Note: Descriptions are shown in the official language in which they were submitted.
:
WO94/04178 ~ 4 1 ~ 9 8 PCT/US93/07582
~T~OD OF IN~IBITING C~L~ PRO~IFERATION
V8ING APOLIPOPROT~IN ~
.~
B~o~.ou~d of th~ Invention
This invention relates to the use of Apolipprotein E in a
method of inhibiting ceil proliferation.
Throughout this specification, various publications are
referenced within parentheses. Full citations for these
references may be found at the end of the specification
immediately prece~ing the claims. The disclosures of these
publications in their entireties are hereby incorporated by
reference into this applciation in order to more fully
describe the state of the art as known to those skilled
therein as of the date of the invention disclosed and
claimed herein.
In normal tissue, cell growth and DNA synthesis are closely
co,.Ltolled by a variety of regulatory factors operating on
both positive and negative levels (Weinberg et al., 1989).
As a normal cell develops into a solid tumor, it undergoes
several changes (Folkman, 1989; Liotta, 1992). At the
physiologic level, growth is stimulated, ;~ n;ty is
decreased, and new blood vessel formation is induced. This
capacity to induce new blood vessels, i.e., angiogenesis and
neovA-clllArization, is characteristic of most malignant
cells and is a prerequisite of solid tumor growth (D'Amore,
1988; Folkman, 1976). Moreover, new blood vessels
Y penetrating the tumor are frequent sites for tumor cell
entry into the circulation. Angiogenesis is also n~CcAry
for expansion of a metastatic colony (Aznavoorian, 1991).
In addition to malignant cell growth, other diseases are
WO94/04178 ~4~598 PCT/US93/07582
also characterized by a~normal neovascularization, including
neovascular glaucoma, diabetic retinopathy, and rheumatoid
arthritis (Folkman, et al., 1989).
Malignant cells produce many factors that stimulate
endothelial cell proliferation and migration and allow new
capillary beds to form within the tumor nodule (D'Amore,
1988; Shing, et al., 1985). A variety of agents have been
suggested as potential modulators of cell proliferation,
including heparin and heparin sulfate (Castellot, et al.,
1987; Clowes, 1977), and growth factors and their inhibitors
(Edelman, et al., 1992; Liu, 1990; Schweigerer, et al.,
1987). Most of these factors also appear to be natural
components of normal tissue. To date, the most studied
growth factor has been basic fibroblast growth factor
(bFGF), a member of the fibroblast growth factor family
(Basilico and Moscatelli, 1992; Schweigerer, et al., 1987;
Thomas and Gimenez-Gallego, 1986). bFGF is a ~LL~1~Y
heparin-binding molecule, present in virtually all t; ~?-~C
and having multiple mitogenic and angiogenic effects (Thomas
and Gimenez-Gallego, 1986). In vascular endothelial cells,
bFGF stimulates a number of functions involved in the
formation of blood vessels and angiogenesis. bFGF is
considered to be one of the most potent angiogenesis
inducers both in vivo and in vitro (Folkman, 1976; Folkman
and Klagsbrun, 1987). Recently it was shown that
intravenous infusion of bFGF stimulated endothelial
regeneration and SMC proliferation (Edelman, et al., 1992;
Ti~ner and Reidy, 1991; Lindner, et al., 1990) after
balloon-induced endothelial denudation. In these studies,
it was confirmed that bFGF was both angiogenic and mitogenic
for SMC in vivo and also demonstrated that these two effects
are coupled. bFGF also binds to heparan sulfate
proteoglycans (HSPG) of both the extracellular matrix (ECM)
and basement membrane (Folkman, et al., 1988 ; Vlodavsky, et
~ WO94/04178 2 1 ~ 1 ~ 9 8 PCT/US93/07582
al., 1987). Thus, it has been postulated that bFGF may play
an important role in the pathogenesis of atherosclerotic
vascular disease.
.
The role of HSPG in regulating cell growth and
differentiation has been described (Burgess and ~si Ag,
1989; Klagsbrun and Baird, 1991; Ruoslahti and Yamaguchi,
1991). Many proteoglycans are constituents of the ECM or
function as a low-affinity cell surface receptor for the
interaction of growth factors, including bFGF and other
heparin-binding growth factor molecules. The role of HSPG
as binders of bFGF appears to protect bFGF from degradation,
and is important in providing a matrix or cell surface bound
reservoir of bFGF. Yayon, et al. (1991) have shown that
bFGF b;n~ing to its high-affinity receptor requires prior
binding either to the heparan sulfate side ~hAi~ of a
membrane ~SPG or to free heparan sulfate (heparin) ~h~n~
and speculate that glycosaminoglycans may change the
conformation of bFGF so that it acquires the ability to bind
to its receptor. Binding of growth factors to proteoglycans
have also been observed with several other growth factors
that bind to heparin or heparan sulfate (Ruoslahti and
Yamaguchi, 1991). Because proteoglycans are abundant and
ubiquitous tissue components, they are likely to attract
most of these growth factors and cytokines that have
affinity for the glycosaminoglycan. It may be that growth
factors and cytokines were meant to act on their target
cells only over a short range, and that their immobilization
at the cell surface and in ECM (through proteoglycan
binding) accomplishes that goal (Ruoslahti and Yamaguchi,
1991) -
Apolipoprotein E (ApoE) is a plasma protein having strongaffinity for heparin and HSPG (Cardin, et al., 1988; Mahley,
3~ 1988; Mahley, et al., 1979; Weisgraber, et al., 1986). ApoE
WO94/04178 PCT/US93/07582
2 1 ~ 4_
participates in plasma li~o~LoLein metabolism through ~ts
high-affinity interaction with cell surface receptors
including the low-density lipoprotein (LDL) and the more
recently identified apoE receptor, LDL receptor-related
protein (LRP) (Hertz, et al., 1988; Lund, et al., 1989;
Yamada, et al., 1989; 1992). The domain of apoE responsible
for b;n~ to the LDL receptor has been identified (Dyer
and Curtiss, 1991; Wilson, 1991). This ~nm~;n is a 20-amino
acid region comprised of residues 140-160 of the apoE
molecule. It has long been known that binding of ApoE to
the LDL receptor depended on its association ~ith lipids
(Innerarity, 1979). However, from results with synthetic
peptides binding to the LDL receptor ;n vitro, one can
assume there is direct b; n~; n~ of the peptide to the LDL
receptor, or one could speculate that the LDL receptor is
not the only b; n~ i ng site on the cell.
It has been shown that intravenous ~; n; ~tration of ApoE
into hyperlipidemic rabbits resulted in reduced plasma
cholesterol levels (Mahley, et al., 1979; Yamada, et al.,
1989)- Recently (Yamada, et al., 1992), following a
sus~ e~ intravenous ~m; n;~tration of ApoE into WatAn~he
heritable hyperlipidemic rabbits, progression of
atherosclerosis was significantly prevented. ~m;~;~tration
of exogenous ApoE had affected both the number and size of
atherosclerotic lesions in the aorta. However, in these
experiments there was no significant difference in plasma
cholesterol levels between ApoE-treated and nontreated
control animals. Based on these and other experiments, the
effect of ApoE on atherogenesis might neither be solely nor
directly related to plasma cholesterol levels.
ApoE is ubiquitously synthesized in many tissues including
liver, intestine, adrenal gland, kidney, lung, spleen,
testes, ovary, and brain (Mahley, 1988). Recently, it has
WO94/04178 2 1 ~ 1 ~ 9 8 PCT/US93/07582
been found in both inflamed and non-inflamed synovial fluid
(Terkeltaub, et al., 1991). ApoE can function in tissue
repair by ~o~ ting lipid redistribution locally (Hui, et
al., 1980; Mahley, 1988). However, it has also been
observed that apoE is synthesized and secreted by a number
of cells that do not nec~c~Arily participate in cholesterol
homeostasis (Boyles, et al. 1989; Hui, et al., 1980).
In addition to its effect on lipoprotein metabolism, apoE
also possesses a variety of functions that are unrelated to
lipid transport (Mahley, 1988). A potent ~ ession of
lymphocyte activation by mitogens and antigens by ApoE-
bearing lipoproteins and ApoE polypeptides has been observed
(Cardin, et al. 1988; Hui, et al., 1980). The present
invention discloses the effect of ApoE on proliferation and
migration of several cell types.
WO94/0417~ PCT/US93/075X2
98 -6-
~ummarY of th~ Inve~t~on
A method of inhibiting actively proliferating cells is
disclosed. This method comprises contacting the cells with
Apoli~o~lo~ein E (ApoE) in an amount effective to inhibit
cell proliferation.
A composition is provided comprising Apolipoprotein E in an
&~ L effective to inhibit cell proliferation.
The invention additionally provides a method of treating a
subject suffering from excessive cell proliferation which
comprises contacting the excessively proliferating cells
with an effective amount of Apolipoprotein E so as to
inhibit the ~c~c-sive cell proliferation.
Further, the present invention provides a method of treating
a subject afflicted with a tumor which comprises contacting
the tumor with an effective amount of an Apolipoprotein E in
conjunction with a chemotherapeutic agent so as to inhibit
proliferation of the tumor cells.
Additionally, the present invention provides a method of
treating a subject afflicted with a tumor which comprises
contacting the tumor with an effective amount of
Apolipoprotein E in conjunction with an amount of
irradiation so as to inhibit proliferation of the tumor
cells.
Additionally, the invention provides a method of treating a
subject suffering from a disorder involving increased
neovascularization. This method comprises administering to
the subject an amount of Apolipoprotein E effective to
normalize neovascularization.
.
W0 94/04178 2 1 4 1 5 9 8 PCr/US93/07582
Brief DQscriPtion of the Fi~ures
Pigure 1: The effect of heparin binding molecules on
the incorporation of [3H]thymidine into bovine aortic
endo~h~ l cell (BAEC) DNA was tested as described in
Examples 1 and 2 using protocol M2, either in the presence
of fetal calf serum (FCS) (1% and 2.5%) alone tcontrol
(ctr)], or with added basic fibroblast growth factor (bFGF).
(All other samples were tested using 10 ng/ml). ~hen
indicated, 0.05 or 0.5 ~M of met-apolipoprotein E (+E.05 or
+E0.5, respectively) or recombinant thrombospondin (rTSP) 18
Kd (~T.05 or +T.5) were added to the wells containing FCS
and bFGF. The mitogenesis assay was terminated after 42 hr.
Figure 2: The time course of the incorporation of
[3H]thymidine into BAEC DNA was obt~ine~. DNA synthesis was
tested on a newly-attached cell culture according to
mitogenesis protocol M2 (see Example l-Methods). Cells were
plated in Dl~lh~cco' 5 modified Eagles's medium (DMEM)-
containing bFGF (10 ng/ml) and 1% FCS alone (ctr) ortogether with 0.5 ~M of the indicated molecules [(rTSP) 18
Kd, recombinant fibronectin (rFN) 33Kd, or met-apoE].
