carrier can be made of polylactides (U.S. Pat. No. 3,773,319; EP
58,481), copolymers of L-glutamic acid and ethyl-L-glutamate
(Sidman et al., Biopolymers, 22, pp. 547-56 (1985));
poly(2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer
et al., J. Biomed. Mater. Res., 15, pp. 167-277 (1981); Langer,
Chem. Tech., 12, pp. 98-105 (1982)).
[0141] The carrier may also serve as a temporary scaffold and
substratum for recruitment of migratory progenitor cells and their
subsequent anchoring and proliferation, until replaced by new bone
or other appropriate tissue. For example, the carrier may contain a
biocompatible matrix made up of particles or otherwise having the
desired porosity or microtexture. The pores will permit migration,
anchoring, differentiation and proliferation of the relevant
progenitor cells. The particle size may be within the range of 70
.mu.m-850 .mu.m, e.g., 70 .mu.m-420 .mu.m or 150 .mu.m-420 .mu.m. A
particulate matrix may be fabricated by close packing particulate
materials into a shape spanning the tissue defect to be treated.
Various matrices known in the art can be employed. See, e.g., U.S.
Pat. Nos. 4,975,526, 5,162,114 and 5,171,574, and WO 91/18558, all
of which are herein incorporated by reference.
[0142] Useful matrix materials include but are not limited to
collagen; celluloses, including carboxymethyl cellulose; homo- or
co-polymers of glycolic acid, lactic acid, and butyric acid,
including derivatives thereof; and ceramics, such as
hydroxyapatite, tricalcium phosphate and other calcium phosphates.
See, e.g., U.S. Pat. No. 5,854,207, col. 25, line 59, through col.
27, line 6. Various combinations of these or other suitable matrix
materials may be useful as determined by the assays set forth
herein. The choice of material depends in part on its in vivo
dissolution rate. In bones, the dissolution rates can vary
according to whether the implant is placed in cortical or
trabecular bone.
[0143] Other useful matrices include particulate, demineralized,
guanidine-extracted, allogenic bone; and specially treated,
particulate, protein-extracted, demineralized xenogenic bone. See,
e.g., Example 6 of U.S. Pat. No. 5,854,207. Such xenogenic bone
powder matrices may be treated with proteases such as trypsin.
Preferably, the xenogenic matrices are treated with one or more
fibril-modifying agents to increase the intraparticle intrusion
volume (porosity) and surface area. Useful modifying agents include
solvents such as dichloromethane, trichloroacetic acid,
acetonitrile and acids such as trifluoroacetic acid and hydrogen
fluoride. A preferred fibril-modifying agent is in the form of a
heated aqueous medium, preferably an acidic aqueous medium having a
pH less than about 4.5, most preferably having a pH between about 2
and 4, inclusive. The acidic aqueous medium can, for instance, be
0.1% acetic acid with a pH of about 3. Heating demineralized,
delipidated, guanidine-extracted bone collagen in an aqueous medium
at elevated temperatures (e.g., at about 37.degree. C.-65.degree.
C., preferably at about 45.degree. C.-60.degree. C.) for
approximately one hour is generally sufficient to achieve the
desired surface morphology. It is hypothesized that the heat
treatment alters the collagen fibrils, resulting in an increase in
the particle surface area.
[0144] Xenogenic bone matrices can be used in a variety of clinical
settings. In addition to its use as a matrix for bone formation in
various orthopedic, periodontal, and reconstructive procedures, the
matrix also may be used as a sustained release carrier, or as a
collagenous coating for orthopedic or general prosthetic
implants.
[0145] Demineralized guanidine-extracted xenogenic bovine bone
contains a mixture of additional materials that may be fractionated
further using standard biomolecular purification techniques. For
example, bone extracts can be fractionated by chromatography, and
the various extract fractions corresponding to the chromatogram
peaks can be added back together to an active matrix. Doing so may
remove inhibitors of bone or tissue-inductive activity, thereby
improving matrix properties.
[0146] Besides morphogenic proteins and hormone/receptor pairs, the
morphogenic devices of this invention may additionally contain
other hormones and trophic agents. The devices may also contain
antibiotics, chemotherapeutic agents, enzymes, enzyme inhibitors
and other bioactive agents. These ingredients may be adsorbed onto
or dispersed within the carrier, and will be released over time at
the implantation site as the carrier material is slowly
absorbed.
