Note: Descriptions are shown in the official language in which they were submitted.
1 3 4 0 2 6 6
This application concerns human pluripotent colony
stimulating factor (P-CSF) also known as pluripoietin.
Background
This work was done in part with government funding
under United States Public Health Service Grants CA-
32516, HL-31780, CA-20194, CA-23766 and CA-00966.
Therefore the United States government has certain rights
in this invention.
Abbreviations
CFU-GEMM:Colony forming unit - granulocyte, erythroid,
macrophage, megakaryocyte.
CFU-GM: Colony forming unit - granulocyte-macrophage,
BFU-E: erythroid burst forming unit,
GM-CSF: Granulocyte-macrophage colony stimulating
factor.
Colony-stimulating factors (CSFs) are hormone-like
glycoproteins produced by a variety of tissues and tumor
cell lines which regulate hematopoiesis and are required
for the clonal growth and maturation of normal bone
marrow cell precursors in vitro (Burgess, A.W., et al.
(1980) Blood 56:947-958; Nicola, N.A., et al. (1984)
Immunology Today 5:76-81). In contrast to the murine
system (Nicola, N.A., et al. (1983) ~. Biol. Chem.
258:9017-9021; Ihle, J.N., et al. (1982) ~. Immunol .
- 2 -
X
13S0266 13~ 66
129:2431-2436; Fung, M.C., et al. (1984) Nature 307:233-
237; Cough, N.M., et al. (1984) Nature 309:763-767),
human CSFs have been less well characteri~ed, both
biologically and biochemically (Nicola, N.A., et al.
(1979) Blood 54:614-627; Wu, M.C., et al. (1980) ~.
Clin. Invest. 65:772-775; Golde, D.W., et al. (1980)
Proc, Nat'l. acad. Sci. USA 77:593-596; Lusis, A.J., et
al. (1981) Blood 57:13-21; Abboud, C.N., et al. (1981)
Blood 58:1148-1154; Okabe, T., et al. (1982) J. Cell.
Phys. 110:43-49). Purification to apparent homogeneity
has only been reported for macrophage active CSF (CSF-l)
(Das, S.K., et al. (1981) Blood 58:630-641; Das, S.K.,
et al. (1982) ~. Biol. Chem. 257:13679-13684) and
erythroid potentiating activity [Westbrook, C.A. et al.
~. Biol. Chem. 259:9992-9996 (1984)] and for
granulocyte-macrophage CSF (GM-CSF) [Gasson, J.C., et
al. Science 226:1339-1342 (1984)], but not for human
pluripotent CSF.
Assays are available to detect human clonogenic
precursors that give rise to cells of the erythroid,
granulocytic, megakaryocytic, macrophage (CFU-GEMM)
(Fauser, A.A., et al. (1978) Blood 52:1243-1248; Fauser,
A.A., et al. (1979) Blood 53:1023-1027) and possibly
lymphoid (Messner, H.A., et al. (1981) Blood 58:402-405)
13~02~
lineages. CSFs with activities on these multipotential
progenitor cells (pluripotent CSF, or P-CSF) are
produced by mitogen- or antigen activated T lymphocytes
(Ruppert, S., et al. (1983) Exp. Hematol. 11:154-161)
and conatitutively by human tumor cell lines such as the
SK-HEP-l human hepatoma cell line (J, Gabrilove,
K.Welte, Li Lu, H. Castro-Malaspina, M.A.S. Moore,
Blood, 66:407-415, August 1985); the 5637 bladder
carcinoma cell line (reported herein and in Proc. Nat.
Acad. sci., 82:1526-1530, 1985); and by the HTLV-
transformed lymphoid cells (Fauser, A.A., et al.(1981)
Stem Cells, 1:73-80; Salahuddin, S.Z., et al., (1984)
Science 223:703-707). Pluripotent CSF is involved in the
proliferation and differentiation of pluripotent
progenitor cells leading to the production of all major
blood cell types. This is therefore a broad spectrum
CSF. It also induces differentiation of leukemic cells.
Su~nary
This application concerns human pluripotent colony
stimulating factor CSF for the stimulation of
proliferation and differentiation of pluripotent
progenitor cells to all major blood cell types which is
purified to apparent homogeneity. Its biological effects
13~02~6
include the induction of functional markers of
differentiation of normal and leukemic cells.
Brief Description of the Drawings
Figure 1 shows ion-exchange chromatography of 5637
conditioned medium (CM) followed by the Gel filtration
chromatograph shown in Figure 2.
Figure 3 shows pooled gel filtration eluants on
HPLC (reverse phase). Figure 4 shows SDS-PAGE whereas
Figure 5 shows preparative SDS-PAGE and figure 6
isoelectrofocusing of the purified pluripotent CSF.
Description o~ the Drawings
Figure 1: Ion exchange chromatography
One liter dialyzed ammonium sulfate-precipitate of
5637 CM was applied in 0.05M Tris/HCl, pH 7.8, on a 1 L
DEAE cellulose (DE 52) column. Bound proteins were
eluted with a linear gradient of NaC1 (0.05 - 0.3 M) in
0.05 - Tris/NCl, pH 7.8, as indicated ( - ). The elution
of proteins was monitored by absorption at 280 nm (o-o)
and each fraction was tested for CSF activities (GM-CSF
activity: ~). Proteins from the first peak of GM-CSF
activity eluted from the column gave rise to mixed
colonies in a CFU-GEMM assay and were used for further
purification (pluripotent CSF).
X
1 3 ~
Fiqure 2: Gel filtration chromatography
The pluripotent CSF containing concentrated pool of
DEAE cellulose chromatography was loaded on an AcA 54
Ultrogel column (2.6x90 cm) and eluted with PBS, Arrows
denote the elution points of bovine serum albumin (MW
68, 000), and chymotrypsinogen (MW 25,000). The elution
of proteins was monitored by absorption at 280 nm (o-o)
and each fraction was tested for pluripotent CSF
activity (GM-CSF activity: ~).
Figure 3: Reverse phase high-performance liquid
chromatography ~HPLC)
The pooled fractions with pluripotent CSF
activities eluted from the gel filtration column were
acidified to pH 4.0 and loaded onto a C 18 (uBondapak,
Waters) column. The bound proteins were eluted with a
linear gradient of 1-propanol in 0. 9M acetic acid/0. 2M
pyridine, pH 4,0. The elution of proteins were monitored
by absorption at 280 nm (-) and each fraction was tested
for pluripotent CSF activity (GM-CSF activity: A ).
Figure 4: SDS-polyacryl~ide qel electrophoresis ~SDS-
PAOE )
The pluripotent CSF eluted from the HPLC column
(200 ng; peak fraction) was lyophilized and treated with
1% SDS in 0. 0625 M Tris/HC1, pH 6 . 8, and 20% glycerol,
13~026
under reducing conditions (5% 2-mercaptoethanol) for one
hour at 37~C and then applied to a 15% polyacrylamide
gel. After electrophoresis, the protein bands were
visualized by the silver staining technique.
Figure 5: Preparati~e SDS-PA~~
Pluripotent CSF eluted from HPLC (Fig. 3) was
treated and processed (under non-reducing conditions) as
shown in Fig. 4. After electrophoresis, the gel was
sliced into 4 mm sections and proteins from each slice
were eluted into RPMI 1640 containing 10% FCS. After 18
hours, eluted proteins were assayed for pluripotent CSF
activity (GM-CSF activity: shaded area).
Figure 6: Isoelectrofo~-~ing
HPLC purified lyophilized pluripotent CSF was
supplemented with 20% (v/v) glycerol and 2% ampholines
(pH 3.5-10) and layered onto the isodense region of an
0-60% gradient of glycerol containing 2% ampholines (pH
3.5-10). After isoelectrofocusing (2,000 V, 24 hours),
5 ml fractions were collected and the pH (o) determined
in each fraction. All fractions were subsequently
dialyzed and tested for pluripotent CSF activity (GM-CSF
activity: ~
We report the purification and biochemical
characterization of a human pluripotent CSF, produced
and released constitutively by human cells especially
. . , ,~ . ..
