WO2008150182A1 - Compositions et procédés de traitement de l'anthrax - Google Patents

Compositions et procédés de traitement de l'anthrax Download PDF

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Publication number
WO2008150182A1
WO2008150182A1 PCT/NZ2008/000131 NZ2008000131W WO2008150182A1 WO 2008150182 A1 WO2008150182 A1 WO 2008150182A1 NZ 2008000131 W NZ2008000131 W NZ 2008000131W WO 2008150182 A1 WO2008150182 A1 WO 2008150182A1
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Prior art keywords
antigen
anthrax
composition according
composition
microparticle
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PCT/NZ2008/000131
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English (en)
Inventor
Frank B. Gelder
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Innate Therapeutics Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/07—Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/5555—Muramyl dipeptides

Definitions

  • the present invention is concerned with compositions and methods for therapeutic and/or prophylactic treatment of anthrax exposure or infection.
  • the present invention is concerned with microparticulate antigen-adjuvant compositions for use as vaccines or in the preparation of passive immuno therapeutics against anthrax exposure and/or infection.
  • anthrax caused by the bacteria B. anthracis, has been recognized since ancient times. It was typically seen in agrarian workers like tanners and wool sorters who had close contact with livestock, the main reservoir for this disease. In more recent times, this endemic pathogen has been developed into one of the most effective biological weapons known to man.
  • the effectiveness of B. anthracis as a weapon is based on its ability to be sporulated and dried, for effective dispersion, combined with significant disease morbidity and mortality.
  • Antibiotics are effective against the bacilli in the early stages of infection but once exotoxin production has begun, antibiotics offer no protection against fatal anthrax toxaemia.
  • One manifestation of anthrax toxaemia is capsule mediated immune dysfunction.
  • anthrax The lack of a safe and effective anthrax vaccine leaves healthcare workers, laboratory workers and first responders with limited means of protection following potential exposures to anthrax spores during response to an event.
  • vaccination against anthrax is an effective way of protecting individuals against infection, vaccinating the general population may not be cost effective or achievable.
  • different approaches may also be required to slow or prevent anthrax toxin induced morbidity and mortality.
  • Inhibiting anthrax toxins early may change the course of infection and may allow for a vigorous immune response against the bacteria and/or time to intervene with antibiotic treatments.
  • the military interest in anthrax centers on countermeasures against its use as an inhalational biological weapon although other clinical forms are more common in natural exposure situations. In the event of inhalation exposure to B.
  • passive immunotherapy is a non-chemical therapeutic, which could provide complete and immediate immunity to the B. anthracis infection.
  • An additional advantage of passive immunotherapy is the ability to create long-term stockpiles and the rapidity of large-scale productions of immunoglobulin preparations for rapid deployment and distribution following a mass event. Most importantly, passive immunotherapy should be successful at preventing death even when administered days after infection with B. anthracis spores.
  • Antibodies are produced by B-lymphocytes.
  • Cell- mediated immunity involves the activation of T-lymphocytes which either act upon infected cells bearing foreign antigens or stimulate other cells to act upon infected cells. Both branches of the mammalian immune system are important in fighting disease.
  • Humoral immunity is the major line of defense against bacterial pathogens and toxins however the induction of helper and cytotoxic T lymphocytes appears to be crucial for long lived protective immunity.
  • an effective vaccine preferably stimulates both branches of the immune system to protect against disease.
  • Vaccines present foreign antigens from disease causing agents to a host so that the host can mount a protective immune response. Often, vaccine antigens are killed or attenuated forms of the microbes which cause the disease.
  • Immune adjuvants are compounds which, when administered to an individual or tested in vitro, increase the immune response to an antigen in a subject to which the antigen is administered, or enhance certain activities of cells from the immune system.
  • a number of compounds exhibiting varying degrees of adjuvant activity have been prepared and tested (see, for example, Shimizu et al. 1985, Bulusu et al. 1992, Ikeda et al. 1993, Shimizu et al. 1994, Shimizu et al. 1995, Miyajima et al. 1996).
  • Shimizu et al. 1985 Bulusu et al. 1992, Ikeda et al. 1993, Shimizu et al. 1994, Shimizu et al. 1995, Miyajima et al. 1996.
  • these and other prior adjuvant systems often display toxic properties, are unstable and/or have unacceptably low immunostimulatory effects.
