Note: Descriptions are shown in the official language in which they were submitted.
13 1029~
NOVEL CELL GROWT~ REGULATORY ~ACTOR
BACKGROUND OF THE INVENTION
Field of the Invention
Leukocytes, ~oth lymphocytes and monocytes,
have been implicated in the inhibition of tumor growth
in several animal tumor models. The increased inci-
dence of malignancies in immunocompromised humans sup-
ports the contention that the white blood cells play a
role in the regulation of neoplastic growth. Protein
factors produced by these white cells that inhibit
tumor growth or modulate immune functions which have
already been isolated and characterized include the
interferons, ~- and Y-, tumor necrosis factor, lympho-
toxin, interleukin-2, and other lymphokines. Since
each of the factors which have been isolated have a
different spectrum of activities and may interact dif-
ferently in conjunction with other factors, there re-
mains a continuing and strong interest in the isolation
of and characterization of all of the factors which
white cells ?roduce in the modulation of cell growth or
immune functions. These compounds individually or to-
gether may find use in the treatment or diagnosis of
cancer, as promoters of wound healing or as
3~ immunomodulators for the treatment of patients with
immunodeficiencies, autoimmunity, organ transplants,
and the like.
There are several difficulties that may be
encountered in the discovery, isolation, purification,
or characterization of naturally occurring factors.
Methodologies must be developed for separating and
purifying a factor of interest from other factors in
~'!10296
the crude starting material without denaturing the ac-
tivity of the desired factor; bioassays must be devel-
oped which allow for identifying the fractions during
separations which concentrate a particular f~ctor; a
novel factor must be distinguished from factors which
are already known or other unknown factors which may be
present and may affect, either negatively or positive-
ly, the activity of the factor being pursued; the puri-
fied factor must be characterized; and the purified
factor must be concentrated in sufficient amount to
permit identification and characterization of the
factor. Therefore, with the increasing number of fac-
tors which have been isolated, each new factor becomes
more difficult to identify, since its role and function
15 may be obscured by the numerous other factors which are
present.
Description of the Prior Art
Beal et al., Cancer Biochem. BiophyS. (1979)
3:93-96 report the presence of peptides in human urine
which inhibit srowth and DNA synthesis more in trans-
formed cells than in normal cells. Holley et al.,
Proc. Natl. Acad. Sci. (1980) 77:5989-5992 describe the
purification of epithelial cell growth inhibitors.
Letansky, Biosci. Rep. (1982) 2:39-45 report that pep-
tides purified from bovine placenta inhibit tumor
growth and thymidine incorporation in DNA to a greater
extent in neoplasms than in normal cells. Chen, Trends
Biochem. Sci. (1982) 7:364-365 reports the isolating of
3~ a peptide from ascites fluid with a cancer suppressing
property. Redding and Schally, Proc. Natl. Acad. Sci.
(1982) 79:7014-7018 report isolation of purified pep-
tide(s) from porcine hypothalmi which exhibit antimito-
genic activity against several normal and tumor cell
lines. Sone _ al., Gann (1984) 75: 920-328 report the
production of a factor(s) produced by human macropha3es
that inhibits the growth of certain tumor cells in
..
134~)296
vitro. Ransom et al., Cancer Res. (1985) 45:851-862,
report the isolation of a factor called leukoregulin
that inhibits replication of certain tumor cell lines
and appears distinct from lymphytoxin, interferon and
5 interleukin 1 and 2. Most of these factors have not
been fully characterized, nor are their primary struc-
tures known.
Aggarwal et al., J. Biol. Chem. (1984) 259:
686-691 purified and characterized human lymphotoxin
0 (LT) produced by a lymphoblastoid cell line and subse-
quently sequenced LT (Aggarwal et al., J. Biol. Chem.
(1985) 260:2334). Gamma interferon (Y-IF) which is
produced by lymphoid cells and has immunomodulatory and
tumor inhibitory activity has been cloned and ex-
5 pressed. (Gray et al., Nature (1982) 2g5:503:508.)
Tumor necrosis factor (TNF), which inhibits growth of
some tumors and is produced by macrophages and certain
leukemia cell lines has been characterized and the TNF
cDNA was cloned and expressed in E. coli (Pennica et
20 al., Nature ( 1984) 312 :724) .
SUMMARY OF THE INVENTION
A novel peptide factor and biologically active
25 fragments thereof are provided, which factor is
available from leukocytes. The factor finds use in
modulating cell growth, such as inhibiting tumor cell
growth and stimulating growth of normal fibrobla~ts
and may modulate immune functions. The factor has an
3~ amino acid sequence distinctively different from the
sequences of other compounds which have been reported
to have analogous properties.
. .
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 represents the amino acid sequence of
fragments of Oncostatin M;
Figure 2 is a series of photomicrographs of
cells treated with Oncostatin M wherein (A-C) are A375
melanoma cells treated with 0, 5 and 100 GIA units,
respectively, and (D-F) are WI38 fibroblasts treated
with 0, 5 and 100 GIA units, respectively; and
Figure 3 is a photograph of an SDS-PAGE
analysis of Oncostatin M.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
A novel polypeptide, polypeptide compositions,
polypeptide fragments and mutations, their preparation
and use are provided, with the compositions demonstrat-
ing activity in the modulation of cell growth, particu-
larly inhibiting tumor cell growth and stimulating
growth of normal fibroblasts. A subject polypeptide,
referred to as Oncostatin M is available from leuko-
cytes; e.g., from conditioned media of stimulated U937cells or conditioned media of stimulated normal human
peripheral blood lymphocytes (PBL). Fragments and
mutants of the polypeptide having the biological
activity of the intact Oncostatin M such as cell growth
modulation activity or immunologic activity are also
provided.
The polypeptide fragments of this invention
are novel polypeptides of at least 8 amino acids that
are biologically active, at least as to being immuno-
logically cross-reactive with naturally occurring
Oncostatin M. By immunologically cross-reactive it is
meant that an antibody induced by a novel polypeptide
of this invention will cross-react with intact Onco-
statin M at least when Oncostatin M is in a denatured
35 state. Those polypeptides are tnerefore useful to
induce antibodies to Oncostatin M which can be used to
determine the concentration of Oncostatin M in a bodily
~'10296
fluid, to bind to Oncostatin M and thus modulate its
activity, and to purify Oncostatin M, as by use in an
affinity column. A portion of the polypeptides may
also retain the cell growth modulatory activity of
intact Oncostatin M, although that activity may be
modulated, usually reduced in comparison to intact
Oncostatin M.
