Note: Descriptions are shown in the official language in which they were submitted.
WO 95~314G7 21 g 0 4 ~ 6 PCrlUS94/05384
.
IL. '~ _ ~"rIC PROTEIN--4
This invention relates to newly identified
polynucleotides, polypeptides encoded by such
polynucleotides, the use of such polynucleotides and
polypeptides, as well as the production of such
polynucleotides and polypeptides. More particularly, the
polypeptide of the present invention is monocyte
chemotactic protein-4 (MCP-4~. The invention also relates
to inhibiting the action of such polypeptides.
There are three forms of monocyte chemotactic protein,
namely, MCP-l, MCP-2 and MCP-3. All of these proteins have
been structurally and functionally characterized and have
also been cloned and expressed. MCP-l and MCP-2 have the
ability to attr~ct leukocytes tmonocytes, and leukocytes ),
while MCP-3 also attracts eosinophils and T lymphocytes
(Dahinderi, E. et al., J. Exp. Med., 179:751-756 (1994)).
Initially, human monocyte-specific attracting factor,
was purified from a glioma cell line and a monocytic cell
line. Matsushima, K. et al, J. Exp. Med., 169:1485-1490
(1989). This factor was originally designated glioma-
derived chemotactic factor (GDCF) and monocyte chemotactic
and activating factor (MCAF) by Matsushima, et al. This
f actor is now ref erred to as MCP-l . Subsequent cloning of
the cD~A for MCP-1 showed it to be highly similar to the
murine JE gene. The JE gene could be massively induced in
--1--
SUBSTITUTE SHEET (RULE 261
WO 95131467 219 0 4 6 ~ PCT/US94/05384
murine fibroblasts by platèlè~derived growth factor.
Cochran, B.H., et al, Cell 33:939-947 (1983). Murine JE i8
highly similar to MCP-1. The MCP-1 protein is 629<~
identical to murine JE in a region of 68 shared N-tPrmin~l
residues. It is widely accepted that JE and MCP-1 are
species homologs. The polypeptide of the present
invention, MCP-4, i5 both structurally and functionally
related to MCP-1 and the murine JE protein, see Figure 2.
A method of = suppressing tumor formation in a
vertebrate by administering JE/MCP-l has been disclosed in
PCT application WO-92/20372, along with methods of treating
localized complicatlons of malignancies and methods of
combatting parasitic infection by admini6tering JE/MCP-1.
Expression of the JE/MCP-1 protein in r~ nAnt cells was
found to suppress the cells ability to form tumor6 tn vivo.
Human MCP-l is a basic peptide of 76 amino acids with
a predicted molecular mass o~ 8,700 daltons. MCP--1 is
inducibly expre6sed mainly in monocytes, endothelial cells
and fibroblasts. Leonard, E.J. and Yoshimura, T., Immunol.
Today, 11:97-101 (1990). The factors which induce this
expression is IL-1, TNF or lipopolysaccharide treatment.
Other properties of MCP-1 include the ability to
strongly activate mature human basophils in a pertussis
toxin-sensitive manner. MCP-1 is a cytokine capable of
directly ;n~ ;ng histamine release by basophils,
(Bischoff, S.C. et al., J. Exp. Med., 175:1271-1275
(1992)). Fur~h- le, MCP-1 promotes the formation of
leukotriene C4 by basophils pretreated with Interleukin 3,
Interleukin 5, or granulocyte/macrophage colony-stimulating
factor. MCP-l induced basophil mediator release may play
an important role in allergic inflammation and other
pathologies expressing MCP-l.
Clones having a nucleotide sequence Pncofl;n~ a human
monocyte chemotactic and activating factor (MCAF) reveal
the primary structure of the MCAF polypeptide to be
composed of a putative signal peptide sequence of 23 amino
acid residues and a mature MCAF sequence of 76 amino acid
residues. Furutani, Y.H., et al, Biochem. Biophys. Res.
-2-
SUBSTITUTE SHEET (RULE 261
W0 95l31467 2 1 ~ 0 4 6 ~ PCr/[lS94/05384
Commu., 159: 249-55 ( 1989 ) . The complete amino aeid
6e~uence c~f human glioma-derived monocyte chemotactic
factor (GDCF-2 ) has also been determined. This peptide
attraets human monocytes but not neutrophils. It was
established that GDCF-2 comprises 76 amino acid residues.
The peptide chain contains 4 half-cysteines, at positions
11, 12, 36 and 52, which create a pair of loops, clustered
at the disulfide bridge6. Further, the MCP-l gene has been
designated to human chromosome 17. Mehrabian, M.R., et al,
G~-n ;rs~ 9:200-3 tl991). Certain data suggests that a
potential role for MCP-1 is mediating monocytic
infiltration of the artery wall. Monocytes appear to be
central to atherogenesis both as the progenitors of foam
cells and as a potential source of growth factors mediating
intimal hyperplasia. Nelken, N.A., et al, J. Clin.
Invest., 88:1121-7 ~1991). It has also been found that
synovial production of MCP-1 may play an important role in
the recruitment of mononuclear phagocytes during
inflammation associated with rheumatoid arthritis and that
synovial tissue macrophages are the dominant source of this
cytokine. MCP-1 levels were found to be significantly
higher in synovial fluid from rheumatoid arthritis patients
compared to synovial fluid from osteoarthritis patients or
from patients with other arthritides. ~och, A.E., et al,
J. Clin . Invest ., 90: 772-9 ( 1992 ) .
MCP-2 and MCP-3 are classified in a subfamily of
proinflainmatory proteins and are functionally related to
MCP-l because they specifically attract monocytes, but not
neutrophils. Van Damme, J., et al, J. Exp. Med., 176:59-65
(1992). MCP-3 shows 71% and 589~ amino aeid homology to
MCP-1 and MCP-2 respectively. ~CP-3 is an ;nfli -tory
eytokine that regulates macrophage functions.
In accordance with one aspect of the present
invention, there is provided a novel mature polypeptide
which is MCP-4, as well as fragments, analogs and
derivatives thereof. The polypeptide of the present
invention is of human origin.
--3--
SU~STITUTE SHEEI ~RULE 26
WO 9~31467 21 g O ~ 6 6 PCT/US94/05384
In accordance with another aspect of the present
invention, there are provided polynucleotides (DNA or R
which encode such polypeptides.
In accordance with yet a fu~her aspect of the present
invention, there i6 provided a proce66 for producing 6uch
polypeptide by r~ i n~nt techniques .