~ 13iO26~
tumor cells such as bladder carcinoma cell line 5637
(ATCC HTB-9) and hepatoma cell SK-HEP-l: (ATCC HTB52).
The cell line (5637) was obtained from Jorgen Fogh at
Sloan-Kettering Institute, 1275 York Avenue, New York,
New York 10021.
Pluripotent CSF biological properties :include
differentiation of progenitor cells to all major blood
types as well as differentiation of leukemic cells.
As~ay for GM-CSF Activity
GM-CSF activity was tested on human bone marrow
(BM) cells cultured with serial dilutions of test
samples in semi-solid agar. BM from healthy human
volunteers, who gave informed consent, was diluted 1:5
in phosphate buffered saline (PBS) and separated by
density gradient centrifugation on Ficoll-Hypaque. 105
separated cells were plated in l ml of 0.3% agar culture
medium that included supplemented McCoy's 5A medium and
10~ heat inactivated fetal calf serum (FCS), as
described (Broxmeyer, H.E. et al. (1977) Exp. ~ematol.
5: 87-102). To this mixture serial dilutions of a
laboratory standard or test samples (10%;v/v) in RPMI
1640 with 10% FCS were added. Cultures were scored for
colonies (greater than 40 cells/aggregate) and
morphology was assessed after 7 and 14 days of
incubation. GM-CSF units were determined from dose
1:~4~26~
response curves and expressed as U/ml, where 50 U is the
CSF concentration stimulating half-maximal colony number
to develop (Nicola, N.A., et al. (1983) ~. Biol. Chem.
258: 9017-9021).
Assay for Colony Stimulating Factor for BFU-E and
CFU-OE MM
The colony assay for human BFU-E and CFU-GEMM was
performed as previously described (Li Lu, et al. (1983)
Bl ood 61: 250-256). Human bone marrow cells were
subjected to a density cut with Ficoll-Hypaque~ (density
1.077 gm/cm3; Pharmacia Fine Chemicals, Piscataway, N.J.)
and the low density cells were suspended in RPM1 1640
containing 10% FCS at 2 x 107 cells/ml and placed for
adherence on Falcon tissue cultures dishes (#3003,
Becton Dickinson and Co., Cockeysville, MD) for 1~ hr.
at 37~C. The nonadherent cells were depleted of T
lymphocytes by rosetting with neuraminidase-treated
sheep erythrocytes. Medium conditioned by leukocytes
from patients with hemochromatosls in the presence of 1%
(v/v) phytohemagglutinin (PHA) (Li Lu, et al., (Bl ood
61:250-256, 1983) as positive control or serial
dilutions of test samples were then added at 5~ (v/v) to
x 104 of these low density, non-adherent and T
lymphocyte depleted bone marrow cells in a 1 ml mixture
X - g _
134026n
of Iscove's modified Dulbecco medium (GIBCO Grand
Island, NY), 0.8% methylcellulose, 30% FCS, 5x10 5 M 2-
mercaptoethanol, 0.2 mM Hemin, and one unit of
erythropoietin {Hyclone, or Connaught Labs., Willowdale,
Ontario, Canada). The addition of Hemin is necessary to
obtain optimal cloning efficiency (Li Lu, et al. (1983)
Exp. Hematol . 11:721-729). Dishes were incubated in a
humidified atmosphere of 5% CO2 in air at 37~C. After 14
days of incubation, colonies were scored and morphology
was assessed.
As shown above, a single protein stimulates colony
formation by CFU-GEMM, BFU-E, and CFU-GM progenitor
cells.
This protein we termed "pluripotent CSF". Due to
the low numbers of mixed colonies per dish attainable in
this assay system, titration of test samples for
determination of pluripotent CSF activity meets with
considerable difficulties. Therefore, we used the GM-
CSF assay units as described above to measure the GM-CSF
aspect of the pluripotent CSF activity in those samples
that supported growth of BFU-E and CFU-GEMM for
calculating the specific activity throughout the
purification procedure.
-- 10 --
~ X
13qO2fi~
.A.
Differentiation Induction Assay
Titrated samples of purified pluripotent CSF were
assayed for differentiation induction of WEHI 3B ~D+) or
HL-60 leukemic cells described (Metcalf. D, (1980) Int.
5 J. Cancer 25:225-233; Fibach, E., et al., ~. Cell
Physiol. 113:(1) 152, (1982) ).
Rossette Assays for Fc Roceptor, OKM1 and LQU M2
antigens
Cell receptors for immunoglobulin Fc were assayed
with IgG (Cappel Laboratories, West Chester, PA) coated
sheep erythrocytes as desribed elsewhere (Ralph, P., et
al. (1983) Blood 62:1169). OKM1 (Ortho Diagnostic
Systems Inc., Raritan, NJ) or Leu M2 (Beckton Dickinson,
Mountain View, CA) reactive antigens were detected by
15 incubating 106 cells/O.1 ml phosphate buffered saline
containing. 1.0 ug/ml monoclonal antibody for 20 min at
24~C, washing, incubating 20 min at 24~C with a a:100
dilution of rabbit anti-rat (Leu M2) or anti-mouse
(OKMl) IgG serum (Cappel Laboratories, Cochranville,
PA), washing and rosetting with protein A-coated
erythrocytes as described previously (Ralph, P. et al.,
Blood 62:1169, 1983).
X
_ .. . . . , . ... _~_ . ... ~_ . , . ... _ .. . . ...
13~02S~
Assays for fMLP Receptor
Receptors for chemotactic peptide, formyl-
Methionyl-Leucyl-Phenylalanine (fMLP), were assayed as
follows: 2 X 106 cells were incubated with 15 nM 3H-fMLP
(New England Nuclear, Boston, MA) in a total volume of
0.2 ml in the presence or absence of 10 uM unlabelled
fMLP (Sigma Corp. St. Louis, MO). After three hours at
4~C the cell suspensions were rapidly filtered onto
glass fiber discs (Whatman Inc., Clifton, N.J.), which
were then washed with 30 ml of 4~C phosphate buffered
saline (Harris, P. et al. (1985) Cancer Res. 45:9).
Radioactivity on the discs were counted by liquid
scintillation spectrophotometry.
Measurement of PMA-Stimulated Hydrogen Peroxide Release
The production of hydrogen peroxide in response to
PMA stimulation was assayed by horse radish peroxidase
(HRPO) (Sigma) mediated H202 dependent oxidation of
homovanillic acid (HVA) (Sigma), as described (Harris,
P., et al. (1985) (Cancer Res., 45:9, (1985)). Briefly,
cells (1 X 106) were suspended in 2 ml of a solution
containing 100 micromolar HVA 5 U/ml HRPO in the absence
or presence of 30 ng/ml PMA. Following 90 min.
incubation at 37~C, the incubation was centrifuged and
0.25 ml of 25 mM EDTA, 0.1 M glycine-NaOH, pH 12 was
added to the supernates. A 30% stock solution of
- 12 -
X
134~261i
hydrogen peroxide (Sigma) was used to prepare H202
standards (0.001 to 50 nmoles/assay) for the
construction of a standard curve. The HVA oxidation
product was measured on a Perkins-Elmer Model MPF-44A
fluorescence spectrophotometer. Excitation and emission
were set at 312 nm and 420 nm, respectively.
Prostaglandin Measurements
Cells for prostaglandin production assay were
washed three times in phosphate-buffered saline and
placed in fresh RPMI 1640 media (without FCS) in the
presence or absence of 10 micrograms/ml Concanvalin - A
(Con-A). Cells were cultured for 24 hours, centrifuged
and the supernates harvested. Supernates were stored at
-20~C until assayed.
