Note: Descriptions are shown in the official language in which they were submitted.
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MORPHOGENIC PROTEINS
This International Application claims priority of United States Provisional
Application No. 60/038,279 filed February 19, 1997. All publications and
patent
applications cited in this specification are herein incorporated by reference
as if
each individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
INTRODUCTION
The field of this invention is proteins which regulate cell function, and in
particular, antagonize bone morphogenic proteins.
Natural regulators of cellular growth, differentiation and function have
provided important pharmaceuticals, clinical and laboratory tools, and targets
for therapeutic intervention. A variety of such regulators have been shown to
15 have profound effects on basic cellular differentiation and developmental
pathways. For example, the recently cloned cerberus protein induces the
formation of head structures in anterior endoderm of vertebrate embryos.
Similarly, the noggin protein induces head structures in vertebrate embryos,
and can redirect mesodermal fates from ventral fates, such as blood and
zo mesenchyme, to dorsal fates such as muscle and notochord and can redirect
epidermal fates to anterior neural fates. The activities of chordin are
similar to
those of noggin, reflecting a common mechanism of action - namely
antagonizing bone morphogenic proteins (BMP) and thereby preventing their
function. BMPs have diverse biological activities in different biological
z5 contexts, including the induction of cartilage, bone and connective tissue,
and
roles in kidney, tooth, gut, skin and hair development.
Different members of the TGF(3 superfamily can instruct cells to follow
different
fates, for example TGF(3 induces neural crest to form smooth muscle, ~~hile
3o BMP2 induces the same cells to became neurons. In Xeno~us experiments,
dissociated animal cap cells (prospective ectoderm) become epidermis in
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response to BMP4 but become mesoderm in response to activin.
Since the sequence identity between activin and BMP4 is low, it is not
surprising that they induce different fates. It is more surprising that
members
of the BMP subfamily, which are quite closely related in sequence, can induce
distinct fates_ A striking example results from implantation of a matrix
impregnated with a BMP into muscle; when the effects are monitored
histologically, BMP2, BMP4 and BMP7 induce endochondral bone formation,
whereas a related molecule BMP12/GDF7 induces connective tissue similar to
tendon. Similarly, BMP4 can induce cell death in the hindbrain neural crest,
1o while the related protein dorsalin does not.
Since different BMP family members can induce different fates, then BMI'
antagonists that have specificity in blocking subsets of BMPs could change the
balance of BMPs that are presented to a cell, thus altering cell fate. In view
of
~5 the importance of relative BMP expression in human health and disease,
regulators of cellular function and BMP function in particular, such as noggin
and cerberus, provide valuable reagents with a host of clinical and
biotechnological applications. The present invention relates to a new family
of
regulators of cellular function.
Relevant Literature
Bouwmeester, et al. (1996) Nature 382: 595-601 describe the cloning of Xenopus
cerberus gene. PCT International Publication No. WO 94/05791 published 17
March 1994 entitled Dorsal Tissue Affecting Factor and Compositions; Lamb, T.
z5 M., et al. (1993) Science 262: 713-718; Smith, W.' C., et al. (1992) Cell
70: 829-840;
Smith, W. C., et al. (1993) Nature 361: 547-549; and Zimmerman, L. B., et al.
(1996) Cell 86: 599-606 describe the isolation and function of the noggin
nucleic
acid and protein. Piccolo, S., et al. (1996) Cell 86: 589-598; Sasai, Y., et
al. (1995)
Nature 376: 333-336; and Sasai, Y., et al. (1994) Cell 79: 779-790 relate to
the
3o chordin protein. Enomoto et al. (1994) Oncogene 9: 2785-2791 and Ozaki, et
al.
(1996) Jpn. J. Cancer Res. 87: 58-61 describe human and marine homologs of the
DAN gene.
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SUMMARY OF THE INVENTION
The in~~ention provides methods and compositions relating to DAN
s (Differential-screening-selected gene Aberrative in Neuroblastoma) and b57
proteins and related nucleic acids. Included are natural DAN and b57 homologs
from different species, as well as proteins comprising a DAN or b57 domain and
having DAN or b57-specific activity, particularly the ability to antagonize a
bone
morphogenic protein such as BMP2 or BMP4. The proteins may be produced
1o recombinantly from transformed host cells with the subject nucleic acids.
The
invention provides isolated hybridization probes and primers capable of
specifically hybridizing with the disclosed genes, specific binding agents
such as
specific antibodies, and methods of making and using the subject compositions
in diagnosis (e.g., genetic hybridization screens for b57 transcripts),
therapy (e.g.,
15 gene therapy to modulate b57 gene expression) and in the biopharmaceutical
industry (e.g., reagents for screening chemical libraries for lead
pharmacological
agents).
Preferred applications of the subject DAN and b57 proteins include modifying
20 the physiology of a cell comprising an extracellular surface by contacting
the cell
or medium surrounding the cell with an exogenous DAN or b57 protein under
conditions whereby the added protein specifically interacts with a component
of
the medium and/or the extracellular surface to effect a change in the
physiology
of the cell. Also preferred are methods for screening for biologically active
2s agents, which methods involve incubating a DAN or b57 protein in the
presence of an extracelluiar DAN or b57 protein-specific binding target and a
candidate agent, under conditions whereby, but for the presence of the agent,
the protein specifically binds the binding target at a reference affinity;
detecting
the binding affinity of the protein to the binding target to determine an
agent-
3o biased affinity, wherein a difference between the agent-biased affinity and
the
reference affinity indicates that the agent modulates the binding of the
protein
to the binding target.
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BRIEF DESCRIPTION OF THE FIGURES
FIGURES 1A - 1B - Demonstration that human b57 binds to BMP2 and BMP4.
FIGURE 2 - Demonstration that human b57 blocks BMP2 biological activity.
FIGURE 3 - Xenopus b57 (also referred to as Gremlin) blocks the activity of
BMP2. BMP2 at 78pM, 156pM, 313 pM, 625 pM, 1.25 nM, 2.5 nM or 5 nM was
to preincubated with a Gremlin COS supernatant at final concentration of 83nM
or 21 nM Gremlin, mock-transfected media, or fresh DMEM prior to addition to
cells. Alkaline phosphatase activity was assayed 24 hours later. Approx. 83nM
Gremlin completely blocks BMP2 activity. Approx. 2lnM Gremlin partially
blocks BMP2 doses tested.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides DAN and b57 proteins which include natural DAN
2o and b57 proteins and recombinant proteins comprising a DAN or b57 amino
acid sequence, or a functional DAN or b57 protein domain thereof having an
assay-discernable DAN or b57-specific activity. Accordingly, the proteins may
be
deletion mutants of the disclosed natural DAN and b57 proteins and may be
provided as fusion products, e.g., with non-b57 polypeptides. The subject DAN
and b57 protein domains have DAN or b57-specific activity or function and are
functionally distinct from each other and from cerberus and noggin homologs.
