Note: Descriptions are shown in the official language in which they were submitted.
WO 95/05190 ~ 1 6 7 9 ~ 2 PCT/US94/09192
--1--
MæT~OD OF INEIBITING ~APOSI'S SARCOMA
5 This application is a continuation-in-part of U.S. Serial
No. 08/105,900, filed August 12, 1993.
Background of the Invention
This invention relates to the use of Apolipoprotein E in a
method of inhibiting Kaposi's sarcoma.
Throughout this specification, various publications are
re~erenced within parentheses. Full citations for these
references may be found at the end of the specification
immediately preceding the claims. The disclosures of these
publications in their entireties are hereby incorporated by
reference into this application in order to more fully
describe the state of art as known to those skilled therein
as of the date of the invention described and claimed
herein.
Kaposi's sarcoma (KS) is a malignant tumor manifest as a
rare skin lesion which was known to occur mainly in older
men of Mediterranean origin (Mcnutt, 1983; Kaposi, 1982
Ensoli, 1991). KS is now known to be the most common
neoplasm associated with hllm~n ;mmllnodeficiency virus (HIV)
infection (Gallo, 1984) and was one of the first indications
that acquired ;mmllnodeficiency syndrome (AIDS) had reached
the USA (Safai, 1985; Safai, 1987; Safai, 1985; Safai, 1987;
Gross, 1989; Palca, 1992; Farizo, 1992). People with
impaired immune systems secondary to organ transplant
medication are particularly susceptible to this skin cancer,
which can be fatal in severe cases.
WO95/OS190 2 1 6 7 ~ ~ 2 PCT~Ss~/09192 ~
--2--
AIDS-related Kaposi's sarcoma (AIDS-KS) is a multifocal
proliferative disorder, characterized by proliferating
spindle shaped cells of probable macrovascular endothelial
cell origin, edema and aniogenesis (Gallo, 1984; Safai,
1985; Safai, 1987). Based on epidemiological studies, it
is believed that AIDS-KS may be caused by an infectious
agent other than HIV (Volberding, 1985; Huang, 1992).
The achievement of long term cell culture of AIDS-KS-derived
spindle cells made possible in vitro and in vivo studies of
the pathogenesis and the regulation of the growth and
development of AIDS-KS cells (Nakamura, 1988; Sal~h~ ; n,
1988). AIDS-KS cells have been shown to share phenotypic
properties with endothelial cells (Nakamura, 1988;
Sal~h~ ;n, 1988; N~;m;, 1988).
The growth of these KS-derived spindle cells requires
inflammatory cytokines and angiogenic factors (Barillari,
1992; Ensoli, 1991). Several factors, including
interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-4
(IL-4), gamma-interferon, transforming growth factor-~ (TGF-
~), platelet factor-4, and tumor necrosis factor-~ (TNF-~),
have been shown to modulate the rate of proliferation of
AIDS-KS cells in culture (Barillari, 1988; Miles, 1990;
Ensoli, 1989; Ensoli, l991). The cytokine Oncostatin M is
one such factor which is p~oduced by activated immune cells.
Oncostatin M is a member of the cytokine family that
includes IL-6 (Tamm, 1989), granulocyte-macrophage colony
stimulating factor (GM-CSF) (Clark, 1987), and lellkPm;~
inhibitory factor (LIF) (Gearing, l991). Oncostatin M,
normally produced by activated lymphoid cells, serves as a
potent regulator of the growth and differentiation of a
number of normal and tu-m~or cells, and has recently been
found to be a very potent exogenous growth factor for AIDS-
KS cells (Miles, 1992; Nair, 1992). Upon exposure to
21~7942
WO95/05190 pcT~ss~lo9ls2
.. ~
--3--
Oncostatin-M, AIDS-KS cells develop the typical spindle-
shaped morphology and characteristics of twmor cells (Gross,
1989). It therefore appears that Oncostatin M plays a role
in the pathogenesis o~ AIDS-KS.
Oncostatin M mP~;~tes the growth stimulation of KS derived
cells via basic fibroblast growth factor (bFGF) (Burgess,
1989). bFGF is a strong heparin-binding protein, present
in virtually all tissues, and having multiple mitogenic and
angiogenic effects (E~e1m~n, 1992). bFGF is considered to
be one of the most potent angiogenic inducers both in vivo
and in vitro (Folkman, 1976; Folkman, 1988; Folkman, 1989).
Therefore, agents that interfere with the activation of bFGF
receptors will inhibit the ability of Oncostatin M to affect
cell growth (Dionne, 1990).
Tumor development is dependent on the adequate provision of
oxygen and nutrients and therefore on extensive
vascularization. The development of blood vessels is known
as angiogenesis. Inter~erence w th angiogenesis is
therefore one approach to inhibitio~ of tumor development
(D'Amore, 1988; Folkman, 1989). FurthermQrel angiogenesis
is also one of the characteristic~manifestations of Kaposi's
sarcoma since it involves endothelial cell proliferation
(Gallo, 1984; Gross, 1989; Safai, 1985, Sa~ai, 1987).
Endothelial cell proliferation is dependent on signal
transduction mP~;~ted by b; n~; ng of bFGF (and other growth
factors) to cell surface specific receptors (Ruoslahti,
1991). Proteoglycans are present in the extracellular
matrix and on the outer surface of the cell. Receptor
binding of bFGF is dependent on heparintheparan-sulfate
proteoglycans (HSPG) which function as an intermediate
receptor for bFGF and other heparin-binding growth ~actors
(Ruoslahti, l991; Yayon, 1991). Protein~ that interact with
WO 95/05190 PCT/US9~/09192
2~-6~2 ~
--4--
HSPG compete with FGF and thus play a major role in the
regulation of angiogenesis.
Kaposi's sarcoma is currently treated with cytotoxic agents
such as vinblastine, and bleomycin (Volberding, 1985; Gill,
1990), or a combination of low doses of doxorubicin,
bleomycin, and vincristine (Heagy, 1989; Gill, 1991; Gill,
1992). Other experimental drugs such as suramin (Levine,
1986), alpha interferon (Krown, 1983; Merigan, 1988),
platelet factor 4 (Maione, 1990), and pentozan-polysulfate
(Biesert, 1989), have also been evaluated. Recently, a
sulfated polysaccharide-peptidoglycan compound (SP-PG)
produced in bacteria, has been found effective in control
of in vitro and in vivo proliferation of AIDS-KS cells
(Nakamura, 1992,1988).
SP-PG may exert its inhibitory effect on KS cells either by
immobilizing bFGF in the extracellular matrix or by
inhibiting binding of free bFGF to the FGF receptor.
