Note: Descriptions are shown in the official language in which they were submitted.
-
CA 02221821 1997-11-21
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k~.~G~ T~T-MONOC~rE ACTIVATlNG poLyr~r~lv~ III
This invention relates to newly identified
polynucleotide~, polypeptides ~ncoded 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 has been putatively
identified as an endoth~ monocyte activating polypeptide
III, sometimes hereinafter referred as "EMAP III". The
invention also relates to ;nh; h; ting the action of such
polypeptides.
Tmmllnogenic tumors such as the ~urine meth A
fibrosarcoma, characteristically have a peripheral zone which
~ontA~n~ a chronic inflammatory infiltry ~Dvorak, H., New
~ngl. J. Med., 315:1650-1658 (1986)). The presence of these
inflammatory cells, often embedded in a mesh work of fibrin
which can extend throughout the tumor stroma, contributes to
the concept that tumors might be considered wounds that do
not heal (Id.). There have been ;~nt; f ied tumor-derived
mediators which prime the host response, altering endoth~
properties, and attracting inflammatory cells to the tumor.
Empa I is a trypsin-sensitive, d~' o~imately 40 kda
polypeptide distinct from other cytokines and growth factors,
and which could activate endoth~ cells and monocytes.
--1--
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Another polypeptide has been identified which is the murine
homologue of vpf/vegf, a factor which had previously been
shown to increase vascular pe~ h;l; ty and to be mytogenic
for endothel;~l cells. Recently, another polypeptide has
been identified in supernatants of meth A tumor cells (EMAP
II). EMAP II activates endothelial cells (ECs) and
onllClear cells potentiating their participation in
procoagulant reactions through induction of tissue factor,
promoting migration of monocytes and polymorphonllclear
leukocytes, and leading to a phlogogenic response when
injected into murine foot pads. EMAP II is an apparently
unique polypeptide which runs as a broad band.
The polypeptide of the present invention has been
putatively identified as a EMAP III as a result o~ amino acid
sequence homology to EMAP II.
In accordance with one aspect of the present invention,
there is provided a novel mature 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.
In accordance with another aspect of the present
invention, there are provided isolated nucleic acid molecules
~ncoA; ng a polypeptide of the present invention including
mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and
biologically active and diagnostically or therapeutically
useful fragments thereof.
In accorA~nce with yet a further aspect of the present
invention, there is provided a process for proAllc~ ng such
polypeptide by recombinant techniques comprising culturing
recombinant prokaryotic and/or eukaryotic host cells,
contA;n;ng a nucleic acid sequence encoding a polypeptide of
the present invention, under conditions promoting expression
of said protein and subsequent recovery of said protein.
In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such
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polypeptide, or polynucleotide encoding such polypeptide for
therapeutic purposes, for example, neoplasia such as tumors
in cancer.
In accordance with yet a further aspect of the present
invention, there are provided antibodies against such
polypeptides.
In accordance with another aspect of the present
invention, there are provided agonists which mimic EMAP III
and bind to the BMAP III receptors to elicit responses.
In accordance with yet a further aspect of the present
invention, there is also provided nucleic acid probes
comprising nucleic acid molecules of suf ficient length to
specifically hybridize to a nucleic acid sequence of the
present invention.
In accordance with still another aspect o~ the present
invention, there are provided diagnostic assays for de~ecting
diseases or susceptibility to diseases related to mutations
in the nucleic acid sequences encoding a polypeptide of the
present invention.
In acco~allce 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, for
example, 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.
The following drawings are illustrative of embod;m~nt~
of the invention and are not meant to limit the scope of the
invention as encompassed by the rl ~
Figure 1 is an illustration Of the cDNA and
corresponding deduced amino acid sequence of the polypeptide
of the present invention. Se~l~nci ng was performed using a
373 automated DNA sequencer (Applied Biosystems, Inc.).
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Figure 2 is an amino acid sequence comparison between
the polypeptide of the present invention (top line) and EMAP
II (bottom line).
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 seguence of Figure 1 (SEQ ID NO:2) or for the
mature polypeptide encoded by the cDNA of the clone deposited
as ATCC Deposit No. on May 26, 1995.
The polynucleotide of this invention was discovered in
a cDNA library derived from resting T-cells. It cont~inC an
open reading frame encoding a protein of 168 amino acid
residues. The 168 amino acid sequence represents the active
~n~; n of EMAP III which is derived from a prosequence which
has been proteolytically cleaved. The protein exhibits the
highest degree of h ~ology to EMAP II with 60 % identity and
75 ~ similarity over a 150 amino acid stretch. The coding
sequence of Figure 1 (SEQ ID NO:2) illustrates the active
~n~-; n of the polypeptide and the polypeptide may comprise
additional amino acid residues. Although the polypeptide of
the present invention is not thought to have a leader
sequence, it is a secreted protein.
The polynucleotide 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 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 ~ncoAes the mature polypeptide may be
identical to the ~oAi ng sequence shown in Figure 1 (SEQ ID
NO:1) or that of the deposited clone or may be a different
~o~; ng sequence which coding se~uence, as a result of the
r~AllnA~ncy or degeneracy of the genetic code, ~nCoAe~ the
same mature polypeptide as the DNA of Figure 1 (SEQ ID NO:1)
or the deposited cDNA.
