Note: Descriptions are shown in the official language in which they were submitted.
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CONNECTTVE TISSUE GROWTH FACTOR-2
INTRODUCTION
[0001] This invention relates to newly identified pol~ynucleotides,
polypeptides
encoded by such polynucleotides, the use of such polynucleotides and
polypeptides, as well as
the production of such polynucleotides and polypeptides. More particularly,
the polypeptide
of the present invention is connective tissue growth factor-2 sometimes
hereinafter referred to
as "CTGF-2". The invention also relates to inhibiting the action of such
polypeptides. In
addition, the invention encompasses methods of using CTGF-2 polynucleotides
and
polypeptides to stimulate angiogenesis, i.e., formation of new blood vessels.
The invention
also encompasses methods of treatment of atherosclerosis, ischemia,
restenosis.
BACKGROUND OF THE INVENTION
[0002] The CTGF polypeptides are structurally and functionally related to a
family of
growth factors which include IGF (insulin-like growth factor), PDGF (platelet-
derived growth
factor), and FGF (fibroblast growth factor). This emerging family of secreted
proteins are a
group of cysteine-rich proteins. This group of growth factors are important
for normal
growth, differentiation, morphogenesis of the cartilaginous skeleton of an
embryo and cell
growth. Among some of the functions that have been discovered for these growth
factors are
wound healing, tissue repair, implant fixation and stimulating increased bone
mass.
[0003] The extended superfamily of growth factors include TGF (transforming
growth factor), bone morphogenic factors, and activins, among others.
[0004] The most well-known growth factor, TGF exerts a number of different
effects
on a variety of cells. For example, TGF-alpha can inhibit the differentiation
of certain cells of
mesodermal origin (Florini, J.R. et al., T. Rill_ C',hem_, 261:1659-16513
(1986) induced the
differentiation of others (Seyedine, S.M. et al., PNA~ TTRA, 82:2267-2271
(1987) and
potently inhibit proliferation of various types of epithelial cells, (Tucker,
R.F., ~,
226:705-705 (1984)). This last activity has led to the speculation that one
important
physiological role for TGF-" is to maintain the repressed growth state of many
types of cells.
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Accordingly, cells that lose the ability to respond to TGF-" are more likely
to exhibit
uncontrolled growth and become tumorigenic.
[0005] Accordingly, due to amino acid sequence homology the polypeptide of the
present invention is a member of this extended family of growth factors which
has many
effects on a variety of different tissues.
SUMMARY OF THE INVENTION
[0006] In accordance with one aspect of the present invention, there is
provided a
novel CTGF-2 polypeptide, as well as biologically active and diagnostically or
therapeutically
useful fragments, analogs and derivatives thereof. The polypeptide of the
present invention is
of human origin. .
[0007] In accordance with another aspect of the present invention, there are
provided
isolated nucleic acid molecules encoding such CTGF-2 polypeptide and
polypeptide
fragments, analogs, and derivatives of the present invention, including mRNAs,
DNAs,
cDNAs, genomic DNAs as well as analogs and biologically active and
diagnostically or
therapeutically useful fragments thereof.
[0008] In accordance with yet a further aspect of the present invention, there
is
provided a process for producing such polypeptide by recombinant techniques
comprising
culturing recombinant prokaryotic and/or eukaryotic host cells, containing a
nucleic acid
sequence encoding a polypeptide of the present invention, under conditions
promoting
expression of said protein and subsequent recovery of said protein.
[0009] In accordance with yet a further aspect of the present invention, there
is
provided a process for utilizing such polypeptide, or polynucleotide encoding
such
polypeptide for therapeutic purposes, for example, enhancing the repair of
connective and
support tissue, promoting the attachment, fixation and stabilization of tissue
implants and
enhancing wound healing. In addition methods for stimulating angiogenesis,
i.e., formation
of new blood vessels, using the polynucleotides and polypeptides of the
invention are
provided. Such methods include, but are not limited to, gene therapy of
patients in need of
new blood vessel formation. Thus, the polynucleotides and polypeptides of the
invention are
useful in the treatment of cardiovascular disease, including but not limited
to atherosclerosis,
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restenosis, reperfusion injury Such disorders include, but are not limited to,
heart failure,
angina, blood vessel (e.g. coronary artery) blockage and ischemia, inlcuding
critical limb
ischemia and refractory myocardial ischemia.
[0010] In accordance with yet a further aspect of the invention, vectors are
provided
for use in administering the polynucleotides of the invention.
[0011] In accordance with yet a further aspect of the present invention, there
are
provided antibodies against such polypeptides.
[OOIZ] In accordance with yet a further aspect of the present invention, there
is also
provided nucleic acid probes comprising nucleic acid molecules of sufficient
length to
specifically hybridize to a nucleic acid sequence of the present invention.
(0013] In accordance with yet a further aspect of the present invention, there
are
provided antibodies against such polypeptides.
(0014] In accordance with another aspect of the present invention, there are
provided
agonists which mimic the polypeptide of the present invention and binds to the
receptors.
[0015] In accordance with yet another aspect of the present invention, there
are
provided antagonists to such polypeptides, which may be used to inhibit the
action of such
polypeptides, for example, in the treatment of CTGF dependent tumor growth.
[0016] In accordance with still another aspect of the present invention, there
are
provided diagnostic assays for detecting diseases or susceptibility to
diseases related to
mutations in the nucleic acid sequences encoding a polypeptide of the present
invention.
[0017] In accordance with yet a further aspect of the present invention, there
is
provided a process for utilizing such polypeptides, or polynucleotides
encoding such
polypeptides, for ih vitro purposes related to scientific research, for
example, synthesis of
DNA and manufacture of DNA vectors.
[0018] These and other aspects of the present invention should be apparent to
those
skilled in the art from the teachings herein.
[0019] The following drawings are illustrative of embodiments of the invention
and
are not meant to limit the scope of the invention as encompassed by the
claims.
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BRIEF DESCRIPTION OF THE FIGURES
[0020] Figures lA-B: Figures lA-B depict the cDNA sequence and corresponding
deduced amino acid sequence of CTGF-2. The standard one-letter abbreviation
for amino
acids is used.
[0021] Figure 2: Migration of HDMEC across 5-~,m pore size Transwell migration
chamber incubated with supernatant of cells infected with Ad-Null, Ad-VEGFms.
or Ad-CTGF-
2. In a comparable manner, Ad-CTGF-2 and Ad-VEGFI6s-infected cells supernatant
significantly stimulated the migration of HDMEC compared with Ad-Null.
*P < 0.05 vs Ad-Null ; Non significant (NS) vs Ad-VEGFiss
[0022] Figure 3: Calculated blood flow at rest (A) and after papaverine
injection (B) in
the ischemic limb. Ad-VEGFI6s and Ad-CTGF-2-treated animals had a significant
increase in
flows when day 40 is compared with day 10 both at rest and after papaverine
infusion. At day
40, rest flow as well as maximum flow were significantly improved in Ad-CTGF-
2' animals
compared with Ad-Null group. Although a trend toward greater blood flows
improvement
appeared in Ad-CTGF-2 in comparison with Ad-VEGFi6s-treated rabbits, there was
no
statistical difference between these two groups.
NS vs day 10 in the same group
*P< 0.01 vs day 10 in the same group
?P<0.05 vs Ad-Null-treated rabbits
[0023] Figure 4: Serial assessement of internal iliac artery luminal diameter
in the
ischemic limb.
[0024] During the 30 days of the follow-up period, a significant increase in
angiographic luminal diameter was recorded only for Ad-VEGFi6s and Ad-CTGF-2-
treated
animals. At day 40, mean diameter of the Ad-CTGF-2 group appeared higher
compared to the
Ad-Null group.
*P< 0.001 vs day 10 in the same group
?P< 0.05 vs Ad-Null-treated rabbits
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[0025] Figure 5: Representative angiograms of the ischemic limb recorded on
day 10
(upper views) and on day 40 (lower views) from A) Ad-Null, B) Ad-VEGFiss and
C) Ad-
CTGF-2-treated animals showing collateral vessel formation through the 30 days
follow-up
period. The morphology of the augmented collateral circulation was typically
made of fine
networks of so-called midzone collateral vessels. In some cases, serial
angiograms disclosed
progressive linear extension of the stem artery to the reentry (popliteal or
saphenous arteries),
predominently for Ad-CTGF-2-treated rabbits.
[0026] Figure 6: Quantitative angiographic analysis of collateral vessel
development in
the medial thigh of ischemic hindlimb evaluated by an angiographic score. The
vascular density
was significantly improved for each group at day 40 compared to day 10.
Angiographic scores
at day 40 were significantly higher in both treated groups compared to control
animals and, in
CTGF-2-treated animals it exceeded that of the VEGF,es-treated group.
*P<0.01 vs day 40 in the same group
**P<0.001 vs day 40 in the same group
?P< 0.0001 vs Ad-Null-treated rabbits
#P< 0.01 vs Ad-VEGF,6s-treated rabbits
[0027] Figure 7: Capillary density evaluated on histological sections of
adductor (A)
and (B) semimembranous muscles harvested at the ime of sacrifice (day 40). In
the adductor
muscle, the capillary-to-myocyte ratio was statistically increased in the Ad-
CTGF-2 group
compared with the Ad-Null and Ad-VEGFiss-treated animals. In the
semimembranous muscle,
the ratio was increased for the Ad-VEGFi6s compared to the Ad-Null whereas the
increase seen
for Ad-CTGF-2 did not attain the level of significance.
NS vs Ad-Null-treated rabbits
*P<0.01 vs Ad-Null-treated rabbits
?P<0.001 vs Ad-VEGFi6s-treated rabbits
#P<0.05 vs Ad-Null-treated rabbits
[0028] Figure 8: Figure 8 shows the vector map of the transfer vector
pTG13387. This
transfer vector contains the Ad5 1-458 region followed by the CMV enhancer/
promoter and a
chimeric intron generated by combining the splice donor from the human (3-
globin intron 1
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and the splice acceptor from the IgG intervening sequence obtained from pCI
plasmid
(Promega, Charbonnieres, France).
[0029] Figure 9: Figure 9 shows the vector map of the CTGF-2 transfer vector
ptgI4488.
[0030] Figure 10: Figure 10 shows the vector map of the adenoviral vector
ptg14550.
[0031] Figures 11A-C: Figure 11 depicts an alternative cDNA sequence and
corresponding deduced amino acid sequence of CTGF-2. The standard one-letter
abbreviation for amino acids is used.
DETAILED DESCRIPTION OF THE INVENTION
[0032] In accordance with an aspect of the present invention, there is
provided an
isolated nucleic acid (polynucleotide) (SEQ ID NO:l) which encodes for the
polypeptide
having the deduced amino acid sequence of Figures lA-B (SEQ ID N0:2) or as
encoded by
the cDNA of the clone deposited as with the American Type Culture Collection
(ATCC) on
June 7, 1994 as Deposit No. 75804. An alternative cDNA sequence (SEQ ID N0:6)
and
corresponding deduced amino acid sequence (SEQ ID N0:7) of CTGF-2 is
demonstrated in
Figures 11A-C.
[0033] Tn accordance with an additional aspect of the present invention, there
is
provided a nucleic acid vector for gene therapy-based methods of delivering
polynucleotides
encoding the CTGF-2 polypeptide having the amino acid sequence of Figures lA-B
as
described in Example 12, which was deposited with the Pasteur Institute
Depository on July
9, 2001, having received the registration number CNCM I-2695. The Pasteur
Institute
Depository is located at the following address: Collection Nationale de
Cultures de
Microorganismes, INSTITUT PASTEUR, 25, rue du DOCTEUR ROUX, F-74724 Paris
Cedex 15, France.
[0034] The polynucleotide of this invention was discovered in a cDNA library
derived
from human fetal lung. It is structurally related to the IGF and PDGF family.
It contains an
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open reading frame encoding a protein of 382 amino acid residues of which
approximately
the first 23 or 24 amino acids residues are the putative leader sequence such
that the putative
mature protein comprises 358 amino acids (residues 24-382 or 25-382). In
addition to the
leader sequence, CTGF-2 contains an IGF Binding Domain that extends from about
amino
acid Ser-24 to about Ala-93; a von Willebrand Factor Type C Repeat Domain from
about
amino acid Arg-98 to about amino acid Asp-164; a Sulfated Glycoconjugate
Binding Motif
from about amino acid Cys-229 to about amino acid Gly-273; and a C-Terminal
Dimerization
and Receptor Binding Domain from about amino acid Cys-286 to about amino acid
His-361.
In this context "about" includes the particularly recited ranges, laxger or
smaller by several (5,
4, 3, 2, or 1 ) nucleotides, at either terminus or at both termini.
[0035] The protein exhibits a high degree of homology to Mouse CTGF with 49%
identity and 67% similarity and to Cyr61 with 89% identity and 93% similarity.
Cyr61 is a
growth factor-inducible immediate early gene initially identified in serum-
stimulated mouse
fibroblasts. It encodes a member of an emerging family of cysteine-rich
secreted proteins that
includes a connective tissue growth factor (O'Brien and Lau, L.F., Cell Growth
Differ., 3:64~-
54 (1992)).
[0036] In accordance with the invention, CTGF-2 is active in stimulating
angiogenesis. Thus, novel methods for stimulating angiogenesis, i.e.,
formation of new blood
vessels, using the polynucleotides and polypeptides of the invention are
provided. Such
methods include, but are not limited to, gene therapy of patients in need of
new blood vessel
formation. Thus, the polynucleotides and polypeptides of the invention are
useful in the
treatment of cardiovascular disease, including but not limited to
atherosclerosis, restenosis,
reperfusion injury Such disorders include, but are not limited to, heart
failure, angina, blood
vessel (e.g. coronary artery) blockage and ischemia, including critical limb
ischemia and
refractory myocardial ischernia.
Nucleic. Acid Rmh~dimentfi
[0037] The CTGF-2 polynucleotides and derivatives described herein below are
useful, for example, in the production of CTGF-2 polypeptide and polypeptide
derivatives
(described below), as well as as agents in gene therapy to stimulate
angiogenesis, as described
in detail below. The polynucleotides of the present invention may be in the
form of RNA or
in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
The
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DNA may be double-stranded or single-stranded, and if single stranded may be
the coding
strand or non-coding (anti-sense) strand. The coding sequence which encodes
the mature
polypeptide may be identical to the coding sequence shown in Figures lA-B or
that of the
deposited clone in ATCC accession no. 75804, or may be a different coding
sequence which
coding sequence, as a result of the redundancy or degeneracy of the genetic
code; encodes the
same mature polypeptide as the DNA of Figures lA-B or the deposited cDNA.
[0038] The polynucleotide which encodes for the mature polypeptide of Figures
lA-B
or for the mature polypeptide encoded by the deposited cDNA may include: only
the coding
sequence for the mature polypeptide; the coding sequence for the mature
polypeptide and
additional coding sequence such as a leader or secretory sequence or a
proprotein sequence;
the coding sequence for the mature polypeptide (and optionally additional
coding sequence)
and non-coding sequence, such as introns or non-coding sequence 5' and/or 3'
of the coding
sequence for the mature polypeptide.
[0039] Thus, the term "polynucleotide encoding a polypeptide" encompasses a
polynucleotide which includes only coding sequence fox the polypeptide as well
as a
polynucleotide which includes additional coding and/or non-coding sequence.
[0040] The present invention further relates to variants of the hereinabove
described
polynucleotides which encode for fragments, analogs and derivatives of the
polypeptide
having the deduced amino acid sequence of Figures lA-B or the polypeptide
encoded by the
cDNA of the deposited clone. The variant of the polynucleotide may be a
naturally occurring
allelic variant of the polynucleotide or a non-naturally occurnng variant of
the polynucleotide.
[0041] Thus, the present invention includes. polynucleotides encoding the same
mature polypeptide as shown in Figures lA-B or the same mature polypeptide
encoded by the
cDNA of the deposited clone as well as variants of such polynucleotides which
variants
encode for a fragment, derivative or analog of the polypeptide of Figures lA-B
or the
polypeptide encoded by the cDNA of the deposited clone. Such nucleotide
variants include
deletion variants, substitution variants and addition or insertion variants.
[0042] As hereinabove indicated, the polynucleotide may have a coding sequence
which is a naturally occurring allelic variant of the coding sequence shown in
Figures lA-B
or of the coding sequence of the deposited clone. As known in the art, an
allelic variant is an
alternate form of a polynucleotide sequence which may have a substitution,
deletion or
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addition of one or more nucleotides, which does not substantially alter the
function of the
encoded polypeptide.
[0043] The polynucleotides of the present invention may also have the coding
sequence fused in frame to a marker sequence which allows for purification of
the
polypeptide of the present invention. The marker sequence may be a hexa-
histidine tag
supplied by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the
marker in the case of a bacterial host, or; for example, the marker sequence
may be a
hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The
HA tag
corresponds to an epitope derived from the influenza hemagglutinin protein
(Wilson, L, et al.,
Cell, 37:767 (1984)).
[0044] The term "gene" means the segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following the coding
region (leader and
trailer) as well as intervening sequences (introns) between individual coding
segments
(exons).
[0045] Fragments of the full length gene of the present invention may be used
as a
hybridization probe for a cDNA library to isolate the full length cDNA and to
isolate other
cDNAs which have a high sequence similarity to the gene or similar biological
activity.
Probes of this type preferably have at least 30 bases and may contain, for
example, 50 or more
bases. The probe may also be used to identify a cDNA clone corresponding to a
full length
transcript and a genomic clone or clones that contain the complete gene
including regulatory
and promoter regions, exons, and introns. An example of a screen comprises
isolating the
coding region of the gene by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary to that of
the gene of the present invention are used to screen a library of human cDNA,
genomic DNA
or mRNA to determine which members of the library. the probe hybridizes to.
[0046] The present invention further relates to polynucleotides which
hybridize to the
hereinabove-described sequences if there is at least 90%, and more preferably
at least 95%
identity between the sequences. The present invention particularly relates to
polynucleotides
which hybridize under stringent conditions to the hereinabove-described
polynucleotides.
The polynucleotides which hybridize to the hereinabove described
polynucleotides in a
preferred embodiment encode polypeptides which either retain substantially the
same
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biological function or activity as the mature polypeptide encoded by the cDNAs
of Figures
lA-B or the deposited cDNA.
[0047] Alternatively, the polynucleotide may have at least 20 bases,
preferably 30
bases, and more preferably at least SO bases which hybridize to a
polynucleotide of the
present invention and which has an identity thereto, as hereinabove described,
and which may
or may not retain activity. For example, such polynucleotides may be employed
as probes for
the polynucleotide of Figures 1A-B, for example, for recovery of the
polynucleotide or as a
diagnostic probe or as a PCR primer.
[0048] Thus, the present invention is directed to polynucleotides having at
least a
90% and more preferably at least a 95% identity to a polynucleotide which
encodes the
polypeptide of Figures lA-B as well as fragments thereof, which fragments have
at least 30
bases and preferably at least 50 bases and to polypeptides encoded by such
polynucleotides.
[0049] 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 complement of the CTGF-2 coding polynucleotide sequence
disclosed herein or
the cDNA clone contained in ATCC Deposit No. 75804. By "stringent
hybridization
conditions" is intended overnight incubation at 42EC in a solution comprising:
50%
formamide, Sx SSC (750 mM NaCI, 7~SmM trisodium citrate), 50 mM sodium
phosphate (pH
7.6), Sx Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured,
sheared salmon
sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.
[0050] 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. In
this context
"about" includes the particularly recited size, larger or smaller by several
(5, 4, 3, 2, or 1)
nucleotides, at either terminus or at both termini.
[0051] 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 Figures lA-
B).
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[0052] Of course, a polynucleotide which hybridizes only to a poly A sequence
(such
as the 3' terminal poly(A) tract of the CTGF-2 cDNA, or to a complementary
stretch of T (or
I~ 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).
[0053] In specific embodiments, the polynucleotides of the invention are less
than
100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300 kb, 250
kb, 200 kb,
175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15
kb, 10 kb, 7.5
kb, or 5 kb in length.
[0054] In further embodiments, polynucleotides of the invention comprise at
least 15,
at least 30, at least 50, at least 100, or at least 250, at least 500, or at
least 1000 contiguous
nucleotides of CTGF-2 coding sequence, but consist of less than or equal to
1000 kb, 500 kb,
250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10
kb, or 5 kb of
genomic DNA that flanks the 5' or 3' coding nucleotide set forth in Figures lA-
B. In further
embodiments, polynucleotides of the invention comprise at least 15, at least
30, at least 50, at
least 100, or at least 250, at least 5.00, or at least 1000 contiguous
nucleotides of CTGF-2
coding sequence, but do not comprise all or a portion of any CTGF-2 intron. In
another
embodiment, the nucleic acid comprising CTGF-2 coding sequence does not
contain coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the CTGF-2 gene in the
genome). In
other embodiments, the polynucleotides of the invention do not contain the
coding sequence
of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic
flanking gene(s).
[0055] As indicated, nucleic acid molecules of the present invention which
encode a
CTGF-2 polypeptide may include, but are not limited to the coding sequence for
the mature
polypeptide, by itself; the coding sequence for the mature polypeptide and
additional
sequences, such as those encoding a 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 firanscribed, non-translated sequences that play a role in transcription,
mRNA processing -
including splicing and polyadenylation signals, for example - ribosome binding
and stability
of mRNA; additional coding sequence which codes for additional amino acids,
such as those
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which provide additional functionalities. Thus, for instance, the polypeptide
may be fused to
a marker sequence, such as a peptide, which facilitates purification of the
fused polypeptide.
In certain preferred embodiments of this aspect of the invention, the marker
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., PYOG.
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 37:767-778 (1984). As discussed below, other
such fusion
proteins include the CTGF-2 receptor fused to Fc at the N- or C-terminus.
[0056] The present invention further relates to variants of the nucleic acid
molecules
of the present invention, which encode portions, analogs, or derivatives of
the CTGF-2
receptor. 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 occurnng variants may be produced using art-known
mutagenesis
techniques.
[0057] 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
or 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 CTGF-2 or portions thereof. Also especially
preferred in this
regard are conservative substitutions.
[00S&) Further embodiments of the invention include isolated nucleic acid
molecules
comprising or, alternatively, consisting of a polynucleotide having a
nucleotide sequence at
least 80%, 85%, or 90% identical, and more preferably at least 95%, 96%, 97%,
98%, or 99°/q
identical to: (a) a nucleotide sequence encoding the polypeptide having the
amino acid
sequence in Figures lA-B; (b) a nucleotide sequence encoding the polypeptide
having, the
amino acid sequence in Figures lA-B, but lacking the amino terminal
methionine; (c) a
nucleotide sequence encoding the polypeptide having the amino acid sequence in
Figures lA-
B, but lacking the N-terminal leader peptide (i.e. the mature protein); (d) a
nucleotide
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sequence encoding the polypeptide having the amino acid sequence encoded by
the cDNA
clone contained in ATCC Deposit No. 75804; (e) a nucleotide sequence encoding
the mature
CTGF-2 polypeptide having the amino acid sequence encoded by the cDNA clone
contained
in ATCC Deposit No. 75804; (f) a nucleotide sequence encoding the CTGF-2 IGF
Binding
Domain; (g) a nucleotide sequence encoding the CTGF-2 von Willebrand Factor
Type C
Repeat Domain; (h) a nucleotide sequence encoding the CTGF-2 Sulfated
Glycoconjugate
Binding Motif; (i) a nucleotide sequence encoding the CTGF-2 C-Terminal
Dimerization and
Receptor Binding Domain; and (j) a nucleotide sequence complementary to any of
the
nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.
[0059] By a polynucleotide having a nucleotide sequence at least, for example,
95%
"identical" to a reference nucleotide sequence encoding a CTGF-2 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 mismatches per
each 100
nucleotides of the reference nucleotide sequence encoding the CTGF-2
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 mismatches 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. The reference (query) sequence may be
the entire
CTGF-2 encoding nucleotide sequence shown in Figures lA-B or any CTGF-2
polynucleotide fragment (e.g., a polynucleotide encoding the amino acid
sequence of any of
the CTGF-2 N- and/or C- terminal deletions described herein), variant,
derivative or analog,
as described herein.
(0060] As a practical matter, whether any particular nucleic acid molecule is
at least
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the
nucleotide
sequence shown in Figures lA-B or to the nucleotide 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, WI 53711).
Bestfit uses the
13
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
local homology algorithm of Smith and Waterman, Advances ira Applied
Mathen2atics 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.
[0061] In a specific embodiment, the identity between a reference (query)
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, is determined using the FASTDB computer program based on
the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred
parameters
used in a FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,
Randomization Group
Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window
Size=500 or the
length of the subject nucleotide sequence, whichever is shorter. According to
this
embodiment, if the subject sequence is shorter than the query sequence because
of 5' or 3'
deletions, not because of internal deletions, a manual correction is made to
the results to take
into consideration the fact that the FASTDB program does not account for 5'
and 3'
truncations of the subject sequence when calculating percent identity. For
subject sequences
truncated at the 5' or 3' ends, relative to the query sequence, the percent
identity is corrected
by calculating the number of bases of the query sequence that are 5' and 3' of
the subject
sequence, which are not matched/aligned, as a percent of the total bases of
the query
sequence. A determination of whether a nucleotide is matchedlaligned is
determined by
results of the FASTDB sequence alignment. This percentage is then subtracted
from the
percent identity, calculated by the above FASTDB program using the specified
parameters, to
arrive at a final percent identity score. This corrected score is what is used
for the purposes of
this embodiment. Only bases outside the 5' and 3' bases of the subject
sequence, as displayed
by the FASTDB alignment, which are not matched/aligned with the query
sequence, are
calculated for the purposes of manually adjusting the percent identity score.
For example, a
90 base subject sequence is aligned to a 100 base query sequence to determine
percent
identity. The deletions occur at the 5' end of the subject sequence and
therefore, the FASTDB
alignment does not show a matched/alignment of the first 10 bases at 5' end.