However, the use of heparin-like molecules, such as SP-PG
is likely to increase the risk of prolonged bleeding and
thus engender risk of hemorrhage. In addition, due to
various hematologic and immunologic complications associated
with HIV infection, most AIDS patients are unable to
tolerate aggressive chemotherapy. It is therefore necessary
to provide a therapeutic agent with ~;n;m~l or no side
ef~ects ~or treatment of Kaposi's sarcoma.
Apolipoprotein E (apoE) is a plasma protein having high
affinity for heparin and HSPG (Mahley, 1988). The most well
studied functions of apoE include its role in cholesterol
and plasma lipoprotein metabolism (Mahley, 1988). ApoE
interacts with the low density lipoprotein (LDL) receptor
and the LDL-related receptor-protein (LRP) (Beisiegel, 1988;
Lund, 1989; Herz, 1988). HSPG is now known to play a major
~ W095/05190 2~67~42 PCT~s94/09l92
role in the binding and uptake of apoE-enriched lipoprotein
particles by cultured cells (Zhong-Sheng, 1993). It has
also been observed that apoE is synthesized by a number of
cells that have no known role in cholesterol homeostasis
(Hui, 1980; Boyles, 1989; Boyles, 1985).
The cloning and expression of ApoE in E. coli (Vogel, 1985)
has now provided an inexpensive and readily available source
of recombinant protein making possible the investigation of
the role of ApoE in cellular functions. The cloning and
expression of ApoE in E. coli has been disclosed in
coassigned U.S. Patent No. 5,126,252.
The present application discloses the use of Apolipoprotein
E in treatment of AIDS-KS and ~mon~trates its activity in
in vitro and in vivo KS models (Nakamura, 1988; Sal~h~ n~
1988).
WO 95/05190 PCT/US9-1/09192
21679~2 1~
--6--
SummarY of the Invention
A method is provided of inhibiting Kaposi's sarcoma
comprising contacting the Kaposi's sarcoma with an amount
of Apolipoprotein E effective to inhibit the Kaposi's
sarcoma.
Additionally, a composition is disclosed for treating
Kaposi~s sarcoma comprising ApoE and a ph~rm~ceutically
acceptable carrier.
A method is also provided a method of treating a subject
suffering from Kaposi~s sarcoma comprising a~m1n;stering to
the subject an amount of the compositior comprising ApoE and
a pharmaceutically acceptable carrier effective to treat the
Kaposi's sarcoma.
~ wO gs,Os,g~ 2 ¦ 6 7 a 4 2 PCT~594/~9l9~
Brief Description of ~he Fi~ures
Figure 1 shows the effect of serum concentration on in vitro
inhibition of mitogenesis of Kaposi's sarcoma cells by ApoE.
The incorporation of 3H-thymidine into DNA of human Kaposi's
sarcoma KS3 cells was tested as described in Example 2 with
varying concentrations of serum (FCS) and met-apoE. At 1~
(lA) and 5~ (lB) FCS, ~m;n; ~tration of Q.3~M met-apoE
resulted in 35~ and 55~ inhibition of DNA synthesis
respectively.
Figure 2 shows the inhibition of mitogenesis of Kaposi's
sarcoma cells by ApoE in the presence of growth promoting
substrates. The incorporation of 3H-thymidine into DNA of
human Kaposi's sarcoma RW248 cells was tested as described
in E~ample 2, in the presence o~ fetal calf serum (FCS)
either alone (1.25~ (2A) and 2.25~ t2B?), or in combination
with conditioned media (CM) ~1.25~ FCS and 20~ CM (2C)).
Met-apoE was added at the indicated concentrations. Using
the data shown in the Figure, the following were determined
to be the ApoE concentration~ with which 50~ inhibition o~
mitogenesis (IC50) was obtained: 0.28~, 0.45~M, and 0.37~M
in 2A, 2B and 2C, respectively.
Figure 3 shows the inhibition o~ mitogenesis o~ Kaposi's
sarcoma cells by ApoE in the presence of growth promoting
substrates. This experiment was similar to that described
in Figure 2, but included addition of the growth promoting
substrate Oncostatin M. ~llm~n Kaposi's sarcoma RW248 cells
were assayed in the presence of 1~ FCS, and either 20~
conditioned medium (3A) or 50ng/ml Oncostatin M (3B), and
~arying concentrations of met-apoE. Using the data shown
in the Figure, the following met-apoE concentrations were
detPrm;nPd to inhibit mitogenesis by 50~: 0.069~M and
0.995~M (3A and 3B respectively).
WO95/05190 2 ~ ~ 7 ~ PCT~S94/09192 ~
Figure 4 shows the inhibition of proliferation of Kaposi's
sarcoma cells by ApoE. The proliferation of Kaposi's
sarcoma KS3 cells was assayed as described in protocol Pl
in Example 2, in the presence of 0.5~ FCS and 20~
conditioned media, either alone (ctr), or together with the
indicated concentrations of met-apoE, either non-heated or
heated for 20 minutes at 100C.
Figure 5 shows the inhibition of proliferation of Kaposi's
sarcoma cells by ApoE. The proliferation of hllm~n Kaposi's
sarcoma RW248 cells was assayed as described in Example 2,
protocol P2, in the presence of 2.5~ FCS and 3Ong/ml
Oncostatin M, either alone or together with the indicated
concentrations o~ met-apoE (-), met-apoE heated 30' at 100C
(-), or buffer control (-) (lO~ ~ormulation buffer i.e.
O.lmM cysteine, 0.2mM sodium-bicarbona~e, lXPBS).
Figure 6 shows the effect of heparin binding molecules on
proliferation of Kaposi's sarcoma cells. The inhibition of
proliferation of human Kaposi~s sarcoma KSY-l cells by met-
apoE, the fibronectin cell binding ~om~;n (FN33), and
thrombospondin (TSP) (obtained from human platelets,
Roberts, 1985) was compared as described in Example 2. The
concentration units shown in the figure are ~M for FN33 and
met-apoE, and l0-2~M for TSP. Of the three substances
tested, only met-apoE caused a consistent and dose-dependent
inhibition of proliferation.
Figure 7 shows the inhibition of chemotaxis of Kaposi's
sarcoma cells by ApoE. Chemotaxis (directed migration) in
response to conditioned medium or fibronectin was measured
as described in Example 2. Trypsinized human Kaposi's
sarcoma KSY-l cells were resuspended in complete ISCOV
medium and allowed to equilibrate for 2 hours. The cells
were recovered by centrifugation and suspended in ISCOV,
2~7~2
~ WO95/OS190 PCT~S94/09l92
g
0.1~ BSA, at one million cells per ml. The cells were mixed
with the indicated concentrations of met-apoE and allowed
to equilibrate for 15 minutes at room temperature prior to
adding to the upper well of the chemotaxis chamber.
Migration towards chemoattractants in the lower chamber [Ctr
(0.1~ BSA), CM (conditioned m~;A, 20~), or FN (hnm~n
fibronectin, 0.1 ~M)], in the presence of the indicated
concentrations of met-apoE was measured following incubation
for 4.5 hours.
