WO1996040243A1 - Liposomes contenant une glycoproteide du virus de l'immunodeficience humaine et leurs procedes d'utilisation - Google Patents

Liposomes contenant une glycoproteide du virus de l'immunodeficience humaine et leurs procedes d'utilisation Download PDF

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Publication number
WO1996040243A1
WO1996040243A1 PCT/US1996/008680 US9608680W WO9640243A1 WO 1996040243 A1 WO1996040243 A1 WO 1996040243A1 US 9608680 W US9608680 W US 9608680W WO 9640243 A1 WO9640243 A1 WO 9640243A1
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liposome
lipid
gpl20
antibodies
liposomes
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PCT/US1996/008680
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English (en)
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Carl L. Alving
Nabila Wassef
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U.S. Department Of The Army
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Priority to AU59785/96A priority Critical patent/AU5978596A/en
Publication of WO1996040243A1 publication Critical patent/WO1996040243A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention provides liposomes containing the HIV glycoprotein gp!20 .
  • the liposomes When injected in mammals the liposomes elicit an immune response, and may be used to produce anti-grpl20 antibodies or in formulating a vaccine against HIV.
  • AIDS acquired immunodeficiency syndrome
  • HIV-I human immunodeficiency virus type I
  • env or gpl20 a single-stranded RNA virus of the family Retroviridae.
  • CD4 a host cell determinant
  • the affinity of HIV for the CD4 determinant is very high, on the order of 10 *9 M. Deen et al . , Nature 331:84- 86 (1988) .
  • the domains which are responsible for the interaction have been identified in both the gpl20 and CD4 molecules.
  • the CD4 site lying near the N-terminus, is linear, non-glycosylated and resides with the first immunoglobulin-like domain.
  • gpl20 is known to exhibit considerable variability between isolates, the CD4 binding site is one of at least four, distinct conserved regions.
  • F. Wong-Staal "Human Immunodeficiency Viruses and Their Replication, " in VIROLOGY, ed. B. Fields and D. Knipe, Raven Press, Ltd., (New York, 1990) pp. 1529-1544.
  • CD4 a soluble form of CD4 has been show to inhibit HIV infection competitively. Deen et al . , supra; Fisher e ⁇ al . , Nature 334:444-447 (1988); Traunecker et al . , Nature 331:84-86 (1988) .
  • a chimeric antibody-like molecule containing the gpl20-binding domain of CD4 was found to be effective at blocking HIV-1 infection of T cells. Capon et al . , Nature 337:525-531 (1989) .
  • the gpl20 binding site for CD4 apparently forms a pocket and, therefore, lacks significant antigenic capacity in its natural conformation.
  • Kowalski et al . Science 237:1351-1355 (1987) ; Lasky et al . , Cell 50:975- 985 (1987) ; Matthews and Bolognesi, Sci . Am. 259:120-127 (1988) .
  • a major neutralizing epitope has been located at the C-terminus of gpl20, but this epitope is part of a hypervariable region and, therefore, antibodies specific for this region are highly site-specific.
  • grpl20 remains an attractive target in the development of an anti-HIV vaccine.
  • Liposomes have been used as drug carriers for in vivo drug delivery.
  • Liposome-encapsulated drugs have the following advantages: the drugs are encapsulated within a relatively impermeable bilayer membrane where the drug is protected from the environment; liposomes can be taken up by cells without overt cytotoxic effects, thus enhancing the cellular uptake of the encapsulated material; encapsulation alters pharmacokinetics; and liposomes are natural, biodegradable and non-toxic. Mayhew et al . , "Therapeutic Applications of Liposomes" in Liposomes, M.J.
  • liposomes may or may not provide enhancement of immunogenicity. Additional modifications such as the addition of a liposomal adjuvant or the utilization of a separate, non-liposomal adjuvant may or may not result in a superior or practical immune response, again depending on the particular epitope. Numerous instances can be cited in which liposomes per se did not serve as an effective adjuvant. For example, Allison and Gregoriadis, U.S. patent No.
  • the liposome-conjugated peptide became immunogenic, however, when the liposomes used during the priming immunization also contained monophosphoryl lipid A ("MPL") . Even when the liposomes contained MPL, however, the peptide was not immunogenic when it was encapsulated within the liposomes rather than being present on the surface of the liposomes.
