U.S. patent application number 13/172664 was filed with the patent office on 2011-10-20 for connective tissue growth factor-3 (ctgf-3).
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Arvind Chopra, Reinhard Ebner, Steven M. Ruben.
Application Number | 20110256145 13/172664 |
Document ID | / |
Family ID | 46300418 |
Filed Date | 2011-10-20 |
United States Patent
Application |
20110256145 |
Kind Code |
A1 |
Ebner; Reinhard ; et
al. |
October 20, 2011 |
Connective Tissue Growth Factor-3 (CTGF-3)
Abstract
The present invention relates to a novel connective tissue
growth factor-3 protein which is a member of the growth factor
superfamily. In particular, isolated nucleic acid molecules are
provided encoding the human connective tissue growth factor-3
protein. Connective tissue growth factor-3 polypeptides are also
provided as are vectors, host cells and recombinant methods for
producing the same. Also provided are diagnostic and therapeutic
methods for detecting and treating connective tissue related
disorders.
Inventors: |
Ebner; Reinhard;
(Gaithersburg, MD) ; Chopra; Arvind;
(Gaithersburg, MD) ; Ruben; Steven M.;
(Brookeville, MD) |
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
46300418 |
Appl. No.: |
13/172664 |
Filed: |
June 29, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12761083 |
Apr 15, 2010 |
7994284 |
|
|
13172664 |
|
|
|
|
11962282 |
Dec 21, 2007 |
7737257 |
|
|
12761083 |
|
|
|
|
10721336 |
Nov 26, 2003 |
|
|
|
11962282 |
|
|
|
|
09712142 |
Nov 15, 2000 |
|
|
|
10721336 |
|
|
|
|
08966020 |
Nov 7, 1997 |
|
|
|
09712142 |
|
|
|
|
60030720 |
Nov 8, 1996 |
|
|
|
Current U.S.
Class: |
424/139.1 ;
435/320.1; 435/325; 435/440; 435/6.1; 435/69.1; 514/7.6; 530/324;
530/326; 530/327; 530/350; 530/387.9; 536/23.5; 536/24.5 |
Current CPC
Class: |
A61P 19/04 20180101;
C07H 21/04 20130101; A61P 5/00 20180101; A61P 43/00 20180101; Y10S
530/866 20130101; C07K 14/475 20130101 |
Class at
Publication: |
424/139.1 ;
536/23.5; 536/24.5; 435/320.1; 435/440; 435/325; 435/69.1; 530/350;
530/327; 530/324; 530/326; 530/387.9; 514/7.6; 435/6.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; C12P 21/06 20060101 C12P021/06; A61P 19/04 20060101
A61P019/04; C07K 7/08 20060101 C07K007/08; C07K 16/22 20060101
C07K016/22; A61K 38/18 20060101 A61K038/18; C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C07K 14/475 20060101
C07K014/475 |
Claims
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding a polypeptide comprising amino acids from about -19 to
about 231 in SEQ ID NO:2; (b) a nucleotide sequence encoding a
polypeptide comprising amino acids from about -18 to about 231 in
SEQ ID NO:2; (c) a nucleotide sequence encoding a polypeptide
comprising amino acids from about 1 to about 231 in SEQ ID NO:2;
(d) a nucleotide sequence encoding a polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 97756; (e) a nucleotide sequence encoding the mature
connective tissue growth factor-3 polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC.TM. Deposit
No. 97756; and (f) a nucleotide sequence complementary to any of
the nucleotide sequences in (a), (b), (c), (d), or (e).
2. The nucleic acid molecule of claim 1, wherein said
polynucleotide has the nucleotide sequence of the cDNA clone
contained in ATCC.TM. Deposit No. 97756.
3. The nucleic acid molecule of claim 1, wherein said
polynucleotide has the nucleotide sequence encoding the connective
tissue growth factor-3 polypeptide having the amino acid sequence
encoded by the cDNA clone contained in ATCC.TM. Deposit No.
97756.
4. The nucleic acid molecule of claim 1, wherein said
polynucleotide has the nucleotide sequence encoding the mature
connective tissue growth factor-3 polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC.TM. Deposit
No. 97756.
5. An isolated nucleic acid molecule, comprising a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence identical to a
nucleotide sequence in (a), (b), (c), (d), (e), or (f) of claim 1,
wherein said polynucleotide which hybridizes does not hybridize
under stringent hybridization conditions to a polynucleotide having
a nucleotide sequence consisting of only A residues or of only T
residues.
6. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a connective tissue growth factor-3 polypeptide having an amino
acid sequence in (a), (b), (c), (d), (e), or (f) of claim 1.
7. The isolated nucleic acid molecule of claim 6, which encodes an
epitope-bearing portion of a connective tissue growth factor-3
polypeptide selected from the group consisting of: a polypeptide
comprising amino acid residues from about 36 to about 49 in SEQ ID
NO:2; a polypeptide comprising amino acid residues from about 75 to
about 109 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about 115 to about 139 in SEQ ID NO:2; and a
polypeptide comprising amino acid residues from about 196 to about
230 in SEQ ID NO:2.
8. A method for making a recombinant vector, comprising inserting
an isolated nucleic acid molecule of claim 1 into a vector.
9. A recombinant vector produced by the method of claim 8.
10. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 9 into a host cell.
11. A recombinant host cell produced by the method of claim 10.
12. A recombinant method for producing a connective tissue growth
factor-3 polypeptide, comprising culturing the recombinant host
cell of claim 11 under conditions such that said polypeptide is
expressed and recovering said polypeptide.
13. An isolated connective tissue growth factor-3 polypeptide
having an amino acid sequence at least 95% identical to a sequence
selected from the group consisting of: (a) amino acids from about
-19 to about 231 in SEQ ID NO:2; (b) amino acids from about -18 to
about 231 in SEQ ID NO:2; (c) amino acids from about 1 to about 231
in SEQ ID NO:2; (d) the amino acid sequence of the connective
tissue growth factor-3 polypeptide having the amino acid sequence
encoded by the cDNA clone contained in ATCC.TM. Deposit No. 97756;
(e) the amino acid sequence of the mature connective tissue growth
factor-3 polypeptide having the amino acid sequence encoded by the
cDNA clone contained in ATCC.TM. Deposit No. 97756; and (f) the
amino acid sequence of an epitope-bearing portion of any one of the
polypeptides of (a), (b), (c), (d), or (e).
14. An isolated polypeptide of claim 13, comprising an
epitope-bearing portion of connective tissue growth factor-3,
wherein said portion is selected from the group consisting of: a
polypeptide comprising amino acid residues from about 36 to about
49 in SEQ ID NO:2; a polypeptide comprising amino acid residues
from about 75 to about 109 in SEQ ID NO:2; a polypeptide comprising
amino acid residues from about 115 to about 139 in SEQ ID NO:2; and
a polypeptide comprising amino acid residues from about 196 to
about 230 in SEQ ID NO:2.
15. The isolated polypeptide of claim 13, which is produced or
contained in a recombinant host cell.
16. The isolated polypeptide of claim 15, wherein said recombinant
host cell is mammalian.
17. An isolated antibody or antibody fragment that binds
specifically to a connective tissue growth factor-3 polypeptide of
claim 13.
18. A method for treating an individual in need of a decreased
level of connective tissue growth factor-3 activity, comprising
administering to said individual a composition comprising the
isolated antibody or antibody fragment of claim 17.
19. A method for treating an individual in need of an increased
level of connective tissue growth factor-3 activity, comprising
administering to said individual a composition comprising the
isolated connective tissue growth factor-3 polypeptide of claim
13.
20. A diagnostic method, comprising: (a) assaying connective tissue
growth factor-3 gene expression level in mammalian cells or body
fluid; and (b) comparing said connective tissue growth factor-3
gene expression level with a standard connective tissue growth
factor-3 gene expression level, whereby an increase or decrease in
said connective tissue growth factor-3 gene expression level
compared to said standard is indicative of a connective
tissue-related disorder.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of Ser. No. 12/761,083 filed
Apr. 15, 2010, which is a division of U.S. application Ser. No.
11/962,282, filed Dec. 21, 2007, which is a continuation of U.S.
application Ser. No. 10/721,336, filed Nov. 26, 2003, which is a
continuation of U.S. application Ser. No. 09/712,142, filed Nov.
15, 2000, which is a continuation of U.S. application Ser. No.
08/966,020, filed Nov. 7, 1997, which claims the benefit of the
filing date of U.S. provisional Application No. 60/030,720, filed
Nov. 8, 1996. Each of said applications is incorporated herein by
reference it its entirety.
STATEMENT UNDER 37 C.F.R. .sctn.1.77(b)(5)
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as a text document. The document is
entitled "PF319C3D2_SequenceListing.txt" (16,384 bytes, created
Jun. 29, 2011), and is hereby incorporated by reference in its
entirety herein.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to a novel connective tissue
growth factor. More specifically, isolated nucleic acid molecules
are provided encoding a human connective tissue growth factor-3.
Connective tissue growth factor-3 polypeptides are also provided,
as are vectors, host cells, and recombinant methods for producing
the same. Also provided are diagnostic and therapeutic methods for
detecting and treating connective tissue related disorders.
[0005] 2. Background Art
[0006] Growth factors are a class of secreted cysteine-rich
polypeptides that stimulate target cells to proliferate,
differentiate, and organize in developing tissues. The action of
growth factors is dependent on their binding to specific receptors,
which stimulate a signaling event within the cell. Examples of some
well-studied growth factors include platelet-derived growth factor
(PDGF), insulin-like growth factor (IGF-I), transforming growth
factor beta (TGF-.beta.), transforming growth factor alpha
(TGF-.alpha.), epidermal growth factor (EGF), and fibroblast growth
factor (FGF). This group of growth factors is important for normal
growth, differentiation, morphogenesis of the cartilaginous
skeleton of an embryo, and cell growth. Among some of the functions
that have been reported for these growth factors are wound healing,
tissue repair/regeneration, implant fixation, and stimulating bone
mass increase.
[0007] PDGF is a cationic, heat-stable protein found in the
alpha-granules of circulating platelets and is known to be a
mitogen and chemotactic agent for connective tissue cells such as
fibroblasts and smooth muscle cells. Because of the activities of
this molecule, PDGF is believed to be a major factor involved in
the normal healing of wounds and pathologically contributes to such
diseases as atherosclerosis and fibrotic diseases. PDGF is a
dimeric molecule consisting of an A chain and a B chain. The chains
form heterodimers or homodimers and all combinations isolated to
date are biologically active.
[0008] Studies on the role of various growth factors in tissue
regeneration and repair have led to the discovery of PDGF-like
proteins. These proteins share both immunological and biological
activities with PDGF and can be blocked with antibodies specific to
PDGF.
[0009] U.S. Pat. No. 5,408,040 to Grotendorst et al. (1995)
discloses a PDGF-like protein called Connective Tissue Growth
Factor (CTGF) that reportedly plays a significant role in the
normal development, growth, and repair of human tissue. The
discovery of the CTGF protein and the cloning of the cDNA encoding
the protein was reportedly significant in that it was a previously
unknown growth factor having mitogenic and chemotactic activities
for connective tissue cells. Although the biological activity of
CTGF was similar to that of PDGF, CTGF is the product of a gene
unrelated to the A or B chain genes of PDGF.
[0010] Since CTGF is produced by endothelial and fibroblastic
cells, both of which are present at the site of a wound, it is
probable that CTGF functions as a growth factor in wound healing.
Accordingly, it is believed that the CTGF polypeptide could be used
as a therapeutic in cases in which there is impaired healing of
skin wounds or where there is a need to augment the normal healing
process.
[0011] Pathologically, CTGF may also be involved in diseases in
which there is an overgrowth of connective tissue cells or an
enhanced production of extracellular matrix components. Such
diseases include cancer, fibrosis, and atherosclerosis. For
example, it has been shown that CTGF gene expression is elevated in
the skin of patients with systemic sclerosis (SSc). Igarashi et
al., J. Invest. Dermatol. 105:280-284 (1995). CTGF gene expression
has also recently been demonstrated in several fibrotic skin
diseases, such as localized scleroderma, keloid scars, nodular
fasciatus, and eosinophilic fasciatus, suggesting a pathogenic role
for this molecule in skin fibrosis. Igarashi et al., J. Invest.
Dermatol. 106:729-733 (1996). Oemar et al., Circulation 92(8),
Supp't 1, Abstract 0811 (October 1995) have reported that human
CTGF is expressed at 5-10 fold higher levels in the aorta, a tissue
prone to develop atherosclerosis, as compared to expression levels
in internal mammary arteries, which are resistant to
atherosclerosis. Their results suggest that hCTGF may play an
essential role in the development and progression of
atherosclerosis. Therapeutically, it has been reported in U.S. Pat.
No. 5,408,040 to Grotendorst et al. (1995) that CTGF antibodies or
fragments of the antibody could be used to neutralize the
biological activity of CTGF in diseases where CTGF is inducing the
overgrowth of tissue. Additionally, antibodies to CTGF polypeptide
or fragments could be valuable as diagnostic tools to aid in the
detection of diseases in which CTGF is a pathological factor.
Id.
[0012] Due to the important role of CTGF in the development and
repair of human tissue, as well as its role in the development and
progression of various connective-tissue related disorders, there
is a clear need in the art for the identification of new connective
tissue growth factors that can be utilized in the development of
diagnostics and therapeutics for various connective tissue related
disorders.
BRIEF SUMMARY OF THE INVENTION
[0013] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding the connective
tissue growth factor-3 polypeptide having the amino acid sequence
shown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNA
clone deposited in a bacterial host as ATCC.TM. Deposit Number
97756 on Oct. 10, 1996.