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The polynucleotide which encodes for the mature
polypeptide of Figure 1 (SEQ ID NO:2) or ~or the mature
polypeptide encoded by the deposited cDNA may include, but is
not limited to: only the coding sequence for the mature
polypeptide; the coding sequence for the mature polypeptide
and additional coding sequence such as a se~retory sequence
or a ~lo~otein sequence; the co~i n~ 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 ~or 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
her~n~hove described polynucleotides which encode for
fragmen~s, analogs and derivatives of the polypeptide having
the ~le~ ce~ amino acid sequence of Figure 1 ( SEQ ID NO:2) or
the polypeptide PnCo~e~ by the cDNA of the deposited clone.
= The variant of the polynucleotide may be a natural~y
occurring allelic variant of the polynucleotide or a non-
naturally oc~ling variant of the polynucleotide.
Thus, the pre~ent invention includes polynucleotides
encoding the same mature polypeptide as shown in Figure 1
(SEQ ID NO:2) 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 Figure 1 ( SEQ ID
NO:2) or the polypeptide encoded by the cDNA of the deposited
clone. Such nucleotide variants include deletion variants,
substitution variants and addition or insertion variants.
As her~;nAhove 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
..
--5--
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NO:1) 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 addition of one or more nucleotides, which does
not substantially alter the ~unction of the encoded
polypeptide.
The present invention also includes polynucleotides,
wherein the coding seauence for the mature polypeptide may be
fused in the same reading frame to a polynucleotide sequence
which aids in expression and secretion of a po~ypeptide from
a host cell, for example, a secretory sequence for
controlling transport of a polypeptide from the cell. The
polynucleotides may also encode for a proproteln 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 remains. T hus, for
example, the polynucleotide o~ the present invention may
encode for a m~ature protein, or for a protein having a
prosequence.
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 tHA) tag when a m-mm~ n
host, e.g. COS-7 cells, is used. The HA tag corresponds to
an epitope derived from the influenza hA~-ggll-t;nin protein
(Wilson, I., et al., Cell, 37:767 (1984)).
The present invention further relates to
polynucleotides which hybridize to the herein~hove-described
sequences if there is at least 70%, preferably at least 90%,
and more preferably at least 95% identity between the
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WO 96/40719 PCTrUS95/07328
sequences. The present invention particularly relates to
polynucleotides which hybridize under stringent conditions to
the her~tnAhove-described polynucleotides. As herein used,
the term ~stringent conditions~ 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 herP~n~hove described polynucleotides
in a preferred em.bo~;~~nt encode polypeptides which either
retain subst~nti~lly the same biological function or activity
as the mature polypeptide encoded by the cDNAs of Figure 1
(SEQ ID NO:1) or the deposited cDNA(s).
Alternatively, the polynucleotide ,m,~y have at least 20
bases, preferably 30 bases, and more preferably at least 50
bases which hybridize to a polynucleotide of the preseht
invention and which has an identity thereto, as hereinabove
described, and which may or may not retain activity. For
example, such polynucleotides ,m,~y be employed as probes for
the polynucleotide of SEQ ID NO:l, for exam.ple, for recovery
of the polynucleotide or as a diagnostic probe or as a PCR
primer.
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
as well as fragments thereof, which fragm~nts have at least
- 30 bases and preferably at least 50 bases and to polypeptides
encoded by such polynucleotides.
The deposit(s) referred to herein will be m~;nt~inP~
under the terms of the Budapest Treaty on the International
Recognition of the Deposit of Micro-or~nt~m~ for purposes of
Patent Procedure. 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 cont~ i n~ in the
deposited materials, as well as the amino acid sequence of
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the polypeptides encoded thereby, are incorporated herein by
reference and 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.
The present invention further relates to a polypeptide
which has the ~ P~ amino acid sequence of Figure 1 (SEQ ID
NO:2) or which has the amino acid sequence encoded by the
deposited cDNA, as well as fragments, analogs and derivatives
of such polypeptide.
The terms "fragment," ''derivativell and 'lanalog'l when
referring to the polypeptide of Figure 1 (SEQ ID NO:2) or
that encoded by the deposited cDNA, means a polypeptide which
retains essentially the same biological function or activ~ty
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
reco~h;n~nt polypeptide, a natural polypeptide or a synthetic
polypeptide, preferably a rec ~n~nt polypeptide.
The fragment, derivative or analog of the polypeptide
of Figure 1 (SEQ ID NO:2) or that encoded by the deposited
cDNA may be (i) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved
amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may
not be one encoded by the genetic code, or (ii) one in which
one or more of the amino acid residues includes a substituent
group, or (iii) one in which the mature polypeptide is fused
with another l_ lo~.d, such as a c~,.,~ound 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 secretory sequence
or a sequence which is employed for purification of the
mature polypeptide or a ~.otein sequence. Such fra~r~nts,
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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 homogeneity.
The term "isolated" means that the material is removed
from its original envi~ t (e.g., the natural environm~nt
if it is naturally occurring). For example, a naturally-
occurring polynucleotide or polypeptide present in a living
~nim~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 envi-o~ Lel~t.
The present invention also relates ~o 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 rers~hin~nt techniques.
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 a~L~Liate 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.
~The polynucleotides of the present invention may be
= employed for pro~llr; n~ polypeptides by reromhinAnt
_g_
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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,
non~hromosomal 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.
The d~~ iate DNA sequence may be inserted into the
vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction
~n~onnclease 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 is 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 ~ Ler, the E. coli. lac or trD, the phage l~mh~ 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 b; n~; ng site
for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for
amplifying expression.