Figure 8 shows the inhibition of chemotaxis of Kaposi's
sarcoma cells by ApoE. Chemotaxis was measured as described
in Example 2. Trypsinized hnm~n Kaposi's sarcoma RW248
cells were resuspended in complete RPMI medium and allowed
to equilibrate for 2 hours. The cells were recovered by
centrifugation and suspended in RPMI, 0.1~ BSA , at 0.5
million cells per ml, prior to adding to the upper well of
the chemotaxis chamber. Migration towards BSA (0,1~), or
Oncostatin M in the presence of the indicated concentrations
of met-apoE in the lower cha-m-ber was measured following
incubation for 3 hours.
Figure 9 shows the inhibition of KS-induced tumors by ApoE.
Human Kaposi's sarcoma RW248 cells (4 x 106) were
transplanted subcutaneously into BALB/c nu/nu athymic mice.
Met-apoE was ~m~n;stered intravenously for 5 days at the
indicated doses. On day 6, the An;m~ls were sacrificed and
the size of the tumors measured. Each value is the mean of
10 ~n;m~ls
Figure 10 shows the histology of angiogenic lesions induced
by KS cells. Histological sections of angiogenic lesions
induced by RW248 cells in the absence ~A) or presence (B)
of met-apoE treatment were obtained as described in Figure
WO 95/05190 PCT/US9~/09192
21~7~2
-10-
9, fixed with 10~ formalin, stained with hematoxylin-eosin,
and photographed. The bar is 100 microns.
Figure 11 shows plasmid pTVR 590-4. Plasmid pTVR 590-4,
deposited in E. coli W1485 under ATCC Accession No. 67360,
is a good expressor of met-apoE under control of the APL
promoter as is described in Example 1. (E. soli W1485 is
freely available from ATCC under Accession No. 12435.)
Figure 12 shows plasmid pTVR6-2. Plasmid pTVR6-2 expresses
a polypeptide fragment of ApoE containing the first 217
Amino acids of naturally occurring apoE; it is not yet known
if an additional N-t~rm; n~ 1 methionine is present.
Production and purification of this polypeptide has been
carried out essentially as described in Example 1 for met-
apoE except that ultrafiltration was performed with a 50K
cassette and the purified polypeptide was treated with 6M
urea. Expression of the polypeptide fragment is under
control of the ~PL promoter and production of the
polypeptide is essentially as described in Example 1.
Plasmid pTVR6-2 was deposited in E. coli 4300 on July 26,
1993 under ATCC Accession No. 69364.
Figure 13 shows the inhibitory effect of peptide 348 on
mitogenesis of human Kaposi's sarcoma RW248 cells as
measured by 3H-thymidine- incorporation as described in
Example 2.
Figure 14 shows the inhibitory effect of intravenous ~iv)
ApoE on the size of tumors induced in mice by KSY-1 cells
as described in Example 2.
Figure 15 shows the inhibitory effect of ApoE on KS induced
vascular hyperp~rmeAhility as described in Example 2.
2~679~
WO95/05190 -ll- PCT~S94/09192
Detailed Description of the In~ention
A method is provided of inhibiting the proliferation of
Kaposi's sarcoma cells comprising contacting the Kaposi's
sarcoma cells with an amount of Apolipoprotein E (ApoE)
effective to inhibit proliferation of the Kaposi's sarcoma
cells.
Inhibition of proliferation of Kaposi's sarcoma cells means
reducing the rate of proliferation of the cells.
A composition is disclosed for inhibiting proliferation of
Kaposi's sarcoma cells comprising Apolipoprotein E in an
amount effective to inhibit the proliferation of the cells
and a suitable carrier. The use of ApoE in the making of
such a composition is also provided.
Additionally, a method is provided of treating a subject
suffering from Kaposi's sarcoma comprising a~m;n;stering to
the subject an amount of Apolipoprotein E effective to treat
the Kaposi's sarcoma.
Treating the Kaposi's sarcoma means preventing the growth
of, or reducing the size or rate of growth of the Kaposi's
sarcoma.
Additionally, a method is provided of treating edema in a
subject suffering from Kaposi's sarcoma comprising
~m;n; ~tering to the subject an amount of Apolipoprotein E
effective to treat the edema.
The Apolipoprotein E may be ~m; n;stered by any means known
to those skilled in the art. In particular embo~;m~nts, the
Apolipoprotein E is ~m;n;stered intravenously (i.v.) or
- 35 subcutaneously (s.c.).
WO95/05190 2 ~ ~ 7 9~ 2 PCT~S94/09192 ~
-12-
Also disclosed is a ph~rm~ceutical composition comprising
Apolipoprotein E in an amount effective to treat Kaposi's
sarcoma and a ph~ rm~ ceutically acceptable carrier.
The amount effective to treat Kaposi's sarcoma is O.lmg -
lg Apolipoprotein E. The precise amount, and the frequency
of ~m~ n; stration of the dose, will be readily determined
by one skilled in the art, based on the characteristics of
the formulation, body weight and condition of the subject,
tumor size, route of ~m;n;stration~ and the characteristics
of the particular Apolipoprotein E polypeptide to be used.
In an additional embodiment the invention encompasses an
article of manufacture comprising packaging material and a
ph~ rmA ceutical agent contained within the packaging
material, wherein the ph~rm~ceutical agent is
therapeutically effective for inhibi~ing proliferation of
Kaposi' 9 sarcoma cells and wherein the packaging material
comprises a label which indicates that the pharmaceutical
agent can be u~ed for the inhibition of proliferation of
Kaposi~s sarcoma cells and wherein the p~rm~ceutical a~ent
comprises Apolipoprotein E.
In additional embodiments, the invention encompasses the use
of Apolipoprotein E for treating edema, a composition
comprising Apolipoprotein E for treatment of edema; and the
use of Apolipoprotein E in the manufacture of a composition
for the treatment of edema.
The term "Apolipoprotein E" (ApoE) as used herein
encompasses any polypeptide, regardless of source e.g.
naturally occurring or recombinant, which includes the
sequence of naturally occurring apoE necessary for the
biological activity of inhibiting proliferation of Kaposi's
sarcoma cells, and mutants whose sequence varies by one or
WO95/05190 PCT~S94109192
2~6~2
-13-
more, typically less than ten amino acids, provided that
such mutants have the biological activity of inhibiting
proliferation of Kaposi's sarcoma 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.
Recombinant 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 se~uence encoding the recombinant ApoE in
a vector DNA molecule such as a plasmid which may be
constructed by recombinant DNA techniques so that the
sequence encoding the recombinant ApoE is incorporated at
a suitable position in the ~ector. The cells are preferably
bacterial cells or other unicellular organisms, but
eucaryotic cells such as yeast, insect or m~mm~1ian cells
may also be used to produce recombinant ApoE.