Figure 1 represents the amino acid sequence of
poly(amino acid)s cross-reactive with Oncostatin M with
the first sequence representing the N-terminus of
Oncostatin M.
Poly(amino acid)s of the present invention
contain an amino acid sequence having at least 8 con-
secutive amino acids that correspond to an amino acid
sequence depicted in Figure 1 and differing from that
sequence by no more than 3, usually no more than 1
amino acid. That difference can be either the inser-
tion of an amino acid, the deletion of an amino acid or
the substitution of one amino acid for another, partic-
ularly a conservative substitution. Usually thepoly(amino acid)s will contain at least 10, more
usually at least 12, consecutive amino acids that
correspond to sequences depicted in the figure and
differ by no more than one amino acid.
For purposes of the subject invention, the
various amino acids will be divided into a number of
subclasses. The following table indicates the sub-
classes:
3~ aliphatic
neutral
non-polar G A P V L I
polar S T C M N Q
acidic D E
basic K R
aromatic F H Y W
2 9 6
By conservative substitution, it is meant that
amino acids from the same subclass (i.e., either
neutral aliphatic, acidic aliphatic, basic aliphatic or
aromatic), more particularly the same polarity, will be
5 substituted for each other. Desirably, amino acids of
two to four carbon atoms or five to six carbon atoms
will define monomer groupings in the aliphatic
subclass.
The poly(amino acid)s will not exceed about
10 1000 amino acids in length. Usually they will have
fewer than one hundred amino acids, more usually fewer
than fifty amino acids. Thus, the poly(amino acid)s
can be readily synthesized. Usually when poly(amino
acid)s exceed 100 amino acids in length, those
15 poly(amino acid)s may be polymers of fragments of
Oncostatin M having fewer than 100 amino acids each, or
fusion proteins where the fragment is fused to an
antigen, enzyme, enzyme fragments, etc. Particularly,
the higher molecular weight poly(amino acid)s can be at
20 least one polypeptide fragment of fewer than about 100
amino acids joined covalently to a large immunogenic
polypeptide carrier to provide for immunogenicity.
Exemplary of such protein carriers are bovine serum
albumin, keyhole limpet hemocyanin (KLH) and the like.
25 Those conjugated polypeptides will be useful for
inducing antibodies in an appropriate host organism.
U937 cells are a cell line derived from a
histiocytic lymphoma cell line (Sundstrom and Nilsson,
Int. J. Cancer (19760 17:565-577) that can be induced
30 to differentiate into cells having characteristics of
macrophages following treatment with a variety of
agents (Harris et al., Cancer Res. (1985) 45:9-13).
For production of Oncostatin M, the U937 cells may be
srown in a conventional nutrient medium with serum and
35 treated with an appropriate inducer. Conveniently,
phorbols or ingenols may be employed, particularly 12-
O-tetradecanoylphorbol-13-acetate (TPA). Usually, from
~402~6
about 5-20ng/ml of the inducer may be employed. The
initial number of cells is ~rom about 105 _1o6
cells/ml.
After allowing cells to be treated with the
inducer for a sufficient time, generally three to six
days, the supernatant is removed, the cells washed with
serum-free nutrient medium, attached cells wasned again
with serum-free medium and the cells allowed to incu-
bate for at least 12 hours, usually not more than about
48 hours, in serum-free nutrient medium, e.g. RPMI-1640
medium. Supernatants are then collected and cells are
removed by centrifugation. Cell-free supernatants were
tested for cell growth inhibitory activity (GIA) as
described in the experimental plan. The supernatant
contains about 50 to 500 units of GIA/ml (see Experi-
mental for definition of GIA units.)
Oncostatin M can also be obtained from
mitogen-stimulated normal human peripheral blood lym-
phocytes (PBL's). PBL's can be isolated from leuko-
fractions by diluting the fractions and centrifugingthem over Ficoll gradients. Cells collected from the
gradient interface are washed and shock-lysed to remove
red blood cells. Remaining cells are collected from
that solution by centrifugation, resuspended in buffer
containing serum and thrombin, agitated and the plate-
let aggregate allowed to settle for a short period of
time. The suspended cells are transferred, recovered
by centrifugation, resuspended in serum and transferred
to a column containing nylon wool. The column is incu-
3~ bated to allow the attachments of monocytes and B-
lymphocytes and then washed. Most peripheral blood T
lymphocytes do not adhere and are eluted from the
column. Those cells were cultured at 37~ in culture
medium, e.g. RPMI-1640 medium, and treated with an
appropriate inducer, e.g. phytohemagglutinin (about 1
to 5 mg/l), for about 100 hours and then supernatants
were collected. The supernatants were centrifuged to
~ 3 ~ 0 2 9 b
remove cells and concentrated as by ultraiiltration or
dialysis.
After isolating cell-free supernatant from
either U937 cells or normal P8L's, the conditioned
medium is concentrated, conveniently using a hollow
fiber system or an ultrafiltration membrane, followed
by dilution with acetic acid (to a concentration of
0.1N acetic acid) followed by concentrating about ten-
fold and the dilution and concentration repeated. The
concentrate may be lyophilized and used directly or the
lyophilized product can be used for further
purification.
The subject Oncostatin M can be purified by a
procedure of gel permeation chromatography using
aqueous 40d acetonitrile-0.1% trifluoroacetic acid as
an isocratic mobile phase on a Bio-sil TS'~250 column,
monitoring activity for each of the fractions. Purifi-
cation provides for a composition having at least about
0.5-5x104 GIA units/ml in the active fractions.
The partially purified product from the gel
permeation chromatography may be further purified by
employing reverse phase high-pressure liquid chromatog-
raphy employing a linear gradient, where the primary
solvent is 0.1% trifluoroacetic acid in water and the
secondary solvent is acetonitrile containing 0.1% tri-
fluoroacetic acid. The schedule can be varied, ~en-
erally the chromatographing requiring about 3-4 hours,
with the major portion of the time, greater than about
50p of the time and not more than about 80p of the
3~ time, in the range of 30-45% of the secondary solvent.
Under these conditions the active fractions elute at
about 41-42p acetonitrile.
The pooled active fractions may be further
purified by repeating the reverse phase HPLC, employing
a more rapid change in the gradient conditions and a
slower flow.rate. Under these conditions, the activity
emerges at about 40.5-41.5p acetonitrile.
* Trade Mark
Bl
.. . . ... . . . .