Such domains include at least 6 and preferably at least 8 consecutive residues
of
a natural DAN or b57 protein (See DAN sequence reported by Enomoto, et al.
(1994) Oncogene 9: 2785-2791 and human b57 sequence disclosed herein).
3o Preferred b57 proteins comprise a b57 sequence conserved across species.
Note that contrary to prior art teachings which state that DAN is an
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intracellular zinc finger protein, applicants disclose that the natural DAN
protein is structurally and functionally related to b57 and that both it and
DAN
proteins as described herein are extracellularly active as antagonists of
certain
morphogenic proteins such as BMPs. DAN or b57-specific activity or function
may be determined by convenient in vitro, cell-based, or in vivo assays -
e.g., in
vitro binding assays, cell culture assays, in animals (e.g., immune response,
gene therapy, transgenics, etc.), etc. Binding assays encompass any assay
where
the specific molecular interaction of a DAN or b57 protein with a binding
target
is evaluated. The binding target may be a natural binding target such as a
TGF(3
1o protein, a morphogenic protein, preferably a bone morphogenic protein such
as
BMP2 or BMP4, chaperone, or other regulator that directly modulates DAN or
b57 activity or its localization; or non-natural binding target such as a
specific
immune protein such as an antibody, or a DAN or b57 specific agent such as
those identified in assays described below. Generally, binding specificity is
assayed by bioassay (e.g., the ability to induce neuronal tissue from injected
embryonic ectoderm), TGFj3 protein binding equilibrium constants (usually at
least about 10~ M-~, preferably at least about 208 M-~, more preferably at
least
about 109 Mr), by the ability of the subject protein to function as negative
mutants in DAN or b57-expressing cells, to elicit DAN or b57 specific antibody
2o in a heterologous host (e.g., a rodent or rabbit), etc.
The claimed proteins may be isolated or pure - an "isolated" protein is one
that
is no longer accompanied by some of the material with which it is associated
in
its natural state, and that preferably constitutes at least about
0.5°/", and more
preferably at least about 5% by weight of the total protein in a given sample;
a
"pure" protein constitutes at least about 90'%, and preferably at least about
99"/"
by weight of the total protein in a given sample. The subject proteins and
protein domains may be synthesized, produced by recombinant technology, or
purified from cells. A wide variety of molecular and biochemical methods are
3o available for biochemical synthesis, molecular expression and purification
of
the subject compositions, see e.g., Molecular Cloning, A Laboratory Manual
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(Sambrook, et al., Cold Spring Harbor Laboratory), Current Protocols in
Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-
Interscience,
NY). An exemplary method for isolating natural DAN and b57 proteins
involves expressing a cDNA library (e.g., one derived from Xenopus ovarian
s cells) and assaying expression products for embryonic axis formation. This
method and other suitable bioassays amenable to detecting DAN and b57
proteins have been described by Lemaire, P., et al., (1995) Cell 81:85-94;
Smith,
W. C., and Harland, R. M. (1992) Cell 70: 829-40; Smith, W. C., and Harland,
R.
M. (1991) Cell 67: 753-765; Piccolo, S., et al., (1996) Cell 86: 589-98; and
1o Zimmerman, L. B., et al., (1996) Cell 86: 599-606.
The subject proteins find a wide variety of uses including use as immunogens,
targets in screening assays, bioactive reagents for modulating cell growth,
differentiation and/or function, etc. For example, the invention provides
15 methods for modifying the physiology of a cell comprising an extracellular
surface by contacting the cell or medium surrounding the cell with an
exogenous DAN or b57 protein under conditions whereby the added protein
specifically interacts with a component of the medium and/or the extracellular
surface to effect a change in the physiology of the cell. According to these
2o methods, the extracellular surface includes plasma membrane-associated
receptors; the exogenous DAN or b57 refers to a protein not made by the cell
or,
if so, expressed at non-natural levels, times or physiologic locales; and
suitable
media include in vitro culture media and physiological fluids such as blood,
synovial fluid, etc. Effective administrations of subject proteins can be used
to
25 reduce undesirable (e.g., ectopic) bone formation, inhibit the growth of
cells that
require a morphogenic protein (e.g., BMP-dependent neuroblastomas and
gliomas), alter morphogen-dependent cell fate/differentiation in culture, such
as with cells for transplantation or infusion, etc. The proteins may be may be
introduced, expressed, or repressed in specific populations of cells by anv
3o convenient way such as microinjection, promoter-specific expression of
recombinant enzyme, targeted delivery of lipid vesicles, etc.
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The in~~ention provides natural and non-natural DAN and b57-specific binding
agents, methods of identifying and making such agents, and their use in
diagnosis, therapy and pharmaceutical development. DAN or b57-specific
binding agents include b57-specific ligands such as BMPs, and receptors, such
as
somatically recombined protein receptors like specific antibodies or T-cell
antigen receptors (See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Laboratory) and also includes other natural
binding agents identified with assays such as one-, two- and three-hybrid
screens, and non-natural binding agents identified in screens of chemical
1o libraries such as described below. Agents of particular interest modulate
DAN
or b57 function.
The invention provides b57 and DAN nucleic acids, which find a wide variety
of applications including use as translatable transcripts, hybridization
probes,
PCR primers, diagnostic nucleic acids, etc., as well as use in detecting the
presence of DAN and b57 genes and gene transcripts and in detecting or
amplifying nucleic acids encoding additional DAN and b57 homologs and
structural analogs. For example, Xenopus and chick b57 sequence data was used
to search the EST database of the LM.A.G.E. consortium and a human cDNA
2o clone 272074 was discerned to contain homologous sequence homologous by
DNA sequencing. The insert was cloned into CS105, a suitable vector for
synthesis of synthetic mRNA (Turner, D. L., and Weintraub, H. (1994) Genes
Dev 8, 1434-47; Baker, J. C., and Harland, R. M. (1996) Genes & Development
10).
b57-specific function of the human gene product was determined by injection of
purified, synthetic transcripts of cDNA clones' into Xeno~us embryos. This
assay provides a bioassay for antagonists of BMP activity, as exemplified by
the
induction of ectopic body axes or enlarged heads. Embryos injected with
transcript from the human b57 clone had enlarged heads and partial ectopic
body axes similar to the Xenopus b57 injected embryos, indicating a b57
specific
3o biological function for the human gene product.