  • alum a group of aluminum salts (e.g., aluminum hydroxide, aluminum phosphate) in which vaccine antigens are formulated.
  • Particulate carriers like alum reportedly promote the uptake, processing and presentation of soluble antigens by macrophages.
  • Alum is not without side-effects and is unfortunately limited to humoral (antibody) immunity only.
  • MDP murumyl dipeptide
  • an MDP composition which lacks the unwanted side effects attributed to MDP while achieving enhanced immuno stimulatory properties (Australian Patent No. 732809).
  • the micro particle was used in the context of an adjuvant coupled to certain viral peptides that were not immunogenic in humans, but when coupled to this adjuvant, were immunogenic in other species.
  • the antigen-adjuvant complex was primarily developed to raise antibodies for passive treatment of HIV infection. The discovery and development of effective adjuvant systems is essential for improving the efficacy and safety of existing and future vaccines.
  • the present invention provides an immunogenic composition comprising an antigen derived from Bacillus anthracis in association with a muramyl dipeptide cross-linked into a microparticle.
  • the present invention provides a vaccine composition comprising an antigen derived from Bacillus anthracis in association with a muramyl dipeptide cross-linked into a microparticle, for the prophylactic or therapeutic treatment of anthrax exposure and/or infection.
  • the present invention provides a composition comprising one or more antibodies, or antigen binding fragments thereof, specific for one or more antigens derived from Bacillus anthracis wherein said antibody is derived by immunising a mammal with a composition comprising an antigen derived from Bacillus anthracis in association with a muramyl dipeptide cross-linked into a microparticle.
  • the composition preferably comprises antibodies capable of binding to B. anthracis antigens PA, LF and EF.
  • the composition comprising one or more antibodies preferably includes antibodies against all three antigens.
  • the B. anthracis antigen is selected from PA, LF or EF, or any combination thereof, and for preference the antigens are used in pure or recombinant form or a combination of pure and recombinant forms. Even more preferred is a combination of all three antigens. Most preferred is pure or recombinant PA83 antigen (rPA83). It will be understood that more than one antigen (i.e. PA, LF or EF) may be associated with the same NT-MDP microparticle. Preferably one or more antigens is each associated with different microparticles.
  • the microparticle has the size range of about 0.1 to 0.2 microns, or 0.01 to 2.0 microns or 0.01 to 1.0 microns diameter.
  • Other suitable particles size ranges are about 0.01 to 0.2 microns, 0.01 to 0.1 microns, 0.01 to 0.5, 0.05 to 1 or 0.1 to 0.5 microns diameter.
  • the route of delivery will also influence the selection of desired particle size.
  • aerosol dry powdered delivery using inhalational methodology is more suited for microparticles in the size range of 0.5 to 2.0 microns.
  • the antigen may be covalently linked to the surface of the NT-MDP particle for stability and appropriate delivery. It will be understood that other methods of coupling of the antigen to the NT-MDP may also be employed. The preferred linkage is via a Schiff base intermediate.
  • the preferred antibody preparation for passive immunisation of subjects is a human or humanised antibody, or antigen binding fragments thereof. It will be understood however that antisera and antibody preparations obtained from other species will also be useful in compositions of the present invention.
  • Antibodies may be prepared using conventional monoclonal antibody technology but may also be prepared using recombinant expression systems (Current Protocols In Immunology; Series Editor: Richard Coico (Cornell University) Published by John Wiley & Sons, Inc. Using Antibodies: A Laboratory Manual, Ed Harlow and David Lane, Published by Cold Spring Harbour Laboratory Press).
  • the present invention provides a pharmaceutical composition comprising a composition according to any one of the first to third aspects.
  • the present invention provides a pharmaceutical composition comprising a composition according to any one of the first to third aspects and one or more other therapeutic agents effective in the treatment of anthrax lethal toxin.
  • the present invention provides a method of prophylactic or therapeutic treatment of a subject exposed to, or infected by, Bacillus anthracis comprising the administration of a composition of any one of the first to fifth aspects to a subject in need thereof.
  • the present invention provides a method of prophylactic or therapeutic treatment of a subject exposed to, or contaminated with, anthrax lethal toxin comprising the administration of a composition of any one of the first to fifth aspects to a subject in need thereof.
  • the present invention provides use of a composition according to any one of the first to fifth aspects, for the manufacture of a medicament for the treatment of a subject exposed to, or infected by, Bacillus anthracis.