In accordance with yet a further aspect of the present
invention, there is provided a process for u~;l;7;nq such
polypeptide, or polynucleotide encoding 6uch polypeptide
for therapeutic purpo6e6, for example, to treat tumor6, to
promote wound healing, to combat para6itic infection and to
regulate hematopoie6i6.
In accordance with yet a further a6pect of the pre6ent
invention, there i6 provided an antibody again6t 6uch
po lypeptide 6 .
In accordance with yet another a6pect of the pre6ent
invention, there are provided antagoni6t/inhibitor6 to 6uch
polypeptides, which may be u6ed to inhibit the action of
such polypeptide6 for therapeutic purpo6e6, for example, to
treat rheumatoid arthriti6, lung inflammation, allergy,
inf ectiou6 di6eases and to prevent inf lammation and
atherosclerosis .
The8e and other aspect6 of the present invention
should be apparent to those 6killed in the art from the
teaching6 herein.
The following drawing6 are illu6trative of: ofl;~ Ls
of the invention and are not meant to limit the scope of
the invention as Isn~ 6ed by the claim6.
FIG. 1 depict6 the cDNA 6equence and corre6ponding
deduced amino acid 6equence of MCP-4. The 119 amino acid
6equence 6hown i6 the full length protein, with
approximately the first 22 amino acid6 representing a
leader sequence such that the mature form of the protein is
97 amino acids in length. The standard one letter
ab~reviation for amino acids is used.
FIG. 2 illustrates the cD~A sequence homology between
MCP-4 and the murine JE protein. The top sequence in each
-4-
SUB~TJ~UTE SHEET (RULE 26)
W0 95/31467 21 9 ~ 4 6 ~ PCTIUS94/05384
three segments is MCP-4 and the bottom sequence i8 murine
JE protein.
FIG. 3 shows the results of a Northern blot analysis
of the mRNA transcript for MCP-4 in human cells.
Figure 4 shows the banding pattern of human MCP-4
following bacterial expression and purif ication .
Figure 5 is a 6chematic representation of the pQE-9
vector .
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 l or for the mature
polypeptide encoded by the cDNA of the clone deposited as
ATCC Deposit No. 75703 on ~arch lO, 1994.
The polynucleotide of this invention was discovered
from an activated monocyte cDNA library. It contains an
open reading frame encoding a protein of approximately 119
amino acids in length of which the f irst 22 amino residues
comprise a putative leader sequence. The mature protein
therefore is predicted to be 97 amino acids in length. It
is structurally related to mouse monocyte chemotactic
protein (MCP-l or JE), showing 27% identity, and 56%
similarity over the entire human MCP-l protein sequence.
The polypeptide contains all four cysteine residues that
occur in all chemokines in a characteri6tic motif. The
sp~cing between these cysteines is conserved compared with
the murine MCP-l/JE which strongly suggests that the new
gene i6 a rlll k; n~ .
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-6tranded 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 l
or that of the deposited clone or may be a different coding
sequence which coding sequence, as a result of the
redundancy or degeneracy of the genetic code, encodes the
--5--
SUBSTITUTE StlEET ~RULE 26
Wo 95/31467 2 1 9 0 4 6 ~ 1~, 5 ~ PCr/US94/05384
same, mature polypeptide as the DNA of Figure 1 or the
deposited cDNA.
The polynucleo~ide which encodes for the mature
polypeptide of Figure 1 or for the mature polypeptide
encoded by the deposited cDNA may include: only the coding
sequence for the mature polypeptide; the coding sequence
for the mature polypeptide and additional coding sequence
such as a leader or secretory sequence or a proprotein
aequence; the coding 6equence 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 8 polypeptide"
P~ ~~ses a polynucleotide which includes only coding
sequence for the polypeptide as well as a polynucleotide
which i n~ rlP~ additional coding and/or non-coding
sequence .
The present invention f urther 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 or the
polypeptide encoded by the cDNA of the deposited clone.
The variant of the polynucleotide may be a naturally
occurring allelic variant of the polynucleotide or a non-
naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides
Pncorl;n~ the same mature polypeptide as shown in Figure 1
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 or the polypeptide encoded
by the cDNA of the deposited clone. Such nucleotide
variants include deletion variants, substitution variants
and addition or insertion variant6.
As hereinabove indicated, the polynucleotide may have
a coding sequence which is a naturally occurring allelic
variant of the coding sequence shown in Figure 1 or of the
coding sequence of the deposited clone. As known in the
--6--
SLIBSTITUTE SHEET (RULE 26)
~ WO 95l31467 2 1 ~ ~ ~ 6 6 PCrlUSg4/05384
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 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 reading frame to a
polynucleotide sequence which aids in expres6ion and
secretion of a polypeptide from a host cell, for example,
a leader sequence which functions as a secretory sequence
for controlling 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 encode 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 remains.
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
r 1 ;An 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 present invention further relate8 to
polynucleotides which hybridize to the hereinabove-
--7--
SUSSTITUTE SHEET (ilULE 26)
Wo 95/31467 2 ~ ~ ~ 4 ~ ~ PCrlUS9~105384
described sequences if there is at least 50% and preferably7O9~ identity between the sequence8. The present invention
particularly relates to polynucleotides which hybridize
under stringent conditions to the herein~bove-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
hereinabove described polynucleotides in a pref erred
embodiment encode polypeptides which retain substantially
the same biological f unction or activity as the mature
polypeptide encoded by the cDNA of Figure 1 or the
deposited cDNA .
The deposit(s) referred to herein will be maintained
under the terms of the Budapest Treaty on the International
Recognition of the Deposit of Micro-organisms for purposes
of Patent Procedure. These deposit6 are provided merely as
convenience to those of 6kill 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 materials, as well as the amino acid sequence of
the polypeptides encoded thereby, ~re incorpor~ted 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 MCP-4
polypeptide which has the deduced amino acid sequence of
Figure l or which has the amino acid sequence encoded by
the deposited cDNA, as welI as fragments, analogs and
derivatives of such polypeptide.
The terms "fragment, " "derivative" and "analog" when
referring to the polypeptide of Figure l or that encoded by
the deposited cDNA, means a polypeptide which retains
essentially the same biological function or activity as
such polypeptide. Thus, an analog includes a proprotein
which can be activated by cleavage of the proprotein
portion to produce an active mature polypeptide.
