Note: Descriptions are shown in the official language in which they were submitted.
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~TP~rFT-T~ /EPIDER~AL GROWT~ F~CTOR ~I~E PROTEIN
This invention relates to newly identified polynucleotides,
polypeptides PnCoAe~ by such polynucleotides, the use of such
polynucleotides and polypeptides, as well as the production of such
polynucleotides and polypeptides. The polypeptide of the present
invention has been putatively identified as a human extracellular
protein-like/Epidermal Growth Factor-like protein, hereafter
referred to as "EEGF". The invention also relates to i nhihi ting
the action of such polypeptides.
Cellular growth and differentiation appear to be initiated,
promoted, ~-int~ine~ and regulated by a multlplicity of
stimulatory, i nhihi tory and synergistic factors and hor~L~nes. The
alteration and/or breakdown of the cellular ho~leostasls mechanism
seems to be a fl~nA~m~ntal cause of growth rel~ed diseases,
including neoplasia. Growth moAl~l~tory factors are implicated in
a wide variety of pathological and physiological processes
including signal transduction, cell communication, growth and
development, embryogenesis, immune response, hematopoiesis cell
survival and differentiation, inflammation, tissue repair and
remodeling, atherosclerosis and cancer. Epidermal growth factor
(BGF), transforming growth factor alpha (TGF~), betacellulin,
hiregulin, and vaccinia growth factor among other factors are
growth and differentiation moA~ tory proteins produced by a
variety of cell types either under normal physiological conditions
or in response to exogenous stimuli and are members of the EGF
family.
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These peptide growth factors influence influence epithelial
and epidermal cells through autocrine and paracrine mechanisms.
They also play important roles in normal wound heAl ing in tissues
such as skin, cornea and gastrointestinal tract and all share
substantial amino acid sequence homology including the conserved
placement of three intra-chain disulfide bonds. In addition, all
the factors of this family bind to a 170,000 molecular weight
transmembrane glycoprotein receptor and activate the tyrosine
kinase acti~ity in the receptor's cytoplasmic ~ i n (Buhrow, S.A.
et al., J. Bio.Chem., 258:7824-7826 (1983)).
The receptors are expressed by many types of cells including
skin keratinocytes, fibroblasts, vascular endothelial cells, and
epithelial cells of the gastro-intestinal tract (GI) tract. These
peptide growth factors are synthesized by several cells in~olved
in wound healing including platelets, keratinocytes, and activated
macrophages. These growth factors have also been implicated in
both the st~ tion of growth and differentiation of certain
cells, for example, neoplasia, and the i nhihi tion of other types
of cells.
Betacellulin is a 32-kilodalton glycoprotein that appears to
be processed from a larger transmembrane precursor by proteolytic
cleavage. The cArhq~cyl-terminal ~ i n of betacellulin has 50%
sequence similarity with that of rat transforming growth factor a.
~etacellulin is a potent mitogen for retinal pigment epithelial
cells and vascular smooth muscle cells.
Amphiregulin is a bifunctional cell growth regulatory factor
which exhibits potent i nhihi tory activity on DNA synthesis in
neoplastic cells, yet promotes the growth of certain normal cells.
A wide variety of uses for amphiregulin have been assigned
including the treatment of wounds and cancers. For example,
amphiregulin has potent anti-proliferative effects in vitro on
several human cancer cell lines of epithelial origin. Amphiregulin
also induces the proliferation of human foreskin fibroblasts as
shown in United States Patent Application No. 5,115,096.
TGF~ has pleiotropic biological effects. The production of
certain m~mh~rs of TGF~ is synthesized by a number of oncogenically
transformed fibroblasts (Ciardiello et al., J. Cell. Biochem.,
42:45-57 (1990)), as well as by a variety of tumors, including
renal, breast and squamous carcino~a.s, melAnomA.s and glioblastomas
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W097/38002
~Derynck, R. et al., Cancer Res., 47:707-712 (1987)). There is
direct evidence that TGF~ expression can be a contributing factor
in the conversion of a normal cell to its tumorigenic counterpart
by analyzing transgenic mice in which tumor cells express high
~ levels of TGF~. TGF~ transgenic ~nim~l S display a variety of
neoplastic lesions, depending on the strain of mouse and the choice
of promotor regulating TGF~ expression ~Sandgren, et al., Cell,
61:1121-1135 (1990)).
TGF~ also plays a role in normal embryonic development and
adult physiology (Derynck, R. Adv. Cancer Res., 58:27-5 (1992)).
TGF~ has been expressed in many tissues including skin, brain,
gastrointestinal mucosa and activating macrophages. Accordingly,
TGF~ is an important factor in controlling growth of epithelial
cells and has a role in wound ~eAli~g. TGF~ has also been found
to be angiogenic (Schreiber, et al., Science, 232:1250-1253
(1986)).
The polypeptide of the present invention has been putatively
identified a~ an Extracellular/Bpidermal Growth Factor. This
identification has been made a~ a result of amino acid sequence
homology to human extracellular protein which is a secreted protein
with EGF-like ~nm~inS that is abundant in heart tissue.
In accordance with one aspect of the present invention, there
are provided novel mature polypeptides, as well as biologically
active and diagnostically or therapeutically useful fragments,
analogs and derivatives thereof. The polypeptides of the present
invention are of human origin.
In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules encoding the
polypeptides of the present invention, including mRNAs, cDNAs,
genomic DNAs as well as analogs and biologically active and
diagnostically or therapeutically useful fragments thereof.
In accordance with another aspect of the present invention
there is provided an isolated nucleic acid molecule encoding a
mature polypeptide expressed by the human cDNA contained in ATCC
Deposit No. 97285.
In accordance with yet a further aspect of the present
invention, there are provided processes for producing such
polypeptide by recombinant technigues comprising culturing
rec~inAnt prokaryotic and~or eukaryotic host cells, cont~i ni ng
CA 02249213 1998-09-17
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WO97/38002
a nucleic acid sequence encoding a polypeptide of the present
invention.
In accordance with yet a further aspect of the present
invention, there are provided processes for utilizing such
polypeptides, or polynucleotides encoding such polypeptides for
therapeutic purposes, for example, to regulate vascular smooth
muscle cell proliferation, to treat Marfan syndrome, to stimulate
wound healing, to restore normal neurological functioning after
trauma or AIDS dementia, to treat ocular disorders, to treat kidney
and liver disorders, to promote hair follicular development, to
sti~nl~te growth and differentiation of various epidermal and
epithP~ ~Al cells in vivo and in vitro and for the treatment of
burns, ulcers and cor~e~l incisions, to stimulate embryogenesis.
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 nucleic acid sequences of the present invention.
In accordance with yet a further aspect of the present
invention, there are provided antibodies against such polypeptides.
In accordance with yet a further a~pect of the present
invention, there are provided agonists to the polypeptide of the
present invention.