Similarly, Xenopus cerberus sequence data was used to search the EST database
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of the LM.A.G.E. consortium and human cDNA clone 272074 was discerned to
contain homologous sequence. This clone was obtained from Genome
Systems, Inc. (St. Louis, MO) and sequenced using the ABI 373A DNA sequencer
and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc.,
Foster City, CA). The nucleotide sequence encoding human b57 is set forth
herein as SEQ. NO. 1 and the deduced amino acid sequence of human b57 is set
forth herein as SEQ. NO. 2.
The subject nucleic acids are of synthetic/non-natural sequences and/or are
1o isolated, i.e., no longer accompanied by some of the material with which it
is
associated in its natural state, preferably constituting at least about 0.5%,
more
preferably at least about 5°/o by weight of total nucleic acid present
in a given
fraction, and usually recombinant, meaning they comprise a non-natural
sequence or a natural sequence joined to nucleotides) other than that which it
is joined to on a natural chromosome. Nucleic acids comprising the nucleotide
sequence of SEQ. NO. 1 or fragments thereof, contain such sequence or
fragment at a terminus, immediately flanked by a sequence other than that to
which it is joined on a natural chromosome, or flanked by a native flanking
region fewer than 10 kb, preferably fewer than 2 kb, which is immediately
2o flanked by a sequence other than that to which it is joined on a natural
chromosome. While the nucleic acids are usually RNA or DNA, it is often
advantageous to use nucleic acids comprising other bases or nucleotide analogs
to provide modified stability, etc.
The amino acid sequences of the disclosed DAN and b57 proteins are used to
back translate DAN or b57 protein-encoding nucleic acids optimized for
selected
expression systems (Holler, et al. (1993) Gene 136: 323-328; Martin, et al.
(1995)
Gene 154: 150-166) or used to generate degenerate oligonucleotide primers and
probes for use in the isolation of natural b57 encoding nucleic acid sequences
("GCG" software, Genetics Computer Group, Inc., Madison, WI). DAN and b57
encoding nucleic acids may be part of expression vectors and may be
incorporated into recombinant host cells, e.g., for expression and screening,
for
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transgenic animals, for functional studies such as the efficacy of candidate
drugs
for disease associated with b57 mediated signal transduction, etc. Expression
systems are selected and/or tailored to effect b57 protein structural and
functional variants through alternative post-translational processing.
The invention also provides for nucleic acid hybridization probes and
replication/amplification primers having a DAN or b57 cDNA specific sequence
and sufficient to effect specific hybridization with SEQ. NO. 1. Demonstrating
specific hybridization generally requires stringent conditions, for example,
lU hybridizing in a buffer comprising 30°/'~ formamide in 5 x SSPE
(0.18 M NaCI,
0.01 M NaP04, pl-~7.7, 0.001 M EDTA) buffer at a temperature of 42°C
and
remaining bound when subject to washing at 42°C with 0.2 x SSPE;
preferably
hybridizing in a buffer comprising 50'% formamide in 5 x SSPE buffer at a
temperature of 42°C and remaining bound when subject to washing at
42°C
with 0.2x SSPE buffer at 42°C. DAN and b57 cDNA homologs can also be
distinguished from other protein using alignment algorithms, such as BLASTX
(Altschul, et al. (1990) Basic Local Alignment Search Tool, J. Mol. Biol. 215:
403-
410).
2U DAN and b57 hybridization probes find use in identifying wild-type and
mutant
alleles in clinical and laboratory samples. Mutant alleles are used to
generate
allele-specific oligonucleotide (ASO) probes for high-throughput clinical
diagnoses. DAN and b57 nucleic acids are also used to modulate cellular
expression or intracellular concentration or availability of active DAN or
b57.
DAN and b57 inhibitory nucleic acids are typically antisense - single stranded
sequences comprising complements of the disclosed natural b57 coding
sequences. Antisense modulation of the expression of a given DAN or b57
protein may employ antisense nucleic acids operably linked to gene regulatory
sequences. Cells are transfected with a vector comprising a DAN or b57
3U sequence with a promoter sequence oriented such that transcription of the
gene
yields an antisense transcript capable of binding to endogenous DAN or b57
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encoding mRNA. Transcription of the antisense nucleic acid may be
constitutive or inducible and the vector may provide for stable
extrachromosomal maintenance or integration. Alternatively, single-stranded
antisense nucleic acids that bind to genomic DNA or mRNA encoding a given
DAN or b57 protein may be administered to the target cell, in or temporarily
isolated from a host, at a concentration that results in a substantial
reduction in
expression of the targeted protein. An enhancement in DAN or b57 expression
is effected by introducing into the targeted cell type DAN or b57 nucleic
acids
which increase the functional expression of the corresponding gene products.
1o Such nucleic acids may be DAN or b57 expression vectors, vectors which
upregulate the functional expression of an endogenous allele, or replacement
vectors for targeted correction of mutant alleles. Techniques for introducing
the
nucleic acids into viable cells are known in the art and include retroviral-
based
transfection, viral coat protein-liposome mediated transfection, etc.
The invention provides efficient methods of identifying agents, compounds or
lead compounds for agents active at the level of DAN or b57 modulatable
cellular function. Generally, these screening methods involve assaying for
compounds which modulate DAN or b57 interaction with a natural DAN or
2o b57 binding target. A wide variety of assays for binding agents are
provided
including protein-protein binding assays, immunoassays, cell based assays,
etc.
Preferred methods are amenable to automated, cost-effective high throughput
screening of chemical libraries for lead compounds.
In vitro binding assays employ a mixture of components including a DAN or
b57 protein, which may be part of a fusion product with another peptide or
polypeptide, e.g., a tag for detection or anchoring, etc. The assay mixtures
comprise a natural DAN or b57 binding target, e.g., a TGF~3 protein such as a
BMP. While native binding targets may be used, it is frequently preferred to
use
portions thereof as long as the portion provides binding affinity and avidity
to
the subject DAN or b57 conveniently measurable in the assay. The assay
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mixture also comprises a candidate pharmacological agent. Candidate agents
encompass numerous chemical classes, though typically they are organic
compounds, preferably small organic compounds, and are obtained from a wide
variety of sources including libraries of synthetic or natural compounds. A
variety of other reagents such as salts, buffers, neutral proteins, e.g.,
albumin,
detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents,
etc.,
may also be included. The mixture components can be added in any order that
provides for the requisite bindings and incubations may be performed at any
temperature which facilitates optimal binding. The mixture is incubated under
to conditions whereby, but for the presence of the candidate pharmacological
agent, the DAN or b57 specifically binds the cellular binding target, portion
or
analog with a reference binding affinity. Incubation periods are chosen for
optimal binding but are also minimized to facilitate rapid, high throughput
screening.