~t 10296
- 9
The reverse phase HPLC may then be repeated,
changing the solvent system, where the secondary sol-
vent is n-propanol-0.1% trifluoroacetic acid. A linear
gradient is employed, where the gradient is changed
slowly in the range of 23-35% n-propanol. The major
activity is observed in the range of 25.5-27.5% pro-
panol, to sive a substantially homogeneous product,
having a specific activity of greater than 10 GIA
units/ng protein. Usually, the product is purified to
provide a specific activity of at least about 100 GIA
units/ng protein, more usually 150 GIA units/ng.
The subject compounds are characterized by
having a molecular weight of about 17 to 13 kiloDaltons
(kD), particularly about 18kD, as determined by size
exclusion chromatography The subject compounds are
further characterized as having an apparent molecular
weight of approximately 28kD as determined by poly-
acrylamide gel electrophoresis under reducing or non-
reducing conditions.
The amino acid sequence of fragments of puri-
fied Oncostatin M was analyzed. Oncostatin has sub-
stantially the amino acid sequences represented in
Figure 1. Referring to Figure 1, the first sequence
illustrates the N-terminus of Oncostatin M, while the
remaining sequences illustrate internal fragments of
the polypeptide.
Active preparations of isolated Oncostatin M
contained a mixture of high mannose and complex N-
linked oligosaccharide. However, non-glycosylated
preparations of Oncostatin M retained cell growth
modulatory activity.
Oncostatin M is further characterized by its
activity toward certain cell strains. The subject
polypeptide lacks cytotoxic activity against WI26 and
WI38 human fibroblasts, and mouse L929 cells which are
sensitive to tumor necrosis factor, and a Y-interferon-
sensitive human tumor cell line. It also is found not
_ _. . ....
3 '~L 0 2 ~ b
l o
to inhibit proliferation of normal human T-lymphocytes
or to inhibit granulocytic/myelocytic colony formation
from bone marrow cells at concentrations up to 100 GIA
units/ml. Further, Oncostatin M stimulates prolifera-
tion of normal human fibroblasts as exemplified by WI38and WI2~ cells and inhibits proliferation of tumor
cells such as A375, H3T10, A549 and SK-MEL28 and may
augment growth of colony forming cells from normal bone
marrow. Oncostatin M did not suQpress human prolifera-
0 tive or cytotoxic T cell responses in mixed leukocyteculture reactions (MLC) at concentrations of 500 GIA
units/ml.
The subject polypeptide is found to be stable
to moderate acid and base and to heat treatment at
5~~C.
The amino acid sequence of the subject poly-
peptide may be completely determined using commercially
available sequencers. The polypeptide may then be syn-
thesized in accordance with known techniques, employing
automated synt'nesizers which are also commercially
available.
Alternatively, the subject polypeptide may be
produced by recombinant DNA techniques. From a partial
amino acid sequence, probes can be deduced which can
then be used for screening a human genomic library.
The library may be a cDNA library or a chromosomal
library. Once the clone(s) have been identified as
annealing to the probe, the fragments containing the
gene of interest may be identified in a number of ways
3~ and manipulated in a number of ways. The fragment may
be reduced in size by endonuclease restriction, with
the resulting fragments cloned and probed for the
presence of the desired gene. Cells which produce the
desired peptide or produce enhanced amounts of the de-
sired peptide may be employed for production of messen-
ger RNA. From the messenger RNA, sinsle stranded cDNA
may be prepared. The cDNA may then be annealed to
-
1~40296
. .
total messenger RNA from a cell which produces little,
if any, of the polypeptide. The unannealed cDNA may
then be isolated and used to prepare ds cDNA, which may
be screened with the probes.
Alternatively, DNA fragments may be inserted
into Agt11, so that coding fragments may be downstream
from and in frame with the ~-galactosidase gene. Anti-
bodies may be prepared to the Oncostatin M polypeptide
and used for screening the resulting fused proteins for
cross-reactivity. In this manner, fragments coding for
the subject polypeptide or fragment thereof may be
identified and used for identifying the desired gene.
Once a complete gene has been identified,
either as cDNA or chromosomal DNA, it may then be
manipulated in a variety of ways to provide for expres-
sion. Where the gene is to be expressed in a host
which recognized the wild-type transcriptional and
translational regulatory regions of Oncostatin M, the
entire gene with its wild-type 5'- and 3'-regulatory
regions may be introduced into an appropriate expres-
sion vector. Various expression vectors exist employ-
ing replication systems from mammalian viruses, such as
Simian Virus 40, adenovirus, bovine papilloma virus,
vaccinia virus, insect baculovirus, etc. These repli-
cation systems have been developed to provide formarkers which allow for selection of transfectants, as
well as providing for convenient restriction sites into
which the gene may be inserted.
Where the gene is to be expressed in a host
which does not recognize the naturally occurring wild-
type transcriptional and translational regulatory
regions, further manipulation will be required. Con-
veniently, a variety of 3'-transcriptional regulatory
regions are known and may be inserted downstream from
the stop codons. The non-coding 5'- region upstream
from the structural gene may be removed by endonuclease
restriction, Bal31 resection, or the like. Alterna-
-
~?, -1029~
12
tively, where a convenient restriction site is present
near the 5'-terminus of the structural gene, tne struc-
tural gene may be restricted and an adaptor employed
for linking the structural gene to the promoter region,
where the adaptor provides for the lost nucleotides of
the structural gene. Various strategies may be em-
ployed for providing for an expression cassette, which
in the 5'-, 3'-direction of transcription has a tran-
scriptional and translational initiation region, which
may also include regulatory sequences allowing for the
induction of regulation, the structural gene under the
transcriptional and translational control of the initi-
ation region, and a transcriptional and translational
termination region.
Illustrative transcriptional initiation re-
gions or promoters include, for bacteria, the ~-gal
promoter, the TAC promoter, lambda left and right pro-
moters, etc.; for yeast, glycolytic enzyme promoters,
such as ADH-I and -II promoters, GPK promoter, and PGI
promoter, TRP promoter, etc.; for mammalian cells, SV40
early and late promoters, adenovirus major late promo-
ters, etc. As already indicated, the expression cas-
sette may be included within a replication system for
episomal maintenance in an appropriate cellular host or
may be provided without a replication system, where it
may become integrated into the host genome. The DNA
may be introduced into the host in accordance with
known techniques, such a~ transformation, using calcium
phosphate-precipitated DNA, transfection by contacting
the cells with the virus, microinjection of the DNA
into cells or the like.