[0147] General Consideration of Matrix Properties
[0148] Factors influencing the performance of a matrix include
matrix geometry, particle size (if the matrix is made up of
particles), the methodology for combining the matrix and
morphogenic proteins, the degree of both intra- and inter-particle
porosity, the presence of mineral, and the presence of surface
charge. For example, studies have shown that, in bone induction
using OP-1 and a morphogenic protein stimulating factor,
perturbation of the matrix charge by chemical modifications can
abolish bone inductive responses. Particle size also influences the
quantitative response of new bone, with sizes between 701 .mu.m and
420 .mu.m capable of eliciting the maximum response. Further,
contamination of the matrix with bone mineral may inhibit bone
formation. Individual heavy metal concentrations in a bone matrix
can be reduced to less than about 1 ppm by the methods described
herein.
[0149] The sequential cellular reactions at the interface of the
bone matrix and an osteogenic protein implant are complex. The
multi-step cascade includes: binding of fibrin and fibronectin to
the implanted matrix, migration and proliferation of mesenchymal
cells, differentiation of the progenitor cells into chondroblasts,
cartilage formation, cartilage calcification, vascular invasion,
bone formation, remodeling, and bone marrow differentiation. A
successful matrix is capable of accommodating each of these
steps.
[0150] The matrix may be shaped as desired in anticipation of
surgery or shaped by the physician or technician during surgery. It
has been shown that new bone is formed essentially with the
dimensions of the implanted device. In the case where the matrix
material is biodegradable in vivo, the matrix material is slowly
absorbed by the body and is replaced by new bone in the shape of,
or very nearly the shape of, the implant. Thus, the matrix is
preferably shaped to span a tissue defect and to take the desired
form of the new tissue. For example, in the case of bone repair of
a non-union defect, it is desirable to use dimensions that span the
non-union, and the new bone will eventually fill the defect.
[0151] The matrix may be a shape-retaining solid made of
loosely-adhered particulate material, e.g., collagen.
Alternatively, the matrix may be a molded, porous solid, or an
aggregation of close-packed particles held in place by surrounding
tissue. Masticated muscle or other tissue may also be used. Large
allogenic bone implants can act as a carrier for the matrix if
their marrow cavities are cleaned and packed with particles
containing dispersed osteogenic protein and hormone/receptor
pair.
[0152] The matrix may also take the form of a paste or a hydrogel.
"Hydrogel" refers to a three dimensional network of cross-linked
hydrophilic polymers in the form of a gel. The gel is substantially
composed of water, for instance, greater than 90% water. Hydrogel
matrices can carry a net positive or net negative charge, or may be
neutral. A typical net negative charged matrix is alginate.
Hydrogels carrying a net positive charge are, for example,
extracellular matrix components such as collagen and laminin.
Examples of commercially available extracellular matrix components
include MATRIGEL.TM. and VITROGEN.TM.. Example of a net neutral
hydrogel are highly cross-linked polyethylene oxide and polyvinyl
alcohol.
[0153] Prosthetic Devices
[0154] This invention also features an implantable prosthetic
device comprising at least one morphogenic protein and at least one
hormone/receptor pair at therapeutic amounts and ratios. The device
can be used in conjunction with a composition containing the same
or other morphogenic protein or hormone/receptor pair. The
prosthetic device may be made from a material containing metal or
ceramic. Exemplary prosthetic devices are hip devices, screws, rods
and titanium cages for spine fusion. The device is implanted in a
mammal (e.g., a human) at a locus where the target tissue and the
surface of the prosthetic device are maintained at least partially
in contact for a time sufficient to permit enhanced tissue growth
between the target tissue and the device.
[0155] The osteogenic composition may be disposed on the prosthetic
implant on a surface region that is to be positioned next to a
target tissue in the mammal. Preferably, the mammal is a human
patient. The composition is disposed in an amount sufficient to
promote enhanced tissue growth into the implant or onto its
surface. The amount of the composition to be used may be determined
empirically by using bioassays such as those described herein and
in Rueger et al., U.S. Pat. No. 5,344,654, which is incorporated
herein by reference. Preferably, animal studies are performed to
optimize the concentration of the ingredients in the device before
a similar prosthetic device is used in a human patient. The
prosthetic devices will be useful for repairing orthopedic defects,
injuries or anomalies in the treated mammal.