Figure 3: Incorporation Of t3H]thymidine into pre-
attached BAEC culture was tested according to protocol Ml,at either 0.5~ FCS alone or together with bFGF (10 ng/ml).
Met-apoE at the indicated concentrations was added to the
cells 5 days after plating, and t3H]thymidine incorporation
was tested the next day after pulsing the cells for 5 hr and
as indicated in protocol Ml.
F~gure 4: Incorporation of t3H]thymidine into a pre-
attached, dense culture of BAEC was studied in a preattached
culture. Mitogenesis was tested according to protocol M1,
3~ using 0% or 0.5% FCS (0 and .5, respectively), together with
W094/04178 ~ PCT/US93/07582
2141~98 -8-
bFGF, 10 ng/ml (OF and .5F, respectively), or in combination
with 0.5 ~M met-apoE (OFE and .SFE, respectively). The
mitogenesis assay was terminated after 40 hr.
Figure 5: Incorporation of [3H]thymidine into BAEC
culture was tested in the presence of bFGF (10 ng/ml) and
one of two concentrations of FCS (as indicated in the
figure) either alone (control), or together with met-apoE
t0-5 ~M), added to the newly plated culture at time zero
(designated as 0-40), or at 15 hr or 22 hr after plating
(designated as 15-40 and 22-40, respectively).
t3H]thymidine was also added at time zero to all wells as
indicated in protocol M2, and the assay was terminated after
40 hr. Incorporation is expressed as a percent of ~G~.L.ol
labelled with t3H]thymidine for the same time in the Ah~en~e
of ApoE.
Figure 6: The effect of bFGF on inhibition by ApoE was
studied. The proliferation assay was performed with culture
of BAEC-1, as indicated in protocol P1, in the presence of
5% FCS either alone (ctr) or together with 20 ng/ml of bFGF.
Where indicated, various concentrations of met-apoE were
added 1 day after cell plating. The cell number was
monitored as indicated in protocol P1.
Figure 7: Incorporation of [3H]thymidine into bovine
corneal endothelial cell (CBEC) culture was studied using
various concentrations of FCS (ctr) as indicated (0%, 1%, or
2%). To this, growth factors were added either alone (bFGF
[10 ng/ml] or EGF [50 ng/ml]), or together with met-apoE (E,
0.5 ~M) or FN 33 Kd (FN, 0.5 ~M). The mitogenesis assay was
performed as described in protocol M2, and terminated after
44 hr.
2~598
WO 94/04178 PCI`/US93/07582
e
_9_
Figure 8: The proliferation assay was performed with a
culture of CBEC as indicated in protocol P2, in the pr~rAn~e
of FCS and bFGF alone (control) at 5% and lO ng/ml,
respectively. When indicated, various concentrations of
either met-apoE or FN 33 (rFN 33 Kd) were added to the cells
at time zero. The average absorbancy of triplicate samples
was calculated and expressed as percent of control.
.
Figur~ 9: The reversibility of ApoE inhibition was
studied. CBEC culture in 1% FCS and 10 ng/ml bFGF was
tested for [3H]thymidine incorporation after 40 hr of
labeling using protocol M2 (control). To parallel cultures,
met-apoE was added at time zero at the indicated
concentrations and either left for the entire lAh~lin~
period (0-40) or for only 22 hr (0-22). For all tested
cultures, media was replaced after 22 hours with the
appropriate combination, including the starting
concentrations of FCS, bFGF, and t3H]thymidine.
Figure lo: Incorporation of t3H]thymidine into culture
of A2058 human melanoma cells was tested as described in
protocol M2, and in the presence of 0.5% FCS and bFGF (10
ng/ml) either alone (ctr) or together with 0.5 ~M and 1.5 ~M
met-apoE (0.5E and 1.5E, respectively), 0.5 ~M TSP 18 Kd
(0.5T18), 0.5 ~M TSP 28 Kd (0.5T28), or 0.5 ~M FN 33 Kd
(0.5FN33). The assay was terminated after 32 hr.
Figure 11: Incorporation of [3H~thymidine into a culture
of h~ n mammary tumor (MDA-435) cells was carried out
according to protocol M2, and in the presence of 0.5% FCS
and bFGF (10 ng/ml) alone (ctr) or with the addition of 0.5
~M met-apoE (E) or TSP 18Kd or with 75 ~g/ml heparin (Hep).
The assay was terminated after 42 hr.
WO94/04178 ~ PCT/US93/07582
214159~ -10-
Figure 12: Proliferation of smooth muscle cells (SMC)
was performed as indicated in protocol P1, in the presence
of either 0.5% or 5% FCS alone (ctr), or combined with bFGF
(~F, 20 ng/ml). Where indicated, met-apoE (E) (4 ~M) was
added to the culture at time zero.
Figure 13: The incorporation of 35S-methionine was
e~m;ned as published (D. Blake 1990) with a slight
modification. Briefly, CBEC cells were plated into 6-well
tissue culture dishes in DMEM + 10% FCS at 105 cells/well.
After 24 hr, cells were washed 3 times with PBS and the
media was changed to DMEM minus methionine (for methionine
depletion) either alone (ctr) or together with met-apoE (0.5
~m). After 1 hr, 0.25 ~Ci [35S]methionine (1268 Ci/mmol,
Amersham) was added, and the culture was incubated at
37C/5~ C02 for 6 hr. Cells were washed twice with
phosphate buffered saline (PBS) contAining 10 mM EDTA and 1
mM phenyl-methyl-sulfonyl and then suspended in the same
buffer. The cells were lysed with 20 mM ammonium hydroxide
(NH40H), then aliquots of the cell lysate were precipitated
with 5 volumes of 20% trichloracetic acid (TCA). After 10
minutes in ice the TCA solution was filtered on glass
microfiber filters (GF/C, Whatman), then the filters were
washed 3 times with 5% cold TCA and 1 time with 70% ethanol.
The radioactivity of the TCA-insoluble [35S]methionine-
labeled protein was monitored by ~ counts.
Figur~ Chemotaxis of BAEC to bFGF was carried out in
a modified Boyden chamber as previously described
tTaraboletti, 1990) using 5 ~m pore size polycarbonate PVP-
free nucleopore filters. Semiconfluent cells were
trypsinized, washed with 10~ FCS, allowed to equilibrate in
10% FCS-DMEM for 2 hours at room temperature while shaking,
and then pelleted, and resuspended in medium containing 0.1%
BSA. Cells were used ;~iately at a concentration of
~ WO94/04178 2 1 ~ ~ ~ 9 8 PCT/US93/07~82
106/ml and c~ecke~ for migration either alone or together
with the indicated concentration of met-apoE (E) toward a
gradient of 0.1% BSA alone (ctr) or together with bFGF (33
ng/ml). Migrated cells in triplicate samples were monitored
after 4 . 5 hr of incubation at 37C, and are expressed as a
percent of the migration toward 0.1% BSA (ctr).
Figure ~5: This figure shows plasmid pTVR 590-4 which
was deposited in E. coli W1485 under ATCC Acceccion No.
67360. Plasmid pTVR 590-4 is a good expressor of met-apoE
under control of the APL promoter as is described in Example
1. (~. coli W1485 is freely available from ATCC under
A~C ssion No. 12435. )
F~gure lC: The construction of plasmid pE2-5 encoding
ApoE cont~;n;ng amino acids 1-217 is described. Plasmid pTV
194-80 (disclosed in coassigned U.S. Patent No. 5,126,252,
Figure 22) was digested with restriction enzymes BssHII and
BglII. The large fragment was isolated and ligated to the
synthetic linkers shown in the Figure. The resulting
plasmid was designated pE2-5.
Figure 17: The construction of plasmid pTVR6-2
expressing ApoE having amino acids 1-217 is described. A
1200bp fragment containing the A CI gene under control of a
portion of the deoP1 promotor sequence was isolated from
ClaI digestion of plasmid pFSAL-B27 (ATCC Accession No.
67071; also disclosed in European Patent Application
Publication No. 303,972). The 1200bp fragment was then
ligated to ClaI digested plasmid pE2-5 (Figure 16). The
resulting plasmid, designated pTVR6-2, contains both the
ApoE structural gene and the A CI repressor gene, and is
therefore an independent plasmid, not limited to use in a
host containing the A CI repressor but able to express the
ApoE polypeptide fragment in a wide variety of hosts. It is
WO94/04178 2 ~ 41~ 9 8 PCT/US93/07582 ~
-12-
not presently known whether an additional N-terminal
methionine is present. Plasmid pTVR6-2 was deposited in
~oli 4300 with the American Type Culture Collection, 12301
Parklawn Drive, Rockville, Maryland on July 26, 1993 under
Accession No. 69364.
Figure 18: The effect of ApoE on proliferation of mouse
endothelioma-END2 cells in culture was studied. The
proliferation assay of END2 cells was performed as Example
7 using 20,000 cells per well and in the presence of 0.5%
FCS, and the indicated concentrations of met-apoE.
Figure 19: The effect of ApoE on the proliferation of
BAEC and C~0 cells was studied. The proliferation of BAEC
and CH0 cells performed with 30,000 cells/ml in the
presence of 0.5% FCS and the indicated concentrations of
met-apoE as described in Example 2.
Figure 20: The effect of ApoE on the proliferation of
BAEC and neuroblastoma cells was studied. The proliferation
of BAEC and neuroblastoma N18TG2 was performed with 20,000
cells per ml in the presence of 0.5% FCS and the indicated
concentrations of met-apoE (20A) and the ApoE-peptide 348
(20B) as described in Example 2.
Figuse 21: The heat-stability of the anti-proliferative
activity of ApoE was ~Y~mined. The proliferation of BAEC
cells (30,000 cells/ml) was tested in the presence of 0.5%
FCS and the indicated concentrations of unheated or heated
(100C for lhr) met-apoE as described in Example 2.
Figure 22: The effect of serum concentration on the
anti-proliferative activity of met-apoE was studied. The
proliferation of BAEC cells was measured at 0.5% and 2.5%
FCS and in the presence of the indicated concentrations of
WO94/04178 2141~g 8 PCT/US93/07582
-13-
ApoE without the addition of exogenous growth factors. Met-
apoE exhibits a lower degree of inhibition at the higher
serum concentration as described in Example 2.
S Figure 23: The effect of the ApoE designated apoE6-2 on
the proliferation of END-2 cells was studied. The
proliferation of END-2 was measured as indicated in Figure
18 in the pre~enc~ of O.5% FCS and the indicated amounts of
the ApoE polypeptide fragment encoded by plasmid pTVR6-2
(Figure 17) as described in Example 8.
Figure 24: The effect of serum concentration on the
anti- proliferative activity of an ApoE polypeptide was
studied. The proliferation of BAEC cells was measured at
0.5% and 2.5% FCS and the indicated concentrations of the
ApoE polypeptide apoE6-2 as described in Example 8.