Once the structural gene has been introduced
into the appropriate host, the host may be grown and
will express the structural gene. In some instances,
it may be desirable to provide for a signal sequence
(secretory leader) upstream from and in reading frame
with the structural gene, which provides for secretion
~ . ~
029~
13
of the structural gene and cleavage of the secretory
leader, so as to provide for the mature polypeptide in
the supernatant. Where secretion is not provided for,
then the host cells may be harvested, lysed in accor-
dance with conventional conditions, and the desiredproduct isolated and purified in accordance with known
techniques, such as chromatography, electrophoresis,
solvent extraction, or the like.
The subject compounds can be used in a wide
0 variety of ways, both in vivo and in vitro. The sub-
ject compounds can be used for making antibodies to the
subject compounds, which may find use in vivo or in
vitro. The antibodies can be prepared in conventional
ways, either by using the subject polypeptide as an
immunogen and injecting the polypeptide into a mamma-
lian host, e.g. mouse, cow, goat, sheep, rabbit, etc.,
particularly with an adjuvant, e.g. complete Freunds
adjuvant, aluminum hydroxide gel, or the like. The
host may then be bled and-the blood employed for iso-
lation of polyclonal antibodies, or in the case of themouse, the peripheral blood lmphocytes or splenic lym-
phocytes (B-cells) employed for fusion with an appro-
priate myeloma cell to immortalize the chromosomes for
monoclonal expression of antibodies specific for the
subject compounds.
Either polyclonal or monoclonal antibodies may
be prepared, which may then be used for diagnosis or
detection for the presence of the subject polypeptide
in a sample, such as cells or a physiological fluid,
3~ e.g. blood. The antibodies may also be used in affini-
ty chromatography for purifying the subject polypeptide
and isolating it from natural or synthetic sources.
The antibodies may also find use in controlling the
amount of the subject polypeptide associated with cells
in culture or in vivo, whereby growth of the cells may
be modified.
~ . .. . . . ..
1~ 102~6
14
The subject compound may be used as a ligand
for detecting the presence of receQtors for tne subject
compound. In this way, cells may be distinguished in
accordance with the presence of and the density of re-
ceptors for the subject compound, monitoring the effectof various compounds on the presence of such receptors.
The subject compound may be used in in vitro
cultures to inhibit the growth of cells or cell lines
sensitive to the subject polypeptide as distinguished
0 from cells which are not sensitive. Thus, heterogene-
ous cell mixtures or cell lines can be freed of unde-
sirable cells, where the undesirable cells are sensi-
tive to the subject polypeptide. The subject polypep-
tide may be administered in vivo in the case of neo-
plastic conditions, for example, by injection, intra-
lesionally, peritoneally, subcutaneously, or the like.
The subject compound may be used in vitro to eliminate
malignant cells from marrow for autolosous marrow
transpla~ts or to inhibit proliferation or eliminate .
malignant cells in other tissue, e.g. blood, prior to
reinfusion.
The subject polypeptide may also be used in a
method for treating wounds, such as cutaneous wounds,
corneal wounds, and various other epithelial and
stromal disruptions, such as chronic ulcers, burns,
surgical incisions, traumatic wounds, and injuries to
the hollow, epithelial-lined organs, such as the esoph-
agus, stomach, large and small bowels, mouth, genital,
and urinary tract. The method relies on the topical
3~ application of a treatment composition including Onco-
statin M in a physiologically-accepable carrier.
The composition of the present invention may
be used for treating a wide variety of wounds including
substantially all cutaneous wounds, corneal wounds, and
injuries to the epithelial-lined hollow organs of the
body. Wounds suitable for treatment include those re-
sulting from trauma such as burns, abrasions, cuts, and
1~ 10~6
the li'~e as well as from sur3ical procedures such as
surgical incisions and skin grafting. Other conditions
suitable for treatment with the compositions of the
present invention include chronic conditions, such as
chronic ulcers, diabetic ulcers, and other non-healing
(trophic) conditions.
Oncostatin M may be incorporated in physio-
103ically-acceptable carriers for application to the
affected area. The nature of t'ne carriers may vary
0 widely and will depend on the intended location of
application. For application to the skin, a cream or
ointment base is usually preferred, suitable bases in-
clude lanol'in, Silvadene (Marion) (particularly for the
treatment of burns), Aquaphor ;Duke Laboratories, South
Norwalk, Connecticut), and the like. If desired, it
will be possible to incorporate Oncostatin M containing
compositions in bandages and ot'ner wound dressings to
provide for continuous exposure of the wound to the
~ peptide. Aerosol applications may also find use.
The concentration of polypeptide in the treat-
ment composition is not critical. The polypeptide will
be present in an epithelial cell proliferation-inducing
amount. The compositions will be applied topically to
the affected area, typically as eye drops to the eye or
as creams, ointments or lotions to the skin. In the
case of eyes, frequent treatment is desirable, usually
being applied at intervals of ~ hours or less. On the
skin, it is desirable to continually maintain the
treatment composition on the affected area during the
3~ healing, with applications of the treatment composition
from two to four times a day or more f~equently.
The amount employed of the subject polypeptide
will vary with the manner of administration, the em-
ployment of other active compounds, and the like, gen-
erally being in the range of about lyg to lOOyg. Thesubject polypeptide may be employed with a physiolog-
ically acceptable carrier, such as saline, phosphate-
* Trade Mark
B
10296
16
buffered saline, or the like. The amount of compoundemployed will be determined empirically, based on the
response of cells in vitro and response of experimental
animals to the subject polypeptides or formulations
containing the subject polypeptides. The subject com-
pounds find use by themselves or in combination with
other growth factors or inhibitors or immunomodulators,
such as TNF, IL-2, Y-interferon, monoclonal antibodies,
etc. The amounts of these other compounds will gener-
ally be in the range of 1~g to 100~g. Conjugates ofthe subject compounds to site directing moieties, e.g.,
antibodies may be prepared, where the antibodies may be
specific for particular malignant cells or organs.
The following examples are offered by way of
illustration and not by way of limitation.
EXPERIMENTAL
Materials and Methods
Oncostatin.M Isolated from U937 Cells
Production of a Tumor Cell Growth Inhibitor
From a Histiocyctic Lymphoma Cell Line
U937 cells, a histiocyctic lymphoma cell line
(Sundstrom and Nilsson, Int. J. Cancer (1976) 17:565-
577), were cultured in 850cm2 roller bottles (Corning
C2540) at a concentration of 4x105 cells/ml in a total
volume of 300ml RPMI 1640 medium supplemented with lod
fetal calf serum (FCS), penicillin/streptomycin (PS),
L-glutamine and 1Ong/ml 12-0-tetradecanoylphorbol 13-
acetate (TPA). Four days later, the; supernatants con-
3~ taining the FCS and TPA were removed, the rollerbottles were washed five times with serum-free RPMI-
1640, and the cells which detached (1x105 cells/ml)
were washed 3 times with serum-free medium and added
back to the bottles, resulting in a final volume of
125ml serum-free RPMI-1640 medium per roller bottle.
