WO1994004178A1 - Procede d'inhibition de la proliferation cellulaire au moyen de l'apolipoproteine e - Google Patents

Procede d'inhibition de la proliferation cellulaire au moyen de l'apolipoproteine e Download PDF

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Publication number
WO1994004178A1
WO1994004178A1 PCT/US1993/007582 US9307582W WO9404178A1 WO 1994004178 A1 WO1994004178 A1 WO 1994004178A1 US 9307582 W US9307582 W US 9307582W WO 9404178 A1 WO9404178 A1 WO 9404178A1
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cells
apoe
proliferation
apolipoprotein
tumor
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PCT/US1993/007582
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English (en)
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Tikva Vogel
Amos Panet
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Bio-Technology General Corp.
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Priority to AU50072/93A priority Critical patent/AU673543B2/en
Priority to JP6506399A priority patent/JPH08502730A/ja
Priority to EP93919995A priority patent/EP0659085A4/fr
Priority to KR1019950700386A priority patent/KR950702430A/ko
Publication of WO1994004178A1 publication Critical patent/WO1994004178A1/fr
Priority to NO950491A priority patent/NO950491L/no

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to the use of Apolipprotein E in a method of inhibiting ceil proliferation.
  • Angiogenesis is also necessary for expansion of a metastatic colony (Aznavoorian, 1991) .
  • other diseases are also characterized by abnormal neovasculariza ion, including neovascular glaucoma, diabetic retinopathy, and rheumatoid arthritis (Folkman, et al., 1989).
  • Malignant cells produce many factors that stimulate endothelial cell proliferation and migration and allow new capillary beds to form within the tumor nodule (D'Amore, 1988; Shing, et al., 1985).
  • a variety of agents have been suggested as potential modulators of cell proliferation, including heparin and heparin sulfate (Castellot, et al., 1987; Clowes, 1977) , and growth factors and their inhibitors (Edelman, et al., 1992; Liu, 1990; Schweigerer, et al., 1987) . Most of these factors also appear to be natural components of normal tissue.
  • bFGF basic fibroblast growth factor
  • Basilico and Moscatelli 1992; Schweigerer, et al., 1987; Thomas and Gimenez-Gallego, 1986
  • bFGF is a strong heparin-binding molecule, present in virtually all tissues and having multiple mitogenic and angiogenic effects (Thomas and Gimenez-Gallego, 1986) .
  • Thomas and Gimenez-Gallego 1986
  • bFGF stimulates a number of functions involved in the formation of blood vessels and angiogenesis.
  • bFGF is considered to be one of the most potent angiogenesis inducers both in vivo arid in vitro ' (Folkman, 1976; Folkman and Klagsbrun, 1987) . Recently it was shown that intravenous infusion of bFGF stimulated endothelial regeneration and SMC proliferation (Edelman, et al., 1992; Lindner and Reidy, 1991; Lindner, et al. , 1990) after balloon-induced endothelial denudation. In these studies, it was confirmed that bFGF was both angiogenic and mitogenic for SMC in vivo and also demonstrated that these two effects are coupled.
  • bFGF also binds to heparan sulfate proteoglycans (HSPG) of both the extracellular matrix (ECM) and basement membrane (Folkman, et al., 1988; Vlodavsky, et al., 1987) .
  • HSP heparan sulfate proteoglycans
  • HSPG The role of HSPG in regulating cell growth and differentiation has been described (Burgess and Maciag, 1989; Klagsbrun and Baird, 1991; Ruoslahti and Yamaguchi, 1991) .
  • Many proteoglycans are constituents of the ECM or function as a low-affinity cell surface receptor for the interaction of growth factors, including bFGF and other heparin-binding growth factor molecules.
  • the role of HSPG as binders of bFGF appears to protect bFGF from degradation, and is important in providing a matrix or cell surface bound reservoir of bFGF. Yayon, et al.
  • bFGF binding to its high-affinity receptor requires prior binding either to the heparan sulfate side chains of a membrane HSPG or to free heparan sulfate (heparin) chains, and speculate that glycosaminoglycans may change the conformation of bFGF so that it acquires the ability to bind to its receptor. Binding of growth factors to proteoglycans have also been observed with several other growth factors that bind to heparin or heparan sulfate (Ruoslahti and Yamaguchi, 1991) .
  • proteoglycans are abundant and ubiquitous tissue components, they are likely to attract most of these growth " factors and cytokines that have affinity for the glycosaminoglycan. It may be that growth factors and cytokines were meant to act on their target cells only over a short range, and that their immobilization at the cell surface and in ECM (through proteoglycan binding) accomplishes that goal (Ruoslahti and Yamaguchi, 1991) .
  • Apolipoprotein E is a plasma protein having strong affinity for heparin and HSPG (Cardin, et al., 1988; Mahley, 1988; Mahley, et al., 1979; Weisgraber, et al., 1986).
  • ApoE participates in plasma lipoprotein metabolism through its high-affinity interaction with cell surface receptors including the low-density lipoprotein (LDL) and the more recently identified apoE receptor, LDL receptor-related protein (LRP) (Hertz, et al., 1988; Lund, et al., 1989; Yamada, et al., 1989; 1992).
  • LDL low-density lipoprotein
  • LRP LDL receptor-related protein
  • the domain of apoE responsible for binding to the LDL receptor has been identified (Dyer and Curtiss, 1991; Wilson, 1991). This domain is a 20-amino acid region comprised of residues 140-160 of the apoE molecule. It has long been known that binding of ApoE to the LDL receptor depended on its association with lipids (Innerarity, 1979) . However, from results with synthetic peptides binding to the LDL receptor in vitro. one can assume there is direct binding of the peptide to the LDL receptor, or one could speculate that the LDL receptor is not the only binding site on the cell.
  • ApoE is ubiquitously synthesized in many tissues including liver, intestine, adrenal gland, kidney, lung, spleen, testes, ovary, and brain (Mahley, 1988) . Recently, it has been found in both inflamed and non-inflamed synovial fluid (Terkeltaub, et al., 1991). ApoE can function in tissue repair by modulating lipid redistribution locally (Hui, et al., 1980; Mahley, 1988). However, it has also been observed that apoE is synthesized and secreted by a number of cells that do not necessarily participate in cholesterol ho eostasis (Boyles, et al. 1989; Hui, et al., 1980).