[0156] Utility of Morphogenic Compositions and Devices
[0157] The compositions, devices and methods of this invention will
permit a physician to treat a variety of tissue injuries, tissue
degenerations, and other diseased tissue conditions. The
compositions and devices can ameliorate or remedy these conditions
by stimulating local tissue formation or regeneration.
[0158] The devices of this invention may be implanted at the
desired locus in a mammal such that the implant is accessible to
the appropriate progenitor cells of this mammal. The devices may be
used alone or in combination with other therapies for tissue repair
and regeneration.
[0159] The morphogenic devices of this invention may also be
implanted in or surrounding a joint for use in cartilage and soft
tissue repair, or in or surrounding nervous system-associated
tissue for use in neural regeneration and repair. The tissue
specificity of the particular morphogenic protein--or combination
of morphogenic proteins with other biological factors--will
determine the cell types or tissues that will be amenable to such
treatments and can be selected by one skilled in the art. The
ability to enhance morphogenic protein-induced tissue regeneration
by co-administering a hormone/receptor pair according to the
present invention is thus not believed to be limited to any
particular cell-type or tissue.
[0160] The osteogenic compositions and devices of this invention
will permit the physician to obtain predictable bone, ligament
and/or cartilage formation using less osteogenic protein to achieve
at least about the same extent of bone or cartilage formation. The
osteogenic compositions and devices of this invention may be used
to treat more effectively the injuries, anomalies and disorders
that have been described in the prior art of osteogenic devices.
These include, for example, forming local bone in fractures,
non-union fractures, fusions and bony voids such as those created
in tumor resections or those resulting from cysts; treating
acquired and congenital craniofacial and other skeletal or dental
anomalies (see e.g., Glowacki et al., Lancet, 1, pp. 959-63
(1981)); performing dental and periodontal reconstructions where
lost bone replacement or bone augmentation is required such as in a
jaw bone; and supplementing alveolar bone loss resulting from
periodontal disease to delay or prevent tooth loss (see e.g.,
Sigurdsson et al., J Periodontol., 66, pp. 511-21 (1995)).
[0161] An osteogenic device of this invention that comprises a
matrix comprising allogenic bone may also be implanted at a site in
need of bone replacement to accelerate allograft repair and
incorporation in a mammal. Another potential clinical application
of the improved osteogenic devices of this invention is in
cartilage repair, for example, following joint injury or in the
treatment of osteoarthritis. The ability to enhance the
cartilage-inducing activity of morphogenic proteins by
co-administering a hormone/receptor pair may permit faster or more
extensive tissue repair and replacement using the same or lower
levels of morphogenic proteins.
[0162] The morphogenic compositions and devices of this invention
will be useful in treating certain congenital diseases and
developmental abnormalities of cartilage, bone and other tissues.
For example, homozygous OP-1-deficient mice die within 24 hours
after birth due to kidney failure (Luo et al., J Bone Min. Res., 10
(Supp. 1), pp. S163 (1995)). Kidney failure in these mice is
associated with the failure to form renal glomeruli due to lack of
mesenchymal tissue condensation. OP-1-deficient mice also have
various skeletal abnormalities associated with their hindlimbs, rib
cage and skull, are polydactyl, and exhibit aberrant retinal
development. These results, in combination with those discussed
above concerning the ability of OP-1 to induce differentiation into
dorsal neural cell fates, indicate that OP-1 plays an important
role in epithelial-mesenchymal interactions during development. It
is anticipated that the compositions, devices and methods of this
invention will be useful for ameliorating these and other
developmental abnormalities.
[0163] Developmental abnormalities of the bone may affect isolated
or multiple regions of the skeleton or of a particular supportive
or connective tissue type. These abnormalities often require
complicated bone transplantation procedures and orthopedic devices.
The tissue repair and regeneration required after such procedures
may occur more quickly and completely with the use of morphogenic
compositions, devices and methods of this invention.
[0164] Examples of heritable conditions, including congenital bone
diseases, for which use of the morphogenic compositions and devices
of this invention will be useful include osteogenesis imperfecta,
the Hurler and Marfan syndromes, and several disorders of
epiphyseal and metaphyseal growth centers such as is presented in
hypophosphatasia, a deficiency in alkaline phosphatase enzymatic
activity.