--8--
SUBSTITUTE SHEET ~RULE 261
Wo 9~/31467 ~ 4 ~ 6 PCTIUS9410~384
The polypeptide of the present invention may be a
recombinant polypeptide, a natural polypeptide or a
synthetic polypeptide, preferably a recombinant
polypeptide .
- The fragment, derivative or analog of the polypeptide
of Figure 1 or that encoded by the deposited cDNA may be
(i) one in which one or more of the amino acid residues are
6ubstituted 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 f used to the mature polypeptide, such as a leader or
secretory sequence or a sequence which is employed for
purif ication of the mature polypeptide or a proprotein
se~uence. Such fragments, derivatives and analogs are
deemed to be within the scope of those skilled in the art
f rom 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
f rom 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 animal is not isolated, but the same
polynucleotide or polypeptide, separated from some or all
of the coexisting materials in the natural system, is
isolated. Such polynucleotides could be part of a vector
and/or such polynucleotides or polypeptides could be part
of a composition, and still be isolated in that such vector
or composition is not part of its natural environment.
The present invention also relates to vectors which
include polynucleotides of the present invention, host
_g _
SUESTITUTE SHEEt (RULE 261
WO 95131~67 219 0 4 6 6 PCrNS9~105384
cells which are genetically ~113i n~F-red with vectors o~ the
invention and the productlon of ~~ polypeptldes o~ the
invention by re~ ; nAnt techn-i~ue6 .
Host cells are genetically ~n~ine-ored (tr~n~ ed 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 'if;ed as appropriate for activating
promoters, selecting transformants or amplifying the MCP-4
genes. 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 producing polypeptides by r~: ` i n~nt
techniques. Thus, for example, the polynucleotide may be
included in any one of a variety of expression vectors _or
expressing a polypeptide. Such vectors include
chromosomal, nonchL, s~ 1 and synthetic DNA sequences,
e . g ., derivatives of SV40 ; bacterial plasmids ; phage DNA;
baculovirus; yeast plasmids; vectors derived f rom
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 appropriate DNA sequence may be inserted into the
vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction
endonuclease site(s) by procedures known in the art. Such
procedures and others are deemed to be within the scope of
those skilled in the art.
'rhe DNA sequence in the expression vector is
operatively linked to an appropriate expression control
6equence~s) (promoter) to direct mRNA synthesis. As
representative examples of such promoters, there may be
mentioned: LTR or SV40 promoter, the E. CQli. lac or tr~,
--10-
SIJBSTITUTE SHEET (RUL E 261
~ WO95/31467 21 9 ~ ~ 6 6 PCIIUS94/05384
the phage lambda P~ promoter and other promoters known to
control expression o~ gene6 in prokaryotic or eukaryotic
cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation
and a transcription terminator. The vector may also
include appropriate sequences for amplifying expres8ion.
In addition, the expression vectors preferably contain
one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells such as
dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture, or such as tetracycline or ampi-
cillin resistance in E. coli.
The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter
or control sequence, may be employed to transform an
appropriate host to permit the host to express the protein.
As representative examples of appropriate hosts, there
may be mentioned: bacterial cells, such as E. coli,
Streptomyces, Salmonella typh; ~ m: fungal cells, such as
yeast; insect cells such as Drosophila and Sf9; animal
cells such as CHO, COS or Bowes melanoma; plant cells, etc.
The selection of an appropriate host is deemed to be within
the scope of those skilled in the art from the te~-h;n~c
herein .
~ ore particularly, the present invention also includes
recombinant constructs comprising one or more of the
sequences as broadly described above. The constructs
comprise a vector, such as a plasmid or viral vector, into
which a 8equence of the invention has been inserted, in a
forward or reverse orientation. In a preferred aspect of
this embodiment, the construct further comprises regulatory
sequences, including, for example, a promoter, operably
linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and
are commercially available. The following vectors are
provided by way of example. Bacterial: pQE70, pQE60, pQE-9
(Qiagen), pbs, pDlO, phagescript, psiXl74, pbluescript SR,
-11-
SU8STITUTE SHEET ~RULE Z6~
WO95131467 ~ 4 ~ 6 PCrlllS94/05384
pb6ks, pNH8A, pNH16a! pNH18A,~ E~NH46A (Stratagene); ptrc99a,
pKK223--3, pKK233--3, pDR54~, pRI~ (ph;~r~^~-ii9) . I~ukaryotic:
pWLNEO, 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 (chloL h~on;col transferase) vectors or other
vectors with selectable markers. Two appropriate vectors
are PKR232-8 and PCM7. Particular named bacterial
promoters include lacI, lacZ, T3, T7, gpt, lambda PR~ PL and
trp. Eukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from
retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector ,Ind promoter is well within the level of
ordinary skill in the art.
In a further P~lhQri;--nt~ the present invention relates
to host cell6 containing the above-described constructs.
The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as
a bacterial cell. Introduction of the construct in~o 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 manner to produce the gene product encoded by
the re~ ;n~nt gequence. Alternatively, the polypeptide8
of the invention can be synthetically produced by
conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells,
yeast, bacteria, or other cells under the control of
appropriate promoters. Cell-free translation systems can
also be employed to produce such proteins using RNAs
derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with
prokaryotic and eukaryotic hosts are described by Sambrook,
--12--
SUBSTITUTE SHEET (RULE 261
~ Wo 95/314G7 21 9 8 ~ 6 ~ PCTIUS94/05384
et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring ~arbor, N.~., (1989), the di6closure
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 PnhAnc~r sequence into the vector. Enhancers
are cis-acting elements of DNA, usually about from 10 to
300 bp that act on a promoter to increase its
transcription. Examples including the SV40 F~nhAnc~r on the
late side of the replication origin bp 100 to 270, a
cyt~ virus early promoter enhancer, the polyoma
~nhAnc~r on the late 5ide of the replication origin, and
adenovirus enhancers.
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 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 (PG~ factor,
acid phosphatase, or heat shock proteins, among others.
The heterologous structural sequence is assembled in
~p~Lu~liate phase with tran61ation initiation and
termination sequences, and preferably, a leader sequence
capable of directing secretion of translated protein into
the periplasmic space or extrAcel 1111Ar medium. Optionally,
the heterologous sequence can encode a fusion protein
including an N-tPrm;nAl identification peptide imparting
desired characteristics, e.g., st~hil;7ation or simplified
purif ication 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 markers and an origin of
replication to ensure maintenance of the vector and to, if
--13--
SUBSTITUTE SHEET (RULE 26)
Wo 95/31467 21 g ~ ~ 6 6 rcr/uss4/o~384
desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include ~. coli,
3acillus subtilis, Salmonella typhimurium and various
species within the genera Pse~ ~ -c, Streptomyces, and
Staphylococcus, although others may also be employed as iY
matter of choice.
