U.S. patent application number 10/323268 was filed with the patent office on 2003-07-10 for modified dorsal tissue affecting factor and compositions.
Invention is credited to Economides, Aris, Harland, Richard M., Stahl, Neil E..
Application Number | 20030129703 10/323268 |
Document ID | / |
Family ID | 25407592 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030129703 |
Kind Code |
A1 |
Economides, Aris ; et
al. |
July 10, 2003 |
Modified dorsal tissue affecting factor and compositions
Abstract
Described are modified Noggin muteins, compositions comprising
the muteins, and DNA or RNA sequences comprising coding (sense) or
antisense sequences for the muteins.
Inventors: |
Economides, Aris; (New York,
NY) ; Stahl, Neil E.; (Carmel, NY) ; Harland,
Richard M.; (Moraga, CA) |
Correspondence
Address: |
Linda O. Palladino
Regeneron Pharmaceuticals, Inc.
777 Old Saw Mill River Road
Tarrytown
NY
10591
US
|
Family ID: |
25407592 |
Appl. No.: |
10/323268 |
Filed: |
December 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10323268 |
Dec 19, 2002 |
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09500253 |
Feb 8, 2000 |
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6500640 |
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09500253 |
Feb 8, 2000 |
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08897236 |
Jul 17, 1997 |
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6075007 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/19.1; 514/7.6; 514/8.3; 530/350;
536/23.5 |
Current CPC
Class: |
C07K 14/475 20130101;
A61K 38/00 20130101; A61P 19/08 20180101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 514/12; 530/350; 536/23.5 |
International
Class: |
A61K 038/17; C07K
014/705; C12P 021/02; C12N 005/06 |
Claims
1. Human Noggin modified by a deletion of amino acid residues
138-144.
2. An isolated nucleic acid molecule encoding the modified human
Noggin of claim 1.
3. An isolated nucleic acid molecule of claim 2, which is a
recombinant DNA molecule operatively linked to an expression
control sequence.
4. A host cell transformed with the recombinant DNA molecule of
claim 3.
5. A method for producing a modified Noggin molecule comprising:
(a) growing a recombinant host cell containing the DNA molecule of
claim 3, so that the DNA molecule is expressed by the host cell to
produce the modified Noggin molecule and (b) isolating the
expressed, modified Noggin molecule.
6. The method according to claim 5, wherein said host cell is a
eukaryotic cell.
7. The method according to claim 5, wherein said host cell is a
prokaryotic cell.
8. A composition comprising human Noggin according to claim 1, and
a pharmaceutical carrier.
9. A method of treatment comprising administering to a patient an
effective amount of the composition of claim 8.
10. Human Noggin modified by a deletion of amino acid residues
133-144.
11. An isolated nucleic acid molecule encoding the modified human
Noggin of claim 10.
12. An isolated nucleic acid molecule of claim 11, which is a
recombinant DNA molecule operatively linked to an expression
control sequence.
13. A host cell transformed with the recombinant DNA molecule of
claim 12.
14. A method for producing a modified Noggin molecule comprising:
(a) growing a recombinant host cell containing the DNA molecule of
claim 12, so that the DNA molecule is expressed by the host cell to
produce the modified Noggin molecule and (b) isolating the
expressed, modified Noggin molecule.
15. The method according to claim 14, wherein said host cell is a
eukaryotic cell.
16. The method according to claim 14, wherein said host cell is a
prokaryotic cell.
17. A composition comprising human Noggin according to claim 2 and
a pharmaceutical carrier.
18. A method of treatment comprising administering to a patient an
effective amount of the composition of claim 17.
19. Biologically active noggin polypeptide modified by the deletion
of the amino acid sequence KKLRRK.
20. A nucleic acid molecule that encodes the noggin polypeptide of
claim 19.
Description
[0001] This invention generally relates to a growth factor with
dorsal growth inducing activity, and more particularly to a
modified form of the Spemann organizer signal Noggin, to
compositions comprising the modified Noggin, and to DNA or RNA
sequences comprising coding (sense) or antisense sequences for the
modified Noggin.
[0002] Throughout this application, various publications are
referenced. The disclosures of those publications, in their
entireties, are hereby incorporated by reference into the subject
application.
BACKGROUND OF THE INVENTION
[0003] Growth factors are substances, such as polypeptide hormones,
which affect the growth of defined populations of animal cells in
vivo or in vitro, but which are not nutrient substances. Proteins
involved in the growth or differentiation of tissues may promote or
inhibit growth or differentiation, and thus the general term
"growth factor" includes cytokines and trophic factors.
[0004] Growth factors, their receptors, DNA or RNA coding or
antisense sequences therefore, and fragments thereof, are useful in
a number of therapeutic, clinical, research, diagnostic, and drug
design applications. See, for example, U.S. Pat. No. 4,857,637,
issued Aug. 15, 1989 (method for immunizing an animal against its
growth hormone receptor); U.S. Pat. No. 4,933,294, issued Jun. 12,
1990 (assays and therapies involving the human EGF receptor); U.S.
Pat. No. 5,030,576, issued Jul. 9, 1991 (the role of receptors and
receptor hybrids in drug design and drug screening by the
pharmaceutical industry); U.S. Pat. No. 5,087,616, issued Feb. 11,
1992 (method for destroying tumor cells using a composition
comprising a growth factor conjugate); U.S. Pat. No. 5,098,833,
issued Mar. 24, 1992 (expression systems useful in therapeutic or
diagnostic compositions); and International Application Publication
No. W092/05254, published Apr. 2, 1992 (various aspects of
isolation, preparation, and applications for a novel neurotrophic
factor); each of which is incorporated herein by reference.
[0005] The Spemann organizer induces neural tissue from dorsal
ectoderm and dorsalizes lateral and ventral mesoderm in Xenopus.
The first molecule to have the properties expected of a Spemann
organizer signal was identified in an expression screen for
activities that induce dorsal structures in Xenopus embryos and was
called Noggin (Smith, W. C. and Harland, R. M. Cell 70: 829-840
(1992)). Organizer signals such as Noggin may be antagonized by
members of the bone morphogenetic protein (BMP) class of the
transforming growth factor beta (TGF-.beta.) gene superfamily. It
was recently reported that Noggin protein binds BMP-4 with high
affinity and can abolish BMP-4 activity by blocking binding to
cognate cell surface receptors (Zimmerman, L. B., et al., Cell 86:
599-606 (1996)).
[0006] 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.
DESCRIPTION OF THE DRAWINGS
[0007] FIGS. 1A and 1B--Nucleotide sequence (SEQ ID NO: 1) for the
human noggin gene and deduced amino acid sequence (SEQ ID NO: 2).
The KKLRRK deletion that is described in Example 9
(hNG.DELTA.138-144) as adequate for reducing the interaction with
heparin to that expected for ionic effects is encoded beginning at
nucleotide 415 (AAG . . . ).
[0008] FIG. 2A--Experimental design: competent animal cap (AC)
ectoderm was dissected from staged embryos as shown. St10.5 dorsal
and ventral AC and ventral marginal zones (VMXZ) also dissected as
shown. Explants were washed once in low Ca/Mg Ringers (LCMR)
solution and then placed in treatment medium containing factor
diluted in LCMR+0.5%BSA. Explants cultured to late stages (St20+)
were removed from treatment medium 6-16 hours after the start of
treatment and placed in LCMR. When explants reached the desired
stage they were either harvested for RNA, or they were fixed for
whole mount in situ hybridization or antibody staining.
[0009] FIG. 2B--Neural induction by noggin in the absence of
muscle. Lanes 1-3 show specific fragments protected by N-CAM,
.beta.-tubulin, and XIF-3 probes respectively in whole St24 embryo
RNA. Lanes 4-8 show protection by the mixture of these three probes
while lanes 9-13 show protection by an actin probe on tRNA(t), St24
embryo RNA (E), and RNA collected from St9 AC treated with 50 pM
activin (A), 25% of 20 fold concentrated control CHO cell medium
(C) or 25% of 20 fold concentrated noggin conditioned CHO cell
medium (N). Ubiquitously expressed cytoskeletal actin used as a
loading control shows that RNA levels in all treatments are
comparable (lanes 11-13).
[0010] FIG. 3--SDS-PAGE (12%) run under reducing conditions.
Proteins were visualized by silver staining. Lane 1 shows molecular
size standards. Lanes 2-7 show 0.0, 0.1, 0.2, 0.5, 1.0 and 2.0
.mu.g of purified human noggin.
[0011] FIG. 4A--Time course of animal caps treated with purified
noggin vs. activin; direct vs indirect neural induction. Animal
caps were dissected as shown in FIG. 2A and treated with LCMR+0.5%
BSA (U), a 20% dilution of activin conditioned COS cell medium (A),
or 1 .mu.g/ml purified human noggin (N). RNA isolated from treated
animal caps (lanes 2-13) along with St22 whole embryo RNA (lane 1)
and tRNA (lane 14) was probed for N-CAM, .beta.-tubulin, muscle and
cytoskeletal actins, collagen type II, and EF-1a.
[0012] FIG. 4B--Expression of early mesoderm markers in activin but
not noggin induced animal caps. Animal caps were dissected from St8
embryos, treated as described in (FIG. 4A), and harvested at St11.
Lanes 1 and 2 respectively show goosecoid and Xbra probe protection
by St10.5 whole embryo RNA. Lanes 3-6 show protection by a mix of
these two probes. Relative RNA levels are demonstrated by separate
EF-1.alpha. probe protection.
[0013] FIG. 4C--Plasmid directed gastrula stage noggin expression
directly induces neural tissue. One cell stage embryos were
injected with 20 pg of pCSKAlacZ or pCSKAnoggin into the animal
pole. Animal caps from injected embryos were dissected at St8-9 and
cultured until St20, when they were harvested for analysis by RNase
protection.
[0014] FIG. 5--Responsiveness of dorsal and ventral animal caps to
neural induction by noggin. St 105 ventral and dorsal animal caps
were dissected as shown in FIGS. 2A-2B. Dorsal and ventral animal
caps were treated with activin medium (DA,VA) or 1 .mu.g/ml human
noggin (DN, VN) and harvested at St26 for RNase protection analysis
using N-CAM, .beta.-tubulin, and actin as probes.
[0015] FIG. 6--Dose response of ventral marginal zones and animal
caps to human noggin protein. St 10.5 VMZs and St9 animal caps were
dissected as shown in FIG. 2A, and treated with 0, 1, 10, 50, 200,
and 1000 ug/ml of human noggin (lanes 3-8 and 10-15 respectively).
RNA from treated explants and control whole embryos aged to St26
was then analyzed by RNase protection, using the probes N-CAM,
.beta.-tubulin, actin and collagen type II. In this experiment,
muscle induction at the dose of 1 ng/ml is stronger than at ng/ml,
and there is a low level of muscle actin expression in the
uninduced VMZs. This could be due to experimental variability since
in repeated experiments we saw muscle induction only at the doses
of 50 ng/ml and above.
[0016] FIGS. 7A to 7L--In situ hybridization and antibody staining.
Tailbud embryos stained for NCAM showing side and dorsal views (7A,
7B); NCAM RNA is only detected in the neural tube, and not the
somites. For comparison, somites of a tailbud embryo stain for
muscle actin, dorsal view (7C). Neural specific 6F11 antibody
staining at St30 (7D-7F). Some cement gland pigment remained in
these embryos after bleaching as seen in (7D), however this pigment
is distinct from antibody staining. The inner mass of staining in
the noggin treated animal caps is due to the 6F11 antibody
detection. Cement gland specific XAG-1 transcripts detected at St23
(7G-7i), and anterior brain otxA trasncripts detected at St35
(7J-7L) in whole embryos at (7D,7G,7J), human noggin treated (1
.mu.g/ml) animal caps (7E,7H,7K), and untreated animal caps
(7F,7i,7L).
[0017] FIG. 8--Reverse phase HPLC profile of two refolded isoforms
of noggin. The refolded noggin solution was applied onto a Brownlee
Aquapore AX-300, 0.46.times.22 cmHPLC column at a flow rate of 1
ml/min. The column was equilibrated with solvent A containing 0.1%
TFA in water. Solvent B was 0.1% TRA in acetonitrile. The column
was developed according to the following protocol: a) 2 min
isocratically at 95% of solvent A-5% of solvent B; 60 min linear
gradient to 65% of solvent B and 35% of solvent A. Correctly
refolded noggin elutes earlier at 44%-46% solvent B.
[0018] FIG. 9--Reverse-phase HPLC chromatography characterization
of recombinant noggin refolded and purified from E. col. Conditions
as in the legend to FIG. 8.
[0019] FIG. 10--Recombinant noggin produced in E. coli and in
insect cells analyzed by 12.5% SDS-PAGE. Lanes H, L: High and low
molecular weight markers of the indicated size, respectively. Lanes
1,2: Recombinant noggin produced in E. coli and in insect cells
respectvely, treated with 2-mercaptoethanol before electrophoresis.
The slower mobility of noggin from insect cells correponds to the
size increase that would occur due to N-linked glycosylation at the
single consensus site. Lanes 2,3: Recombinant noggin produced in E.
coli and in insect cells respectvely, not treated with
2-mercaptoethanol before electrophoresis.
[0020] FIG. 11--Circular dichroism spectra of recombinant noggin
produced in E. coli (--), and in insect cells (-).
[0021] FIG. 12--Ventral marginal zone assay showing induction of
muscle actin mRNA after exposure to human noggin (0.01, 0.05,
0.2,.mu.g/ml) produced in baculovirus, a mock transfected culture
of baculovirus (0.02, 1 .mu.g/ml) or human noggin produced in E.
coli (0.1, 0.5, 2, or 10 .mu.g/ml).
[0022] FIGS. 13A and 13B--Nucleotide sequence (SEQ ID NO: 25) for
the mouse noggin gene and deduced amino acid sequence (SEQ ID NO:
26).
[0023] FIG. 14--Amino acid sequence of hNG.DELTA.133-144Fc
[0024] The putative signal peptide sequence of human noggin (hNG)
is shown in italics.
[0025] (.dagger.) marks the position of cysteines (C), except as
noted below.
[0026] (.Yen.) marks the position of N-linked glycosylation
sites.
[0027] (.DELTA.) marks the position in which the .DELTA.133-144
deletion was created.
[0028] The sequence of amino acids 133 to 144 in the wild-type hNG
is KKQRLSKKLRRK and may be referred to as the `basic region` of
hNG.
[0029] The CYS involved in inter-chain disulfide bridges in hNG is
marked in bold (. . . SECKCSC. . . ).
[0030] The position of the Ser-Gly bridge that connects the
hNG.DELTA.133-144 sequence with the constant region of human IgG1
(Fc) is shown in bold (SG). The sequence of the Fc domain is shown
underlined.
[0031] (.diamond.) marks the two cysteines (amino acids number 371
and 374) of the IgG hinge preceding the Fc that form the
inter-chain disulfide bridges that link two Fc domains of human
IgG1.
[0032] (.circle-solid.) shows the position of the STOP codon.
[0033] FIG. 15--hNG.DELTA.133-144Fc binds to human BMP4
[0034] The activity of either hNG or hNG.DELTA.133-144Fc was tested
by comparing their ability to bind to human Bone Morphogenetic
Protein 4 (hBMP4). hBMP4 (2 .mu.g/ml) was coated on ELISA plates
(CORNING) by passive binding. Unbound hBMP4 was removed by washing
four times with PBS, and the plates were blocked with 1% BSA in
PBS. A standard curve of hNG-Fc was performed to show dose-depended
binding of hNG-Fc to hBMP4 and compared to identical amounts of
hNG.DELTA.133-144Fc. After a 1 hour incubation unbound hNG-Fc or
hNG.DELTA.133-144Fc was removed by washing four times with PBS, and
0.5 .mu.g/ml anti-human IgG.circle-solid.Alkalin- e Phosphatase
conjugate (anti-Fc.circle-solid.AP) was added to each well. After a
1 hour incubation unbound anti-Fc.circle-solid.AP was removed by
washing four times with TBS+0.1% Tween and then Alkaline
Phosphatase substrate (para-nitrophenyl phosphate; Sigma) was
added. Alkaline Phosphatase converts this substrate to a product
whose production can be monitored by measuring Absorbance at 405
nm. The ability of hNG or hNG.DELTA.133-144Fc to bind to BMP4 was
visualized by comparing A405 units.
[0035] Note: There is no binding of hNG-Fc or hNG.DELTA.133-144Fc
to the plates if hBMP4 is omitted.
[0036] FIG. 16--hNG.DELTA.133-144Fc binds to heparin with reduced
affinity Approximately 1 .mu.g of hNG-Fc, hNG.mu.138-144Fc, or
hNG.DELTA.133-144Fc each were incubated with 50 .mu.l
heparin.circle-solid.agarose (Pierce) in 1 ml 1% BSA in PBS. After
1 hour of incubation at room temperature the
heparin.circle-solid.agarose beads were precipitated by
centrifugation, resuspended in PBS and moved to new tubes.
Subsequently each pellet was sequentially washed with 100 .mu.l of
0.1, 0.25, 0.5, 0.75, and 1.0 M NaCl and the supernatant derived
from each of these steps was kept for loading on a 4 to 12%
NuPAGE/MES gels (Novex) under reducing conditions. 1/5 of each
supernatant and the resuspended heparin.circle-solid.agarose pellet
was loaded onto the gels. The Fc-tagged hNGs were visualized by
western blotting using a Horse Radish Peroxidase conjugated
anti-human IgG antibody (Rockland, Inc.) followed by
chemiluminescent detection (Pierce).
[0037] FIG. 17A--hNG.DELTA.133-144Fc pharmacokinetics in mice 0.1
mg of hNG.DELTA.133-144Fc were injected into six week old Balb/c
male mice (2 mice, 0.1 mg/mouse) intra-peritoneously (ip). Prior to
injection a sample of blood was collected from each-mouse
(`pre-bleed`). Subsequently, blood was drawn from the mice at 1, 3,
6 and 24 hours post-injection. Serum was prepared from these
samples by following standard serological procedures, and the
levels of biologically active hNG.DELTA.133-144Fc available in the
sera of Bab/c mice after the ip-injection were determined by using
a functional ELISA.