[0165] Inflammatory joint diseases may also benefit from the
improved methods, compositions and devices of this invention. These
diseases include but are not limited to rheumatoid and psoriatic
arthritis, bursitis, ulcerative colitis, regional enteritis,
Whipple's disease, ankylosing spondylitis (also called Marie
Strumpell or Bechterew's disease), and the so-called "collagen
diseases" such as systemic lupus erythematosus (SLE), progressive
systemic sclerosis (scleroderma), polymyositis (dermatomyositis),
necrotizing vasculitides, Sjogren's syndrome (sicca syndrome),
rheumatic fever, amyloidosis, thrombotic thrombocytopenic purpura
and relapsing polychondritis. Heritable disorders of connective
tissue include Marfan's syndrome, homocystinuria, Ehlers-Danlos
syndrome, osteogenesis imperfecta, alkaptonuria, pseudoxanthoma
elasticum, cutis laxa, Hurler's syndrome, and myositis ossificans
progressiva.
EXAMPLES
[0166] The following are examples which illustrate the morphogenic
compositions and devices of this invention, and methods used to
characterize them. These examples should not be construed as
limiting. They are included for purposes of illustration and the
present invention is limited only by the claims.
Example 1
[0167] FIG. 1 shows the effects of IL-6, sIL-6R, and mixtures of
recombinant human IL-6 and recombinant human sIL-6R ("IL-6/R"),
respectively, on the OP-1-induced AP activity in FRC cells.
Confluent FRC cells were treated with the indicated agent(s) for 24
hrs. The concentrations of agent(s) used (ng/ml) are indicated in
parentheses. For IL-6/R, the molar ratio of the two was maintained
at about 1.2, and their respective amounts are indicated also in
parentheses. Total AP activity was determined
spectrophotometrically. Total cellular protein was determined by
the Bradford Assay. Specific AP activity was calculated as
AP/protein unit. AP activity values were normalized to that of 150
ng/ml OP-1 (=1) and represent the means of 8-12 independent
determinations using 3 different FRC cell preparations.
[0168] As shown in FIG. 1, IL-6 alone in the concentration range
tested did not stimulate the basal AP activity. SIL-6R alone
stimulated the basal AP activity slightly; however, the stimulation
did not seem to be sIL-6R dose-dependent.
[0169] FIG. 1 further demonstrates that IL-6 potentiates the
OP-1-induced AP activity in a dose-dependent manner. A maximum of
about 2-fold stimulation was observed (p<0.05). SIL-6R also
potentiated the OP-1-induced AP activity in a dose-dependent
manner. A higher fold (about 3.5-fold; p<0.02) of stimulation
than observed with IL-6 was achieved.
[0170] The effect of IL-6/R on the OP-1-induced AP activity was
also examined. At the highest tested dose of IL-6 plus its soluble
receptor (10 ng/ml IL-6 and 125 ng/ml sIL-6R), the OP-1-induced AP
activity was synergistically enhanced by about 10-fold. This
enhancement was reproducible. However, at the lower dose range, the
IL-6/R combination did not appear to stimulate beyond what was
achieved by either IL-6 or its receptor alone; on the contrary, the
combination appeared to suppress AP activity.
Example 2
[0171] FIG. 2 shows that IL-6 alone enhanced OP-1 action in a
mineralized bone nodule formation assay. FRC cells were grown in
.alpha.MEN (supplemented with 5% FBS, 30 .mu.g/ml gentamycin, 100
.mu.g/ml ascorbic acid and 5 mM .beta.-glycerolphosphate), and
treated for various durations of time with (1) solvent vehicle, (2)
200 ng/ml OP-1, or (3) 200 ng/ml OP-1 plus IL-6 at various
concentrations. The culture media were replenished with the same
treatment agent(s) every three days. Progress of nodule formation
was monitored every three days. After a total of 15 days, cells
were fixed with formalin and photographed. As seen in FIG. 3,
IL-6/R also enhanced OP-1's ability to induce the formation of
mineralized bone nodules.