In addition, the expression vectors preferably C~nt~ n
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 ~. coli.
The vector cont~; n; ng the a~Lu~riate DNA sequence as
herP;n~hove described, as well as an appropriate promoter or
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control sequence, may be emyloyed to trans~orm an appropriate
host to permit the host to express the protein.
A8 representative examples of appropriate hosts, there
may be mentioned: bacterial cells, such as E. coli,
Stre~tomYces, Salmonell~ tV~him~lrium; fungal cells, such as
yeast; insect cells such as Droso~hila S2 and S~odoptera Sf9;
~ni m~ 1 cells ~uch as CH0, 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 ~rom the teachings herein.
More particularly, the present invention also includes
recombinant constructs comprising one or more of the
sequences as broadly de~cribed 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
~orward or reverse orientation. In a preferred aspect o~ this
embodiment, the construct ~urther comprises regulatory
seguences, 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
c~ ~rcially av~ hle~ 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); ptrc99a, pKK223-
3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNE0,
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
ho~t.
Promoter regions can be selected from any desired gene
using CAT (chloramphenicol transferase) vectors or other
vector~ with selectable markers. Two ayyloy iate vectors are
pK~232-8 and pCM7. Particular named bacterial promoters
include lacI, lacZ, T3, T7, gpt, l~mh~l~ PR, PL and trp.
Eukaryotic promoters include CMV ;~ te early, HSV
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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 o~ ordinary
skill in the art.
In a further Pmho~imPnt~ the present invention relates
to host cells C~ntAt n; ng the above-described constructs. The
host cell can be a higher eukaryotic cell, such as a
m~m~l ian cell, or a lower eukaryotic cell, such as a yeast
cell, or the host cell can be a pro~aryotic 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 electroporatlon (Davis, L.,
Dibner, M., Battey, I., Basic Methods in Molecular Biology,
(1986)).
The constructs in host cells can be used in a
conventional m~nn~r to produce the gene product encoded by
the recombinant sequence. Alternatively, the polypeptides of
the invention can be synthetically produced by conventional
peptide synthesizers.
Mature proteins can be expressed in mAmm~A1; An cells,
yeast, bacteria, or other cells under the control of
a~lu~Liate ~l~...oLers. Cell-free translation systems can
also be employed to produce such proteins using RNAs derived
from the DNA constructs of the present invention.
A~l~riate cloning and expression vectors for use with
prokaryotic and eukaryotic hosts are described by Sa,.~.ook,
et al., Molecular Cloning: A Laboratory MA~11~A 1~ Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclûsure of
which is hereby incorporated by reference.
Transcription of the DNA F~n~orl; ng the polypeptides of
the present invention by higher eukaryotes is increased by
inserting an Pnh~Ancer sequence into the vector. Fnh~ncers
are cis-acting eli- nts of DNA, usually about from 10 to 300
bp that act on a promoter to increase its transcription.
Examples include the SV40 PnhAncP~ on the late side of the
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replication origin bp 100 to 270, a-cytomegalovirus early
promoter ~nh~ncer~ the polyoma ~nhAncer on the late side of
the replication origin, and adenovirus ~nh~ncers~
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. coli and S. cerevisiae TRP1 gene, and
a promoter derived ~rom 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
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
rec~;nAnt product.
Useful expression vectors for bacterial use are
constructed by inserting a structural DNA sequence ~nCo~;ng
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 E. coli,
Bacillus subtilis, SA1m~7ne11A tvr-h;~l~ium and various species
within the genera Pse~l~nmonAc, Streptomyces, and
Staphylococcus, although others may also be employed as a
O matter of choice.
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As a representative but nonl ;mi ting example, useful
expression vectors for bacterial use can comprise a
selectable marker and bacterial origin of replication derived
from c -rcially available plasmids comprising genetic
el~m~nts of the well known cloning vector pBR322 (ATCC
37017). Such cor~ercial vectors include, for example,
pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1
(Promega Biotec, Madison, WI, USA). These pBR322 ~backbone~
sections are combined with an appropriate 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 centri~ugation,
disrupted by physical or chemical means, and the resulting
crude extract ret~;n~ 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 m~mm~l ian cell culture systems can also be
employed to express reromh;n~nt protein. Examples of
~m~-l ian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described by Gluzman, Cell, 23:175
(1981), and other cell lines ~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 ~nh~ncer~
and also any necessary ribo~ome h; n~l;ng sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5~ fl~nk;ng
nontranscribed se~l~nc~. DNA sequences derived from the
-14-
.
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SV40 splice, and polyadenylation sites may be used to provide
the required nontranscribed genetic elements.
The polypeptide can be recovered and purified from
- recomh;n~nt cell cultures by methods including ~mmoni um
sulfate or ethanol precipitation, acid extraction, anion or
cation exchange chromatography, phosphocellulose
chromatography, hyJl~h~;c interaction chromatography,
affinity chromatography, l~ydLux~lapatite chromatography and
lectin chromatography. Protein refolding steps can be used,
as necessary, in co~pleting configuration o~ the mature
protein. Finally, high performance liquid chromatography
(HPLC) can be em.ployed for final purification steps.
The polypeptides of the present invention may be a
naturally puri~ied product, or a product of chemical
synthetic procedures, or produced by recom.binant techniques
from a prokaryotic or eukaryotic host (~or example, by
bacterial, yeast, higher plant, insect and m~mm~lian cells in
culture). Depending upon the host employed in a reco~mhi n~nt
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 BMAP III polypeptide of the present invention may be
employed to regress neoplasia, such as tumors in cancers.
