JP2001518449A - Apolipoprotein E / growth factor complex and uses thereof - Google Patents

Abstract

  (57) [Summary] Provided herein is a composition comprising a complex of apolipoprotein E and nerve growth factor, neurotrophin 4 or gamma interferon. Apolipoprotein E may be of any isoform, but apolipoprotein E3 is preferred. Further, a covalent complex of apolipoprotein E and nerve growth factor, γ-type interferon or neurotrophin 4 is also preferable. Further, there is provided a method for promoting the survival of nerve cells by administering a composition comprising a complex of apolipoprotein E and nerve growth factor or neurotrophin 4. Also, a method for administering a composition containing a complex of apolipoprotein E and γ-type interferon to protect against viral infection, treat diabetes, treat bone loss, treat vascular trauma, and generate an antitumor effect Are also disclosed. The method of the present invention can be practiced in vitro or in vivo.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] RELATED APPLICATION information This application claims the right of US Provisional Application No. 60 / 060,533, filed Sep. 30, 1997, the provisional application is incorporated herein by reference in its entirety Shall be.

[0002] report the invention of federal funding, the grant number from the United States National Institute of Health (NIH) (grant
number) US Federal funding under R01AG12532-01 (G
It was carried out in response to an average support. The United States Federal Government has certain rights in this invention.

FIELD OF THE INVENTION The present invention relates to apolipoprotein E and the nerve growth factor neurotrophin (n
The present invention relates to a composition comprising a complex containing (Eurotropin) 4 or γ-type interferon and a method of administering the composition.

[0004] background apolipoprotein E of the invention (protein: apo E; alleles: APOE), the brain [For a review, Marley (Mahley), Science 240,62
2, 1988] and cerebrospinal fluid (CSF) [Pitas
J. et al. Biol. Chem. 262, 14352, 1987]. Several observations indicate that ApoE is involved in the damaged nervous system. After lesions of the olfactory sulcus endothelium,
Increased expression of apoE mRNA by hippocampal astrocytes [Poirie (P
Oirier) et al., Mol. Brain Res. 11, 97 pages, 1991
Year〕. Oligodendrocytes and macrophages increase apoE expression after optic nerve injury and after sciatic nerve injury [optic nerve: Stoll
GLIA 2, p. 170, 1989; sciatic nerve: Skene and Shoter, Proc. Nat. Acad
. Sci. ScL USA 80, 4169, 1983; Stoll and Mueller, Neurosci. Lett.
72, 233, 1986]. In addition, the apoE protein is found in the peripheral nervous system (
It increases to 5% of total extracellular protein after PNS) injury (Skene and Shooter, supra). APOE is a familial and late-onset Alzheimer's disease susceptibility gene [Alzheimer's disease: Strittmatte
r) et al., Proc. Nat. Acad. Sci. USA 90, 1977, 1993; for a review, see Stritt Matter and Roses (Strrit).
tmatter and Roses), Proc. Nat. Acad. Sci
. USA 92, p. 4725, 1995]. The abundance of APOE4, one of the three major alleles of human APOE, has been correlated with an increased risk of developing Alzheimer's disease and a reduced average age of onset. These observations indicate that apoE is involved in the damaged or affected nervous system.

[0005] The major isoforms of human apoE, apoE2, apoE3 and apoE4, are distinguished by cysteine-arginine substitutions at positions 112 and 158. ApoE3, the most common isoform, is secreted as a 299 amino acid protein with one cysteine at position 112 and one arginine at position 158, and apoE2
It contains one cysteine at position 58 and apoE4 contains one arginine at position 112. Apo E contains two structurally and functionally distinct regions, a hydrophobic region and a hydrophilic receptor binding region [Weisgraber.
Adv. Prot. Chem. 45, 249, 1994]. The crystal structure of the hydrophilic region of Apo E is homologous to the 4 helix bundle growth factor family, including ciliary neurotrophic factor, but the sequences of these proteins differ greatly [
CNTF; review by Bazan Neuron 7, p. 197;
1991; Mott and Campbell
Curr. Opin. Struc. Biol. 5, p. 114, 1995; Wilson et al., Apo E Crystal Structure, Science 252,
1817, 1991; McDonald et al., CNTF.
Crystal structure, EMBO J. 14, 2689, 1995].

[0006] Nerve growth factor (NGF) includes brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4/5 (NT4 / 5) and neurotrophin 6 (NT6) Belongs to the neurotrophin family. All of these proteins bind to the low affinity NGF receptor p75 and certain members of the trk family of tyrosine kinase receptors. In the case of NGF, this is the trkA receptor. Binding of a 26 kDa homodimer of NGF to trkA triggers an intracellular signaling cascade that begins with autophosphorylation of trkA. Eventually, trkA is activated, altering gene expression, resulting in nerve survival, outgrowth, excitability and differentiation.

[0007] It has been demonstrated that NGF promotes the survival of sensory cells and neurite growth from developing sympathetic and neural crests [for review, see Snider and Johnson, Annual Neuro.
org. 26, p. 489, 1989]. This is NGF or t
Mice that are homozygous mutants for rkA,
> 90%) and severe cell loss in the dorsal root ganglion (DRG, 70-80%) [Crowley et al., Cell 78, 1001, p. 1994; Smeyne et al., Nature. 368, 246
, 1994], and the fact that they die within three weeks of birth. The ability of NGF to promote neurite outgrowth is one of the most dramatic activities of NGF. Indeed, neurite outgrowth from explanted chick embryo DRG is a standard bioassay for testing NGF activity. Dissociated cultures of DRG and SCG also respond to NGF and increase neurite growth. PC12 from rat adrenal cortex
Cell lines have provided an excellent system to study the neurite outgrowth and survival effects of NGF. When PC12 cells are cultured in a medium supplemented with fetal calf serum and horse serum, the PC12 cells proliferate and have a small, spherical, bright phase (phase-bris).
ght) Appear as cells. When NGF is added to the medium, these cells stop dividing and differentiate into a sympathetic neuron phenotype with multiple neurites and large cell bodies. NGF is also required for survival of PC12 cells in a serum-free environment.
Because this paradigm is readily available, most studies that seek to elucidate the mechanism of NGF action use PC12 cells.

As DRG neurons mature, their NGF-dependent time course and sensitivity for their survival change. To illustrate this, there is 45% cell loss in the corresponding DRG 24 hours after transection of the sciatic nerve of neonatal rats [Yip et al. Neurosci. 4, p. 2986, 1984
Year〕. Systemic administration of NGF reduces cell death by 18% (supra). In contrast, in adult rats, transection without regeneration results in the death of 22% of DRG axotomized cells 3 weeks after axotomy. Applying NGF directly to the proximal stump prevents all cell death due to axotomy [Rich et al.
J. Neuro. Cytol. 16, 569, 1987].

[0009] Exogenous NGF can promote the survival and regeneration of transected sciatic nerves. Numerous studies have confirmed that silastic tube implants filled with NGF enhance the rate and degree of recovery of sensory function [Ricchi et al. Neuro. Cytol. 16, 1987; Rich et al. Ner
uro. Cytol. 18, 569, 1989; Derby
) Et al., Exp. Neurol. 119, 176, 1993].

