Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN VASCULAR PERMFsABILITY FACTOR
Background of the Invention
This invention relates to a novel human
vascular permeability factor and its in vitro
production in a highly purified form from the human
histiocytic lymphoma cell line; U-937.
Vascular permeabilii~y factors (VPFs) are
proteins originally obtained from a variety of tumors
which cause a rapid and reversible increase in blood
vessel permeability when nanogram amounts are
injected under the skin of a warm blooded mammal.
VPF activity has been found in tumor ascites fluid
from guinea pigs, hamsters and mice and is secreted
by these tumors and a variety of tumor cell lines in
vitro according to Senger et al., Science 219,
983-985 (1983).
In U.S. Patent 4,45~~,550, a purified
VPF is described which has the following
characteristics:
(a) in an aqueous solution (0.01 M Na3P04,
pH 7) whose concentration of lVaC1 is varied linearly,
VPF is eluted from a heparin-~Sepharose chromatography
column in a peak centered at ~0.4 NaCl;
(b) in an aqueous solution of Na3P04, pH
7.0, whose concentration is varied linearly, VPF is
eluted from a hydroxylapatite column in a peak
centered at 0.25 M Na3P04; and
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(c) when subjected to SDS gel
electrophoresis in a polyacrylamide slab gel (0.375 M
tris-HC1, pH 8.8, 0.1% SDS) at 35 milliamps and 4°C.,
VPF is localized to a region .corresponding to a
molecular weight between 34,000 and 45,000 daltons.
VPF of the foregoing characteristics was
thus purified about 1800 fold from serum-free
conditioned medium of guinea pig tumor cell culture or
10,000 fold from ascites fluid by a series of steps
consisting of:
(a) affinity chromatography with a
column of heparin-*Sepharose~
(b) chromatography with a column
of hydroxylapaptite; and
(c) sodium dodecylsulfate/
polyacrylamide gel electrophoresis.
According to said patent, as :little as 200 ng (5 x
10 12 moles) of this purified VPF increased the
vascular permeability equivalE:nt to 1.25 Ng (4 x 10 9
moles) of histamine. Histamine is a standard
permeability mediator describf:d by Miles and Miles,
J. Physiol. 118, 228-257 (195:?). The VPF is said to
have therapeutic value insofar as it enables blood
nutrients to reach tissue with increased need for
nutrients, as in wound healin<~.
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According to Folkman and Klagsbrun, Science
235, 442-447 (1987), VPF causes leakage of proteins,
including fibrinogen, from blood vessels, thereby
initiating the formation of a fibrin gel which, in
turn, may play a role in angiogenesis. See also
Dvorak et al., J. Immunol. 1.?2(1), 166-174 (1979);
Dvorak, NEngl. J. Med. 315, 1650-1659 (1986);
Kadish et al., Tissue & Cell _11, 99 (1979); and
Dvorak et al., J. Natl: Cancer Inst. 62, 1459-1472
(1979).
. A method of stimu~Lating endothelial cell
growth is known which compr~.ses subjecting said
cells to a growth stimulating amount of a highly
Purified VPF. The highly purified VPF derived from
guinea pig tumor cells has t:he following
characteristics:
(a) it has a Mr about 34,000 - 40,000
as determined by sodium dodecyl-
sulfate polyacrylamide gel
electrophoresis (SDS/PAGE);
(b) it is a disulfide-linked protein
dimer;
(c) it has a N-terminal amino acid
sequence as.follows:
AlaProMetAlaGluGlyGluGlnLy;sProArgGluValValLys
PheMetAspValTyrLysArgSerTy:rCysArgProIleGluMet
31 35
LeuValAspIlePheGln; and
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(d) it exhibits substantial mitogenic
activity to endothelial cells in culture.
The foregoing highly purified guinea pig
VPF, also referred to as gVPF,, was isolated from
, serum-free conditioned culturE~ medium of guinea pig
tumor cells in a series of stf:ps comprising:
(a) affinity chromatography of said
conditioned culture medium with a
column of heparin-Sepharose CL-6B;
(b) cation exchange chromatography
of the VPF active fractions from
said affinity chromatography
with a TSK SP-5-~PW column;
(c) high performance: liquid
chromatography (HPLC) of the VPF
active fractions from said cation
exchange chromatography with a
Vydac C4 reversed phase HPLC column;
and
(d) HPLC of the VPF active fractions
from said C9 HPhC with a Vydac C18
reversed phase EIPLC column.
A method of producing antibodies against
gVPF is known in which certain peptide fragments
.25 of gVPF are used as immunogens .
