WO2008133595A1 - Compositions and methods for stimulating an immune response in-vivo and in-vitro - Google Patents

Compositions and methods for stimulating an immune response in-vivo and in-vitro Download PDF

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Publication number
WO2008133595A1
WO2008133595A1 PCT/SG2008/000139 SG2008000139W WO2008133595A1 WO 2008133595 A1 WO2008133595 A1 WO 2008133595A1 SG 2008000139 W SG2008000139 W SG 2008000139W WO 2008133595 A1 WO2008133595 A1 WO 2008133595A1
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Prior art keywords
antigen
immunogenic composition
cell
cells
cell population
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PCT/SG2008/000139
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French (fr)
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Lie Tao Li
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Newbiomed Pika Pte Ltd
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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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2

Definitions

  • the immune system has evolved over time as a result of exposure to a wide variety of pathogens that occur in the environment. On exposure to pathogens the human immune system will trigger a chain of cellular responses that result in both an innate and an adaptive immune response.
  • TLRs toll-like receptors
  • PAMPs pathogen associated molecular patterns
  • TLR Toll-like receptors
  • the immunogenic composition according to any aspect of the invention may further comprise at least one antigen.
  • the immunogenic composition according to any aspect of the invention may be for use in medicine.
  • the immunogenic composition may be for use in inducing activation of a dendritic cell (DC) in an individual.
  • the immunogenic composition may also be for use in increasing an immune response to an antigen in an individual.
  • DC dendritic cell
  • an immune cell population enriched in a selected immune cell type for increasing an immune response to at least one antigen in an individual wherein the enriched immune cell population is prepared by contacting a starting cell population with a population of polyriboinosinic- polyribocytidylic acid (PIC) molecules, at least one antibiotic and at least one positive ion,
  • PIC polyriboinosinic- polyribocytidylic acid
  • the invention further contemplates the use of cells and/or substances resulting from an immune response in-vitro, for example, immune cells, co-stimulatory molecules, cytokines and/or chemokines and/or antibodies, and the like, including the use of such cells and/or substances, for example, to elicit, induce, enhance and/or potentiate an immune response in a host.
  • the invention further contemplates administration of P ⁇ CA in combination with cells and/or substances resulting from an immune response in-vitro.
  • the PIC molecules according to any aspect of the invention may have a molecular weight in a range of from about 66,000 to about 1,200,000 Dalton.
  • Fig. 1 Titers of IL-6 observed in murine blood serum post in-vivo stimulation with
  • Fig. 3 Titers of INF-gamma observed in murine blood serum post in-vivo stimulation with PIKA
  • PIKA Fig.5 Production of CD25 observed post in-vitro stimulation of murine natural killer cells with PIKA
  • adjuvant refers to any substance or mixture of substances that increases or diversifies the immune response of a host to an antigenic compound.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • immune response refers to any response to an antigenic or immunogenic compound by the immune system of a vertebrate subject.
  • exemplary immune responses include, but are not limited to local and systemic cellular as well as humoral immunity, such as cytotoxic T lymphocytes (CTL) responses, including antigen-specific induction of CDS + CTLs, helper T-cell responses including T-cell proliferative responses and cytokine release, and B-cell responses including antibody response.
  • CTL cytotoxic T lymphocytes
  • inducing an immune response refers to an immune response that is stimulated, initiated, or induced.
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • One aspect the present invention provides for a substance comprising polyinosinic acid - polycytidylic acid that acts as an immune regulator when administered to a host.
  • the substance comprising polyinosinic acid - polycytidylic acid may facilitate an immune response in-vitro, for example, the activation, proliferation and/or maturation of natural killer cells and/or antigen presenting cells including but not limited to, dendritic cells and B cells.
  • the invention provides for a substance comprising polyinosinic acid - polycytidylic acid together with an antigen or antigens that acts as an immune regulator in-vitro.
  • the resulting composition is a PIC-containing composition that further contains an antibiotic and a positive ion.
  • Li a particular embodiment, where the antibiotic is kanamycin and the ion is calcium the product may be described as PICKCa.
  • the PICKCa composition may contain molecules without restriction of different physical characteristics.
  • the PDCA composition molecules in the composition have an average molecular weight equal or equal to or greater than 150,000 Daltons, or equal to or greater than 250,000 Daltons, or equal to or greater than 350,000 Daltons, or equal to or greater than 500,000 Daltons, or equal to or greater than 650,000 Daltons, or equal to or greater than 750,000 Daltons, or equal to or greater than 1,000,000 Daltons, or equal to or greater than 1,200,000 Daltons, or equal to or greater than 1,500,000 Daltons, or equal to or greater than 2,000,000 Daltons.
  • Example 2 demonstrates that PIKA markedly induces the expression of CD25 ( Figure 5) and indicative of the proliferation of NK cells in vitro.
  • B cells are lymphocytes that are instrumental in the humoral immune response. B cells undergo a maturing process as a result of which various types of B cells are produced including plasma B cells that on exposure to an antigen will produce and secrete large amounts of antibodies and memory B cells post exposure to an antigen will remain in the body and are able to respond quickly to a second exposure to the same antigen.
  • Example 3 demonstrates that PIKA markedly induces the expression of CD25 ( Figure 6) and indicative of the proliferation of B cells in vitro.
  • Activated dendritic cells may be used to activate T-cells ex- vivo.
  • Activated T- cells e.g. cytotoxic T-cells (e.g., CDS + T cells) and/or helper T-cells (e.g., CD4 + T cells) may be administered to patients alone, or in combination with other immunostimulatory substances, for the prevention or treatment of chronic or infectious diseases.
  • cytotoxic T-cells e.g., CDS + T cells
  • helper T-cells e.g., CD4 + T cells
  • the antigen is a polypeptide or peptide epitope associated with an intracellular pathogen.