  • apoE In addition to its effect on lipoprotein metabolism, apoE also possesses a variety of functions that are unrelated to lipid transport (Mahley, 1988) . A potent suppression of lymphocyte activation by mitogens and antigens by ApoE- bearing lipoproteins and ApoE polypeptides has been observed (Cardin, et al. 1988; Hui, et al., 1980). The present invention discloses the effect of ApoE on proliferation and migration of several cell types.
  • a method of inhibiting actively proliferating cells comprises contacting the cells with Apolipoprotein E (ApoE) in an amount effective to inhibit cell proliferation.
  • Apolipoprotein E Apolipoprotein E
  • a composition comprising Apolipoprotein E in an amount effective to inhibit cell proliferation.
  • the invention additionally provides a method of treating a subject suffering from excessive cell proliferation which comprises contacting the excessively proliferating cells with an effective amount of Apolipoprotein E so as to inhibit the excessive cell proliferation.
  • the present invention provides a method of treating a subject afflicted with a tumor which comprises contacting the tumor with an effective amount of an Apolipoprotein E in conjunction with a chemotherapeutic agent so as to inhibit proliferation of the tumor cells.
  • the present invention provides a method of treating a subject afflicted with a tumor which comprises contacting the tumor with an effective amount of Apolipoprotein E in conjunction with an amount of irradiation so as to inhibit proliferation of the tumor cells.
  • the invention provides a method of treating a subject suffering from a disorder involving increased neovascularization. This method comprises administering to the subject an amount of Apolipoprotein E effective to normalize neovascularization.
  • Figure 1 The effect of heparin binding molecules on the incorporation of [ 3 H]thymidine into bovine aortic endothelial cell (BAEC) DNA was tested as described in Examples 1 and 2 using protocol M2, either in the presence of fetal calf serum (FCS) (1% and 2.5%) alone [control (ctr) ] , or with added basic fibroblast growth factor (bFGF) . (All other samples were tested using 10 ng/ml) .
  • FCS fetal calf serum
  • bFGF basic fibroblast growth factor
  • 0.05 or 0.5 ⁇ M of met-apolipoprotein E (+E.05 or +E0.5, respectively) or recombinant thrombospondin (rTSP) 18 Kd (+T.05 or +T.5) were added to the wells containing FCS and bFGF.
  • the mitogenesis assay was terminated after 42 hr.
  • FIG. 2 The time course of the incorporation of [ 3 H]thymidine into BAEC DNA was obtained. DNA synthesis was tested on a newly-attached cell culture according to mitogenesis protocol M2 (see Example 1-Methods) . Cells were plated in Dulbecco's modified Eagles , s medium (DMEM)- containing bFGF (10 ng/ml) and 1% FCS alone (ctr) or together with 0.5 ⁇ M of the indicated molecules [(rTSP) 18 Kd, recombinant fibronectin (rFN) 33Kd, or met-apoEJ .
  • DMEM Dulbecco's modified Eagles , s medium
  • bFGF 10 ng/ml
  • FCS alone 1% FCS alone
  • Figure 3 Incorporation of [ 3 H]thymidine into pre- attached BAEC culture was tested according to protocol Ml, at either 0.5% FCS alone or together with bFGF (10 ng/ml). Met-apoE at the indicated concentrations was added to the cells 5 days after plating, and [ 3 H]thymidine incorporation was tested the next day after pulsing the cells for 5 hr and as indicated in protocol Ml.
  • FIG. 4 Incorporation of [ 3 H]thymidine into a pre- attached, dense culture of BAEC was studied in a preattached culture. Mitogenesis was tested according to protocol Ml, using 0% or 0.5% FCS (0 and .5, respectively), together with bFGF, 10 ng/ml (OF and .5F, respectively), or in combination with 0.5 ⁇ M met-apoE (OFE and .5FE, respectively). The mitogenesis assay was terminated after 40 hr.
  • FIG. 5 Incorporation of [ 3 H]thymidine into BAEC culture was tested in the presence of bFGF (10 ng/ml) and one of two concentrations of FCS (as indicated in the figure) either alone (control) , or together with met-apoE (0.5 ⁇ M) , added to the newly plated culture at time zero (designated as 0-40) , or at 15 hr or 22 hr after plating (designated as 15-40 and 22-40, respectively). [ 3 H]thymidine was also added at time zero to all wells as indicated in protocol M2, and the assay was terminated after 40 hr. Incorporation is expressed as a percent of control labelled with [ 3 H]thymidine for the same time in the absence of ApoE.
  • Figure 6 The effect of bFGF on inhibition by ApoE was studied.
  • the proliferation assay was performed with culture of BAEC-l, as indicated in protocol PI, in the presence of 5% FCS either alone (ctr) or together with 20 ng/ml of bFGF. Where indicated, various concentrations of met-apoE were added 1 day after cell plating. The cell number was monitored as indicated in protocol Pi.
  • FIG. 7 Incorporation of [ 3 H]thymidine into bovine corneal endothelial cell (CBEC) culture was studied using various concentrations of FCS (ctr) as indicated (0%, 1%, or 2%) . To this, growth factors were added either alone (bFGF [10 ng/ml] or EGF [50 ng/ml]) , or together with met-apoE (E, 0.5 ⁇ M) or FN 33 Kd (FN, 0.5 ⁇ M) . The mitogenesis assay was performed as described in protocol M2, and terminated after 44 hr.
  • FCS ctr
  • Figure 8 The proliferation assay was performed with a culture of CBEC as indicated in protocol P2, in the presence of FCS and bFGF alone (control) at 5% and 10 ng/ml, respectively. When indicated, various concentrations of either met-apoE or FN 33 (rFN 33 Kd) were added to the cells at time zero. The average absorbancy of triplicate samples was calculated and expressed as percent of control.
  • Figure 9 The reversibility of ApoE inhibition was studied. CBEC culture in 1% FCS and 10 ng/ml bFGF was tested for [ 3 H]thymidine incorporation after 40 hr of labeling using protocol M2 (control) . To parallel cultures, met-apoE was added at time zero at the indicated concentrations and either left for the entire labeling period (0-40) or for only 22 hr (0-22) . For all tested cultures, media was replaced after 22 hours with the appropriate combination, including the starting concentrations of FCS, bFGF, and [ 3 H]thymidine.