Lobb et al., Int. J. Cancer 36, 473-478
(1985), describe a partially purified a VPF from a
human adenocarcinoma cell line: HT-29 having a
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molecular weight of 45,000. 'this VPF, however, does
not bind to immobilized heparin as does the VPF
derived from guinea pig tumor cell material by Senger
and Dvorak.
Senger et al., Cancer Res. 46, 5629-5632
(1986), describe the production of VPF from a variety
of human tumor cell lines, namely human osteogenic
sarcoma, bladder sarcoma, cervical carcinoma and
fibrosarcoma cell lines. However, none of these
human cell lines were found to be as active as the
guinea pig cell line 10 for the producton of VPF.
The finding of an improved human cell line
and method for the production of human VPF therefrom
in a highly purified form would have significant
advantages.
Brief Description o:f the Invention
The present inventors have investigated
numerous human cell lines for the production of VPF
by in vitro cell culture methods but most of them
have been eliminated as unsuitable candidates in view
of their relatively poor VPF production or failure to
produce VPF.
A cell line that ha;a unexpectedly been
found by the inventors to have good growth
characteristics in cell cultu:re and to be able to
elaborate the desired human V7?F in suitable quantities
is the human histiocytic lymphoma cell line U-937.
This cell line was originally established from cells
from the pleural effusion of a patient with diffuse
histiocytic lymphoma as reported by Sundstrom and
Nilsson, Int. J. Cancer 17, 565-577 (1976). These
cells are widely distributed as evidenced by
publications and are also readily available to the
public in an unrestricted culture deposit from the
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American Type Culture Collection, Rockville, Maryland,
under accession number ATCC fRL 1593. Further
background on these cells can. be had by reference to
J. Exp. Med. 143, 1528-1533 (1976); Nature 279,
328-331 (1979); and J. Immunol. 125, 463-465 (1980).
A recent report on the use of U-937 cells
to produce VPF-like activity was made by Beck and
Habicht, J. Leukocyte Biol. 42, 568 Absts., Dec.
1987. However, the activity was not purified and
chemical characterization or identity was not
disclosed.
The human VPF produced by U-937 cells
herein was identified by its inhibition and binding
by rabbit polyclonal antibodies to guinea pig VPF. A
rabbit polyclonal antiserum to guinea pig VPF
inhibited the permeability activity produced by U-937
cells as determined in the assay of Miles and Miles,
supra, (hereinafter also referred to as the Miles
assay). This U-937 generated VPF activity was about
70% to 80% removed by binding to immunoabsorbents
produced with protein A-Sepharose~ which had been
reacted with the rabbit polyclonal antiserum to guinea
pig VPF.
The process for the production of human VPF
in accordance with the invention comprises growing
cells derived from the human histiocytic lymphoma cell
line U-937 in serum-free nutrient culture medium at
about 35° to 38°C for a sufficient time to elaborate
VPF and isolating the resulting VPF from the spent
cells or the cell culture conditioned medium.
A preferred method of isolating the human
VPF from the cell culture conditioned medium of the
U-937 cells comprises the following steps:
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(a) cation exchange chromatography of said
conditoned cell. culture medium, for
example with a column of CM-cellulose,
CM-Sephadex~, Amberlite~ IR-120H or
S-Sepharose Fart Flow cation exchanger;
(b) metal affinity chromatography of the
VPF active fractions from said cation
exchange chromatography, for example
with a Cu2+, Zn2+ or Ni2+/iminodi-
acetic acid(IDA,)/Sepharose column; and
(c) reverse phase H:PLC of the active
VPF fractions from said method affinity
chromatography, for example with a C4
or C1$ reverse phase HPLC column.
The thus purified human VPF is a protein of
Mr 34,000-42,000. When subjected to N-terminal
amino-acid sequence analysis, it was found to have a
distinct and novel structure whereby it differed from
gVPF in four of the first ten amino acid positions.
This novel human VPF or hVPF has the following
N-terminal amino acid sequence:
1 5 10
Ala-Pro-Met-Ala-Glu-Gly-Gl:y-Gly-Gln- Asn
As described hereinafter, the hVPF of this
invention also contains several novel internal
sequences.
Purified hVPF of the present invention was
active in promoting vessel leakage at a dose of 22 ng
(5.5 x 10 13 Moles) upon intr,adermal injection into
guinea pigs. This highly purified hVPF thus is about
9 times more potent than the gVPF described in U.S.
G
F
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Patent 4,456,550. Another advantage of hVPF of this
invention is its human origin which is indicative of
potential use as a human therapeutic compared to other
agents of lesser purity or derived from guinea pig or
other animals such as would cause immunological
reactions.