  • Polypeptides and peptide epitopes associated with intracellular pathogens are known in the art and include, but are not limited to, antigens associated with human immunodeficiency virus (HIV), e.g., HIV g ⁇ l20, or an antigenic fragment thereof; cytomegalovirus antigens; Mycobacterium antigens (e.g., Mycobacterium avium, Mycobacterium tuberculosis, and the like); Pneumocystis carinii (PCP) antigens; malarial antigens, including, but not limited to, antigens associated with Plasmodium falciparum or any other malarial species, such as 41-3, AMA-I, CSP, PFEMP-I, GBP-130, MSP-I, PFS- 16, SERP, etc.; fungal antigens; yeast antigens (e.g., an antigen
  • Tumor-specific antigens include, but are not limited to, any of the various MAGEs (Melanoma-Associated Antigen E), including MAGE 1 (e.g., GenBank Accession No. M77481), MAGE 2 (e.g., GenBank Accession No. U03735), MAGE 3, MAGE 4, etc.; any of the various tyrosinases; mutant ras; mutant p53 (e.g., GenBank Accession No. X54156 and AA494311); and ⁇ 97 melanoma antigen (e.g., GenBank Accession No. M12154).
  • MAGE 1 e.g., GenBank Accession No. M77481
  • MAGE 2 e.g., GenBank Accession No. U03735
  • MAGE 3 MAGE 4
  • any of the various tyrosinases mutant ras
  • mutant p53 e.g., GenBank Accession No. X54156 and AA494311
  • Allergens include, but are not limited to, environmental aeroallergens; weed pollen allergens; grass pollen allergens; tree pollen allergens; house dust mite allergens; storage mite allergens; mold spore allergens; animal allergens (examples by species - cat, dog, guinea pig, hamster, gerbil, rat, mouse); animal allergens (examples by source - epithelial, salivary, urinary proteins); food allergens, including but not limited to the following common examples: crustaceans, nuts, such as peanuts, and citrus fruits; insect allergens (other than mites listed above); venoms, including, but not limited to, hymenoptera, yellow jacket, honey bee, wasp, hornet, and fire ant venoms; other environmental insect allergens from cockroaches, fleas, mosquitoes, etc.; bacteria such as streptococcal antigens; parasites such as as
  • a peptide can be produced through the sequential additional of activated monomelic units to a solid phase bound growing peptide chain.
  • Well-established recombinant DNA techniques can be employed for production of polypeptide antigens.
  • the antigen is pure, e.g., at least about S0%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%, or greater than 98%, pure.
  • the subject composition of the invention can be administered before and/or after, and/or simultaneously with the adjuvant.
  • the subject composition of the invention may be administered with the initial administration of the antigen, followed by a boost dose of vaccine comprising subject composition of the invention and/or the adjuvant.
  • the initial dose of vaccine administered may exclude the subject composition of the invention but an immunogenic substance comprising the subject composition of the invention is subsequently administered to the patient.
  • the subject immunogenic composition may be administered with antibodies, cytokines, chemokines or other co- stimulatory molecules for example: an interleukin, e.g., IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-IO, IL- 12, IL- 15, and the like; a colony stimulating factor (e.g., Neupogen® (filgrastim; G-CSF); Neulasta (pegfilgrastk ⁇ ); granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor); an interferon (e.g., IFN- ⁇ , EFN- ⁇ , IFN- ⁇ , EFN- ⁇ ; IFN- ⁇ ); a chemokine (e.g., IP-IO; Mig; Gro ⁇ /IL-8, RANTES; MIP-I ⁇ ; MlP-l ⁇ ; MCP-I;
  • PIKA may be used in conjunction with a delivery complex.
  • the delivery complex is a substance associated with a targeting means e.g. a molecule that results in higher affinity binding to target cell such as dendritic cell surfaces and/or increased cellular uptake by target cells.
  • delivery complexes include but are not limited to; nucleic acid delivery acids associated with: a sterol (e.g. cholesterol), a lipid (e.g. cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by a target cell specific receptor).
  • Preferred complexes may be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex may be cleavable under appropriate conditions within the cell.
  • the invention provides a kit comprising a subject immunogenic composition, which includes an enriched immune cell population and/or one or more co- stimulatory molecules (e.g., a co-stimulatory molecule produced by a cell in the enriched immune cell population, e.g., a cytokine, a chemokine, an antibody, etc.) and may further be provided in combination with PIKA (e.g., in the same or different compositions), hi some embodiments, a subject kit comprises an enriched immune cell population, one or more factors (e.g., a cytokine, a chemokine, and antibody, etc.), where the one or more factors can be factors produced by the enriched immune cell population; PIKA; and an antigen. In some embodiments, the kit further provides a delivery system (e.g., one or more of a syringe; a needle; a system for intravenous infusion; etc.) for delivering the immunogenic composition to an individual.
  • a delivery system e
  • a subject kit comprises a mature dendritic cell, e.g., an enriched mature DC population.
  • a subject kit comprises a mature, antigen- primed dendritic cell, e.g., an enriched, mature, antigen-primed DC population.
  • a subject kit comprises a mature dendritic cell (which may be antigen primed); and PIKA.
  • a subject kit comprises a mature, antigen- primed dendritic cell.
  • a subject kit comprises a mature dendritic cell (which may be antigen primed); and an antigen.
  • a subject kit comprises a mature, antigen-primed dendritic cell.
  • the invention provides a kit comprising PIKA and an antigen in separate fo ⁇ nulations.
  • a subject kit comprises a subject immunogenic composition in a sterile liquid (e.g., aqueous) formulation, where the formulation is sterile, and is provided in a sterile container, a sterile vial, or a sterile syringe.
  • a sterile liquid e.g., aqueous
  • a subject immunogenic composition may be microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized aerosols, pellets for implantation into the skin, or dried onto a sharp object (e.g., a needle) to be scratched into the skin.
  • a sharp object e.g., a needle
  • a subject immunogenic composition which includes an enriched immune cell population (e.g., isolated mature dendritic cells, which are optionally antigen-primed) and may further be provided in combination with PIKA (e.g., in the same or different compositions), is provided in any of a variety of formulations.
  • a subject immunogenic composition may be prepared as an injectable, liquid solution, for example: aqueous or saline solution or as: a suspension, cream, emulsion, microcapsule, drops, gel, syrup or slurry.
  • the subject immunogenic substance may be delivered alone or in conjunction with a dispersion system.
  • the dispersion system is selected from the group consisting of for example: macromolecular complexes, nanocapsules, microspheres, beads and lipid based systems.
