U.S. patent application number 10/958942 was filed with the patent office on 2005-03-24 for human pluripotent hematopoietic colony stimulating factor, method of production and use.
Invention is credited to Gabrilove, Janice L., Mertelsmann, Roland, Moore, Malcolm A.S., Platzer, Erich, Welte, Karl.
Application Number | 20050063946 10/958942 |
Document ID | / |
Family ID | 27384261 |
Filed Date | 2005-03-24 |
United States Patent
Application |
20050063946 |
Kind Code |
A1 |
Welte, Karl ; et
al. |
March 24, 2005 |
Human pluripotent hematopoietic colony stimulating factor, method
of production and use
Abstract
Highly purified Pluripotent hematopoietic colony-stimulating
factor (pluripotent CSF), a glycoprotein (MW 19,600) constitutively
produced by human tumor cells has been highly purified from low
serum-containing conditioned medium to apparant homogeneity.
Pluripotent CSF supports the growth of human mixed colonies
(CFU-GEMM), granulocyte-macrophage colonies (CFU-GM), early
erythroid colonies (BFU-E) and induces differentiation of human
leukemic cells. The specific activity of the purified pluripotent
CSF in the CFU-GM assay is 1.5.times.10.sup.8 U/mg protein.
Inventors: |
Welte, Karl; (New York,
NY) ; Platzer, Erich; (Erlangen, DE) ;
Gabrilove, Janice L.; (New York, NY) ; Mertelsmann,
Roland; (Mainz, DE) ; Moore, Malcolm A.S.;
(Larchmont, NY) |
Correspondence
Address: |
Bell, Boyd & Lloyd LLC
P.O. Box 1135
Chicago
IL
60690-1135
US
|
Family ID: |
27384261 |
Appl. No.: |
10/958942 |
Filed: |
October 5, 2004 |
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10958942 |
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10117423 |
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6838549 |
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6419918 |
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09587476 |
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6114166 |
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08996051 |
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08816159 |
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5808008 |
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08816159 |
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08481946 |
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5662895 |
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08481946 |
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08280582 |
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5532341 |
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08132240 |
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06835270 |
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Current U.S.
Class: |
424/85.1 ;
435/320.1; 435/325; 435/69.5; 530/351; 536/23.5 |
Current CPC
Class: |
C07K 14/53 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
424/085.1 ;
530/351; 536/023.5; 435/069.5; 435/320.1; 435/325 |
International
Class: |
A61K 038/19; C07H
021/04; C07K 014/535; C12P 021/02 |
Goverment Interests
[0002] 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 government has
certain rights in this invention.
Claims
1. A highly purified glycoprotein human pluripotent colony
stimulating factor.
2. The factor of claim 1 wherein the factor is purified to
homogenity.
3. The factor of claim 1 wherein the factor is derived from human
cells.
4. The factor of claim 1 wherein the factor is derived from human
tumor cells.
5. The factor of claim 1 wherein the factor is derived from human
bladder cell line 5637, subclone 1A6 from 5637, and hepatoma cell
line SK-HEP-1.
6. The factor of claim 1 characterized by a) a molecular weight of
19,600 daltons under reducing and non-reducing conditions as
determined by SDS-PAGE; b) a molecular weight of 32,000 daltons as
determined by gel filtration; c) having a specific activity of at
least 1.5.times.10.sup.8 U/Mg as measured in the GM-CSF activity
assay; d) having the ability to stimulate in vitro growth of early
hematopoietic progenitor cells as mixed colony progenitor cells,
early erythroid progenitor cells, megakaryocytic cells and
granulocyte-macrophage progenitors; e) isoelectric point of 5.5; f)
having the pharmacological activity to induce differentiation of
leukemic cells; and g) having a partial amino acid composition as
determined from the amino-terminal end as Threo, Pro, Leu, Gly,
Pro, Ala, Ser, Ser Leu, Pro, Gln, Ser, Phe, Leu, Leu, Lys, Cys,
Leu, Glu, Gln, Val, Arg, Lys, Ile, Gln, Gly, Asp, Gly, Ala, Ala,
Leu, Gln, Phe, Lys, Leu, Gly, Ala, Thr, Tyr, Lys, Val, Phe, Ser,
Thr, (Arg), (Phe), (Met), X.
7. The factor of claim 6 wherein the leukemic cells induced are
leukemic cell lines.
8. The factor of claim 1 having the ability to induce the
acquistion of increased receptors for chemotactic peptide and
increased glycoconjugate synthesis.
9. Highly purified glycoprotein human pluripotent colony
stimulating factor derived from human tumor cells from the group
consisting of bladder tumor cell line 5637, subclone 1A6 from 5637
and hepatoma cell line SK-Hep-1.
10. A method for preparing highly purified glycoprotein pluripotent
colony stimulating factor from human cells which comprises: a)
high-salt precipitation of protein from a cell-free medium; b) ion
exchange chromatography of the precipitate from a) above; c) gel
filtration of active fractions from step b); and d) reverse-phase
high performance liquid chromatography of active fractions from
step c) above.
11. Highly purified glycoprotein human pluripotent colony
stimulating factor prepared by the method comprising: a) high-salt
precipitation of protein from a cell-free medium; b) ion exchange
chromatography of the precipitate from a) above; c) gel filtration
of active fractions from step b); and d) reverse-phase high
performance liquid chromatography of active fractions from step c)
above.
12. Method of inducing differentiation of human leukemic cells
which comprise contacting human leukemic cells with
pharmacologically active doses of highly purified glycoprotein
human pluripotent colony stimulating factor.
13. Method of claim 12 wherein therapeutically active doses of
highly purified glycoprotein human pluripotent colony stimulating
factor are used to treat leukemia.
14. A method of enhancing bone marrow recovery in allogeneic or
autologous transplantation and in treatment, radiation, chemically,
or chemotherapeutically induced bone marrow aplasia or
myelosuppression which comprises treating bone marrow transplant or
aplasia patients with therapeutically effective doses of highly
purified glycoprotein pluripotent CSF.
15. A method of treating conditions requiring optimum neutrophil or
macrophage function such as wounds, wound infection or burn wounds
which comprises systemically or topically contacting the affected
area with therapeutically effective doses of highly purified
glycoprotein pluripotent CSF.