One day later, the supernatants were collected, centri-
fuged to remove the cells, filtered through 0.45 micron
0296
17
(~) Nalgene filter and concentrated using a hollow
fiber system (Amicon cartridge HIP10-20)~to a volume of
150ml (initial volume 1500ml). Oncostatin M was also
isolated from supernatants of serum-free TPA-treated
U937 cells in 150 CM2 tissue cultur~ flasks. The
supernatant was concentrated with an Amicon Diaflo
membrane PM-10,lOkD cut-off and dialyzed. Following
dialysis, the concentrate was diluted with acetic acid
resulting in a final concentration of 0.1~ acetic acid
0 in 500ml and concentrated to 50ml using an Amicon PM 10
filter. The 50ml concentrate was diluted to 400ml with
0.1N acetic acid and concentrated to 40ml with the same
filter. The concentrate was diluted with 1N acetic
acid and the resulting precipitate was removed by
centrifugation. The resulting concentrate was frozen
and lyophilized. The lyophilized material was used for
purification steps.
Gel Permeation Chromatography
A 3io-sil TSK-250 column (600x21.5mm) (Bio-
Rad) was attached to a high pressure liquid chromato-
graphic system. The crude fraction (10mg/ml) was dis-
solved in 40% acetonitrile in 0.1~ aqueous trifluoro-
acetic acid (0.1p TFA). A 2ml aliquot of the mixture
was injected and elution was performed isocratically
with a mobile phase of 40p acetonitrile in 0.1~ TFA.
The flow rate was 2.5ml/min and chart speed was set at
0.25cm/min. 5ml fractions were collected. The chroma-
tography was performed at room temperature. An aliquot
3~ from each fraction was evaporated and assayed in trip-
licate for growth inhibitory activity (GIA) of A375
cells.
The active fractions (Fractions 21 and 22)
from six runs were pooled. The pooled material had a
total of approximately 4.8x105 GIA units. The factor
was found to have an apparent molecular weight of 18kD
as determined by size exclusion chromatography (Bio-Sil
TS'~-250 column).
*Trade-mark
,
.... .. . . . .. . ...
?~ ~'1029~
18
. Reverse Phase High Pressure Chromatography (~PLC)
~f TSK-250 Fractions
Pooled TSK-250 fractions 21 and 22 described
above were diluted two-fold with O.lq TF.4. This mix-
ture was injected isocratically on a ~-Bondapak-C18 *
column (7.8x300mm) (referred to as C181) at room
temperature. The flow rate was set at 2.0 ml/min and
the chart speed was 0.25 cm/min. The linear gradient
was used between primary solvent 0.1d TFA and the sec-
ondary solvent acetonitrile-TFA 0.1~. Tne gradient
conditions were 0-30p in 20min; then 30-45~ in 150min;
45_55d in 20min; and 55-lOOp in lOmin. All solvents
were HPLC grade. 4ml fractions were collected and
aliquots of each fraction were assayed for growth
inhiDitory activity. Fractions 72-75 were found to
contain the majority of activity. The a~tive fractions
eluted between 41-52p of acetonitrile concentration.
Fractions 72-75 were pooled. 16ml of 0.1~ TFA
was zdded to the pooled fractions. The mixture was in-
jected into a ~-Bondap2k-Ct8 column (3.9x300mm) (re-
ferred to as C182) at room temperature. The flow rate
was set a lml/min and chart speed was 0.25cm/min. The
gradient conditions were 0-35~ in lOmin; 35_45d in
lOOmin; and 45-100~ in lOmin. Fractions were collected
and aliquots wer~ taken and assayed for GIA. Most of
the activity emer8ed from the column getween 40.7 to
~1.3~ acetonitrile concentration (retention time 83-
86min).
Active fractions were pooled 2nd diluted two-
fold with O.ld TFA and injected isocratically on a ~-
Bondapa~-C18~column (3.9x300mm) (referred to as C?83)
at room temperature. The flow rate was 1ml/min and
ch2rt speed was 0.25 cm/min. A linear gradient was
used betweem ?rim2ry solvent 0.1~ TFA 2nd the secondary
solvent n-propanol-TFA (0.1~). The gr2dient conditions
were 0-23d in 20min and 23-35~o in 120min. Fr2ctions
were collected 2nd aliquots of each fr2ction were
* Trade Mark
4 ~-
.. . . ~, . . . . .
0 2 9 6
, 9
assayed for GIA. Most of the activity appeared between
25-26.5% propanol concentration (retention time 59min).
This apparently homogeneous fraction contained approxi-
mately 300ng protein and about 40,000 GIA units.
Cell Growth Modulatory Assay Using ,
3H-Thymidine Incorporation into DNA (GIA)
The assays were Derformed in 96 flat well
plates (Costar 3596). Human melanoma cells (A375) were
used as a sensitive indicator cell line. Cells (3x103)
in 0.1ml Dulbecco's modified Eagles medium (DMEM) sup-
plemented with 10p FCS and PS were placed in each well.
Three hours later, 0.1ml of test samples was added to
each well. Plates were incubated at 37~C for 3 days.
Then 0.025ml (0.5~Ci) of a solution of 3H-thymidine
(specific activity 27~Ci/~g) was added to each well for
the final 6 hours of incubation. The cells were then
transferred to glass filter strips by using a multiwell
harvester (PHD Cell Harvester, Cambridge Technology,
Inc.). The filters were transferred to scintillation
vials to which were added 2ml of scintillation fluid
(ScientiVerse II,* Fisher Scientific Co.) prior to
counting in a scintillation counter for quantitating
3H-thymidine incorporation.
Soft Agar Colony Inhibition Assay (TGI)
A 0.5ml base layer of 0.5p agar (Agar Noble;
Difco Laboratories, Detroit, Michigan) in DMEM contain-
ing 10p fetal calf serum (FCS) was added to 24 well
3~ Costar tissue culture plates. 0.5ml 0.3p agar contain-
ing the same medium-FCS mixture, 1-2.5x103 A375 cells
and the factor to be tested at various concentrations
were overlaid on the base layer of agar. The plates
were incubated at 37~C in a humidified atmosphere of 5%
C~2 in air and refed after 7 days by addition of 0.5ml
of 0.3% agar containing the same medium and concentra-
tions of factor. Colonies were enumerated unfixed and
* Trademark
F
.. . . . . . ...