Example 3
[0172] To determine whether IL-6/R effects its synergy with OP-1 by
directly stimulating OP-1 responsive cells or by increasing the
number of OP-1 responsive cells, primary cultures of FRC were used
as a model system, in which AP activity levels were used as a
biochemical marker of OP-1 responsiveness. Histochemical data
showed that the number of AP positive cells in cultures treated
with IL-6/R (40 ng/ml IL-6 and 50 ng/ml sIL-6R) and OP-1 (200
ng/ml) was similar to that in cultures treated with OP-1 alone (200
ng/ml). However, the AP activity level was higher in the former
cultures than the latter cultures. IL-6 alone (40 ng/ml) did not
stimulate AP positive cells; and sIL-6R alone (50 ng/ml) or IL-6R
(40 ng/ml IL-6 and 50 ng/ml sIL-6R) stimulated AP positive cells to
a smaller extent, as compared to the combination of IL-6R and OP-1.
These data suggest that IL-6/R's synergistic effect on OP-1 results
from IL-6/R's direct stimulation of OP-1 responsive cells.
Example 4
[0173] To investigate whether IL-6/R stimulates the expression of
OP-1 receptors on FRC cells, the mRNA levels of three BMP type I
receptors (BMPR-IA, BMPR-IB, and ActR-I) and one BMP type II
receptor (BMPR-II) were measured by Northern blot analysis.
[0174] Briefly, confluent FRC cells were treated for 48 hours with
(1) a vehicle; (2) 200 ng/ml OP-1; (3) 40 ng/ml IL-6 and 50 ng/ml
sIL-6R; or (4) 200 ng/ml OP-1, 40 ng/ml IL-6, and 50 ng/ml sIL-6R.
Total RNA was isolated using the TR1 reagent (Sigma) and loaded
onto an AGAROSE GTG (FMC) gel containing formaldehyde. Northern
blots were prepared and probed with .sup.32P-labeled cDNA encoding
for the various BMPRs. These probes hybridized only to mRNA. The
radioactive bands were detected and quantified using a
PHOSPHORIMAGER (Molecular Dynamics, Sunnyvale, Calif.). To
normalize the band intensity of the BMPR bands, the blots were also
probed with an oligonucleotide for the 18S rRNA.
[0175] The mRNA levels in control FRC cells, OP-1-treated cells,
IL-6/R-treated cells, and (OP-1+IL-6/R)-treated cells were compared
(FIG. 4). The data showed that OP-1 did not affect the mRNA level
of the type I receptors, but stimulated the BMPR-II mRNA level by
about 2.2 fold. Likewise, IL-6/R did not alter the mRNA expression
level of the type I receptors, but increased the BMPR-II mRNA level
by about 1.5-fold. In the presence of OP-1 and IL-6/R, the mRNA
level of the type I receptors was not significantly changed;
however, the BMPR-II mRNA level was almost 3-fold higher than the
control. These results suggest that IL-6/R can stimulate the
osteogenic activity of OP-1 by elevating BMPR-II mRNA
expression.
Example 5
[0176] The OP-1 protein used in Examples 1-5 was provided
exogenously to the test cells. To investigate whether the same
IL-6/R synergistic effect would be observed when the OP-1 protein
was expressed intracellularly in test cells, FRC cells were
transfected with pW24, a plasmid carrying an OP-1 coding sequence
under the control of the CMV promoter.
[0177] Briefly, confluent FRC cells were transfected with pW24 (2
.mu.g/ml). After recovery, the transfected cells were treated with
exogenous sIL-6R alone or IL-6/R for 24 hours. Then the total AP
activity levels were determined (FIG. 5).
[0178] The data showed that the levels of OP-1-induced AP activity
in pW24transfected cells were enhanced by sIL-6R in a
dose-dependent manner. At a concentration of 75 ng/ml, sIL-6R
stimulated the OP-1-induced AP activity by as much as 4 fold.
[0179] The data also showed that the levels of OP-1-induced AP
activity in pW24transfected cells were also enhanced by IL-6/R in a
dose-dependent manner. A 2.5-fold stimulation of the OP-1-induced
AP activity was observed when IL-6/R was applied to the test cells
at concentrations of 60 ng/ml for IL-6 and 75 ng/ml for sIL-6R.