Detailed Description of the Invention
While the specification concludes with
claims particularly pointing out and distinctly
claiming the subject matter regarded as forming the
present invention, it is believed that the invention
will be better understood from the following detailed
description of preferred embodiments of the invention
taken in conjunction with the accompanying drawings
in which:
FIG. 1 is a schematic representation which
shows the stepwise isolation of the human VPF (hVPF)
from the conditioned cell culture medium of U-937
cells in one embodiment of the invention.
FIG. 2 is a graphical representation which
shows the elution patterns in the stepwise
purification of the hVPF in four panels A, B, C and D
in the embodiment of FIG. 1 as follows:
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A.) Cation Exchange Chromatography of hVPF.
Serum-free conditioned medium from U-937 cells (6 L of
6-fold concentrate) was adjusted to pH 7.0 and loaded
at a flow rate of 60 ml/hr onto an S-Sepharose column
(5 x 45 cm) equilibrated in 0.01 M sodium phosphate,
pH 7Ø A linear gradient from 0.2 M to 0.8 M NaCl in
the same buffer was used to elute hVPF.
B.) Metal Affinity Chromatography of hVPF.
The active eluate from the cation exchange column was
concentrated to 20 ml by ultrafiltration and loaded
onto a Sepharose (Fast Flow)/IDA/Cu+2 column
equilibrated in 0.01 M sodium phosphate, pH 7.0, 2 M
sodium chloride, 0.5 M imidaz~ole. hVPF was
eluted with a linear gradient of imidazole as shown.
C.) RP-HPLC of hVPF. The active
eluate from the metal affinity column was loaded onto
a C1$ RP-HPLC column equilibrated with 0.05%
trifluoroacetic acid (TFA) in water and eluted at 1
ml/min with a gradient of acetonitrile as shown.
D.) Sodium Dodecyls~ulfate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE) of RP-HPLC Fractions.
Aliquots were removed from fractions around the
activity peak and analyzed by electrophoresis without
reducing agent. Standards were first reduced with
~-mercaptoethanol.
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FIG. 3 shows the ELISA of hVPF and
gVPF. Rabbit anti-gVPF IgG was used to coat the
polystyrene wells of microtiter plates. Various
amounts of either hVPF (lower panel) or gVPF
(upper panel) were allowed to bind overnight, and the
amount of bound antigen detected with
biotin-anti-gVPF IgG followed by HRP-avidin. Wells
were developed with HRP substrate, the absorbance was
read at 490 nm. Wells containing readings that were
off scale were diluted and re~-read in the linear
range of the Bio-Tek microplate reader; the
absorbances given are corrected for dilution.
Different x-axis scales were used for hVPF
and gVPF. Early and late bleed refers to both
primary and secondary biotin-conjugated IgGs prepared
from sera collected at the fourth bleed (after 3
immunizations) and at the ele~Tenth bleed (after 5
immunizations), respectively.
The U-937 cells can be cultured in
well-known cell culture media such as basal medium
Eagle's (BME), Dulbecco's modified Eagle medium
(DMEM), medium 199, RPMI 1640 medium, and the like
cell culture media such as described in detail by H.
J. Morton, In Vitro 6, 89-108 (1970). These
conventional culture media contain known amino acids,
mineral salts, vitamins, hormones and carbohydrates.
They are also frequently fortified with mammalian
sera such as fetal bovine sertun (FBS). Other
components which can be used p'.n the media are bovine
serum albumin (BSA), growth factors such as trans-
ferrin and insulin, protein h5rdrolysates such as
lactalbumin hydrolysate, trypt~one, tryptose and
peptone, as well as lipids, surfactants and the like
materials. The U-937 cells preferably are cultured in
serum-free media for the production of hVPF.
20 03361 ~'
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Methods for the large scale growth of
mammalian cells are well-known and these methods can
be used for the culture of the U-937 cells defined
herein. Such methods are described, for example, by
Tolbert et al., Biotech. Bioenq_ XXIV, 1671-1679
(1982); Tolbert and Feder, An.n. Rept. Ferm. Proc.
Vol. 6, Ch. 3, pp. 35-74 (1983); Harakas, Ibid. Vol.
7, Ch. 7, pp. 159-211 (1984); and references cited in
said publications. U.S. Pat. Nos. 4,166,768;
4,289,854; and 4,537,860 disclose particularly useful
methods and apparatus for the large scale growth and
maintenance of cells for the production of protein-
aceous materials. The methods and apparatus
disclosed therein can be usE;d for the culture
of the U-937 cells defined herein.
The cells also can be cultured on a large
scale basis in nutrient medium at 37°C. in agitated
suspension culture as described in U.S.
Pat. No. 4,289,854 and, after' a suitable growth
period, can be maintained in the static maintenance
reactor described in U.S. Pat.. No. 4,537,860 in which
the medium is supplemented with 0.5% lactalbumin
hydrolysate.