~.3~0296
unstained and the number of colonies 3reater than 6
cells were scored between days 7 and 14.
Results
Sequences of Oncostatin M Isolated from U937 Cells
The N-terminal sequence and internal fragments
of Oncostatin M were determined by microsequence
analysis of the reduced and S-pyridinethylated poly-
peptide and of peptides obtained from enzymatic digests
of reduced and S-pyridinethylated Oncostatin M with the
endoproteinase Lys-C and Staphylococcus aureus V8
protease. The peptide fragments were purified by
reverse phase HPLC, using volatile solvents. The
peptides were subjected to automated repetitive Edman
degradation in the Model 470A protein sequencer
(Applied Biosystems, Inc.). The phenylthiohydantoin
amino acids were analyzed by reverse phase HPLC
(Applied Biosystems, Inc.) with a PTH-C18 column
(2.1x220mm, ABI), usins a sodium acetate buffer/t2tra-
hydrofuran/acetonitrile gradient for elution.
The resultant amino acid sequences aresubstantially as illustrated in Figure 1.
A comparison of these sequences wit,h those
stored in the curent protein data base (PIR Release
g.o, May 1986), revealed no significant sequence homol-
ogies with any other known sequence. In addition,
there is no homology with tumor necrosis factor, lymph-
otoxin, colony stimulating factor, interleukin 1 or 2
or ~-transforming growth factor.
Inhibition of Proliferation of Tumor Cells and Augmen-
tation of Proliferation of Normal Human Fibroblasts
Employing the soft agar colony inhibition
assay described above, the following results were
obtained:
*Trade-mark
~-'
Table l 13 ~0296
Inhibition of A375 Melanoma Cell Colony
Formation in Soft Agar by Purified Oncostatin X
Isolated from U937 Calls*
% Inhibition of
GIA UNits/Well% of Colonies Colony Formation
250 4 96
83 6 94
27 11 89
32 69
0 106
';A375 cells were plated in soft agar, with or without
factor in a final volume of 2ml, as described above. The factor
used was from a Cl8 propanol column fraction with peak tumor
growth inhibitory activity (GIA). Eleven days later the number
of colonies were enumerated. A colony was defined as a cluster
of at least six cells. One GIA unit was defined as the amount to
cause 50% inhibition of H-thymidine incorporation into A375
cells in micro wells as detailed above.
Table 2
Effects of Various Treatments of Supernatants
of TPA-Induced U937 Cells on Tumor
Growth Inhibitory Activity '~
Final Dilution of Supernatant
1:4 1:8 1:16
Media Control 39,780
Untreated Super 7,206-';*13,896 16,000
IN Acetic Acid 6,670 17,07318,783
INH40H 6,956 15,01613,923
-'-U937 cells were treated with TPA (lOng/ml) for 3 days
and then the cells were washed with media and incubated for 24
hr in serum-free media before collecting the supernatants. The
supernatants were treated with lH acetic acid or lN ammonium
hydroxide (NH40H). They were then displayed against medium and
tested for their ability to inhibit H-thymidine incorporation
into A375 cells. A375 cells were labeled with H-thymidine
(3H-TdR) for the last 6 hr of a 3-day incubation.
~ ;-';Data shown are H-TdR incorporation counts per
minute.
*Trade-mark - 21 -
.. . ,, . .. _ . . . . . .
.~3 102~6
- 22
The above results demonstrate that Onco-
statin M in the dialyzed supernatant is substantially
resistant to inactivation by one normal acetic acid and
one normal ammonium hydroxide. Thus, the subject com-
pounds are relatively insensitive to both moderatelystrong acid and moderately strong base. The subject
compound is stable to heat treatment at 56~C. for 1
hour but not to 90~C. for 30 minutes.
The subject compound was also tested for heat
stability and was found to retain its activity after
exposure at 56~C for lhr, but to lose substantially all
its activity after exposure to 95~C for 30min.
In the next study, the ability of the subject
polypeptides to inhibit tumor cell re~lication of a
variety of neoplastic cells was investigated. The fol-
lowing table indicates the results.
Table 3. Inhibition of Replication of Tumor Cells by
Purified Oncostatin M from U937 Cells
Units GIA to Cause
30% Inhibition
Tumor Cells 3H-TdR Incorporation
A549 (lung cancer) 21
HTB10 (neuroblastoma) 81
A375 (melanoma) 0.3
Tumor cells were plated in microwells 3hr prior to the addi-
tion of various dilutions of Oncostatin M, purified by re-
verse phase HPLC as detailed above. For the final 6hr of a
3-day incubation, cells in 0.2ml medium were labeled with
3H-thymidine (3H-TdR) (0.5~Ci/well). One unit tumor growth
inhibitory activity (GIA) is defined in the legend to Table
1 as that amount which causes 50~ inhibition of 3H-TdR
incorporation into A375 melanoma cells. One unit was
determined to be approximately 10pg of purified protein,
therefore the concentration (ng/ml) to cause 30% inhibition
3H-TdR into A549, HTB10 and A375 cells was approximately
1.4, 4.0, and .015ng/ml, respectively. 3H-TdR incorporation
into WI26 normal human fibroblasts was not suppressed by
U937 factor in any experiment.
102g6
23
The above results demonstrate that Onco-
statin M is selective in its ability to inhibit repli-
cation, having widely varying effects depending upon
the nature of the cell. The subject compound is effec-
tive against melanoma cells, such as A375 melanomacells, squamous lung cancer cells, such as A549, and
neuroblastoma cells, such as HTB10.
Tumor cells were seeded at 3x103 cells/well
10 and normal fibroblasts at 1.5x103 cells/well in 96 well
pIates for 3 hours. Various concentrations of purified
Oncostatin M, obtained from the fraction from the C183
column with peak antiproliferative activity against
A375 cells were added and three days later 3H-thymidine
incorporation into cells was measured in triplicate
wells at each concentration. The results are shown in
Table 4.
3o
...