[0014] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of connective tissue growth factor-3
polypeptides or peptides by recombinant techniques.
[0015] The invention further provides an isolated connective tissue
growth factor-3 polypeptide having an amino acid sequence encoded
by a polynucleotide described herein.
[0016] The invention further provides methods for isolating
antibodies that bind specifically to a connective tissue growth
factor-3 polypeptide having an amino acid sequence as described
herein. Such antibodies are useful diagnostically or
therapeutically as described below.
[0017] The present invention also provides a screening method for
identifying compounds capable of enhancing or inhibiting a cellular
response induced by connective tissue growth factor-3, which
involves contacting cells that express connective tissue growth
factor-3 with the candidate compound, assaying a cellular response,
and comparing the cellular response to a standard cellular
response, the standard being assayed when contact is made in the
absence of the candidate compound; whereby, an increased cellular
response over the standard indicates that the compound is an
agonist and a decreased cellular response over the standard
indicates that the compound is an antagonist.
[0018] In another aspect, a screening assay for agonists and
antagonists is provided, which involves determining the effect a
candidate compound has on connective tissue growth factor-3 binding
to the connective tissue growth factor-3 receptor. In particular,
the method involves contacting the connective tissue growth
factor-3 receptor with a connective tissue growth factor-3
polypeptide and a candidate compound and determining whether
connective tissue growth factor-3 polypeptide binding to the
connective tissue growth factor-3 receptor is increased or
decreased due to the presence of the candidate compound.
[0019] The present inventors have discovered that connective tissue
growth factor-3 is expressed in multiple human tissues, including,
for example, ovary, heart, lung, skeletal muscle, adrenal medulla,
adrenal cortex, thymus, prostate, small intestine, and colon, as
well as in Hela cells. It is also expected that connective tissue
growth factor-3 will be expressed in fibrotic human skin and liver.
For a number of connective tissue disorders or clinical states, it
is believed that significantly higher or lower levels of connective
tissue growth factor-3 gene expression can be detected in certain
tissues (e.g., ovary, testis, fibrotic skin and liver) or bodily
fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid)
taken from an individual having such a disorder, relative to a
"standard" connective tissue growth factor-3 gene expression level,
i.e., the connective tissue growth factor-3 expression level in
tissue or bodily fluids from an individual not having the
connective tissue related disorder. Thus, the invention provides a
diagnostic method useful during diagnosis of a connective tissue
related disorder, which involves: (a) assaying connective tissue
growth factor-3 gene expression level in cells or body fluid of an
individual; (b) comparing the connective tissue growth factor-3
gene expression level with a standard connective tissue related
growth factor-3 gene expression level, whereby an increase or
decrease in the assayed connective tissue growth factor-3 gene
expression level compared to the standard expression level is
indicative of a connective tissue related disorder.
[0020] An additional aspect of the invention is related to a method
for treating an individual in need of an increased level of
connective tissue growth factor-3 activity in the body comprising
administering to such an individual a composition comprising a
therapeutically effective amount of an isolated connective tissue
growth factor-3 polypeptide of the invention or an agonist
thereof.
[0021] A still further aspect of the invention is related to a
method for treating an individual in need of a decreased level of
connective tissue growth factor-3 activity in the body comprising,
administering to such an individual a composition comprising a
therapeutically effective amount of a connective tissue growth
factor-3 antagonist. Preferred antagonists for use in the present
invention are connective tissue growth factor-3-specific
antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0022] FIG. 1A and 1B show the nucleotide (SEQ ID NO:1) and deduced
amino acid (SEQ ID NO:2) sequences of connective tissue growth
factor-3. The protein has a leader sequence of about 19 amino acid
residues (underlined) and a deduced molecular weight of about 26
kDa.
[0023] FIG. 2 shows the regions of similarity between the amino
acid sequences of the connective tissue growth factor-3 protein
(SEQ ID NO:2) and connective tissue growth factor-1 (SEQ ID
NO:3).
[0024] FIG. 3 shows an analysis of the connective tissue growth
factor-3 amino acid sequence. Alpha, beta, turn, and coil regions;
hydrophilicity and hydrophobicity; amphipathic regions; flexible
regions; antigenic index and surface probability are shown. In the
"Antigenic Index--Jameson-Wolf" graph, amino acid residues 55-68,
94-128, 134-158, and 215-249 in FIG. 1 correspond to the shown
highly antigenic regions of the connective tissue growth factor-3
protein. These highly antigenic fragments in FIG. 1 correspond to
the following fragments, respectively, in SEQ ID NO:2: amino acid
residues about 36 to about 49, about 75 to about 109, about 115 to
about 139, and about 196 to about 230.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a connective tissue
growth factor-3 polypeptide having the amino acid sequence shown in
SEQ ID NO:2, which was determined by sequencing a cloned cDNA. The
connective tissue growth factor-3 protein of the present invention
shares sequence homology with connective tissue growth factor-1
(FIG. 2) (SEQ ID NO:3). The nucleotide sequence shown in FIG. 1
(SEQ ID NO:1) was obtained by sequencing a cDNA clone, which was
deposited Oct. 10, 1996 at the American Type Culture Collection,
10801 University Blvd., Manassas, Va. 20110-2209, USA, and given
accession number 97756. The deposited clone is contained in the
Uni-Zap XR vector (Stratagene, LaJolla, Calif.).
[0026] Nucleic Acid Molecules
[0027] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0028] Using the information provided herein, such as the
nucleotide sequence in SEQ ID NO:1, a nucleic acid molecule of the
present invention encoding a connective tissue growth factor-3
polypeptide may be obtained using standard cloning and screening
procedures, such as those for cloning cDNAs using mRNA as starting
material. Illustrative of the invention, the nucleic acid molecule
described in SEQ ID NO:1 was discovered in a cDNA library derived
from human osteoblasts. The gene was also identified in cDNA
libraries from the following tissues: ovary, testis, heart, lung,
skeletal muscle, adrenal medulla, adrenal cortex, thymus, prostate,
small intestine, and colon.
[0029] The determined nucleotide sequence of the connective tissue
growth factor-3 cDNA of SEQ ID NO:1 contains an open reading frame
encoding a protein of 250 amino acid residues, a predicted leader
sequence of about 19 amino acid residues, and a deduced molecular
weight of about 26 kDa. The connective tissue growth factor-3
protein shown in SEQ ID NO:2 is about 44% identical and about 59%
similar to human connective tissue growth factor-1 (SEQ ID NO:3)
(FIG. 2).
[0030] The present invention also provides the mature form of the
connective tissue growth factor-3 protein of the present invention.
According to the signal hypothesis, proteins secreted by mammalian
cells have a signal or secretory leader sequence which is cleaved
from the mature protein once export of the growing protein chain
across the rough endoplasmic reticulum has been initiated. Most
mammalian cells and even insect cells cleave secreted proteins with
the same specificity. However, in some cases, cleavage of a
secreted protein is not entirely uniform, which results in two or
more mature species on the protein. Further, it has long been known
that the cleavage specificity of a secreted protein is ultimately
determined by the primary structure of the complete protein, that
is, it is inherent in the amino acid sequence of the polypeptide.
Therefore, the present invention provides a nucleotide sequence
encoding the mature connective tissue growth factor-3 polypeptide
having the amino acid sequence encoded by the cDNA clone contained
in the host identified as ATCC.TM. Deposit No. 97756 and as shown
in SEQ ID NO:2. By the mature connective tissue growth factor-3
protein having the amino acid sequence encoded by the cDNA clone
contained in the host identified as ATCC.TM. Deposit 97756 is meant
the mature form of the connective tissue growth factor-3 protein
produced by expression in a mammalian cell (e.g., COS cells, as
described below) of the complete open reading frame encoded by the
human DNA sequence of the clone contained in the vector in the
deposited host. As indicated below, the mature connective tissue
growth factor-3 having the amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No. 97756 may or may not differ
from the predicted "mature" connective tissue growth factor-3
protein shown in SEQ ID NO:2 (amino acids from about 1 to about
231) depending on the accuracy of the predicted cleavage site based
on computer analysis.
[0031] Methods for predicting whether a protein has a secretory
leader as well as the cleavage point for that leader sequence are
available. For instance, the methods of McGeoch (Virus Res.
3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690
(1986)) can be used. The accuracy of predicting the cleavage points
of known mammalian secretory proteins for each of these methods is
in the range of 75-80%. von Heinje, supra. However, the two methods
do not always produce the same predicted cleavage point(s) for a
given protein.
[0032] In the present case, the predicted amino acid sequence of
the complete connective tissue growth factor-3 polypeptides of the
present invention were analyzed by a computer program ("PSORT") (K.
Nakai and M. Kanehisa, Genomics 14:897-911 (1992)), which is an
expert system for predicting the cellular location of a protein
based on the amino acid sequence. As part of this computational
prediction of localization, the methods of McGeoch and von Heinje
are incorporated. The analysis by the PSORT program predicted the
cleavage site between amino acids -1 and 1 in SEQ ID NO:2.
Thereafter, the complete amino acid sequences were further analyzed
by visual inspection, applying a simple form of the (-1,-3) rule of
von Heinje. von Heinje, supra. Thus, the leader sequence for the
connective tissue growth factor-3 protein is predicted to consist
of amino acid residues from about -19 to about -1 in SEQ ID NO:2,
while the mature connective tissue growth factor-3 protein is
predicted to consist of residues from about 1 to about 231.
[0033] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors discussed above, as well as the
variability of cleavage sites for leaders in different known
proteins, the actual connective tissue growth factor-3 polypeptide
encoded by the deposited cDNA comprises about 250 amino acids, but
may be anywhere in the range of 235 to 265 amino acids; and the
actual leader sequence of this protein is about 19 amino acids, but
may be anywhere in the range of about 15 to about 25 amino
acids.
[0034] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
[0035] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0036] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) shown
in SEQ ID NO:1; DNA molecules comprising the coding sequence for
the mature connective tissue growth factor-3 protein; and DNA
molecules which comprise a sequence substantially different from
those described above but which, due to the degeneracy of the
genetic code, still encode the connective tissue growth factor-3
protein. Of course, the genetic code is well known in the art.
Thus, it would be routine for one skilled in the art to generate
such degenerate variants.
[0037] In addition, the present inventors have identified the
following cDNA clones related to extensive portions of SEQ ID NO:1:
HSNAA66R (SEQ ID NO:10) and HSVAF4OR (SEQ ID NO:11). The following
public ESTs, which relate to portions of SEQ ID NO:1, have also
been identified: AA385680 (SEQ ID NO:12) and C01967 (SEQ ID
NO:13).
[0038] In another aspect, the invention provides isolated nucleic
acid molecules encoding the connective tissue growth factor-3
polypeptide having an amino acid sequence encoded by the cDNA clone
contained in the plasmid deposited as ATCC.TM. Deposit No. 97756 on
Oct. 10, 1996. In a further embodiment, nucleic acid molecules are
provided encoding the mature connective tissue growth factor-3
polypeptide or the full length connective tissue growth factor-3
polypeptide lacking the N-terminal methionine. The invention also
provides an isolated nucleic acid molecule having the nucleotide
sequence shown in SEQ ID NO:1 or the nucleotide sequence of the
connective tissue growth factor-3 cDNA contained in the
above-described deposited clone, or a nucleic acid molecule having
a sequence complementary to one of the above sequences. Such
isolated molecules, particularly DNA molecules, are useful as
probes for gene mapping, by in situ hybridization with chromosomes,
and for detecting expression of the connective tissue growth
factor-3 gene in human tissue, for instance, by Northern blot
analysis.
[0039] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated nucleic acid molecule having the nucleotide sequence
of the deposited cDNA or the nucleotide sequence shown in SEQ ID
NO:1 is intended fragments at least about 15 nt, and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably, at least about 40 nt in
length which are useful as diagnostic probes and primers as
discussed herein. Of course, larger fragments 50, 75, 100, 125,
150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,
475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775,
800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075,
1100, 1125, 1150, 1175, 1200, 1225, 1250, or 1275 nt in length are
also useful according to the present invention as are fragments
corresponding to most, if not all, of the nucleotide sequence of
the deposited cDNA or as shown in SEQ ID NO:1. By a fragment at
least 20 nt in length, for example, is intended fragments which
include 20 or more contiguous bases from the nucleotide sequence of
the deposited cDNA or the nucleotide sequence as shown in SEQ ID
NO:l.
[0040] Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding epitope-bearing portions of
the connective tissue growth factor-3 protein. In particular, such
nucleic acid fragments of the present invention include nucleic
acid molecules encoding: a polypeptide comprising amino acid
residues from about 55 to about 68 in FIG. 1 (about 36 to about 49
in SEQ ID NO:2); a polypeptide comprising amino acid residues from
about 94 to about 128 in FIG. 1 (about 75 to about 109 in SEQ ID
NO:2); a polypeptide comprising amino acid residues from about 134
to about 158 in FIG. 1 (about 115 to about 139 in SEQ ID NO:2); and
a polypeptide comprising amino acid residues from about 215 to
about 249 in FIG. 1 (about 196 to about 230 in SEQ ID NO:2). The
inventors have determined that the above polypeptide fragments are
antigenic regions of the connective tissue growth factor-3 protein.
Methods for determining other such epitope-bearing portions of the
connective tissue growth factor-3 protein are described in detail
below.
[0041] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clone contained in ATCC.TM.
Deposit 97756. By "stringent hybridization conditions" is intended
overnight incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0042] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide. These are useful as diagnostic probes and
primers as discussed above and in more detail below.
