Note: Descriptions are shown in the official language in which they were submitted.
2~72~
B~HRINGNERKE AKTIENGESELLSCHAFT HOE 90/B 019 - Ma 830
Dr. HaJsd
Description
Monoclonal antibodie~ again6t PP4, proce~ses for the
preparation thereof and the ~se thereof
The invention relates to monoclonal antibodies specifi-
cally directed against the protein PP4, to hybridoma cell
lines producing such antibodies, and to the use thereof.
The isolation and physicochemical characterization of
placental protein 4 (PP4) was carried out for the first
time by Bohn (EP 0123 307). This protein was also found
subsequently by other groups of re earchers, who did not
initially recognize its identity and called it annexin V,
lipocortin V or VAC alpha. Sequencing of the correspond-
ins cDNA (Grundmann et al. (1988) Proc. Natl. Acad. Sci.85, 3708-3712) showed that PP4 belongs to a family of
proteins which are now called lipocortins or annexins.
The annexins play a crucial regulatory part in the
release of mediators of inflammation. It is possible by
administering annexins to inhibit the release of media-
tors of inflammation.
The annexins are valuable therapeutics for coagulation
disturbances such as disseminated intravascular coagula-
tion tDIC).
Human placenta, which is available in adequate quantity,
represents a suitable source for obtaining PP4. The
established processes for purifying PP4 from natural
sources are, however, elaborate and associated with
losses of PP4. A similar statement applies to genetically
engineered PP4 which can be obtained in biologically
active form from cultures of E. coli or animal cells.
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There is thus a need for a rapid purification process
which is associated with minimal losses.
~he use of monoclonal antibodies for purification pur-
poses has already been described and established for some
other proteins. The conditions and the agents which can
be used to elute aga.in the protein which is bound to
immobilized monoclonal antibodies are crucial for the
yield and the native properties of the purified protein.
It is often possible to achieve dissociation of
monoclonal antibody and protein only by extreme pH values
or chaotropic agents, which frequently leads to denatura-
tion of the protein. This is why not any monoclonal
antibody is suitable for rapid and non-deleterious
purification.
The ob~ect of the invention was therefore to develop
monoclonal antibodies against PP4 which, when bound to
suitable supports, permit non-deleterious and efficient
purification of PP4 from various starting materials.
The invention relates to monoclonal antibodies aqainst
PP4, to a process for the preparation thereof and the use
thereof, preferably for the purification or diagnosis of
PP4.
Surprisingly, monoclonal antibodies which bind the
annexin PP4 only in the presence of divalent cations,
preferably calcium, have been found. Solutions which
do not contain calcium or contain chelating agents are able to
desorb PP4 in biologically active form and with high
yield and purity from the antibody. Such monoclonal
antibodies, bound to suitable support materials, are very
suitable for purifying PP4 from human cells or organs
as well as for the purification of a recombinant PP4
from cultures of E. coli or animal cells. Such antibodies
can likewise be used for diagnostic purposes.
As has been shown, PP4 can be regarded as a marker for
& ~ ~ ?
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certain diseases. Normally, PP4 is detectable only in
very low concentrations (O - 10 ng/ml) in human body
fluids such as, for example, plasma. The concentration of
this protein is significantly increased in certain
diseases, for example some tumors, and can be used as a
parameter for rapid diagnosis. This includes both the
guantitative determination of PP4 with the aid of mono-
clonal antibodies in body fluids, for example u~ing a
radioimmunoassay or enzyme immunoassay, and the qualita-
tive or quantitative determination on cell surfaces, in
tissue extracts or on histological specimens, ~uch as
tissue sections.
Antibodies suitable for these purposes can be prepared in
the following manner:
Obtaining antibody-producing cell~
Mammals, for example BALB/c mice, are immuni~ed by
subcutaneous injection of an emulsion of 50 ~g of PP4
(purified from human placenta as described in Example 1)
in complete Freund's adjuvant (day 1). On each of days 28
and 56, 50 ~g of PP4, emulsified in incomplete Freund~s
adjuvant, are likewise in~ected subcutaneously. This is
followed on day 92 by an intraperitoneal injection of
100 ~g of PP4 in 0.5 ml of physiological saline. On day
95 lym~hoc~tes are obtained by mechanical disintegration
of the s?leen.