In one embodiment, the ApoE is a mutant differing from the
naturally occurring polypeptide by the addition, deletion,
or substitution of one or more non-essential amino acid
residues typically less than lO, provided that the resulting
polypeptide retains the KS-inhibitory acti~ity 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 procedures, including,
for example, conventional methods for the design and
manufacture of DNA se~uences coding for bacterial expression
of mutants of the subject polypeptide, the modification of
WO 95/05190 ~ PCT/US94/09192 ~
2~ 67~2
-14-
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 detenm~ n~ tion of the biochemical
activity of the polypeptides using conventional biochemical
assays.
Examples of mutants of apoE are deletion mutants ~ont~;ning
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 KS-inhibitory activity of
naturally occurring apoE.
Polypeptides having substantially the same amino acid
sequence as naturally occurring apolipoprotein E encompass
the addition or deletion of fewer than four amino acids at
the N-term;nll~ of the amino acid sequence of the
polypeptide. There may be additional substitutions and/or
deletions in the ~equence which do not eliminar~ the KS-
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 equivalent
groups described by e.g. Lehninger, Biochemistry, 2nd ed.
Worth Pub., N.Y. (1975); Creighton, Protein Structure, a
Practical Approach, IR~ Press at Oxford Univ. Press, Oxford,
England (1989); and Dayhoff, Atlas of Protein Sequence and
Structure 1972, National Biomedical Research Foundation,
Maryland (1972).
In a particular em~odiment, the ApoE is recombæ~nt met-
apoE, e.g. recombinant apoE with an additional methionine
~ W095/05190 2 1 ~ ~ ~ 4 2 PCT~S94/09192
-15-
at the N-t~rm;nlls of the sequence of naturally occurring
apoE.
Also encompassed by the term "Apolipoprotein E" are
polypeptide fragments of recombinant ApoE and of naturally
occurring apoE which exhibit the KS-inhibitory activity of
apoE. One example of such a fragment is a 30-mer fragment,
designated peptide 348, disclosed in U.S. Patent No.
5,177,189, issued January 5, 1993 (see also Dyer, Smith, and
Curtiss (1991), and Dyer and Curtiss, (1991)).
Additional examples of such polypeptide fragments have amino
acids 1-217, 1-187 or 1-185 of naturally occurring apoE.
A particular embodiment of a polypeptide fragment having
amino acids 1-217 of naturally occurring apoE is encoded by
plasmid pTVR6-2 (Figure 12) which was deposited in E. coli
4300 on July 26, 1993 with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland under
Accession No. 69364.
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
obtaining such polypeptides are well known to those skilled
in the art and are described in numerous publications
including Sambrook, Fritsch and Maniatis, Molecular Cloning:
A ~aboratory ~nn~l, 2nd edition, Cold Spring Harbor
Laboratory Press, USA ~1989).
Preferably, the ApoE is ~m; n; stered in a ph~rm~ceutically
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,
WO95/05190 ~ 7 ~ 4 ~ pcT~ss~losls2
-16-
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.
In sum, the method of the present invention may be practiced
with any Apolipoprotein E having substantially the same KS-
inhibitory activity as naturally occurring apoE.
21~7942
WO95/05190 .-~ PCT~S94/09192
.
-17-
Exam~le~
The following examples are presented to illustrate the
invention. They are specific embodiments which are set
forth to aid in underst~n~;ng the invention, but are not
intended and should not be construed to limit in any way the
scope of the invention as set forth in the claims which
follow.
WO95/05190 ~ 9~;~ ` ' pcT~s9~losls
-18-
xample 1: Method of Production of a Recombinant ApoE
Polypeptide
Met-apolipoprotein E was produced by the recombinant host-
plasmid system comprising E. coli W1485 harboring plasmidpTVR 590-4 which has been deposited in the ATCC under
Accession No. 67360. Plasmid pTVR 590-4, shown in Figure
11, produces, as insoluble inclusion bodies, recombinant
met-apoE having the sequence of naturally occurring apoE
plus an additional N-term;n~l methionine. The inclusion
bodies may be isolated and the recombinant met-apoE
recovered and purified. The description of a specific
embodiment of the production and recovery of purified
recombinant met-apoE follows.
I. Production of E. coli cont~;n;nq recombinant met-apoE
1. Seed Flask Development
The contents of frozen vials cont~;n;ng ATCC No. 67360 were
used to inoculate seed flasks cont~;n;ng 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
Ampicillin, sodium salt 0.l g
Deionized water 1 L
WO95/05190 216 7 9 4 2 PCT~S94109192
.
-19 -
The trace elements stock solution contains:
MnS04 .H20 1 g
ZnSO4 .7H20 2.78 g
Cocl2~6H2o 2 g
Na~MoO4 2H20 2 g
CaCl2 2H2
CuS04 5H20 1.85 g
H3BO3 0 5 g
Concentrated HCl 100 m~
Deionized Water 900 mL
The glucose and ampicillin are added from sterile
concentrated stock solutions after autoclaving the other
components of the medium. The cultures are incubated at
30C overnight on a rotary shaker at 250 rpm, and reach an
OD660 of about 3.5-5Ø
2. Seed Fermenter
The content~ o the seed ~lask were used to inoculate a 50
L seed fermenter cont~;n;ng 25-30 L of the following
production medium, which contains per liter:
K2HPO4 8 g
KH2P04 2 g
Sodium citrate 2 g
NH4Cl 2 g
FeN~ citrate 0.02 g
CaCl2.2H2O 0.04 g
K2S04 0.6 g
Trace elements solution 3 mL
- (as in Section 1)
Antifoam
- 35 (Silicolapse 5000) 2 mL
WO95/05190 ~ 6 7 ~ 4 2 `; - - PCT~S94/09192
-20-
Added after sterilization (per liter of medium)
MgSO4 . 7H20
Sodium ampicillin 0.1 g
Glucose 40-60 g
NH3 (25-28~ in water) approx. 40 mL
Glucose is added batchwise at inoculation; ammonia is
automatically added as needed to maintain the pH at about
7 during growth (set point of controller pH = 7.0).
The culture is cultivated at 30C for 15-20 hours. The OD660
generally reaches 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 were used to inoculate
a 750 L (nomt n~l volume) fermenter contA;n;ng about 360 L
of the same production medium described for the seed
fermenter, but excluding ampicillin. The culture is
cultivated at 30C until an OD~o Of about 10 i8 obtA;ne~.
Induction of ApoE expression is then achieved by raising the
temperature of the fermenter to 42C. At the start of
induction, the following are added to the fermenter:
D~-methionine 0.6 g per L of medium
Sodium acetate 5 g per L of medium
The sodium acetate (0.1~ ) is added to protect cells
from the "toxic e~fect" caused by ApoE.