The 10 unpaired
14
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
bases represent 10% of the sequence (number of bases at the 5' and 3' ends not
matchedltotal
number of bases in the query sequence) so 10% is subtracted from the percent
identity score
calculated by the FASTDB program. If the remaining 90 bases were perfectly
matched the
final percent identity would be 90%. In another example, a 90 base subject
sequence is
compared with a 100 base query sequence. This time the deletions are internal
deletions so
that there are no bases on the 5' or 3' of the subject sequence which are not
matched/aligned
with the query. In this case the percent identity calculated by FASTDB is not
manually
corrected. Once again, only bases 5' and 3' of the subject sequence which are
not
matchedlaligned with the query sequence are manually corrected for. No other
manual .
corrections are made for the purposes of this embodiment.
[0062] The present application is directed to nucleic acid molecules at least
80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence
for example,
shown in Figures 1A-B, or to the nucleic acid sequence of the deposited cDNA,
irrespective
of whether they encode a polypeptide having CTGF-2 activity. This is because
even where a
particular nucleic acid molecule does not encode a polypeptide having CTGF-2
functional
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
CTGF-2 activity include, ifZter alias (1) isolating the CTGF-2 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 CTGF-2 gene, as described in
Verma et al.,
Hunzah Chro»zosomes: A Mafzual of Basic Techniques, Pergamon Press, New York
(1988);
and (3) Northern Blot analysis for detecting CTGF-2 rnRNA expression in
specific tissues.
[0063] Preferred, however, are nucleic acid molecules having sequences at
least 90%,
80%, 85%, 95%, 96%, 97%, 98% or 99% identical to for example, the nucleic acid
sequence
shown in Figures lA-B, or to the nucleic acid sequence of the deposited cDNA,
which do, in
fact, encode a polypeptide having CTGF-2 functional activity. By "a
polypeptide having
CTGF-2 functional receptor activity" is intended polypeptides exhibiting
activity similar, but
not necessarily identical, to an activity of the CTGF-2 of the invention
(either the full-length
protein or, preferably, the mature protein), as measured in a particular
biological assay. For
example CTGF-2 functional receptor activity can be measured using the cell
proliferation or
angiogenesis assays performed essentially as previously described in the
Examples, below.
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
[0064] 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 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for
example,
the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence
shown in
Figures lA-B will encode a polypeptide "having CTGF-2 functional 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
CTGF-2
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).
[0065] For example, guidance concerning how to make phenotypically silent
amino
acid substitutions is provided in J.U. Bowie 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.
Pr»tein/Pe~lvne»tie~e Rmhediments
[0066] The present invention further relates to a polypeptide which has the
deduced
amino acid sequence of Figures lA-B or which has the amino acid sequence
encoded by the
deposited cDNA, as well as fragments, analogs and derivatives of such
polypeptide.
[0067) The terms "fragment," "derivative" and "analog" when referring to the
polypeptide of Figures lA-B or that encoded by the deposited cDNA, means a
polypeptide
which retains essentially the same biological function or activity as such
polypeptide. Thus,
an analog includes a proprotein which can be activated by cleavage of the
proprotein portion
to produce an active mature polypeptide.
[0068] The polypeptide of the present invention may be a recombinant
polypeptide, a
natural polypeptide or a synthetic polypeptide, preferably a recombinant
polypeptide.
[0069] The fragment, derivative or analog of the polypeptide of Figures lA-B
or that
encoded by the deposited cDNA may be (i) one in which one or mare 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
16
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
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 a 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.
[0070] The polypeptides and polynucleotides of the present invention are
preferably
provided in an isolated form, and preferably are purified to homogeneity.
[0071] The term "isolated" means that the material is removed from its
original
environment (e.g., the natural environment if it is naturally occurring). For
example, a
naturally-occurnng polynucleotide or polypeptide present in a living animal is
not isolated,
but the same polynucleotide or polypeptide, separated from some or all of the
coexisting
materials in the natural system, is isolated. Such polynucleotides could be
part of a vector
and/or such polynucleotides or polypeptides could be part of a composition,
and still be
isolated in that such vector or composition is not part of its natural
environment.
[0072] The polypeptides of the present invention include the full-length
polypeptide,
the mature polypeptide, as well as polypeptides which have at least 70%
similarity (preferably
at least 70% identity) to the polypeptide of Figures lA-B and more preferably
at least 90%
similarity (more preferably at Ieast 90% identity) to the polypeptide of
Figures lA-B and still
more preferably at least 95% similarity (still more preferably at least 95%
identity) to the
polypeptide of Figures lA-B and also include portions of such polypeptides
with such portion
of the polypeptide generally containing at least 30 amino acids and more
preferably at least 50
amino acids.
[0073] As known in the art "similarity" between two polypeptides is determined
by
comparing the amino acid sequence and its conserved amino acid substitutes of
one
polypeptide to the sequence of a second polypeptide.
[0074] Fragments or portions of the polypeptides of the present invention may
be
employed for producing the corresponding full-length polypeptide by peptide
synthesis;
therefore, the fragments may be employed as intermediates for producing the
full-length
17
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
polypeptides. Fragments or portions of the polynucleotides of the present
invention may be
used to synthesize full-length polynucleotides of the present invention.
[0075) To improve or alter the characteristics of CTGF-2 polypeptides, protein
engineering may be employed. Recombinant DNA technology known to those skilled
in the
art can be used to create novel mutant proteins or muteins including single or
multiple amino
acid substitutions, deletions, additions or fusion proteins. Such modified
polypeptides can
show, e.g., enhanced activity or increased stability. In addition, they may be
purified in
higher yields and show better solubility than the corresponding natural
polypeptide, at least
under certain purification and storage conditions. For instance, for many
proteins, including.
the extracellular domain or the mature forms) of a secreted protein, it is
known in the art that
one or more amino acids may be deleted from the N-terminus or C-terminus
without
substantial loss of biological function. For instance, Ron et al., J. Biol.
Chem.,
268:2984-2988 (1993) reported modified KGF proteins that had heparin binding
activity even
if 3, 8, or 27 amino-terminal amino acid residues were missing.
[0076] In the present case, deletions of N-terminal amino acids up to the
Ile(.I) residue
at position 376 in Figures lA-B may retain some biological activity. The amino
acids
constituting the IGF-Binding Domain, von Willebrand Factor Type C Repeat
Domain,
Sulfated Glycoconjugated Binding Motif, and C-Terminal Dimerization and
Receptor
Binding Domain of the CTGF-2 protein are described above. However, even if
deletion of
one or more amino acids from the N-terminus of a protein results in
modification of loss of
one or more biological functions of the protein, other functional activities
may still be
retained. Thus, the ability of the shortened protein to induce and/or bind to
antibodies which
recognize the complete or extracellular domain of the protein generally will
be retained when
less than the majority of the residues. of the complete or extracellular
domain of the protein
are removed from the N-terminus. Whether a particular polypeptide lacking N-
terminal
residues of a complete protein retains such immunologic activities can readily
be determined
by routine methods described herein and otherwise known in the art.
[0077] Accordingly, the present invention further provides polypeptides having
one or
more residues from the amino terminus of the amino acid sequence of the CTGF-2
shown in
Figures lA-B, up to the isoleucine residue at position 376 (Ile-376 residue
from the amino
terminus), and polynucleotides encoding such polypeptides. In particular, the
present
invention provides polypeptides having the amino acid sequence of residues n1-
381 of
18
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
Figures lA-B, where n1 is an integer in the range of the amino acid position
of amino acid
residues 2-376 of the amino acid sequence in Figures lA-B. More in particular,
the invention
provides polynucleotides encoding polypeptides comprising, or alternatively
consisting of, an
amino acid sequence selected from the group consisting of residues S-2 to D-
381; S-3 to D-
3 81; R-4 to D-3 81; I-5 to D-3 81; A-6 to D-3 81; R-7 to D-3 81; A-8 to D-3
81; L-9 to D-3 81;
A-10 to D-381; L-11 to D-381; V-12 to D-381; V-13 to D-381; T-14 to D-381; L-
15 to D-
381; L-16 to D-381; H-17 to D-381; L-18 to D-381; T-19 to D-381; R-20 to D-
381; L-21 to
D-3 81; A-22 to D-3 81; L-23 to D-3 81; S-24 to D-381; T-25 to D-3 81; C-26 to
D-3 81; P-27 to
D-381; A-28 to D-381; A-29 to D-381; C-30 to D-381; H-31 to D-381; C-3.2 to D-
381; P-33
to D-381; L-34 to D-38I; E-35 to D-381; A-36 to D-381; P-37 to D-381; K-38 to
D-381; C-
3 9 to D-3 81; A-40 to D-3 81; P-41 to D-3 81; G-42 to D-3 81; V-43 to D-3 81;
G-44 to D-3 81;
L-45 to D-3 81; V-46 to D-3 81; R-47 to D-3 81; D-48 to D-3 81; G-49 to D-3
81; C-50 to D-
381; G-51 to D-381; C-52 to D-381; C-53 to D-381; K-54 to D-381; V-55 to D-
381; C-56 to
D-3 81; A-5 7 to D-3 81; K-5 8 to D-3 81; Q-5 9 to D-3 81; L-60 to D-3 81; N-
61 to D-3 81; E-62
to D-381; D-63 to D-381; C-64 to D-381; S-65 to D-381; K-66 to D-381; T-67 to
D-381; Q-
6 8 to D-3 81; P-69 to D-3 81; C-70 to D-3 81; D-71 to D-3 81; H-72 to D-3 81;
T-73 to D-3 81;
K-74 to D-3 8 I ; G-75 to D-3 81; L-76 to D-3 81; E-T7 to D-3 81; C-78 to D-3
81; N-79 to D-
381; F-80 to D-381; G-81 to D-381; A-82 to D-381; S-83 to D-381; S-84 to D-
381; T-85 to
D-381; A-86 to D-381; L-87 to D-38I; K-S8 to D-381; G-89 to D-381; I-90 to D-
38I; C-91
to D-3 81; R-92 to D-3 81; A-93 to D-3 81; Q-94 to D-3 81; S-95 to D-3 81; E-
96 to D-3 81; G-
97 to D-381; R-98 to D-381; P-99 to D-381; C-100 to D-381; E-101 to D-381; Y-
I02 to D-
381; N-103 to D-381; S-104 to D-38.1; R-105 to D-381; I-106 to D-381; Y-107 to
D-381; Q-
108 to D-381; N-109 to D-381; G-110 to D-381; E-111 to D-381; S-112 to D-38I;
F-113 to
D-381; Q-I 14 to D-381; P-l I5 to D-38I; N-I I6 to D-38I; C-117 to D-381; K-
118 to D-381;
H-119 to D-381; Q-120 to D-381; C-121 to D-381; T-122 to D-381; C-123 to D-
381; I-124 to
D-381; D-125 to D-381; G-126 to D-381; A-127 to D-381; V-128 to D-381; G-129
to D-381;
C-130 to D-381; I-131 to D-381; P-132 to D-381; L-133 to D-381; C-134 to D-
381; P-135 to
D-381; Q-136 to D-381; E-137 to D-381; L-138 to D-381; S-139 to D-381; L-140
to D-381;
P-141 to D-381; N-142 to D-381; L-143 to D-381; G-144 to D-381; C-145 to D-
381; P-146
to D-381; N-147 to D-381; P-148 to D-381; R-149 to D-381; L-150 to D-381; V-
151 to D-
381; K-I52 to D-381; V-153 to D-381; T-I54 to D-381; G-155 to D-381; Q-156 to
D-381; C-
157 to D-381; C-158 to D-381; E-159 to D-381; E-160 to D-381; W-161 to D-381;
V-162 to
19
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
D-381; C-163 to D-381; D-164 to D-381; E-165 to D-381; D-166 to D-381; S-167
to D-381;
I-168 to D-381; K-169 to D-381; D-170 to D-381; P-171 to D-381; M-172 to D-
381; E-173
to D-381; D-174 to D-381; Q-175 to D-381; D-176 to D-381; G-177 to D-381; L-
178 to D-
381; L-179 to D-381; G-180 to D-381; K-181 to D-381; E-182 to D-381; L-183 to
D-381; 6-
184 to D-381; F-185 to D-381; D-186 to D-381; A-187 to D-381; S-188 to D-381;
E-189 to
D-381; V-190 to D-381; E-191 to D-381; L-192 to D-381; T-193 to D-381; R-194
to D-381;
N-195 to D-381; N-196 to D-381; E-197 to D-381; L-198 to D-381; I-199 to D-
381; A-200 to
D-3 81; V-201 to D-3 81; G-202 to D-3 81; K-203 to D-3 81; G-204 to D-3 81; S-
205 to D-3 81;
S-206 to D-381; L-207 to D-381; K-208 to D-381; R-209 to D-381; L-210 to D-
381; P-211 to
D-3 81; V-212 to D-3 81; F-213 to D-3 81; G-214 to D-3 81; M-215 to D-3 81; E-
216 to D-3 81;
P-217 to D-3 81; R-218 to D-3 81; I-219 to D-3 81; L-220 to D-3 81; Y-221 to D-
3 81; N-222 to
D-381; P-223 to D-381; L-224 to D-381; Q-225 to D-381; G-226 to D-381; Q-227
to D-381;
K-228 to D-381; C-229 to D-381; I-230 to D-381; V-231 to D-381; Q-232 to D-
381; T-233 to
D-381; T-234 to D-381; S-235 to D-381; W-236 to D-381; S-237 to D-381; Q-238
to D-381;
C-23 9 to D-3 81; S-240 to D-3 81; K-241 to D-3 81; T-242 to D-3 81; C-243 to
D-3 81; G-244
to D-381; T-245 to D-381; G-246 to D-381; I-247 to D-381; S-248 to D-381; T-
249 to D-
381; R-250 to D-381; V-251 to D-381; T-252 to D-381; N-253 to D-381; D-254 to
D-381; N-
255 to D-381; P-256 to D-381; E-257 to D-381; C-258 to D-381; R-259 to D-381;
L-260 to
D-381; V-261 to D-381; K-262 to D-381; E-263 to D-381; T-264 to D-381; R-265
to D-381;
I-266 to D-381; C-267 to D-381; E-268 to D-381; V-269 to D-381; R-270 to D-
381; P-271 to
D-381; C-272 to D-381; G-273 to D-381; Q-274 to D-381; P-275 to D-381; V-276
to D-38.1;
Y-277 to D-381; S-278 to D-381; S-279 to D-381; L-280 to D-381; K-281 to D-
381; K~282
to D-381; G-283 to D-381; K-284 to D-381; K-285 to D-381; C-286 to D-381; S-
287 to D-
381; K-288 to D-381; T-289 to D-381; K-290 to D-381; K-291 to D-381; S-292 to
D-381; P-
293 to D-381; E-294 to D-381; P-295 to D-381; V-296 to D-381; R-297 to D-381;
F-298 to
D-381; T-299 to D-381; Y-300 to D-381; A-301 to D-381; G-302 to D-381; C-303
to D-381;
L-304 to D-381; S-305 to D-381; V-306 to D-381; K-307 to D-381; K-308 to D-
38I; Y-309
to D-3 81; R-310 to D-3 81; P-311 to D-3 81; K-312 to D-3 81; Y-313 to D-3 81;
C-314 to D-
3 81; G-315 to D-3 81; S-316 to D-3 81; C-317 to D-3 81; V-318 to D-3 81; D-
319 to D-3 81; 6-
320 to D-381; R-321 to D-381; C-322 to D-381; C-323 to D-381; T-324 to D-381;
P-325 to
D-381; Q-326 to D-381; L-327 to D-381; T-328 to D-381; R-329 to D-381; T-330
to D-381;
V-331 to D-381; K=332 to D-381; M-333 to D-381; R-334 to D-381; F-335 to D-
381; R-336
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
to D-381; C-337 to D-381; E-338 to D-381; D-339 to D-381; G-340 to D-381; E-
341 to D-
381; T-342 to D-381; F-343 to D-381; S-344'to D-381; K-345 to D-381; N-346 to
D-381; V-
347 to D-381; M-348 to D-381; M-349 to D-381; I-350 to D-381; Q-351 to D-381;
S-352 to
D-381; C-353 to D-381; K-354 to D-381; C-355 to D-381; N-356 to D-381; Y-357
to D-381;
N-358 to D-381; C-359 to D-381; P-360 to D-381; H-361 to D-381; A-362 to D-
381; N-363
to D-381; E-364 to D-381; A-365 to D-381; A-366 to D-381; F-367 to D-381; P-
368 to D-
381; F-369 to D-381; Y-370 to D-381; R-371 to D-381; L-372 to D-381; F-373 to
D-381; N-
374 to D-381; D-375 to D-381; I-376 to D-381 of Figures lA-B. The present
application is
also directed to nucleic acid molecules comprising, or alternatively,
consisting of, a
polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical
to the
polynucleotide sequence encoding, the CTGF-2 polypeptides described above. The
present
invention also encompasses the above polynucleotide sequences fused to a
heterologous
polynucleotide sequence. Polypeptides encoded by these nucleic acids and/or
polynucleotide
sequences are also encompassed by the invention, as are polypeptides.
comprising an amino
acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid
sequence described above, and polynucleotides that encode such polypeptides.
[0078] Similarly, many examples of biologically functional C-terminal deletion
muteins are known. For instance, Interferon gamma shows up to ten times higher
activities
by deleting 8-10 amino acid residues from the carboxy terminus of the protein
(Dobeli et al.,
J. Bioteclzhology 7:199-216 (1988). However, even if deletion of one or more
amino acids
from the C-terminus of a protein results in modification of loss of one or
more biological
functions of the protein, other functional activities may still be retained.
Thus, the ability of
the shortened protein to induce and/or bind to antibodies which recognize the
complete or
mature protein generally will be retained when less than the majority of the
residues of the
complete or mature protein are removed from the C-terminus. Whether a
particular
polypeptide lacking C-terminal residues of a complete protein retains such
immunologic
activities can readily be determined by routine methods described herein and
otherwise
known in the art.
[0079] Accordingly, the present invention further provides polypeptides having
one or
more residues from the carboxy terminus of the amino acid sequence of the CTGF-
2 shown in
Figures lA-B, up to the histidine residue at position 361 (His-361) and
polynucleotides
encoding such polypeptides. In particular, the present invention provides
polypeptides having
21
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
the amino acid sequence of residues 1-mt of the amino acid sequence in Figures
lA-B, where
ml is any integer in the range of the amino acid position of amino acid
residues 6 to 380 in
Figures lA-B. More in particular, the invention provides polynucleotides
encoding
polypeptides comprising, or alternatively consisting of, an amino acid
sequence selected from
the group consisting of residues M-1 to R-380; M-1 to F-379; M-1 to K-378; M-1
to H-377;
M-1 to I-376; M-1 to D-375; M-1 to N-374; M-1 to F-373; M-1 to L-372; M-1 to R-
371; M-1
to Y-370; M-1 to F-369; M-1 to P-368; M-1 to F-367; M-1 to A-366; M-1 to A-
365; M-1 to
E-364; M-1 to N-363; M-1 to A-362; M-1 to H-361; M-1 to P-360; M-1 to C-359; M-
1 to N-
358; M-1 to Y-357; M-1 to N-356; M-1 to C-355; M-1 to K-354; M-1 to C-353; M-1
to 5-
352; M-1 to Q-351; M-1 to I-350; M-1 to M-349; M-1 to M-348; M-1 to V-347; M-1
to N-
346; M-1 to K-345; M-1 to S-344; M-1 to F-343; M-1 to T-342; M-1 to E-341; M-1
to 6-
340; M-1 to D-339; M-1 to E-338; M-1 to C-337; M-1 to R-336; M-1 to F-335; M-1
to 8-
334; M-1 to M-333; M-1 to K-332; M-1 to V-331; M-1 to T-330; M-1 to R-329; M-I
to T-
328; M-1 to L-327; M-1 to Q-326; M-1 to P-325; M-1 to T-324; M-1 to C-323; M-1
to C-
322; M-1 to R-321; M-1 to G-320; M-1 to D-319; M-1 to V-318; M-1 to C-317; M-1
to 5-
316; M-1 to G-315; M-1 to C-314; M-1 to Y-313; M-1 to K-312; M-1 to P-311; M-1
to 8-
310; M-1 to Y-309; M-1 to K-308; M-1 to K-307; M-1 to V-306; M-1 to S-305; M-1
to L-
304; M-1 to C-303; M-1 to G-302; M-1 to A-301; M-1 to Y-300; M-1 to T-299; M-1
to F-
298; M-1 to R-297; M-1 to V-296; M-1 to P-295; M-1 to E-294; M-1 to P-293; M-1
to 5-
292; M-1 to K-291; M-1 to K-290; M-1 to T-289; M-1 to K-288; M-1 to S-287; M-1
to C-
286; M-1 to K-285; M-1 to K-284; M-1 to G-283; M-1 to K-282; M-1 to K-28I; M-1
to L-
280; M-1 to S-279; M-1 to S-278; M-1 to Y-277; M-1 to V-276; M-1 to P-275; M-1
to Q-
274; M-1 to G-273; M-1 to C-272; M-1 to P-271; M-1 to R-270; M-1 to V-269; M-1
to E-
268; M-1 to C-267; M-1 to I-266; M-1 to R-265; M-1 to T-264; M-1 to E-263; M-1
to K-262;
M-1 to V-261; M-1 to L-260; M-1 to R-259; M-1 to C-258; M-I to E-257; M-1 to P-
256; M-
1 to N-255; M-1 to D-254; M-1 to N-253; M-1 to T-252; M-1 to V-251; M-1 to R-
250; M-1
to T-249; M-1 to S-248; M-1 to I-247; M-1 to G-246; M-1 to T-245; M-I to G-
244; M-1 to
C-243; M-1 to T-242; M-1 to K-241; M-1 to S-240; M-1 to C-239; M-1 to Q-238; M-
1 to 5-
237; M-1 to W-236; M-1 to S-235; M-1 to T-234; M-1 to T-233; M-1 to Q-232; M-1
to V-
231; M-1 to I-230; M-1 to C-229; M-1 to K-228; M-1 to Q-227; M-1 to G-226; M-1
to Q-
225; M-1 to L-224; M-1 to P-223; M-1 to N-222; M-1 to Y-221; M-1 to L-220; M-1
to I-219;
M-1 to R-218; M-I to P-217; M-1 to E-216; M-1 to M-215; M-1 to G-2I4; M-1 to F-
213; M-
22
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
1 to V-212; M-1 to P-2I l; M-1 to L-210; M-1 to R-209; M-1 to K-208; M-1 to L-
207; M-I to
S-206; M-1 to S-205; M-1 to G-204; M-1 to K-203; M-1 to G-202; M-1 to V-201; M-
1 to A-
200; M-1 to I-199; M-1 to L-198; M-1 to E-197; M-1 to N-196; M-1 to N-195; M-1
to R-194;
M-1 to T-193; M-1 to L-192; M-1 to E-191; M-1 to V-190; M-1 to E-189; M-1 to S-
188; M-1
to A-187; M-1 to D-186; M-1 to F-185; M-1 to G-184; M-1 to L-183; M-1 to E-
182; M-1 to
K-181; M-1 to G-180; M-1 to L-179; M-1 to L-178; M-1 to G-177; M-1 to D-176; M-
1 to Q-
175; M-1 to D-174; M-1 to E-173; M-1 to M-172; M-1 to P-171; M-I to D-170; M-1
to K-
169; M-I to I-168; M-1 to S-167; M-1 to D-166; M-1 to E-165; M-1 to D-164; M-1
to C-163;
M-1 to V-162; M-1 to W-161; M-1 to E-160; M-1 to E-159; M-1 to C-158; M-1 to C-
157; M-
1 to Q-156; M-1 to G-ISS; M-1 to T-154; M-1 to V-153; M-1 to K-152; M-1 to V-
151; M-1
to L-150; M-1 to R-149; M-1 to P-148; M-1 to N-147; M-1 to P-146; M-1 to C-
145; M-1 to
G-144; M-1 to L-143; M-1 to N-142; M-1 to P-141; M-1 to L-140; M-1 to S-139; M-
1 to L-
138; M-1 to E-137; M-1 to Q-136; M-1 to P-135; M-1 to C-134; M-1 to L-133; M-1
to P-
132; M-1 to I-131; M-1 to C-130; M-1 to G-129; M-1 to V-128; M-1 to A-127; M-1
to 6-
126; M-1 to D-125; M-1 to I-124; M-1 to C-123; M-1 to T-122; M-1 to C-121; M-1
to Q-
120; M-1 to H-119; M-1 to K-118; M-1 to C-117; M-1 to N-116; M-1 to P-115; M-1
to Q-
114; M-1 to F-113; M-1 to S-112; M-1 to E-111; M-1 to G-110; M-1 to N-109; M-1
to Q-
108; M-1 to Y-107; M-1 to I-106; M-I to R-105; M-1 to S-104; M-1 to N-103; M-I
to Y-
102; M-1 to E-101; M-1 to C-100; M-1 to P-99; M-1 to R-98; M-1 to G-97; M-1 to
E-96; M-
1 to S-95; M-1 to Q-94; M-1 to A-93; M-1 to R-92; M-1 to C-91; M-1 to I-90; M-
1 to G-89;
M-1 to K-88; M-1 to L-87; M-1 to A-86; M-1 to T-85; M-1 to S-84; M-1 to S-83;
M-1 to A-
82; M-1 to G-81; M-1 to F-80; M-I to N-79; M-1 to C-78; M-1 to E-77; M-1 to L-
76; M-1 to
G-75; M-1 to K-74; M-1 to T-73; M-1 to H-72M-1 to D-71; M-1 to C-70; M-1 to P-
69; M-1
to Q-68; M-1 to T-67; M-1 to K-66; M-1 to S-65; M-1 to C-64; M-I to D-63; M-1
to E-62;
M-1 to N-61; M-1 to L-60; M-1 to Q-59; M-1 to K-58; M-1 to A-57; M-1 to C-56;
M-1 to V-
55; M-1 to K-54; M-1 to C-53; M-I to C-52; M-1 to G-51; M-1 to C-50; M-1 to G-
49; M-1 to
D-48; M-1 to R-47; M-I to V-46; M-1 to L-45; M-1 to G-44; M-1 to V-43; M-1 to
G-42; M-1
to P-4I; M-I to A-40; M-1 to C-39; M-I to K-38; M-1. to P-37; M-1 to A-36; M-1
to E-35;
M-1 to L-34; M-1 to P-33; M-1 to C-32; M-1 to H-31; M-1 to C-30; M-1 to A-29;
M-1 to A-
28; M-1 to P-27; M-1 to C-26; M-I to T-25; M-1 to S-24; M-1 to L-23; M-1 to A-
22; M-1 to
L-21; M-1 to R-20; M-I to T-19; M-1 to L-18; M-1 to H-17; M-1 to L-16; M-1 to
L-15; M-1
to T-14; M-1 to V-13; M-1 to V-12; M-1 to L-11; M-1 to A-10; M-1 to L-9; M-1
to A-8; M-1
23
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
to R-7; M-1 to A-6 of Figures lA-B. The present application is also directed
to nucleic acid
molecules comprising, or alternatively, consisting of, a polynucleotide
sequence at least 90%,
92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence
encoding the
CTGF-2 polypeptides described above. The present invention also encompasses
the above
polynucleotide sequences fused to a heterologous polynucleotide sequence.
