U.S. patent application number 09/791946 was filed with the patent office on 2002-03-07 for methods and compositions for producing morphogen analogs.
Invention is credited to Carlson, William D., Griffith, Diana L., Keck, Peter C., Rueger, David C., Sampath, Kuber T..
Application Number | 20020028453 09/791946 |
Document ID | / |
Family ID | 24358486 |
Filed Date | 2002-03-07 |
United States Patent
Application |
20020028453 |
Kind Code |
A1 |
Keck, Peter C. ; et
al. |
March 7, 2002 |
Methods and compositions for producing morphogen analogs
Abstract
The invention disclosed herein provides methods and compositions
for the computer-assisted design of morphogen analogs. Practice of
the invention is enabled by the use of at least a portion of the
atomic co-ordinates defining the three-dimensional structure of
human osteogenic protein-1 (hOP-1) as a starting point in the
design of the morphogen analogs. In addition, the invention
provides methods for producing morphogen analogs of interest, and
methods for testing whether the resulting analogs mimic or agonize
human OP-1-like biological activity. The invention also provides a
family of morphogen analogs produced by such methods.
Inventors: |
Keck, Peter C.; (Millbury,
MA) ; Griffith, Diana L.; (Weston, MA) ;
Carlson, William D.; (Weston, MA) ; Rueger, David
C.; (Hopkinton, MA) ; Sampath, Kuber T.;
(Medway, MA) |
Correspondence
Address: |
c/o MINTZ, LEVIN
One Financial Center
Boston
MA
02111
US
|
Family ID: |
24358486 |
Appl. No.: |
09/791946 |
Filed: |
February 22, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09791946 |
Feb 22, 2001 |
|
|
|
08786284 |
Jan 22, 1997 |
|
|
|
6273598 |
|
|
|
|
08786284 |
Jan 22, 1997 |
|
|
|
08589552 |
Jan 22, 1996 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
514/8.8; 514/8.9; 702/19 |
Current CPC
Class: |
C07K 1/00 20130101; C07K
14/51 20130101 |
Class at
Publication: |
435/6 ; 702/19;
514/2 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50; A61K 038/00 |
Claims
What is claimed is:
1. A computer system comprising: (a) a memory having disposed
therein atomic X-ray crystallographic co-ordinates defining at
least a portion of human OP-1; and (b) a processor in electrical
communication with the memory; the processor comprising a process
which generates a molecular model having a three-dimensional shape
representative of at least a portion of human OP-1.
2. The system of claim 1, wherein the processor further comprises a
process which generates the molecular model having a solvent
accessible surface representative of at least a portion of human
OP-1.
3. The system of claim 1, wherein said co-ordinates are stored on a
computer readable diskette.
4. The system of claim 1, wherein the molecular model is
representative of at least a portion of human OP-1 finger 1
region.
5. The system of claim 1 or 4, wherein the molecular model is
representative of at least a portion of the human OP-1 heel
region.
6. The system of claim 1 or 4, wherein the molecular model is
representative of at least a portion of the human OP-1 finger 2
region.
7. The system of claim 6, wherein the molecular model is
representative of at least a portion of the human OP-1 heel
region.
8. The system of claim 1, wherein the processor further identifies
a morphogenic analog having a three-dimensional shape and a solvent
accessible surface corresponding to at least a portion of the
three-dimensional shape and the solvent accessible surface of human
OP-1.
9. The system of claim 1, wherein the processor further identifies
at least one candidate amino acid defined by the co-ordinates,
which upon modification enhances water solubility or stability of
human OP-1.
10. A method of producing a morphogenic analog having osteogenic
protein-1 (OP-1) like biological activity, the method comprising
the steps of: (a) providing a molecular model defining a three
dimensional shape representative of at least a portion of human
OP-1; (b) identifying a candidate analog having a three dimensional
shape corresponding to the three dimensional shape representative
of at least a portion of human OP-1; and (c) producing the
candidate analog identified in step (b).
11. The method of claim 10, further comprising the step of
determining whether the compound produced in step (c) has an
OP-1-like biological activity.
12. The method of claim 10, wherein the molecular model provided in
step (a) is representative of at least a portion of a finger 1
region of human OP-1.
13. The method of claim 10 or 12, wherein the molecular model
provided in step (a) is representative of at least a portion of a
heel region of human OP-1.
14. The method of claim 10 or 12, wherein the model provided in
step (a) is representative of at least a portion of a finger 2
region of human OP-1.
15. The method of claim 14, wherein the molecular model provided in
step (a) is representative of at least a portion of a heel region
of human OP-1.
16. The method of claim 10, wherein the analog comprises a
plurality of charged moieties spaced about the solvent accessible
surface thereof and disposed in a spaced-apart relation
corresponding to charged moieties spaced about a portion of the
solvent accessible surface of human OP-1.
17. The method of claim 10, wherein steps (a) and (b) are performed
by means of an electronic processor.
18. The method of claim 17, wherein step (a) comprises storing a
representation of at least a portion of the atomic co-ordinates of
human OP-1 in a computer memory.
19. A method of producing a morphogen analog that modulates an
osteogenic protein-1 (OP-1) mediated biological effect, the method
comprising the steps of: (a) providing in a computer memory atomic
X-ray crystallographic co-ordinates defining at least a portion of
human OP-1; (b) generating with a processor a molecular model
having a three-dimensional shape and a solvent accessible surface
representative of at least a portion of human OP-1, (c) identifying
a candidate morphogen analog having a three-dimensional structure
shape and a solvent accessible surface corresponding to the
three-dimensional shape and the solvent accessible surface of at
least a portion of human OP-1; (d) producing the candidate
morphogen analog identified in step (c); and (e) determining
whether the candidate morphogen analog produced in step (d)
modulates the OP-1 mediated biological effect.
20. The method of claim 11 or 19, further comprising the additional
step of producing the compound in a commercially useful
quantity.
21. The method of claim 11 or 19, wherein said compound is a
peptide.
22. A compound that modulates an OP-1 mediated biological effect
produced by the method of claim 11 or 19.
23. The compound of claim 22, wherein said compound agonizes the
biological activity of human OP-1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of copending
application U.S. Ser. No. 08/589,552, filed Jan. 22, 1996, the
disclosure of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to methods and
compositions for designing, identifying, and producing compounds
useful as tissue morphogenic protein analogs. More specifically,
the invention relates to structure-based methods and compositions
useful in designing, identifying, and producing molecules which act
as functional mimetics of the tissue morphogenic protein osteogenic
protein-1 (OP-1).
BACKGROUND OF THE INVENTION
[0003] Cell differentiation is the central characteristic of tissue
morphogenesis which initiates during embryogenesis, and continues
to various degrees throughout the life of an organism in adult
tissue repair and regeneration mechanisms. The degree of
morphogenesis in adult tissue varies among different tissues and is
related, among other things, to the degree of cell turnover in a
given tissue.
[0004] The cellular and molecular events which govern the stimulus
for differentiation of cells is an area of intensive research. In
the medical and veterinary fields, it is anticipated that discovery
of the factor or factors which control cell differentiation and
tissue morphogenesis will advance significantly the ability to
repair and regenerate diseased or damaged mammalian tissues and
organs. Particularly useful areas for human and veterinary
therapeutics include reconstructive surgery, the treatment of
tissue degenerative diseases including, for example, arthritis,
emphysema, osteoporosis, cardiomyopathy, cirrhosis, degenerative
nerve diseases, inflammatory diseases, and cancer, and in the
regeneration of tissues, organs and limbs. In this and related
applications, the terms "morphogenetic" and "morphogenic" are used
interchangeably.
[0005] A number of different factors have been isolated in recent
years which appear to play a role in cell differentiation.
Recently, a distinct subfamily of the "superfamily" of structurally
related proteins referred to in the art as the "transforming growth
factor-.beta. (TGF-.beta.) superfamily of proteins" have been
identified as true tissue morphogens.
[0006] The members of this distinct "subfamily" of true tissue
morphogenic proteins share substantial amino acid sequence homology
within their morphogenetically active C-terminal domains (at least
50% identity in the C-terminal 102 amino acid sequence), including
a conserved six or seven cysteine skeleton, and share the in vivo
activity of inducing tissue-specific morphogenesis in a variety of
organs and tissues. The proteins apparently contact and interact
with progenitor cells e.g., by binding suitable cell surface
molecules, predisposing or otherwise stimulating the cells to
proliferate and differentiate in a morphogenetically permissive
environment. These morphogenic proteins are capable of inducing the
developmental cascade of cellular and molecular events that
culminate in the formation of new organ-specific tissue, including
any vascularization, connective tissue formation, and nerve
innervation as required by the naturally occurring tissue. The
proteins have been shown to induce morphogenesis of both bone
cartilage and bone, as well as periodontal tissues, dentin, liver,
and neural tissue, including retinal tissue.
[0007] True tissue morphogenic proteins identified to date include
proteins originally identified as bone inductive proteins. These
include OP-1, (osteogenic protein-1, also referred to in related
applications as "OP1"), its Drosophila homolog, 60A, with which it
shares 69% identity in the C-terminal "seven cysteine" domain, and
the related proteins OP-2 (also referred to in related applications
as "OP2") and OP-3, both of which share approximately 65-75%
identity with OP-1 in the C-terminal seven cysteine domain, as well
as BMP5, BMP6 and its murine homolog, Vgr-1, all of which share
greater than 85% identity with OP-1 in the C-terminal seven
cysteine domain, and the BMP6 Xenopus homolog, Vg1, which shares
approximately 57% identity with OP-1 in the C-terminal seven
cysteine domain. Other bone inductive proteins include the CBMP2
proteins (also referred to in the art as BMP2 and BMP4) and their
Drosophila homolog, DPP. Another tissue morphogenic protein is
GDF-1 (from mouse). See, for example, PCT documents US92/01968 and
US92/07358, the disclosures of which are incorporated herein by
reference. Members of the BMP/OP subfamily and the amino acid
sequence identities (expressed as percentages) between selected
members of the TGF-.beta. superfamily are shown in FIG. 6.
[0008] As stated above, these true tissue morphogenic proteins are
recognized in the art as a distinct subfamily of proteins different
from other members of the TGF-.beta. superfamily in that they share
a high degree of sequence identity in the C-terminal domain and in
that the true tissue morphogenic proteins are able to induce, on
their own, the full cascade of events that result in formation of
functional tissue rather than merely inducing formation of fibrotic
(scar) tissue. Specifically, members of the family of morphogenic
proteins are capable of all of the following in a morphogenetically
permissive environment: stimulating cell proliferation and cell
differentiation, and supporting the growth and maintenance of
differentiated cells. The morphogenic proteins apparently also may
act as endocrine, paracrine or autocrine factors.
[0009] The morphogenic proteins are capable of significant species
"crosstalk." That is, xenogenic (foreign species) homologs of these
proteins can substitute for one another in functional activity. For
example, dpp and 60A, two Drosophila proteins, can substitute for
their mammalian homologs, BMP2/4 and OP-1, respectively, and induce
endochondral bone formation at a non-bony site in a standard rat
bone formation assay. Similarly, BMP2 has been shown to rescue a
dpp.sup.- mutation in Drosophila. In their native form, however,
the proteins appear to be tissue-specific, each protein typically
being expressed in or provided to one or only a few tissues or,
alternatively, expressed only at particular times during
development. For example, GDF-1 appears to be expressed primarily
in neural tissue, while OP-2 appears to be expressed at relatively
high levels in early (e.g., 8-day) mouse embryos. The endogenous
morphogens may be synthesized by the cells on which they act, by
neighboring cells, or by cells of a distant tissue, the secreted
protein being transported to the cells to be acted on.
[0010] A particularly potent tissue morphogenic protein is OP-1.
This protein, and its xenogenic homologs, are expressed in a number
of tissues, primarily in tissues of urogenital origin, as well as
in bone, mammary and salivary gland tissue, reproductive tissues,
and gastrointestinal tract tissue. It is expressed also in
different tissues during embryogenesis, its presence coincident
with the onset of morphogenesis of that tissue.
[0011] The morphogenic protein signal transduction across a cell
membrane appears to occur as a result of specific binding
interaction with one or more cell surface receptors. Recent studies
on cell surface receptor binding of various members of the
TGF-.beta. protein superfamily suggests that the ligands mediate
their activity by interaction with two different receptors,
referred to as Type I and Type II receptors to form a
hetero-complex. A cell surface bound beta-glycan also may enhance
the binding interaction. The Type I and Type II receptors are both
serine/threonine kinases, and share similar structures: an
intracellular domain that consists essentially of the kinase, a
short, extended hydrophobic sequence sufficient to span the
membrane one time, and an extracellular domain characterized by a
high concentration of conserved cysteines.
[0012] Morphogenic proteins are disulfide-linked dimers which are
expressed as large precursor polypeptide chains containing a
hydrophobic signal sequence, a long and relatively poorly conserved
N-terminal pro region of several hundred amino acids, a cleavage
site and a mature domain comprising an N-terminal region which
varies among the family members and a more highly conserved
C-terminal region. The C-terminal region, which is present in the
processed mature proteins of all known morphogen family members,
contains approximately 100 amino acids with a characteristic motif
having a conserved six or seven cysteine skeleton. Each of the
morphogenic proteins isolated to date are dimeric structures
wherein the monomer subunits are held together by non-covalent
interactions or by one or more disulfide bonds. The morphogenic
proteins are active as dimeric proteins but are inactive as
individual monomer subunits.
[0013] As a result of their biological activities, significant
effort has been directed toward the development of morphogen-based
therapeutics for treating injured or diseased mammalian tissue,
including, for example, therapeutic compositions for inducing
regenerative healing of bone defects such as fractures, as well as
therapeutic compositions for preserving or restoring healthy
metabolic properties in diseased bone tissue, e.g., osteopenic bone
tissue. Complete descriptions of efforts to develop and
characterize morphogen-based therapeutics for non-chondrogenic
tissue applications in mammals, particularly humans, are set forth,
for example, in: EP 0575,555; WO93/04692; WO93/05751; WO94/06399;
WO94/03200; WO94/06449; WO94/10203; and WO94/06420, the disclosures
of each of which are incorporated herein by reference.
[0014] Certain difficulties may be experienced upon administration
of naturally isolated or recombinantly produced morphogenic
proteins to a mammal. These difficulties may include, for example,
loss of morphogenic activity due to disassociation of the
biologically active morphogen dimer into its inactive monomer
subunits, and/or handling problems due to low solubility under
physiological conditions.
[0015] Accordingly, a need remains for the identification of
morphogen analogs, which mimic or enhance the physiological effects
of a morphogenic protein, for example OP-1. The analogs may be
modified, morphogenically active hOP-1 protein dimers, or fragments
or truncated analogs thereof, peptides or small organic molecules.
Preferably the analogs have enhanced therapeutic value, for
example, by being more stable and/or more soluble under
physiological conditions than naturally occurring hOP-1, or, for
example, by having enhanced tissue targeting specificity, enhanced
biodistribution or a reduced clearance rate in the body.
[0016] It is an object of the present invention to provide a
database defining the atomic co-ordinates of the three-dimensional
structure of mature hOP-1, all or a portion of which can be used as
part of a computer system for designing and/or identifying a
functional analog of hOP-1. Another object is to provide means for
designing and/or identifying a molecule having enhanced solubility
and/or stability under physiological conditions as compared with
hOP-1 and which is capable of mimicking or enhancing the biological
activity of hOP-1 in a mammal. Another object of the invention is
to provide a therapeutic composition comprising an analog designed
and/or identified, and produced by the methods of the invention,
and suitable for administration to a mammal in need thereof, such
as a mammal afflicted with a metabolic bone disease, e.g., a
disease characterized by osteopenia. Another object of the
invention is to provide methods and compositions useful for
designing and/or identifying, and producing an hOP-1 antagonist
capable of, for example, competing with hOP-1 for receptor binding,
but incapable of inducing a receptor-mediated downstream biological
effect.
[0017] These and other objects and features of the invention will
be apparent from the description, drawings, and claims which
follow.
SUMMARY OF THE INVENTION
[0018] The present invention is based, in part, upon the X-ray
crystallographic determination of the three-dimensional structure
of mature, dimeric human osteogenic protein-1 (hOP-1). The
three-dimensional structure of hOP-1 has been resolved to 2.3.ANG..
Provided herein are two sets of atomic X-ray crystallographic
co-ordinates for hOP-1, one set defining a hOP-1 structure resolved
to a resolution of 2.8.ANG., and the other set defining a hOP-1
structure resolved to a resolution of 2.3.ANG.. With this
disclosure, the skilled artisan is provided with sets of atomic
co-ordinates for use in conventional computer aided design (CAD)
methodologies to identify or design protein or peptide analogs of
OP-1, or alternatively, to identify or design small organic
molecules that functionally mimic OP-1.
[0019] In one aspect, the invention provides a computer system
comprising a memory and a processor in electrical communication
with the memory. The memory has disposed therein, atomic X-ray
crystallographic co-ordinates which together define at least a
portion of the three-dimensional structure of hOP-1. In a preferred
embodiment, the atomic co-ordinates are defined by either a portion
or all of the atomic co-ordinates set forth in FIG. 15 or FIG.
16.
[0020] The processor, in electrical communication with the memory,
comprises a process which generates a molecular model having a
three-dimensional shape representative of at least a portion of
human OP-1. In a preferred embodiment, the processor is capable of
producing a molecular model having, in addition to the
three-dimensional shape, a solvent accessible surface
representative of at least a portion of human OP-1.
[0021] As used herein, the term "computer system" is understood to
mean any general or special purpose system which includes a
processor in electrical communication with both a memory and at
least one input/output device, such as a terminal. Such a system
may include, but is not limited to, personal computers,
workstations or mainframes. The processor may be a general purpose
processor or microprocessor or a specialized processor executing
programs located in RAM memory. The programs may be placed in RAM
from a storage device, such as a disk or preprogrammed ROM memory.
The RAM memory in one embodiment is used both for data storage and
program execution. The term computer system also embraces systems
where the processor and memory reside in different physical
entities but which are in electrical communication by means of a
network.
[0022] In the present invention, the processor executes a modeling
program which accesses data representative of the X-ray
crystallographic co-ordinates of hOP-1 thereby to construct a
three-dimensional model of the molecule. In addition, the processor
also can execute another program, a solvent accessible surface
program, which uses the three-dimensional model of hOP-1 to
construct a solvent accessible surface of at least a portion of the
hOP-1 molecule and optionally calculate the solvent accessible
areas of atoms. In one embodiment the solvent accessible surface
program and the modeling program are the same program. In another
embodiment, the modeling program and the solvent accessible surface
program are different programs. In such an embodiment the modeling
program may either store the three-dimensional model of hOP-1 in a
region of memory accessible both to it and to the solvent
accessible surface program, or the three-dimensional model may be
written to external storage, such as a disk, CD ROM, or magnetic
tape for later access by the solvent accessible surface
program.
[0023] The memory may have stored therein the entire set of X-ray
crystallographic co-ordinates which define mature biologically
active human OP-1, or may comprise a subset of such co-ordinates
including, for example, one or more of: a finger 1 region; a finger
2 region; and a heel region. The protein structures which
correspond to the finger and heel regions are described in detail
below.
[0024] In another preferred embodiment, the processor also is
capable of identifying a morphogen analog, or a morphogen
antagonist for example, a protein, peptide or small organic
molecule, having a three-dimensional shape and preferably, in
addition, a solvent accessible surface corresponding to at least a
portion of human OP-1 and competent to mimic an OP-1 specific
activity.