Figure 25: The reversal of heparin activity by ApoE was
studied. ApoE has a high affinity for heparin, and this
property of ApoE was studied by the effect of met-apoE on a
complex consisting of heparin, antithrombin III and
thrombin. Addition of ApoE to the complex reverses the
heparin acti~ity resulting in inhibition of the antithrombin
activity of heparin. A nonreactive short peptide designated
peptide 185 was used as a negative control. A second
negative control [ctrl (-)] did not include any ApoE. A
positive control [ctrl (+)] did not contain heparin. The
results indicate that at the ApoE concentration at which
there is 50~ inhibition of thrombin acrtivity, there is
about a 3:1 ratio of heparin:ApoE, i.e. 2-3 molecules of
heparin are bound by each molecule of ApoE.
; ~ ~
WO94/04178 214 l S 9 8 PCT/US93/07S82
-14-
~etailed Description of the Invention
The present invention provides a method of inhibiting
actively proliferating cells comprising contacting actively
proliferating cells with an amount of Apolipoprotein E
(ApoE) effective to inhibit proliferation. Inhibition of
proliferation means reduction of the rate of proliferation
of the cells.
~he cells may be smooth muscle cells, endothelial cells e.g.
aortic or corneal endothelial cells, or tumor cells, e.g.
human melanoma cells, m~ ry tumor cells, human sarcoma
cells, or carcinoma cells. Other actively proliferating cell
types known to those skilled in the art are also encomp~
by the methods of the invention.
A composition for inhibiting the proliferation of actively
proliferating cells comprising Apolipoprotein E and a
suitable carrier is also provided.
Additionally, a method is provided of treating a subject
suffering from excessive cell proliferation which comprises
~min;~tering to the subject a amount of Apolipoprotein E
effective to inhibit the ~Yc~sive cell proliferation. Such
a method may involve a~inictration of the Apolipoprotein E
in conjunction with other therapeutic means such as a
chemotherapeutic agent or irradiation treatment, i.e.
a~;nistration of ApoE prior to, during or after the other
therapeutic means. Other therapeutic means for use in
conjunction with Apolipoprotein E are known to those skilled
in the art and are also encompassed by the methods of the
invention.
A pharmaceutical composition is provided which comprises
Apolipoprotein E in an amount effective to inhibit excessive
WO94/04178 2l 41~ 8 PCT/US93/07582
proliferation and a pharmaceutically acceptable carrier.
In one embodiment, e~ce~sive cell proliferation is a tumor.
The term "Apolipoprotein E" (ApoE) as used herein
encompA~ses any polypeptide, regardless of source e.g.
naturally occurring or recombinant which includes the
sequence of naturally occurring apoE necec~ry for the
biological activity of inhibiting proliferation of cells,
and mutants whose sequence varies by one or more, typically
less than ten amino acids provided that such mutants have
the biological activity of inhibiting proliferation of
cells.
Naturally occurring apoE may be obtained from plasma or
serum by methods known to those skilled in the art and is
available commercially e.g. Calbiochem cat. no. 178466.
Rec~mhinant ApoE may be obtained from genetically engineered
cells which produce recombinant ApoE. The cells may be of
any strain in which a DNA sequence encoding recombinant ApoE
has been introduced by recombinant DNA techniques so long as
the cells are capable of expressing the DNA sequence and
producing the recombinant ApoE polypeptide. The cells may
contain the DNA sequence encoding the recombinant ApoE in a
vector DNA molecule such as a plasmid which may be
constructed by recombinant DNA t~chn;ques so that the
sequence encoding the recombinant ApoE is incorporated at a
suitable position in the vector. The cells are preferably
bacterial cells or other unicellular org~ni~mc, but
eucaryotic cells such as yeast, insect or m~mm~lian cells
may also be used to produce recombinant ApoE.
In one emho~;ment the ApoE is a mutant of recombinant apoE
differing from the naturally occurring polypeptide by the
WO94/04178 PCT/US93/07582
2 1 ~ 8
-16-
addition, deletion, or substitution of one or more non-
essential amino acid residues, typically less than 10,
provided that the resulting polypeptide retains the cell
proliferation inhibitory activity of apoE. Persons skilled
in the art can readily determine which amino acids residues
may be added, deleted, or substituted (including with which
amino acids such substitutions may be made) using
established well known proce~llres, including, for example,
conventional methods for the design and manufacture of DNA
sequences coding for bacterial expression of mutants of the
subject polypeptide, the modification of cDNA and genomic
sequences by site-directed mutagenesis techniques, the
construction of recombinant proteins and expression vectors,
the bacterial expression of the polypeptides, and the
determination of the biochemical activity of the
polypeptides using conventional biochemical assayc.
Examples of mutants of apoE are deletion mutants cont~; n i n~
less than all the amino acid residues of naturally occurring
apoE, substitution mutants wherein one or more residues are
replaced by other residues, and addition mutants wherein one
or more amino acids residues are added to the polypeptide.
All such mutants share the cell proliferation inhibitory
activity of naturally occurring apoE.
Polypeptides having substantially the same amino acid
sequence as naturally occurring apolipoprotein E enComp~cs
the addition or deletion of fewer than four amino acids at
the N-terminus of the amino acid sequence of the
polypeptide. There may be additional substitutions and/or
deletions in the sequence which do not eliminate the cell
proliferation inhibiting biological activity of the
polypeptide. Such substitutions and deletions are known to
those skilled in the art. Substitutions may encompass up to
about 10 residues in accordance with the homologous or
WO94/04178 ~1 4 1 ~ 9 8 PCT/US93/07S82
-17-
equivalent ~ uu~ described by e.g. T~hninger, Biochemistry,
2nd ed. Worth Pub., N.Y. (1975); Creighton, Protein
Structure a Practical A~roach, IRL Press at Oxford Univ.
Press, Oxford, England (1989); and Dayhoff, Atlas of Prote;n
Sequence and Structure 1972, National Biomedical Research
Foundation, Maryland (1972).
In a particular embodiment, the ApoE is recombinant met-
apoE, e.g. a recombinant polypeptide comprising the se~uence
of naturally occurring apoE with an additional methionine at
the N-terminus.
Also encompassed by the term "Apolipoprotein E" are
polypeptide fragments of recombinant ApoE and of naturally
occurring apoE which exhibit the cell proliferation
inhibitory activity of apoE. One example of such a fragment
is a 15-mer fragment disclosed in U.S. Patent No. 5,177,189,
issued January 5, 1993.
Additional examples of such polypeptide fragments have amino
acids 1-217 or 1-185 of naturally occurring apoE. A
particular embodiment of an ApoE polypeptide having amino
acids 1-217 of naturally occurring apoE is e~co~ by
plasmid pTVR6-2 (Figure 17) which was deposited in E. coli
4300 on July 26, 1993 with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland under
~e~cion No. 69364. Another fragment is the 22KD N-
terminal ApoE polypeptide produced by thrombin digestion of
naturally occurring apoE or recombinant met-apoE.
Similar ApoE polypeptides may be obtained by those skilled
in the art from plasmids constructed on the basis of any of
the above described plasmids and their use is encompassed by
the claims defining the invention. Procedures for
constructing such plasmids and obtaining such polypeptides
WO94/04178 ~ 9 PCT/US93/07S8
-18-
are well known to those ~killed in the art and are described
in numerous publications including Sambrook, Fritsch and
Maniatis, MO1~C111A~ Cloning: A Laboratory ~A~ 2nd
edition, Cold Spring Harbor Laboratory Press, USA (1989).
s
In sum, the method of the present invention may be practiced
with any ApoE having at least substantially the same cell
proliferation inhibitory activity as naturally occurring
apo~.
Further, the invention provides in vitro methods of
inhibiting DNA synthesis of proliferating cells. This
method comprises contacting the cells with ApoE in an amount
effective to inhibit DNA synthesis. The invention also
provides in vit~o methods of inhibiting the chemotactic
response of endothelial cells. This method comprises
contacting cells with ApoE in an amount effective to inhibit
the chemotactic response.
As used herein, chemotactic response means the migration of
cells in response to a stimuli. The present invention
provides a method whereby cells induced to migrate by
exposure to a growth factor are inhibited by the contacting
of ApoE with the migrating cells. The amount effective to
inhibit a chemotactic response is any amount effective to
inhibit the migration of cells stimulated in response to a
growth factor.
It is herein disclosed that ApoE inhibits proliferation of
various m~mm~lian cells including aortic and corneal
endothelial cells and mammary carcinoma cells, me1~nom~, and
smooth muscle cells. As used herein, carcinoma refers to a
malignant epithelial tumor. Furthermore, since ApoE
inhibits aortic endothelial cells, it will likely inhibit
neovascularization. Without neovascularization, i.e., the
2~41~98
WO94/04178 PCT/US93/07582
--19--
formation of new blood vessels, cells cannot actively
proliferate. In the case of tumor cells, inhibition of
neovascularization results in inhibition of proliferation
and thus interferes with tumor growth. Consequently,
5 ~mi n; ~tration of ApoE is a novel treatment for the
inhibition of the formation of new blood vessel growth
(angiogenesis), with concommitant inhibition of tumor cell
growth, and metastasis. Thus ~oth indirectly by inhibition
of angiogenesis, and directly, ApoE inhibits proliferation
of a wide range of tumor cells including melanoma, sarcoma,
ly~rho~ and leukemia cells.
It is herein disclosed that ApoE inhibits proliferation of
smooth muscle cells. The formation and ~lo~,ession of
plaques and metastatic tissue, due to abnormal
neov~cc~ ~ization, is accompanied by the migration of
smooth muscle cells to the site of the plaque or the
metastatic tissue. Inhibition of proliferation of smoo~h
muscle cells may interfere with the progression of formation
of pla~ues and metastatic tissue.
Furthermore, abnormal neovascularization of smooth muscle
cells may lead to the formation of a sarcoma. As used
herein, sarcoma refers to a soft tissue tumor. Thus,
treatment by ApoE for the inhibition of proliferating smooth
muscle cells is also provided, thus constituting a treatment
for the modulation of the formation and progression of
plaques, metastatic tissue, and sarcoma tumors.
It is additionally provided that ApoE may ~e used
therapeutically as an inhibitor of blood vessel formation
for treatment of subjects suffering from disorders
characterized by abnormal neovascularization. Abnormal
neovascularization means the increased and enhanced ability
to form ~lood vessels. Examples of abnormal
WO94/04178 PCT/US93/07582
2~41~98
-20-
neovascularization include such disorders as neovacc~
glaucoma, diabetic retinopathy, rheumatoid arthritis
~Folkman, et al., 1989) and hemangioma.
In the mammalian body, cells may be actively proliferating
because they are tumor cells or because they are being
stimulated by endogenous or naturally occurring growth
factors, or serum factors, or for other reasons.
Preferably, the ApoE is a~m; n; stered in a pharmaceutically
acceptable carrier. Pharmaceutically acceptable carrier
encompasses any of the st~n~rd pharmaceutical carriers such
as sterile solution, tablets, coated tablets and capsules.
Typically such carriers contain excipients such as starch,
milk, sugar, certain types of clay, gelatin, stensic acid,
talc, vegetable fats or olis, gums, glycols, or other known
excipients. Such carriers may also include flavor and color
additives and other ingredients.