In accordance with yet another aspect of the present
invention, there are provided antagonists to such polypeptides,
which may be used to i nhl hl t the action of such polypeptides, for
example, in the treatment of corneal inflammation, neoplasia, for
example, tumors and cancers and for psoriasis.
In accordance with still another aspect of the present
invention, there are provided diagnostic assays for detecting
diseases related to overexpression of the polypeptide of the
present invention and mutations in the nucleic acid sequences
PncoAl ng such polypeptide.
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
in vitro purposes related to scientific research, synthesis of DNA
and manufacture of DNA vectors.
These and other aspects of the present invention should be
apparent to those skilled in the art from the teachings herein.
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W097/38002
The following drawings are illustrative of PmhoAi~ents of the
invention and are not meant to limit the scope of the invention as
encompassed by the claims.
Figure 1 depicts the cDNA sequence and correspon~i ng deduced
amino acid sequence of EEGF. Both the st~n~Ard one letter and
three letter ab~reviations for amino acids are used.
Figure 2 is an illustration of comparative amino acid sequence
homology between the polypeptide of the present invention (lower
line) and human extracellular protein (upper line) (SEQ ID NO:9).
In accordance with an aspect of the present invention, there
is provided an isolated nucleic acid (polynucleotide) which encodes
for the mature polypeptide having the deduced amino acid sequence
of Figure 1 (SEQ ID NO:2).
A polynucleotide encoding a polypeptide of the present
invention may be obtained from human brain and early stage brain
tissue. The polynucleotide of this invention was discovered in a
human fetal heart cDNA li~rary. It has homology to the
characteristic EGF domains. It contains an open reading frame
encoding a polypeptide of 392 amino acids. EEGF eyhih~ts the
highest degree of homology at the amino acid level to human
extracellular protein with 45% identity and 34~ similarity over a
392 amino acid stretch. Northern blot analysis of this protein
shows high levels of expression in heart tissue with the transcript
being approximately 2 kb. In accordance with another aspect of
the present invention there are provided isolated polynucleotides
encoding a mature polypeptide expressed by the human cDNA contained
in ATCC Deposit No. 97285, deposited with the American Type Culture
Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, USA,
on September 26, 1995. The deposited material is an a plasmid that
contAin~ the full-length EEGF cDNA which has been transformed into
a viable host.
The deposit(s) have been made under the terms of the Budapest
Treaty on the International Recognition of the Deposit of Micro-
org~n~s~c for purposes of Patent Procedure. The strain will be
irrevocably and without restriction or condition released to the
public upon the issuance of a patent. These deposits are provided
merely as convenience to those of skill in the art and are not an
admission that a deposit is required under 35 U.S.C. 112. The
sequence of the polynucleotides contained in the deposited
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materials, as well as the amino acid sequence of the polypeptides
encoded thereby, are controlling in the event of any conflict with
any description of sequences herein. A license may be required to
make, use or sell the deposited materials, and no such license is
hereby granted. References to "polynucleotides" throughout this
specification includes the DNA of the deposit referred to above.
The polynucleotide of the present invention may be in the form
of RNA or in the form of DNA, which DNA includes cDN~, genomic DNA,
and synthetic DNA. The 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 Figure 1 ~SEQ ID NO:l) or may be a different coding sequence
which coding sequence, as a result of the reA-~n~ncy or degeneracy
of the genetic code, encodes the same mature polypeptide as the DNA
of Figure 1 ~SEQ ID NO:1).
The polynucleotide which ~ncq~es for the mature polypeptide
of Figure 1 (SBQ ID NO:2) may include, but is not limited to: only
the coAin~ sequence for the mature polypeptide; the coding sequence
for the mature polypeptide and additional ro~ ng 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.
Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequence
for the polypeptide as well as a polynucleotide which includes
additional coding and/or non-coding sequence.
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 Figure 1 (SEQ ID NO:2). The variant of the
polynucleotide may be a naturally occurring allelic variant of the
polynucleotide or a non-naturally occurring variant of the
polynucleotide.
Thus, the present invention includes polynucleotides PncoAing
the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) as
well as variants of such polynucleotides which variants encode for
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a fragment, derivative or analog of the polypeptide of Figure 1
(SEQ ID NO:2). Such nucleotide variants include deletion variants,
substitution variants and addition or insertion variants.
As her~in~hove indicated, the polynucleotide may have a coding
sequence which is a naturally occurring allelic variant of the
coding sequence shown in Figure 1 (SEQ ID NO:1). As known in the
art, an allelic variant is an alternate form of a polynucleotide
sequence which may have a substitution, deletion or addition of one
or more nucleotides, which does not subst~nti Al ly alter the
function of the encoded polypeptide.
The present invention also includes polynucleotides, wherein
the coding sequence for the mature polypeptide may be fused in the
same r~A~i ng frame to a polynucleotide sequence which aids in
expression and secretion of a polypeptide from a host cell, for
example, a eader sequence which functions as a secretory sequence
for contro].ling transport of a polypeptide from the cell. The
polypeptide having a leader sequence is a preprotein and may have
the leader sequence cleaved by the host cell to form the mature
form of the polypeptide. The polynucleotides may also enco~e for
a proprotein which is the mature protein plus additional 5' amino
acid residues. A mature protein having a prosequence is a
proprotein and is an inactive form of the protein. Once the
prosequence is cleaved an active mature protein r~m~i n~ Thus,
for example, the polynucleotide of the present invention may encode
for a mature protein, or for a protein having a prosequence or for
a protein having both a prosequence and a presequence (leader
sequence).
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 ~ ian
host, e.g. COS-7 cells, is used. The HA tag corresponds to an
epitope derived from the influenza hemagglutinin protein (Wilson,
I., et al., Cell, 37:767 (1984)).
The term "gene" means the segment of DNA involved in producing
a polypeptide chain; it includes regions preceding and following
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the coding region (leader and trailer) as well as intervening
sequences (introns) between individual coding segments (exons).
Fragments of the full length EEGF gene may be used as a
hybridization probe for a cDNA library to isolate the full length
~ene and to isolate other genes which have a high sequence
similarity to the gene or S jmi 1 ~r 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 EEGF gene
including regulatory and promotor 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 ~equence
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.
The present invention further relates to polynucleotides
which hybridize to the hereinabove-described sequences if there is
at least 70%, preferably 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 her~inAhove-described polynucleotides.
As herein used, the term "stringent conditionsll means hybridization
will occur only if there is at least 95~ and preferably at least
97% identity between the sequences. The polynucleotides which
hybridize to the hereinabove described polynucleotides in a
preferred embodiment encode polypeptides which either retain
substantially the same biological function or activity as the
mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO:1).
Alternatively, the polynucleotide may have at least 15 bases,
preferably at least 30 bases, and more preferably at least 50 bases
which hybridize to a polynucleotide of the present invention and
which has an identity thereto, as hereinAhove described, and which
may or may not retain activity. ~or example, such polynucleotides
may be employed as probes for the polynucleotide of SEQ ID NO:1,
for example, for recovery of the polynucleotide or as a dia~nostic
probe or as a PCR primer.