Polypeptides
encoded by these nucleic acids and/or polynucleotide sequences are also
encompassed by the
invention, as are polypeptides comprising an amino acid sequence at least 90%,
92%, 95%,
96%, 97%, 98% or 99% identical to the amino acid sequence described above, and
polynucleotides that encode such polypeptides.
[0080] Also provided are polypeptides having one or more amino acids deleted
from
both the amino and the carboxyl termini, which may be described generally as
having
residues nt-ml of Figures lA-B, where n1 and ml are integers as defined above.
Also
included are a nucleotide sequence encoding a polypeptide consisting of a
portion of the
complete CTGF-2 amino acid sequence encoded by the deposited cDNA clone
contained in
ATCC Accession No. 75804 where this portion excludes from 1 to 376 amino acids
from the
amino terminus or from 1 to 380 amino acids from the C-terminus of the
complete amino
acid sequence (or any combination of these N-terminal and C-terminal
deletions) encoded by
the cDNA clone in the deposited clone. Polynucleotides encoding all of the
above deletion
polypeptides are encompassed by the invention.
Rn'
[0081] In specific embodiments polypeptides of the present invention comprise,
or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, eleven, twelve,
thirteen, fourteen, or all fourteen of the irntnunogenic epitopes of the CTGF-
2 protein shown in
Figures lA-B as residues: Glu-35 to Pro-41, Arg-47 to Gly-51, Gln-59 to Gly-
75, Cys-91 to
His-119, Cys-145 to Leu-150, Asp-164 to Asp-176, Gly-202 to Lys-208, Pro-223
to Lys-228,
Cys-239 to Gly-244, Arg-250 to Glu-257, Ser-279 to Val-296, Lys-307 to Cys-
314, Val-318 to
Cys-323, and Cys-337 to Phe-343. Fragments andlor variants of these
polypeptides, such as, for
example, fragments and/or variants as described herein, are encompassed by the
invention.
Polynucleotides encoding these polypeptides (including fragments and/or
variants) are also
encompassed by the invention, as are antibodies that bind these polypeptides.
24
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
[0082] The present invention encompasses polypeptides comprising, or
alternatively
consisting of, an epitope of the polypeptide having an amino acid sequence of
Figures lA-B,
or an epitope of the polypeptide sequence encoded by a polynucleotide sequence
contained in
deposited clone (ATCC accession no. 75804) or encoded by a polynucleotide that
hybridizes
to the complement of the sequence of Figures lA-B or contained in deposited
clone (ATCC
accession no. 75804) under stringent hybridization conditions or lower
stringency
hybridization conditions as defined supra. The present invention further
encompasses
polynucleotide sequences encoding an epitope of a polypeptide sequence of the
invention
(such as, for example, the sequence disclosed in Figures 1A-B), polynucleotide
sequences of
the complementary strand of a polynucleotide sequence encoding an epitope of
the invention,
and polynucleotide sequences which hybridize to the complementary strand under
stringent
hybridization conditions or lower stringency hybridization conditions defined
supra.
[0083] The term "epitopes," as used herein, refers to portions of a
polypeptide having
antigenic or immunogenic activity in an animal, preferably a mammal, and most
preferably in
a human. In a preferred embodiment, the present invention encompasses a
polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An
"immunogenic epitope," as used herein, is defined as a portion of a protein
that elicits an
antibody response in an animal, as determined by any method known in the art,
for example,
by the methods for generating antibodies described infra. (See, for example,
Geysen et al.,
Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term "antigenic epitope,"
as used
herein, is defined as a portion of a protein to which an antibody can
immunospecifically bind
its antigen as determined by any method well known in the . art, for example,
by the
immunoassays described herein. Tmmunospecific binding excludes non-specific
binding but
does not necessarily exclude cross-reactivity with other antigens. Antigenic
epitopes need not
necessarily be immunogenic.
[0084] Fragments that function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985),
further
described in U.S. Patent No. 4,631,211).
[0085] In the present invention, antigenic epitopes preferably contain a
sequence of at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10, at least
15, at least 20, at least 25, and, most preferably, between about IS to about
30 amino acids.
Preferred polypeptides comprising immunogenic or antigeuc epitopes are at
least 10, 15, 20,
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid
residues in length.
Antigenic epitopes are useful, for example, to raise antibodies, including
monoclonal
antibodies, that specifically bind the epitope. Antigenic epitopes can be used
as the target
molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778
(1984);
Sutcliffe et al., Scieyzce 219:660-666 (1983)).
[0086] Similarly, immunogenic epitopes can be used, for example, to induce
antibodies according to methods well known in the art. (See, for instance,
Sutcliffe et al.,
supra; Wilson et al., supra; Chow et al., Proc: Natl. Acad. Sci. USA 82:910-
914; and Bittle et
al., J. Gen. Virol. 66:2347-2354 (1985). The polypeptides comprising one or
more
immunogenic epitopes may be presented for eliciting an antibody response
together with a
carrier protein, such as an albumin, to an animal system (such as, for
example, rabbit or
mouse), or, if the polypeptide is of sufficient length (at least about 25.
amino acids), the
polypeptide may be presented without a carrier. However, immunogenic epitopes
comprising
as few as 8 to 10 amino acids have been shown to be sufficient to raise
antibodies capable of
binding to, at the very least, linear epitopes in a denatured polypeptide
(e.g., in Western
blotting).
(0087] Epitope-bearing polypeptides of the present invention may be used to
induce
antibodies according to methods well known in the art including, but not
limited to, iya vivo
immunization, irr vitro immunization, and phage display methods. See, e.g.,
Sutcliffe et al.,
sup~~a; Wilson et al., supYa, and Bittle et al., J. Gen. Virol., 66:2347-2354
(1985). If in vivo
immunization is used, animals may be immunized with free peptide; however,
anti-peptide
antibody titer may be boosted by coupling the peptide to a macromolecular
Garner, such as
keyhole limpet hemacyanin (I~L,H) or tetanus toxoid. For instance, peptides
containing
cysteine residues may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS), while other peptides may be coupled to
carriers, using a
more general linking agent such as glutaraldehyde. Animals such as, for
example, rabbits,
rats, and mice are immunized with either free or Garner-coupled peptides, for
instance, by
intraperitoneal and/or intradermal injection of emulsions containing about 100
micrograms of
peptide or carrier protein and Freund's adjuvant or any other adjuvant known
for stimulating
an immune response. Several booster injections may be needed, for instance, at
intervals of
about two weeks, to provide a useful titer of anti-peptide antibody that can
be detected, for
example, by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-
26
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
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.
[0088] As one of skill in the art will appreciate, and as discussed above, the
polypeptides of the present invention comprising an immunogenic or antigenic
epitope can be
fused to other polypeptide_sequences. For example, the polypeptides of the
present invention
may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),
or portions
thereof (CH1, CH2, CH3, or any combination thereof and portions thereof)
resulting in
chimeric polypeptides. Such fusion proteins may facilitate purification and
may increase
half life in vivo. This has been shown 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. See, e.g., EP 394,827;
Traunecker et
al., Nature, 331:84-86 (1988). IgG Fusion proteins that have a disulfide-
linked dimeric
structure due to the IgG portion desulfide bonds have also been found to be
more efficient in
binding and neutralizing other molecules than monomeric polypeptides or
fragments thereof
alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995).
Nucleic acids
encoding the above epitopes can also be recombined with a gene of interest as
an epitope tag
(e.g., the hemagglutinin ("HA") tag or flag tag) to aid in detection and
purification of the
expressed polypeptide. For example, a system described by Janknecht et al.
allows for the
ready purification of non-denatured fusion proteins expressed in human cell
lines (Janknecht
et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897). In this system, the
gene of interest is
subcloned into a vaccinia recombination plasmid such that the open reading
frame of the gene
is translationally fused to an amino-terminal tag consisting of six histidine
residues. The tag
serves as a matrix-binding domain for the fusion protein. Extracts from cells
infected with
the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose
column and
histidine-tagged proteins can be selectively eluted with imidazole-containing
buffers.
[0089] Additional fusion proteins of the invention may be generated through
the
techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be employed
to modulate
the activities of polypeptides of the invention, such methods can be used to
generate
polypeptides with altered activity, as well as agonists and antagonists of the
polypeptides.
See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252;
and 5,837,458,
27
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends
Biotechnol.
16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and
Lorenzo and
Blasco, Biotechniques 24(2):308- 13 (1998) (each of these patents and
publications are
hereby incorporated by reference in its entirety). In one embodiment,
alteration of
polynucleotides corresponding to Figures lA-B and the polypeptides encoded by
these
polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the
assembly of
two or more DNA segments by homologous or site-specific recombination to
generate
variation in the polynucleotide sequence. In another embodiment,
polynucleotides of the
invention, or the encoded polypeptides, may be altered by being subjected to
random
mutagenesis by error-prone PCR, random nucleotide insertion or other methods
prior to
recombination. In another embodiment, one or more components, motifs,
sections, parts,
domains, fragments, etc., of a polynucleotide coding a polypeptide of the
invention may be
recombined with one or more components, motifs, sections, parts, domains,
fragments, etc. of
one or more heterologous molecules. .
[0090] The present invention further relates to antibodies and T-cell antigen
receptors
(TCR) which immunospecifically bind a polypeptide, preferably an epitope, of
the present
invention (as determined by immunoassays well known in the art for assaying
specific
antibody-antigen binding). Antibodies of the invention include, but are not
limited to,
polyclonal, monoclonal, multispecific, human, humanized or chimeric
antibodies, single
chain antibodies, Fab fragments, Flab?) fragments, fragments produced by a Fab
expression
library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id
antibodies to antibodies of
the invention), and epitope-binding fragments of any of the above. The term
"antibody," as
used herein, refers to immunoglobulin molecules and immunologically active
portions of
immunoglobulin molecules, i.e., molecules that contain an antigen binding site
that
immunospecifically binds an antigen. The immunoglobulin molecules of the
invention can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl,
IgG2, IgG3, IgG4,
IgAl and IgA2) or subclass of immunoglobulin molecule.
[0091] Most preferably the antibodies are human antigen-binding antibody
fragments
of the present invention and include, but are not limited to, Fab, Fab' and
F(ab')2, Fd, single-
chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and
fragments
28
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
comprising either a VL or VH domain. Antigen-binding antibody fragments,
including
single-chain antibodies, may comprise the variable regions) alone or in
combination with the
entirety or a portion of the following: hinge region, CHl, CH2, and CH3
domains. Also
included in the invention are antigen-binding fragments also comprising any
combination of
variable regions) with a hinge region, CH1, CH2, and CH3 domains. The
antibodies of the
invention may be from any animal origin including birds and mammals.
Preferably, the
antibodies are human, marine, donkey, ship rabbit, goat, guinea pig, camel,
horse, or chicken.
As used herein, "human" antibodies include antibodies having the amino acid
sequence of a
human immunoglobulin and include antibodies isolated from human
irmnunoglobulin
libraries or from animals transgenic for one or more human immunoglobulin and
that do not
express endogenous immunoglobulins, as described infra and, for example in,
U.S. Patent
No. 5,939,598 by Kucherlapati et al.
[0092] The antibodies of the present invention may be monospecific,
bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a polypeptide of the present invention or may be
specific for both a
polypeptide of the present invention as well as for a heterologous epitope,
such as a
heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. hnmunol.
147:60-69
(1991); U.S. Patent Nos. 4,474,893; 4,714,68.1; 4,925,648; 5,573,920;
5,601,819; Kostelny et
al., J. Immunol. 148:1547-1553 (1992).
(0093] Antibodies of the present invention may be described or specified in
terms of
the epitope(s) or portions) of a polypeptide of the present invention that
they recognize or
specifically bind. The epitope(s) or polypeptide portions) may be specified as
described
herein, e.g., by N-terminal and C-terminal positions, by size in contiguous.
amino acid
residues, or listed in the Tables and Figures. Antibodies that specifically
bind any epitope or
polypeptide of the present invention may also be excluded. Therefore, the
present invention
includes antibodies that specifically bind polypeptides of the present
invention, and allows for
the exclusion of the same.
[0094] Antibodies of the present invention may also be described or specified
in terms
of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog
of a polypeptide of the present invention are included. Antibodies that bind
polypeptides with
at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least
70%, at least 65%,
29
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
at least 60%, at least 55%, and at least 50% identity (as calculated using
methods known in
the art and described herein) to a polypeptide of the present invention are
also included in the
present invention. Antibodies that do not bind polypeptides with less than
95%, less than
90%, less than 85%, less than 80%, less than 75%, less than 70%, less than
65%, less than
60%, less than 55%, and less than 50% identity (as calculated using methods
known in the art
and described herein) to a polypeptide of the present invention are also
included in the present
invention. Further included in the present invention are antibodies that bind
polypeptides
encoded by polynucleotides which hybridize to a polynucleotide of the present
invention
under stringent hybridization conditions (as described herein). Antibodies of
the present
invention may also be described or specified in terms of their binding
affinity to a polypeptide
of the invention. Preferred binding affinities include those with a
dissociation constant or I~.d
less than SX10-ZM, 10-2M, 5X10-3M, 10-3M, 5X10-4M, 10-4M, SX10-sM, 10-sM, SX10-
6M, 10-
6M, 5X10-~M, 10-'M, 5X10-8M, 10-8M, SX10-9M, 10-9M, SX10-'°M, 10-
1°M, SX10-11M, 10-
11M, SX10-IZM, 10-12M, 5X10-13M, 10-13M, SX10-14M, 10-14M, 5X10-lsM, and 10-
lsM.
[0095] The invention also provides antibodies that competitively inhibit
binding of an
antibody to an epitope of the invention as determined by any method known in
the art for
determining competitive binding, for example, the immunoassays described
herein. In
preferred embodiments, the antibody competitively inhibits binding to the
epitope by at least
90%, at least 80%, at least 70%, at least 60%, or at least 50%.
[0096] Antibodies of the present invention may act as agonists or antagonists
of the
polypeptides of the present invention. For example, the present invention
includes antibodies
which disrupt the receptor/ligand interactions with the polypeptides of the
invention either
partially or fully. The invention features both receptor-specific antibodies
and ligand-specific
antibodies. The invention also features receptor-specific antibodies which do
not prevent
ligand binding but prevent receptor activation. Receptor activation (i.e.,
signaling) may be
determined by techniques described herein or otherwise known in the art. For
example,
receptor activation can be determined by detecting the phosphorylation (e.g.,
tyrosine or
serine/threonine) of the receptor or its substrate by immunoprecipitation
followed by western
blot analysis (for example, as described supra). In specific embodiments,
antibodies are
provided that inhibit ligand or receptor activity by at least 90%, at least
80%, at least 70%, at
least 60%, or at least 50% of the activity in absence of the antibody.
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
(0097] The invention also features receptor-specific antibodies which both
prevent
ligand binding and receptor activation as well as antibodies that recognize
the receptor-ligand
complex, and, preferably, do not specifically recognize the unbound receptor
or the unbound
ligand. Likewise, included in the invention are neutralizing antibodies which
bind the ligand
and prevent binding of the ligand to the receptor, as well as antibodies which
bind the ligand,
thereby preventing receptor activation, but do not prevent the ligand from
binding the
receptor. Further included in the invention are antibodies which activate the
receptor. These
antibodies may act as receptor agonists, i.e., potentiate or activate either
all or a subset of the
biological activities of the ligand-mediated receptor activation. The
antibodies may be
specified as agonists, antagonists or inverse agonists for biological
activities comprising the
specific biological activities of the peptides of the invention disclosed
herein. The above
antibody agonists can be made using methods known in the art. See, e.g., PCT
publication
WO 96/40281; LT.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988
(1998); Chen,
et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794
(1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon, et al., J.
Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. I11(Pt2):237-247 (1998);
Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997);
Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al.,
Neuron
14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bariunek
et al.,
Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in
their
entireties).
[0098] Antibodies of the present invention may be used, for example, but not
limited
to, to purify, detect, and target the polypeptides of the present invention,
including both in
vitro and in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in
immunoassays for qualitatively and quantitatively measuring levels of the
polypeptides of the
present invention in biological samples. See, e.g., Harlow et al., Antibodies:
A Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by
reference
herein in its entirety).
[0099] As discussed in more detail below, the antibodies of the present
invention may
be used either alone or in combination with other compositions. The antibodies
may further
be recombinantly fused to a heterologous polypeptide at the N- or C-terminus
or chemically
conjugated (including covalently and non-covalently conjugations) to
polypeptides or other
31
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
compositions. For example, antibodies of the present invention may be
recombinantly fused
or conjugated to molecules useful as labels in detection assays and effector
molecules such as
heterologous polypeptides, drugs, or toxins. See, e.g., PCT publications WO
92/08495; WO
91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
[0100] The antibodies of the invention include derivatives that are modified,
i.e, by
the covalent attachment of any type of molecule to the antibody such that
covalent attachment
does not prevent the antibody from generating an anti-idiotypic response. For
example, but
not by way of limitation, the antibody derivatives include antibodies that
have been modified,
e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation,
derivatization by
known protecting/blocking groups, proteolytic cleavage, linkage to a cellular
ligand or other
protein, etc. Any of numerous chemical modifications may be carried out by
known
techniques, including, but not limited to specific chemical cleavage,
acetylation, formylation,
metabolic synthesis of tunicamycin, etc. Additionally, the derivative may
contain one or
more non-classical amino acids.
[0101] The antibodies of the present invention may be generated by any
suitable
method known in the art. Polyclonal antibodies to an antigen-of interest can
be produced by
various procedures well known in the art. For example, a polypeptide of the
invention can be
administered to various host animals including, but not limited to, rabbits,
mice, rats, etc. to
induce the production of sera containing polyclonal antibodies specific for
the antigen.
Various adjuvants may be used to increase the immunological response,
depending on the
host species, and include but are not limited to, Freund's (complete and
incomplete), mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium parvum. Such adjuvants are also well known in the art.
[0102] Monoclonal antibodies can be prepared using a wide variety of
techniques
known in the art including the use of hybridoma, recombinant, and phage
display
technologies, or a combination thereof. For example, monoclonal antibodies can
be produced
using hybridoma techniques including those known in the art and taught, for
example, in
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd
ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas
563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference in their
entireties). The term
32
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
"monoclonal antibody" as used herein is not limited to antibodies produced
through
hybridoma technology. The term "monoclonal antibody" refers to an antibody
that is derived
from a single clone, including any eukaryotic, prokaryotic, or phage clone,
and not the
method by which it is produced.
[0103] Methods for producing and screening for specific antibodies using
hybridoma
technology are routine and well-known in the art. Briefly, mice can be
immunized with a
polypeptide of the invention or a cell expressing such peptide. Once an immune
response is
detected, e.g., antibodies specific for the antigen are detected in the mouse
serum, the mouse
spleen is harvested and splenocytes isolated. The splenocytes are then fused
by well-known
techniques to any suitable myeloma cells, for example cells from cell line
SP20 available
from the ATCC. Hybridomas are selected and cloned by limited dilution. The
hybridoma
clones are then assayed by methods known in the art for cells that secrete
antibodies capable
of binding a polypeptide of the invention. Ascites fluid, which generally
contains high levels
of antibodies, can be generated by immunizing mice With positive hybridoma
clones.
[0104] Accordingly, the present invention provides ' methods of generating
monoclonal antibodies as well as antibodies produced by the method comprising
culturing a
hybridoma cell secreting an antibody of the invention wherein, preferably, the
hybridoma is
generated by fusing splenocytes isolated from a mouse immunized with an
antigen of the
invention with myeloma cells and then screening the hybridomas resulting from
the fusion for
hybridoma clones that secrete an antibody able to bind a polypeptide of the
invention.
[0105] Antibody fragments that recognize specific epitopes may be generated by
known techniques. For example, Fab and F(ab')2 fragments of the invention may
be
produced by proteolytic cleavage of immunoglobulin molecules, using enzymes
such as
pepsin (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2
fragments contain the variable region, the light chain constant region and the
CHl domain of
the heavy chain.
[0106] For example, the antibodies of the present invention can also be
generated
using various phage display methods known in the art. In phage display
methods, functional
antibody domains are displayed on the surface of phage particles which carry
the
polynucleotide sequences encoding them. In a particular, such phage can be
utilized to
display antigen-binding domains expressed from a repertoire or combinatorial
antibody
library (e.g., human or murine). Phage expressing an antigen binding domain
that binds the
33
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
antigen of interest can be selected or identified with antigen, e.g., using
labeled antigen or
antigen bound or captured to a solid surface or bead. Phage used in these
methods are
typically filamentous phage including fd and M13 binding domains expressed
from phage
with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused
to either the
phage gene IB or gene VIII protein. Examples of phage display methods that can
be used to
make the antibodies of the present invention include those disclosed in
Brinkman et al., J.
Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-
186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene
187 9-18
(1997); Burton et al., Advances in Itn~munology 57:191-280 (1994); PCT
application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos.
5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated
herein by reference in its entirety.
(0107] As described in the above references, after phage selection, the
antibody
coding regions from the phage can be isolated and used to generate whole
antibodies,
including human antibodies, or any other desired antigen binding fragment, and
expressed in
any desired host, including mammalian cells, insect cells, plant cells, yeast,
and bacteria, e.g.,
as described in detail below. For example, techniques to recombinantly produce
Fab, Fab'
and F(ab')2 fragments can also ~be employed using methods known in the art
such as those
disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques
12(6):864-869
(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science
240:1041-1043
(1988) (said references incorporated by reference in their entireties).
[0108] Examples of techniques which can be used to produce single-chain Fvs
and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al.,.
Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and
Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo
use of
antibodies in humans and in vitro detection assays, it may be preferable to
use chimeric,
humanized, or human antibodies. A chimeric antibody is a molecule in which
different
portions of the antibody are derived from different animal species, such as
antibodies having
a variable region derived from a murine monoclonal antibody and a human
immunoglobulin
constant region. Methods for producing chimeric antibodies are known in the
art. See e.g.,
34
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986);
Gillies et al.,
(1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567;
and
4,816397, which are incorporated herein by reference in their entireties.
Humanized
antibodies are antibody molecules from non-human species antibody that binds
the desired
antigen having one or more complementarity determining regions (CDRs) from the
non-
human species and framework regions from a human immunoglobulin molecule.
Often,
framework residues in the human framework regions will be substituted with the
corresponding residue from the CDR donor antibody to alter, preferably
improve, antigen
binding. These framework substitutions are identified by methods well known in
the art, e.g.,
by modeling of the interactions of the CDR and framework residues to identify
framework
residues important for antigen binding and sequence comparison to identify
unusual
framework residues at particular positions. (See, e.g., Queen et al., U.S.
Patent No.
5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by
reference in their entireties.) Antibodies can be humanized using a variety of
techniques
known in the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO
91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing
(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991);
Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al.,
PNAS 91:969-973
(1994)), and chain shuffling (U.S. Patent No. 5,565,332).
[0109] Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods known
in the art including phage display methods described above using antibody
libraries derived
from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and
4,716,111; and PCT publications WO 98/46645, WO 98150433, WO 98/24893, WO
98/I6654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is
incorporated
herein by reference in its entirety.
[0110] Human antibodies can also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can
express
human immunoglobulin genes. For example, the human heavy and light chain
immunoglobulin gene complexes may be introduced randomly or by homologous
recombination into mouse embryonic stem cells. Alternatively, the human
variable region,
constant region, and diversity region may be introduced into mouse embryonic
stem cells in
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
addition to the human heavy and light chain genes. The mouse heavy and light
chain
immunoglobulin genes may be rendered non-functional separately or
simultaneously with the
introduction of human immunoglobulin loci by homologous recombination. In
particular,
homozygous deletion of the JH region prevents endogenous antibody production.
The
modified embryonc stem cells are expanded and microinjected into blastocysts
to produce
chimeric mice. The chimeric mice are then bred to produce homozygous offspring
that
express human antibodies. The transgenic mice are immunized in the normal
fashion with a
selected antigen, e.g., all or a portion of a polypeptide of the invention.
Monoclonal
antibodies directed against the antigen can be obtained from the immunized,
transgenic mice
using conventional hybridoma technology. The human immunoglobulin transgenes
harbored
by the transgenic mice rearrange during B cell differentiation, and
subsequently undergo class
switching and somatic mutation. Thus, using such a technique, it is possible
to produce
therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of
this technology
for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev.
Immunol. 13:65-
93). For a detailed discussion of this technology for producing human
antibodies and human
monoclonal antibodies and protocols for producing such antibodies, see, e.g.,
PCT
publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Patent Nos.
5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and
5,939,598, which are
incorporated by reference herein in their entirety. In addition, companies
such as Abgenix,
Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide
human
antibodies directed against a selected antigen using technology similar to
that described
above.