As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a
selectable marker and bacterial origin of replication
derived from commercially available plasmids comprising
genetic elements of the well known cloning vector psR322
(ATCC 37017). Such commercial vectors include, for
example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,
Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These
pBR322 "backbone" sections are ' in~d with an appropriate
promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and
growth of the host strain to an eppropriate cell density,
the selected promoter is induced by appropriate means
(e.g., temperature shift or chemical inductionl and cells
are cultured f or an additional period .
Cells are typically harvested by centrifugation,
disrupted by physical or chemical means, and the resulting
crude extract retained 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 know to those
skilled in the art.
Various l; An cell culture systems can also be
employed to express re~ ' inAnt protein. Examples of
r l iAn expresgion systems include the COS-7 lines of
monkey kidney fibroblasts, described by Gluzman, Cell,
23:175 (1981), and other cell lines capable of expressing
a compatible vector, for example, the C127, 3T3, CH0, HeLa
and BHK cell lines . Mi l; An expression vectors will
comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
--14--
~UBSrl~UTE SHEET (RULE 26
219~66
o95l31467 PCrlUSs4l05384
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5~ ~lanking
nontranscribed sequences. DNA sequences derived from the
SV40 splice, and polyadenylation sites may be used to
provide the required nontranscribed genetic elements.
The polypeptide can be lecuv~:Led and purified from
reL ;nAnt cell cultures by method6 including ~ illm
sulfate or ethanol precipitation, acid extraction, anion or
cation f.Yrh~n~e chromatography, phosphocellulose
chromatography, hydrophobic interaction chromatography,
affinity chromatography, hydroYylapatite chromatography and
lectin chromatography . It is pref erred to have low
concentrations tapproximately 0.15-5 mM) of calcium ion
present during purification. (Price et al., J. Biol.
Chem., 244:917 (1969) ) . Protein refolding steps can be
used, as necessary, in completing configuration of the
mature protein. Finally, high performance liquid
chromatography (HPLC~ can be employed for final
purif ication steps .
The polypeptides of the present invention may be a
naturally purified product, or a product of chemical
synthetic ~Luce~uLes, or produced by re ;nAnt techniques
from a prokaryotic or eukaryotic host (for example, by
bacterial, yeast, higher plant, insect and -1 iAn cells
in culture). r~ep~n-lin~ 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 meth;nn;n~ amino ~cid residue.
The polypeptides of the present invention, in
particular MCP-4, may be used for the promotion of wound
healing. Since MCP-4 is a rh lc; nQ~ it is a chemo-
attractant for leukocytes (such as monocytes, T
lymphocytes, basophils, etc.); therefore, it causes
infiltration of target immune cells to a wound area.
The ~CP-4 polypeptides may also be used as an anti-
tumor treatment and for treating localized complications of
a malignancy, such as pleural effusions or ascites.
--15--
SUBSIITUTE SHEE~ (RULE
Wo 95131-167 2 1~ d ~ 6 ~ PCTIUS94/053X4
Instilling MCP-4 into the involved anatomic space can lead
to local monocyte accumulation and activation.
The presence of MCPs in vivo is ac -nied by a local
increase in the presence of eosinophils which have the
distinctive function of killing the larvae of parasites
that invade tissues, as in schistosomiasis, trichinosis and
ascariasis. Therefore, MCP-4 may be used for combatting
parasitic infections.
MCP--4 polypeptides may also play a role in the
regulation of hematopoiesis, by regulating various
hematopoietic progenitor cell activation and
dif f erentiation .
The polypeptides may also be employed in accordance
with the present invention by expression of such
polypeptides in vivo, which is often referred to as "gene
therapy . "
For example, cells from a patient may be F~n~i nP~red
with a polynucleotide (DNA or RNA) encoding an MCP-4
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. For example, cells
may be Pr~i ne~ored by procedures known in the art by use of
a retroviral particle containing RNA encoding a polypeptide
of the present invention.
Similarly, cells may be engineered in vivo for
expression of an MCP-4 polypeptide in vivo by, for example,
procedures known in the art. As known in the art, a
producer cell for producing a retroviral particle
containing RNA encoding the polypeptide of the present
invention may be administered to a patient for f~n~; nf~rring
cells in vivo and eYpression 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 ~Arh;n~ of the present
invention. For example, the expression vehicle for
~n~inf.Pring cells may be other than a retrovirus, for
eYample, an adenovirus which may be used to ~n~inf~r cells
in vivo after combination with a suitable delivery vehicle.
--16--
SUBSrITUTE SHEET (RULE 26)
~ Wo 951314G7 21 g O ~ 6 6 PCrlUss4/os384
The polypeptides of the present invention may be
employed in combination with a suitable rhArr~ eutical
carrier. Such compositions comprise a therapeutically
effectiv~ amount of the polypeptide, and a pharmaceutically
acceptable carrier or excipient. Such a carrier includes
but is not limited to aaline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The
formulation should suit the mode of administration.
The invention also provides a pharmaceutical pack or
kit comprising one or more containers filled with one or
more of the ingredients of the rhArr--eutical compositions
of the invention. Associated with such container(s) can be
a notice in the form prescribed by a governmental agency
regulating the manufacture, use or sale of rh_rr-~-euticals
or biological products, which notice ref lects approval by
the agency of manufacture, use or sale for human
administration. In addition, the polypeptides of the
pre6ent invention may be employed in conjunction with other
therapeutic compounds.
The pharmaceutical compositions may be administered in
a convenient manner such as by the topical, intr~venous,
intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes. ~CP-4 is administered in an amount
which is effective for treating and/or prophylaxis of the
specif ic indication . The amounts and dosage regimens of
MCP-4 administered to a subject will depend on a number of
factors such as the mode of administration, the nature of
the condition being treated ~nd the judgment of the
prescribing physician. In general, the ~CP-4 will be
administered in an amount of at least about lO ~lg/kg body
weight and in most cases they will be administered in an
amount not in excess of about 8 mg/Kg body weight per day.
n most cases, the dosage is from about lO yg/kg to about
l mg/kg body weight daily, taking into account the routes
of administr~tion, symptoms, etc.