  • Lipid based systems optionally include oil-in-water emulsions, micelles, mixed micelles or liposomes.
  • RBCs red blood cells
  • Removal of RBCs may be accomplished by standard methods known to those skilled in the art.
  • Kits for isolating DCs are commercially available and can be used to obtain DCs.
  • StemSepTM is an example of a DC isolation kit.
  • an "effective amount" of a subject immunogenic composition is an amount that provides for an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or an at least about 1000-fold, or greater, increase in the number of antigen-primed APCs, compared to the number of antigen- primed APCs in the starting cell population.
  • an "effective amount" of a subject immunogenic composition is an amount that provides for an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or an at least about 1000-fold, or greater, increase in the number of antigen-primed B cells, compared to the number of antigen- primed B cells in the starting cell population.
  • isolated antigen-primed DCs can be at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, pure, e.g., free of cells other than DCs, and/or at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about S0%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% free of non-antigen-primed DCs.
  • compositions required will vary from subject to subject, depending on the species, age, weight, and general conditions of the subject, the severity of the disease, infection, or condition that is being treated or prevented, the particular compound used, its mode administration, and the like. An appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. Following an initial administration, subjects may receive one or several booster immunizations adequately spaced.
  • serial doses of a subject immunogenic composition are administered.
  • the second dose of a subject immunogenic composition is administered to the individual after the individual has been immunologically primed by exposure to the first dose.
  • the booster may be administered days, weeks or months after the initial immunization, depending upon the patient's response and condition.
  • the booster dose is administered from about 2 days to about 12 months after the initial dose, e.g., from about 2 days to about 7 days, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 8 weeks, from about 8 weeks to about 6 months, or from about 6 months to about 12 months after the initial dose.
  • the present invention further contemplates the use of a third, fourth, fifth, sixth or subsequent booster immunization, using, e.g., a third, fourth, fifth, sixth, or subsequent dose.
  • the invention provides for a method for eliciting and/or enhancing an immune response to an immunogenic compound in an individual (a "recipient” or a "recipient individual”), comprising administering to the individual an enriched cell population and/or factors produced by an enriched cell population, where the enriched cell population and/or factors are produced by a subject in vitro method.
  • a method for increasing an immune response to an antigen in an individual comprising administering an immunogenic composition comprising an enriched immune cell population enriched in a selected immune cell type to the individual, where the enriched cell population is generated by a method comprising contacting a starting cell population obtained from a donor individual with PIKA composition.
  • Subjects suitable for treatment with a subject method for treating cancer include individuals who have been infected with a carcinogenic substance, individuals who are susceptible to cancer but who have not yet been diagnosed with cancer; and individuals who are at risk of contracting cancer, but who have not yet been diagnosed with cancer. Suitable subjects include infants, children, adolescents, and adults.
  • Subjects suitable for treatment with the formulations and methods of the instant invention for treating allergy include any individual who has been diagnosed as having an allergy.
  • Subjects amenable to treatment using the methods and agents described herein include individuals who are known to have allergic hypersensitivity to one or more allergens.
  • Subjects amenable to treatment include those who have any of the above-mentioned allergic disorders.
  • Also amenable to treatment are subjects that are at risk of having an allergic reaction to one or more allergens.
  • individuals who failed treatment with one or more standard therapies for treating an allergic disorder are also referred to treat the allergic disorder.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.
  • Example 1 - PIKA Induces an Innate Immune Response In-vivo
  • TNF- ⁇ reached the peak level after 1 hour, IL-6, IL-12p40 and IFN- ⁇ after 2 hours.
  • the cells were harvested and washed twice with PBS, and the Fc receptors were blocked with anti-CD 16/CD32 monoclonal antibodies at 4 0 C for 20 minutes and stained with fluorescence labeled antibodies. All incubations were conducted on ice and washed twice with cold staining buffer after each step (PBS containing 0.1% BSA and 0.05% NaN 3 ). The cells were collected on a FACSCalibur (BD Bioscience, Mountain View, CA, USA), and the data was analyzed using CellQuest software (BD Bioscience, Mountain View, CA, USA).

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Abstract

The present invention provides an immunogenic composition comprising polyinosinic acid - polycytidylic acid and methods of use in eliciting an immune response in-vivo. The present invention also provides an immunogenic composition comprising polyinosinic acid - polycytidylic acid and methods of use in eliciting an immune response in-vitro, e.g., activation and maturation of natural killer cells, antigen presenting cells, and T-cells. The present invention further contemplates methods of use of such activated and matured natural killer cells, antigen presenting cells and T-cells, particularly in eliciting an immune response when administered to a host.

Description

COMPOSITIONS AND METHODS FOR STIMULATING AN IMMUNE RESPONSE IN-VΓVO AND
IN-VITRO
Background
The immune system has evolved over time as a result of exposure to a wide variety of pathogens that occur in the environment. On exposure to pathogens the human immune system will trigger a chain of cellular responses that result in both an innate and an adaptive immune response.
Recent investigations have demonstrated that toll-like receptors "TLRs" are the critical links between the innate and the adaptive immunity (Ref. 1, 2). The recognition of specific pathogen associated molecular patterns (PAMPs) is mediated primarily by members of the TLR family. Stimulation through these receptors results in quantitative and qualitative changes in antigen presentation and cellular activation, thereby linking innate and adaptive immunity. The secretion of cytokines and regulation of the expression of co-stimulatory molecules induced by the innate response shape the magnitude and quality of the adaptive immune response.
In recent years, there has been interest in developing in-vitro techniques that result in the activation, proliferation and /or maturation of natural killer cells, antigen presenting cells and T-cells and the production of associated co-stimulatory molecules, cytokines chemokines, antibodies, and the like. Medical application of such in vzϊro-produced cells and co-stimulatory molecules includes the administration to a host of compositions resulting from the in-vitro simulation thereby complementing the host's own immune response.
There remains a clinical need for methods of inducing such immune activity in-vitro that is reliable, efficient and overcomes the drawbacks of the current methods. Literature
1. Jiang ZH, Koganty RR. Synthetic vaccines: the role of adjuvants in immune targeting. Curr Med Chem. 2003; 10: 1423 -1439.