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Thus, the present invention is directed to polynucleotides
having at least a 70% identity, preferably at least 90~ and more
preferably at least a 95~ identity to a polynucleotide which
encodes the polypeptide of SEQ ID NO:2 and polynucleotides
complementary thereto as well as portions thereof, which portions
have at least 15 consecutive bases preferably at least 30
consecutive bases and preferably at least 50 consecutive bases and
to polypeptides encoded by such polynucleotides.
The present invention further relates to a polypeptide which
has the deduced amino acid sequence of Figure 1 (SEQ I~ NO:2), as
well as fragments, analogs and derivatives of such polypeptide.
The terms "fragment," "derivative" and "analog" when referring
to the polypeptide of Figure 1 (SEQ ID NO:2), 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.
The polypeptide of the present invention may be a recombinant
polypeptide, a natural polypeptide or a synthetic polypeptide,
preferably a reComhin~nt polypeptide.
The fragment, derivative or analog of the polypeptide of
Figure 1 (SEQ ID NO:2) may be (i) one in which one or more of the
amino acid residues are substituted with a conserved or non-
conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be
one encoded by the genetic code, or (ii) one in which one or more
of the amino acid residues includes a substituent group, or ~iii)
one in which the mature polypeptide is fused with another compound,
such as a compound to increase the half-life of the polypeptide
(for example, polyethylene glycol), or ~iv) one in which the
additional amino acids are fused to the mature polypeptide, such
as 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.
The polypeptides and polynucleotides of the present invention
are preferably provided in an isolated form, and preferably are
purified to hu...oyel,eity.
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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-occurring
polynucleotide or polypeptide present in a living ~ni~-l 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.
The polypeptides of the present invention include the
polypeptide of SEQ ID NO:2 (in particular the mature polypeptide)
as well as polypeptides which have at least 70% similarity
(preferably at lea~t 70% identity) to the polypeptide of SEQ ID
NO:2 and mor-_ preferably at least 90~ similarity (more preferably
at least 90% identity) to the polypeptide of SBQ ID NO:2 and still
more preferably at least 95~ simil~rity (still more preferably at
least 95~ identity) to the polypeptide of SBQ ID NO:2 and also
include portions of such polypeptides with such portion of the
polypeptide generally cont~ning at least 30 amino acids and more
preferably at least 50 amino acids.
As known in the art "s~milArity" 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.
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
polypeptides. Fragments or portions of the polynucleotides of the
present invention may be used to synthesize full-length
polynucleotides of the present invention.
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
techni~ues.
Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this invention
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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 a~pLGpriate for
activating promoters, selecting transformants or am.plifying 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.
The polynucleotides of the present invention m~y be employed
for producing polypeptides by recomhin~nt 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;
baculovirusi yeast plasmids; vectors derived from com~binations 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.
The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA se~uence is
inserted into an ~ o~Liate restriction Pn~ lease 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.
The DNA sequence in the expression vector s operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or tr~, the phage l~mh~ P~ promoter and other promoters
known to control expression of genes in prokaryotic or eukaryotic
cells or their viruses. The expression vector also cont~tn~ a
ribosome htn~tng site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
In addition, the expression vectors prefera~ly cont~tn one or
more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resifitance for eukaryotic cell culture, or such as
tetracycline or am~icillin resistance in E. coli.
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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 a~-u~iate host
to permit the host to express the protein.
As representative examples of appropriate hosts, there may be
mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella tY~himllrium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and S~odo~tera Sf9; ~nim~l 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.
More particularly, the present invention also includes
recomh~n~nt 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 em~o~im~nt, 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, pD10,
phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A,
pNH46A (Stratagene)i ptrc99a, pKK223-3, pKK233-3, pDR540, pRlT5
(Pharmacia); Eukaryotic: pWLN~O, pSV2CAT, pOG44, pXT1, 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.
Promoter regions can be selected from any desired gene using
CAT (chlor~m~h~nicol 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, l;~mh~l~ PRI 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.
In a further emhoAiment, the present invention relates to host
cells contAining the above-described constructs. The host cell can
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be a higher eukaryotic cell, such as a m~ l ian 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 effected by calcium phosphate
transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional
m~nner to produce the gene product encoded by the recomhinAnt
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in m;~ ian 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. A~Lo~iate cloning and expression
vectors for u~e with prokaryotic and eukaryotic hosts are described
by Sambrook, et al., Molecular Cloning: A Laboratory ~ml~l, Second
Bdition, Cold Spring Harbor, N.Y., (1989), the disclosure of which
is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting
an Pnh~ncer sequence into the vector. Rnh~ncers are cis-acting
elements of DNA, u~ually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples including the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter Pnh~ncer, the polyoma Pnh~ncer on
the late side of the replication origin, and adeno~irus Pnh~ncers.
Generally, reCo~hln~nt expression vectors will include origins
of replication and selecta~le markers permitting transformation of
the host cell, e.g., the ampicillin resistance gene of E. coli and
S. cerevisiae 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), ~-
factor, acid phosphatase, or heat shock proteins, among others.
The heterologous structural sequence is assembled in appropriate
pha~e with translation initiation and termination sequences, and
preferably, a leader sequence capable of directing secretion of
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W O 97/38002 PCT/U~ v3~47
translated protein into the periplasmic space or extracellular
medium. Optionally, the heterologous sequence can ~nco~ a fusion
protein including an N-terminal identification peptide imparting
desired characteristics, e.g., stabilization or simplified
purification of expressed recombinant product.
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 ~rk~rs 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 E. coli, Bacillus
subtilis, S~?~nella tY~himl~rium and various species within the
genera Pse~l~nmon~.s, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression
vectors for bacterial use can cu..~lise a selectable marker and
bacterial origin of replication derived from commercially av~ hle
plasmids comprising genetic elements of the well known cloning
vector pBR322 (ATCC 37017). Such commercial vectors include, for
example, pKR223-3 tPharmacia Fine Chemicals, Ur~s~l~, Sweden) and
GEMl (Plo...e~d Biotec, Madison, WI, USA). These pBR322 "backbone"
sections are combined with an a~lo~iate promoter and the
structural seguence to be expressed.
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.
Cells are typically harvested by centrifugation, disrupted by
physical or chemical means, and the resulting crude extract
ret~in~ for further purification.
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.
Various ~ ian cell culture systems can also be employed
to express reco~hin~nt protein. Examples of mammalian expression
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systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell, 23:175 (1981), and other cell lines
c~p~hle of expressing a compatible vector, for example, the C127,
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 ~ i n~ sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination se~uences, 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.
The polypeptides can be recovered and purified from
reCo~hinAnt cell cultures by methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation ~rh~nge
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used, a~ necessary, in completing
configuration of the mature protein. Finally, high performance
liquid chromatography tHPLC) can be employed for final purification
steps.
The polypeptides of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures,
or produced by reco~ nt 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. Polypeptides of the invention may also include an
initial methionine amino acid residue.