[0111) Completely human antibodies which recognize a selected epitope can be
generated using a technique referred to as "guided selection." In this
approach a selected non-
human monoclonal antibody, e.g., a mouse antibody, is used to guide the
selection of a
completely human antibody recognizing the same epitope. (Jespers et al.,
Biotechnology
12:899-903 (1988)).
[0112] Further, antibodies to the polypeptides of the invention can, in turn,
be utilized
to generate anti-idiotype antibodies that "mimic" polypeptides of the
invention using
techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona, FASEB J.
7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For
example,
antibodies which bind to and competitively inhibit polypeptide multimerization
and/or
36
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
binding of a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes
that "mimic" the polypeptide multimerization and/or binding domain and, as a
consequence,
bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-
idiotypes or Fab
fragments of such anti-idiotypes can be used in therapeutic regimens to
neutralize polypeptide
Iigand. For example, such anti-idiotypic antibodies can be used to bind a
polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block its
biological activity.
Pelvnncle~ticle~ Fncedin~ Antiheclie~
(0113) The invention further provides polynucleotides comprising a nucleotide
sequence encoding an antibody of the invention and fragments thereof. The
invention also
encompasses polynucleotides that hybridize under stringent or lower stringency
hybridization
conditions, e.g., as defined supra, to polynucleotides that encode an
antibody, preferably, that
specifically binds to a polypeptide of the invention, preferably, an antibody
that binds to a
polypeptide having the amino acid sequence of Figures lA-B.
[0114] Such polynucleotides are used in accordance with the invention both to
produce antibodies for isolation, and as components for use in antibody-based
gene therapy
regimens. Thus, these polynucleotides can be used in according to the
procedures for using
CTGF-2 encoding polynucleotides in gene therapy as described in detail below.
[0115] The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the nucleotide
sequence of the antibody is known, a polynucleotide encoding the antibody may
be assembled
from chemically synthesized oligonucleotides (e.g., as described in I~utmeier
et al.,
BioTeclmiques 17:242 (1994)), which, briefly, involves the synthesis of
overlapping
oligonucleotides containing portions of the sequence encoding the antibody,
annealing and
ligation of those oligonucleotides, and then amplification of the ligated
oligonucleotides by
PCR.
[0116] Alternatively, a polynucleotide encoding an antibody may be generated
from
nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding a particular
antibody is not available, but the sequence of the antibody molecule is known,
a nucleic acid
encoding the immunoglobulin may be obtained from a suitable source (e.g., an
antibody
cDNA library, or a cDNA library generated from, or nucleic acid, preferably
poly A+ RNA,
isolated from, any tissue or cells expressing the antibody, such as hybridoma
cells selected to
37
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
express an antibody of the invention) by PCR amplification using synthetic
primers
hybridizable to the 3' and 5' ends of the sequence or by cloning using an
oligonucleotide
probe specific for the particular gene sequence to identify, e.g., a cDNA
clone from a cDNA
library that encodes the antibody. Amplified nucleic acids generated by PCR
may then be
cloned into replicable cloning vectors using any method well known in the art.
[0117] Once the nucleotide sequence and corresponding amino acid sequence of
the
antibody is determined, the nucleotide sequence of the antibody may be
manipulated using
methods well known in the art for the manipulation of nucleotide sequences,
e.g.,
recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for
example; the
techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory
Manual, 2d
Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al.,
eds., 1998,
Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both
incorporated by reference herein in their entireties ), to generate antibodies
having a different
amino acid sequence, for example to create amino acid substitutions,
deletions, and/or
insertions.
[0118] In a specific embodiment, the amino acid sequence of the heavy and/or
light
chain variable domains may be inspected to identify the sequences of the
complementarity
determining regions (CDRs) by methods that are well know in the art, e.g., by
comparison to
known amino acid sequences of other heavy and light chain variable regions to
determine the
regions of sequence hypervariability. Using routine recombinant DNA
techniques, one or
more of the CDRs may be inserted within framework regions, e.g., into human
framework
regions to humanize a non-human antibody, as described supra. The framework
regions may
be naturally occurring or consensus framework regions, and preferably human
framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 2?8: 45?-4?9 (1998) for a
listing of human
framework regions). Preferably, the polynucleotide generated by the
combination of the
framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of the
invention. ~ Preferably, as discussed supra, one or more amino acid
substitutions may be made
within the framework regions, and, preferably, the amino acid substitutions
improve binding
of the antibody to its antigen. Additionally, such methods may be used to make
amino acid
substitutions or deletions of one or more variable region cysteine residues
participating in an
intrachain disulfide bond to generate antibody molecules lacking one or more
intrachain
38
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
disulfide bonds. Other alterations to the polynucleotide are encompassed by
the present
invention and within the skill of the art.
[0119] In addition, techniques developed for the production of "chimeric
antibodies"
(Mornson et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al.,
1984, Nature
312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a
mouse
antibody molecule of appropriate antigen specificity together with genes from
a human
antibody molecule of appropriate biological activity can be used. As described
supra, a
chimeric antibody is a molecule in which different portions are derived from
different animal
species, such as those having a variable region derived from a marine mAb and
a human
immunoglobulin constant region, e.g., humanized antibodies.
[0120] Alternatively, techniques described for the production of single chain
antibodies (U.S. Patent No. 4,694,778; Bird, 1988, Science 242:423- 42; Huston
et al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-
54) can be
adapted to produce single chain antibodies. Single chain antibodies are formed
by linking
the heavy and light chain fragments of the Fv region via an amino acid bridge,
resulting in a
single chain polypeptide. Techniques for the assembly of functional Fv
fragments in E. coli
may also be used (Skerra et al., 1988, Science 242:1038- 1041).
Methods nfPr~ducin~ Antihedies
[0121] The antibodies of the invention can be produced by any method known in
the
art for the synthesis of antibodies, in particular, by chemical synthesis or
preferably, by
recombinant expression techniques.
[0122] Recombinant expression of an antibody of the invention, or fragment,
derivative or analog thereof, e.g., a heavy or light chain of an antibody of
the invention,
requires construction of an expression vector containing a polynucleotide that
encodes the
antibody. Once a polynucleotide encoding an antibody molecule or a heavy or
light chain of
an antibody, or portion thereof (preferably containing the heavy or light
chain variable
domain), of the invention has been obtained, the vector for the production of
the antibody
molecule may be produced by recombinant DNA technology using techniques well
known in
the art. Thus, methods for preparing a protein by expressing a polynucleotide
containing an
antibody encoding nucleotide sequence are described herein. Methods which are
well known
to those skilled in the art can be used to construct expression vectors
containing antibody
39
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
coding. sequences and appropriate transcriptional and translational control
signals. These
methods include, for example, in vitro recombinant DNA techniques, synthetic
techniques,
and in vivo genetic recombination. The invention, thus, provides replicable
vectors
comprising a nucleotide sequence encoding an antibody molecule of the
invention, or a heavy
or light chain thereof, or a heavy or light chain variable domain, operably
linked to a
promoter. Such vectors may include the nucleotide sequence encoding the
constant region of
the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication
WO
89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the
antibody may be
cloned into such a vector for expression of the entire heavy or light chain.
[0123] The expression vector is transferred to a host cell by conventional
techniques
and the transfected cells are then cultured by conventional techniques to
produce an antibody
of the invention. Thus, the invention includes host cells containing a
polynucleotide
encoding an antibody of the invention, or a heavy or light chain thereof,
operably linked to a
heterologous promoter. In preferred embodiments for the expression of double-
chained
antibodies, vectors encoding both the heavy and light chains may be co-
expressed in the host
cell for expression of the entire immunoglobulin molecule, as detailed below.
(0124] A variety of host-expression vector systems may be utilized to express
the
antibody molecules of the invention. Such host-expression systems represent
vehicles by
which the coding sequences of interest may be produced and subsequently
purified, but also
represent cells which may, when transformed or transfected with the
appropriate nucleotide
coding sequences, express an antibody molecule of the invention in situ. These
include but
are not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia)
transformed with
recombinant yeast expression vectors containing antibody coding sequences;
insect cell
systems infected with recombinant virus expression vectors (e.g., baculovirus)
containing
antibody coding sequences; plant cell systems infected with recombinant virus
expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed
with recombinant plasmid expression vectors (e.g., Ti plasmid) containing
antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells)
harboring
recombinant expression constructs containing promoters derived from the genome
of
mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g., the
CA 02412124 2002-12-13
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adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,
bacterial cells such
as Escherichia coli, and more preferably, eukaryotic cells, especially for the
expression of
whole recombinant antibody molecule, are used for the expression of a
recombinant antibody
molecule. For example, mammalian cells such as Chinese hamster ovary cells
(CHO), in
conjunction with a vector such as the major intermediate early gene promoter
element from
human cytomegalovirus is an effective expression system for antibodies
(Foecking et al.,
1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).
[0125] In bacterial systems, a number of expression vectors may be
advantageously
selected depending upon the use intended for the antibody molecule being
expressed. For
example, when a large quantity of such a protein is to be produced, for the
generation of
pharmaceutical compositions of an antibody molecule, vectors which direct the
expression of
high levels of fusion protein products that are readily purified rnay be
desirable. Such vectors
include, but are not limited, to the E. coli expression vector pUR278 (Ruther
et al., 1983,
EMBO J. 2:1791), in which the antibody coding sequence may be ligated
individually into the
vector in frame with the lac Z coding region so that a fusion protein is
produced; pJN vectors
(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,
1989, J.
Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to
express foreign
polypeptides as fusion proteins with glutathione S-transferase (GST). In
general, such fusion
proteins are soluble and can easily be purified from lysed cells by adsorption
and binding to a
matrix glutathione-agarose beads followed by elution in the presence of free
glutathione. The
pGEX vectors are designed to include thrombin or factor Xa protease cleavage
sites so that
the cloned target gene product can be released from the GST moiety.
[0126] In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera
frugiperda cells. The antibody coding sequence may be cloned individually into
non-essential
regions (for example the polyhedrin gene) of the virus and placed under
control of an AcNPV
promoter (for example the polyhedrin promoter).
[0127] In mammalian host cells, a number of viral-based expression systems may
be
utilized. In cases where an adenovirus is used as an expression vector, the
antibody coding
sequence of interest may be ligated to an adenovirus transcription/translation
control
complex, e.g., the late promoter and tripartite leader sequence. This chimeric
gene may then
be inserted in the adenovirus genome by in vitro or in vivo recombination.
Insertion in a non-
41
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
essential region of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus
that is viable and capable of expressing the antibody molecule in infected
hosts. (e.g., see
Logan & Shenk, 1984., Proc. Natl. Acad. Sci. USA 81:355-359). Specific
initiation signals
may also be required for efficient translation of inserted antibody coding
sequences. These
signals include the ATG initiation codon and adjacent sequences. Furthermore,
the initiation
codon must be in phase with the reading frame of the desired coding sequence
to ensure
translation of the entire insert. These exogenous translational control
signals and initiation
codons can be of a variety of origins, both natural and synthetic. The
efficiency of expression
may be enhanced by the inclusion of appropriate transcription enhancer
elements,
transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol.
15.3:51-544).
[0128] In addition, a host cell strain may be chosen which modulates the
expression
of the inserted sequences, or modifies and processes the gene product in the
specific . fashion
desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of protein
products may be important for the function of the protein. Different host
cells have
characteristic and specific mechanisms for the post-translational processing
and modification
of proteins and gene products. Appropriate cell lines or host systems can be
chosen to ensure
the correct modification and processing of the foreign protein expressed. To
this end,
eukaryotic host cells which possess the cellular machinery for proper
processing of the
primary transcript, glycosylation, and phosphorylation of the gene product may
be used.
Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela,
COS,
MDCK, 293, 3T3, WT38, and in particular, breast cancer cell lines such as, for
example,
BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such
as, for
example, CRL7030 and Hs578Bst. '
[0129] For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express the
antibody molecule
may be engineered. Rather than using expression vectors which contain viral
origins of
replication, host cells can be transformed with DNA controlled by appropriate
expression
control elements (e.g., promoter, enhancer, sequences, transcription
terminators,
polyadenylation sites, etc.), and a selectable marker. Following the
introduction of the
foreign DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media,
and then are switched to a selective media. The selectable marker in the
recombinant plasmid
confers resistance to the selection and allows cells to stably integrate the
plasmid into their
42
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
chromosomes and grow to form foci which in turn can be cloned and expanded
intp cell lines.
This method may advantageously be used to engineer cell lines which express
the antibody
molecule. Such engineered cell lines may be particularly useful in screening
and evaluation
of compounds that interact directly or indirectly with the antibody molecule.
[0130] A number of selection systems may be used, including but not limited to
the
herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223),
hypoxanthine-
guanine phosphoribosyltransferase (Szybalski & Szybalski, 192, Proc. Natl.
Acad. Sci. USA
48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell
22:817) genes can
be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite
resistance can be
used as the basis of selection for the following genes: dhfr, which confers
resistance to
methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al.,
1981, Proc.
Natl. Acad. Sci: USA 78:1527); gpt, which confers resistance to mycophenolic
acid
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which
confers resistance
to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, 1991,
Biotherapy
3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,
1993,
Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-
217;
May; 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to
hygromycin
(Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of
recombinant
DNA technology which can be used are described in Ausubel et al. (eds.), 1993,
Current
Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene
Transfer and
Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and
13,
Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley
& Sons, NY.;
Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated by
reference
herein in their entireties.
[0131] The expression levels of an antibody molecule can be increased by
vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based on gene
amplification for the expression of cloned genes in mammalian cells in DNA
cloning, Vol.3.
(Academic Press, New York, 1987')). When a marker in the vector system
expressing
antibody is amplifiable, increase in the level of inhibitor present in culture
of host cell will
increase the number of copies of the marker gene. Since the amplified region
is associated
with the antibody gene, production of the antibody will also increase (Crouse
et al., 1983,
Mol. Cell. Biol. 3:257).
43
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
[0132] The host cell may be co-transfected with two expression vectors of the
invention, the first vector encoding a heavy chain derived polypeptide and the
second vector
encoding a light chain derived polypeptide. The two vectors may contain
identical selectable
markers which enable equal expression of heavy and light chain polypeptides.
Alternatively,
a single vector may be used which encodes both heavy and light chain
polypeptides. In such
situations, the light chain should be placed before the heavy chain to avoid
an excess of toxic
free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl.
Acad. Sci. USA
77:2197). The coding sequences for the heavy and light chains may comprise
cDNA or
genomic DNA.
[0133] Once an antibody molecule of the invention has been recombinantly
expressed, it may be purified by any method known in the art for purification
of an
immunoglobulin molecule, for example, by chromatography (e.g., ion exchange,
affinity,
particularly by affinity for the specific antigen after Protein A, and sizing
column
chromatography), centrifugation, differential solubility, or by any other
standard technique for
the purification of proteins.
Antih~dy c~r~n
[0134] The present invention encompasses antibodies recombinantly fused or
chemically conjugated (including both covalently and non-covalently
conjugations) to a
polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids
of the
polypeptide) of the present invention to generate fusion proteins. The fusion
does not
necessarily need to be direct, but may occur through linker sequences. The
antibodies may be
specific for antigens other than polypeptides (or portion thereof, preferably
at least 10, 20 or
50 amino acids of the polypeptide) of the present invention. For example,
antibodies may be
used to target the polypeptides of the present invention to particular cell
types, either in vitro
or in vivo, by fusing or conjugating the polypeptides of the present invention
to antibodies
specific for particular cell surface receptors. Antibodies fused or conjugated
to the
polypeptides of the present invention may also be used in in vitro
immunoassays and
purification methods using methods known in the art. See e.g., Harbor et al.,
supra, and PCT
publication WO 93/21232; EP 439,095; Naramura et al., Itnmunol. Lett. 39:91-99
(1994);
U.S. Patent 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol.
146:2446-2452(1991), which are incorporated by reference in their entireties.
44
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
[0135] Polypeptides of the invention (including antibodies of the invention,
see
below) may also be fused to albumin (including but not limited to recombinant
human serum
albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent
0 413 622,
and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by
reference in their
entirety)), resulting in chimeric polypeptides. In a preferred embodiment,
polypeptides
(including antibodies) of the present invention (including fragments or
variants thereof) are
fused with the mature form of human serum albumin (i.e., amino acids 1-585 of
human serum
albumin as shown in Figures 1 and 2 of EP Patent 0 322 094) which is herein
incorporated by
reference in its entirety. In another preferred embodiment, polypeptides
and/or antibodies of
the present invention (including fragments or variants thereof) are fused with
polypeptide
fragments comprising, or alternatively consisting of, amino acid residues 1-z
of human serum
albumin, where z is an integer from 369 to 419, as described in U.S. Patent
5,766,883 herein
incorporated by reference in its entirety. Polypeptides and/or antibodies of
the present
invention (including fragments or variants thereof) may be fused to either the
N- or C-
terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide
or human
serum albumin polypeptide). Such human serum albumin-CTGF-2 fusion proteins
may be
used therapeutically in accordance with the invention to stimulate, for
example, angiogenesis
as indicated below with respect to adenoviral-vector delivered DNA encoding
CTGF-2.
[0136] The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other than the .
variable regions. For example, the polypeptides of the present invention may
be fused or
conjugated to an antibody Fc region, or portion thereof. The antibody portion
fused to a
polypeptide of the present invention may comprise the constant region, hinge
region, CH1
domain, CH2 domain, and CH3 domain or any combination of whole domains or
portions
thereof. The polypeptides may also be fused or conjugated to the above
antibody portions to
form multimers. For example, Fc portions fused to the polypeptides of the
present invention
can form dimers through disulfide bonding between the Fc portions. Higher
multimeric
forms can be made by fusing the polypeptides to portions of IgA and IgM.
Methods for
fusing or conjugating the polypeptides of the present invention to antibody
portions are
known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046;
5,349,053;
5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO
91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991);
Zheng et
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
al., J. hnmunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci.
USA 89:11337-
11341(1992) (said references incorporated by reference in their entireties).
[0137] As discussed, supra, the polypeptides of the present invention may be
fused or
conjugated to the above antibody portions to increase the in vivo half life of
the polypeptides
or for use in immunoassays using methods known in the art. Further, the
polypeptides of the
present invention may be fused or conjugated to the above antibody portions to
facilitate
purification. One reported example describes 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. (EP 394,827; Traunecker et
al.,
Nature 331:84-86 (1988). The polypeptides of the present invention fused or
conjugated to
an antibody having disulfide- linked dimeric structures (due to the IgG) may
also be more
efficient in binding and neutralizing other molecules, than the monomeric
secreted protein or
protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964
(1995)). In many .
cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis,
and thus can result
in, for example, improved pharmacokinetic properties. (EP A 232,262).
Alternatively,
deleting the Fc part after the fusion protein has been expressed, detected,
and purified, would
be desired. For example, the Fc portion may hinder therapy and diagnosis if
the fusion
protein is used as an antigen for immunizations. In drug discovery, for
example, human
proteins, such as hIL-5, 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., J.
Molecular
Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-947-1
(1995)0.
[0138] Moreover, the antibodies or fragments thereof of the present invention
can be
fused to marker sequences, such as a peptide to facilitates their
purification. In preferred
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,
91311),
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. Other peptide tags useful for
purification
include, but are not limited to, the HA tag, which corresponds to an epitope
derived from the
influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
flag tag.
[0139] The present invention further encompasses antibodies or fragments
thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically to,
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CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
for example, monitor the development or progression of a tumor as part of a
clinical testing
procedure to, e.g., determine the efficacy of a given treatment regimen.
Detection can be
facilitated by coupling the antibody to a detectable substance. Examples of
detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent
materials, bioluminescent materials, radioactive materials, positron emitting
metals using
various positron emission tom~ographies, and nonradioactive paramagnetic metal
ions. See,
for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated
to antibodies
for use as diagnostics according to the present invention. Examples of
suitable enzymes
include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent
material includes.
luminol; examples of bioluminescent materials include luciferase, luciferin,
and aequorin;
and examples of suitable radioactive material include lzsh 1311 n1~ or 99Tc.
[0140] Further, an antibody or fragment thereof may be conjugated to a
therapeutic
moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a
therapeutic agent or a
radioactive metal .ion. A cytotoxin or cytotoxic agent includes any agent that
is detrimental to
cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,
colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, Iidocaine,
propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents include, but are
not limited
to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,
cytarabine, 5-
fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil,
melphalan, carmustine (BSNC>] and lomustine (CCNU), cyclothosphamide,
busulfan,
dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine
platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and
doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and
anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
(0141] The conjugates of the invention can be used for modifying a given
biological
response, the therapeutic agent or drug moiety is not to be construed as
limited to classical
47
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
chemical therapeutic agents. For example, the drug moiety may be a protein or
polypeptide
possessing a desired biological activity. Such proteins may include, for
example, a toxin such
as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such
as tumor necrosis
factor, a-interferon, 13-interferon, nerve growth factor, platelet derived
growth factor, tissue
plasminogen activator, a thrombotic agent or an anti- angiogenic agent, e.g.,
angiostatin or
endostatin; or, biological response modifiers such as, for example,
lymphokines, interleukin-1
("IL-1 "), interleukin-2 ("TL-2"), interleukin-6 ("IL-6"), granulocyte
macrophase colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-
CSF"), or other
growth factors.
[0142] Antibodies may also be attached to solid supports, which are
particularly
useful for immunoassays or purification of the target antigen. Such solid
supports include,
but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,
polyvinyl chloride
or polypropylene.
[0143] Techniques for conjugating such therapeutic moiety to antibodies are
well
known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of
Drugs In
Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
(eds.), pp.
243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug
Delivery", in
Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel
Dekker, Inc.
1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in
Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et
al. (eds.), pp.
475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic
Use Of
Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer
Detection
And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and
Thorpe et al.,
"The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev.
62:119-58 (1982).
[0144] Alternatively, an antibody can be conjugated to a second antibody to
form an
antibody heteroconjugate as described by Segal in U.S. Patent No. 4,6?6,980,
which is
incorporated herein by reference in its entirety.
[0145] An antibody, with or without a therapeutic moiety conjugated to it,
administered alone or in combination with cytotoxic factors) and/or
cytokine(s) can be used
as a therapeutic.
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CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
Ay s Fir Antih~ciy Ry
[0146] The antibodies of the invention may be assayed for immunospecific
binding by
any method known in the art. The immunoassays which can be used include but
are not
limited to competitive and non-competitive assay systems using techniques such
as western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich"
immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin
reactions, immunodiffusion assays, agglutination assays, complement-fixation
assays,
immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to
name but
a few. Such assays are routine and well known in the art (see, e.g., Ausubel
et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
York, which
is incorporated by reference herein in its entirety). Exemplary immunoassays
are described
briefly below (but are not intended by way of limitation).
[0147] Immunoprecipitation protocols generally comprise lysing a population of
cells
in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium
deoxycholate,
0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol)
supplemented
with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium
vanadate), adding the antibody of interest to the cell lysate; incubating for
a period of time
(e.g., 1-4 hours) at 4°C, adding protein A and/or protein G sephaxose
beads to the cell lysate,
incubating for about an hour or more at 4°C, washing the beads in lysis
buffer and
resuspending the beads in SDS/sample buffer. The ability of the antibody of
interest to
immunoprecipitate a particular antigen can be assessed by, e.g., western blot
analysis. One of
skill in the art would be knowledgeable as to the parameters that can be
modified to increase
the binding of the antibody to an antigen and decrease the background (e.g.,
pre-clearing the
cell lysate with sepharose beads). For further discussion regarding.
immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular
Biology, Vol. 1,
John Wiley & Sons, Inc., New York at 10.16.1.
[0148] Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein
sample from the
polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon,
blocking the
membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing
the
membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with
primary
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CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
antibody (the antibody of interest) diluted in blocking buffer, washing the
membrane in
washing buffer, blocking the membrane with a secondary antibody (which
recognizes the
primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic
substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I)
diluted in blocking buffer, washing the membrane in Wash buffer, and detecting
the presence
of the antigen. One of skill in the art would be knowledgeable as to the
parameters that can
be modified to increase the signal detected and to reduce the background
noise. For further
discussion regarding western blot protocols see, e.g., Ausubel et al, eds,
1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
(0149] ELISAs comprise preparing antigen, coating the well of a 96 well
microtiter
plate with the antigen, adding the antibody of interest conjugated to a
detectable compound
such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline
phosphatase) to the
well and incubating for a period of time, and detecting the presence of the
antigen. In
ELISAs the antibody of interest does not have to be conjugated to a detectable
compound;
instead, a second antibody (which recognizes the antibody of interest)
conjugated to a
detectable compound may be added to the well. Further, instead of coating the
well with the
antigen, the antibody may be coated to the well. In this case, a second
antibody conjugated to
a detectable compound may be added following the addition of the antigen of
interest to the
coated well. One of skill in the art would be knowledgeable as to the
parameters that can be
modified to increase the signal detected as well as other variations of ELISAs
known in the
art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds,
1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
11.2.1.
(0150] The binding affinity of an antibody to an antigen and the off rate of
an
antibody-antigen interaction can be determined by competitive binding assays.
One example
of a competitive binding assay is a radioimmunoassay comprising the incubation
of labeled
antigen (e.g., 3H or lzsl) with the antibody of interest in the presence of
increasing amounts of
unlabeled antigen, and the detection of the antibody bound to the labeled
antigen. The
affinity of the antibody of interest for a particular antigen and the binding
off rates can be
determined from the data by scatchard plot analysis. Competition with a second
antibody can
also be determined using radioimmunoassays. In this case, the antigen is
incubated with
antibody of interest is conjugated to a labeled compound (e.g., 3H or l2sn in
the presence of
increasing amounts of an unlabeled second antibody.