2. Majewska M, Szczeparήk M. The role of Toll-like receptors (TLR) in innate and adaptive immune responses and then' function in immune response regulation. Postepy Hig Med Dosw. 2006;60: 52-63.
3. Datta et al. (2003) J. Immunol 170:4102-4110.
4. WO 2006/131023
Summary of the Invention hi general, the present invention relates to novel immunogenic compositions comprising a polyinosinic acid — polycytidylic acid composition and methods of use in eliciting an immune response in a host. hi particular, there is provided an immunogenic composition comprising a population of polyriboinosinic-polyribocytidylic acid (PIC) molecules, at least one antibiotic, and at least one positive ion.
According to another aspect, there is also provided an immunogenic composition comprising: a) a population of polyriboinosinic-polyribocytidylic acid (PIC) molecules, at least one antibiotic, and at least one positive ion; and b) at least one enriched immune cell population.
The enriched immune cell population may comprise mature dendritic cells. The immunogenic composition comprising the enriched immune cell population may further comprise at least one co-stimulatory molecule. The co-stimulatory molecule may be a cytokine, for example at least one cehmokine.
The immunogenic composition according to any aspect of the invention may further comprise at least one antigen. The immunogenic composition according to any aspect of the invention may be for use in medicine. In particular, the immunogenic composition may be for use in inducing activation of a dendritic cell (DC) in an individual. The immunogenic composition may also be for use in increasing an immune response to an antigen in an individual.
The invention further contemplates the use of novel immunogenic compositions comprising a polyinosinic acid - polycytidylic acid composition alone or with at least one antigen and methods of use in eliciting an immune response in-vitro and/or in vivo. According to another aspect, there is provided the use of a population of polyriboinosinic-polyribocytidylic acid (PIC) molecules, at least one antibiotic, and at least one positive ion, in the preparation of an immunogenic composition for inducing activation of a dendritic cell (DC) in an individual.
According to another aspect, there is provided the use of an immune cell population enriched in a selected immune cell type, for increasing an immune response to at least one antigen in an individual wherein the enriched immune cell population is prepared by contacting a starting cell population with a population of polyriboinosinic- polyribocytidylic acid (PIC) molecules, at least one antibiotic and at least one positive ion,
The invention further contemplates the use of cells and/or substances resulting from an immune response in-vitro, for example, immune cells, co-stimulatory molecules, cytokines and/or chemokines and/or antibodies, and the like, including the use of such cells and/or substances, for example, to elicit, induce, enhance and/or potentiate an immune response in a host. In addition, the invention further contemplates administration of PπCA in combination with cells and/or substances resulting from an immune response in-vitro.
According to another aspect, the present invention provides a method of inducing activation of a dendritic cell (DC) in an individual, the method comprising: administering to the individual an effective amount of an immunogenic composition, wherein the immunogenic composition comprises a population of polyriboinosinic-polyribocytidylic acid (PIC) molecules, at least one antibiotic, and at least one positive ion.
According to another aspect, there is provided a method of increasing an immune response to an antigen in an individual, the method comprising: administering an immunogenic composition comprising an immune cell population enriched in a selected immune cell type to the individual, wherein the enriched immune cell population is generated by a method comprising contacting a starting cell population with an immunogenic composition in vitro, wherein the immunogenic composition comprises a population of polyriboinosinic-polyribocytidylic acid (PIC) molecules, at least one antibiotic, and at least one positive ion, wherein said administering of the enriched immune cell population increases the immune response to the antigen.
In the method of the invention, the immunogenic composition may further comprise at least one antigen, and wherein the DC is primed with the antigen. The antigen may be (but not limited to) an autoantigen, a tumor-associated antigen, an allergen, and/or an antigen associate with a microbial pathogen.
The PIC molecules according to any aspect of the invention may have a molecular weight in a range of from about 66,000 to about 1,200,000 Dalton.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 — Titers of IL-6 observed in murine blood serum post in-vivo stimulation with
PIKA
Fig. 2 - Titers of TNF-alpha observed in murine blood serum post in-vivo stimulation with PIKA
Fig. 3 - Titers of INF-gamma observed in murine blood serum post in-vivo stimulation with PIKA
Fig. 4 - Titers of IL-12p40 observed in murine blood serum post in-vivo stimulation with
PIKA Fig.5 - Production of CD25 observed post in-vitro stimulation of murine natural killer cells with PIKA
Fig. 6 - Production of CD25 observed post in-vitro stimulation of murine B-cells with
PΠCA
Fig. 7 - Production of CDSO observed post in-vitro stimulation of bone marrow derived dendritic cells with PIKA
Fig. 8 - Production of CD86 observed post in-vitro stimulation of bone marrow derived dendritic cells with PIKA
Fig. 9 — Production of CD40 observed post in-vitro stimulation of bone marrow derived dendritic cells with PIKA
Fig. 10 - Production of IL-12p40 observed post in-vitro stimulation of bone marrow derived dendritic cells with PIKA
Fig. 11 - Production of IL12p70 observed post in-vitro stimulation of bone marrow derived dendritic cells with PIKA
Fig. 12 - Production of IL-6 observed post in-vitro stimulation of bone marrow derived dendritic cells with PtKA
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION
The present invention may be understood more readily by reference to the following detailed description of certain embodiments of the invention and the Examples included herein.
Throughout this application, where publications are referenced, the disclosures of these publications are hereby incoiporated by reference, in their entireties, into this application in order to describe more fully the state of art to which this invention pertains.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the puipose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an immunogenic composition" includes a plurality of such compositions and reference to "the antigen" includes reference to one or more antigens and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.
DEFINITIONS OF TERMS
Prior to setting forth details of the present invention it may be useful to an understanding thereof to set forth definitions of several terms that are used herein.
The term "adjuvant," as used herein, refers to any substance or mixture of substances that increases or diversifies the immune response of a host to an antigenic compound.
The term "PICKCa" generally refers to a composition of poly LC, kanamycin and calcium irrespective of particular physical and immunogenic properties. "PIKA" refers to a composition of the invention comprising poly I:C, an antibiotic (e.g., kanamycin), and a positive ion (e.g., calcium), where the PIKA is characterized by specific physical characteristics (e.g., molecular weight, size, and the like) such that upon administration, PIKA exhibits distinct immunogenic characteristics including for example, greater potency (e.g., stimulates an enhanced immune response) and/or reduced adverse side effects (e.g., reduced toxicity) that differentiate PIKA from other forms of PICKCa.