  • MPL monophosphoryl lipid A
  • the peptide was not immunogenic when it was encapsulated within the liposomes rather than being present on the surface of the liposomes.
  • the foregoing account illustrates that liposomes are not inherently capable of inducing an immune response against any liposome-associated epitope. Even when such a response is induced by the addition of an adjuvant such as lipid A, it appears that the physical state of the liposome-associated epitope, e . g.
  • Liposomes also have been used to encapsulate antiviral drugs for the treatment of HIV infections. Pontani et al . , Antiviral Res . 11:119-126 (1989); Szebeni et al . , AIDS Res . Human Retroviruses 6:691-702 (1990) .
  • a formulation that can elicit an immune response that targets HIV-1 and there is particular need for a formulation that can generate a protective immune response to HIV-1.
  • gpl20 Because of the highly variable nature of gpl20, there also is a need for a rapid and reliable method for the production of anti- gpl20 antibodies for the characterization and diagnosis of HIV-1.
  • compositions for use in the generation of an anti-HIV immune response in a mammal.
  • the compositions comprise liposomes and an adjuvant and further include HIV-1 gpl20.
  • An additional objective of the present invention is to provide methods for the generation of an anti-HIV response, in particular, an anti-HIV-1 gpl20 response.
  • a liposome comprising at least one liposomal lipid, a least one HIV gpl20 epitope, and lipid A, wherein the epitope is encapsulated in the liposome and is capable of inducing an immune response when administered to a mammalian subject.
  • the liposome may further comprise alum.
  • the liposome may comprise the grpl20 molecule, or the V3 region or CD4-binding region of the gpl20 molecule.
  • an anti-HIV vaccine comprising (i) liposomes comprising at least one liposomal lipid, an HIV gpl20 epitope and lipid A, wherein the epitope is encapsulated in the liposome and is capable of inducing an immune response when administered to a mammalian subject; and (ii) a pharmaceutically acceptable carrier or diluent.
  • the present invention provides a liposome formulation comprising a liposomal lipid, an antigenic HIV gpl20 epitope, and lipid A.
  • liposome means a group of lipid molecules that self-associate into a smectic mesophase.
  • the lipid molecules are, in general, longer than they are wide, and have a polar or aromatic group somewhere along the length of the molecule.
  • the molecular shape and polar-polar or aromatic interaction permit the molecules to self-associate in partially ordered arrays, such that the self-associated molecules have a long-range order in the direction of the long axis of the molecule, which is called a smectic mesophase.
  • the smectic mesophases may be in single or double layers.
  • an antigenic HIV gpl20 epitope is encapsulated in the liposome and is capable of inducing an immune response when administered to a mammalian subject.
  • liposomes are non-toxic and biodegradable.
  • they can serve as an adjuvant for a non-immunogenic epitope incorporated therein.
  • the amount of antigenic epitope in a single liposome, i.e., the epitope density can be varied. It also may prove beneficial to include other components in the liposomes such as different antigens, cytokines, drugs, adjuvants and/or anti-inflammatory agents.
  • the liposome can act as a timed release vehicle, the half-life of the epitope in the subject may be increased.
  • the liposome formulation of the present invention induces an immune response in mammals, including humans.
  • One aspect of the immune response is the humoral response, the generation of anti-gpl20 antibodies.
  • Another aspect of the immune response is the cellular response, the induction of cytotoxic T-lymphocytes which recognize and kill virus-infected cells.
  • the induction of cytotoxic T-lymphocytes is a useful strategy in developing a vaccine. We have reported previously the ability of liposome-encapsulated antigens to induce cytotoxic T-lymphocytes. Alving et al . AIDS Res . and Human Retroviruses 10 (4) : S91-S94 (1994) .
  • the liposome formulation of the present invention induces cytotoxic T-lymphocytes specific for HIV-infected cells in humans. After two injections of the liposome formulation a 12% lysis of HIV target cells compared to a 1% lysis of control target cells was reported; after three injections of the liposome formulation, a 47% lysis of HIV target cells compared to a 27% lysis of control target cells was reported. These data are particularly notable because other anti-HIV vaccines studied were not found to induce cytotoxic T-lymphocytes.