16. A purified subclonal cell line 1A6 isolated from a parent human
tumor bladder cell line 5637.
17. The cell line 1A6 of claim 16 wherein 1A6 produces between 2-10
fold higher amounts of p-CSF than the parent cell line.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/117,423, filed on Apr. 4, 2002, now pending, which is a
continuation of application Ser. No. 09/587,476, filed on Jun. 5,
2000, now U.S. Pat. No. 6,419,918, which is a continuation of
application Ser. No. 08/996,051, filed on Dec. 22, 1997, now U.S.
Pat. No. 6,114,166, which is a division of application Ser. No.
08/816,159, filed on Mar. 12, 1997, now U.S. Pat. No. 5,808,008,
which is a division of application Ser. No. 08/481,946, filed on
Jun. 7, 1995, now U.S. Pat. No. 5,662,895, which is a continuation
of application Ser. No. 08/280,582, filed on Jul. 26, 1994, now
U.S. Pat. No. 5,532,341, which is a continuation of application
Ser. No. 08/132,240, filed on Oct. 6, 1993, now abandoned, which is
a continuation of application No. 06/835,270, filed on Mar. 7,
1986, now abandoned, which is a continuation-in-part of application
No. 06/716,844, filed on Mar. 28, 1985, now abandoned; each of
which is incorporated herein in their entirety by reference.
[0003] This application concerns human pluripotent colony
stimulating factor (P-CSF) also known as pluripoietin.
BACKGROUND OF THE INVENTION
[0004] Abbreviations:
[0005] CFU-GEMM: Colony forming unit--granulocyte, erythroid,
macrophage, megakaryocyte.
[0006] CFU-GM: Colony forming unit--granulocyte-macrophage
[0007] BFU-E: erythroid burst forming unit
[0008] GM-CSF: Granulocyte-macrophage colony stimulating factor
[0009] 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) J. Biol. Chem. 258: 9017-9021;
Ihle, J. N., et al. (1982) J. Immunol. 129: 2431-2436; Fung, M. C.,
et al. (1984) Nature 307: 233-237; Gough, N. M., et al. (1984)
Nature 309: 763-767), human CSFs have been less well characterized,
both biologically and biochemically (Nicola, N. A., et al. (1979)
Blood 54: 614-627; Wu, M. C., et al. (1980) J. 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-1) (Das, S.
K., et al. (1981) Blood 58: 630-641; Das, S. K., et al. (1982) J.
Biol. Chem. 257: 13679-13684) and erythroid potentiating activity
[Westbrook, C. A. et al. J. Biol. Chem. 259: 9992-9-996 (1984)] and
for granulocyte-macrophage CSF (GM-CSF) [Gasson, J. C., et al.
Science 226: 1339-1342 (1984)], but not for human pluripotent
CSF.
[0010] 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) 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 constitutively by
human tumor cell lines such as the SK-HEP-1 human hepatoma cell
line (J. Gabrilove, K. Welte, Li Lu, H. Castro-Malaspina, M. A. S.
Moore, Blood, in Press and hereby incorporated by reference); the
5637 bladder carcinoma cell line (reported herein and in Proc.
Nat'l. Acad. Sci. 82: 1526-1530 1985 hereby incorporated by
reference); 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 spectum CSF. It also induces differentiation of
leukemic cells.
SUMMARY OF THE INVENTION
[0011] This application concerns human puripotent 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 include the induction of functional markers of
differentiation of normal and leukemic cells.
[0012] Additional features and advantages of the present invention
are described in, and will be apparent from, the following Detailed
Description of the Invention and the figures.
BRIEF DESCRIPTION OF THE FIGURES
[0013] Figure one shows ion-exchange chromatography of 5637
conditioned medium (CM) followed by the Gel filtration
chromatograph shown in figure two.
[0014] Figure three shows pooled gel filtration eluants on HPLC
(reverse phase). FIG. 4 shows SDS-PAGE whereas FIG. 5 shows
preparative SDS-PAGE and FIG. 6 isoelectrofocusing of the purified
pluripotent CSF.
DESCRIPTION DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1: Ion Exchange Chromatography
[0016] 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
VaCl (0.05-0.3 M) in 0.05 M Tris/HCl, pH 7.8, as indicated (-). The
elution of proteins was monitored by absorption at 280 nm
(.cndot.-.cndot.) and each fraction was tested for CSF activities
(GM-CSF activity: .DELTA.). 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).
[0017] FIG. 2: Gel Filtration Chromatography
[0018] The pluripotent CSF containing concentrated pool of DEAE
cellulose chromatography was loaded on an AcA 54 Ultrogel column
(2.6.times.90 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 (.cndot.-.cndot.) and each fraction was tested for
pluripotent CSF activity (GM-CSF activity: .DELTA.).
[0019] FIG. 3: Reverse Phase High-performance Liquid Chromatography
(HPLC)
[0020] 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 (.mu.Bondapak, 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: .DELTA.).
[0021] FIG. 4: SDS-Polyacrylamide Gel Electrophoresis
(SDS-PAGE)
[0022] The pluripotent CSF eluted from the HPLC column (200 ng;
peak fraction) was lyophilized and treated with 1% SDS in 0.0625 M
Tris/HCl, pH 6.8, and 20% glycerol, under reducing conditions (5%
2-mercaptoethanol) for one hour at 37.degree. C. and then applied
to a 15% polyacrylamide gel. After electrophoresis, the protein
bands were visualized by the silver staining technique.
[0023] FIG. 5: Preparative SDS-PAGE
[0024] 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).
[0025] FIG. 6: Isoelectrofocusing
[0026] 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 isoelctrofocusing
(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:).
[0027] We report the purification and biochemical characterization
of a human pluripotent CSF, produced and released constitutively by
human cells especially tumor cells such as bladder carcinoma cell
line 5637 (ATCC HTB-9) and hepatoma cell line SK-HEP-1 (ATCC
HTB52). The cell line (5637) was obtained from Jorgen Fogh at
Sloan-Kettering Institute, 1275 York Avenue, New York, N.Y.
10021.
[0028] Pluripotent CSF biological properties include
differentiation of progenitor cells to all major blood types as
well as differentiation of leukemic cells.