[0043] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in FIG. 1
(SEQ ID NO:1)). Of course, a polynucleotide which hybridizes only
to a poly A sequence (such as the 3' terminal poly(A) tract of the
connective tissue growth factor-3 cDNA shown in FIG. 1 (SEQ ID
NO:1)), or to a complementary stretch of T (or U) resides, would
not be included in a polynucleotide of the invention used to
hybridize to a portion of a nucleic acid of the invention, since
such a polynucleotide would hybridize to any nucleic acid molecule
containing a poly (A) stretch or the complement thereof (e.g.,
practically any double-stranded cDNA clone).
[0044] As indicated, nucleic acid molecules of the present
invention which encode a connective tissue growth factor-3
polypeptide may include, but are not limited to those encoding the
amino acid sequence of the mature polypeptide, by itself; the
coding sequence for the mature polypeptide and additional
sequences, such as those encoding the about 19 amino acid leader or
secretory sequence, such as a pre-, or pro- or prepro-protein
sequence; the coding sequence of the mature polypeptide, with or
without the aforementioned additional coding sequences, together
with additional, non-coding sequences, including for example, but
not limited to introns and non-coding 5' and 3' sequences, such as
the transcribed, non-translated sequences that play a role in
transcription, mRNA processing, including splicing and
polyadenylation signals, for example--ribosome binding and
stability of mRNA; an additional coding sequence which codes for
additional amino acids, such as those which provide additional
functionalities. Thus, the sequence encoding the polypeptide may be
fused to a marker sequence, such as a sequence encoding a peptide
which facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (Qiagen, Inc.), among others, many of
which are commercially available. As described in Gentz et al.,
Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,
hexa-histidine provides for convenient purification of the fusion
protein. The "HA" tag is another peptide useful for purification
which corresponds to an epitope derived from the influenza
hemagglutinin protein, which has been described by Wilson et al.,
Cell 37: 767 (1984). As discussed below, other such fusion proteins
include the connective tissue growth factor-3 fused to Fc at the N-
or C-terminus.
[0045] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs, or derivatives of the connective tissue growth
factor-3 protein. Variants may occur naturally, such as a natural
allelic variant. By an "allelic variant" is intended one of several
alternate forms of a gene occupying a given locus on a chromosome
of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,
New York (1985). Non-naturally occurring variants may be produced
using art-known mutagenesis techniques.
[0046] Such variants include those produced by nucleotide
substitutions, deletions, or additions, which may involve one or
more nucleotides. The variants may be altered in coding regions,
non-coding regions, or both. Alterations in the coding regions may
produce conservative or non-conservative amino acid substitutions,
deletions, or additions. Especially preferred among these are
silent substitutions, additions, and deletions, which do not alter
the properties and activities of the connective tissue growth
factor-3 protein or portions thereof. Also especially preferred in
this regard are conservative substitutions.
[0047] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 95% identical, and more preferably at
least 96%, 97%, 98%, or 99% identical to (a) a nucleotide sequence
encoding the polypeptide having the amino acid sequence in SEQ ID
NO:2; (b) a nucleotide sequence encoding the polypeptide having the
amino acid sequence in SEQ ID NO:2, but lacking the N-terminal
methionine; (c) a nucleotide sequence encoding the polypeptide
having the amino acid sequence at positions from about 1 to about
231 in SEQ ID NO:2; (d) a nucleotide sequence encoding the
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No. 97756; (e) a nucleotide
sequence encoding the mature connective tissue growth factor-3
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No. 97756; or (f) a nucleotide
sequence complementary to any of the nucleotide sequences in (a),
(b), (c), (d), or (e).
[0048] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a connective tissue growth factor-3 polypeptide is
intended that the nucleotide sequence of the polynucleotide is
identical to the reference sequence except that the polynucleotide
sequence may include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence encoding the
connective tissue growth factor-3 polypeptide. In other words, to
obtain a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0049] As a practical matter, whether any particular nucleic acid
molecule is at least 95%, 96%, 97%, 98%, or 99% identical to, for
instance, the nucleotide sequence shown in FIG. 1 or to the
nucleotides sequence of the deposited cDNA clone can be determined
conventionally using known computer programs such as the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711). Bestfit uses the local homology
algorithm of Smith and Waterman, Advances in Applied Mathematics 2:
482-489 (1981), to find the best segment of homology between two
sequences. When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for
instance, 95% identical to a reference sequence according to the
present invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0050] The present application is directed to nucleic acid
molecules at least 95%, 96%, 97%, 98%, or 99% identical to the
nucleic acid sequence shown in SEQ ID NO:1 or to the nucleic acid
sequence of the deposited cDNA, irrespective of whether they encode
a polypeptide having connective tissue growth factor-3 activity.
This is because even where a particular nucleic acid molecule does
not encode a polypeptide having connective tissue growth factor-3
activity, one of skill in the art would still know how to use the
nucleic acid molecule, for instance, as a hybridization probe or a
polymerase chain reaction (PCR) primer. Uses of the nucleic acid
molecules of the present invention that do not encode a polypeptide
having connective tissue growth factor-3 activity include, inter
alia: (1) isolating the connective tissue growth factor-3 gene or
allelic variants thereof in a cDNA library; (2) in situ
hybridization (e.g., "FISH") to metaphase chromosomal spreads to
provide precise chromosomal location of the connective tissue
growth factor-3 gene, as described in Verma et al., Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York
(1988); and Northern Blot analysis for detecting connective tissue
growth factor-3 mRNA expression in specific tissues.
[0051] Preferred, however, are nucleic acid molecules having
sequences at least 95%, 96%, 97%, 98%, or 99% identical to the
nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1) or to the
nucleic acid sequence of the deposited cDNA which do, in fact,
encode a polypeptide having connective tissue growth factor-3
protein activity. By "a polypeptide having connective tissue growth
factor-3 activity" is intended polypeptides exhibiting connective
tissue growth factor-3 activity in a particular biological assay.
For example, it is believed that connective tissue growth factor-3
will have chemotactic and mitogenic activity for connective tissue
cells, similar to platelet-derived growth factor (PDGF). Assays to
test these activities are described in DiCorleto, P. E., Exp. Cell.
Res. 153:167-172 (1984). In addition, it is believed that
connective tissue growth factor-3 activity will include an
increased synthesis of extracellular matrix/connective tissue
components, such as, e.g., collagen, fibronectin, PA1-1, syndecan,
and elastin. This activity can be tested by Northern and Western
blot or ELISA analyses after treatment of cultured cells with
CTGF-3 protein.
[0052] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence
of the deposited cDNA or the nucleic acid sequence shown in FIG. 1
(SEQ ID NO:1) will encode a polypeptide "having connective tissue
growth factor-3 protein activity." In fact, since degenerate
variants of these nucleotide sequences all encode the same
polypeptide, this will be clear to the skilled artisan even without
performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that
are not degenerate variants, a reasonable number will also encode a
polypeptide having connective tissue growth factor-3 protein
activity. This is because the skilled artisan is fully aware of
amino acid substitutions that are either less likely or not likely
to significantly effect protein function (e.g., replacing one
aliphatic amino acid with a second aliphatic amino acid).
[0053] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U. et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that proteins are surprisingly tolerant of amino
acid substitutions.
[0054] Vectors and Host Cells
[0055] The present invention also relates to vectors that include
the isolated DNA molecules of the present invention, host cells
that are genetically engineered with the recombinant vectors, and
the production of connective tissue growth factor-3 polypeptides or
fragments thereof by recombinant techniques.
[0056] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0057] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will preferably include a translation
initiating at the beginning and a termination codon (UAA, UGA or
UAG) appropriately positioned at the end of the polypeptide to be
translated.
[0058] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS, and Bowes
melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in the
art.
[0059] Among vectors preferred for use in bacteria include pQE70,
pQE60, and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 available from Pharmacia. Among preferred eukaryotic vectors
are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene;
and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other
suitable vectors will be readily apparent to the skilled
artisan.
[0060] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0061] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to
solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobin molecules together with
another human protein or part thereof. In many cases, the Fc part
in a fusion protein is thoroughly advantageous for use in therapy
and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected, and purified
in the advantageous manner described. This is the case when the Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example, when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as, the hIL5-receptor, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
of hIL-5 See, D. Bennett et al., Journal of Molecular Recognition
8:52-58 (1995) and K. Johanson et al., The Journal of Biological
Chemistry 270:9459-9471 (1995).
[0062] The connective tissue growth factor-3 protein can be
recovered and purified from recombinant cell cultures by well-known
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography ("HPLC") is employed for
purification. Polypeptides of the present invention include
naturally purified products, products of chemical synthetic
procedures, and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial,
yeast, higher plant, insect and mammalian cells. Depending upon the
host employed in a recombinant production procedure, the
polypeptides of the present invention may be glycosylated or may be
non-glycosylated. In addition, polypeptides of the invention may
also include an initial modified methionine residue, in some cases
as a result of host-mediated processes.
[0063] Connective Tissue Growth Factor-3 Polypeptides and
Fragments
[0064] The invention further provides an isolated connective tissue
growth factor-3 polypeptide having the amino acid sequence encoded
by the deposited cDNA, or the amino acid sequence in SEQ ID NO:2,
or a peptide or polypeptide comprising a portion of the above
polypeptides.
[0065] It will be recognized in the art that some amino acid
sequences of the connective tissue growth factor-3 polypeptide can
be varied without significant effect of the structure or function
of the protein. If such differences in sequence are contemplated,
it should be remembered that there will be critical areas on the
protein which determine activity.
[0066] Thus, the invention further includes variations of the
connective tissue growth factor-3 polypeptide that show substantial
connective tissue growth factor-3 polypeptide activity or that
include regions of connective tissue growth factor-3 protein, such
as the protein portions discussed below. Such mutants include
deletions, insertions, inversions, repeats, and type substitutions.
As indicated above, guidance concerning which amino acid changes
are likely to be phenotypically silent can be found in Bowie, J. U.
et al., "Deciphering the Message in Protein Sequences: Tolerance to
Amino Acid Substitutions," Science 247:1306-1310 (1990).
[0067] Thus, the fragment, derivative or analog of the polypeptide
of SEQ ID NO: 2, or that encoded by the deposited cDNA, may be: (i)
one in which one or more of the amino acid residues are substituted
with a conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the
mature polypeptide, such as an IgG Fc fusion region peptide or
leader or secretory sequence or a sequence which is employed for
purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0068] Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the
connective tissue growth factor-3 protein. The prevention of
aggregation is highly desirable. Aggregation of proteins not only
results in a loss of activity but can also be problematic when
preparing pharmaceutical formulations, because they can be
immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967);
Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit.
Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).
[0069] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions
Aromatic Phenylalinine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Blutamic Acid Small Alanine Serine
Threonine Glycine
[0070] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of amino acid
substitutions for any given connective tissue growth factor-3
polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5, or
3.
[0071] Amino acids in the connective tissue growth factor-3
polypeptide of the present invention that are essential for
function can be identified by methods known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, Science 244:1081-1085 (1989)). The latter
procedure introduces single alanine mutations at every residue in
the molecule. The resulting mutant molecules are then tested for
biological activity such as receptor binding or in vitro
proliferative activity. Sites that are critical for protein
activity can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos
et al. Science 255:306-312 (1992)).
[0072] The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides that
have been purified, partially or substantially, from a recombinant
host cell or a native source. For example, a recombinantly produced
version of the connective tissue growth factor-3 polypeptide can be
substantially purified by the one-step method described in Smith
and Johnson, Gene 67:31-40 (1988).
[0073] The polypeptides of the present invention include the
polypeptide encoded by the deposited cDNA including the leader; the
mature polypeptide encoded by the deposited cDNA minus the leader
(i.e., the mature protein); a polypeptide comprising amino acids
about -19 to about 231 in SEQ ID NO:2; a polypeptide comprising
amino acids about -18 to about 231 in SEQ ID NO:2; a polypeptide
comprising amino acids about 1 to about 231 in SEQ ID NO:2; as well
as polypeptides which are at least 95% identical, more preferably
at least 96%, 97%, 98%, or 99% identical to those described above,
and also include portions of such polypeptides with at least 30
amino acids and more preferably at least 50 amino acids.
[0074] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
connective tissue growth factor-3 polypeptide is intended that the
amino acid sequence of the polypeptide is identical to the
reference sequence except that the polypeptide sequence may include
up to five amino acid alterations per each 100 amino acids of the
reference amino acid of the connective tissue growth factor-3
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0075] As a practical matter, whether any particular polypeptide is
at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the
amino acid sequence shown in SEQ ID NO:2 or to the amino acid
sequence encoded by the deposited cDNA clone can be determined
conventionally using known computer programs such as the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711). When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0076] The polypeptide of the present invention could be used as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0077] As described in detail below, the polypeptides of the
present invention can also be used to raise polyclonal and
monoclonal antibodies, which are useful in assays for detecting
connective tissue growth factor-3 protein expression as described
below or as agonists and antagonists capable of enhancing or
inhibiting connective tissue growth factor-3 protein function.
Further, such polypeptides can be used in the yeast two-hybrid
system to "capture" connective tissue growth factor-3 protein
binding proteins which are also candidate agonist and antagonist
according to the present invention. The yeast two hybrid system is
described in Fields and Song, Nature 340:245-246 (1989).
[0078] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide described herein.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
[0079] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe et al., "Antibodies That React With Predetermined Sites
on Proteins," Science 219:660-666 (1983). Peptides capable of
eliciting protein-reactive sera are frequently represented in the
primary sequence of a protein, can be characterized by a set of
simple chemical rules, and are confined neither to the
immunodominant regions of intact proteins (i.e., immunogenic
epitopes) nor to the amino or carboxyl terminals. Peptides that are
extremely hydrophobic and those of six or fewer residues generally
are ineffective at inducing antibodies that bind to the mimicked
protein; longer, peptides, especially those containing proline
residues, usually are effective. Sutcliffe et al., supra, at 661.