  • the present invention provides use of a composition of any one of the first to fifth aspects, for the manufacture of a medicament for the treatment of a subject exposed to, or contaminated with, anthrax lethal toxin.
  • Figure 1 The chemical composition of the monomeric subunit of MDP.
  • Bacillus anthracis Ames strain spores and treated 1 hour after challenge with anti- anthrax toxin antibody, with or without Ciprofloxacin (b.i.d for 6 days).
  • Figure 4 Survival of Swiss Webster mice challenged intranasally with 5LD 50
  • Bacillus anthracis Ames strain spores and treated 48 hours after challenge with anti- anthrax toxin antibody, with or without Ciprofloxacin (b.i.d for 6 days).
  • Day 0 is 1 st immunization
  • asterix indicate 2 nd (day 14), 3 rd (day 28) and 4 th (day 56) booster immunizations.
  • Sera was obtained by plasmapheresis from goats on day 94 (time point designated as a square).
  • anthrax antigen is intended to encompass any component or fragment of the Bacillus anthracis organism, soluble or particulate, that is capable of causing or potentiating disease in a mammal and that can on its own or in conjunction with an adjuvant induce an immune response in a mammal.
  • this phrase encompasses protective antigen, PA; lethal factor, LF; oedema factor, EF and includes Anthrax lethal toxin (LeTx) of Bacillus anthracis.
  • the Anthrax antigen may be purified from a natural source or may be in recombinant form.
  • muramyl dipeptide encompasses any natural or synthetic source of muramyl dipeptide.
  • MDP-microparticle In the context of the present invention the terms "MDP-microparticle”, “NT- MDP” or “non-toxic muramyl dipeptide” may be used interchangeably.
  • antibody as used in the context of the present invention is intend to encompass polyclonal as well as monoclonal antibodies. It also encompasses antibodies of various species, fragments of antibodies, recombinant and humanised antibodies or recombinantly produced fragments thereof.
  • antibody and antibodyum or “antisera” may be used interchangeably ' herein.
  • the present invention is motivated by the lack of a safe and efficacious prevention or treatment for anthrax infection and toxicity, and is in part based on the unique and advantageous properties of a micro particulate adjuvant.
  • the vaccines and passive immunotherapeutic compositions of the present invention represent a significant improvement in immunotherapy of anthrax exposure and/or infection, without the drawbacks of the presently available therapies.
  • B. anthracis carries two virulence factors: a poly-D- glutamate capsule and two bipartite exotoxins, which are the actual cause of disease and death.
  • Anthrax toxin which consists of three bacterially encoded polypeptides, protective antigen (PA), lethal factor (LF) and edema factor (EF), is a major virulence factor of B. anthracis.
  • the LF and EF components are enzymes that are carried into the cell by PA.
  • PA protective antigen
  • LF lethal factor
  • EF edema factor
  • the combination of PA and LF forms lethal toxin (LT or LeTx) while PA combines with edema factor (EF) to form edema toxin (ET or EdTx) [1-3].
  • Anthrax toxin enters cells via a receptor-mediated endocytosis [4, 5]. Exotoxin secretion can begin within a few hours of infection, concomitant with bacterial replication. PA plays an elaborate yet critical role in virulence and has been the main target for eliciting immunity against the anthrax toxins.
  • the present invention makes use of one or more of PA, EF and LF as antigenic components to induce the production of antibodies in an animal host against a virulent strain of Bacillus anthracis (B. anthracis) or its various antigens and toxins.
  • the immunogenic compositions comprise one or more of PA, EF and LF in association with an adjuvant that is a non-toxic muramyl dipeptide (NT-MDP) in microparticulate form.
  • N-MDP non-toxic muramyl dipeptide
  • such compositions represent effective vaccines but may also be used in the context of the present invention to generate passive immunotherapeutic compositions for treatment of anthrax exposure and infection. It will be understood however, that any other antigenic components of B.
  • anthracis may be used in combination with the NT-MDP microparticulate adjuvant for preparation of suitable immunogens. It will also be clear to those skilled in the art that crude preparations of B. anthracis antigens may be as useful as purified or recombinant antigens for purposes of the present invention.
  • the MDP adjuvant is cross-linked so that it forms a stable, non-toxic particle (NT-MDP) of relatively uniform size.