[0029] Assay for GM-CSF Activity
[0030] 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. 10.sup.5
separated cells were plated in 1 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. Hematol. 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 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) J. Biol. Chem.
258:9017-9021).
[0031] Assay for Colony Stimulating Factor for BFU-E and
CFU-GEMM
[0032] The colony assay for human BFU-E and CFU-GEMM was performed
as previously described (Li Lu, et al. (1983) Blood 61: 250-256).
Human bone marrow cells were subjected to a density cut with
Ficoll-Hypaque (density 1.077 gm/cm.sup.3; Pharmacia Fine
Chemicals, Piscataway, N.J.) and the low density cells were
suspended in RPMI 1640 containing 10% FCS at 2.times.10.sup.7
cells/ml and placed for adherence on Falcon tissue cultures dishes
(#3003, Becton Dickinson and Co., Cockeysville, Md.) for 11/2 hr.
at 37.degree. C. The nonadherent cells were depleted of T
lymphocytes by rosetting with neuraminidase-treated sheep
erythrocytes. Medium conditioned by leukocytes from patients with
hemochromatosis in the presence of 1% (v/v) phytohemagglutinin
(PHA) (Li Lu, et al. (1983) Supra) as positive control or serial
dilutions of test samples were then added at 5% (v/v) to
5.times.10.sup.4 of these low density, non-adherent and T
lymphocyte depleted bone marrow cells in a 1 ml mixture of Iscove's
modified Dulbecco medium (GIBCO Grand Island, N.Y.), 0.8%
methylcellulose, 30% FCS, 5.times.10.sup.-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% CO.sub.2 in air at 37.degree. C. After
14 days of incubation, colonies were scored and morphology was
assessed.
[0033] 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.
[0034] Differentiation Induction Assay
[0035] Titrated samples of purified pluripotent CSF were assayed
for differentiation induction of WEHI 3B (D+) or HL-60 leukemic
cells as described (Metcalf, D. (1980) Int. J. Cancer 25:225-233;
Fibach, E., et al., J. Cell Physiol. 113:(1) 152 (1982)).
[0036] Rossette Assays for Fc Receptor, OKM1 and Leu M2
Antigens
[0037] Cell receptors for immunoglobulin Fc were assayed with IgG
(Cappel Laboratories, West Chester, Pa.) coated sheep erythrocytes
as described elsewhere (Ralph, P., et al. (1983) Blood 62:1169).
OKM1 (Ortho Diagnostics Systems Inc., Raritan, N.J.) or Leu M2
(Beckton Dickinson, Mountain View, Calif.) reactive antigens were
detected by incubating 10.sup.6 cells/0.1 ml phosphate buffered
saline containing 1.0 .mu.g/ml monoclonal antibody for 20 min at
24.degree. C., washing, incubating 20 min at 24.degree. C. with a
a: 100 dilution of rabbit anti-rat (Leu M2) or anti-mouse (OKM1)
IgG serum (Cappel Laboratories, Cochranville, Pa.), washing and
rosetting with protein A-coated erythrocytes as described
previously (Ralph, P. et al., Supra).
[0038] Assays for fMLP Receptor
[0039] Receptors for chemotactic peptide,
formyl-Methionyl-Leucyl-Phenylal- -anine (fMLP), were assayed as
follows; 2.times.10.sup.6 cells were incubated with 15 nM.
.sup.3H-fMLP (New England Nuclear, Boston, Mass.) in a total volume
of 0.2 ml in the presence or absence of 10 .mu.M unlabelled fMLP
(Sigma Corp, St. Luis, Mo.). After three hrs at 4.degree. C., the
cell suspensions were rapidly filtered onto glass fiber dics
(Whatman Inc., Clifton, N.J.), which were then washed with 30 mls
of 4.degree. C. phosphate buffered saline (Harris, P. et al. (1985)
Cancer Res. 45:9). Radioactivity on the discs were counted by
liquid scintillation spectrophotometry.
[0040] Measurement of PMA-Stimulated Hydrogen Peroxide Release
[0041] The production of hydrogen peroxide in response to PMA
stimulation was assayed by horse radish peroxidase (HRPO) (Sigma)
mediated H.sub.2O.sub.2 dependant oxidation of homovanillic acid
(HVA) (Sigma), as described (Harris, et al. Supra (1985). Briefly,
cells (1.times.10.sup.6) 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.degree.
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 hydrogen peroxide (Sigma) was used to prepare
H.sub.2O.sub.2 standards (0.001 to 50 mmoles/assay) for the
construction of a standard curve. The EVA 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.
[0042] Prostaglandin Measurements
[0043] 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 hrs, centrifuged
and the supernates harvested. Supernates were stored at -20.degree.
C. until assayed.
[0044] Prostaglandin standards PGE.sub.2, 6-keto-PGF1.sub.a and
TBX.sub.2 were kindly supplied by Dr. J. Pike (Upjohn Company,
Kalamazoo, Mich.). Tritium labeled compounds were purchased from
New England Nuclear (Boston, Mass.). Rabbit antisera to PGE.sub.2
were obtained from the Pasteur Institute (Paris, France).
Antibodies to 6-keto PGF1.sub.a 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 PGE.sub.2
antisera which cross reacted 10% with PGE.sub.1 standard. The
procedure for extracting the prostaglandins has been described
earlier (Karmali, R. A., et al. (1982) Prostagl. Leukotr. Med. 8:
565). Briefly, a trace of [.sup.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.degree. C. for
8-12 hours. Bound and free [.sup.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 10 pg. The intra-assay coefficient of
variation was 9.0%.
[0045] Alkaline and Acid Phosphatase, b-Glucuronidase and N-Acetyl
Glucuronidase Assays
[0046] Cell extracts were prepared in 0.5 ml of PBS 1% NP-40,
incubated 5 min at 24.degree. C., then spun for 10 min at
3.times.10.sup.4 g. 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 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.
[0047] Glycoconjugate Assay
[0048] Cytokine preparations were assayed in a
[.sup.3H]-Glucosamine incorporation assay. Replicate wells were
plated with 100 microliters of inducing agent to be tested.