The sequences of the present invention are also
valuable for chromosome identification. The sequence is
specif ically targeted to and can hybridize with a
--17--
SUBSTtTLlTE SHEET (RULE 261
o 95~31467 ;~ 0 4 ~ 6 PCT/US94/05384
particular location on an individual human chromosome.
I1o~ v~:r, there i6 ~' current need ~or identi~ying
particular sites on the chl~ ~ ~. Few chromosome marking
reagents based on actual sequence data ( repeat
polymorphisms ) are presently available for marking
chromosomal location. The mapping of DNAs to chL~
according to the present invention is an important f irst
step in correlating those sequences with genes associated
with disease.
Briefly, sequences can be mapped to chL~ - - by
preparing PCR primers (preferably l5-25 bp) from the cDNA.
Computer analysis o~ the cDNA is used to rapidly select
primers that do not span more than one exon in the genomic
DNA, thus complicating the amplification process. These
primers are then used for PCR screening of somatic cell
hybrids containing individual human ~IIL I { ~ - . Only
those hybrids containing 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 invention with the same
oligonucleotide primers, sublocalization can ~e achieved
with panels of fragments from specific chL~ s~ -, or pools
of large genomic clQnes in an analogous manner. Other
mapping strategies thAt can similarly be used to map to its
ch~ ~ - include ill situ hybridization, prescreening with
labeled flow-sorted Cl1L~ _ ~ and preselection by
hybridization to construct chromosome specif ic-cDNA
lib}aries .
Fluorescence in sltu hybridization (FISH) of a cDNA
clones to a metaphase chL, - 1 spread can be used to
provide a precise chromosomal location in one step. This
technique can be used with cDNA as short as 500 or 600
bases; however, clones larger than 2,000 bp have a higher
1 ;k~l ihos~d of binding to a unique chll 6~ '1 location with
sufficient signal intensity for simple detection. FISH
requires use of the clones from which the EST was derived,
and the longer the better. For example, 2, 000 bp is good,
--18--
SUBS11TUTE SHEET (RUI~ 26
W095/314G7 f ~ PCrlUS94/05384
4,000 is better, and more than 4,000 i8 probably not
neces6ary to get good results a reasonable percentage of
the time. For a review of this technique, see Verma et
al., Human Chromosomes: a Manual of Basic Techniques,
Pergamon Press, New York ( I988 ) .
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, M~nc~ 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 sequence between af f ected 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 potential causative genes. tThis
assumes 1 megabase mapping resolution and one gene per 20
kb) .
Comparison of affected and unaffected individuals
generally involves first looking for structural alterations
in the chromosomes, such as deletions or translocations
that are visible from ch~ - spreads or detectable
using PCR based on that cDNA sequence. Ultimately,
complete sequencing of genes from several individuals is
required to conf irm the presence of a mutation and to
dist;n~li~h mutations from polymorphisms.
The polypeptides, their f ragments or other
derivatives, or analogs thereof, or cells expressing them
can be used as an; ~, ~~ to produce antibodies thereto.
These antibodies can be, for example, polyclonal or
--19--
SUSSTITUTE SHEET ~RULE 261
Wo 9S131467 21~ 0 4 6 6 PCrNS94/OS384
monoclonal an~;ho~ s. The presen~imYention also includes
chimeric, single chain, and h~ n;7ed antibodie6, as well
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 fragments.
Antibodies generated against the polypeptides
corresponding to a sequence of the present invention can be
obtained by direct injection of the polypeptides into an
~nimal or by administering the polypeptides to an animal,
preferably a nonhuman. The antibody so obtained will then
bind the polypeptides itself. In this manner, even a
sequence Pncntl;n~ only a fragment of the polypeptides can
be used to generate antibodies binding the whole native
polypeptide6. 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. Examples include the
hybridoma technique (Rohler and Milstein, 1975, Nature,
256:495-497), the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today
4: 72 ), and the EBV-hybridoma technique to produce human
monoclonal an~;ho~l;ec (Cole, et al., 1985, in Monoclonal
An~; ho~ and Cancer 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 antibodies to immunogenic polypeptide
products of this invention.
The present invention also relates to a diagnostic
assay for detectin~ the level of MCP-4 both quantitatively
and qualitatively. Such assays are well known in the art
and include an ELISA assay and the radio; ~ say. The
levels of MCP-4 detected in the assay can be useful for the
elucidation of the 6ignificance of MCP-4 in various
diseases and for the diagnosis of disea8es in which MCP-4
may play a role.
-20-
SUBSTITUTE SHEET (RULE 26
~ WO 9~/3l467 2 ~ ~ 4 ~6 PCT/US94/05384
This invention provides a method for identification of
MCP--4 receptor6 . The gene ~n--o~l i n~ an MCP--4 receptor can
be identified by expression cloning. Briefly,
polyadenylated RNA i6 prepared from a cell responsive to
MCP-4 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 MCP-4. Transfected cells which
are grown on glass slides are exposed to labeled MCP-4.
MCP-4 can be labeled by a variety of means including
iodidation or inclusion of a recognition site for a site-
specif ic protein kinase . Following f ixation and
incubation, the slides are subjected to autoradiographic
analysis. Positive pools are identified and sub-pools are
prepared and retransfected using an iterative sub-pooling
and rescreening process, eventually yielding a single clone
that encodes the putative receptor. As an alternative
approach for receptor identification, labeled MCP-4 can be
photoaffinity linked with cell membrane or extract
preparations that express an MCP-4 receptor molecule.
Cross-linked material is resolved by PAGB and exposed to x-
ray film. The labeled complex containing the MCP-4
receptor can be excised, resolved into peptide fragments,
and subjected to protein microsequencing. The amino acid
sequence obtained from microsequencing would be used to
design a set of generate oligonucleotide probes to screen
a cDNA library to identify a gene encoding the putative receptor.
This invention also provides a method of screening
drugs to identify those which enhance (agonists) or block
( antagonists ) interaction of MCP-4 to its receptor . An
agonist increases the biological fllncf it~nl: of MCP-4, while
an antagonist reduces or eliminates such f unctions . As an
example, a l; Rn cell or membrane preparation
expressing an MCP-4 receptor would be incubated with
labeled MCP-4 in the presence of drug. The ability of drug
to enhance or block this interaction could then be
measured. Alternatively, the response of a known second
messenger system following interaction of MCP-4 and it8
receptor would be measured compared in the presence or
--21--
SUBSTITIJT~ SHEET (RULE 26)
Wo 95/31467 ~ 6 ~ PCT/US94/0538
absence of drug. Such second messenger systems include but
~re not limited to, cAMP guanylate cyclase, ion rhAnnt~l c or
phosphoinositide hydrolysis.