The term "Poly I:C" or "PIC" refers to a composition comprising polyriboinosinic and polyribocytidylic nucleic acids, which may also be referred to as polyinosinic acid- polycytidylic acid or polyinosinic acid-polycytidilic, respectively.
"PIC-containing molecule" or "PIC-containing compound" refers to, without limitation, PIC, which may be optionally complexed or otherwise combined with at least one or both of an antibiotic (e.g., kanamycin) and a positive ion (e.g., calcium) present in a composition comprising the PIC-containing molecule. In one embodiment, the PIC- containing molecule does not include poly-L-lysine or a derivative thereof in the complex.
"Heterogeneous" as used herein in the context of compositions of the invention indicates that components of the composition, e.g., the PIC-containing molecules, are not uniform with respect to a physical characteristic of molecular weight, size, or both. Where a composition is described as heterogeneous for a given physical characteristic, and is further described by a range of values for that physical characteristic, the composition is said to be composed substantially of molecules characterized by molecules having a physical characteristic that is distributed within and across the recited range. While the composition may not contain a molecule representative of every physical characteristic value within the upper and lower limits of a recited range, the composition will generally include at least one molecule having the physical characteristic of the upper value and of the lower value. The composition in certain embodiments may include molecules outside the stated range of physical characteristics used to describe the composition. The molecules that are present in the composition outside the prescribed range do not materially affect the basic and novel characteristics of the composition.
The term "individual," used interchangeably herein with "host," "subject," and "animal," includes humans, non-human primates, all domestic livestock and pets, wild mammals and fowl, including, without limitation, cattle, horses, cows, swine, sheep, goats, dogs, cats, rabbits, deer, mink, chickens, ducks, geese, turkeys, game hens, and the like.
The term "antibody" includes polyclonal and monoclonal antibodies, as well as antigenic compound binding fragments of such antibodies including Fab, F(ab')2, Fd, Fv fragments, and single chain derivatives of the same. In addition, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."
As used herein, the term "antigenic compound" refers to any substance that can be recognized by the immune system (e.g., bound by an antibody or processed so as to elicit a cellular immune response) under appropriate conditions.
An "antigen" as used herein includes but is not limited to cells; cell extracts; proteins; lipoproteins; glycoproteins; nucleoproteins; polypeptides; peptides; polysaccharides; polysaccharide conjugates; peptide mimics of polysaccharides; lipids; glycolipids; carbohydrates; viruses; viral extracts; bacteria; bacterial extracts; fungi; fungal extracts; multicellular organisms such as parasites; and allergens. Antigens may be exogenous (e.g., from a source other than the individual to whom the antigen is administered, e.g., from a different species) or endogenous (e.g., originating from within the host, e.g., a diseased element of body, a cancer antigen, a virus infected cell producing antigen, and the like). Antigens may be native (e.g., naturally-occurring); synthetic; or recombinant. Antigens include crude extracts; whole cells; and purified antigens, where "purified" indicates that the antigen is in a form that is enriched relative to the environment in which the antigen normally occurs and/or relative to the crude extract, for example, a cultured form of the antigen.
An "immunogenic composition" as used here in refers to a substance that elicits an immune response when administered to a host.
The term "polypeptide," "peptide," "oligopeptide," and "protein," are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
An "effective amount of an antigenic compound" refers to an amount of antigenic compound which, alone or in optional combination with an adjuvant, will cause the subject to produce a specific immunological response to the antigenic compound.
The term "immune response" refers to any response to an antigenic or immunogenic compound by the immune system of a vertebrate subject. Exemplary immune responses include, but are not limited to local and systemic cellular as well as humoral immunity, such as cytotoxic T lymphocytes (CTL) responses, including antigen-specific induction of CDS+ CTLs, helper T-cell responses including T-cell proliferative responses and cytokine release, and B-cell responses including antibody response.
The term "eliciting an immune response" is used herein generally to encompass induction and/or potentiation of an immune response.
The term "inducing an immune response" refers to an immune response that is stimulated, initiated, or induced.
The term "potentiating an immune response" refers to a pre-existing immune response that is improved, furthered, supplemented, amplified, enhanced, increased or prolonged. The expression "enhanced immune response" or similar means that the immune response is elevated, improved or enhanced to the benefit of the host relative to the prior immune response status, for example, before the administration of an immunogenic composition of the invention.
The terms "humoral immunity" and "humoral immune response" refer to the form of immunity in which antibody molecules are produced in response to antigenic stimulation.
The teπns "cell-mediated immunity" and "cell-mediated immune response" are meant to refer to the immunological defense provided by lymphocytes, such as that defense provided by T cell lymphocytes when they come into close proximity to their victim cells. A cell-mediated immune response normally includes lymphocyte proliferation. When "lymphocyte proliferation" is measured, the ability of lymphocytes to proliferate in response to a specific antigen is measured. Lymphocyte proliferation is meant to refer to B cell, T-helper cell or cytotoxic T-lymphocyte (CTL) cell proliferation.
The term "immunogenic amount" refers to an amount of antigenic compound sufficient to stimulate an immune response.
The term "immunopotentiating amount" refers to the amount of a substance needed to effect an increase in antibody titer and/or cell-mediated immunity when administered to a host.
The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a subject, e.g., a mammalian subject, e.g., a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, e.g., arresting its development; or relieving the disease symptom, i.e., causing regression of the disease or symptom (c) reduction of a level of a product produced by the infectious agent of a disease (e.g., a toxin, an antigen, and the like); and (d) reducing an undesired physiological response to the infectious agent of a disease (e.g., fever, tissue edema, and the like).
As used herein, the term "mixing" includes any method to combine the components of the composition; such methods include, but are not limited to, blending, dispensing, dissolving, emulsifying, coagulating, suspending, or otherwise physically combining the components of the composition.
A "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naρhthalenesulfonic acid, 4- toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3- hydroxy-2-ene-l-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, and the like. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically/physiologically acceptable diluent, carrier or vehicle.