Prostaglandin standards PGE2 6-keto-PGFla and TBX2
were kindly supplied by Dr. J. Pike (Upjohn Company,
Kalamazzo, Mch.). Tritium labelled compounds were
purchased from New England Nuclear (Boston, MA). Rabbit
antisera to PGE2 were obtained from the Pasteur Institute
(Paris, France). Antibodies to 6-keto PGFla were raised
in the laboratory (Rashida Karmali). The cross
reactivity of these antibodies for the non-targeted PGs
were to greater than 4% except for the PGE2 antisera
which cross reacted 10% with PGE1 standard. The
procedure for extracting the prostaglandins has been
13~02~
described earlier (Karmali, R.A., et al. (1982)
Prostagl. Leukotr. Med. 8:565). Briefly, a trace of
[3H]-PG was added to aliquots of standard and samples
before being extracted once with petroleum ether. After
acidification to pH 3.5, the samples were extracted
twice with diethyl ether, dried under nitrogen and
reconstitution in assay buffer. The efficiency of this
extraction procedure to this point was 85-95~. Standard
quantities of each prostaglandin (0-1000 pg) or the
extracted sample to be measured were prepared in 0.1 ml
aliquots of assay buffer. Antisera and label were added
successively in 0.1 ml aliquots and incubated at 4~C for
8-12 hours. Bound and free [3H]-PG were separated by 0.5
ml dextran-coated charcoal (0.5-1.0% w/v) to estimate
the amount of each compound in the unknown sample. The
detection limit of this assay has been found to be lOpg.
The intra-assay coefficient of variation was 9.0%.
Alkaline and Acid pho~rh~tase~ b-Glucuronidase and N-
Acetyl Glucuronidase Assays
Cell extracts were prepared in 0.5 ml of PBS 1% NP-
40, incubated 5 min at 24~C, then spun for 10 min at
3X104g. Supernates were collected and assayed. Extracts
were assayed of their alkaline and acid phosphatase, b-
glucuronidase and N-acetyl glucuronidase activity using
the respective Sigma kits. Activities of extracts are
- 14 -
~' X
~ , .... . _ . ......... ... ..
02~6
expressed as change in absorbance per unit time per unit
sample volume divided by the cell concentration in the
culture or in the extract and compared to control
activity. Measurements were made in a Beckman ACTA-CV
spectrophotometer.
Glycoconjugate assay
Cytokine preparations were assayed in a [3H]-
Glucosamine incorporation assay. Replicate wells were
plated with 100 microliters of inducing agent to be
tested. Previously washed (3X in PBS) HL-60 or U937
cells (1 X 107 cells/ml) in RPMI 1640 without FCS were
added (50 microliters). After a four hour incubation 20
microliters of 25uCi/ml [3H]-Glucosamine in 1~ BSA (w/v)
in PBS was added to the culture and plates were
incubated for an additional 16 hours. Cells were
harvested (Mini-Mash, Microbiological Associates, MD)
onto glass filter paper with water wash (x4, 0.1 ml
each), followed by 0.4N Perchlorate wash (x4, 0.1 ml)
and water (2x, 0.1 ml). Radioactivity on glass discs
was determined by liquid scintillation
spectrophotometry.
Statistical Analysis
Students' T test to compare means was carried out
using the significance limits of a two tailed test.
- 15 -
X
~ 13~026~
Pr~paration of 5637 cell line conditioned mQdium (5637
CM)
The human bladder carcinoma cell line 5637 has been
reported to produce a colony stimulating factor for
granulocytes and macrophages (Svet-Moldavsky, G.J., et
al. (1980) Exp. Hematol. 8 (Suppl. 7):76). The cell
line has been maintained at Sloan-Kettering Institute
(New York, N.Y.) for several years. It is serially
passaged by trypsinization in the presence of EDTA and
grows rapidly to form an adherent monolayer in plastic
tissue culture flasks. Routinely, cells are cultured in
RPMI 1640, supplemented with 2 mM L-glutamine,
antibiotics and 10% FSC. For purification of
pluripotent CSF activity from 5637 conditioned medium
(5637 CM), confluent cell cultures were intermittently
cultured in medium containing 0.2% FSC. After 48-72
hours, 5637 CM was harvested, cells and cell debris
removed by centrifugation (20 min, 10,000 x g), and
stored at -20~C until use.
5637 cells also contain a multitude of subclones
which either produce p-CSF in better yield and/or have
less inhibitor present. Over 120 subclones have been
isolated. One such subclone lA6 was found to produce at
least twice as much as the parent cell line and possibly
5-10 fold more resulting in a range of between 2-10
- 16 -
X
13~0~6S
times more p-CSF from the lA6 subclone than from the
parent 5637 cell line as determined by the assay methods
outlined. This subclone or the parent cell line 5638
can be used to isolate p-CSF in good yield. Subclones
are isolated by limiting single dilution techniques to
produce a single cell per well in order to grow up a
pure cell line from each well. Best results are obtained
if the cells are distributed such that 37% of the wells
(one out of every three) show growth at a certain
dilution. There is then a good mathematical chance of
obtaining subcloning to obtain outgrowth of only one
cell from the one of three wells showing growth.
Subclone lA6 cell line is on deposit and available at
Sloan-Kettering Institute for Cancer Research 1275 York
Avenue, New York, N.Y. 10021. We refer to use of the
lA6 in yielding sequence data on the protein p-CSF with
subsequent preparation of the recombinant p-CSF from
such a sequenced probe (P11 - top P14) as follows:
sequence data on the protein p-CSF with subsequent
preparation of recombinant p-CSF from such a sequenced
probe (P11 - top P14) as follows:
"(B)Sequencing of Materials Provided by Revised
Methods
In order to obtain a sufficient amount of pure
material to perform suitably definitive amino acid
- 17 -
X
134~2~fi
sequence analysis, cells of a bladder carcinoma cell line
5637 (subclone lA6) as produced at Sloan-Kettering were
obtained from Dr. E. Platzer. Cells were initially
cultured Iscove's medium (GIBCO, Grand Island, New York)
in flasks to confluence. When confluent, the cultures
were trypsinized and seeded into roller bottles (1-1/2
flasks/bottle) each containing 25 ml of preconditioned
Iscove's medium under 5% CO2. The cells were grown
overnight at 37~C at 0.3 rpm.
- 17a -
.. . . . ..
0266
Cytodex-l* beads (Pharmacia, Uppsala, Sweden) were
washed and sterili~ed using the following procedures. Eight
grams of beads ~ere introduced into a bottle and 400 ml of
Pss was added. Beads were su~pend~d by swirling gen~ly fo~
3 hours. ~fter allo~inq the bead8 to set~le~ the PBS was
drawn off, the bead~ were rinsed in PBS and fresh pBS was
added. The ~eads ~ere autoclaved for 15 minutes, Prior to
use, the bead~ wer~ wa~hed in Iscove'S mediu~ plus 10~ fe~al
calf serum (FCS) before adding fresh medium plu~ 10% FCS to
obtain trea~ed bead6.
~ f~er removing all but 30 ml of the medium from
each roller bo~tle, 3~ ml of fre~h medium plus 10~ F~S and
40 ml of treated bead6 were added to the bottles. The
bottles were ~assed with 5% Co2 ~nd all bubbles were removed
by ~uction. The bottles were pl~ced in rolle~ racks at 3
rpm for 1/~ hour before reducing the speed to 0.3 rpm.
After 3 hour~, an additional flask ~a~ trypsinized and added
to each roller bottle oontaining ~eads.
~ t 40~ to 50~ of confluence the roller bottle
~ultures were washed with 50 ml PBS and rolled for 10 min.
before re~oving the PB~. The cells were cultured for 48
hours in medium A [Iscove I 8 medium containin~ 0.2% FCS,
10 8~ hydrocortisone, ~mM glutamine, 10~ units/ml
Trademark
- 18 -
. , .
13~02E~
penicillin, and 100 ug/~l ~trepto~ycin]. ~ext, the culture
supernatant was harve~ted by centrifugation at 3000 rpm for
15 ~in., and stored at -70~C, The cùltures were refed with
~edium A ~ontaining 10% FCS and were cultured for 48 hour~.