Previously washed (3.times. in PBS) HL-60 or U937 cells
(1.times.10.sup.7 cells/ml) in RPMI 1640 without FCS were added (50
microliters). After a four hour incubation 20 microliter of 25
.mu.Ci/ml [.sup.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 (.times.4, 0.1 ml each),
followed by 0.4N Perchlorate wash (.times.4, 0.1 ml) and water
(2.times., 0.1 ml). Radioactivity on glass discs was determined by
liquid scintillation spectrophotometry.
[0049] Statistical Analysis
[0050] Student's T-test to compare means was carried out using the
significance limits of a two tailed test.
[0051] Preparation of 5637 Cell Line Conditioned Medium (5637
CM)
[0052] 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% FCS. For
purification of pluripotent CSF activity from 5637 conditioned
medium (5637 CM), confluent cell cultures were intermittently
cultured in medium containing 0.2% FCS. After 48-72 hours, 5637 CM
was harvested, cells and cell debris removed by centrifugation (20
min, 10,000.times.g), and stored at -20.degree. C. until use.
[0053] 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
1A6 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 times more p-CSF from the 1A6 subclone than from the parent
5637 cell line as determined by the assay methods outlined. This
subclone or the parent cell line 5637 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
1A6 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 1A6 in a U.S. patent application
filed Aug. 23, 1985 Ser. No. 768,959 entitled "Production of
Pluripotent Granulocyte colony-stimulating Factor" by Lawrence M.
Souza to yield sequence data on the protein p-CSF with subsequent
preparation of recombinant p-CSF from such a sequenced probe
(P11-top P14) as follows:
[0054] "(B) Sequencing of Materials Provided by Revised Methods
[0055] In order to obtain a sufficient amount of pure material to
perform suitably definitive amino acid sequence analysis, cells of
a bladder carcinoma cell line 5637 (sublcone 1A6) as produced at
Sloan-Kettering were obtained from Dr. E. Platzer. Cells were
initially cultured Iscove's medium (GIBCO, Grand Island, N.Y.) in
flasks to confluence. When confluent, the cultures were trypsinized
and seeded into roller bottles (11/2 flasks/bottle) each containing
25 ml of preconditioned Iscove's medium under 5% CO.sub.2. The
cells were grown overnight at 37.degree. C. at 0.3 rpm.
[0056] Cytodex-1 beads (Pharmacia, Uppsala, Sweden) were washed and
sterilized using the following procedures. Eight grams of beads
were introduced into a bottle and 400 ml of PBS was added. Beads
were suspended by swirling gently for 3 hours. After allowing the
beads to settle, the PBS was drawn off, the beads were rinsed in
PBS and fresh PBS was added. The beads were autoclaved for 15
minutes. Prior to use, the beads were washed in Iscove's medium
plus 10% fetal calf serum (FCS) before adding fresh medium plus 10%
FCS to obtain treated beads.
[0057] After removing all but 30 ml of the medium from each roller
bottle, 30 ml of fresh medium plus 10% FCS and 40 ml of treated
beads were added to the bottles. The bottles were gassed with 5%
CO.sub.2 and all bubbles were removed by suction. The bottles were
placed in roller racks at 3 rpm for {fraction (1/2)} hour before
reducing the speed to 0.3 rpm. After 3 hours, an additional flask
was trypsinized and added to each roller bottle containing
beads.
[0058] At 40% to 50% of confluence the roller bottle cultures were
washed with 50 ml PBS and rolled for 10 min. before removing the
PBS. The cells were cultured for 48 hours in medium A [Iscove's
medium containing 0.2% FCS, 10.sup.-8M hydrocortisone, 2 mM
glutamine, 100 units/ml penicillin, and 100 .mu.g/ml streptomycin].
Next, the culture supernatant was harvested by centrifugation at
3000 rpm for 15 min., and stored at -70.degree. C. The cultures
were refed with medium A containing 10% FCS and were cultured for
48 hours. After discarding the medium, the cells were washed with
PBS as above and cultured for 48 hours in medium A. The supernatant
was again harvested and treated as previously described.
[0059] Approximately 30 liters of medium conditioned by 1A6 cells
were concentrated to about 2 liters on a Millipore Pellicon unit
equipped with 2 cassettes having 10,000 M.W. cutoffs at a filtrate
rate of about 200 ml/min. and at a retentate rate of about 1000
ml/min. The concentrate was diafiltered with about 10 liters of 50
mM Tris (pH 7.8) using the same apparatus and some flow rates. The
diafiltered concentrate was loaded at 40 ml/min. onto a 1 liter DE
cellulose column equilibrated in 50 mM Tris (pH 7.8). After
loading, the column was washed at the same rate with 1 liter of 50
mM Tris (pH 7.8) and then with 2 liters of 50 mM Tris (pH 7.8) with
50 mM NaCl. The column was then sequentially eluted with six 1
liter solutions of 50 mM Tris (pH 7.5) containing the following
concentrations of NaCl: 75 mM; 100 mM; 125 mM; 150 mM; 100 mM; and
300 mM. Fractions (50 ml) were collected, and active fractions were
pooled and concentrated to 65 ml on an Amicon ultrafiltration
stirred cell unit equipped with a YM5 membrane. This concentrate
was loaded onto a 2 liter AcA54 gel filtration column equilibrated
in PBS. The column was run at 80 ml/hr. and 10 ml fractions were
collected. Active fractions were pooled and loaded directly onto a
C4 high performance liquid chromatography (HPLC) column.
[0060] Samples, ranging in volume from 125 ml to 850 ml and
containing 1-8 mg of protein, about 10% of which was hpG-CSF.
Samples were loaded onto the column at a flow rate ranging from 1
ml to 4 ml per minute. After loading and an initial washing with
0.1M ammonium acetate (pH 6.0-7.0) in 80% 2-propanol at a flow rate
of 1/ml/min. One milliliter fractions were collected and monitored
for proteins at 220 run, 260 nm and 280 nm.