Fu~ion of lymphocytes with ~yeloma cells
Hybridoma cells are obtained by standard processes
(Kohler and Milstein, 1975; Nature 256:495-497): for
example, the myeloma cell line SP2/0-Agl4 i8 cultivated
in Dulbecco's modified Eagle's medium (DMEM) containing
10% fetal calf serum (FCS). For a fusion to generate
hybridoma cells, typically the spleen cells from one
mouse (about 108j are mixed with 5 x 107 myeloma cells,
washed in serum-free DMEM and spun down. After the
supernatant has been completely r0moved, 0.5 ml of a 50~
stren~th solution of polyethylene glycol 4000 in ~1are
2 ~ 2 rJ
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added dropwise over the course of 1 minute to the cell
pellet. The suspension is incubated at 37C for 90
seconds and then diluted by addition of 7.5 ml of DMEM
over a period of 5 minutes. After incubation at room
temperature for 10 minutes, DMEM is added to make up to
40 ml, and the cells are spun down. After the supernatant
has been aspirated off, the cells are resuspended in DMEM
containing 20% FCS and dis~ed on 6 microtiter plates
(2nO ~1 per cavity). Hybridoma cells are selected by
adding 13.6 mg/ml ~ypoxanthine, 0.18 mg/ml aminopterin
and 3.9 mg/ml thymidine (HAT medium). The medium is
replaced by fresh one at intervals of 3 - 4 days and, after
10 days, HAT medium i8 replaced by HT medium.
Biotinylation of PP4
Biotinylated PP4 is required for the assay for PP4-
specific antibodies. This can be prepared in the follow-
ing way: PP4 is dialyzed against 0.1 M NaHC03, pH 8Ø To
the solution is added 0.1 mg of D-biotin N-hydroxy-
succinimide ester (0.1% strength solution in dLmethyl
sulfoxide) per milligram of protein. The reaction mixture
is incubated at room temperature for 4 h and then di-
alyzed against 0.01 M Na2HP04, 0.01 M NaH2P04, pH 7.2,
0.15 M NaCl.
Assay for PP4 antibodies
14 days after the fusion, the cell culture supernatants
of the fused cells are assayed for antibodies against PP4
using an enzyme immunoassay:
Polystyrene ~icrotest plates are incubated with
0.5 ~g/ml goat anti-mouse IgG in 0.1 M NaHC03, pH 9.6
(24 h, 4C). Subsequently, cell culture supernatants are
applied (2 h, 37C), followed by incubation with biotiny-
lated PP4 (1 ~g/ml). This is followed by incubation at
37C for 30 min with a complex of avidin and biotinylated
peroxidase (1 ~g/ml each). The substrate used is a
solution of 0.1% (weight/volume) 2,2'-azino-di-(3-ethyl-
benzothiazoline-6-sulfonate) and 0.012~ (volume/volume)
H202 in 0.1 M citric acid, 0.1 M Na2HP04, pH 4.5. After
~ 2.~J
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incubation at 37C for 30 min, the absorption at 405 nm
is measured. Washing between the individual incubation
steps was carried out with 0.01 M Na2HPO4, 0.01 N NaH2PO4,
pH 7.2, 0.15 M NaCl, 0.05% Tween 20 (PBS/Tween). A11 the
reagents are diluted in 0.02 M Tris/HCl, pH 7.4, 0.15 M
NaCl, 0.02 M CaClzr 2~ bovine serum albumin (TBS/BSA). The
enzyme immunoassay i8 carried out in parallel
using the same cell culture ~upernatants, but replacing
the calcium chloride in the dilution buffer by 0.02 M
EDTA. Antibodies which bind PP4 only in the presence of
CaCl2 bu~ not of EDTA are selected. These are particularly
suitable for purifying PP4 by immunoaffinity chroma-
tography.