After discarding the medium, the cells were washed with PBS
as above and cultured ~or 4 8 hou~s in medium A . The
supernatant was again har~e~ted and ~reated as pr~viously
described,
~ pproximately 30 liters of medium condi~ioned by
lA6 cel~s were concentrated to abou~ 2 liters on a Millipore
Pellicon ~ni~ equipped with 2 cassettes ha~ing 10,0~0 M.W.
cutoffs at a filtra~e rate of about ~00 ml/min. and a~ a
~etentate rate of about 1000 ml/min. The concentrate was
diafiltered with about 10 liters ~f 50mM Tris (pH 7.8) u~n~
the same apparatus and some flow rates. The diafiltered
concentrate was loaded ~t 40 ml/mi~. onto a 1 liter DE
cellulose colu~n equilibrated in 50 m~ Tris (pH 7.8). After
loadinq, the column was washed at the same rate with 1 liter
of 50mM Tris (pH 7.8) and then wit~ 2 liters of 50 ~M Tri~
(pH 7.8) with 50 mM Na~1, The col~mn was then sequentially
eluted with s~x 1 liter solutio~s of 50~M Tris (pH 7 . 5~
containing the following concentrations of NaCl: 75~M;
lOOmM; 125mM; 150~M; l~OmM; and 300m~. Fractions ~50 ~1)
were collected, and active fractions were pooled and
.. .
13~02~
c~ncentrated to 65 ~1 on an Amicon ultrafiltration stirred
cell unit equipped ~ith a YM5 membrane~ This ~oncentrate
was loaded onto a ~ liter AcA54 g~l filtra~ion column
equilibrated in PBS. The column was run at 80 ml/hr. and 10
ml fractions were collected. Activ~ fraction~ were pooled
and loaded directly onto a C4 high performance li~uid
chromatography (HPLC) column.
Sa~ples, ranging in volume from 125 ml to 850 ml
and containin~ 1-8 ~g of protein, about 10% of which ~as
hpG-CSF. Sample~ were lo~ded onto the column at a flo~ rate
ran~ing fro~ 1 ml to 4 ml per minute. After loading and an
initial washin~ w~th 0.lM am~oniu~ ace~ate (p~ 6~0-7.0) in
80~ 2-propanol at a flow rate of 1/ml~min. One ~illilite~
fraction~ were collected and monito~ed for protein~ at
2~0nm, 2~0nm and 280 n~.
As a re~ult Of purification, fractions containing
hp~-CSF were ~learly separated la~ fractions ~2 and 73 of
B0) from other protein-containing fraction~. HpG-CSF wa~
isolated (1~0-30 ~) at a purity of about 85~5~ and at a
yield of about 50~. From thic purifi~d materlal 9~4g was
~ed in Run #4, an a~ino a¢~d sequence analysis wherein the
protein sample was ~pplied ~o a TFA-activated ~lass fiber
dis~ without polybrene, Se~uence analysi~ wa~ c~rried out
- 20 -
. . _ ~
13402fi~
wi~h an AB 470A sequencer according to the methods of
Hewick, et al., J. Biol. Chem., 256, 7990-7Y97 ~1981) and
Lai, Anal. Chem. Acta. 163, 243-248 (19~4). The results of
Run #4 appear in Table ~II.
TABLE III
1 5 10
Thr - P~o - ~eu - Gly - Pro - Ala - Ser - Ser - Leu - P~o-
~0
Gln - Ser - Phe - Leu - Leu - Lys - ~Lys) - Le~l - (Glu) - Glu -
~0
Val - Arg - Lys - Ile -(Gln~- ~ly - Val - Gly - Ala - Ala-
~eu - X - X
In Run #~, ~eyond 31 cycle~ (~orresponding to
residue 31 in Ta)~le III, no further significant se~uen~e
infor~ation was obtained. In order to obtain a longer
unambiguous sequence, in a Ru~ ~5, 14~g of hpG-CSF purified
fro~ conditioned medium were reduced with 10~ of
-~ercaptoethanol for one hour at 45~C, then thoroughly dried
under a vacuum. ~he protein residue was then redi~solved in
5~ formi~ acid before being applied to a polybrenized glass
fiber disc. Se~uenoe analysis was carried out as for Run ~4
- 21 -
._, .
13~02~
a~ove. The results of ~un #5 are given ~n Table IV.
TABLE IV
1 5 10
~hr-Pro-Leu-Gly-Pro-Ala-Ser-Ser- Leu - P~o - Gln -Ser-
Phe-Leu-Leu-Lys-Cys-Leu-Glu-Gln- Val - Arg - ~y6 -Ile-
Gln-~ly-~sp-Gly-Ala-Ala-Leu-Gln- Phe - ~ys - Leu -Gly-
~5
Ala-Thr-Tyr-Lys-Val-Phe-Ser-Thr-(Arg)-(Phe)-(~et)-X-
The amino acid sequence given in Table IV W~8
sufficiently long ~44 residues) and unambiguous to conRt~uct
probes for obtaining hpG-CSF cDNA." (end quote).
quote)
A~monlum Sulfate Precipitation, Ion-exchange-chromatography,
Gel filtration
The firfit three puri~ication ~eps
((NH4)2SO4~precipitat~on, Ion-exchange-Chromatography on
DE~E cellulose, DE 52, Whatman, Clifton, ~.J., and gel
filtrati~n on A~A 54 Ultrogel, LKB, Inc. Rockland, MD) were
performed as described (Wel~e, K., et al. ~1982) J. Exp.
Med, 156;4$4-464) except that AcA 54 was used instead of A~
, . ... ,. ,, ,, ~ ,. ~
134026~
44 (see also Descriptions of Fi~. 1 and 2).
Re~erse Pha~e High-Performance ~iquid ~hromatography
~RP-HPLC)
RP-HPLC was performed with a Waters HPLC system (M
6, 000 solvent del~ very pump~, Inodel 400 variable wa~elength
detector, data module and data p~ocessor, ~aters,
Associates, Milford, MA). The separation was performed on a
~Bondapak ~18 column (Waters). The buffers used ~ere:
Buffer ~: 0.~ M acetic acid/0.2M pyridine, p~ 4.0; buffer ~:
buffe~ A in S096 l-p~opanol (Burdick and ~c)cson, IJab.,
Muskegon, MI). Acetio acid and py~idine were purcha~ed from
Fisher, Scientifi~ Co.. T~e plurip~tent CSF con~aining pool
obtained f~om gel ~iltration was acidified with aeetie acid
to pH 4 . O and injected onto the uBond~pak C1~ col~mn without
regard to sample volume. The column was was~ed with buffer
A (10 min) and bound proteins were eluted using a steep
gradient 0-40~ buffer B within the fir~t 20 mln and ~ 40 -
10~ gradient of buffer B in 120 Tnin. THe flow ~te wa~
adjusted to lml/nlin and 3 ml fraction~ were collected. ~rom
each f~action a 0. 5 ml aliquot wa~ supplemented with 10%
FCS, dialy~ed ~gainst PBS and tested for pluripotent CSF
activity .
-- 23 --
.
13~26~
Isoelectrofoc~ing
One ml of the purified pluripotent CSF wa~
supplemented wit~ 20~ glycerol (vol/vol) and 2% Ampholines
(vol/vol), pR 3.5-10 (L~ Produets, Inc.). A 5-60% glycerol
density gradient containing ~ A~pholines, p~ 3.5-10, ~as
layered in~o a isoelectrofocusing colum~ (LR~ ~100). The
pluripoten~ CSF sample was applied onto the isodense region
of the gradient, followed by isoelecSrofocusing (2,000 V, 24
hour~). Fi~e ml fra~tions were collected and the p~I
determined in each fraetion~ The ~ractions were dialyzed
against PBS and subsequently tested for pluripotent CSF
a¢tivity,
Sodium Dodeeylsulfate-Polyacrylamide ~el Electrophoresis
(SDS-PAGE)
The diQcontinuous Tris-glyCine ~ystem of Laemmli
(Laem~ .K. (1970) Nature 227:6~0-685) wa~ used for 1.5
mm slab gel~ of 15% a~rylamide. The samples (200 ng
lyophilized protein eluted from HPLC) were treated with 1
SDS in 0.0625 M Tris-~Cl, p~ 6,8 at ~7~C for 1 hour under
both reducin~ (5~ 2-mercaptoethanol~ and non-reducing
condition~ and then loaded on the gel. After
elec~rophoresis, gel~ were ~tain~d ~y the Biorad silver
staining method ~Biorad Labora~ories, Ro~kville Centre, NY).