[0061] As a result of purification, fractions containing hpG-CSF
were clearly separated (as fractions 72 and 73 of 80) from other
protein-containing fractions. HPG-CSF was isolated (150-300.mu.) at
a purity of about 85.+-.5% and at a yield of about 50%. From this
purified material 9 .mu.g was used in Run #4, an amino acid
sequence analysis wherein the protein sample was applied to a
TFA-activated glass fiber disc without polybrene. Sequence analysis
was carried out with an AB 470A sequencer according to the methods
of Hewick, et al., J. Biol. Chem., 256, 7990-7997 (1981) and Lai,
Anal. Chem. Acta. 163, 243-248 (1984). The results of Run #4 appear
in Table III.
1TABLE III 1 5 10 Thr - Pro - Leu - Gly - Pro - Ala - Ser - Ser -
Leu - Pro- 15 20 Gln - Ser - Phe - Leu - Leu - Lys -(Lys)- Leu
-(Glu)- Glu - 25 30 Val - Arg - Lys - Ile -(Gln)- Gly - Val - Gly -
Ala - Ala- Leu - X - X
[0062] In Run #4, beyond 31 cycles (corresponding to residue 31 in
Table III, no further significant sequence information was
obtained. In order to obtain a longer unambiguous sequence, in a
Run #5, 14, .mu.g of hpG-CSF purified from conditioned medium were
reduced with 10 .mu.l of-mercaptoethanol for one hour at 45.degree.
C., then thoroughly dried under a vacuum. The protein residue was
then redissolved in 5% formic acid before being applied to a
polybrenized glass fiber disc. Sequence analysis was carried out as
for Run #4 above. The results of Run #5 are given in Table IV.
2TABLE IV 1 5 10 Thr-Pro-Leu-Gly-Pro-Ala-Ser-Ser- Leu - Pro - Gln
-Ser- 15 20 Phe-Leu-Leu-Lys-Cys-Leu-Glu-Gln- Val - Arg - Lys -Ile
25 30 35 Gln-Gly-Asp-Gly-Ala-Ala-Leu-Gln- Phe - Lys - Leu -Gly- 40
45 Ala-Thr-Tyr-Lys-Val-Phe- -Ser-Thr-(Arg)-(Phe)-(Met)-x-
[0063] The amino acid sequence give in Table IV was sufficiently
long (44 residues) and unambiguous to construct probes for
obtaining hpG-CSF cDNA as described infra." (end quote)
[0064] Ammonium Sulfate Precipitation, Ion-Exchange-Chromatography,
Gel Filtration
[0065] The first three purification steps
((NH.sub.4).sub.2SO.sub.4-precip- itation,
Ion-exchange-chromatography on DEAE cellulose, DE 52, Whatman,
Clifton, N.J., and gel filtration on AcA 54 Ultrogel, LKB, Inc.
Rockland, Md.) were performed as described (Welte, K., et al.
(1982) J. Exp. Med. 156: 454-464) except that AcA 54 was used
instead of AcA 44 (see also Descriptions of FIGS. 1 and 2).
[0066] Reverse Phase High-Performance Liquid Chromatography
(RP-HPLC)
[0067] RP-HPLC was performed with a Waters HPLC system (M 6,000
solvent delivery pumps, model 400 variable wavelength detector,
data module and data processor, Waters Associates, Milford, Mass.).
The separation was performed on a .mu.Bondapak C18 column (Waters).
The buffers used were: Buffer A: 0.9 M acetic acid/0.2M pyridine,
pH 4.0; buffer B: buffer A in 50% 1-propanol (Burdick and Jackson,
Lab., Muskegon, Mich.). Acetic acid and pyridine were purchased
from Fisher, Scientific Co. The pluripotent CSF containing pool
obtained from gel filtration was acidified with acetic acid to pH
4.0 and injected onto the .mu.Bondapak C18 column without regard to
sample volume. The column was washed with buffer A (10 min) and
bound proteins were eluted using a steep gradient 0-40% buffer B
within the first 20 min and a 40-100% gradient of buffer B in 120
min. The flow rate was adjusted to 1 ml/min and 3 ml fractions were
collected. From each fraction a 0.5 ml aliquot was supplemented
with 10% FCS, dialyzed against PBS and tested for pluripotent CSF
activity.
[0068] Isoelectrofocusing
[0069] One ml of the purified pluripotent CSF was supplemented with
20% glycerol (vol/vol) and 2% Ampholines (vol/vol), pH 3.5-10 (LKB
Products, Inc.). A 5-60% glycerol density gradient containing 2%
Ampholines, pH 3.5-10, was layered into a isoelectrofocusing column
(LKB 8100). The pluripotent CSF sample was applied onto the
isodense region of the gradient, followed by isoelectrofocusing
(2,000 V, 24 hours). Five ml fractions were collected and the pH
determined in each fraction. The fractions were dialyzed against
PBS and subsequently tested for pluripotent CSF activity.
[0070] Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE)
[0071] The discontinuous Tris-glycine system of Laemmli (Laemmli,
U. K. (1970) Nature 227: 680-685) was used for 1.5 mm slab gels of
15% acrylamide. The samples (200 ng lyophilized protein eluted from
HPLC) were treated with 1% SDS in 0.0625 M Tris-HCl, pH 6.8 at
37.degree. C. for 1 hour under both reducing (5% 2-mercaptoethanol)
and non-reducing conditions and then loaded on the gel. After
electrophoresis, gels were stained by the Biorad silver staining
method (Biorad Laboratories, Rockville Centre, N.Y.). Apparent
molecular weights were determined using protein standards ovalbumin
(MW 43,000), chymotrypsinogen (MW 25,700), beta-lactoglobulin (MW
18,400), lysozyme (MW 14,300) and cytochrome C (MW 12,300)
(Bethesda Research Laboratories, Inc. Gaithersburg, Md.) or from
Pharmacia Fine Chemicals, Piscataway, N.J. After treatment (see
above) of lyophilized pluripotent CSF under non-reduced conditions
and subsequent electrophoresis, parallel gels were sliced in 4 mm
or 2 mm sections, respectively and proteins from each slice eluted
either into 0.5 ml RPMI 1640 containing 10% FCS or into phosphate
buffered saline (PBS; 20 mM phosphate, 0.15 M NaCl). After
extensive dialysis, the eluted material was assayed for pluripotent
CSF activity.