For instance, 18 of 20 peptides designed according to these
guidelines, containing 8-39 residues covering 75% of the sequence
of the influenza virus hemagglutinin HA1 polypeptide chain, induced
antibodies that reacted with the HA protein or intact virus; and
12/12 peptides from the MuLV polymerase and 18/18 from the rabies
glycoprotein induced antibodies that precipitated the respective
proteins. Antigenic epitope-bearing peptides and polypeptides of
the invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. Thus, a high proportion of hybridomas, obtained by
fusion of spleen cells from donors immunized with an antigen
epitope-bearing peptide, generally secrete antibody, reactive with
the native protein. Sutcliffe et al., supra, at 663. The antibodies
raised by antigenic epitope-bearing peptides or polypeptides are
useful to detect the mimicked protein, and antibodies to different
peptides may be used for tracking the fate of various regions of a
protein precursor which undergo post-translational processing. The
peptides and anti-peptide antibodies may be used in a variety of
qualitative or quantitative assays for the mimicked protein, for
instance, in competition assays since it has been shown that even
short peptides (e.g., about 9 amino acids) can bind and displace
the larger peptides in immunoprecipitation assays. See, for
instance, Wilson et al., Cell 37:767-778 (1984) at 777. The
anti-peptide antibodies of the invention also are useful for
purification of the mimicked protein, for instance, by adsorption
chromatography using methods well known in the art.
[0080] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine, and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention. However, peptides or polypeptides
comprising a larger portion of an amino acid sequence of a
polypeptide of the invention, containing about 30 to about 50 amino
acids, or any length up to and including the entire amino acid
sequence of a polypeptide of the invention, also are considered
epitope-bearing peptides or polypeptides of the invention and also
are useful for inducing antibodies that react with the mimicked
protein.
[0081] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate connective tissue growth
factor-3-specific antibodies include: a polypeptide comprising
amino acid residues from about 55 to about 68 in FIG. 1 (about 36
to about 49 in SEQ ID NO:2); a polypeptide comprising amino acid
residues from about 94 to about 128 in FIG. 1 (about 75 to about
109 in SEQ ID NO:2); a polypeptide comprising amino acid residues
from about 134 to about 158 in FIG. 1 (about 115 to about 139 in
SEQ ID NO:2); and a polypeptide comprising amino acid residues from
about 215 to about 249 in FIG. 1 (about 196 to about 230 in SEQ ID
NO: 2). As indicated above, the inventors have determined that the
above polypeptide fragments are antigenic regions of the connective
tissue growth factor-3 protein.
[0082] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. Houghten, R.
A., "General Method for the Rapid Solid-Phase Synthesis of Large
Numbers of Peptides: Specificity of Antigen-Antibody Interaction at
the Level of Individual Amino Acids," Proc. Natl. Acad. Sci. USA
82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Pat. No. 4,631,211 to
Houghten et al. (1986).
[0083] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, for instance, Sutcliffe et al., supra; Wilson et al.,
supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914
(1985); and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354
(1985). Generally, animals may be immunized with free peptide;
however, anti-peptide antibody titer may be boosted by coupling of
the peptide to a macromolecular carrier, such as keyhole limpet
hemocyanin (KLH) or tetanus toxoid. For instance, peptides
containing cysteine may be coupled to carrier using a linker such
as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carrier using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats, and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 g peptide or carrier protein and
Freund's adjuvant. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0084] Immunogenic epitope-bearing peptides of the invention, i.e.,
those parts of a protein that elicit an antibody response when the
whole protein is the immunogen, are identified according to methods
known in the art. For instance, Geysen et al., supra, disclose a
procedure for rapid concurrent synthesis on solid supports of
hundreds of peptides of sufficient purity to react in an
enzyme-linked immunosorbent assay. Interaction of synthesized
peptides with antibodies is then easily detected without removing
them from the support. In this manner, a peptide bearing an
immunogenic epitope of a desired protein may be identified
routinely by one of ordinary skill in the art. For instance, the
immunologically important epitope in the coat protein of
foot-and-mouth disease virus was located by Geysen et al. with a
resolution of seven amino acids by synthesis of an overlapping set
of all 208 possible hexapeptides covering the entire 213 amino acid
sequence of the protein. Then, a complete replacement set of
peptides in which all 20 amino acids were substituted in turn at
every position within the epitope were synthesized, and the
particular amino acids conferring specificity for the reaction with
antibody were determined. Thus, peptide analogs of the
epitope-bearing peptides of the invention can be made routinely by
this method. U.S. Pat. No. 4,708,781 to Geysen (1987) further
describes this method of identifying a peptide bearing an
immunogenic epitope of a desired protein.
[0085] Further still, U.S. Pat. No. 5,194,392 to Geysen (1990)
describes a general method of detecting or determining the sequence
of monomers (amino acids or other compounds) which is a topological
equivalent of the epitope (i.e., a "mimotope"), which is
complementary to a particular paratope (antigen binding site) of an
antibody of interest. More generally, U.S. Pat. No. 4,433,092 to
Geysen (1989) describes a method of detecting or determining a
sequence of monomers which is a topographical equivalent of a
ligand which is complementary to the ligand binding site of a
particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971
to Houghten, R. A. et al. (1996) entitled "Peralkylated
Oligopeptide Mixtures" discloses linear C.sub.1-C.sub.7-alkyl
peralkylated oligopeptides and sets and libraries of such peptides,
as well as methods for using such oligopeptide sets and libraries
for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
[0086] The entire disclosure of each document cited in this section
on "Polypeptides and Peptides" is hereby incorporated herein by
reference.
[0087] As one of skill in the art will appreciate, connective
tissue growth factor-3 polypeptides of the present invention and
the epitope-bearing fragments thereof described above can be
combined with parts of the constant domain of immunoglobulins
(IgG), resulting in chimeric polypeptides. These fusion proteins
facilitate purification and show an increased half-life in vivo.
This has been shown, e.g., for chimeric proteins consisting of the
first two domains of the human CD4-polypeptide and various domains
of the constant regions of the heavy or light chains of mammalian
immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86
(1988)). Fusion proteins that have a disulfide-linked dimeric
structure due to the IgG part can also be more efficient in binding
and neutralizing other molecules than the monomeric connective
tissue growth factor-3 protein or protein fragment alone
(Fountoulakis et al., J Biochem 270:3958-3964 (1995)).
[0088] Diagnosis and Prognosis of Connective Tissue Related
Disorders
[0089] It is believed that certain tissues in mammals with various
connective-tissue related disorders express significantly altered
levels of the connective tissue growth factor-3 protein and mRNA
encoding the connective tissue growth factor-3 protein, when
compared to a corresponding "standard" mammal, i.e., a mammal of
the same species not having the disorder. By "connective tissue
related disorders" is intended any disease or condition that is
caused by, associated with, or characterized by an over or under
growth of connective tissue cells. Some non-limiting examples of
such disorders include cancer, arthritis, fibrosis,
atherosclerosis, and osteoporosis.
[0090] For example, it is believed that enhanced levels of the
connective tissue growth factor-3 protein can be detected in
certain body fluids (e.g., sera, plasma, urine, and spinal fluid)
or tissues from mammals with cancer, fibrosis, arthritis, or
atherosclerosis when compared to sera from mammals of the same
species not having these diseases. Thus, the invention provides a
diagnostic method useful during diagnosis of connective-tissue
related disorders, such as cancer, fibrosis, arthritis, or
atherosclerosis, which involves assaying the expression level of
the gene encoding the connective tissue growth factor-3 protein in
mammalian cells or body fluid and comparing the gene expression
level with a standard connective tissue growth factor-3 gene
expression level, whereby an increase in the gene expression level
over the standard is indicative of these diseases.
[0091] Where a diagnosis of any of these diseases has already been
made according to conventional methods, the present invention is
also useful as a prognostic indicator, whereby patients exhibiting
enhanced connective tissue growth factor-3 gene expression will
experience a worse clinical outcome relative to patients expressing
the gene at a lower level.
[0092] It is also believed that decreased levels of the connective
tissue growth factor-3 protein can be detected in certain body
fluids (e.g., sera, plasma, urine, and spinal fluid) or tissues
from mammals with certain connective-tissue related disorders, such
as osteoporosis, when compared to sera from mammals of the same
species not having the disease. Thus, the invention provides a
diagnostic method useful during diagnosis of connective-tissue
related disorders, such as osteoporosis, which involves assaying
the expression level of the gene encoding the connective tissue
growth factor-3 protein in mammalian cells or body fluid and
comparing the gene expression level with a standard connective
tissue growth factor-3 gene expression level, whereby a decrease in
the gene expression level over the standard is indicative of the
disease.
[0093] By "assaying the expression level of the gene encoding the
connective tissue growth factor-3 protein" is intended
qualitatively or quantitatively measuring or estimating the level
of the connective tissue growth factor-3 protein or the level of
the mRNA encoding the connective tissue growth factor-3 protein in
a first biological sample either directly (e.g., by determining or
estimating absolute protein level or mRNA level) or relatively
(e.g., by comparing to the connective tissue growth factor-3
protein level or mRNA level in a second biological sample).
[0094] Preferably, the connective tissue growth factor-3 protein
level or mRNA level in the first biological sample is measured or
estimated and compared to a standard connective tissue growth
factor-3 protein level or mRNA level, the standard being taken from
a second biological sample obtained from an individual not having
the connective-tissue related disorder. As will be appreciated in
the art, once a standard connective tissue growth factor-3 protein
level or mRNA level is known, it can be used repeatedly as a
standard for comparison.
[0095] By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other
source which contains connective tissue growth factor-3 protein or
mRNA. Biological samples include mammalian body fluids (such as
sera, plasma, urine, synovial fluid and spinal fluid) which contain
secreted mature connective tissue growth factor-3 protein, and
ovarian, testicular, prostate, heart, placenta, pancreas liver,
spleen, lung, breast and umbilical tissue. Methods for obtaining
tissue biopsies and body fluids from mammals are well known in the
art. Where the biological sample is to include mRNA, a tissue
biopsy is the preferred source.
[0096] The present invention is useful for detecting cancer in
mammals. In particular, the invention is useful during diagnosis of
the following types of cancers in mammals: breast, ovarian,
cervical, prostate, bone, liver, lung, pancreatic, and splenic.
Preferred mammals include monkeys, apes, cats, dogs, cows, pigs,
horses, rabbits and humans. Particularly preferred are humans.
[0097] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels
of mRNA encoding the connective tissue growth factor-3 protein are
then assayed using any appropriate method. These include Northern
blot analysis, S 1 nuclease mapping, the polymerase chain reaction
(PCR), reverse transcription in combination with the polymerase
chain reaction (RT-PCR), and reverse transcription in combination
with the ligase chain reaction (RT-LCR).
[0098] Northern blot analysis can be performed as described in
Harada et al., Cell 63:303-312 (1990). Briefly, total RNA is
prepared from a biological sample as described above. For the
Northern blot, the RNA is denatured in an appropriate buffer (such
as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected
to agarose gel electrophoresis, and transferred onto a
nitrocellulose filter. After the RNAs have been linked to the
filter by a UV linker, the filter is prehybridized in a solution
containing formamide, SSC, Denhardt's solution, denatured salmon
sperm, SDS, and sodium phosphate buffer. Connective tissue growth
factor-3 protein cDNA labeled according to any appropriate method
(such as the .sup.32P-multiprimed DNA labeling system (Amersham))
is used as probe. After hybridization overnight, the filter is
washed and exposed to x-ray film. cDNA for use as a probe according
to the present invention is described in the sections above, and
will preferably be at least 15 bp in length.
[0099] S1 mapping can be performed as described in Fujita et al.,
Cell 49:357-367 (1987). To prepare probe DNA for use in S1 mapping,
the sense strand of the above-described cDNA is used as a template
to synthesize labeled antisense DNA. The antisense DNA can then be
digested using an appropriate restriction endonuclease to generate
further DNA probes of a desired length. Such antisense probes are
useful for visualizing protected bands corresponding to the target
mRNA (i.e., mRNA encoding the connective tissue growth factor-3
protein). Northern blot analysis can be performed as described
above.
[0100] Preferably, levels of mRNA encoding the connective tissue
growth factor-3 protein are assayed using the RT-PCR method
described in Makino et al., Technique 2:295-301 (1990). By this
method, the radioactivities of the "amplicons" in the
polyacrylamide gel bands are linearly related to the initial
concentration of the target mRNA. Briefly, this method involves
adding total RNA isolated from a biological sample in a reaction
mixture containing a RT primer and appropriate buffer. After
incubating for primer annealing, the mixture can be supplemented
with a RT buffer, dNTPs, DTT, RNase inhibitor and reverse
transcriptase. After incubation to achieve reverse transcription of
the RNA, the RT products are then subject to PCR using labeled
primers. Alternatively, rather than labeling the primers, a labeled
dNTP can be included in the PCR reaction mixture. PCR amplification
can be performed in a DNA thermal cycler according to conventional
techniques. After a suitable number of rounds to achieve
amplification, the PCR reaction mixture is electrophoresed on a
polyacrylamide gel. After drying the gel, the radioactivity of the
appropriate bands (corresponding to the mRNA encoding the
connective tissue growth factor-3 protein)) is quantified using an
imaging analyzer. RT and PCR reaction ingredients and conditions,
reagent and gel concentrations, and labeling methods are well known
in the art. Variations on the RT-PCR method will be apparent to the
skilled artisan.