  • FIG. 10 Incorporation of [ 3 H]thymidine into culture of A2058 human melanoma cells was tested as described in protocol M2, and in the presence of 0.5% FCS and bFGF (10 ng/ml) either alone (ctr) or together with 0.5 ⁇ M and 1.5 ⁇ M met-apoE (0.5E and 1.5E, respectively), 0.5 ⁇ M TSP 18 Kd (0.5T18), 0.5 ⁇ M TSP 28 Kd (0.5T28), or 0.5 ⁇ M FN 33 Kd (0.5FN33). The assay was terminated after 32 hr.
  • FCS and bFGF 10 ng/ml
  • FIG 11 Incorporation of [ 3 H]thymidine into a culture of human mammary tumor (MDA-435) cells was carried out according to protocol M2, and in the presence of 0.5% FCS and bFGF (10 ng/ml) alone (ctr) or with the addition of 0.5 ⁇ M met-apoE (E) or TSP 18Kd or with 75 ⁇ g/ l heparin (Hep) . The assay was terminated after 42 hr.
  • Figure 12 Proliferation of smooth muscle cells (SMC) was performed as indicated in protocol PI, in the presence of either 0.5% or 5% FCS alone (ctr), or combined with bFGF (+F, 20 ng/ml) . Where indicated, met-apoE (E) (4 ⁇ M) was added to the culture at time zero.
  • SMC smooth muscle cells
  • Figure 13 The incorporation of 35 S-methionine was examined as published (D. Blake 1990) with a slight modification. Briefly, CBEC cells were plated into 6-well tissue culture dishes in DMEM + 10% FCS at 10 5 cells/well. After 24 hr, cells were washed 3 times with PBS and the media was changed to DMEM minus methionine (for methionine depletion) either alone (ctr) or together with met-apoE (0.5 ⁇ m) . After 1 hr, 0.25 ⁇ Ci [ 35 S]methionine (1268 Ci/mmol, Amersham) was added, and the culture was incubated at 37°C/5% C0 2 for 6 hr.
  • FIG 14 Chemotaxis of BAEC to bFGF was carried out in a modified Boyden chamber as previously described (Taraboletti, 1990) using 5 ⁇ m pore size polycarbonate PVP- free nucleopore filters. Semiconfluent cells were trypsinized, washed with 10% FCS, allowed to equilibrate in 10% FCS-DMEM for 2 hours at room temperature while shaking, and then pelleted, and resuspended in medium containing 0.1% BSA. Cells were used immediately at a concentration of 10 6 /ml and checked for migration either alone or together with the indicated concentration of met-apoE (E) toward a gradient of 0.1% BSA alone (ctr) or together with bFGF (33 ng/ml) . Migrated cells in triplicate samples were monitored after 4.5 hr of incubation at 37°C, and are expressed as a percent of the migration toward 0.1% BSA (ctr).
  • FIG. 15 This figure shows plasmid pTVR 590-4 which was deposited in E. coli W1485 under ATCC Accession No. 67360. Plasmid pTVR 590-4 is a good expressor of met-apoE under control of the *P L promoter as is described in Example 1. (E. coli W1485 is freely available from ATCC under Accession No. 12435.)
  • FIG 16 The construction of plasmid pE2-5 encoding ApoE containing amino acids 1-217 is described.
  • Plasmid pTV 194-80 (disclosed in coassigned U.S. Patent No. 5,126,252, Figure 22) was digested with restriction enzymes BssHII and Bglll. The large fragment was isolated and ligated to the synthetic linkers shown in the Figure. The resulting plasmid was designated pE2-5.
  • Figure 17 The construction of plasmid pTVR6-2 expressing ApoE having amino acids 1-217 is described.
  • a 1200bp fragment containing the ⁇ Cl gene under control of a portion of the deoPl promotor sequence was isolated from Clal digestion of plasmid pFSAL-B27 (ATCC Accession No. 67071; also disclosed in European Patent Application Publication No. 303,972).
  • the 1200bp fragment was then ligated to Clal digested plasmid pE2-5 ( Figure 16) .
  • the resulting plasmid designated pTVR6-2, contains both the ApoE structural gene and the * ⁇ Cl repressor gene, and is therefore an independent plasmid, not limited to use in a host containing the * ⁇ Cl repressor but able to express the ApoE polypeptide fragment in a wide variety of hosts. It is not presently known whether an additional N-terminal methionine is present. Plasmid pTVR6-2 was deposited in f.. coli 4300 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland on July 26, 1993 under Accession No. 69364.
  • Figure 18 The effect of ApoE on proliferation of mouse endothelioma-END2 cells in culture was studied.
  • the proliferation assay of END2 cells was performed as Example 7 using 20,000 cells per well and in the presence of 0.5% FCS, and the indicated concentrations of met-apoE.
  • Figure 19 The effect of ApoE on the proliferation of BAEC and CHO cells was studied.
  • the proliferation of BAEC and CHO cells performed with 30,000 cells/ml in the presence of 0.5% FCS and the indicated concentrations of met-apoE as described in Example 2.
  • Figure 20 The effect of ApoE on the proliferation of BAEC and neuroblastoma cells was studied.
  • the proliferation of BAEC and neuroblastoma N18TG2 was performed with 20,000 cells per ml in the presence of 0.5% FCS and the indicated concentrations of met-apoE (20A) and the ApoE-peptide 348 (20B) as described in Example 2.
  • Figure 21 The heat-stability of the anti-proliferative activity of ApoE was examined.
  • the proliferation of BAEC cells (30,000 cells/ml) was tested in the presence of 0.5% FCS and the indicated concentrations of unheated or heated (100°C for lhr) met-apoE as described in Example 2.
  • Figure 22 The effect of serum concentration on the anti-proliferative activity of met-apoE was studied.
  • the proliferation of BAEC cells was measured at 0.5% and 2.5% FCS and in the presence of the indicated concentrations of ApoE without the addition of exogenous growth factors.
  • Met- apoE exhibits a lower degree of inhibition at the higher serum concentration as described in Example 2.
  • Figure 23 The effect of the ApoE designated apoE6-2 on the proliferation of END-2 cells was studied.
  • the proliferation of END-2 was measured as indicated in Figure 18 in the presence of 0.5% FCS and the indicated amounts of the ApoE polypeptide fragment encoded by plasmid pTVR6-2 ( Figure 17) as described in Example 8.
  • Figure 24 The effect of serum concentration on the anti- proliferative activity of an ApoE polypeptide was studied. The proliferation of BAEC cells was measured at 0.5% and 2.5% FCS and the indicated concentrations of the ApoE polypeptide apoE6-2 as described in Example 8.