Cloning of antibody-producing cell lines
Cell lines which ~how a positive reaction in the assay
for PP4 antihodies in the presence of EDTA but not in the
presence of calcium are cloned by the limitinq dilution
method. For this, about 60 cells in 20 ml DL~'~I contaLning 20~
FCS and 5~ human endothelial culture supernatant (Costar)
are distributed over the 96 cavities of a cellculture plate.
Single clones are identified under the microscope and
assayed for antibody production. The cloning i~ repeated
twice.
Purification of monoclonal antibodies
For the production of antibodies, clonal cell lines are
transferred into roller bottles and cultivated in
Iscove's modified Dulbecco's medium. Cell supernatant is
obtained by centrifugation and concentrated about 10
times by ultrafiltration. The concentrate is passed
through protein A-RSepharose CL-4B (Pharmacia), and bound
IgG is eluted -~ith 0.2 M glycine/HCl, pH 3Ø The
protein-containing fractions are dialyzed against 0.1 M
citrate, pH 6.5, and are concentrated to about 5 mg/ml by
ultrafiltration. This results, for example, in an anti-
body of the IgGl kappa subtype which, on isoelectric
focusing, appears in several bands in the pH range
6.2 - 6.5 and to which PP4 binds in the presence of
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calcium ions.
Suitable immunoaffinity gels can be prepared in the
following way: one of the monoclonal antibodies whose
preparation is described above is immobilized on a
suitable insoluble support material (for example agarose,
cellulose, polyacrylamide and its derivatives) by pro-
cesses familiar to the person skilled in the art (for
example ac ivation by cyanogen bromide, epoxides or
carbodiimides). In a preferred procedure, it is possible
for such monoclonal antibodies to be coupled to RSepharose
4B tPharmacia) which has been activated with cyanogen
bromide in accordance with the manufacturer~ 8 instruc-
tions. For example, 5 mg of antibody can be bound per
1 ml of gel. These gels can then be used for the immuno-
affinity chromatography of PP4.
The examples which follow illustrate the invention:
~xample 1
Purification of PP4 from human placenta and deteTminationof the biological acti~ity
36 kg of human placenta were comminuted, washed several
times with 0.15 M NaCl and subsequently lyophilized. The
lyophilisate (3 kg) was extracted with 50 l of 0.02 M
Tris/HCl, pH 7.5, 0.15 M NaCl, 0.1 M trisodium citrate,
mixed with ammonium sulfate (33% saturation) and incu-
bated with 4 l of phenyl-RSepharose in a batch process.
After the resin had been washed, PP4 was eluted all at
once with water, precipitated by adding ammonium sulfate
to 80% saturation, pelleted by centrifugation, taken up
in 0.02 M Tris/HCl, pH 8.0 (buffer A) and dialyzed
against the same buffer. After addition of CaCl2 to a
final concentration of 0.005 M, the dialysate was mixed
in a batch process with 1 l of a heparin-RSepharose and
stirred at room temperature for 60 min, and the super-
natant solution was separated off. The adsorbent was then
2~4~72 -~
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washed with buffer A with the addition of 0.005 M CaCl2.
PP4 was eluted by a linear gradient from 0 to 0.5 M NaCl
in calcium-containing buffer A. After dialysis of the
PP4-containing fractions, EDTA was added to a final
concentration of 0.001 M, and the dialysate was contacted
with 200 ml of a heparin-RSepharose equilibrated in buffer
A containing 0.001 M EDTA and stirred at room temperature
for 60 min, and the supernatant PP4-containing ffolution
was separated off~ If necessary, ~he PP4-containing
olution was contacted with a DEAE-RSepharose, and
the protein was eluted with a linear NaCl gradient. The
PP4 purified in this way appeared as a band with a
molecular weight of 33 kDa in S~S polyacrylamide gel
electrophoresis ~PAGE). PP4 showed no cross-reactions
with polyclonal antibodies against five other annexins.