Although purification of the hVPF from the
spent culture media can employ various known
procedures for the separation. of proteins such as, for
example, salt and solvent fractionation, adsorption
with colloidal materials, gel filtration, ion exchange
chromatography, affinity chromatography, immuno-
affinity chromatography, electrophoresis and high
performance liquid chromatography (HPLC), the above
described three-step chromatographic method is
preferred. Suitable metal affinity chromatography
procedures are illustrated by Sulkowski, Trends
Biotech. 3, 1-7 (1985).
20 03361
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In a preferred embodiment of the invention
the U-937 cells are subcloned. or passaged through
nude mice to improve the yield of hVPF. Such
treatment of the cells has provided for the
production of hVPF in quantities of up to 200 to 800
ng/ml of the conditioned medium. This is equivalent
to or greater than the gVPF levels reported for the
guinea pig cell Line 10.
The U-937 cells have been passaged through
nude mice by injection of 1 x 10~ cells into the
mouse peritoneum. Nude mice are an immunodeficient
species in which human cells can grow and not be
rejected. After about 3 to 4 weeks, the mice were
sacrificed and soft tumors were removed from their
abdomens. The tumors were mechanically dissociated
into cells and these U-937 cells were again put into
culture.
The following examples will further
illustrate the invention although it will be
appreciated that the invention is not limited to these
specific examples or the details described therein.
Example 1
MATERIALS AND METHODS
Growth of U-937 Cells. U-937 cells
originally obtained from the i~merican Type Culture
Collection (ATCC) were subcloned in soft agar and
selected for fast growth. One of these clones
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was selected for scale-up, but other clones, and even
uncloned ATCC cells, also produced hVPF. The serum-
free medium used contained the following components:
RPMI 1640, DME (high glucose), Ham's F12 in a 1:1:1
ratio; HEPES (25 mM, pH 7.10-'7.15); glutathione (1
mM); ethanolamine (20 NM); selenium (30 nM); NaHC03 (2
mM); CuS04 (5 nM); NH4VO3 (5 ~aM}; ZnS04 (0.5 NM);
MnS04 (0.5 nM); FeS04 (4 NM); bovine serum albumin,
Miles "Pentex" (100 Ng/ml); iron rich transferrin,
Miles (5 ~g/ml); bovine insulin (10 ~g/ml); ExCyte,
Miles-lipid fraction (0.1% v/v); F-68 Pluracol~ (0.05%
w/v). The volume was adjusted to yield an osmolarity
of 280 mOsm. The doubling tune was about 50-60 hours
in this medium, whereas it was only 35-40 hours in
medium containing 2% fetal bovine serum (FBS).
Cells were scaled-up in the serum-free
medium from T-flasks into rol7.er bottles and then
into small spinners. A 12 L :>pinner was then used to
innoculate a 14 L perfusion chemostat which was
perfused at a rate of approximately 3 ml
medium/hour/ml of total packed cells. The culture
was subsequently transferred t:o a 100 L perfusion
chemostat, which was perfused under limiting nutrient
conditions (1.5-2.0 ml medium~'hour/ml packed cells, or
0.1-0.15 ml/day/million cells). Cells were recycled
to the reactor using an AG Tec:hnology hollow
fiber-cartridge (model CFP-4-E;-6, 0.4 micron). Cell
density ranged from 1.0 x 106 to 4.6 x 106 viable
cells/ml, 3.0 to 23 ml/L of packed cells, and
viability ranged from 64% to 84%. The production run
in the 100 L reactor lasted 24 days, during which
time a total of 1000 L of serum free conditioned
medium was produced.
The permeate from the perfusion reactor was
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collected and stored at 4°C. Concentration was
performed in 200-300 L lots on an Amicon DC-30
ultrafiltration apparatus with three low protein
binding 10 kDa cutoff spiral cartridges (Amicon
S10Y10) operated in parallel. A concentration of
about 6 fold was achieved, including a phosphate
buffered saline (PBS) wash of the cartridges and
equipment that was pooled with the concentrate. The
concentrate was stored at -20°C.
Vascular Permeability Assay. A Miles-type
permeability assay (Miles and Miles, supra) was used
to detect hVPF. Hairless guinea pigs (IAF/HA-H0,
Charles River, Wilmington MA) were anesthetized by
inhalation of methoxyflurane (Metofane~,
Pitman-Moore, Inc.). A 1 ml volume of 0.5% (w/v)
Evan's blue dye (Sigma Chemical Co.) prepared in
sterile saline for injection (Abbott Laboratories)
was injected intracardially into the circulation.