  • Figure 25 The reversal of heparin activity by ApoE was studied.
  • ApoE has a high affinity for heparin, and this property of ApoE was studied by the effect of met-apoE on a complex consisting of heparin, antithrombin III and thrombin. Addition of ApoE to the complex reverses the heparin activity resulting in inhibition of the antithrombin activity of heparin.
  • a nonreactive short peptide designated peptide 185 was used " as a negative control.
  • a second negative control [Ctrl (-) ] did not include any ApoE.
  • a positive control [Ctrl (+) ] did not contain heparin.
  • the present invention provides a method of inhibiting actively proliferating cells comprising contacting actively proliferating cells with an amount of Apolipoprotein E (ApoE) effective to inhibit proliferation.
  • Inhibition of proliferation means reduction of the rate of proliferation of the cells.
  • the cells may be smooth muscle cells, endothelial cells e.g. aortic or corneal endothelial cells, or tumor cells, e.g. human melanoma cells, mammary tumor cells, human sarcoma cells, or carcinoma cells.
  • endothelial cells e.g. aortic or corneal endothelial cells
  • tumor cells e.g. human melanoma cells, mammary tumor cells, human sarcoma cells, or carcinoma cells.
  • Other actively proliferating cell types known to those skilled in the art are also encompassed by the methods of the invention.
  • a composition for inhibiting the proliferation of actively proliferating cells comprising Apolipoprotein E and a suitable carrier is also provided.
  • a method is provided of treating a subject suffering from excessive cell proliferation which comprises administering to the subject a ' amount of Apolipoprotein E effective to inhibit the excessive cell proliferation.
  • Such a method may involve administration of the Apolipoprotein E in conjunction with other therapeutic means such as a chemotherapeutic agent or irradiation treatment, i.e. administration of ApoE prior to, during or after the other therapeutic means.
  • Other therapeutic means for use in conjunction with Apolipoprotein E are known to those skilled in the art and are also encompassed by the methods of the invention.
  • a pharmaceutical composition which comprises Apolipoprotein E in an amount effective to inhibit excessive proliferation and a pharmaceutically acceptable carrier.
  • excessive cell proliferation is a tumor.
  • Apolipoprotein E 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 cells, and mutants whose sequence varies by one or more, typically less than ten amino acids provided that such mutants have the biological activity of inhibiting proliferation of cells.
  • Naturally occurring apoE may be obtained from plasma or serum by methods known to those skilled in the art and is available commercially e.g. Calbiochem cat. no. 178466.
  • 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 sequence 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 vector.
  • the cells are preferably bacterial cells or other unicellular organisms, but eucaryotic cells such as yeast, insect or mammalian cells may also be used to produce recombinant ApoE.
  • the ApoE is a mutant of recombinant apoE differing from the naturally occurring polypeptide by the addition, deletion, or substitution of one or more non- essential amino acid residues, typically less than 10, provided that the resulting polypeptide retains the cell proliferation inhibitory activity of apoE.
  • mutants of apoE are deletion mutants containing less than all the amino acid residues of naturally occurring apoE, substitution mutants wherein one or more residues are replaced by other residues, and addition mutants wherein one or more amino acids residues are added to the polypeptide. All such mutants share the cell proliferation inhibitory activity of naturally occurring apoE.
  • Polypeptides having substantially the same amino acid sequence as naturally occurring apolipoprotein E encompass the addition or deletion of fewer than four amino acids at the N-terminus of the amino acid sequence of the polypeptide. There may be additional substitutions and/or deletions in the sequence which do not eliminate the cell proliferation inhibiting biological activity of the polypeptide. Such substitutions and deletions are known to those skilled in the art. Substitutions may encompass up to about 10 residues in accordance with the homologous or equivalent groups described by e.g. Lehninger, Biochemistry. 2nd ed. Worth Pub., N.Y. (1975); Creighton, Protein Structure, a Practical Approach. IRL Press at Oxford Univ. Press, Oxford, England (1989); and Dayhoff, Atlas of Protein Sequence and Structure 1972. National Biomedical Research Foundation, Maryland (1972) .
  • the ApoE is recombinant met- apoE, e.g. a recombinant polypeptide comprising the sequence of naturally occurring apoE with an additional methionine at the N-terminus.
  • Apolipoprotein E polypeptide fragments of recombinant ApoE and of naturally occurring apoE which exhibit the cell proliferation inhibitory activity of apoE.
  • One example of such a fragment is a 15-mer fragment disclosed in U.S. Patent No. 5,177,189, issued January 5, 1993.
  • polypeptide fragments have amino acids 1-217 or 1-185 of naturally occurring apoE.
  • a particular embodiment of an ApoE polypeptide having amino acids 1-217 of naturally occurring apoE is encoded by plasmid pTVR6-2 ( Figure 17) which was deposited in E. coli 4300 on July 26, 1993 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland under Accession No. 69364.
  • Another fragment is the 22KD N- terminal ApoE polypeptide produced by thrombin digestion of naturally occurring apoE or recombinant met-apoE.
  • the method of the present invention may be practiced with any ApoE having at least substantially the same cell proliferation inhibitory activity as naturally occurring apoE.
  • the invention provides in vitro methods of inhibiting DNA synthesis of proliferating cells. This method comprises contacting the cells with ApoE in an amount effective to inhibit DNA synthesis.
  • the invention also provides in vitro methods of inhibiting the chemotactic response of endothelial cells. This method comprises contacting cells with ApoE in an amount effective to inhibit the chemotactic response.
  • chemotactic response means the migration of cells in response to a stimuli.
  • the present invention provides a method whereby cells induced to migrate by exposure to a growth factor are inhibited by the contacting of ApoE with the migrating cells.
  • the amount effective to inhibit a chemotactic response is any amount effective to inhibit the migration of cells stimulated in response to a growth factor.
  • ApoE inhibits proliferation of various mammalian cells including aortic and corneal endothelial cells and mammary carcinoma cells, melanoma, and smooth muscle cells.
  • carcinoma refers to a malignant epithelial tumor.
  • ApoE inhibits aortic endothelial cells, it will likely inhibit neovascularization. Without neovascularization, i.e., the for ation of new blood vessels, cells cannot actively proliferate. In the case of tumor cells, inhibition of neovascularization results in inhibition of proliferation and thus interferes with tumor growth.
  • ApoE is a novel treatment for the inhibition of the formation of new blood vessel growth (angiogenesis) , with concommitant inhibition of tumor cell growth, and metastasis.