Compositions comprising such carriers are formulated by well
known conventional methods. However, a composition
comprising ApoE in an amount effective to inhibit
proliferation of actively proliferating cells was previously
unknown.
In the method of this invention, the administration of the
ApoE-containing composition may be effected by any of the
well known methods, including but not limited to, oral,
intravenous, intramuscular, andsubcutaneous a~m; n; stration.
In the practice of the method of this invention, the amount
of Apolipoprotein E incorporated in the composition may vary
widely. The amount of ApoE effective to inhi~it cell
proliferation is O.lmg - lg ApoE. The precise amount and
the frequency of administration of the dose will readily be
WO94/04178 2 1 ~ t 5 9 ~ PCT/US93/07582
-21-
determined by one skilled in the art, based on the
characteristics of the formulation, body weight and
condition of the subject, tumor size, route and frequency of
;stration, and the characteristics of the particular
Apolipoprotein E to be used.
2~4159~
WO94/04178 PCT/US93/0758
-22-
~MPLE~
This invention is illustrated by the Examples which follow.
These Examples present specific ~ho~; ments and are set
forth to aid in an underst~n~ing of the invention but are
not intended to and should not be construed to limit in any
way the invention as set forth in the claims which follow.
W O 94/04178 ~ t 4 1 5 ~ 8 PC~r/US93/07582
~mPle 1. Methods and Materials
A. Methods and Materials used to measure cellular arowth
Cellular growth was evaluated under different growth
conditions and by various methods, including DNA synthesis
(mitogenesis) by measuring incorporation of 3H-thymidine
into DNA, and proliferation, by directly quantitating cell
n-~h~rs by their associated enzymatic activity.
PolyPe~tides
Recombinant met-apoE contains the amino acid sequence of the
human ApoE3 isoform with an additional N-terminal methionine
(Vogel, et al. PNAS 1985) and was produced as described in
section B below.
Plasmatic apoE was kindly provided by S. Ei~C ~h~g
(Laboratory of Lipids, ~c~a Medical School, Ein Kerem,
Jerusalem). It was isolated from plasma lipoproteins
derived from healthy human volunteers homozygous for the E3
isoform and as described earlier (Rall S.C. et al , Methods
In Enzymology 128:273 (1986)).
The ApoE designated ApoE6-2, spanning amino acids 1-217 of
apoE was produced as described in Example 8.
The ApoE designated peptide 348, a tandem dimeric peptide
Sp~nn;ng amino acids 141-155 of apoE was prepared as
described in Example 2.
The ApoE produced by thrombin digestion of apoE which
removes the C-terminus, may be produced as described in
Example 8.
WO94/04178 21~15 ~ ~ PCT/US93/07582
R~c~hinant TSPl8 is an 18 Kd polypeptide fragment, and
recombinant TSP28 a 28 Kd polypeptide fragment, both of
which contain the heparin binding ~nm~ i n comprising amino
acids 1-174 and 1-242, respectively of human thrombospondin.
rFN33 is a recombinant 33 kD polypeptide fragment of the
cell binding ~om~in of human fibronectin comprising amino
acids 1329 - 1722, but deleted of amino acids 1600-1689.
The plasmid designated pFN 137-2 which encodes for the 33 kD
polypeptide fragment has been fully described in co-assigned
patent application U.S. Serial No. 2gl,951, filed December
29, 1988, and has been deposited in Escherichia coli strain
A4255 under ATCC Accession No. 67910.
The recombinant polypeptides listed above were stored
lyophilized at -70C.
Met-apoE was reconstituted by first dissolving in distilled
water at a concentration of 2mg/ml followed by the addition
of 0.1 volumes of 10X PBS. rFN33 was dissolved in distilled
water at a concentration of 3.5-5 mg/ml. rTSP 18 and 28
were first dissolved in distilled water at 0.5 mg/ml,
desalted on a PD10 column (Pharmacia #170851-01) that was
equilibrated with 10 mM sodiumbicarbonate pH 9.5, and then
eluted with the same buffer. One tenth the volume of a
solution of 10X PBS was then added. Polypeptide samples
were stored in small aliquots at -20C and used within one
month. Heparin (Sodium, injection USP, 1000 U/mlO, 6.25
mg/ml) was supplied by Lilly.
Cell T-; nes and Reaqents
Bovine aortic endothelial cells (BAEC), were kindly provided
by Dr. E. Gallin (AFRY, Bethesda, MD), and were used at
WO94/04178 21 4 1 ~ 9 8 PCT/US93/07582
-25-
passages 5-10. BAEC culture was routinely maintA;ne~ in low
glucose DMEM, cont~ining 10~ FCS, 4 mM glutamine, 0.5 mg/ml
ascorbic acid and 500 u/ml penicillin and 500 u/ml
~Lr~Lomycin. (Biofluids Inc., Rockville, MD).
s
Bovine corneal endothelial cells (CBEC), were kindly
provided by Dr. D. Blake. (Maharry Medical College,
Nashville, TN), and used at passages 2-8. CBEC culture was
routinely maintained in low glucose DMEM cont~;ning 10% FCS,
4 mM glutamine, 500 u/ml penicillin, 500 u/ml streptomycin,
and 2.5~g/ml Fungison (BioFluids, Rockville, MD, 20850).
The media was changed every 2-3 days.
The human melanoma cell line A2058 cells (Todaro, et al.
Proc. Natal. Acad. Sci. U.S.A., 77, 5258-5262, 1980) and the
h-lm~n mammary tumor cell line MDA-MB 435 (Coillean, et al.,
(1978) In Vitro 14, 911-915), were maint~ A in high
glucose DMEM, containing 10% FCS, 4mM glutamine and 500 u/ml
penicillin and 500 u/ml streptomycin purchased from
Biofluids, Inc., Rockville, MD 20850, USA. The human
smooth muscle fibroblasts (SMC) were obtained from Dr.
Philip Browning of the National Cancer Institute, National
Institute of Health, Bethesda, Maryland. The cells were
cultured in RPMI medium containing 10% FCS, 4mM glut~in~,
500 u/ml penicillin, and 500 u/ml streptomycin (purchased
from BioFluids).
Mouse endothelioma cells (END2) (Williams et al., Cell
57:1053 (1989)) expressing the polyoma middle-T antigen were
provided by I. Voldavsky (Hadassa Medical School, Ein Kerem,
Jerusalem). The END2 cells were routinely maintained in low
glucose DMEM containing 10% FCS, 4mM glutamine, 500U/ml each
of penicillin and streptomycin. END2 cells were normally
used at 70-80% confluency after 5-8 days culture. Medium
was replaced every 3-4 days. Medium components were obtained
21~1598
WO94/04178 PCT/US93/07582
-26-
from Kibbutz Beit Haemek, Israel.
~h;n~ce hamster ovary cells (CH0) and neuroblastoma N18TG2
cells (Z. Vogel, Weizmann Institute) were routinely
maintAin~ in high glucose DMEM cont~;ning 10% FCS, 2mM
glutAm;ne, and 500U/ml each of penicillin and ~LLe~Lomycin.
Medium cu~u~ents were obtained from Kibbutz Beit Haemek,
Israel.
AssaYs
Both ApoE and bFGF are heparin-binding molecules, exhibiting
high affinity for heparin and HSPG both on the cell surface
and the ECM. The effect of ApoE on bFGF-st;~~ ted growth
was examined in several cell types in two separate systems:
mitogenesis-by following the incorporation of [3H]thymidine
into DNA; and proliferation-by measuring the actual number
of cells in the culture.
1. Inhi~ition of Mitoqenesis
Protocol Ml: Pre-attached, dense culture.
Cells were seeded at 105/well in 0.5 ml of medium
supplemented with 10% FCS in the inner well of a 24-well
tissue culture plate. After 3 days, the cells were washed
with PBS and fed with 0.5 ml of medium cont~; n; ng 0.5% FCS
and r~;ne~ in this medium for 48 hr (starvation
conditions). Serum-free medium cont~;n;ng the tested growth
effectors was then added to the wells and 22 hours later the
cells were labeled for 5 hours with [methyl-3H]thymidine
(86.1 ci/mmol, NEN; 2.5 ~Ci/well). To terminate the
mitogenesis assay, the cells were washed twice with 1 ml
PBS, fixed with 0.3 ml solution of methanol/acetic acid
(3:1), washed twice with 0.5 ml ethanol (80%), and air-
WO94/04178 ~ 8 PCT/US93/07582
dried. The cells were extracted from the wells by
inCllh~tion with 300 ~1 trypsin/EDTA (1 hr at 37C and 30 min
at room temperature), and by the addition of 100 ~1 1% SDS.
The radioactivity of the extracted material was measured in
a scintillation counter.
~rotoco~ M2: Newly-attached, medium-dense culture.
Confluent monolayers of cells in T-150 cm2 flasks were
washed once in PBS, and then incubated in 0.5% FCS-
cont~;~; ng medium for 48 hours (starvation conditions).
Cells were trypsinized, washed in 10% FCS-containing medium,
resuspended in medium cont~;n;~ 0.1~ BSA, and seeded in 24-
well plates at 2 x 104 cells/well in the presence of various
~oncentrations of FCS, growth effectors, and [3H]thymidine
(at the concentration indicated in protocol Ml). At
different time points (indicated in the figure legends),
mitogenesis was terminated as described in protocol Ml.
2. Inhibition of Proliferation
Protocol Pl: 105 cells were plated into 35 mm culture
~;~h~, in 10% Fcs-cont~ining medium, allowed to attach for
24 hours, and then refed with media cont~ini~g 5% FCS and
the tested growth effectors. After 72 hours, cells were
detached from the plates by trypsinization, and their number
monitored by a Coulter counter.
Protocol P2: This was carried out using the cell titer
96TM nonradioactive cell assay (Promega #G4000) based on the
methods described in Denizot et al., J. Immunol. Meth.
89:271 (1986). 5-30x103 cells were plated into each well of
a 96-well culture dish in medium containing 5% FCS together
with the indicated concentrations of growth effectors.
After 72 hours, 15 ~1 of dye solution was added to each well
WO94/04178 2 1 4 1 5 9~8~ PCT/US93/07582
-28-
and the plates were incubated for an additional 4 hours.
Then 100 ~1 of solubilization solution was added and after
24 hours, the amount of dye ret~inp~ by the well was
~Y~r;ne~ by recording absorbency at 570 nm using an ELISA
plate reader.
3. The F~D2 hemanqioma model
END2 cells, grown as described above, were harvested by
trypsinization at mid-confluency after 5 days in culture.
The cells were then susp~n~ in DMEM containing 10% FCS,
centrifuged, respended in basal DMEM without serum at 6X106
cells/ml (or as otherwise indicated) and kept on ice. The
cells were then diluted 1:1 with an extracellular matrix
composition (Matrigel, H. Kleinman, Dental Institute,
National Institutes of Health). Aliquots of 0.lml were
injected into the hind leg of female Balb/C mice (20-25g).