[0038] hBMP4 (2 .mu.g/ml) was coated on ELISA plates (Immulon4 from
Dynatech) by passive binding. Unbound hBMP4 was removed by washing
four times with PBS, and the plates were blocked with 1% BSA in
PBS. A standard curve of hNG.DELTA.133-144Fc was performed. After a
1 hour incubation unbound hNG-Fc or hNG.DELTA.133-144Fc was removed
by washing four times with PBS, and 0.5 .mu.g/ml anti-human
IgG.circle-solid.Alkalin- e Phosphatase conjugate
(anti-Fc.circle-solid.AP) was added to the plate and processed as
described above (FIG. 16). The level of hNG.DELTA.133-144Fc in the
sera collected at each time point was determined by performing
serial dilutions of each serum sample on the hBMP4-coated ELISA
plates, and detecting Fc-immunoreactivity using the assay described
above. A405 units were then converted to concentrations of
hNG.DELTA.133-144Fc and plotted as a function of time.
[0039] Note: The amount of serum proteins present in these assays
did not interfere with the binding and detection of
hNG.DELTA.133-144Fc.
[0040] FIG. 17B--hNG.DELTA.133-144Fc pharmacokinetics in rats 1.0
mg of hNG.DELTA.133-144Fc was injected into a 250 gram adult male
rat intravenously (iv). Prior to injection, a sample of blood was
collected (`pre-bleed`). Subsequently, blood was drawn from the rat
at 10, 30, 60, 120, and 240 minutes post-injection. Serum was
prepared from these samples by following standard serological
procedures and the levels of biologically active
hNG.DELTA.133-144Fc available in the serum samples were determined
by using the functional ELISA described in FIG. 17A.
SUMMARY OF THE INVENTION
[0041] Polypeptides of the invention induce dorsal growth in
vertebrates and can be prepared in soluble, physiologically active
form for a number of therapeutic, clinical and diagnostic
applications.
[0042] In a preferred embodiment, human noggin protein as set forth
in FIGS. 1A-1B (SEQ ID NO: 2) or modified noggin protein as set
forth, for example, in FIG. 14, is prepared for use in therapeutic,
clinical and diagnostic applications.
[0043] In another aspect of the present invention an
oligonucleotide, such as cDNA, is provided having substantial
similarity to (or being the same as) SEQ ID NO: 8 (deduced amino
acid sequence, SEQ ID NO: 9), SEQ ID NO: 10 (deduced amino acid
sequence, SEQ ID NO: 11), or SEQ ID NO: 1. This oligonucleotide can
be single or double stranded, be formed of DNA or RNA bases, and
can be in the antisense direction with respect to SEQ ID NOS: 8, 10
or 1. SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 1 each code for a
functional polypeptide that we have designated "noggin," which is
capable of inducing dorsal development in vertebrates when
expressed.
[0044] Noggin or fragments or muteins thereof (which also may be
synthesized by in vitro methods) may be fused (by recombinant
expression or in vitro covalent methods) to an immunogenic
polypeptide and this, in turn, may be used to immunize an animal in
order to raise antibodies against a noggin epitope. Anti-noggin is
recoverable from the serum of immunized animals. Alternatively,
monoclonal antibodies may be prepared from cells to the immunized
animal in conventional fashion. Antibodies identified by routine
screening will bind to noggin but will not substantially
cross-react with "wnt" or other growth factors. Immobilized
anti-noggin antibodies are useful particularly in the diagnosis (in
vitro or in vivo) or purification of noggin. Substitutional,
deletional, or insertional mutants of noggin may be prepared by in
vitro or recombinant methods and screened for
immuno-crossreactivity with noggin and for noggin antagonist or
agonist activity.
[0045] Noggin or fragments or muteins thereof also may be
derivatized in vitro in order to prepare immobilized noggin and
labelled noggin, particularly for purposes of diagnosis of noggin
or its antibodies, or for affinity purification of noggin
antibodies.
[0046] The present invention further provides for expression of
biologically active noggin molecules in prokaryotic and eukaryotic
expression systems.
[0047] The present invention further provides for the production of
noggin or fragments or muteins thereof in quantities sufficient for
therapeutic and diagnostic applications. Likewise, anti-noggin
antibodies may be utilized in therapeutic and diagnostic
applications. For most purposes, it is preferable to use noggin
genes or gene products from the same species for therapeutic or
diagnostic purposes, although cross-species utility of noggin may
be useful in specific embodiments of the invention.
[0048] In additional embodiments, the noggin nucleic acids,
proteins, and peptides of the invention may be used to induce
neural tissue formation or block BMP activity in mammals.
DETAILED DESCRIPTION OF THE INVENTION
[0049] Throughout this application, when nucleic acid sequences
that encode polypeptides are set forth, it is understood that the
complementary strand of the coding sequence is thereby taught as
well.
[0050] We have discovered a structurally unique growth factor that
is readily available in substantially pure, soluble form. We have
named the inventive polypeptide "noggin." This growth factor
induces dorsal development and blocks BMP activity in
vertebrates.
[0051] An earlier described family of proteins that also induces
dorsal development are the "wnt" proteins. These, however, in
contrast to noggin remain tenaciously bound to cell surfaces. Our
initial work with noggin has been in Xenopus embryos; however,
noggin is highly conserved among vertebrates, as our work with
mouse noggin has demonstrated. The prior known FGF growth factor
family is also known to be involved in early embryonic induction,
but both the FGF proteins and their receptors are distinctly
different from noggin. Noggin modifies the actions of FGF (and also
activin), for example by potentiating growth, and is thus
particularly suggested in therapeutic compositions for use in
combination with other growth factors (as therapeutic adjuvants),
such as to modify or potentiate their effects.
[0052] We have cloned cDNA for noggin. The noggin cDNA contains a
single reading frame encoding a 26 kDa protein with a hydrophobic
amino-terminal sequence. Noggin is secreted. Noggin's cDNA encodes
the protein as a 26 kDa protein, but we have determined that noggin
is secreted in vivo, apparently as a dimeric glycoprotein with a
starting apparent molecular weight of about 33 kDa (as the
wild-type subunit). When not glycosylated, the monomeric unit has
an apparent molecular weight on SDS PAGE of about 25-30 kDa.
[0053] We have cloned the gene for human noggin (FIGS. 1A-1B; SEQ
ID NO: 1). The sequence codes for a protein which has noggin
activity (SEQ ID NO: 2). The carboxy terminal region of noggin
shows homology to a Kunitz-type protease inhibitor, indicating that
noggin protein, or fragments thereof, may exhibit activities of a
protease inhibitor.
[0054] We have been able to express biologically active noggin in
both eukaryotic and prokaryotic host cells. Two expression systems
we have successfully used to express biologically active noggin
have been mammalian cell lines (COS and mouse 293). A third
expression system is injection of synthetic mRNA into Xenopus
oocytes. In addition, we have successfully expressed biologically
active human noggin in a prokaryotic system, E. coli, and in
baculovirus.
[0055] Expression in these several different systems also
illustrates the high degree of conservation for noggin. We have
found, for example, substantial sequence similarity between frog
noggin and mouse noggin with a number of completely conserved
stretches. Thus, the following amino acid sequences represent
completely conserved portions as between frog noggin and mouse
noggin:
1 QMWLWSQTFCPVLY; (SEQ ID NO:3) RFWPRYVKVGSC; (SEQ ID NO:4)
SKRSCSVPEGMVCK; (SEQ ID NO:5) LRWRCQRR; (SEQ ID NO:6) and,
ISECKCSC. (SEQ ID NO:7)
[0056] There is about 87% overall conservation between the mouse
and frog sequences, and we have also observed a unique cysteine
distribution between the two.
[0057] Noggin nucleic acids, or oligonucleotides, encode a noggin
polypeptide or hybridize to such DNA and remain stably bound to it
under stringent conditions and are greater than about 10 bases in
length; provided, however, that such hybridizing nucleic acid is
novel and unobvious over any prior art nucleic acid including that
which encodes or is complementary to nucleic acid encoding other
growth factors.
[0058] By "stringent conditions" is meant those which (1) employ
low ionic strength and high temperature for washing, for example,
0.15 M NaCl/0.015 M sodium citrate/0.1% NaDodSo.sub.4 at 50.degree.
C., or (2) use during hybridization a denaturing agent such as
formamide, for example, 50% (vol/vol) formamide with 0.1% bovine
serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate
at 42.degree. C.
[0059] By "substantial similarity," when we are referring to a
nucleotide sequence, is meant cross hybridization of sequences
under conditions of moderate stringency using a probe greater than
100 nucleotides long at 30.degree. C. in a standard buffer (Wahl et
al., PNAS 76: 3683) and washes at 37.degree. C. in 300 mM NaCl, 30
mM sodium citrate, 0.2% SDS at pH 7. Alternatively, one is able to
isolate, by polymerase chain reaction, a fragment of DNA coding for
noggin or noggin family members when using primers of degenerate
sequence that encode those SEQ ID NOS: 3-7.
[0060] By "substantial similarity" when reference is made to
proteins is that noggin from different species, or noggin family
members within a species, will preserve the positions of cysteine
residues in at least 80% of positions throughout the protein. Like
the neurotrophin family, the sequence of the mature form of noggin
and noggin related polypeptides will be identical in at least 40%
of positions. Substantial similarity at the protein level includes
an ability of a subject protein to compete with noggin for binding
to receptors and some (but not all) monoclonal antibodies raised
against noggin epitopes.
[0061] The cloned cDNA for noggin (derived from frog) is designated
herein as SEQ ID NO: 8, partial sequence from mouse as SEQ ID NO:
10 or full sequence of mouse noggin as shown in FIGS. 13A-13B (SEQ
ID NO: 25). The human sequence is designated herein as SEQ ID NO:
1. We have used RNA transcripts from the SEQ ID NO: 8 clone to
rescue embryos and return them to substantially normal development
when the noggin RNA is injected into ventralized embryos. In high
doses-this results in excessive head development and it is for this
reason we named the protein "noggin." In Northern blot analysis,
the noggin cDNA hybridizes to two mRNAs that are expressed both
maternally and zygotically.
[0062] When using nucleotide sequences coding for part or all of
noggin in accordance with this invention, the length of the
sequence should be at least sufficient in size to be capable of
hybridizing with endogenous mRNA for the vertebrate's own noggin.
Typically, sufficient sequence size (for example, for use as
diagnostic probes) will be about 15 consecutive bases (DNA or RNA).
In some diagnostic and therapeutic applications, one may wish to
use nucleotide noggin coding sequences (analogous to all or a
portion of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 25 or SEQ ID NO:
1 or FIG. 14) in the anti-sense direction with respect to either
SEQ ID NOS: 8, 10, 25, or 1 or FIG. 14.
[0063] We suggest as a few preferred primers for amplifying noggin
from other species (e.g. human):
2 5' Primer 1 SEQ ID NO:12 C A A/G A C N T T C/T T G C/T C C N G T
N 5' Primer 2 SEQ ID NO:13 T T C/T T G G C C N C/A G N T A C/T G T
N A A A/G G T N G G 5' Primer 3 SEQ ID NO:14 C C N G A A/G G G N A
T G G T N T G 3' Primer 1 SEQ ID NO:15 C A N C/G T/A A/G C A C/T T
T A/G C A C/T T C 3' Primer 2 SEQ ID NO:16 C A N A C C A T N C C
C/T T C N G G 3' Primer 3 SEQ ID NO:17 C G/T N C G/T T/C T G G/A C
A N C G/T C C A
[0064] where N represents a mixture of all four nucleotides and
mixtures of two nucleotides are represented by alternates (e.g.
A/G).
[0065] Although noggin transcript is not localized in the oocyte
and cleavage stage embryo, zygotic transcripts are initially
restricted to the presumptive dorsal mesoderm, and reach their
highest levels at the gastrula stage in the dorsal lip of the
blastopore (Spemann's organizer). In the neurula, noggin is
transcribed in the notochord and prechordal mesoderm.
[0066] Without being bound by theory, we have formulated hypotheses
about the embryological effects of noggin based on where it is
expressed, and on the effects of RNA injection in embryos. Since
noggin is expressed in the Spemann organizer, we believe noggin to
be a mediator of the effects of the Spemann organizer, namely
neural induction and dorsalization of the mesoderm. We have shown
that noggin is able to directly induce neural tissue formation.
Since noggin is expressed in the notochord and head mesoderm, we
believe noggin to influence either the dorsal-ventral pattern or
anterior-posterior pattern of the neural plate. Since noggin is
expressed in the branchial arch neural crest, we believe it may
therefore influence whether neural crest cells deposit cartilage
and also to influence later branchial arch growth and remodelling.
Noggin is expressed in the tail fin neural crest, and since neural
crest is required for growth of the fin, noggin may act as a growth
factor for epidermis or mesenchyme.
[0067] Although much of our experimental work has involved rescue
of embryonic development, because expression in the notochord
persists in the growing tail bud and a discontinuous line of
stained cells (indicating expression of noggin initiated at new
sites) runs the length of the roof plate of the neural tube (and is
also apparent in the head mesoderm), we believe noggin is expressed
as an adult cell function also.
[0068] A number of applications for noggin are suggested from its
properties. The noggin cDNA should be useful as a diagnostic tool
(such as use of oligonucleotides as primers in a PCR test to
amplify those with sequence similarities to the oligonucleotide
primer, and to see how much noggin is present, e.g. primers such as
5' Primers 1-3 and 3' Primers 1-3).
[0069] Because noggin has a pattern of expression that suggests it
is used to regulate cartilage production in the embryonic head,
clinical uses to regulate cartilage and bone growth are suggested
for noggin in therapeutic compositions and particularly in
combination with other growth factors due to a property of noggin
to potentiate at least some growth factors. Since neural crest
cells are required for the tadpole fin to grow, noggin seems to be
a growth factor for the tissue matrix and epidermis and should
prove useful, for example, in wound healing compositions.
[0070] When one views noggin as ligand in complexes, then complexes
in accordance with the invention include antibody bound to noggin,
antibody bound to peptides derived from noggin, noggin bound to its
receptor, or peptides derived from noggin bound to its receptor.
Mutant forms of noggin, which are either more potent agonists or
antagonists, or have improved propeerties such as increased
bioavailability, are believed to be clinically useful. Such
complexes of noggin and its binding protein partners will find uses
in a number of applications.
[0071] Practice of this invention includes use of an
oligonucleotide construct comprising a sequence coding for noggin
and for a promoter sequence operatively linked to noggin in a
mammalian, bacterial or a viral expression vector. Expression and
cloning vectors contain a nucleotide sequence that enables the
vector to replicate in one or more selected host cells. Generally,
in cloning vectors this sequence is one that enables the vector to
replicate independently of the host chromosomes, and includes
origins of replication or autonomously replicating sequences. The
well-known plasmid pBR322 is suitable for most gram negative
bacteria, the 2.mu. plasmid origin for yeast and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0072] Expression and cloning vectors should contain a selection
gene, also termed a selectable marker. Typically, this is a gene
that encodes a protein necessary for the survival or growth of a
host cell transformed with the vector. The presence of this gene
ensures that any host cell which deletes the vector will not obtain
an advantage in growth or reproduction over transformed hosts.
Typical selection genes encode proteins that (a) confer resistance
to antibiotics or other toxins, e.g. ampicillin, neomycin,
methotrexate or tetracycline, (b) complement auxotrophic
deficiencies.
[0073] Examples of suitable selectable markers for mammalian cells
are dihydrofolate reductase (DHFR) or thymidine kinase. Such
markers enable the identification of cells which were competent to
take up the noggin nucleic acid. The mammalian cell transformants
are placed under selection pressure in which only the transformants
are uniquely adapted to survive by virtue of having taken up the
marker.
[0074] Selection pressure is imposed by culturing the transformants
under conditions in which the concentration of selection agent in
the medium is successively changed. Amplification is the process by
which genes in greater demand for the production of a protein
critical for growth are reiterated in tandem within the chromosomes
of successive generations of recombinant cells. Increased
quantities of noggin can therefore be synthesized from the
amplified DNA.
[0075] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium which contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell in this case is the
Chinese hamster ovary (CHO) cell line deficient in DHFR activity,
prepared and propagated as described by Urlaub and Chasin, Proc.
Nat. Acad. Sci., 77, 4216 (1980). The transformed cells then are
exposed to increased levels of Mtx. This leads to the synthesis of
multiple copies of the DHFR gene and, concomitantly, multiple
copies of other DNA comprising the expression vectors, such as the
DNA encoding noggin. Alternatively, host cells transformed by an
expression vector comprising DNA sequences encoding noggin and
aminoglycoside 3' phosphotransferase (APH) protein can be selected
by cell growth in medium containing an aminoglycosidic antibiotic
such as kanamycin or neomycin or G418. Because eukarotic cells do
not normally express an endogenous APH activity, genes encoding APH
protein, commonly referred to as neo resistant genes, may be used
as dominant selectable markers in a wide range of eukaryotic host
cells, by which cells transformed by the vector can readily be
identified.
[0076] Expression vectors, unlike cloning vectors, should contain a
promoter which is recognized by the host organism and is operably
linked to the noggin nucleic acid. Promoters are untranslated
sequences located upstream from the start codon of a structural
gene (generally within about 100 to 1000 bp) that control the
transcription and translation of nucleic acid under their control.
They typically fall into two classes, inducible and constitutive.
Inducible promoters are promoters that initiate increased levels of
transcription from DNA under their control in response to some
change in culture conditions, e.g. the presence or absence of a
nutrient or a change in temperature. At this time a large number of
promoters recognized by a variety of potential host cells are well
known. These promoters can be operably linked to noggin encoding
DNA by removing them from their gene of origin via restriction
enzyme digestion, followed by insertion 5' to the start codon for
noggin.