~34~296
24
Table 4. Inhibition of Proliferation of Tumor Cells and Aug-
mentation of Proliferation of Normal Fibroblasts by
Oncostatin M
GIA
units/well % inhibition % stimulation
A375 WI38
Exp. 1 16 ~3 25
4 62 30
1 46 46
A375 HTB10 WI26
Exp. 2 27 NT 28 46
9 87 22 36
3 76 11 52
A375 A549
Exp. 3 75 ~9 30
22
8 71 16
A375 SK-MEL28
Exp. 4 20 ~7 44
1 59 11
Results shown are ~ inhibition or % stimulation of 3H-thy-
midine incorporated into tumor cells (A375, HTB10, A549, and
SK-MEL28) and normal fibroblasts (WI26 and WI38~, respec- -
tively. One GIA unit is defined in the legend to Table 1 as
the amount of Oncostatin M that causes 50% inhibition of 3H-
thymidine incorporation into A375 celIs.
In addition to the observed differential ef-
fect on 3H-thymidine incorporation into tumor cells and
normal human fibroblasts, there is also an observed
differential effect of morphology and cell number fol-
lowing 3 days of treatment of the two cell types with
Oncostatin M as shown in Figure 2.
The Oncostatin M used was from the HPLC-C183
3o column fraction with peak activity for inhibiting pro-
liferation of A375 cells. Figure 2 is a series of
photomicrographs of A375 melanoma cells that were un-
treated (A), treated with 5 growth inhibitory activity
(GIA) units of Oncostatin M (B), or 100 units (C).
Photomicrographs of WI38 fibroblasts that were un-
treated (D), treated with 5 units GIA (E), or 100 units
.. ~
..... .
~ ~402~6
(F). The cells were stained with crystal violet in
0.5~ methanol. Magnification = 63X.
NaDodSO~/PAGE of Oncostatin M
Purified Oncostatin M, subjected to NaDodS04
performed under reducing conditions, was found to have
an apparent molecular weight of approximately 28,kD as
shown in Figure 3. The following proteins were used as
standards (lane A): ovalbumin, Mr = 43 kD chymotryp-
sinogen a, Mr = 25.7 kD; lactoglobulin ~, Mr = 18.4 kD;
lysozyme, Mr - 14.2 kD; bovine trypsin inhibitor = 6.2
kD; insulin A and B chain, Mr = 2.3 kD and 3.4 kD re-
spectively. Oncostatin M was applied to lane a.
Oncostatin M, subjected to PAGE under non-
reducing conditions, also has an apparent molecular
weight of 28 kD and protein electroeluted from the band
was found to inhibit proliferation of A375 cells.
Antibody to a Synthetic Peptide of Oncostatin M Reacts
in 125I-labeled Oncostatin M in Radioimmune
Precipitations
a) Peptide Synthesis: The peptide corresponds to
residues 6-19 of the Oncostatin M protein and was syn-
thesized by solid phase techniques on a Beckman auto-
mated instrument as described (Gentry et al., J. Biol.
Chem. (1983) 258:11219). The peptide was cleaved fromthe resin using the "low-high" HF procedure (Tam et
al., J. Amer. Chem. Soc. (1983) 105:6442-6445).
Purification was accomplished by preparative HPLC.
b) Production of Antibodies: The peptide was
coupled to bovine Y-globulin as described (Gentry and
Lawton, Virology (1986) 152:421-431). New Zealand
white rabbits were primed and boosted (5 times) at 4
sites subcutaneously as described (Gentry and Lawton,
Virology (1986) 152:421-431). Antisera used were ob-
tained 2 weeks after the fifth boost.
-
~' 3~0~96
26
c) Iodination of Oncostatin M: A sample of par-
tially purified Oncostatin M was radiolabeled with
iodine-125 using published procedures (Linsley et al.,
PNAS (1985) 82:356-350). An aliquot of the labeled
preparation containing 100,000 cpm was mixed ~ith
rabbit antiserum directed against the N-terminal 17
amino acids of Oncostatin M (final dilution of 1:20),
in the absence or presence of the N-terminal peptide
(the N-terminal 17 amino acids of Oncostatin M) (2 ~g)
and subjected to immune precipitation analysis as
described (Linsley et al., Biochemistry (1986) 25:2978-
2986.
Specifically, one tube containing 5 ~l was
preincubated with 2 ~g of the N-terminal peptide in 10
Ml TNEN (20mM Tris pH 7.5, 5mM EDTA, 150mM NaCl, 0.05p
Nonidet P-40~ containing 0.1~ BSA for 30 minutes at 4~C
prior to tne addition of I125 Oncostatin M in 851 TNEN
con~aining 0.1~ BSA and 40 mM dithiothreitol (DTT).
Seven tubes containing 5 ~l antisera were incubated
with I125 Oncostatin M in 85 ~l TNEN containing 0.1%
BSA and 40 mM DTT for 30 minutes at 4~C prior to the
addition of 50 yl of 10p formalin-fixed Staphylococcus
aureus (Pansorbin,* Calbiochem).
Following an additional incubation for 30 min.
at 4~C, the tubes ~ere microfuged and the pellets were
washed 4 times with 1 ml TNEN prior to subjecting them
to PAGE analysis. A diffuse band of Mr ~ 32 kD was ob-
served after SDS/PAGE analysis of the immune precipi-
tates. Precipitation of this species was inhibited by
the inclusion of an excess of unlabeled peptide corres-
ponding to the N-terminal 17 amino acids of Onco-
statin M, indicating that the precipitation was spe-
cific for this peptide.
The carbohydrate composition of Oncostatin M
was examined by testing for glycosidase sensitivity.
Immune precipitates prepared as in c above were treated
with buffer, endoglycosidase H, or neuraminidase, as
* Trademark
F
.. ....
0~96
27
described by Linsley et al. (1986). Treatment with
endoglycosidase H, an enzyme with specificity for N-
linked high mannose oligosaccharides resulted in the
appearance of a lower molecular weight s~ecies of ~r =
24 kD. Only a portion of the radiolabeled material ~as
sensitive to this enzyme, indicating that not all mole-
cules contained high mannose oligosaccharides. Treat-
ment with neuraminidase resulted in the appearance of a
sin~le band of Mr = 27 kD, indicating that the hetero-
geneity in size of untreated 125I-labeled Oncostatin ~
was due to molecular heterogeneity in sialyation of the
glycoprotein core. The results indicated that active
preparations of Oncostatin M contained a mixture of
high mannose and complex N-linked oligosaccharide side
chains.
Oncostatin M Isolated from
Normal Human Peripneral Blood Lymphocytes (P~L)
Production of a Tumor Cell Growth Inhibitor from PBL's
Leucofractions containing P3L's, obtained from
the Blood Bank, were diluted 1:1 with phosphate buf-
fered saline, pH 7.4 (PBS). Thirty five ml of diluted
blood were underlayed with 10 ml of a solution con-
sisting of 9~ Ficoll containing 20d by volume of 50p
sodium diatrizoate (final specific gravity of 1.080).