[0010] Neurotrophin 4 (NT4) is another member of the neurotrophin family. NT4 promotes survival of neonatal rat corticospinal motoneurons [Junger and Varon, Bra
in Res. 762, p. 56, 1997], increasing the survival of rat septum neurons cultured under normal and stress conditions [Nonner (No.
Nner) et al. Neurosci. 16, 6665 pages, 1996],
Promoting survival and ecological and biochemical differentiation of rat embryonic striatal neurons in culture [Ventimiglia et al., Eur. J. Ne
urosci. 7, 213, 1995], and promoting survival of rat vestibular ganglion neurons in culture and protecting these cells from neurotoxins [Zheng et al., J. Am. Neurobiol. 28, 330 pages,
1995].

[0011] Interferon (IFN) is a protein secreted from eukaryotic cells after viral infection, which in turn protects cells from viral infection.
At present, three types of interferons are known. These are IFNα,
Called IFNβ and IFNγ, each has a different structure and biological effect. All of the IFNγs identified to date have been glycosylated, but glycosylation does not appear to affect biological activity [Keller et al., J. Am. Biol
. Chem. 258, 8010, 1983]. Gamma-type interferons are considered lymphokines. This is because gamma-type interferon is produced by lymphocytes following specific or non-specific stimulation by antigens. Gamma-type interferons are well known as antiviral and antitumor agents [US Patent No. 5 issued to Hauptmann et al.
, 602, 010]. Other studies have shown that IFNγ increases the concentration of intravascular Cl inhibitors in subjects exhibiting or at risk for Cl inhibitor deficiency [US Pat. No. 5,271,933 to Lotz et al.
No. 1]. Cl inhibitors are serine protease inhibitors involved in the regulation of several proteolytic systems, including the complement system, the contact system, the coagulation system and the fibrinolytic system [Davis et al., Ann. Rev .. Immunol. 6,
595, 1988]. Other activities of IFNγ include promoting multinucleated giant cell generation and activating macrophages [Weinberg et al., Pro.
c. Natl. Acad. Sci. USA 81, p. 4554, 1984], inhibition of bone resorption (U.S. Pat. No. 4,921,697 to Peterlik et al.), And prevention of type I diabetes [Sobe
U.S. Patent No. 5,624,895 to I).

[0012] Thus, NGF, NT4 and IFNγ have biologically important activities. Accordingly, there is a need in the art for methods to enhance the activity of these factors. In particular, it was an object of the present invention to provide the neurotrophic growth factors NGF and NGF.
To provide a means to promote the activity of T4, to slow the progression of neurodegenerative diseases, to protect against neurodegeneration after injury, and to promote nerve regeneration.

SUMMARY OF THE INVENTION [0013] Disclosed herein is a novel discovery that apoE binds to the neurotrophic factor NGF and enhances the biological effects of NGF. "Enhance" (potentia
te) means that apoE increases the achieved maximal response of growth factors and / or
Or increasing the potency (ie, shifting the dose response curve to the left), thereby promoting at least one biological effect of NGF. Or,
The term "enhance" refers to the fact that apoE stabilizes the NGF molecule and reduces its degradation. Also disclosed is the finding that apoE advantageously binds to NT4 and IFNγ.

The present invention provides, as a first aspect, a composition comprising a complex of ApoE and NGF. Also disclosed is a composition comprising a complex of ApoE and NT4. Also disclosed are compositions comprising a complex of Apo E and IFNγ. The Apo E component may be Apo E2, Apo E3 or Apo E4, and may be defatted (de
(lipidated) state. Further, the complex is composed of ApoE and NG
It may be generated by covalent or non-covalent interactions with F, NT4 or IFNγ molecules.

The present invention provides, as a second aspect, a method for promoting the survival of nerve cells. The method comprises administering to the nerve cells a survival promoting amount of a complex of ApoE and NGF or NT4.

The present invention provides, in a third aspect, a method of administering to a subject in need of treatment a therapeutically effective amount of a composition comprising a complex of ApoE and NGF or NT4.

The present invention provides, as a fourth aspect, a method for protecting cells from viral infection. The method comprises administering to the cells an antiviral effective amount of a composition comprising a complex of ApoE and IFNγ. Further, there is provided a method of administering to a subject in need of treatment an effective antiviral amount of IFNγ.

In another aspect, the present invention provides a method of administering an anti-tumor effective amount of IFNγ to a subject in need of treatment.

In the following description of the invention, these and other aspects of the invention will be described in more detail.

[0020] DETAILED DESCRIPTION OF THE INVENTION A. Neurotrophic factor-NGF and NT4 nervous system coordinately express many proteins, including growth and survival factors, cell surface receptors, and secreted extracellular proteins, against acute injury and chronic neurodegenerative diseases To respond. Many of these proteins, particularly neurotrophic factors, are involved in recovery from injury and protection from neurodegenerative diseases. In the complex environment of the traumatized nervous system, it is unlikely that only one factor works. The coordinated interaction of growth factors with other proteins may play an important role in growth factor stability, localization or presentation. Optimal functioning of growth and survival factors released at the site of injury is important for reversing nerve trauma.
ApoE increases under acute and chronic stress in both the peripheral and central nervous systems.

Disclosed herein is the discovery that Apo E binds to the neurotrophic factors NGF and NT4. In addition, it discloses the discovery that apoE enhances the biological activity of NGF. As described above, "enhancing" means that ApoE increases the achieved maximal response of growth factors and / or increases potency (ie, shifts the dose response curve to the left) By promoting at least one biological effect of NGF. Alternatively, the term "enhancing" refers to the fact that Apo E stabilizes the NGF molecule and reduces its degradation.

Although it is not intended to stick to any particular theorem of the present invention, Apo E
The finding that it binds to T4 suggests that apoE may act as an accessory protein for these growth factors and modulate growth factor metabolism or biological activity. Some mechanisms may allow the growth factor to be localized to the extracellular matrix by binding the growth factor via the apoE molecule. It has previously been demonstrated that apoE binds to the extracellular matrix protein laminin, thereby increasing neuronal attachment and altering growth cone elongation [Huan
g) et al., Exp. Neurology 136, 251 pages, 1995].
ApoE also binds to the growth factor, and by altering the ability of the growth factor to interact with cell surface receptors, such as by inhibiting the proteolytic inactivation of the growth factor, or by altering the ability of the growth factor to interact with cell surface receptors. May alter activity. Apo E and NGF
And / or interaction with NT4 may be important for the regulation of the role of these growth factors in the neurodegenerative response, both in acute and chronic diseases of the nervous system.

Disclosed herein are compositions comprising an ApoE: NGF complex and an ApoE: NT4 complex. The complex of the present invention can be formed simply by mixing Apo E with a neurotrophic factor (generally an aqueous solution) or by any other suitable method known in the art. The composition may include both a complex of Apo E and NGF and a complex of Apo E and NT4. Furthermore, Apo E: NG
Compositions comprising the F complex and / or the apoE: NT4 complex and / or the complex of apoE and other neurotrophic factors such as ciliary neurotrophic factor are also encompassed by the present invention.