The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials
for discovery of treatm~ntc and diagnostics to human disease.
This invention provides a method for identification of
the receptor for BMAP III. The gene ~nco~; ng the receptor
can be identified by numerous methods known ~o those of skill
in the art, for example, ligand p~nn; ng and FACS sorting
(Coligan, et al., Current Protocols in Immun., 1(2), Chapter
5, (1991)). Preferably, expression cloning is employed
wherein polyadenylated RNA is prepared from a cell responsive
to EMAP III, and a cDNA library created from this RNA is
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divided into pools and used to transfect COS cells or other
cells that are not responsive to EMAP III. Transfected cells
which are grown on glass slides are exposed to labeled EMAP
III. EMAP III 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 vielding a single clone that
encodes the putative receptor. As an alternative approach
for receptor itlf~nt; fication, 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 ligand-receptor can
be excised, resolved into peptide fragments, and subjected to
protein miCroSe~lPn-~ing The amino acid sequence obtained
from microse~l~nctng would be used to design a set of
degenerate oligonucleotide probes to screen a cDNA library to
identify the gene encoding the putative receptor.
This in~ention provides a method of screening compounds
to identify those which ~nhAnce (agonists) the biological
action EMAP III. As an example, a , -lian cell or membrane
preparation expressing the EMAP III receptor is incubated
with a lAh~lled compound. The ability of the compound to
bind to the EMAP III receptor is then measured. The ability
to bind to the receiptors is measured by the response of a
known second messenger system following interaction of the
cmpound and the receiptor. Such second messenger systems
include but are not limited to cAMP guanylate cyclase, ion
chAnnel~ or phosphoinositide hydrolysis.
The polypeptides and agonists of the present invention
may be employed in co~;nAtion with a suitable pharmaceutical
carrier. Such compositions co...~lise a therapeutically
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effective amount of the polypeptide or agonist, 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
~mi n i ~tration-
The invention also provides a pharmaceutical pack or kit
comprising one or more cont~in~rS 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 governmental agency regulating
the manufacture, use or sale o~ pharmaceuticals or biological
products, which notice reflects approval by the agency of
manufacture, use or sale for human ~mi ni stration. In
addition, the polypeptides or agonists of the present
invention may be employed in conjunction with other
therapeutic cc _,o~ds.
The pharmaceutical compositions may be a~m;nistered in
a convenient ~-nn~r such as by the oral, direct injection,
parenterally, intravenous, intraperitoneal, intramuscular,
subc~lt~neous, intr~n~l or intradermal routes. The
pharmaceutical compositions are ~m; ni ~tered in an amount
which is effecti~e for treating and/or prophylaxis o~ the
specific indication. In general, they are ~m;ni~tered in an
amount of at least about 10 ~g/kg body weight and in most
cases they will be ~mi n; ctered 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 ~min; stration, symptoms,
etc.
The EMAP III polypeptide~ and agonists 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."
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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 t~Achings herein. For example, cells may be engineered
by the use of a retroviral plasmid vector cont~ining RNA
encoding a polypeptide o~ the present invention.
S;m; l~rly, cells may be engineered in vivo for
expression of a polypeptide in vivo by, f or example,
procedures known in the art. For example, a packaging cell
is transduced with a retroviral plasmid vector containing RNA
encoding a polypeptide of the present lnvention such that the
packaging cell now produces infectlous vlral particles
cont~ining the gene of interest. These producer cells may be
AAmi ni ~tered to a patient for engineering cells in vivo and
expression of the polypeptide in vivo. These and other
methods for ~Aministering 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
her~in~hove 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 immllnodeficienCy virus, adenovirus,
Myeloproliferative Sarcoma Virus, and ~-mm~ry tumor virus.
In one emboAi - t, the retroviral plasmid vector is derived
~rom Moloney Murine Leukemia Virus.
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.,
Biotechniaues, Vol. 7, No. 9, 980-990 (1989), or any other
promoter (e.g., cellular promoters such as eukaryotic
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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 B19 parvovirus promoters. The selection o~ a suitable
promoter will be apparent to those skilled in the art ~rom
the teachin~s rontAine~ 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 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 ~-hllmin promoter; the ApoAI promoter; human
globin promoters; viral thymidine kinase promoters, such as
the Herpes Simplex thymidine kinase promoter; retroviral LTRs
(including the modi~ied retroviral LTRs here;nAhove
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 em~loyed to trAn~Allce
packaging cell lines to ~orm producer cell lines. Examples
o~ packaging cells which may be trans~ected 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 Therapy, Vol. 1,
pgs. 5-14 ~1990), which is incorporated herein by re~erence
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, electl~o~dtion, the use o~
liposomes, and CaPO4 precipitation. In one alternative, the
retroviral plA~mi~ vector may be encapsulated into a
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liposome, or coupled to a lipid, and then ~mi ni stered to a
host.
The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles
then may be employed, to transduce eukaryotic cells, either
in vitro or in vivo. 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,
e~ yOlliC carr; nom~ cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothPl;?l cells, and bronchiAl epithelial cells.
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 EMAP III.
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
obt~n~ 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
compl- - t~y to the nucleic acid encoding EMAP III can be
used to i-l~nt~fy and analyze mutations. For example,
deletions and insertions can be detected by a change in size
of the amplified product in comparison to the nonmal
genotype. Point mutations can be identified by hybridizing
amplified DNA to radiolabeled RNA or alternatively,
radiolabeled antisense DNA sequences. Perfectly matched
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~equences can be distin~lishe~ from mismatched duplexes by
RNase A digestion or by differences in melting temperatures.