Samples for hVPF determination were prepared
at appropriate dilutions in saline or phosphate
buffered saline, and 200 ~1 volumes injected
intradermally into sites on the back of the guinea
pig. The presence of hVPF was indicated by an intense
blue spot at the site of the :injection where dye
(bound to serum protein) had :Leaked from the
circulation into the tissues.
Cation Exchange Chromatography. Six liters
of six-fold concentrated conditioned medium was
adjusted to pH 7.0 with acetic acid and passed
through a column (5 cm X 44 cm) of S Sepharose~ Fast
Flow (Pharmacia) cation exchange gel equilibrated
with 0.01 M sodium phosphate, pH 7Ø At 4°C a
significant portion of the permeability enhancing
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activity passed through the column, but at ambient
temperature (25°C) 50 to 70% of the activity became
bound to the column. This step was therefore
regularly performed at room temperature. Sodium
azide (0.01% w/v) was added to all buffers. Flow
rates for loading and elution were 10 ml per minute.
After loading, the column was washed with 900 ml of
0.01 M sodium phosphate, pH 7.0, and then eluted with
a 2.3 L linear gradient containing from 0.2 M to 0.8
M sodium chloride in the same buffer. Between runs,
the column was washed with 0.1. M sodium hydroxide
before re-equilibrating with 0.01 M sodium phosphate,
pH 7Ø
Metal Affinity Chromatography. Metal
affinity chromatography was performed using a
copper-iminodiacetic acid complex covalently linked
to agarose via a spacer arm. The gel was synthesized
in two steps by first adding a,n epoxide-containing
spacer arm to the agarose, and then reacting the
activated gel with iminodiacet:ic acid.
Highly cross-linked agarose (Sepharose Fast
Flow, Pharmacia) was repeatedly washed with distilled
water to remove all buffers and preservatives and
then dried by suction. About 100 g (100 ml) of this
damp gel was suspended in 60 mil distilled water, and
then 40 ml freshly prepared 2.5 M NaOH solution was
added to the gently stirring a.garose suspension.
Then 100 ml diethyleneglycol d.iglycidyl ether,
prepared as described by Gu et. al., Synthesis,
649-651 (1983), was added, and. the mixture was gently
stirred at 30°C for 16 hours. The activated gel was
repeatedly washed with distilled water to remove the
excess epoxide and base. The washed, suction-dried
gel contained 70 micromoles active epoxide groups per
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mL gel. The activated gel was stored in distilled
water at 4°C and generally used within 24 hours of
preparation.
About 100 ml (100 g) of-the activated
Sepharose Fast Flow was washed with distilled water,
dried by suction, and suspended in 100 ml of 1.0 M
Na2NH(CHZCOZ)-H20 solution, which was adjusted to pH
- 11Ø This mixture was gently stirred at 65°C for
24 hours and then repeatedly washed with distilled
water to remove excess ligand. The functionalized
gel was stored in ethanol/water (25/75 v/v) at 4°C
until ready for use. Titration with thiosulfate
showed the absence of epoxide groups, so capping with
ethanolamine was deemed unnecessary. To determine
the metal binding capacity of the gel, 10 ml of
suction-dried gel was saturate=d with excess 50 mM
Cu(Clo4)2 and then carefully washed with distilled
water. Finally, the bound copper was removed with an
excess of 50 mM Na2H2EDTA. Using standardized
copper-EDTA solutions for comparison, the copper
content was photometrically determined to be 43 micro-
moles Cu per milliliter of damp, suction-dried gel.
Chromatography was performed in a glass
column (1.0 x 13 cm.) containing 10 ml of gel. The
gel was charged with a 50 mM ~~olution of Cu(C104)2,
pH = 4.5, and then saturated with a buffered
imidazole solution (20 mM imidazole + 2 M NaCl + 50
mM NaH2P04, pH 7.0). After thoroughly washing with
1.0 M NaCl, the column was then equilibrated with the
starting buffer (50 mM NaH2P04, pH 7.0, 2 M NaCl, 0.5
mM imidazole). The fractions from the cation
exchange step containing permeability enhancing.
activity were pooled and concentrated from a volume
of about 700 ml to 20 ml using an *Amicon YM5
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membrane. Upon concentration, a protein precipitate
usually formed that could be removed by filtration.
The sample was applied to the column at ambient
temperature (25°C) and eluted at a flow rate of 0.5
ml/min over 500 minutes with .a linear gradient of
imidazole (0.5 mM to 60 mM) in 50 mM NaH2P04, pH 7.0,
2 M NaCl.
Reverse-Phase HPLC. Reverse-phase HPLC
(RP-HPLC) was performed using a 4.6 mm x 25 cm Vydac
column (Separations Group) containing 5 ~M packing
with 330 angstrom pore size. The mobile phases
were: "A", 0.05% trifluoroacE_tic acid (TFA) in water
and "B", 0.05% TFA in acetonii~rile. After loading
the sample, the column was washed with "A" until the
absorbance again reached baseline, and then eluted
with the following linear gradients: 0% to 20% "B"
over 20 minutes, 20% to 40% "B" over the next 80
minutes, and then 40% to 100% "B" over the next 20
minutes. All flow rates were 1 ml/min. Fractions
were collected in siliconized glass tubes.