[0077] Nucleic acid is operably linked when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
which-participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, operably linked means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist, then synthetic oligonucleotide adapters or
linkers are used in accord with conventional practice.
[0078] Transcription of noggin-encoding DNA in mammalian host cells
is controlled by promoters obtained from the genomes of viruses
such as polyoma, cytomegalovirus, adenovirus, retroviruses,
hepatitis-B virus, and most preferably Simian Virus 40 (SV40), or
from heterologous mammalian promoters, e.g. the actin promoter. Of
course, promoters from the host cell or related species also are
useful herein.
[0079] In particular embodiments of the invention expression of
noggin in E. coli is preferably performed using vectors which
comprise the following: a lac UV5 promoter which may be controlled
by the lactose operon repressor; a strong ribosome binding site,
for example, the ribosome binding site of bacteriophage T7; a
mutation in the replication control region of the plasmid which may
increase copy number; and a mutation which limits the expression of
the antibiotic resistance protein.
[0080] In a preferred embodiment, noggin is expressed in a high
copy number kanamycin resistant pBR322-derived plasmid under the
control of a lac UV5 promoter. In an additional preferred
embodiment, noggin is expressed in baculovirus under the control of
the polyhedrin promoter of Autographa californica Multiple Nuclear
Polyhedrosis virus in insect host cells.
[0081] An object of the present invention is to provide novel
modified Noggin molecules for the treatment of diseases or
disorders including, but not limited to, Fibrodysplasia Ossificans
Progressiva (FOP), as well as for treating abnormal bone growth,
such as the pathological growth of bone following hip replacement
surgery, trauma, burns or spinal cord injury.
[0082] A further object of the present invention is to provide a
method for producing modified Noggin molecules, other than those
specifically described herein, that have improved therapeutic
properties.
[0083] These and other objects are achieved in accordance with the
invention, whereby amino acid deletions in human Noggin protein
enhance its therapeutic properties.
[0084] Thus, according to the invention, certain amino acid
deletions in the human Noggin protein result in a modified human
Noggin protein that exhibits improved bioavailability in animal
sera while retaining the ability to bind to a Bone Morphogenetic
Protein. Such a modified Noggin protein would be expected to have
enhanced therapeutic properties.
[0085] The present invention also provides for pharmaceutical
compositions comprising a modified Noggin molecule, as described
herein and a suitable pharmaceutical carrier.
[0086] The active ingredient, which may comprise the modified
Noggin, should be formulated in a suitable pharmaceutical carrier
for systemic or local administration in vivo by any appropriate
route including, but not limited to injection (eq., intravenous,
intraperitoneal, intramuscular, subcutaneous, endoneural,
perineural, intraspinal, intraventricular, intravitreal,
intrathecal etc.), by absorption through epithelial or
mucocutaneous linings (e, oral mucosa, rectal and intestinal
mucosa, etc.); or by a sustained release implant, including a
cellular or tissue implant.
[0087] 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.
[0088] The concentration of the active ingredient used in the
formulation will depend upon the effective dose required and the
mode of administration used. The 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.
[0089] Practice of this invention includes preparation and uses of
a diagnostic or therapeutic agent comprising a nucleotide sequence
of at least about 15 DNA or RNA bases analogous to all or a portion
of either FIG. 14 or SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 25, or
SEQ ID NO: 1 or of the nucleic acid sequences contained in
bacteriophages, hnog.lambda.-9 or hnog.lambda.-10. That is, noggin
preparations are useful as standards in assays for noggin and in
competitive-type receptor binding assays when labelled with
radioiodine, enzymes, fluorophores, spin labels, and the like.
Therapeutic formulations of noggin are prepared for storage by
mixing noggin having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers, in
the form of lyophilized cake or aqueous solutions. Acceptable
carriers, excipients or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and include buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid; low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin or
immunoglobulins. Other components can include glycine, glutamine,
asparagine, arginine, or lysine; monosaccharides,disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as Tween, Pluronics or PEG.
[0090] Noggin may be used according to the invention as described
supra. The concentration of the active ingredient used in the
formulation will depend upon the effective dose required and the
mode of administration used. The 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.
[0091] By referring to noggin, the present invention also
contemplates the use of fragments, derivatives, muteins, agonists
or antagonists of noggin molecules.
[0092] Noggin may be administered in any pharmaceutically
acceptable carrier. The administration route may be any mode of
administration known in the art, including but not limited to
intravenously, intrathecally, subcutaneously, by injection into
involved tissue, intraarterially, intranasally, orally, or via an
implanted device. The present invention provides for pharmaceutical
compositions comprising noggin in a pharmaceutically acceptable
carrier.
[0093] Administration may result in the distribution of noggin
throughout the body or in a localized area. For example, in some
conditions which involve distant regions of the nervous system,
intravenous or intrathecal administration of noggin may be
desirable. Alternatively, and not by way of limitation, when
localized regions of the nervous system are involved, local
administration may be desirable. In such situations, an implant
containing noggin may be placed in or near the lesioned area.
Suitable implants include, but are not limited to, gelfoam, wax, or
microparticle-based implants.
[0094] Inventive complexes comprise a ligand characterized by one
or more of the SEQ ID NOS: 3-7. The ligand can be bound to a
protein, such as antibody. Such antibodies can be polyclonal or
monoclonal. Polyclonal antibodies to noggin generally are raised in
animals by multiple subcutaneous (sc) or intraperitoneal (ip)
injections of noggin and an adjuvant. It may be useful to conjugate
noggin or a fragment containing the target amino acid sequence to a
protein which is immunogenic in the species to be immunized, e.g.,
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing
agent, for example, maleimidobenzoyl sulfosuccinimide ester
(coniugation through cysteine residues), N-hydroxy-succinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR.
[0095] Animals can be immunized against the immunogenic conjugates
or derivatives by combining 1 mg or 1 .mu.g of conjugate (for
rabbits or mice, respectively) with 3 volumes of Freund's complete
adjuvant and injecting the solution intradermally in multiple
sites. One month later the animals are boosted with 1/5 to
{fraction (1/10)} the original amount of conjugate in Fruend's
complete adjuvant by subcutaneous injection at multiple sites.
Seven to 14 days later animals are bled and the serum is assayed
for anti-noggin titer. Animals are boosted until the titer
plateaus. Preferably, the animal is boosted with the conjugate of
the same noggin polypeptide, but conjugated to a different protein
and/or through a different cross-linking agent. Conjugates also can
be made in recombinant cell culture as protein fusions. Also,
aggregating agents such as alum are used to enhance the immune
response.
[0096] Monoclonal antibodies are prepared by recovering spleen
cells from immunized animals and immortalizing the cells in
conventional fashion, e.g. by fusion with myeloma cells or by EB
virus transformation and screening for clones expressing the
desired antibody.
[0097] In a preferred embodiment, a rat monoclonal antibody such as
RP57-16, prepared after immunization of a rat with recombinant
human noggin, reacts specifically with both Xenopus and human
noggin, but not with the neurotrophins BDNF, NT-3 and NT-4.
[0098] Noggin antibodies are useful in diagnostic assays for noggin
or its antibodies. In one embodiment of a receptor binding assay,
an antibody composition which binds to all of a selected plurality
of members of the noggin family is immobilized on an insoluble
matrix, the test sample is contacted with the immobilized antibody
composition in order to adsorb all noggin family members, and then
the immobilized family members are contacted with a plurality of
antibodies specific for each member, each of the antibodies being
individually identifiable as specific for a predetermined family
member, as by unique labels such as discrete fluorophores or the
like. By determining the presence and/or amount of each unique
label, the relative proportion and amount of each family member can
be determined.
[0099] Noggin antibodies also are useful for the affinity
purification of noggin from recombinant cell culture or natural
sources. Noggin antibodies that do not detectably cross-react with
other growth factors can be used to purify noggin free from these
other family members.
[0100] Aspects of the invention will now be illustrated by the
following examples.
EXAMPLES
[0101] Production of Xenopus Embryos
[0102] Xenopus embryos were prepared by the protocol described by
Condie and Harland (Development, 101, 93-105, 1987). Embryos were
staged according to the table of Nieuwkoop and Faber ("Normal Table
of Xenopus laevis"(Daubin), Amsterdam: North Holland, 1967).
Ventralized embryos were produced by irradiation with a Statalinker
(Stratagene), and dorsalized embryos were produced by treatment
with LiCl as described by us in our paper on certain "wnt" proteins
(designated "Xwnt-8"), Smith and Harland, Cell, Vol. 67, pp.
753-765 (1991) (incorporated by reference and occasionally referred
to hereinafter as "S&H, supra").
Example 1
[0103] Isolation and Sequencing of Noggin cDNA
[0104] The construction of the size-selected plasmid cDNA library
from stage 11 LiCl-treated embryos was as follows. Sixty micrograms
of poly(A) RNA from stage 11 LiCl-treated embryos was size
fractionated on a 10% to 30% sucrose gradient in the presence of
methylmercuric hydroxide. First strand cDNA was synthesized from 2
.mu.g of the size-fractionated poly(A) RNAs primed with oligo(dT)
oligonucleotide containing the recognition site for NotI. After
synthesis of the second strand, cDNAs were treated with EcoRI
methylase, ligated with EcoRI linkers, digested with EcoRI and
NotI, and finally ligated to 125 ng of modified pGEM-5Zf(-)
(Promega). The pGEM-5Zf(-) used here was modified by the addition
of an oligonucleotide into the NsiI site to create an EcoRi site.
The vector was not treated with alkaline phosphatase, but the
excised polylinker sequence was removed on a sepharose 4BCL column.
The ligated products were used to transform XL-I Blue cells
(Stratagene), and plated to give 100,000 colonies per 10 cm plate.
Plasmid DNAs were isolated from plate cultures by the
alkaline-lysis/polyethylene glycol precipitation protocol.
[0105] Dorsalizing activity in the library was assayed by injecting
RNA transcripts made from pooled plasmid DNA. Single clones were
isolated by a process of sib selection. In this procedure the
plasmid library was replated on 12 plates with 10-fold fewer
colonies per plate. RNA was synthesized from pooled plasmid DNAs
isolated from each plate and tested for dorsalizing activity by
injection into UV-ventralized embryos. Those pools with dorsalizing
activity were replated and screened as described above. This
process was repeated until single clones were isolated.
[0106] In vitro RNA synthesis, injection assay for dorsal axis
rescue and sib-selections were also done, as described by us in
S&H, supra.
[0107] The nucleotide sequence of both strands of the isolated
noggin cDNA clone was determined by the dideoxy termination method
using modified T7 DNA polymerase (US Biochem). Deletions were
prepared in sequencing templates by both restriction enzyme and
exonuclease III digestion (Henikoff, Meth. Enzymol, 155, 156-165,
1987).
[0108] In vitro Translation
[0109] One-half fig of in vitro synthesized noggin, Xwnt-8, and
goosecoid mRNAs were translated in a nuclease treated rabbit
reticulocyte lysate (Promega) with added .sup.35S-methionine
according to the manufacturer's instructions. The translation
products were visualized by SDS-polyacrylamide gel electrophoresis
(12% gels) followed by fluorography. Noggin protein had the
molecular weight predicted by the open reading frame.
[0110] RNA Isolation and Analysis
[0111] Total RNA was isolated from embryos and oocytes by a small
scale protocol as described by Condie and Harland, supra. Dorsal
lips were dissected from 30 unfixed stage 10.5 embryos and pooled
for total RNA preparation. Samples containing either the total RNA
equivalent of 2.5 embryos or approximately 2 .mu.g of poly A+ RNA
were analyzed by northern blotting. Random primed DNA probes were
prepared from a 1,323 bp fragment of noggin cDNA from the EcoRI
site at nucleotide -83 to an EcoRV site that lies in the vector
immediately 3' to the end of the cDNA.
[0112] RNAse protection assays were done using a protocol as
detailed by Melton et al. (Nuc. Acids Res., 12, 7035-7056, 1984)
with minor modifications (C. Kintner, Salk Institute, La Jolla,
Calif.). A noggin cDNA exonuclease III deletion clone, illustrated
by SEQ ID NO: 8 but having a deletion from the 3' end to nucleotide
383, was used as a template for synthesizing RNA probes. The
template DNA was linearized by EcoRI restriction enzyme digestion
and a 463 base antisense RNA incorporating .sup.32p was synthesized
with T7 RNA polymerase. A 387 base antisense EFI.alpha. RNA probe
was used as a control for amount of RNA per sample. Probes were gel
purified prior to use.
[0113] In situ Hybridization
[0114] After fixation and storage, the embryos were checked to
ensure the blastocoel and archenteron were punctured. Care was
taken to puncture the residual blastocoel of neurulae and tadpoles
as well as the archenteron. Embryos were rewashed at room
temperature in 100% ethanol for two hours to remove residual lipid.
After hybridization, staining was allowed to develop overnight and
the embryos were then fixed in Bouin's. Newly stained embryos have
a high background of pink stain but most of this washes out,
leaving the specific stain. Following overnight fixation, the
embryos were washed well with 70% ethanol, 70% ethanol buffered
with PBS and methanol. Embryos were cleared in Murray's mix and
photographed with Kodak Ektar 25 film, using a Zeiss axioplan
microscope (2.5 or 5.times.objective with 3.times.12B telescope to
assist with focusing).
[0115] Lineage Tracing
[0116] Lineage tracing with mRNA that encodes nuclear localized
B-galactosidase was as we described in S&H, supra. Ventralized
embryos were coinjected at the 32 cell stage with 0.5 ng
B-galactosidase and 25 pg noggin.DELTA.5' mRNAs. Embryos were fixed
and stained with X-gal at approximately stage 22.
[0117] Results
[0118] Noggin cDNA Encodes a Novel Polypeptide
[0119] The 1833 nucleotide sequence of the selected clone is shown
by SEQ ID NO: 8 and sometimes also referred to as "clone A3." The
sequence contains a single long open reading frame encoding a 222
amino acid polypeptide with a predicted molecular weight of 26 kDa.
At the amino terminus, the hydrophobic stretch of amino acids
suggests that the polypeptide enters the secretory pathway. There
is a single potential site for N-linked glycosylation at amino acid
61. Extensive untranslated regions are located both 5' and 3' to
the reading frame (593 and 573 bp, respectively). The 3'
untranslated region is particularly rich in repeated dA and dT
nucleotides, and contains, in addition to a polyadenylation signal
sequence located 24 bp upstream from the start of the poly A tail,
a second potential polyadenylation sequence 147 bp further
upstream.
[0120] Sense RNA synthesized from clone A3 with SP6 RNA polymerase
was translated in a rabbit reticulocyte lysate system. The
3S-labeled products were fractionated on a 12% SDS-polyacrylamide
gel and visualized by fluorography. The major protein product had
the expected molecular weight of approximately 26 kDa.
[0121] Comparison of the amino acid sequence of the predicted
polypeptide to the National Center for Biotechnology Information
BLAST network (non-redundant data base) did not identify any
similar sequence. Thus, this clone encodes the new type of protein
we have named "noggin" which is secreted, and which has dorsal
inducing activity in Xenopus.
[0122] Noggin mRNA can Rescue a Complete Dorsal-Ventral Axis
[0123] Injection of noggin RNA into a single blastomere of a four
cell stage UV-ventralized embryo can restore the complete spectrum
of dorsal structures. The degree of axis rescue was dependent upon
the amount of RNA injected, with embryos receiving low doses having
only posterior dorsal structures, while embryos receiving higher
doses had excess dorsal-anterior tissue. RNA transcripts from two
noggin plasmids were tested. The first contained the full cDNA. The
second (pNoggin.DELTA.5') had a deletion removing the first 513
nucleotides of the 5' untranslated region up to the EcoRI site. The
resulting embryos from injection of RNA transcripts of these two
plasmids, as well as Xwnt-8 RNA for comparison, were scored
according to the dorsoanterior index (DAI) scale of Rao and Elinson
(Dev. Biol., 127, 64-77, 1988). In this scale, a completely
ventralized embryo is scored as zero, a normal embryo is scored as
5, and the most severely dorsoanteriorized embryos, those having
radial dorsoanterior structures, were scored as 10. RNA synthesized
from pNoggin.DELTA.5' (noggin.DELTA.5' mRNA) repeatedly gave a
higher DAI than the equivalent amount of mRNA synthesized from the
complete cDNA. The dose-dependency of axis rescue by
noggin.DELTA.5' mRNA was very similar to that of Xwnt-8 mRNA.
[0124] UV treated embryos were also injected with a higher doses
(1,000 pg) of the noggin mRNAs. Injection of this dose of noggin
mRNA into one blastomere at the four cell stage resulted in embryos
with very severe hyperdorsalization (DAI>7). However, most of
these embryos died at the late gastrula/early neurula stage.
Apparently excessively strong gastrulation movements resulted in
the thinning and rupture of the blastocoel roof. We have also
observed this effect with high doses of injected Xwnt-8 mRNA.
[0125] The rescue of dorsal development by both noggin.DELTA.5' and
Xwnt-8 mRNAs followed a consistent pattern in which increasing
amounts of the mRNAs lead to progressively more anterior structures
being rescued. For example, embryos that received 1 pg of the RNAs
had primarily the posterior and trunk dorsal structures rescued,
and for the most part lacked head structures. Higher doses (10 or
100 pg) of both of the RNAs resulted in embryos with more anterior
development, and many had either nearly normal or hyperdorsalized
phenotypes.