The polynucleotides and polypeptides of the present invention
may be employed as research reagents and materials for discovery
of treatments and diagnostics for human disease.
The polypeptide of the present invention may be employed to
to regulate vascular smooth muscle cell proliferation.
The polypeptide of the present invention may also be employed
for characterization of receptors. The EGF family of receptors
~UL ' e~.tly includes four EGF receptors, denoted as EGFR1, EGFR2,
EGFR3 and EGFR4. The EGFR2 receptor may also be referred to as
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ERB-2 and this molecule is useful for a variety of diagnostic and
therapeutic indications (Prigent, S.A., and Lemoine, N.R., Proq
Growth Factor Res., 4:1-24 (1992)). The EEGF polypeptide is likely
a ligand for one or more of these receptors as well as for an
unidentified new EGF-type receptor. Use of the EEGF can assist
with the identification, characterization and cloning of such
receptors. For example, the EG~ receptor gene represents the
cellular homolog of the v-erb-B oncogene of avian erythroblastosis
virus. Over expression of the E~F-receptor or deletion of kinase
regulatory segments of the protein can bring about tumorigenic
transformation of cells (Manjusri, D. et al., Human CYtokines, 364
and 381 ~1991)).
The polypeptides of the present invention may also be employed
for restoration or ~nh~ncement of neurological functions ~i~inished
as a result of trauma or other damaging pathologies (such as AIDS
~ , senile dementia, etc). TGF~ and its homologs have been
found to be the most Ahl~n~nt ligand for the EGF/TGF~ receptor in
most parts of the brain (Raser, et al., Mol Brain Res: 16:316-322,
(1992)). EBGF or soluble form thereof may also be employed to
treat ocular disorders, for example, corneal inflammation. A
variety of experiments have implicated m~mh~rs of the TGFa gene
family in such pathologies. A recent paper summarizes some of the
data related to the role these growth factors play in eye disease
(Mann, et al, Cell, 73:249-261 (1993)). Recent experiments have
shown that a number of mice lacking the TGF~ gene displayed corneal
inflammation due to an infiltration of leukocytes and other cells
to the substantia propria of the eyes.
In addition, the specificity of certain growth factors for
their target cells can be exploited as a me~h~ni~~ to destroy the
target cell. For example, EEGF or soluble forms thereof can be
coupled, by a wide variety of methods known in the art, to toxic
molecules: for example, a radiopharmaceutical which inactivate
target cells. These growth factor-toxin fusions kill the target
cell (and in certain cases neighboring cells by a variety of
"byst~nA~n effects). A recent example of such toxin-fusion genes
is pllhli~hed by Mesri, et al., J. Biol. Chem. 268:48~3-62 (1993).
~EGF and related molecules may also be encapsulated in liposomes
and may be conjugated to antibodies which recognize and bind to
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tumor or cell specific antigens, thereby provided a means for
"targeting~ cells.
The EBGF polypeptide may also be employed to treat certain
kidney disorders, since it has been found that there has been
expression of these growth factors in the kidney. Thus, these
factors may be necessary for the proper physiological maintenance
of this organ. Treatments may also be related to liver
regeneration or liver dysfunction.
A significant treatment involving EEGF relates to wound
healing. The compositions of the present invention may be employed
for treating a wide variety of wounds including substantially all
cutaneous wounds, corneal wounds, and injuries to the epithelial-
lined hollow organs of the body. Wounds suitable for treatment
include those resulting from trauma such as burns, abrasions and
cuts, as well as from surgical procedures such as surgical
incisions and skin grafting. Other conditions suitable for
treatment with the polypeptide of the present invention include
chronic conditions, such as chronic ulcers, diabetic ulcers, other
non-h~Al ;ng ~trophic) conditions, to treat Marfan syndrome, to
promote hair follicular development, to stimulate growth and
differentiation of various epidermal and epit~eli~l cells in vivo
and in vitro and to st~ te embryogenesis.
E~GF or soluble fragment thereof may be incorporated in
physiologically-acceptable carriers for application to the affected
area. The nature of the carriers may vary widely a.d will depend
on the intended location of application. For application to the
skin, a cream or o~ntm~nt base is usually preferred; suitable
bases include lanolin, Silvadene (Marion) (particularly for the
treatment of burns), Aquaphor (Duke Laboratories, South Norwalk,
Conn.), and the like. If desired, it will be possible to
incorporate EEGF cont~ ni ng compositions in bandages and other
wo~nd dressings to provide for continuous exposure of the wound to
the peptide. Aerosol applications may also find use.
The concentration of EEGF in the treatment composition is not
critical but should be enough to induce epithelial cell
proliferation. ~he compositions may be applied topically to the
affected area, typically as eye drops to the eye or as creams,
ointments or lotions to the skin. In the case of the eyes,
frequent treatment is desirable, usually being applied at intervals
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of 4 hours or less. On the skin, it is desirable to continually
maintain the treatment composition on the affected area during the
healing, with applications of the treatment composition from two
to four times a day or more frequently.
The amount employed of the subject polypeptide will vary with
the m~nner of ~mi ni stration, the employment of other active
compounds, and the like, generally being in the range of about l
~g to 100 ~g. The subject polypeptide may be employed with a
physiologically acceptable carrier, such as saline, phosphate-
buffered saline, or the like. The amount of compound employed will
be determined empirically, based on the response of cells in vitro
and response of ex~perimental ~ni~-l S to the subject polypeptides
or formulations ront~i~tn~ the subject polypeptides.
The EEGF or soluble fragment thereof m~y be employed in the
moA~ tion cr angiogenesis, bone resorption, im~ln~ response, and
synaptic and neuronal effector functions. EEGF may also be used
in the moiulation of the arachidonic acid cascade.
Applic-.ions are also related to alopecia, hair loss and to
other skin conditions which affect hair follicular development.
Several lines of evidence implicate the involvement of growth
factors in such conditions. As described above, "knockoutll mice
engineered to CQntA i n a null mutation in the TGF~ gene display
abnormalities related to quantitative and qualitative hair
synthesis. In addition, mapping studies in mice have shown that
some mutations affecting hair growth map to the TGF~ gene locus
(Mann et al, Cell, 73:249-261~1993)). Topical or systemic
applications of EEGF or derivatives thereof may be employed to
treat some forms of alopecia and hair loss and these claims fall
within the scope of this invention.
Certain disease pathologies may be partially or completely
ameliorated by the systemic clinical ~mi ni stration of the EEGF
growth factor. This ~mtnistration can be in the form of gene
therapy (see below); or through the ~mi~istration of peptides or
proteins synthesized from recomhin~nt constructs of EEGF DNA or
from peptide chemical synthesis (Woo, et al., Protein Engineering
3:29-37 (1989).