  • angiogenesis new blood vessel growth
  • ApoE inhibits proliferation of a wide range of tumor cells including melanoma, sarcoma, lymphoma and leukemia cells.
  • ApoE inhibits proliferation of smooth muscle cells.
  • the formation and progression of plaques and metastatic tissue, due to abnormal neovascularization, is accompanied by the migration of smooth muscle cells to the site of the plaque or the metastatic tissue. Inhibition of proliferation of smooth muscle cells may interfere with the progression of formation of plaques and metastatic tissue.
  • sarcoma refers to a soft tissue tumor.
  • treatment by ApoE for the inhibition of proliferating smooth muscle cells is also provided, thus constituting a treatment for the modulation of the formation and progression of plaques, metastatic tissue, and sarcoma tumors.
  • ApoE may be used therapeutically as an inhibitor of blood vessel formation for treatment of subjects suffering from disorders characterized by abnormal neovascularization.
  • Abnormal neovascularization means the increased and enhanced ability to form blood vessels. Examples of abnormal neovascularization include such disorders as neovascular glaucoma, diabetic retinopathy, rheumatoid arthritis (Folkman, et al., 1989) and hemangioma.
  • cells may be actively proliferating because they are tumor cells or because they are being stimulated by endogenous or naturally occurring growth factors, or serum factors, or for other reasons.
  • the ApoE is administered in a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers such as sterile solution, tablets, coated tablets and capsules. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stensic acid, talc, vegetable fats or olis, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives and other ingredients.
  • compositions comprising such carriers are formulated by well known conventional methods. However, a composition comprising ApoE in an amount effective to inhibit proliferation of actively proliferating cells was previously unknown.
  • the administration of the ApoE-containing composition may be effected by any of the well known methods, including but not limited to, oral, intravenous, intramuscular, and subcutaneous administration.
  • the amount of Apolipoprotein E incorporated in the composition may vary widely.
  • the amount of ApoE effective to inhibit cell proliferation is O.lmg - lg ApoE.
  • the precise amount and the frequency of administration of the dose will readily be determined by one skilled in the art, based on the characteristics of the formulation, body weight and condition of the subject, tumor size, route and frequency of administration, and the characteristics of the particular Apolipoprotein E to be used.
  • Cellular growth was evaluated under different growth conditions and by various methods, including DNA synthesis (mitogenesis) by measuring incorporation of 3 H-thymidine into DNA, and proliferation, by directly quantitating cell numbers by their associated enzymatic activity.
  • DNA synthesis mitogenesis
  • proliferation by directly quantitating cell numbers by their associated enzymatic activity.
  • Recombinant met-apoE contains the amino acid sequence of the human ApoE3 isoform with an additional N-terminal methionine (Vogel, et al. PNAS 1985) and was produced as described in section B below.
  • Plasmatic apoE was kindly provided by S. Eiesenberg (Laboratory of Lipids, Hadassa Medical School, Ein Kerem, Jerusalem) . It was isolated from plasma lipoproteins derived from healthy human volunteers homozygous for the E3 isoform and as described earlier (Rail S.C. et al , Methods In Enzymology 128:273 (1986)).
  • the ApoE designated ApoE6-2, spanning amino acids 1-217 of apoE was produced as described in Example 8.
  • the ApoE designated peptide 348 a tandem dimeric peptide spanning amino acids 141-155 of apoE was prepared as described in Example 2.
  • the ApoE produced by thrombin digestion of apoE which removes the C-terminus may be produced as described in Example 8.
  • Recombinant TSP18 is an 18 Kd polypeptide fragment, and recombinant TSP28 a 28 Kd polypeptide fragment, both of which contain the heparin binding domain comprising amino acids 1-174 and 1-242, respectively of human thrombospondin.
  • rFN33 is a recombinant 33 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1329 - 1722, but deleted of amino acids 1600-1689.
  • the plasmid designated pFN 137-2 which encodes for the 33 kD polypeptide fragment has been fully described in co-assigned patent application U.S. Serial No. 291,951, filed December 29, 1988, and has been deposited in Escherichia coli strain A4255 under ATCC Accession No. 67910.
  • the recombinant polypeptides listed above were stored lyophilized at -70°C.
  • Met-apoE was reconstituted by first dissolving in distilled water at a concentration of 2mg/ml followed by the addition of 0.1 volumes of 10X PBS.
  • rFN33 was dissolved in distilled water at a concentration of 3.5-5 mg/ml.
  • rTSP 18 and 28 were first dissolved in distilled water at 0.5 mg/ml, desalted on a PD10 column (Pharmacia #170851-01) that was equilibrated with 10 mM sodiumbicarbonate pH 9.5, and then eluted with the same buffer.
  • PD10 column Pharmacia #170851-01
  • Bovine aortic endothelial cells were kindly provided by Dr. E. Gallin (AFRY, Bethesda, MD) , and were used at passages 5-10. BAEC culture was routinely maintained in low glucose DMEM, containing 10% FCS, 4 mM glutamine, 0.5 mg/ml ascorbic acid and 500 u/ml penicillin and 500 u/ml streptomycin. (Biofluids Inc., Rockville, MD) .
  • Bovine corneal endothelial cells were kindly provided by Dr. D. Blake. (Maharry Medical College, Arlington, TN) , and used at passages 2-8. CBEC culture was routinely maintained in low glucose DMEM containing 10% FCS, 4 mM glutamine, 500 u/ml penicillin, 500 u/ml streptomycin, and 2.5 ⁇ g/ml Fungison (BioFluids, Rockville, MD, 20850). The media was changed every 2-3 days.
  • the human melanoma cell line A2058 cells (Todaro, et al. Proc. Natal. Acad. Sci. U.S.A., 77, 5258-5262, 1980) and the human mammary tumor cell line MDA-MB 435 (Coillean, et al., (1978) In Vitro 14, 911-915), were maintained in high glucose DMEM, containing 10% FCS, 4mM glutamine and 500 u/ml penicillin and 500 u/ml streptomycin purchased from Biofluids, Inc., Rockville, MD 20850, USA.
  • the human smooth muscle fibroblasts (SMC) were obtained from Dr. Philip Browning of the National Cancer Institute, National Institute of Health, Bethesda, Maryland.
  • the cells were cultured in RPMI medium containing 10% FCS, 4mM glutamine, 500 u/ml penicillin, and 500 u/ml streptomycin (purchased from BioFluids) .