The anticoagulant activity of PP4 was examined in a
modified prothrombin time test: 50 ~1 of citrated plasma
(standard human plasma) were mixed with 150 ~1 of a
solution of 0.05 M Tris/~CL, pH 7.5, 0.15 M NaCl, and
25 ~1 of buffer or sample and 25 ~1 of calcium-free
thromboplastin. This mixture was incubated at 37C for
3 min. Coagulation was initiated by adding 25 ~1 of a
solution of 0.02 M CaCl2. The coagulation time was deter-
mined using a Schnitger and Gross coagulometer. A calib-
ration cur~e was constructed using the PP4 purified from
placenta as described above.
Example 2
..
Puri~ication of PP4 from humun placenta by i unoaffinity
chromatography
Mature human placenta was washed, lyophilized and extrac-
ted with citrate buffer as described in Example 1. This
extract was dialyzed against 0.02 M Tris/HCl, pH 7.5. A
sample of the dialysate which contained 4.5 mg of PP4
(determined by the enzyme immunoassay described in
Example 4) was mixed with CaCl2 (final concentration
- 8 - 2~7~
0.02 M) and pumped through an affinity gel (20 mg of
monoclonal antibodies coupled to 4 ml of CNBr-activated
RSepharose 4B). Unbound protein was subsequently washed
off the gel with 100 ml of TBS/calcium tO.02 M Tris,
0.15 M NaCl, 0.02 M CaCl2, pH 7.4). The gel was then
washed with 30 ml of TBS without calcium, whereupon PP4
was eluted from the antibody. The yield of antigen and
activity from the immunoaffinity chromatography were 92
and 90% respectively. PP4 appeared as a band with a
molecular weight of 33 kDa in SDS PAGE.
E~ample 3
Purification of recombinant PP4 from ~. coli by immuno-
affinity chromatography
The cDNA coding for PP4, isolated and characterized from
a human placental gene bank, was cloned and used for the
expression in E. coli by means of the vector pTrc99A.
Recombinant PP4 (r-PP4) was expressed by fermentation of
the E. coli strain W31101acIQ. The E. coli was fermented
by procedures known to the person skilled in the art.
After completion of the fermentation, the cell~ were
harvested by centrifugation. The pellet was resuspended
in 0.02 M Tris/HCl, pH 7.5, and 0.01 M EDTA, and the
cells were lysed using a French press. Cell fragm0nts
were removed by centrifugation, and ~he supernatant was
2S then purified by filtration through a 0.45 ~m filter. ~he
r-PP4-containing 601ution wa~ mixed with RTriton X-100 and
dialyzed against a buffer of 0.02 M Tris/HCl, pH 7.5. A
sample of the dialysate which contained 3.8 mg of PP4
(determined by the enzyme immunoassay described in
Example 4) was mixed with CaCl2 (final concentration
0.02 M), the resulting precipitate was spun down, and the
supernatant was purified by immunoaffinity chromatography
as described in Example 2. The yield of PP4 antigen and
its activity (determined by the enzyme immunoassay
described in Example 4 and by the activity assay desc-
ribed in Example 1 respectively) were 94 and 93%
- 9 2~ 2~
respectively. The protein appeared homogeneous with a
molecular weight of 33 kDain an SDS gel.
Example 4
San~wich ~LISA for quantification of PP4 antigen
A sandwich ELISA was developed for the quantification of
PP4 in biological fluids:
Microtiter plates were coated with polyclonal antibodies
against PP4 (1 - 20 ~g/ml). The PP4-containing sample
(for example placental ti~sue extract, E. coli lysates or
plasma) was incubated on the antibody-coated plate3.
Incubation was subsequently carried out with a biotiny-
lated monoclonal antibody (2 ~g/ml) followed by a complex
of avidin and biotinylated peroxidase. The procedures for
the enzyme Lmmunoassay and the detection of the peroxi-
dase activity wera as described under "Assay for PP4antibodies". A calibration line was constructed using the
PP4 purified from placenta as described in Example 1. It
was possible to determine PP4 in a concentration range
between 125 ng/ml and 2 ng/ml (Table 1).
Table 1
PP4 concentration IAborption at 405 nm
(ng/ml)
125 11O601
62.5 11.393
31.25 11.067
15.63 10.812
7.82
3.91 10.298
1.96 10.076