[0025] As used herein, with respect to OP-1 (or related
morphogens), or with respect to a region of OP-1, the phrase "at
least a portion of the three-dimensional structure of" or "at least
a portion of" is understood to mean a portion of the
three-dimensional surface structure of the morphogen, or region of
the morphogen, including charge distribution and
hydrophilicity/hydrophobicity characteristics, formed by at least
three, more preferably at least three to ten, and most preferably
at least ten contiguous amino acid residues of the OP-1 monomer or
dimer. The contiguous residues forming such a portion may be
residues which form a contiguous portion of the primary structure
of the OP-1 molecule, residues which form a contiguous portion of
the three-dimensional surface of the OP-1 monomer, residues which
form a contiguous portion of the three-dimensional surface of the
OP-1 dimer, or a combination thereof. Thus, the residues forming a
portion of the three-dimensional structure of OP-1 need not be
contiguous in the primary sequence of the morphogen but, rather,
must form a contiguous portion of the surface of the morphogen
monomer or dimer. In particular, such residues may be
non-contiguous in the primary structure of a single morphogen
monomer or may comprise residues from different monomers in the
dimeric form of the morphogen. As used herein, the residues forming
"a portion of the three-dimensional structure of" a morphogen, or
"a portion of" a morphogen, form a contiguous three-dimensional
surface in which each atom or functional group forming the portion
of the surface is separated from the nearest atom or functional
group forming the portion of the surface by no more than 40 .ANG.,
preferably by no more than 20 .ANG., more preferably by no more
than 5-10 .ANG., and most preferably by no more than 1-5 .ANG..
[0026] As used herein the term "X-ray crystallographic
co-ordinates" refers to a series of mathematical co-ordinates
(represented as "X", "Y" and "Z" values) that relate to the spatial
distribution of reflections produced by the diffraction of a
monochromatic beam of X-rays by atoms of an hOP-1 molecule in
crystal form. The diffraction data are used to generate electron
density maps of the repeating units of a crystal, and the resulting
electron density maps are used to define the positions of
individual atoms within the unit cell of the crystal.
[0027] As will be apparent to those of ordinary skill in the art,
the hOP-1 structure presented herein is independent of its
orientation, and that the atomic co-ordinates listed in FIGS. 15
and 16 merely represent one possible orientation of the hOP-1
structure. It is apparent, therefore, that the atomic co-ordinates
listed in FIGS. 15 and 16, may be mathematically rotated,
translated, scaled, or a combination thereof, without changing the
relative positions of atoms or features of the hOP-1 structure.
Such mathematical manipulations are intended to be embraced herein.
Furthermore, it will be apparent to the skilled artisan that the
X-ray atomic co-ordinates defined herein have some degree of
uncertainty in location (see, for example, column ".delta." in FIG.
16 which shows the thermal uncertainty in location of each atom, as
expressed in .ANG.). Accordingly, for purposes of this invention, a
preselected protein or peptide having the same amino acid sequence
as at least a portion of hOP-1 is considered to have the same
structure as the corresponding portion of hOP-1, when a set of
atomic co-ordinates defining backbone C.alpha. atoms of the
preselected protein or peptide can be superimposed onto the
corresponding C.alpha. atoms for hOP-1 (as listed in FIG. 16) to a
root mean square deviation of preferably less than about 1.5 .ANG.,
and most preferably less than about 0.75 .ANG..
[0028] As used herein, the term "morphogen analog", is understood
to mean any molecule capable of mimicking OP-1's receptor binding
activity and/or and inducing a receptor mediated downstream
biological effect characteristic of a morphogenic protein. Inducing
alkaline phosphatase activity is a characteristic biological
effect. The analog may be a protein, peptide, or non-peptidyl based
organic molecule. Accordingly, the term morphogen analog embraces
any substance having such OP-1 like activity, regardless of the
chemical or biochemical nature thereof. The present morphogen
analog can be a simple or complex substance produced by a living
system or through chemical or biochemical synthetic techniques. It
can be a large molecule, e.g., a modified hOP-1 dimer produced by
recombinant DNA methodologies, or a small molecule, e.g., an
organic molecule prepared de novo according to the principles of
rational drug design. It can be a substance which is a mutein (or
mutant protein) of hOP-1, a substance that structurally resembles a
solvent-exposed surface epitope of hOP-1 and binds an OP-1 specific
receptor, or a substance that otherwise stimulates an OP-1 specific
receptor displayed on the surface of an OP-1 responsive cell.
[0029] As used herein, the terms "OP-1 or OP-1-like biological
activity" are understood to mean any biological activities known to
be induced or enhanced by OP-1. OP-1 and OP-1-like biological
activities include, but are not limited to, stimulating
proliferation of progenitor cells; stimulating differentiation of
progenitor cells; stimulating proliferation of differentiated
cells; and supporting growth and maintenance of differentiated
cells. The term "progenitor cells" includes uncommitted cells,
preferably of mammalian origin that are competent to differentiate
into one or more specific types of differentiated cells, depending
on their genomic repertoire and the tissue specificity of the
permissive environment where morphogenesis is induced.
Specifically, with regard to bone, cartilage, nerve, and liver
tissue, the OP-1 stimulated morphogenic cascade culminates in the
formation of new or regenerative differentiated tissue appropriate
to the selected local environment. OP-1 mediated morphogenesis,
therefore, differs significantly from simple reparative healing
processes in which scar tissue (e.g., fibrous connective tissue) is
formed and fills a lesion or other defect in differentiated
functional tissue.
[0030] As used herein a "morphogen antagonist" is a molecule
competent to mimic OP-1 receptor binding activity but which cannot
induce a receptor-mediated downstream effect.
[0031] In yet another preferred embodiment, the processor is
capable of identifying amino acids defined by the co-ordinates,
which upon site-directed modification, either by chemical
modification or amino acid substitution, enhance the solubility
and/or stability of human OP-1.
[0032] In a related aspect, the invention provides a method of
producing a morphogen analog that mimics or enhances an OP-1 or
OP-1-like biological activity. The method comprises the steps of:
(a) providing a molecular model defining a three-dimensional shape
representative of at least a portion of human OP-1, (b) identifying
a compound having a three-dimensional shape corresponding to the
three-dimensional shape representative of at least the portion of
human OP-1; and (c) producing the compound identified in step (b).
The method can comprise the additional step of testing the compound
in a biological system to determine whether the resultant candidate
compound mimics or agonizes the biological activity of OP-1. It is
contemplated that, in the aforementioned method, step (a) and/or
(b) may be performed by means of an electronic processor using
commercially available software packages.
[0033] It is contemplated that, upon determination of whether the
candidate compound modulates OP-1 activity, the candidate compound
can be iteratively improved using conventional CAD and/or rational
drug design methodologies, well known and thoroughly documented in
the art. Furthermore, it is contemplated that the resultant
compound identified thus far, may be produced in a commercially
useful quantity for administration into a mammal.
[0034] In another embodiment, the morphogen analog is created using
atomic co-ordinates set forth in either FIGS. 15 or 16. By
reviewing the atomic co-ordinates set forth in FIGS. 15 and 16, the
skilled artisan can observe the three-dimensional structure of
particular amino acid sequences located in situ within the
three-dimensional structure of hOP-1. Preferred amino acid
sequences are defined by one or more of the peptides selected from
the group consisting of: H1, H-n2, H-c2, F1-2, F2-2 and F2-3, as
discussed hereinbelow. The peptides provide templates which can be
used in the production of more effective morphogen analogs. In a
preferred embodiment, the C.alpha. atoms of amino acid residues in
the morphogen analog are located within 6.ANG., preferably within
3.ANG., and most preferably within 2.ANG. of the corresponding
C.alpha. atom as defined by the respective atomic co-ordinates in
FIGS. 15 or 16. In another preferred embodiment, the C.alpha. atoms
of amino acid residues in the morphogen analog are located within
6.ANG., preferably within 3.ANG., and most preferably within 2.ANG.
of the corresponding C.alpha. atoms of at least three amino acids
in the peptide sequences H1, H-n2, H-c2, F1-2, F2-2 and F2-3,
wherein each of the C.alpha. atoms in the peptides are defined by
the respective atomic co-ordinates set forth in FIGS. 15 or 16.
[0035] In another embodiment, the invention provides morphogen
analogs having greater solubility and/or stability in aqueous
buffers than native dimeric hOP-1. In yet another embodiment, the
invention provides a morphogen analog which is a modified form of
dimeric hOP-1, in which the modification eliminates an epitope or
region on OP-1 normally recognized by an antibody or by a cellular
scavenging protein for clearing OP-1 from the body.
[0036] In another embodiment, the invention provides means for
creating an analog with altered receptor binding characteristics.
For example, provided with the structure, charge distribution, and
solvent accessible surface information pertaining to the putative
receptor binding site, one can alter or modify receptor binding
specificity and avidity. In one embodiment, amino acid replacements
in this region are made with reference to the corresponding amino
acids of other known morphogens, disclosed for example, in WO
94/06449 or WO 93/05751.
[0037] After having determined the three-dimensional structure of
human OP-1, a skilled artisan, in possession of the atomic
co-ordinates defining the OP-1 structure is hereby enabled to use
conventional CAD and/or rational drug design methodologies to
identify or design protein or peptide analogs, or other small
organic molecules which, after having been produced using
conventional chemistries and methodologies, can be tested either in
vitro or in vivo to assess whether they mimic or enhance the
biological activity of human OP-1.
[0038] The foregoing and other objects, features and advantages of
the present invention will be made more apparent from the following
detailed description of preferred embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0040] The objects and features of the invention may be better
understood by reference to the drawings described below, wherein
like referenced features identify common features in corresponding
figures.
[0041] FIG. 1A is a simplified line drawing useful in describing
the structure of a monomeric subunit of hOP-1. See the Summary of
the Invention, infra, for explanation. FIGS. 1B, 1C, and 1D are
monovision ribbon tracings of the respective peptide backbones of
hOP-1 finger-1, heel, and finger-2 regions. FIGS. 1E and 1F are
schematic representations of monomeric and dimeric forms of hOP-1,
respectively, as represented by a left hand motif.
[0042] FIG. 2 is a schematic drawing of a monomeric subunit of
hOP-1. The hOP-1 cysteine knot comprising three disulfide bonds
constitutes the core of the hOP-1 monomer subunit. Two disulfide
bonds which connect residues Cys 67-Cys 136 and Cys 71-Cys 138
produce an eight residue ring through which the third disulfide
bond which connects residues Cys 38-Cys 104 passes. Four strands of
antiparallel .beta.-sheet, which emanate from the knot, form the
two finger like projections. An .alpha.-helix located on the
opposite end of the knot, lies perpendicular to the axis of the two
fingers thereby forming the heel. The N-terminus of the monomer
subunit remains unresolved. The .beta.-sheets are displayed as
arrows and labeled from .beta.1 through .beta.8. The .alpha.-helix
is displayed as a tube and labeled .varies.1. The intra-subunit
disulfide bonds that constitute the cysteine knot are shown in
solid lines. Starting from Gln 36 ("N.sub.36"), the first residue
shown in this figure, the amino acid residues which produce
secondary structure in the Finger 1 region include: Lys 39-His 41
(.beta.1), Tyr 44-Ser 46 (.beta.2), Glu 60-Ala 63 (.beta.3), Tyr
65-Glu 70 (.beta.4); the amino acid residues which produce
secondary structure in the Finger 2 region include: Cys 103-Asn 110
(.beta.5); Ile 112-Asp 118 (.beta.6); Asn 122-Tyr 128 (.beta.7);
Val 132-His 139 (.beta.8); and the amino acid residues which
produce secondary structure in the heel region include: Thr 82-Ile
94(.alpha.1).
[0043] FIG. 3 is a structure-based sequence alignment of the hOP-1
and TGF-.beta.2 finger-1, heel, and finger-2 regions. Amino acid
residues in the heel regions which constitute inter-chain contacts
in the dimers of hOP-1 and TGF-.beta.2 are highlighted as white on
black. Amino acid residues in the finger-1 and finger-2 regions
which contact the other chain are highlighted as black on gray. In
hOP-1 and TGF-.beta.2, the amino acids located at the same residue
positions constitute the inter-chain contacts.
[0044] FIGS. 4A and 4B are stereo peptide backbone ribbon trace
drawings illustrating the three-dimensional shape of hOP-1: A) from
the "top" (down the two-fold axis of symmetry between the subunits)
with the axes of the helical heel regions generally normal to the
paper and the axes of each of the finger 1 and finger 2 regions
generally vertical, and B) from the "side" with the two-fold axis
between the subunits in the plane of the paper, with the axes of
the heels generally horizontal, and the axes of the fingers
generally vertical. The hOP-1 monomer has an accessible non-polar
surface area of approximately 4394A.sup.2, while that for the dimer
is approximately 6831A.sup.2 resulting in a hidden area upon
dimerization of approximately 979A.sup.2 per monomer. The reader is
encouraged to view the stereo alpha carbon trace drawings in
wall-eyed stereo, for example, using a standard stereo viewer
device, to more readily visualize the spatial relationships of
amino acids sequences in the morphogen analog design.
[0045] FIG. 5A is a backbone ribbon trace drawing illustrating the
hOP-1 dimer comprising the two hOP-1 monomer subunits resolved to
2.8.ANG.. One monomer subunit is shown in green and the other
monomer subunit is shown in gold. Amino acid residues disposed
within the purported receptor binding domain having solvent
accessible side chains are shown as atomic spheres. The tips of the
finger 1 and finger 2 regions of one OP-1 monomeric subunit and a
loop at the C-terminal end of the heel of the other OP-1 monomeric
subunit are believed to constitute the receptor binding domain.
Amino acids located at positions of variable amino acid sequence
shown in white while amino acids located at more conserved
positions are shown in red. FIGS. 5B and 5C are pictures showing
the respective solvent accessible surfaces of OP-1 and TGF-.beta.2
dimers colored based on their electrostatic potential. Surface
regions having an electrostatic potential of -3 kT or less are
shown in red while surface regions of +3 kT or greater are shown in
blue. Neutral regions are shown in green or gold to correspond to
the backbone ribbons shown in 5A.
[0046] FIG. 6 is a table showing an identity matrix for the
TGF-.beta. superfamily. The matrix comprises members of the
TGF-.beta. superfamily having an amino acid sequence identity
relative to OP-1 of greater than 36%. In the matrix, the TGF-.beta.
superfamily members are placed in order of decreasing amino acid
identity relative to OP-1. TGF-.beta.2 has an amino acid sequence
of identity of 36% relative to OP-1 and is positioned the bottom of
the matrix. Boxes enclose families of sequences having 50% or
higher identity with a majority of the other members of the family;
with sequences having identities of 75% or higher are shown in
gray. Recombinantly expressed OP/BMP family members which have been
shown to make bone are denoted by a "+" in the left margin. In the
left margin, TGF-.beta. superfamily members with three-dimensional
structures determined are highlighted white on black. The sequences
are referenced in Kingsley (Kingsley. (1994) Genes and Development
8:133-146), except for the following: (UNIVIN (Stenzel et al.
(1994) Develop. Biol. 166:149-158.), SCREW (Arora, et al.(1994)
Genes and Dev. 8:2588-2601.), BMP-9 (Wozney, et al.(1993) PCT/WO
93/00432, SEQ. ID. NO.9), BMP-10 (Celeste et al. (1994) PCT/WO
94/26893, SEQ. ID. NO. 1), GDF-5 (Storm et al. (1994) Nature
368:639-643) (also called CDMP-1 (Chang et al. (1994) J. Biol.
Chem. 269: 28227-28234.), GDF-6 (Storm, et al. (1994) Nature
368:639-643), GDF-7 (Storm et al. (1994) Nature 368:639-643),
CDMP-2 (Chang et al. (1994) J. Biol. Chem. 269: 28227-28234.), OP-3
(zkaynak et al. (1994) PCT/WO 94/10203, SEQ. ID. NO. 1), Inhibin
.beta.c (Hotten, et al. (1995) Bioch. Biophys. Res. Comm.
206:608-613), and GDF-10 (Cunningham, et al. (1995) Growth Factors
12:99-109.). The disclosures of the aforementioned citations are
incorporated herein by reference. Several sequences in the matrix
have alternate names: OP-1 (BMP-7), BMP-2 (BMP-2a), BMP-4 (BMP-2b),
BMP-6 (Vgr1), OP-2 (BMP-8), 60A (Vgr-D), BMP-3 (osteogenin), GDF-5
(CDMP-1, MP-52), GDF-6 (CDMP-2, BMP-13) and GDF-7 (CDMP-3,
BMP-12).
[0047] FIG. 7 is a summary of amino acid residues which, according
to the 2.8.ANG. resolution structure, together define the solvent
accessible surfaces of dimeric hOP-1. FIGS. 7A, 7B, and 7C show the
amino acid sequences defining the human OP-1 finger 1, heel, and
finger 2 regions, respectively. The amino acid residues having 40%
or greater of their sidechain exposed to solvent are boxed, wherein
the solvent accessible amino acid residues that are highly variable
among the BMP/OP family of the TGF-.beta. superfamily are
identified by shaded boxes.
[0048] FIG. 8 is a table, based on the 2.8.ANG. structure, which
summarizes the percentage surface accessibility of the amino acid
side chains in a hOP-1 monomer subunit and in a hOP-1 dimer. Amino
acid residues believed to constitute putative epitopes are
designated "EPITOPE" and amino acid residues which are potential
candidates as surface modifiable amino acids are marked with an
asterisk. In addition, surface modifiable amino acids which are
preferred candidates for enhancing solubility are marked with an
asterisk.
[0049] FIG. 9 is a table, based on the 2.8.ANG. structure, which
summarizes amino acid residues believed to define the ridge. Amino
acid residues believed to constitute the receptor binding domain in
the ridge are marked with an asterisk.
[0050] FIG. 10 is a schematic representation of a computer system
useful in the practice of the invention.
[0051] FIGS. 11A and 11B are tables, produced by reference to the
2.8.ANG. structure, which summarize amino acid pairs believed to be
useful as sites for introducing additional inter-chain (11A) or
intra-chain (11B) disulfide bonds in the hOP-1 dimer.
[0052] FIG. 12 is an amino acid sequence alignment showing the
amino acid sequence of mature human OP-1, and peptides defining the
finger-1, finger-2 and heel regions of human OP-1.
[0053] FIGS. 13A-13D are bar graphs illustrating the effect of
finger-2 and heel peptides on the alkaline phosphatase activity of
ROS cells incubated in either the presence or absence of soluble
OP-1. FIGS. 13A, 13B, 13C, and 13D show the effect of peptides
F2-2, F2-3, Hn-2 and Hn-3, respectively, on the alkaline
phosphatase activity of ROS cells incubated in the presence (shaded
bars) or absence of soluble OP-1 (unshaded bars).
[0054] FIGS. 14A and 14B are graphs showing the displacement of
radiolabelled soluble OP-1 from ROS cell membranes by finger 1,
finger 2, and heel peptides. FIG. 14A shows the displacement of
radiolabelled OP-1 from ROS cell membranes by unlabeled soluble
OP-1 (open circles and triangles), finger 2 peptide F2-2 (closed
circles) and finger 2 peptide F2-3 (closed triangles). FIG. 14B
shows the displacement of radiolabelled OP-1 from ROS cell
membranes by unlabeled soluble OP-1 (open triangles), finger 1
peptide F1-2 (closed boxes), heel peptide H-n2 (open diamonds) and
heel peptide H-c2 (open circles).
[0055] FIG. 15 is a table summarizing the atomic co-ordinates of
hOP-1 resolved to 2.8.ANG..
[0056] FIG. 16 is a table summarizing the atomic co-ordinates of
hOP-1 resolved to 2.3.ANG..
[0057] Further particulars concerning the drawings are disclosed in
the following description which discloses details of the
three-dimensional structure of hOP-1, methods for identifying
morphogen analogs, and methods for making, testing and using such
morphogen analogs.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0058] I. Introduction
[0059] As described hereinbelow, the three-dimensional crystal
structure of mature hOP-1 now has been solved to 2.3.ANG.. The
disclosure provides two sets of atomic co-ordinates for hOP-1,
wherein one set of co-ordinates (see FIG. 15) represents the
structure of hOP-1 resolved to 2.8.ANG., and the other set of
co-ordinates (see FIG. 16) represents the structure of hOP-1
resolved to 2.3.ANG.. This disclosure thus provides, the atomic
co-ordinates defining the relative positions, in three-dimensional
space, of at least the C-terminal 104 amino acids of human OP-1
which are sufficient for imparting biological activity. The
disclosure provides also an analysis of the structural features of
hOP-1. The skilled artisan now can use some or all of these
co-ordinates in a database for making morphogenic protein analogs,
particularly OP-1 analogs. Specifically, the artisan can select
part or all of the database to create templates of part, or all of
the hOP-1 structure in three-dimensions, and using this template,
create a desired analog or variant which may be amino acid-based,
or alternatively composed, in whole or in part, by non-amino
acid-based organic components.