This invention provides a method for identification of EEGF
receptors. The gene encoding a receptor can be identified by
numerous methods known to those of skill in the art, for example,
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ligand pAnning and FACS sorting (Coligan, et al., Current Protocols
in Immun., 1~2), Chapter 5, tl991)). Preferably, expression
cloning is employed wherein polyadenylated RNA is prepared from a
cell responsive to EEGF, 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 EEGF. Transfected cells which are
grown on glass slides are exposed to labeled EEGF, which can be
labeled by a variety of means including iodination or inclusion of
a recognition site for a site-specific protein kinase. Following
fixation and incubation, the slides are subjected to auto-
radiographic analysis. Positive pools are identified and sub-pools
are prepared and re-transfected using an iterative sub-pooling and
re-screening 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 l,-e-.~ne or extract preparations that express the
receptor molecule. Cross-linked material is resolved by PAGE and
exposed to X-ray film. The labeled complex cont~ining the ligand-
receptor can be excised, resolved into peptide fragments, and
subjected to protein microse~Pnri n~. The amino acid sequence
obtained from microse~lencing would be used to design a set of
degenerate oligonucleotide probes to screen a cDNA library to
identify the gene ~nCo~in~ the putative receptor.
This invention also provides a method of screening compounds
to identify antagonist compounds to the polypeptide of the present
invention. As an example, a mammalian cell or membrane preparation
expressing a EEGF receptor is incubated with EEGF and a potential
antagonist compound and the ability of the compound to inhihi t a
second signal from the receptor is measured to determine if it is
an effective antagonist. Such second messenger systems include but
are not limited to, cAMP guanylate cyclase, ion ch~nnels or
phosphoinositide hydrolysis.
Another assay for identifying potential antagonists specific
to the receptors to the polypeptide of the present invention is a
competition assay which comprises isolating plasma l.le..~anes which
over-express a receptor to the polypeptide of the present
invention, for example, human A431 carcinoma cells. Serially
diluted test sample in a medium (volume is approximately 10
microliters) containing 10 nM '25I-EEGF is added to five micrograms
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of the plasma membrane in the presence o~ the potential antagonist
compound and incubated for 4 hours at 4~C. The reaction ~ixtures
are diluted and immediately passed throu~h a millipore filter. The
filters are then rapidly washed and the bound radioactivity is
measured in a gamma counter. The amount of bound EEGF is then
measured. A control assay is also performed in the absence of the
compound to determine if the antagonists reduce the amount of bound
EEGF.
Potential antagonist compounds include an antibody, or in some
cases, an oligopeptide, which binds to the polypeptide.
Alternatively, a potential antagonist may be a closely related
protein which binds to the receptor which is an inactive forms of
the polypeptide and thereby prevent the action of the polypeptide
of the present invention.
Another antagonist compound is an antisense construct prepared
using antisense technology. 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 desi~n 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
~ee et al., Nucl. Acids Res., ~:3073 (1979); Cooney et al, Science,
241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),
thereby preventing transcription and the production of the
polypeptide of the present invention. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into the polypeptide of the
present invention (Antisense - Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynuc~eotides 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 DN~ may be expressed in vivo to inhihi t production of the
polypeptide of the present invention.
Antagonist compounds include a small molecule which binds to
the polypeptide of the present invention and blocks its action at
the receptor such that normal biological activity is prevented.
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The small molecules may also bind the receptor to the polypeptide
to prevent hi n~i n~. Examples of small molecules include but are
not limited to small peptides or peptide-like molecules.
The antagonists may be employed to treat neoplasia, for
example, cancers and tumors. It is known that inhibition of
secretion or production of me~hers of the EGF family by tumor cells
in mice causes regression of tumors, since these proteins stimulate
induction of DNA synthesis in all cells including neoplastic cells.
The antagonists to the polypeptides of the present invention
may also be used therapeutically for the treatment of certain skin
disorders, for example, psoriasis. ~levated levels of expression
of mPmhers of this family of growth factors in skin biopsies taken
from diseases such as psoriatic lesions have been found to be
elevated (Cook, et al., Cancer Research, 52:3224-3227 (1992)). The
antagonists may be employed in a composi.~sn with a
pharmaceutiCally acceptable carrier, e.g., as hereinafter
described.
The polypeptides of the present invention or agonist or
antagonist compounds may be employed in comhin~tion with a suitable
pharmaceutical carrier. Such compositions cG...~lise a
therapeutically effective amount of the polypeptide or compound,
and a pharmaceutically acceptable carrier or excipient. Such a
carrier includes but is not limited to sAlinp~ buffered saline,
dextrose, water, glycerol, ethanol, and com~in~tions thereof. The
~ormulation should suit the mode of ~dmi ni stration.
The invention also provides a pharmaceutical pac~ or kit
comprising one or more cont~iners filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such cont~in~r(s) can be a notice in the form
prescribed by a governm~ntAl 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 a~mi ni stration. In addition, the polypeptides or compounds
of the present invention may be employed in conjunction with other
therapeutic compounds.
The pharmaceutical compositions may be administered in a
convenient m~nn~r such as by the oral, topical, intravenous,
intraperitoneal, intramuscular, subcutaneous, intr~n~R~l or
intradermal routes. The pharmaceutical compositions are
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administered in an amount which is effective for treating and~or
prophylaxis of the specific indication. In general, they are
~mi ni stered in an amount of at least about 10 ~g/kg body weight
and in most cases they will be ~mi n i stered in an amount not in
excess of about 8 mg/Kg body weight per day. In most cases, the
dosage is from about 10 ~g/kg to about 1 mg/kg body weight daily,
taking into account the routes of administration, symptoms, etc.
The polypeptides, and agonists and antagonists which are
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.ll
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 in the art and
are apparent from the teachings herein. For example, cells may be
engineered by the use of a retroviral plasmid vector cont~ining RNA
Pnco~i n~ a polypeptide of the present invention.
si~i 1 ~rly, cells may be engineered in VlVO for expression of
a polypeptide in vivo by, for example, procedures known in the art.
~or example, a packaging cell is transduced with a retroviral
rl~mid vector con~nin~ RNA Pnco~in~ a polypeptide of the present
invention such that the packaging cell now produces infectious
viral particles cQntA~ning the gene of interest. These producer
cells may be ~mj nistered to a patient for engineering cells in
vivo and expression of the polypeptide in 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.
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 em~odiment, the retroviral
plasmid vector is derived from Moloney Murine Leukemia Virus.
The vector includes one or more promoters. Suitable promoters
which may be employed include, but are not limited to, the
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retroviral LTR; the SV40 promoter; and the human cytomegalovirus
(CMV) promoter described in Miller, et al., Biotechniques, 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 ~-actin promoters). Other
viral promoters which may be employed include, but are not limited
to, adenovirus promoters, thymidine kinase (TK) promoters, and Bl9
parvovirus promoters. The selection of a suitable promoter will
be apparent to those skilled in the art from the teachings
contained herein.