Sequence differences between the reference gene and
- genes having mutations may be revealed by the direct DNA
se~lPnc;ng method. In addition, cloned DNA segments may be
employed as probes to detect speci~ic DNA segments The
8ensitivity of this method is greatly Pnh~nced 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
detenmination 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 fra~nts in gels with or without denaturing
agents. Small sequence deletions and insertions can ~e
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 a~ 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 Sl
protection or the chemical cleavage method (e.g., Cotton et
al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA se~l~ncing or the use of
restriction enzymes, (e.g., Restriction Fragment Length
Polymorphisms (RFLP)) and Sollth~rn blotting of genomic DNA.
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In addition to more conventional gel-electrophoresis and
DNA se~7~ncing~ mutations can also be detected by in situ
analysis.
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 co~r~Ared to normal control tissue samples can
detect the presence of EMAP III 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 radioimmunoassays, competitive-binding
assays, Western Blot analysis and preferably an ELISA assay.
An ELISA assay initially comprises preparing an antibody
specific to the EMAP III 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 ~1 hl-m;n, Next, the monoclonal
Ant;hoAy iS incubated in the dish during which time the
monoclonal Ant~hoA;es attached to any of the polypeptide of
the present invention attached to the polystyrene dish. All
unbound monoclonal antibody is washed out with buffer. The
reporter antibody l; nk~A to horseradish peroxidase is now
placed in the dish resulting in htnA~ng of the reporter
antibody to any monoclonal antibody bound to the polypeptide
of the present invention. UnattAch~A 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
CA 02221821 1997-ll-21
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present invention present in a given volume of patient sample
when compared against a stAn~Ard curve.
A competition assay may be employed wherein antibodies
specific to the polypeptide of the present invention are
attached to a solid support and labeled EMAP III and a sample
derived from the host are passed over the s~lid support and
the amount of label detected attAch~ to the solid support
can be correlated to a quantity of the polypeptide of the
present invention in the sample.
The se~lPnr~c o$ the present invention are also valuable
for chromosome identification. The seguence 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
c~ rome marking reagents based on actual sequence data
(repeat polymor~hismc) are presently a~ailable 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.
Briefly, se~lenc~s can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than
one exon in the genomic DNA, thus complicating the
amplification process. These primers are then used $or PCR
screening of somatic cell hybrids cont~ining individual human
chromosomes. Only those hybrids contAining the human gene
corresponA;ng to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure
for asr-igning a particular DNA to a particular chromosome.
Using the present invention with the same oligonucleotide
primers, sublocalization can be achieved with pAnPls of
fragments from specific chromosomes or pools of large genomic
clones in an analogous ~~nn~ Other mapping strategies that
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can s;m;l~rly 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.
Fluorescence in situ hybridization (FISH) of a cDNA
clone to a met~phA~e chromosomal spread can be used to
provide a precise ch ~somal 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 Venma et al.,
Human Chro~fiomP~: a Manual of Basic Techniques, Pely
Press, New York (1988).
Once a se~uence has been mapped to a preclse chromosomal
location, the physical position of the sequence on the
chromosome can be correlated with yenetic map data. Such
data are found, for example, in V. McKusick, MPn~elian
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 identi~ied through linkage
analysis (coinheritance of physically adjacent genes).
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.
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 potPnt;~l causative genes. (This
assumes 1 megabase mapping resolution and one gene per 20
kb).
The polypeptides, their fra~m~nts or other derivatives,
or analogs thereof, or cells expressing them can be used as
an ;mmllnogen to produce antibodies thereto. These Ant;ho~;es
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can be, for example, polyclonal or monoclonal antibodies.
The pre~ent invention also includes ~him~ric~ single chain,
and h~ n;zed Antiho~;es, as well as Fab fragments, or the
~ product of an Fab expression library. various procedures
known in the art may be used for the production of such
antibodies and fra~ - t~.
Antibodies generated against the polypeptides
corresponding to a sequence of the present invention can be
obtA;n~A by direct injection of the polypeptides into an
An~m~l or by ~m;n~ stering the polypeptides to an ~n;m~l,
preferably a nonhllman. The antibody so obtained will then
bind the polypeptides itself. In this manner, even a
sequence ~nco~tng 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.
For preparation of monoclonal antibodies, any technique
which provides ~nt;hodies produced by continuous cell line
cultures can be used. Examples include the hybridoma
technique (Kohler and Milstein, 1975, Nature, 256:49s-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 Ant;ho~tes
(Cole, et al., 1985, in Monoclonal ~nt;ho~;es and r~nc~
Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain
antibodies (U.S. Patent 4,946,778) can be adapted to produce
single chain Ant; hodies to ;~lnngenic polypeptide products
of this invention. Also, transgenic mice may be used to
expres~; h- nt zed ~nt;ho~l;es to i ~lnogenic polypeptide
products of this invention.
The present invention will be further described with
re~erence to the following examples; however, it is to be
understood that the present invention is not limited to such
CA 02221821 1997-11-21
W O 96/40719 PCTAJS95/07328
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 pla~mids herein are either commercially available,
publicly aVAi 1 ~hl e on an unrestricted basis, or can be
constructed from av~ hl e 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.
~ 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 ~g of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ~l
of buf$er solution. For the purpose of isolating DNA
fra_ ts 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).