[0101] Any set of oligonucleotide primers that will amplify reverse
transcribed target mRNA can be used and can be designed as
described in the sections above.
[0102] Assaying connective tissue growth factor-3 protein levels in
a biological sample can occur using any art-known method. Preferred
for assaying connective tissue growth factor-3 protein levels in a
biological sample are antibody-based techniques. For example,
connective tissue growth factor-3 protein expression in tissues can
be studied with classical immunohistological methods. In these, the
specific recognition is provided by the primary antibody
(polyclonal or monoclonal) but the secondary detection system can
utilize fluorescent, enzyme, or other conjugated secondary
antibodies. As a result, an immunohistological staining of tissue
section for pathological examination is obtained. Tissues can also
be extracted, e.g., with urea and neutral detergent, for the
liberation of connective tissue growth factor-3 protein for
Western-blot or dot/slot assay (Jalkanen, M., et al., J. Cell.
Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol.
105:3087-3096 (1987)). In this technique, which is based on the use
of cationic solid phases, quantitation of connective tissue growth
factor-3 protein can be accomplished using isolated connective
tissue growth factor-3 protein as a standard. This technique can
also be applied to body fluids. With these samples, a molar
concentration of connective tissue growth factor-3 protein will aid
to set standard values of connective tissue growth factor-3 protein
content for different body fluids, like serum, plasma, urine,
spinal fluid, etc. The normal appearance of connective tissue
growth factor-3 protein amounts can then be set using values from
healthy individuals, which can be compared to those obtained from a
test subject.
[0103] Other antibody-based methods useful for detecting connective
tissue growth factor-3 protein gene expression include
immunoassays, such as the enzyme linked immunosorbent assay (ELISA)
and the radioimmunoassay (RIA). For example, a connective tissue
growth factor-3 protein-specific monoclonal antibody can be used
both as an immunoabsorbent and as an enzyme-labeled probe to detect
and quantify the connective tissue growth factor-3 protein. The
amount of connective tissue growth factor-3 protein present in the
sample can be calculated by reference to the amount present in a
standard preparation using a linear regression computer algorithm.
Such an ELISA for detecting a tumor antigen is described in
Iacobelli et al., Breast Cancer Research and Treatment 11:19-30
(1988). In another ELISA assay, two distinct specific monoclonal
antibodies can be used to detect connective tissue growth factor-3
protein in a body fluid. In this assay, one of the antibodies is
used as the immunoabsorbent and the other as the enzyme-labeled
probe.
[0104] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. The "one-step" assay involves
contacting connective tissue growth factor-3 protein with
immobilized antibody and, without washing, contacting the mixture
with the labeled antibody. The "two-step" assay involves washing
before contacting the mixture with the labeled antibody. Other
conventional methods may also be employed as suitable. It is
usually desirable to immobilize one component of the assay system
on a support, thereby allowing other components of the system to be
brought into contact with the component and readily removed from
the sample.
[0105] Suitable enzyme labels include, for example, those from the
oxidase group, which catalyze the production of hydrogen peroxide
by reacting with substrate. Glucose oxidase is particularly
preferred as it has good stability and its substrate (glucose) is
readily available. Activity of an oxidase label may be assayed by
measuring the concentration of hydrogen peroxide formed by the
enzyme-labelled antibody/substrate reaction. Besides enzymes, other
suitable labels include radioisotopes such as iodine (.sup.125I,
.sup.131I, carbon (.sup.14C), sulphur (.sup.35S), tritium
(.sup.3H), indium (.sup.112In), and technetium (.sup.99mTc), and
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0106] In addition to assaying connective tissue growth factor-3
protein levels in a biological sample obtained from an individual,
connective tissue growth factor-3 protein can also be detected in
vivo by imaging. Antibody labels or markers for in vivo imaging of
connective tissue growth factor-3 protein include those detectable
by X-radiography, NMR, or ESR. For X-radiography, suitable labels
include radioisotopes such as barium or cesium, which emit
detectable radiation, but are not overtly harmful to the subject.
Suitable markers for NMR and ESR include those with a detectable
characteristic spin, such as deuterium, which may be incorporated
into the antibody by labeling of nutrients for the relevant
hybridoma.
[0107] A connective tissue growth factor-3 protein-specific
antibody or antibody fragment which has been labeled with an
appropriate detectable imaging moiety, such as a radioisotope (for
example, .sup.131I, .sup.112In, .sup.99mTc), a radio-opaque
substance, or a material detectable by nuclear magnetic resonance,
is introduced (for example, parenterally, subcutaneously or
intraperitoneally) into the mammal to be examined for cancer. It
will be understood in the art that the size of the subject and the
imaging system used will determine the quantity of imaging moiety
needed to produce diagnostic images. In the case of a radioisotope
moiety, for a human subject, the quantity of radioactivity injected
will normally range from about 5 to 20 millicuries of .sup.99mTc.
The labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain connective tissue
growth factor-3 protein. In vivo tumor imaging is described in S.
W. Burchiel et al. , "Immunopharmacokinetics of Radiolabelled
Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, eds., S. W. Burchiel and B. A.
Rhodes, Masson Publishing, Inc. (1982)).
[0108] Connective tissue growth factor-3-protein specific
antibodies for use in the present invention can be raised against
the intact connective tissue growth factor-3 protein or an
antigenic polypeptide fragment thereof, which may be presented
together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or mouse) or, if it is long enough (at least
about 25 amino acids), without a carrier.
[0109] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody fragments (such as, for example, Fab and F(ab').sub.2
fragments) which are capable of specifically binding to connective
tissue growth factor-3 protein. Fab and F(ab').sub.2 fragments lack
the Fc fragment of intact antibody, clear more rapidly from the
circulation, and may have less non-specific tissue binding of an
intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
Thus, these fragments are preferred.
[0110] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, cells expressing the
connective tissue growth factor-3 protein or an antigenic fragment
thereof can be administered to an animal in order to induce the
production of sera containing polyclonal antibodies. In a preferred
method, a preparation of connective tissue growth factor-3 protein
is prepared and purified to render it substantially free of natural
contaminants Such a preparation is then introduced into an animal
in order to produce polyclonal antisera of greater specific
activity.
[0111] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or connective tissue growth
factor-3 protein binding fragments thereof). Such monoclonal
antibodies can be prepared using hybridoma technology (Kohler et
al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511
(1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et
al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier,
N.Y., pp. 563-681 (1981)). In general, such procedures involve
immunizing an animal (preferably a mouse) with a connective tissue
growth factor-3 protein antigen or, more preferably, with a
connective tissue growth factor-3 protein-expressing cell. Suitable
cells can be recognized by their capacity to bind anti-connective
tissue growth factor-3 protein antibody. Such cells may be cultured
in any suitable tissue culture medium; however, it is preferable to
culture cells in Earle's modified Eagle's medium supplemented with
10% fetal bovine serum (inactivated at about 56.degree. C.), and
supplemented with about 10 g/l of nonessential amino acids, about
1,000 U/ml of penicillin, and about 100 g/ml of streptomycin. The
splenocytes of such mice are extracted and fused with a suitable
myeloma cell line. Any suitable myeloma cell line may be employed
in accordance with the present invention; however, it is preferable
to employ the parent myeloma cell line (SP.sub.2O), available from
the American Type Culture Collection, Manassas, Va. After fusion,
the resulting hybridoma cells are selectively maintained in HAT
medium, and then cloned by limiting dilution as described by Wands
et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells
obtained through such a selection are then assayed to identify
clones which secrete antibodies capable of binding the connective
tissue growth factor-3 protein antigen.
[0112] Alternatively, additional antibodies capable of binding to
the connective tissue growth factor-3 protein antigen may be
produced in a two-step procedure through the use of anti-idiotypic
antibodies. Such a method makes use of the fact that antibodies are
themselves antigens, and that, therefore, it is possible to obtain
an antibody which binds to a second antibody. In accordance with
this method, connective tissue growth factor-3-protein specific
antibodies are used to immunize an animal, preferably a mouse. The
splenocytes of such an animal are then used to produce hybridoma
cells, and the hybridoma cells are screened to identify clones
which produce an antibody whose ability to bind to the connective
tissue growth factor-3 protein-specific antibody can be blocked by
the connective tissue growth factor-3 protein antigen. Such
antibodies comprise anti-idiotypic antibodies to the connective
tissue growth factor-3 protein-specific antibody and can be used to
immunize an animal to induce formation of further connective tissue
growth factor-3 protein-specific antibodies.
[0113] It will be appreciated that Fab and F(ab').sub.2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce
F(ab').sub.2 fragments). Alternatively, connective tissue growth
factor-3 protein-binding fragments can be produced through the
application of recombinant DNA technology or through synthetic
chemistry.
[0114] Where in vivo imaging is used to detect enhanced levels of
connective tissue growth factor-3 protein for tumor diagnosis in
humans, it may be preferable to use "humanized" chimeric monoclonal
antibodies. Such antibodies can be produced using genetic
constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric
antibodies are known in the art. See, for review, Morrison, Science
229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et
al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison
et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al.,
WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et
al., Nature 314:268 (1985).
[0115] Further suitable labels for the connective tissue growth
factor-3 protein-specific antibodies of the present invention are
provided below. Examples of suitable enzyme labels include malate
dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,
yeast-alcohol dehydrogenase, alpha-glycerol phosphate
dehydrogenase, triose phosphate isomerase, peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase, and acetylcholine esterase.
[0116] Examples of suitable radioisotopic labels include .sup.3H,
.sup.111In, .sup.125I, .sup.131I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sub.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu,
.sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc,
.sup.109Pd, etc. .sup.111In is a preferred isotope where in vivo
imaging is used since it avoids the problem of dehalogenation of
the .sup.125I or .sup.131I-labeled monoclonal antibody by the
liver. In addition, this radionucleotide has a more favorable gamma
emission energy for imaging (Perkins et al., Eur. J. Nucl. Med.
10:296-301 (1985); Carasquillo et al., J. Nucl. Med. 28:281-287
(1987)). For example, .sup.111In coupled to monoclonal antibodies
with 1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in
non-tumorous tissues, particularly the liver, and therefore
enhances specificity of tumor localization (Esteban et al., J.
Nucl. Med. 28:861-870 (1987)).
[0117] Examples of suitable non-radioactive isotopic labels include
.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, and .sup.56Fe.
Examples of suitable fluorescent labels include an .sup.152Eu
label, a fluorescein label, an isothiocyanate label, a rhodamine
label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine
label.
[0118] Examples of suitable toxin labels include diphtheria toxin,
ricin, and cholera toxin.
[0119] Examples of chemiluminescent labels include a luminal label,
an isoluminal label, an aromatic acridinium ester label, an
imidazole label, an acridinium salt label, an oxalate ester label,
a luciferin label, a luciferase label, and an aequorin label.
[0120] Examples of nuclear magnetic resonance contrasting agents
include heavy metal nuclei such as Gd, Mn, and iron.
[0121] Typical techniques for binding the above-described labels to
antibodies are provided by Kennedy et al., Clin. Chim. Acta 70:1-31
(1976), and Schurs et al., Clin. Chim. Acta 81:1-40 (1977).
Coupling techniques mentioned in the latter are the glutaraldehyde
method, the periodate method, the dimaleimide method, the
m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which
methods are incorporated by reference herein.
[0122] Therapeutics: Connective Tissue Growth Factor-3 Protein
[0123] It will be appreciated by those skilled in the art that
individuals with conditions characterized by a decrease in the
standard or normal level of connective tissue growth factor-3
activity, can be treated by administration of connective tissue
growth factor-3 protein. For example, it is believed that
individuals in need of wound healing, tissue repair, or increased
bone mass (i.e., patients with osteoporosis) would benefit from
such treatment. Thus, the invention further provides a method of
treating an individual in need of an increased level of connective
tissue growth factor-3 activity comprising administering to such an
individual a pharmaceutical composition comprising an effective
amount of an isolated connective tissue growth factor-3 polypeptide
of the invention, particularly a mature form of the connective
tissue growth factor-3, effective to increase the connective tissue
growth factor-3 activity level in such an individual.
[0124] The connective tissue growth factor-3 polypeptide
composition will be formulated and dosed in a fashion consistent
with good medical practice, taking into account the clinical
condition of the individual patient (especially the side effects of
treatment with connective tissue growth factor-3 polypeptide
alone), the site of delivery of the connective tissue growth
factor-3 polypeptide composition, the method of administration, the
scheduling of administration, and other factors known to
practitioners. The "effective amount" of connective tissue growth
factor-3 polypeptide for purposes herein is thus determined by such
considerations.
[0125] As a general proposition, the total pharmaceutically
effective amount of connective tissue growth factor-3 polypeptide
administered parenterally per dose will be in the range of about 1
.mu.g/kg/day to 10 mg/kg/day of patient body weight, although, as
noted above, this will be subject to therapeutic discretion. More
preferably, this dose is at least 0.01 mg/kg/day, and most
preferably for humans between about 0.01 and 1 mg/kg/day for the
hormone. If given continuously, the connective tissue growth
factor-3 polypeptide is typically administered at a dose rate of
about 1 .mu.g/kg/hour to about 50 .mu.g/kg/hour, either by 1-4
injections per day or by continuous subcutaneous infusions, for
example, using a mini-pump. An intravenous bag solution may also be
employed. The key factor in selecting an appropriate dose is the
result obtained, as measured by increases in antibody production,
increases in splenocyte or thymocyte number, increase in splenic
B-cells, etc. The length of treatment needed to observe changes and
the interval following treatment for responses to occur appears to
vary depending on the desired effect.