Apparent molecular weight~ ~ere determined using protein
6 6
standards ovalbumin (Mw 43,000), chymotrypsinogen ~MW
~5,700~, beta-lactoglobulin (MW 18,400), lysozy~e ~
14,300) and cytochrome C (M~ 12,3001 (Bethesda Research
Laboratories, Inc. ~a~ther~burg, MP~ or from Pharmacia Fine
Chemical~, Pis~ataway, New Jersey. After treatment (see
above) of lyophilized pluripotent CSF under non-reduced
conditions and subsequent electrophoresis, parallel gels
were sliced in ~ mm or 2 mm sections, respeetively and
proteins from ea~h ~ e eluted either in~o 0.5 ml RPMI 1~40
containin~ 10% FCS or into phosphate bu~fered saline (P~S;
20 mM phosphate, O.lS M ~aCl). Af~er exten~ive dialysis,
the eluted material was a~sayed for pluripotent CSF
a~tivity.
Protein Assay
T~e protein content of samples we~e measured using
the ~owry technique (Lo~ry, O,~., et al. ~19~1) J. Biol
Chem. 193:265-275)~ For protein ~on~entration$ lower than 2
microgra~/ml, samples were subjected to S~S-PAGE, the
protein bands were visualized by the silver staining
technique and the protein concentration es~ima~ed ~y
~omparison wi~h a serial dilution of known amounts of
proteins .
The example~ shown serve to illustrate the
invention without limiting s~me.
.
13402~
Example I
Pluri~tent ~SF activity in 5~37 CM
Confluent layers of 5~37 human bladder carcinoma
cells, when cultured for 48-72 hour~ in the presence of 10
~CS, released into the culture medium 3,0~0 - 10,000
uni~s/ml of G~-CSF activity. ~edia conditioned in the
presence of 0.~ FC5 ~till con~ained 10 - 30% ~f ~his
ac~i~ity, ~hereas in ~erum free 5637 CM the activity drop6
below 5~ of the activity o~tained in the pre~ence of 10%
~CS. Although GM-C~F activity in 5~37 CM i~ readily
detec~able in soft ~gar bone marrow cultures, no~ all
batches o~ unfractionated 5637 CM support in vitro grow~h of
BFU-E and CFU-GEMM. Four to ten times concentrated 5~37 CM
support in vitro growth of BFU-E and CFU-GE~M, Four to ten
~imes concentrate~ 5637 ~M reduced colony formation ~y
CFU-~ 30-70~ indica~ing the presen~e of inhibitor (s) in
5637 CM. In~ibitor~ were removed after ion-exchange
chromatography.
Example II
Puri~ication of pluripotent CS~
~ ~0-fold concentration of proteins from the 5637
CM was achieved by precipitation with ~m~onium culfate at
- 2~ -
~ ~ . .. ... .... . ..
1~3~2~
80~ ~at~ration. The dialyzed precipitate was loaded on to a
DEAE cellulose (DE 52) column~ Bound protein~ were eluted
with a salt gradient from 0.05-0~3M NaCl in 0.05 M Tris-~Cl,
p~ 7.8. GM-C~F acti~ity eluted as peak l between 0.075 M
and O.lM NaCl and with a ~econd peak at 0.13 M NaCl (Fig.
1). Since only peak 1 revealed pluripotent CSP activity,
was used only this pool for further purifications, Pea~ 2
included proteins with only ~-CSF activity. We calculated
the "fold" purific~tion by measurin~ the GM-CSF ~ctivity of
pluripotent CSF. In the unfractionated CM we could not
di~criminate between GM-CSF activity a~ part of pluripotent
CSF activity and GM-CSF activity without pluripotent
properties. Therefore we considered the GM-CSF activity
contained in peak 1 from DE 52 as the starting a~tivity
(Table l).
Since in the subsequent purification schedule
GM-CSF, BFU-E and CFU-GE~M activities copurified in all
steps, we named these combined activitie~ "pluripotent CSF"
and have used this ~er~ thereafter. The protein~ of peak 1
of DE 52 chromatography ~including pluripotent C~F activlty)
were co~centrated by dialyzing against ~0~ (w/v)
Polye~hylenglyCol in PBS and purified further by ACA 54
Ultrogel gel filtration. The pluripotent CS~ activity
eluted in fraction~ 42 -4~ as a single peak corresponding to
_ 3"~_,
~" C.3
_ . _ _ , . . . _ . , . , . . _ _ , , , " ~ . , _ _ _ , . , _
13 ~02 G~
a molec~lar weight of 32,000 daltons (Fig. 2). This ~tep
resulted in a 65~ recovery of activ~tie~ and a 15 fold
increase of cpecific activities (Table 1).
The final ~tep involved chromatography on a
reverse phase HPLC column (ubondapak* C 18). Tlle majority of
proteins did not bind to this col~mn or eluted at low
l-propanol con¢ent~ation~ (less th~n 20% l-propanol; Fig.
3). A minor peak of GM-CSF activity without activity in the
CFU-GEMM and BFU-E assays but differentation inaucing
activi~y on HL-60 leukemic cells was eluted at around ~0
l-propanol. Pluripotent CSF ~ctivity eluted as a single
sharp peak at 42 ~ l-propanol (Fi~, 3). This purifica~ion
step resulted in a 600-fold increase of specific ~ctivity
and a 25 % recovery of activity. The protein content of the
H~LC fra~tion was measured by comparing the density in
silver stained SDS-PAG~ with protein standards of known
concentrationS. Using this meas~rement, we obtained a
specific~ activity of 1.$ x 10~ U/mg protein and a final
purification of ~,00~-fold, calculated from the fir~t peak
of DEAE cellulo$e chromatography. The overall yield ~a~
6.2%. The puriflc-ation table ~ith the degree of
purification of pluripotent CSF as measured by GM-CS~
act~vi~y, protein content, specific activity and y~eld is
detailed in Table 1.
* Trademark - 28 -
,~ ,.
13~026~
The final preparation obtained after HPLC(pluripotent CSF activity peak fraction) was analyzed on
a 15~ SDS-PAGE gel followed by the sensitive silver
staining technique (Fig. 4). Only one major protein band
with a molecular weight of 18,000 was seen under both,
reducing (5% 2-mercaptoethanol) (Fig. 4) and non-reducing
conditions. Since the buffer system used for HPLC did
not allow monitoring the protein elution pattern by
measuring the optical density at 280 nm, we applied
proteins of all active fractions on SDS-PAGE. The
density of the stained protein band at 18,000 MW in the
peak and side fractions was proportional to the amount of
biological pluripotent CSF activity. After
electrophoresis under non-reducing conditions, a parallel
gel was sliced into 4 mm sections and proteins eluted
from each slice into RPMI 1640 containing 5~ FCS.
Pluripotent CSF activity was found to be localized in the
slice number corresponding to 18,000 MW (Fig. 5).
In three additional, independent purification runs,
pluripotent CSF had the same properties and specific
activity as described above. In all three runs, parallel
gels were sliced into 2 mm sections, proteins eluted into
PBS and tested for pluripotent CSF activity.
Re-electrophoresis of the proteins eluted from the
slices with pluripotent CSF activity again revealed one
1~02~
single band in a silver stained gel with a molecular
weight of 18,000, identical to that shown in Fig. 4.
However, further work using markers from Pharmacia
shows the molecular weight of the glycosylated p-CSF to
be 19,600. The unglycosylated recombinant protein shows
a MW of 18,800.
The purified CSF was also subjected to
isoelectrofocusing analysis using a 5-60% glycerol
gradient in an IEF column and 2% Ampholines, pH 3.5 - 10.