[0060] Provided below is a detailed description of the
three-dimensional crystal structure of hOP-1, along with a detailed
description on how to use co-ordinates in a database to design a
morphogen analog or structural variant of interest. Amino acid
sequences as exemplary templates are provided as examples for
designing, identifying, and producing an OP-1 analog using one of
the OP-1 atomic co-ordinate databases. Specifically contemplated
herein as useful analogs include: small amino molecules which mimic
the receptor binding region of the protein; analogs having enhanced
stability or solubility; analogs having reduced clearance rates
from the body; or enhanced target tissue specificity. The reader
will appreciate that these examples are merely exemplary. Given the
disclosure of the co-ordinates, the three-dimensional structure,
the use of the co-ordinates in a database, and the level of skill
in the art today, still other analogs, not specifically recited
herein, are contemplated and enabled by this disclosure. In
particular, it will be appreciated that, given the disclosure
herein, and the known amino acid sequences for other, closely
related morphogens, the methods can be used to create other
morphogen analogs of, for example, BMP2, BMP4, OP2, BMP5 and
BMP6.
[0061] II. Structural Determination of hOP-1
[0062] A. Determination of the 2.8.ANG. Structure
[0063] Crystals of mature hOP-1 were grown by mixing equal volumes
of purified protein (zkaynak et al. (1990) EMBO J. 9:2085-20893;
and Sampath et al.(1992) J. Biol. Chem. 267:20352-20362) at 10
mg/ml, with 8% saturated ammonium sulfate in 50 mM sodium acetate
buffer (pH 5.0) (Griffith et al. (1994) J. Mol. Biol. 244:657-658).
The crystals have the symmetry of space group P3.sub.221 with unit
cell dimensions a=b=99.46.ANG., and c=42.09.ANG.. One crystal was
used to collect a complete native data set to 2.8.ANG. resolution
at 4.degree. C. Two heavy atom derivative data sets were collected
at 4.degree. C., one from a crystal soaked for seven days in 0.3 mM
uranyl nitrate and the other from a crystal soaked for eight hours
in 0.5 mM sodium gold (II) tetra chloride (Griffith et al (1994)
supra).
[0064] The native and derivative data sets were integrated and
reduced with the R-AXIS-IIC software suite (Higashi (1990) A
Program for Indexing and Processing R-AXIS IIC Imaging Plate Data,
Rigaku Corp.) and scaled together using the CCP4 program ANSC
(Collaborative Computation Project (1994) Acta Cryst. D50:760-763).
Inspection of the Harker sections of the difference Patterson map
reveals a single uranyl site. The position of the single gold site
was determined by using cross-Fourier techniques using the uranyl
position as the phasing site. The heavy atom x,y,z parameters and
occupancy were refined with the program TENEYCK (Ten Eyck et al.
(1976) J. Mol. Biol. 100:3-11). Using these two derivatives and
their anomalous signals, an initial phase set was calculated to
4.0.ANG. resolution with a mean figure of merit of 0.72. The phases
were improved and extended to 3.5.ANG. resolution by cycles of
solvent flattening (Wang (1985) Meth. Enzymol. 115:90-112) and
phase combination (Reed (1986) Acta Cryst. A42:140-149) using the
CCP4 (Collaborative Computation Project (1994) supra)
crystallographic package. A completely interpretable 3.5.ANG.
resolution electron density map permitted the unambiguous tracing
of the polypeptide chain and identification of the amino acids from
Gln 36 to His 139 using the graphic program "O" (Jones et al.
(1991) Acta Crystallogr. A47:110-119). The model was refined with
the program XPLOR (Brunger et al. (1987) Science 235:458-460) by
using all reflections between 10.ANG. and 2.8.ANG. resolution for
which F.sub.obs>2.O.sigma.(F.sub.obs). There were no water
molecules included in the refinement. The root mean square (rms)
deviation from ideality is 0.02 .ANG. for bond lengths, 3.2.degree.
for bond angles. Good stereochemistry was observed for backbone
torsion angles. The current R factor is 22.8%.
[0065] The atomic co-ordinates defining the 2.8.ANG. resolution
structure are listed in FIG. 15. In FIG. 15, the columns entitled
"Atom" denote atoms whose co-ordinates have been measured. The
first letter in the column defines the element. The columns
entitled "Residue" denote the amino acid residues in the hOP-1
monomer which contain an atom whose co-ordinates have been
measured. The column entitled "Chain" denotes whether the atom of
interest is located within the first (A) or second (B) monomer
subunit of the hOP-1 dimer. The columns "X, Y, Z" are the Cartesian
co-ordinates that define the atomic position of the atom
measured.
[0066] B. Determination of the 2.3.ANG. Structure
[0067] Crystals of mature hOP-1 were produced as described in the
previous section. One crystal, frozen in liquid nitrogen, was used
to collect a data set to 2.3.ANG. resolution that was 91% complete.
The data were collected on imaging plates at beam line X12C
(National Synchrotron Light Source) with an oscillation range of
0.5 degrees (overlap of 0.1 degrees) and exposure times of 60-90
seconds. The digitalized data were processed, merged and scaled
with DENZO and SCALEPACK (available from Molecular Structure
Corporation, Texas). An initial 2Fo-FC map, calculated after X-PLOR
rigid-body refinement using the 2.8.ANG. model, was readily
interpretable. Portions of the model were manually refitted to the
electron-density map with the interactive graphics programs "O" and
"Chain". Subsequent cycles of refinement (XPLOR/PROFFT) and manual
rebuilding (QUANTA) rapidly converged to the present model.
[0068] The current model yielded a conventional crystallographic R
factor of 23.5% for data from 10 to 2.3.ANG. (1.5.sigma. cutoff)
and a R.sub.free of 27%. The refined structure was analyzed using
the PROCHECK (available from Protein Data Bank, Brookhaven, N.Y.)
algorithm and corrected where appropriate. The root mean square
(rms) deviation from ideality is 0.015.ANG. for bond distances,
0.034.ANG. for angle distances, and 0.142.ANG. for planar 1-4
distances. The rms deviation from ideality is 1.7.degree. for bond
angles. The upper estimate of the error in the atomic positions
from the Luzzati plots (EXPLOR) using the free R factor is
0.25-0.33.ANG.. The final model, comprising one monomer subunit,
consists of 828 protein atoms (i.e., all non-hydrogen atoms) and 33
water molecules. The average temperature (B) factor is
33.ANG..sup.2 for protein atoms and 37.ANG..sup.2 for solvent
atoms.
[0069] The atomic co-ordinates defining the 2.3.ANG. resolution
structure are listed in FIG. 16. In FIG. 16, the columns entitled
"Atom" denote atoms whose co-ordinates have been measured. The
first letter in the column defines the element. The columns
entitled "Residue" denote the amino acid residues in the hOP-1
monomer which contain an atom whose co-ordinates have been
measured. The column entitled "Chain" denotes whether the atom of
interest is located within the first (A) or second (B) monomer
subunit of the hOP-1 dimer. The columns "X, Y, Z" are the Cartesian
co-ordinates that define the atomic position of the atom measured.
The column denoted ".delta." represents the uncertainty in the
position of the co-ordinate as derived from the temperature factor
(B) of each corresponding atom. The uncertainty of each co-ordinate
was derived from the 1 formula = B 8 2
[0070] (see "Protein Crystallography" (1976) T. L. Blundell and L.
N. Johnson, Academic Press, p. 121) and is expressed in units of
.ANG..
[0071] III. Structural Features of hOP-1 Monomer Subunits
[0072] Human OP-1, like TGF-.beta.2, is a dimeric protein having a
unique folding pattern involving six of the seven C-terminal
cysteine residues, as illustrated in FIG. 1A. Each of the subunits
in OP-1, like TGF .beta.2 (See Daopin et al. (1992) Science
257:369-373; and Schulnegger et al (1992) Nature 358:430-434) have
a characteristic folding pattern, illustrated schematically in FIG.
1A, that involves six of the seven C-terminal cysteine
residues.
[0073] Referring to FIG. 1A, four of the cysteine residues in each
subunit form two disulfide bonds which together create an eight
residue ring, while two additional cysteine residues form a
disulfide bond that passes through the ring to form a knot-like
structure (cysteine knot). With a numbering scheme beginning with
the most N-terminal cysteine of the 7 conserved cysteine residues
assigned number 1, the 2nd and 6th cysteine residues are disulfide
bonded to close one side of the eight residue ring while the 3rd
and 7th cysteine residues are disulfide bonded to close the other
side of the ring. The 1st and 5th conserved cysteine residues are
disulfide bonded through the center of the ring to form the core of
the knot. Amino acid sequence alignment patterns suggest this
structural motif is conserved between members of the TGF-.beta.
superfamily. The 4th cysteine is semi-conserved and when present
typically forms an inter-chain disulfide bond (ICDB) with the
corresponding cysteine residue in the other subunit.
[0074] Each hOP-1 monomer subunit comprises three major tertiary
structural elements and an N-terminal region. The structural
elements are made up of regions of contiguous polypeptide chain
that possess over 50% secondary structure of the following types:
(1) loop, (2) helix and (3) .beta.-sheet. Furthermore, in these
regions the N-terminal and C-terminal strands are not more than 7
.ANG. apart.
[0075] The amino acid sequence between the 1st and 2nd conserved
cysteines (FIG. 1A) form a structural region characterized by an
anti-parallel .beta.-sheet finger, referred to herein as the finger
1 region (F1). A ribbon trace of the human OP-1 finger 1 peptide
backbone is shown in FIG. 1B. Similarly the residues between the
5th and 6th conserved cysteines in FIG. 1A also form an
anti-parallel .beta.-sheet finger, referred to herein as the finger
2 region (F2). A ribbon trace of the human OP-1 finger 2 peptide
backbone is shown in FIG. 1D. A .beta.-sheet finger is a single
amino acid chain, comprising a .beta.-strand that folds back on
itself by means of a .beta.-turn or some larger loop so that the
entering and exiting strands form one or more anti-parallel
.beta.-sheet structures. The third major structural region,
involving the residues between the 3rd and 4th conserved cysteines
in FIG. 1A, is characterized by a three turn .alpha.-helix referred
to herein as the heel region (H). A ribbon trace of the human OP-1
heel peptide backbone is shown in FIG. 1C.
[0076] The organization of the monomer structure is similar to that
of a left hand (see FIG. 1E) where the knot region is located at
the position equivalent to the palm (16), the finger 1 region is
equivalent to the index and middle fingers (12 and 13,
respectively), the .alpha.-helix, or heel region, is equivalent to
the heel of the hand (17), and the finger 2 region is equivalent to
the ring and small fingers (14 and 15, respectively). The
N-terminal region (undefined in the 2.8 .ANG. resolution map
disclosed herein) is predicted to be located at a position roughly
equivalent to the thumb (11).
[0077] Monovision ribbon tracings illustrating the alpha carbon
backbones of each of the three major independent structural
elements of the monomer are illustrated in FIGS. 1B-1D.
Specifically, the finger 1 region comprising the first
anti-parallel .beta.-sheet segment is shown in FIG. 1B, the heel
region comprising the three turn .alpha.-helical segment is shown
in FIG. 1C, and the finger 2 region comprising second and third
anti-parallel .beta.-sheet segments is shown in FIG. 1D.
[0078] For the sake of comparison, FIG. 3 shows an alignment of the
amino acid sequences defining the finger 1, finger 2 and heel
regions of hOP-1 and TGF-.beta.2. In FIG. 3, the OP-1 and
TGF-.beta.2 amino acid sequences were aligned according to the
corresponding regions of local structural identity in the OP-1 and
TGF-.beta.2 structures. Alignment gaps were positioned in loop
regions, which is where the local conformational homology of the
.alpha.-carbon traces tends to be the lowest.
[0079] The structure-based alignment of OP-1 and TGF-.beta.2 then
was used as a template for the alignment of the 7-cysteine domain
sequences of other TGF-.beta. superfamily members (other members of
the TGF-.beta. superfamily are set forth in FIG. 6). Alignment gaps
were positioned in regions which are loops in both the OP-1 and
TGF-.beta.2 structures. Percent identity between pairs of sequences
was calculated as the number of identical aligned sequence
positions, excluding gaps, normalized to the geometric mean of the
lengths of the sequences and multiplied by 100. FIG. 6 is a matrix
of the resulting pair wise present identities between super family
sequences so aligned. Using such principles, it is contemplated
that the hOP-1 and TGF-.beta.2 structures, either alone or in
combination, may be used for homology modeling of other proteins
belonging to the TGF-.beta. superfamily whose three-dimensional
structures have not yet been determined (see, for example, the
other members of the TGF-.beta. superfamily listed in FIG. 6). It
is contemplated that such models may be useful in designing
morphogen analogs for the particular candidate morphogens of
interest, however, for simplicity, the disclosure hereinbelow
refers specifically the design, identification, and production of
morphogen analogs of hOP-1.
[0080] FIG. 3 also shows, based on an analysis of the 2.8.ANG.
resolution structure, a comparison of interchain contact residues
in OP-1 and TGF-.beta.2. Residues were designated as contact
residues if the distance between the centers of at least one
non-hydrogen atom from each side chain was less than the sum of
their Van der Waals radii plus 1.1.ANG.. Despite the low level of
sequence identity between OP-1 and TGF-.beta.2, the inter chain
contacts between residues in the heel of one chain and residues in
finger 1 and finger 2 of the other chain are well conserved.
[0081] Upon detailed inspection of the 2.8.ANG. resolution
structure of hOP-1, the finger 1 region of hOP-1 is an antiparallel
.beta.-sheet containing a thirteen residue omega loop (Phe 47-Glu
60) (FIG. 2). The structural alignment of the OP-1 and TGF-.beta.2
sequences in FIG. 3 places two gaps in the omega loop. The first
gap represents a deletion in hOP-1 that aligns with Arg 26 in the
.alpha.2 helix of TGF-.beta.2. This deletion results in a tighter,
non-.alpha.-helical turn in OP-1 as compared with TGF-.beta.2. The
second gap corresponds to the insertion of Gln 53 in OP-1, which
has the result of directing both Gln 53 and Asp 54 side chains into
the solvent. By comparison, in the corresponding region of
TGF-.beta.2, only Lys 31 is in contact with the solvent. These
differences in the conformation of the omega loop also result in
the conserved proline (Pro 59) adopting a trans conformation in
hOP-1 rather than cis, as in TGF-.beta.2. The conformation of the
omega loop orients six non-polar residues so they can contribute to
a solvent inaccessible interface with Finger 2. Of these six, four
are aromatic (Phe 47, Trp 55, Tyr 62 and Tyr 65), and two are
aliphatic (Ile 56 and Ile 57). In all, the conformation of the
omega loop backbone places five polar residues (Arg 48, Asp 49, Gln
53, Asp 54, and Glu 60) in contact with solvent. The net surface
charge in this region is -2 whereas it is +2 for TGF-.beta.2 (FIG.
5).
[0082] According to the 2.8.ANG. structure, the only a helix in the
monomer is located between the third and fifth cysteines (Cys 71
and Cys 104). This helix extends for three and one-half turns from
residues Thr 82 to Ile 94, is amphipathic, and contains a number of
hydrophobic residues which in the dimer make contact with residues
from Finger 1 and Finger 2 of the other monomer (FIG. 3). Several
hydrophilic residues (Thr 82, His 84, and Gln 88) form one wall of
an internal solvent pocket near the 2-fold axis of the dimer, while
others (Asn 83, His 92, and Asn 95) are in contact with the
external solvent. The conformation of the loop leading from the
C-terminal end of the helix back to the cysteine knot is similar in
OP-1 and TGF-.beta.2. By comparison, the loop located at the
N-terminal end of the helix is 3 residues longer in OP-1, resulting
in a different fold than in TGF-.beta.2. In this loop of OP-1, it
is believed that an N-linked sugar moiety is attached to Asn 80,
however, no such corresponding glycosylation site exists in
TGF-.beta.2. Further, this loop is uncharged in OP-1 whereas it is
negatively charged in TGF-.beta.2.
[0083] According to the 2.8.ANG. structure, Finger 2 is the second
antiparallel .beta.-sheet in OP-1 (FIG. 2). The polypeptide chain
reverses direction between segments .beta.6 and .beta.7 through a
3:5 turn (Sibanda, et al. (1991) Methods in Enzymol. 202:59-82)
beginning at residue Asp 118 and ending at residue Asn 122. In
contrast, TGF-.beta.2 has one less residue in this loop and adopts
a 2:2 turn (Sibanda et al. (1991) supra). Residues Arg 129 to Val
132, located between segments .beta.7 and .beta.8, form a peptide
bridge that crosses over the C-terminal end of strand .beta.5 and
produces a 180.degree. twist in the Finger 2 antiparallel
.beta.-structure. A similar structure is observed in other cysteine
knot growth factors, however the peptide bridge length varies
(McDonald et al. (1991) Nature 354:411-414). Within the monomer,
Finger 2 makes intra-chain contacts with Finger 1 by contributing
aromatic residues Tyr 116, Phe 117 and Tyr 128, and aliphatic
residues Val 114, Leu 115, Val 123, Met 131 and Val 133 to a
solvent inaccessible interface. OP-1 and TGF-.beta.2 differ by
three charges in the region of the Finger 2 turn; OP-1 has two
negative charges while TGF-.beta.2 has one positive charge. In the
region between the turn and the peptide bridge, OP-1 has a net
charge of +3 while TGF-.beta.2 is neutral (FIG. 5).
[0084] The N-terminus of each monomeric subunit is believed to be
highly mobile and has not been resolved in the 2.8.ANG. resolution
structure of hOP-1. The N-terminal region can be deleted without
affecting biological activity and, therefore, it is contemplated
that this portion of mature hOP-1 may be removed and replaced with
other protein or peptide sequences, such as antibodies, and/or
radiolabel binding sites for enhancing targeting to a particular
locus in vivo or for use in in vivo imaging experiments. In
addition, the N-terminal region may be replaced with an ion
chelating motif (e.g., His.sub.6) for use in affinity purification
schemes, or replaced with proteins or peptides for enhancing
solubility in aqueous solvents.
[0085] IV. Structural Features of the hOP-1 Dimer
[0086] FIG. 4 shows stereo ribbon trace drawings representative of
the peptide backbone of the hOP-1 dimer complex, based on the
2.8.ANG. structure. The two monomer subunits in the dimer complex
are oriented symmetrically such that the heel region of one subunit
contacts the finger regions of the other subunit with the knot
regions of the connected subunits forming the core of the molecule.
The 4th cysteine forms an inter-chain disulfide bond with its
counterpart on the second chain thereby equivalently linking the
chains at the center of the palms. The dimer thus formed is an
ellipsoidal (cigar shaped) molecule when viewed from the top
looking down the two-fold axis of symmetry between the subunits
(FIG. 4A). Viewed from the side, the molecule resembles a bent
"cigar" since the two subunits are oriented at a slight angle
relative to each other (FIG. 4B).
[0087] As shown in FIG. 4, each of the structural elements which
together define the native monomer subunits of the dimer are
labeled 43, 43', 44, 44', 45, 45', 46, and 46', wherein, elements
43, 44, 45, and 46 are defined by one subunit and elements 43',
44', 45', and 46' belong to the other subunit. Specifically, 43 and
43' denote the finger 1 regions; 44 and 44' denote heel regions; 45
and 45' denote the finger 2 regions; and 46 and 46' denote
disulfide bonds which connect the 1 st and 5th conserved cysteines
of each subunit to form the knot-like structure. From FIG. 4, it
can be seen that the heel region from one subunit, e.g., 44, and
the finger 1 and finger 2 regions, e.g., 43' and 45', respectively
from the other subunit, interact with one another. These three
elements are believed to cooperate with one another to define a
structure interactive with the ligand binding interactive surface
of the cognate receptor.