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 heterologous promoters, such as the cytomegalovirus
tCMV) promoter; the respiratory syncytial virus (RSV) promoter;
inAllcihle promoters, such as the MMT promoter, the metallothionein
promoter; heat shock promoters; the albumin promoter; the ApoAI
promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thymidine kinase promoter; retroviral
LTRs (including the modified retroviral LTRs her~ln~hove
described); the ~-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-19-17-
H2, ~CRE, ~CRIP, GP+E-86, GP+envAml2, and DAN cell lines as
described in Miller, Human Gene Thera~Y, Vol. 1, pgs. 5-14 (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 CaPO4 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.
The producer cell line generates infectious retroviral vector
particles which include the nucleic acid sequence(s) encoding the
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polypeptides. Such retroviral vector particles then may be
employed, to transduce eukaryotic cells, either in vitro or in
vlvo. The transduced eukaryotic cells will express the nucleic
acid sequence(s) 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, f ibroblasts, myoblasts, keratinocytes,
endothelial cells, and broncht~l epithPli~l cells.
This invention is also related to the use of the gene of the
present invention as a diagnostic. Detection of a mutated fonm of
the gene of the present invention will allow a diagnosis of a
disease or a susceptibility to a disease which results f rom
underexpression of the polypeptide of the present invention, for
example, improper wound heAl ing, improper neurological functioning,
ocular disorders, kidney and li~er disorders, hair f ollicular
development, angiogenesis and en~Lyoy~lesis.
Individuals carrying mutations in the human 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, such as from 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 ~n~o~ing a polypeptide of the
present invention can be used to identify and analyze mutations
thereof. For example, deletions and insertions can be detected by
a change in size 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.
Seguence differences between the reference gene and genes
having mutations may be revealed by the direct DNA se~pncing
method. In addition, cloned DNA segments may be employed as probes
to detect specific DNA se~Pnts~ The sensitivity of this method
is greatly ~nh~nced when cQ~ined with PCR. For example, a
seqllencing primer is used with double-stranded PCR product or a
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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.
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
se~l~nce~ may be distinguished on denaturing formamide gradient
gels in which the mn~i ~ i ties 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)).
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 (19~5)).
Thus, the detection of a specific DNA sequence may be achieved
by methods such as hybridization, RNase protection, chemical
cleavage, direct DNA se~)~ncin~ or the use of restriction enzymes,
(e.g., Restriction Fragment Length Polymorph~ F~ (RFLP)) and
SollthPrn blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations can also be detected by in situ analysis.
The present invention also relates to ~ nostic assays for
detecting altered levels of the polypeptide of the present
invention in various tissues since an over-expression of the
proteins comr~red to normal control tissue samples can detect the
presence of certain disease conditions such as neoplasia, skin
disorders, ocular disorders and inflammation. 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 radioimm~lno~csays, competitive-binding assays,
Western Blot analysis and preferably an ELISA assay. An ELISA
assay initially comprises preparing an antibody specific to an
antigen of the polypeptide of the present invention, preferably a
monoclonal antibody. In addition a reporter antibody is prepared
against the monoclonal antibody. To the reporter antibody is
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attached a detectable reagen~ 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 suy~or L, e.g. a
polystyrene dish, that binds the proteins in the sam~le. Any free
protein binding sites on the dish are then covered by incubating
with a non-specific protein such as bovine serum Alhnmin Next,
the monoclonal antibody is incubated in the dish during which time
the monoclonal antibodies attach to any polypeptides of the present
invention attached to the polystyrene dish. All un~ound monoc~Qn~
antibody is washed out with buffer. The reporter Antihody l;nke~
to horseradish peroxidase is now placed in the dish resulting in
hintli ng of the reporter antibody to any monoclonal Antihody bound
to polypeptides of the present invention. UnattAcheA Le~o~Ler
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 protein present in a given
volume of patient sample when compared against a st~n~Ard cur~e.
A competition assay may also be employed to determine levels
of the polypeptide of the present invention in a sample derived
from the hosts. Such an assay comprises isolating plasma membranes
which over-express the receptor for the polypeptide of the present
invention. A test sample cnntAin~ng the polypeptides of the
present invention which have been labeled, are then added to the
plasma membranes and then incubated for a set period of time. Also
added to the reaction mixture is a sample derived from a host which
is suspected of containing the polypeptide of the present
invention. The reaction mixtures are then passed through a filter
which is rapidly washed and the bound radioacti~ity is then
measured to determine the amount of competition for the receptors
and therefore the amount of the polypeptides of the present
invention in the sample.
Antibodies specific to EEGF may be used for cancer diagnosis
and therapy, since many types of cancer cells up-regulate various
members of the TGF~ family during the process of neoplasia or
hyperplasia. These antibodies bind to and inactivate EEGF.
Monnrlonal Antiho~ies against EEGF (and/or its family members) are
in clinical use for both the diagnosis and therapy of certain
disorders including (~ut not limited to) hyperplastic and
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neoplastic growth abnormalities. Upregulation of growth factor
expression by neoplastic tissues forms the basis for a variety of
serum assays which detect increases in growth factor in the blood
of affected patients. These assays are typically applied not only
in diagnostic settings, but are applied in prognostic settings as
well (to detect the presence of occult tumor cells following
surgery, chemotherapy, etc).
In addition, malignant cells expressing the EEGF receptor may
be detected by using labeled ~EGF in a receptor binding assay, or
by the use of antibodies to the EEGF receptor it~elf. Cells may
be distinguished in accordance with the presence and density of
receptors for EEGF, thereby providing a means for predicting the
susceptibility of such cells to the biological activities of EEGF.
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 seguence data (repeat
polymorrhi ,eTnC) are presently av~ hl e for marking chromosomal
location. The mapping of DNAs to chromosomes according to the
present inYention is an important first step in correlating those
sequences with genes associated with disease.
Briefly, sequences can be mapped to c},l~...osomes by preparing
PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis
of the 3' untranslated region of the gene is used to rapidly select
primers that do not span more than one exon in the genomic DNA,
thus complicating the amplification process. These primers are
then used for PCR screening of somatic cell hybrids cont~inin~
individual human chromosomes. Only those hybrids cont~ining the
human gene corresponding to the primer will yield an amplified
fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present in~ention 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 ~-nner. Other mapping strategies that can similarly be
used to map to its chromosome include in si tu hybridization,
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prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to
a meeAph~se chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 50 or 60 bases. For a review of this technique,
see Verma et al., Human Chromosomes: a ~Aml~l of Basic Techniques,
PeL~dl..oll Press, New York (1988).
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, Men~Pl i ~n Inheritance in Man (available
on line through Johns Hopkins University Welch Medical Library).
~he 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).
Next, it is necessary to determine the differences in the cDNA
or genomic se~uence between affected and unaffected individuals.
If a m.utation 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.
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).