On the ninth day, the mice were sacrificed and tumor
development in the injected leg was observed. The tumor
appeared as a hematomatous lump of purplish color the size
of which varied in correlation with the number of cells
initially injected. It was found that while a full size
tumor developed after injection of 106 cells, the size was
markedly smaller when 105 cells were injected, and only very
small tumors developed following injection with 3x104 cells.
Therefore, the experiments were routinely performed with
3x105 cells/mouse.
4. Thrombin activitY
Throm~in activity may be measured by the hydrolysis of a
chromogenic substrate resulting in release of a colored
compound essentially as described by Lotenberg (BBA, 142:556
(1983)). Briefly, thrombin is able to cleave the synthetic
substrate Tos-Gly-Pro-Arg-paranitroaniline resulting in the
WO94/04178 PCT/US93/07582
~141~
-29-
release of paranitroaniline (PNA) whose ~oncentration may be
deterr; n~ by absorbance at 405nm.
B. Production of Apo~
I. Host-Vector Svstem For Ex~ression of Recom~inant A~oE
The preferred host-vector system used for production of
met-ApoE is E. coli strain W1485 (ATCC No. 12435) harboring
plasmid pTVR 590-4; the host-vector system has been
deposited with the American Type Culture Collection (ATCC)
in Rockville, Maryland under ATCC Accession No. 67360~
The construction of plasmid pTVR 590-4 which is described
below has been fully described in co-assigned cop~
patent application, EPO Publication No. 303,972 (see Figure
15).
Plasmid pTVR 590-4 contains the following elements:
a) Origin of replication.
b) The AmpR gene in counter clockwise orientation.
c) In clockwise orientation and in 5' to 3' order, a
truncated deo P1 promoter sequence and the lambda cI~7
temperature-sensitive repressor coding sequence.
d) In counterclockwise orientation and in 5' to 3'
order, the lambda promoter, the beta lactamase
promoter-ribosomal binding site, the coding sequence
for ApoE and the T1T2 transcription termination
sequences.
This plasmid is a high level expressor of ApoE under the
control of the strong leftward promoter of bacteriophage
WO94/04178 ~ PCT/US93/07582
4~
-30-
1 A~hA~ (PL) which is thermoinducibly CGil~ olled by the
constitutively expressed cI857 temperature-sensitive
repressor also situated on the plasmid. Production of ApoE
from this plasmid takes place upon heat- induction at 42C.
This is a so-called "host independent" expression system
since the plasmid can thermoinducibly produce met-ApoE
in~p~n~nt of prior insertion of the l~mh~ cI857 gene into
the host E. coli chromosome. This plasmid can therefore be
used to transform a wide variety of host bacterial cells.
The host described, E. coli W1485 is a prototrophic
wild-type strain of E. coli freely obtainable from the ATCC
under ~CceC~ion No. 12435.
II. Growth of ~. coli W1485 harborin~ ~lasmid ~TVR 590-4
and ~roduction of a bacterial cake containing ApoE
The following description of the fermentation of E. coli
W1485 harboring plasmid pTVR 590-4 is a preferred emho~iment
for production of a cell cake containing ApoE.
1. Seed Flask Develo~ment
The contents of frozen vials containing E. coli ATCC No.
12435/pTVR 590-4 are used to inoculate seed flasks
containing the following medium:
K2HPO4
KH2PO4 1 g
NaCl 5 g
MgSO4.7H20 0.2 g
NH4Cl 1 g
FeNH4 citrate 0.01 g
Trace elements solution 1 ml
Biotin 0.5 mg
Glucose 5 g
~ WO94/04178 ~ 4 1 ~;9 ~ - PCT/US93/07582
Ampicillin, sodium salt 0.1 g
Deionized water 1 L
Trace elements stock solution:
MnSO4 H20 1 g
ZnSO4 .7H2O 2.78 g
CoCl2.6H2O 2 g
Na2MoO4-2H2O 2 g
CaC12 2H2
CUS04 5H20 1. 85 g
H3BO3 0-5 g
Concentrated HCl 100 mL
Deionized Water 900 mL
Glucose and ampicillin are added from sterile concentrated
stock solutions after autoclaving the other components of
the medium. The cultures are ;cl~h~ted at 30C overnight on
a rotary shaker at 250 rpm, and reach an OD~ of 3.5-5Ø
2. Seed Fermenter
The contents of the seed flask are used to inoculate a 50 L
seed fermenter containing 25-30 L of the following
production medium, which contains per liter:
KzHPO4 8 g
KH2PO4 2 g
Sodium citrate 2 g
NH~Cl 2 g
FeNH4 citrate 0.02 g
CaCl2.2H2O 0.04 g
K2SO4 0.6 g
Trace elements solution 3 mL
(as in Section 1)
Antifoam
(Silicolapse 5000) 2 mL
W O 94/04178 2 ~ 4 1 S 9 ~ PC~r/US93/07582
Added after sterilization (per liter of medium)
~IgSO4 7H20
Sodium ampi~ 0.1 g
Glucose 40-60 g
NH3 (25-28% in water) approx. 40 mL
Glucose is added batchwise at i no~ll 1 Ation; ammonia is
automatically added for p~ control (set point pH = 7.0)
during growth.
The culture is cultivated at 30C for 15-20 hours in order
to achieve growth; the OD~o generally re~che~c 20-30 during
this time. This is equivalent to a dry cell weight (DCW) of
7.5-12 g/L.
3. Production Fermenter
The contents of the seed fermenter are used to inoculate a
750 L (nominal volume) fermenter containing about 360 L
production medium as described for seed fermenter, but
excluding ampicillin. The cuIture is cultivated at 30C
until an OD~o Of 10 is obtained. Induction of ApoE
expression is then achieved by raising the fermenter
temperature to 42C. At induction, the following are added
to the fermenter:
DL-methionine 0.6 g per L of medium
Sodium acetate 5 g per L of medium
The sodium acetate (0.1% - 1%) is added to protect cells
from the "toxic effect" caused by the ApoE analog.
The fermenter temperature is maintained at 42C for three
hours, at which time the cells are harvested. The OD~o of
WO94/04178 2 1 ~ 1 5 9 8 PCT/US93/07582
-33-
the cell suspension at harvest is generally 16-20, the
volume is 400-430 L and the DCW is 5.0-6.5 g/L.
4. Harvest of Cells
The cell suspension is centrifuged at 14,000 rpm (16,000 g)
in a CEPA 101 tubular bowl centrifuge at a feed rate of
250L!hr, and a cell cake weighing about 10 Kg is pro~llcP~
and saved. Alternatively, the cell suspension is
centrifuged in a Westfalia CSA-19 continuous centrifuge at
500 L/hr. The sludge is either disrupted immediately or
frozen.
In both cases, the supernatant contains no detectable ApoE
as measured by SDS-polyacrylamide gel electrophoresis.
III. Purification of Recombinant A~oE
The following method is suitable for scale-up for industrial
application and yields very pure ApoE. The general scheme
of the downstream process (Scheme 1) consists of steps A
through G as follows:
A CELL DI~u~llON IN PRESENCE OF MAGNESIUM
IONS.
B EXTRACTION OF CELL PELLET WITH TRITONR.
C 100K ULTRAFILTRATION.
D DEAE CHROMATOGRAPHY
E Q SEPHAROSE ~OM~TOGRAPHY
F CM SEPHAROSE CHROMATOGRAPHY
G 100K ULTRAFILTRATION - TRITONR REMOVAL.
The following detailed example of the steps in purification
of ApoE was performed on 3 Kg cell cake. In addition we
have sl-cçe~fully processed a 15 Kg cell cake using the
WO94/04178 PCT/US93/07582
~ 98 _34_
methods described below with only minor modifications
involving scale-up in the size of the equipment used.
Steps A through D were performed on 2 batches of bacterial
cake, each weighing l.5 Kg. After step D, the two batches
were combined and processed as one batch through steps E to
G. Steps A, B, C were performed at 4C - 10C, except where
otherwise indicated. All other activities were performed at
room temperature.
A. ~rT. DI~ ON IN PRESENCE OF MAGNESIUM IONS
l.5 Kg of wet cell cake was suspended in 6 L of buffer A
which consists of 50 mM tris/HCl, 30 mM MgC12, 0.25% beta
hydroxybutyrate sodium salt, pH=7.5. (The beta
hydroxybutyrate was added as a protease inhibitor) This was
then homogenized using a Rinematica homogenizer yielding 7.5
L of homogenate. Disruption was then performed using a
Dynomill KDL bead mill disrupter (Willy A. Bachofen, Basel)
at 5 L/hr (in two cycles). Three-fold dilution of the
resulting suspension using buffer A yielded a volume of 22.5
L. This lysate contained about 6 g ApoE, i.e. about 4 g
ApoE per Kg of original bacterial cake.
Centrifugation was then performed in a continuous ~EPA-41
tubular bowl centrifuge, (Carl Padberg, Lahr/Schwarzwald)
with a feed rate of 9 L/hr at 20,000 rpm (17,000 g). The
pellet, weighing approximately 700 g and cont~; n; ng
insoluble ApoE was saved and the supernatant was discarded.
(Note that the ApoE is insoluble due to the presence of Mg~
ions.)
B. EXTRACTION OF CELL PELLET WITH TRITONR
Six liters ~l:l0) of extraction buffer were added to the
W094/04178 2 1 4 1 ~ 9 8 PCT/US93/07582
-35-
pellet. (Extraction buffer: 50 mM tris/HCl, 20 mM EDTA,
0.3% TritonR, pH adjusted to 3.0 with HCl). Suspension was
achieved using a homogenizer (Kinematica) at low ~peed.
Then another 6 L extraction buffer was added (giving a final
pellet:buffer ratio of 1:20) and the pH ~as adjusted to 4.5
with 1 N NaOH. The resulting 12 L suspension was ;nc~lhAted
for 10 minutes at room temperature with stirring.
After incubation, the suspension was centrifuged on the CEPA
41 Centrifuge at a feed rate of 20 L/hr. The pellet
weighing about 450 g was discarded and the supernatant
solution containing ApoE was titrated to pH=7.5 with 1 N
NaOH and saved.
Note: TritonR is present in all following steps and is
removed in step G.
C. 100 K ULTRAFILTRATION
The purpose of this step is to remove low molecular weight
contaminants by ultrafiltration/dialysis.
A Millipore Pellicon ultrafiltration system using one 100 K
cassette type PTHK was utilized to concentrate the
supernatant of the previous step (about 12 L) to about 2 L.
The feed pressure was 20 psig and the filtrate flow rate was
20 L/hr. The dialysis buffer was 50 mM tris HCl, lO mM EDTA
and 0.1% TritonR, pH=7.5. The 2 L retentate con~ining
about 2-3 mg ApoE /ml was kept cool with ice.
The retentate was dialyzed using the recirculating mode of
the Pellicon ultrafiltration system until a filtrate
conductivity equivalent to that of the dialysis buffer was
obtained; this was the criterion used throughout the
purification for termination of dialysis.
WO94/04178 ~ PCT/US93/075X2 -
. .. .