[0088] The helical axis is defined as the line equi-distant from
the alpha carbons in the helical region. A sequence of four points
is needed to define the dihedral angle between the axes of the
helices in the dimer. The two inner points were chosen to lie on
the helical axes adjacent to the .alpha.-carbon of residue His 84
in OP-1 or His 58 in TGF-.beta.2, respectively. The two outer
points were chosen to lie on their respective helical axes, but
their location is arbitrary. To measure the angle between the
helices, the first two points used to define the dihedral angle
were translated so as to superimpose the inner points. The
resulting three points define the angle.
[0089] A major difference between the OP-1 and TGF-.beta.2 dimers
is the relative orientation of the helices in the heel region. The
angle between the axes of the helices in the heel region of OP-1 is
43.degree. which is 10.degree. larger than that measured for
TGF-.beta.2. The measured dihedral angle between the helices is
-20.degree. for OP-1 which is 14.degree. more negative than for
TGF-.beta.2. Despite these differences in helical orientation, the
same helix and finger residue positions are involved in making
inter-chain contacts, as evidenced by the shaded residues in FIG.
3.
[0090] A. Differences in the hOP-1 Dimer Relative To Individual
Monomer Subunits
[0091] During dimerization of the monomer subunits, several amino
acids on the surface of each monomer subunit become buried in the
hOP-1 dimer. FIG. 8 highlights differences in the surface
accessibility of particular amino acid residues located in the
hOP-1 monomer subunit relative to those in the hOP-1 dimer, as
determined from the 2.8 .ANG. structure.
[0092] Loss of non-polar surface area during dimerization was
calculated using ACCESS (version 2.1) with a 1.4.ANG. probe (Lee et
al. (1971) J. Mol. Biol. 55:379-400). Non-polar surface area is
defined as the contribution to the total accessible surface from
carbon and sulfur atoms. The surface area measurement algorithm in
ACCESS slices the structure into 0.25.ANG. slabs perpendicular to
the Z-axis. As a consequence, the results are sensitive to the
orientation of a structure relative to the Z-axis (Lee et al.
(1971) supra). In order to minimize this effect, we evaluated three
perpendicular and one intermediate orientations of each structure.
The results of these calculations were combined by accepting, for
each non-polar atom, the largest accessible area measured among the
four orientations. The values for TGF-.beta.2 reported here were
calculated using coordinates from entry 2TG1 (Daopin et al. (1992)
supra) and entry ITFG (Schlunegger et al. (1992) supra) obtained
from the January 1994 release of the Protein Data Bank (Bernstein
et al. (1977) J. Mol. Biol. 112:535-542) at Brookhaven National
Laboratory.
[0093] In FIG. 8, the column entitled "Residue" denotes an amino
acid of interest. The column entitled "Monomer % Area" denotes the
percentage of the amino acid that is exposed on the surface of the
hOP-1 monomer, the column entitled "Dimer % Area" denotes the
percentage of the amino acid that is exposed on the surface of the
hOP-1 dimer, and the column denoted "Hidden % Area" denotes amount
of surface area for each amino acid that is lost upon dimerization
of each monomer subunit to produce the hOP-1 dimer. This analysis
reveals amino acids which become buried during dimerization and,
thus, likely are located at the interface of the two monomer
subunits. For example, 70.75% of the surface area of His 84 becomes
hidden upon dimerization. A review of the structure of dimeric
hOP-1 reveals that His 84 is located at the interface between the
two monomers.
[0094] B. Solution Electrostatic Potentials on the Surface of OP-1
and TGF-.beta.2
[0095] The solution electrostatic potentials surrounding the OP-1
and TGF-.beta.2 (ITFG) (Schlunegger et al. (1992) supra) dimers
were calculated using DELPHI (Gilson et al. (1987) Nature
330:84-86; and Nicholls et al. (1991) J. Comput. Chem. 12:435-445)
(Biosym Technologies, Inc., San Diego, Calif.). The calculations
were performed using a solvent dielectric constant of 80, a solvent
radius of 1.4.ANG., an ionic strength of 0.145M and an ionic radius
of 2.0 .ANG.. The interior of the protein was modeled using a
dielectric constant of 2.0. Formal charges were used and
distributed as follows: atoms OD1 and OD2 of Asp were each charged
-0.5, atoms OE1 and OE2 of Glu were each charged -0.5, atoms ND1
and NE2 of His were each charged 0.25, atom NZ of Lys was charged
+1.0, atoms NH1 and NH2 of Arg were each charged +0.5, and atom OXT
of the C-terminal carboxyl group was charged -1.0.
[0096] The differences in charge distribution on the surfaces of
OP-1 and TGF-.beta.2 can be observed by comparing the color
distributions of FIGS. 5B and 5C, respectively. Surface regions
having an electrostatic potential of -3 kT or less are shown in red
while surface regions of +3 kT or greater are shown in blue.
Neutral regions are shown in green or gold to correspond to the
backbone ribbons shown in FIG. 5A. As mentioned in the following
section, the differences in electrostatic potential on the surfaces
of OP-1 and TGF-.beta.2 may play an important role in the specific
interactions of the TGF-.beta.3 superfamily members with their
cognate receptors.
[0097] C. Receptor Binding Domain
[0098] Without wishing to be bound by theory, it is contemplated
that the receptor binding regions of hOP-1 includes amino acids
that are both solvent accessible and lie at positions of
heterogeneous composition, as determined from the amino acid
sequence of hOP-1 when aligned with other members of the TGF-.beta.
superfamily (See FIG. 3). Divergent structural features in hOP-1,
like TGF-.beta.2, occur primarily in the external loops of finger 1
and finger 2, the loops bordering the helix in the heel region, and
the residues in the N-terminal domain preceding the first cysteine
of the cysteine knot. These regions are solvent accessible. In both
the OP-1 and TGF-.beta.2 dimer structures, the tip of finger 2 and
the omega loop of finger 1 from one chain, and the C-terminal end
of the .alpha.-helix in the heel of the other chain form a
contiguous ridge approximately 40 .ANG. long and 15 .ANG. wide
(FIG. 5A). It is contemplated that this ridge contains the primary
structural features that interact with the cognate receptor, and
that the binding specificity between different TGF-.beta.
superfamily members derives from conformational and electrostatic
variations on the surface of this ridge.
[0099] Differences in the conformation of the finger 1 omega loop,
which constitutes the mid section of the ridge, and in the turn at
the end of finger 2, which forms one end of the ridge are noted.
However, there are striking differences in the surface charge of
the ridge in hOP-1 relative to TGF-.beta.2 (see FIGS. 5B and 5C).
In hOP-1, the ends of the finger regions are negatively charged
whereas in TGF-.beta.2, the ends of the finger regions are
positively charged. This results in a net charge of -4 for the
receptor binding ridge of hOP-1 versus +3 for TGF-.beta.2.
Conversely, the .beta.-strand located C-terminal to the turn of
finger 2 (.beta.7, FIG. 2) is positively charged in OP-1 whereas it
is negatively charged in TGF-.beta.2 (FIGS. 5B and 5C). These
features suggests that electrostatic charge distribution plays an
important role in the specific interactions of the TGF-.beta.
superfamily members with their cognate receptors.
[0100] FIG. 9 summarizes the amino acid residues which, according
to the 2.8 .ANG. structure, are believed to constitute the ridge,
and also indicates whether each amino acid residue is disposed
within the heel, finger 1, or finger 2 domains. FIG. 9 also
provides a list of amino acid residues which are believed to
constitute at least part, if not all of the receptor binding domain
of hOP-1.
[0101] V. Design of Morphogen Analogs
[0102] Although it is contemplated that the design of morphogen
analogs can be facilitated by conventional ball and stick type
modeling procedures, it is contemplated that the ability to design
morphogen analogs is enhanced significantly using modern
computer-driven modeling and design procedures.
[0103] It is contemplated that the design of morphogen analogs, as
discussed in detail hereinbelow, is facilitated using conventional
molecular modeling computers or workstations, commercially
available from, for example, Silicon Graphics, Inc. or Evans and
Sutherland Computer Corp., which implement equally conventional
computer modeling programs, for example, INSIGHTII, DISCOVER, and
DELPHI, commercially available from Biosym, Technologies Inc., and
QUANTA, and CHARMM commercially available from Molecular
Simulations, Inc.
[0104] Furthermore, it is understood that any computer system
having the overall characteristics set forth in FIG. 10 may be
useful in the practice of the instant invention. More specifically,
FIG. 10, is a schematic representation of a typical computer work
station having in electrical communication (100) with one another
via, for example, an internal bus or external network, a processor
(101), a RAM (102), a ROM (103), a terminal (104), and optionally
an external storage device, for example, a diskette, CD ROM, or
magnetic tape (105).
[0105] It is contemplated, that the co-ordinates can be used not
only to provide a basis for re-engineering hOP-1 dimers by using,
for example, site-directed mutagenesis methodologies, to enhance,
for example, the solubility and or/stability of the active hOP-1
dimer in physiological buffers, but also to provide a starting
point for the de novo design and production of peptides or other
small molecules which mimic the bioactivity of hOP-1. Set forth
below are illustrative examples demonstrating the usefulness of
hOP-1 atomic co-ordinates in the design of morphogen analogs,
however, it is understood the examples below are illustrative and
not meant to be limiting in any way.
[0106] A. Engineering hOP-1 Dimers
[0107] In one aspect, the availability of the atomic co-ordinates
for hOP-1, enables the artisan to perform theoretical amino acid
replacements and to determine by calculation, in advance of
actually making and testing the candidate molecule in a laboratory
setting, whether a particular amino acid substitution disrupts the
packing of the OP-1 dimer and whether a morphogen analog is likely
to be more stable and/or soluble than the template OP-1 molecule.
Such procedures assist the artisan to eliminate non viable
replacements and to focus efforts on more promising candidate
analogs.
[0108] (i) Enhancing the Stability of hOP-1 Dimers
[0109] It is contemplated that the skilled artisan in possession of
the atomic co-ordinates defining hOP-1 can introduce additional
inter- or intra-chain covalent and/or non-covalent interactions
into the hOP-1 dimer to stabilize the dimer by preventing
disassociation or unfolding of each monomer subunit. Preferred
engineered covalent interactions include, for example, engineered
disulfide bonds, and preferred engineered non-covalent interactions
include, for example, hydrogen bonds, salt bridges, and hydrophobic
interactions.
[0110] For example, in order to introduce additional disulfide
bonds, the skilled artisan can identify sites suitable for the
introduction of a pair of cysteine amino acid residues by using
standard molecular modeling programs, for example, INSIGHT,
DISCOVER, CHARMM and QUANTA. Another program useful in identifying
pairs of amino acids as potential sites for introducing stabilizing
disulfide bonds is described in U.S. Pat. No. 4,908,773, the
disclosure of which is incorporated herein by reference.
[0111] For example, the skilled artisan using the INSIGHT program
can screen for pairs of amino acids, where the distance between the
C.beta. atoms of each amino acid is in the range of about 3.0 to
about 5.0, or more preferably about 3.5 to about 4.5 .ANG. apart.
For this purpose, glycines, which contain no C.beta.--C.beta. bond,
are first converted to alanines on the computer. The possible range
of C.beta.--C.beta. distances in a disulfide bond are 3.1 .ANG. to
4.6 .ANG., but separations outside this range can be accommodated
by small shifts in the neighboring atoms. Searching C.beta., rather
than C.alpha. distances, ensures both reasonable spacing as well as
proper orientation of the C.alpha.--C.beta. bond. The effects of
adding such an additional linkage on protein structure are
determined by mutating the two candidates residues to Cys; rotating
each new Cys about the C.alpha.--C.beta. bond to bring the two
.gamma. sulfurs as close to within 2.ANG. as possible; creating a
disulfide between the .gamma. sulfurs; and energy minimizing
structural regions within 5 .ANG. of the disulfide bond. Any
deformation of the structure caused by introduction of the
additional disulfide bond is revealed by inspection when the
minimized, mutated model structure is superimposed on the native
structure.
[0112] It is contemplated that the introduction of additional
linkages will improve solubility by preventing transient exposure
of non-polar interface or buried residues. FIG. 11A lists amino
acid residues, based on the 2.8.ANG. structure, which may be
mutated to cysteine residues for introducing additional inter-chain
disulfide bonds, based upon the selection criteria presented above.
For reference purposes, Table 11A includes also the length of the
naturally occurring inter-chain disulfide linkage in wild type
hOP-1, that is, the disulfide linkage connecting Cys-103 of one
monomer subunit with the counterpart Cys-103 of the other monomer
subunit.
[0113] A preferred pair of residues suitable for modification
include the residue at position 83 of one chain and the residue at
position 130 of the other chain. It is contemplated that the
additional inter-chain linkage stabilizes the dimeric structure by
connecting the N-terminal end of the Heel helix of the first
subunit to the middle of the Finger 2 region in the second subunit.
A disulfide bond between position 82 on one chain and position 130
of the other chain also is geometrically feasible, but because Thr
82 is part of the NAT glycosylation site in OP-1, its modification
may inhibit proper glycosylation.
[0114] FIG. 11B summarizes amino acid residues which can be mutated
to cysteine residues for introducing additional intra-chain
disulfide bonds, based upon the selection criteria presented above.
As noted previously, the putative receptor binding region comprises
at least two physically proximal, but sequentially separate
regions, namely the tips of Finger 1 and Finger 2. It is
contemplated that the structural integrity of the putative receptor
binding ridge can be stabilized by engineering an intra-chain
disulfide link between residues of Finger 1 and Finger 2. In a
preferred embodiment, the residue at position 58 in Finger 1 can be
disulfide bonded with the residue at position 114 in Finger 2. It
is contemplated that a link between the residues at positions 58
and 115 also would be viable, however, this would move the
disulfide bond nearer to the putative receptor binding region on
Finger 2. Also a link between positions 65 and 133 is possible,
however, this would be located near to the knot region of each
chain and, thus may have little effect on stabilizing the putative
receptor binding regions at the tips of Finger 1 and Finger 2.
Additionally, the proximity of such a linkage to the disulfide
bonds in the knot region might interfere with the proper formation
of those structures.
[0115] With regard to non-covalent interactions, it is contemplated
that the structural stability of the hOP-1 dimer can be enhanced by
increasing inter-chain hydrogen bonding.
[0116] The electrostatic potential (due to other charges in a
protein) in the region of a charged residue affects the pK of that
residue. Because the pK's of both histidine and the N-terminal
primary amino group are near neutrality, it may be possible to
modify their pKs through the placement of charges on the surface of
the molecule. It is contemplated that the buried His at position
His 84 in hOP-1 helps stabilize the structure of the dimer by
participating in hydrogen bonds with backbone carboxyl groups of
residues Ala 64 and Tyr 65 of the other chain. Accordingly, it is
contemplated that the introduction of surface charges may enhance
this effect and thereby further stabilize the structure of the
molecule. For example, mutating Tyr 65 or Val 132 to Asp may
further polarize the carbonyl bonds of the amino acid residues at
positions 64 and 65, as well as raise the pK of His 84. The pK of
His 84 may further be affected by replacing residues Tyr 44, Ala
63, or Asn 110 by an Asp. It is contemplated that the preferred
modification for this purpose is Tyr 65->Asp 65.
[0117] Using the same basic principles, the skilled artisan
likewise can identify pairs of amino acids whose replacement can
facilitate the introduction of an inter-chain salt bridge, internal
hydrogen bond, or hydrophobic interaction. Such determinations are
within the scope of an artisan having an ordinary level of skill in
the field of molecular modeling.
[0118] Once a pair of target amino acids has been identified, the
site-directed replacement of the target amino acids with the
desirable replacements can be facilitated by the use of
conventional site-directed mutagenesis procedures, for example, by
cassette mutagenesis or oligonucleotide-directed mutagenesis. Such
techniques are thoroughly documented in the art and so are not
discussed herein. The effect of the site-specific replacements on
the stability of resulting modified hOP-1 dimers or muteins can be
measured, after production and purification, using standard
methodologies well known in the art, for example, circular
dichroism, analytical centrifugation, differential scanning
calorimetry, fluoresence or other spectroscopic techniques.
[0119] (ii) Enhancing Water Solubility of hOP-1 Dimer
[0120] OP-1 has limited solubility in aqueous solvents. It is
contemplated, however, that by using the hOP-1 atomic co-ordinates
that the artisan can replace amino acids at the solvent accessible
surface of the dimer thereby to increase the dielectric properties
of dimeric hOP-1. For example, solvent accessible hydrophobic amino
acid residues, such as, glycine, alanine, valine, leucine and
isoleucine may be replaced by more polar residues, such as, lysine,
arginine, histidine, aspartate, asparagine, glutamate and
glutamine.
[0121] The solvent accessible amino acids can be identified using a
computer program, such as ACCESS (version 2.1) using a 1.4 .ANG.
probe (Lee et al. (1972) supra). In FIG. 7 amino acid residues
having at least 20% of their side chain areas exposed to solvent
are boxed. When modifying surface residues it is important not to
produce new epitopes that can be recognized as non-host especially,
if the hOP-1 analogs are to be used as injectable molecules. It is
believed that, amino acid side chains seen by a 10 .ANG. spherical
probe likely are part of surface epitopes. One skilled in the art
can use ACCESS with a 10 .ANG. spherical probe to identify
potential epitopes, however this process can be carried out
manually using a graphics package, such as, INSIGHT II. In FIG. 8,
residue side chains so identified as potential epitopes are
highlighted. Residue positions that are candidates for modification
so as to improve the solubility of the dimer are highlighted.
Preferred candidate amino acids for replacement include, for
example, Ala 63, Ala 72, Ala 81, Ala 111 and Ala 135, Ile 86, Ile
112, Tyr 52, Tyr 65, and Tyr 128.
[0122] Once solvent accessible hydrophobic or non polar amino acids
have been identified (see FIG. 9), these amino acids theoretically
may be replaced, via a computer, with more polar amino acids. The
effect of the amino acid replacements on the solution electrostatic
potentials surrounding the modified hOP-1 dimer as well as the free
energy of the dimer can calculated using the program DELPHI (Gilson
et al (1987) supra; Nicholls et al. (1991) supra). Preferred amino
acid substitutions lower the free energy of the hOP-1 dimer without
introducing potential antigenic sites. As mentioned above, such
antigenic sites may be detected by implementing a computer program
like ACCESS (version 2.1) using a 10 .ANG. probe. In addition, it
is contemplated that preferred surface residues suitable for
replacement do not constitute part of the receptor binding
domain.
[0123] The resulting candidate morphogen analogs can be produced
using conventional site-directed mutagenesis methodologies and the
effect of the site-directed modification on the solubility of the
hOP-1 dimer can be measured, for example, by comparing the
partition coefficient or "salting out properties" of the modified
hOP-1 dimer versus the native hOP-1 dimer. See for example, Scopes
(1987) in Protein Purification: Principles and Practice, 2nd
Edition (Springer-Verlag); and Englard et al. (1990) Methods in
Enzymology 182: 285-300.
[0124] (iii) Engineering Glycosylation Sites
[0125] In addition to replacing single, solvent accessible amino
acid residues with more polar or hydrophobic amino acid residue,
one or more solvent accessible amino acid residues may be replaced
so as to create a new eukaryotic glycosylation site or
alternatively to eliminate or alter an existing glycosylation site.
Glycosylation sites are well known and are thoroughly described in
the art. Addition of a new glycosylation site or alteration of an
existing site may result in the addition of one or more glycosyl
groups, e.g., N-acetyl-sialic acid, which may enhance the
solubility of the morphogen analog. As described herein, such sites
can be introduced by site-directed mutagenesis methodologies which
are well known in the art. Preferably, such sites do not create new
antigenic determinants (although these may be tolerable for short
duration therapeutic uses). Reference to Table 8 identifies surface
accessible amino acid residues, based on the 2.8.ANG. structure,
which likely are not part of an antigenic epitope and which may be
used as candidates for introducing an additional glycosylation
site.