  • Phospholipids may be used for preparing the liposomes according to the present invention.
  • liposomes carrying a net positive charge, a net negative charge or that are neutral all are effective as adjuvants. Accordingly, the liposomes to be used in accordance with the present invention can carry a net positive charge, a net negative charge or be neutral.
  • Phosphatidyl glycerol or dicetyl phosphate can be employed to confer a net negative charge on the liposomes, and stearylamine can be used to confer a net positive charge on the liposomes.
  • the phospholipids may be naturally occurring or synthetic phospholipids, and may have one or more unsaturated bonds in the fatty acyl lipid group.
  • the phospholipids to be used are diacylglycerols in which at least one acyl group comprises at least twelve carbon atoms, preferably between about fourteen to about twenty-four carbon atoms. It also is preferred that at least one head group of the phospholipids (the portion of the molecule containing the phosphate-group) is a phosphocholine , a phosphoethanolamine, a phosphoglycerol a phosphoserine or a phosphoinositol .
  • Lipids suitable for use according to the present invention can be purchased from commercial sources.
  • dimyristoyl phosphatidylcholine (“DMPC”) can be purchased from Sigma Chemical Co., dicetyl phosphate
  • DCP is available from K & K Laboratories (Plainview,
  • DMPG dimyristoyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/metha ⁇ ol can be stored at about -20°C.
  • chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • Phospholipids from natural sources such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin and plant or bacterial phosphatidylethanolamine also may be used. These naturally occurring phospholipids are readily available and inexpensive. However, if they are used as the primary phosphatide, i . e . , constituting 50% or more of the total phosphatide composition, the resulting liposomes may be leaky and relatively unstable.
  • the liposomes prepared in accordance with the present invention can comprise lipids in any molar ratio, and can, but need not, contain cholesterol.
  • dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and cholesterol, respectively are combined in respective molar ratios of about 0.9:0.1:0.75.
  • Non-phospholipid liposomes also may be used in the present invention, such as amphipathic molecules that have the ability to form smectic mesophases. Such non- phospholipid liposomes that may be useful in the present invention include long-chain fatty acids.
  • Adjuvants An additional component of the liposomes according to the present invention is an adjuvant, preferably aluminum hydroxide formulations or lipid A. Exemplary of the commercially-available aluminum hydroxide formulations that can be used is an aluminum hydroxide absorptive gel ("alum"), which may be obtained in a form that has been prepared in accordance with the Good Manufacturing Practices (GMP) prescribed by the U.S. Food and Drug Administration.
  • the aluminum hydroxide to be used for adsorption of liposomes may be diluted in phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • Lipid A is a lipoidal constituent of lipopoly- saccharide ("LPS") from Gram-negative bacteria such as E. coli , Salmonella and Shigella, and can be prepared in the manner described in Alving et al . , LIPOSOME TECHNOLOGY, G. Gregoriadis, ed. , CRC Press (1993) pp.317-343, the contents of which are incorporated by reference. In essence, LPS is prepared by phenol extraction or by trichloroacetic acid treatment of suitable bacterial cells. Lipid A also can be purchased from available commercial sources, such as Calbiochem-Behring (La Jolla, CA) , List Biological Laboratories, Inc.
  • LPS lipopoly- saccharide
  • LPS also is available from commercial sources, such as Difco (Detroit, MI), List Biological Laboratories, Inc. and Ribi ImmunoChem Research, Inc.
  • lipid A For extraction of lipid A from LPS, the LPS is heated in a boiling water bath for about two hours in about 1% acetic acid in an amount of 10 mg of LPS per ml. The precipitate formed is washed three times with distilled water by centrifugation at 4°C and then lyophilized. To further purify the lipid A thus obtained, an aqueous solution of about 0.5% triethylamine ("TEA") is used for solubilization of lipid A in the aqueous phase. This solution is allowed to stand for about 10 to 30 minutes. The phases are separated, with or without centrifugation at about 12,000 x g for about 10 minutes. The resulting purified lipid A is chloroform-soluble and may be analyzed for phosphate content.
  • TAA triethylamine
  • the other essential component of the liposome formulations of the present invention is the antigenic epitope.
  • HIV epitopes suitable for use in the present invention are gag, pol , nef, tat, rev, vif, vpr and vpu, among others.