The fermenter temperature is maintained at 42C for three
hours, at which time the cells are harvested. The OD~o of
WO95/05190 % ~ 9 4 2 PCT~S94/09192
-21-
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 was centrifuged at about 14,000 rp~
(16,000 g) in a CEPA 101 tubular bowl centrifuge at a feed
rate of 250L/hr, and the resulting cell cake weighing about
Kg was stored frozen until further processing.
Alternatively, the cell suspension may be centrifuged in a
Westfalia CSA-l9 continuous centrifuge at 500 L/hr. The
sludge cont~; n; ng the recombinant met-apoE may either be
processed immediately or stored frozen.
In either case, the supernatant obtained following
centrifugation by either method cont~;ne~ no detectable met-
apoE as detPrm;ne~ by SDS-polyacrylamide gel
electrophoresis.
II. Recovery of purified recombinant met-apoE
The following method is suitable for scale-up for industrial
application and yields very pure ApoE. The general scheme
of the downstream process consists of steps A through G as
follows:
Downstream Processinq of Recombinant Human Met-apoE
~ ~ 6~4~
WO 95/05190 PCT/US9 V09192
-22 -
A CELL DISRUPTION IN PRESENCE OF MAGNESIUM IONS.
B EXTRACTION OF CELL PE~LET WITH TRIT~N~.
C 100K ULTRAEILTRATION.
D DEAE CHROMATOGRAPHY
E Q SEPHAROSE CHROMATOGRAPHY
F CM SEPHAROSE CHROMATOGRAPHY
G 100K ULTRAFILTRATION - TRITON~ REMOVAL.
The following procedure was performed on 3 Kg cell cake
obtained as described above. In addition, 15 Kg cell cake
were processed using the same method with only minor
modifications involving scale-up of the size of the
e~uipment used.
Steps A through D were performed on 2 batches of bacterial
cake, each weighing 1.5 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. A11 other activities were performed
at room temperature.
A. Cell Disru~tion in Presence of Maqnesium Ions
l.S Kg of wet cell cake was suspended in 6 L of buffer A
which consists of 50 mM Tris-HCl, 30 mM MgCl2, 0.25~ beta
hydroxybutyrate sodium salt, pH=7.5. (The beta
hydroxybutyrate was added as a protease inhibitor). This
was then homogenized using a ~in~m~tica homogenizer yieldins
7.5 L of homogenate. Disruption was then performed using
a Dynomill KDL bead mill disrupter (Willy A. Bachofen,
Basel) at 5 ~/hr (in two cycles). Three-fold dilution of
the resulting suspension using buffer A yielded a volume of
22.5 L. This lysate cont~'neA about 6 g ApoE, i.e. about
4 g ApoE per Kg of original bacterial cake.
WO95/05l90 21~ ~ 9 4 2 PCT~594/09l9~
Centrifugation was then performed in a continuous CEPA-41
tubular bowl centrifuge, (Carl Padberg, Lahr/S~hwarzwald)
with a feed rate of 9 ~/hr at 20,000 rpm (17,000 g). The
pellet, weighing approximately 700 g and containing the
insoluble ApoE was saved and the supernatant was discarded.
(Note that the ApoE is insoluble due to the presence of Mg+t
ions.)
B. Extraction of Cell Pellet with Triton~
Six liters (1:10) of extraction buffer were added to the
pellet. (Extraction buffer: 50 mM Tris-HCl, 20 mM EDTA,
0.3~ Triton~, pH adjusted to 3.0 with HCl). Suspension was
achieved using a homogenizer (Kinematica) at low speed.
Then another 6 L extraction buffer was aaded (giving a final
pellet:buffer ratio of 1:20) and the pH was adjusted to 4.5
with 1 N NaOH. The resulting 12 L suspension was incubated
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
weighin~ about 450 g was discarded and the supernatant
solution cont~;n;ng the ApoE was titrated to pH=7.5 with 1
N NaOH and saved.
Note: Triton~ 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
cont~m;n~nts by ultrafiltration/dialysis.
.
A Millipore Pellicon ultrafiltration system using one 100
- 35 K cassette type PTHK was utilized to concentrate the
WO 95/OSI90 , PCT/US94/09192
-24-
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 ~/hr. The dialysis buffer was 50 mM Tris-HCl, 10 mM EDTA
and 0.1~ Triton~, pH=7.5. The 2 L retentate cont~' n; ng
about 2-3 mg ApoE per ml was kept cool with ice.
The retentate was dialyzed using the recirculating mode o~
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 t~rm;nAtion of dialysis.
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/hour (CV/hr). The capacity of the column under
these conditions was detPrm;ne~ to be 4 mg ApoE/ml. The
column was first equilibrated with DEAE equilibration
buffer: 20 mM Tris-HCl, 1 mM EDTA, 0.5~ Triton~, 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 equilibration buffer. The first elution was
performed using 3 CV of equilibration buffer contAln;ng 120
mM NaCl. 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 st~ne~ by Coomassie Blue
and the trailing edge of the peak (3.1 CV) was saved.
21~7~2
Wo95/05190 - -- PCT~S94/09192
-25-
The second elution was performed using the equilibration
buffer cont~;n;ng 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 le~el of endotoxin was
3 ~g per mg ApoE.
Concentration and dialysis after DEAE-Sepharose
The fractions indicated from the first and second eluates
were pooled and dialyzed using the Pell con ultrafiltration
system, with one lOOK cassette; the dialysis buffer was 20
mM Tris-HCl, 1 mM EDTA, 0.1~ Triton~, pH=7.5. The sample
was concentrated to 2 L (about 2-3 m~ ApoE/ml) and dialyzed.
E. O-Sepharose (OS) ChromatographY
The purpose of this step is to separate active ApoE from
inactive ApoE and to remove additional 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~ Triton~, pH=7.8. After equilibr&~ion, 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 cont~;n;ng 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 cont~;n;ng 20 mM NaCl and the second elution was
performed with about 5.5 CV of equilibration buffer
WO 95/05190 2 1 ~ 7 9 4 ~ PCT~S9~/09192
-26 -
cont~;n;ng 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 analog.
Two subsequent elutions using buffer cont~;n;ng 70 mM NaCl
and 350 mM NaCl respectively eluted the inactive 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.
The dialysis buffer was lO mM Tris-HCl, 1 mM EDTA, 0.1~
Triton~, pH-7.5. The sample was dialyzed using the
recirculating mode whilst maint~;n;ng the ApoE concentration
at 2-3 mg/ml. The final retentate volume was about 500 ml.
20 F. CM-Sepharose ChromatographY
The purpose of this step is to further remove endotoxins and
to lower the concentration of Triton~ to 0.05~.
In this step a 120 ml CM-Sepharose Fast Flow (Pharmacia)
column was used. The equilibration buffer was 20 mM Na
acetate, 1 mM EDTA, 0.2~ Triton~, 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 lO CV/hr.