D. DEAE CHROMATOGRAPHY
The purpose of this step is to separate the ApoE from
cont~m;nAnts such as proteins and other cellular materials.
In this step a 1.6 L DEAE Sepharose Fast Flow column
(Pharmacia) was used. The flow rate was 10 column
volumes/hr. The capacity of the column under these
conditions was determined to be 4 mg ApoE/ml. The col G
was first equilibrated with DEAE equilibration buffer: 20 mM
tris/HCl, 1 mM EDTA, 0.5% TritonR, pH=7.5.
The retentate solution from the previous step (about 3 L)
was then loaded on the column and washed with 3 column
volumes (CV) of eguilibration buffer. The first elution was
performed using 3 CV of equilibration buffer containing 120
mM NaC1. Fractions were collected and the progress of the
run was monitored by continuously following the absorbance
of the eluate at 280 nm. The fractions were analyzed by SDS
polyacrylamide gel electrophoresis stained by Coomassie Blue
and the trailing edge of the peak (3.1 CV) was saved.
The second elution was performed using the equilibration
buffer containing 150 mM NaCl. Fractions were collected and
analyzed by SDS gel electrophoresis and most of the peak
(3.9 CV) was saved. Endotoxins were measured by the Limulus
Amebocyte Lysate (LAL) assay described in U.S. Pharmacopeia
(U.S.P.) XXI, 1165-1166 (1985). The level of endotoxin was
3 ~g per mg ApoE.
Concentration and dialvsis after DEAE-Se~harose
The fractions indicated from the first and second eluates
were pooled and dialyzed using the Pellicon ultrafiltration
system, with one 100K cassette; the dialysis buffer was 20
W O 94/04178 2 1 4 1 5 9 8 PC~r/US93/07582
-37-
mM tris/HCl, 1 mM EDTA, 0.1% TritonR, pH=7.5. The sample
was concentrated to 2 L (about 2-3 mg ApoE/ml) and dialyzed.
E. O-S~PHAROSE (OS) CHROMATOGR~PHY
The purpose of this step is to separate active from inactive
ApoE and to further remove endotoxins.
In this step a 1.6 L QS Fast Flow Column (Pharmacia) was
used; the column capacity under these conditions was about
7 mg ApoE/ml and the flow rate was about 10 CV/hr.
The QS equilibration buffer was 20 mM tris/HCl, 1 mM EDTA,
0.2% TritonR, pH=7.8. After equilibration, the retentate
solutions from two batches of the previous step were
combined and loaded on to the column, i.e. a total volume of
about 5 L of buffer containing about 5 g ApoE. The column
was then washed with 2.8 CV of equilibration buffer. The
first elution was performed with 3 CV of equilibration
buffer containing 20 mM NaCl and the second elution was
performed with about 5.5 CV of equilibration buffer
cont~ining 40 mM NaCl. Fractions were collected, monitored
and analyzed as described above, and 2.0 CV were combined
and saved. The level of endotoxin was measured by the LAL
assay and was now less than 250 pg/mg ApoE.
Two subsequent elutions using buffer containing 70 mM NaCl
and 350 mM NaCl respectively eluted the inert ApoE.
Concentration and dialysis after O-Sepharose
The QS-derived saved pooled fractions were concentrated and
dialyzed by ultrafiltration through a Millipore Pellicon
Ultrafiltration system using one lOOK cassette.
WO94/04178 2 1 4 1 ~ 9 8 PCT/US93/07582 -
-38-
The dialysis buffer was 10 mM tris/HCl, 1 mM EDTA, 0.1%
TritonR, pH=7.5. The sample was dialyzed using the
recirculating mode whilst maint~;ning the ApoE concentration
at 2-3 mg/ml. The final retentate volume was about 500 ml.
F. CM-S~PHAROS~ CHROMATOGRAPHY
The purpose of this step is to further remove endotoxins and
to lower the concentration of TritonR to 0.05%.
In this step a 120 ml CM-Sepharose Fast Flow (Pharmacia)
column was used. The eguilibration buffer was 20 mM Na
acetate, 1 mM EDTA, 0.2% TritonR, pH=4.8. After
equilibration, the retentate solution from the previous step
was loaded on to the CM-Sepharose column. The capacity of
the column was 10 mg ApoE/ml and the flow rate was 10 CV/hr.
The column was then washed with the following solutions: 4
CV of equilibration buffer followed by 5 CV of equilibration
buffer containing 70 mM NaCl followed by 2 CV of 20 mM Na
acetate, 1 mM EDTA, 0.05% TritonR, 70 mM NaCl pH=4.8. The
eluate from the loading and washing steps was discarded.
The column was then eluted. The eluent was 8 CV of 20 mM Na
acetate, 1 mM EDTA, 0.05% TritonR, 300 mM NaCl, pH=5Ø The
progress of the elution was monitored by continuously
following the absorbance of the eluate at 280 nm. (Two
~ifferent base lines are used during the elution: one is the
high U.V. absorbance buffer containing 0.2~ Triton, the
other is the low U.V. absorbance buffer cont~;n;ng 0.05%
Triton. The use of a sensitivity scale of a~out 1.0 OD
allows both buffers to appear on the chart column, the low
at the foot and the high at a~out 0.5 O~.)
WO94/04178 2 1 4 1 ~ 9 ~ PCT/US93/07582
-39-
The sample contA; n i ng ApoE was immediately titrated to pH
7.8 and saved. The endotoxin level in this sample was below
50 pg per mg ApoE as measured by the LAL assay.
S G. l00K ULTRAFILTRATION - TRITONR REMOVAL
The ~u~o~e of this step is to remove the TritonR.
This step was carried out at 4C using the Millipore
Pellicon Ultrafiltration System, containing one l00K
cassette, pre-washed with 0.5 M NaOH overnight. The flow
rate was 9-12 L/hr and the inlet/pressure was 5-l0 psig.
(This low flow rate is used to prevent aggregation of the
ApoE as the TritonR is being removed.) The ApoE sample from
the previous step (960 ml containing about 600 mg ApoE) was
diluted to 0.5 mg/ml with l0 mM NaH~03 buffer pH=7.7.
The sample was then treated in the ultrafiltration system
and the following conditions were applied throughout this
TritonR removal step:
a) The TritonR concentration must be lower than 0.02% i.e.
the TritonR concentration must be below its critical
micelle concentration in order to achieve effective
TritonR removal across the l00K membrane.
b) The ApoE must not be diluted below 0.5 mg/ml or
dissociation of the ApoE molecule will occur and it may
cross the l00 K membrane.
c) The ApoE must not be concentrated above l.5 mg/ml or
aggregation of the ApoE molecules may occur.
The dialysis buffer used in the ultrafiltration system was
l0 mM NaHC03, 150 mM NaCl, pH=7.8.
WO94/04178 ; PCT/US93/07582
2 ~ 9 8 -40-
After concentration and dilution steps in accordance with
the above conditions, the dialysis ~as performed at constant
volume and constant flow rate and the dialysis was completed
when the absorbance at 280 nm of the filtrate was O.Ol
units. (TritonR solution absorbs at 280 nm and an
absorbance of O.Ol is equivalent to 0.0005% TritonR.) The
total volume of final retentate was 770 ml and the total
volume of the filtrate was 9.5 L.
The solution containing the ApoE was then filtered (0.2
micron filter) and stored at -70C in 80 ml glass bottles.
Overall Yield:
0.3 g of highly purified met-apoE were recovered from 3 Kg
of bacterial cake. The ApoE, approximately 97% pure, was in
the same aggregation state as plasmatic apoE when tested
under the same conditions of gel permeation analysis. The
ApoE sample contained less then 50 pg of endoto~;n~/mg
protein.
T vo~hilization
If the ApoE is to be lyophilized the dialysis buffer in the
TritonR removal step is 2 mM NaHCO3 pH=7.8, lmM cystein/mg
apoE,, and after lyophilization the samples of ApoE are
stored at -20C.
After lyophilization, the ApoE can be redissolved and it
retains its normal biological activity. The lyophilized
ApoE is very stable for at least 5 years.
WO94/04178 ~14 1 5 ~ 8 PCT/US93/07582
-41-
~xam~le 2. Effect of ApoE on bovine aortic endothelial
cell cultures
A . ~f fect of A~oE on incorporation of 3H thvmidine
Addition of bFGF to a freshly plated 48 hr serum-starved
culture of bovine aortic endothelial cells (BAEC) stimulated
the incorporation of [3H]thymidine by several-fold compared
to the control culture, both at 1% and 2.5% FCS, as shown in
Figure l. This stimulation of DNA synthesis is strongly
inhibited by addition of ApoE to the culture, and was both
dose- and serum-dependent. At 1% FCS, inhibition by 0.05 ~M
and 0.5 ~M ApoE was approximately 85~ and 98~, respectively.
However, at 2.5% FCS, the inhibition was lower, i.e., in the
range of 55% and 85%, respectively.
Addition of another heparin-binding molecule such as the
recombinant TSP fragment (rTSP 18 ) resulted in much less
inhibition of DNA synthesis at 1% FCS. At 2.5% FCS, the
inhibition by ApoE and rTSP was identical.
B. ~ffect of A~oE on time course of thvmidine
incor~oration
The effect of ApoE on the time-course of t3H]thymidine
incorporation in a freshly plated culture of BAEC in the
presence of both bFGF and 1% FCS is demonstrated in Figure
2. [3H]thymidine incorporation is time-dependent, reaching
m~;m11m capacity around 42 hrs after cessation of cell
starvation. In the presence of 0.5 ~M ApoE, incorporation
of t3H]thymidine was reduced by more than 95%. the residual
activity ~2-3%) of t3H]thymidine incorporation had also
reached a m~;m11~ after 42 hrs. In parallel cultures, 0.5
~M rTSPl8 or rFN33 gave either 30% inhibition or no
inhibition, respectively.
WO94/04178 PCT/US93/07582
9 8
-42-
C. ~ffect of various concentrations of APoE
The effect of various concentrations of ApoE on
[3H]thymidine incorporation of pre-attached cultures of BAEC
was measured. The example in Figure 3 demonstrates that the
inhibition obtained by 5 nM ApoE in the presence of 0.5% FCS
and bFGF is approximately 45%, while in the presence of FCS
alone, the inhibition by the same amount of ApoE is 10-fold
lower (- 4%). It should be noted that throughout the
ongoing experiments, the stimulation of cell growth by
exogenously added bFGF was variable. Generally, when cells
display high basal activity of [3H]thymidine incorporation
at low serum concentrations (e.g., 0-0.5%), the cells are
not further stimulated by added bFGF.
D. ~ffect of A~oE on qrowth factor de~endent ~roliferation
Pre-attached cultures of BAEC plated at high density
(105/well) demonstrate a very high level of t3H]thymidine
incorporation at both 0% and 0.5% FCS (as shown in Figure
4). The addition of bFGF to the cultures did not show any
substantial stimulation of the t3H]thymidine incorporation.
However, only cultures contA;n;ng the exogenous bFGF
displayed a remarkable inhibition (~ 65%) by added ApoE.