[0024] One of skill in the art will appreciate that a composition comprising an ApoE complex of the invention thus produced typically comprises ApoE, NGF and / or NT4 monomers, homodimers and multimers. It will be understood that it includes. Generally, 35%, 25%, 20%,
15% or lower low percentage of NGF molecules, or 40%, 50%, 70%
, 85%, 90%, 95%, 99% or higher of NGF molecules will be complexed with ApoE. As used herein, the term “Apo E
: NGF "and" ApoE: NT4 "refer to the ApoE: NGF and ApoE: NT4 complexes containing NGF or NT4 monomers, dimers, trimers and larger multimers, respectively. Include. Alternatively, an apoE monomer, homodimer, homotrimer, or the like may be associated with an NGF or NT4 monomer, homodimer, homotrimer, or the like. Accordingly, the present invention provides for one or more apoE molecules and one or more N
Includes complexes with GF or NT4 molecules.

While not wishing to adhere to any particular theorem of the present invention, it is believed that the active form of NGF is a homodimer, and that the NGF homodimer forms a complex with the apoE monomer . Heterodimeric ApoE: NGF complex and heterotrimeric ApoE: (NGF
) _Two complexes are preferred. Similarly, heterodimers of ApoE and NT4 are also preferred. 1
Complexes of one or more apoE molecules and one or more NT4 molecules are also an aspect of the invention.
One of skill in the art will appreciate that the ApoE: NGF and ApoE: NT4 complexes may be more loosely associated with additional molecules.

Alternatively, Apo E is first bound to a substrate such as a polymer surface (ie, tissue culture plate, test tube) and then exposed to NGF or NT4 to form a complex with NGF or NT4. You may. Such an ApoE binding substrate is useful for collecting NGF or NT4 from solution. Another alternative is ApoE: N
A GF complex or an apoE: NT4 complex can be formed before binding to a substrate. Such substrates are useful for culturing cells in vitro.

The binding interaction between Apo E and NGF or NT4 is strong. Dissociation constant of apo E complex, ^{10 -7} or more, preferably ^{10 -8} or more, more preferably 10 ^{- 9} or more. The complex can be formed by covalent or non-covalent interactions, but a covalent complex of Apo E with NGF or NT4 is preferred. Typically, the ApoE: NGF and ApoE: NT4 complexes
Stable in solutions containing 1% or more SDS. ApoE3 and NGF or N
Complexes formed with T4 are also preferred.

[0028] The Apo E component of the complex of the invention may be Apo E2, Apo E3, Apo E4 or a combination thereof. Preferably, the complex comprises ApoE3. ApoE variants and apoE fragments that bind to the apoE receptor and / or form a complex with NGF and / or NT4 to enhance the biological effects of NGF and / or NT4 are also included in the invention. Is done. Apo E may be from any source species, but is preferably derived from mammals, and more preferably from human.
The Apo E molecule may be in a natural lipid-bound state or a defatted state, but is preferably in a defatted state.

Apo E can be purified from a natural source (ie, blood, serum or peritoneal fluid). U.S. Pat. No. 5,672,685 describes a method for separating native apo E from peritoneal fluid, the disclosure of which is incorporated herein by reference in its entirety. Most serum-derived apoE is associated with lipoprotein particles.
Purification of ApoE from serum requires delipidation with organic solvents or detergents, which causes significant protein denaturation. A method of ultracentrifuging lipoproteins, then lyophilizing, defatting and chromatographically separating apoE is described by Rall et al. Methods Enzymol. 128, 2
73, 1986. An alternative method for separating apoE from an apolipoprotein mixture utilizes gel electrophoresis. Purification of the Apo E isoform can be performed using isoelectric focusing (Roll et al., Supra).

Apo E may be separated from contaminating proteins using heparin-Sepharose chromatography utilizing the heparin binding properties of Apo E (Roll et al., Supra).
. Apo E may be separated and / or purified and optionally homogenized using conventional methods such as affinity chromatography, size exclusion chromatography, gas chromatography, HPLC and combinations thereof. To separate the cysteine-free E4 isoform of apo E from mixed cysteine-containing proteins, thiopropyl chromatography on thiopropyl sepharose is used [Weisgraber et al. Biol. Chem. 2
58, 2508, 1983].

[0031] Recombinant Apo E can be produced using methods known in the art, and human recombinant Apo E is commercially available. However, recombinant proteins are not in their native glycosylated form and therefore tend to be denatured or oxidized during purification.

[0032] The NGF component of the complex may also be derived from natural or recombinant sources and may be produced by any means known in the art. Natural form: Della Barre (Dell
a Valley) et al., US Pat. Nos. 5,210,185 and 5
U.S. Pat. No. 4,407, issued to Young.
744. Recombinant: U.S. Patent No. 5,27, issued to Chan et al.
U.S. Pat. No. 5,288,62 to Gray et al.
No. 5,082, issued to Heinrich.
No. 774. Further, the NGF component of the apoE: NGF complex of the present invention may be derived from any source species, but is preferably derived from mammals, and more preferably human. The term “NGF” encompasses NGF variants, analogs and derivatives. For example, US Pat. No. 5,349,055 to Persson et al. Discloses that binding to the low affinity p75 receptor is significantly reduced but binding to the trk receptor is not substantially altered. Discloses NGF analogs. In addition, this reference also discloses NGF analogs with increased stability. In the art, chimeric NGF molecules in which the NGF peptide region is substituted with the corresponding residue of brain-derived growth factor (BDGF) are also known [Ibanez (Ibanez)
z) et al., EMBO J .; 10, 2105, 1991]. Finally, the term "
"NGF" also encompasses NGF fragments that bind to the NGF receptor and / or elicit the neurotrophic effect of NGF. For example, US Pat. No. 5,134,121 to Mobley et al. Discloses NGF peptides and analogs thereof that elicit an NGF-associated biological response.

Similarly, the NT4 component of the disclosed apoE: NT4 complex may be derived from natural or recombinant sources and may be produced using any means known in the art. Recombinant: US Patent No. 5,364,769 to Rosenthal. Further, NT4 of the apoE: NT4 complex of the present invention
The components may be from any source species, but are preferably of mammalian origin, and more preferably of human origin. The term "NT4" refers to variants of NGF, such as those disclosed in US Patent No. 5,364,769 to Rosenthal and US Patent No. 5,349,055 to Person.
Also encompasses analogs and derivatives. The term "NT4" also encompasses NT4 fragments that bind to the NGF receptor and / or elicit the neurotrophic effects of NT4.

[0034] A method of promoting neuronal cell survival by administering an ApoE: NGF complex and / or an ApoE: NT4 complex is also an aspect of the present invention. The term "promoting survival" of neural cells is intended to be broadly interpreted and encompasses the neurotrophic and neuroregenerative effects of the compositions of the present invention. Or, "
The expression "promoting survival" refers to the effect of the composition of the present invention in protecting neurons from damage and / or enhancing recovery from nerve damage. By "promoting survival" is meant that the conjugate of the invention, as defined above, improves neuronal survival to some extent. 5%, 10%, 25%, 50%
, 75%, 100% or more. "Nerve cells" include in vitro
The cells and tissues of the central and peripheral nervous systems of the tro and in vivo are included. Compositions comprising an ApoE: NGF complex and / or an ApoE: NT4 complex for use in the method of the invention are as described above in more detail.

