CA2167942A1 - Method of inhibiting kaposi's sarcoma - Google Patents

Abstract

Methods and compositions comprising Apolipoprotein E are provided for inhibiting the proliferation of Kaposi's sarcoma cells. Additionally, a method is provided of treating a subject suffering from Kaposi's sarcoma comprising administering to the subject an amount of a composition comprising an amount of Apolipoprotein E effective to treat the Kaposi's sarcoma and a pharmaceutically acceptable carrier.

Description

WO 95/05190 ~ 1 6 7 9 ~ 2 PCT/US94/09192 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 ~

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 .. ~

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 ~

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).

21679~2 1~

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 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 ~

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 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 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 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 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 .

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 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:

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 .

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 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 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~

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 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 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 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 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 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 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 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 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 .

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 ~

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 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

2~ ~7~42 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 (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 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~
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Claims (14)

Claims

1. A method of inhibiting proliferation of Kaposi's sarcoma cells comprising contacting the Kaposi's sarcoma cells with an amount of Apolipoprotein E
effective to inhibit proliferation of the Kaposi's sarcoma cells.

2. A composition 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.

3. A method of treating a subject suffering from Kaposi's sarcoma comprising administering to the subject an amount of Apolipoprotein E effective to treat the Kaposi's sarcoma.

4. A method of claim 3 wherein the Apolipoprotein E is administered intravenously.

5. A method of claim 3 wherein the Apolipoprotein E is administered subcutaneously.

6. A pharmaceutical composition comprising Apolipoprotein E in an amount effective to treat Kaposi's sarcoma and a pharmaceutically acceptable carrier.

7. The composition of claim 6 suitable for intravenous administration.

8. The composition of claim 6 suitable for subcutaneous administration.

9. Use of Apolipoprotein E in the manufacture of a composition for the inhibition of proliferation of Kaposi's sarcoma cells.

10. Apolipoprotein E for use in the inhibition of proliferation of Kaposi's sarcoma cells.

11. An article of manufacture comprising packaging material and a pharmaceutical agent contained within the packaging material, wherein the pharmaceutical agent is therapeutically effective for inhibiting proliferation of Kaposi's sarcoma cells and wherein the packaging material comprises a label which indicates that the pharmaceutical agent can be used for the inhibition of proliferation of Kaposi's sarcoma cells and wherein the pharmaceutical agent comprises Apolipoprotein E.

12. The use of Apolipoprotein E for treating edema.

13. A composition comprising Apolipoprotein E for treatment of edema.

14. The use of Apolipoprotein E in the manufacture of a composition for the treatment of edema.