  • N-MDP non-toxic particle
  • uniformity of size, distribution and absolute particle size are not critical, the preferred size is about 0.1 to 0.2 micron in diameter, however other suitable particle size ranges include 0.1 to 0.2 microns, 0.01 to 2.0 microns or 0.01 to 1.0 microns, 0.01 to 0.2 microns, 0.01 to 0.1 microns, 0.01 to 0.5 microns, 0.05 to 1 microns or 0.1 to 0.5 microns diameter.
  • the adjuvant is capable of stimulating the relevant cellular components of the immune system, to achieve maximal response to the antigen while minimising stimulation of unwanted cell-types.
  • the preferred particle size can accommodate sufficient amount of antigen to ensure an adequate and timely immune response.
  • Conjugation of antigens to the micro particulate NT-MDP makes for a stable and highly immunogenic composition.
  • One of the advantages of these vaccine compositions is that protective titres are achieved in a relatively short time (typically within 28 days).
  • the prior art vaccine requires months to achieve protective titres and is associated with a number of significant side effects.
  • the administration of the immunogen comprising recombinant anthrax antigen rPA83 conjugated to microparticulate NT-MDP (MDP+PA83) to goats and mice had no appreciable side effects.
  • the observed lack of adverse reactions in the animal models used, which is expected to translate into humans, is in part associated with the low toxicity of the microparticulate adjuvant, NT-MDP, and in part from the selective delivery of immunogen to antigen processing cells. This contrasts with the prior art immunogen/adjuvant combinations where local and diffusible delivery of immunogen with aluminum based adjuvants results in toxicity secondary to adjuvant dose and subsequently local Arthus reaction at the injection site.
  • the present invention also encompasses passive immunotherapeutics for treating B. anthracis infection and/or toxaemia, based on the use of NT-MDP+anthrax antigen- immunogen for the preparation of anti-anthrax antibodies in one species that are effective in treating anthrax infection/challenge in another species.
  • passive immunotherapeutics for treating B. anthracis infection and/or toxaemia based on the use of NT-MDP+anthrax antigen- immunogen for the preparation of anti-anthrax antibodies in one species that are effective in treating anthrax infection/challenge in another species.
  • recombinant anthrax antigen rPA83 conjugated to microparticulate NT-MDP (MDP+PA83) was used to prepare anti-sera in goats, which anti-sera were used to protect mice against challenge with anthrax lethal toxin (LeTx).
  • human antibody For passive immunotherapy in humans, human antibody is preferred however in the absence of immunized human serum donors any mammal can be used for preparation of anti-serum, antibodies, or Fab fragments there of.
  • a bioterrorist threat agent such as anthrax
  • animal size, response to immunogen, availability, ease of handling among others are considerations.
  • horse, sheep, goat, and rabbits are frequently used however any species capable of producing the desired neutralising antibody would be acceptable.
  • the present invention makes use of animal models and in vitro test systems to demonstrate different embodiments of the invention with respect to vaccine and passive immunotherapeutic composition utility and efficacy.
  • the murine and caprine models are used to demonstrate the rapid increase in titres of relevant antibodies following immunisation with the vaccine compositions of the present invention. These models also demonstrate the lack of any appreciable side-effects usually associated with anthrax vaccine administration.
  • the murine model was also used to demonstrate the efficacy of the vaccine compositions of the present invention in protecting the animals following challenge with anthrax LeTx. Protective efficacy of passive immunotherapy was also demonstrated both in vivo (caprine anti-sera into murine model).
  • the vaccine compositions are highly effective in eliciting a protective immune response as evidenced by protection from mortality and morbidity following anthrax lethal toxin challenge 14 days post 3 immunizations over a 21 -day period while causing no adverse side-effects.
  • the anthrax antisera are shown to be effective passive immunotherapy for anthrax challenge and/or infection.
  • the present invention also encompasses methods for treatment of subjects exposed to, or infected with, B. anthracis.
  • the methods contemplated make use of the immunogenic compositions described herein either as vaccines to protect against anthrax infection or for preparation of anti-sera for passive immunotherapy of subjects exposed to or infected with B. anthracis.
  • the vaccine compositions may comprise any of the anthrax antigens in combination with NT-MDP or indeed a combination of such antigens. Similar compositions may be used to raise anti-sera in one species for use as passive immunotherapeutics in another species.
  • the passive immunotherapy is particularly useful as post-exposure treatment of subjects with confirmed anthrax infection.