N-Terminal Amino Acid Sequence Analysis.
Automated Edman degradation chemistry was used to
determine N-terminal amino acid sequence. An Applied
Biosystems, Inc., model 470A c~as phase sequencer
(Foster City, CA) was employed for the degradations
[Hunkapiller, et al, Methods E,nzymol. 91, 399-413
(1983}]. The respective PTH-amino acid derivatives
were identified by RP-HPLC analysis in an on-line
fashion employing an Applied E~iosystems, Inc., Model
120A PTH Analyzer fitted with a Brownlee 2.1 mm I.D.
PTH-C,Q column. Yields of PTH: amino acids wPrP
determined by comparison with an external standard
mixture. The average repetitive yield was calculated
~o033s~
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by linear regression analysis of the log pmolar yield
versus cycle number plot.
Similarities between the obtained sequence
and known sequences were investigated using the
computer program FAST A [Pearson and Lipman,
Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988)].
Similarity searches were also performed against the
National Biomedical Research ;Foundation (NBRF)
protein sequence data base [S:idney et al.,
Nucleic Acids Res. 16, 1869-1:371 (1988), Release 17,
June 1988] and the translated GENBANK DNA base
[Bilofsky and Burks, Nucleic Acids Res. _16, 1861-1864
(1988), Release 56, June 1988].
Tryptic peptides were prepared from reduced
and alkylated hVPF. hVPF (1 iunole) was dissolved in
100 ~1 of 0.5 M Tris HC1, pH 8.5, 6 M guanidine-HC1, 1
mM EDTA, and 5 mM dithiothreii:ol. The solution was
incubated for 30 minutes at 37°C before the addition
of sodium iodoacetate (to a final concentration of 5
mM) and incubation at 4°C overnight. After dialyzing
against 2 M guanidine HC1, O.C)1 M Tris-HC1, pH 8.5,
and then 0.1 M ammonium bicarbonate, 1 ~g of TPCK
treated trypsin (Sigma Chemical Co., St. Louis, MO)
was added and the solution incubated overnight at
37°C. Peptides were separatecl by RP-HPLC using a
°.
Nucleosil C18, 5 micron, 100 F,, 4.6 x 250 mm column
(Macherey-Nagel, Inc.). The f.'low rate was 1 ml/min at
room temperature. A linear gradient of 0 to 90%
acetonitrile in 0.1% trifluoro~acetic acid run over 270
minutes was used for elution.
Electrophoresis. Sodium dodecyl
sultate-polyacrylamide gel electrophoresis (SDS-PAGE)
was carried out on 10-15% gradient polyacrylamide
gels using a Pharmacia PhastSystem. The buffer
systems and the silver staining protocol were those
that were recommended by the manufacturer.
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Antibodies and Immunoassays. Rabbit
polyclonal antiserum to gVPF (designated F001) was
prepared by immunizing a New «ealand White (NZW)
rabbit with repeated injections of gVPF. The first
injection was in Complete Freund's Adjuvant followed
by boosts in Incomplete Freund's Adjuvant. gVPF was
purified by preparative SDS-PAGE [Senger et al,
Science 219 983-985 (1983)] following purification by
the method of Senger et al. _Fe~d. Proc. _46, 2102
(1987).
Sandwich enzyme linl~;ed immunosorbant
assays (ELISA) for human and guinea pig VPF were
performed as described below. The IgG fraction of
F001 antiserum was purified using adsorption onto
protein A-Sepharose. 500 ng/well of the IgG obtained
from an 11th bleed serum was coated onto polystyrene
microtiter plates for 3 hours. Then various known
concentrations of human and guinea pig VPF were
allowed to bind overnight. Th.e concentrations were
estimated from the maximal dilution which still
produced a detectable response in the Miles
permeability assay. This concentration was 50 ng/ml
of VPF. The amount of bound antigen was
detected with 500 ng/well biotin-a-gVPF IgG (11th
bleed F001 antiserum) for 2 hours followed by 1/2000
dilution of horseradish peroxidase (HRP)-avidin
(Cappel Labs) for 90 minutes. Wells were developed
with the HRP substrate o-phenylenediamine-2HC1 plus
H202 and the absorbance was read at 490 nm in a
BioTek reader.