When ApoE was added to the culture containing only 0.5% FCS,
the inhibitory effect of ApoE was many fold lower (- 5%),
i.e. ApoE inhibits the growth factor dependent proliferation
of these cells.
0 E. Ef~ect of time of addition of ApoE to bovine aortic
endothelial cells
Newly attached BAEC cultures were allowed to grow in the
presence of bFGF and either 0.5% or 1% FCS. The addition of
ApoE at time zero together with bFGF caused the m~;m~l
WO94/04178 PCT/US93/07582
21~598
-43-
effect of inhibition. The addition of ApoE 15 or 22 hr
later caused much less inhibition at both serum
concentrations. The results are shown in Figure 5. Thus,
the first 15 hr of growth are most effective for ApoE
inhibition. After 15 hr, ApoE was considerably less
effective in reducing DNA synthesis in these cultures. It
should be noted that the cells became refractory to ApoE at
a time prior to initiation of DNA synthesis based on the
time course presented in Figure 2. Therefore, ApoE probably
inhibits entry into S-phase but does not significantly
arrest DNA synthesis once it is initiated.
The effect of ApoE on the proliferation of bovine aortic
endothelial cells was determined in the presence of 5% FCS.
Following 3 days of growth, the number of cells was ~ ned
by trypsinization and direct counting. When cells are
plated in the absence of bFGF, the total increase in cell
number was 10-15%, and there was no effect on cell number
upon addition of ApoE. However, when bFGF was added to
parallel cultures, the number of cells was increased by 2.5-
fold. The addition of ApoE together with bFGF inhibited
growth in a dose-dependent manner. At ApoE concentrations
of 0.5 ~M, 2.5 ~M, and 5 ~M, cell growth inhibition was
approximately 10%, 40%, and 50%, respectively. These
proliferation results are consistent with the thymidine
incorporation results in Example 2D.
The effect of ApoE on the proliferation of vascular
endothelial cells from bovine aorta (BAEC) was further
studied without added exogenous bFGF. The results
demonstrate the inhibition by ApoE of proliferation at 0.5%
with the ICso around 0.15~M. When heated at 100C for 60
minutes, more than 95% of the antiproliferative activity of
ApoE is lost, indicating the dependence of the inhibitory
activity on the native form of the ApoE molecule (Figure
~VO 94/04178 2~ 98 P~r/~S93/07582
21). A similar degree of inhibition of proliferation of
BAEC was also observed with apoE isolated from human plasma.
The specificity of the inhibition by ApoE was further
demonstrated using peptide 348, a tandem dimeric peptide
fragment of apoE derived from the LDL-receptor binding
region and the strong heparin binding consensus sequence
spAnn;~g amino acids 141-155 (rRKr~KRTTRn~nDL)2 (Dyer et
al., J. Biol. Chem. 266:15009 (1991) and disclosed in U.S.
Patent No. 5,177,189, issued January 5. 1993. The peptide,
kindly provided by H. C. Krutzsch (Laboratory of Pathology,
NCI, NIH) was synthesized by the stAn~Ard solid phase method
of Merrifield and purified as described earlier (Guo et al.,
PNAS 89:3040 (1992)).
Thus, the proliferation of BAEC cells is inhibited by met-
apoE with an IC50 of 0.1-0.3~M (Figure 20A), and by peptide
348 with an ICso of peptide 348 of 10-15~M (Figure 20B).
The inhibition of proliferation by met-apoE indicates
selectivity for cell type and seems to favor v~c~ r
endothelial cells, since BAEC cells are more sensitive to
inhibition than CH0 cells (Figure 19) and neuroblastoma
cells (Figure 20).
Moreover, the inhibition of proliferation of endothelial
cells in the absence of exogenous bFGF is also observed at
both low and high serum concentrations (Figure 22), thus
indicating that the anti-proliferative activity of ApoE is
effectively achieved in the presence of the endogenous
naturally occurring growth factors normally present in
serum. Furthermore, the observation of inhibition of serum
dependent proliferation in the presence of other growth
factors such as EGF (Figure 7) which are not heparin
dependent, suggests that there are other mech~ni ~m~ of
214~9~
W094/04178 PCT/US93/0~582
r
~45~
inhibition of proliferation by ApoE.
In another experiment, reco_binant met-apoE was comr~red
with plasmatic apoE in the BAEC cell proliferation assay at
0.5% serum conc ntration. The degree of inhibition was
similar except for a slightly higher degree of inhibition
exhibited by recombinant met-apoE.
In a further experiment, the effect in the BAEC cell
proliferation assay of prior treatment of purified met-apoE
with guanidine chloride (GuCl) was tested. The results
showed that at 0.5% FCS, the GuCl treatment caused a slight
increase in the inhibitory activity of met-apoE, while at 5%
FCS, GuCl treatment induced a large increase in the degree
of inhibition.
WO94/04178 ~ PCT/US93/07582
-46-
~mnle 3. ~ffect of A~oE on Bovine Cornea endothelial
Cells
A. ~ffect of A~oE where CBEC are arowth factor stimulated
The effect of ApoE on the mitogenesis and proliferation of
a second type of endothelial dells, derived from bovine
corneal endothelial cells (CBEC), was ~Amined. The results
are demonstrated in Figures 7 and 8, respectively. The
results in Figure 7 describe the incorporation of
[3H]thymidine into CBEC, as was measured on a newly attached
culture following 2 days serum starvation. At 0~ FCS, the
incorporation of t3H]thymidine is relatively low. ~he
incorporation is simulated several-fold when bFGF was added
to the culture. The addition of met-apoE (at 0.5 ~M) had
dramatically reduced this bFGF-dependent DNA synthesis
yielding inhibition of more than 90%. When cells are
allowed to grow in the presence of 1% or 2~ FCS, their
initial growth was much greater and they were not stimulated
by the addition of bFGF to the serum. With increasing serum
concentrations, the ApoE-induced inhibition was reduced to
about 50% and 35% at 1% and 2% FCS, respectively.
Surprisingly, a similar mode of inhibition by ApoE was
obtained with EGF-stimulated CBEC culture which is
considered to be a non-heparin-binding growth factor. Here
again, ApoE inhibited (>90%) the EGF-dependent growth, and
to much less extent, the serum-dependent growth (45% and 25%
at 1% and 2.5% FCS, respectively).
Based on the above results, ApoE will inhibit corneal
endothelial cells from other mammalian (including human)
sources, especially when the cells are actively
proliferating.
WO94/04178 2 1 4 ~ 5 ~ 8 PCT/US93/07582
B. Effect of A~oE when CBEC are serum stimulated
Higher concentrations of ApoE inhibit serum-stimulated
proliferation in CBEC culture. The effect of met-apoE on
CBEC proliferation was measured and is shown in Figure 8.
CBEC were tested for proliferation at 5% FCS and in the
presence of bFGF. The proliferation by bFGF was inhibited
in a dose-dependent manner, with an ICso -1.5 ~M. It is not
clear whether the inhibition of proliferation in this case
is dependent on the presence of bFGF. However, it is clear
that ApoE inhibits proliferation of actively proliferating
corneal endothelial cells.
C. ~eversibilitY of APoE inhibition
The inhibition of t3H~thymidine incorporation by ApoE of
CBEC culture is reversible, as shown in Figure 9. This
reversibility is dependent upon the amount and time at which
ApoE is given to the culture. When the newly attached CBEC
cultures, in the presence of 1% FCS and bFGF, are maintA;~e~
for the entire length of the mitogenesis experiment (40 hr)
at 1.0 and 1.5 ~M met-apoE, inhibition was 30% and 50%,
respectively. However, when ApoE was given only for the
first 22 hr, the inhibition by 1.0 and 1.5 ~M Apo~ following
40 hr incorporation, was much lower, e.g., in the order of
5% and 15%, respectively.
D. The effect of ApoE on Protein SYnthesis of CBEC
Protein synthesis in CBEC was measured following a 1 hour
starvation for methionine, and administration of S35-
methionine (6 hours), as shown in Figure 13. When met-apoE
(0.5 ~M) was added to a parallel culture, the incorporation
of S35-methionine into protein was identical to the control
without ApoE. For both, the incorporation was approximately
2 1 ~ 8
WO94/04178 PCT/US93/07582
-48-
60%. Thus, addition of ApoE has no direct effect on the
synthesis of proteins.
21~1598
WO94/04178 PCT/US93/07582
Exam~le 4. ~ffect of ApoE on Melanoma Cells
DNA synthesis in human A2058H melanoma cells was tested in
a newly attached culture and in the presence of 0.5% FCS and
bFGF, and following the addition of met-apoE, rTSPl8
(rTSPl8) and rFN33, as shown in Figure lO. Addition of ApoE
at 0.5 and l.0 ~M caused inhibition of approximately 40% and
75%, respectively. No such inhibition was observed with the
other molecules added in parallel. Indeed, addition of 0.5
~M of either TSP or FN stimulated t3H]thymidine
incorporation by 2-2.5-fold.
2141~98
W O 94/04178 PC~r/US93/07582
-50-
~mple 5. ~ffect of APoE on Carcinoma Cells
DNA synthesis in the human ~m~ry carcinoma (MDA) cells was
measured in a newly attached culture in 0.5% FCS and bFGF,
S as shown in Figure 11. Addition of 0.5 ~M met-apoE or
rTSP18 inhibited [3H]thY~;~;ne incorporation by
approximately 60% and 45%, respectively. Inhibition of D~A
synthesis by heparin was lower (in the range of 30%).
~ 2141~98
WO94/04178 PCT/US93/07582
~xample 6. ~ffect of ApoE on Smooth Muscle Cells
The effect of ApoE on the proliferation of h~n smooth
muscle cells (SMC) was tested at 0.5% and 5% FCS. In the
presence of 0.5% FCS and a high concentration of met-apoE (4
~M), a strong inhibition of both serum and bFGF-dependent
proliferation of SMC was obt~inD~ (approximately 80%
inhibition in both cases). When serum ronrDntration was
increased to 5~ the inhibition obtained by the same
concentration of met-apoE was lower (approximately 30% for
both serum and bFGF-dependent proliferation). The results
are shown in Figure 12. Thus ApoE inhibits proliferation of
actively proliferating human smooth muscle cells.
WO94/04178 PCT/US93/07582 ~
z~ 98
-52-
~xam~le 7. Effect of ApoE on Mouse Endothelioma Cells
The antiproliferative and antiangiogenic activity of ApoE
was further demonstrated in vitro and in vivo using mouse
endothelioma cells END2 (Williams, Cell 1989). END2 are
polyoma middle T-antigen transformed mouse endothelial
cells, which develop spindle shaped cells in vitro and
vascular tumors and hemangiomas in vivo. These cells have
been used in vivo as a model system for studying
angiogenesis. Cell proliferation was assayed as described
in Example 1 according to protocol P1. Met-apoE inhibited
the proliferation of END2 cells in culture with an ICso of
about 0.25~M at 0.5~ FCS (Figure 18). Inhibition of
proliferation by another ApoE is described in Example 8.