The gradients were centrifuged at room temperature for
20 minutes at 850 x g. Cells were collected from the
gradient interface and washed with PBS. Red blood
cells were shoc'~-lysed for 3-4 minutes with 10-20 ml of
a solution containing 0.8p ammonium chloride and 0.1p
Na4-EDTA.
Cells were collected from red blood cell
lysing solution by centrifugation at 600 x g for 10
~inutes and resuspended in 10 ml of RPMI 1640 medium
(GIBCO) containing 5~ fetal bovine serum. Thrombin was
added to a final concentration of 0.5 U/ml. The cell
suspension was agitated for 5 minutes at 37~C and the
B~ * Trade Mark
, 10296
28
platelet aggregate allowed to settle for 5 minutes.
The suspended cells were transferred to new tubes, re-
covered by centrifugation, resuspended in 1 ml fetal
bovine serum, and tr~nsferred into a column containin3
0.5 g of brushed, prewetted nylon wool, type 200
(Fenwal).
The nylon wool column was incubated at 37~C
for 60 minutes to allow attachment of monocytes and B
lymphocytes. The column was th-en washed with 3 x vol-
umes of RPMI 1640 medium (37~C) containing 5~ fetalbovine serum and tne eluted nonadherent cells (~BL)
collected.
PBL (2 x lo6 cells/ml) were cultured at 37~C,
5% C02-95P air for 96 hours in RPMI-1640 medium
(10.4g/l) containin3 FeS04 7H20 (1 mg/l), ZnS04 7H20 (2
mg/l) Na2SeO3-5H20 (0.017 mg/l), 1-aminoethanol (1
mg/l) human transferrin (5 mg/l), bovine serum albumin-
linoleic acid conjugate (Sigma) (200 mg/l), L-glutamins
(300 mg/l), penicillin/streptomycin (100,000 units/l)
and phytohemagglutinin-P (Wellcome) (2 mg/l). The
supernatants were collected, centrifuged to remove the
cells, concentrated by ultrafiltr~tion (Amicon Diaflo
membrane YM-10, 10 Kd cut-off), and dialyzed against
0.1 M acetic acid (Spectrapore 3*dialy~is tubing). The
clarified retentate was lyophilized.
Gel Permeation Chromatosraphy
The crude fraction was reconstituted in 20 ml
of 1 M acetic acid (50 mg/ml) and applied to a BioGel*
3~ P-100 column (2.6 x 88 cm) equilibrated with 1 M acetic
acid at a flow rate of 0.5 ml/min. Twelve ml fractlons
were collected. An aliquot from each fraction was
evaporated and assayed in triplicate for growth inhibi-
tory activity (GIA) of A375 cells. The active frac-
tions were pooled, lyophilized, and rechromatographed
on a Bio-Sil TSK-250 column (600 x 21.5 mm), as
described.
.
* Trademark
. . , - .. , . ~ ...... . .
Iq29~
29
Reverse Phase High Pressure Chromatography of TS~-250
Fractions:
The final purification of pooled TSK-250
fractions was achieved by reverse phase HPLC essen-
tially as described. PBL-derived tumor cell inhibitor
eluted from ~Bondapak C18 support at 40-41% ace-
tonitrile and at 26.5% n-propanol concentrations,
respectively.
Cell Growth Modulatory Assay Using 125I-Iodo-
deoxyuridine Incorporation into DNA (GIA):
These assays were performed in flat-bottomed
96-well tissue culture trays (Costar 3596). Human
melanoma cells, A375 (4x103), in 50 ~l of test sample
was added to each well and incubated for 3 days at
37~C. Cells were labeled for 24 hours with 125I-IdU
(0.05 ~Ci/well) and incubated an additional 24 hours.
Cells were washed three times, ha~vested with a
multiple sample harvester, and radioactivity was
counted in a gamma counter.
Results:
One preparation of PBL-derived tumor cell
inhibitor was subjected to automated repetitive Edman
degradation. The aminoterminal amino acid sequence is
as follows:
1 5 10 15
A-A-I-G-X-X-X-K-E-Y-X-V-L-X-X-Q-L-Q-K
X represents an amino acid that has not been
3~ identified.
A comparison of this sequence with that of
U937 factor clearly indicates indentity with the N-
terminus of PBL-derived factor.
1 5 10 15
PBL-factor A-A-I-G-X-X-X-K-E-Y-X-V-L-X-X-Q-L-Q-K
U937-factor A-A-I-G-S-C-S-K-E-Y-R-V-L-L-G-Q-L-Q-K
.
~ 3~0296
3o
In the next study, the ability of PBL derived
Oncostatin M to effect replication of a variety of
cells was investigated. It was found that mouse L929
cells were insenstive to PBL-derived Oncostatin M using
up to 1000 GIA units/ml. Human fibroblasts, WI26, were
stimulated to grow by treatment with 1000 GIA
units/ml. Normal human T-lymphocyte proliferation at
72 hours post mitogenesis was not affected by up to 500
GIA units/ml.
It is evident from the above results, that a
novel polypeptide and polypeptide fragments are
provided, which can be used for modulating cellular
growth. The compound is found to have varying activity
depending upon the nature of the cell line involved, so
that it may be used by itself or in conjunction with
other compounds in modulating cellular srowth. The
subject polypeptides therefore add an additional poly-
peptide which may be used with mixtures of cells, both
in vivo and in-vitro, to selectively reduce or enhance
cellular proliferation of a particular type of cell.
In particular, the factor can be used to treat
cells for autologous bone marrow transplants. Use of
the factor inhibits the growth of tumor cells in the
marrow and may stimulate colony cell formation.
Oncostatin M may also be used to stimulate growth of
epithelial cells thereby promoting wound healing. In
addition, the intact polypeptide or fragments thereof
can be used as immunogens to induce antibody forma-
tion. The induced antibodies find use to titer
3~ Oncostatin M present in a bodily fluid or to modulate
the activity of the factor by binding to it. Further,
those antibodies together with purified Oncostatin M or
fragments thereof serve as a component of diagnostic
kits in conjunction with other reagents, particularly
antibodies to detect and quantitate Oncostatin M.
~ 3402~6
Although the foregoing invention has been
described in some detail by way of illustration and
example for purposes of clarity of understanding, it
will be obvious that certain changes and modificatlons
may be practiced within the scope of the appended
claims.
1 0
3o