[0126] Pharmaceutical compositions containing the connective tissue
growth factor-3 of the invention may be administered orally,
rectally, parenterally, intracistemally, intravaginally,
intraperitoneally, topically (as by powders, ointments, drops or
transdermal patch), bucally, or as an oral or nasal spray. By
"pharmaceutically acceptable carrier" is meant a non-toxic solid,
semisolid or liquid filler, diluent, encapsulating material or
formulation auxiliary of any type. The term "parenteral" as used
herein refers to modes of administration which include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion.
[0127] The connective tissue growth factor-3 polypeptide is also
suitably administered by sustained-release systems. Suitable
examples of sustained-release compositions include semi-permeable
polymer matrices in the form of shaped articles, e.g., films, or
mirocapsules. Sustained-release matrices include polylactides (U.S.
Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556
(1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.
12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.)
or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
connective tissue growth factor-3 polypeptide compositions also
include liposomally entrapped connective tissue growth factor-3
polypeptide. Liposomes containing connective tissue growth factor-3
polypeptide are prepared by methods known per se: DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985);
Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP
52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.
Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP
102,324. Ordinarily, the liposomes are of the small (about 200-800
Angstroms) unilamellar type in which the lipid content is greater
than about 30 mol. percent cholesterol, the selected proportion
being adjusted for the optimal connective tissue growth factor-3
polypeptide therapy.
[0128] For parenteral administration, in one embodiment, the
connective tissue growth factor-3 polypeptide is formulated
generally by mixing it at the desired degree of purity, in a unit
dosage injectable form (solution, suspension, or emulsion), with a
pharmaceutically acceptable carrier, i.e., one that is non-toxic to
recipients at the dosages and concentrations employed and is
compatible with other ingredients of the formulation. For example,
the formulation preferably does not include oxidizing agents and
other compounds that are known to be deleterious to
polypeptides.
[0129] Generally, the formulations are prepared by contacting the
connective tissue growth factor-3 polypeptide uniformly and
intimately with liquid carriers or finely divided solid carriers or
both. Then, if necessary, the product is shaped into the desired
formulation. Preferably the carrier is a parenteral carrier, more
preferably a solution that is isotonic with the blood of the
recipient. Examples of such carrier vehicles include water, saline,
Ringer's solution, and dextrose solution. Non-aqueous vehicles such
as fixed oils and ethyl oleate are also useful herein, as well as
liposomes.
[0130] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, mannose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0131] The connective tissue growth factor-3 polypeptide is
typically formulated in such vehicles at a concentration of about
0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3
to 8. It will be understood that the use of certain of the
foregoing excipients, carriers, or stabilizers will result in the
formation of connective tissue growth factor-3 polypeptide
salts.
[0132] Connective tissue growth factor-3 polypeptide to be used for
therapeutic administration must be sterile. Sterility is readily
accomplished by filtration through sterile filtration membranes
(e.g., 0.2 micron membranes). Therapeutic connective tissue growth
factor-3 polypeptide compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0133] Connective tissue growth factor-3 polypeptide ordinarily
will be stored in unit or multi-dose containers, for example,
sealed ampoules or vials, as an aqueous solution or as a
lyophilized formulation for reconstitution. As an example of a
lyophilized formulation, 10-ml vials are filled with 5 ml of
sterile-filtered 1% (w/v) aqueous connective tissue growth factor-3
polypeptide solution, and the resulting mixture is lyophilized. The
infusion solution is prepared by reconstituting the lyophilized
connective tissue growth factor-3 polypeptide using bacteriostatic
Water-for-Injection.
[0134] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the polypeptides of the present
invention may be employed in conjunction with other therapeutic
compounds.
[0135] Therapeutics: Antibodies to Connective Tissue Growth
Factor-3 Protein
[0136] The present invention is further directed to antibody-based
therapies, which involve administering a connective tissue growth
factor-3 antibody to a mammalian patient for treating disorders
characterized by an overgrowth of connective tissue cells and most
likely an over-expression of CTGF-3. Such disorders include cancer,
arthritis, atherosclerosis, fibrositis (muscles, joints), fibrosis
of vital organs, such as the liver and kidney, or fibrotic
conditions (e.g., scleroderma, keloids). Methods for producing
anti- connective tissue growth factor-3 polyclonal and monoclonal
antibodies are described in detail above. Such antibodies may be
provided in pharmaceutically acceptable compositions as known in
the art or as described herein.
[0137] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding connective
tissue growth factor-3 locally or systemically in the body or by
direct cytotoxicity of the antibody, e.g., as mediated by
complement (CDC) or by effector cells (ADCC). Some of these
approaches are described in more detail below. Armed with the
teachings provided herein, one of ordinary skill in the art will
know how to use the antibodies of the present invention for
diagnostic, monitoring, or therapeutic purposes without undue
experimentation.
[0138] The CTGF-3 antibody of the present invention may be
administered by any means that achieve their intended purpose.
Amounts and regimens for the administration of antibodies, their
fragments or derivatives can be determined readily by those with
ordinary skill in the clinical art of treating
connective-tissue-related diseases.
[0139] For example, administration may be by parenteral,
subcutaneous, intravenous, intramuscular, intraperitoneal,
transdermal, or buccal routes. Alternatively, or concurrently,
administration may be by the oral route. The dosage administered
will be dependent upon the age, health, and weight of the
recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired.
[0140] Compositions within the scope of this invention include all
compositions wherein the antibody, fragment or derivative is
contained in an amount effective to achieve its intended purpose.
While individual needs vary, determination of optimal ranges of
effective amounts of each component is within the skill of the art.
The effective dose is a function of the individual chimeric or
monoclonal antibody, the presence and nature of a conjugated
therapeutic agent (see below), the patient and his clinical status,
and can vary from about 10 .mu.g/kg body weight to about 5000 mg/kg
body weight. The preferred dosages comprise 0.1 to 500 mg/kg body
wt. The composition may contain suitable pharmaceutically
acceptable carriers comprising excipients and auxiliaries which
facilitate processing of the active compounds into preparations
which can be used pharmaceutically. Preferably, the preparations,
contain from about 0.01 to 99 percent, preferably from about 20 to
75 percent of active compound(s), together with the excipient.
[0141] Similarly, preparations of a connective tissue growth
factor-3 antibody or fragment of the present invention for
parenteral administration, such as in detectably labeled form for
imaging or in a free or conjugated form for therapy, include
sterile aqueous or non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media, parenteral
vehicles including sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, such
as those based on Ringer's dextrose, and the like. Preservatives
and other additives may also be present, such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like. See, generally, Remington's Pharmaceutical Science,
16th ed., Mack Publishing Co., Easton, Pa., 1980.
[0142] In particular, the antibodies, fragments and derivatives of
the present invention are useful for treating a subject having or
developing connective tissue related disorders, as described
herein, which are characterized by uncontrolled tissue growth. Such
treatment comprises parenterally administering a single or multiple
doses of the antibody, fragment or derivative, or a conjugate
thereof.
[0143] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hemopoietic growth factors,
etc., which serve to increase the number or activity of effector
cells which interact with the antibodies.
[0144] Preferred for human therapeutic use are high affinity murine
and murine/human or human/human chimeric antibodies, and fragments,
regions and derivatives having in vivo CTGF-3-inhibiting and/or
neutralizing activity.
[0145] Chromosome Assays
[0146] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0147] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a connective
tissue growth factor-3 protein gene. This can be accomplished using
a variety of well known techniques and libraries, which generally
are available commercially. The genomic DNA then is used for in
situ chromosome mapping using well known techniques for this
purpose. Typically, in accordance with routine procedures for
chromosome mapping, some trial and error may be necessary to
identify a genomic probe that gives a good in situ hybridization
signal.
[0148] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Only those
hybrids containing the human gene corresponding to the primer will
yield an amplified portion.
[0149] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of portions from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0150] Fluorescence in situ hybridization ("FISH") of a cDNA clone
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
probes from the cDNA as short as 50 or 60 bp. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual Of Basic
Techniques, Pergamon Press, New York (1988).
[0151] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance In Man, available
on-line through Johns Hopkins University, Welch Medical Library.
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0152] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0153] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Expression and Purification of Connective Tissue Growth Factor-3 in
E. Coli
[0154] The bacterial expression vector pQE9 (pD10) is used for
bacterial expression in this example. (QIAGEN, Inc., 9259 Eton
Avenue, Chatsworth, Calif., 91311). pQE9 encodes ampicillin
antibiotic resistance ("Amp") and contains a bacterial origin of
replication ("ori"), an IPTG inducible promoter, a ribosome binding
site ("RBS"), six codons encoding histidine residues that allow
affinity purification using nickel-nitrilo-tri-acetic acid
("Ni-NTA") affinity resin sold by QIAGEN, Inc., supra, and suitable
single restriction enzyme cleavage sites. These elements are
arranged such that an inserted DNA fragment encoding a polypeptide
expresses that polypeptide with the six His residues (i.e., a
"6.times. His tag")) covalently linked to the amino terminus of
that polypeptide.
[0155] The DNA sequence encoding the desired portion of the
connective tissue growth factor-3 protein lacking the hydrophobic
leader sequence is amplified from the deposited cDNA clone using
PCR oligonucleotide primers which anneal to the amino terminal
sequences of the desired portion of the connective tissue growth
factor-3 protein and to sequences in the deposited construct 3' to
the cDNA coding sequence. Additional nucleotides containing
restriction sites to facilitate cloning in the pQE9 vector are
added to the 5' and 3' primer sequences, respectively.
[0156] For cloning the mature protein, the 5' primer has the
sequence 5' CACCACGGATCCAAGGTGCGTACCCAGCTGTGCCCG 3' (SEQ ID NO:4)
containing the underlined BamH1 restriction site, which encodes 24
nucleotides of the connective tissue growth factor-3 protein coding
sequence in FIG. 1 (SEQ ID NO:1) beginning immediately after the
signal peptide. One of ordinary skill in the art would appreciate,
of course, that the point in the protein coding sequence where the
5' primer begins may be varied to amplify a DNA segment encoding
any desired portion of the complete connective tissue growth
factor-3 protein shorter or longer than the mature form.
[0157] The 3' primer has the sequence 5'
GATGTAAGCTTCGTGTCCCCATTCCCAGCCCG 3' (SEQ ID NO:5) containing the
underlined HindIII restriction site followed by 21 nucleotides
complementary to the sequence immediately downstream from the
connective tissue growth factor-3 protein coding sequence in FIG.
1.
[0158] The amplified connective tissue growth factor-3 DNA fragment
and the vector pQE9 are digested with BamHI and HindIII and the
digested DNAs are then ligated together. Insertion of the
connective tissue growth factor-3 DNA into the restricted pQE9
vector places the connective tissue growth factor-3 protein coding
region downstream from the IPTG-inducible promoter and in-frame
with an initiating AUG and the six histidine codons.
[0159] The ligation mixture is transformed into competent E. coli
cells using standard procedures, such as those described in
Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989). E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses lac repressor and confers kanamycin
resistance ("Kan.sup.r"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing connective tissue growth factor-3
protein, is available commercially from QIAGEN, Inc., supra.
Transformants are identified by their ability to grow on LB plates
in the presence of ampicillin and kanamycin. Plasmid DNA is
isolated from resistant colonies and the identity of the cloned DNA
confirmed by restriction analysis, PCR, and DNA sequencing.
[0160] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of
approximately 1:100 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-b-D-thiogalactopyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from lac
repressor sensitive promoters, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
are then harvested by centrifugation.
[0161] The cells are then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH8. The cell debris is removed by
centrifugation, and the supernatant containing the connective
tissue growth factor-3 is loaded onto a nickel-nitrilo-tri-acetic
acid ("NiNTA") affinity resin column (available from QIAGEN, Inc.,
supra). Proteins with a 6.times. His tag bind to the NI-NTA resin
with high affinity and can be purified in a simple one-step
procedure (for details see: The QlAexpressionist, 1995, QIAGEN,
Inc., supra). Briefly the supernatant is loaded onto the column in
6 M guanidine-HCl, pH8, the column is first washed with 10 volumes
of 6 M guanidine-HCl, pH8, then washed with 10 volumes of 6 M
guanidine-HCl pH6, and finally the connective tissue growth
factor-3 is eluted with 6 M guanidine-HCl, pH5.
[0162] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl
pH7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation,
the proteins can be eluted by the addition of 250 mM imidazole.
Imidazole is removed by a final dialyzing step against PBS or 50 mM
sodium acetate pH6 buffer plus 200 mM NaCl. The purified protein is
stored at 4.degree. C. or frozen at -80.degree. C.
Example 2
Cloning and Expression of Connective Tissue Growth Factor-3 Protein
in a Baculovirus Expression System
[0163] In this illustrative example, the plasmid shuttle vector pA2
is used to insert the cloned DNA encoding the complete protein,
including its naturally associated secretary signal (leader)
sequence, into a baculovirus to express the mature connective
tissue growth factor-3 protein, using standard methods as described
in Summers et al., A Manual of Methods for Baculovirus Vectors and
Insect Cell Culture Procedures, Texas Agricultural Experimental
Station Bulletin No. 1555 (1987). This expression vector contains
the strong polyhedrin promoter of the Autographa californica
nuclear polyhedrosis virus (AcMNPV) followed by convenient
restriction sites such as BamHI and Asp718. The polyadenylation
site of the simian virus 40 ("SV40") is used for efficient
polyadenylation. For easy selection of recombinant virus, the
plasmid contains the beta-galactosidase gene from E. coli under
control of a weak Drosophila promoter in the same orientation,
followed by the polyadenylation signal of the polyhedrin gene. The
inserted genes are flanked on both sides by viral sequences for
cell-mediated homologous recombination with wild-type viral DNA to
generate viable virus that express the cloned polynucleotide.