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 ~nti~odies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes rhim~ric, single chain, and hnm~nized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures ~nown in the art m.ay be
used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides correspon~i n~
to a se~uence of the present invention can be obtained by direct
injection of the polypeptides into an ~ni ~1 or by administering
the polypeptides to an ~ni~l, preferably a nnnhllm~n. The antibody
so obt~ine~ will then bind the polypeptides itself. In this
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m~nner~ even a seguence encoding only a fragment of the
polypeptides can be used to generate antibodies bi~i n~ the whole
native polypeptides. Such antibodies can then be used to isolate
the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which
provides antibodies produced by continuous cell line cultures can
be used. Bxamples include the hybridoma technique (Kohler and
Milstein, 1975, Nature, 256:49~-497), the trioma technigue, the
human B-cell hybridoma technique (Kozbor et al., 1983, Jm~lnology
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).
Technigues described for the production of single chain
antibodies (U.S. Patent 4,946,778) can be adapted to produce single
chain antibodies to t mmllnogeniC polypeptide products of this
invention. Also, transgenic mice may be used to express humanized
antibodies to immunogenic polypeptide products of this invention.
The present invention will be further described with reference
to the following examples; howe~er, 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.
In order to facilitate underst~n~ng of the following examples
certain frequently occurring methods and/or terms will be
described.
"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
estricted basis, or can be constructed from available plasmids
in accord with published procedures. In addition, eguivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain seguences 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 ~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
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fragments for plasmid construction, typically 5 to 50 ~g 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 37 C 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.
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).
IlOligonucleotides'' 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.
"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 pro~ided, ligation may
be accompl;~;hP~l using known buffers and conditions with 10 units
of T4 DNA ligase ("ligase") per 0.5 ~g of approximately equimolar
amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as
described in the method of Graham, F. and Van der Eb, A., Virology,
52:456-457 (1973).
~xample 1
Bacterial ExDression and Purification of EEGF
The DNA sequence encoding EBGF, ATCC # 97285, is
initially amplified using PCR oligonucleotide primers corresponding
to the 5' sequences of the EEGF protein and the sequences 3' of the
EEGF gene. The 5' oligonucleotide primer has the sequence 5'
GACTTCATGA~ lAACCAAAATGG 3' ~SEQ ID NO:3) contains a Bsp HI
restriction enzyme site followed by 19 nucleotides of E~GF coding
sequence starting from the presumed terminal amino acid. The 3'
sequence 5' GACTGGATCCGAATGGGTACTGCGACACATATATC 3' (SEQ ID NO:4)
contains complementary sequences to a BamHI site and is followed
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by 25 nucleotides of EEGF. The restriction enzyme sites correspond
to the restriction enzyme sites on the bacterial expression vector
pQE-60 (Qiagen, Inc. Chatsworth, CA). pQE-60 ~n~oA~ antibiotic
resistance (Ampr), a bacterial origin of replication (ori), an IPTG-
regulatable promoter operator (P/O), a ribosome binding site (RBS),
a 6-His tag and restriction enzyme sites. pQE-60 is then digested
with BamHI. The amplified sequences are ligated into pQE-60 and
are inserted in frame with the sequence encoding for the histidine
tag and the RBS. The ligation mixture is then used to transform
E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described
in Sambrook, J. et al., Molecular Cloning: A Laboratory ~n~
Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple
copies of the plasmid pREP4, which expresses the lacI repressor and
also confers kanamycin resistance (Kanr). Transformants are
identified by their ability to grow on ~3 plates and
ampicillin/kanamycin resistant colonies are selected. Plasmid DNA
is isolated and confirmed by restriction analysis. Clones
cont~i ni n~ the desired constructs are grown o~ernight (OJN) in
liquid culture in LB media supplemented with both Amp (100 ug/ml)
and Kan (25 ug/ml). The O/N culture is used to inoculate a large
culture at a ratio of 1:100 to 1:250. The cells are grown to an
optical density 600 (O.D.~) of between 0.4 and 0.6. IPTG
("Isopropyl-B-D-thiogalacto pyranoside"~ is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O le~i n~ to increased gene ~-xpression.
Cells are grown an extra 3 to 4 hours. Cells are then harvested
by centrifugation. The cell pellet is solubilized in the
chaotropic agent 6 Molar Guanidine HCl. After clarification,
solubilized EEGF is purified from this solution by chromatography
on a Nickel-Chelate column under conditions that allow for tight
bin~i~g by proteins cont~ining the 6-His tag (Hochuli, E. et al.,
J. Chromatography 411:177-134 (1984)). EEGF is eluted from the
column in 6 molar guanidine HCl pH 5.0 and for the purpose of
renaturation adju~ted to 3 molar guanidine HCl, 100mM sodium
phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione
(oxidized). After incubation in this solution for 12 hours the
protein is dialyzed to 10 mmolar sodium phosphate.
Exam~le 2
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W O 97/38002 PCT~US96/OS247
Cloninq and exDression of EBGF usin~ the baculovirus ex~ression
sYstem
The DNA sequence encoding the EEGF protein, ATCC X 97285, is
amplified using PCR oligonucleotide primers corresp~n~i n~ to the
5' and 3' sequences of the gene.
The primer sequences are as follows: 5' CAGTGGATCCGCCATC
ATGAl~lb~ AACCAAAATGGCG 3' (SEQ ID NO:5), has a BamHI restriction
enzyme site (in bold) followed by 6 nucleotides res~m~ling an
efficient signal for the initiation of translation in eukaryotic
cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) (the initiation
codon for translation is "ATGn).
The 3' primer 5' GCATGGT~CrC~l-C~A&GCTCCAGCCCGAGG 3~ (SEQ ID
NO:6) contains the cleavage site for the re~triction Pn~nnllrlea5e
Asn718 (bold) and 22 nucleotides complementary to the 3~ end of the
~G~ gene. The amplified sequences are isolated from a 1% agarose
gel using a ~om~rcially av~ hle kit ("Geneclean,'l BIO 101 Inc.,
La Jolla, Ca.). The fragment is then digested with the
Pn~on~lCleaseS BamHI and Asp718 and then purified again on a 1%
agarose gel. This fragment is designated F2.
The vector pA2 is used (modification of pVL941 vector,
discussed below) for the expression of the EEGF protein using the
baculovirus expression system (for review see: Summer~ M.D. and
Smith, G.E. 1987, A manual of methods for baculo~ir_~ vectors and
insect cell culture procedures, Texas Agricultural ~xperimental
Station Bulletin No. 1555). This expression ve~tor ~ -nt~i ns the
strong polyhedrin promoter of the Autographa cali__rnica nuclear
polyhedrosis virus (AcMNPV) followed by the recognition sites for
the restriction ~ leases. The polyadenylation site of the
~ n virus (SV)40 is used for efficient polyadenylation. For an
ea~y selection of recombinant virus 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 sequPnces are flanked at both sides by viral
sequences for the cell-mediated homologous recom~in~tion of co-
transfected wild-type viral DNA. Many other baculovirus vectors
could be used such as pAc373, pRG1, pVL941 and pAcIM1 (Luckow, V.A.
and Summers, M.D., Virology, 170:31-39).