  • the present invention contemplates veterinary as well as human immunotherapeutic uses of the immunogenic compositions.
  • compositions may comprise crude or partially purified anti-sera and immunoglobulin preparations or they may comprise specific monoclonal antibodies or combination of monoclonal antibodies. Further, purified or recombinant fragments of antibodies may be used. Methods for derivation of monoclonal antibodies, fragments or recombinant antibodies are well known in. the art and are described for example in Current Protocols In Immunology; Series Editor: Richard Coico (Cornell University) Published by John Wiley & Sons, Inc.
  • compositions of the present . invention may be used in conjunction with conventional antibiotic therapy regimes and may be administered simultaneously with, prior to, or following antibiotic treatment.
  • Vaccine composition of the present invention may be administered by any suitable means.
  • the method of immunizing a subject against a disease according to the present invention may employ a number of methods to administer a liquid solution formed by the vaccine composition.
  • Exemplary methods of administration are intramuscular injection, subcutaneous injection, intravenous injection, intra peritoneal injection, eye drop, via drinking water, aerosol, or nasal spray.
  • any suitable veterinary formulation may be used.
  • formulations may be in the form of powders or pastes and may be added to feed or administered orally in the usual manner. Suitable formulation protocols and excipients can be found in standard texts such as Remington: The Science and Practice of Pharmacy, 19 th Ed, 1995 (Mack Publishing Co. Pennsylvania, USA), British Pharmacopoeia, 2000, and the like.
  • the immuno therapeutic compositions of the present invention are useful for administration to mammals, particularly humans, to treat and/or prevent anthrax infection.
  • Vaccine compositions containing the anthrax antigens are administered to a patient infected with B. anthracis or to an individual susceptible to, or otherwise at risk of, being infected with B. anthracis, to elicit an immune response against one or more anthrax antigens and thus enhance the patient's own immune response capabilities.
  • the immunotherapeutic compositions are administered to a patient in an amount sufficient to elicit an effective cellular and humoral immune response to the anthrax antigen and to cure or at least partially arrest or slow symptoms and/or complications of the infection.
  • Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • the immuno therapeutic compositions of the present invention may also be used purely as prophylactic agents.
  • the dosage for an initial prophylactic immunization generally occurs in a dosage range of about 1, 5, 50, 500, or 5000 ⁇ g of immunogen and preferably in a dose range of 5 ⁇ g to 500 ⁇ g immunogen.
  • Dosage values for. a human typically range from about 10 ⁇ g to about 100 ⁇ g per 70 kilogram patient.
  • the maximum dose expected to be given to a human may be up to 1 mg per 70 kilogram, however those skilled in the art will recognize dose is dependent on immunogenicity and will vary to achieve the desired effect. This is followed by boosting immunizations at defined intervals from about two weeks to about twelve months to achieve the desired level of protection.
  • the immunogenicity of the vaccine may be assessed by measuring the specific antibody concentration and cellular immune response from a sample of the patient's blood.
  • a multiple repeat of muramyl dipeptide (MDP) isolated from Propionibacterium acini as described in the following text formed the core structure of the MDP microparticle carrier complex of this example.
  • the chemical composition of the preferred monomeric subunit is as shown in Figure 1.
  • Other synthetic and/or natural and/or semi-synthetic muramyl dipeptides may also be used in the inventive compositions.
  • MDP has well known immunostimulatory properties which have been extensively evaluated in studies designed to determine its effect on increasing immune function. To date, both MDP isolated from natural sources and synthetic MDP have been associated with significant toxicity when administered to mammals. This toxicity has limited the effectiveness of MDP as an adjuvant.
  • a method for the isolation of MDP free from toxic components is provided herein.
  • Propionibacterium acnes was grown to a mid-stationary growth phase and washed to remove contaminants of bacterial culture origin employing techniques well known to those in the art. Hydrophobic components contained in the cell walls and cytoplasm were sequentially extracted by successive washes with increasing concentrations of ethanol/isopropano I/water (10%:10%:80%, 25%:25%:50% and 40%:40%:20%) at elevated temperatures.
  • the isopropyl alcohol is then removed with successive washes with decreasing concentrations (80%, 50%, 40% and 20%) of ethanol at elevated temperatures.
  • the resulting MDP microparticle is then suspended in 20% ethanol and its concentration measured by relating its absorbance at 540 nm to the absorbance of turbidity standards. The concentration of the MDP microparticle was adjusted to 10 mg/ml for storage and later use.