[0126] Noggin Injected Blastomeres Act as a Nieuwkoop Center
[0127] The effect of varying the site of noggin mRNA injection was
investigated. Thirty-two cell stage UV-treated embryos were
injected with either 0.5 ng of B-galactosidase mRNA alone or 0.5 ng
B-galactosidase mixed with 25 pg noggin.DELTA.5' mRNA. Injection of
noggin mRNA into blastomeres of the vegetal tier gave the most
strongly dorsoanteriorized embryos. In both of the vegetal injected
embryos the nuclear X-Gal staining was found almost exclusively in
the endoderm (the mRNA encodes a B-galactosidase that translocates
to the nucleus, allowing distinction from the diffuse background
stain). One of the embryos shown was strongly hyperdorsalized (DAI
approximately 7) as a result of the noggin mRNA injection, and had
a severely truncated tail and enlarged head structures. Embryos
were also rescued by noggin mRNA injections into the marginal
zone.
[0128] In these embryos B-galactosidase staining was observed
primarily in the axial and head mesoderm. Injection of noggin mRNA
into the animal pole had very little effect on axis formation.
Likewise, B-galactosidase mRNA alone was without effect.
[0129] Noggin mRNA is Expressed Both Maternally and Zygotically
[0130] In northern blot analysis of RNA from Xenopus embryos two
noggin mRNA species of approximate sizes 1.8 and 1.4 kb were
observed. A relatively low level of noggin mRNA was detected in
oocytes. By stage 11 the level of noggin mRNA was significantly
higher, reflecting zygotic transcription (as opposed to the
maternally deposited transcripts seen in oocytes). Noggin mRNA
remained at the elevated level up to the latest stage examined
(stage 45).
[0131] We expect that the primary dorsalizing RNA in our library to
be elevated in LiCl-treated embryos relative to normal or
UV-treated embryos. Lithium ion treatment resulted in a large
increase in the amount of noggin mRNA expressed, relative to
untreated embryos. UV treatment had the opposite effect. Noggin
mRNA expression was essentially undetectable in total RNA samples
from these embryos. Thus, the abundance of noggin mRNA in
manipulated embryos parallels the rescuing activity.
[0132] We analyzed the distribution of noggin in oocytes and
cleavage stage embryos. Since the amount of maternally deposited
noggin RNA is too low for in situ hybridization to detect above
background, we used an RNAse protection assay. Oocytes were
dissected into animal and vegetal halves. No enrichment of noggin
mRNA was seen in either hemisphere relative to total oocyte RNA.
Four-cell stage embryos were dissected into dorsal and ventral
halves, as well as animal and vegetal halves. Noggin transcripts
were found to be distributed evenly between dorsal and ventral
hemispheres as well as animal and vegetal hemispheres. The same
result was obtained with embryos that were tilted 90.degree.
immediately following fertilization and then marked with a vital
dye on their uppermost side to indicate the future dorsal side.
Older (32 cell stage) blastula embryos were also dissected into
dorsal-ventral and animal-vegetal halves. No enrichment of noggin
mRNA in any of the hemispheres was seen relative to the total
embryo. In addition, treatment did not alter the abundance of
maternally deposited noggin RNA, indicating no preferential
degradation in ventral tissues. Samples with known amounts of in
vitro synthesized noggin mRNA were included in the RNAase
protection assay. From these and other data we estimate that there
is approximately 0.1 pg of noggin mRNA per blastula stage embryo
and 1 pg per gastrula stage embryo.
[0133] The localization of noggin transcripts was investigated in
early gastrula stage embryos. Dorsal lips were dissected from stage
10.5 embryos. A northern blot of equal amounts of total RNA from
intact embryos, dissected dorsal lips, and from the remaining
embryo after dissection of the dorsal lip was hybridized with a
noggin probe and then re-hybridized with an EFIa probe, as a
control for amount of RNA loaded per sample. The autoradiograph of
the blot showed that noggin mRNA at this stage is enriched in the
dorsal lip.
[0134] In situ Hybridization: Zygotic Expression of Noggin in the
Spemann Organizer
[0135] The localization of noggin transcripts in developing embryos
was examined in greater detail using whole mount in situ
hybridization. Whole fixed embryos were hybridized with digoxigenin
containing RNA probes.
[0136] Hybridized RNA probe was then visualized with an alkaline
phosphatase-conjugated anti-digoxigenin antibody. The specificity
of hybridization seen with antisense noggin probes was tested both
by hybridizing embryos with sense noggin probes, and by using two
non-overlapping antisense probes. Due both to the low level of
expression, and to background staining, noggin mRNA could not be
detected unequivocally before the late blastula stage. The
increased level of noggin mRNA that was detected by northern blot
following activation of zygotic transcription was apparent in in
situ hybridization at stage 9 as a patch of staining cells on the
dorsal side of the embryo. Viewed from the vegetal pole, this patch
of cells was restricted to a sector of about 600. A side view of
the same embryo shows that the staining cells were located within
the marginal zone (i.e., between the animal and vegetal poles and
within the presumptive dorsal mesoderm forming region). Transcripts
are largely restricted to the nucleus at this stage.
[0137] A side view of an early gastrula stage embryo 30
(approximate stage 10.5) shows specific hybridization primarily in
the involuting mesoderm at the dorsal lip. A vegetal view of the
same embryo (blastopore lip arrowed) shows that noggin mRNA is most
abundant on the dorsal side, but expression extends at the lower
level to the ventral side of the embryo. This method of in situ
hybridization does not detect transcripts in the most yolky
endodermal region of embryos, therefore we cannot rule out
expression in more vegetal regions than those seen in the Figure.
Treatments which are known to affect the size of the dorsal lip
(LiCl treatment, UV irradiation) had a profound effect on the
pattern of noggin in situ hybridization. In LiCl treated embryos
the staining is intense throughout the marginal zone. UV treatment
reduced the hybridization signal to low levels. This result is
consistent with amounts of noggin mRNA seen by northern blot
analysis. The UV treated embryo also is a negative control for
specificity of hybridization.
[0138] As gastrulation proceeds, noggin mRNA staining follows the
involuting dorsal mesoderm, and is highest in the presumptive
notochord. By the late neurula stage (approximately 18) noggin mRNA
expressing cells are clearest in the most dorsal mesoderm,
primarily in the notochord but also extending more anteriorly into
the pre-chordal mesoderm. The anterior tip of the notochord is
arrowed. During tailbud stages expression of noggin in the dorsal
mesoderm declines, through expression in the notochord persists in
the growing tailbud. Expression of noggin initiates at several new
sites, which become progressively clearer as the tadpole matures. A
discontinuous line of stained cells runs the length of the roof
plate of the neural tube. Staining is also apparent in the head
mesoderm, primarily in the mandibular and gill arches. We suspect
that this expression corresponds to skeletogenic neural crest
cells. Furthermore, subsets of these cells express homeobox genes
that mark different anterior-posterior levels of the head neural
crest, for example En-2 in the mandibular arch is seen by antibody
staining. Cells with stellate morphology stained from noggin mRNA
in the tail fin. These stellate cells are also likely to be derived
from the neural crest. None of these patterns were seen with the
sense probe, or with a number of other probes.
Example 2
Noggin cDNA Transfected into COS Cells Produces Active Conditioned
Medium
[0139] For COS cells the noggin cDNA was inserted into a COS cell
expression vector. COS cells were transfected, and medium harvested
after allowing expression of the introduced noggin genes. This
medium has been tested in an animal cap assay for mesoderm inducing
or dorsalizing activity. We have tested two transfection protocols,
a standard one, where cells recover and then are transferred to
serum-free medium, and an alternate where cells are transferred to
a defined medium lacking serum but containing transferrin, insulin,
and BSA. Cells remain healthy in the supplemented medium and a
cotransfected .beta.-galactosidase gene gives 100 fold more
activity than in the unsupplemented medium. The results of treating
cells with these media is shown below in Table 1. Animal caps were
taken from ventralized animals, treated and at the end of
neurulation they were scored for elongation, usually a sign that
notochord or neural tissues have been induced. Elongation is
indicated in Table 1 by a "+" and even greater elongation a "++."
In addition, they are scored for a molecular marker by Northern
blotting.
[0140] As shown by the data of Table 1, the noggin cDNA has a large
effect on the COS cell conditioned medium. However, noggin is
probably interacting with something else in the medium, since
COS-cell conditioned medium alone has some activity. It is possible
that noggin is causing the cells to secrete something that they
normally would not, but the experiments do indicate that noggin is
secreted and is responsible for some of the activity.
3TABLE 1 Cos Cell Conditioned Medium: Effects on Animal Caps
Elongation N-CAM expression Transferred to serum free medium +
transferrin, BSA, and insulin 1. Vector only +/- + 2. Noggin cDNA
++ ++ Transferred to serum free medium without supplements 1.
Vector only -- -- 2. Noggin cDNA -- --
[0141] Noggin mRNA Injected into Oocytes Produces Active Secreted
Noggin Protein
[0142] A second approach to studying whether protein can be
secreted in active form is to inject oocytes with mRNA and take
material secreted by the oocyte. A particular advantage of this
method is that the injected mRNA is efficiently translated, and
most of the translation of the oocyte can be taken up by the
injected mRNA. A new protein, whose synthesis is directed by
injected noggin mRNA is secreted into the medium. Noggin clearly
synergizes with activin to produce elongated explants that express
elevated levels of muscle actin.
[0143] Biochemical Properties of Noggin
[0144] Injected oocytes are injected with mRNA, and labelled with
.sup.35S methionine. Most of the radioactive protein secreted into
the medium is from the injected mRNA. The noggin protein, which is
almost isotopically pure, can then be analyzed. From this analysis
we have determined that noggin is a dimeric glycoprotein. When run
under reducing conditions, and treated with N-glycanase to remove
sugar residues, noggin migrates only slightly slower than its
predicted molecular weight of 26 kDa. The removal of sugar side
chains results in a loss of about 4 kDa from a starting apparent
molecular weight of 33 kDa. When run under non-reducing conditions
it migrates at double this value.
[0145] We do not yet know if the dimer of the protein is the active
species, or if there is a proteolytically processed form which is
active. In a control experiment with activin mRNA, oocytes produce
activin activity, but the bulk of the radiolabelled protein
migrates as the precursor form. Only a small amount of processed
protein (15 kDa) was detected. It is possible that noggin injected
oocytes secrete predominantly unprocessed protein and a trace of
extremely active processed protein that we have not detected.
Despite the caveats, the main point from analysis of injected
oocytes and transfected COS cells is that active noggin can be
obtained as a freely soluble secreted polypeptide. This sets it
apart from the other group of genes with dorsalizing activity, the
wnts. Wnt proteins have not been available in soluble form and this
has greatly hampered the analysis of their biological activities,
and of the receptor that binds to them.
Example 3
[0146] Cloning of the Mouse Noggin Homolog
[0147] It is currently impossible to eliminate zygotic noggin
transcription from developing Xenopus embryos. In contrast, it
should be possible to generate homozygous null mutations in the
mouse. We have cloned the mouse noggin cDNA (SEQ ID NO: 10). This
is useful to generate mutant mice. In addition to generating the
probes and tools to make mutant mice, a comparison of the noggin
sequences should be a useful predictor of conserved domains and
functions. The C-terminal 80 amino acids are 87% identical between
SEQ ID NOS: 8 and 10.
[0148] Mouse noggin was isolated from an embryonic cDNA library by
probing with a radiolabelled frog noggin cDNA under conditions of
moderate stringency (as defined earlier). Subsequently a genomic
clone was isolated by probing a genomic library with the mouse
noggin cDNA 15 under conditions of high stringency (as defined, but
hybridized at 42.degree. C. and washed at 50.degree. C. in 15 mM
NaCl, 1.5 mM sodium citrate). The full nucleotide sequence of mouse
noggin cDNA (SEQ ID NO: 25) as well as the deduced amino acid
sequence (SEQ ID NO: 26) are shown in FIGS. 13A-13B. There are only
two amino acid differences between mouse noggin and human
noggin.
Example 4
[0149] Cloning of the Human Noggin Homolog
[0150] Materials and Methods
[0151] Probe Preparation
[0152] Two oligonucleotides were synthesized based on the mouse
noggin sequence (supra). The sequence of the oligonucleotides is
noggin 5': 5'-CAG ATG TGG CTG TGG TCA-3' (SEQ ID NO: 18)
corresponding to amino acids QMWLWS (SEQ ID NO: 19) and noggin 3':
5'-GCAGGAACACTTACACTC-3' (SEQ ID NO: 20) corresponding to amino
acids ECKCSC (SEQ ID NO: 21) of the mouse noggin protein.
[0153] The oligonucleotides were used for PCR amplification of a
segment of DNA of 260 nucleotides using as a template a mouse cDNA
clone prepared as set forth in Example 3. The amplified fragment
had a nucleotide sequence that corresponds to nucleotides 2 through
262 of the mouse sequence as set forth in SEQ ID NO: 10. After
amplification, the PCR reaction was electrophosed in agarose gels,
the DNA band of 260 nts purified by Magic PCR (Promega), and used
as template for the probe labeling reaction. The probe was labeled
using a standard PCR reaction (Perkin-Elmer) on 20 ng of DNA
template and 0.2 m Curie of alpha 32P-dCTP (Du Pont 3000 Ci/mmol)
instead of dCTP. Unincorporated label was separated from the probes
on a G50 NICK column (Pharmacia). The excluded volume of the
reaction contained a total of 1.8.times.108 cpm.
[0154] In addition, one degenerated oligonucleotide, named noggin
D, corresponding to conserved mouse and Xenopus noggin sequences,
was synthesized as follows: Noggin D: 5'- GARGGIATGGTITGYAARCC-3'
(SEQ ID NO: 22). Noggin D (SEQ ID NO: 22) was labeled by kinase
reaction using T4 polynucleotide kinase and gamma 32P ATP. The
labeled oligonucleotide was purified by NAPS (Pharmacia) column and
used for library hybridization.
[0155] Library Screening
[0156] A human placental genomic library (Clontech Cat#HL1067J,
average insert size 15 kb) in vector EMBL-3 was plated according to
manufacturer specifications in NM 538 E. coli. Approximately 3
million plaques were transferred to nitrocellulose filters (BA-85
Schleicher and Schuell) in three replicas (named A, B and C) and
screened according to Maniatis, et al.[Sambrook, et a., Molecular
cloning a laboratory manual, CSH Lab Press, New York (1989)]. The
replica filters A and C were hybridized in a buffer containing 0.5
M sodium phosphate, pH 7.2, 7% sodium dodecyl sulphate, 1%
crystalline BSA, 1 mM EDTA, 40 m g/ml denaturated salmon sperm DNA
and about 1.times.106 cpm/ml of the PCR probe (supra). After
hybridization for 12 h at 65.degree. C., the filters were washed
twice at room temperature in 2.times.SSC (30 mM sodium citrate, 0.3
M NaCl), 0.1% SDS and then at 65.degree. C. in 2.times.SSC, 0.1%
SDS for 20 min and exposed to Kodak X-OMAT AR film. The filter
replica B were hybridized with the labeled oligonucleotide noggin D
in 6.times.SSC , 0.1% SDS at 51.degree. C. for 12 h followed by
wash at 2.times.SCC, 0.1% SDS at room temperature, and in
6.times.SSC, 0.1% SDS at 50.degree. C. and exposed to Kodak X-OMAT
AR film. Positive plaques from all replicas were isolated and
purified by re-screening as above. Purified positive plaques were
suspended in 500 .mu.l SM (100 mM NaCl, 10 mM MgSO4.times.7H2O, 50
mM Tris HCl pH 7.5, 0.01% gelatin). 160 .mu.l of phage suspension
was mixed with 0.5 ml saturated NM538 culture, incubated for 20 min
at 37.degree. C. and then inoculated into 250 ml LB containing 10
mM Mg SO4, 0.2% maltose. The cultures were incubated until cell
lysis (7-8 hr) at 37.degree. C. The phage lysates were used for
phage DNA purification by the Qiagen procedure according to the
manufacturers recommendations (Qiagen).
[0157] Sequencing
[0158] Sequencing was performed by using the Applied Biosystems
Model 373A automatic sequencer and Applied Biosystems Taq
DyeDeoxy.TM. Terminator Cycle Sequencing Kit.
[0159] Results
[0160] Filters hybridized to the PCR mouse noggin probes (SEQ ID
NOS: 18 and 20) showed two strong signals corresponding to phage
plaques named hnog.lambda.-9 and hnog-10. These plaques also
hybridized to degenerate oligonucleotide probe nogginD (SEQ ID NO:
22) revealed that these clones correspond to the human noggin gene.
In addition, two other plaques named hnog.lambda.-5 and
hnog.lambda.-7 produced slightly weaker signals when hybridized to
the PCR probes. These clones correspond to either human noggin or
related gene(s). All of the human DNA inserts can be excised from
the vectors using known restriction sites as described in the
literature regarding each particular library.
[0161] A 1.6 kb Sacl fragment from clone hnog.lambda.-9 containing
the human noggin gene was subcloned and the nucleotide sequence
determined as set forth in FIGS. 1A-1B. The amino acid sequence for
human noggin, as deduced from the nucleotide sequence, is also set
forth in FIGS. 1A-1B. The gene or cDNA may be expressed in various
eukaryotic or prokaryotic expression systems to produce
biologically active human noggin protein. It is expected that the
human protein will exhibit neurotrophic activity similar to that
exhibited by Xenopus noggin protein.
Example 5
[0162] Tissue Localization of Message for Human Noggin
[0163] Materials and Methods
[0164] Probe Preparation
[0165] Probes were prepared as set forth in Example 4. The oligos
used are as follows:
4 SEQ ID NO:23 5' GAC.TCG.AGT.CGA.CAT.CGC.AGA.TGT.GGC.TGT.G- GT.CAC
SEQ ID NO:24 5' CCA.AGC.TTC.TAG.AAT.TCG.CA- G.GAA.CAC.TTA.CAC.
TCG.G
[0166] (The underlined sequence represent mouse noggin sequence;
the rest of the sequence are tails containing restriction sites for
cloning.)
[0167] A DNA fragment of approximately 300 bp was obtained by PCR
amplification of a mouse cDNA clone prepared as described in
Example 3.