  • the preferred antigenic epitope is derived from the HIV-1 env glycoprotein, in particular gpl20.
  • epitopes can be used to produce monoclonal antibodies according to the present invention, including epitopes purified from transformed or transfected cells, recombinantly-produced epitopes and synthetic epitopes. Recombinant and synthetic peptides are most useful in eliciting antibodies to "continuous" or “linear” epitopes. Smaller peptide epitopes are typically attached to carrier molecules such as bovine serum albumin and keyhole limpet hemocyanin to improve their immunogenicity.
  • carrier molecules such as bovine serum albumin and keyhole limpet hemocyanin to improve their immunogenicity.
  • epitopes are used as epitopes. Again, these epitopes can be synthesized chemically but it may be more feasible to isolate whole antigens from transfected or transformed cells.
  • Bacterial hosts are desired where post- translational modifications of the protein to be produced are minimal. Where post-translational modifications are significant, it may be necessary to use eukaryotic hosts such as yeast or even human cells.
  • the gpl20 epitopes used in the present invention may consist of the gpl20 molecule itself, or may consist of an immunogenic region of the grpl20 molecule.
  • the gp!20 molecule has been extensively studied, and immunogenic regions of the molecule will be known to, or be readily determined by, those skilled in the art.
  • the VI, V2, V3 or V4 regions of the gpl20 molecule may be immunogenic, and can be used in a liposome of the present invention.
  • the V3 region is comprised of a 36 amino acid disulfide crossbridged loop (amino acids 296-331 of gpl20) that appears on the surface of the gpl20 molecule.
  • the CD4-binding region of gpl20 also is an immunogenic region of the molecule. Putney, "How antibodies block HIV infection: paths to an AIDS vaccine," Trends in Bioche . Sci . 17: (1992) ; and Broliden et al. , "Identification of human neutralization-inducing regions of the human immunodeficiency virus type 1 envelope glycoproteins, " Proc. Natl . Acad. Sci . , USA, 89: 461-465
  • Liposomes used according to the present invention can be made by different methods.
  • the size of the liposomes varies depending on the method of synthesis.
  • a liposome suspended in an aqueous solution is generally in the shape of a spherical vesicle, having one or more concentric layers of lipid bilayer molecules. Each layer consists of a parallel array of molecules represented by the formula XY, wherein X is a hydrophilic moiety and Y is a hydrophobic moiety.
  • the concentric layers are arranged such that the hydrophilic moieties tend to remain in contact with an aqueous phase and the hydrophobic regions tend to self-associate.
  • liposomes within the scope of the present invention can be prepared in accordance with known laboratory techniques.
  • liposomes are prepared by mixing liposomal lipids, including lipid A, in a solvent in a container, e . g. , a glass, pear-shaped flask.
  • the container should have a volume ten-times greater than the volume of the anticipated suspension of liposomes.
  • the solvent is removed at approximately 40°C under negative pressure.
  • the solvent normally is removed within about 5 min to 2 hours, depending on the desired volume of the liposomes.
  • the composition can be dried further in a desiccator under vacuum.
  • the dried lipids generally are discarded after about 1 week because of a tendency to deteriorate with time.
  • Dried lipids can be hydrated at approximately 25-50 mM phospholipid in sterile, pyrogen-free water by shaking until all the lipid film is resuspended. The aqueous suspension of liposomes are then lyophilized and sealed under vacuum.
  • liposomes can be prepared in accordance with other known laboratory procedures: the method of Bangham et al . , J. Mol . Biol . 13:238-52 (1965), the contents of which are incorporated herein by reference; the method of Gregoriadis, as described in
  • the dried lipids or lyophilized liposomes prepared as described above may be dehydrated and reconstituted in a solution of the gp!2Q epitope and diluted to an appropriate concentration with an suitable solvent, e . g. , Dulbecco's phosphate buffered saline (DPBS) .
  • DPBS Dulbecco's phosphate buffered saline
  • the mixture is then vigorously shaken in a vortex mixer. Unencapsulated epitope is removed by centrifugation at 29,000 x g and the liposomal pellets are washed.
  • the washed liposomes are resuspended at an appropriate total phospholipid concentration, e . g. , about 50-200 mM.