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~ Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two com.plementary 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 ~ragment ~hat has not been
APp~nsphorylated.
"Ligation" refers to the process of forming
phosphodiester bonds between two double stranded nucleic acid
fragm~n~s (Maniatis, T., et al., Id., p. 146). Unless
otherwise provided, ligation may be accomplished using known
buffers and conditions with 10 units o~ T4 DNA ligase
("ligase") per 0.5 ~g of approximately equimolar amounts of
the DNA fra3~~nts 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).
Example 1
Bacterial Ex~ression and Purification of EMAP III
The DNA sequence encoding EMAP III, ATCC # _, is
initially amplified using PCR oligonucleotide primers
corresponAing to the 5' seqn~nc~s of the processed EMAP III
protein (minus the signal peptide sequence) and the vector
sequences 3' to the EMAP III gene. Additional nucleotides
corresron~;ng to EMAP III were added to the 5' and 3'
sequences respectively. The 5' oligonucleotide primer has
the sequence 5' GATCGGATCCGAGr~Gr-TCATCCCATCC 3' (SEQ ID NO:3)
r~nt~;n~ a BamHI restriction enzyme site followed by EMAP III
ro~;ng ~equence starting from the initial amino acid (Glu) of
the processed protein. The 3' sequence 5' GATCAAGCTTC
T~r.~T~A~ 3~ (SEQ ID NO:4) cnnt~;nc compl~m~nt~ry
sequences to HindIII and is followed by nucleotides of EMAP
III coding sequence starting from the terminal amino acid.
c
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The restriction enzyme sites correspond to the restriction
enzyme sites on the bacterial expression vector pQE-g
(Qiagen, Inc., Chatsworth, CA, 91311). pQE-9 encodes
antibiotic resistance (Ampr), a bacterial origin of
replication (ori), an IPTG-regulatable promoter operator
(P/O), a ribosome h~n~;ng site (RBS), a 6-His tag and
restriction enzyme sites. pQE-9 was then digested with BamHI
and HindIII. The amplified sequences were ligated into pQE-9
and were inserted in frame with the sequence encoding for the
histidine tag and the RBS. The ligation mixture was 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 Manual, Cold Spring Laboratory Press,
(1989). M15/rep4 contains multiple copies of the plas~id
pREP4, which expresses the lacI repressor and also confers
kanamycin resistance (Kanr). Transformants are identified by
their ability to grow on LB plates and ampicillin/kanamycin
resistant colonies were selected. Plasmid DNA was isolated
and confirmed by restriction analysis Clones contAining the
desired constructs were grown overnight (O/N) in liquid
culture in LB media supplemented with both Amp (100 ug/ml)
and Kan (25 ug/ml). The 0/N culture is used to inoculate a
large culture at a ratio of 1:100 to 1:250. The cells were
grown to an optical density 600 (o.D.600) of between 0.4 and
0.6. IPTG ("Iso~L~l-B-D-thiogalacto pyranoside") was then
added to a final concentration of 1 mM. IPTG induces by
inactivating the lacI repressor, clearing the P/0 leading to
increased gene expression. Cells were grown an extra 3 to 4
hours. Cells were then harvested by centrifugation. The
cell pellet was ~olubilized in the chaotropic agent 6 Molar
Guanidine HCl. After clarification, solubilized EMAP III was
purified from this solution by chromatography on a Nickel-
Chelate column under conditions that allow for tight b; n~; ng
by proteins contA~n~ng the 6-His tag (Hochuli, E. et al., J.
~hromatography 411:177-184 (1984)). EMAP III protein (~90%
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W O 96/~0719 PCT~US9~/07328
pure) was eluted from the column at pH 5.0 and was pooled and
dialyzed versus decreasing concentrations of GnHCl, and then
finally into a buffer contAinlng 20 mM Tris HCl pH 8.0; 59 mM
NaCl; 0.1 ~ w/v octyl-~-glucoside. The concentration of
soluble protein was determined using a BioRad protein assay,
and bacterial LPS c~nt~m~n~tion assayed.
Rxam~le 2
Cloninq and exPression of EMAP III usina the baculovirus
expression system
The DNA sequence encoding the ~ull lenyth EMAP III
protein, ATCC # , iS amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5~
GATCGGATrCr-A~J--AG~,TCATCCCATCC 3' (SEQ ID NO:5) and contains a
BamHI restriction enzyme site (in bold) the first 18
nucleotides of the EMAP III gene.
The 3' primer has the sequence 5' GATCGGATCCCTA
GATAA~ C 3' (SEQ ID NO:6) and contains the cleavage
site for the restriction ~n~onllclease BamHI and 18
nucleotides compl; ~ t~ry to the 3' sequence of the EMAP III
gene. The am~lified sequences are isolated from a 1~ agarose
gel using a commercially av~ hle kit ("Geneclean,ll BIO 101
Inc., La Jolla, Ca.). The fragment is then digested with the
en~onllclease BamHI and purified again on a 1~ agarose gel.
This fragment i8 designated F2.
The vector pRG1 (modification of pVL941 vector,
discussed below) is used for the expression of the EMAP III
protein using the baculovirus expression system (for review
see: Summers, M.D. and Smith, G.E. 1987, A mAnll~l of methods
for baculovirus vectors and insect cell culture procedures,
Texas A~ricultural Exper~ -nt~l Station Bulletin No. 1555).