It had been observed that the earlier
bleeds of rabbit F001 contained a higher
concentration and/or higher affinity of antibodies
cross-reactive with hVPF compared to later
2003361
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bleeds. Therefore, an ELISA for hVPF was also
performed with an early bleed (4th bleed) antiserum.
It was performed under the same conditions as
described above except that 1500 ng/well of a
biotin-F001 IgG (4th bleed) w<~s used to detect the
amount of bound antigen.
RESULTS
U-937 Permeability Enhancing Activity. The
serum-free conditioned medium from U-937 cells in
culture produced a positive response when tested in
the Miles permeability assay. These results were
unexpected since many cells do not produce a VPF-like
activity as seen from Example 2,~below. The Miles
assay measures extravassation of Evan's blue dye-serum
albumin complexes from the circulation after
intradermal injection of a te~;t material. However,
the assay is non-specific and could measure positive
response from a variety of substances, including, for
example, histamine. It was therefore not known
initially if the permeability enhancing activity
produced by U-937 cells was related to guinea pig
tumor VPF. To test this, the medium was mixed with an
immunoadsorbent composed of protein A-Sepharose~ and
IgG obtained from polyclonal anti-sera against gVPF.
Most, but not all, of the permeability enhancing
activity present in the U-937 medium was adsorbed
using this procedure, but not when control IgG
obtained from rabbits not immunized with gVPF was
used instead. Most of the permeability enhancing
activity secreted into the medium of U-937 cells
therefore appeared to be related to guinea pig tumor
derived VPF. However, as discussed below, even
though hVPF shares some immunocrossreactivity with
gVPF, it is immunologically distinct from gVPF.
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hVPF Purification. Initial
attempts at purification of U-937 cell derived
permeability enhancing activity employed the
purification method previously used for gVPF as
described by Senger et al, _Fe~d. Proc. 46, 2102
(1987). The application of this method, or minor
modifications thereof, did not produce homogeneous
protein from U-937 cell conditioned medium, even
though the chromatographic behaviour of the
permeability enhancing activity was very similar to
that of gVPF. A novel purification method was
therefore developed that incorporated cation exchange
chromatography, metal affinity chromatography, and
RP-HPLC-(Fig. 1). In the first step, concentrated
conditioned medium was passed over an S Sepharose
cation exchange column (Fig. :?A). About 50-70% of
the permeability enhancing aci~ivity was bound to the
column at pH 7Ø The non-adsorbed activity was not
characterized. The bound activity was eluted with a
gradient of sodium chloride, and after concentration
by ultrafiltration, loaded onto a metal affinity
column (Fig. 2B). All detectable activity was tightly
bound by the copper/IDA/Sepharose column, and was
eluted after most of the other proteins in a gradient
of imidazole. The final step utilized RP-HPLC (Fig.
2C) and resulted in elution of a group of
Mr ~40 kDa proteins in the fractions associated with
the peak of permeability enhancing activity (Fig.
2D). This method has been repeated numerous times
and it reproducibly yielded Mr. ~40 kDa protein of
about 90% or greater purity as analyzed by SDS-PAGE
with silver staining or by N-terminal sequence
analysis. Approximately 1-2 Ng of pure protein can
be obtained per liter of U-937 cell conditioned
medium.
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Dose Response of hVPF Induced
Permeability Enhancement. Different amounts of hVPF
were tested in the Miles permeability assay. The
lowest dilution producing a positive response was at a
hVPF concentration of about 2.75 nM. This corresponds to
an injection dose of 22 ng, or 0.55 picomoles of Mr 40
kDa hVPF. This is equivalent to only one-ninth the
200 ng required for a similar response by the gVPF
described in U.S. Patent 4,456,550.
Amino Acid Sequence of hVPF.
hVPF was subjected to N-terminal amino acid
sequence analysis (Table 1). Complete identity was
observed between hVPF and the guinea pig
tumor derived gVPF for the first 6 positions, but the
sequence diverged for the next 4 amino acids
sequenced. The identity is tlZUS only 60% in this
N-terminal region.
Table :L
2 0 Comparison of N-Terminal Sequences of hVPF and gVPF
Residue (pMole Yield)
Cycle hVPF gVPF
1 Ala (627) Ala (838)
2 5 2 Pro (427) Pro (598)
3 Met (406) Met (358)
4 Ala (130) Ala (463)
5 Glu (85) Glu (456)
6 Gly (55) Gly (434)
3 0 7 Gly (76) Glu (537)
8 Gly (150) Gln (276)
9 Gln (34) Lys (179)
10 Asn (29) Pro (354)
3 5 Approximately 1 nmole (about 40 Ng as estimated by
SDS-PAGE and silver staining) of hVPF was sequenced. The amino
acids detected and the yield at each cycle (in parentheses) are
shown. The average repetitive yield was 73%. These data are
representative of several other runs on similar hVPF preparations.