  • the amount of protein epitope encapsulated can be determined in accordance with the method of Lowry et al . , J. Biol . Chem. 193: 265-275 (1951), the contents
  • any of the methods of making liposomes discussed above are adapted such that a single lipid or hydrated liposome formulation is divided into a plurality of portions before being hydrated with the epitope, each portion is individually hydrated with the gpl20 epitope, and the portions are combined to form a single batch of the liposome formulation.
  • the liposomes may be diluted to about 100 micrograms of antigen per 0.5 ml and may be either bottled immediately or incubated with alum, for example, at approximately 2.0 mg/ml of aluminum for 1 hour at room temperature, before bottling in vials and storing at 4°C until use.
  • lipid A may be added to the flask along with the other lipids.
  • lipid A from S. minnesota R595, herein designated “native lipid A” is included in the liposomes at a concentration of about 0.2 moles to about 20 moles of lipid A phosphate per mole of liposomal phospholipid.
  • nontoxic monophosphoryl lipid A hereafter “MPL” is used in place of native lipid A at 0.5 moles of lipid A phosphate per mole of liposomal phospholipid.
  • MPL nontoxic monophosphoryl lipid A
  • “Monophosphoryl lipid A” as used herein denotes a monophosphoryl fraction isolated from native lipid A that has reduced pyrogenicity when tested in rabbits via established techniques.
  • the epitope may be mixed with liposomes comprising lipid A prior to encapsulation and then mixed with aluminum hydroxide for adsorption, as described in Wassef et al . , Immunomethods 4: 217-222 (1994), the contents of which are incorporated by reference. III. Use of Liposome Compositions
  • Administration of a liposome-based pharmaceutical to a subject may be via oral, nasal, buccal, rectal, vaginal, or topical routes or by subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Injection is preferred.
  • polyclonal or monoclonal antibodies with the requisite properties, produced by immunization with a liposome-based pharmaceutical may be isolated and administered by any of these routes.
  • a typical pharmaceutical composition for administration to a mammal comprises a pharmaceutically acceptable carrier such as aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • a pharmaceutically acceptable carrier such as aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • a pharmaceutically acceptable carrier such as aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • a pharmaceutically acceptable carrier such as aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art.
  • the dose of epitope suitable for injection can be determined via routine experimentation. For example, by injecting various groups of animals or humans with different doses of the alum-adsorbed, liposome/lipid encapsulated epitope, and monitoring the immune response of each group, the dosage inducing the highest antibody titer or cellular immune response for the longest period may be determined.
  • the amount of protein or peptide epitope to be injected is less than about 800 micrograms, and more preferably in the range of about 1 to about 200 micrograms per dose.
  • the number of doses of formulation to inject also can be determined by routine experimentation. Typically, the dose can vary depending upon the level of protection desired. For example, after an initial injection at 0 week and a booster at 4 weeks, further booster doses can be injected at monthly, semi-yearly, or yearly intervals.
  • the liposome compositions described above are capable of generating both humoral and cellular immune responses in vivo in mammals, including humans.
  • the compositions can induce antibodies specific for gpl20, including neutralizing antibodies, and can induce cytotoxic T lymphocytes specific for HIV-infected cells.
  • the compositions also can induce antibody-dependent cytotoxic T cells (ADCC's) .
  • Antibodies specific for HIV are valuable not only as effectors of an immune response in a vaccine context, but also as valuable diagnostic tools.
  • the antibodies may be made in vivo in suitable laboratory animals by injecting the animal with the liposome formulation of the present invention and harvesting the antibodies produced by the animal in response.
  • references to antibodies herein is meant to include whole polyclonal and monoclonal antibodies, and parts thereof, either alone or conjugated with other moieties.
  • Antibody parts include Fab and F(ab) 2 fragments and single chain antibodies.
  • Monoclonal antibodies and fragments thereof according to this invention may be obtained according to in vi tro and in vivo methods well-known in the art.
  • a mouse can be injected with a formulation comprising the liposome of the present invention, and the mouse can be bled for polyclonal antibodies.
  • Monoclonal antibodies can be obtained from spleen cells harvested from the mouse. Harlow and Lane, ANTIBODIES. A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press (New York, 1988) .
  • Multiplication in vi tro may be carried out in suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e . g. , feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
  • suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e . g.
  • feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
  • In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies.
  • Techniques for large scale hybridoma cultivation under tissue culture conditions are known in the art and include homogenous suspension culture
  • Monoclonal antibody of the present invention also may be obtained by multiplying hybridoma cells in vivo.
  • Cell clones are injected into mammals which are histocompatible with the parent cells, e . g. , syngeneic mice, to cause growth of antibody- producing tumors.
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
  • a "humanized” monoclonal antibody may be produced by techniques well-known in the art. Carter et al . , Proc . Na t ' l Acad. Sci . USA 89:4285- 4289 (1992); Singer et al . , J. Immun. 150:2844-2857 (1992) ; Mountain et al . , Biotechnol . Genet . Eng. Rev. 10:1-142 (1992), the contents of each of which are incorporated by reference. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V-chains of the mouse Ig into a human V-domain, followed by the replacement of some human residues in the framework regions of their murine counterparts. "Humanized" monoclonal antibodies are suitable for use in in vivo diagnostic and therapeutic methods.
  • CDRs mouse complementary determining regions
  • Fragments of the monoclonal antibodies can be obtained from the monoclonal antibodies described above by methods which include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction.
  • monoclonal antibody fragments can be synthesized using an automated peptide synthesizer, or they may be produced manually using techniques well known in the art. Geysen, et al . J. Immunol . Methods 102:259-274 (1978), the contents of which are incorporated by reference.
  • Monoclonal conjugates can be prepared by methods known in the art, e . g. , by reacting a monoclonal antibody prepared as described above with, for instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced.
  • a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • Conjugates with metal chelates are similarly produced.
  • Radioactively labeled monoclonal antibodies are produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • Monoclonal antibodies according to the invention may be labeled with technetium-"m by a ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labelling techniques, e . g. , by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium-potassium phthalate solution, and the antibody.
  • a reducing agent such as SNC1 2
  • a buffer solution such as sodium-potassium phthalate solution
  • the antibodies made in accordance with the present invention can be used, for example, in immunoassays such as those described in U.S. Pat. No. 4,367,110 (double monoclonal antibody sandwich assay) , Wide et al . , Radio immunoassay Methods, Kirkham and Hunter, eds., E. & S. Livingstone, Edinburgh, IK (1970), U.S. Patent No. 4,452,901 (western blot), Brown et al . , J. Biol . Chem. 255:4980-4983 (1980) (immunoprecipitation of labeled ligand), and Brooks et al . , Clin . Exp. Immunol . 39:477 (1980) (immunocytochemistry) , the contents of each of which are incorporated herein by reference.
  • These immunoassays can be used, for example, in the detection of HIV.
  • kits may comprise a receptacle that is compartmentalized to receive one or more containers such as vials, tubes and the like, for holding separate elements of the invention.
  • a container may contain a first antibody bound to an insoluble or partly soluble carrier and a second container may contain soluble, detectably-labeled second antibody in lyophilized form or in solution.
  • the receptacle may also contain a third container holding a detectably labeled third antibody in lyophilized form or in solution.
  • a kit of this nature can be used in a diagnostic assay.
  • Example 1 Encapsulation of qrpl20 in 100 mM Liposomal Phospholipids
  • a lipid mixture was prepared with the following composition in chloroform:
  • the mixture was dried on a rotary evaporator, placed under high vacuum overnight, and resuspended at a concentration of 25 mM phospholipid using 16 ml of sterile deionized water.
  • the suspension was transferred to four 20 ml capacity bottles at 4 ml per bottle and lyophilized.
  • Each of the bottles of dried liposomes was reconstituted with 1 ml of the following concentrations of gpl20 (Chiron) in DPBS: 0.125 mg/ml, 0.25 mg/ml, 0.75 mg/ml, and 1.0 mg/ml.
  • the liposomes were reconstituted at a concentration of 100 mM of phospholipids.
  • Liposomes reconstituted with 1.0 mg/ml of gpl20 encapsulated about 0.250 mg/ml or about 25%.
  • Each of the bottles of dried liposomes was reconstituted with 1 ml of the following concentrations of gpl20 (Chiron) in DPBS: 0.125 mg/ml, 0.25 mg/ml, 0.75 mg/ml, and 1.0 mg/ml.