This expression vector contains the strong polyhedrin
promoter of the Autographa californica nuclear polyhedrosis
virus (~cMNPV) followed by the recognition sites for the
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restriction ~n~nnllclease BamHI. The polyadenylation site of
the simian virus (SV)40 is used for efficient
polyadenylation. For an easy selection of recombinant virus
the beta-galactosidase gene from E.coli is inserted in the
same orientation as the polyhedrin promoter ~ollowed by the
polyadenylation signal of the polyhedrin gene. The
polyhedrin se~l~nc~ are flanked at both sides by viral
sequences for the cell-mediated homologous recombination of
co-transfected 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).
The plasmid is digested with the restriction enzyme
BamEI and dephosphorylated using calf intestinal phosphatase
by procedures known in the art. The DNA is then isolated
from a 1~ agarose gel using the commercially available kit
("Geneclean" BIO 101 Inc., La 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 cont~;ne~ the
plasmid (pBac EMAP III) with the EMAP III gene using the
enzyme BamHI. The sequence of the cloned fragment is
confirmed by DNA se~enc~ ng,
5 ~g of the plasmid pBac EMAP III is co-transfected with
1.0 ~g of a r_ -rcially available linearized baculovirus
("BaculoGold~ baculovirus DNA", Phanmingen, 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
pBac EMAP III are mixed in a sterile well of a microtiter
plate ~ont~n~ng 50 ~l of serum free Grace's medium (hife
Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l
hipofectin plus 90 ~l Grace's medium are added, mixed and
incubated for 15 minutes at room temperature. Then the
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WO 96/40719 PCTAJS95/07328
transfection mixture i~ 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 ~rom the plate and l ml of
Grace's insect medium supplemented with 10~ fetal calf serum
is added. The plate i8 put back into an incubator and
cultivation contin~le~ at 27~C ~or ~our 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 stained pla~ues. (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 addedto the cells and blue stAine~ plaques are picked with the tip
of an Eppendorf pipette. The agar contA;n;n~ the re~nm~;nAnt
viruses is then resuspended in an Eppendorf tube CQntA;ning
200 ~l of Grace's medium. The agar is removed by a brief
centrifugation and the supernatant contA;n;ng the recomh;nAnt
baculovirus is used to infect Sf9 cells seeded in 35 mm
~;shes~ 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-EMAP III at a multiplicity of
infection (MOI) of 2. Six hours later the medium is ~ ved
and replaced with SF900 II medium minu methionine and
cysteine (Life Technologies 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
.
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W O 96/40719 PCTrUS95/07328
hours before they are harvested by centrifugation and the
labelled proteins visualized by SDS-PAGE and autoradiography.
Exam~le 3
~xPression of Recombinant EMAP III in COS cells
The expression of plasmid, EMAP III HA is derived from
a vector pcDNAI/Amp (Invitrogen) rontAining: 1) SV40 origin
of replication, 2) ampicillin resistance gene, 3) E.coli
replication origin, 4) CMV promoter followed by a polylinker
region, an SV40 intron and polyadenylation site. A DNA
fragment encoding the entire EMAP III precursor and a HA tag
fused in frame to its 3' end is cloned into the polylinker
region of the vector, therefore, the recombinant protein
expression is directed under the CMV promoter The HA tag
corresponds to an epitope derived from the influenza
hemaggl~ltinin protein as previously described (I. Wilson; H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner,
1984, Cell 37:767, (1984)). The infusion of HA tag to the
target protein allows easy detection of the recombinant
protein with an ~ntihody that recognizes the HA epitope.
The plasmid construction strategy is described as
follows:
The DNA sequence encoding EMAP III, ATCC # , is
constructed by PCR using two primers: the 5' primer 5'
GATCGGATCCG~ TCATCCCATCC 3' cont~i n~ a BamHI site
followed by 18 nucleotides of EMAP III coding sequence
starting from the initiation codon; the 3' sequence 5'
GATCAAG~-l-L~-lAGATAAl~l-L~C~ 3 ' contA; nc complementary
sequences to a BamHI site, translation stop codon, HA tag and
the last 18 nucleotides of the EMAP III coding sequence.
Therefore, the PCR product contAinC a BamHI site, ~MAP III
~o~;ng sequence followed by HA tag fused in frame, and a
translation termination stop codon next to the HA tag. The
PCR amplified DNA fragment and the vector, pcDNAI/Amp, are
digested with BamHI restriction enzyme and ligated. The
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W O 96/40719 PCT/US95/07328
ligation mixture is transformed into E. coli strain SURE
(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
~rom transformants and F~ minP~l by restriction analysis for
the presence of the correct fragment. For expression of the
recomh;nAnt EMAP III, COS cells are transfected with the
expression vector by DEA~-DEXTRAN method (J. Sambrook, E.
Fritsch, T. Maniatis, Molecular Cloning: A Laboratory ~mlAl,
Cold Spring Laboratory Press, (1989)). The expression of the
EMAP III HA protein is detected by radiolabelling and
i~nnoprecipitation method (E. Harlow, D. Lane, Antibodies:
A Laboratory ~nll~l, Cold Spring Harbor Laboratory Press,
(1988)). Cells are labelled 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, 196 NP-40, 0.596 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.
ExamDle 5
Ex~ression via Gene Thera~Y
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 rhllnk~ of tissue remain fixed
to the bottom of the flask and fresh media (e.g., Ham's F12
media, with 10~ FBS, penic;ll ;n and streptomycin, is added.