Pluripotent CSF activity was localized in one fraction
(5 ml) with an isoelectric point of 5.5 (Fig. 6). The
total recovery of pluripotent CSF activity applied to the
column was approximately 20%.
Pluripotent CSF activity did not b:ind to a
Concanavalin A agarose. Treatment with neuraminidase did
not abolish the biological activity and did not change
the IEP. However, the isoelectrofocusing under our
conditions did not allow judgment of minor changes of the
IEP. These findings suggest that glycosylation might not
be a major structural feature.
The partial amino acids sequence was determined by
Applied Molecular Genetics (Thousand Oaks, California) on
an AB 1470A- Beatrice microsequencer. From the amino
terminal end the sequence is Thr, Pro, Leu, Gly, Pro,
- 30 -
X
134~2~
.
Ala, Ser, Ser, Leu, Pro. Also see the extended 44
residue sequence above.
Example III
Biological activity of pluripotent CSF: Progenitor cell
stimulation and effect on leukemic cells.
1. Progenitor cells:
Fifty units of GM-CSF activity, enough to support
the half m~x;m~l growth of CFU-GM, had no clear effect in
a CFU-GEMM assay; however, 500 U/ml (GM-CSF activity) of
pluripotent CSF clearly supported the growth of human
mixed colonies (CFU-GEMM), megakaryocytic colonies, and
early erythroid colonies (BFU-E) under our experimental
conditions (Table IIA & IIB).
Pluripotent-CSF supports the growth of colony
forming progenitors of the granulocyte, mixed
granulocyte, macrophage, eosinophil and megakaryocytic
cell types. These results can be seen for example in
vitro.
We show the results of comparison of 5637-CM and
lA6-CM in Table IIC at dilutions of 1/10 through 1/1600.
The 1/10 dilution of lA6 shows an inhibitor to be present
in the CM. Essentially this table serves as an example
that the lA6 subclone of 5637 has 8.7 times more p-CSF in
U/ml under growing conditions containing FCS.
When purifying p-CSF the FCS is reduced to 0.2%.
13~2~
2. Pluripotent CSF also induces the
differentiation of leukemic cells. For example, leukemic
cell lines HL-60 and WEHI-3B (D+) are induced to
differentiate along the granulocytic and/or macrophage
pathway. The human leukemic cell line KG-1 responds to
pluripotent CSF by increased colony formation in agar and
proliferation in liquid suspension culture.
As for mature cells, pluripotent CSF induces
increased protein content, for example, in macrophages,
whereas IL-3 is not reported to be active on macrophages
~Table III). 50 U/ml and 200 U/ml of GM-CSF activity of
the pluripotent CSF were needed to induce half-maximal
differentiation of the leukemic cell lines WEHI-3B (D+)
and HL-60, respectively. These cells were used in a test
system (Metcalf Int. J. Cancer (1980) 25:225 and Fibach
et al. (1982) J. Cell. Physiol. 113:152) Table IV(A&B) to
show the effect of pluripotent-CSF on leukemic cells.
U937 was obtained from Dr. Nilsson and HL-6() from Dr.
Gallo as freeze-backs of early passages. HL-60 is a
myeloid cell line from an acute promyelocytic leukemia
[Gallagher et al. Blood 54:713 (1979)]. U937 is a
histiocytic lymphoma cell line (Sundstrom and Nilsson
(1976) Int. J. Cancer 17:565).
Differentiation of leukemic cells lines in vitro
can be achieved by a variety of nonphysiologic (e.g.
X - 32 -
... . _ .. .
13iO~6
DMSO, phorboldiesters) and physiologic (e.g. retinoic
acid, vitamin D3) inducers (Koeffler et al. (1983) Blood
62:709). Murine G-CSF is known to be a potent inducer of
differentiation of WEHI-3B (D+) murine myelomonocytic
leukemia cells, whereas Interleukin 3 lacks this activity
(Nicola et al. (1984) Immunol Today 5:76) See Table V).
Pluripotent-CSF was tested for leukemia
differentiating activity (GM-DF) in a clonal assay system
described by Metcalf (1980) Int. J. Cancer 25:~25; Fibach
E., et al., J. Cell. Physiol. 113:152, (1'382) using
murine WEHI-3B (D+) and human HL-60 promyelocytic
leukemia cell lines (Platzer et al., J. Exp. Med.
_ :1788-1801, (1985). Quantitation of GM-DF was
obtained by incubation of leukemic cells in agar with
serial dilutions of pluripotent CSF. Pluripotent CSF had
GM-DF activity on both cell lines. However, HL-60
required approximately 2.5-5X higher concentrations of
Pluripotent CSF to achieve 50~ differentiated, spreading
colonies versus undifferentiated tight blast cell
colonies, than did WEHI-3B (D+) (Platzer et al., J. Exp.
Med. 162:1788-1801, (1985).
Morphological and cytochemical analysis of HL-60
colonies were performed using alpha-naphthylacetate
esterase (ANAE) and luxol fast blue (LFB) stains, as
X
l~4o2~
markers of the monocyte, macrophage and eosinophil
granulocyte lineage respectively. In the presence of
pluripotent CSF there is observed an increase in the
number of colonies containing polymorphonuclear cells (by
hematoxylin stain), LFB cells and in intensit;y of ANAE
stain. Therefore, pluripotent CSF induces differentiation
along the macrophage as well as granulocyte pathway. The
human leukemia cell line KGl (courtesy Dr. H.P. Koeffler)
responded to pluripotent CSF in a dose dependent manner
with increased colony formation in agar and increased 3H-
thymidine incorporation after 24-48 hours in suspension
culture. This might indicate that the GM-DF activity of
pluripotent CSF reflects the differentiating capacity of
a given leukemic cell line rather than an intrinsic
property of the factor.
CM from SK-HEP and cell line 5637 containing
pluripotent CSF (free of Interferon) has also shown
acquisition of immunoglobulin Fc receptor, growth
inhibition, increased expression of monocyte related
surface antigens and an increase in lysosomal enzyme
content as well as (to distinguish P-CSF from Interferon-
gamma) increased receptors for chemotactic peptide,
increased hydrogen peroxide release in response to
phorbol myristic acetate (PMA) stimulation and the
- 34 -
X
1302~
release of prostaglandins (PGE2 and 6-keto PGF1A) as
features of differentiation of human promyelocytic
leukemia cell line HL-60 and monoblastic leukemia cell
line U937. These broad range differentiation factors are
thus different from Interferon and conventional colony
stimulating activity (CSA). Highly purified Fluripotent
CSF increased the receptors for chemotactic peptide and
increased glycoconjugate synthesis as a feature of
differentiation in both the human promyelocytic leukemia
cell line HL-60 and monoblastic leukemia cell llne U937.
3. Pluripotent CSF shows species cross-reactivity
on normal murine bone marrow and leukemic cells.
Normal mouse bone marrow cells cultured in agar for
7 days in the presence of saturating concentrations of
Pluripoietin formed approximately 10% of the colonies
supported by WEHI-3B conditioned media as standard source
of CSF('S). All colonies formed in the presence of
Pluripotent CSF were of similar morphology, not staining
for alpha-naphtyl-acetate esterase or Kaplow's
myeloperoxidase; this suggests that a subpopulation of
murine colony forming progenitors is responsive to
Pluripotent CSF. Weak cross-species activity was found
on continuous murine mast cell lines, established as
described from murine long-term bone marrow cultures
(Tertian et al. (1980) J. Immunol . 127:788). 5,000
A
13~2fi~
cells/well of a mast cell growth factor (MCGF) dependent
murine mast cell line were incubated for 24 hours at 37~C
in 96 well plates with serial dilutions of growth
factors, and then assayed for H-thymidine uptake as
described (Yung et al. (1981) J. Immunol . 127:794).
Results demonstrate little more than 10% murine MCGF
activity of Pluripotent CSF as compared to ConA-LBRM CM,
which was used as a standard preparation of murine MCGF.