[0126] B. Engineering Small Molecules Based Upon The hOP-1
Structure
[0127] The availability of atomic co-ordinates for hOP-1 enables
the skilled artisan to design small molecules, for example,
peptides or non-peptidyl based organic molecules having certain
chemical features, which mimic the biological activity of hOP-1.
Chemical features of interest may include, for example, the
three-dimensional structure of a particular protein domain, solvent
accessible surface of a particular protein domain, spatial
distribution of charged and/or hydrophobic chemical moieties,
electrostatic charge distribution, or a combination thereof. Such
chemical features may readily be determined from the
three-dimensional representation of hOP-1.
[0128] (i) Peptides
[0129] After having determined which amino acid residues contribute
to the receptor binding domain (supra), it is possible for the
skilled artisan to design synthetic peptides having amino acid
sequences that define a pre-selected receptor binding motif. A
computer program useful in designing potentially bioactive
peptido-mimetics is described in U.S. Pat. No. 5,331,573, the
disclosure of which is incorporated by reference herein.
[0130] In addition to choosing a desirable amino acid sequence, the
skilled artisan using standard molecular modeling software
packages, infra, can design specific peptides having, for example,
additional cysteine amino acids located at pre-selected positions
to facilitate cyclization of the peptide of interest. Oxidation of
the additional cysteine residues results in cyclization of the
peptide thereby constraining the peptide in a conformation which
mimics the conformation of the corresponding amino acid sequence in
native hOP-1. It is contemplated, that any standard covalent
linkage, for example, disulfide bonds, typically used to cyclize
synthetic peptides maybe useful in the practice of the instant
invention. Alternative cyclization chemistries are discussed in
International Application PCT/WO 95/01800, the disclosure of which
is incorporated herein by reference.
[0131] In addition, it is contemplated that a single peptide
containing amino acid sequences derived from separate hOP-1 subunit
domains, for example, a single peptide having an amino acid
sequence defining the tip of the finger 1 region linked by means of
a polypeptide linker to an amino acid sequence defining the tip of
the finger 2 region. The amino sequence defining each of the finger
regions may further comprise a means, for example, disulfide bonds
for cyclizing each finger region motif. The resulting peptide
therefore comprises a single polypeptide chain having a first amino
acid sequence defining a three-dimensional domain mimicking the tip
of the finger 1 region and a second said sequence defining a
three-dimensional domain mimicking the tip of the finger 2
region.
[0132] Such peptides may be synthesized and screened for OP-1 like
activity using any of the standard protocols described below.
[0133] (ii) Organic Molecules
[0134] As discussed above, upon determination of the receptor
binding domain of hOP-1, it is contemplated that the skilled
artisan, can design non-peptidyl based small molecules, for
example, small organic molecules, whose structural and chemical
features mimic the same features displayed on at least part of the
surface of the receptor binding domain of hOP-1.
[0135] Because a major contribution to the receptor binding surface
is the spatial arrangement of chemically interactive moieties
present within the sidechains of amino acids which together define
the receptor binding surface, a preferred embodiment of the present
invention relates to designing and producing a synthetic organic
molecule having a framework that carries chemically interactive
moieties in a spatial relationship that mimics the spatial
relationship of the chemical moieties disposed on the amino acid
sidechains which constitute the receptor binding site of hOP-1.
Preferred chemical moieties, include but are not limited to, the
chemical moieties defined by the amino acid side chains of amino
acids believed to constitute the receptor binding domain of hOP-1
(See FIG. 9). It is understood, therefore, that the receptor
binding surface of the morphogen analog need not comprise amino
acid residues but the chemical moieties disposed thereon.
[0136] For example, upon identification of relevant chemical
groups, the skilled artisan using a conventional computer program
can design a small molecule having the receptor interactive
chemical moieties disposed upon a suitable carrier framework.
Useful computer programs are described in, for example, Dixon
(1992) Tibtech 10: 357-363; Tschinke et al. (1993) J. Med. Chem.
36: 3863-3870; and Eisen et al. (1994) Proteins: Structure,
Function, and Genetics 19: 199-221, the disclosures of which are
incorporated herein by reference.
[0137] One particular computer program entitled "CAVEAT" searches a
database, for example, the Cambridge Structural Database, for
structures which have desired spatial orientations of chemical
moieties (Bartlett et al. (1989) in "Molecular Recognition:
Chemical and Biological Problems" (Roberts, S. M., ed) pp 182-196).
The CAVEAT program has been used to design analogs of tendamistat,
a 74 residue inhibitor of .alpha.-amylase, based on the orientation
of selected amino acid side chains in the three-dimensional
structure of tendamistat (Bartlett et al. (1989) supra).
[0138] Alternatively, upon identification of a series of analogs
which mimic the biological activity of OP-1, as determined by in
vivo or in vitro assays, the skilled artisan may use a variety of
computer programs which assist the skilled artisan to develop
quantitative structure activity relationships (QSAR) and further to
assist in the de novo design of additional morphogen analogs. Other
useful computer programs are described in, for example,
Connolly-Martin (1991) Methods in Enzymology 203:587-613; Dixon
(1992) supra; and Waszkowycz et al. (1994) J. Med. Chem. 37:
3994-4002.
[0139] Thus, for example, one can begin with a portion of the three
dimensional structure of OP-1 (or a related morphogen)
corresponding to a region of known or suspected biological
importance. One such region is the solvent accessible loop or "tip"
of the finger 2 region between the .beta.6 and .beta.7 sheets
(i.e., from approximately residues 118-122). Synthetic, cyclic
peptides (i.e., F2-2 and F2-3) were produced including this region
(and several flanking residues) and were shown to possess OP-1-like
biological activity (see Examples below). Based upon the
three-dimensional structure of this region, disclosed herein, one
is now enabled to produce more effective OP-1-like (or, generally,
morphogen-like) analogs. For example, as shown in great detail in
FIGS. 7-9 and 15, the charged .gamma.-carboxy groups of Asp 118 and
Asp 119, and the relatively hydrophilic hydroxyl groups of Ser 120
and Ser 121, are solvent accessible and believed to be involved in
OP-1 receptor binding. The relative positions of these groups in
three dimensions in OP-1 are given in FIGS. 15 and 16. These
functional groups define a contiguous portion of the three
dimensional structure of the OP-1 surface. The peptide backbone of
these residues, however, is not solvent accessible and, therefore,
is not believed to form a portion of the three-dimensional surface
of the OP-1 molecule. Thus, one of ordinary skill in the art, when
choosing or designing an OP-1 or morphogen analog, can choose or
design a molecule having the same or substantially equivalent
(e.g., thiol v. hydroxyl) functional groups in substantially the
same (e.g., .+-.1-3 .ANG.) three-dimensional conformation. The same
is true for other regions of interest in the OP-1 monomers or
dimers (e.g., the receptor binding domain, the finger 1, finger 2,
or heel regions, or solvent accessible portions thereof). By using
the three-dimensional structures disclosed herein, including the
disclosure of the positions of solvent accessible and probable
receptor contact residues, one of ordinary skill in the art can
choose a portion of the three-dimensional structure of the OP-1 (or
a related morphogen) molecule and, using this "portion" as a
template select or design an analog which functionally mimics the
template structure.
[0140] The molecular framework or backbone of the morphogen analog
can be freely chosen by one of ordinary skill in the art so that it
(1) joins the functional groups which mimic the portion of the
morphogen's contiguous three-dimensional surface, including charge
distribution and hydrophobicity/hydrophilicity characteristics, and
(2) maintains or, at least, allows the functional groups to
maintain the appropriate three-dimensional surface interaction and
spatial relationships, including any hydrogen bonding and
electrostatic interactions. As described above, peptides are
obvious choices for the production of such morphogen analogs
because they can provide all of the necessary functional groups and
can assume appropriate three-dimensional structures. Several
examples of peptide analogs of the finger regions are described
herein, below. The peptides are cyclized to maintain hydrogen bonds
and create a structure which mimics that of the template. These
peptides are synthesized from a linear primary sequence of amino
acids in finger 2. An alternative peptide can be created, for
example, which combines portions of finger 1 and finger 2,
constructed to mimic the structure of the tips of fingers 1 and 2
together as they occur in the folded OP1 monomer. Biologically
active peptides such as F2, F3 or others, then can be used as is
or, more preferably, become lead compounds for iterative
modification to create a compound that is more stable or more
active in vivo. For example, the peptide backbone can be reduced or
replaced to reduce hydrolysis in vivo. Alternatively, structural
modifications can be introduced to the backbone or by amino acid
substitutions which more accurately mimic the protein's structure
when bound to the receptor. These second generation structures then
can be tested for enhanced binding. In addition, iterative amino
acid replacements with alanines, ("alanine scan") can be used to
determine the minimum residue contacts required for binding.
[0141] Once these minimum functional groups are known, a fully
synthetic molecule can be created which mimics the charge or
electrostatic distribution of the minimum required functional
groups, and provides the appropriate bulk and structure to
functionally mimic a second generation molecule having the desired
binding affinity.
[0142] VI. Production of Morphogen Analogs
[0143] As mentioned above, the morphogen analogs of the invention
may comprise modified hOP-1 dimeric proteins or small molecules,
for example, peptides or small organic molecules. It is
contemplated that any appropriate methods can be used for producing
a pre-selected morphogen analog. For example, such methods may
include, but are not limited to, methods of biological production
from suitable host cells or synthetic production using synthetic
organic chemistries.
[0144] For example, modified hOP-1 dimeric proteins or hOP-based
peptides may be produced using conventional recombinant DNA
technologies, well known and thoroughly documented in the art.
Under these circumstances, the proteins or peptides may be produced
by the preparation of nucleic acid sequences encoding the
respective protein or peptide sequences, after which, the resulting
nucleic acid can be expressed in an appropriate host cell. By way
of example, the proteins and peptides may be manufactured by the
assembly of synthetic nucleotide sequences and/or joining DNA
restriction fragments to produce a synthetic DNA molecule. The DNA
molecules then are ligated into an expression vehicle, for example
an expression plasmid, and transfected into an appropriate host
cell, for example E. coli. The protein encoded by the DNA molecule
then is expressed, purified, folded if necessary, tested in vitro
for binding activity with an OP-1 receptor, and subsequently tested
to assess whether the morphogen analog induces or stimulates
hOP-1-like biological activity.
[0145] The processes for manipulating, amplifying, and recombining
DNA which encode amino acid sequences of interest generally are
well known in the art, and therefore, are not described in detail
herein. Methods of identifying and isolating genes encoding hOP-1
and its cognate receptors also are well understood, and are
described in the patent and other literature.
[0146] Briefly, the construction of DNAs encoding the biosynthetic
constructs disclosed herein is performed using known techniques
involving the use of various restriction enzymes which make
sequence specific cuts in DNA to produce blunt ends or cohesive
ends, DNA ligases, techniques enabling enzymatic addition of sticky
ends to blunt-ended DNA, construction of synthetic DNAs by assembly
of short or medium length oligonucleotides, cDNA synthesis
techniques, polymerase chain reaction (PCR) techniques for
amplifying appropriate nucleic acid sequences from libraries, and
synthetic probes for isolating OP-1 genes or genes encoding other
members of the TGF-.beta. superfamily as well as their cognate
receptors. Various promoter sequences from bacteria, mammals, or
insects to name a few, and other regulatory DNA sequences used in
achieving expression, and various types of host cells are also
known and available. Conventional transfection techniques, and
equally conventional techniques for cloning and subcloning DNA are
useful in the practice of this invention and known to those skilled
in the art. Various types of vectors may be used such as plasmids
and viruses including animal viruses and bacteriophages. The
vectors may exploit various marker genes which impart to a
successfully transfected cell a detectable phenotypic property that
can be used to identify which of a family of clones has
successfully incorporated the recombinant DNA of the vector.
[0147] One method for obtaining DNA encoding the biosynthetic
constructs disclosed herein is by assembly of synthetic
oligonucleotides produced in a conventional, automated,
oligonucleotide synthesizer followed by ligation with appropriate
ligases. For example, overlapping, complementary DNA fragments may
be synthesized using phosphoramidite chemistry, with end segments
left unphosphorylated to prevent polymerization during ligation.
One end of the synthetic DNA is left with a "sticky end"
corresponding to the site of action of a particular restriction
endonuclease, and the other end is left with an end corresponding
to the site of action of another restriction endonuclease. The
complementary DNA fragments are ligated together to produce a
synthetic DNA construct.
[0148] After the appropriate DNA molecule has been synthesized, it
may be integrated into an expression vector and transfected into an
appropriate host cell for protein expression. Useful prokaryotic
host cells include, but are not limited to, E. coli, and B.
subtilis. Useful eukaryotic host cells include, but are not limited
to, yeast cells, insect cells, myeloma cells, fibroblast 3T3 cells,
monkey kidney or COS cells, chinese hamster ovary (CHO) cells,
mink-lung epithelial cells, human foreskin fibroblast cells, human
glioblastoma cells, and teratocarcinoma cells. Alternatively, the
genes may be expressed in a cell-free system such as the rabbit
reticulocyte lysate system.
[0149] The vector additionally may include various sequences to
promote correct expression of the recombinant protein, including
transcriptional promoter and termination sequences, enhancer
sequences, preferred ribosome binding site sequences, preferred
mRNA leader sequences, preferred protein processing sequences,
preferred signal sequences for protein secretion, and the like. The
DNA sequence encoding the gene of interest also may be manipulated
to remove potentially inhibiting sequences or to minimize unwanted
secondary structure formation. The morphogenic protein analogs
proteins also may be expressed as fusion proteins. After being
translated, the protein may be purified from the cells themselves
or recovered from the culture medium and then cleaved at a specific
protease site if so desired.
[0150] For example, if the gene is to be expressed in E. coli, it
is cloned into an appropriate expression vector. This can be
accomplished by positioning the engineered gene downstream of a
promoter sequence such as Trp or Tac, and/or a gene coding for a
leader peptide such as fragment B of protein A (FB). During
expression, the resulting fusion proteins accumulate in refractile
bodies in the cytoplasm of the cells, and may be harvested after
disruption of the cells by French press or sonication. The isolated
refractile bodies then are solubilized, and the expressed proteins
folded and the leader sequence cleaved, if necessary, by methods
already established with many other recombinant proteins.
[0151] Expression of the engineered genes in eukaryotic cells
requires cells and cell lines that are easy to transfect, are
capable of stably maintaining foreign DNA with an unrearranged
sequence, and which have the necessary cellular components for
efficient transcription, translation, post-translation
modification, and secretion of the protein. In addition, a suitable
vector carrying the gene of interest also is necessary. DNA vector
design for transfection into mammalian cells should include
appropriate sequences to promote expression of the gene of interest
as described herein, including appropriate transcription
initiation, termination, and enhancer sequences, as well as
sequences that enhance translation efficiency, such as the Kozak
consensus sequence. Preferred DNA vectors also include a marker
gene and means for amplifying the copy number of the gene of
interest. A detailed review of the state of the art of the
production of foreign proteins in mammalian cells, including useful
cells, protein expression-promoting sequences, marker genes, and
gene amplification methods, is disclosed in Bendig (1988) Genetic
Engineering 7:91-127.
[0152] The best characterized transcription promoters useful for
expressing a foreign gene in a particular mammalian cell are the
SV40 early promoter, the adenovirus promoter (AdMLP), the mouse
metallothionein-I promoter (mMT-I), the Rous sarcoma virus (RSV)
long terminal repeat (LTR), the mouse mammary tumor virus long
terminal repeat (MMTV-LTR), and the human cytomegalovirus major
intermediate-early promoter (hCMV). The DNA sequences for all of
these promoters are known in the art and are available
commercially.
[0153] The use of a selectable DHFR gene in a dhfr.sup.- cell line
is a well characterized method useful in the amplification of genes
in mammalian cell systems. Briefly, the DHFR gene is provided on
the vector carrying the gene of interest, and addition of
increasing concentrations of the cytotoxic drug methotrexate, which
is metabolized by DHFR, leads to amplification of the DHFR gene
copy number, as well as that of the associated gene of interest.
DHFR as a selectable, amplifiable marker gene in transfected
chinese hamster ovary cell lines (CHO cells) is particularly well
characterized in the art. Other useful amplifiable marker genes
include the adenosine deaminase (ADA) and glutamine synthetase (GS)
genes.
[0154] The choice of cells/cell lines is also important and depends
on the needs of the experimenter. COS cells provide high levels of
transient gene expression, providing a useful means for rapidly
screening the biosynthetic constructs of the invention. COS cells
typically are transfected with a simian virus 40 (SV40) vector
carrying the gene of interest. The transfected COS cells eventually
die, thus preventing the long term production of the desired
protein product but provide a useful technique for testing
preliminary analogs for binding activity.
[0155] The various cells, cell lines and DNA sequences that can be
used for mammalian cell expression of the single-chain constructs
of the invention are well characterized in the art and are readily
available. Other promoters, selectable markers, gene amplification
methods and cells also may be used to express the proteins of this
invention. Particular details of the transfection, expression, and
purification of recombinant proteins are well documented in the art
and are understood by those having ordinary skill in the art.
Further details on the various technical aspects of each of the
steps used in recombinant production of foreign genes in mammalian
cell expression systems can be found in a number of texts and
laboratory manuals in the art, such as, for example, Ausubel et
al., ed., Current Protocols in Molecular Biology, John Wiley &
Sons, New York, (1989).
[0156] Alternatively, morphogen analogs which are small peptides,
usually up to 50 amino acids in length, may be synthesized using
standard solid-phase peptide synthesis procedures, for example,
procedures similar to those described in Merrifield (1963) J. Am.
Chem. Soc., 85:2149. For example, during synthesis,
N-.alpha.-protected amino acids having protected side chains are
added stepwise to a growing polypeptide chain linked by its
C-terminal end to an insoluble polymeric support, e.g., polystyrene
beads. The peptides are synthesized by linking an amino group of an
N-.alpha.-deprotected amino acid to an .alpha.-carboxy group of an
N-.alpha.-protected amino acid that has been activated by reacting
it with a reagent such as dicyclohexylcarbodiimide. The attachment
of a free amino group to the activated carboxyl leads to peptide
bond formation. The most commonly used N-.alpha.-protecting groups
include Boc which is acid labile and Fmoc which is base labile.
[0157] Briefly, the C-terminal N-.alpha.-protected amino acid is
first attached to the polystyrene beads. Then, the
N-.alpha.-protecting group is removed. The deprotected
.alpha.-amino group is coupled to the activated .alpha.-carboxylate
group of the next N-.alpha.-protected amino acid. The process is
repeated until the desired peptide is synthesized. The resulting
peptides are cleaved from the insoluble polymer support and the
amino acid side chains deprotected. Longer peptides, for example
greater than about 50 amino acids in length, typically are derived
by condensation of protected peptide fragments. Details of
appropriate chemistries, resins, protecting groups, protected amino
acids and reagents are well known in the art and so are not
discussed in detail herein. See for example, Atherton et al. (1963)
Solid Phase Peptide Synthesis: A Practical Approach (IRL Press,),
and Bodanszky (1993) Peptide Chemistry, A Practical Textbook, 2nd
Ed, Springer-Verlag, and Fields et al. (1990) Int. J. Peptide
Protein Res. 35:161-214, the disclosures of which are incorporated
herein by reference.
[0158] Purification of the resulting peptide is accomplished using
conventional procedures, such as preparative HPLC, e.g., gel
permeation, partition and/or ion exchange chromatography. The
choice of appropriate matrices and buffers are well known in the
art and so are not described in detail herein.
[0159] With regard to the production of non-peptide small organic
molecules which induce OP-1 like biological activities, these
molecules can be synthesized using standard organic chemistries
well known and thoroughly documented in the patent and other
literatures.
[0160] VII. Screening For Binding and Biological Activity
[0161] As a first step in determining whether a morphogen analog
induces an OP-1 like biological activity, the skilled artisan can
use a standard ligand-receptor assay to determine whether the
morphogen analog binds preferentially to OP-1 receptor. For
standard receptor-ligand assays, the artisan is referred to, for
example, Legerski et al. (1992) Biochem. Biophys. Res. Comm. 183:
672-679; Frakar et al. (1978) Biochem. Biophys. Res. Comm.