This is then incubated at 37~C for a~o~imately one week.
-
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CA 02221821 1997-11-21
WO 96/40719 PCT~US9~ 28
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 tPrminAl repeats of the Moloney murine
sarcoma virus, is digested with EcoRI and HindIII and
subsequently treated with calf intestinal phosphatase. The
l~ne~r vector is fractionated on agarose gel and purified,
using glass beads.
The cDNA PnCo~;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 contains an
EcoRI site and the 3' primer includes a HindIII site. Equal
quantities of the Moloney murine sarcoma virus linear
backbone and the amplified EcoRI and HindIII fragment are
added together, in the presence o~ 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 B 101, which are then plated onto
agar-cont~ini~g 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),
penirill;n and streptomycin. The MSV vector cont~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~ining 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~;n;ng the
infectious viral particles, is filtered through a ~illipo~e
filter to le.l.~vè det~ch~ producer cells and this media is
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CA 02221821 1997-ll-21
W O 96/40719 PCTrUS95/07328
then used to in~ect fibroblast cells. Media is re~oved 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 o~ 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 u~e a retroviral vector that has a selec~able
marker, such as B~ 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 ~ibroblasts 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 clAim-~, the
invention may be practiced otherwise than as particulariy
described.
CA 02221821 1997-11-21
W O 96/40719 PCT~US9;~7~28
SEQu~N~ LISTING
(1) ~RNRRAR INFORMATION:
(i) APPLICANT: COLEMAN, ET AL.
(ii) TITLE OF lNV~N-llON: Endothelial-Monocyte Activating
Polypeptide III
(iii) NUMBER OF SEQUENCES:
(iv) CORRESPONDEN OE ADDRESS:
(A) ADDR~SSEE: CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) ~O~ ~Y: USA
(F) ZIP: 07068
(V) CoM~u-L~t RE.Pn.~RT-R FORM:
(A) MEDIUM TYPE: 3.5 INCH DISK~-l-
(B) CO~U-1~K: IBM PS/2
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1
(vi) rURRR~T APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: Concurrently
(C) CLASSIFICATION:
~vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
-36-
CA 02221821 1997-11-21
WO 96/40719 PCT~US9~/07328
(viii) Al-lOKN~Y/AGENT lN~-O,~TION:
(A) NAME: F8RRARO, GREGORY D.
(B) REGISTR~TION NUMBER: 36,134
(C) R~ OE /DOCK~T NUMBER: 325800-
(viii) TELECOMMUNICATION INFORMATION:
(A) TEL~ 201-994-1700
(B) TELEFAX: 201-994-1744
(2) lN~O.~TION FOR SEQ ID NO:1:
(i) SEQ~N~ ACTERIsTIcs
(A) LENGTH: 636 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRAN~ SS: SINGLE
(D) TOPOLOGY: T.TNRZ~l~
(ii) MOLECULE TYPE: CDNA
(xi) SE~u~ DESCRIPTION: SEQ ID NO:1:
TACCC~,CC cTrAAAAAAr TGGCCAGCGC TGccTArrrA GAl~L~l~AA AnrArAAr~cc 60
AATGGcrAAA w C~l~AA GAATT Q GAA cr~nArrAnc TCATCCCATC C~G~l~,aT 120
A~ , ~ ~, GrAAAATcAT Q ~l~.~,AG AAGrArCrAG ATGrAr-ArAr CCTGTATGTA 180
r.ArAA~.ATTG AC~ GCK~A AGCTGAACCA CGGACTGTGG Tr~AncGGr~l GGTACAGTTC 240
~,lC7CC~A~, A w AACTGCA Gr-ArArGcTG GTA~ ,l~C TGTGCA~ACCT rAAArCcr~r, 300
AAGATGAGAG GAGTCGAGTC CrAAr~rATG ~11~1.71~1-7 CTTCTATAGA AGGr.ATAAAc 360
CGCCAGGTTG AAC~1~1~7A CC~1CC(7~A GG~l~l~l~ CTGGTGAGCA ~71~7111~1~7 420
AAGGGCTATG AAAAr~G~ccA AcrAnATGAG GAGCTCAAGC crAArAArAA A~l~.l~AG 480
AAGTTGCAGG CTGACTTCAA AAlll~l~AG GAGTGCATCG CACAGTGGAA GrAAArrAAc 540
TTCATGACCA AGCTGGGCTC CAlll~l-,l AAAlCG~l-,A AA~GGI:~A CATTAGCTAG 600
crArccrAr.c A ~ll~CCCC ~ll~ll~AC Q CTGA 636
CA 02221821 1997-11-21
W O 96/40719 PCT~US9J1~7~28
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQu~ CHARACTERISTICS
~A) LENGTH: 168 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STR~
(D) TOPOLOGY T.T~R~
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQu~ DESCRIPTION: SEQ ID NO:2:
Glu Glu Val Ile Pro Ser Arg Leu Asp Ile Arg Val Gly Lys Ile
Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser Leu Tyr Val Glu
Lys Ile Asp Val Gly Glu Ala Glu Pro Arg Thr Val Val Ser Gly
Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln Asp Arg Leu Val
Val Val Leu Cys Asn heu Lys Pro Gln Lys Met Arg Gly Val Glu
Ser Gln Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile Asn Arg
Gln Val Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly Glu
100 105
His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu
110 115 120
Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe
125 130 135
Lys Ile Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe
140 145 150
Met Thr Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly
155 160 165
Asn Ile Ser