  • the liposomes were reconstituted at a concentration of 200 mM of phospholipids.
  • Liposomes reconstituted with 1.0 mg/ml of gp!20 encapsulated about 0.160 mg/ml or about 16%.
  • mice Two groups of five mice each were immunized twice at five days interval.
  • the first group composed of mice no. 801, 802, 803, 804, and 805, received liposomes with gpl20 and MPL.
  • the second group composed of mice 806, 807, 808, 809, and 810, received liposomes with gpl20, MPL and alum.
  • the injection volume was 0.08 ml injected intramuscularly in the thigh.
  • I consisted of 10-12 ⁇ g of gpl20 and 0.35 mg MPL.
  • the inoculum for.Group II contained twice the concentration of liposomes, i.e., 20-24 ⁇ g gpl20 and 0.7 mg MPL, in 0.04 ml which were adsorbed to 0.04 ml alum (Amphogel; 2 mg/ml) so that the final concentration of gp!20 and lipid A was the same for both groups.
  • the alum and the liposomes were mixed thoroughly and incubated at room temperature for at least 30 min before injection. Total volume per injection was 0.08 ml, consisting of 0.04 ml liposomes and 0.04 ml alum.
  • mice were bled from the tail vein before immunization and on days 5, 12, and 16.
  • the mouse sera were tested for antibody activity using an enzyme-linked immunosorbent assay (ELISA) as described above. Values reported were adjusted by subtracting values in blank wells that lacked both antigen and mouse serum.
  • ELISA enzyme-linked immunosorbent assay
  • a phase I, randomized, double-blind, placebo controlled clinical trial was conducted in humans to compare the safety and immunogenicity in healthy HIV-1 uninfected individuals of the recombinant envelope protein, SF-2 rgpl20/HIV (from Biocine Corp.) combined with one of seven adjuvants.
  • One of the seven adjuvants was a liposome comprising gp!20 and lipid A, in accordance with the present invention.

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Abstract

L'invention porte sur des compositions de liposomes contenant la glycoprotéide gp120 du VIH et un lipide A ainsi que des procédés d'utilisation de ces compositions. Celles-ci s'avèrent efficaces pour stimuler une réponse immunitaire à titre élevé contre la molécule gp120.
PCT/US1996/008680 1995-06-07 1996-06-07 Liposomes contenant une glycoproteide du virus de l'immunodeficience humaine et leurs procedes d'utilisation WO1996040243A1 (fr)

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Cited By (3)

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US20100047331A1 (en) * 2007-04-13 2010-02-25 Haynes Barton F Method of inducing neutralizing antibodies to human immunodeficiency virus
US9402893B2 (en) * 2005-04-12 2016-08-02 Duke University Liposome-peptide conjugate and method of using same to induce production of anti-HIV antibodies
US10076567B2 (en) 2013-09-27 2018-09-18 Duke University MPER-liposome conjugates and uses thereof

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WO1993019775A1 (fr) * 1992-03-31 1993-10-14 Medimmune, Inc. Administration de liposomes contenant des peptides ou des proteines comportant des determinants antigeniques de ctl de proteines vih
WO1993022343A1 (fr) * 1992-05-01 1993-11-11 The Rockfeller University Systeme antigenique a plusieurs peptides possedant des proprietes d'adjuvant, vaccins prepares a partir dudit systeme

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9402893B2 (en) * 2005-04-12 2016-08-02 Duke University Liposome-peptide conjugate and method of using same to induce production of anti-HIV antibodies
US9717789B2 (en) 2005-04-12 2017-08-01 Duke University Liposome-peptide conjugate and method of using same to induce production of anti-HIV antibodies
US10588960B2 (en) 2005-04-12 2020-03-17 Duke University Liposome-peptide conjugate and method of using same to induce production of anti-HIV antibodies
US20100047331A1 (en) * 2007-04-13 2010-02-25 Haynes Barton F Method of inducing neutralizing antibodies to human immunodeficiency virus
US8956627B2 (en) * 2007-04-13 2015-02-17 Duke University Method of inducing antibodies to human immunodeficiency virus involving the administration of MPER peptide-liposome conjugates
US10076567B2 (en) 2013-09-27 2018-09-18 Duke University MPER-liposome conjugates and uses thereof

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