[0168] RNA Preparation and Northern Blots
[0169] Selected tissues were dissected from Sprague-Dawley rats and
immediately frozen in liquid nitrogen. RNAs were isolated by
homogenization of tissues in 3 M LiCl, 6 M urea, as described in
Bothwell, et al. 1990 (Methods of Cloning and Analysis of
Eukaryotic Genes, Boston, M S, Jones and Bartlett). RNAs (10 .mu.g)
were fractionated by electrophoresis through quadruplicate 1%
agarose-formaldehyde gels (Bothwell, et al., 1990, Methods of
Cloning and Analysis of Eukaryotic Genes, Boston, M S, Jones and
Bartlett) followed by capillary transfer to nylon membranes
(MagnaGraph, Micron Separations Inc.) with 10.times.SSC (pH7). RNAs
were UV-cross-linked to the membranes by exposure to ultraviolet
light (Stratalinker, Stratagen, Inc.) and hybridized at 68.degree.
C. with radiolabled probes in the presence of 0.5 M NaPO.sub.4 (pH
7), 1% bovine serum albumin (fraction V, Sigma, Inc.) 7% SDS, 1 mM
EDTA [Mahoudi, et al., Biotechniques 7:331-333 (1989)], 100
.mu.g/ml sonicated, denatured salmon sperm DNA. Filters were washed
at 68.degree. C. with 3.times.SSC, 0.1% SDS and subjected to
autoradiography for 1 day to 2 weeks with one or two intensifying
screens (Cronex, DuPont) and X-ray film (AR-5, Kodak) at 70.degree.
C. Ethidium bromide staining of the gels demonstrated that
equivalent levels of total RNA were being assayed for the different
samples [as in Maisonpierre, et al., Science 247:1446-1451
(1990)].
[0170] RNA was prepared from the following human cell lines:
5 Neuroblastoma Neuroepithelioma CHP-134 SK-N-MC LA-N-1 CHP-100
LA-N-5 IARC-EWI IMR-32 SK-N-LO SHSY5Y SK-ES SKNSH DADY SHEP Small
Cell Hematopoetic Lung Carcinoma Cervical Carcinoma k562 Calu 3
HeLa U937 SKLu M1 NCI-H69 TF1 SKMES BAF B9 Sympathodrenal Precursor
Hepatoblastoma Medulloblastoma MAH HEPG2 Madsen Med U266
Pheochromocytoma PC12
[0171] Results
[0172] We have amplified a DNA fragment from the mouse noggin
plasmid, corresponding to the region conserved between Xenopus and
mouse noggin.
[0173] The amplified fragment of approximately 300 bp was used as
probe to hybridize to northerns, with RNAs prepared from adult and
embryonic tissues, as well as from various cell lines. Noggin
transcript of about 2 kb in size was detected in adult rat brain,
and in a cell line, SKMES, a small cell lung carcinoma.
[0174] Expression of noggin transcripts was examined in various
tissues from rat and mouse at different stages of development and
in adult. In the mouse, noggin transcripts can be detected in
embryos or head from E9 to E12, as well as in newborn brain and
adult brain. There was no detectable signal in peripheral tissues
examined except in skeletal muscle. Abundant level of expression
was also found in hippocampal astrocytes isolated from postnatal
mouse.
[0175] In the rat, noggin transcripts were detectable in embryos or
head from E9 to E18, as well as in brain from P1, P19 and adult
brain. In the cerebellum, expression of noggin appeared to be
higher in E18 and P1; in the spinal cord, expression of noggin mRNA
peaked at P1. Examination of noggin expression in all of the CNS
regions, especially the olfactory bulb, midbrain, hindbrain and
cerebellum. In the adult, noggin mRNA could be detected in all CNS
regions, especially the olfactory bulb and cerebellum. There also
appeared to be low levels in the skin.
Example 6
[0176] Neural Induction by Noggin
[0177] Materials and Methods
[0178] Preparation of Xenopus Noggin CHO Cell Conditioned
Medium
[0179] Xenopus noggin CHO conditioned medium was made by selecting
for stably transfected CHO cells. Dihydrofolate reductase (DHFR)
deficient CHO parental cells (J. Papkoff, Syntex Research) were
transfected with a Xenopus noggin expression plasmid containing
noggin in tandem with the dihydrofolate reductase gene. Growth in
nucleoside free medium was used to select for successfully
transfected cells. Nine colonies of transfectants were picked and
grown up individually. The noggin gene in these cells was amplified
by slowly increasing the dose of methotrexate, an inhibitor of
DHFR. The presence of noggin transcripts was first tested by
Northern analysis. Subsequently, two clones, B3 and C3, were shown
to secrete noggin protein, since conditioned medium from these
lines was capable of dorsalizing ventral marginal zones.
Furthermore, by labeling B3 cellular proteins with 35S-methionine,
noggin protein could be identified as a band of about 30 kD on
reducing SDS-PAGE, and a band of 60 kD on non-reducing SDS-PAGE
indicating it forms the expected dimer. These properties matched
those of the noggin protein previously produced in Xenopus oocytes
supra, (Smith et al., Nature 361, 547-49, 1993). B3 conditioned
medium was collected in a mixture of 1 part alpha MEM and 9 parts
CHO-S-SFMII (Gibco-BRL). The cells were allowed to condition the
medium for 3 days. Control medium from parental cells (CHO dhfr-)
was collected identically. Twenty fold concentrated medium was made
using Centriprep 10 concentrators, where the 20 fold change is
measured by volume.
[0180] Purification of Human Noggin from COS Cells
[0181] Human noggin protein was purified by a cationic exchange
column. COS/M5 cells were transiently transfected with a human
noggin expression plasmid, pCAE11. Cells were allowed to condition
DMEM (Specialty Media) for two to three days, after which the
medium was removed. Particulates from the medium were removed by a
centrifugation step and subsequent passage through a 0.2 um
cellulose acetate filter. This cleared medium was pumped onto a
MonoS (Pharmacia) column which was washed with several volumes 40
mM sodium phosphate (pH 7.3), 150 mM NaCl, 1 mM EDTA. Proteins were
then eluted in a linear gradient with 40 mM sodium phosphate (pH
8.5), 1.8M NaCl, 1 mM EDTA. Noggin protein elutes at 0.8M NaCl and
is .gtoreq.90% pure by SDS-PAGE.
[0182] Xenopus Otx Isolation
[0183] To isolate Xenopus Otx clones a tadpole head cDNA library
(Hemmati-Brivanlou, et al., Development 106, 611-617, 1989) was
screened with a mouse otx cDNA (S-L Ang and Rossant, Toronto) at
low stringency. The clones that were picked fell into two classes.
One class, which we have designated otxA, included pXOT21.2, the
probe used here. By in situ hybridization, transcripts are first
detected prior to gastrulation in the superficial layer on the
dorsal side. During neurulation a large anterior domain expressed
the gene, and includes both neural and non-neural tissues. After a
decline in expression in the tailbud tadpole, the gene is
reexpressed specifically in the brain and eyes.
[0184] Ventral Marginal Zone Assay
[0185] Embryo Preparation
[0186] Xenopus laevis embryos are fertilized and de-jellied as
described (Condie and Harland, 1987. Development 101, 93-105),
routinely the evening before dissections. Embryos are cultured
overnight at 15.degree. C. The vitelline membrane surrounding each
developing embryo is manually removed the following morning at the
late blastula stage. Until dissection, the embryos are maintained
in 1/3.times.modified ringers in agarose coated dishes.
[0187] Ventral Marginal Zone Dissection
[0188] Embryos are oriented with their yolky vegetal hemisphere up
so the dorsal side can be identified. The dorsal side of the early
gastrula is marked by the presence of a small arc of dense pigment
called the "dorsal lip" which marks the start of involution of
dorsal mesoderm. The ventral marginal zone (VMZ) is found directly
opposite the dorsal lip, and is dissected. Since the vitelline
membrane has been removed, the embryo tends to flatten. Using a
specially constructed knife made of an eyebrow, mounted onto a
glass pipet with wax, two cuts are made through the flattened
embryo from the top facing vegetal pole through to the animal pole.
The cuts are made such that they isolate approximately 30-60
degrees of the ventral side away from more lateral tissues. A third
cut which is perpendicular to the first two cuts completely
isolates the ventral marginal zone tissue away from the rest of the
embryo. This third cut is at the level of approximately two thirds
of the radius of the embryo from the center. Prior to treatment the
VMZ is washed 1.times. in the culture medium.
[0189] Assay
[0190] Approximately between 5 to 10 VMZs are used per assay. The
washed VMZs are dropped gently (trying to minimize transfer of
liquid) into eppendorf tubes containing the desired treatment
protein medium for assay. The VMZs are allowed to develop to the
late neurula or early tailbud stage as assessed by control whole
embryo development. At this time RNA is isolated from the VMZs and
control whole embryos as described (Condie and Harland, ibid). The
expression of muscle actin in VMZs indicates a dorsalization event
(Lettice and Slack, 1933. Development, 117, 263-72). RNA from each
sample is run on a formaldehyde-agarose gel and blotted to gene
screen. The blot is then hybridized with a Xenopus muscle actin
probe (Dworkin-Rastl et al., 1986. J. Embryol. exp. Morph. 91,
153-68). Quantitation of dorsalization can be carried out by
normalizing muscle actin signal to that of the ubiquitously
expressed EF-1.alpha. (Krieg et al., 1989. Devl. Biol. 133,
93-100). Quantitation is done using phosphor imaging.
[0191] RNase Protection Assay
[0192] RNase protection was carried out as described (D. A. Melton
et al., Nucleic Acids Res 12,7035-56, 1984), with the modification
that digestion was carried out at room temperature (22 C.) using
RNase T1 only (Calbiochem 556785) at 10 units/ml. 20-30 animal caps
were harvested for each lane, of this 80% was used for neural
markers and 10% for muscle actin and collagen type II. For
goosecoid and brachyury 20 caps were used. Exposures ranged from 12
hours to 5 days. In all cases, films were preflashed. In cases
where a marker was not expressed, the result was confirmed with
greater sensitivity using phosphor imaging.
[0193] Results
[0194] The development of vertebrate embryos requires several
inductive interactions. Mesoderm, which eventually forms tissues
such as notochord, muscle, heart, mesenchyme and blood, is induced
in the equatorial region of the embryo (Nieuwkoop, Wilhelm Roux'.
Arch. EntwMech. Org, 162, 341-373, 1969). This inductive event is
well studied, and there are several candidates for the endogenous
inducer(s) including members of the fibroblast growth factor(FGF)
family and activin (Jessell and Melton, Cell 68, 257-70 1992; Sive,
Genes Dev 7, 1-12, 1993) and TGFb family (Asashima, et al., Roux's
Arch. Dev. Biol. 198, 330-335, 1990; Asashima, et al.,
Naturwissenschaften 77, 8, 389-91, 1990; Green and Smith, Nature
347, 391-394, 1990; Smith, et al., Nature 345, 6277, 729-31, 1990;
Thomsen, et al., Cell 63, 485-493, 1990; van, et al., Nature 345,
6277, 732-4, 1990). The use of dominant negative receptors for both
FGF (Amaya, et al., Cell 66, 257-270, 1991) and activin
(Hemmati-Brivanlou and Melton, Nature 359, 609-614, 1992) in
Xenopus embryos strongly suggests that the signaling pathways
activated by these molecules are essential for proper mesoderm
formation. Molecules such as wnts (Christian, et al., Development
111, 1045-1055, 1991; McMahon and Moon, Cell 58, 1075-84, 1989;
Smith and Harland, Cell 67, 753-765, 1991; Sokol, et al., Cell 67,
741-752, 1991) and noggin (Smith, et al., Nature 361, 547-49, 1993)
modify the kinds of mesoderm made without inducing mesoderm
directly.
[0195] In a subsequent induction, the dorsal mesoderm of the
Spemann organizer signals nearby lateral mesoderm to take on a more
dorsal fate (Dale and Slack, Development 100, 2, 279-95, 1987;
Lettice and Slack, Development, 117, 263-271, 1993; Spemann and
Mangold, Arch. mikrosk. Anat. EntwMech. 100, 599-638, 1924; Stewart
and Gerhart, Development 109, 363-372, 1990). The only known factor
which is expressed in the organizer and can mimic its dorsalizing
activity is noggin.
[0196] Dorsal mesoderm of the Spemann organizer also signals nearby
ectoderm to become neural tissue. Neural induction by dorsal
mesoderm has been demonstrated in amphibians (Dixon and Kintner,
Development 106, 749-757, 1989; Doniach, et al., Science 257, 5069,
542-5, 1992; Hamburger, The Heritage of Experimental Embryology:
Hans Spemann and the Organizer, 1988; Kintner and Melton,
Development 99, 311-25, 1987; Spemann, Arch. mikrosk. Anat.
EntwMech. 100, 599-638, 1938), birds (Kintner and Dodd, Development
113, 1495-1506, 1991; Tsung, et al., Acta Biol exp Sinica 10,
69-80, 1965), and recently in mice (Ang and Rossant, Development
118, 139-149, 1993). Despite decades of effort, little is known
about the molecular nature of the factors responsible for this
induction. Among known inducers, activin can promote formation of
neural tissue, but this is due to a secondary induction by the
dorsal mesoderm that activin induces (Green, et al., Development
108, 1, 173-83, 1990; Green and Smith, Nature 347, 391-394, 1990;
Kintner and Dodd, Development 113, 1495-1506, 1991). Thus, activin
cannot promote formation of neural tissue when added to gastrula
ectoderm; however, such ectoderm remains competent to be neuralized
by dorsal mesoderm until the end of gastrulation (Sharpe and
Gurdon, Development 109, 765-74, 1990).
[0197] Direct Neural Induction by Noggin
[0198] Candidates for the endogenous inducer are expected to induce
neural tissue in the absence of dorsal mesoderm. Competent animal
cap ectoderm from late blastula stage embryos (St9) was used to
test noggin's neural inducing capacity. Xenopus noggin protein
conditioned medium was collected from stably transfected CHO cells
and twenty fold concentrated medium was used to treat St 9 animal
caps. Markers used in an RNase protection assay were N-CAM
(Jacobson and Rutishauser, Developmental Biology 116, 524-31, 1986;
Kintner and Melton, Development 99, 311-25, 1987), a neural cell
adhesion molecule, a neural specific isoform of b-tubulin (Good, et
al. Nucleic Acids Res 17, 8000, 1989; Good, et al., Dev Biol 137,
414-8, 1990; Richter, et al., Proc Natl Acad Sci USA 85, 8086-90,
1988) that is expressed in the hind brain and spinal cord, and
XIF3, a neurally expressed intermediate filament gene (Sharpe, et
al., Development 107, 701-14, 1989) to assay for neural induction.
All these markers are restricted to neural tissue, however, only
NCAM is expressed throughout the nervous system. We found that
Xenopus-noggin conditioned medium induces high levels of N-CAM and
XIF3 expression[FIG. 2.; lane8] in treated animal caps, without
inducing muscle actin(lane 13) (Dworkin-Rastl, et al., J. Embryol.
exp. Morph. 91, 153-168, 1986; Mohun, et al., Nature 311, 716-721,
1984). Control CHO cell medium induces neither muscle nor neural
tissues (lanes 7,12). St 9 activin treated animal caps express
muscle actin(lane11) and all three neural markers(lane 6),
demonstrating activin's ability to generate neural tissue
indirectly. It is interesting to note that noggin induces very
little, if any b-tubulin expression, while inducing high levels of
N-CAM, but activin induction has nearly the converse effect.
[0199] To determine whether noggin protein is sufficient to induce
neural tissue, COS cells were transfected with pCAE11, a human
noggin expression plasmid, and the conditioned medium was purified
by cation exchange chromatography resulting in noggin preparations
that were 90% pure [FIG. 3.]. Such purified human noggin protein is
also able to induce neural tissue in animal caps [FIG. 4a., see
below].
[0200] We have shown that noggin does not induce muscle in late
blastula stage animal caps, however, it is possible that noggin
induces other types of dorsal mesoderm. To address this concern, we
asked whether noggin could induce the expression of the early
mesoderm markers goosecoid (Blumberg, et al., Science 253, 194-6,
1991; Cho, et al., Cell 67, 1111-20, 1991), a marker of organizer
tissue and subsequently head mesoderm or X-brachyury (Smith, et
al., Cell 67, 79-87, 1991), which appears to be expressed in all
mesodermal precursors early, and subsequently is expressed in
posterior mesoderm and notochord. Animal caps were treated at stage
9 and collected at stage 11, when expression of goosecoid and
brachyury in the normal embryo is high. Neither marker is turned on
by purified human noggin (FIG. 4b. lane 5) at a dose with
demonstrated neural inducing activity (FIG. 6 lane 15); in contrast
animal caps treated in the same fashion with activin show both
goosecoid and X-bra expression (FIG. 4b. lane 4) as expected for
this mesoderm inducing factor (Cho, et al., Cell 67, 1111-20, 1991;
Smith, et al., Cell 67, 79-87, 1991). Untreated animal caps show no
expression of these mesodermal markers (lane 3), and RNA levels in
the collected animal caps are shown to be comparable using EF-1a
levels (Krieg, et al., Dev Biol 133, 93-100, 1989)
[0201] Since purified human noggin is capable of driving neural
induction, no additional factors which may have been present in the
crude conditioned medium are required. Furthermore, Xenopus and
human noggin, with 80% amino acid identity, can both act to induce
neural tissue in Xenopus, suggesting a conserved function for these
two proteins. However, for noggin to be a candidate endogenous
neural inducer it must be able to induce neural tissue at a stage
when neural induction occurs in normal whole embryos. It is unclear
when the first instructive signals are sent from dorsal mesoderm to
ectoderm in embryos. However, it is known that by early gastrula
stages, dorsal ectoderm has already been specified to become neural
tissue (Jones and Woodland, Development 107, 785-91, 1989). The
neural inducing signal is therefore likely to start before this
stage. The latest stage at which animal caps have been shown to be
competent to respond to neural-inducing mesoderm is the early
neurula (St13-14) (Sharpe and Gurdon, Development 109, 765-74,
1990). Thus, a candidate endogenous neural inducer must be able to
induce neural tissue from gastrula stage competent ectoderm.