[0164] Many other baculovirus vectors could be used in place of the
vector above, such as pAc373, pVL941 and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, in
Luckow et al., Virology 170:31-39 (1989).
[0165] The cDNA sequence encoding the full length connective tissue
growth factor-3 protein in the deposited clone, including the AUG
initiation codon and the naturally associated leader sequence shown
in FIG. 1 (SEQ ID NO:2), is amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene.
[0166] The 5' primer has the sequence 5'
CGGCAGGATCCGCCATCATGAGAGGCACACCGAAGACCC 3' (SEQ ID NO:6) containing
the underlined BamHI restriction enzyme site, an efficient signal
for initiation of translation in eukaryotic cells, as described by
Kozak, M., J. Mol. Biol. 196:947-950 (1987), followed by 22 bases
of the sequence of the complete connective tissue growth factor-3
protein shown in FIG. 1, beginning with the AUG initiation
codon.
[0167] The 3' primer has the sequence 5'
GATGTGGTACCCGTGTCCCCATTCCCAGCCCG 3' (SEQ ID NO:7) containing the
underlined Asp718 restriction site followed by 21 nucleotides
complementary to the 3' noncoding sequence in FIG. 1.
[0168] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment is then digested with BamHI and
Asp718, and again purified on a 1% agarose gel. This fragment is
designated herein "F1."
[0169] The plasmid is digested with the restriction enzymes BamHI
and Asp718 and optionally, can be dephosphorylated using calf
intestinal phosphatase, using routine procedures known in the art.
The DNA is then isolated from a 1% agarose gel using a commercially
available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This
vector DNA is designated herein "V1."
[0170] Fragment F1 and the dephosphorylated plasmid V1 are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla,
Calif.) cells are transformed with the ligation mixture and spread
on culture plates. Bacteria are identified that contain the plasmid
with the human connective tissue growth factor-3 gene using the PCR
method, in which one of the primers that is used to amplify the
gene and the second primer is from well within the vector so that
only those bacterial colonies containing the connective tissue
growth factor-3 gene fragment will show amplification of the DNA.
The sequence of the cloned fragment is confirmed by DNA sequencing.
This plasmid is designated herein pBacconnective tissue growth
factor-3.
[0171] Five .mu.g of the plasmid pBacconnective tissue growth
factor-3 is co-transfected with 1.0 .mu.g of a commercially
available linearized baculovirus DNA ("BACULOGOLD.TM. baculovirus
DNA", Pharmingen, San Diego, Calif.), using the lipofection method
described by Felgner et al., Proc. Natl. Acad. Sci. USA
84:7413-7417 (1987). One .mu.g of BACULOGOLD.TM. virus DNA and 5 gg
of the plasmid pBacconnective tissue growth factor-3 are mixed in a
sterile well of a microtiter plate containing 50 .mu.l of
serum-free Grace's medium (Life Technologies Inc., Gaithersburg,
Md.). Afterwards, 10 .mu.l Lipofectin plus 90 .mu.l Grace's medium
are added, mixed and incubated for 15 minutes at room temperature.
Then the transfection mixture is added drop-wise to Sf9 insect
cells (ATCC.TM. CRL 1711) seeded in a 35 mm tissue culture plate
with 1 ml Grace's medium without serum. The plate is rocked back
and forth to mix the newly added solution. The plate is then
incubated for 5 hours at 27.degree. C. After 5 hours the
transfection solution is removed from the plate and 1 ml of Grace's
insect medium supplemented with 10% fetal calf serum is added. The
plate is put back into an incubator and cultivation is continued at
27.degree. C. for four days.
[0172] After four days, the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg,
Md.) is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
Md., pages 9-10). After appropriate incubation, blue stained
plaques are picked with the tip of a micropipettor (e.g.,
Eppendorf). The agar containing the recombinant viruses is then
resuspended in a microcentrifuge tube containing 200 .mu.l of
Grace's medium and the suspension containing the recombinant
baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes are
harvested and then they are stored at 4.degree. C. The recombinant
virus is called V-connective tissue growth factor-3.
[0173] To verify the expression of the connective tissue growth
factor-3 gene, Sf9 cells are grown in Grace's medium supplemented
with 10% heat inactivated FBS. The cells are infected with the
recombinant baculovirus V-connective tissue growth factor-3 at a
multiplicity of infection ("MOI") of about 2. Six hours later the
medium is removed and is replaced with SF900 II medium minus
methionine and cysteine (available from Life Technologies Inc.,
Gaithersburg, Md.). If radiolabeled proteins are desired, 42 hours
later, 5 .mu.Ci of .sup.35S-methionine and 5 .mu.Ci
.sup.35S-cysteine (available from Amersham) are added. The cells
are further incubated for 16 hours and then they are harvested by
centrifugation. The proteins in the supernatant as well as the
intracellular proteins are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled). Microsequencing of the amino
acid sequence of the amino terminus of purified protein may be used
to determine the amino terminal sequence of the mature protein and
thus the cleavage point and length of the secretory signal
peptide.
Example 3
Cloning and Expression of Connective Tissue Growth Factor-3 in
Mammalian Cells
[0174] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences, and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI, and the early
promoter of the cytomegalovirus (CMV). However, cellular elements
can also be used (e.g., human actin promoter). Suitable expression
vectors for use in practicing the present invention include, for
example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,
Sweden), pRSVcat (ATCC.TM. 37152), pSV2dhfr (ATCC.TM. 37146) and
pBC12MI (ATCC.TM. 67109). Mammalian host cells that could be used
include, human HeLa 293, H9 and Jurkat cells, mouse NIH3T3 and C127
cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells, and
Chinese hamster ovary (CHO) cells.
[0175] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, or hygromycin allows the identification and isolation of
the transfected cells.
[0176] The transfected gene can also be amplified to express large
amounts of the encoded protein. Dihydrofolate reductase (DHFR) is a
useful marker to develop cell lines that carry several hundred or
even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy et al., Biochem J, 227:277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of
proteins.
[0177] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology, 438-447 (March 1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites
BamHI, XbaI, and Asp718, facilitate the cloning of the gene of
interest. In addition, vectors contain the 3' intron and the
polyadenylation and termination signal of the rat preproinsulin
gene.
Example 3(a)
Cloning and Expression in COS Cells
[0178] The expression plasmid, pCTGF-3 HA, is made by cloning a
cDNA encoding connective tissue growth factor-3 into the expression
vector pcDNAI/Amp or pcDNAIII (which can be obtained from
Invitrogen, Inc.).
[0179] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron; (5) several codons
encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) followed by a termination codon and polyadenylation
signal arranged so that a cDNA can be conveniently placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al., Cell 37:767 (1984). The fusion of the HA tag to the
target protein allows easy detection and recovery of the
recombinant protein with an antibody that recognizes the HA
epitope. pcDNAIII contains, in addition, the selectable neomycin
marker.
[0180] A DNA fragment encoding the connective tissue growth
factor-3 protein is cloned into the polylinker region of the vector
so that recombinant protein expression is directed by the CMV
promoter. The plasmid construction strategy is as follows. The
connective tissue growth factor-3 cDNA of the deposited clone is
amplified using primers that contain convenient restriction sites,
much as described above for construction of vectors for expression
of connective tissue growth factor-3 in E. coli. Suitable primers
include the following, which are used in this example.
[0181] The 5' primer, containing the underlined BamHI site, a Kozak
sequence, an AUG start codon, and 7 codons of the 5' coding region
of the complete connective tissue growth factor-3 has the following
sequence:
TABLE-US-00002 (SEQ ID NO: 8) 5'
GCTCGGATCCGCCATCATGAGAGGCACACCGAAGACCCAC 3'.
[0182] The 3' primer, containing the underlined XbaI site, a stop
codon, and 32 by of 3' coding sequence has the following sequence
(at the 3' end):
TABLE-US-00003 (SEQ ID NO: 9) 5'
GATGTTCTAGAAGAAGGCACTGTTTTGTGGACTGCGACCCCTG 3'.
[0183] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with BamHI and XbaI and then ligated. The ligation
mixture is transformed into E. coli strain SURE (available from
Stratagene Cloning Systems, 11099 North Torrey Pines Road, La
Jolla, Calif. 92037). The transformed culture is then plated on
ampicillin media plates that are then incubated to allow growth of
ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis or other
means for the presence of the connective tissue growth
factor-3-encoding fragment.
[0184] For expression of recombinant connective tissue growth
factor-3, COS cells are transfected with an expression vector, as
described above, using DEAE-DEXTRAN, as described, for instance, in
Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold
Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989). Cells are
incubated under conditions for expression of connective tissue
growth factor-3 by the vector.
[0185] Expression of the connective tissue growth factor-3 HA
fusion protein is detected by radiolabeling and
immunoprecipitation, using methods described in, for example Harlow
et al., Antibodies: a Laboratory Manual, 2nd Ed.; Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To this
end, two days after transfection, the cells are labeled by
incubation for 8 hours in media containing .sup.35S-cysteine. The
cells and the media are collected, and the cells washed and then
lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40,
0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by
Wilson et al. cited above. Proteins are precipitated from the cell
lysate and from the culture media using an HA-specific monoclonal
antibody. The precipitated proteins are then analyzed by SDS-PAGE
gels and autoradiography. An expression product of the expected
size is seen in the cell lysate, which is not seen in negative
controls.
Example 3(b)
Cloning and Expression in CHO Cells
[0186] The vector pC4 is used for the expression of connective
tissue growth factor-3 protein. Plasmid pC4 is a derivative of the
plasmid pSV2-dhfr [ATCC.TM. Accession No. 37146]. The plasmid
contains the mouse DHFR gene under control of the SV40 early
promoter. Chinese hamster ovary- or other cells lacking
dihydrofolate activity that are transfected with these plasmids can
be selected by growing the cells in a selective medium (alpha minus
MEM, Life Technologies) supplemented with the chemotherapeutic
agent methotrexate (MTX). The amplification of the DHFR genes in
cells resistant to methotrexate has been well documented (see,
e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978);
Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta 1097:107-143
(1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68
(1991)). Cells grown in increasing concentrations of MTX develop
resistance to the drug by overproducing the target enzyme, DHFR, as
a result of amplification of the DHFR gene. If a second gene is
linked to the DHFR gene it is usually co-amplified and
over-expressed. It is known in the art that this approach may be
used to develop cell lines carrying more than 1,000 copies of the
amplified gene(s). Subsequently, when the methotrexate is
withdrawn, cell lines are obtained that contain the amplified gene
integrated into one or more chromosome(s) of the host cell.
[0187] For expressing the gene of interest, plasmid pC4 contains
the strong promoter of the long terminal repeat (LTR) of the Rous
Sarcoma Virus (Cullen et al., Molecular and Cellular Biology
5:438-447 (March 1985)), plus a fragment isolated from the enhancer
of the immediate early gene of human cytomegalovirus (CMV) (Boshart
et al., Cell 41:521-530 (1985)). Downstream from the promoter are
restriction enzyme cleavage sites that allow integration of the
genes. Behind these cloning sites, the plasmid contains the 3'
intron and polyadenylation site of the rat preproinsulin gene.
Other high efficiency promoters can also be used for the
expression, e.g., the human (.beta.-actin promoter, the SV40 early
or late promoters or the long terminal repeats from other
retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On
gene expression systems and similar systems can be used to express
the connective tissue growth factor-3 in a regulated way in
mammalian cells (Gossen, M., & Bujard, H., Proc. Natl. Acad.
Sci. USA 89: 5547-5551 (1992)). For the polyadenylation of the
mRNA, other signals, e.g., from the human growth hormone or globin
genes can be used as well. Stable cell lines carrying a gene of
interest integrated into the chromosomes can also be selected upon
co-transfection with a selectable marker such as gpt, G418, or
hygromycin. It is advantageous to use more than one selectable
marker in the beginning, e.g., G418 plus methotrexate.
[0188] The plasmid pC4 is digested with the restriction enzymes
BamHI and Asp718, and then dephosphorylated using calf intestinal
phosphates by procedures known in the art. The vector is then
isolated from a 1% agarose gel.
[0189] The DNA sequence encoding the complete connective tissue
growth factor-3 protein including its leader sequence is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3'
sequences of the gene.
[0190] The 5' primer has the sequence 5'
CGGCAGGATCCGCCATCATGAGAGGCACACCGAAGACCC 3' (SEQ ID NO:6) containing
the underlined BamH1 restriction enzyme site followed by an
efficient signal for initiation of translation in eukaryotes, as
described by Kozak, M., J. Mol. Biol. 196:947-950 (1987), and 22
bases of the coding sequence of connective tissue growth factor-3
shown in FIG. 1 (SEQ ID NO:1).
[0191] The 3' primer has the sequence 5'
GATGTGGTACCCGTGTCCCCATTCCCAGCCCG 3' (SEQ ID NO:7) containing the
underlined Asp718 restriction site followed by 21 nucleotides
complementary to the non-translated region of the connective tissue
growth factor-3 gene shown in FIG. 1 (SEQ ID NO:1).
[0192] The amplified fragment is digested with the endonucleases
BamHI and Asp718, and then purified again on a 1% agarose gel. The
isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then
transformed and bacteria are identified that contain the fragment
inserted into plasmid pC4 using, for instance, restriction enzyme
analysis.