In addition, it was also demonstrated that ApoE inhibits the
development of angiogenic lesions and he~An~iomas in vivo in
the endothelioma model. Thus, intravenous (i.v.)
administration of met-apoE at a concentration of 0.4
mg/mouse, daily, for 8 days, resulted in a 40-60~ reduction
in the size of the hemangioma compared to the non-ApoE
treated control.
Groups of ten mice were injected with the Matrigel
suspension of END2 cells described above. The groups were
then treated either with 0.2ml saline (control group) or
0.2ml saline cont~i ni ng 2mg/ml met-apoE (experimental
group). The animals were sacrificed on the ninth day and
the tumors were scored on a scale of 0-3 (0=no tumor). The
results are shown in Table l.
WO94/04178 2 1 4 1 ~ 9 8 PCT/US93/07582
-53-
Table 1
Group n~ Score2
Control 10 2.75+0.42
Experimental 10 1.7+0.82
1 n=number of mice
2 score: mean+st~n~d deviation
lo The results were found to be significant (P<0.005) according
to the Wilcoxon Rank Sum Test.
Based on the above results, ApoE will be effective in
treating hemangiomas in humans.
.
W094/04l78 2~ ~1598 PCT/US93/07582 -
-54-
~Amnle 8. APO E Fraqments
As mentioned earlier, ApoE has a specific ability to bind to
lipoprotein particles for directing their removal from the
plasma, "reverse cholesterol homeostasis", and for mediating
receptor dependent endocytosis to the liver. Due to the high
numbers of li~u~Lein particles cont~;ne~ in plasma, ApoE,
upon delivery in small amounts to the site of a tumor, will
bind to li~o~-o~ein particles and will be unavailable to
function as an anti-proliferative agent. Therefore, full
length ApoE (e.g. met-apoE) in the presence of serum or
plasma is able to inhibit proliferation of cells only upon
delivery in large amounts to the tumor.
This deficiency may be overcome by cleavage of the apoE
se~uence to remove the lipid binding domain present at the
carboxy terminus. Upon cleavage of apoE with a proteolytic
enzyme and elimination of the carboxyl end lipophilic
domain, the remaining amino terminal fragment no longer has
normal affinity for binding to lipoprotein particles but
still readily binds to heparin and heparan sulfate
proteoglycans. Therefore smaller amounts of ApoE will
suffice to obtain inhibition of cell proliferation.
In a particular embodiment, such an ApoE fragment may be
produced from apoE containing the complete se~uence of
naturally occurring apoE by cleavage of the carboxyl
terminus with a proteolytic enzyme generating, for example,
a 22 kD fragment containing the amino terminus (Thuren,
1992). One example of a proteolytic enzyme that may be used
is thrombin. Upon digestion of recombinant met-apoE by
thrombin , one 22 kD and one 10 kD fragment may be obtained.
The 22 kD fragment, containing the first l91 amino terminal
residues, has the binding sites for manganese heparan
WO94/04178 2 1 4 1 ~ 9 8 PCT/US93/07582
sulfate and heparin and the low density lipoprotein tLDL)
receptors, but has reduced ability to interact with natural
lipoprotein particles.
An improved method of obt~; ni n~ an increased amount of the
free form of ApoE in the plasma and the reduction of ~ts
sequestration by lipid particles, is by production of an
ApoE fragment deleted of the major lipid binding domain of
apoE from the carboxyl terminus by means of recombinant DNA
techn;ques.
In a specific ~ho~;ment, an ApoE polypeptide fragment,
ApoE6-2, spanning amino acids 1-217 of apoE is encoAe~ and
expressed by plasmid pTVR6-2 (Figure 17). This 28KD MW
ApoE was also tested for its effect on cell proliferation.
The purification of this ApoE is similar to the purification
of met-apoE as described in Example 1 with minor
modifications, one of which was ultrafiltration with a 50K
instead of lOOK cutoff membrane. The purified polypeptide
was then treated with 6M urea and dialyzed to remove the
urea.
ApoE6-2 inhibited proliferation of BAEC culture with an IC50
around 0.3 ~M at both 0.5% and 2.5% FCS respectively (Figure
24). Thus, in contrast to met-apoE, which displays a
substantial reduction in activity at higher serum
~once~trations, ApoE6-2 is much less affected by increasing
serum concentration.
This ApoE was also found to inhibit the proliferation of
END-2 cells. The proliferation of END-2 was measured as
indicated in Figure 18 in the presence of 0.5% FCS and the
indicated amounts of ApoE6-2.
Based on these results, this and similar ApoE fragments will
WO94/04178 PCT/US93/07582
21~98
-56-
provide high antiproliferative and antiangiogenic activity
in vivo (where serum conc ntration is 100%).
~ WO94/04178 ~141~ 9 8 PCT/US93/07582
.
-57-
~ample 9. Reversal of antithrombin activitv of he~Arin
bY A~oE
- In this example, the effect of ApoE on the activity of
heparin in a heparin-antithrombinIII-thrombin complex was
demonstrated. Heparin activates antithrombinIII which in
turn inhibits thrombin activity. Sequestration of heparin
from this complex by ApoE results in the inactivation of
antithrombinIII and a conco~;tant increase in thrombin
activity. The effect of ApoE is dose depen~nt (Figure 25).
The stoichiometry of the reaction indicates that ApoE
displays high affinity for heparin, and that one mole of
ApoE can bind 2-3 moles of heparin.
Thrombin activity was assayed as described in Example 1.
To be more specific, in a total volume of 140~1,
antithrombin (50~1 at 2.9U/ml, Sigma), heparin (15 ~1, Eli
Lilly 14KD, 0.4 USP/mI; 2~M final ronc~ntration in the
reaction) and met-apoE (15~1 of the indicated dilutions of
a 60~M solution) were preincubated for 30 seconds. Thrombin
(10~1, 20U/ml, Sigma) was then added and the reaction mix
further incubated for an additional 30 seconds folowing
which the chromogenic substrate (2~M, Sigma) was added. The
results are shown in Figure 25. Under the reaction
conditions used, no decrease in thrombin activity by heparin
was observed until the met-apoE was diluted 64 fold. 50%
inhibition of thrombin activity was achieved at
approximately 1:100 dilution of ApoE (0.6~M) Therefore one
molecule of ApoE can bind about 2-3 molecules of heparin.
WOg4/04178 PCT/US93/07582 ~
21 ll598 -58-
Refere~ae~
Aznavoorian, S., Liotta, L.A., Triangle 30, 89, 1991.
Basilico, C., Moscatelli, D., Adv. ~nc~r, Res., 1992, in
press.
Boyles, J.K., et al., J. Clin. Invest., 83: 1015, 1989.
Burgess, W.H., Maciag, T., Annu. Rev. Biochem., 58: 575,
1989.
Cardin, A.D., Bowlin, T.L., and Krsten~ncky~ J.L., Biochem.
Biophys. Res. Commun., 154: 741, 1988.
Castellot, J.J., Jr., Wright, T.C., Karnovsky, M.J., Semin.
Thromb. Hemostasis, 13: 489, 1987.
Clowes, A.W., Karnovsky, M.J., Nature (Lond.), 265: 625,
1977.
D'Amore, P.A., Semin. Thromb. Hemostasis, 14: 73, 1988.
Dyer, C.A., Curtiss, L.K., J. Biol. Chem. 266: 22803, 1991.
Edelman, R., Nugent, M.A., Smith, L.T., Karnovsky, M.J., J.
Clin. Invest., 89, 465, 1992.
Folkman, J. Sci. Am., 234: 58, 1976.
Folkman, J., Klagsbrun, M., Sasse, J., et al., Am. J.
Pathol., 130: 393, 1988.
Folkman, J., Klagsbrun, M., Sasse, J., et al., Am. J.
Pathol., 130: 393, 1989.
~ WO94/04178 21415 9 8 PCT/US93/07582
-59-
Folkman, J., Watson, K., Ingber, D., ~AnhAh~n, D. Nature
339, 58, 1989.
Hertz, J., Hamann, U., Rogne, S., et al., EMBO J., 7:4118,
1988.
Hui, D.Y., Harmony, A.K., Innerarity, T.L., M~hley, R.W., J.
Biol. Chem., 255, 11775, 1980.
Innerarity, T.L., J. Biol. Chem., 254: 4186, 1979.
Klagsbrun, M., Baird, A., Cell, 67: 22g, 1991.
Tin~ner, V., Reidy, M.A., Proc. Natl. Acad. Sci. U.S.A., 88:
3739, 1991.
Tin~er, V., Majack, R.A., Reidy, M.A., J. Clin. Invest.,
85:2004, l99Q.
Liotta, L., Sci. Am., 266(2): 54, 1992.
Liu, M.W., Roubin, G.S., Robinson, K.A., et al.,
Circulation, 81: 1089, 1990.
Lund, H., TA~Ah~ch;, K., Hamilton, R.L., et al., Proc. Natl.
Acad. Sci. U.S.A., 86: 9318, 1989.
Mahley, R.W., Science, 240, 622, 1988.
Mahley, R.W., Weisgraber, K.H., Hussain, M.M., et al., J.
Clin. Invest., 83: 2125, 1989.
.
Mahley, R.W., Weisgraber, K.H., Innerarity, T.L., Biochim.,
Biophys. Acta, 575,81, 1979.
WO94/04178 ~ 9 ~ PCT/US93/07582
-60-
Ruoslahti, E., Yamaguchi, Y., Cell, 64: 867, 1991.
Schweigerer, L., Nuefeld, G., Friedman, J., et al., Nature
(Lond.) 325: 257, 1987.
Shing, Y., Folkman, J., Haudenschild, D., et al., J. Cell.
Biochem., 29: 275, 1985.
Terkeltaub, R.A., Dyer, C.A., Martin, J., et al., J. Clin.
Invest. 87: 20, 1991.
Thomas, K.A., Gimenez-Gallego, G., Trends Biochem. Sci. 11:
81, 1986.
Thuren et al., Biochemistry 31: 2332 (1992)
Vlodavsky, I., Folkman, J., Sullivan, R., et al., Proc.
Natl. Acad. Sci., USA, 84: 2292, 1987.
Vogel, T., et al., Proc. Natl. Acad. Sci., USA 82: 8696,
1985.
Weisgraber, K.H., Rall, S.C., Jr., Mahley, R.W., et al., J.
Biol. Chem. 261: 2068, 1986.
Wienberg, et al., Biochemistry 28: 8263, 1989.
Wilson, C., Wardell, M.R., Weisgraber, K.H., et al., Science
252: 1817, 1991.
Yayon, A., Klagsbrun, M., Esko, J.D., et al., Cell 64: 841,
1991 .
Yamada, N., Inoue, I., Kawamura, M., et al., J. Clin.
Invest. 89: 706, 1992.
WO94/04178 2 1 4 1 5 9 ~ PCT/US93/07~82
-61-
Yamada, N., Shimano, H., Mokuno, H., et al., Proc. Natl.
Acad. Sci., USA, 86: 665, 1989.