  • particle size ranges that can be obtained and may be suitable include the ranges of about 0.1 to 0.2 microns, or 0.01 to 1.0 microns, or 0.01 to 0.2 microns, 0.01 to 0.1 microns, 0.01 to 0.5 microns, 0.05 to 1 microns or 0.1 to 0.5 microns diameter.
  • the terminal dipeptide amino-linked L-alanine-D-isoglutamine corresponded to the monomeric structure shown in Figure 1.
  • the MDP microparticles contain muramic acid with amino-linked L-alanine-D-isoglutamine dipeptide as the bioactive component.
  • Such a microparticle can be isolated from natural sources, as above, or synthesized using well-known synthetic procedures (e.g.
  • the muramyl dipeptide microparticles generated by the present methods can have a broad range of sizes (e.g. 0.01-2.0 microns) but the preferred size is in the range of 0.05-0.2 microns.
  • Example 2 Covalent attachment of immunogen to NT-MDP
  • a NT-MDP micro particle is used to deliver immunogen to antigen presenting cells resulting in both a rapidly occurring and vigorous cellular and humoral immune response. Quantitation of these immune responses demonstrates 10 to 100 fold increases in antibody concentration as compared to other adjuvants.
  • the covalent attachment of immunogen to the NT-MDP can be made through bi-functional cross linkers, or to the aldehyde oxidation product of the carbohydrate moiety as disclosed in this examples as described in Current Protocols In Immunology; Series Editor: Richard Coico (Cornell University) Published by John Wiley & Sons, Inc. Recombinant PA and LF were obtained from Wadsworth Center of the New
  • NT-MDP (20 mg) in 20% ethanol is pelleted by centrifugation, resuspended in and extensively washed with water.
  • the NT-MDP is then pelleted and resuspended at a concentration of 50 mg NT-MDP / ml in sodium metaperiodate (0.5M) and the oxidation reaction is carried out for 1 hour at room temperature. Following activation with sodium metaperiodate the NT-MDP microparticle suspension is pelleted by centrifugation, resuspended in and extensively washed with water. The concentration of the sodium metaperiodate and the reaction time can be varied to regulate the number of activated sites produced within the NT-MDP microparticle during oxidation.
  • An activated NT-MDP microparticle should react with and covalently attach at least one molecule of the subject immunogen per NT-MDP microparticle, preferably 10-100 molecules of subject peptide per NT-MDP microparticle and most preferably 100 to 1000 subject peptides per NT-MDP microparticle.
  • a final concentration of 0.5 M sodium metaperiodate is used and the oxidation reaction is carried out for one hour.
  • NT-MDP is then pelleted and washed extensively to removal the sodium metaperiodate.
  • the activated NT-MDP is then re-suspended in the desired immunogen (anthrax toxin PA, LF and EF respectively all at >1 mg/ml at a 20:1 w/w ratio) -in sodium bicarbonate buffer (0.1 M pH 9.5) and incubated (ambient temperature) for 18-24 hours.
  • the reactants are centrifuged and the pellet which now contains immunogen linked to the NT-MDP particles through an intermediate Schiff base is reduced forming a stable covalent linkage between the microparticle and immunogen.
  • reducing agents can be employed and sodium bo ro hydride is an example of a reducing agent typically used for this purpose.
  • sodium bo ro hydride is an example of a reducing agent typically used for this purpose.
  • the NT-MDP microparticle-immunogen composition affects immunogenicity by influencing preferential cell uptake, protein half-life, and antigen presentation through MHC immunological events.
  • One or more different subject immunogens can be linked to the same microparticle but preferably a single subject immunogen is attached to the NT-MDP microparticle.
  • a cocktail of subject immunogen NT-MDP microparticle conjugates can be prepared by mixing individual conjugates at ratios to optimize immunogenicity of each subject peptide introduced in the cocktail.
  • immunogen processing by the antigen presenting cell results in a high density, usually more than 100 and most frequently more than 500 peptides, presented at the cell surface of the antigen presenting cell through MHC interactions.
  • the Schiff s base intermediate is converted to a stable covalent linkage.
  • the number of immunogens per microparticle can be controlled by varying oxidation conditions and quantified as required by employing a radioactive tracer. These methods are well known in the art (e.g. Current Protocols In Immunology; Series Editor: Richard Coico (Cornell University) Published by John Wiley & Sons, Inc.). The method of attachment and attachment configuration can vary from immunogen to immunogen as needed to achieve the desired response.