Sequence CWU 1
1
14 1 1822 DNA Homo sapiens CDS (49)..(1341) 1 ggtgcgggcc cggagcccgg
agcccgggta gcgcgtagag ccggcgcg atg cac gtg 57 Met His Val 1 cgc tca
ctg cga gct gcg gcg ccg cac agc ttc gtg gcg ctc tgg gca 105 Arg Ser
Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala 5 10 15 ccc
ctg ttc ctg ctg cgc tcc gcc ctg gcc gac ttc agc ctg gac aac 153 Pro
Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn 20 25
30 35 gag gtg cac tcg agc ttc atc cac cgg cgc ctc cgc agc cag gag
cgg 201 Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu
Arg 40 45 50 cgg gag atg cag cgc gag atc ctc tcc att ttg ggc ttg
ccc cac cgc 249 Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu
Pro His Arg 55 60 65 ccg cgc ccg cac ctc cag ggc aag cac aac tcg
gca ccc atg ttc atg 297 Pro Arg Pro His Leu Gln Gly Lys His Asn Ser
Ala Pro Met Phe Met 70 75 80 ctg gac ctg tac aac gcc atg gcg gtg
gag gag ggc ggc ggg ccc ggc 345 Leu Asp Leu Tyr Asn Ala Met Ala Val
Glu Glu Gly Gly Gly Pro Gly 85 90 95 ggc cag ggc ttc tcc tac ccc
tac aag gcc gtc ttc agt acc cag ggc 393 Gly Gln Gly Phe Ser Tyr Pro
Tyr Lys Ala Val Phe Ser Thr Gln Gly 100 105 110 115 ccc cct ctg gcc
agc ctg caa gat agc cat ttc ctc acc gac gcc gac 441 Pro Pro Leu Ala
Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp 120 125 130 atg gtc
atg agc ttc gtc aac ctc gtg gaa cat gac aag gaa ttc ttc 489 Met Val
Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe 135 140 145
cac cca cgc tac cac cat cga gag ttc cgg ttt gat ctt tcc aag atc 537
His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile 150
155 160 cca gaa ggg gaa gct gtc acg gca gcc gaa ttc cgg atc tac aag
gac 585 Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys
Asp 165 170 175 tac atc cgg gaa cgc ttc gac aat gag acg ttc cgg atc
agc gtt tat 633 Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile
Ser Val Tyr 180 185 190 195 cag gtg ctc cag gag cac ttg ggc agg gaa
tcg gat ctc ttc ctg ctc 681 Gln Val Leu Gln Glu His Leu Gly Arg Glu
Ser Asp Leu Phe Leu Leu 200 205 210 gac agc cgt acc ctc tgg gcc tcg
gag gag ggc tgg ctg gtg ttt gac 729 Asp Ser Arg Thr Leu Trp Ala Ser
Glu Glu Gly Trp Leu Val Phe Asp 215 220 225 atc aca gcc acc agc aac
cac tgg gtg gtc aat ccg cgg cac aac ctg 777 Ile Thr Ala Thr Ser Asn
His Trp Val Val Asn Pro Arg His Asn Leu 230 235 240 ggc ctg cag ctc
tcg gtg gag acg ctg gat ggg cag agc atc aac ccc 825 Gly Leu Gln Leu
Ser Val Glu Thr Leu Asp Gly Gln Ser Ile Asn Pro 245 250 255 aag ttg
gcg ggc ctg att ggg cgg cac ggg ccc cag aac aag cag ccc 873 Lys Leu
Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys Gln Pro 260 265 270
275 ttc atg gtg gct ttc ttc aag gcc acg gag gtc cac ttc cgc agc atc
921 Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser Ile
280 285 290 cgg tcc acg ggg agc aaa cag cgc agc cag aac cgc tcc aag
acg ccc 969 Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys
Thr Pro 295 300 305 aag aac cag gaa gcc ctg cgg atg gcc aac gtg gca
gag aac agc agc 1017 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val
Ala Glu Asn Ser Ser 310 315 320 agc gac cag agg cag gcc tgt aag aag
cac gag ctg tat gtc agc ttc 1065 Ser Asp Gln Arg Gln Ala Cys Lys
Lys His Glu Leu Tyr Val Ser Phe 325 330 335 cga gac ctg ggc tgg cag
gac tgg atc atc gcg cct gaa ggc tac gcc 1113 Arg Asp Leu Gly Trp
Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala 340 345 350 355 gcc tac
tac tgt gag ggg gag tgt gcc ttc cct ctg aac tcc tac atg 1161 Ala
Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 360 365
370 aac gcc acc aac cac gcc atc gtg cag acg ctg gtc cac ttc atc aac
1209 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile
Asn 375 380 385 ccg gaa acg gtg ccc aag ccc tgc tgt gcg ccc acg cag
ctc aat gcc 1257 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr
Gln Leu Asn Ala 390 395 400 atc tcc gtc ctc tac ttc gat gac agc tcc
aac gtc atc ctg aag aaa 1305 Ile Ser Val Leu Tyr Phe Asp Asp Ser