  • END2 Mouse endothelioma cells (Williams et al., Cell 57:1053 (1989)) expressing the polyoma middle-T antigen were provided by I. Voldavsky (Hadassa Medical School, Ein Kere , Jerusalem) .
  • the END2 cells were routinely maintained in low glucose DMEM containing 10% FCS, 4mM glutamine, 500U/ml each of penicillin and streptomycin.
  • END2 cells were normally used at 70-80% confluency after 5-8 days culture. Medium was replaced every 3-4 days. Medium components were obtained from Kibbutz Beit Haemek, Israel.
  • CHO Chinese hamster ovary cells
  • N18TG2 cells Z. Vogel, Weizmann Institute
  • FCS 10% FCS
  • 2mM glutamine 2mM glutamine
  • 500U/ml 500U/ml each of penicillin and streptomycin.
  • Medium components were obtained from Kibbutz Beit Haemek, Israel.
  • ApoE and bFGF are heparin-binding molecules, exhibiting high affinity for heparin and HSPG both on the cell surface and the ECM.
  • the effect of ApoE on bFGF-stimulated growth was examined in several cell types in two separate systems: mitogenesis-by following the incorporation of [ 3 H]thymidine into DNA; and proliferation-by measuring the actual number of cells in the culture.
  • Protocol Ml Pre-attached, dense culture.
  • Protocol M2 Newly-attached, medium-dense culture.
  • Protocol PI 10 5 cells were plated into 35 mm culture dishes, in 10% FCS-containing medium, allowed to attach for 24 hours, and then refed with media containing 5% FCS and the tested growth effectors. After 72 hours, cells were detached from the plates by trypsinization, and their number monitored by a Coulter counter.
  • Protocol P2 This was carried out using the cell titer 96TM nonradioactive cell assay (Promega #G4000) based on the methods described in Denizot et al., J. Immunol. Meth. 89_:271 (1986). 5-30xl0 3 cells were plated into each well of a 96-well culture dish in medium containing 5% FCS together with the indicated concentrations of growth effectors. After 72 hours, 15 ⁇ l of dye solution was added to each well and the plates were incubated for an additional 4 hours. Then 100 ⁇ l of solubilization solution was added and after 24 hours, the amount of dye retained by the well was examined by recording absorbency at 570 nm using an ELISA plate reader.
  • END2 cells grown as described above, were harvested by trypsinization at mid-confluency after 5 days in culture. The cells were then suspended in DMEM containing 10% FCS, centrifuged, respended in basal DMEM without serum at 6xl0 6 cells/ml (or as otherwise indicated) and kept on ice. The cells were then diluted 1:1 with an extracellular matrix composition (Matrigel, H. Kleinman, Dental Institute, National Institutes of Health). Aliquots of 0.1ml were injected into the hind leg of female Balb/C mice (20-25g) . On the ninth day, the mice were sacrificed and tumor development in the injected leg was observed.
  • an extracellular matrix composition Matrigel, H. Kleinman, Dental Institute, National Institutes of Health
  • the tumor appeared as a hematomatous lump of purplish color the size of which varied in correlation with the number of cells initially injected. It was found that while a full size tumor developed after injection of 10 6 cells, the size was markedly smaller when 10 5 cells were injected, and only very small tumors developed following injection with 3xlO cells. Therefore, the experiments were routinely performed with 3xl0 5 cells/mouse.
  • Thrombin activity may be measured by the hydrolysis of a chromogenic substrate resulting in release of a colored compound essentially as described by Lotenberg (BBA, 142:556 (1983)). Briefly, thrombin is able to cleave the synthetic substrate Tos-Gly-Pro-Arg-paranitroaniline resulting in the release of paranitroaniline (PNA) whose concentration may be determined by absorbance at 405nm.
  • PNA paranitroaniline
  • the preferred host-vector system used for production of met-ApoE is E. coli strain W1485 (ATCC No. 12435) harboring plasmid pTVR 590-4; the host-vector system has been deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland under ATCC Accession No. 67360.
  • Plasmid pTVR 590-4 contains the following elements: a) Origin of replication.
  • This plasmid is a high level expressor of ApoE under the control of the strong leftward promoter of bacteriophage lambda (P L ) which is thermoindueibly controlled by the constitutively expressed cl 857 temperature-sensitive repressor also situated on the plasmid. Production of ApoE from this plasmid takes place upon heat- induction at 42°C.
  • P L strong leftward promoter of bacteriophage lambda
  • E. coli W1485 is a prototrophic wild-type strain of E. coli freely obtainable from the ATCC under Accession No. 12435.
  • Glucose and ampicillin are added from sterile concentrated stock solutions after autoclaving the other components of the medium.
  • the cultures are incubated at 30°C overnight on a rotary shaker at 250 rpm, and reach an OD ⁇ of 3.5-5.0.
  • the contents of the seed flask are used to inoculate a 50 L seed fermenter containing 25-30 L of the following production medium, which contains per liter:
  • the culture is cultivated at 30°C for 15-20 hours in order to achieve growth; the OD ⁇ o generally reaches 20-30 during this time. This is equivalent to a dry cell weight (DCW) of 7.5-12 g/L.
  • DCW dry cell weight
  • the contents of the seed fermenter are used to inoculate a 750 L (nominal volume) fermenter containing about 360 L production medium as described for seed fermenter, but excluding ampicillin.
  • the culture is cultivated at 30 ⁇ C until an OD ⁇ o of 10 is obtained.
  • Induction of ApoE expression is then achieved by raising the fermenter temperature to 42°C. At induction, the following are added to the fermenter:
  • the sodium acetate (0.1% - 1%) is added to protect cells from the "toxic effect" caused by the ApoE analog.
  • the fermenter temperature is maintained at 42°C for three hours, at which time the cells are harvested.
  • the OD ⁇ Q of 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.
  • the cell suspension is centrifuged at 14,000 rp (16,000 g) in a CEPA 101 tubular bowl centrifuge at a feed rate of 250L/hr, and a cell cake weighing about 10 Kg is produced and saved.
  • the cell suspension is centrifuged in a Westfalia CSA-19 continuous centrifuge at 500 L/hr. The sludge is either disrupted immediately or frozen.
  • the supernatant contains no detectable ApoE as measured by SDS-polyacrylamide gel electrophoresis.
  • 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 4°C - 10°C, except where otherwise indicated. All other activities were performed at room temperature.