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
Th raise stir. TTY .~
[0151] The present invention is further directed to antibody-based therapies
which
involve administering antibodies (e.g., CTGF-2 antagonists) of the invention
to an animal,
preferably a mammal, and most preferably a human, patient for treating one or
more of the
described disorders. Therapeutic compounds of the invention include, but are
not limited to,
antibodies of the invention (including fragments, analogs and derivatives
thereof as described
herein) and nucleic acids encoding antibodies of the invention (including
fragments, analogs
and derivatives thereof as described herein). The antibodies of the invention
can be used to
treat, inhibit or prevent diseases and disorders associated with aberrant
expression and/or
activity of a polypeptide of the invention, including, but not limited to,
inhibition of
angiogenesis, as discussed in detail below. The treatment and/or prevention of
diseases and
disorders associated with aberrant expression and/or activity of a polypeptide
of the invention
includes, but is not limited to, alleviating symptoms associated with those
diseases and
disorders. Antibodies of the invention may be provided in pharmaceutically
acceptable
compositions as known in the art or as described herein.
[0152] A summary of the ways in which the antibodies of the present invention
may
be used therapeutically includes binding polynucleotides or polypeptides of
the present
invention 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.
[0153] The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for
example, which serve to
increase the number or activity of effector cells which interact with the
antibodies.
[0154] The antibodies of the invention may be administered alone or in
combination
with other types of treatments (e.g., radiation therapy, chemotherapy,
hormonal therapy,
immunotherapy and anti-tumor agents). Generally, administration of products of
a species
origin or species reactivity (in the case of antibodies) that is the same
species as that of the
patient is preferred. Thus, in a preferred embodiment, human antibodies,
fragments
51
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
derivatives, analogs, or nucleic acids, are administered to a human patient
for therapy or
prophylaxis.
[0155] It is preferred to use high affinity andlor potent .in vivo inhibiting
and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention,
fragments or regions thereof, for both immunoassays directed to and therapy of
disorders
related to polynucleotides or polypeptides, including fragments thereof, of
the present
invention. Such antibodies, fragments, or regions, will preferably have an
affinity for
polynucleotides or polypeptides, including fragments thereof. Preferred
binding affinities
include those with a dissociation constant or Kd less than 5 X 10-g M, 10-6 M,
5 X 10-~ M, 10-
M, 5 X 10-8 M, 10-8 M, 5 X 10-9 M, 10-9 M, 5 X 10-1 ° M, 10-' °
M, 5 X 10-11 M, 10-11 M, 5 X
10-1 z M, 10-1 a M, 5 X 10-i 3 M, 10-13 M, S X 10-~ 4 M, 10-I a M, S X 10-1 s
M, and 10-1 s M. .
(~Tene Therapy
[0156] The novel use of CTGF-2 encoding DNA in the stimulation of angiogenesis
by
gene therapy, in accordance with the invention, is disclosed herein. (See,
e.g., Example 12
below).
[0157] In accordance with the invention, nucleic acids comprising sequences
encoding CTGF-2 or functional derivatives thereof, are administered to
stimulate
angiogenesis. Gene therapy refers to therapy performed by the administration
to a subject of
an expressed or expressible nucleic acid. In this embodiment of the invention,
the nucleic
acids produce their encoded protein that mediates a therapeutic effect.
[0158] Any of the methods for gene therapy available in the art can be used
according
to the present invention., Exemplary methods are described below.
[0159] For general reviews of the methods of gene therapy, see Goldspiel et
al., 1993,
Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev,
1993,
Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932;
and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH
11 (5):155-215). Methods commonly known in the art of recombinant DNA
technology which
can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in
Molecular
Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and
Expression, A
Laboratory Manual, Stockton Press, NY.
52
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
[0160] In a preferred aspect, the compound comprises nucleic acid sequences
encoding CTGF-2 or polypeptide having CTGF-2 activity as described above, said
nucleic
acid sequences being part of expression vectors that express the CTGF-2
protein or
derivative in a suitable host. In particular, such nucleic acid sequences have
promoters
operably linked to the antibody coding region, said promoter being inducible
or constitutive,
and, optionally, tissue- specific. In another particular embodiment, nucleic
acid molecules are
used in which the CTGF-2 coding sequences and any other desired sequences are
flanked by
regions that promote homologous recombination at a desired site in the genome,
thus
providing for intrachromosomal expression of the antibody nucleic acids
(Koller and
Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al.,
1989, Nature
. 342:435-438). The expressed polynucleotides include the polynucleotides of
the invention
described in detail, above.
[0161] Delivery of the nucleic acids into a patient may be either direct, in
which case
the patient is directly exposed to the nucleic acid or nucleic acid- carrying
vectors, or indirect,
in which case, cells are first transformed with the nucleic acids in vitro,
then transplanted into
the patient. These two approaches are known, respectively, as in vivo or ex
vivo gene
therapy.
[0162] In a specific embodiment, the nucleic acid sequences are directly
administered
in vivo, where it is expressed to produce the encoded product. This can be
accomplished by
any of numerous methods known in the art, e.g., by constructing them as part
of an
appropriate nucleic acid expression vector and administering it so that they
become
intracellular, e.g., by infection using defective or attenuated retrovirals or
other viral vectors
(see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by
use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating
with lipids or
cell-surface receptors or transfecting agents, encapsulation in liposomes,
microparticles, or
microcapsules, or by administering them in linkage to a peptide which is known
to enter the
nucleus, by administering it in linkage to a ligand subject to receptor-
mediated endocytosis
(see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used
to target
cell types specifically expressing the receptors), etc. In another embodiment,
nucleic acid-
ligand complexes can be formed in which the ligand comprises a fusogenic viral
peptide to
disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
In yet another
embodiment, the nucleic acid can be targeted in vivo for cell specific uptake
and expression,
53
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180
dated April 16,
1992 (Wu et al.); WO 92/22636 dated December 23, 1992 (Wilson et al.);
W092/20316
dated November 26, 1992 (Findeis et al.); W093/14188 dated July 22, 1993
(Clarke et al.),
WO 93/20221 dated October 14, 1993 (Young)). Alternatively, the nucleic acid
can be
introduced intracellularly and incorporated within host cell DNA for
expression, by
homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
USA 86:8932-
8935; Zijlstra et al., 1989, Nature 342:435-438).
[0163] In a specific embodiment, viral vectors that contains nucleic acid
sequences
encoding a CTGF-2 polypeptide of the invention are used. For example, a
retroviral vector
can be used (see Miller et al., 1993, Meth. Enzymol. 217:681-599). These
retroviral vectors
have been to delete retroviral sequences that are not necessary for packaging
of the viral
genome and integration into host cell DNA. The nucleic acid sequences encoding
the
antibody to be used in gene therapy are cloned into one or more vectors, which
facilitates
delivery of the gene into a patient. More detail about retroviral vectors can
be found in
Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a
retroviral vector to
deliver the mdrl gene to hematopoietic stem cells in order to make the stem
cells more
resistant to chemotherapy. Other references illustrating the use of retroviral
vectors in gene
therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-661; Kiem et al.,
1994, Blood
83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and
Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110'-114.
[0164] Adenoviruses are other viral vectors that can be used in gene therapy
as
detailed in a preferred embodiment in Example 12, below. Adenoviruses are
especially
attractive vehicles for delivering genes to respiratory epithelia.
Adenoviruses naturally infect
respiratory epithelia where they cause a mild disease. Other targets for
adenovirus-based
delivery systems are liver, the central nervous system, endothelial cells, and
muscle.
Adenoviruses have the advantage of being capable of infecting non-dividing
cells. Kozarsky
and Wilson, 1993, Current Opinion in Genetics and Development 3:499-603
present a review
of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 6:3-10
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of
rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can
be found in
Rosenfeld et al., 1991, Science 262:431-434; Rosenfeld et al., 1992, Cell
68:143- 166;
Mastrangeli et al., 1993, J. Clin. Invest. 91:226-234; PCT Publication
W094/12649; and
64
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
Wang, et al., 1995, Gene Therapy 2:775-783, each of which is hereby
incorporated by
reference in its entirety. In a preferred embodiment, adenovirus vectors are
used.
[0165] In a specific, preferred embodiment, the CTGF-2 cDNA of ATCC deposit
no.
75804 is cloned into the BamHI restriction site of Bluescript vector
(Stratagene); and then is
EcoRI/XbaI subcloned in a transfer vector containing the CMV promoter. An
E1/E3 deleted
AdCTGF-2 vector is obtained by homologous recombination in 293 cells between
the transfer
vector and the genomic CIaI DNA fragment isolated from the HSd1324 virus. The
recombinant virus is plaque purified and amplified on 293 cells, and genome
virus integrity is
analyzed by restriction enzyme digestion and Southern blot. Titres of
infectious viral progeny
are determined as infectious units (IUD by quantitative DBP
immunofluorescence.
[0166] Adeno-associated virus (AAV) has also been proposed for use in gene
therapy
(Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Patent No.
5,436,146).
[0167] Another approach to gene therapy involves transferring a gene to cells
in tissue
culture by such methods as electroporation, lipofection, calcium phosphate
mediated
transfection, or viral infection. Usually, the method of transfer includes the
transfer of a
selectable marker to the cells. The cells are then placed under selection to
isolate those cells
that have taken up and are expressing the transferred gene. Those cells are
then delivered to a
patient.
[0168] In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be carried out
by any method known in the art, including but not limited to transfection,
electroporation,
microinjection, infection with a viral or bacteriophage vector containing the
nucleic acid
sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated
gene
transfer, spheroplast fusion, etc. Numerous techniques are known in the art
for the
introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993,
Meth. Enzymol.
217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985,
Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention, provided
that the
necessary developmental and physiological functions of the recipient cells are
not disrupted.
The technique should provide for the stable transfer of the nucleic acid to
the cell, so that the
nucleic acid is expressible by the cell and preferably heritable and
expressible by its cell
progeny.
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
[0169] The resulting recombinant cells can be delivered to a patient by
various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor
cells) are preferably administered intravenously. The amount of cells
envisioned for use
depends on the desired effect, patient state, etc., and can be determined by
one skilled in the
art.
[0170] Cells into which a nucleic acid can be introduced for purposes of gene
therapy
are prefereably endothelial cells, but encompass any desired, available cell
type, and include
but are not limited to epithelial cells, keratinocytes, fibroblasts, muscle
cells, hepatocytes;
blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages,
neutrophils,
eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells,
in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow,
umbilical cord
blood, peripheral blood, fetal liver, etc.
[0171] In a preferred embodiment, the cell used for gene therapy is autologous
to the
patient.
[0172] In an embodiment in which recombinant cells are used in gene therapy,
nucleic
acid sequences encoding an antibody are introduced into the cells such that
they are
expressible by the cells or their progeny, and the recombinant cells are then
administered in
vivo for therapeutic effect. In a specific embodiment, stem or progenitor
cells are used. Any
stem and/or progenitor cells which can be isolated and maintained in vitro can
potentially be
used in accordance with this embodiment of the present invention (see e.g. PCT
Publication
WO 94/08598, dated April 28, 1994; Stemple and Anderson, 1992, Cell 71:973-
985;
Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo
Clinic
Proc. 61:771).
[0173] W a specific embodiment, the nucleic acid to be introduced for purposes
of
gene therapy comprises an inducible promoter operably linked to the coding
region, such that
expression of the nucleic acid is controllable by controlling the presence or
absence of the
appropriate inducer of transcription.
F~-e~~icn_ Prcdncticn, end screening
[0174] The present invention also relates to vectors which include
polynucleotides of
the present invention, host cells which are genetically engineered with
vectors of the
invention and the production of polypeptides of the invention by recombinant
techniques.
56
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
[0175) Host cells are genetically engineered (transduced or transformed or
transfected) with the vectors of this invention which may be, for example, a
cloning vector or
an expression vector. The vector may be, for example, in the form of a
plasmid, a viral
particle, a phage, etc. The engineered host cells can be cultured in
conventional nutrient
media modified as appropriate for activating promoters, selecting
transformants or amplifying
the genes of the present invention. -The culture conditions, such as
temperature, pH and the
like, are those previously used with the host cell selected for expression,
and will be apparent
to the ordinarily skilled artisan.
[0176) The polynucleotides of the present invention may be employed for
producing
polypeptides by recombinant techniques. Thus, for example, the polynucleotide
may be
included in any one of a variety of expression vectors for expressing a
polypeptide. Such
vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g.,
derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast
plasmids; vectors
derived from combinations of plasmids and phage DNA, viral DNA such as
vaccinia,
adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be
used as
long as it is replicable and viable in the host.
[0177] The appropriate DNA sequence may be inserted into the vector by a
variety of
procedures. In general, the DNA sequence is inserted into an appropriate
restriction
endonuclease site(s). by procedures known in the art. Such procedures and
others are deemed
to be within the scope of those skilled in the art.
[0178) The DNA sequence in the expression vector is operatively linked to an
appropriate expression control sequences) (promoter) to direct mRNA synthesis.
As
representative examples of such promoters, there may be mentioned: LTR or SV40
promoter,
the F~i lac or trp., the phage lambda PL promoter and other promoters known to
control
expression of genes in prokaryotic or eukaryotic cells or their viruses. The
expression vector
also contains a ribosome binding site for translation initiation and a
transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
[0179] In addition, the expression vectors preferably contain one or more
selectable
marker genes to provide a phenotypic trait for selection of transformed host
cells such as
dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or
such as
tetracycline or ampicillin resistance in F~oli.
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CA 02412124 2002-12-13
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[0180] The vector containing the appropriate DNA sequence as hereinabove
described, as well as an appropriate promoter or control sequence, may be
employed to
transform an appropriate host to permit the host to express the protein. .
[0181] As representative examples of appropriate hosts, there may be
mentioned:
bacterial cells, such as E.~li, ~tre~t~m3~, salmonella ~mhimurium; fungal
cells, such as
yeast; insect cells such as Dr~s~la S2 and ~ ~; animal cells such as CHO, COS
or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an
appropriate host is
deemed to be within the scope of those skilled in the art from the teachings
herein.
(0182] More particularly, the present invention also includes recombinant
constructs
comprising one or more of the sequences as broadly described above. The
constructs
comprise a vector, such as a plasmid or viral vector, into which a sequence of
the invention
has been inserted, in a forward or reverse orientation. In a preferred aspect
of this
embodiment, the construct further comprises regulatory. sequences, including,
for example, a
promoter, operably linked to the sequence. Large numbers of suitable vectors
and promoters
are known to those of skill in the art, and are commercially available. The
following vectors
are provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS,
pDlO,
phagescript, psiX174, pbluescript SK, pbsks, pNHBA, pNHl6a, pNHl8A, pNH46A
(Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia);
Eukaryotic:
pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL
(Pharmacia). However, any other plasmid or vector may be used as long as they
are
replicable and viable in the host.
[0183] Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable
markers. Two
appropriate vectors are pKK232-8 and pCM7. Particular named bacterial
promoters include
lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include
CMV immediate
early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and
mouse
metallothionein-I. Selection of the appropriate vector and promoter is well
within the level of
ordinary skill in the art.
[0184] In a further embodiment, the present invention relates to host cells
containing
the above-described constructs. The host cell can be a higher eukaryotic cell,
such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host
cell can be a
prokaryotic cell, such as a bacterial cell. Introduction of the construct into
the host cell can be
58
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
effected by calcium phosphate transfection, DEAE-Dextran mediated
transfection, or
electroporation (Davis, L., Dibner, M., Battey, L, Basic Methods in Molecular
Biology,
(1986)).
[0185] The constructs in host cells can be used in a conventional manner to
produce
the gene product encoded by the recombinant sequence. Alternatively, the
polypeptides of
the invention can be synthetically produced by conventional peptide
synthesizers.
[0186] Mature proteins can be expressed in mammalian cells, yeast, bacteria,
or other
cells under the control of appropriate promoters. Cell-free translation
systems can also be
employed to produce such proteins using RNAs derived from the DNA constructs
of the
present invention. Appropriate cloning and expression vectors for use with
prokaryotic and
eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A
Laboratory Manual,
Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is
hereby
incorporated by reference.
[0187] Transcription of the DNA encoding the polypeptides of the present
invention
by higher eukaryotes is increased by inserting an enhancer sequence into the
vector.
Enhancers are cis-acting elements of DNA, usually about from 10 to 300 by that
act on a
promoter to increase its transcription. Examples include the SV4.0 enhancer on
the late side of
the replication origin by 100 to 270, a cytomegalovirus early promoter
enhancer, the polyoma
enhancer on the late side of the replication origin, and adenovirus enhancers.
[0188] Generally, recombinant expression vectors will include origins of
replication
and selectable markers permitting transformation of the host cell, e.g., the
ampicillin
resistance gene of E.~li and ~- cerevi~iae TRP1 gene, and a promoter derived
from a highly-
expressed gene to direct transcription of a downstream structural sequence.
Such promoters
can be derived from operons encoding glycolytic enzymes such as 3-
phosphoglycerate kinase
(PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The
heterologous
structural sequence is assembled in appropriate phase with translation
initiation and
termination sequences, and preferably, a leader sequence capable of directing
secretion of
translated protein into the periplasmic space or extracellular medium.
Optionally, the
heterologous sequence can encode a fusion protein including an N-terminal
identification
peptide imparting desired characteristics, e.g., stabilization or simplified
purification of
expressed recombinant product.
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CA 02412124 2002-12-13
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[0189] Useful expression vectors for bacterial use are constructed by
inserting a
structural DNA sequence encoding a desired protein together with suitable
translation
initiation and termination signals in operable reading phase with a functional
promoter. The
vector will comprise one or more phenotypic selectable markers and an origin
of replication
to ensure maintenance of the vector and to, if desirable, provide
amplification within the host.
Suitable prokaryotic hosts for transformation include F,~oli, Racill"~
~nhtilis, ~almc,nella
t~im~a~~m. and various species within the genera Pseudomonas, Streptomyces,
and
Staphylococcus, although others may also be employed as a matter of choice.
[0190] As a representative but nonlimiting example, useful expression vectors
for
bacterial use can comprise a selectable marker and bacterial origin of
replication derived from
commercially available plasmids comprising genetic elements of the well known
cloning
vector pBR322 (ATCC 37017). Such commercial vectors include, for example,
pKK223-3
(Phannacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison,
WI,
USA). These pBR322 "backbone" sections are combined with an appropriate
promoter and
the structural sequence to be expressed.
[0191] Following transformation of a suitable host strain and growth of the
host strain
to an appropriate cell density, the selected promoter is induced by
appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for an
additional period.
[0192] Cells are typically harvested by centrifugation, . disrupted by
physical or
chemical means, and the resulting crude extract retained. for further
purification.
[0193] Microbial cells employed in expression of proteins can be disrupted by
any
convenient method, including freeze-thaw cycling, sonication, mechanical
disruption, or use
of cell lysing agents, such methods are well known to those skilled in the
art.
[0194] Various mammalian cell culture systems can also be employed to express
recombinant protein. Examples of mammalian expression systems include the COS-
7 lines
of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and
other cell lines
capable of expressing a compatible vector, for example, the C I27, 3T3, CHO,
HeLa and
BHK cell lines. Mammalian expression vectors will comprise an origin of
replication, a
suitable promoter and enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites, transcriptional
termination sequences,
and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40
splice, and
polyadenylation sites may be used to provide the required nontranscribed
genetic elements.
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
[0195] The polypeptide can be recovered and purified from recombinant cell
cultures
by 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. Protein refolding steps can be used, as necessary, in
completing
configuration of the mature protein. Finally, high performance liquid
chromatography
(HPLC) can be employed for final purification steps.
[0196] The polypeptides of the present invention may be a naturally purified
product,
or a product of chemical synthetic procedures, or produced by recombinant
techniques from a
prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher
plant, insect and
mammalian cells in culture). Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may be
glycosylated or may
be non-glycosylated.
[0197] Polypeptides of the invention may also include an initial methionine
amino
acid residue.
[0198] This invention provides a method for identification of the receptor for
the
CTGF-2 polypeptide. The gene encoding the receptor can be identified by
expression
cloning. Briefly, polyadenylated RNA is prepared from a cell responsive to
CTGF-2, and a
cDNA library created from this RNA is divided into pools and used to transfect
COS cells or
other cells that are not responsive to CTGF-2. Transfected cells which are
grown on glass
slides are exposed to labeled CTGF-2. The CTGF-2 can be labeled by a variety
of means
including iodidation or inclusion of a recognition site for a site-specific
protein kinase.
Following fixation and incubation, the slides are subjected to
autoradiographic analysis.
Positive pools are identified and sub-pools are prepared and retransfected
using an iterative
sub-pooling and rescreening process, eventually yielding a single clone that
encodes the
putative receptor. As an alternative approach for receptor identification,
labeled ligand can be
photoaffinity linked with cell membrane or extract preparations that express
the receptor
molecule. Cross-linked material is resolved by PAGE and exposed to x-ray film.
The labeled
complex containing the CTGF-2-receptor can be excised, resolved into peptide
fragments,
and subj ected to protein microsequencing. The amino acid sequence obtained
from
microsequencing would be used to design a set of generate oligonucleotide
probes to screen a
cDNA library to identify the gene encoding the putative receptor.
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CA 02412124 2002-12-13
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[0199] This invention also provides a method of screening compounds to
identify
those which bind to the CTGF-2 receptor and elicit a second messenger response
(agonists) or
do not elicit a second messenger response (antagonists). As an example, a
mammalian cell or
membrane preparation expressing the CTGF-2 receptor would be incubated with a
labeled
compound. The response of a known second messenger system following
interaction of the
compound and the CTGF-2 receptor is then measured. Such second messenger
systems
include but are not limited to, cAMP guanylate cyclase, ion channels or
phosphoinositide
hydrolysis.
Methods ~f Treatment
Stimnlatien ef Angi~g,~n
[0200] . CTGF-2 stimulates angiogenesis. Thus, the CTGF-2 polynucleotides and
polypeptides of the invention, described in detail above, are useful in the
treatment of
disorders in which stimulation of new blood vessel development would
ameliorate the
disorder. Such disorders include, but are not limited to, heart failure,
angina, blood vessel
(e.g. coronary artery) blockage and ischemia, inlcuding critical limb ischemia
and refractory
myocardial ischemia. CTGF-2 encoding polynucleotides can be delivered to
individuals to
using the gene therapy embodiments described above.
[0201] ~ CTGF-2 polynucleotides or polypeptides, or agonists CTGF-2 of the
present
invention, as a result of the ability to stimulate vascular endothelial cell
growth, may be
employed in treatment for stimulating re-vascularization of ischemic tissues
due to various
disease conditions such as thrombosis, arteriosclerosis, and other
cardiovascular conditions.
The polypeptide, polynucleotide, agonist, or antagonist of the present
invention may also be
employed to stimulate angiogenesis and limb regeneration, as discussed above.
R
(0202] CTGF-2 polynucleotides or polypeptides, or agonists CTGF-2 can be used
to
differentiate, proliferate, and attract cells, leading to the regeneration of
tissues. (See, Science
276:59-87 (1997).) The regeneration of tissues could be used to repair,
replace, or protect
tissue damaged by congenital defects, trauma (wounds, burns, incisions, or
ulcers), age,
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disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver
failure), surgery,
including cosmetic plastic surgery, fbrosis, reperfusion injury, or systemic
cytokine damage.
[0203] Tissues that could be regenerated using the present invention include
organs
(e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or
cardiac), vasculature (including vascular and lymphatics), nervous,
hematopoietic, and
skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,
regeneration occurs
without or decreased scarring. Regeneration also may include angiogenesis.
[0204] Moreover, CTGF-2 polynucleotides or polypeptides, or agonists CTGF-2,
may
increase regeneration of tissues difficult to heal. For example, increased
tendon/ligament
regeneration would quicken recovery time after damage. CTGF-2 polynucleotides
or
polypeptides, or agonists CTGF-2, of the present invention could also be used
prophylactically in an effort to avoid damage. Specific diseases that could be
treated include
of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A
further
example of tissue regeneration of non-healing wounds includes pressure ulcers,
ulcers
associated with vascular insufficiency, surgical, and traumatic wounds.
[0205] Similarly, nerve and brain tissue could also be regenerated by CTGF-2
polynucleotides or polypeptides, or agonists CTGF-2, to proliferate and
differentiate nerve
cells. Diseases that could be treated using this method include central and
peripheral nervous
system diseases, neuropathies, or mechanical and traumatic disorders (e.g.,
spinal cord
disorders, head trauma, cerebrovascular disease, and stoke). Specifically,
diseases associated
with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from
chemotherapy or
other medical therapies), localized neuropathies, and central nervous system
diseases (e.g.,
Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic
lateral sclerosis,
and Shy-Drager syndrome), could all be treated using the CTGF-2
polynucleotides or
polypeptides, or agonists CTGF-2.
Tnhihitie~n ~f Angi~g Y1PR1C
[0206] As detailed above, CTGF-2 stimulates angiogenesis. Thus, antagonists to
CTGF-2 may be useful in the treatment disorders in which inhibition of
angiogenesis would
amelioriate the disorder. Such antagonists include, but are not limited to,
antibodies,
peptides, and other molecules that bind to CTGF-2, as well as ribozymes,
antisense
molecules, and triple helix forming oligonucleotides that interfere with the
transcription and
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translation of the CTGF-2 genes, and are described in further detail, below.
For example
such antagonists may be used in accordance with the invention to treat
cancers, such as solid
tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx,
esophagus, testes,
liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,
kidney, bladder,
thyroid cancer; primary tumors and metastases; melanomas; glioblastoma;
Kaposi's sarcoma;
leiomyosarcoma; non- small cell lung cancer; colorectal cancer; advanced
malignancies;
blood born tumors (such as leukemias); benign tumors, for example hemangiomas,
acoustic
neuromas, neurofibzomas, trachomas, and pyogenic granulomas; artheroscleric
plaques;
ocular angiogenic diseases, for example diabetic retinopathy, retinopathy of
prematurity,
macular degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia,
rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth)
of the eye;
rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis;
vasculogenesis;
granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma;
.trachoma;
vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral
collaterals;
arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber
Syndrome; plaque
neovascularization; telangiectasia; hemophiliac joints; angiofibroma;
fibromuscular
dysplasia; wound granulation; Crohn's disease; and atherosclerosis.