Ser Asn Val Ile Leu Lys Lys 405 410 415 tac aga aac atg gtg gtc cgg
gcc tgt ggc tgc cac tagctcctcc 1351 Tyr Arg Asn Met Val Val Arg Ala
Cys Gly Cys His 420 425 430 gagaattcag accctttggg gccaagtttt
tctggatcct ccattgctcg ccttggccag 1411 gaaccagcag accaactgcc
ttttgtgaga ccttcccctc cctatcccca actttaaagg 1471 tgtgagagta
ttaggaaaca tgagcagcat atggcttttg atcagttttt cagtggcagc 1531
atccaatgaa caagatccta caagctgtgc aggcaaaacc tagcaggaaa aaaaaacaac
1591 gcataaagaa aaatggccgg gccaggtcat tggctgggaa gtctcagcca
tgcacggact 1651 cgtttccaga ggtaattatg agcgcctacc agccaggcca
cccagccgtg ggaggaaggg 1711 ggcgtggcaa ggggtgggca cattggtgtc
tgtgcgaaag gaaaattgac ccggaagttc 1771 ctgtaataaa tgtcacaata
aaacgaatga atgaaaaaaa aaaaaaaaaa a 1822 2 431 PRT Homo sapiens 2
Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5
10 15 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe
Ser 20 25 30 Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg
Leu Arg Ser 35 40 45 Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu
Ser Ile Leu Gly Leu 50 55 60 Pro His Arg Pro Arg Pro His Leu Gln
Gly Lys His Asn Ser Ala Pro 65 70 75 80 Met Phe Met Leu Asp Leu Tyr
Asn Ala Met Ala Val Glu Glu Gly Gly 85 90 95 Gly Pro Gly Gly Gln
Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser 100 105 110 Thr Gln Gly
Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr 115 120 125 Asp
Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys 130 135
140 Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu
145 150 155 160 Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu
Phe Arg Ile 165 170 175 Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn
Glu Thr Phe Arg Ile 180 185 190 Ser Val Tyr Gln Val Leu Gln Glu His
Leu Gly Arg Glu Ser Asp Leu 195 200 205 Phe Leu Leu Asp Ser Arg Thr
Leu Trp Ala Ser Glu Glu Gly Trp Leu 210 215 220 Val Phe Asp Ile Thr
Ala Thr Ser Asn His Trp Val Val Asn Pro Arg 225 230 235 240 His Asn
Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser 245 250 255
Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn 260
265 270 Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His
Phe 275 280 285 Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln
Asn Arg Ser 290 295 300 Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met
Ala Asn Val Ala Glu 305 310 315 320 Asn Ser Ser Ser Asp Gln Arg Gln
Ala Cys Lys Lys His Glu Leu Tyr 325 330 335 Val Ser Phe Arg Asp Leu
Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu 340 345 350 Gly Tyr Ala Ala
Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 355 360 365 Ser Tyr
Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His 370 375 380
Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln 385
390 395 400 Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn
Val Ile 405 410 415 Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys
Gly Cys His 420 425 430 3 102 PRT Artificial Sequence Description
of Artificial Sequence OPX 3 Cys Xaa Xaa His Glu Leu Tyr Val Ser
Phe Xaa Asp Leu Gly Trp Xaa 1 5 10 15 Asp Trp Xaa Ile Ala Pro Xaa
Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 20 25 30 Glu Cys Xaa Phe Pro
Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 35 40 45 Ile Xaa Gln
Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 50 55 60 Xaa
Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 65 70
75 80 Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys Xaa Arg Asn Met Val
Val 85 90 95 Xaa Ala Cys Gly Cys His 100 4 97 PRT Artificial
Sequence Description of Artificial Sequence Generic- Seq-7 4 Leu
Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro
20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa
Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Cys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Val Xaa Leu Xaa Xaa Xaa Xaa Xaa
Met Xaa Val Xaa Xaa Cys Xaa Cys 85 90 95 Xaa 5 102 PRT Artificial
Sequence Description of Artificial Sequence Generic- Seq-8 5 Cys
Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa 1 5 10
15 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly
20 25 30 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn
His Ala 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 50 55 60 Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu
Xaa Xaa Xaa Xaa Xaa Met Xaa Val 85 90 95 Xaa Xaa Cys Xaa Cys Xaa
100 6 97 PRT Artificial Sequence Description of Artificial Sequence
Generic- Seq-9 6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa
Gly Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 85 90 95
Xaa 7 102 PRT Artificial Sequence Description of Artificial
Sequence Generic- Seq-10 7 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85
90 95 Xaa Xaa Cys Xaa Cys Xaa 100 8 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Peptide 8 Cys Xaa Xaa
Xaa Xaa 1 5 9 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Peptide 9 Cys Xaa Xaa Xaa Xaa 1 5 10 102 PRT
Artificial Sequence Description of Artificial Sequence Generic
amino acid sequence 10 Cys Xaa Xaa Xaa Xaa Leu Xaa Val Xaa Phe Xaa
Asp Xaa Gly Trp Xaa 1 5 10 15 Xaa Trp Xaa Xaa Xaa Pro Xaa Gly Xaa
Xaa Ala Xaa Tyr Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa Pro Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 35 40 45 Xaa Xaa Gln Xaa Xaa
Val Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Pro Xaa 50 55 60 Xaa Cys Cys
Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Tyr Xaa Xaa Met Xaa Val 85 90
95 Xaa Xaa Cys Xaa Cys Xaa 100 11 96 PRT Artificial Sequence
Description of Artificial Sequence Synthetic amino acid (COP5) 11
Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp Asp Trp Ile Val Ala 1 5
10 15 Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly Glu Cys Pro Phe
Pro 20 25 30 Leu Ala Asp His Phe Asn Ser Thr Asn His Ala Val Val
Gln Thr Leu 35 40 45 Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala
Cys Cys Val Pro Thr 50 55 60 Glu Leu Ser Ala Ile Ser Met Leu Tyr
Leu Asp Glu Asn Glu Lys Val 65 70 75 80 Val Leu Lys Tyr Asn Gln Glu
Met Val Val Glu Gly Cys Gly Cys Arg 85 90 95 12 96 PRT Artificial
Sequence Description of Artificial Sequence Synthetic amino acid
(COP7) 12 Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile
Val Ala 1 5 10 15 Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu
Cys Pro Phe Pro 20 25 30 Leu Ala Asp His Leu Asn Ser Thr Asn His
Ala Val Val Gln Thr Leu 35 40 45 Val Asn Ser Val Asn Ser Lys Ile
Pro Lys Ala Cys Cys Val Pro Thr 50 55 60 Glu Leu Ser Ala Ile Ser
Met Leu Tyr Leu Asp Glu Asn Glu Lys Val 65 70 75 80 Val Leu Lys Tyr
Asn Gln Glu Met Val Val Glu Gly Cys Gly Cys Arg 85 90 95 13 97 PRT
Artificial Sequence Description of Artificial Sequence Generic
amino acid sequence 13 Leu Xaa Val Xaa Phe Xaa Asp Xaa Gly Trp Xaa
Xaa Trp Xaa Xaa Xaa 1 5 10 15 Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys
Xaa Gly Xaa Cys Xaa Xaa Pro 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Asn His Ala Xaa Xaa Gln Xaa Xaa 35 40 45 Val Xaa Xaa Xaa Asn
Xaa Xaa Xaa Xaa Pro Xaa Xaa Cys Cys Val Pro 50 55 60 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Val
Xaa Leu Xaa Xaa Tyr Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 85 90
95 Xaa 14 102 PRT Artificial Sequence Description of Artificial
Sequence Generic amino acid sequence 14 Cys Xaa Arg Xaa Xaa Leu Xaa
Val Xaa Phe Xaa Asp Xaa Gly Trp Xaa 1 5 10 15 Xaa Trp Xaa Xaa Xaa
Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 20 25 30 Xaa Cys Xaa
Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 35 40 45 Xaa
Xaa Gln Xaa Xaa Val Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Pro Xaa 50 55
60 Xaa Cys Cys Val Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa
65 70 75 80 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Tyr Xaa Xaa Met
Xaa Val 85 90 95 Xaa Xaa Cys Xaa Cys Xaa 100
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