80:849-857; Chio et al. (1990) Nature 343: 266-269; Dahlman et al.
(1988) Biochem 27: 1813-1817; Strader et al. (1989) J. Biol. Chem.
264: 13572-13578; and D'Dowd et al. (1988) J. Biol. Chem. 263:
15985-15992.
[0162] In a typical ligand/receptor binding assay useful in the
practice of this invention, purified OP-1 having a known,
quantifiable affinity for a pre-selected OP-1 receptor (see, for
example, Ten Dijke et al. (1994) Science 264:101-103, the
disclosure of which is incorporated herein by reference) is labeled
with a detectable moiety, for example, a radiolabel, a chromogenic
label, or a fluorogenic label. Aliquots of purified receptor,
receptor binding domain fragments, or cells expressing the receptor
of interest on their surface are incubated with labeled OP-1 in the
presence of various concentrations of the unlabeled morphogen
analog. The relative binding affinity of the morphogen analog may
be measured by quantitating the ability of the candidate (unlabeled
morphogen analog) to inhibit the binding of labeled OP-1 with the
receptor. In performing the assay, fixed concentrations of the
receptor and the OP-1 are incubated in the presence and absence of
unlabeled morphogen analog. Sensitivity may be increased by
pre-incubating the receptor with the candidate morphogen analog
before adding labeled OP-1. After the labeled competitor has been
added, sufficient time is allowed for adequate competitor binding,
and then free and bound labeled OP-1 are separated from one
another, and one or the other measured.
[0163] Labels useful in the practice of the screening procedures
include radioactive labels (e.g., .sup.125I, .sup.131I, .sup.111In
or .sup.77Br), chromogenic labels, spectroscopic labels (such as
those disclosed in Haughland (1994) "Handbook of Fluorescent and
Research Chemicals 5 ed." by Molecular Probes, Inc., Eugene,
Oreg.), or conjugated enzymes having high turnover rates, for
example, horseradish peroxidase, alkaline phosphatase, or
.beta.-galactosidase, used in combination with chemiluminescent or
fluorogenic substrates.
[0164] The biological activity, namely the agonist or antagonist
properties of the resulting morphogen analogs subsequently may be
characterized using any conventional in vivo and in vitro assays
that have been developed to measure the biological activity of
OP-1. A variety of specific assays believed to be useful in the
practice of the invention are set forth in detail in Example 1,
hereinbelow.
[0165] Furthermore, it is appreciated that many of the standard
OP-1 assays may be automated thereby facilitating the screening of
a large number of morphogen analogs at the same time. Such
automation procedures are within the level of skill in the art of
drug screening and, therefore, are not discussed herein.
[0166] Following the identification of useful morphogen analogs,
the morphogenic analogs may be produced in commercially useful
quantities (e.g., without limitation, gram and kilogram
quantities), for example, by producing cell lines that express the
morphogen analogs of interest or by producing synthetic peptides
defining the appropriate amino acid sequence. It is appreciated,
however, that conventional methodologies for producing the
appropriate cell lines and for producing synthetic peptides are
well known and thoroughly documented in the art, and so are not
discussed in detail herein.
[0167] VIII. Formulation and Bioactivity
[0168] Morphogen analogs, including OP-1 analogs, can be formulated
for administration to a mammal, preferably a human in need thereof
as part of a pharmaceutical composition. The composition can be
administered by any suitable means, e.g., parenterally, orally or
locally. Where the morphogen analog is to be administered locally,
as by injection, to a desired tissue site, or systemically, such as
by intravenous, subcutaneous, intramuscular, intraorbital,
ophthalmic, intraventricular, intracranial, intracapsular,
intraspinal, intracistemal, intraperitoneal, buccal, rectal,
vaginal, intranasal or aerosol administration, the composition
preferably comprises an aqueous solution. The solution preferably
is physiologically acceptable, such that administration thereof to
a mammal does not adversely affect the mammal's normal electrolyte
and fluid volume balance. The aqueous solution thus can comprise,
e.g., normal physiologic saline (0.9% NaCl, 0.15M), pH 7-7.4.
[0169] Useful solutions for oral or parenteral systemic
administration can be prepared by any of the methods well known in
the pharmaceutical arts, described, for example, in "Remington's
Pharmaceutical Sciences" (Gennaro, A., ed., Mack Pub., 1990, the
disclosure of which is incorporated herein by reference).
Formulations can include, for example, polyalkylene glycols such as
polyethylene glycol, oils of vegetable origin, hydrogenated
naphthalenes, and the like. Formulations for direct administration,
in particular, can include glycerol and other compositions of high
viscosity. Biocompatible, preferably bioresorbable polymers,
including, for example, hyaluronic acid, collagen, tricalcium
phosphate, polybutyrate, polylactide, polyglycolide and
lactide/glycolide copolymers, may be useful excipients to control
the release of the morphogen analog in vivo.
[0170] Other potentially useful parenteral delivery systems for the
present analogs can include ethylene-vinyl acetate copolymer
particles, osmotic pumps, implantable infusion systems, and
liposomes. Formulations for inhalation administration can contain
as excipients, for example, lactose, or can be aqueous solutions
containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate or deoxycholate, or oily solutions for administration
in the form of nasal drops or as a gel to be applied
intranasally.
[0171] Alternatively, the morphogen analogs, including OP-1
analogs, identified as described herein may be administered orally.
For example, liquid formulations of morphogen analogs can be
prepared according to standard practices such as those described in
"Remington's Pharmaceutical Sciences" (supra). Such liquid
formulations can then be added to a beverage or another food
supplement for administration. Oral administration can also be
achieved using aerosols of these liquid formulations.
Alternatively, solid formulations prepared using art-recognized
emulsifiers can be fabricated into tablets, capsules or lozenges
suitable for oral administration.
[0172] Optionally, the analogs can be formulated in compositions
comprising means for enhancing uptake of the analog by a desired
tissue. For example, tetracycline and diphosphonates
(bisphosphonates) are known to bind to bone mineral, particularly
at zones of bone remodeling, when they are provided systemically in
a mammal. Accordingly, such components can be used to enhance
delivery of the present analogs to bone tissue. Alternatively, an
antibody or portion thereof that binds specifically to an
accessible substance specifically associated with the desired
target tissue, such as a cell surface antigen, also can be used. If
desired, such specific targeting molecules can be covalently bound
to the present analog, e.g., by chemical crosslinking or by using
standard genetic engineering techniques to create, for example, an
acid labile bond such as an Asp-Pro linkage. Useful targeting
molecules can be designed, for example, according to the teachings
of U.S. Pat. No. 5,091,513.
[0173] It is contemplated also that some of the morphogen analogs
may exhibit the highest levels of activity in vivo when combined
with carrier matrices i.e., insoluble polymer matrices. See for
example, U.S. Pat. No. 5,266,683 the disclosure of which is
incorporated by reference herein. Currently preferred carrier
matrices are xenogenic, allogenic or autogenic in nature. It is
contemplated, however, that synthetic materials comprising
polylactic acid, polyglycolic acid, polybutyric acid, derivatives
and copolymers thereof may also be used to generate suitable
carrier matrices. Preferred synthetic and naturally derived matrix
materials, their preparation, methods for formulating them with the
morphogen analogs of the invention, and methods of administration
are well known in the art and so are not discussed in detailed
herein. See for example, U.S. Pat. No. 5,266,683.
[0174] Still further, the present analogs can be administered to
the mammal in need thereof either alone or in combination with
another substance known to have a beneficial effect on tissue
morphogenesis. Examples of such substances (herein, cofactors)
include substances that promote tissue repair and regeneration
and/or inhibit inflammation. Examples of useful cofactors for
stimulating bone tissue growth in osteoporotic individuals, for
example, include but are not limited to, vitamin D.sub.3,
calcitonin, prostaglandins, parathyroid hormone, dexamethasone,
estrogen and IGF-I or IGF-II. Useful cofactors for nerve tissue
repair and regeneration can include nerve growth factors. Other
useful cofactors include symptom-alleviating cofactors, including
antiseptics, antibiotics, antiviral and antifungal agents,
analgesics and anesthetics.
[0175] Analogs preferably are formulated into pharmaceutical
compositions by admixture with pharmaceutically acceptable,
nontoxic excipients and carriers. As noted above, such compositions
can be prepared for systemic, e.g., parenteral, administration,
particularly in the form of liquid solutions or suspensions; for
oral administration, particularly in the form of tablets or
capsules; or intranasally, particularly in the form of powders,
nasal drops or aerosols. Where adhesion to a tissue surface is
desired, the composition can comprise a fibrinogen-thrombin
dispersant or other bioadhesive such as is disclosed, for example,
in PCT US91/09275, the disclosure of which is incorporated herein
by reference. The composition then can be painted, sprayed or
otherwise applied to the desired tissue surface.
[0176] The compositions can be formulated for parenteral or oral
administration to humans or other mammals in therapeutically
effective amounts, e.g., amounts which provide appropriate
concentrations of the morphogen analog to target tissue for a time
sufficient to induce the desired effect. Preferably, the present
compositions alleviate or mitigate the mammal's need for a
morphogen-associated biological response, such as maintenance of
tissue-specific function or restoration of tissue-specific
phenotype to senescent tissues (e.g., osteopenic bone tissue).
[0177] As will be appreciated by those skilled in the art, the
concentration of the compounds described in a therapeutic
composition will vary depending upon a number of factors, including
the dosage of the drug to be administered, the chemical
characteristics (e.g., hydrophobicity) of the compounds employed,
and the route of administration. The preferred dosage of drug to be
administered also is likely to depend on such variables as the type
and extent of a disease, tissue loss or defect, the overall health
status of the particular patient, the relative biological efficacy
of the compound selected, the formulation of the compound, the
presence and types of excipients in the formulation, and the route
of administration. In general terms, the therapeutic molecules of
this invention may be provided to an individual where typical doses
range from about 10 ng/kg to about 1 g/kg of body weight per day;
with a preferred dose range being from about 0.1 mg/kg to 100 mg/kg
of body weight.
[0178] IX. Examples
[0179] Practice of the invention will be more fully understood from
the following examples, which are presented herein for illustrative
purposes only, and should not be construed as limiting the
invention in any way.
Example 1
Introduction of Inter-chain Disulfide Bonds to Stabilize the hOP-1
Dimer
[0180] As discussed in section V.A.(i) it is contemplated that
introduction of one or more additional inter-chain disulfide may
stabilize further the hOP-1 dimer. The introduction of additional
inter-chain disulfide bonds is described here.
[0181] A Sma I to Bam HI fragment of the human OP-1 cDNA as
described in Ozkaynak et al. (1990) supra is cloned into Bluescript
KS+ (available from Stratagene Cloning Systems, La Jolla, Calif.),
previously cleaved with Eco RV and Bam HI. Upon transformation into
E. coli, the resulting colonies are screened by a blue-white
selection process wherein the desired colonies containing the OP-1
cDNA insert are blue. The correct clone may be identified by
restriction screening to give the following expected restriction
fragments.
1 Restriction Enzyme Fragment size (bp) EcoR I 84, 789, 3425 Xho I
161, 1223, 2914 Sac II 97, 650, 3551
[0182] In order to introduce two additional inter-chain disulfide
bridges, a double cysteine mutant containing Asn 83 to Cys and Asn
130 to Cys replacements is produced. The cysteine mutant can be
prepared by site-directed mutagenesis using synthetic
oligonucleotides and either PCR or the site-directed mutagenesis
methods, see for example, Kunkel et al. (1985) Proc. Natl. Acad.
Sci. USA 822: 488; Kunkel et al. (1985) Meth. Enzymol. 154: 367 and
U.S. Pat. No. 4,873,192. Neither mutation causes a frameshift and,
therefore, E. coli transformed with mutagenesis products that give
white colonies indicate an error in the sequence. The presence of
the appropriate mutation is verified by conventional dideoxy
sequencing.
[0183] Then, linkers are introduced into the N- and C-termini of
the mutant gene by oligonucleotide-directed mutagenesis using
appropriate oligonucleotides. A preferred N terminal linker
introduces a unique Not I site and a preferred C terminal linker
introduces a non-suppressible stop codon TAA at the end of the
mutein gene followed by a unique Bgl II site (AGATCT). Each of the
resulting mutant genes are excised from the cloning vector by the
restriction enzymes Nde I and Bgl II, isolated, and ligated
independently into pET vector (New England Biolabs, Beverly, Mass.)
previously cleaved with Nde I and Bam HI. The ligation products
then are transformed into E. coli and transformants containing, and
expressing each individual mutant protein are identified.
[0184] Expression of the double cysteine containing mutant analog
is induced after the expression of T7 RNA polymerase (initiated
through infected with .lambda.CE6 phage). During expression, the
mutant analog is produced as inclusion granules which are harvested
from the cell paste. Then, the mutant protein is dissolved in 6M
guanidine-HCl, 0.2M Tris-HCl, pH 8.2 and 0.1 M 2-mercaptoethanol,
and the mixture dialyzed exhaustively against 6M urea, 2.5 mM
Tris-HCl, pH 7.5 and 1 mM EDTA. 2-mercaptoethanol is added to a
final concentration of 0.1M and the solution incubated at room
temperature. The mixture is dialyzed exhaustively against buffer
containing 2.5 mM Tris-HCl, pH 7.5 and 1 mM EDTA. Folded mutant
protein is purified by affinity chromatography on a column packed
with surface immobilized OP-1 receptor. Unbound material is removed
by washing as described above and the specific OP-1 receptor
binding material eluted.
[0185] Following purification the stabilizing effect of the
additional bond is determined by fluorescence polarization. For
example, the rotational rates of morphogen analog (mutein) and
natural hOP-1 are determined as a function of temperature using a
fluorescence spectrophotometer modified for fluoresence anisotropy
(Photon Technology International). It is anticipated that the
mutein dimer will exhibit a lower rational rate upto a higher
temperature than natural hOP-1 dimer, thereby indicating that the
mutein dimer remains as a dimer and is more stable upto a higher
temperature than is the wild type protein.
[0186] The biological activity of the resulting mutant protein or
mutein can be tested using any of the bioassays developed to date
for determining the biological activity of native hOP-1. A variety
of such exemplary assays are described below. The assays which
follow are recited for ease of testing. Specific in vivo assays for
testing the efficacy of a morphogenic protein or analog in an
application to repair or regenerate damaged bone, liver, kidney, or
nerve tissue, periodontal tissue, including cementum and/or
periodontal ligament, gastrointestinal and renal tissues, and
immune-cell mediated damages tissues are disclosed in publicly
available documents, which include, for example, EP 0575,555;
WO93/04692; WO93/05751; WO/06399; WO94/03200; WO94/06449; and
WO94/06420. The skilled artisan can test an analog in any of these
assays without undue experimentation.
[0187] A. Mitogenic Effect on Rat and Human Osteoblasts
[0188] The following example is a typical assay useful in
determining whether an OP-1 morphogen analog induces proliferation
of osteoblasts in vitro. It is contemplated that in this, and all
other examples using osteoblast cultures, preferably uses rat
osteoblast-enriched primary cultures. Although these cultures are
heterogeneous in that the individual cells are at different stages
of differentiation, the culture is believed to more accurately
reflect the metabolism and function of osteoblasts in vivo than
osteoblast cultures obtained from established cell lines. Unless
otherwise indicated, all chemicals referenced are standard,
commercially available reagents, readily available from a number of
sources, including Sigma Chemical, Co., St. Louis; Calbiochem,
Corp., San Diego and Aldrich Chemical Co., Milwaukee.
[0189] Briefly, rat osteoblast-enriched primary cultures are
prepared by sequential collagenase digestion of newborn rat
calvaria (e.g., from 1-2 day-old animals, Long-Evans strain,
Charles River Laboratories, Wilmington, Mass.), following standard
procedures, such as are described, for example, in Wong et al.
(1975) Proc. Natl. Acad. Sci. USA 72: 3167-3171. Rat osteoblast
single cell suspensions then are plated onto a multi-well plate
(e.g., a 24 well plate at a concentration of 50,000 osteoblasts per
well) in alpha MEM (modified Eagle's medium, Gibco, Inc., NY)
containing 10% FBS (fetal bovine serum), L-glutamine and
penicillin/streptomycin. The cells are incubated for 24 hours at
37.degree. C., at which time the growth medium is replaced with
alpha MEM containing 1% FBS and the cells incubated for an
additional 24 hours so that cells are in serum-deprived growth
medium at the time of the experiment.
[0190] The cultured cells are divided into four groups: (1) wells
which receive, for example, 0.1, 1.0, 10.0, 40.0 and 80.0 ng of the
OP-1 morphogen analog (mutein), (2) wells which receive 0.1, 1.0,
10.0 and 40.0 ng of wild type OP-1; (3) wells which receives 0.1,
1.0, 10.0, and 40.0 ng of TGF-.beta., and (4) the control group,
which receive no growth factors. The cells then are incubated for
an additional 18 hours after which the wells are pulsed with 2
mCi/well of .sup.3H-thymidine and incubated for six more hours. The
excess label then is washed off with a cold solution of 0.15 M NaCl
and then 250 ml of 10% tricholoracetic acid is added to each well
and the wells incubated at room temperature for 30 minutes. The
cells then are washed three times with cold distilled water, and
lysed by the addition of 250 ml of 1% sodium dodecyl sulfate (SDS)
for a period of 30 minutes at 37.degree. C. The resulting cell
lysates are harvested using standard means and the incorporation of
.sup.3H-thymidine into cellular DNA determined by liquid
scintillation as an indication of mitogenic activity of the cells.
In the experiment, it is contemplated that the OP-1 morphogen
analog construct (mutein), like natural OP-1, will stimulate
.sup.3H-thymidine incorporation into DNA, and therefore promote
osteoblast cell proliferation. In contrast, the effect of the
TGF-.beta. is expected to be transient and biphasic. Furthermore,
it is contemplated that at higher concentrations, TGF-.beta. will
have no significant effect on osteoblast cell proliferation.
[0191] The in vitro effect of the OP-1 morphogen analog on
osteoblast proliferation also may be evaluated using human primary
osteoblasts (obtained from bone tissue of a normal adult patient
and prepared as described above) and on human osteosarcoma-derived
cell lines.
[0192] B. Progenitor Cell Stimulation
[0193] The following example is designed to demonstrate the ability
of OP-1 morphogen analogs to stimulate the proliferation of
mesenchymal progenitor cells. Useful naive stem cells include
pluripotent stem cells, which may be isolated from bone marrow or
umbilical cord blood using conventional methodologies, (see, for
example, Faradji et al. (1988) Vox Sang. 55 (3): 133-138 or
Broxmeyer et al. (1989) Proc. Natl. Acad. Sci. USA. 86: 3828-3832),
as well as naive stem cells obtained from blood. Alternatively,
embryonic cells (e.g., from a cultured mesodermal cell line) may be
used.
[0194] Another method for obtaining progenitor cells and for
determining the ability of OP-1 morphogen analogs to stimulate cell
proliferation is to capture progenitor cells from an in vivo
source. For example, a biocompatible matrix material able to allow
the influx of migratory progenitor cells may be implanted at an in
vivo site long enough to allow the influx of migratory progenitor
cells. For example, a bone-derived, guanidine-extracted matrix,
formulated as disclosed for example in Sampath et al. (1983) Proc.
Natl. Acad. Sci. USA 80: 6591-6595, or U.S. Pat. No. 4,975,526, may
be implanted into a rat at a subcutaneous site, essentially
following the method of Sampath et al. After three days the implant
is removed, and the progenitor cells associated with the matrix
dispersed and cultured.
[0195] Progenitor cells, however obtained, then are incubated in
vitro with the candidate OP-1 morphogen analog under standard cell
culture conditions, such as those described hereinbelow. In the
absence of external stimuli, the progenitor cells do not, or only
minimally, proliferate on their own in culture.
[0196] However, progenitor cells cultured in the presence of a
biologically active OP-1 morphogen analog, like OP-1, will
proliferate. Cell growth can be determined visually or
spectrophotometrically using standard methods well known in the
art.