[0072] Protein Assay
[0073] The protein content of samples were measured using the Lowry
technique (Lowry, O. H., et al. (1951) J. Biol. Chem. 193:
265-275). For protein concentrations lower than 2 microgram/ml,
samples were subjected to SDS-PAGE, the protein bands were
visualized by the silver staining technique and the protein
concentration estimated by comparison with a serial dilution of
known amounts of proteins.
[0074] The examples shown serve to illustrate the invention without
limiting same.
EXAMPLE I
Pluripotent CSF Activity in 5637 CM
[0075] Confluent layers of 5637 human bladder carcinoma cells, when
cultured for 48-72 hours in the presence of 10% FCS, released into
the culture medium 3,000-10,000 units/ml of GM-CSF activity. Media
conditioned in the presence of 0.2% FCS still contained 10-30% of
this activity, whereas in serum free 5637 CM the activity drops
below 5% of the activity obtained in the presence of 10% FCS.
Although GM-CSF activity in 5637 CM is readily detectable in soft
agar bone marrow cultures, not all batches of unfractionated 5637
CM support in vitro growth of BFU-E and CFU-GEMM. Four to ten times
concentrated 5637 CM support in vitro growth of BFU-E and CFU-GEMM.
Four to ten times concentrated 5637 CM reduced colony formation by
CFU-GM 30-70% indicating the presence of inhibitor(s) in 5637 CM.
Inhibitors were removed after ion-exchange chromatography.
EXAMPLE II
Purification of Pluripotent CSF
[0076] A 20-fold concentration of proteins from the 5637 CM was
achieved by precipitation with ammonium sulfate at 80% saturation.
The dialyzed precipitate was loaded on to a DEAE cellulose (DE 52)
column. Bound proteins were eluted with a salt gradient from
0.05-0.3M NaCl in 0.05 M Tris-HCl, pH 7.8. GM-CSF activity eluted
as peak 1 between 0.075 M and 0.1M NaCl and with a second peak at
0.13 M NaCl (FIG. 1). Since only peak 1 revealed pluripotent CSF
activity, was used only this pool for further purifications. Peak 2
included proteins with only GM-CSF activity. We calculated the
"fold" purification by measuring the GM-CSF activity of pluripotent
CSF. In the unfractionated CM we could not discriminate between
GM-CSF activity as part of pluripotent CSF activity and CM-CSF
activity without pluripotent properties. Therefore we considered
the GM-CSF activity contained in peak 1 from DE 52 as the starting
activity (Table 1).
[0077] Since in the subsequent purification schedule GM-CSF, BFU-E
and CFU-GEMM activities copurified in all steps, we named these
combined activities "pluripotent CSF" and have used this term
thereafter. The proteins of peak 1 of DE 52 chromatography
(including pluripotent CSF activity) were concentrated by dialyzing
against 50% (w/v) Polyethylenglycol in PBS and purified further by
ACA 54 Ultrogel gel filtration. The pluripotent CSF activity eluted
in fractions 42-49 as a single peak corresponding to a molecular
weight of 32,000 daltons (FIG. 2). This step resulted in a 65%
recovery of activities and a 15 fold increase of specific
activities (Table 1).
[0078] The final step involved chromatography on a reverse phase
HPLC column (.mu.Bondapak C 18). The majority of proteins did not
bind to this column or eluted at low 1-propanol concentrations
(less than 20% 1-propanol; FIG. 3). A minor peak of GM-CSF activity
without activity in the CFU-GEMM and BFU-E assays but
differentation inducing activity on HL-60 leukemic cells was eluted
at around 30% 1-propanol. Pluripotent CSF activity eluted as a
single sharp peak at 42% 1-propanol (FIG. 3). This purification
step resulted in a 600-fold increase of specific activity and a 25%
recovery of activity. The protein content of the HPLC fraction was
measured by comparing the density in silver stained SDS-PAGE with
protein standards of known concentrations. Using this measurement,
we obtained a specific activity of 1.5.times.10.sup.8 U/mg protein
and a final purification of 9,000-fold, calculated from the first
peak of DEAE cellulose chromatography. The overall yield was 6.2%.
The purification table with the degree of purification of
pluripotent CSF as measured by GM-CSF activity, protein content,
specific activity and yield is detailed in Table 1.
[0079] 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).
[0080] 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 single band
in a silver stained gel with a molecular weight of 18,000,
identical to that shown in FIG. 4.
[0081] 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 M.W. of 18,800.
[0082] 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%.
[0083] Pluripotent CSF activity did not bind 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.
[0084] The partial amino acid sequence was determined by Applied
Molecular Genetics (Thousand Oaks, Calif.) on an AB 1470A-Beatrice
microsequencer. From the amino terminal end the sequence is ThrEO,
Pro, Leu, Gly, Pro, 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
[0085] 1. Progenitor Cells:
[0086] Fifty unit of GM-CSF activity, enough to support the half
maximal 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).
[0087] 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.
[0088] We show the results of comparison of 5637-CM and 1A6-CM in
Table IIC at dilutions of 1/10 through 1/1600. The 1/10 dilution of
1A6 shows an inhibitor to be present in the CM. Essentially this
table serves as an example that the 1A6 subclone of 5637 has 8.7
times more p-CSF in U/ml under growing conditions containing
FCS.
[0089] When purifying p-CSF the FCS is reduced to 0.2%.
[0090] 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.
[0091] 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) 113:152) Table IV(A&B) to show the effect of
pluripotent-CSF on leukemic cells. U937 was obtained from Dr.
Nilsson and HL-60 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).
[0092] Differentiation of leukemic cells lines in vitro can be
achieved by a variety of nonphysiologic (e.g. DMSO,
phorboldiesters) and physiologic (e.g. retinoic acid, vitamin
D.sub.3) 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).
[0093] Pluripotent-CSF was tested for leukemia differentiating
activity (GM-DF) in a clonal assay system described by Metcalf
((1980) Int. J. Cancer 25: 225; Fibach, E., et al. J. Cell.
Physiol. Supra) using murine WEHI-3B (D+) and human HL-60
promyelocytic leukemia cell lines (Platzer et al. (in press)
(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 2.5-5.times.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. 1985, Supra).