The column was then washed with the following solutions: 4
CV of equilibration buffer followed by 5 CV of equilibration
buffer cont~;nlng 70 mM NaCl followed by 2 CV of 20 mM Na
WO9S/05190 ~ 9 4 2 PCT~S94/09192
-27-
acetate, 1 mM EDTA, 0.05~ Triton~, 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~ Triton~, 300 mM NaCl, pH=5Ø
The progress of the elution was monitcred by continuously
following the absorbance of the eluate at 280 nm. (Two
different base lines are used during the elution: one is the
high U.V. absorbance buffer cont~;n;ng 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 about 1.0 OD
allows both buffers to appear on the chart column, the low
at the foot and the high at about 0.5 OD.)
The sample containing the ApoE was ;mm~ately titrated to
pH 7.8 and saved. The endotoxin level in this sample was
below 50 pg per mg ApoE analog as measured by the LAL assay.
G. 100K Ultrafiltration - Triton~ Removal
The purpose of this step is to remove the Triton~.
This step was carried out at 4C using the Millipore
Pellicon Ultrafiltration System, cont~;n;ng one 100K
cassette, pre-washed with 0.5 M NaOH overnight. The ~low
rate was 9-12 L/hr and the inlet/pressure was 5-10 psig.
(This low flow rate is used to prevent aggregation of the
ApoE as the Triton~ is being removed.) The ApoE sample from
the previous step (960 ml cont~;n;ng about 600 mg ApoE) was
diluted to 0.5 mg/ml with 10 mM NaHCO3 bu~er pH=7.7.
The sample was then treated in the ultrafiltration system
- and the following conditions were applied throughout this
Triton~ removal step:
wo95/osl9o ~1 6 7 9 ~ 2 PCT~S94109192
-28-
a) The Triton~ concentration must be lower than 0.02~
i.e. the Triton~ concentration must be below its
critical micelle concentration in order to achieve
effective Triton~ removal across ~he 100K 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 100 K membrane.
0 c) The ApoE analog must not be concentrated above 1.5
mg/ml or aggregation of the ApoE may occur.
The dialysis buf~er used in the ultrafiltration system was
10 mM NaHCO3, 150 mM NaCl, pH=7.8.
A~ter concentration and dilution steps in accordance with
the above conditions, the dialysis was performed at constant
volume and constant flow rate and the dialysis was completed
when the absorbance at 280 nm of the filtrate was 0.01
units. (Triton~ solution absorbs at 280 nm and an
absorbance of 0.01 is equivalent to 0.0005~ Triton~.) The
total volume of final retentate was 770 ml and the total
volume of the filtrate was 9.5 L.
The solution cont~ining ApoE was then filtered (0.2 micron
filter) and stored at -70~C in 80 ml glass bottles.
Overall Yield:
0.3 g of highly purified ApoE were recovered from 3 Kg of
bacterial cake. The ApoE, approximately 97~ pure, was in
the same aggregation state as plasma apoE when tested under
the same conditions of gel permeation analysis. The ApoE
sample contained less then 50 pg of endotoxins/mg protein.
WO95/05190 216 ~ 9 ~ 2 PCT~S9~/09192
-29-
ophilization
If the recombinant ApoE is to be lyophilized, the dialysis
buffer in the Triton~ removal step is 2 mM NaHCO3, pH=7.8
and lmg cystein/mg ApoE. After lyophilization, the ApoE is
stored at -20C.
Lyophilized ApoE has been found to retain its normal
biological activity upon dissolution as long as five years
after lyophilization.
WO 95/05190 PCTIUS9~/09192 ~
21~79~2
-30-
EXAMPLE 2: Inhibition of the Development of Kaposi's
Sarcoma Lesions
I. Materials.
The Apolipoprotein E, met-apoE, was produced as described
in Example 1. Met-apoE solutions ~lmg/ml-Smg/ml), are in
lXPBS [PBS: NaCl 80g/l, KCl 2g/l, Na2HPO4, KH2PO4 2g/l]
cont~;n;ng 2mM sodium bicarbonate-lmM cystein per 1 mg apoE.
The ApoE, peptide 348, is a 30-mer t~n~m dimeric peptide
comprising the receptor binding region of apoE (amino acids
141-155) as described in U.S. Patent 5,177,189, issued
January 5, 1993.
FN 33 is a recombinant 33KD cell binding ~om~;n polypeptide
of human fibronectin consisting of the amino acid sequence
1329-1722, but deleted of amino acids 1600-1689 as disclosed
in coassigned International Publication No. WO 90/07577.
Oncostatin M is obtained from Peprotech, Inc., Rocky Hill,
N.J.
Conditioned medium (CM38) prepared from activated
lymphocytes was obtained from ABL, Inc. Rockville, Md.
Human Kaposi's sarcoma cell lines: KS3 is a human diploid
cell line which has been described by Nakamura (1988) and
Salahuddin (1988). The RW248 cell line was isolated from
the pleural effusion o~ a HIV-1+ homosexual male with KS.
RW248 has a normal human diploid karyotype and a phenotype
the same as KS3. KSY-1 is a human tetraploid cell line
isolated ~rom the pleural e~usion of a HIV-1+ homosexual
male with KS.
wo 95/oSl9O 2 ~ ~ 7 9 4 ~ PCT~Sg~/09192
-31-
KS3 cells and RW248 cells were grown in Iscove's DMEM, 10
FBS, 20~ 38CM, 10~M hydrocortisone, and lx ~l7m~n Nutridoma.
KSY-1 cells were maintained in RPMI 1640 with 10~ FBS.
II. Methods and Results
.
The ability of ApoE to inhibit the growth of AIDS-KS derived
cells is exemplified by Kaposi's sarcoma cell lines KS3 r
KSY-1, and RW248 as shown in Figures 1-6 and 13-14. The
effect of ApoE on other KS cell characteristics sucn as
migration, tumor development and vascular hyperpermeability
is shown in Figures 7-10 and 15.
A. In vitro
A1. DNA SYnthesis Assay
DNA synthesis (mitogenesis) is one parameter which provides
a means of measuring cell growth and proliferation.
Human AIDS-KS cell strains were grown to con~luence in
complete growth medium at which time the culture medium was
replaced with fresh basal RPMI 1640 (GIBCO-BRL,
Gaithersburg, MD) cont~;ning 0.5~ fetal bovine serum (FBS)
(GIBCO-BRL). The cells were cultured for 72 hours to arrest
cell growth at the Go stage of the cell cycle, after which
they were trypsinized and plated at a density of 2 x 104
cells/well in 24-well tissue culture plates (Falcon). The
cells were cultured in RPMI 1640 cont~;n;ng 1~ fetal bovine
serum, either recombinant Oncostatin M (30 ng/ml)
(PeproTech, Rocky Hill, N.J.) or 20~ activated lymphocyte
conditioned medium (CM) (Barillari, 1992), and varying
concentrations of ApoE (Vogel, 1985). Each concentration
of ApoE was assayed in triplicate. After 24 hours the cells
were pulsed with 1 uCi/ml of 3H-thymidine (New England
WO 95/05190 ' ; .: PCT/US94/09192 ~
2 ~ 4 2 -32 -
Nuclear, Boston, MA) for 6-12 hours and the incorporation
of 3H-thymidine into cellular DN~ assayed.