EXEMPLARY EMBOPΓMENTS OF THE INVENTION
The present invention is directed to immunogenic compositions and methods useful for the eliciting, inducing, enhancing and/or potentiating an immune response, which may be an innate or adaptive immune response. The innate immune response may be in a human, a non-human animal, or cell culture. The adaptive immune response may be humoral and/or cell-mediated, in a human, a non-human animal, or cell culture.
In some embodiments, a subject immunogenic composition comprises an enriched immune cell population, where the enriched immune cell population is generated in vitro, as described in more detail below, hi other embodiments, a subject immunogenic composition comprises an enriched immune cell population, and PIKA, and dose not include an antigen (e.g., a polypeptide antigen). In other embodiments, a subject immunogenic composition comprises an enriched immune cell population, PIKA, and one or more co-stimulatory molecules (e.g., a co-stimulatory molecule produced by a cell in the enriched immune cell population, e.g., a cytokine, a chemokine, an antibody, etc.). hi other embodiments, a subject immunogenic composition comprises an enriched immune cell population, PIKA, and an antigen. In other embodiments, a subject immunogenic composition comprises an enriched immune cell population, PIKA, an antigen, and one or more co-stimulatory molecules (e.g., a co-stimulatory molecule produced by a cell in the enriched immune cell population).
One aspect the present invention provides for a substance comprising polyinosinic acid - polycytidylic acid that acts as an immune regulator when administered to a host.
More specifically, one aspect the present invention provides for a substance comprising polyinosinic acid - polycytidylic acid that acts as an immune regulator of the host's innate immune response.
In a related embodiment of the invention the substance comprising polyinosinic acid - polycytidylic acid may act as an anti-viral, anti-bacterial, anti-fimgal, anti-cancer, anti- allergy for the prevention and/or treatment of disease when administered to a host.
In an embodiment of particular interest the invention provides for a substance comprising polyinosinic acid - polycytidylic acid that acts as an immune regulator in-vitro.
In a related embodiment of the invention the substance comprising polyinosinic acid - polycytidylic acid may facilitate an immune response in-vitro, for example, the activation, proliferation and/or maturation of natural killer cells and/or antigen presenting cells including but not limited to, dendritic cells and B cells.
A related embodiment of the invention contemplates the use of substances resulting from such an in-vitro stimulation, for example, immune cells, co-stimulatory molecules, cytokines and/or chemokines and/or antibodies and the like, including the use of such substances as an immunotherapy, for example, to elicit, induce, enhance and/or potentiate an immune response in a host.
In a related embodiment the invention provides for a substance comprising polyinosinic acid - polycytidylic acid together with an antigen or vaccine where the source of the antigen is a human antigen, a animal antigen, a plant antigen, one or more agents from infectious agents from any vims, bacteria including mycobacterium, fungus or parasite, cancer antigen, allergenic agents, autoantigens and the like.
In a related embodiment the invention provides for a substance comprising polyinosinic acid - polycytidylic acid together with an antigen or antigens that acts as an immune regulator in-vitro. In a related embodiment of the invention the substance comprising polyinosinic acid - polycytidylic acid together with an antigen that may facilitate the activation of an immune response in-vitro including the activation, proliferation and/or maturation of antigen presenting cells, including but not limited to, dendritic cells and B cells.
In a related embodiment of the invention the substance comprising polyinosinic acid - polycytidylic acid together with an antigen that may facilitate the activation of an immune response in-vitro including the activation, proliferation and/or maturation of antigen presenting cells, including but not limited to, dendritic cells and B cells together with associated co-stimulatory molecules, cytokines, chemokines, antibodies and the like, which may subsequently be used as an immunotherapy in a host.
In a related embodiment of the invention the substance comprising polyinosinic acid - polycytidylic acid together with an antigen that may facilitate the activation of an immune response in-vitro including the activation, proliferation and/or maturation of antigen presenting cells, including but not limited to, dendritic cells and B cells which may subsequently be used to activate, proliferate and/or mature T-cells.
In a related embodiment of the invention the substance comprising polyinosinic acid - polycytidylic acid together with an antigen that may facilitate the activation of an immune response in-vitro including the activation, proliferation and/or maturation of antigen presenting cells, including but not limited to, dendritic cells and B cells which may subsequently be used to activate, proliferate and/or mature T-cells together with associated co-stimulatory molecules, cytokines, chemokines, antibodies, and the like, that may then be used as an immunotherapy in a host.
PIKA Composition
The PπCA composition, is generally composed of polyinosinic acid, polycytidylic acid, an antibiotic (e.g., kanamycin), and a divalent cation (e.g., calcium). It will be understood that reference to PIKA herein is exemplary of such PIC-containing compositions. PIC-containing compositions of interest can be manufactured using methods available in the art. The PIC-containing composition can be manufactured through any appropriate process. For example the polynucleotide composition can be manufactured by mixing of polyinosinic acid, polycytidylic acid, an antibiotic and the source of a positive ion in a sodium chloride/phosphate buffer solution that has a pH between pH6 and pH8. The polyinosinic acid and polycytidylic acid are generally provided at a concentration of 0.1 mg/ml to 10 mg/ml, e.g., from about 0.1 mg/ml to about 0.5 mg/ml, from about 0.5 mg/ml to about 5 mg/ml, from about 5 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 2.5 mg/ml. The hyperchromicity value should be greater than 10%, e.g., greater than 10%, greater than 15%, greater than 20%, or greater than 50%. The preparation of the PIC and the combination with the antibiotic (e.g., kanamycin) and the positive ion (e.g., calcium) is generally conducted under quality standards consistent with international Good Manufacturing Process.
In certain embodiments of the present invention, the antibiotic component of the subject composition of the invention is kanamycin. Where the antibiotic is kanamycin, in some embodiments, the kanamycin in the subject composition of the invention is used together with or substituted by one or more antibiotics selected from the group including tobramycin, anthracyclines, butirosin sulfate, gentamicins, hygromycin, amikacin, dibekacin, nebramycin, metrzamide, neomycin, puromycin, streptomycin and streptozocin. The antibiotic (e.g., Kanamycin or the like) in the subject composition of the invention is generally provided at a concentration of from about 10 units/ml to 100,000 units/ml, from about 100 units/ml to 10,000 units/ml, or from about 500 units/ml to 5,000 units/ml.