The present invention is also directed to
antagonist/inhibitor molecules to the polypeptides of the
present invention. Antagonists include negative dominant
mutants of MCP-4. MCP-4 is a tetrameric polypeptide
wherein one mutated unit will cause the entire polypeptide
to be non-functional. A negetive dominant mutant of MCP-4
binds to the MCP-4 receptor but fails to activate cells
( leukocytes and monocytes ) to which it binds . An assay to
detect negative dominant mutants of MCP-4 is an in vitro
chemotaxis assay wherein a multiwell chemotaxis chamber
equipped with polyvinylpyrrolidone-free polycarbonate
membranes is used to measure the chemoattractant ~bility of
MCP-4 for leukocytes in the presence ~nd absence of
potential antagonist/inhibitor or agonist molecules.
An example of an inhibitor is an antisense DNA or RNA
construct. Antisense technology can be used to control
gene expression throuy-h triple-helix formation or antisense
DNA or RNA, both of which methods Are based on binding of
a polynucleotide to DNA or RNA. For example, the 5 ' coding
portion of the polynucleotide sequence, which encodes for
the mature polypeptides of the present invention, is used
to design an antisense RNA oligonucleotide of from about 10
to 40 base pairs in length. A DNA ol; ~t~n~ leotide is
designed to be complementary to a region of the gene
involved in transcription (triple helix -see Lee et al.,
Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science,
241:456 (1988); and Dervan et al., Science, 251: 1360
(1991~ ), thereby preventing transcription and the
production of MCP-~. The antisense RNA oligonucleotide
hybridizes to the mRNA i~ vivo and blocks translation of
the mRNA molecule into the MCP-4 (antisense - Okano, J.
Neurochem., 56:560 (l991~; Oliyudev,~yllucleotides as
Antisense Inhibitors of Gene Expression, CRC Press, Boca
Raton, FL ( 1988 ) ) . Alternatively, antisense RNA and DNA
--22--
SU8~TITUTE ~HEET (RULE 26
Wo 95131467 21~ ~ 4 6 6 PCT/U~94/OS384
may be delivered to cell6 such that they are expressed i~
vivo to inhibit production of MCP-4.
Another example of an antagonist is a peptide
derivative of MCP-4 which are naturally or synthetically
modif ied analogs of MCP-4 that h~ve lost biological
function yet still recognize and bind to receptors thereby
effectively blocking the receptors.
The antagonist/inhibitors may be used to treat
inf lammation by preventing the attraction of monocytes to
a wound or a site of trauma, and to regulate normal
plll Ary macrophage populations, since acute and chronic
inflammatory p~ ry diseases are associated with
sequestration of mononuclear phagocytes in the lung. They
may also be used to treat rheumatoid arthritis, since MCP
levels were found to be significantly elevated in synovial
f luid f rom rheumatoid arthritis patients which suggests
that synovial production of MCP attracts monocytes whose
influx and activation are important in the pathogenesis of
both degenerative and inf lammatory arthropathies .
The antagonist/inhibitors may also be used for
treating atherosclerosis, since MCPs mediate monocyte
infiltration in the artery wall which infiltration leads to
atherosclerosis, and to prevent allergies, since it has
been shown that MCPs directly induce histamine release by
basophi 18 .
Antagonist/inhibitors may also be used to treat
infectious diseases such as tuberculosis, since
tuberculosis targets cells, usually monocytes, causing the
monocytes to release MCPs which attracts more monocytes to
the lungs causing severe ; nf l; tion . The
antagonist/inhibitors may be employed in a composition with
a pharmaceutically acceptable carrier, e.g., as hereinabove
described .
The present invention also relates to an assay for
identifying potential antagonist/inhibitors specific to
MCP-4 . An example of such an assay ~ i n~8 MCP-4 and a
potential antagonist/inhibitor with membrane-bound MCP-4
receptors or recombinant MCP-4 receptors under ~ u~iate
--23--
S118STITUTE SHEET (RULE 2~)
W0 9~/31467 ~ PCTIUS94/0~384
condition6 for a competitive inhibition assay. MCP-4 can
be la`oeled, such as by radioactivity, such that the number
of MCP-4 molecules bound to the receptor can determine the
ef f ectiveness of the potential antagonist/inhibitor .
The present invention will be further described with
reference to the following ~ ; however, it is to be
understood that the present invention is not limited to
such examples. All parts or amounts, unless otherwise
specified, are by weight.
In order to facilitate understanding of the following
examples certain frequently occurring methods and/or terms
will be described.
"Plasmids" are designated by a lower case p preceded
~nd/or f ollowed by capit~l letters ~nd/or numbers . The
starting plasmids herein are either commercially available,
publicly available on an unrestricted basis, or can be
constructed from available pl~cm; rl~ 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
~nalytical purposes, typically 1 yg of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20
yl of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~g of
D~A 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
directly on a polyacrylamide gel to isolate345Xelectr
the desired fragment.
--24-
SUBâTITUTE SHEET (RULE 26
W095l31467 ~ 5 Pcrlu~94105384
Size separation of the cleaved fragments is performed
u~ing 8 percent polyacrylamide gel described by Goeddel, D.
et ql., Nucleic Acids Re6., 8:4057 (1980).
~ oligonucleotides~ refers to either a single stranded
polydeoxynucleotide or two complementary
polydeoxynucleotide strands which may be chemically
synthesized. Such synthetic ol; gonllcleotides have no 5 '
phosphate and thus will not ligate to another
ol ;gonllr~eotide without ~Idding 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 provided, ligation may be accomplished using
known buffers and conditions with 10 units to T4 DNA ligase
( ligase ) per 0.5 ,ug of approximately equimolar amounts of
the DNA f ragments 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).