After incubation, the agent-biased binding between the DAN or b57 and one or
more binding targets is detected by any convenient way. For cell-free binding
type assays, a separation step is often used to separate bound from unbound
components. Separation may be effected by precipitation, immobilization, etc.,
2o followed by washing by, e.g., membrane filtration or gel chromatography.
For
cell-free binding assays, one of the components usually comprises or is
coupled
to a label. The label may provide for direct detection as radioactivity,
luminescence, optical or electron density, etc., or indirect detection such as
an
epitope tag, an enzyme, etc. A variety of methods may be used to detect the
label depending on the nature of the label anci other assay components, e.g.,
through optical or electron density, radiative emissions, nonradiative energy
transfers, or indirectly detected with antibody conjugates, etc. A difference
in
the binding affinity of the DAN or b57 protein to the target in the absence of
the
agent as compared with the binding affinity in the presence of the agent
indicates that the agent modulates the binding of the DAN or b57protein to the
corresponding binding target. A difference, as used herein, is statistically
significant and preferably represents at least a 50°/«, more preferably
at least a
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90°ra difference.
The invention provides for a method for modifying the physiology of a cell
comprising an extracellular surface in contact with a medium, said method
comprising the step of contacting said medium with an exogenous DAN or b57
protein under conditions whereby said protein specifically interacts with at
least
one of a component of said medium and said extracellular surface to effect a
change in the physiology of said cell.
The invention further provides for a method for screening for biologically
active agents, said method comprising the steps of a) incubating a DAN or b57
protein in the presence of an extracellular DAN or b57 protein specific
binding
target and a candidate agent, under conditions whereby, but for the presence
of
said agent, said protein specifically binds said binding target at a reference
affinity; b) detecting the binding affinity of said protein to said binding
target to
determine an agent-biased affinity, wherein a difference between the agent-
biased affinity and the reference affinity indicates that said agent modulates
the
binding of said protein to said binding target.
2o One embodiment of the invention is an isolated b57 protein comprising the
amino acid sequence as set forth in SEQ NO. 2 or a fragment thereof having b57-
specific activity.
Another embodiment of the invention is a recombinant nucleic acid encoding
b57 protein comprising the amino acid sequence as set forth in SEQ NO. 2 or a
fragment thereof having b57- specific activity.
Still another embodiment is an isolated nucleic acid comprising a nucleotide
sequence as set forth in SEQ NO. 1 or a fragment thereof having at least 18
consecutive bases of SEQ NO. 1 and sufficient,to specifically hybridize with a
nucleic acid having the sequence of SEQ NO. 1 in the presence of natural DAN
and cerberus cDNA.
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The present invention also provides for antibodies to the b57 protein
described
herein which are useful for detection of the protein in, for example,
diagnostic
applications. For preparation of monoclonal antibodies directed toward this
b57
protein, any technique which provides for the production of antibody
molecules by continuous cell lines in culture may be used. For example, the
hybridoma technique originally developed by Kohler and Milstein (1975,
Nature 256:495-497), as well as the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the
1o EBV-hybridoma technique to produce human monoclonal antibodies (Cole et
al., 1985, in "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc.
pp.
77-96) and the like are within the scope of the present invention.
The monoclonal antibodies for diagnostic or therapeutic use may be human
monoclonal antibodies or chimeric human-mouse (or other species)
monoclonal antibodies. Human monoclonal antibodies may be made by any of
numerous techniques known in the art (~ Teng et al., 1983, Proc. Natl. Acad.
Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79;
Olsson
et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be
2o prepared containing a mouse antigen-binding domain with human constant
regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda
et al.,
1985, Nature 314:452).
Various procedures known in the art may be used for the production of
polyclonal antibodies to epitopes of the b57 protein described herein. For the
production of antibody, various host animals can be immunized by injection
with the b57 protein, or a fragment or derivative thereof, including but not
limited to rabbits, mice and rats. Various adjuvants may be used to increase
the
immunological response, depending on the host species, and including but not
limited to Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
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dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum.
A molecular clone of an antibody to a selected b57 protein epitope can be
prepared by known techniques. Recombinant DNA methodology (see e.g.,
Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct
nucleic acid sequences which encode a monoclonal antibody molecule, or
antigen binding region thereof.
io
The present invention provides for antibody molecules as well as fragments of
such antibody molecules. Antibody fragments which contain the idiotype of
the molecule can be generated by known techniques. For example, such
fragments include but are not limited to: the F(ab')., fragment which can bc:
produced by pepsin digestion of the antibody molecule; the Fab' fragments
which can be generated by reducing the disulfide bridges of the F(ab'),
fragment,
and the Fab fragments which can be generated by treating the antibody molecule
with papain and a reducing agent. Antibody molecules may be purified by
known techniques, ~, immunoabsorption or immunoaffinity
2o chromatography, chromatographic methods such as HPLC (high performance
liquid chromatography), or a combination thereof.
The invention further provides for a method of using a DAN or b57 protein or
fragment thereof as an antagonist of the activity of a bone morphogenic
protein
(BMP). Preferably, the invention provides for a method of antagonizing the
function of a Bone Morphogenic Protein (BMP) which comprises contacting
said BMP with b57. The method of the invention is carried out under
conditions whereby the b57 binds to the BMP. In a preferred embodiment of the
invention, the b57 is mammalian b57, and more preferably, human b57, or a
3o fragment thereof capable of binding to the BMP. In further preferred
embodiments of the invention, the human b57 is used to antagonize the
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function of BMP2 or BMP4.
Antagonists to BMP's may be useful for preventing and treating BMP-related
disorders of animals, especially of humans. It was, therefore, an object of
this
invention to identify substances which effectively antagonize the function of
BMP's in disease states in animals, preferably mammals, especially in humans.
It was another object of this invention to prepare novel compounds which
inhibit BMP. It was still another object of this invention to develop a method
of antagonizing the functions of BMP's in disease states in mammals. It was
1o also an object of this invention to develop a method of preventing or
treating
disorders relating to the function of BMP's.
In addition to their roles in normal bone formation, the BMPs appear to be
involved in diseases in which they promote abnormal bone growth. For
example, BMPs have been reported to play a causative role in the disease
known as Fibrodysplasia Ossificans Progressiva (FOP), in which patients grow
an abnormal "second skeleton" that prevents any movement.