  • Centrifugation was then performed in a continuous CEPA-41 tubular bowl centrifuge, (Carl Padberg, Lahr/Schwarzwald) with a feed rate of 9 L/hr at 20,000 rpm (17,000 g) .
  • the pellet, weighing approximately 700 g and containing insoluble ApoE was saved and the supernatant was discarded. (Note that the ApoE is insoluble due to the presence of Mg** ions.)
  • extraction buffer 50 mM tris/HCl, 20 mM EDTA, 0.3% Triton", pH adjusted to 3.0 with HC1
  • extraction buffer 50 mM tris/HCl, 20 mM EDTA, 0.3% Triton", pH adjusted to 3.0 with HC1
  • Suspension was achieved using a homogenizer (Kinematica) at low speed.
  • another 6 L extraction buffer was added (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.
  • Triton R is present in all following steps and is removed in step G.
  • the purpose of this step is to remove low molecular weight contaminants by ultrafiltration/dialysis.
  • a Millipore Pellicon ultrafiltration system using one 100 K cassette type PTHK was utilized to concentrate the supernatant of the previous step (about 12 L) to about 2 L.
  • the feed pressure was 20 psig and the filtrate flow rate was 20 L/hr.
  • the 2 L retentate containing about 2-3 mg ApoE /ml was kept cool with ice.
  • This step is to separate the ApoE from contaminants such as proteins and other cellular materials.
  • a 1.6 L DEAE Sepharose Fast Flow column (Pharmacia) was used.
  • the flow rate was 10 column volumes/hr.
  • the capacity of the column under these conditions was determined to be 4 mg ApoE/ml.
  • the 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 containing 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 stained by Coomassie Blue and the trailing edge of the peak (3.1 CV) was saved.
  • the second elution was performed using the equilibration buffer containing 150 mM NaCl. Fractions were collected and analyzed by SDS gel electrophoresis and most of the peak (3.9 CV) was saved. Endotoxins were measured by the Limulus Amebocyte Lysate (LAL) assay described in U.S. Pharmacopeia (U.S.P.) XXI, 1165-1166 (1985). The level of endotoxin was 3 ⁇ g per mg ApoE.
  • LAL Limulus Amebocyte Lysate
  • the sample was concentrated to 2 L (about 2-3 mg ApoE/ml) and dialyzed.
  • the purpose of this step is to separate active from inactive ApoE and to further remove endotoxins.
  • the retentate solutions from two batches of the previous step were combined and loaded on to the column, i.e. a total volume of about 5 L of buffer containing about 5 g ApoE.
  • the column was then washed with 2.8 CV of equilibration buffer.
  • the first elution was performed with 3 CV of equilibration buffer containing 20 mM NaCl and the second elution was performed with about 5.5 CV of equilibration buffer containing 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.
  • the QS-derived saved pooled fractions were concentrated and dialyzed by ultrafiltration through a Millipore Pellicon Ultrafiltration system using one 100K cassette.
  • the sample was dialyzed using the recirculating mode whilst maintaining the ApoE concentration at 2-3 mg/ml.
  • the final retentate volume was about 500 ml.
  • This step is to further remove endotoxins and to lower the concentration of Triton R to 0.05%.
  • CM-Sepharose Fast Flow (Pharmacia) column was used.
  • the retentate solution from the previous step was loaded on to the CM-Sepharose column.
  • the capacity of the column was 10 mg ApoE/ml and the flow rate was 10 CV/hr.
  • the column was then eluted.
  • the progress of the elution was monitored 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 containing 0.2% Triton, the other is the low U.V. absorbance buffer containing 0.05% Triton.
  • the purpose of this step is to remove the Triton".
  • This step was carried out at 4°C using the Millipore Pellicon Ultrafiltration System, containing one 100K cassette, pre-washed with 0.5 M NaOH overnight.
  • the flow rate was 9-12 L/hr and the inlet/pressure was 5-10 psig.
  • ApoE as the Triton is being removed.
  • 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 the 100K membrane.
  • 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.
  • the ApoE must not be concentrated above 1.5 mg/ml or aggregation of the ApoE molecules may occur.
  • the solution containing the ApoE was then filtered (0.2 micron filter) and stored at -70°C in 80 ml glass bottles.
  • 0.3 g of highly purified met-apoE were recovered from 3 Kg of bacterial cake.
  • the ApoE approximately 97% pure, was in the same aggregation state as plasmatic apoE when tested under the same conditions of gel permeation analysis.
  • the ApoE sample contained less then 50 pg of endotoxins/mg protein.
  • peptide 348 a tandem dimeric peptide fragment of apoE derived from the LDL-receptor binding region and the strong heparin binding consensus sequence spanning amino acids 141-155 (LRKI-RKRLLRDADDL) 2 (Dyer et al., J. Biol. Chem. 266:15009 (1991) and disclosed in U.S. Patent No. 5,177,189, issued January 5. 1993.
  • the peptide kindly provided by H. C. Krutzsch (Laboratory of Pathology, NCI, NIH) was synthesized by the standard solid phase method of Merrifield and purified as described earlier (Guo et al., PNAS 89:3040 (1992) ) .
  • ApoE will inhibit corneal endothelial cells from other mammalian (including human) sources, especially when the cells are. actively proliferating.
  • Protein synthesis in CBEC was measured following a 1 hour starvation for methionine, and administration of S 35 - methionine (6 hours) , as shown in Figure 13.
  • met-apoE 0.5 ⁇ M
  • the incorporation of S 35 -methionine into protein was identical to the control without ApoE.
  • the incorporation was approximately 60%.
  • addition of ApoE has no direct effect on the synthesis of proteins.
  • DNA synthesis in the hviman mammary carcinoma (MDA) cells was measured in a newly attached culture in 0.5% FCS and bFGF, as shown in Figure 11.
  • Addition of 0.5 ⁇ M met-apoE or rTSP18 inhibited [ 3 H]thymidine incorporation by approximately 60% and 45%, respectively.
  • Inhibition of DNA synthesis by heparin was lower (in the range of 30%) .
  • END2 mouse endothelioma cells
  • END2 are polyoma middle T-antigen transformed mouse endothelial cells, which develop spindle shaped cells in vitro and vascular tumors and hemangiomas in vivo. These cells have been used in vivo as a model system for studying angiogenesis.
  • Cell proliferation was assayed as described in Example 1 according to protocol PI.