In certain embodiments of the present invention, the subject composition of the invention further comprises a positive ion (cation), usually a divalent cation, normally a cation of an alkali metal. The positive ion is generally provided in the composition of the invention as a source of positive ions such as a salt or complex, e.g., an organic or inorganic salt or complex, usually an inorganic salt or organic complex. Exemplary positive ions include, but are not necessarily limited to, calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, or zinc.
The positive ion can be provided in the form of any suitable salt or organic complex, including, but not necessarily limited to chloride, fluoride, hydroxide, phosphate, or sulfate salts. For example, where the positive ion is calcium, the ion can be in the form of calcium carbonate, calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphates, or calcium sulfate.
The positive ion (e.g. calcium) can be provided in the composition of the invention at a concentration in the range of from about 10 umol to about 10 mmol/ml, e.g., from about 10 umol/ml to about 50 umol/ml, from about 50 umol/ml to about 5 mmol/ml, or from about 100 umol to 1 mmol/ml. The term "umol" is used throughout to refer to micromole.
Where the positive ion in the subject composition of the invention is calcium, it can be in combination with or substituted by other positive ions, including cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, and zinc, wherein the ions can be hi the form of inorganic salts or organic complexes.
The resulting composition is a PIC-containing composition that further contains an antibiotic and a positive ion. Li a particular embodiment, where the antibiotic is kanamycin and the ion is calcium the product may be described as PICKCa. hi a related embodiment the PICKCa composition may contain molecules without restriction of different physical characteristics.
In a particular exemplary embodiments, the PIC-containing composition is PIKA. PIKA may be produced in a variety of ways, with production from PICKCa being of particular interest. PIKA can be produced from PICKCa through additional manufacturing processes that involve the isolation and/or concentration of molecules of a defined molecular size and/or weight. The separation and concentration of polynucleotide molecules of particular characteristics using filtration} chromatography, thermal treatment, centrifugal separation, electrophoresis, and similar methods that are standard processes and are known to those skilled in the art.
In embodiments of particular interest, the invention features a composition generally referred to as PIKA comprising a polyriboinosinic-polyribocytidylic acid (PIC), an antibiotic (e.g., kanamycin), and a positively charged ion (e.g., a calcium ion), wherein the composition contains molecules of the subject composition of the invention heterogeneous for molecular weight having a molecular weight of from about 66,000 to 1,200,000 Daltons. That is, the subject composition of the invention comprises molecules with a weight distribution in the range of from about 66,000 to 1,200,000 Daltons.
In related embodiments, the PIKA composition molecules in the composition are heterogeneous, that is the weight of the molecules are distributed within a range of molecular weight, where the molecular weight is from about 300,000 to 1,200,000 Daltons, or from about 66,000 to 660,000 Daltons, or from about 300,000 to 660,000 Daltons, or from about 300,000 to 2,000,000 Daltons, or from about 66,000 Daltons to about 100,000 Daltons, 100,000 to 200,000 Daltons, from about 300,000 Daltons to about 4,000,000 Daltons, or from about 500,000 Daltons to 1,000,000 Daltons, or from about 1,000,000 Daltons to 1,500,000 Daltons, or from about 1,500,000 Daltons to 2,000,000 Daltons, or from about 2,000,000 Daltons to 2,500,000 Daltons, or from about 2,500,000 Daltons to 3,000,000 Daltons, or from about 3,000,000 Daltons to 3,500,000 Daltons, or from about 3,500,000 Daltons to 4,000,000 Daltons, or from about 4,000,000 Daltons to 4,500,000 Daltons, or from about 4,500,000 Daltons to 5,000,000 Daltons.
In related embodiments, the PDCA composition molecules in the composition have an average molecular weight equal or equal to or greater than 150,000 Daltons, or equal to or greater than 250,000 Daltons, or equal to or greater than 350,000 Daltons, or equal to or greater than 500,000 Daltons, or equal to or greater than 650,000 Daltons, or equal to or greater than 750,000 Daltons, or equal to or greater than 1,000,000 Daltons, or equal to or greater than 1,200,000 Daltons, or equal to or greater than 1,500,000 Daltons, or equal to or greater than 2,000,000 Daltons.
In embodiments of particular interest, the invention features a composition generally referred to as PIKA comprising a polyriboinosinic-polyribocytidylic acid (PIC), an antibiotic, and a positive ion wherein the composition contains molecules of the composition are heterogeneous, that is the size of the molecules are distributed within a range of molecular size, for molecular size having a sediment co-efficient Svedbergs (S) of from about 6.43S to 24.03S.
In related embodiments, the PIKA composition molecules in the composition are heterogeneous, that is the size of the molecules are distributed within a range of molecular size, where the molecular size is from about 12.8S to 24.03S, or from about 3S to 12S or from about 6.43 to 18.31S, or from about 12.8 to 18.31S, or from about 12.8S to 30.31S, or from about 12.8S to 41.54S, or from about 13.5S, to 18.31S, or from about 13.5S to 24.03S, or from about 16.14 to 22.12S, or from about 22.12S to 26.6S, or from about 26.6S to 30.31S, or from about 30.31S to 33.55S, or from about 33.55S to 36.45S, or from about 36.45S to 39.1S, or from about 39.1S to 41.54S, or from about 41.54S to 43.83S, or from about 43.83S to 45.95S.
In further related embodiments, the PEKA composition has an average sedimentation coefficient (Svedbergs) greater than 9, or greater than 12, or greater than 13.5, or greater than 15, or greater than 16, or greater than 17, or greater than 18, or greater than 19, or greater than 20, or greater than 21, or greater than 22 or greater than 25, or greater than 30.