Therefore, an object of the present invention is to provide a novel molecule
for
2o the treatment of diseases or disorders including, but not limited to,
Fibrodysplasia Ossificans Progressiva (FOP). Since b57 is a Mocker of BMP's,
it
offers hope as a therapeutic agent for this disease. Additionally, abnormal
bone
growth can occur after hip replacement surgery and thus ruin the surgical
outcome. This is a more common example of pathological bone growth and a
situation in which Mockers of BMP's such as b57 may be therapeutically useful.
b57 may be useful as well as for treating other forms of abnormal bone growth,
such as the pathological growth of bone following trauma, burns or spinal cord
injury. In addition, b57 may be useful for treating or preventing the
undesirable actions of BMP's associated with the abnormal bone growth seen in
3o connection with metastatic prostate cancer or osteosarcoma.
In additional embodiments, the b57 nucleic acids, proteins, and peptides of
the
CA 02282302 1999-08-18
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invention may be used to block BMP activity in mammals.
The present invention also provides for compositions comprising a b57
molecule, as described herein and a suitable carrier. The active ingredient,
which may comprise the b57, should be formulated in a suitable carrier for
systemic or local administration in vivo by any appropriate route including,
but
not limited to injection (e.~., intravenous, intraperitoneal, intramuscular,
subcutaneous, endoneural, perineural, intraspinal, intraventricular,
intravitreal, intrathecal etc.), by absorption through epithelial or
1o mucocutaneous linings (e.~., oral mucosa, rectal and intestinal mucosa,
etc.); or
by a sustained release implant, including a cellular or tissue implant.
Depending upon the mode of administration, the active ingredient may be
formulated in a liquid carrier such as saline, incorporated into liposomes,
microcapsules, polymer or wax-based and controlled release preparations, or
formulated into tablet, pill or capsule forms.
The concentration of the active ingredient used in the formulation will depend
upon the effective dose required and the mode of administration used. The
2o dose used should be sufficient to achieve circulating plasma concentrations
of
active ingredient that are efficacious. Effective doses may be extrapolated
from
dose-response curves derived from in vitro or animal model test systems.
In addition, the invention further provides for the use of yon Willebrand
factor to regulate or modulate the activity of a' BMP. By aligning the carboxy-
terminal domains of chicken (chB57), human (hB57) and Xenopus b57 (xB57),
human and mouse DAN, mouse and Xenopus cerberus and human yon
Willebrand factor (VWF), (see Table 1) applicants have discovered a striking
homology among these various proteins, including the conservation of nine
3o separate cysteine residues. Given this striking homology, it is expected
that yon
Willebrand factor may also be useful for regulating or modulating the activity
of a BMP.
16
_ T ___ t _ .. _._ T
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The following protocol may be used for a high throughput human b57 - BMP
binding assay:
A. Reagents:
- Neutralite Avidin: 20 ug/ml in PBS.
Blocking buffer: 5'%'~ BSA, 0.5% Tween 20 in PBS; 1 hour at room
temperature.
- Assay Buffer: 100 mM KC1, 20 mM HEPES pH 7.6, 1 mM MgCl2, 1'%
glycerol, 0.5% NP-40, 50 mM ~i-mercaptoethanol, 1 mg/ml BSA, cocktail of
protease inhibitors.
- s3P human b57 protein lOx stock: 10-A - 10-6M "cold" human b57
supplemented with 200,000-250,000 cpm of labeled human b57 (Beckman
counter). Place in the 4°C microfridge during screening.
- Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB #
109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma # B-
6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB # 917575),
and 2mM NaV03 (Sigma # S-6508) in 10 ml of PBS.
- BMP: 10-~ - 10-4M biotinylated BMP in PBS.
B. Preparation of assay plates:
- Coat with 120 ~l of stock N-Avidin per well overnight at 4°C.
- Wash 2 times with 200 ~1 PBS.
- Block with 150 Ill of blocking buffer.
- Wash 2 times with 200 Ill PBS.
C. Assay:
- Add 40 ftl assay buffer/well.
- Add 10 ul compound or extract
- .Add 10 ftl ~~P- b57 protein (20-25,000 cpm/0.1-10 pmoles/well =10-'~- 10-~
17
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WO 98/37195 PCTNS98/03283
M final conc).
- Shake at 25°C for 15 minutes.
- Incubate additional 45 minutes at 25°C.
- Add 40 ~tl biotinylated BMP (0.1-10 pmoles/40 ~! in assay buffer)
- Incubate 1 hour at room temperature.
- Stop the reaction by washing 4 times with 200 ~l PBS.
- Add 150 ~tl scintillation cocktail.
- Count in Topcount.
to D. Controls for all assays (located on each plate):
i. Non-specific binding
ii. Soluble (non-biotinylated b57} at 80"/° inhibition.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLES
1. Sequencing of Human b57 clone
As stated previously, Xenopus cerberus sequence data was used as a probe to
z5 search the EST database of the LM.A.G.E. consortium and human cDNA clone
272074 was discerned to contain homologous sequence. This clone ~n~as
obtained from Genome Systems, Inc. (St. Louis, MO) and sequenced using the
ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit
(Applied Biosystems, Inc., Foster City, CA). The nucleotide sequence encoding
3o human b57 is set forth as SEQ. NO. 1 and the deduced amino acid sequence of
human b57 is set forth as SEQ. NO. 2.