[0202] Neural Induction at the Gastrula Stage
[0203] In order to assess the competence of ectoderm to respond to
noggin we treated animal caps taken from blastula (St8), late
blastula (St9), early gastrula (St10) and ventral animal caps from
mid-gastrula (St10.5) stage embryos with purified human noggin[FIG.
2.]. We also treated similarly staged animal caps with activin to
demonstrate its mesoderm inducing and secondary neural inducing
activities, and to contrast activin's effects with those of
noggin[FIG. 4a.]. Activin treated animal caps show neural induction
only in conjunction with induction of dorsal mesoderm, such as
muscle and notochord (lanes 3,6,9). In a number of experiments, we
confirmed that activin's ability to induce dorsal mesoderm, and
consequently neural tissue, declines rapidly at the gastrula stage
(lane 12) (Green, et al., Development 108, 173-83, 1990; Kintner
and Dodd, Development 113, 1495-1506, 1991). In the experiment
shown here a larger than usual dose of activin was given. Under
these conditions, only a small amount of neural tissue is made,
perhaps because so much mesoderm is induced that there is not much
competent ectoderm left in the explant to be neuralized. In
contrast noggin can induce neural tissue in animal caps taken from
all of these stages without inducing the notochord and somite
marker, collagen type II (Amaya, et al., Development 118, 477-87,
1993; Bieker and Yazdani-Buicky, J Histochem Cytochem 40, 1117-20,
1992), or muscle actin (lanes 4,7,10,13). This gives additional
support to the proposal that noggin is a direct neural inducer,
since it can act in the absence of both early and late mesoderm
markers. Furthermore, we have shown that noggin can induce neural
tissue in competent ectoderm at a time when mesoderm inducers are
inactive.
[0204] In some experiments, noggin addition to gastrula (but not
blastula) animal caps resulted in induction of muscle (data not
shown). This occurred at stages when activin could no longer induce
muscle. We interpret this as a result of a dorsalizing action by
noggin on tissues that have received a weak mesoderm-inducing
signal. The mesoderm-inducing signal which spreads into the
gastrula animal cap is not enough to induce mesoderm, but in the
presence of Xwnt-8 or noggin, muscle is formed. One interesting
corollary of the induction of muscle is that the kinds of neural
tissue seen in the explant are modified. Induction in explants that
contain no muscle usually yields N-CAM expression, but if muscle is
present, expression of both N-CAM and b-tubulin is seen. This
phenomenon is demonstrated in the secondary neural induction by
activin in St. 9 animal caps[FIG. 2.] and in the comparison of
neural tissue induced by noggin in ventral marginal zones versus
animal caps [FIG. 6.]. In the ventral marginal zones and animal
caps in which muscle is present, both N-CAM and b-tubulin are
expressed, whereas induced animal caps without muscle, show only
N-CAM expression.
[0205] Neural Induction after Injection of DNA Coding for
Noggin
[0206] To confirm our conclusions using a different experimental
approach, we have directed noggin expression to gastrula stage
animal caps by injecting the plasmid pCSKA-noggin into the animal
pole of a one cell stage embryo. This plasmid, in which noggin is
under the control of the cytoskeletal actin promoter, turns on the
expression of noggin mRNA at the onset of gastrulation (Smith, et
al., Nature 361, 547-49, 1993). At the blastula stage, the animal
caps are dissected and then matured to tailbud stages for
molecular-analysis. Animal caps injected with the noggin plasmid
show expression of N-CAM in the absence of muscle or notochord
markers (FIG. 4c. lane 2). A control plasmid directing the
expression of lac Z showed no neural or mesodermal induction as
expected (lane 1). This experiment demonstrates that ectopic noggin
expression can directly induce neural tissue in gastrula stage
ectoderm, a stage when neural induction is taking place in whole
embryos.
[0207] Differences in Competence between Dorsal and Ventral Animal
Caps
[0208] Animal caps taken from the dorsal side of gastrula stage
embryos show greater competence to form neural tissue than ventral
animal caps (Otte and Moon, Cell 68, 1021-29, 1992; Sharpe, et al.,
Cell 50, 749-58, 1987), when involuted anterior mesoderm is used as
the inducer. This type of mesoderm, however, has weaker inducing
capacity than the rest of the involuted mesoderm (Sive, et al.,
Cell 58, 171-180, 1989). Furthermore, the ventral side of an embryo
can support the formation of a complete secondary axis when the
organizer is placed on that side (Gimlich and Cooke, Nature 306,
471-3, 1983; Smith and Slack, J. Embryol. Exp. Morph. 78, 299-317,
1983; Spemann, Arch. mikjrosk. Anat. EntwMech. 100, 599-638, 1938),
indicating that there is no qualitative difference in competence.
Thus, while a weak inducer might unmask slight differences in
competence of the ectoderm, it has been suggested that a robust
neural inducer would show little difference in its effects on
dorsal and ventral ectoderm (Servetnick and Grainger, Development
112, 177-88, 1991). Therefore we tested noggin's effects on dorsal
and ventral ectoderm from the early gastrula. No difference in
N-CAM expression is detected (FIG. 5, lanes 4,6), while the ventral
animal caps treated with noggin show a greater amount of muscle
actin expression (presumably through dorsalization of tissues that
received a low-grade mesoderm induction). Activin treated dorsal
caps show induction of roughly the same level of muscle actin
expression (lane 5) as the ventral noggin treated caps, however,
activin treatment did not induce detectable neural specific
transcripts (lanes 3,5). This indicates that muscle tissue induced
at this stage is not sufficient to secondarily induce neural
tissue, and that noggin must be present to induce neural
tissue.
[0209] We conclude that there is no dorsal-ventral difference in
noggin mediated neural induction, suggesting that noggin behaves
like the robust neural inducing signal of the Spemann organizer,
not like the weaker signal from early anterior mesoderm.
[0210] Dose Dependence
[0211] To determine what levels of noggin protein are required for
neural inducing activity, we carried out a dose response
experiment. In addition to determining the doses required for
neural induction in animal caps, we have also carried out a dose
response of the dorsalization of ventral marginal zones in order to
compare the doses required for these two types of inductions. Stage
9 animal caps or St. 10.5 VMZ were treated with purified human
noggin, and N-CAM and .beta.-tubulin were used to assay neural
induction, while muscle actin was used as a marker of dorsal
mesoderm. This experiment shows that neural induction occurs at a
dose of 1 .mu.g/ml, which is a twenty fold higher dose than
required for dorsalization of VMZ [FIG. 5]. There are several
observations that may account for the apparently high dose
requirement. First, to get a maximal neural response from dorsal
mesoderm, the tissues must be left in contact through most of
neurulation (Sharpe and Gurdon, Development 109, 765-74, 1990); in
contrast, the animal caps treated with noggin close up rapidly,
this inhibits factor access, and consequently they receive only a
brief effective dose. Second, it is likely that noggin is not the
only neural inducer active in the embryo; it has been shown in a
variety of amphibians that the somites (Hemmati-Brivanlou, et al.,
Science 250, 800-802, 1990; Jones and Woodland, Development 107,
785-91, 1989) and the neural plate have neural inducing activity
(Hamburger, The Heritage of Experimental Embryology: Hans Spemann
and the Organizer, 1988; Servetnick and Grainger, Dev Biol 147,
73-82, 1991) and noggin transcripts are not detected there. Thus it
is plausible that noggin is one of several neural-inducing
activities. In this connection it is worth noting that noggin is
equally potent in inducing neural tissue in ventral marginal zones
as in dorsalizing them to generate muscle. Numerous other
experiments (see FIG. 5) show that induction of a similar amount of
muscle at this stage by activin does not result in neural
induction. Fourth, it may be that only a small fraction of the
purified protein is active, and that the experiment overestimates
the amount of protein needed for neural induction. Finally, it is
possible that the accessibility of exogenously added soluble noggin
is significantly lower than noggin protein being secreted
endogenously.
[0212] Patterning
[0213] Embryonic neural tissue develops an anteroposterior (A-P)
pattern, with various brain structures, eyes, and the spinal cord.
It is thought that A-P neural pattern requires the presence of
dorsal mesoderm, whether it be adjacent to the responding ectoderm
in a planar configuration (Dixon and Kintner, Development 106,
749-757, 1989; Doniach, et al., Science 257, 542-5, 1992; Kintner
and Melton, Development 99, 311-25, 1987; Ruiz i Altaba,
Development 108,595-604, 1990), or directly beneath it in a
vertical interaction (Dixon and Kintner, Development 106, 749-757,
1989) (Hemmati-Brivanlou, et al., Science 250, 800-802, 1990;
Sharpe and Gurdon, Development 109, 765-74, 1990; Sive, et al.,
Cell 58, 171-180, 1989). Both of these types of interactions occur
in normal development, and both probably contribute to the
resulting pattern. To determine if noggin induces patterned neural
tissue, and if so, what neural regions are represented, we used
Xenopus otx as a marker of forebrain and mid brain; En-2
(Hemmati-Brivanlou, et al., Development 111, 715-724, 1991) as a
marker of the mid brain-hind brain boundary, and Krox-20
(Wilkinson, et al., Nature 337, 461-4, 1989) as a marker of the
third and fifth rhombomeres of the hind brain in in situ
hybridization (Harland, Methods in Cell Biology, 36, 675-685,
1991). Antibodies directed against XIHbox 6 (Wright, et al.,
Development 109, 225-34, 1990) mark posterior hind brain and spinal
cord structures. Prior to the use of these markers, we observed the
formation of cement glands in noggin treated animal caps. Since
cement glands are induced organs of ectodermal origin found
anterior to the neural plate, this result suggests that noggin
induces anterior structures. In situ hybridization confirms this by
showing the presence of a cement gland specific transcript, XAG-1
(Sive, et al., Cell 58, 171-180, 1989) in noggin treated animal
caps, but not in control treated animal caps[FIG. 7A-L.]. In situ
hybridization with the region specific neural markers[FIG. 7A-L.]
show that noggin induces forebrain type tissue as seen by the
expression of otx in noggin treated animal caps. We have not
detected En-2, Krox20, or XIHbox, suggesting that these more
posterior markers are not induced by noggin.
[0214] Expression of Neural Antigens
[0215] We have demonstrated that noggin directly induces the
expression of neural specific transcripts. A further demonstration
is to use antibodies against neural specific antigens to show that
the noggin induced tissue is phenotypically neural. To this end, we
have treated animal cap tissue with noggin and cultured them to a
late stage (St 35) for antibody staining. We have used the 6F11
anti-N-CAM antibody, which stains the entire neural tube of a
normal embryo. Noggin treated animal caps express this antigen
[FIGS. 7A-L.] while control untreated animal caps do not. This
indicates that noggin can induce the production of neural specific
proteins in treated animal caps. We have failed to detect the
expression of numerous other antigens that are characteristic of
various subclasses of differentiated neural cells. These included
2G9, which stains most neural tissue, including peripheral neurons,
Tor 24.55, which stains sensory neurons, and Tor 23, which stains a
variety of neurons including motor neurons.
Example 7
[0216] Production of recombinant human noggin from E. coli and
baculovirus
[0217] Materials and Methods
[0218] Genetic Engineering and Cell Culture
[0219] A lactose inducible expression plasmid was constructed by
replacing the Swa1/Bsm1 region of pRPN40 (Masiakowski et al, J.
Neurochem. 57, 1003-1012, 1991) with the Swa1/Bsm1 region of the
human noggin gene obtained by PCR and spanned by the same
restriction sites, resulting in plasmid pRG301. pRG301 is a high
copy number kanamycin resistant plasmid derived from pBR322 with
the human noggin gene under the control of the lacUV5 promoter. A
plasmid containing the high copy number kanamycin resistant gene
was deposited with the Agricultural Research Collection (NRRL),
Peoria, Ill., and bears accession number B-18600. This plasmid was
described in U.S. patent application Ser. No. 07/478,338, which is
incorporated by reference herein in its entirety. E. coli
W3110laclq cells transformed with pRG301 were grown at 37.degree.
C., induced with lactose, harvested by centrifugation, washed once
with 100 mM Tris-HCl, 50 mM EDTA pH 8 and stored frozen,
essentially as described (Masiakowski et al, ibid.).
[0220] Recovery from Inclusion Bodies
[0221] E. coli cell paste (32 g) was suspended in ten volumes (v/w)
of 50 mM TrisHCl-pH 8.0-5 mM EDTA, lysed in a French Press at 8,000
psi and 80.degree. C. and centrifuged at 8,000.times.g for 30 min
at 4.degree. C. The pellet containing noggin was suspended in the
original volume of 2 M urea-20 mM TrisHCl, pH 8.0 and stirred for
30 min. The suspension was centrifuged at 8,000.times.g at
4.degree. C. for 30 min and the pellet consisting mostly of
inclusion bodies (IB) was suspended in 20 volumes (v/w) of 6 M
guanidine HCl, 50 mM TrisHCl,1 mM EDTA, 50 mM DTT and stirred for
one hour at room temperature. After centrifugation at 8,000.times.g
for 30 min, the supernatant containing 0.45-0.50 g denatured and
reduced noggin was diafiltered against 10 volumes of 6 M urea-50 mM
sodium acetate pH 4.5-1 mM EDTA-0.1 mM DTT using Omega 10,000 MW
cut-off membranes. The diafiltrate containing 0.4-0.44 g noggin was
loaded at a flow rate of 30 ml/min onto a 2.6.times.10 cm column of
S-Sepharose (Pharmacia), equilibrated in 6 M urea-50 mM sodium
acetate-1 mM EDTA-0.1 mM DTT pH 4.5 The column was first washed
with the same buffer and then with a one liter gradient (0-1M NaCl)
at a flow rate of 30 ml/min. Fractions containing noggin were
identified by gel electrophoresis and pooled. The yield was
0.2-0.25 g noggin.
[0222] Refolding
[0223] The denatured and reduced noggin solution was adjusted to
0.05-0.2 mg/ml protein concentration and brought to 1.5-2.5 M
guanidineHCl-0.1 M TrisHCl pH 8.0-0.1 mM EDTA-0.2-2 mM reduced
glutathione-0.02-0.2 mM oxidized glutathione (preferably at a ratio
of 10:1 reduced to oxidized glutathione) at 4.degree. C. under slow
stirring. After 24-72 hours, two refolded noggin isoforms were
identified by RP-HPLC chromatography (FIG. 8). The refolded noggin
solution was diafiltered against 20 volumes of 0.05 M sodium
acetate pH 4.5, brought to 50 mM potassium phosphate pH 7.2 and
stirred slowly at 4.degree. C. for 1 hour minimum. Misfolded noggin
precipitated and was removed by centrifugation for 30 min at
8,000.times.g.
[0224] Reverse Phase HPLC Chromatography
[0225] Refolded noggin can be purified by chromatography on a 12 mm
C8, 1.times.25 cm Dynamax 300 A column equilibrated in solvent A
(0.1% TFA in water). After loading, the column was washed with
solvent A and was developed at a flow rate of 4 ml/min according to
the following protocol: (a) 10 min isocratically at 70% of solvent
A; 30% of solvent B (0.1% TFA in acetonitrile); 30 min linear
gradient to 60% solvent B and 40% solvent A. Correctly refolded
noggin elutes earlier at 44%-46% solvent B. The yield was 0.07-0.1
g noggin.
[0226] Production of human noggin in Baculovirus cell culture
[0227] The SF21 line of Spodoptera frugiperda was routinely
maintained as cell monolayers in Grace's Insect Cell medium
supplemented with lactalbumin hydrolysate and yeastolate (Gibco).
This medium completed with 10% v/v heat-inactivated fetal calf
serum (Irvine Scientific) is identified as TMNFH-10. Cells were
also cultured in serum-free medium (SF-900-II; Gibco) after
adaptation. Suspension cultures in either medium were raised in
microcarrier culture flasks (Bellco) using a stirring speed of 80
rpm. All cultures were maintained at >96% viability, as judged
by trypan blue exclusion.
[0228] Construction of Recombinant Baculovirus
[0229] Sequences corresponding to human noggin were excised as a
5'-BamH1-Pst1-3' fragment from an expression plasmid containing the
human noggin gene. This fragment was inserted into BamH1-Pst1
digested pVL1393 (Invitrogen). The resulting plasmid, pTR 1009, has
the human noggin sequence immediately downstream of the polyhedrin
promoter of Autrographa californica Multiple Nuclear Polyhedrosis
Virus (AcMNPV), and this promoter-heterologous gene fusion is
flanked in turn by recombination targets derived from the AcMNPV
polyhedrin region. Recombinant plasmid DNA was purified by alkaline
lysis and CsCl centrifugation. SF21 cells were co-transfected with
plasmid and viral DNA by the following method: Plasmid DNA (3 mg)
was mixed with 0.5 mg linearized, deleted viral DNA (Baculo
Gold.TM., Pharminigen), and precipitated with ethanol. Dried DNA
was then resuspended in water (50 ml), mixed with 1.5 ml Grace's
medium, and 30 ml Lipofectin.TM. cationic liposomes (BRL). The
DNA-liposome mixture was vortexed, allowed to stand at room
temperature for 15 minutes and added dropwise to a monolayer of
SF21 cells (2.times.106 cells/60 mm plate). After incubation at
27.degree. C. for four hours, 2 ml TMNFH-10 was added and the
culture returned to incubation for 5 days. Tissue culture medium
was harvested and used as a source of virus for plaque
isolation.