The apoE complex of the present invention can be administered to nerve cells in vitro. Usually, in vitro administration will involve administration of the ApoE: NGF complex and / or ApoE
: It is only necessary to add a solution containing the NT4 complex (ie, an aqueous solution) to the medium.
Alternatively, the apoE component and the NGF or NT4 component can be added individually, simultaneously or sequentially to the medium. As another alternative, the ApoE molecule or ApoE: growth factor complex can be bound to a substrate, preferably prior to culturing the cells on the substrate (ie, a tissue culture dish or dish). It is well known in the art that ApoE binds to macromolecular surfaces or coated surfaces (with extracellular matrix proteins such as laminin) of tissue culture plates.

The complex of Apo E and NGF or NT4 enhances the activity of these neurotrophic factors, and therefore, the in vitro culture of neurons such as immortalized PC12 cells (see, eg, US Pat. No. 5,349,055) and primary culture of neurons [see, eg, Varon, Brain Res. 76
2, 56, 1997; Barrett and Barrett,
d Barrett), J.A. Neurosci. 16, 6665 pages, 199
6 years; Abiru et al., Brain Res. Dev. Brain R
es. 91, 260, 1996; Snyder and Johnson (Sni)
der and Johnson), Annual Neurology 26
489, 1989; Yip et al. Neurosci. 4
, P. 2986, 1984].

As provided by US Pat. No. 5,134,121 to Mobley, NGF is also utilized as a component of a medium that promotes neuronal survival. It is also used in the treatment of Alzheimer's disease, Huntington's disease and other neurodegenerative diseases.
A method using GF is also disclosed. NGF is also used in the treatment of drug-induced neuropathy, as demonstrated in U.S. Pat. No. 5,604,202 to Kessler et al. Therefore, the NGF complex of the present invention having enhanced neurotrophic activity can be used in the same manner as known NGF compositions.

As evidenced by US Pat. No. 5,364,769 to Rosenthal, NT4 promotes neuronal cell survival in vitro,
It is used as a component of a medium that induces growth. Also disclosed is the use of NT4 in the treatment of neurodegenerative diseases. Therefore, the NT4 complex of the present invention having enhanced neurotrophic activity can be used in the same manner as the known NT4 composition.

The apoE: NGF complex and the apoE: NT4 complex of the present invention are also used in tissue culture since the complexed growth factor is protected from proteolytic destruction.

Finally, the conjugates of the present invention are used in methods for quantifying or purifying NGF or NT4 receptors or other proteins that bind to these neurotrophic factors. Apo E
Easily binds to surfaces such as test tubes and microtiter plates. Therefore, N
GF or NT4 can be bound to a surface for use in sandwich assays and affinity purification methods well known to those skilled in the art.

The apoE: NGF complex or apoE: NT4 complex of the present invention can be administered to a subject in vivo.
It can also be administered in vivo. The administration method and the pharmaceutical preparation of the composition of the present invention include:
It is described in further detail below. The method of the present invention is useful for treating a subject suffering from a neurodegenerative disease or a subject suffering from damage or trauma to nerve tissue. Such subjects include those suffering from Alzheimer's disease, Parkinson's disease, peripheral nerve injury, peripheral neuropathy (particularly diabetes-induced peripheral neuropathy), amyotrophic lateral sclerosis, head injury and stroke. But not limited to them. The invention is particularly useful for treating Alzheimer's disease and diabetes-induced peripheral neuropathy.

Subjects suitable for practicing the present invention generally include humans, monkeys, horses, goats, cows, sheep, pigs, dogs, cats, rabbits, rats, hamsters, mice, quails, chickens and turkeys, and the like. (But not limited to) mammals and birds. For in vivo administration, human subjects are preferred. Similarly, cultured neural tissues / cells for use in the present invention include human, monkey, horse, goat, cow, sheep, pig, dog, cat, rabbit, rat, hamster, mouse,
Including nervous tissue and cells from quail, chicken and turkey (however,
These include, but are not limited to, mammalian and avian tissues and cells.

[0043] The disclosed compositions can be contained in a physiologically acceptable carrier, preferably a sterile carrier, which is not harmless to the cells and pharmaceutically acceptable. The resulting carrier is included.

The following are pharmaceutical preparations:
In the manufacture of a medicament of the invention, referred to as a pharmaceutical composition, the composition of the invention is typically mixed with a pharmaceutically acceptable carrier. For injections, the carrier will typically be liquid. For other modes of administration, the carrier can be a solid or a liquid such as pyrogen-free sterile water or pyrogen-free phosphate buffered saline. Alternatively, the conjugate of the present invention may be incorporated or encapsulated in a suitable polymer matrix, liposome or membrane to provide a sustained release device suitable for implantation near the site to be treated locally.

When preparing these compositions for administration to a subject, the pharmaceutical carriers are compatible with conventional dosage forms, for example, injection solutions, tablets, capsules, sugars, syrups, solvents, suspensions and the like. Can be used. As an injection medium, it is preferable to use water containing an additive useful in the case of an injection liquid such as a stabilizer, a salt or a salt solution and / or a buffer. The active agent or a pharmaceutical preparation thereof may be included in a nutrient medium, for example, in a nutrient. Oral preparations may be sustained release formulations or enteric coatings that facilitate delivery of the peptide to the small intestine.

When administering the apoE conjugate of the invention to a subject in need of treatment, any suitable route of administration may be used. Suitable routes of administration include parenteral injection (eg, subcutaneous, intramuscular or intradermal), or oral, rectal, topical, intranasal, intraocular, intrathecal, and intracerebral administration. The ApoE: NGF complex and the ApoE: NT4 complex are included in an amount effective to carry out the treatment of interest. Generally, the apoE complex of the invention is present in an amount effective to promote neuronal cell survival. ApoE: NGF complex and ApoE: NT
The 4-complex may be administered simultaneously with or in combination with the other agent. In particular, the Apo E: NGF complex and / or the Apo E: NT4 complex
It can be administered with other neurotrophic factors such as F.

The exact dosage of the ApoE: NGF conjugate or ApoE: NT4 conjugate is routinely determined and depends on the age and species of the subject, the desired effect, the isoform of ApoE and the route of administration. . The preferred dose is simply a known amount of apoE, as known to those skilled in the art.
: A composition containing an NGF complex or an apoE: NT4 complex is administered to a subject in vivo.
Administered tro or in vivo, the cells, tissues or subjects may be monitored for the desired effect and determined.

There are no specific upper or lower limits for the dose of the ApoE: NGF conjugate or ApoE: NT4 conjugate administered in accordance with the present invention. For in vivo administration, the dose may be as low as 10, 3, 1, 0.5 or 0.1 g or more per kg body weight. The in vivo dose is 10 g / 30 g / kg body weight,
It may be as high as 50 g or 100 g or more. Generally, the in vivo dose of the administered ApoE: NGF or ApoE: NT4 complex will be:
About 1 × ^{10 -1} per liter, an amount sufficient to obtain a 1 × 10 ⁰ or complex peak plasma concentrations of from 1 × 10 ¹ pmol to about ^{1 × 10 2, 1 × 10} 3 or 1 × 10 ⁴ pmol Will.

[0049] Similarly, there is no particular lower or upper limit for the dose of the conjugate of the invention administered in vitro. For in vitro administration, the dose may be as low as 10, 5, 1, 0.5 or 0.1 ng or less per ml of vehicle. In vitro doses are 10, 50, 100, 500 per ml of medium.
, 1,000 or 1,500 ng or more.