~0033fi1
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In order to obtain internal sequence
information, hVPF was reduced, carboxymethylated with
iodoacetic acid, and treated 'with trypsin. The
resulting peptides were then separated using RP-HPLC.
Several of the isolated peptides were sequenced, as
shown in Table 2. None of these sequences, nor the
N-terminal sequences, showed significant homology to
proteins present in published data bases. The novel
hVPF of the present invention thus is substantially
different from previously described proteins and from
gVPF.
Table :>_
Sequence of hVPF Tryptic Peptides
Peptide
(Gln)GlnGlnLys Pro
27 (Arg)GlnGluGln Arg(Pro Lys)
53 Phe AspVal Tyr Gln Arg(Arg)
Met
2 0 71 Ile LysPro Ser Cys Val F'ro Leu Met
Phe Arg
93 Val IlePhe G5n Glu Tyr F'ro Asp Glu
Asp Ile Glu Tyr
io
Reduced and carboxymethylated hVPF was treated with
trypsin and the peptides separated by RP-HPLC. Peptides that
were well isolated were sequenced. The number designation
corresponds to the fraction number in which the peptide appeared.
Residues in parentheses are designated with a fairly low degree
of confidence.
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Reactivity of hVPF ~W_ith Anti-gVPF
Antibodies. Polyclonal rabb it antibody against gVPF
was tested for cross-reactivity with hVPF
using a sandwith ELISA. Figure 3 shows that
. hVPF is recognized by anti-gVl?F IgG, but that
the concentration of hVPF required for
detection is about 10 fold higher than that for
gVPF. Furthermore, although the maximum absorbance
attained for hVPF (1.5 absorbance units above
background) was significant, it was about 3 fold
lower than that attained usinct gVPF as antigen.
These results imply that the t:wo proteins are
related, but that hVPF is immunologically distinct
from gVPF.
Example 2
hVPF derived from U-937 cells was compared
with VPF from several other cell lines by the Miles
assay with the following results. The Mnng HOS human
osteogenic sarcoma cell line was used as
representative of the prior art human cell lines for
production of VPF described by Senger et al., Cancer .
Res. 46, 5629-5632 (1986). The cells were extracted
at a density of 1.5 to 2.0 x 106 cells/ml for 48
hours in serum-free basal medium, either RPMI 1640 or
DME. The extract was collected, centrifuged to
remove cells and debris, and then concentrated if
necessary using *Centricon 10 membranes (Amicon
Corp.). The extracts were then tested in the Miles
assay. The results are set forth in Table 3.
*Trade-mark
20 0336 1
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Table 3
Cell Line Conditioned Miles Estimates
Media of
Concn.(a) Assay VPF Concn.
(units)(b) (ng/ml)
(c)
U-937(ATCC)
Uncloned 1X 1 50
U-937(ATCC) 1X 2-8 100-400
Cloned (d)
U-937(ATCC)
Uncloned-
Passaged through 1X 4-8 200-400
Nude mouse
Mnng HOS
Human osteogenic lOX +++ (e) 30 (e)
sarcoma
JURKAT
Human acute
lymphoblastic 12.3X 1-2 4-8
leukemia
F2.11-2X
Human T cell-T cell lOX 0 <5
hybridoma
2 5 HFF
Neonatal human 26.6X 0 <2
foreskin fibroblasts
1MR 90
Human fetal
3 0 lung fibroblasts 26.6X 5 10
HEK
Human embryonic 16.9X 4 10
kidney
8387-M15
3 5 Human sarcoma 36X >16 >20
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(a) Conditioned media were concentrated X-fold as indicated
prior to testing by the Miles assay.
(b) Activity of sample = greatest dilution of sample
at which blue spot was detectable.
1 unit (u) = the amount of VPF in a sample
producing the smallest detectable blue
spot discernible from control injections
without VPF.
(c) Estimate of VPF concentration in the
unconcentrated spent media. in ng/ml based on
1 unit activity= 50 ng/ml, a relationship
established with the guinea pig Line 10
VPF of U.S. Patent 4,456,550.
(d) Several clones, obtained by limiting
dilution cloning procedures, produced
activities in the 100-800 ng/ml range.
(e) This is an estimate based on the previously
determined activity of Mnng HOS cells, since
dilutions of the samples were not performed
in this assay.
Various other examples will be apparent to
the person skilled in the art .after reading the
present disclosure without departing from the spirit
and scope of the invention. It is intended that all
such examples be included within the scope of the
appended claims.