The murine Interleukin-3 dependent cell line FDC-P2
(courtesy Dr. M. Dexter) did not respond with increased
H-thymidine uptake to concentrations of Pluripoietin as
high as 2,000 U/ml.
We herein describe the purification of a
pluripotent CSF, which is constitutively produced by the
human bladder carcinoma cell line 5637, its lA6 subclone
or SK-HeP-1. This protein is capable of stimulating the
in vitro growth of mixed colony progenitor cells (CFU-
GEMM), early erythroid progenitor cells (BFU-E),
megakaryocytic (CFU-Mega), granulocyte- macrophage
progenitors (CFU-GM) and in addition induces
differentiation of both the murine myelomonocytic (WEHI-
3B (D+)) and the human promyelocytic (HL-60) leukemic
cell lines. The purified pluripotent CSF had a specific
activity in the GM-CSF assay of 1.5 X 10 U/mg protein.
- 36 -
X
. . . .... ~
13~02~fi
To our knowledge, this is the highest specific activity
for a human pluripotent CSF reported to date.
Pluripotent CSF has a molecular weight of 32,000 by gel
filtration and 18,000 by SDS-PAGE under both, reduced and
non-reduced conditions and an isoelectric point of 5.5.
Pluripotent CSF activities could be eluted from gel
slices representing the same molecular weight range as
the stained protein band.
The purified protein shown in SDS-PAGE is
consistent with pluripotent CSF because:
1) the profile of protein elution visualized in
SDS-PAGE and elution of pluripotent CSS
activity (Fig. 3) from reverse phase HPLC
columns is equivalent in the major fraction and
side fractions;
2) additional chromatography of the purified
protein on diphenyl or octyl reverse phase HPLC
coll~mns using acetonitrile or ethanol as
organic solvents for elution did not lead to a
separation of protein and pluripotent CSF
activity;
3) identical localization of protein band and
pluripotent CSF activity in a preparative SDS-
PAGE;
- 37 -
X
. . . ~ .,
1~026~
4) high specific GM-CSF activity (1.5 X 10 U/mg
protein).
Since purified pluripotent CSF is apparently homogeneous,
amino acid sequence analysis of the purified protein has
been initiated and is partially determined.
Based on the molecular weight of pluripotent CSF as
18,000 it could be calculated that 1 U of pluripotent CSF
was equivalent to 6.7 pg protein or 3.7 X 10 moles. A
pluripotent CSF concentration of 50 U/ml or 1.85 X 10 M
was required for half maximal colony formation for CFU-GM
activity in normal human bone marrow cells.
A ten-fold increase in the amount of pluripotent
CSF (500 U/ml GM-CSF activity) was required for clear
detection of human CFU-GEMM and erythroid BFU-E
activities (Table II); a 1-2 or 1-2.5 fold increase in
pluripotent CSF (e.g. 50-200 U GM-CSF) was needed to
induce the differentiation of either WEHI-3B (D+) or HL-
60 leukemic cells respectively. These data suggest that
the particular action(s) of pluripotent CSF are
determined by its concentration as first suggested by
Burgess and Metcalf (Blood, 56:947-958) in the murine
system. The fact that human pluripotent CSF is able to
induce differentiation of leukemic cell lines makes it a
protein with unique properties, since for the murine
- 38 -
13~2~fi
multi CSF (Interleukin 3) no differentiation activity on
leukemic cells has been reported (Ihle, J.N. et al., J.
Immunol. 129:2431-2436 (1982); Nicola, et al.r Immunol.
Today, 5:76, (1984); Watson, et al., Immunol. Ioday, 5:76
(1983); and Fung et al., Nature, 307:233, (1984). (Table
V compares the two entities). The murine IL-3 dependent
cell line FDC-P2 (Dr. M. Dexter) did not respond with
increased 3H-thymidine uptake to Pluripotent-CSF as high
as 2,000 U/ml.
Several human CSFs (GM-CSF, G-CSF, eosinophilic
CSF, erythroid potentiating activity) have molecular
weights between 30,000 and 40,000 on gel filtration
(Nicola, N.A. et al., Blood, 54:614-627, (1979); Gold,
D.W. et al., Proc. Natl. Acad. Sci. U. S.A., 77:593-596,
(1980); Lusis, A.J. et al., Blood 57:13-21, (1981);
Abboud, C.N. et al., Blood, 58:1148-1154, (19~1); Okabe,
T. et al., J. Cell. Phys., 110:43-49, (1982), which is
similar to the native molecular weight of the pluripotent
CSF described here. However, only partially purified
erythroid-potentiating activity has been report:ed to have
activity in a CFU-GEMM assay (Fauser, A.A., et al., Stem
Cells 1: 73-80, (1981).
Constitutive production of pluripotent CSF by the
bladder carcinoma cell line 5637 and its lA6 subclone or
- 39 -
X
- 134026~
other 5637 subclones suggests that these are valuable
sources for large scale production and for isolation and
cloning of the gene which codes for pluripotent CSF. The
availability of purified human pluripotent CSF has
important and far reaching implications in the management
of clinical diseases involving hematopoietic derangement
or failure, either alone or in combination with other
lymphokines or chemotherapy. Such disorders include
leukemia and white cell disorders in general. It is
useful in transplantation, whether allogeneic or
autologous, to augment growth of bone marrow progenitor
cells. It can be used in induced forms of bone marrow
aplasia or myelosuppression, in radiation therapy or
chemotherapy-induced bone marrow depletion, wound
healing, burn patients, and in bacterial inflammation.
Here the action of pluripotent-CSF may possibly be due to
enhancement of chemotactic peptide receptors or by
functioning as a chemo-attractant. It is also found in
saliva so may prevent tooth decay and oral infection.
p-CSF may be used alone or together with
recombinant material or in conjunction with
erythropoietin for treatment in hematopoietic disorders.
- 40 -
X
134026~
TABLE 1
Table I: Purification of human pluripotent CSF
Fraction Protein Total Specific Purifica- Yield
activity activity tion
(UX10 )(U/mg)(fold) (9~)
5637 CM 2g 12 6 X 103 - 100
DEAE 300 mg 5 4 1b 42
cellulose 1.7X10
AcA 54 13 mg 3.1 14 26
Ultrogel 2.4X10
RP--HPLC 5 ~lg 0.74 9 000 6.2
1 . 5X108
GM-CSF activity of pluripotent CSF; U=Units;
estimate of fold purification based on starting activity
of peak 1 of DEAE cellulose chromatography.
TABLE IIA
Table IIA: Comparison of CFU-GEMM and BFU-E activities of
pluripotent CSF (500 U/ml GM-CSF activity)
CFR-GEMM BFU-Ea
(Colonies + 1 SEM) (Colonies + 1 SEM)
Experiment 1 2 3 1 2 3
#
Medium 0.3+0.3 0 0 42+6 17+3 17+2
PHA--LCM7 + 1 3+0 3.3+0.3 67+1 65+3 34+3
7 7+2.1 4+0. 82.3+0.9 85+6 31+1 28+2
Plurlpotent ' -- -- -- -- -- --
CSF
aTarget cells were 5 X 104/ml low density, non-adherent
and T cell depleted normal human bone marrow cells.
Experiment 3 was done in the absence of Hemin.
- 41 -
X
13~2~6
bMedium conditioned by leukocytes from patients with
hemochromatosis in the presence of 1% PHA.
(positive control)
TABLE IIB
Table IIB: Activity of Pluripoietin on pre-CFU
Pluripoietin Exp.1 Exp. 2 Exp. 3
concentration 7 days in 7 days in 5 days in 9 days in
suspension suspension suspension suspension
culture culture culture culture
1000 U/ml416il8 20i4 32i5 80i8
500 U/ml 367i57 39i4 n.t. n.t.
100 U/ml n.t. 29i6 73i4 30i5
10 U/ml n.t. 12i3 52i3 34i4
Control 200il6 8i2 26i5 20i4
medium
~,.