The plasmid is digested with the restriction enzymes BamHI and
Asp718 and then dephosphorylated using calf intestinal phosphatase
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PCTAUS96/05247
W O 97/38002
by procedures known in the art. The DNA is then isolated from a
1~ agarose gel using the commercially available kit ("Geneclean~
BI0 ~01 Inc., ~a Jolla, Ca.). This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 are ligated
with T4 DNA ligase. E.coli HB101 cells are then transformed and
bacteria identified that contained the plasmid (p~acEEGF) with the
~EGF gene using the restriction enzymes BamHI and Asp718. The
sequence of the cloned fragment is confirmed by DNA seqllPncing.
5 ~g of the plasmid pBacEEGF is co-transfected 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. USA, 84:7413-7417
(1987)).
l~g of BaculoGold~ virus DNA and 5 ~g of the plasmid pBacEEGF
are mixed in a sterile well of a microtiter plate cont~ining 50 ~l
of serum free Grace's medium (Life Technologies Inc., Gaithersburg,
MD). Afterwards 10 ~l Lipofectin plu~ 90 ~l Grace~s medium are
added, mixed and incubated for 15 minutes at room temperature.
Then the transfection mixture is added drop-wise to the Sf9 insect
cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with
1 ml Grace's medium without serum. The plate is rocked back and
forth to mix the newly added solution. The plate is then incubated
for 5 hours at 27~C. After 5 hours the transfection solution is
removed from the plate and 1 ml of Grace's insect medium
supplemented with 10~ fetal calf serum is added. The plate is put
back into an incubator and cultivation continued at 27~C for four
days.
After four days the supernatant is 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) is used which allows an easy isolation of blue
st~inP~ 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).
Four days after the serial dilution, the virus is added to the
cells and blue st~i nP~ plaques are picked with the tip of an
Bppendorf pipette. The agar cont~i ni n~ the recombinant viruses is
then resuspended in an Eppendorf tube cont~ining 200 ~l of Grace~s
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W O 97/38002 PCTAUS96/05247
medium. The agar is removed by a brief centrifugation and the
supernatant cont~ining the recombinant baculovirus is used to
infect Sf9 cells seeded in 35 mm dishes. Four days later the
supernatants of these culture dishes are harvested and then stored
at 4~C.
Sf9 cells are grown in Grace's medium supplemented with 10~
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus V-EEGF at a multiplicity of infection (MOI) of 2. Six
hours later the medium is removed and replaced with SF900 II medium
minus methionine and cysteine (Life Technoloyies Inc.,
Gaithersburg). 42 hours later 5 ~Ci of 35S-methionine and 5 ~Ci 35S
cysteine (Amersham) are added. The cells are further incubated for
16 hours before they are harvested by centrifugation and the
labelled proteins visualized by SDS-PAGE and autoradiography.
Exam~le 3
Bx~ression of Recombinant REGF in COS cells
The expression of plasmid, EEGF HA is deri~ed from a vector
pcDNA3/Amp (In~itrogen) cont~inin~ 1) SV40 origin of replication,
2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV
promoter followed by a polylinkPr region, an SV40 intron and
polyadenylation site. A DNA fragment ~nco~;ng the entire EBGF
precursor and a HA tag fused in frame to its 3' end is cloned into
the polylinkpr region of the vector, therefore, the reco~inAnt
protein expression is directed under the CMV promoter. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein as previously described (I. Wilson, H. Niman, R. Heighten,
A Cherenson, M. Connolly, and R. T~ern~r~ 1984, Cell 37:767,
(1984)). The infusion of HA tag to the target protein allows easy
detection of the recor~in~nt protein with an antibody that
recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding EEGF, ATCC # 97285, is constructed
by PCR using two primers: the 5~ primer 5'
GACTGGATCCGCCACCATGA~ lAACCAAAATG 3' ~SEQ ID NO:7) contains
a BamHI site (in bold) followed by 6 nucleotides resembling an
efficient signal for the initiation of translation in eukaryotic
cells and 22 nucleotides of EEGF coding sequence starting from the
i n i t i a t i o n c o d o n ; t h e 3 ' s e q u e n c e 5 '
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W O 97/38002 PCTAUS96/05247
GA~-~ r.~A~ ACTGCGACACATAT 3~ (SEQ ID NO:B) contains
complementary sequences to an XbaI site, 22 nucleotides of the EEGF
gene followed by sequences encoding the HA tag. The pcDNA3JAmp
vector contains BamHI/XbaI cloning sites which bring the PCR insert
in frame with the 3' HA tag followed by a stop codon. The PCR
amplified DNA fragment and the vector, pcDNA3/Amp, are digested
with BamHI and XbaI restriction enzyme and ligated. The ligation
mixture is transformed into E. coli strain SURE (avAilAhle from
Stratagene Cloning Systems, La Jolla, CA 92037) the transformed
culture is plated on ampicillin media plates and resistant colonies
are selected. Plasmid DNA is isolated from transformants and
P~AminP~ by restriction analysis for the presence of the correct
fragment. For expression of the reComhinAnt EFGF, COS cells are
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 EBGF HA protein is detected by radiolabelling and
i~mllnoprecipitation method (E. Harlow, D. Lane, Antibodies: A
Laboratory ~Anll~l, Cold Spring Harbor Laboratory Press, ~1988)).
Cells are lAhPlled for 8 hours with 35S-cysteine two days post
transfection. Culture media is then collected and cells are lysed
with detergent (RIPA buffer (150 mM NaCl, 1~ NP-40, 0.1% SDS, 1%
NP-40, 0.5% DOC, 50mM Tris, pH 7.5) ~Wilson, I. et al., Id. 37:767
(1984)). Both cell lysate and culture media are precipitated with
an HA specific monoclonal antibody. Proteins precipitated are
analyzed on 15% SDS-PAGE gels.
Exam~le 4
ExDression via Gene Thera~
Fibroblasts are obtAinP~ from a subject by skin biopsy. The
resulting tissue is placed in tis~ue-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~ F~S, penicillin and streptomycin, is
added. This is then incubated at 37~C for approximately one week.
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W O 97/38002 PCT~US96/05247
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.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked
by the long terminal repeats of the Moloney murine sarcoma virus,
is digested with EcoRI and ~indIII and subsequently treated with
calf intestinal phosphatase. The ~n~r vector is fractionated on
agarose gel and purified, using glass beads.
The cDNA ~nco~i ng 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 HindIII site. Equal quantities
of the Moloney murine sarcoma virus ~lne~r backhonP and the
amplified EcoRI and HindIII fragment are added together, in the
presence of T4 DNA ligase. The resulting mixture is maint~ine~
under conditions appropriate for ligation of the two fragments.
The ligation mixture is used to transform bacteria H~101, which are
then plated onto agar-contAjning kanamycin for the purpose of
confirming that the vector had the gene of interest properly
inserted.
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 c~nt~ining 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
cont~ning the gene (the packaging cells are now referred to as
producer cells).
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, cont~ini ng 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
CA 02249213 1998-09-17
W097t38~2 PCT~S96/05247
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his.
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.
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.