[0207] Moreover, antagonists (i.e., peptides, antibodies, antisense, ribozyme,
triple
helix forming molecules and other CTGF-2 inhibitory molecules) that inhibit
CTGF-2
activity are useful in inhibiting the angiogenesis of proliferative cells or
tissues, either alone,
as a protein fusion, or in combination with other polypeptides directly or
indirectly, as
described elsewhere herein. In a most preferred embodiment, such anti-
angiogenic effect may
be achieved indirectly, for example, through the inhibition of hematopoietic,
tumor-specific
cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl
Cancer Inst,
90(21):1648-53 (1998), which is hereby incorporated by reference). Antibodies
and peptides
that bind and inhibit CTGF-2 may also result in inhibition of angiogenesis
directly, or
indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998),
which is hereby
incorporated by reference)).
[0208] The naturally occurnng balance between endogenous stimulators and
inhibitors of angiogenesis is one in which inhibitory influences predominate.
Rastinejad et
al., Cell 56:345-355 (1989). In those rare instances in which
neovascularization occurs under
normal physiological conditions, such as wound healing, organ regeneration,
embryonic
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development, and female reproductive processes, angiogenesis is stringently
regulated and
spatially and temporally delimited. Under conditions of pathological
angiogenesis such as that
characterizing solid tumor growth, these regulatory controls fail. Unregulated
angiogenesis
becomes pathologic and sustains progression of many neoplastic and non-
neoplastic diseases.
A number of serious diseases are dominated by abnormal neovascularization
including solid
tumor growth and metastases, arthritis, some types of eye disorders, and
psoriasis. See, e.g.,
reviews by Moses et al., Biotech. 9:630-634 (1991); Foll~man et al., N. Engl.
J. Med.,
333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985);
Folkman,
Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New
York, pp.
175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al.,
Science
221:719-725 (1983). In a number of pathological conditions, the process of
angiogenesis
contributes to the disease state. For example, significant data have
accumulated which
suggest that the growth of solid tumors is dependent on angiogenesis. Folkman
and
Klagsbrun, Science 235:442-447 (1987).
[0209] The present invention provides for treatment of diseases or disorders
associated with neovascularization by administration of CTGF-2 antagonists
(e.g., antibodies
or peptides) of the present invention. Malignant and metastatic conditions
which can be
treated with the antagonists of the invention include, but are not limited to,
malignancies,
solid tumors, and cancers described herein and otherwise known in the art (for
a review of
such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co.,
Philadelphia
(1985)). Thus, the present invention provides a method of treating an
angiogenesis-related
disease and/or disorder, comprising administering to an individual in need
thereof a
therapeutically effective amount of a CTGF-2 antagonist of the invention. For
example
CTGF-2 antagonists may be utilized in a variety of additional methods in order
to
therapeutically treat a cancer or tumor. Cancers which may be treated with
CTGF-2
antagonists include, but are not limited to solid tumors, including prostate,
lung, breast,
ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary
tract, colon,
rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary
tumors and
metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-
small cell
lung cancer; colorectal cancer; advanced malignancies; and blood born tumors
such as
leukemias. For example, polynucleotides, polypeptides, antagonists and/or
agonists may be
CA 02412124 2002-12-13
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delivered topically, in order to treat cancers such as skin cancer, head and
neck tumors, breast
tumors, and Kaposi's sarcoma.
[0210] ~ Within yet other aspects, CTGF-2 antagonists may be utilized to treat
superficial forms of bladder cancer by, for example, intravesical
administration. CTGF-2
antagonists may be delivered directly into the tumor, or near the tumor site,
via injection or a
catheter. Of course, as the artisan of ordinary skill will appreciate, the
appropriate mode of
administration will vary according to the cancer to be treated. Other modes of
delivery are
discussed herein.
[0211] For example, within one aspect of the present invention methods are
provided
for treating hypertrophic scars and keloids, comprising the step of
administering a CTGF-2
antagonist of the invention to a hypertrophic scar or keloid. Within one
embodiment of the
present invention antagonists are directly injected into a hypertrophic scar
or keloid, in order
to prevent the progression of these lesions. This therapy is of particular
value in the
prophylactic treatment of conditions which are known to result in the
development of
hypertrophic scars and keloids (e.g., burns), and is preferably initiated
after the proliferative
phase has had time to progress (approximately 14 days after the initial
injury), but before
hypertrophic scar or keloid development.
[0212] The present invention also provides methods for treating neovascular
diseases
of the eye, including for example, corneal neovascularization, neovascular
glaucoma,
proliferative diabetic retinopathy, retrolental fibroplasia and macular
degeneration.
[0213] Moreover, ocular disorders associated with neovascularization which can
be
treated with the polynucleotides and polypeptides of the present invention
(including agonists
and/or antagonists) include, but are not limited to: neovascular glaucoma,
diabetic
retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of
prematurity
macular degeneration, corneal graft neovascularization, as well as other eye
inflammatory
diseases, ocular tumors and diseases associated with choroidal or iris
neovascularization. See,
e.g., reviews by Waltman et al., Ana. J. Ophthal. 85:704-710 (1978) and
Gartner et al., Surv.
OphtlZal. 22:291-312 (1978).
[0214] Thus, within one aspect of the present invention methods are provided
for
treating neovascular diseases of the eye such as corneal neovascularization
(including corneal
graft neovascularization), comprising the step of administering to a patient a
therapeutically
effective amount of a CTGF-2 antagonist to the cornea, such that the formation
of blood
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vessels is inhibited. Briefly, the cornea is a tissue which normally lacks
blood vessels. In
certain pathological conditions however, capillaries may extend into the
cornea from the
pericorneal vascular plexus of the limbus. When the cornea becomes
vascularized, it also
becomes clouded, resulting in a decline in the patient's visual acuity. Visual
loss may become
complete if the cornea completely opacitates. A wide variety of disorders can
result in
corneal neovascularization, including fox example, corneal infections (e.g.,
trachoma, herpes
simplex keratitis, leishmaniasis and onchocerciasis), immunological processes
(e.g., graft
rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation
(of any
cause), toxic and nutritional deficiency states, arid as a complication of
wearing contact
lenses.
[0215] Within particularly preferred embodiments of the invention, may be
prepared
for topical administration in saline (combined with any of the preservatives
and ahtimicrobial
agents commonly used in ocular preparations), and administered in eyedrop
form. The
solution or suspension may be prepared in its pure form and administered
several times daily.
Alternatively, anti-angiogenic composition, prepared as described above, may
also be
administered directly to the cornea. Within preferred embodiments, the anti-
angiogenic
composition is prepared with a muco-adhesive polymer which binds to cornea.
Within
further embodiments, the anti-angiogenic factors or anti-angiogenic
compositions may be
utilized as an adjunct to conventional steroid therapy. Topical therapy may
also be useful
prophylactically in corneal lesions which are known to have a high probability
of inducing an
angiogenic response (such as chemical burns). In these instances the
treatment, likely in
combination with steroids, may be instituted immediately to help prevent
subsequent
complications.
[0216] Within other embodiments, the compounds described above may be injected
directly into the corneal stroma by an ophthalmologist under microscopic
guidance. The
preferred site of injection may vary with the morphology of the individual
lesion, but the goal
of the administration would be to place the composition at the advancing front
of the
vasculature (i.e., interspersed between the blood vessels and the normal
cornea). In most
cases this would involve perilimbic corneal injection to "protect" the cornea
from the
advancing blood vessels. This method may also be utilized shortly after a
corneal insult in
order to prophylactically prevent corneal neovascularization. In this
situation the material
could be injected in the perilimbic cornea interspersed between the corneal
lesion and its
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undesired potential limbic blood supply. Such methods may also be utilized in
a similar
fashion to prevent capillary invasion of transplanted corneas. In a sustained-
release form
injections might only be required 2-3 times per year. A steroid could also be
added to the
injection solution to reduce inflammation resulting from the injection itself.
[0217] Within another aspect of the present invention, methods are provided
for
treating neovascular glaucoma, comprising the step of administering to a
patient a
therapeutically effective amount of a CTGF-2 antagonists to the eye, such that
the formation
of blood vessels is inhibited. In one embodiment, the compound may be
administered
topically to the eye in order to treat early forms of neovascular glaucoma.
Within other
embodiments, the compound may be implanted by injection into the region of the
anterior
chamber angle. Within other embodiments, the compound may also be placed in
any location
such that the compound is continuously released into the aqueous humor. Within
another
aspect of the present invention, methods are provided for treating
proliferative diabetic
retinopathy, comprising the step of administering t~ a patient a
therapeutically effective
amount of a polynucleotide, polypeptide, antagonist and/or agonist to the
eyes, such that the
formation of blood vessels is inhibited.
[0218] Within particularly preferred embodiments of the invention,
proliferative
diabetic retinopathy may be treated by injection into the aqueous humor or the
vitreous, in
order to increase the local concentration of the CTGF-2 antagonist in the
retina. Preferably,
this treatment should be initiated prior to the acquisition of severe disease
requiring
photocoagulation.
[0219] Within another aspect of the present invention, methods are provided
for
treating retrolental fibroplasia, comprising the step of administering to a
patient a
therapeutically effective amount of a CTGF-2 antagonist to the eye, such that
the formation of
blood vessels is inhibited. The compound may be administered topically, via
intravitreous
injection and/or via intraocular implants.
[0220] Additionally, disorders which can be treated with CTGF-2 antagonists
include,
but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma,
atherosclerotic plaques,
delayed wound healing, granulations, hemophilic joints, hypertrophic scars,
nonunion
fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma,
and vascular
adhesions.
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[0221] Moreover, disorders and/or states, which can be treated with be treated
with
the the CTGF-2 antagonists include, but are not limited to, solid tumors,
blood born tumors
such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for
example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic
granulomas,
rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example,
diabetic retinopathy,
retinopathy of prematurity, macular degeneration, corneal graft rejection,
neovascular
glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis,
delayed wound
healing, endometriosis, vascluogenesis, granulations, hypertrophic scars
(keloids), nonunion
fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis,
coronary
collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb
angiogenesis,
Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac
joints,
angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease,
atherosclerosis,
birth control agent by preventing vascularization required for embryo
implantation controlling.
menstruation, diseases that have angiogenesis as a pathologic consequence such
as cat .scratch
disease (Rochele minalia quintosa), ulcers (Helicobacter pylori),
Bartonellosis and bacillary
angiomatosis.
[0222] In one aspect of the birth control method, an amount of the compound
sufficient to block embryo implantation is administered before or after
intercourse and
fertilization have occurred, thus providing an effective method of birth
control, possibly a
"morning after" method. CTGF-2 antagonists may also be used in controlling
menstruation or
administered as either a peritoneal lavage fluid or for peritoneal
implantation in the treatment
of endometriosis.
(0223] CTGF-2 antagonists of the present invention may be incorporated into
surgical
sutures in order to prevent stitch granulomas.
[0224] CTGF-2 antagonists may be utilized in a wide variety of surgical
procedures.
For example, within one aspect of the present invention a compositions (in the
form of, for
example, a spray or film) may be utilized to coat or spray an area prior to
removal of a tumor,
in order to isolate normal surrounding tissues from malignant tissue, and/or
to prevent the
spread of disease to surrounding tissues. Within other aspects of the present
invention,
compositions (e.g., in the form of a spray) may be delivered via endoscopic
procedures in
order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet
other aspects of
the present invention, surgical meshes which have been coated with anti-
angiogenic
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compositions of the present invention may be utilized in any procedure wherein
a surgical
mesh might be utilized. For example, within one embodiment of the invention a
surgical
mesh laden with an anti-angiogenic composition may be utilized during
abdominal cancer
resection surgery (e.g., subsequent to colon resection) in order to provide
support to the
structure, and to release an amount of the anti-angiogenic factor.
[0225] Within further aspects of the present invention, methods are provided
for
treating tumor excision sites, comprising administering a CTGF-2 antagonists
to the resection
margins of a tumor subsequent to excision, such that the local recurrence of
cancer and the
formation of new blood vessels at the site is inhibited. Within one embodiment
of the
invention, the anti-angiogenic compound is administered directly to the tumor
excision site
(e.g., applied by swabbing, brushing or otherwise coating the resection
margins of the tumor
with the anti-angiogenic compound). Alternatively, the anti-angiogenic
compounds may be
incorporated into known surgical pastes prior to administration. Within
particularly preferred
embodiments of the invention, the anti-angiogenic compounds are applied after
hepatic .
resections for malignancy, and after neurosurgical operations.
[0226] Within one aspect of the present invention CTGF-2 antagonists may be
administered to the resection margin of a wide variety of tumors, including
for example,
breast, colon, brain and hepatic tumors. For example, within one embodiment of
the
invention, anti-angiogenic compounds may be administered to the site of a
neurological
tumor subsequent to excision, such that the formation of new blood vessels at
the site are
inhibited.
[0227] The CTGF-2 antagonists of the present invention may also be
administered
along with other anti-angiogenic factors. Representative examples of other
anti-angiogenic
factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof,
paclitaxel,
Suramin, Tissue Inhibitor of Metalloproteinase-l, Tissue Inhibitor of
Metalloproteinase-2,
Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and
various forms of
the lighter "d group" transition metals.
[0228] Lighter "d group" transition metals include, for example, vanadium,
molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition
metal
species may form transition metal complexes. Suitable complexes of the above-
mentioned
transition metal species include oxo transition metal complexes.
CA 02412124 2002-12-13
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[0229] Representative examples of vanadium complexes include oxo vanadium
complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes
include
metavanadate and orthovanadate complexes such as, for example, ammonium
metavanadate,
sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes
include, for
example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate
hydrates such
as vanadyl sulfate mono- and trihydrates.
[0230] Representative examples of tungsten and molybdenum complexes also
include
oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten
oxide
complexes. Suitable tungstate complexes include ammonium tungstate, calcium
tungstate,
sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten (IV)
oxide and tungsten (VI] oxide. Suitable oxo molybdenum complexes include
molybdate,
molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes
include
ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium
molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VT)
oxide,
molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes
include, for
example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes
include hydroxo derivatives derived from, for example, glycerol, tartaric
acid, and sugars.
(0231] A wide variety of other anti-angiogenic factors may also be utilized
within the
context of the present invention. Representative examples include platelet
factor 4;
protamine sulphate; sulphated chitin derivatives (prepared from queen crab
shells), (Murata et
al., Cancer Res. S 1:22-26, 1991); Sulphated Polysaccharide Peptidoglycan
Complex (SP- PG)
(the function of this compound may be enhanced by the presence of steroids
such as estrogen,
and tamoxifen citrate); Staurosporine; modulators of matrix metabolism,
including. for
example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline,
Thiaproline,
alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-S-(4-pyridinyl)-
2(3H)-
oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-
serum;
ChIMP-3 (Pavloff et aL, J. Bio. Chem. 267:17321-17326, 1992); Chymostatin
(Tomkinson et
al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin;
Camptothecin; Fumagillin (Ingber et al., Nature 348:SSS-SS7, 1990); Gold
Sodium
Thiomalate ("GST"; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987);
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem.
262(4):1659-1664,
1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-
carboxyphenyl-4-
7I
CA 02412124 2002-12-13
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chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions
36:312-316,
1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and
metalloproteinase inhibitors such as BB94.
Antag~ni its
[0232] The present invention is also directed to antagonists molecules of the
polypeptides of the present invention, and their use in reducing or
eliminating the function of
CTGF-2.
(0233] An example of an antagonist is an antibody or in some cases, an
oligonucleotide, which binds to the CTGF-2 polypeptide. Alternatively,
antagonists include
closely related proteins that have lost biological function and thereby
prevent the action of
CTGF-2 since receptor sites are occupied.
(0234] Antisense technology may be employed to decrease the level of i~ vivo
circulation of CTGF-2. Antisense technology can be used to control gene
expression through
triple-helix formation or antisense DNA or RNA, both of which methods are
based on
binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion
of the
polynucleotide sequence, which encodes for the mature polypeptides of the
present invention,
is used to design an antisense RNA oligonucleotide of from about 10 to 40 base
pairs in
length. A DNA oligonucleotide is designed to be complementary to a region of
the gene
involved in transcription (triple helix -see Lee et al., Nucl. Acids Res.,
6:3073 (1979); Cooney
et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),
thereby
preventing transcription and the production of CTGF-2. The antisense RNA
oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule
into CTGF-2
(antisense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as
Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The
oligonucleotides
described above can also be delivered to cells such that the antisense RNA or
DNA may be
expressed if2 vivo to inhibit production of CTGF-2.
[0235] Another example of an antagonist is a small molecule which binds to the
CTGF-2 receptors such that normal biological activity is prevented. Examples
of small
molecules include but are nat limited to small peptides or peptide-like
molecules.
[0236] The antagonists may be employed to prevent scar formation due to excess
proliferation of connective tissues and to prevent CTGF-2 dependent tumor
growth. The
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WO 02/04480 PCT/USO1/21799
antagonists may be employed in a composition with a pharmaceutically
acceptable carrier,
e.g., as hereinabove described.
F~rmulatiens and Administration
[0237] The polypeptides and antagonists and agonists of the present invention
may be
employed in combination with a suitable pharmaceutical carrier. Such
compositions
comprise a therapeutically effective amount of the polypeptide, agonist or
antagonist, and a
pharmaceutically acceptable carrier or excipient. Such a carrier includes but
is not limited to
saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations
thereof. The
formulation should suit the mode of administration.
[0238]. 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 containers) 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, agonists and antagonists of the
present
invention may be employed in conjunction with other therapeutic compounds.
[0239] The pharmaceutical compositions may be administered in a convenient
manner
such as by the topical, intravenous, intraperitoneal, intramuscular,
subcutaneous, intranasal or
intradermal routes. CTGF-2 is administered in an amount which is effective for
treating
and/or prophylaxis of the specific indication. In general, CTGF-2 will be
administered in an
amount of at least about 10 Fg/kg body weight and in most cases CTGF-2 will be
administered in an amount not in excess of about 8 mg/Kg body weight per day.
In most
cases, the dosage is from about 10 Fglkg to about 1 mg/kg body weight daily,
taking into
account the routes of administration, symptoms, etc.
[0240] The polypeptides may also be employed in accordance with the present
invention by expression of such polypeptides in vivo, which is often referred
to as "gene
therapy."
[0241] Thus, for example, cells from a patient may be engineered with a
polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the
engineered cells then
being provided to a patient to be treated with the polypeptide. Such methods
are well-known
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CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
in the art. For example, cells may be engineered by procedures known in the
art by use of a
retroviral particle containing RNA encoding a polypeptide of the present
invention.
[0242] Similarly, cells may be engineered ira vivo for expression of a
polypeptide in
vivo by, for example, procedures known in the art. As known in the art, a
producer cell for
producing a retroviral particle containing RNA encoding the polypeptide of the
present
invention may be administered to a patient for engineering cells in vivo and
expression of the
polypeptide irt vivo. These and other methods for administering a polypeptide
of the present
invention by such method should be apparent to those skilled in the art from
the teachings of
the present invention. For example, the expression vehicle for engineering
cells may be other
than a retrovirus, for example, an adenovirus which may be used to engineer
cells in vivo
after combination with a suitable delivery vehicle.
[0243] Retroviruses from which the retroviral plasmid vectors hereinabove
mentioned
may be derived include, but are not limited to, Moloney Murine Leukemia Virus,
spleen
necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,
avian
leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus,
adenovirus,
Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment,
the
retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
[0244) The vector includes one or more promoters. Suitable promoters which may
be
employed include, but are not limited to, the retroviral LTR; the SV40
promoter; and the
human cytomegalovirus (CMV) promoter described in Miller, et al.,
Ri~technianes, Vol. 7,
No. 9,. 980-990 (1989), or any other promoter (e.g., cellular promoters such
as eukaryotic
cellular promoters including, but not limited to, the histone, pol III, and a-
actin promoters).
Other viral promoters which may be employed include, but are not limited to,
adenovirus
promoters, thymidine kinase (TK) promoters, and B 19 parvovirus promoters. The
selection
of a suitable promoter will be apparent to those skilled in the art from the
teachings contained
herein.
[0245) The nucleic acid sequence encoding the polypeptide of the present
invention is
under the control of a suitable promoter. Suitable promoters which may be
employed include,
but are not limited to, adenoviral promoters, such as the adenoviral major
late promoter; or
hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the
respiratory
syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter,
the
metallothionein promoter; heat shock promoters; the albumin promoter; the
ApoAI promoter;
74
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
human globin promoters; viral thymidine kinase promoters, such as the Herpes
Simplex
thymidine kinase promoter; retroviral LTRs (including the modified retroviral
LTRs
hereinabove described); the a-actin promoter; and human growth hormone
promoters. The
promoter also may be the native promoter which controls the gene encoding the
polypeptide.
The retroviral plasmid vector is employed to transduce packaging cell lines to
form producer
cell lines. Examples of packaging cells which may be transfected include, but
are not limited
to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-1T-H2, oCRE, oCRIP, GP+E-
86,
GP+envAml2, and DAN cell lines as described in Miller, unman (T n . T_h
.~rany, Vol. 1, pgs.
S-14 (1990), which is incorporated herein by reference in its entirety. The
vector may
transduce the packaging cells through any means known in the art. Such means
include, but
are not limited to, electroporation, the use of liposomes, and CaP04
precipitation. In one
alternative, the retroviral plasmid vector may be encapsulated into a
liposome, or coupled to a
lipid, and then administered to a host.
[0246] The producer cell line generates infectious retroviral vector particles
which
include the nucleic acid sequences) encoding the polypeptides. Such retroviral
vector
particles then may be employed, to transduce eukaryotic cells, either ih vitro
or in vivo. The
transduced eukaryotic cells will express the nucleic acid sequences) encoding
the
polypeptide. Eukaryotic cells which may be transduced include, but are not
limited to,
embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem
cells,
hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and
bronchial epithelial
cells.
[0247] This invention is also related to the use of the gene of the present
invention as
a diagnostic. Detection of a mutated form of the gene will allow a diagnosis
of a disease or a
susceptibility to a disease which results from underexpression of CTGF-2.
[0248] Individuals carrying mutations in the gene of the present invention may
be
detected at the DNA level by a variety of techniques. Nucleic acids for
diagnosis may be
obtained from a patient's cells, including but not limited to blood, urine,
saliva, tissue biopsy
and autopsy material. The genomic DNA may be used directly for detection or
may be
amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166
(1986)) prior to
analysis. RNA or cDNA may also be used for the same purpose. As an example,
PCR
primers complementary to the nucleic acid encoding CTGF-2 can be used to
identify and
analyze mutations. For example, deletions and insertions can be detected by a
change in size
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
of the amplified product in comparison to the normal genotype. Point mutations
can be
identified by hybridizing amplified DNA to radiolabeled RNA or alternatively,
radiolabeled
antisense DNA sequences. Perfectly matched sequences can be distinguished from
mismatched duplexes by RNase A digestion or by differences in melting
temperatures.
[0249] Sequence differences between the reference gene and genes having
mutations
may be revealed by the direct DNA sequencing method. In addition, cloned DNA
segments
may be employed as probes to detect specific DNA segments. The sensitivity of
this method
is greatly enhanced when combined with PCR. For example, a sequencing primer
is used
with double-stranded PCR product or a single-stranded template molecule
generated by a
modified PCR. The sequence determination is performed by conventional
procedures with
radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-
tags.
[0250] Genetic testing based on DNA sequence differences may be achieved by
detection of alteration in electrophoretic mobility of DNA fragments in gels
with or without
denaturing agents. Small sequence deletions and insertions can be visualized
by high
resolution gel electrophoresis. DNA fragments of different sequences may be
distinguished
on denaturing formamide gradient gels in which the mobilities of different DNA
fragments
are retarded in the gel at different positions according to their specific
melting or partial
melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)).
[0251] Sequence changes at specific locations may also be revealed by nuclease
protection assays, such as RNase and S1 protection or the chemical cleavage
method (e.g:,
Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
[0252] Thus, the detection of a specific DNA sequence may be achieved by
methods
such as hybridization, RNase protection, chemical cleavage, direct DNA
sequencing or the
use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms
(RFLP)) and
Southern blotting of genomic DNA.
(0253] In addition to more conventional gel-electrophoresis and DNA
sequencing,
mutations can also be detected by in situ analysis.
[0254] The present invention also relates to a diagnostic assay for detecting
altered
levels of the polypeptide of the present invention in various tissues since an
over-expression
of the proteins compared to normal contxol tissue samples can detect the
presence of disorders
of the host. Assays used to detect levels of the polypeptide of the present
invention in a
sample derived from a host are well-known to those of skill in the art and
include
76
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
radioimmunoassays, competitive-binding assays, Western Blot analysis and
preferably an
ELISA assay. An ELISA assay initially comprises preparing an antibody specific
to the
CTGF-2 antigen, preferably a monoclonal antibody. In addition a reporter
antibody is
prepared against the monoclonal antibody. To the reporter antibody is attached
a detectable
reagent such as radioactivity, fluorescence or in this example a horseradish
peroxidase
enzyme. A sample is now removed from a host and incubated on a solid support,
e.g. a
polystyrene dish, that binds the proteins in the sample. Any free protein
binding sites on the
dish are then covered by incubating with a non-specific protein such as bovine
serum
albumin. Next, the monoclonal antibody is incubated in the dish during which
time the
monoclonal antibodies attached to the polypeptide of the present invention
attached to the
polystyrene dish. All unbound monoclonal antibody is washed out with buffer.
The reporter
antibody linked to horseradish peroxidase is now placed in the dish resulting
in binding of the
reporter antibody to any monoclonal antibody bound to the polypeptide of the
present
invention. Unattached reporter antibody is then washed out. Peroxidase
substrates are then
added to the dish and the amount of color developed in a given time period is
a measurement
of the amount of the polypeptide of the present invention present in a given
volume of patient
sample when compared against a standard curve.