  • mice Female Balb/c mice (7 weeks, average weight 17g, Taconic Farms, NY), 3 mice per group, were immunized with 2.5 ⁇ g PA83 attached to 50 ⁇ g NT-MDP
  • mice were bled via the tail vein on days 17 and 28, the serum was collected and tested in serial dilutions against PA83 via EIA. Suitable EIA methods are described in Current Protocols In Immunology; Series Editor: Richard Coico (Cornell
  • mice in groups #1 and #2 showed significant serum titers of anti-PA83 antibodies by the second immunization
  • mice were challenged with 200 ⁇ g
  • mice in group #3 all succumbed to LeTx within 24 hours as expected.
  • Mice in groups #1 and #2 were fully protected against the lethal effects of LeTx challenge until the end of the study at day 49 (14 days post LeTx challenge). Results of these studies are shown in Figure 2.
  • Example 4A Immunization of goats with NT-MDP+rPA83 and purification of caprine IgG.
  • Six goats were immunized with lOO ⁇ g NT-MDP+rPA83 conjugates then boosted three times with 50 ⁇ g NT-MDP+rPA83 immunogen over a thirteen week period.
  • Hyper-immune plasma was collected by plasmapheresis from each animal two weeks following the last immunization. Plasma was pooled, IgG was purified using standard techniques and subsequently analyzed by SDS-PAGE (non reduced) and ELISA using standard techniques (e.g. Current Protocols In Immunology; Series
  • the IgG fraction from the plasma was purified using standard techniques including octanoic acid precipitation of non- immunoglobulin plasma proteins, ion exchange, and size chromatography (eg. Handbook of Experimental Immunology, Weir et al, 1986, or similar text).
  • Example 4B A Caprine anti-anthrax toxins IgG cocktail protects mice against anthrax spore challenge.
  • mice Female Swiss Webster mice (average weight 25.2 g, Taconic Farms) were infected with approximately 5 x 10 4 B. anthracis Ames spores (5 LD50) by 20 ⁇ l instillations in each nares. Mice received either PBS (Group #1, 10 mice) or an irrelevant antibody (Group #2, 6 mice) at 1 hour post-infection or an anti-anthrax toxins IgG cocktail (an antibody cocktail comprising 80% ⁇ PA83, 10% ⁇ LF; 10% ⁇ EF - referred to in figures 4, 5 and 6 as "Trithrax") (Group #3, 10 mice) at 24 hours post-infection (32 mg/kg) by intraperitoneal injection. Mice were monitored twice daily for 14 days for signs of illness and death.
  • PBS Group #1, 10 mice
  • an irrelevant antibody Group #2, 6 mice
  • an anti-anthrax toxins IgG cocktail an antibody cocktail comprising 80% ⁇ PA83, 10% ⁇ LF; 10% ⁇ EF - referred to in figures 4, 5 and 6 as
  • Ciprofloxacin was administered twice daily at 0.9 mg/day via intraperitoneal injection for the first six days post spore challenge (Group #4, 10 mice).
  • a Cipro floaxcin control group (Group #5, 10 mice) received only Ciprofloaxcin with no concomitant immunoglobulin treatment.
  • mice treated with an anti-anthrax toxins IgG cocktail had survived. By day 6 this survival rate stabilized at 60% out to the end of the study (day 14 p.i.). A 10% survival rate was observed for control mice (Groups #1 & 2) by day 14. Concomitant administration of Ciprofloxacin and an anti- anthrax toxins IgG cocktail enhanced survival, protecting 100% of the mice for 6 days (Group #4), however this rate dropped to 60% once antibiotic treatment was halted. Mice receiving only low-dose Ciprofloaxcin (Group #5) demonstrated a 50% survival rate at day 14 post-infection. See Figures 4 to 6.
  • caprine anti-sera against recombinant components of the anthrax lethal toxin are capable of producing a protective effect in vivo and that caprine anti-sera can be a successful therapeutic treatment for human intoxication due to Bacillus anthracis infection.
  • Example 5 Cellular immunity in caprine animal model
  • NT-MDP plus antigen vaccine formulations also induce strong cellular immunity in goats, against immunizing, but not irrelevant antigens, as measured by the blast transformation assay.