  • Met-apoE inhibited the proliferation of END2 cells in culture with an IC 50 of about 0.25 ⁇ M at 0.5% FCS ( Figure 18). Inhibition of proliferation by another ApoE is described in Example 8.
  • ApoE inhibits the development of angiogenic lesions and hemangiomas in vivo in the endothelioma model.
  • intravenous (i.v.) administration of met-apoE at a concentration of 0.4 mg/mouse, daily, for 8 days resulted in a 40-60% reduction in the size of the hemangioma compared to the non-ApoE treated control.
  • ApoE will be effective in treating hemangiomas in humans.
  • ApoE has a specific ability to bind to lipoprotein particles for directing their removal from the plasma, "reverse cholesterol homeostasi ⁇ ", and for mediating receptor dependent endocytosis to the liver. Due to the high numbers of lipoprotein particles contained in plasma, ApoE, upon delivery in small amounts to the site of a tumor, will bind to lipoprotein particles and will be unavailable to function as an anti-proliferative agent. Therefore, full length ApoE (e.g. met-apoE) in the presence of serum or plasma is able to inhibit proliferation of cells only upon delivery in large amounts to the tumor.
  • ApoE e.g. met-apoE
  • This deficiency may be overcome by cleavage of the apoE sequence to remove the lipid binding domain present at the carboxy terminus. Upon cleavage of apoE with a proteolytic enzyme and elimination of the carboxyl end lipophilic domain, the remaining amino terminal fragment no longer has normal affinity for binding to lipoprotein particles but still readily binds to heparin and heparan sulfate proteoglycans. Therefore smaller amounts of ApoE will suffice to obtain inhibition of cell proliferation.
  • such an ApoE fragment may be produced from apoE containing the complete sequence of naturally occurring apoE by cleavage of the carboxyl terminus with a proteolytic enzyme generating, for example, a 22 kD fragment containing the amino terminus (Thuren, 1992) .
  • a proteolytic enzyme that may be used is thrombin. Upon digestion of recombinant met-apoE by thrombin , one 22 kD and one 10 kD fragment may be obtained.
  • the 22 kD fragment containing the first 191 amino terminal residues, has the binding sites for manganese heparan sulfate and heparin and the low density lipoprotein (LDL) receptors, but has reduced ability to interact with natural lipoprotein particles.
  • LDL low density lipoprotein
  • An improved method of obtaining an increased amount of the free form of ApoE in the plasma and the reduction of its sequestration by lipid particles is by production of an ApoE fragment deleted of the major lipid binding domain of apoE from the carboxyl terminus by means of recombinant DNA techniques.
  • an ApoE polypeptide fragment, ApoE6-2, spanning amino acids 1-217 of apoE is encoded and expressed by plasmid pTVR6-2 ( Figure 17) .
  • This 28KD MW ApoE was also tested for its effect on cell proliferation.
  • the purification of this ApoE is similar to the purification of met-apoE as described in Example l with minor modifications, one of which was ultrafiltration with a 50K instead of 100K cutoff membrane. The purified polypeptide was then treated with 6M urea and dialyzed to remove the urea.
  • ApoE6-2 inhibited proliferation of BAEC culture with an IC 50 around 0.3 ⁇ M at both 0.5% and 2.5% FCS respectively ( Figure 24) .
  • IC 50 around 0.3 ⁇ M at both 0.5% and 2.5% FCS respectively
  • Figure 24 shows that in contrast to met-apoE, which displays a substantial reduction in activity at higher serum concentrations, ApoE6-2 is much less affected by increasing serum concentration.
  • Thrombin activity was assayed as described in Example 1.
  • antithrombin 50 ⁇ l at 2.9U/ml, Sigma
  • heparin 15 ⁇ l, Eli Lilly 14KD, 0.4 USP/ml; 2 ⁇ M final concentration in the reaction
  • met-apoE 15 ⁇ l of the indicated dilutions of a 60 ⁇ M solution
  • Yamada, N. Inoue, I., Kawamura, M. , et al., J. Clin. Invest. 89: 706, 1992.
  • Yamada, N. Shimano, H. , Mokuno, H. , et al., Proc. Natl. Acad. Sci., USA, 86: 665, 1989.

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Abstract

La présente invention se rapporte à l'utilisation de l'ApoE visant à inhiber la prolifération des cellules tumorales renfermant des cellules proliférant activement. Ce procédé consiste à mettre en contact les cellules avec une quantité d'apolipoprotéine E (ApoE) efficace dans l'inhibition de la prolifération cellulaire.
PCT/US1993/007582 1992-08-12 1993-08-12 Procede d'inhibition de la proliferation cellulaire au moyen de l'apolipoproteine e WO1994004178A1 (fr)

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AU50072/93A AU673543B2 (en) 1992-08-12 1993-08-12 Method of inhibiting cell proliferation using apolipoprotein E
JP6506399A JPH08502730A (ja) 1992-08-12 1993-08-12 アポリポタンパクｅを用いた細胞増殖を阻害する方法
EP93919995A EP0659085A4 (fr) 1992-08-12 1993-08-12 Procede d'inhibition de la proliferation cellulaire au moyen de l'apolipoproteine e.
KR1019950700386A KR950702430A (ko) 1992-08-12 1993-08-12 아포리포프로테인 이(Apolipoprotein E)를 이용한 세포증식 억제방법
NO950491A NO950491L (no) 1992-08-12 1995-02-09 Fremgangsmåte for hemming av celleproliferasjon ved bruk av apolipoprotein E

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EP0715521A1 (fr) * 1993-08-12 1996-06-12 Biotechnology General Corp. Procede d'inhibition du sarcome de kaposi
US6171609B1 (en) 1995-02-15 2001-01-09 Neorx Corporation Therapeutic inhibitor of vascular smooth muscle cells
WO1998030233A1 (fr) * 1997-01-14 1998-07-16 Ramot University Authority For Applied Research & Industrial Development Ltd. Composition pharmaceutique pour le traitement des yeux
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CA2141598A1 (fr) 1994-03-03
ZA935879B (en) 1994-03-11
EP0659085A4 (fr) 1996-06-12
JPH08502730A (ja) 1996-03-26
AU673543B2 (en) 1996-11-14
EP0659085A1 (fr) 1995-06-28
IL106664A0 (en) 1993-12-08
NZ255732A (en) 1997-05-26
KR950702430A (ko) 1995-07-29
AU5007293A (en) 1994-03-15

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