18
T _ ___ _ T __- i
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WO 98137195 PCT/US98/03283
SEQ. NO. 1 - Nucleotide Sequence encoding human b57
20 30 40 50 60
ATGAGCCGCA.CAGCTTACAC GGTGGGAGCC CTGCTTCTCC TCTTGGGGAC CCTGCTGCCG
70 80 90 100 110 120
GCTGCTGAAG GGAAAAAGAA AGGGTCCCAA GGTGCCATCC CCCCGCCAGA CAAGGCCCAG
130 140 150 160 170 180
CACAATGACT CAGAGCAGAC TCAGTCGCCC CAGCAGCCTG GCTCCAGGAA CCGGGGGCGG
190 200 210 220 230 240
GGCCAAGGGC GGGGCACTGC CATGCCCGGG GAGGAGGTGC TGGAGTCCAG CCAAGAGGCC
250 260 270 280 290 300
*
CTGCATGTGA CGGAGCGCAA ATACCTGAAG CGAGACTGGT GCAAAACCCA GCCGCTTAAG
310 320 330 340 350 360
CAGACCATCC ACGAGGAAGG CTGCAACAGT CGCACCATCA TCAACCGCTT CTGTTACGGC
370 380 390 400 410 420
* * * * *
CAGTGCAACT CTTTCTACAT CCCCAGGCAC ATCCGGAAGG AGGAAGGTTC CTTTCAGTCC
430 440 450 460 470 480
* *
TGCTCCTTCT GCAAGCCCAA GAAATTCACT ACCATGATGG TCACACTCAA CTGCCCTGAA
490 500 510 520 530 540
CTACAGCCAC CTACCAAGAA GAAGAGAGTC ACACGTGTGA AGCAGTGTCG TTGCATATCC
550
ATCGATTTGG ATTAR
19
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WO 98/37195 PCT/US98/03283
SEQ. NO. 2 - Deduced Amino Acid Sequence of human b57
20
MetSerArgThrAlaTyrThrValGlyAla LeuLeuLeuLeuLeuGlyThrLeuLeuPro
30 40
AlaAlaGluGlyLysLysLysGlySerGln GlyAlaIleProProProAspLysAlaGln
50 60
HisAsnAspSerGluGlnThrGlnSerPro GlnGlnProGlySerArgAsnArgGlyArg
70 80
GlyGlnGlyArgGlyThrAlaMetProGly GluGluValLeuGluSerSerGlnGluAla
90 100
LeuHisValThrGluArgLysTyrLeuLys ArgAspTrpCysLysThrGlnProLeuLys
110 120
GlnThrIleHisGluGluGlyCysAsnSer ArgThrIleIleAsnArgPheCysTyrGly
130 140
GlnCysAsnSerPheTyrIleProArgHis IleArgLysGluGluGlySerPheGlnSer
150 160
CysSerPheCysLysProLysLysPheThr ThrMetMetValThrLeuAsnCysProGlu
170 180
LeuGlnProProThrLysLysLysArgVal ThrArgValLysGlnCysArgCysIleSer
IleAspLeuAsp
_._ __T T __- _ __ T
CA 02282302 1999-08-18
WO 9S/37195 PCT/US98/03283
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21
CA 02282302 1999-08-18
WO 98/37195 PCT/US98/03283
Human b57 belongs to a family of proteins that includes cerberus
(Bouwmeester et al., 1996) and DAN (Enomoto et al., 1994) - see Table 1. These
b57 relatives have been postulated to function as antagonists for different
members of the bone morphogenetic protein (BMP) family. The BMP family
has many different members displaying varying degrees of homology to each
other and it includes not only the BMPs, but also the growth differentiation
factors (GDFs), transforming growth factor beta and its homologues (TGF(3s),
the
activins, the inhibins, the dorsalins, as well as nodal, vegetal, vegetal-
related,
and several new members (Furuta et ai., 1997). BMPs have been shown to play
to important role in many different biological processes and thus the
existence of
naturally occurring antagonists of their activity is of great interest and
pharmacological potential.
Other than the cerberus/b57/DAN family of BMP antagonists, several other
antagonists to BMPs are known namely noggin (Smith and Harland, 1992;
Zimmerman et al., 1996), chordin (Piccolo et al., 1996), and follistatin
(Hemmati-Brivanlou et al., 1994). These do not belong to the
cerberus/b57/DAN family, and they also do not share any homology with each
other. Nonetheless, noggin and chordin have been shown to bind to BMP2 and
2o BMP4 and inhibit their biological actions by blocking the interaction with
the
BMP receptors.
We describe here the expression of human b57 using mammalian expression
systems and have generated, using standard laboratory techniques (See, e.g.,
z5 Molecular Cloning, A Laboratory Manual (Sambrook, et al., Cold Spring
Harbor
Laboratory), several tagged forms of hb57, such as hb57-Fc~Cl, hb57-FLAG, and
hb57myc3. hb57 has also been expressed in E. cofi and refolded, and rabbit
anti-
b57 polyclonal antisera have been raised against it (Antisera # Q-1523-1 and Q-
1523-2 prepared under contract by Quality Controlled Biochemicals, Inc.,
3o Hopkiaton, Massachusetts, 01748 USA). Using recombinant hb57 proteins, we
have tested hb57 for binding to hBMP2 and hBMP4, and the ability of noggin to
22
__ t T _ I
CA 02282302 1999-08-18
WO 98/37195 PCT/US98/03283
antagonize this interaction. We have also tested the ability of hb57 to block
the
biological activity of hBMP2 in a cell-based assay.
2. Construction of hb57 expression plasmid pRG622
A DNA fragment encoding the gene for human b57 (hb57) was PCR amplified
from an EST clone using the primers
N1-hb57 (5'-GAGAGTCATGAAAAAGAAAGGGTCCCAAGGTGC-3') and
io C1-hb57 (5'-GAGAGGCGGCCGCTCATTAATCCAAATCGATGGATATGCAAC-
3'). The resulting 509 by fragment was digested with BspH 1 and Not 1 then
ligated into the Nco 1-Not I sites of pRG536. A clone was identified and named
pRG622, then transformed into E. coli strain RFJ143. The construct was
confirmed by DNA sequence analysis.
3. Purification of hb57
E. coli strain RFJ143 containing pRG622 was grown in LB medium and
2o expression of hb57 was induced by the addition of 1 mM IPTG. Induced cells
were collected by centrifugation, resuspended in 10 volumes of 100 mM Tris-
HCI, pH 8.5, 20 mM EDTA, and lysed by passage through a Niro-Soave Panda
cell disrupter. The cell lysate was centrifuged and the pellet was resuspended
in
10 volumes of 9 M urea, 50 mM Tris-HCI, pH 8.5, 1 mM EDTA, 100 mM
z5 Na2S03, 10 mM Na2S4O6 and stirred for 16 hr at room temperature. The
solubilized inclusion bodies were fractionated on a Sephacryl S-300 column
equilibrated in 8 M urea, 20 mM MES, pH 6.0, 200 mM NaCI, 1 mM EDTA.
Fractions containing hb57 were pooled and diluted 10-fold into 1 M urea, 50
mM Tris-HCI, pH 8.0, 2 M NaCI, 0.1 mM EDTA, 0.5 mM cysteine. After 1-2 days
3o incubation at 4°C, the refolded hb57 was purified by reverse phase
chromatography on a Jupiter C5 column. Properly refolded protein was eluted
from the column by a 1.3"/«/min. gradient from 30'%'~ to 50'%« acetonitrile in
0.1'%'~
23
CA 02282302 1999-08-18
WO 98/37195 PCT/US98/03283
TFA. Fractions containing hb57 c~~ere pooled, dried under vacuum, and
resuspended in 20 mM Tris-HCI, pH 8.0, 150 mM NaCI, 0.1 mM EDTA.