[0197] C. Morphogen-Induced Cell Differentiation
[0198] A variety of assays also can be used to determine OP-1 based
morphogen analog-induced cellular differentiation.
[0199] (1) Embryonic Mesenchyme Differentiation
[0200] As with natural OP-1, it is contemplated that the OP-1
morphogen analog (mutein) can induce cell differentiation. The
ability of OP-1 morphogen analogs to induce cell differentiation
can be demonstrated by culturing early mesenchymal cells in the
presence of OP-1 morphogen analog and then studying the histology
of the cultured cells by staining with toluidine blue using
standard cell culturing and cell staining methodologies well
described in the art. For example, it is known that rat mesenchymal
cells destined to become mandibular bone, when separated from the
overlying epithelial cells at stage 11 and cultured in vitro under
standard tissue culture conditions, e.g., in a chemically defined,
serum-free medium, containing for example, 67% DMEM (Dulbecco's
modified Eagle's medium), 22% F-12 medium, 10 mM Hepes pH 7, 2 mM
glutamine, 50 mg/ml transferrin, 25 mg/ml insulin, trace elements,
2mg/ml bovine serum albumin coupled to oleic acid, with HAT (0.1 mM
hypoxanthine, 10 mM aminopterin, 12 mM thymidine, will not continue
to differentiate. However, if these same cells are left in contact
with the overlying endoderm for an additional day, at which time
they become stage 12 cells, they will continue to differentiate on
their own in vitro to form chondrocytes. Further differentiation
into osteoblasts and, ultimately, mandibular bone, requires an
appropriate local environment, e.g., a vascularized
environment.
[0201] It is anticipated that, as with natural OP-1, stage 11
mesenchymal cells, cultured in vitro in the presence of OP-1
morphogen analog (mutein), e.g., 10-100 ng/ml, will continue to
differentiate in vitro to form chondrocytes just as they continue
to differentiate in vitro if they are cultured with the cell
products harvested from the overlying endodermal cells. This
experiment can be performed with different mesenchymal cells to
demonstrate the cell differentiation capability of OP-1 morphogen
analog in different tissues.
[0202] As another example of morphogen-induced cell
differentiation, the ability of OP-1 morphogen analogs to induce
osteoblast differentiation can be demonstrated in vitro using
primary osteoblast cultures, or osteoblast-like cells lines, and
assaying for a variety of bone cell markers that are specific
markers for the differentiated osteoblast phenotype, e.g., alkaline
phosphatase activity, parathyroid hormone-mediated cyclic AMP
(cAMP) production, osteocalcin synthesis, and enhanced
mineralization rates.
[0203] (2) Induction of Alkaline Phosphatase Activity in
Osteoblasts
[0204] Cultured osteoblasts in serum-free medium are incubated with
a range of OP-1 morphogen analog concentrations, for example, 0.1,
1.0, 10.0, 40.0 or 80.0 ng OP-1 morphogen analog/ml medium; or with
a similar concentration range of natural OP-1 or TGF-.beta.. After
a 72 hour incubation the cell layer is extracted with 0.5 ml of 1%
Triton X-100. The resultant cell extract is centrifuged, and 100 ml
of the extract is added to 90 ml of para-nitroso-phenylphosphate
(PNPP)/glycerine mixture and incubated for 30 minutes in a
37.degree. C. water bath and the reaction stopped with 100 ml NaOH.
The samples then are run through a plate reader (e.g., Dynatech
MR700 plate reader, and absorbance measured at 400 nm, using
p-nitrophenol as a standard) to determine the presence and amount
of alkaline phosphate activity. Protein concentrations are
determined by the BioRad method. Alkaline phosphatase activity is
calculated in units/mg protein, where 1 unit=1 nmol p-nitrophenol
liberated/30 minutes at 37.degree. C.
[0205] It is contemplated that the OP-1 morphogen analog, like
natural OP-1, will stimulate the production of alkaline phosphatase
in osteoblasts thereby promoting the growth and expression of the
osteoblast differentiated phenotype. The long term effect of OP-1
morphogen analog on the production of alkaline phosphatase by rat
osteoblasts also can be demonstrated as follows.
[0206] Rat osteoblasts are prepared and cultured in multi-well
plates as described above. In this example six sets of 24 well
plates are plated with 50,000 rat osteoblasts per well. The wells
in each plate, prepared as described above, then are divided into
three groups: (1) those which receive, for example, 1 ng of OP-1
morphogen analog per ml of medium; (2) those which receive 40 ng of
OP-1 morphogen analog per ml of medium; and (3) those which receive
80 ng of OP-1 morphogen analog per ml of medium. Each plate then is
incubated for different lengths of time: 0 hours (control time), 24
hours, 48 hours, 96 hours, 120 hours and 144 hours. After each
incubation period, the cell layer is extracted with 0.5 ml of 1%
Triton X-100. The resultant cell extract is centrifuged, and
alkaline phosphatase activity determined using
para-nitroso-phenylphosphate (PNPP), as above. It is contemplated
that the OP-1 morphogen analog, like natural OP-1, will stimulate
the production of alkaline phosphatase in osteoblasts in a
dose-dependent manner so that increasing doses of OP-1 morphogen
analog will further increase the level of alkaline phosphatase
production. Moreover, it is contemplated that the OP-1 morphogen
analog-stimulated elevated levels of alkaline phosphatase in the
treated osteoblasts will last for an extended period of time.
[0207] (3) Induction of PTH-Mediated cAMP
[0208] This experiment is designed to test the effect of OP-1
morphogen analogs on parathyroid hormone-mediated cAMP production
in rat osteoblasts in vitro. Briefly, rat osteoblasts are prepared
and cultured in a multiwell plate as described above. The cultured
cells then are divided into four groups: (1) wells which receive,
for example, 1.0, 10.0 and 40.0 ng OP-1 morphogen analog/ml
medium); (2) wells which receive for example, natural OP-1, at
similar concentration ranges; (3) wells which receive for example,
TGF-.beta., at similar concentration ranges; and (4) a control
group which receives no growth factors. The plate then is incubated
for another 72 hours. At the end of the 72 hours the cells are
treated with medium containing 0.5% bovine serum albumin (BSA) and
1 mM 3-isobutyl-1-methylxanthine for 20 minutes followed by the
addition into half of the wells of human recombinant parathyroid
hormone (hPTH, Sigma, St. Louis) at a concentration of 200 ng/ml
for 10 minutes. The cell layer then is extracted from each well
with 0.5 ml of 1% Triton X-100. The cAMP levels then are determined
using a radioimmunoassay kit (e.g., Amersham, Arlington Heights,
Ill.). It is contemplated that OP-1 morphogen analog alone, like
OP-1, will stimulate an increase in the PTH-mediated cAMP response,
thereby promoting the growth and expression of the osteoblast
differentiated phenotype.
[0209] (4) Induction of Osteocalcin Production
[0210] Osteocalcin is a bone-specific protein synthesized by
osteoblasts which plays an integral role in the rate of bone
mineralization in vivo. Circulating levels of osteocalcin in serum
are used as a marker for osteoblast activity and bone formation in
vivo. Induction of osteocalcin synthesis in osteoblast-enriched
cultures can be used to demonstrate OP-1 morphogen analog efficacy
in vitro.
[0211] Rat osteoblasts are prepared and cultured in a multi-well
plate as above. In this experiment the medium is supplemented with
10% FBS, and on day 2, cells are fed with fresh medium supplemented
with fresh 10 mM .beta.-glycerophosphate (Sigma, Inc.). Beginning
on day 5 and twice weekly thereafter, cells are fed with a complete
mineralization medium containing all of the above components plus
fresh L(+)-ascorbate, at a final concentration of 50 mg/ml medium.
OP-1 morphogen analog then is added to the wells directly, e.g., in
50% acetonitrile (or 50% ethanol) containing 0.1% trifluoroacetic
acid (TFA), at no more than 5 ml morphogen analog/ml medium.
Control wells receive solvent vehicle only. The cells then are
re-fed and the conditioned medium sample diluted 1:1 in standard
radio immunoassay buffer containing standard protease inhibitors
and stored at -20.degree. C. until assayed for osteocalcin.
Osteocalcin synthesis is measured by standard radioimmunoassay
using a commercially available osteocalcin-specific antibody.
[0212] Mineralization is determined on long term cultures (13 day)
using a modified von Kossa staining technique on fixed cell layers:
cells are fixed in fresh 4% paraformaldehyde at 23.degree. C. for
10 min, following rinsing cold 0.9% NaCl. Fixed cells then are
stained for endogenous alkaline phosphatase at pH 9.5 for 10 min,
using a commercially available kit (Sigma, Inc.). Purple stained
cells then are dehydrated with methanol and air dried. After 30 min
incubation in 3% AgNO.sub.3 in the dark, H.sub.2O-rinsed samples
are exposed for 30 sec to 254 nm UV light to develop the black
silver-stained phosphate nodules. Individual mineralized foci (at
least 20 mm in size) are counted under a dissecting microscope and
expressed as nodules/culture.
[0213] It is contemplated that the OP-1 morphogen analog, like
natural OP-1, will stimulate osteocalcin synthesis in osteoblast
cultures. Furthermore, it is contemplated that the increased
osteocalcin synthesis in response to OP-1 morphogen analog will be
in a dose dependent manner thereby showing a significant increase
over the basal level after 13 days of incubation. Enhanced
osteocalcin synthesis also can be confirmed by detecting the
elevated osteocalcin mRNA message (20-fold increase) using a rat
osteocalcin-specific probe. In addition, the increase in
osteocalcin synthesis correlates with increased mineralization in
long term osteoblast cultures as determined by the appearance of
mineral nodules. It is contemplated also that OP-1 morphogen
analog, like natural OP-1, will increase significantly the initial
mineralization rate as compared to untreated cultures.
[0214] (5) Morphogen-Induced CAM Expression
[0215] Members of the BMP/OP family (see FIG. 6) induce CAM
expression, particularly N-CAM expression, as part of their
induction of morphogenesis (see copending U.S. Ser. No. 922,813).
CAMs are morphoregulatory molecules identified in all tissues as an
essential step in tissue development. N-CAMs, which comprise at
least 3 isoforms (N-CAM-180, N-CAM-140 and N-CAM-120, where "180",
"140" and "120" indicate the apparent molecular weights of the
isoforms as measured by SDS polyacrylamide gel electrophoresis) are
expressed at least transiently in developing tissues, and
permanently in nerve tissue. Both the N-CAM-180 and N-CAM-140
isoforms are expressed in both developing and adult tissue. The
N-CAM-120 isoform is found only in adult tissue. Another neural CAM
is L1.
[0216] The ability of OP-1 based morphogen analogs to stimulate CAM
expression may be demonstrated using the following protocol, using
NG108-15 cells. NG108-15 is a transformed hybrid cell line
(neuroblastoma x glioma, America Type Culture Collection (ATCC),
Rockville, Md.), exhibiting a morphology characteristic of
transformed embryonic neurons. As described in Example D, below,
untreated NG108-15 cells exhibit a fibroblastic, or minimally
differentiated, morphology and express only the 180 and 140
isoforms of N-CAM normally associated with a developing cell.
Following treatment with members of the vg/dpp subgroup these cells
exhibit a morphology characteristic of adult neurons and express
enhanced levels of all three N-CAM isoforms.
[0217] In this example, NG108-15 cells are cultured for 4 days in
the presence of increasing concentrations of either the OP-1
morphogen analog or natural OP-1 using standard culturing
procedures, and standard Western blots are performed on whole cell
extracts. N-CAM isoforms are detected with an antibody which
crossreacts with all three isoforms, mAb H28.123, obtained from
Sigma Chemical Co., St. Louis, the different isoforms being
distinguishable by their different mobilities on an electrophoresis
gel. Control NG108-15 cells (untreated) express both the 140 kDa
and the 180 kDa isoforms, but not the 120 kDa, as determined by
Western blot analyses using up to 100 mg of protein. It is
contemplated that treatment of NG108-15 cells with OP-1 morphogen
analog, like natural OP-1 may result in a dose-dependent increase
in the expression of the 180 kDa and 140 kDa isoforms, as well as
the induction of the 120 kDa isoform. In addition, it is
contemplated that the OP-1 morphogen analog, like natural
OP-1-induced CAM expression may correlate with cell aggregation, as
determined by histology.
[0218] (D) OP-1 Morphogen Analog-Induced Redifferentiation of
Transformed Phenotype
[0219] It is contemplated that OP-1 morphogen analog, like natural
OP-1, also induces redifferentiation of transformed cells to a
morphology characteristic of untransformed cells. The examples
provided below detail morphogen-induced redifferentiation of a
transformed human cell line of neuronal origin (NG108-15); as well
as mouse neuroblastoma cells (N1E-115), and human embryo carcinoma
cells, to a morphology characteristic of untransformed cells.
[0220] As described above, NG108-15 is a transformed hybrid cell
line produced by fusing neuroblastoma x glioma cells (obtained from
ATCC, Rockville, Md.), and exhibiting a morphology characteristic
of transformed embryonic neurons, e.g., having a fibroblastic
morphology. Specifically, the cells have polygonal cell bodies,
short, spike-like processes and make few contacts with neighboring
cells. Incubation of NG108-15 cells, cultured in a chemically
defined, serum-free medium, with 0.1 to 300 ng/ml of morphogen
analog or natural OP-1 for four hours induces an orderly,
dose-dependent change in cell morphology.
[0221] For example, NG108-15 cells are subcultured on poly-L-lysine
coated 6 well plates. Each well contains 40-50,000 cells in 2.5 ml
of chemically defined medium. On the third day, 2.5 ml of OP-1
morphogen analog or natural OP-1 in 60% ethanol containing 0.025%
trifluoroacetic is added to each well. The media is changed daily
with new aliquots of morphogen. It is contemplated that OP-1
morphogen analog, like OP-1, may induce a dose-dependent
redifferentiation of the transformed cells, including a rounding of
the soma, an increase in phase brightness, extension of the short
neurite processes, and other significant changes in the cellular
ultrastructure. After several days it is contemplated also that
treated cells may begin to form epithelioid sheets that then become
highly packed, multi-layered aggregates, as determined visually by
microscopic examination.
[0222] Moreover, it is contemplated that the redifferentiation may
occur without any associated changes in DNA synthesis, cell
division, or cell viability, making it unlikely that the
morphologic changes are secondary to cell differentiation or a
toxic effect of the morphogen. In addition, it is contemplated that
the morphogen analog-induced redifferentiation may not inhibit cell
division, as determined by .sup.3H-thymidine uptake, unlike other
molecules such as butyrate, DMSO, retinoic acid or Forskolin, which
have been shown to stimulate differentiation of transformed cells
in analogous experiments. Thus, it is contemplated that the OP-1
morphogen analog, like natural OP-1, may maintain cell stability
and viability after inducing redifferentiation.
[0223] The morphogen described herein would, therefore, provide
useful therapeutic agents for the treatment of neoplasias and
neoplastic lesions of the nervous system, particularly in the
treatment of neuroblastomas, including retinoblastomas, and
gliomas.
[0224] (E) Maintenance of Phenotype
[0225] OP-1 morphogen analogs, like natural OP-1, also may be used
to maintain a cell's differentiated phenotype. This application is
particularly useful for inducing the continued expression of
phenotype in senescent or quiescent cells.
[0226] (1) In Vitro Model for Phenotypic Maintenance
[0227] The phenotypic maintenance capability of morphogens is
determined readily. A number of differentiated cells become
senescent or quiescent after multiple passages in vitro under
standard tissue culture conditions well described in the art (e.g.,
Culture of Animal Cells: A Manual of Basic Techniques, C. R.
Freshney, ed., Wiley, 1987). However, if these cells are cultivated
in vitro in association with a morphogen such as OP-1, cells are
stimulated to maintain expression of their phenotype through
multiple passages. For example, the alkaline phosphatase activity
of cultured osteoblasts, such as cultured osteosarcoma cells and
calvaria cells, is significantly reduced after multiple passages in
vitro. However, if the cells are cultivated in the presence of
OP-1, alkaline phosphatase activity is maintained over extended
periods of time. Similarly, phenotypic expression of myocytes also
is maintained in the presence of a morphogen. In the experiment,
osteoblasts are cultured as described in Example A. The cells are
divided into groups, incubated with varying concentrations of
either OP-1 morphogen analog or natural OP-1 (e.g., 0-300 ng/ml)
and passaged multiple times (e.g., 3-5 times) using standard
methodology. Passaged cells then are tested for alkaline
phosphatase activity, as described in Example C as an indication of
differentiated cell metabolic function. It is contemplated that
osteoblasts cultured in the absence of OP-1 morphogen analog may
have reduced alkaline phosphatase activity, as compared to OP-1
morphogen analog, or natural OP-1-treated cells.
[0228] (2) In Vivo Model for Phenotypic Maintenance
[0229] Phenotypic maintenance capability also may be demonstrated
in vivo, using a standard rat model for osteoporosis. Long Evans
female rats (Charles River Laboratories, Wilmington, Mass.) are
sham-operated (control animals) or ovariectomized using standard
surgical techniques to produce an osteoporotic condition resulting
from decreased estrogen production. Following surgery, e.g., 200
days after ovariectomy, rats are systemically provided with
phosphate buffered saline (PBS) or morphogen, (e.g., OP-1 morphogen
analog, or natural OP-1, 1-100 mg) for 21 days (e.g., by daily tail
vein injection.) The rats then are sacrificed and serum alkaline
phosphatase levels, serum calcium levels, and serum osteocalcin
levels are determined, using standard methodologies as described
therein and above. It is contemplated that the OP-1 morphogen
analog treated rats, like the OP-1 treated rats may exhibit
elevated levels of osteocalcin and alkaline phosphatase activity.
It is contemplated also that histomorphometric analysis on the
tibial diaphyseal bone may show improved bone mass in OP-1
morphogen analog-treated animals as compared with untreated,
ovariectomized rats.
[0230] F. Proliferation of Progenitor Cell Populations
[0231] Progenitor cells may be stimulated to proliferate in vivo or
ex vivo. It is contemplated that cells may be stimulated in vivo by
injecting or otherwise providing a sterile preparation containing
the OP-1 morphogen analog into the individual. For example, the
hematopoietic pluripotential stem cell population of an individual
may be stimulated to proliferate by injecting or otherwise
providing an appropriate concentration of OP-1 morphogen analog to
the individual's bone marrow.
[0232] Progenitor cells may be stimulated ex vivo by contacting
progenitor cells of the population to be enhanced with a
morphogenically active OP-1 morphogen analog under sterile
conditions at a concentration and for a time sufficient to
stimulate proliferation of the cells. Suitable concentrations and
stimulation times may be determined empirically, essentially
following the procedure described in Example A, above. It is
contemplated that a OP-1 morphogen analog concentration of between
about 0.1-100 ng/ml and a stimulation period of from about 10
minutes to about 72 hours, or, more generally, about 24 hours,
typically should be sufficient to stimulate a cell population of
about 10.sup.4 to 10.sup.6 cells. The stimulated cells then may be
provided to the individual as, for example, by injecting the cells
to an appropriate in vivo locus. Suitable biocompatible progenitor
cells may be obtained by any of the methods known in the art or
described hereinabove.
[0233] G. Regeneration of Damaged or Diseased Tissue
[0234] It is contemplated that OP-1 morphogen analogs may be used
to repair diseased or damaged mammalian tissue. The tissue to be
repaired preferably is assessed first, and excess necrotic or
interfering scar tissue removed as needed, e.g., by ablation or by
surgical, chemical, or other methods known in the medical arts.
[0235] OP-1 morphogen analog then may be provided directly to the
tissue locus as part of a sterile, biocompatible composition,
either by surgical implantation or injection. The morphogen analog
also may be provided systemically, as by oral or parenteral
administration. Alternatively, a sterile, biocompatible composition
containing progenitor cells stimulated by a morphogenically active
OP-1 morphogen analog may be provided to the tissue locus. The
existing tissue at the locus, whether diseased or damaged, provides
the appropriate matrix to allow the proliferation and
tissue-specific differentiation of progenitor cells. In addition, a
damaged or diseased tissue locus, particularly one that has been
further assaulted by surgical means, provides a morphogenically
permissive environment. Systemic provision of OP-1 morphogen analog
may be sufficient for certain applications (e.g., in the treatment
of osteoporosis and other disorders of the bone remodeling
cycle).