[0094] Morphological and cytochemical analysis of HL-60 colonies
were performed using alpha-naphthylacetate esterase (ANAE) and
luxol fast blue (LFB) stains, as markers of the monocyte,
macrophage and eosinophil granulocyte lineage respectively
(Platzer, E., et al. J. Immunol. in press). 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 intensity of ANAE stain. Therefore pluripotent CSF
induces differentiation along the macrophage as well as granulocyte
pathway. The human leukemia cell line KG1 (courtesy Dr. H. P.
Koeffler) responded to Pluripotent CSF in a dose dependent manner
with increased colony formation in agar and increased
.sup.3H-thymidine incorporation after 24-48 hrs. in suspension
culture. This might indicate that the GM-DF activity of Pluripotent
CSF reflects the differentiating capacity of a given leukemic cell
lines rather than an intrinsic property of the factor.
[0095] 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 release of prostaglandins (PGE.sub.2 and 6-keto
PGF.sub.1A) 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)
(Harris et al. submitted). Highly purified pluripotent 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
leukemic cell line U937.
[0096] 3. Pluripoietin CSF shows species crossreactivity on normal
murine bone marrow and leukemic cells:
[0097] 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-naphthyl-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
cells/well of a mast cell growth factor (MCGF) dependent murine
mast cell line were incubated for 24 hrs. at 37.degree. C. in 96
well plates with serial dilutions of growth factors, and then
assayed for .sup.3H-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 .sup.3H-thymidine
uptake to concentrations of Pluripoietin as high as 2,000 U/ml.
[0098] We herein describe the purification of a pluripotent CSF,
which is constitutively produced by the human bladder carcinoma
cell line 5637, its 1A6 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-macrophoage
progenitors (CFU-GM) and in addition induces differentiation of
both the murine myelomoncytic (WEHI-3B(D+)) and the human
promyelocytic (HL-60) leukemic cell lines (E. Platzer, K. Welte, J.
Gabrilove, Li Lu, M. A. S. Moore, manuscript in preparation). The
purified pluripotent CSF had a specific activity in the GM-CSF
assay of 1.5.times.10.sup.8 U/mg protein. 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.
[0099] 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 CSF activity
(FIG. 3) from reverse phase EPLC columns is equivalent in the major
fraction and side fractions; 2) additional chromatography of the
purified protein on diphenyl or octyl reverse phase HPLC columns
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; 4) high specific GM-CSF
activity (1.5.times.10.sup.8 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.
[0100] 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.times.10.sup.-16 moles. A pluripotent CSF
concentration of 50 U/ml or 1.85.times.10.sup.-11 M was required
for half maximal colony formation form CFU-GM activity in normal
human bone marrow cells.
[0101] 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-3 B (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,
Supra) 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 multi CSF
(Interleukin 3) no differentiation activity on leukemic cells has
been reported (Ihle, J. N., et al. (1982) J. Immunol. 129:
2431-2436; Nicola, et al. (1984) Immunol. Today 5: 76, Watson, et
al. (1983) Immunol. Today 5: 76, and Fung et al. (1984) Nature 307:
233). (Table V compares the two entities). The murine IL-3
dependent cell line FDC-P2 (Dr. M. Dexter) did not respond with
increased .sup.3H-thymidine uptake to Pluripotent-CSF as high as
2,000 U/ml.
[0102] 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. (1979)
Blood 54: 614-627; 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) which is similar to the native
molecular weight of the pluripotent CSF described here. However,
only partially purified erythroid-potentiating activity has been
reported to have activity in a CFU-GEMM assay (Fauser, A. A., et
al. (1981) Stem Cells 1: 73-80).
[0103] Constitutive production of pluripotent CSF by the bladder
carcinoma cell line 5637 and its 1A6 subclone or other 5637
subclones suggests that these are valuable source 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 myelosuppresion, 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.
[0104] p-CSF may be used alone or together with recombinant
material or in conjunction with erythropoietin for treatment in
hematopoietic disorders.
3TABLE I Purification of human pluripotent CSF Total Specific
activity.sup.M activity Purification Yield Fraction Protein (U
.times. 10.sup.-6) (U/mg) (fold) (%) 5637 CM 2 g 12 6 .times.
10.sup.3 -- 100 DEAE- 300 mg 5 1.7 .times. 10.sup.4 1.sup.b 42
Cellulose AcA 54 13 mg 3.1 2.4 .times. 10.sup.5 14 26 Ultrogel
RP-HPLC 5 .mu.g 0.74 1.5 .times. 10.sup.8 9,000 6.2 .sup.aGM-CSF
activity of pluripotent CSF: U = Units .sup.bestimate of fold
purification based on starting activity of peak 1 of DEAE cellulose
chromatography
[0105]
4TABLE IIA Comparison of CFU-GEMM and BFU-E activities of
pluripotent CSF (500 U/ml GM-CSF activity) Experi- CFU-GEMM.sup.a
BFU-E.sup.a ment (Colonies .+-. 1 SEM) (Colonies .+-. 1 SEM) #: 1 2
3 1 2 3 Medium 0.3 .+-. 0.3 0 0 42 .+-. 6 17 .+-. 3 17 .+-. 2 PHA-
7 .+-. 1 3 .+-. 0 3.3 .+-. 0.3 67 .+-. 1 65 .+-. 3 34 .+-. 3
LCM.sup.b Pluri- 7.7 .+-. 2.1 4 .+-. 0.8 2.3 .+-. 0.9 85 .+-. 6 31
.+-. 1 28 .+-. 2 Potent CSF .sup.aTarget cells ware 5 .times.
10.sup.4/ml low density, non-adherent and T cell depleted normal
human bone marrow cells. Experiment 3 was done in the absence of
Hemin. .sup.bMedium conditioned by leukocytes from patients with
hemochromatosis in the presence of 1% PHA. (positive control)
[0106]
5TABLE IIB Activity of Pluripoietin on pre-CFU Exp. 1 Exp. 2 Exp. 3
7 days in 7 days in 5 days in 9 days in Pluripoietin suspension
suspension suspension suspension Concentration culture culture
culture culture 1000 U/ml 416 .+-. 18 20 .+-. 4 32 .+-. 5 80 .+-. 8
500 U/ml 367 .+-. 57 39 .+-. 4 n.t. n.t. 100 U/ml n.t. 29 .+-. 6 73
.+-. 4 30 .+-. 5 10 U/ml n.t. 12 .+-. 3 52 .+-. 3 34 .+-. 4 Control
medium 200 .+-. 16 8 .+-. 2 26 .+-. 5 20 .+-. 4
[0107]
6TABLE IIC COMPARISON OF ACTIVITY OF 5637 AND 1A6 IN GM CFU ASSAY
Dilution Cells 1/10 1/100 1/200 1/400 1/800 1/16000 U/ml 5637-
Colonies % 190 .+-. 17 75 .+-. 1 18 .+-. 6 0 0 0 2750 .+-. 350 CM
100% 39% 9% (max) of max 1A6 Colonies % 0 .+-. 0 93 .+-. 7 130 .+-.