TABLE llC
COMPARISON OF ACTIVITY OF 5637 AND lA6 IN GM CFU ASSAY
Dilution
Cells 1/10 1/100 1/200 1/400 1/800 1/16000 U/ml
Colonies 190 + 17 75 + 1 18 + 6 0 0 0 2750
5637-CM +
%100~ (max)39~ of 9% 350
max.
Colonies 0 + 0 93 + 7 130 + 11 124 + 0 64 + 0 30 + 6 24,000
lA6
0~ 49 69 65 34 16 3,000
2'3
cr~
02~
Legend Table II
Normal human bone marrow cells were separated by
Ficoll, adherence to plastic and depletion of T cells by
rosetting with neuraminidase treated sheep red blood
cells. Quadruplicate cultures of 25,000 cells in 100
microliters/well were incubated in 96 well flat bottom
tissue culture plates in Iscove's modified Dulbecco's
medium supplemented with 30% fetal bovine serum (FBS),
5X10 M 2-mercapto-ethanol and serial dilutions of
purified Pluripoietin or control medium for 5, 7 or 9
days at 37~C in 5% CO2 in air. Contents of each well were
then resuspended and incorporated into 1 ml agar system
in supplemented McCoy's with saturating concentrations
(10% v/v) of 5637 CM. Colonies were scored after 7 days
of incubation at 37~C in a humidified atmosphere of 5% CO2
in air. Results are expressed as mean colony number per
well + 1 standard deviation. CFU input on day 0 were 79
+ 5 (exp. 1), 26 + 1 (exp. 2) and 22 + 3 (exp. 3) per
well. Bone marrow cells from the donor for experiment 1
grew high numbers of CFU-GM in two unrelated experiments;
no pathophysiological situation was recognized.
A
134026~
TABLE III
Influence of Pluropoietin on protein content in cultures
of human macrophages
Time in culture Adherent cell Adherent cell
protein in response protein in response
to control medium to Pluripoietin
~g/coverslip ~g/coverslip
Day 1-210.0 + 2.0 28.6 + 7.7
Day 1-320.4 + 1.6 26.8 + 2.5
Day 1-428.4 + 1.6 41.2 + 1.9
Day 4-528.8 + 1.6 28.1 + 3.6
Day 4-643.1 + 4.7 28.1 + 3.6
Day 4-738.2 + 6.1 44.~ + 0.7
Legend Table III
Normal human monocytes/macrophages were isolated
from peripheral blood mononuclear cells by adherence to
glass surfaces . Two X 10 cells were plated per 13 mm
diameter coverslips in 0.1 ml of supplemented RPMI 1640
containing 25% fresh frozen human serum. After 2 hours
at 37~C, nonadherent cells were removed by rinsing, and
coverslips transferred to 24 well tissue culture trays
(day 0). On day 1 and 4, supernatants were replaced by
fresh culture medium containing 500 U/ml of purified
Pluripoietin or control medium. Protein content was
determined 1 to 3 days thereafter by rinsing coverslips
13~q26~
free of culture medium, solubilizing adherent cell
protein in 0.5 N NaOH and measuring protein concentration
according to the method of Lowry . Results are expressed
as mean + 1 standard deviation, from triplicate cultures.
X
TABLE IVA
Table IVA. Leukemia differentiation (GM-DF) activity of purified Pluripoietin
Purification CM-CSF activity GM-DF activity GM-DF activity
WEH1-3B (D+) HL-60
Specific U/ml U/ml Ratio U/ml Ratio
activity DF/CSF DF/CSF
U/mg protein
I 1.5X1084,000 246,000 2.9 54,000 0.6
II 201,000 502,000 2.5 80,000 0.4
1.25X10
C~
~3
- 47 -
13ql~266
TABLE IVB
Glycoconjugate Synthesis
Inducer HL-60 U937
CPM/5X10 cells
media 465 210
gIFN500 U/ml 1029a :1500a
100 U/ml 800a 537a
50 U/ml 410 258
LK 50%
(500 U/ml gIFN) 427 910a
5637 CM (GM-CSA)
2 kU/ml 1828a 1200a
1 kU/ml 980a 780a
500 U/ml 670a 490a
Pluripoetin 1 kU 4235a 2400a
430 306
pp aCSF 1 kU
1439a 604a
SK-Hep CM 50% 420 200
GCT-CM100% 490a 250
PMA 3.0 ng/ml
2000a 1700a
50.0 ng/ml 420 240
aIFN5000 U/ml 425 230
IL-2 100 U/ml
Glycoconjugate synthesis was measured as follows, cells
(5X105) were incubated with inducers for 4 hours then
- 48 ~
X
... .. , .. . _ _ . .. . . .. .. . . .
13~026~
glucosamine incorporation was evaluated after an
additional 16 hours.
Results are mean values from three or more experiments.
a, Significantly different from control, p less than 0.05
by Students T test; b, human P-CSF, units assigned by CFUC
activity; c, partially purified aCSF-like activity,
units assigned by CFUc activity.
Legend Table IVa
For determination of specific activity, protein
concentration of purified Pluripoietin was estimated by
comparison with serial dilutions of known amounts of
protein in SDS-PAGE, visualized by silver stain. Due to
the low frequency of CFU-GEMM in normal human bone marrow
cells, the biological activity of Pluripoietin had to be
measured using the GM-CSF assay. We compared the ability
of serial dilutions of Pluripoietin and a previously
determined laboratory standard of 5637 CM to support GM-
colony formation in 1 ml semi-solid agar cultures
containing 10 low density, normal human bone marrow
cells. Fifty units of GM-CSF activity were arbitrarily
defined as inducing 50% of maximal colony growth on day 7
of culture. Concentrations of 500 U/ml of P~uripoietin
were sufficient to stimulate colony growth from CFU-GEMM
and BFU-E comparable to that supported by optimal amounts
of phytohemagglutinin-activated lymphocyte conditioned
media. Two independent purifications (I and II) resulted
- 49 -
y
13~266
in very similar specific activity. Due to different
amounts of starting material, the final concentration of
biological activity differs between I and II, but is
useful for comparison of GM-CSF and leukemia
differentiating activity (GM-DF) of Pluripoietin. GM-DF
activity was determined by incubating 3X10 /ml WEHI-3B
(D+) or 10 /ml HL-60 leukemic cells in 0.3% agar in
McCoy's medium containing 12,5% FBS with serial dilutions
of Pluripoietin. Cultures were scored on day 7 (WEHI-3B)
and day 14 (HL-60) for induction of dispersed,
differentiated colonies vs. tight, blast cell colonies
(Metcalf, et al. (1980) Int. J. Cancer 25:225 and Fibach,
et al., (1982) J. Cell. Physiol. 113:152). Fifty units
of GM-DF activity were defined as inducing 50%
differentiated colonies.
- 50 -
, . . ~ ... . . .. . . . . . ..
13~6~
Table V. Biological activities of purified human
Pluripoietin and murine Interleukin-3.
Activity Pluripoietin ) Interleukin-3
Clonal growth of
hemopoietic progenitors:
CFU-GEMM + +
BFU-E + +
CFU-G,M,GM + +
CFU-EOS + +
CFU-MEG n.t. +
pre-CFU-c (~GPA) + n.t.
stem cell multi-
plication (CFU-s) +
c ) +
Species crossreactlv1ty
Leukemia differentiating
activity (GM-DF) on: +
WEHI-3B (D+)
HL-60
H-TdR uptake in cell +
lines: _ +
KG1
FDC-P2
Murine mast cell + +
lines
(MCGF activity) n.t. +
Histamine production n.t.
Protein synthesis of
mature macrophages +
Induction of 2D-SDH
Growth of: +
natural cytotoxic cells n.t.
pre-B cell clones
a) Pluripoietin was tested on human target cells, if
not noted otherwise.
b) Interleukin-3 activity on murine target cells, if
not noted otherwise.
X
, . . . .. ~ . . ......
- i3~0~
Data derived from literature, except GM-DF and
acvivity on KG1.
c) Activity on bone marrow derived colony formation in
agar cultures.
No human test system available.
n.t. Not tested.
- 51a -
X
" , . _ . . . ..