B. Another aspect of the gamma interferon present invention apo E: is IFNγ complex. In a preferred embodiment, the biological activity of IFNγ is increased by forming a complex with ApoE (NGF and NGF).
(As defined for T4). ApoE: IFNγ complex is apoE
: NGF complex and apoE: NT4 complex can be formed as described above.

[0051] One of skill in the art will appreciate that compositions comprising the ApoE: IFNγ complex of the present invention will typically comprise an ApoE
And also includes IFNγ monomers, homodimers and multimers. In general, low percentages of 35%, 25%, 20%, 15% or less, or 40%, 50%, 70%, 85%, 90%, 95%, 99% or more in the composition Of IFNγ molecules form a complex with ApoE. As used herein, the term “apoE: IFNγ” encompasses an apoE: IFNγ complex that includes monomers, dimers, trimers and larger multimers of IFNγ. Alternatively, an apoE monomer, homodimer, homotrimer, or the like, may be associated with an IFNγ monomer, homodimer, homotrimer, or the like. Accordingly, the present invention includes a complex of one or more apoE molecules and one or more IFNγ molecules. While not wishing to adhere to the particular principles of the present invention, the active form of IFNγ is a homodimer,
It is believed that the gamma homodimer forms a complex with the apoE monomer. Such apoE: IFNγ heterotrimeric complexes are preferred. One skilled in the art will also appreciate that the apoE: IFNγ complex may be more loosely associated with additional molecules.

Alternatively, first, ApoE was bound to a substrate and then exposed to IFNγ to
A complex with γ may be formed. As another alternative, the apoE: IFNγ complex can be formed and then bound to a substrate.

The binding interaction force between Apo E and IFNγ is determined by the Apo E: NGF complex and Apo E
: As described above for the NT4 complex.

The Apo E component of the disclosed conjugate comprises an Apo E: NGF conjugate and an Apo E:
As described above for the NT4 complex. The IFNγ component of the complex can be from a natural or recombinant source and can be produced by any means known in the art. Natural Form: U.S. Patent No. 5,518,899 to Kurimoto; U.S. Patent No. 4,723,000 to Georgiades et al .; Fleischman
US Pat. No. 5,132,110 to H. Ann et al .; Recombinant: US Pat. No. 5,602,010 to Hauptmann et al.
U.S. Patent No. 4,970,161 to Kakutani et al .; U.S. Patent No. 4,889,803 to Revel et al. further,
The IFNγ component of the apoE: IFNγ complex of the present invention may be from any source species, but is preferably of mammalian origin, more preferably of human origin. The term “IFNγ” also refers to IFNγ that retains the biological activity of IFNγ.
Also encompasses variants, analogs and derivatives of gamma. For example, cowling (
U.S. Patent No. 4,845,196 to Cowling; PCT Application No. 83/04053; U.S. Patent No. 4,898, issued to Itoh et al.
931 and 4,758,656; Franke et al., DN.
A 1, 223, 1982; King et al. Gen. Vi
rol. 64, 1815, 1983, variants, analogs and derivatives of IFNγ. The term “IFNγ” further encompasses fragments of an IFNγ molecule that bind to an IFNγ receptor and / or elicit a biological effect of IFNγ.

Another aspect of the present invention is a method of administering a composition comprising an apoE: IFNγ complex to a cell in a biologically effective amount. Compositions comprising an apoE: IFNγ complex are used in a method of protecting cells from viral infection, wherein such a method comprises providing the cell with an antiviral effective amount of a complex of apolipoprotein E and IFNγ. Administering the composition. For example, Wheelock, Science
149, p. 310, 1965. By "protecting" a cell from a viral infection is meant reducing or eliminating the rate of viral infection, or reducing or eliminating an already existing infection, by the disclosed methods. Using such a method, cultured cells can be protected in vitro and test subjects can be protected in vivo.

Another aspect of the invention is a method of administering an anti-tumor effective amount of a composition comprising an ApoE: IFNγ complex. An “anti-tumor effective amount” of a conjugate of the invention is a dose effective to reduce tumor incidence or reduce tumor size, growth or metastasis. Also, administering apoE: IFNγ of the invention to treat a subject suffering from a tumor (eg, by reducing the size, growth or metastasis of the tumor), or treating a subject at risk of developing a tumor. The present invention can be used to reduce the incidence.

The apoE complex of the present invention can be administered to cells in vitro. Typically, in vitro administration is simply performed using a solution containing the ApoE: IFNγ complex (
That is, it is only necessary to add the aqueous solution) to the medium. Alternatively, Apo E component and IF
The Nγ component can be separately, simultaneously or sequentially added to the medium. As another alternative, the ApoE molecule or ApoE complex can be bound to a substrate, preferably prior to culturing the cells on the substrate (ie, a tissue culture dish or dish). It is well known in the art that ApoE binds to macromolecular surfaces or coated surfaces (with extracellular matrix proteins such as laminin) of tissue culture plates.

The disclosed apoE: IFNγ complexes can also be administered to a subject in vivo. Suitable subjects, modes of administration, pharmaceutical preparations of the ApoE: IFNγ-containing composition and appropriate dosages thereof are as described above for the ApoE: NGF complex and the ApoE: NT4 complex. The disclosed apoE: IFNγ conjugates can be administered alone or in combination with other therapeutic agents.

The composition containing the ApoE: IFNγ complex can be administered in vivo to a subject in need of antiviral and antitumor treatment. The conjugates of the invention can be used, for example, for the treatment of bone degradation and bone resorption in the elderly, postmenopausal women, and women suffering from or at risk of developing osteoporosis. It can also be administered. Finally, IFNγ can be administered to stimulate blood coagulation by stimulating blood Cl inhibitor levels or to treat type I diabetes.

ApoE: IFNγ complexes that enhance the activity of IFNγ are useful for the in vitro culture of IFNγ-responsive cells, including, but not limited to, bone cells [provided by Peterlik et al. United States Patent No. 4,921
, 697].

[0061] US Patent No. 6,268,1 to Brandelly et al.
As demonstrated by No. 69, IFNγ is useful in treating ovarian cancer. It is also known to use IFNγ for the treatment of type I diabetes [Sober (Sobbel)
e.g., U.S. Pat. No. 5,624,895). Furthermore, IFNγ
Are disclosed in U.S. Pat. No. 5,271,933 issued to Lotz et al.
No. 1] and bone loss (Peterlik, supra). Therefore, IF of the present invention having enhanced biological activity
Nγ complexes are used in the same applications as known IFNγ compositions.

The apoE: IFNγ complex of the present invention is also used in tissue culture, since the complexed IFNγ molecule is protected from proteolytic destruction.

Finally, the apoE: IFNγ complex is used for quantification or purification of IFNγ receptor or other proteins that bind to IFNγ. ApoE binds easily to plastic surfaces such as test tubes and microtiter plates. Therefore, IFNγ
Can be attached to a plastic surface for use in sandwich assays or affinity purification methods well known to those skilled in the art.

The following examples are provided to illustrate the present invention and should not be construed as limiting the invention.