Demonstration That hb57 Binds to BMP2 and BMP4
In one example, hb57myc3 (1 ml of COS7-derived serum-free conditioned
media) was co-incubated with hBMP2 (1 ~tg/ml) or hBMP4 (1 ~tg/ml) in the
absence or in the presence of human noggin protein (hNG~B2, 10 llg/ml). The
formation of a stable complex between hb57 and the BMPs was determined by
1o immunoprecipitating hb57 and associated proteins using an anti-myc
monoclonal antibody (9E10; 1 ~g/ml) bound to Protein G-Sepharose beads
(Pharmacia). The binding reaction was carried out in the serum-free
conditioned media after it was made 20 mM Tris pl-~ 7.6, 150 mM NaCI, 0.1%
Tween 20 (TBST), 1 mg/ml bovine serum albumin (BSA), by addition of a 10x
concentrate of these reagents. Binding was allowed to proceed for 1 hour, at
25°C, in a reaction volume of I.1 ml, with continuous mixing to keep
the
Protein G-Sepharose in suspension, after which point the beads were spun
down, washed once with TBST, moved to new eppendorf tubes, and washed 3
more times with TBST. Proteins bound to the beads were solubilized by
2o addition of 25 ~l of Laemli SDS-PAGE sample buffer and loaded onto ~ to 12%
NuPAGE/MES gradient gels (Novex), which were run under reducing
conditions. The proteins were subsequently transferred on Immobilon P and
western blotted for the presence of BMP2 or BMP4 using polvclonal antisera
raised against the respective proteins.
As can be seen in Figures lA-1B, hb57myc3 binds to both hBMP2 (Fig. lA, lane
1) and hBMP4 (figure 1B, lane 1) . This interaction appears to be stable in 1
M
NaCI (Fig. 1A, lane 2; and Fig. 1B, lane 2), although some reduction of
binding is
seen under those conditions. Addition of 10 ~g hNG completely blocks this
so interaction (Fig. lA, lane 3; and Fig. 1B, lane 3), presumably by binding
to
hBMP2 or hBMP4 and blocking their ability to bind to hb57myc3. Further more,
there was no binding of hBMP2 or hBMP4 to the beads if hb57myc3 was omitted
24
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WO 98/37195 PCT/US98/03283
from the reaction (Fig. 1A, lane 6; and Fig. 1B, lane 6), indicating that
there is no
non-specific binding of hBMP2 or hBMP4 to the beads and that the observed
binding is hb57-dependent. It should be noted that identical results have been
obtained using different tagged forms of hb57, and also using a different
buffer
system containing 20 mM Tris pH 7.6, 200 mM KCI, 0.1 '%. Nonidet P-40, 1
mg/ml bovine serum albumin (BSA). For comparison and as a positive
control, hBMP2 and hBMP4 were also tested for their ability to bind to
hNG~B2Fc (an Fc-tagged form of the hNG mutein hNGOB2}. Both hBMP2 and
hBMP4 bound to hNGOB2Fc (Fig. lA, lane 4; and Fig. 1B, lane 4), in agreement
to with results obtained previously (Zimmerman et al., 1996), and the
interaction
was blocked by the addition of untagged hNG (Fig. lA, lane 5; and Fig. 1B,
lane
5). Taken together, these results indicate that the epitope recognized by hb57
on
hBMP2 and hBMP4 is the same or overlaps with the epitope recognized by
noggin, or alternatively that binding of noggin to BMP2 and BMP4 sterically
hinders the binding of hb57.
5. Demonstration That hb57 Blocks BMP2 Biological Activity
In another example, hb57 that had expressed in E. coli and refolded, was
tested
2o for its ability to block the biological activity of hBMP2 in the C2C12
mouse
pluripotent mesenchymal precursor cell line. The C2C12 cells have been
shown to respond to BMP2 and BMP4 (Katagiri et al., 1994). One of the
hallmarks of the response is upregulation of expression of Alkaline
Phoshatase,
the activity of which can easily be measured in cells or cell lysates using a
colorimetric substrate. As shown in figure 2, C2C12 cells respond to hBMP2
with a maximal response obtained at 200 ng/ml hBMP2, and a minimal
response obtained with ~10 ng/ml hBMP2, with an apparent EC50 of ~70 ng/ml.
The ability of hb57 to block this response was tested by co-incubating
different
amounts of hb57 (3 ~g/ml, or 1 ~g/ml, or 0.3 ~tg/ml) while performing a dose
3o response with hBMP2 starting at i ~g/ml. As can be seen in figure 2,
inclusion
of 3 ~tg/ml hb57 leads to complete blocking of the hBMP2 response when used
at 0.5 ~g/ml. With hb57 at 1 ~g/ml, there is ~-50°/'> inhibition of the
hBMP2
CA 02282302 1999-08-18
WO 98/37195 PCT/US98/03283
response when hBMP2 is at ~ 0.3 ug/ml and complete inhibition when hBMP2
is at 0.12 ~tg/ml. In order to make certain that the blocking of Alkaline
Phosphatase induction in BMP2-stimulated C2C12 was not due to inhibition of
their proliferation by b57, an identically-treated plate was subjected to an
MTT
assay (Mosmann, 1983) which measures the proliferation of cells. There was no
effect on cell proliferation by the hb57 treatment, indicating that inhibition
of
the Alkaline Phosphatase expression is due to blocking of hBMP2 activity in
this assay. Thus, hb57 is a potent antagonist of BMP2 activity, and it appears
to
mediate this effect by directly binding to BMP2 and blocking its biological
1o actions. Taken together with the binding results, it is postulated that
hb57
should also block the activity of hBMP4.
Xenopus b57 (Gremlin) was also examined for its ability to antagonize the
activity of purified BMP-2 in a cytokine assay. The murine bone marrow
stromal cell line W-20-17 provides a direct, quantitative bioassay for BMP
activity by induction of alkaline phosphatase in response to BMP treatment.
(Thies, et al. Endocrinology 130: 1318-24 (1992}). Preincubation of purified
BMP-
2 with a Gremlin COS supernatant at a final concentration of approx. 83nM
Gremlin completely blocked BMP-2 activity at doses from 78pM to 5nM. At
approx. 2lnM Gremlin, BMP-2 activity was reduced, but not eliminated (see
Figure 3). Mock-transfected COS supernatant had no effect. Similar results
were obtained with BMP-4.
6. Tissue Expression of hb57
We have examined the expression of human b57 by analysis of RNA prepared
from different adult human tissues. Table 2 lists the tissues tested and the
level
of expression of hb57 detected in these tissues. The expression of hb57 in so
many different tissues indicates that it may play important biological roles.