The results are shown in Figures 1-3 and show that the
addition of increasing concentrations of ApoE to the culture
inhibited the synthesis of DNA in the treated cells in a
dose dependent m~nner~ The concentrations of ApoE required
for 50~ inhibition of DNA synthesis (ICso) was 0.069 ~M in
the presence of activated lymphocyte conditioned medium
(20~), and 0.995 ~M in the presence of Oncostatin M
(5Ong/ml) (Fig 3). The reason for the nearly 10-~old
difference in IC50 values between conditioned medium and
Oncostatin M is not understood. However, since Oncostatin
M upregulates the LDL receptor (Grove, 1991), the higher
ApoE concentrations needed for inhibiting DNA synthesis in
the presence of Oncostatin M suggest that ApoE m~ tes
inhibition of mitogenesis through otner receptor(s) in
addition to the LDL receptor.
In an additional experiment, the effect of the ApoE, peptide
348 on mitogenic activity of RW248 cell was studied. The
incorporation of 3H-thymidine into DNA of the cells was
measured in the presence of 1.25~ FCS and 20~ CM and the
indicated concentrations of peptide 348. The ICso in a
prel;m;n~ry experiment was det~rm;npd to be between 20-40
~g/ml (Figure 13).
A2. Cell Proliferation Assays
Protocol P1: Cell proliferation was assayed using the cell
titer 96TM nonradioactive cell assay supplied by Promega
(#G4000) (Denizot, 1986). Briefly, the assay is based on
the cellular conversion of a tetrazo ium blue salt into a
blue form~n product by the mitochondrial enzyme succinate
dehydrogenase. The colored product is formed in an amount
wo 95/oSI9O ~1 ~ 7 9 4 2 PCT~S94/09192
.
-33-
proportional to the cell concentration and may be determined
by absorbance at 570nm. 2 X 104 cells/well were seeded in
96 well flat bottom plates (Falcon, Franklin Lakes, NJ) that
contained basal medium with FCS (0.5~-5~), either alone or
together with activated lymphocytes conditioned media (20~),
or Oncostatin M (50ng/ml) and appropriate amounts of ApoE
as shown in figures 4-6. The culture plates were incubated
for 48 hours at 37C in a CO2 incubator; 15 ~l of Promega dye
solution I was added to each well and incubation continued
for an additional 4 hrs, followed by the addition of 100~1
of Promega solution II. Absorbance at 570nm was determined
after 20 hours using an ELISA plate reader.
Protocol P2: The proliferation assay described above was
slightly modified. Following cell growth, the medium was
aspirated and replaced by 100~1 of basal medium (witho~t any
additions), and the assay was developed with the Promega
reagents as in protocol 1.
The inhibition of cell proliferation by ApoE is shown in
Figures 4-6. This inhibitory e~fect was abolished if the
ApoE was heat inactivated (Figures 4-5), proving that the
inhibitory effect is in fact attributable to the biological
activity of ApoE.
A3. Mi~ration Assay
The affect of ApoE on chemotaxis (directed migration towards
an increasing concentration gradient of a chemoattractant)
and chemokinesis (r~n~sm migration in response to a
stimulus) of AIDS-KS cells in culture was measured by a
modified Boyden chamber assay (Taraboletti, 1990), using 8~m
- Nucleopore filters coated with gelatin.
WO 95tOS190 21 6 7 9 4 2 PCT/US94/09192 ~
-34-
The migratory properties of the AIDS-KS derived cells we~e
highly inhibited by ApoE (~igs 7-8). Chemotaxis of RW248
cells from the upper chamber to increasing concentrations
of Oncostatin M in the lower chamber was more than two-fold
stimulated over the basal migration us~ng BSA as a control.
This Oncostatin M stimulated chemotaxis was greatly
inhibited by the addition of ApoE (O.1-O.4~M) to the lower
chamber together with the Oncostatin M. Moreover, this
inhibition by ApoE is highly specific, since the basal
migration toward BSA was not effected by the addition of
ApoE.
The chemotactic response of AIDS-KS cells to conditioned
media and to fibronectin (FN) was also tested. The addition
of conditioned me~i A or FN to the lower chamber stimulated
the directed migration of KSY-1 cells two and three fold
respectively, in comparison to the basal migration in
response to BSA. Addition of ApoE (O.l~M or O.3~M) to the
cells in the upper chamber inhibited the migration of the
cells to the conditioned media (approximately 30~ and 70~,
respectively) in a dose dependent ~ashion. This inhibition
of migration by ApoE was specific to migration stimulated
by conditioned medium, since migration towards BSA and FN
was not affected.
These results (summ-~rized in Figurea 7 and 8) are an
indication that ApoE does not affect ch~mokinesis (rAn~nm
migration) but rather specifically inhibits chemotaxis in
response to particular components in the conditioned medium
and to Oncostatin M.
B. In-vivo: Inhibition of Anqioqenic lesions induced by
AIDS-KS cells
wo 95/oSI9O 21 6 7 ~ 4 2 PCT1594/09192
Based on the in vi~ro results, the KS-inhibitory activity
of ApoE was tested in vivo.
Bl. 2 X 1o6 KS3 cells were suspended in phosphate buffered
saline (PBS) and mixed in the presence and absence of met-
apoE with an equal volume of an extracellular matrix
composition, Matrigel (Collaborative Research). The
suspension was then transplanted subcutaneously (s.c.) into
the backs of Balb/c nu/nu athymic mice (day 0). The ~nim~ls
were ~mlnlstered a daily intravenous (i.v.) dose of various
concentrations of met-apoE or PBS (as control) from day 1-5.
On day 6 the angiogenic lesions were observed and measured,
fixed in 10~ formalin, and stained with hematoxylin-eosin.
The results of three different experiments performed with
KS-3 cells are summarized in Table 1.