[0230] Recombinant virus was isolated by multiple rounds of plaque
purification on SF21 cells. Diluted virus (0.5 ml) was adsorbed to
cell monolayers (2.times.106 cells/60 mm plate) for a period of one
hour at 27.degree. C., aspirated, and virus plaques were allowed to
develop with an overlay of 0.5% agarose in TMNFH-10 medium for a
period of 6 days. Virus plaques were picked after microscopic
inspection, and eluted into 2 ml SF900-II medium. Virus stocks were
amplified by low multiplicity (0.1 pfu/cell) infection. Virus
clones expressing noggin were identified by metabolic labeling of
infected cultures with 35S-methionine and 35S-cysteine and
analysing total labeled protein by polyacrylamide gel
electrophoresis and autoradiography. A labeled protein of the
expected apparent Mr of 20,000-30,000 was detected by this method
in candidate clones but not in control cultures.
[0231] Expression and Purification of Baculovirus-Derived
Noggin
[0232] SF21 cells were cultured in suspension flasks to a density
of approximately 1.8.times.106/ml in SF900-l1 medium. Cultures (500
ml) were collected by centrifugation at 1000.times.g for 10 min and
resuspended in 20 ml of growth medium containing 5-10 pfu/cell
recombinant virus. Virus was allowed to adsorb for 1 hour at room
temperature with gentle mixing. Infected cells were then diluted to
their original volume with fresh growth medium, and incubated at
27.degree. C. for 3 days. Cells and debris were subsequently
clarified from the growth medium by centrifugation at 1.times.g for
20 min.
[0233] Cell supernatants were brought to pH 8.0, passed through a
0.45 mm Millipak 60 filter and applied to a Fast S column that had
been equilibrated in 25 mM HEPES pH 8.0. The column was washed with
the same buffer and developed with a linear NaCl gradient to remove
other medium components. Noggin eluted from this column at 1 M
NaCl.
[0234] Results
[0235] Characterization of Human Noggin Produced in E. coli and in
Baculovirus
[0236] Reverse-phase HPLC chromatography shows that recombinant
noggin refolded and purified from E. coli elutes in a single sharp
peak, indicating the presence of one predominant isoform (FIG.
9).
[0237] Electrophoresis on 15% polyacrylamide-SDS-reducing gels
shows that noggin from either E. coli or insect cells is better
than 95% pure and migrates in a single band corresponding to a
protein of 20-30 kD. Noggin from insect cells shows slightly slower
mobility, apparently due to additional mass from N-linked
glycosylation at the single consensus site (FIG. 10). Treatment
with Endo F converts the mobility of insect-produced noggin to that
of the bacterially produced protein (data not shown).
[0238] In the absence of reducing agents, noggin produced either in
E. coli or in baculovirus behaves as a disulfide-linked oligomeric
protein (FIG. 10). However, by gel filtration analysis and mass
spectroscopy noggin is primarily a dimeric protein (data not
shown).
[0239] Circular dichroism studies show that recombinant noggin
refolded and purified from E. coli as well as noggin purified from
insect cells have very similar conformations (FIG. 11). Secondary
structure determined by this method indicates that noggin consists
of 48% alpha-helix, 0% beta-structure, and 52% random coil.
[0240] Biological Activity of Human Noggin Produced in E. coli and
in Baculovirus
[0241] Biological activity of human noggin produced in E. coli or
in baculovirus was determined by assay of muscle actin expression
in the ventral marginal zone assay, as described supra. Results
shown in FIG. 12 indicate a positive dose response for induction of
muscle actin mRNA in VMZ exposed to either bacterially produced
human noggin, or baculovirus produced human noggin.
Example 8
[0242] Production and Characterization of Rat Monoclonal Antibody
RP57-16 Reactive with Human Noggin
[0243] Materials and Methods
[0244] Production of Antibody
[0245] RP57-16 rat monoclonal antibody reactive with recombinant
human and Xenopus noggin was produced by the immunization of a
female Lewis rat with four 35 .mu.g injections of purified
recombinant human noggin (produced in E. coli) over a two month
period. For the initial immunization, the protein was injected in
the rear foot pad in Freund's complete adjuvant. Subsequent
injections were given in the same foot pad in Freund's incomplete
adjuvant. The rat was euthanized 3 days after the fourth
injection.
[0246] Lymph node cells from the immunized rat were mixed with
SP2/0-E.O. mouse myeloma cells at a ratio of 2:1. After
centrifugation, the cell mixture was resuspended in 0.25 ml of 42%
(w/v) PEG 3350 (Baker) in phosphate-buffer-saline with 10% (v/v)
dimethylsulfoxide (Sigma) for a total of 3 minutes in a 37.degree.
C. water bath. Cells were plated at a density of 5.times.10.sup.4
lymphocytes per well in 96-well plates (Falcon 3072) in DMEM/F-12
(Mediatech, Inc.) containing 10% FBS (supplemented with
streptomycin, penicillin, pyruvate, and glutamine) and HMGT
(1.6.times.10-3 M thymidine, 4.0.times.10-4 methotrexate,
1.3.times.10-3 sodium bicarbonate and 1.0.times.10-2 hypoxanthine).
After 10 days in culture, supernatants were harvested and assayed
for antibody activity against recombinant human noggin by indirect
ELISA. Supernatant from COS-M5 cells transfected with the plasmid
containing the human noggin gene was air dried overnight in Probind
96-well assay plates (Falcon 3915). Non-specific binding was
eliminated by 2 hour incubation at ambient temperature with PBS/1%
BSA (Sigma). Plates were washed 2 times with PBS/0.02% Tween 20.
Culture supernants were then added and incubated at ambient
temperature for 1 hour. Plates were washed 4 times with PBS/0.02%
Tween 20. Secondary antibody, Goat anti-Rat IgG (H+L) alkaline
phosphatase conjugate(Caltag) diluted 1:2000 in PBS/1% BSA was
added to each well and the plates incubated at ambient temperature
for 1 hour. Plates were again washed 4 times with PBS/0.02% Tween
20. Antibody binding was visualized by 1 hour incubation at ambient
temperature in the dark with pNPP (p-nitrophenyl phosphate, Sigma)
1 mg/ml in diethanolamine buffer, pH 9.8. The reaction was stopped
by the addition of an equal volume of 100 mM EDTA. Absorbance was
read at 405 nm on a Thermomax Microplate Reader (Molecular
Devices). A reaction was considered positive if the absorbance was
2 times that of the negative control (diluent alone followed by
secondary antibody and substrate). Positive clones were expanded
and culture supernatant containing monoclonal antibody was
collected for specificity analysis.
[0247] RP57-16 was cloned in soft agar. Cloned hybrid cells were
expanded in DMEM/F-12 (Mediatech, Inc.) containing 10% FBS
(supplemented with streptomycin, penicillin, pyruvate, and
glutamine). Supernatant containing antibody was aliquoted and
stored at -70.degree. C. until use.
[0248] Specificity Analysis
[0249] ELISA
[0250] 100 ng of purified recombinant human noggin, Xenopus noggin,
BDNF, NT-3, and NT4 protein was individually passively adsorbed to
Probind 96-well assay plates by overnight incubation at 4.degree.
C. in 50 mM bicarbonate buffer, pH 9.6. BDNF, NT-3 and NT-4 were
used to assess non-specific binding of rat monoclonal antibody
RP57-16. Supernatants from COS-M5 cells transfected with either the
plasmid containing the human noggin gene or the plasmid containing
the fig C-terminal tagged Xenopus noggin gene were air dried to
Probind 96-well plates overnight. Non-specific binding was
eliminated by 2 hour incubation at ambient temperature with PBS/1%
BSA (Sigma). Plates were washed 2 times with PBS/0.02% Tween 20.
Undiluted RP57-16 was added and incubated at ambient temperature
for 1 hour. Plates were washed 4 times with PBS/0.02% Tween 20.
Secondary antibody, Goat anti-Rat IgG (H+L) alkaline phosphatase
conjugate(Caltag) diluted 1:2000 in PBS/1% BSA was added to each
well and the plates incubated at ambient temperature for 1 hour.
Plates were again washed 4 times with PBS/0.02% Tween 20. Antibody
binding was visualized by 1 hour incubation at ambient temperature
in the dark with pNPP (p-nitrophenyl phosphate, Sigma) 1 mg/ml in
diethanolamine buffer, pH 9.8. The reaction was stopped by the
addition of an equal volume of 100 mM EDTA. Absorbance was read at
405 nm on a Thermomax Microplate Reader (Molecular Devices). A
reaction was considered positive if the absorbance was 2 times that
of the negative control (diluent alone followed by secondary
antibody and substrate).
[0251] Electrophoresis and Western Blotting
[0252] Rat monoclonal antibody RP57-16 was also analyzed by Western
blotting. 50 ng of recombinant human noggin, non-reduced and
reduced, were electrophoresed on 12.5% SDS-polyacrylamide gels and
electroblotted on nitrocellulose membranes. Membranes were blocked
with PBS/1% Casein/0.1% Tween 20, and then incubated for 2 hours
with undiluted RP57-16 culture supernatant. Following 4 washes in
PBS/0.02% Tween 20, the membranes were incubated with a 1:5000
dilution of Goat anti-Rat IgG (H+L) horseradish peroxidase
conjugate (Pelfreeze) in PBS/1% BSA/0.1% Tween 20. Membranes were
washed 4 times with PBS/0.02% Tween 20. Proteins were visualized
with ECL Western Blotting Reagents (Amersham) according to the
manufacturer's instructions. Membranes were then exposed to XAR 5
Scientific Imaging film (Kodak) for 5 seconds.
[0253] Results
[0254] Rat monoclonal antibody RP57-16 reacts with both recombinant
human and Xenopus noggin and with recombinant human noggin produced
in E. coli, in insect cells, and in COS-M5 cells. The antibody does
not react with the neurotrophins BDNF, NT-3 and NT-4. Western
blotting showed that the antibody detects both reduced and
non-reduced protein.
Example 9
Creation of Noggin Deletion Muteins
[0255] The subject invention further concerns the discovery that
native human noggin can be modified to create new compounds with
highly desirable characteristics.
[0256] The basic region of Xenopus noggin corresponds to amino
acids 123-135 (KKHRLSKKLRRKL) of the molecule. (See Smith, W. C.
and Harland, R. M. Cell 70: 829-840 (1992) for sequence). Based
upon what was known about the TGF-.beta. family of molecules, the
basic region appeared to be a possible processing site. To better
understand the function of the basic region, constructs of Xenopus
noggin were created that deleted the highly conserved basic region
(.DELTA.123-135) or a larger portion of the peptide including the
basic region (.DELTA.91-135). Both of these deletion mutants were
active, yielding hyperdorsalized embryos when injected as RNA. In
addition, preliminary experiments demonstrated that noggin had an
affinity for heparin, while a modified form with the basic region
deleted did not possess this binding activity.
[0257] Native human noggin (hNG) exhibits low bioavailability in
animal sera, likely due to its binding to extracellular matrix.
Fc-tagged human noggin (hNG-Fc) has been shown to bind to BMP4 with
very high affinity. (Zimmerman, L. B., et al., Cell 86: 599-606
(1996)). Modification of hNG has resulted in the identification of
compounds which show improved bioavailability while retaining the
ability to bind and antagonize Bone Morphogenetic Proteins (BMPs).
The specific modifications resulting in altered biological
properties involve deletion of amino acids identified as
responsible for conferring heparin binding activity to the native
but, unexpectedly, retains the ability to bind and antagonize
BMP4.
[0258] Specifically, applicants have created two molecules, known
as hNG.DELTA.138-144Fc and hNG.DELTA.133-144Fc. These molecules are
Fc-tagged deletion muteins of human noggin lacking either amino
acids 138 to 144 (for hNG.DELTA.138-144Fc) or 133 to 144 (for
hNG.DELTA.133-144Fc) and tagged with the Fc domain of human IgG1 at
their C-termini. To construct Fc-tagged noggins, a BspEl-Notl human
IgG1 fragment (See Davis, S., et al., Science 266: 816-819 (1994);
Economides, A. N., et al., Science 270: 1351-1353 (1995)) may be
fused to human noggin using an oligonucleotide encoding a peptide
bridge sequence as described by Zimmerman, L. B., et al., Cell 86:
599-606 (1996). As described below, the deletion muteins
hNG.DELTA.138-144Fc and hNG.DELTA.133-144Fc displayed identical
activity with hNG-Fc for binding to BMP4, as well as reduced
affinity for heparin and superior pharmacokinetics in animal sera
as compared to hNG.
[0259] Construction of hNG.DELTA.133-144Fc
[0260] hNG.DELTA.133-144Fc consists of the sequence of human noggin
(hNG) with a deletion of amino acids 133 to 144 (numbering begins
with the initiating methionine being amino acid number 1), fused to
the constant region of human immunoglobulin G1 (Fc) via a Ser-Gly
"bridge" (encoded by a genetically engineered Bsp El restriction
enzyme site) (FIG. 14). hNG.DELTA.133-144Fc was constructed by PCR
and was cloned into the mammalian expression vector pMT21
(pMT21.hNG.DELTA.133-144Fc) using standard genetic engineering
techniques. The correctness of the sequence of hNG.DELTA.133-144Fc
was verified by sequencing.
[0261] Subsequently, this deletion mutein was also transferred to
the sequence of hNG.DELTA.133-144Fc was verified by sequencing.
Subsequently, this deletion mutein was also transferred to the
expression vector pSRa, and both the Fc-tagged and the untagged
version of this hNG mutein were constructed
(pSRa.hNG.DELTA.133-144Fc, and pSRa.hNG.DELTA.133-144
respectively).
[0262] Expression of hNG.DELTA.133-144Fc
[0263] hNG.DELTA.133-144Fc was initially expressed in COS7 cells
using a Lipofectamine (GIBCO/BRL) based transfection protocol.
hNG.DELTA.133-144Fc, which like hNG is secreted, was purified from
the conditioned media bypassing through a Protein A-Sepharose
column (Pharmacia) and eluting it with 100 mM acetic acid which was
subsequently neutralized with Tris buffer to pH 7. The purity of
this preparation was checked by SDS-PAGE followed by staining with
the fluorescent dye SYPRO Orange (Molecular Probes, Inc.). The
resulting preparation was more than 90% pure by this criteria and
it was primarily in a disulfide-linked dimeric form, in agreement
with previous observations made both with untagged hNG as well as
with hNG-Fc, both of which also form disulfide-linked dimers.
[0264] Activity of hNG.DELTA.133-144Fc
[0265] Binding to BMP4:
[0266] To determine if this hNG mutein was active, its ability to
bind to hBMP4 was compared with that of hNG-Fc, which binds human
Bone Morphogenetic Protein 4 (hBMP4) with affinity essentially
identical to that of untagged hNG. An ELISA format assay where
hBMP4 is captured on the surface of an ELISA plate was used to
compare the two proteins. The hNG.DELTA.133-144Fc mutein displayed
the same binding to BMP4 as hNG-Fc (FIG. 15), indicating that the
two proteins have the same biological activity.
[0267] Binding to Heparin:
[0268] We examined the heparin binding profile of hNG-Fc and
compared it with that of hNG.DELTA.133-144Fc and another hNG
deletion mutein which bears a shorter deletion in the basic region,
namely, hNG.DELTA.138-144Fc (also referred to as
hNG.DELTA.KKLRRK-Fc). As shown on FIG. 16, hNG-Fc starts to elute
from heparin at about 0.75 M NaCl, whereas hNG.DELTA.133-144Fc and
hNG.DELTA.138-144Fc start eluting at 0.25 M NaCl. Binding to
heparin at above 0.5 M NaCl is considered to be an affinity
interaction with heparin whereas binding to heparin below 0.5 M
NaCl is considered to be due to ionic interactions (heparin
contains sulfate groups which are negatively charged and hNG has
regions which are positively charged). Therefore, it appears that
the two hNG deletion muteins, hNG.DELTA.133-144Fc and
hNG.DELTA.138-144Fc interact with heparin through ionic
interactions whereas hNG-Fc displays an affinity for heparin. Since
the KKLRRK' deletion (i.e. that embodied by hNG.DELTA.138-144Fc) is
adequate for reducing the interaction with heparin to that expected
for ionic effects, we conclude that the sequence KKLRRK (i.e. amino
acids 138 to 144 in hNG) is or contains the heparin binding domain
of hNG. The Fc-domain did not bind to heparin.
[0269] We have also found that hNG and hNG-Fc display identical
binding to heparin by the method described above, whereas a
chemically modified form of hNG (citraconyl-hNG) does not bind to
heparin. We have also examined the heparin binding profile of
another hNG deletion mutein that bears a longer deletion beyond the
basic region, namely hNG.DELTA.133-179Fc. hNG.DELTA.133-179Fc
displays the same binding profile to heparin as hNG.DELTA.133-144Fc
and hNG.DELTA.138-144Fc, indicating that further deletion of the
sequence downstream of the basic region does not further reduce the
ionic component of hNG binding to heparin. From this we conclude
that the ionic component of hNG binding to heparin must reside
within the Kunitz-like domain of hNG. Furthermore, the chemically
modified citraconyl-hNG and the two deletion muteins
hNG.DELTA.138-144Fc and hNG.DELTA.133-144Fc are all biologically
active (i.e. they bind to BMP4 with the same apparent affinity as
hNG-Fc), whereas the deletion mutein hNG.DELTA.133-179Fc is
inactive. Taken together, these results indicate that binding of
hNG to heparin can be separated from binding to BMP4, and that the
heparin binding domain of hNG is not required for its BMP4 blocking
activity.
[0270] Pharmacokinetic Profile of hNG.DELTA.133-144Fc
[0271] The pharmacokinetic properties of hNG.DELTA.133-144Fc were
tested in both mice and rats. It had been previously determined
that unmodified hNG expressed in E. coli and refolded displays very
poor bioavailability in rat serum after intravenous administration,
with an apparent half life of less than 60 minutes. Citraconyl-hNG,
which does not bind to heparin, displays a 30-fold improvement in
bioavailability but also disappears from circulation at about 2
hours post-injection. We reasoned that hNG.DELTA.133-144Fc which
does not bind to heparin and which also has an Fc-tag may have a
longer half-life in vivo. To examine this possibility, we injected
mice and rats with hNG.DELTA.133-144Fc and determined its
bioavailability in sera.