[0255] A competition assay may be employed wherein antibodies specific to the
polypeptide of the .present invention are attached to a solid support and
labeled CTGF-2 and a
sample derived from the host are passed over the solid support and the amount
of label
detected attached to the solid support can be correlated to a quantity of the
polypeptide of the
present invention in the sample.
(0256] The sequences 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.
[0257] Briefly, 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
77
CA 02412124 2002-12-13
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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
fragment.
[0258] 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
fragments 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.
[0259] Fluorescence ih 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 cDNA having at least 50 or 60 bases. For a review
of this
technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques,
Pergamon Press, New York (1988).
[0260] 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).
[0261] 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.
[0262] With current resolution of physical mapping and genetic mapping
techniques,
a cDNA precisely localized to a chromosomal region associated with the disease
could be one
of between 50 and 500 potential causative genes. (This assumes 1 megabase
mapping
resolution and one gene per 20 kb).
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WO 02/04480 PCT/USO1/21799
[0263] The polypeptides, their fragments or other derivatives, or analogs
thereof, or
cells expressing them can be used as an immunogen to produce antibodies
thereto. These
antibodies can be, for example, polyclonal or monoclonal antibodies. The
present invention
also includes chimeric, single chain, and humanized antibodies, as well as Fab
fragments, or
the product of an Fab expression library. Various procedures known in the art
rnay be used
for the production of such antibodies and fragments.
[0264] Antibodies generated against the polypeptides corresponding to a
sequence of
the present invention can be obtained by direct injection of the polypeptides
into an animal or
by administering the polypeptides to an animal, preferably a nonhuman. The
antibody so
obtained will then bind the polypeptides itself. In this manner, even a
sequence encoding
only a fragment of the polypeptides can be used to generate antibodies binding
the whole
native polypeptides. Such antibodies can then be used to isolate the
polypeptide from tissue
expressing that polypeptide.
[0265] For preparation of monoclonal antibodies, any technique which provides
antibodies produced by continuous cell line cultures can be used. Examples
include the
hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the
trioma
technique, the human B-cell hybridoma technique (Kozbor et al., 1983,
Immunology Today
4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies
(Cole, et
al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,
pp. 77-96).
[0266] Techniques described for the production of single chain antibodies
(i~.S.
Patent 4,946,778) can be adapted to produce single chain antibodies to
irnmunogenic
polypeptide products of this invention. Also, transgenic mice may be used to
express
humanized antibodies to immunogenic polypeptide products of this invention.
[0267] The present invention will be further described with reference to the
following
examples; however, it is to be understood that the present invention is not
limited to such
examples. All parts or amounts, unless otherwise specified, are by weight.
[0268] In order to facilitate understanding of the following examples certain
frequently occurring methods and/or terms will be described.
[0269] "Plasmids" are designated by a lower case p preceded and/or followed by
capital letters and/or numbers. The starting plasmids herein are either
commercially
available, publicly available on an unrestricted basis, or can be constructed
from available
79
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
plasmids in accord with published procedures. In addition, equivalent plasmids
to those
described are known in the art and will be apparent to the ordinarily skilled
artisan.
[0270] "Digestion" of DNA refers to catalytic cleavage of the DNA with a
restriction
enzyme that acts only at certain sequences in the DNA. The various restriction
enzymes used
herein are commercially available and their reaction conditions, cofactors and
other
requirements were used as would be known to the ordinarily skilled artisan.
For analytical
purposes, typically 1 p,g of plasmid or DNA fragment is used with about 2
units of enzyme in
about 20 ~,l of buffer solution. For the purpose of isolating DNA fragments
for plasmid
construction, typically 5 to 50 ~,ig of DNA are digested with 20 to 250 units
of enzyme in a
larger volume. Appropriate buffers and substrate amounts for particular
restriction enzymes
are specified by the manufacturer. Incubation times of about 1 hour at 37EC
are ordinarily
used, but may vary in accordance with the supplier's instructions. After
digestion the reaction
is electrophoresed directly on a polyacrylamide gel to isolate the desired
fragment.
[0271] Size separation of the cleaved fragments is performed using 8 percent
polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057
(1980).
[0272] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or
two complementary polydeoxynucleotide strands which may be chemically
synthesized.
Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate
to another
oligonucleotide without adding a phosphate with an ATP in the presence of a
kinase. A
synthetic oligonucleotide will ligate to a fragment that has not been
dephosphorylated.
[0273] "Ligation" refers to the process of forming phosphodiester bonds
between two
double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146).
Unless otherwise
provided, ligation may be accomplished using known buffers and conditions with
10 units to
T4 DNA ligase ("ligase") per 0.5 p,g of approximately equimolar amounts of the
DNA
fragments to be ligated.
[0274] Unless otherwise stated, transformation was performed as described in
the
method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).
Fxamnle 1: C'hning and expression of C'.TCF-2 in a hacLlnvirns .xnrecsion
c~rstem
[0275] The DNA sequence encoding the full length CTGF-2 protein, ATCC
accession
no. 75804, was amplified using PCR oligonucleotide primers corresponding to
the 5' and 3'
sequences of the gene:
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
[0276] The 5' primer has the sequence
CGCGGGATCCTGCGCGACACAATGAGCT (SEQ ID NO: 3) and contains a BamHI
restriction enzyme site (in bold) followed by 18 nucleotides resembling an
efficient signal for
the initiation of translation in eukaryotic cells (J. Mol. Biol. 1987,19f, 947-
950, Kozak, M.).
The initiation codon for translation "ATG" is underlined.
[0277] The ~ 3' primer has the sequence
CGCGGGTACCAGGTAGCATTTAGTCCCTAA (SEQ ID N0:8) and contains the
cleavage site for the restriction endonuclease Asp781 and 20 nucleotides
complementary to
the 3' non-translated sequence of the CTGF-2 gene. The amplified sequences
were isolated
from a 1% agarose gel using a commercially available kit ("Geneclean", BIO 101
Inc., La
Jolla, Ca.). The fragment was then digested with the endonucleases BamHI and
Asp781 and
then purified by isolation on a 1% agarose gel. This fragment is designated
F2.
[0278] The vector pRGl (modification of pVL941 vector, discussed below) is
used
for the expression of the CTGF-2 protein using the baculovirus expression
system (for review
see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus
vectors and
insect cell culture procedures, Texas Agricultural Experimental Station
Bulletin No. 1555).
This expression vector contains the strong polyhedrin promoter of the
Autographa californica
nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the
restriction
endonucleases BamHI and Asp781. The polyadenylation site of the simian virus
(SV)40 is
used for efficient polyadenylation. For an easy selection of recombinant
viruses the beta-
galactosidase gene from E.coli is inserted in the same orientation as the
polyhedrin promoter
followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin
sequences are
flanked at both sides by viral sequences for the cell-mediated homologous
recombination of
cotransfected wild-type viral DNA. Many other baculovirus vectors could be
used in place of
pRGl such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M.D.,
Virology,
170:31-39).
[0279] The plasmid was digested with the restriction enzymes BamHI and Asp781
and then dephosphorylated using calf intestinal phosphatase by procedures
known in the art.
The DNA was then isolated from a 1% agarose gel. This vector DNA is designated
V2.
[0280] Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4
DNA
ligase. E.coli HB101 cells were then transformed and. bacteria identified that
contained the
81
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
plasmid (pBacCTGF-2) with the CTGF-2 gene using the enzymes BamHI and Asp781.
The
sequence of the cloned fragment was confirmed by DNA sequencing.
[0281] 5 ~,g of the plasmid pBacCTGF-2 were cotransfected with 1.0 ~,g of a
commercially available linearized baculovirus ("BaculoGold baculovirus DNA",
Pharmingen,
San Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl. Acad.
Sci. LISA,
84:7413-7417 (1987)).
[0282] 1 ~cg of BaculoGold virus DNA and 5 ~.g of the plasmid pBacCTGF-2 were
mixed in a sterile well of a microtiter plate containing 50 p,1 of serum free
Grace's medium
(Life Teclmologies Inc., Gaithersburg, MD). Afterwards 10 ~,l Lipofectin plus
90 ~,1 Grace's
medium were added, mixed and incubated for 15 minutes at room temperature.
Then the
transfection mixture was added dropwise to the Sf~ insect cells (ATCC CRL
1711) seeded in
a 35 mm tissue culture plate with lml Grace' medium without serum. The plate
was rocked
back and forth to mix the newly added solution. The plate was then incubated
for 5 hours at
27EC. After 5 hours the transfection solution was removed from the plate and 1
ml of
Grace's insect medium supplemented with 10% fetal calf serum was added. The
plate was
put back into an incubator and cultivation continued at 27°C for four
days.
[0283] After four days the supernatant was collected and a plaque assay
performed
similar as described by Summers and Smith (supra). As a modification an
agarose gel with
"Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows an
easy isolation of
blue stained plaques. (A detailed description of a "plaque assay" can also be
found in the
user's guide for insect cell culture and baculovirology distributed by Life
Technologies Inc.,
Gaithersburg, page 9-10).
[0284] Four days after the serial dilution of the viruses was added to the
cells, blue
stained plaques were picked with the tip of an Eppendorf pipette. The agar
containing the
recombinant viruses was then resuspended in an Eppendorf tube containing 200
p,1 of Grace's
medium. The agar was removed by a brief centrifugation and the supernatant
containing the
recombinant baculoviruses was used to insect S~ cells seeded in 35 mm dishes.
Four days
later the supernatants of these culture dishes were harvested and then stored
at 4°C.
[0285] S~ cells were grown in Grace's medium supplemented with 10% heat-
inactivated FBS. The cells were infected with the recombinant baculovirus V-
CTGF-2 at a
multiplicity of infection (M01) of 2. Six hours later the medium was removed
and replaced
with SF900 II medium minus methionine and cysteine (Life Technologies Inc.,
Gaithersburg).
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CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
42 hours later 5 ~,Ci of 3$S-methionine and 5 ~,Ci 35S cysteine (Amersham)
were added. The
cells were further incubated for 16 hours before they were harvested by
centrifugation and the
labelled proteins visualized by SDS-PAGE and autoradiography.
Fxamnle 2: Fx~re~sinn of Recombinant C.'T(iF-2 in COS cells
[0286] The expression of plasmid, CTGF-2 HA is derived from a vector
pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)
ampicillin resistance
gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker
region, a SV40
intron and polyadenylation site. A DNA fragment encoding the entire CTGF-2
precursor and
a HA tag fused in frame to its 3' end was cloned into the polylinker region of
the vector,
therefore, the recombinant protein expression is directed under the CMV
promoter. The HA
tag correspond to an epitope derived from the influenza hemagglutinin protein
as previously
described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.
Lerner,
1984, Cell 37, 767). The infusion of HA tag to our target protein allows easy
detection of the
recombinant protein with an antibody that recognizes the HA epitope.
[0287] The plasmid construction strategy is described as follows:
[0288] The DNA sequence encoding for CTGF-2, ATCC accession no. 75804, was
constructed by PCR on the full-length clone using two primers: the 5' primer
5'
AAAGGATCCACAATGAGCTCCCGAATC 3' (SEQ ID NO: 4) contains a Bam HI site
followed by l 8 nucleotides of CTGF-2 coding sequence starting from the -3
position relative
to the initiation codon; the 3' sequence 5'
CGCTCTAGATTAAGCGTAGTCTGGGACGTCGTATGGGTATTGGAACAGCCTGTAG
AAG 5' (SEQ ID NO: 5) contains complementary sequences to an Xba I site,
translation stop
codon, HA tag and the last 19 nucleotides of the CTGF-2 coding sequence (not
including the
stop codon). Therefore, the PCR product contains a Bam HI site, CTGF-2 coding
sequence
followed by an HA tag fused in frame, a translation termination stop codon
next to the HA
tag, and an Xba I site. The PCR amplified DNA fragment and the vector,
pcDNAI/Amp,
were digested with Bam HI and Xba I restriction enzymes and ligated. The
ligation mixture
was transformed into E. coli strain SURE (available from Stratagene Cloning
Systems, 11099
North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was
plated on
ampicillin media plates and resistant colonies were selected. Plasmid DNA was
isolated from
transformants and examined by restriction analysis for the presence of the
correct fragment.
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WO 02/04480 PCT/USO1/21799
For expression of the recombinant CTGF-2, COS cells were transfected with the
expression
vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis,
Molecular
Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The
expression of
the CTGF-2 HA protein was detected by radiolabelling and immunoprecipitation
method. (E.
Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press,
(1988)). Cells were labelled for 8 hours with 35S-cysteine two days post
transfection. Culture
media were then collected and cells were lysed with detergent (RIl'A buffer
(150 mM NaCl,
1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, SOmM Tris, pH 7.5). (Wilson, I. et
al., Id.
37:767 (1984)). Both cell lysate and culture media were precipitated with a HA
specific
monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.
Fxam,~~le 3: Fxnrescinn via Gene Theran~
[0289] Fibroblasts are obtained from a subject by skin biopsy. The resulting
tissue is
placed in tissue-culture medium and separated into small pieces. Small chunks
of the tissue
are placed on a wet surface of a tissue culture flask, approximately ten
pieces are placed in
each flask. The flask is turned upside down, closed tight and left at room
temperature over
night. After 24 hours at room temperature, the flask is inverted and the
chunks of tissue
remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12
media, with 10%
FBS, penicillin and streptomycin, is added. This is then incubated at
37°C for approximately
one week. At this time, fresh media is added and subsequently changed every
several days.
After an additional two weeks in culture, a monolayer of fibroblasts emerge.
The monolayer
is trypsinized and scaled into larger flasks.
[0290] pMV-7 (I~irschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the
long
terminal repeats of the Moloney marine sarcoma virus, is digested with EcoRI
and HindIll
and subsequently treated with calf intestinal phosphatase. The linear vector
is fractionated on
agarose gel and purified, using glass beads.
[0291] The cDNA encoding a polypeptide of the present invention is amplified
using
PCR primers which correspond to the 5' and 3' end sequences respectively. The
5' primer
containing an EcoRI site and the 3' primer further includes a HindIll site.
Equal quantities of
the Moloney marine sarcoma virus linear backbone and the amplified EcoRI and
HindllI
fragment are added together, in the presence of T4 DNA ligase. The resulting
mixture is
maintained under conditions appropriate for ligation of the two fragments. The
ligation
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mixture is used to transform bacteria HB101, which are then plated onto agar-
containing
kanamycin for the purpose of confirming that the vector had the gene of
interest properly
inserted.
[0292] The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue
culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with
10% calf
serum (CS), penicillin and streptomycin. The MSV vector containing the gene is
then added
to the media and the packaging cells are transduced with the vector. The
packaging cells now
produce infectious viral particles containing the gene (the packaging cells
are now referred to
as producer cells).
[0293] Fresh media is added to the transduced producer cells, and
subsequently, the
media is harvested from a 10 cm plate of confluent producer cells. The spent
media,
containing the infectious viral particles, is filtered through a millipore
filter to remove
detached producer cells and this media is then used to infect fibroblast
cells. Media is
removed from a sub-confluent plate of fibroblasts and quickly replaced with
the media from
the producer cells. This media is removed and replaced with fresh media. If
the titer of virus
is high, then virtually all fibroblasts will be infected and no selection is
required. If the titer is
very low, then it is necessary to use a retroviral vector that has a
selectable marker, such as
n~ or his.
[0294] The engineered fibroblasts are then injected into the host, either
alone or after
having been grown to confluence on cytodex 3 microcarrier beads. The
fibroblasts now
produce the protein product.
Fix m_nle 4: Effect of C.'TGF-2 on Card Forma .ion in An,giogenesiC
[0295] A step in angiogenesis is cord formation, marked by differentiation of
endothelial cells. This bioassay measures the ability of microvascular
endothelial cells to
form capillary-like structures (hollow structures) when cultured in vitro.
[0296] CADMEC (microvascular endothelial cells) are purchased from Cell
Applications, Inc. as proliferating (passage 2) cells and are cultured in Cell
Applications'
CADMEC Growth Medium and used at passage 5. For the in vitro angiogenesis
assay, the
wells of a 48-well cell culture plate are coated with Cell Applications'
Attachment Factor
Medium (200 microliter/well) for 30 min. at 37°C. CADMEC are seeded
onto the coated
wells at 7,500 cells/well and cultured overnight in Growth Medium. The Growth
Medium is
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then replaced with 300 micrograms Cell Applications' Chord Formation Medium
containing
control buffer or CTGF-2 (0.1 to 100 ng/ml) and the cells are cultured for an
additional 48 hr.
The numbers and lengths of the capillary-like chords are quantitated through
use of the
Boeckeler VIA-170 video image analyzer. All assays are done in triplicate.
[0297] Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.
beta-esteradiol (1 ng/ml) is used as a negative control. The appropriate
buffer (without
protein) is also utilized as a control.
[0298] The studies described in this example test the activity in CTGF-2
protein.
However, one skilled in the art could easily modify the exemplified studies to
test the activity
of CTGF-2 polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of CTGF-2.
Exam 1e _ : An,gi_~gPnic Effect ~n C,'hick C.'horioallantoic Membrane
[0299] Chick chorioallantoic membrane (CAM) is a well-established system to
examine angiogenesis. Blood vessel formation on CAM is easily visible and
quantifiable.
The ability of CTGF-2 to stimulate angiogenesis in CAM can be examined.
[0300] Fertilized eggs of the White Leghorn chick (callus gczllus) and the
Japanese
quail (Coturnix coturfaix) are incubated at 37.8°C and 80% humidity.
Differentiated CAM of
16-day-old chick and 13-day-old quail embryos is studied with the following
methods.
[0301] On Day 4 of development, a window is made into the egg shell of chick
eggs.
The embryos are checked for normal development and the eggs sealed with
cellotape. They
are further incubated until Day 13. Thermanox coverslips (Nunc, Naperville,
IL) are cut into
disks of about 5 mm in diameter. Sterile and salt-free growth factors, and the
protein to be
tested, are dissolved in distilled water and about 3.3 mg/ 5 ml are pipetted
on the disks. After
air-drying, the inverted disks are applied on CAM. After 3 days, the specimens
are fixed in
3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate
buffer.
They are photographed with a stereo microscope [Wild M8] and embedded for semi-
and
ultrathin sectioning as described above. Controls are performed with carrier
disks alone.
[0302] The studies described in this example test the activity in CTGF-2
protein.
However, one skilled in the art could easily modify the exemplified studies to
test the activity
of CTGF-2 polynucleotides (e.g., gene therapy), agonists, andlor antagonists
of CTGF-2.
Fxamnle fi: Angiogenesic Acsav ilsing a Matrigel Tmnlant ~n Mm~ce
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[0303] W order to establish an in vivo model for angiogenesis to test CTGF-2
protein
activities, mice and rats are implanted subcutaneously with methylcellulose
disks containing
either 20 mg of BSA (negative control), 1 mg of CTGF-2 or 0.5 mg of VEGF-1
(positive
control). The negative control disks should contain little vascularization,
while the positive
control disks should show signs of vessel formation.
[0304] The studies described in this example test the activity in CTGF-2
protein.
However, one skilled in the art could easily modify the exemplified studies to
test the activity
of CTGF-2 polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of CTGF-2.
Fxamnle 7: Rescue ~f Tcchemia in Rahhit Tower T,imh Model
[0305] To study the in vivo effects of CTGF-2 on ischemia, a rabbit hindlimb
ischemia model is created by surgical removal of one femoral arteries as
described previously
(Takeshita, S. et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the
femoral artery
results in retrograde propagation of thrombus and occlusion of the external
iliac artery.
Consequently, blood flow to the ischemic limb is dependent upon collateral
vessels
originating from the internal iliac artery (Takeshita, S. et al., Am J. Pathol
147:1649-1660
(1995)). An interval of 10 days is allowed for post-operative recovery of
rabbits and
development of endogenous collateral vessels. At IO day post-operatively (day
0), after
performing a baseline angiogram, the internal iliac artery of the ischemic
limb is transfected
with 500 mg naked CTGF-2 expression plasmid by arterial gene transfer
technology using a
hydrogel-coated balloon catheter as described (Riessen, R. et al., Hum Gene
Ther. 4:749-758
(1993); Leclerc, G. et al., J. Clin. Invest. 90: 936-944 (1992)). When CTGF-2
is used in the
treatment, a single bolus of 500 mg CTGF-2 proteimor control is delivered into
the internal
iliac artery of the ischemic limb over a period of 1 min. through an infusion
catheter. On day
30, various parameters are measured in these rabbits: (a) BP ratio - The blood
pressure ratio
of systolic pressure of the ischemic limb to that of normal limb; (b) Blood
Flow and Flow
Reserve - Resting FL: the blood flow during undilated condition and Max FL:
the blood flow
during fully dilated condition (also an indirect measure of the blood vessel
amount) and Flow
Reserve is reflected by the ratio of max FL: resting FL; (c) Angiographic
Score - This is
measured by the angiogram of collateral vessels. A score is determined by the
percentage of
circles in an overlaying grid that with crossing opacified arteries divided by
the total number
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m the rabbit thigh; (d) Capillary density - The number of collateral
capillaries determined in
light microscopic sections taken from hindlimbs.
[0306] The studies described in this example test the activity in CTGF-2
protein.
However, one skilled in the art could easily modify the exemplified studies to
test the activity
of CTGF-2 polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of CTGF-2.
Fxamnle 8: Rat lschemic Skin Flan Model
[0307] The evaluation parameters include skin blood flow, skin temperature,
and
factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction.
CTGF-2
expression, during the skin ischemia, is studied using in situ hybridization.
[0308] The study in this model is divided into three parts as follows:
[0309] Ischemic skin
[0310] Ischemic skin wounds
[0311] Normal wounds
[0312] The experimental protocol includes:
[0313] Raising a 3x4 cm, single pedicle full-thickness random skin flap
(myocutaneous flap over the lower back of the animal).
[0314) An excisional wounding (4-6 mm in diameter) in the ischemic skin (skin-
flap).
[0315] Topical treatment with CTGF-2 of the excisional wounds (day 0, 1, 2, 3,
4
post-wounding) at the following various dosage ranges: lmg to 100 mg.
[0316] Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-
wounding for
histological, immunohistochemical, and in situ studies.
[0317] The studies described in this example test the activity in CTGF-2
protein.
However, one skilled in the art could easily modify the exemplified studies to
test the activity
of CTGF-2 polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of CTGF-2.
Fxamyle 9: Peripheral Arterial nisease Model
[0318] Angiogenic therapy using CTGF-2 is a novel therapeutic strategy to
obtain
restoration of blood flow around the ischemia in case of peripheral arterial
diseases. The
experimental protocol includes:
[0319] One side of the femoral artery is ligated to create ischemic muscle of
the
hindlimb, the other side of hindlimb serves as a control.
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[0320] CTGF-2 protein, in a dosage range of 20 mg - 500 mg, is delivered
intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3
weeks.
(0321] The ischemic muscle tissue is collected after ligation of the femoral
artery at 1,
2, and 3 weeks for the analysis of CTGF-2 expression and histology. Biopsy is
also
performed on the other side of normal muscle of the contralateral hindlimb.
[0322] The studies described in this example test the activity in CTGF-2
protein.
However, one skilled in the art could easily modify the exemplified studies to
test the activity
of CTGF-2 polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of CTGF-2.
Fxamnle 1 fl: Tschemic Mo~ncardial T)isease Model
[0323] CTGF-2 is evaluated as a potent mitogen capable of stimulating the
development of collateral vessels, and restructuring new vessels after
coronary artery
occlusion. Alteration of CTGF-2 expression is investigated iyZ situ. The
experimental
protocol includes:
[0324] The heart is exposed through a left-side thoracotomy in the rat.
hnmediately,
the left coronary artery is occluded with a thin suture (6-0) and the thorax
is closed.
[0325] CTGF-2 protein, in a dosage range of 20 mg - 500 mg, is delivered
intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-4
weeks.
[0326] Thirty days after the surgery, the heart is removed and cross-sectioned
for
morphometric and in situ analyzes.
[0327] The studies described in this example test the activity in CTGF-2
protein.
However, one skilled in the art could easily modify the exemplified studies to
test the activity
of CTGF-2 polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of CTGF-2.
Fxam~]e 11: Rat C'.orneal Wound Healing Model
[0328] This animal model shows the effect of CTGF-2 on neovascularization. The
experimental protocol includes:
[0329] Making a 1-1.5 mm long incision from the center of cornea into the
stromal
layer.
(0330] Inserting a spatula below the lip of the incision facing the outer
corner of the
eye.
[0331] Making a pocket (its base is 1-1.5 mm form the edge of the eye).
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[0332] Positioning a pellet, containing Song- Sug of CTGF-2, within the
pocket.
[0333] CTGF-2 treatment can also be applied topically to the corneal wounds in
a
dosage range of 20mg - SOOmg (daily treatment for five days).
[0334] The studies described in this example test the activity in CTGF-2
protein.
However, one skilled in the art could easily modify the exemplified studies to
test the activity
of CTGF-2 polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of CTGF-2.
Fxamnle 12: Therapeutic Angioue~ is Ry Adenoviral-Mediated C.'T~F-2 C:ene
Transfer
[0335] Connective tissue growth factor 2 (CTGF-2) is a secreted, cystein-rich
heparin-
binding protein that is associated with extracellular matrix and cell surface.
In this Example the
angiogenic effect of the human CTGF-2 was evaluated in comparison with the
vascular
endothelial growth factor (VEGFI~s) in an adenoviral context after
intramuscular administration
(llVI) in the rabbit ischemic hindlimb model.
[0336] Briefly, three randomized groups of New Zealand White rabbits received
IM
injections of 5x108 infectious units (i.u.) of an adenovirus carrying either
the CTGF-2 gene (Ad-
CTGF-2), the VEGFz6s gene (Ad-VEGFi6s) or no transgene (Ad-Null), ten days
after femoral
artery excision in one limb. Perfusion of the ischemic limb was evaluated
before adenoviral
treatment (day 10) and 30 days post-injection (day 40). Angiographic,
hemodynamic and
histologic parameters indicated that atumals in the Ad-CTGF-2 group had a
significantly better
perfusion than in the Ad-Null group. Interestingly, this improvement exceeded
that achieved
with Ad-VEGFIbs .