WO95/05190 : pcT~s94lo9ls2
21~7~2
-36 -
Table 1: Inhibition of KS induced anqiogenic lesions in Balb/c nu/nu
athvmic mice
Expt. ApoE' n2ApoE Tumor size ~mm)
no. (mg) admin3
0 5 - 5 mice: 11x12 ~average)
4day 0 5 3 mlce (75 ~ no tumor
0 3 - 3 mice (100%): 9x6; 5x7: 4x8
0.2 6day 0-5 4 mice (67%): no tumor
1 mouse: 5x6
2 1 mouse: 7x7
6 day 1-5 4 mice (67%): no tumor
1 mouse: 6x8
1 mouse: 4x6
0 7 - 7 rnice (100%) 12x13 (average)
3 0.3 7day 1-5 3 mice (42%): no tumor
4 mice: 4x6
7day 1-5 7 mice (100%): no tumor
o 1 AnoE: 0 = buffer control
2 n: number of mice
3 adm;.~ aliu~:
day 0: subcutaneous ~s.c.) - incGr~,~raled with the KS
cells into Matrigel
daY 1-5: intravenous (i.v.)
B2. In a similar experiment, 5xl06 KS RW248 cells were
suspended in phosphate buffered saline (PBS) and mixed with
an equal volume of an extracellular matrix composition
Matrigel. 0.4 ml of the suspension cont~;n~ng 2 X l06 cells
was injected subcutaneously into male Balb/c athymic nu/nu
mice on day 0. On days 1-6 the mice were ~m;n;Rtered
intravenous doses of either met-apoE, or PBS as a control.
The ~n;m~l S were sacri,iced on day 6. The size and
appearance of the lesion were noted and the lesions fixed
in l0~ formalin for histological e~mln~tion~ The results
are shown in Table 2 and Figures 9 and l0.
wo 95/o5l~0 2 1 6 7 3 4 ~ pcT~ss~lo9l92
Table 2. Inhibition of KS induced angiogenic lesions in Balb/c nu/nu
athymic mice
ApoE nLesion Size Histological Descri~lion of
(mg) (mm2)~ Lesion
0.8 1620.8 + 13.9'' few cells, no angiogenesis
0.4 1237.9 + 18.4^'' few cells, no angiogenesis
0.2 784.2 + 26.2 many cells,
neoangiogenesis
0 (buffer 1675.9 + 33.8 many cells,
control) neoangiogenesis
* Data are means + SD. St~ al analysis was
based upon two-tailed Student's t test.
** p<0.00001 vs buffer control.
**~ p=0.0008 vs buffer control.
In these experiments a dose-dependent decrease in the size
of the KS cell induced angiogenic lesions was ob~erved
following ~m; n~ stration of ApoE to Balb/c nu/nu mice. No
substantial difference in the size of the lesions was
observed whether the ApoE was first ~m; n~ stered on day 0
or on day l (Table l). Furthermore, upon histological
e~m;n~tion of the lesions, ApoE-treated mice had either no
lesions at all (at the highest doses) or lesions devoid of
both inflammatory cells and neoangiogenesis. This was in
sharp contrast to the typical lesions in the control mice
which were characterized by the presence of spindle cells,
inflammatory cells, and neoangiogenesis (Table 2, Figure
10) .
B3. The previous experiments tested the effect of ApoE on
tumors induced by primary AIDS-KS ce ls. The following
experiment tests the effect of ApoE on tumors induced by
an immortalized truly malignant AIDS-KS cell line, KSY-l.
500,000 KSY-l cells in 0.2ml DMEM were injected
subcutaneously into the backs of 6 week old athymic SCID
WO 95/05190 PCT/US91/09192
2~ ~7~42
-38-
mice. Intravenous injections of ApoE (0.8mg in 0.2ml; 10
mice) or PBS (0.2ml; 10 mice) were ~m;n;stered daily for
20 days, starting 30 minutes after the injection of the
cells. The ~n;m~l S were sacrificed on the 21st day and the
lesions were photographed, measured internally and
externally, fixed in 10~ formalin, paraffin-embedded, and
hematoxylin-eosin stained for histological observation.
The gross tumor growth results are sum~.arized in Figure 14.
In general, KSY-1-induced tumors showed considerably less
neoangiogenisis than tumors induced by the primary AIDS-KS
cells. Tumor size was moderately but significantly reduced
by treatment with ApoE (Figure 14). Furthermore,
macroscopic and microscopic analysis revealed a reduction
in vascularization and a dramatic increase in necrotic
regions in the ApoE-treated tumors in comparison to the
nontreated controls; this indicates the therapeutic
potential of ApoE treatment.
C. I~ vivo: Tnh; hition of KS-induced vascular
hyperE;~erme~bility
An additional complication observed in AIDS-KS patients is
late phase vascular hyperp~rm~hility and the resulting
edema (Nakamura, 1994). This effect is believed to be
connected with secretion of particular cellular factors by
AIDS-KS cells. The following experiment was performed to
test the effect of ApoE on KS-induced vascular
hyperp~rm~hility.
Groups of six 8 week old female BA~B/C athymic nude mice
were injected subcutaneously with either 2,000,000 KSY-1 or
70,000 KS-4 cells (in 0.2ml DMEM) per ~n;m~l One hour
before and 6 hours after injection of the KS cells, each
~n;m~l was injected with either lmg ApoE (in 0.2ml) or PBS
2~7~2
WO 95/05190 PCT/US94/09192
-39-
(0.2ml) as control. After 12 hours, the mice were injected
intravenously with Evans blue dye (0.5mg in O.lml). After
3 hours, the ~n;m~ls were sacrificed and the leaked dye from
the region of the injected cells was extracted with
formamide and quantified spectrophotometrically.
The results shown in Figure 15 ~mn~qtrate the large
decrease in extracted dye in the ~n;m~ls treated with ApoE.
Thus, this experiment indicates that ApoE can be used to
treat edema in a subject suffering from Kaposi's sarcoma.
It is also envisaged that Apolipoprotein E might be used
in treating edema not caused by Kaposi's sarcoma, in
particular edema resulting from vascular hyperpermeability,
~capillary leak", or edema mediated by cellular factors such
as VEGF and bFGF.
III. Summary and conclusion
In these studies, the ability of Apolipoprotein E to
function as a negative modulator of AIDS-KS derived cell
growth in vitro and in vivo and as an inhibitor of KS cell
induced neoangiogenenic lesions and vascular
hyperperme~hility in vivo was ~m;ned.
The results of these experiments demonstrated that ApoE is
a potent inhibitor of mitogenesis, proliferation, and
migration of human KS cells, and i9 a potent inhibitor of
the development of hllm~n KS lesions and of resulting edema.
Based on the preceding results, Apolipoprotein E is
inhibitory to KS lesions in m~mm~ls including humans.
The in vitro and in vivo results of our studies indicate
that ApoE also functions in the regulation of endothelial
WO95/05190 PCTf~S94109192 ~
216~42
-40-
cell development and neoangiogenesis, and tumor cell grow~h
and metastasis.
The use of ApoE in these model systems, which were designed
to study the pathogenesis of AIDS associated KS, indicates
that ApoE is an effective therapy for Kaposi's sarcoma.
21~79~
WO95/05190 -41- PCT~S94/09192
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