r le 1
Bacterial E~ression and p1lrification of MCP-4
The DNA sequence ~ncn~i;n~ for MCP-4, ATCC # 75703, is
initially amplified using PCR oligonucleotide primers
corresponding to the 5 ~ and 3 ' sequences of the processed
MCP-4 protein (minus the signal peptide sequence) and the
vector sequences 3 ~ to the MCP-4 gene. Additional
nucleotides ~:u~ Le,,~ollding to MCP-4 were added to the 5 ' and
3 ' sequences respectively. The 5 ' oligonucleotide primer
has the sequence 5 '-TCAGGATCCCCTACGGG~ L~ 3 ' contains
a Bam H1 restriction enzyme site followed by 18 nucleotides
of MCP-4 coding sequence starting f rom the presumed
t~rm;nAl amino acid of the processed protein codon. The 3'
sequence 3 '-CGCTCTAGAG~ rr-~rGGCCAGT-5 ' contains
complementary sequences to the XbaI site and to a
pBluescript SK- vector sequence located 3' to the MCP-4 DNA
--25--
SU8~TITUTE SHEET (RULE 261
4~
Wo 9~/31467 ~ PCr/Uss4/05384
insert. The restriction enzyme sites correspond to the
restriction enzyme sites on th~ bacterial expression vector
pQE-9. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA,
91311 ) . pQE-9 encodes 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 site6. pQE-9 was then
digested with Bam H1 and Xba I . The amplif ied sequences
were ligated into pQE-9 and were inserted in frame with the
3equence encoding for the histidine tag and the RBS.
~igure 5 shows a schematic representation of this
arrangement. The ligation mixture was then used to
transform E. coli strain ml5/rep4 available from Qiagen
under the trademark M15/rep 4 by the procedure described in
Sambrook, J. et al, Molecular Cloning: A Laboratory
Manual, Cold Spring Laboretory Press, 1989. M15/rep4
contains multiple ~copies of the plasmid pREP4, which
expresses the lacI repressor and also confers kanamycin
resistance (Ranr). Transformants are ;rl.ontif;~d by their
~bility to grow on LB plates and ampicillin/kanamycin
resistant colonies were selected. Plasmid DNA was isolated
and confirmed by restriction analysis. Clones ront~in;n~
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 O/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.a~) of
between 0 . 4 and 0 . 6 . IPTG ( " Isopropyl-B-D-thiogalacto
pyranoside" ) was then added to a f inal concentration of
mM. IPTG induces by inactivating the lacI repressor,
clearing the P/O leading to increased gene expression.
Cells were grown an extra 3 to 4 hours. Cells were then
harvested by centrifugation. The cell pellet was
solllhil;7r~rl in the chaotropic agent 6 Molar G1l~n;rl;nF- HCl.
After clarification, solubilized MCP-4 was purified from
this solution by chromatography on a Nickel-Chelate column
under conditions that allow for tight binding by proteins
containing the 6-His tag . Hochuli , E . et al ., J .
-26-
SU9STITUTE SHEET (RULE 261
21~0~6~
WO 95131467 PCrlUS94/05384
Chromatography 411:177-184 (1984). ~CP-4 (954 pure) was
eluted from the column in 6 molar guF~ni~7in~ HC1 pH 5.0 and
for the purpose of renaturation adjusted to 3 molar
g~,~n;.7inP HCl, lOOmM godium phosphate, 10 mmolar
. glutathione (reduced) and 2 mmolar glutathione (oxidized).
After incubation in this solution for 12 hours the protein
was dialyzed to 10 mmolar sodium phosphate. Figure 4.
Example 2
rnreS8ion Pattern of MCP-4 in human cells
Northern blot analysis was carried out to examine the
levels of expression of MCP-4 in human cells. Total
cellular RNA samples were isolated with RNAzol~ B system
(Biotecx Laboratories, Inc. 6023 South Loop East, Hou6ton,
TX 77033 ) . About lO~Lg of total RNA isolated from each
human tissue spec; f i f-d was separated on 1% agarose gel and
blotted onto a nylon filter. (Sambrook, Fritsch, and
Maniatis, Nolecular Cloning, Cold Spring Harbor Press,
( 1989 ) ) . The 7 ~h~l i n~ reaction was done according to the
Stratagene Prime-It kit with 50ng DNA fragment. The
labeled DNA was purified with a Select-G-50 column. (5
Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, C0
80303 ) . The filter was then hybridized with radioactive
labeled full length MCP-4 gene at l,000,000 cpm/ml in 0.5
M NaP0~, pH 7.4 and 796 SDS overnight at 65 C. After wash
twice at room temperature and twice at 60 C with 0.5 x SSC,
0.19~ SDS, the filter was then exposed at -70 C overnight
with an intensifying screen. The message RNA for MCP-4 is
abundant in activated and unactivated T cells, monocytes
and T cell lines. Figure 3.
Numerous modifications and variations of the present
invention are possible in light of the above t~arhin~ and,
therefore, within the scope of the appended claims, the
invention may be practiced otherwise than as particularly
described .
--27--
SU~STITUTE SHEET (RULE 26)
WO 95131467 219 ~ 4 6 ~ PCTIUS94/05384 ~,
~t;UU~ ; LISTING
( 1 ) GENERAL INFORMATION:
( i ) APPLICANT: LI, ET AL .
(ii) TITLE OF INVENTION: Monocyte Chemotactic
Protein-4
( iii ) NUMBER OF ~;uu~ ;S: 2
(iv) C:u~h'~ u~ ; ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
( B ) STREET: 6 BECRER FARM ROAD
(C) CITY: Rn~T.PlNn
( D ) STATE: NEW ~ERSEY
( E ) COUNTRY: USA
(F) ZIP: 07068
(v) C~ ~ R~n~RTT FORM:
(A) MEDIUM TYPE: 3 . 5 INCH DISRETTE
( B ) Cu.~u ~: IBM PS/2
(C) OPERATING SYSTEM: MS--DOS
( D ) SOFTWARE: WORD PERFECT 5 .1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: Submitted herewith
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
( A ) NAME: FERRARO, GREGORY D .
(B) REGISTRATION NUMBER: 36,134
( C ) k~ u~;/DOCRET NUMBER: 3 2 5 8 0 0 -16 0
(iX) T~T.~'nMMTTNICATION INFORMATION:
(A) TELEPHONE: 201--994--1700
(B) TELEFAX: 201--994--1744
( 2 ) INFORMATION FOR SEQ ID NO :1:
;uu~;~u~; CHARACTERISTICS
(A) LENGTH: 360 BASE PAIRS
--28--
SUBS~lhrrE SHEET ~RULE 261
. `~ 21~4~
6EQU~NCE LISTING
( 1 ) GENERAL INFORMAlmION:
(i~ APPLICANT: numan Genome Science8, Inc.
9410 Key We~t Avenue
Rockville, MD 20850
tJnited States of America
APPLICANTS/INVENTORS: Li, Haodong
Ruben, Steven M.
Sutton III, Granger G.