[0337] These results show that CTGF-2 gene transfer demonstrates a stronger
stimulus
for limb revascularization compared to VEGFiss , thereby promoting a greater
improvement in
tissue perfusion in the ischemic limb. These findings show that CTGF-2 is
useful for treating of
severe peripheral ischemic diseases.
[0338] Despite substantial advances in prevention and treatment of ischemic
diseases,
an increasing number of patients with advanced peripheral ischemia remain
untreatable by
angioplasty or surgical revascularization. Therefore, alternative treatments
for these patients are
compelling. Therapeutic angiogenesis, i.e., the clinical use of growth factors
to enhance
revascularization in ischemic tissue, may be a promising strategy in this
setting.
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[0339] Among the numerous angiogenic growth factors that have been tested in
preclinical studies, vascular endothelial growth factor (VEGF)1-s, and
fibroblast growth factor
(FGF) families6'~ are the most widely studied. They can be considered as the
first line of
therapeutic candidates that have been used in clinical setting8'9,1°.
Despite some promising
results, the early clinical trials indicate that the optimal angiogenic factor
has not yet been
identified, stimulating the search for new therapeutic candidates.
[0340] Connective tissue growth factor 2 (also named cyr61) is a member of the
CCN
family which comprises six distinct proteins (Fispl2/~TGF, C.yr6l, Islov, Elin-
1, Cop-1/Wisp2,
and Wisp3) involved in cell growth and differentiationll. CTGF-2 is encoded by
a growth
factor-inducible immediate-early gene that is transcriptionally activated by
serum and growth
factors (bFGF, PDGF and TGF(3)la-14. CTGF-2 is a 42-kDa secreted, cysteine-
rich heparin-
binding protein that is associated with extracellular matrix and cell surfaces
i,is,i6. Recombinant
CTGF-2 protein mediates adhesion of vascular endothelial cells, fibroblasts
and lung epithelial
cellsl~-19, stimulates migration of fibroblasts and vascular endothelial
cells2° and synergizes
bFGF-induced DNA synthesis in both endothelial cells and fibroblasts1~,21. The
potential
interest of CTGF-2 iya vivo is poorly investigated since only one study
performed in a rat corneal
micropocket angiogenesis model demonstrated the angiogenic effect of CTGF-ZZO.
[0341] Therefore, the purpose of the present study was to examine the
angiogenic
effects of adenoviral-mediated gene transfer of human CTGF-2 in the rabbit
ischemic hindlimb
mode122, in comparison with those observed with VEGFi6s. Perfusion of the
ischemic limb was
evaluated before treatment and 30 days after direct intramuscular injection of
an adenovirus
carrying either CTGF-2 gene (Ad-CTGF-2), VEGFi6s gene (Ad-VEGFI6s) or no
transgene (Ad-
Null). Results demonstrated that Ad-CTGF-2 gene transfer promotes a
significant improvement
of tissue perfusion in the ischemic limb. CTGF-2 appeared more prone to
stimulate limb
revascularization in comparison with VEGFI6s, indicating that CTGF-2
represents a promising
candidate for therapeutic gene-based angiogenesis for promote, for example,
perfusion in the
ischemic limb in severe peripheral ischemic diseases.
Methods:
Adenoviral vectors
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[0342] The recombinant El/E3-deleted adenoviral vectors containing the human
CTGF-2 cDNA (cDNA clone ID HLFBE49; Ad-CTGF-2) or the human cDNA sequence
encoding the VEGFi6s protein (RT-PCR from HLTVEC; Ad-VEGFISS) under the
control of the
Cytomegalovirus promoter were obtained by homologous recombinationz3. Virus
propagation,
purification and titration of infectious units (i.u.) by indirect
immunofluorescence of the viral
DNA binding were carried out as described previously 24.
[0343] More specifically, the HLFBE49 cDNA clone contained a BamHI restriction
enzyme site at the 5' and 3' sequences. After digestion, the Bam/Bam fragment
was isolated
from a 1% agarose gel using a commercially avalaible kit ("Qiaquick", Qiagen,
Courtaboeuf,
France) and cloned in the Bam HI site of a pBluescript phagemid vector
(Stratagene, La Jolla,
CA). Ligation mixture was transformed into DHSoc strain. Plasmid DNA was
isolated from
transformants and examined by restriction analysis for the presence of the
correct fragment.
The candidate clone was sequenced, and then, digested with Eco RIIXba I
restriction enzymes
for the ligation with the transfer vector (named pTG13387). This transfer
vector contains the
Ad5 1-458 region followed by the CMV enhancer/ promoter and a a chimeric
intron
generated combining the splice donor from the human (3-globin intron 1 and the
splice
acceptor from the IgG intervening sequence obtained from pCI plasmid (Promega,
Charbonnieres, France). The recognition sites for the restriction
endonucleases XbaI and
EcoRI was inserted upstream of the bovine growth hormone polyadenylation site
followed by
the Ad5 3511-5788 and 3512-5788 regions. This vector contains the ampicillin
resistance
gene.
[0344] The E1/E3-deleted adenoviral vector containing the gene encoding CTGF-2
(named AdTG14550) was obtained by homologous recombination in 293 ce11s23,
between the
CTGF-2 transfer vector (named pTG14488) and the genomic CIaI DNA fragment
isolated
from the HSd1324 virus.
[0345] Virus propagation, purification and titration of infectious units (iu)
by indirect
immunofluorescence of the viral DNA binding protein were carried out as
described
previously24. Purified virus was stored in viral storage buffer (1 M sucrose,
10 mM Tris-HCl
[pH = 8.5], 1 mM MgClz, 150 mM NaCl, 0.005% [vol/vol] Tween 80).
In vitro angiogenesis assay
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[0346] Human dermal microvascular endothelial cells (HDMEC) were purchased
from
PromoCell (Heidelberg, Germany). Cells were grown and maintained in
Endothelial Cell
Growth Medium (ECGM) supplemented with 5% Fetal Calf Serum (FCS), lOng/ml
Epidermal
Growth Factor and 0.4% Endothelial Cell Growth Supplement. Cells were infected
with
recombinant adenovirus at a multiplicity of infection of 50 for 12 hours and
cell supernatants
were then collected. For the migration assay, 1x10s cells were added to the
upper side of S~uxn
pore size Transwell migration chambers (Costar) in ECGM containing 5% FCS.
Adenoviral-
infected HDMEC supernatants were added in the lower compartment and cell
migration was
allowed to proceed at 37°C for 3.5 hours. Cells that migrated to the
underside were stained with
0.5% crystal violet in 70% methanol followed by a brief rinse in PBS and
counted on nine
randomly selected microscopic fields (magnification x 20) for each condition.
Rabbit Model
[0347] The animal experiments were performed in accordance with Guiding
Principles
in the Care and Use ofAnimals approved by the American Physiological Society.
[0348] Male New Zealand White rabbits (3-3.25 kg) were anesthetized with a
mixture
of ketamine (50 mg/kg) and acepromazine (0.8 mg/kg) after premeditation with
xylazine (2
mglkg), all injected intramuscularly. The surgical excision of the femoral
artery was performed
as described previouslyl. Postoperation, prophylactic antibiotics (15 mg/kg
sulfamethoxazole
and 3 mg/kg trimethoprim) were administered subcutaneously and analgesia (1g
paracetamol)
was added in the drinking, water.
Intramuscular gene transfer
[0349] Animals (n=24) were randomly divided into 3 groups. An interval of 10
days
was permitted for postoperative recovery (day 10). Rabbits were injected
either with 5.108 i.u. of
Ad-Null, Ad-VEGFi6s or Ad-CTGF-2 in five muscular sites [medial large (x2),
adductor (x2),
semimembranous], after completion of baseline measurements (angiography,
Doppler-derived
flow).
Angiography and Doppler-derived blood flow at rest and after papaverine
infusion
[0350] In each animal, angiography and blood flow measurement were performed
before adenoviral injection (day 10) and 30 days after injection (day 40) as
described
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previouslyl. A 4-French infusion catheter introduced through the common
carotid artery was
positioned to the aortoiliac bifurcation. After infra-arterial injection of
nitroglycerin (0.25mg), a
bolus of contrast media was injected and serial images of the aortoiliac
bifurcation were
recorded to obtain angiograms. Subsequently, a 0.014-in Doppler guide wire
(Cardiometrics,
Mountain view, CA) was positioned in the proximal segment of either the
internal iliac artery
supplying the ischemic limb or the common iliac artery supplying the non-
ischemic limb.
Average peak velocities (APV) were recorded at rest and after bolus injection
of 2mg of
papaverine (maximum APV). Angiographic luminal diameter of the internal iliac
artery in the
ischemic limb and of the common iliac artery in the normal limb were
determined with an
automated edge-detection system. The luminal diameter was measured at the site
of the Doppler
wire position.
Vascular density
[0351] A morphometric angiographic analysis of collateral vessel development
in the
ischemic limb was performed using the angiograms recorded at day 10 and day
40. A grid
overlay composed of 5-mm2 squares was placed over the angiogram at the level
of the medial
thigh area, positioned always at the same place considering anatomic
references. The number of
contrast-opacified arteries crossing over rows in two sides were counted in a
single blind
fashion. This angiographic score reflects vascular density in the medial
thigh.
Capillary density
[0352] At the time of sacrifice, tissue samples were harvested from the
adductor and
semimembranous muscles of the ischemic limb. Muscles were frozen in iced-
isopentane and
stored at -80°C. Transverse tissue sections (7 ~,m) were stained for
alkaline phosphatase with an
indoxyl-tetrazolium method25 to detect capillary endothelial cells. A total of
20 randomly
selected microscopic fields for each muscle were examined under a lOX
objective to determine
the mean number of capillaries and myofibers to calculate the capillary-to-
muscle fiber ratio.
Statistical analysis
[0353] All results are expressed as means ~ SEM. For parametric data,
statistical
significance was evaluated using unpaired or paired Students t-test for
comparison between two
means and two-way analysis of variance (ANOVA) followed by Fishers test for
more than two
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mean values. Mann-Withney rank sum test was used for comparison of non
parametric data if
the interaction exists with analysis of variance (Kruskall-Wallis). A value of
P < 0.05 was
interpreted to denote statistical significance.
Fxamnle 12 Results
Ifz vitro studies -
[0354] As shown in Fig.2, Ad-CTGF-2 and Ad-VEGFjss-infected cell supernatants
enhanced significantly the migration of HDMEC in comparison with Ad-Null (P<
0.05),
validating adenoviral-mediated gene expression and angiogenic activities of
the gene product.
The stimulation of cell migration by Ad-VEGFiss or by Ad-CTGF-2 was similar.
Ifz vivo studies
Resting and stimulated Doppler-derived blood flows
[0355] In the ischemic limb, rest blood flows or maximal blood flows did not
differ
significantly among groups at day 10. At day 40, however, Ad-VEGFISS and Ad-
CTGF-2-
treated animals had a significant increase in blood flows compared with day 10
both at rest
(Fig.3A; Ad-VEGFISS: +77%; P < 0.01 and Ad-CTGF-2: +125.3%; P < 0.01) and
after
papaverine infusion (Fig.3B; Ad-VEGFi6s: +78.9%; P < 0.01 and Ad-CTGF-2 :
+151.5%; P <
0.01). Rest blood flow as well as maximum blood flow were significantly
improved in Ad-
CTGF-2 animals compared with Ad-Null group (rest flow: P < 0.05 and maximum
flow: P <
0.05). A trend toward greater blood flows appeared in Ad-CTGF-2 in comparison
with Ad-
VEGFiss-treated rabbits, but the difference was not statistically significant
(NS).
[0356] In the non ischemic limb, baseline and hyperemic blood flows determined
at day
were not different from those obtained at day 40 either in each group or among
groups (data
not shown). When ischemic and non-ischemic limb blood flows were compared at
day 10, rest
and stimulated blood flows were significantly higher in the normal limb
whereas at day 40, rest
blood flow was restored in Ad-VEGFI6s and Ad-CTGF-2 groups (Ad-VEGFiss
:13.1~1.4
versus 16.4~2.5 ml/min, NS ; Ad-CTGF-2: 18.6.8 versus 19.6~4.5 ml/min, NS) but
remained altered in Ad-Null-treated rabbits (Ad-Null: 10.8~1.7 versus 17.9~2.3
ml/min, P <
0.01). Moreover, maximum blood flow was also restored in Ad-CTGF-2 group
(34.2~S.5
versus 50.8~10.7m1/min, NS) whereas it was not in Ad-VEGFi6s or in Ad-Null
groups
(P<0.05).
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Vessel diameter
[0357] In the ischemic limb (Fig.4), a significant increase in angiographic
luminal
diameter of the internal iliac artery was observed during the 30 days of the
follow-up period in
Ad-VEGFi6s (P < 0.001) and Ad-CTGF-2-treated animals (P < 0.001) but not in Ad-
Null group,
reflecting the expansive remodeling of the artery supplying the ischemic limb
in response to the
chronic increase of blood flow. At day 40 the diameter was significantly
higher in Ad-CTGF-2-
treated rabbits than in Ad-Null group (P < 0.05).
[0358] Angiographic luminal diameters of the common iliac artery showed no
significant modification in diameter at day 10 and day 40 between the three
groups (data not
shown).
Vascular density
[0359] Representative angiograms of rabbit ischemic hindlimb showed that
collateral
vessel development during the 30 days of the follow-up period was more marked
in the Ad-
VEGFiss and the Ad-CTGF-2-treated groups, over control groups (Fig.S). The
quantitative
analysis of collateral blood vessel development in rabbit hindlimb is
summarized in Fig.6.
Angiographic scores did not differ significantly among groups before treatment
at day 10. An
increase in vascular density was measured in each group from day 10 to day 40
(Ad-Null:
+26.3%; P < 0.01; Ad-VEGFI6s: +76.4%; P < 0.0001 and Ad-CTGF-2: +100.7%; P c
0.001).
At day 40, vascular densities were significantly higher in both treated groups
compared with
control animals (P < 0.0001) as suggested on angiograms. Interestingly, the
angiographic score
in CTGF-2-treated animals exceeded that of the VEGFi6s-treated group (P <
0.01), indicating a
marked effect of CTGF-2 on arteriogenesis.
Capillary density
[0360] In the adductor muscle (Fig.7A), capillary density as capillary-to-
myocyte ratio
was increased in the Ad-CTGF-2 group compared with Ad-Null (P< 0.01) and Ad-
VEGFi6s-
treated animals (P<0.001) whereas there was no difference between these two
last groups. In the
semimembranous muscle (Fig.7B), the capillary density was significantly higher
in Ad-
VEGFt6s compared with Ad-Null group (P < 0.05). An increase was also observed
in the Ad-
96
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
CTGF-2 group in comparison to the Ad-Null group but the level of significance
was not
reached.
[0361] These results demonstrate in vivo, the angiogenic potency of human CTGF-
2
adenoviral-mediated gene transfer. Indeed, experiments performed on rabbit
ischemic hindlimb
provided evidence that Ad-CTGF-2 improves limb perfusion, as suggested by the
augmentation
of angiographically visible collateral vessels, increased capillary density,
and consequent
hemodynamic improvement.
[0362] The choice of VEGFI6s as a reference has been suggested by the
extensive
literature demonstrating its potent angiogenic effect. Nevertheless,
adenoviral-mediated gene
transfer of VEGFISS has never been used in the rabbit ischemic hindlimb model.
[0363] In this study, data obtained in Ad-VEGFiss-treated animals axe in good
general
agreement with previous findings demonstrating that VEGF administered as a
recombinant
protein or as naked DNA increased limb perfusionzz,z6,z~. ~ fact, we showed an
increase in rest
as well as maximal blood flows, angiographic score and capillary density in
the adductor
muscle.
[0364] In animals treated with Ad-CTGF-2, a marked improvement of the blood
flows
in the ischemic limb was obtained and measured. This improvement in
hemodynaxnic status
seems related to the stimulation of angiogenesis and arteriogenesis as
revealed by increased
capillary density in the semimembranous muscle as well as increased
angiographic score in Ad-
CTGF-2-treated animals. Although angiogenesis (capillary sprouting) may
deliver some relief to
the underperfused territory, only true collateral arteries, resulting from in
situ proliferation of
preexisting arteriolar connections, are capable of providing large enough
amounts of blood flow
to the ischemic area to reestablish flow to the more distal arteries in the
legz8. Tlus enhanced
arteriogenesis contributed, at least partly, to the increase in blood flows
between day 10 and day
40 at rest as well as after papaverine-mediated arteriolar vasodilatation.
Interestingly, CTGF-2
appears to be more efficient than VEGFi6s to enhance the collateral growth as
suggested by the
greater angiographic score. This could explain that in the CTGF-2 group, the
maximal blood
flow was restored in the ischemic limb compared with the non ischemic limb,
but not in
VEGFi6s-treated animals.
[0365] This result strengthens the idea that a pleiotropic factor is more
prone than an
endothelium-specific mitogen to trigger arteriogenesis which is a complex
morphogenic
process. CTGF-2 may stimulate the revascularization by acting as an angiogenic
inducer upon
97
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
endothelial cells as VEGF does, and additionally by acting as a chemotactic,
mitogenic and
matrix remodeling factor upon fibroblastsl~'zo. Indeed, a potential function
of CTGF-2 in matrix
remodeling has been recently suggested through the activation of matrix
metalloproteinases 1
and 3z9, enzymes known to play a role in angiagenesis. Involvement of CTGF-2
in
angiogenesis can also be attributed to its interaction with different
integrins: av(33-dependent
pathway for adhesion and migration of endothelial cellsl9,zo, as(31 and av(33.
for adhesion and
migration of fibroblasts, respectively3°. Moreover, even if CTGF-2 is
not mitogenic by itself, it
may synergize with growth factors like bFGF to enhance mitogenesis of
endothelial cells or
fibroblastsl~°zo,zi.
[0366] Interestingly, CTGF2 angiogenic properties appears closely related to
bFGF,
with respect to induction of MMP-1 and MMP-3, to its interaction with integrin
av(33,
implicated in bFGF-mediated angiogenesis3l, and also because bFGF, a major
inducer of the
CTGF-2 gene, has been presented as the main angiogenic factor implicated in
arteriogenesisz8.
The interplay between the actions of bFGF and CTGF-2 is likely to be complex
and remains to
be more extensively investigated.
[0367] In conclusion, we have established that Ad-CTGF-2 gene transfer
promotes a
significant improvement of tissue perfusion in the ischemic limb. The results
revealed that
CTGF-2 appeared more prone to stimulate limb revascularization in comparison
with VEGFi6s,
indicating that CTGF-2 is a useful as a therapeutic agent for treatment of
severe peripheral
ischemic disease.
R .f re e~.nre.,~ fir
[0368] 1. Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N,
Symes JF, Isner JM. Physiological assessment of augmented vascularity induced
by VEGF in
ischemic rabbit hindlimb. Ana JPhysiol. 1994;267:H1263-71.
[0369] 2. Takeshita S, ZVeir L, Chen D, Zheng LP, Riessen R, Bauters C, Symes
JF, Ferrara N, Isner JM. Therapeutic angiogenesis following arterial gene
transfer of vascular
endothelial growth factor in a rabbit model of hindlimb ischemia. Bioclaem
Biophys Res
Commun. 1996;227:628-35.
[0370] 3. Rivard A, Silver M, Chen D, Kearney M, Magner M, Annex B, Peters
K, Isner JM. Rescue of diabetes-related impairment of angiogenesis by
intramuscular gene
therapy with adeno-VEGF. Arra JPatlzol. 1999;154:355-63.
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[0371) 4. Mack CA, Patel SR, Schwarz EA, Zanzonico P, Hahn RT, Ilercil A,
Devereux RB, Goldsmith SJ, Christian TF, Sanborn TA, Kovesdi I, Hackett N,
Isom OW,
Crystal RG, Rosengart TK. Biologic bypass with the use of adenovirus-mediated
gene
transfer of the complementary deoxyribonucleic acid for vascular endothelial
growth factor
121 improves myocardial perfusion and function in the ischemic porcine heart.
J Tho~ac
Cardiovasc SuYg. 1998;115:168-76; discussion 176-7.
[0372] 5. Witzenbichler B, Asahara T, Murohara T, Silver M, Spyridopoulos I,
Magner M, Principe N, Kearney M, Hu JS, Isner JM. Vascular endothelial growth
factor-C
(VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia. Am J
Pathol.
1998;153:381-94.
[0373] 6. Garcia-Martinez C, Opolon P, Trochon V, Chianale C, Musset K, Lu
H, Abitbol M, Perricaudet M, Ragot T. Angiogenesis induced in muscle by a
recombinant
adenovirus expressing functional isoforms of basic fibroblast growth factor.
Gene They.
1999;6:1210-21.
[0374) 7. Safi J, DiPaula AF, Riccioni T, Kajstura J, Ambrosio G, Becker LC,
Anversa P, Capogrossi MC. Adenovirus-mediated acidic fibroblast growth factor
gene
transfer induces angiogenesis in the nonischemic rabbit heart. Microvasc Res.
1999;58:238-
49.
[0375] 8. Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K,
Isner JM. Constitutive expression of phVEGF165 after intramuscular gene
transfer promotes
collateral vessel development in patients with critical limb ischemia [see
comments].
Circulation. 1998;97:1114-23.
[0376] 9. Lazarous DF, Unger EF, Epstein SE, Stine A, Arevalo JL, Chew EY,
Quyyumi AA. Basic fibroblast growth factor in patients with intermittent
claudication: results
of a phase I trial. JAm Coll Cardiol. 2000;36:1239-44.
[0377) 10. Hammond HK, McKirnan MD. Angiogenic gene therapy for heart
disease: a review of animal studies and clinical trials. Cardiovasc Res.
2001;49:561-7.
[0378] 11. Lau LF, Lam SC. The CCN family of angiogenic regulators: the
integrin connection. Exp Cell Res. 1999;248:44-57.
[0379] 12. Brunner A, Chum J, Neubauer M, Purchio AF. Identification of a gene
family regulated by transforming growth factor-beta. DNA Cell Biol.
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[0380] 13. Lau LF, Nathans D. Identification of a set of genes expressed
during
the GO/Gl transition of cultured mouse cells. Embo J. 1985;4:3145-51.
[0381] 14. Lau LF, Nathans D. Expression of a set of growth-related immediate
early genes in BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc.
PYOC Natl Acad
Sci U S A. 1987; 84:1182-6.
[0382] 15. O'Brien TP, Yang GP, Sanders L, Lau LF. Expression of cyr6l, a
growth factor-inducible immediate-early gene. Mol Cell Biol. 1990;10:3569-77.
(0383] 16. Yang GP, Lau LF. Cyr6l, product of a growth factor-inducible
immediate early gene, is associated with the extracellular matrix and the cell
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[0384] 17. Kireeva ML, Mo FE, Yang GP, Lau LF. Cyr6l, a product of a growth
factor-inducible immediate-early gene, promotes cell proliferation, migration,
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Mol Cell Biol. 1996;16:1326-34.
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AS, Lau LF. Cyr61 and Fispl2 are both ECM-associated signaling molecules:
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metabolism, and localization during development. Exp Cell Res. 1997;233:63-77.
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endothelial cells to the immediate-early gene product Cyr61 is mediated
through integrin
alphavbeta3. JBiol Chem. 1998;273:3090-6.
[0387] 20. Babic AM, Kireeva ML, Kolesnikova TV, Lau LF. CYR61, a product .
of a growth factor-inducible immediate early gene, promotes angiogenesis and
tumor growth.
Proc Natl Acad Sci USA. 1998;95:6355-60.
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bFGF-induced DNA synthesis in human umbilical vein endothelial cells.
Oncogene.
1998;16:747-54.
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revascularization of the ischemic limb by angiogenic therapy. Circulation.
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M.
Efficient generation of recombinant adenovirus vectors by homologous
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Escherichia coli. J Virol. 1996;70:4805-10.
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[0399] Numerous modifications and variations of the present invention are
possible in
light of the above teachings and, therefore, within the scope of the appended
claims, the
invention may be practiced otherwise than as particularly described.
[0400] The entire disclosure of each document cited (including patents, patent
applications, journal articles, abstracts, laboratory manuals, books, or other
disclosures) in the
Background of the Invention, Detailed Description, and Examples is hereby
incorporated
101
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
herein by reference. Further, the hard copy of the sequence listing submitted
herewith and the
corresponding computer readable form are both incorporated herein by reference
in their
entireties.
[0401] Certain CTGF-2 polynucleotides and polypeptides of the present
invention,
including antibodies, were disclosed in U.S. provisional application numbers
60/217,402,
filed July 11, 2000, and 60/291642, filed May 18, 2001, the specifications and
sequence
listings of which are herein incorporated by reference in their entireties.
102
CA 02412124 2002-12-13
WO 02/04480 PCT/USO1/21799
1
SEQUENCE LISTING
<110> Human Genome Sciences, Inc. et al.
<120> Connective Tissue Growth Factor-2
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<140> Unassigned
<14l> 2001-07-11
<150> 60/217,402
<151> 2000-07-11
<150> 60/291,642
<151> 2001-05-18
<160> 8
<170> PatentIn version 3.0
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CA 02412124 2002-12-13
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CA 02412124 2002-12-13
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CA 02412124 2002-12-13
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CA 02412124 2002-12-13
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Gly Ala Cys Ile Pro Leu Cys Pro Gln Glu Leu Ser Leu Pro Asn Leu
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Gly Cys Pro Asn Pro Arg Leu Val Lys Val Thr Gly G1n Cys Cys Glu
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Leu Thr Arg Asn Asn Glu Leu Ile Ala Val Gly Lys Gly Ser Ser Leu
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Lys Arg Leu Pro Val Phe Gly Met Glu Pro- Arg Ile Leu Tyr Asn Pro
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cgcgggtacc aggtagcatt tagtccctaa 30