One
piece of evidence that supports this hypothesis is that the expression of drm
(Topol et al., 1997), which is the rat homolog of hb57, is down-regulated in
26
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transformed cells. A similar observation has been made for DAN (Ozaki et al.,
1995; Ozaki et al., 1996; Enomoto et al., 1994), which is related by homology
to
b57.
_ TABLE 2 - Tissue Expression of hb57
Tissue Relative Level of Expression
heart very low
brain medium
placenta undetectable
lung undetectable
liver low
skeletal muscle low
kidney low
~5 pancreas low
spleen undetectable
thymus undetectable
prostate low
testis very low
ovary very low
small intestine high
colon (mucosa lining) high
peripheral blood leukocytes undetectable
stomach high
thyroid very low
spinal chord medium
lymph node high
trachea low
adrenal gland low
bone marrow very low
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7. Materials and Methods For Examples 2 through 6
a. Bindin assays:
Bindings were carried out in 20 mM Tris pH 7.6, 150 mM NaCI, 0.1'%. Tween 20
(TBST), 1 mg/ml bovine serum albumin (BSA}, at 25°C, with continuous
mixing. Protein G-Sepharose {G-Se) was used to capture either the anti-myc
9E10 monoclonal antibody or the Fc-tagged hNGOB2Fc. Non-specifically bound
proteins were removed from the beads by washing once with TBST, then
moving the G-Se beads to new tubes and washing three more times with TBST.
1o Bound hBMP2 and hBMP4 were visualized by western blotting with anti-
hBMP2 or anti-hBMP4 polyclonal antisera.
b. Anti-hBMP2 and anti-hBMP4 westerns:
The antibodies were derived from rabbits immunized with recombinant
hBMP2 or recombinant hBMP4. The antibody preparation was total serum
from the bled rabbits.
1. Block the filters in 5°/'> non-fat dry milk (NFDM) in 20 mM Tris pH
7.6, 150
mM NaCI, 0.1% Tween-20 (TBST) for 1 hour or more.
2. Probe with anti-BMP2 @ 1:20,000 or with the anti-hBMP4 antibody ~ 1:10,000
2o dilution in 2.5% NFDM/TBST for 1 hour.
3. Wash three times with TBST, 10 min. each time.
=I. Probe with the anti-rabbit IgG~HRPO conjugated 2° (Rockland, Inc.)
at 50
ng/ml {1:20,000 dilution) in 2.5% NFDM/TBST for 1 hour.
5. Wash three times with TBST, 10 min. each time.
6. Wash three times with TBS (without Tween-20), 5 to 10 min. each time.
7. Perform ECL (Pierce).
c. C2C12 bioassa,~pro~ (adapted from (Katagiri et al., 1994)):
1. Seed C2C12 ~ 500 cells/well in 96-well plate, in DMEM + 15"/« FBS +
Pen/Str.
+ Glutamine. Important: Cells must be monodispersed during trypsinization
and prior to plating. Clumps of cells will give erroneous results and
variability
in the response.
28
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WO 98/37195 PCT/US98/03283
2. The following day add BMPs and other factors to each well.
Important points:
a. C2C12 will respond to BMP2 and to BMP4. If the cells are incubated with
these factors for three days a maximal response can be obtained at 1 to 2
ug/ml.
A response above background is seen at 10 ng/ml. If the cells are incubated
with the BMPs for 4 days, a response to as little as 1 ng/ml can be seen, and
a
maximal response is obtained at 300 ng/ml. No change of medium is required
during the 4 day incubation period.
b. Incubation to both BMP2 and BMP4 together does not lead to an additive
response above that expected for the equivalent amount of each factor alone.
c. Dilution of BMP2 or BMP4 must be made either directly into the assay media
(as long as the concentration is kept below 10 ~tg/ml).
d. The response to BMP2 and BMP4 can be blocked by addition of an equimolar
amount of human noggin.
3. Incubate for 3 to 4 days, depending on levels of BMPs used.
4. Aspirate media, wash once with PBS, add 0.05 ml of ddH20 per well. Freeze
C~-20°C until next day or proceed to step 5.
5. Freeze-thaw three times to lyse the cells. A dry ice tray may be used for
this.
6. Add 0.05 ml 2x Alk-Phos substrate/buffer mix (Sigma N2720).
7. Follow development - usually it takes about 40 minutes. Stop development
by bringing the pH in each well to 14, by using a 50% w/v solution of NaOH.
8. Measure A405.
d. Northern Analysis:
I. Probe preparation:
1. Restrict 20 Bg of plasmid pCAE304 (pMT21.hb57) with NgoM I and Bgl II.
2. Gel-purify the 486 by fragment (about 2 ug), and elute in 100 ~1 ddH~O.
3. Label the probe using the Prime-It II Random Primer Labeling Kit
(Stratagene), according to the following protocol:
3o a. Mix 4 Itl of the purified hb57 fragment + 20 ~l HBO + 10 ~tl random
oligonucleotide primers in a tube.
29
CA 02282302 1999-08-18
WO 9.8/37195 PCT/US98103283
b. Heat the reaction tube in a boiling water bath for 5 minutes, then leave at
room temperature.
c. Add 10 ~l of 5x dCTP primer buffer + 5 ~ti of [a-32P] dCTP + 1 ~l Exo(-)
Klenow
enzyme (5 u/~1) to the tube.
d. Incubate the tube at 37-40°C for 30 minutes.
=1. Purify the labeled probe with MicroSpin Column (Pharmacia Biotech).
II. Northern Blotting of Human Multiple Tissue Northern (MTN) Blots
(CLONTECH):
1. Prehybridize the blots in 20 ml of PreHyb solution + ssDNA (10 ~l/ml) at
65°C
for more than 2 hours, using Hybridization oven.
2. Denature the labeled probe at 95°C for 5 minutes, immediately put
the tube
on ice.
3. Add the probe directly to the PreHyb solution, hybridize the blots at
65°C,
overnight.
4. Wash the blots twice with 100 ml of 2x SSC + 0.1'% SDS at 65°C, 1
hour each
time.
5. Expose the blots on film.
e. References:
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31
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Topol, L. Z., Marx, M., Laugier, D., Bogdanova, N. N., Boubnov, N. V.,
Clausen,
P. A., Calothy, G., and Blair, D. G. (1997). Identification of drm, a novel
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whose expression is suppressed in transformed cells and which can inhibit
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to Cell 86, 599-606.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be
~5 readily apparent to those of ordinary skill in the art in light of the
teachings of
this invention that certain changes and modifications may be made thereto
without departing from the spirit or scope of the appended claims.
32