  • Blast transformation was measured using a CFSE-dye dilution assay which was performed as follows. Blood samples were taken from 3 goats that had not been immunized or had been immunized with anthrax toxin protective antigen (PA). Goat PBMC were purified from heparinised blood using Ficoll density purification (Lymphoprep 1.077 g/ml, Axis).
  • Cells were diluted to 2 x 10 6 /ml in pre-warmed (37 0 C) PBS/1% w/v BSA and combined with an equal volume of 10 ⁇ m CFSE dye (Invitrogen). Labelling was achieved by incubating the cells in a 37 0 C water bath for precisely 5 min. The reaction was stopped by diluting the labeled cells in 3 volumes of ice-cold complete medium (hepes-buffered RPMI + 10% v/v heat-inactivated FBS).
  • the cells were washed and then cultured at 10 6 /ml (U bottom 96 well plate) in the presence of Phytohaemaglutinin lectin from Phaseolus vulgaris (10 ⁇ g/ml, PHA, Sigma) as an assay positive control and either 0.1 ⁇ g/ml of immunising antigen (PA) or irrelevant antigen (lethal factor, LF) for PA immunised goats.
  • PA immunising antigen
  • LF irrelevant antigen
  • Cells were cultured for 5 days (humidified, 37 0 C, 5% CO2). At the end of this period, the cells were washed once in PBS, and then the cell pellets were resuspended in Propidium Iodide at 1 ⁇ g/ml (Invitrogen) ready for flow cytometric analysis.
  • Live cells were identified based on PI negativity, and the CFSE fluorescent profile was determined.
  • the intensity of CFSE fluorescence associated with non-division was determined by examining the fluorescence of cells cultured in the absence of mitogen. PHA-stimulated samples confirmed the region associated with cell-division associated CFSE-dye dilution. The proportion of live cells which had completed cell division (ie had diluted CFSE) was determined.
  • the results clearly show that PBMC from goats immunized with PA preferentially proliferate in response to the immunizing antigen (PA) as opposed to the non-immunizing antigen (LF). In contrast there was no antigen- induced proliferation by PBMC from non-immunised goats towards either antigen.
  • PA immunizing antigen
  • LF non-immunizing antigen

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Abstract

La présente invention concerne des compositions immunogènes et des procédés destinés au traitement thérapeutique et/ou prophylactique d'une exposition à l'anthrax ou d'une infection due à l'anthrax.
PCT/NZ2008/000131 2007-06-05 2008-06-05 Compositions et procédés de traitement de l'anthrax WO2008150182A1 (fr)

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WO2010147484A1 (fr) * 2009-06-16 2010-12-23 Innate Therapeutics Limited Compositions et procédés pour le traitement de la sclérose en plaques

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WO2007075188A2 (fr) * 2005-05-05 2007-07-05 George Mason University Procedes de traitement d'une infection a bacillus
WO2008054532A2 (fr) * 2006-05-08 2008-05-08 University Of Virginia Patent Foundation Compositions et procédés permettant de traiter une mortalité due à l'anthrax

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AU2006200454A1 (en) * 1996-10-10 2006-02-23 Innate Immunotherapeutics Limited Compositions and methods for treating viral infections
WO2007075188A2 (fr) * 2005-05-05 2007-07-05 George Mason University Procedes de traitement d'une infection a bacillus
WO2008054532A2 (fr) * 2006-05-08 2008-05-08 University Of Virginia Patent Foundation Compositions et procédés permettant de traiter une mortalité due à l'anthrax

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KARGINOV V.A. ET AL.: "Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design", PNAS, vol. 102, no. 42, 2005, pages 15075 - 15080 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010147484A1 (fr) * 2009-06-16 2010-12-23 Innate Therapeutics Limited Compositions et procédés pour le traitement de la sclérose en plaques
CN102458476A (zh) * 2009-06-16 2012-05-16 伊耐特免疫治疗有限公司 用于治疗多发性硬化症的组合物及方法
AU2010260585B2 (en) * 2009-06-16 2013-02-21 Innate Immunotherapeutics Limited Compositions and methods for treatment of Multiple Sclerosis
US8389479B2 (en) 2009-06-16 2013-03-05 Innate Immunotherapeutics Limited Compositions and methods for treatment of multiple sclerosis
CN102458476B (zh) * 2009-06-16 2014-08-20 伊耐特免疫治疗有限公司 用于治疗多发性硬化症的组合物及方法

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