[0193] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSV2-neo using
lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a
dominant selectable marker, the neo gene from Tn5, encoding an
enzyme that confers resistance to a group of antibiotics including
G418. The cells are seeded in alpha minus MEM supplemented with 1
mg/ml G418. After 2 days, the cells are trypsinized and seeded in
hybridoma cloning plates (Greiner, Germany) in alpha minus MEM
supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml
G418. After about 10-14 days, single clones are trypsinized and
then seeded in 6-well petri dishes or 10 ml flasks using different
concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800
nM). Clones growing at the highest concentrations of methotrexate
are then transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 .mu.M, 2 .mu.M, 5 .mu.M, 10 mM,
20 mM). The same procedure is repeated until clones are obtained
which grow at a concentration of 100-200 .mu.M. Expression of the
desired gene product is analyzed, for instance, by SDS-PAGE and
Western blot or by reverse phase HPLC analysis.
Example 4
Tissue Distribution of Connective Tissue Growth Factor-3 Protein
Expression
[0194] Northern blot analysis was carried out to examine connective
tissue growth factor-3 gene expression in human tissues, using
methods described by, among others, Sambrook et al., cited above. A
cDNA probe containing the entire nucleotide sequence of the
connective tissue growth factor-3 protein (SEQ ID NO:1) was labeled
with .sup.32P using the REDIPRIME.TM. DNA labeling system (Amersham
Life Science), according to manufacturer's instructions. After
labelling, the probe was purified using a CHROMA SPIN-100.TM.
column (Clontech Laboratories, Inc.), according to manufacturer's
protocol number PT1200-1. The purified labelled probe was then used
to examine various human tissues for connective tissue growth
factor-3 mRNA.
[0195] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) were obtained from Clontech and were examined
with labelled probe using EXPRESSHYB.TM. hybridization solution
(Clontech) according to manufacturer's protocol number PT 1190-1.
Following hybridization and washing, the blots were mounted and
exposed to film at -70.degree. C. overnight, and films developed
according to standard procedures.
[0196] By Northern expression analysis, CTGF-3 was abundantly
expressed in ovary and Hela cells, as well as other organs, as
shown in the Table below.
TABLE-US-00004 heart ++ lung + skeletal muscle ++ adrenal medulla
++ adrenal cortex +++ Hela cells ++++++++ thymus ++ prostate +++
ovary +++++++ small intestine + colon +++
[0197] It is expected that fibrotic skin or liver would also
express high levels of CTGF-3.
[0198] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0199] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
[0200] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
Sequence CWU 1
1
1311285DNAHomo
sapiensCDS(9)..(758)sig_peptide(9)..(65)mat_peptide(66)..()
1caggggac atg aga ggc aca ccg aag acc cac ctc ctg gcc ttc tcc ctc
50 Met Arg Gly Thr Pro Lys Thr His Leu Leu Ala Phe Ser Leu -15
-10ctc tgc ctc ctc tca aag gtg cgt acc cag ctg tgc ccg aca cca tgt
98Leu Cys Leu Leu Ser Lys Val Arg Thr Gln Leu Cys Pro Thr Pro Cys-5
-1 1 5 10acc tgc ccc tgg cca cct ccc cga tgc ccg ctg gga gta ccc
ctg gtg 146Thr Cys Pro Trp Pro Pro Pro Arg Cys Pro Leu Gly Val Pro
Leu Val 15 20 25ctg gat ggc tgt ggc tgc tgc cgg gta tgt gca cgg cgg
ctg ggg gag 194Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg
Leu Gly Glu 30 35 40ccc tgc gac caa ctc cac gtc tgc gac gcc agc cag
ggc ctg gtc tgc 242Pro Cys Asp Gln Leu His Val Cys Asp Ala Ser Gln
Gly Leu Val Cys 45 50 55cag ccc ggg gca gga ccc ggt ggc cgg ggg gcc
ctg tgc ctc ttg gca 290Gln Pro Gly Ala Gly Pro Gly Gly Arg Gly Ala
Leu Cys Leu Leu Ala60 65 70 75gag gac gac agc agc tgt gag gtg aac
ggc cgc ctg tat cgg gaa ggg 338Glu Asp Asp Ser Ser Cys Glu Val Asn
Gly Arg Leu Tyr Arg Glu Gly 80 85 90gag acc ttc cag ccc cac tgc agc
atc cgc tgc cgc tgc gag gac ggc 386Glu Thr Phe Gln Pro His Cys Ser
Ile Arg Cys Arg Cys Glu Asp Gly 95 100 105ggc ttc acc tgc gtg ccg
ctg tgc agc gag gat gtg cgg ctg ccc agc 434Gly Phe Thr Cys Val Pro
Leu Cys Ser Glu Asp Val Arg Leu Pro Ser 110 115 120tgg gac tgc ccc
cac ccc agg agg gtc gag gtc ctg ggc aag tgc tgc 482Trp Asp Cys Pro
His Pro Arg Arg Val Glu Val Leu Gly Lys Cys Cys 125 130 135cct gag
tgg gtg tgc ggc caa gga ggg gga ctg ggg acc cag ccc ctt 530Pro Glu
Trp Val Cys Gly Gln Gly Gly Gly Leu Gly Thr Gln Pro Leu140 145 150
155cca gcc caa gga ccc cag ttt tct ggc ctt gtc tct tcc ctg ccc cct
578Pro Ala Gln Gly Pro Gln Phe Ser Gly Leu Val Ser Ser Leu Pro Pro
160 165 170ggt gtc ccc tgc cca gaa tgg agc acg gcc tgg gga ccc tgc
tcg acc 626Gly Val Pro Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys
Ser Thr 175 180 185acc tgt ggg ctg ggc atg gcc acc cgg gtg tcc aac
cag aac cgc ttc 674Thr Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn
Gln Asn Arg Phe 190 195 200tgc cga ctg gag acc cag cgc cgc ctg tgc
ctg tcc agg ccc tgc cca 722Cys Arg Leu Glu Thr Gln Arg Arg Leu Cys
Leu Ser Arg Pro Cys Pro 205 210 215ccc tcc agg ggt cgc agt cca caa
aac agt gcc ttc tagagccggg 768Pro Ser Arg Gly Arg Ser Pro Gln Asn
Ser Ala Phe220 225 230ctgggaatgg ggacacggtg tccaccatcc ccagctggtg
gccctgtgcc tgggccctgg 828gctgatggaa gatggtccgt gcccaggccc
ttggctgcag gcaacacttt agcttgggtc 888caccatgcag aacaccaata
ttaacacgct gcctggtctg tctggatccc gaggtatggc 948agaggtgcaa
gacctagtcc cctttcctct aactcactgc ctaggaggct ggccaaggtg
1008tccagggtcc tctagcccac tccctgccta cacacacagc ctatatcaaa
catgcacacg 1068ggcgagcttt ctctccgact tcccctgggc aagagatggg
acaagcagtc ccttaatatt 1128gaggctgcag caggtgctgg gctggactgg
ccatttttct gggggtagga tgaagagaag 1188gcacacagag attctggatc
tcctgctgcc ttttctggag tttgtaaaat tgttcctgaa 1248tacaagccta
tgcgtgaaaa aaaaaaaaaa aaaaaaa 12852250PRTHomo sapiens 2Met Arg Gly
Thr Pro Lys Thr His Leu Leu Ala Phe Ser Leu Leu Cys -15 -10 -5Leu
Leu Ser Lys Val Arg Thr Gln Leu Cys Pro Thr Pro Cys Thr Cys -1 1 5
10Pro Trp Pro Pro Pro Arg Cys Pro Leu Gly Val Pro Leu Val Leu Asp
15 20 25Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Pro
Cys30 35 40 45Asp Gln Leu His Val Cys Asp Ala Ser Gln Gly Leu Val
Cys Gln Pro 50 55 60Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu
Leu Ala Glu Asp 65 70 75Asp Ser Ser Cys Glu Val Asn Gly Arg Leu Tyr
Arg Glu Gly Glu Thr 80 85 90Phe Gln Pro His Cys Ser Ile Arg Cys Arg
Cys Glu Asp Gly Gly Phe 95 100 105Thr Cys Val Pro Leu Cys Ser Glu
Asp Val Arg Leu Pro Ser Trp Asp110 115 120 125Cys Pro His Pro Arg
Arg Val Glu Val Leu Gly Lys Cys Cys Pro Glu 130 135 140Trp Val Cys
Gly Gln Gly Gly Gly Leu Gly Thr Gln Pro Leu Pro Ala 145 150 155Gln
Gly Pro Gln Phe Ser Gly Leu Val Ser Ser Leu Pro Pro Gly Val 160 165
170Pro Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys
175 180 185Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe
Cys Arg190 195 200 205Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg
Pro Cys Pro Pro Ser 210 215 220Arg Gly Arg Ser Pro Gln Asn Ser Ala
Phe 225 2303349PRTHomo sapiens 3Met Thr Ala Ala Ser Met Gly Pro Val
Arg Val Ala Phe Val Val Leu1 5 10 15Leu Ala Leu Cys Ser Arg Pro Ala
Val Gly Gln Asn Cys Ser Gly Pro 20 25 30Cys Arg Cys Pro Asp Glu Pro
Ala Pro Arg Cys Pro Ala Gly Val Ser 35 40 45Leu Val Leu Asp Gly Cys
Gly Cys Cys Arg Val Cys Ala Lys Gln Leu 50 55 60Gly Glu Leu Cys Thr
Glu Arg Asp Pro Cys Asp Pro His Lys Gly Leu65 70 75 80Phe Cys Asp
Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Thr 85 90 95Ala Lys
Asp Gly Ala Pro Cys Ile Phe Gly Gly Thr Val Tyr Arg Ser 100 105
110Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp
115 120 125Gly Ala Val Gly Cys Met Pro Leu Cys Ser Met Asp Val Arg
Leu Pro 130 135 140Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu
Pro Gly Lys Cys145 150 155 160Cys Glu Glu Trp Val Cys Asp Glu Pro
Lys Asp Gln Thr Val Val Gly 165 170 175Pro Ala Leu Ala Ala Tyr Arg
Leu Glu Asp Thr Phe Gly Pro Asp Pro 180 185 190Thr Met Ile Arg Ala
Asn Cys Leu Val Gln Thr Thr Glu Trp Ser Ala 195 200 205Cys Ser Lys
Thr Cys Gly Met Gly Ile Ser Thr Arg Val Thr Asn Asp 210 215 220Asn
Ala Ser Cys Arg Leu Glu Lys Gln Ser Arg Leu Cys Met Val Arg225 230
235 240Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys Lys Gly Lys Lys
Cys 245 250 255Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile Lys Phe Glu
Leu Ser Gly 260 265 270Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys Phe
Cys Gly Val Cys Thr 275 280 285Asp Gly Arg Cys Cys Thr Pro His Arg
Thr Thr Thr Leu Pro Val Glu 290 295 300Phe Lys Cys Pro Asp Gly Glu
Val Met Lys Lys Asn Met Met Phe Ile305 310 315 320Lys Thr Cys Ala
Cys His Tyr Asn Cys Pro Gly Asp Asn Asp Ile Phe 325 330 335Glu Ser
Leu Tyr Tyr Arg Lys Met Tyr Gly Asp Met Ala 340 345436DNAArtificial
Sequence5' primer with BamHI site 4caccacggat ccaaggtgcg tacccagctg
tgcccg 36532DNAArtificial Sequence3' primer with HindIII site
5gatgtaagct tcgtgtcccc attcccagcc cg 32639DNAArtificial Sequence5'
primer with BamHI site 6cggcaggatc cgccatcatg agaggcacac cgaagaccc
39732DNAArtificial Sequence3' primer with Asp718 site 7gatgtggtac
ccgtgtcccc attcccagcc cg 32840DNAArtificial Sequence5' primer with
BamHI site 8gctcggatcc gccatcatga gaggcacacc gaagacccac
40943DNAArtificial Sequence3' primer with XbaI site 9gatgttctag
aagaaggcac tgttttgtgg actgcgaccc ctg 4310264DNAHomo SapiensEST
10ctggtggccc tgtgcctggg ccctgggctg atggaagatg gtccgtgccc aggccnttgg
60ctgcaggcaa cactttagct tgggtccacc atgcagaaca ccaatattaa cacgctgcct
120ggtctgtntg gatcccgagg tatggcagag gtgcaagacc tagtcctctt
tcctctaact 180cactgcctag gaggctggcc aaggtgtcca gggtcctcta
gcccacttcc tgcctacaca 240cacagnctat atcaaacatg caca 26411239DNAHomo
SapiensEST 11ggcanagggn cacaccgaag acccacctcc tggccttctc cctcctctgc
ctcctctcaa 60aggtgcgtac ccagctgtgc ccganaccat gtacctgccc ctgggcacct
ccccnatgcc 120cgctgggagt acccctggtg ctggatggct gtggctgctg
ccggngttat gtgcacggcg 180gctgggggag ccctgcacta nactccacgt
ctgcaaggnc agcnaagggc ctggtntgc 23912313DNAHomo SapiensEST
12ccaatattaa cacgctgcct ggnctgtntg gttcccgagg tatggcagag gtgcaagacc
60tagtcccctt tcctctaact cactgcctag gaggctggcc aaggtgtcca gggtcctcta
120gcccactccc tgcctacaca cacagcctat atcaaacatg cacacgggcg
agctttctct 180ccgacttccc ctgggcaaga gatgggacaa gcagtccctt
aatattgagg ctgcagcagg 240tgctgggctg gactggccat ttttntgggg
gtaggatgaa gagaaggcac acagagattc 300tggatctcct gct 3131360DNAHomo
SapiensEST 13gatctcctgc tgcctttcct ggagtttgta aaattntncc tgaatacaag
cctatgcgtg 60
* * * * *