[0065]

【Example】

Example 1 Gel Shift Assay Previous description [Rall et al., Methods Enzymol. 128
273, 1986], defatted apoE was purified from apoE3 or apoE4 homozygous individuals. Apo E3 or Apo E4 was added to Tris-buffered saline (T
(BS) at 37 ° C for up to 4 hours with growth factors. Incubation was terminated by adding 4 × SDS-Laemmli buffer without adding reducing agent. The proteins were electrophoretically separated by SDS-PAGE and PVD
F membrane [Immobilon P, Millipore (Millipo)
re), Bedford, Mass.]. Blot membrane (B
lotto) (5% milk powder in TBS, pH 7.6, 0.05% Tween (T
ween) [Surfact Amps-20
), Pierce, Rockford, Ill.) For 1 hour and then incubated for 1 hour in primary antibody. The anti-apoE antibody was a polyclonal goat anti-human apoE [1: 2000 diluted in the blot.
Calbiochem, San Diego, CA]. The membrane was washed three times for 10 minutes each in the blot and then incubated for 1 hour with the secondary antibody. All incubations and washes were performed at 25 ° C.

The secondary antibody for detection of anti-apoE antibody was horseradish peroxidase [HRP; Boehringer Mannheim Biochemical Co., Ltd.
Beohringer Mannheim Biochemicals, Indianapolis, IN].
As described previously [Strittmatter et al., Proc. Na
t. Acad. Sci. USA 90, p. 1977, 1993], an ECL chemiluminescent substrate [Amersham (Amersham)
am), Arlington Heights, IL] was added and visualized and exposed to Hyperfilm (Amersham).

Example 2 ApoE3 forms an SDS stable complex with NGF, IFNγ and NT4 . Screening for growth factors that bind to apoE was performed using the gel shift assay described in Example 1. 50 ng of apoE3 or apoE4 and 50 ng of growth factors were incubated together for up to 4 hours. The growth factors evaluated were human recombinant ciliary neurotrophic factor (CNTF), IFNγ, neurotrophin-3 (N
T3), neurotrophin-4 (NT4), NGF, fibroblast growth factor bF
GF and LIF. CNTF, IFNγ and LIF are members of the 4 helix bundle growth factor family. The sources of growth factors are as follows: CN
TF: Regeneron Pharmaceutical Company [(Regeneron Pha
rmaceuticals, Tarrytown, NY
IFNγ: human, recombinant # 40044, Collaborative Biomedical Products [(Collaborative Bio)
medical products, Bedford, Mass .; NT3: Regeneron Pharmaceuticals; NT4: human, recombinant # G1511, Promega (Madison, Wis.); NGF: mouse, natural # 40005, collaborative bio Medical Products (ibid.); BFGF: Collaborative Biomedical Products (ibid.); And LIF: R & D Systems (R & D Systems, Minneapolis, Minn.).

Samples were boiled in non-reducing SDS sample buffer, separated by SDS-PAGE, PV
Transferred to DF paper and probed with a secondary antibody conjugated to anti-apoE and peroxidase. Immunoreactivity was detected using Amersham chemiluminescence detection reagent.

[0069] ApoE3 was expressed in the absence of reducing agents with CNTF, IFNγ, NGF and NT4 and SD.
Form an S-stable complex. No complex was observed between ApoE3 and IL-6, LIF, NT3, bFGF or BDNF. Apo E4 does not form SDS stable complexes with any of these growth factors. The gel mobility of each apoE / growth factor complex is consistent with the bimolecular complex of apoE3 and growth factor (the active forms of NGF and IFNγ are dimers, thus the apoE3 / NGF complex It is thought to be a trimer).

In another experiment, the complex of apoE3 with NGF and CNTF was detected during a 30 minute incubation and was found to reach equilibrium by 3-6 hours.
The SDS-stable apoE3 / CNTF complex is reducible with β-mercaptoethanol, suggesting the formation of disulfide bonds. Exact evidence that one of these bands is a true molecular complex indicates that the CNTF / ApoE complex
It was obtained by demonstrating that it was immunoreactive with both F and ApoE antibodies.

Example 3 ApoE Promotes NGF Activity of PC12 Cells PC12 cells require NGF to survive in serum-free medium. NGF
An experiment was performed to evaluate the survival of PC12 cells in the presence of a serum-free medium containing ApoE3 or NGF + ApoE3.

PC12 cells were plated in 96-well dishes pre-coated with poly-1-lysine and then treated with a solution containing serial dilutions of NGF and ApoE3 that had been pre-incubated together. It is known that apoE3 is absorbed into tissue culture dishes under these conditions. For pre-incubation, serial dilutions of NGF were added to a constant volume of ApoE3 (50 ng / ml), incubated at 37 ° C for 3 hours, and then added to each well. These proteins were allowed to bind to the substrate at 37 ° C. for an additional 3 hours. The final treatment is 50 ng / ml ApoE3 + 0, 100 and 1,000 ng / m
1 NGF. All wells were washed three times with PBS to remove soluble NGF and ApoE. To another set of wells, the same concentration of soluble NGF was added,
All were absorbed into the wells (0, 100 and 1,000 ng / ml).

PC12 cells were incubated with serum-free medium alone or with apoE3 + NGF bound to the substrate or soluble NGF as described above for 72 hours. After 72 hours,
As described in Example 4, Promega CellTiter Aqueous MTS Assay
) Was used to assess cell viability. Each condition was performed in triplicate to subtract non-specific background.

The complex of apoE3 and NGF bound to the substrate promoted the survival of serum-free PC12 cells to a greater extent than that of NGF bound to the substrate or soluble NGF alone (
(Fig. 1). In fact, 50 ng mixed with 100 ng / ml NGF and bound to the substrate
/ Ml of apoE3 promoted the survival of PC12 cells more than twice as compared to that of 100 ng / ml of soluble NGF alone (p <0.001). The difference at 1,000 ng / ml NGF was also significant, at the p <0.001 level. Regardless of whether Apo E is absorbed by the substrate or contained in the medium, Apo E alone (1 μg / ml)
However, there is no survival promoting activity on PC12 cells.

It is the promotion of NGF activity that best describes the apoE / NGF complex's survival promoting activity. Thus, apoE enhances the activity of NGF in maintaining NGF-responsive peripheral neurons.

Example 4 MTS viability assay Promega viability assay [Cell titer 96 Aquaeas (CellTit)
er 96 Aqueous, Promega, Madison, Wis.). Yip and Jankopoulos (
Ip and Yancopoulos), Progress in Grow
th Factor Res. 4, page 1, 1992; Riddul
le) et al., Nature 378, 189, 1995.
This is a colometric assay in which the tetrazolium compound (3- (4,5-dimethylthiazol-2) is used.
Reduction of -yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) 2H-trazolium (MTS) in viable cells gives a soluble formazan product. Formazan absorbance is measured directly from a 96-well plate.
The amount of formazan product measured at 490 nm absorbance is proportional to the number of viable cells. In performing the assay, 100 μl of medium was removed from each well, and then 20 μl of MTS solution was added to each well. After returning the plate to the incubator and incubating for 4 hours, the absorbance was measured by Dynate.
ch) Read with an MR5000 microplate reader (Dynatech Laboratories). Data were collected and for each experiment, the percent difference between each treatment group and the matched control (no apoE / no NGF condition) was calculated. The value in the case of the apoE-free / NGF-free condition was set to 100%. The percent range of each experiment was then averaged.