[0236] In some circumstances, particularly where tissue damage is
extensive, the tissue may not be capable of providing a sufficient
matrix for cell influx and proliferation. In these instances, it
may be necessary to provide progenitor cells stimulated by the OP-1
morphogen analog to the tissue locus in association with a
suitable, biocompatible, formulated matrix, prepared by any of the
means described below. The matrix preferably is in vivo
biodegradable. The matrix also may be tissue-specific and/or may
comprise porous particles having dimensions within the range of
70-850 .mu.m, most preferably 150-420 .mu.m.
[0237] OP-1 morphogen analog also may be used to prevent or
substantially inhibit immune/inflammatory response-mediated tissue
damage and scar tissue formation following an injury. OP-1
morphogen analog may be provided to a newly injured tissue locus,
to induce tissue morphogenesis at the locus, preventing the
aggregation of migrating fibroblasts into non-differentiated
connective tissue. Preferably the OP-1 morphogen analog may be
provided as a sterile pharmaceutical preparation injected into the
tissue locus within five hours of the injury. Where an
immune/inflammatory response is unavoidably or deliberately
induced, as part of, for example, a surgical or other aggressive
clinical therapy, OP-1 morphogen analog preferably may be provided
prophylactically to the patient prior to, or concomitant with, the
therapy.
[0238] Described below is a protocol for demonstrating whether a
OP-1 morphogen analog-induces tissue morphogenesis in bone.
[0239] (1) OP-1 Morphogen Analog-Induced Bone Morphogenesis
[0240] A particularly useful mammalian tissue model system for
demonstrating and evaluating the morphogenic activity of a
morphogen analog is the endochondral bone tissue morphogenesis
model known in the art and described, for example, in U.S. Pat. No.
4,968,590, incorporated herein by reference. The ability to induce
endochondral bone formation includes the ability to induce
proliferation and differentiation of progenitor cells into
chondroblasts and osteoblasts, the ability to induce cartilage
matrix formation, cartilage calcification, and bone remodeling, and
the ability to induce formation of an appropriate vascular supply
and hematopoietic bone marrow differentiation.
[0241] The local environment in which the morphogenic material is
placed is important for tissue morphogenesis. As used herein,
"local environment" is understood to include the tissue structural
matrix and the environment surrounding the tissue. For example, in
addition to needing an appropriate anchoring substratum for their
proliferation, the cells stimulated by morphogens need signals to
direct the tissue-specificity of their differentiation. These
signals vary for the different tissues and may include cell surface
markers. In addition, vascularization of new tissue requires a
local environment which supports vascularization.
[0242] The following sets forth various procedures for evaluating
the in vivo morphogenic utility of OP-1 morphogen analogs and OP-1
morphogen analog containing compositions. The compositions may be
injected or surgically implanted in a mammal, following any of a
number of procedures well known in the art. For example, surgical
implant bioassays may be performed essentially following the
procedure of Sampath et al. (1983) Proc. Natl. Acad. Sci. USA 80:
6591-6595 and U.S. Pat. No. 4,968,590.
[0243] Histological sectioning and staining is preferred to
determine the extent of morphogenesis in vivo, particularly in
tissue repair procedures. Excised implants are fixed in Bouins
Solution, embedded in paraffin, and cut into 6-8 .mu.m sections.
Staining with toluidine blue or hemotoxylin/eosin demonstrates
clearly the ultimate development of the new tissue. Twelve day
implants are usually sufficient to determine whether the implants
contain newly induced tissue.
[0244] Successful implants exhibit a controlled progression through
the stages of induced tissue development allowing one to identify
and follow the tissue-specific events that occur. For example, in
endochondral bone formation the stages include: (1) leukocytes on
day one; (2) mesenchymal cell migration and proliferation on days
two and three; (3) chondrocyte appearance on days five and six; (4)
cartilage matrix formation on day seven; (5) cartilage
calcification on day eight; (6) vascular invasion, appearance of
osteoblasts, and formation of new bone on days nine and ten; (7)
appearance of osteoclastic cells, and the commencement of bone
remodeling and dissolution of the implanted matrix on days twelve
to eighteen; and (8) hematopoietic bone marrow differentiation in
the resulting ossicles on day twenty-one.
[0245] In addition to histological evaluation, biological markers
may be used as markers for tissue morphogenesis. Useful markers
include tissue-specific enzymes whose activities may be assayed
(e.g., spectrophotometrically) after homogenization of the implant.
These assays may be useful for quantitation and for rapidly
obtaining an estimate of tissue formation after the implants are
removed from the animal. For example, alkaline phosphatase activity
may be used as a marker for osteogenesis.
[0246] Incorporation of systemically provided OP-1 morphogen analog
may be followed using labeled protein (e.g., radioactively labeled)
and determining its localization in the new tissue, and/or by
monitoring their disappearance from the circulatory system using a
standard labeling protocol and pulse-chase procedure. OP-1
morphogen analog also may be provided with a tissue-specific
molecular tag, whose uptake may be monitored and correlated with
the concentration of OP-1 morphogen analog provided. As an example,
ovary removal in female rats results in reduced bone alkaline
phosphatase activity, and renders the rats predisposed to
osteoporosis (as described in Example E). If the female rats now
are provided with OP-1 morphogen analog, a reduction in the
systemic concentration of calcium may be seen, which correlates
with the presence of the provided OP-1 morphogen analog and which
is anticipated to correspond with increased alkaline phosphatase
activity.
Example 2
Enhancing the Solubility of a hOP-1 Dimer
[0247] As described in section V.A.(ii), supra, it is contemplated
that the solubility of the hOP-1 dimer can be enhanced by replacing
hydrophobic amino acid residues located at the solvent accessible
surface of hOP-1 dimer with more polar or hydrophilic amino acid
residues. This example provides a description of such an
approach.
[0248] A Sma I to Bam HI fragment of the human OP-1 cDNA as
described in Ozkaynak et al. (1990) supra is cloned into a vector
to produce a plasmid similar to the plasmid called pW24 in
International Application PCT/US94/12063, the disclosure of which
is incorporated herein by reference. The pW24 plasmid contains OP-1
cDNA under the transcriptional control of the CMV (cytomegalovirus)
immediate early promoter. The selective marker on pW24 is the
neomycin gene which provides resistance to the cytostatic drug
G418. The pW24 plasmid also employs an SV40 origin of replication
(ori). The early SV40 promoter is used to drive transcription of
the neomycin marker gene.
[0249] Then, the alanine at position 63 is mutated to a serine by
site-directed mutagenesis using, for example, synthetic
oligonucleotides and either PCR or the site-directed mutagenesis
methods. See, for example, Kunkel et al. (1985) Proc. Natl. Acad.
Sci. USA 822: 488; Kunkel et al. (1985) Meth. Enzymol. 154: 367 and
U.S. Pat. No. 4,873,192. The resulting mutation is confirmed by
dideoxy sequencing.
[0250] Two additional vectors have been developed for use in a
triple transfection procedure along with pW24 to enhance OP-1
expression. One of the vectors employs the adenovirus E1A gene
under the VA1 gene as translation stimulation for the gene DHFR
gene. The other vector employs the adenovirus E1A gene under the
control of the thymidine kinase promoter as a transactivating
transcription activator. Both additional vectors, known as pH1130
and pH1176, as well as preferred transfection and screening
procedures are described in International Application
PCT/US94/12063.
[0251] Briefly, triple transfections are performed using the
calcium phosphate coprecipitation procedure. CHO cells are cultured
in .alpha.MEM, containing 5% or 10% fetal bovine serum (FBS),
non-essential amino acids, glutamine and antibiotics: penicillin
and streptomycin. Stable cell line transfections are carried out by
seeding 1-2.times.10.sup.6 cells in a 9 cm. petri dish. Following
an incubation period of up to 24-hour, each petri dish is
transfected with 10-30 .mu.g total vector DNA in equimolar amounts,
by calcium phosphate coprecipitation followed by glycerol shock
using standard methodology. Cells are incubated at 37.degree. C. in
growth medium for 24 hours, then transferred to selection medium.
All cultures are fed once or twice weekly with fresh selective
medium. After 10-21 days, resistant colonies are picked and assayed
for protein production.
[0252] Approximately 30 individual clones are selected, transferred
to a 24-well petri dish, and allowed to grow to confluence in
serum-containing media. The conditioned media from all surviving
clones is screened for protein production using a standard ELISA
(enzyme-linked immunosorbent assay) or Western blot. The
methodologies for these assay protocols as well as for generating
antibodies for use in these assays are well described in the art
(see e.g., Ausubel, supra).
[0253] Under such conditions, the VA1 and E1A genes typically act
synergistically to enhance OP1 expression in unamplified
transfected CHO cells. Candidate cell lines identified by the
screening protocol, then are seeded on ten 100 mm petri dishes at a
cell density of either 50 or 100 cells per plate, and with a higher
drug concentration (e.g., 1.0-.mu.m).
[0254] After 10-21 days of growth, the clones are isolated using
cloning cylinders and standard procedures, and cultured in 24-well
plates. Then, clones are screened for OP-1 expression by Western
immunoblots using standard procedures, and OP-1 expression levels
compared to parental lines. Candidate cells showing higher protein
production than cells of parental lines then are replated and grown
in the presence of a still higher drug concentration (e.g., 5-20
.mu.m). Generally, no more than 2-3 rounds of these "amplification"
cloning steps are necessary to achieve cell lines with high protein
productivity. Useful high producing cell lines may be further
subcloned to improve cell line homogeneity and product
stability.
[0255] A currently preferred method of large scale protein
production e.g., at least 2 liters, is by suspension culture of the
host Chinese hamster ovary (CHO) cells. CHO cells prefer attachment
but can be adapted to grow in suspension mode of cultivation. The
cells are trypsinized from a culture dish, introduced to growth
media containing 10% FBS and completely suspended to produce a
single cell suspension. The single cell suspension is introduced to
a spinner flask and placed in a 37.degree. C. 95% air/5% CO.sub.2
humidified incubator. Over a period of time the cells are
subcultured in medium with descending concentrations of serum.
[0256] Specifically, the adapted cells are introduced into a 3 L
spinner flask at an initial viable cell density of approximately
2.times.10.sup.5 cells/ml. Preferred culture medium is DMEM/F-12
(1:1) (GIBCO, New York) supplemented with 2% FBS, and preferred
agitation is approximately 50-60 rpm with a paddle impeller. After
7 days, the culture media is harvested, centrifuged at 1500 rpm and
the clarified conditioned media stored at 4.degree. C.
[0257] A representative purification scheme for purifying
recombinant morphogenic protein involves three chromatographic
steps (S-Sepharose, phenyl-Sepharose and C-18 HPLC) and is
described in International Application PCT/US94/12063. Morphogen
analog containing culture media is diluted to 6M urea, 0.05M NaCl,
13 mM HEPES, pH 7.0 and loaded onto an S-Sepharose column, which
acts as a strong cation exchanger. The column subsequently is
developed with two salt elutions. The first elution employs a
solution containing 0.1M NaCl, and the second elution employs a
buffer containing 6M urea, 0.3M NaCl, 20 mM HEPES, pH 7.0.
[0258] Ammonium sulfate is added to the 0.3M NaCl fraction to give
a solution containing 6M urea, 1M (NH.sub.4).sub.2SO.sub.4, 0.3M
NaCl, 20 mM HEPES, pH 7.0. Then, the sample is loaded onto a
phenyl-Sepharose column in the presence of 1M
(NH.sub.4).sub.2SO.sub.4). Then, the column is developed with two
step elutions using decreasing concentrations of ammonium sulfate.
The first elution employs 0.6M (NH.sub.4).sub.2SO.sub.4 and the
second elution employs 6M urea, 0.3M NaCl, 20 mM HEPES, pH 7.0
buffer. The material harvested from the second elution is dialyzed
against water, followed by 30% acetonitrile (0.1% TFA), and then
applied to a C-18 reverse phase HPLC column. Purified morphogen
analog is harvested from the HPLC column.
[0259] The enhanced solubility of the resulting morphogen analog is
measured by comparing the partition coefficient of the Ala
63->Ser 63 mutein versus wild type hOP-1 dimer. It is
contemplated that the Ala 63->Ser 63 mutein has a higher
solubility than native hOP-1. It is contemplated that, additional
muteins having multiple hydrophobic to hydrophilic substitutions
can be produced and characterized using the protocols described in
this Example. The biological activity of the resulting morphogen
analogs can be determined using one or more of the OP-1 activity
assays described Example 1.
Example 3
Biological Activity of Finger 1, Finger 2. and Heel Peptides
[0260] The hOP-1-based peptides described in this example were
produced and characterized prior to determination of the
three-dimensional structure of hOP-1. These peptides either agonize
or antagonize the biological activity of hOP-1. It is contemplated
that, further refinements based upon the hOP-1 crystal structure,
for example, the choice of more suitable sites for cyclizing
peptides which constrain the peptide into a conformation that more
closely mimics the shape of the corresponding region in hOP-1, may
be used to further enhance the agonostic or antagonistic properties
of such hOP-1-based peptides.
[0261] All of the peptides used in the following experiments, as
well as their relationships with the mature hOP-1 amino acid
sequence, are shown in FIG. 12. The finger 1-based peptides are
designated F1-2; the heel-based peptides are designated H-1, H-n2
and H-c2; and the finger 2-based peptides are designated F2-2, and
F2-3. Potential intra-peptide disulfide linkages are shown for each
peptide. All the peptides were synthesized on a standard peptide
synthesizer in accordance with the manufacturer's instructions. The
peptides were deprotected, cyclized by oxidation, and then cleaved
from resin prior to use.
[0262] In a first series of experiments, increasing concentrations
of peptides F2-2 (FIG. 13A), F2-3 (FIG. 13B), Hn-2 (FIG. 13C) and
Hc-2(FIG. 13D) were added to ROS cells either alone (open bars) or
in combination with 40 ng/ml soluble OP-1 (filled bars) and their
effect on alkaline phosphatase activity measured. Soluble OP-1 is
the form of OP-1 in which the pro-domain is still attached to the
mature portion of OP-1 (see WO94/03600). A basal alkaline
phosphatase activity is shown by the line and represents the
alkaline phosphatase activity of cells incubated in the absence of
both soluble OP-1 and peptide.
[0263] In FIG. 13A, peptide F2-2 at a concentration of about 60
.mu.M appears to double the basal alkaline phosphatase level and,
in the presence of soluble OP-1, increases alkaline phosphatase
activity by about 20% relative to soluble OP-1 alone. In FIG. 13B,
peptide F2-3 at a concentration of about 0.01 .mu.M appears to
increase the basal alkaline phosphatase level and, in the presence
of soluble OP-1, increases alkaline phosphatase activity by about
20% relative to soluble OP-1 alone. Accordingly, both peptides F2-2
and F2-3, in the alkaline phosphatase assay, appear to act as weak
OP-1 agonists.
[0264] In FIG. 13C, peptide H-n2 displays little or no effect on
alkaline phosphatase activity either alone or in combination with
soluble OP-1. FIG. 13D, peptide H-c2, at concentrations greater
than about 5 .mu.M, appears to antagonize the activity of soluble
OP-1.
[0265] In a second series of experiments, the ability of unlabeled
soluble OP-1 and unlabeled peptides F1-2, F2-2, F2-3, H-n2 and H-c2
to displace .sup.125I labeled soluble OP-1 from ROS cell membranes
was measured. The activities of peptides F2-2 and F2-3 relative to
soluble OP-1 are shown in FIG. 14A, and the activities of peptides
F1-2, H-n2 and H-c2 relative to soluble OP-1 are shown in FIG. 14B.
OP-1 receptor-enriched plasma membranes of ROS cells were incubated
for 20 hrs at 4.degree. C. with .sup.125 I-labeled soluble OP-1 and
unlabeled peptide. Receptor bound material was separated from
unbound material by centrifugation at 39,500.times.g. The resulting
pellet was harvested and washed with 50 mM HEPES buffer, pH7.4
containing 5 mM MgCl.sub.2 and 1 mM CaCl.sub.2. Radioactivity
remaining in the pellet was determined by means of a gamma
counter.
[0266] In FIG. 14A, peptide F2-2 (filled circles) soluble competes
with soluble OP-1 with an Effective Dose.sub.50 (ED.sub.50) of
about 1 .mu.M, but cannot completely displace soluble OP-1
ED.sub.50 is the concentration of peptide to produce half maximal
displacement of labeled soluble OP-1. Peptide F2-3 (filled
triangles) competes and is able to completely displace soluble OP-1
with an ED.sub.50 of about 5 .mu.M. In FIG. 14B, peptide F1-2
(filled boxes), peptide H-n2 (open diamonds) and peptide H-c2 (open
circles) all appear to exhibit little or no ability to displace
iodinated soluble OP-1 from ROS cell membranes.
[0267] Although the peptide experiments appear promising, it is
contemplated that resolution of the hOP-1 structure will enable the
skilled practitioner to design constrained peptides that more
closely mimic the receptor binding domains of human OP-1 and which
are more effective at agonizing or antagonizing an hOP-1 mediated
biological effect.
Example 4
Elimination of a Binding Site on the Surface of OP-1
[0268] .alpha.-2 macroglobulin, a protease scavenging protein known
to bind proteins in serum and target them to the kidney for
clearance from the body, binds OP-1. As described herein,
.alpha.-2's interaction sites on the OP-1 protein have been mapped.
Accordingly, using the database and structural information provided
herein, one can design an analog of OP-1 which eliminates one or
more .alpha.-2 macroglobulin interaction sites and provide an
analog having enhanced bioavailability in the body. This same
strategy can be applied for identifying and/or eliminating
interaction sites for other binding proteins on the OP-1
surface.
[0269] A. Identifying .alpha.-2 Macroglobulin Binding Sites
[0270] OP-1 was determined to interact specifically with .alpha.-2
macroglobulin in a standard competition binding assay, using
immobilized, commercially available .alpha.-2 macroglobulin, and
labeled and unlabeled OP-1 protein. Truncated mature OP-1, wherein
the first 22 amino acids have been cleaved from the mature form of
OP-1 in a standard trypsin digest, bound .alpha.-2 with 10-fold
less affinity, indicating that the N terminal portion of the mature
protein is involved in binding. This N-terminal portion of the
protein, which is not part of the crystal structure, is positively
charged and likely is highly flexible in solution. Elimination of
this sequence does not interfere with OP-1 activity. Two cyclized
peptides to all or a portion of the heel region, H-n2 and H1
(Cys.sub.71-Pro.sub.102, where Pro.sub.102 has been changed to a
cysteine to allow a disulfide bond between the two cysteines) also
compete for binding; while peptides to the finger regions (F2-2,
F2-3) do not compete.
[0271] .alpha.-2 macroglobulin was determined not to interfere with
OP-1's ability to stimulate alkaline phosphatase activity in a ROS
cell assay. Accordingly, .alpha.-2 macroglobulin binding does not
appear to sterically inhibit OP-1 receptor binding.
[0272] B. Design of Modified OP-1 Analog
[0273] The precise (.alpha.-2 macroglobulin interaction sites on
OP-1 now can be mapped and an analog designed using the structure
information provided herein. For example, the exact contact
residues can be identified by creating model peptides like H-N2
and/or H1 in conjunction with an "alanine scan" mutagenesis
program, wherein each residue is individually changed to an alanine
in turn, and the constructs then tested for their ability to
compete for binding. Once the contact residues are mapped, an
analog can be designed which eliminates the contact residues
without altering the overall structure of the heel region.
Specifically, a template of the region can be called up on the
computer from the database, and candidate replacement residues
tested. The information in Table 8 identifies particularly useful
candidate residues in the heel region which are solvent accessible,
which likely are not available as epitopes and make good candidates
for modification.
Equivalents
[0274] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
Sequence CWU 1
1
* * * * *