11 124 .+-. 0 64 .+-. 0 30 .+-. 6 24,000 .+-. 0% 49 69 65 34 16
3,000
[0108] Legend Table II
[0109] Normal human bone marrow cells were separated by Ficoll,
adherence to plastic and depletion of T cells by resetting with
neuraminidase treated sheep red blood cells, as described (Platzer,
E., et al. J. Immunol./in press). 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), 5.times.10.sup.-5 M
2-mercaptoethanol and serial dilutions of purified Pluripoietin or
control medium for 5, 7 or 9 days at 37.degree. C. in 5% CO.sub.2
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, as described
(Platzer, E., et al. 1985 Supra, Welte, K., et al. (1985) Proc.
Nat'l. Acad. Sci. U.S.A. in press). Colonies were scored after 7
days of incubation at 37.degree. C. in a humidified atmosphere of
5% CO.sub.2 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.
7TABLE III Influence of Pluripoietin on protein content In cultures
of human macrophages Adherent cell protein Adherent cell protein in
response to in response to Time in control medium Pluripoietin
culture .mu.g/coverslip .mu.g/coverslip Day 1-2 10.0 .+-. 2.0 28.6
.+-. 7.7 Day 1-3 20.4 .+-. 1.6 26.8 .+-. 2.5 Day 1-4 28.4 .+-. 1.6
41.2 .+-. 1.9 Day 4-5 28.8 .+-. 1.6 26.1 .+-. 3.6 Day 4-6 43.1 .+-.
4.7 28.1 .+-. 3.6 Day 4-7 38.2 .+-. 6.1 44.8 .+-. 0.7
[0110] Legend Table III
[0111] Normal human monocytes/macrophages were isolated from
peripheral blood mononuclear cells by adherence to glass
surfaces.sup.25. 2.times.10.sup.6 cells were plated per 13 mm
diameter coverslips in 0.1 ml of supplemented RPMI 1640 containing
25% fresh frozen human serum. After 2 hrs. at 37.degree. 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
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. *Lowry et al., J. Biol. Chem., Supra
8TABLE IVA Leukemia differentiating (GM-DF) activity of purified
Pluripoietin GM-CSF activity GM-DF Specific activity activity Ratio
GM-DF activity U/mg DF/ Ratio Purification protein U/ml U/ml CSF
U/ml DF/CSF I 1.5 .times. 10.sup.8 84,000 246,000 2.9 54,000 0.6 II
1.25 .times. 10.sup.8 201,000 502,000 2.5 80,000 0.4
[0112] Legend Table IVA
[0113] 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 105 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
Pluripoietin 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 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 3.times.10.sup.2/ml WEHI-3B(D+) or
10.sup.3/ml HL-60 leukemic cells in 0.31% agar in McCoy's medium
containing 12,5% FBS with serial dilutions of Pluripoietin.
Cultures were scored on day 7 (WEHI-3E) and day 14 (HL-60) for
induction of disperse, 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.
9TABLE IVB Glycoconjugate Synthesis HL-60 U937 Inducer CPM/5
.times. 10.sup.5 cells media 465 210 gIFN 500 U/ml 1029a 1500a 100
U/ml 800a 537a 50 U/ml 410 258 LK 50% 427 910a (500 U/ml gIFN) 5637
CM (GM-CSA) 2 kU/ml 1828a 1200a 1 kU/ml 980a 780a 500 U/ml 670a
490a Pluripoetin.sup.b 1 KU 4235a 2400a pp aCSF.sup.c 1 KU 430 306
SK-Hep CM 50% 1439a 604a GCT-CM 100% 420 200 PMA 3.0 ng/ml 490a 250
50.0 ng/ml 2000a 1700a aIFN 5000 U/ml 420 240 IL-2 100 U/ml 425
230
[0114] Glycoconjugate synthesis was measured as follows, cells
(5.times.10.sup.5) were incubated with inducers for 4 hrs. then
glucosamine incorporation was evaluated after an additional 16
hrs.
[0115] Results were mean values from three or more experiments
[0116] a. Significantly different from control, p less than 0.05 by
Students T test.
[0117] b. Human P-CSF, units assigned by CFU.sub.c activity.
[0118] c. partially purified aCSF-like activity, units assigned by
CFU.sub.c activity.
10TABLE V Biolog activities of purified human pluripoietin and
murine Interleukin 3. Activity Pluripoietin.sup.a) Interleukin
3.sup.b) Clonal growth of hemopoietic progenitors: CFU-GEMM + +
BFU-E + + CFU-G, M, GM + + CFU-EOS + + CFU-MEG n.t. + Pre-CFU-c
(.DELTA.GPA) + n.t. Stem cell multiplacation .sctn. +
(CFU-s).sup.c) Species crossreactivity + - Leukemia differentiating
activity (GM-DF) on: WEHI-3B (D+) + - HL60 + - .sup.3H-TdR uptake
in cell lines: KG1 + - FDC-P2 - + Murine mast cell lines + + (MCGF
activity) Histamine production n.t. + Protein synthesis of + n.t.
mature macrophages Induction of 20xSDH .sctn. + Growth of: natural
cytotoxic cells .sctn. + pre-B cell clones n.t. +
.sup.a)Pluripoietin was tested on human target cells, if not noted
otherwise. .sup.b)Interleukin 3 activity on murine target cells, if
not noted otherwise. Data derived from literature, except GM-DF and
activity on KG1. .sup.c)Activity on bone marrow derived colony
formation in agar cultures. .sctn. No human test system available
n.t. Not tested
* * * * *