Example 5 ApoE Enhances NGF Activity on Neurite Growth Another known activity of NGF is the ability of NGF to promote neurite growth. In this regard, studies evaluating whether apoE promotes NGF activity have been published in PC
Performed on 12 cells. First, PC12 cells were grown in serum and 500 ng / ml NGF for 7 days to sensitize neurite growth. At this point, all cells had neurites larger than one cell body in diameter. These neurites were removed mechanically when the cells were replanted into 96-well plates. By this NGF sensitization,
Allows for rapid growth of neurites under experimental conditions.

100 ng / ml NGF and 1 μg / ml apoE3, apoE4 or BSA
After 3 days of culture in serum-free medium containing (as a control), the phase images of the cells were digitally collected. Neurites were measured in random fields using IMAGE 1 ™ analysis software (Universal Imaging Corp., Westchester, PA). ApoE3 but not apoE4 enhanced neurite outgrowth from sensitized PC12 cells (FIG. 2). Neurite outgrowth of cells grown in ApoE3 + NGF was 1.5 times greater than cells grown in BSA or ApoE4.

Example 6 ApoE "knockout" mice exhibit peripheral neuropathy In three separate experiments, the ultrastructure of sciatic nerve from ApoE knockout mice and control mice was analyzed. ApoE knockout mice were born using gene targeting in embryonic stem cells [Piedrahita]
Proc. Natl. Acad. Sci. USA 89, p. 447, 1
992]. Carefully dissect the sciatic nerve of a 12-week-old wild-type knockout mouse,
They were fixed by immersion in 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide. The sample was rinsed, then dehydrated with an ethanol gradient and final rinsed in propylene oxide. The nerve was embedded in epoxy resin, which was cured at 60 ° C. for 24 hours. 80 nm thin sections were cut with a diamond knife and covered with a formvar slot grid.
ds) Picked up so that the entire cross section could be seen. Phillips
(ps) Photomicrographs were taken with a type 410 electron microscope.

In all three experiments, significant loss of unmyelinated fibers was observed, especially in the perineural area. Although the number of myelinated fibers is substantially equal between knockout and wild-type mice, the number of unmyelinated fibers is as low as 50% in ApoE knockout mice (FIG. 3).
In addition, the morphology of the remaining unmyelinated fibers is extremely abnormal. Wild-type unmyelinated fibers are circular and are clearly separated from each other in the cytoplasm of Schwann cells, whereas fibers in knockout mice have an irregular shape and are surrounded by very little Schwann cytoplasm. Also, in the knockout mice, no clear distinction can be made between axons and Schwann cell cytoplasm.

[0081] These abnormalities seen in ApoE knockout mice were due to NGF in vivo.
Consistent with the role of Apo E in enhancing vo activity. Unmyelinated fibers in the sciatic nerve are trk
Expresses the NGF receptor and is responsive to NGF. These fibers are anti-N
These neurons can be rescued by NGF after transection of the nerve, although lost from animals treated with the GF antibody. These results are also significant for diabetic neuropathy where NGF-sensitive neurons are compromised.

Example 7 Neurite Growth and Schwann Cell Migration of Neonatal Rats DRG is Broken at NGF-treated Distal Stumps of ApoE Knockout Mice Neurite Growth of Neonatal Rat Dorsal Root Ganglia (DRG) And the migration of Schwann cells was measured in NGF-treated distal stumps of ApoE knockout mice and wild type mice. In these experiments, neonatal rat DRGs were explanted on cryostat sections of the distal stump of ApoE knockout or wild-type animals. Five days after the cross section, the distal stump was removed and the O.D. C. T. Fresh frozen in compound, thickness 20
The sections were cut into μm vertical sections. Sections were placed in 100 μl of 100 ng / ml NGF at 37 ° C.
For 3 hours. The nerve is then washed and unbound NGF
Was removed and neonatal DRG explants were placed on the nerve and cultured in serum-free medium. 72
After time, images of neurite growth and Schwann cell migration were obtained using the vital dye carboxyfluorescein and fluorescence microscopy.

NGF-treated distal stumps of cross-sectioned sciatic nerve prepared from ApoE knockout mice reduced neurite growth and Schwann cell migration. It has already been demonstrated that NGF promotes both neurite outgrowth (see Example 3) and Schwann cell migration. Thus, these studies provide further evidence for the involvement of apoE in promoting in vivo NGF action.

Example 8 Apo E Knockout Mice Have Delayed Response to Noxious Heat Stimulation Unmyelinated fibers are those that transmit pain and temperature sensitivity. Thus, the finding that APO E knockout animals have substantially reduced unmyelinated fibers (Example 6)
These animals suggest a reduced response to noxious heat stimuli. To test this, the withdrawal latencies of wild-type and ApoE knockout mice from a warm water bath were measured.

The retraction latencies of wild type mice and knockout mice withdrawing their hind paws from a 55 ° C. water bath were measured. As shown in FIG. 4, Apo E knockout mice show a greater than 50% increase in their response latency (p <0.001). Similar results were observed with the tail withdrawal paradigm (data not shown).

These data further support the role of Apo E in promoting unmyelinated fiber health and survival by interacting with NGF.

Example 9 Summary of Key Findings Supporting the Hypothesis that Apo E Enhances NGF Activity The data presented in the above examples strongly suggest that Apo E enhances NGF activity. The evidence is summarized below.

• Apo E3, but not Apo E4, forms an SDS stable complex with NGF.

• The complex of apoE3 and NGF bound to the substrate enhances NGF's survival promoting activity.

• ApoE3 but not ApoE4 enhances neurite outgrowth activity of NGF.

• NGF-dependent neurite outgrowth and Schwann cell migration are reduced on substrates from ApoE knockout animals.

• ApoE knockout animals have lost 50% of NGF-sensitive unmyelinated sensory fibers.

• Apo E knockout mice have a delayed response to noxious heat stimuli.

While the above invention has been described in considerable detail through the drawings and examples for clarity and understanding, certain changes and modifications may be made within the scope of the appended claims. That will be self-evident.

[Brief description of the drawings]

FIG. 1 shows that apoE3 enhances NGF survival promoting activity in PC12 cells. The data are 1,000 ng / ml N without ApoE.
Expressed as mean survival ± standard deviation compared to controls treated with GF alone.
Using the Student's Tee test for the difference between the means, 100 and 1
P <0.001 for 2,000 ng / ml NGF. Bars with wide hatching:
Substrate-bound apoE3 (50 ng / ml) pre-incubated with 0, 100 and 1,000 ng / ml NGF. Closely spaced bars: 0, 100 and 1, 0
Only soluble NGF added at 00 ng / ml.

FIG. 2 shows that apoE3 but not apoE4 promotes neurite outgrowth from PC12 cells in response to NGF. Neurite growth is 100n
It was measured after culturing for 3 days in a serum-free medium containing g / ml NGF and 1 μg / ml of apoE3, apoE4 or bovine serum albumin (BSA) as a control protein. The phase images of the cells were obtained using IMAGE 1 ™ analysis software [Universal Imaging Corp.].
, Westchester, PA] using digital random acquisition. Neurite length is expressed as percent of control treated growth (NGF + B
SA) Shown as ± SEM. Using a paired Student's T-test, p <0.005 for ApoE3 vs. BSA and p <0.01 for ApoE3 vs. ApoE4.