[0272] When hNG.DELTA.133-144Fc was injected into mice
intraperitoneously (ip), it was detectable even in the latest time
point sampled, which was 24 hours, achieving levels of 2 .mu.g/ml
(FIG. 17A). Intravenous injection in rat also showed favorable
pharmacokinetics, although the latest serum sample was taken at 6
hours. In that experiment approximately 18 .mu.g/ml of
hNG.DELTA.133-144Fc were detected (FIG. 17B). Similar results have
been achieved with hNG.DELTA.138-144Fc, indicating that deletion of
the heparin binding domain of hNG is responsible for the improved
pharmacokinetic profile of these molecules over hNG. Since the
assay used to detect hNG.DELTA.133-144Fc post-ip or post-iv
injection in animal sera relies on the ability of
hNG.DELTA.133-144Fc to bind to BMP4, we also know that the
hNG.DELTA.133-144Fc that is detected is functional and capable of
interacting with BMP4. Furthermore, since hNG displays a very high
affinity for BMP4, we anticipate that the levels of
hNG.DELTA.133-144Fc achieved in these experiments will block BMP4
activity in animal model and in clinical situations.
[0273] Deposit of Microorganisms
[0274] The following were deposited with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Md. 20852 under the
terms of the Budapest Treaty:
6 ATOC No. Date of Deposit phage hnog.lambda.-5 75311 September 23,
1992 phage hnog.lambda.-7 75309 September 23, 1992 phage
hnog.lambda.-9 75310 September 23, 1992 phage hnog.lambda.-10 75308
September 23, 1992 hybridoma RP57-16 CRL 11446 August 25, 1993
[0275] While the invention has been described above in conjunction
with preferred specific embodiments, the description and examples
are intended to illustrate, and not to limit, the scope of the
invention.
Sequence CWU 1
1
27 1 699 DNA human CDS (1)..(696) 1 atg gag cgc tgc ccc agc cta ggg
gtc acc ctc tac gcc ctg gtg gtg 48 Met Glu Arg Cys Pro Ser Leu Gly
Val Thr Leu Tyr Ala Leu Val Val 1 5 10 15 gtc ctg ggg ctg cgg gcg
aca ccg gcc ggc ggc cag cac tat ctc cac 96 Val Leu Gly Leu Arg Ala
Thr Pro Ala Gly Gly Gln His Tyr Leu His 20 25 30 atc cgc ccg gca
ccc agc gac aac ctg ccc ctg gtg gac ctc atc gaa 144 Ile Arg Pro Ala
Pro Ser Asp Asn Leu Pro Leu Val Asp Leu Ile Glu 35 40 45 cac cca
gac cct atc ttt gac ccc aag gaa aag gat ctg aac gag acg 192 His Pro
Asp Pro Ile Phe Asp Pro Lys Glu Lys Asp Leu Asn Glu Thr 50 55 60
ctg ctg cgc tcg ctg ctc ggg ggc cac tac gac cca ggc ttc atg gcc 240
Leu Leu Arg Ser Leu Leu Gly Gly His Tyr Asp Pro Gly Phe Met Ala 65
70 75 80 acc tcg ccc ccc gag gac cgg ccc ggc ggg ggc ggg ggt gca
gct ggg 288 Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly Gly Gly Gly Ala
Ala Gly 85 90 95 ggc gcg gag gac ctg gcg gag ctg gac cag ctg ctg
cgg cag cgg ccg 336 Gly Ala Glu Asp Leu Ala Glu Leu Asp Gln Leu Leu
Arg Gln Arg Pro 100 105 110 tcg ggg gcc atg ccg agc gag atc aaa ggg
cta gag ttc tcc gag ggc 384 Ser Gly Ala Met Pro Ser Glu Ile Lys Gly
Leu Glu Phe Ser Glu Gly 115 120 125 ttg gcc cag ggc aag aag cag cgc
cta agc aag aag ctg cgg agg aag 432 Leu Ala Gln Gly Lys Lys Gln Arg
Leu Ser Lys Lys Leu Arg Arg Lys 130 135 140 tta cag atg tgg ctg tgg
tcg cag aca ttc tgc ccc gtg ctg tac gcg 480 Leu Gln Met Trp Leu Trp
Ser Gln Thr Phe Cys Pro Val Leu Tyr Ala 145 150 155 160 tgg aac gac
ctg ggc agc cgc ttt tgg ccg cgc tac gtg aag gtg ggc 528 Trp Asn Asp
Leu Gly Ser Arg Phe Trp Pro Arg Tyr Val Lys Val Gly 165 170 175 agc
tgc ttc agt aag cgc tcg tgc tcc gtg ccc gag ggc atg gtg tgc 576 Ser
Cys Phe Ser Lys Arg Ser Cys Ser Val Pro Glu Gly Met Val Cys 180 185
190 aag ccg tcc aag tcc gtg cac ctc acg gtg ctg cgg tgg cgc tgt cag
624 Lys Pro Ser Lys Ser Val His Leu Thr Val Leu Arg Trp Arg Cys Gln
195 200 205 cgg cgc ggg ggc cag cgc tgc ggc tgg att ccc atc cag tac
ccc atc 672 Arg Arg Gly Gly Gln Arg Cys Gly Trp Ile Pro Ile Gln Tyr
Pro Ile 210 215 220 att tcc gag tgc aag tgc tcg tgc tag 699 Ile Ser
Glu Cys Lys Cys Ser Cys 225 230 2 232 PRT human 2 Met Glu Arg Cys
Pro Ser Leu Gly Val Thr Leu Tyr Ala Leu Val Val 1 5 10 15 Val Leu
Gly Leu Arg Ala Thr Pro Ala Gly Gly Gln His Tyr Leu His 20 25 30
Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro Leu Val Asp Leu Ile Glu 35
40 45 His Pro Asp Pro Ile Phe Asp Pro Lys Glu Lys Asp Leu Asn Glu
Thr 50 55 60 Leu Leu Arg Ser Leu Leu Gly Gly His Tyr Asp Pro Gly
Phe Met Ala 65 70 75 80 Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly Gly
Gly Gly Ala Ala Gly 85 90 95 Gly Ala Glu Asp Leu Ala Glu Leu Asp
Gln Leu Leu Arg Gln Arg Pro 100 105 110 Ser Gly Ala Met Pro Ser Glu
Ile Lys Gly Leu Glu Phe Ser Glu Gly 115 120 125 Leu Ala Gln Gly Lys
Lys Gln Arg Leu Ser Lys Lys Leu Arg Arg Lys 130 135 140 Leu Gln Met
Trp Leu Trp Ser Gln Thr Phe Cys Pro Val Leu Tyr Ala 145 150 155 160
Trp Asn Asp Leu Gly Ser Arg Phe Trp Pro Arg Tyr Val Lys Val Gly 165
170 175 Ser Cys Phe Ser Lys Arg Ser Cys Ser Val Pro Glu Gly Met Val
Cys 180 185 190 Lys Pro Ser Lys Ser Val His Leu Thr Val Leu Arg Trp
Arg Cys Gln 195 200 205 Arg Arg Gly Gly Gln Arg Cys Gly Trp Ile Pro
Ile Gln Tyr Pro Ile 210 215 220 Ile Ser Glu Cys Lys Cys Ser Cys 225
230 3 14 PRT frog and mouse 3 Gln Met Trp Leu Trp Ser Gln Thr Phe
Cys Pro Val Leu Tyr 1 5 10 4 12 PRT frog and mouse 4 Arg Phe Trp
Pro Arg Tyr Val Lys Val Gly Ser Cys 1 5 10 5 14 PRT frog and mouse
5 Ser Lys Arg Ser Cys Ser Val Pro Glu Gly Met Val Cys Lys 1 5 10 6
8 PRT frog and mouse 6 Leu Arg Trp Arg Cys Gln Arg Arg 1 5 7 8 PRT
frog and mouse 7 Ile Ser Glu Cys Lys Cys Ser Cys 1 5 8 36 DNA
Artificial Sequence Primer 8 gactcgagtc gacatcgcag atgtggctgt
ggtcac 36 9 37 DNA Artificial Sequence Primer 9 ccaagcttct
agaattcgca ggaacactta cactcgg 37 10 1180 DNA Mouse CDS
(421)..(1161) modified_base (16)..(16) n = a,c,g, or t 10
taactcactc attagncacc ccagccttac actttatgct tccggctcgt atgttgtgtg
60 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
tacgccaagc 120 tcgaaattaa ccctcactaa agggaacaaa agctggagct
ccaccgcggt ggcggccgcc 180 ttcccaagta gagcggcggg ggggaattgc
gaccaactcg tgcgcgtctt ctgcnccgcg 240 gcgggagccg gcgctgcgcg
aacggctctc ctcgcagctc atgctgcctg ccctgcgcct 300 gctcagcctc
gggtgagcca cctccggagg gaccggggag cgcggcagcg ccgcggactc 360
ggcgtgctct cctccgggga cgcgggacga agaggcagcc ccggggcgcg cgcgggaggc
420 atg gag cgc tgc ccc agc ctg ggg gtc acc ctc tac gcc ctg gtg gtg
468 Met Glu Arg Cys Pro Ser Leu Gly Val Thr Leu Tyr Ala Leu Val Val
1 5 10 15 gtc ctg ggg ctg cgg gca gca cca gcc ggc ggc cag cac tat
cta cac 516 Val Leu Gly Leu Arg Ala Ala Pro Ala Gly Gly Gln His Tyr
Leu His 20 25 30 atc cgc cca gca ccc agc gac aac ctg ccc ttg gtg
gac ctc atc gaa 564 Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro Leu Val
Asp Leu Ile Glu 35 40 45 cat cca gac cct atc ttt gac cct aag gag
aag gat ctg aac gag acg 612 His Pro Asp Pro Ile Phe Asp Pro Lys Glu
Lys Asp Leu Asn Glu Thr 50 55 60 ctg ctg cgc tcg ctg ctc ggg ggc
cac tac gac ccg ggc ttt atg gcc 660 Leu Leu Arg Ser Leu Leu Gly Gly
His Tyr Asp Pro Gly Phe Met Ala 65 70 75 80 act tcg ccc cca gag gac
cga ccc gga ggg ggc ggg gga ccg gct gga 708 Thr Ser Pro Pro Glu Asp
Arg Pro Gly Gly Gly Gly Gly Pro Ala Gly 85 90 95 ggt gcc gag gac
ctg gcg gag ctg gac cag ctg ctg cgg cag cgg ccg 756 Gly Ala Glu Asp
Leu Ala Glu Leu Asp Gln Leu Leu Arg Gln Arg Pro 100 105 110 tcg ggg
gcc atg ccg agc gag atc aaa ggg ctg gag ttc tcc gag ggc 804 Ser Gly
Ala Met Pro Ser Glu Ile Lys Gly Leu Glu Phe Ser Glu Gly 115 120 125
ttg gcc caa ggc aag aaa cag cgc ctg agc aag aag ctg agg agg aag 852
Leu Ala Gln Gly Lys Lys Gln Arg Leu Ser Lys Lys Leu Arg Arg Lys 130
135 140 tta cag atg tgg ctg tgg tca cag acc ttc tgc ccg gtg ctg tac
gcg 900 Leu Gln Met Trp Leu Trp Ser Gln Thr Phe Cys Pro Val Leu Tyr
Ala 145 150 155 160 tgg aat gac cta ggc agc cgc ttt tgg cca cgc tac
gtg aag gtg ggc 948 Trp Asn Asp Leu Gly Ser Arg Phe Trp Pro Arg Tyr
Val Lys Val Gly 165 170 175 agc tgc ttc agc aag cgc tcc tgc tct gtg
ccc gag ggc atg gtg tgt 996 Ser Cys Phe Ser Lys Arg Ser Cys Ser Val
Pro Glu Gly Met Val Cys 180 185 190 aag cca tcc aag tct gtg cac ctc
acg gtg ctg cgg tgg cgc tgt cag 1044 Lys Pro Ser Lys Ser Val His
Leu Thr Val Leu Arg Trp Arg Cys Gln 195 200 205 cgg cgc ggg ggt cag
cgc tgc ggc tgg att ccc atc cag tac ccc atc 1092 Arg Arg Gly Gly
Gln Arg Cys Gly Trp Ile Pro Ile Gln Tyr Pro Ile 210 215 220 att tcc
gag tgt aag tgt tcc tgc tag aac tcg ggg ggg gcc cct gcc 1140 Ile
Ser Glu Cys Lys Cys Ser Cys Asn Ser Gly Gly Ala Pro Ala 225 230 235
cgc gcc cag aca ctt gat gga tcccccgggc tgagatttt 1180 Arg Ala Gln
Thr Leu Asp Gly 240 245 11 232 PRT Mouse 11 Met Glu Arg Cys Pro Ser
Leu Gly Val Thr Leu Tyr Ala Leu Val Val 1 5 10 15 Val Leu Gly Leu
Arg Ala Ala Pro Ala Gly Gly Gln His Tyr Leu His 20 25 30 Ile Arg
Pro Ala Pro Ser Asp Asn Leu Pro Leu Val Asp Leu Ile Glu 35 40 45
His Pro Asp Pro Ile Phe Asp Pro Lys Glu Lys Asp Leu Asn Glu Thr 50
55 60 Leu Leu Arg Ser Leu Leu Gly Gly His Tyr Asp Pro Gly Phe Met
Ala 65 70 75 80 Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly Gly Gly Gly
Pro Ala Gly 85 90 95 Gly Ala Glu Asp Leu Ala Glu Leu Asp Gln Leu
Leu Arg Gln Arg Pro 100 105 110 Ser Gly Ala Met Pro Ser Glu Ile Lys
Gly Leu Glu Phe Ser Glu Gly 115 120 125 Leu Ala Gln Gly Lys Lys Gln
Arg Leu Ser Lys Lys Leu Arg Arg Lys 130 135 140 Leu Gln Met Trp Leu
Trp Ser Gln Thr Phe Cys Pro Val Leu Tyr Ala 145 150 155 160 Trp Asn
Asp Leu Gly Ser Arg Phe Trp Pro Arg Tyr Val Lys Val Gly 165 170 175
Ser Cys Phe Ser Lys Arg Ser Cys Ser Val Pro Glu Gly Met Val Cys 180
185 190 Lys Pro Ser Lys Ser Val His Leu Thr Val Leu Arg Trp Arg Cys
Gln 195 200 205 Arg Arg Gly Gly Gln Arg Cys Gly Trp Ile Pro Ile Gln
Tyr Pro Ile 210 215 220 Ile Ser Glu Cys Lys Cys Ser Cys 225 230 12
18 DNA Artificial Sequence Primer 12 caracnttyt gyccngtn 18 13 26
DNA Artificial Sequence Primer 13 ttytggccnm gntaygtnaa rgtngg 26
14 17 DNA Artificial Sequence Primer 14 ccngarggna tggtntg 17 15 17
DNA Artificial Sequence Primer 15 canswrcayt trcaytc 17 16 17 DNA
Artificial Sequence Primer 16 canaccatnc cytcngg 17 17 17 DNA
Artificial Sequence Primer 17 cknckytgrc anckcca 17 18 18 DNA
Artificial Sequence Primer 18 cagatgtggc tgtggtca 18 19 6 PRT Mouse
19 Gln Met Trp Leu Trp Ser 1 5 20 18 DNA Mouse 20 gcaggaacac
ttacactc 18 21 6 PRT Mouse 21 Glu Cys Lys Cys Ser Cys 1 5 22 20 DNA
Artificial Sequence Oligonucleotide 22 garggnatgg tntgyaarcc 20 23
449 PRT Human 23 Met Glu Arg Cys Pro Ser Leu Gly Val Thr Leu Tyr
Ala Leu Val Val 1 5 10 15 Val Leu Gly Leu Arg Ala Thr Pro Ala Gly
Gly Gln His Tyr Leu His 20 25 30 Ile Arg Pro Ala Pro Ser Asp Asn
Leu Pro Leu Val Asp Leu Ile Glu 35 40 45 His Pro Asp Pro Ile Phe
Asp Pro Lys Glu Lys Asp Leu Asn Glu Thr 50 55 60 Leu Leu Arg Ser
Leu Leu Gly Gly His Tyr Asp Pro Gly Phe Met Ala 65 70 75 80 Thr Ser
Pro Pro Glu Asp Arg Pro Gly Gly Gly Gly Gly Ala Ala Gly 85 90 95
Gly Ala Glu Asp Leu Ala Glu Leu Asp Gln Leu Leu Arg Gln Arg Pro 100
105 110 Ser Gly Ala Met Pro Ser Glu Ile Lys Gly Leu Glu Phe Ser Glu
Gly 115 120 125 Leu Ala Gln Gly Leu Gln Met Trp Leu Trp Ser Gln Thr
Phe Cys Pro 130 135 140 Val Leu Tyr Ala Trp Asn Asp Leu Gly Ser Arg
Phe Trp Pro Arg Tyr 145 150 155 160 Val Lys Val Gly Ser Cys Phe Ser
Lys Arg Ser Cys Ser Val Pro Glu 165 170 175 Gly Met Val Cys Lys Pro
Ser Lys Ser Val His Leu Thr Val Leu Arg 180 185 190 Trp Arg Cys Gln
Arg Arg Gly Gly Gln Arg Cys Gly Trp Ile Pro Ile 195 200 205 Gln Tyr
Pro Ile Ile Ser Glu Cys Lys Cys Ser Cys Ser Gly Asp Lys 210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225
230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys 24 6
PRT Human 24 Lys Lys Leu Arg Arg Lys 1 5 25 12 PRT Human 25 Lys Lys
Gln Arg Leu Ser Lys Lys Leu Arg Arg Lys 1 5 10 26 7 PRT Human 26
Ser Glu Cys Lys Cys Ser Cys 1 5 27 13 PRT frog and mouse 27 Lys Lys
His Arg Leu Ser Lys Lys Leu Arg Arg Lys Leu 1 5 10
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