(ii) TITLE OF INVENTION: Monocyte Chemctactic Protein-4
(iii) NUMBER OF SEQUENCES: 5
(iV) mUKltl:;:~!'UN~r;N~ ADDRESS:
(A) ~nnR~qq~ Sterne, Ressler, Gcldstein & Fox, P.L.L.C.
(B) STREET: 1100 New York Ave, N.W., Suite 600
(C) CI~Y: Washington
(D) STATE: D.C.
(E) COUNTRY: United States of America
(F) ZIP: 20005-3934
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC hl~
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Relea~e #1.0, Ver~ion #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NOMBER: PCT/US94/05384
(B) FILING DAT~: 16-MAY-1994
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gold~tein, Jorge A.
(B) REGISTRATION NUMBER: 29,021
(C) REFERENCE/DOCRET N[lMB~R: 1488.034PC00
(ix) TEL~i~._. mT~N INFoRMATIoN:
(A) TELEPHONE: 202-371-2600
(B) TEL13FAX: 202-371-2540
(2) INFORMATION FOR SEQ ID NO:1:
(i) Si;:QUENC~ R~-~TFRT.qTICS:
(A) LENGTH: 360 ba~e pairs
(B) TYPE: nucleic acid
(C) ~ ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
,~ t
(ix) FEAT~RE:
(A) NAME/REY: CDS
(B) LOCATION: 1..357
( ix ) FBATURE:
(A~ NAME/RBY: Dig_peptide
(B) LOCATION: 1..66
( ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 67..357
(xi) SEQUBNCE DESCRIPTION: SEQ ID NO:1:
ATG GCA GGC CTG ATG ACC ATA GTA ACC AGC CTT CTG TTC CTT GGT GTC 48
Met Ala Gly Leu Met Thr Ile Val Thr Ser Leu Leu Phe Leu Gly Val
-22 -20 -15 -10
TGT GCC CAC CAC ATC ATC CCT ACG GGC TCT GTG GTC ATA CCC TCT CCC 96 _~
Cy3 Ala His His Ile Ile Pro Thr Gly Ser Val Val Ile Pro Ser Pro
-5 1 5 10
TGC TGC ATG TTC TTT GTT TCC A~G AGA ATT CCT GAG AAC CGA GTG GTC 144
Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn Arg Val Val
15 20 25
AGC TAC CAG CTG TCC AGC AGG AGC ACA TGC CTC AAG GGA GGA GTG ATC 192
Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Gly Gly Val Ile
30 35 40
TTC ACC ACC AAG AAG GGC CAG C~G TTC TGT GGC GAC CCC AAG CAG GAG 240
Phe Thr Thr Ly3 Lys Gly Gln Gln Phe Cys Gly Asp Pro Lys Gln Glu
45 50 55
TGG GTC CAG AGG TAC ATG APG AAC CTG GAC GCC A~G CAG AAG AAG GCT 288
Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln Lys Lys Ala
60 65 70
TCC CCT AGG ~CC AGG GCA GTG GCT GTC APG GGC CCT GTC C~G AGA TAT 336
Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro Val Gln Arg Tyr
75 80 85 9o
CCT GGC AAC CAA ACC ACC TGC TAA 3 6 0
Pro Gly Asn Gln Thr Thr Cys
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE ~Tl~T:~'T~17T~:TICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
. 219~fil~
(ii) MOLECULE TYPE: protein
(xi) SEQVENCE ~ LUI~: SEQ ID NO:2:
Met Ala Gly Leu Met Thr Ile Val Thr Ser Leu Leu Phe Leu Gly Val
-22 -20 -15 -10
Cys Ala Elis E~is Ile Ile Pro Thr Gly Ser Val Val Ile Pro Ser Pro
- -5 1 5 10
ys Cya Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn Arg Val val
15 20 25
er Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Gly Gly Val Ile
30 35 40
he Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro Lys Gln Glu
45 50 55
Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln Lys Lys Ala
60 65 70
Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro Val Gln Arg Tyr
75 80 85 90
ro Gly Asn Gln Thr Thr Cys
2) INFORMATION FO~ SEQ ID NO:3:
(i) SEQUENCE ~7~ 'TR~TCTICS:
(A) LENGTEI: 148 amino acids
(B) TYPE: amino acid
(C) ST~NnRnNRq~: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) 8EQUENC_ DESCRIPTION: SEQ ID NO:3:
Met Gln Val Pro Val Met Leu Leu Gly Leu Leu Phe Thr Val Ala Gly
5 10 15
Trp Ser Ile E~i~ Val Leu Ala Gln Pro Asp Ala Val Asn Ala Pro Leu
20 2s 30
Thr Cys Cys Tyr Ser Phe Thr Ser Lys Met Ile Pro Met Ser Arg Leu
35 40 45
Glu Ser Tyr Lys Arg Ile Thr Ser Ser Arg Cys Pro Lys Glu Ala Val
so ss 60 =
~/
. ,,, 21g~466
Val Phe val Thr Lys Leu Lys Arg Glu Val Cy~ Ala Asp Pro Lys Lys
65 70 75 80
Glu Trp Val Gln Thr Tyr Ile Lys Asn Leu Asp Arg Asn Gln Met Arg
85 90 95
Ser Glu Pro Thr Thr Leu Phe Lys Thr Ala Ser Ala Leu Arg Ser Ser
100 105 110
Ala Pro Leu Asn Val Lys Leu Thr Arg I.ys Ser Glu Ala Asn Ala Ser
115 120 125
Thr Thr Phe Ser Thr Thr Thr Ser Ser Thr Ser Val Gly Val Thr Ser
130 135 140
Val Thr Val A~n
145
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE ~T~R~rT~Rr.qTICS
(A) LENGT~: 26 ba~e pairs
(B) TYPE: nucleic acid
(C) STR~ nN~qq: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: c~NA
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:4:
TCAGGATCCC CTACGGGCTC GTGGTC 26
(2~ INFORMATION FOR SEQ ID NO:5:
(i~ SEQUENCE ~R~rT~RT~TIc8
(~ LENGTEI: 26 3~ase pairs
(B~ TYPE: nucleic acid
( C ~ STR 7~ ~: 8 ingle
(D~ TOPOLOGY: linear
(ii~ MOL.ECUI,E TYPE: cDNA
(xi~ SEQI~ENCE DESCRIPTION: 8EQ ID NO:5:
CGCTCTAGAG TZ~ rr.~rt~ GCCAGT 26
0~ 2/