FIG. 3 shows that apoE knockout (KO) mice have a reduced number of unmyelinated nerve fibers as compared to wild type (WT) mice. The total area covered by high performance electron micrographs of three wild-type and three knockout animals was approximately 18,000 μm ^{2 for} each group (wild-type mice: 17,900, knockout mice: 18,200). Met. The total number of myelinated and unmyelinated axons in this area was measured. The nerves of knockout animals were found to have a 50% reduction in unmyelinated nerve fibers compared to the nerves of wild-type animals, but the number of myelinated nerve fibers was found to be equal.

FIG. 4 shows that ApoE knockout mice have a delayed response to noxious heat stimuli. In this experiment, the withdrawal latency (seconds) of the mouse pulling out its hind paws from a 55 ° C. water bath was measured. The data shown are the mean ± SEM of three measurements per hind paw of each mouse in each group of three mice. Knockout mice have an increase in response latency of 50% or more compared to wild-type mice. Using a paired Student's T-test, the p-value of the difference between the two means is p <
0.001.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 25/16 A61P 35/00 25/28 43/00 111 31/12 A61K 37/02 35/00 37 / 24 43/00 111 37/66 G C12N 5/06 C12N 5/00 E (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA , BB, BG , BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Street Matter Warren J Gutman Catherine Earl United States of America 27701 Durham W Lynch Street 109 (72) Inventor Fullerton Stephanie M United States of America Scarolina 27705 Durham Moraine Road 305 B F-term (reference) 4B065 AA90X AA93X BB19 BB34 CA44 4C084 AA02 AA03 AA23 BA41 BA44 DA24 DB59 NA14 ZA022 ZA162 ZA212 ZB262 ZB332

Claims (57)

[Claims] 1. A composition comprising a complex of apolipoprotein E and nerve growth factor. 2. The composition according to claim 1, further comprising a complex of apolipoprotein E and neurotrophin 4. 3. The composition according to claim 1, wherein said apolipoprotein E is defatted apolipoprotein E. 4. The composition according to claim 1, wherein the complex is a covalent complex. 5. The complex is a non-covalent complex,
The composition according to claim 1. 6. The composition according to claim 1, wherein said complex is bound to a substrate. 7. The composition according to claim 1, wherein said apolipoprotein E is apolipoprotein E2. 8. The composition according to claim 1, wherein said apolipoprotein E is apolipoprotein E3. 9. The composition according to claim 1, wherein said apolipoprotein E is apolipoprotein E4. 10. The composition according to claim 1, wherein said apolipoprotein E is human apolipoprotein E. 11. The composition according to claim 1, wherein said nerve growth factor is human nerve growth factor. 12. The composition according to claim 1, wherein said composition is in a physiologically acceptable carrier. 13. A method for promoting survival of a nerve cell, comprising administering to the nerve cell a survival promoting amount of a composition comprising a complex of apolipoprotein E and nerve growth factor. 14. The method according to claim 13, wherein the composition further comprises a complex of apolipoprotein E and neurotrophin 4. 15. The method according to claim 13, wherein the apolipoprotein E is defatted apolipoprotein E. 16. The method according to claim 13, wherein the complex is a covalent complex. 17. The method according to claim 13, wherein the complex is a non-covalent complex. 18. The method according to claim 13, wherein the apolipoprotein E is apolipoprotein E2. 19. The method according to claim 13, wherein the apolipoprotein E is apolipoprotein E3. 20. The method according to claim 13, wherein the apolipoprotein E is apolipoprotein E4. 21. The method according to claim 13, wherein the apolipoprotein E is human apolipoprotein E. 22. The method according to claim 13, wherein the nerve growth factor is human nerve growth factor. 23. The method according to claim 13, wherein the method is administered in vitro. 24. The method according to claim 23, wherein the complex is bound to a substrate. 25. The method according to claim 24, wherein after the apolipoprotein E is bound to the substrate, a complex with a nerve growth factor is formed. 26. The method according to claim 13, wherein the method is administered in vitro. 27. The method according to claim 26, wherein the nerve cell is a human nerve cell. 28. The method according to claim 27, wherein the nerve cell is a nerve cell of a human subject suffering from a neurodegenerative disease. 29. The method according to claim 27, wherein the nerve cells are nerve cells of a human subject who has suffered damage or trauma to nerve tissue. 30. The nerve of a human subject, wherein the nerve cells suffer from a condition selected from the group consisting of Alzheimer's disease, Parkinson's disease, peripheral nerve injury, peripheral neuropathy, amyotrophic lateral sclerosis, head injury and stroke. 28. The method according to claim 27, wherein the method is a cell. 31. A method of administering to a subject in need of treatment a therapeutically effective amount of a composition comprising a complex of apolipoprotein E and nerve growth factor. 32. The method according to claim 31, wherein the complex is a covalent complex. 33. The method according to claim 31, wherein the complex is a non-covalent complex. 34. The method according to claim 31, wherein the apolipoprotein E is apolipoprotein E2. 35. The method according to claim 31, wherein the apolipoprotein E is apolipoprotein E3. 36. The method according to claim 31, wherein the apolipoprotein E is apolipoprotein E4. 37. The method according to claim 31, wherein the apolipoprotein E is human apolipoprotein E. 38. The method according to claim 31, wherein the nerve growth factor is human nerve growth factor. 39. The method according to claim 31, wherein the subject is a human subject. 40. The method of claim 39, wherein the human subject is suffering from a neurodegenerative disease. 41. The method of claim 39, wherein the human subject has suffered damage or trauma to nerve tissue. 42. A human subject suffering from a condition selected from the group consisting of Alzheimer's disease, Parkinson's disease, peripheral nerve injury, peripheral neuropathy, amyotrophic lateral sclerosis, head injury and stroke. 40. The method of claim 39, wherein the method is characterized by: 43. A composition comprising a complex of apolipoprotein E and neurotrophin 4. 44. The composition according to claim 43, wherein the composition is in a pharmaceutically acceptable carrier. 45. A method for promoting the survival of a nerve cell, comprising administering to the nerve cell a survival promoting amount of a composition comprising a complex of apolipoprotein E and neurotrophin 4. 46. A method of administering to a subject in need of treatment a therapeutically effective amount of a composition comprising a complex of apolipoprotein E and neurotrophin 4. 47. A composition comprising a complex of apolipoprotein E and γ-type interferon. 48. The composition according to claim 47, which is in a physiologically acceptable carrier. 49. The method according to claim 49, wherein the cells comprise an antiviral effective amount of apolipoprotein E and γ.
A method of protecting cells from viral infection, comprising administering a composition comprising a complex with interferon-type. 50. A method of administering to a subject in need of treatment an effective antiviral amount of a composition comprising a complex of apolipoprotein E and γ-type interferon. 51. The subject is afflicted with a viral infection,
A method according to claim 50. 52. The method of claim 50, wherein said subject is at risk of developing a viral infection. 53. A method for administering to a subject in need of treatment, an antitumor effective amount of a composition comprising a complex of apolipoprotein E and γ-type interferon. 54. The method of claim 53, wherein the subject is suffering from a tumor. 55. The method according to claim 54, wherein the tumor is malignant. 56. The method according to claim 53, wherein the subject is at risk of developing a tumor. 57. A method of administering to a subject in need of treatment a therapeutically effective amount of a composition comprising a complex of apolipoprotein E and gamma interferon.