In some embodiments, a subject immunogenic composition comprises an enriched immune cell population, where the enriched immune cell population is generated in vitro, as described in more detail below. In other embodiments, a subject immunogenic composition comprises an enriched immune cell population, and PEBCA, and dose not include an antigen (e.g., a polypeptide antigen). In other embodiments, a subject immunogenic composition comprises an enriched immune cell population, PDvA, and one or more co-stimulatory molecules (e.g., a co-stimulatory molecule produced by a cell in the enriched immune cell population, e.g., a cytokine, a chemokine, an antibody, etc.). In other embodiments, a subject immunogenic composition comprises an enriched immune cell population, PIKA, and an antigen. In other embodiments, a subject immunogenic composition comprises an enriched immune cell population, PIKA, an antigen, and one or more co-stimulatory molecules (e.g., a co-stimulatory molecule produced by a cell in the enriched immune cell population).
Immunogenic Properties dsRNA is a universal viral molecular pattern and activates an immune response through recognition by TLR3. TLRs are a family of pattern-recognition receptors that recognize structural components shared by many bacteria, viruses and fungi.
Administration of PDCA in-vivo has been shown to induce the production of molecules that are characteristic of an innate immune response, (see Example 1 and Figures 1, 2, 3 and 4).
Natural killer "NK" cells are major players in the antiviral immune response. NK cells are able to recognize virally infected cells and tumor cells and are able to induce cell apoptosis prior to the development of antigen specific cyctotoxic T-cells, thereby providing an rapid response to infection.
Example 2 demonstrates that PIKA markedly induces the expression of CD25 (Figure 5) and indicative of the proliferation of NK cells in vitro.
B cells are lymphocytes that are instrumental in the humoral immune response. B cells undergo a maturing process as a result of which various types of B cells are produced including plasma B cells that on exposure to an antigen will produce and secrete large amounts of antibodies and memory B cells post exposure to an antigen will remain in the body and are able to respond quickly to a second exposure to the same antigen. Example 3 demonstrates that PIKA markedly induces the expression of CD25 (Figure 6) and indicative of the proliferation of B cells in vitro.
Example 3 demonstrates that PQCA induced the maturation of dendritic cells (bone marrow derived dendritic cells "BMDCs") as assessed by up-regulation of co-stimulatory molecules CD80, CD86 and CD40, (Figures 7, 8 and 9) and significantly induced the production of cytokines including IL-12p40, IL-12p70 and IL-6 (Figures 10, 11 and 12) in a dose-dependent manner by BMDCs.
The use of PIKA in conjunction with an antigen or antigens may therefore be used to produce activated dendritic cells which are of interest clinically as a therapy administered alone or in combination with other immunostimulatory substances to complement the patient's own immune response.
Further activated dendritic cells may be used to activate T-cells ex- vivo. Activated T- cells e.g. cytotoxic T-cells (e.g., CDS+ T cells) and/or helper T-cells (e.g., CD4+ T cells) may be administered to patients alone, or in combination with other immunostimulatory substances, for the prevention or treatment of chronic or infectious diseases.
Example 5 demonstrates that PKA is able to induce the maturation of dendritic cells and natural killer cells derived from human blood samples.
Antigens
In some embodiments, as noted above, a subject immunogenic composition comprises PIKA and one or more antigens. For example, in some embodiments, a subject immunogenic composition comprises an enriched immune cell population, PIKA, and one or more antigens.
Suitable antigens include, but are not limited to, viral antigens, bacterial antigens, parasite antigens, tumor-associated antigens (also referred to as tumor-specific antigens), allergens, autoantigens, and the like. In some embodiments, the antigen is an antigenic protein derived from or associated with an autoantigen; an allergen; a tumor-associated antigen; a pathogenic organism that infects a mammalian or avian host, e.g., a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or any other pathogenic organism that infects a mammalian or avian host.
Viral antigens
Suitable viral antigens include those derived from known causative agents responsible for diseases, including, but not limited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), and C (e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBank Accession No. M24444), hantavirus, and HIV antigens (e.g., GenBank Accession No. Ul 8552).
Bacteria! antigens, parasite antigens
Suitable bacterial and parasitic antigens include those derived from known causative agents responsible for diseases including, but not limited to, diphtheria, pertussis (e.g., GenBank Accession No. M35274), tetanus (e.g., GenBank Accession No. M64353), tuberculosis, bacterial and fungal pneumonias (e.g., Haemophilus influenzae, Pneumocystis carinii, etc.), cholera, typhoid, plague, shigellosis, salmonellosis (e.g., GenBank Accession No. L03833), Legionnaire's Disease, Lyme disease (e.g., GenBank Accession No. U59487), malaria (e.g., GenBank Accession No. X53S32), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. L08198), trypanosomiasis, leshmaniasis, giardiasis (e.g., GenBank Accession No. M33641), amoebiasis, filariasis (e.g., GenBank Accession No. J03266), borreliosis, and trichinosis.
In some embodiments, the antigen is a polypeptide or peptide epitope associated with an intracellular pathogen. Polypeptides and peptide epitopes associated with intracellular pathogens are known in the art and include, but are not limited to, antigens associated with human immunodeficiency virus (HIV), e.g., HIV gρl20, or an antigenic fragment thereof; cytomegalovirus antigens; Mycobacterium antigens (e.g., Mycobacterium avium, Mycobacterium tuberculosis, and the like); Pneumocystis carinii (PCP) antigens; malarial antigens, including, but not limited to, antigens associated with Plasmodium falciparum or any other malarial species, such as 41-3, AMA-I, CSP, PFEMP-I, GBP-130, MSP-I, PFS- 16, SERP, etc.; fungal antigens; yeast antigens (e.g., an antigen of a Candida spp.); toxoplasma antigens, including, but not limited to, antigens associated with Toxoplasma gondii, Toxoplasma encephalitis, or any other Toxoplasma species; Epstein-Barr virus (EBV) antigens; and the like.
Tumor-associated antigens
Tumor-specific antigens include, but are not limited to, any of the various MAGEs (Melanoma-Associated Antigen E), including MAGE 1 (e.g., GenBank Accession No. M77481), MAGE 2 (e.g., GenBank Accession No. U03735), MAGE 3, MAGE 4, etc.; any of the various tyrosinases; mutant ras; mutant p53 (e.g., GenBank Accession No. X54156 and AA494311); and ρ97 melanoma antigen (e.g., GenBank Accession No. M12154). Other tumor-specific antigens include the Ras peptide and p53 peptide associated with advanced cancers, the HPV 16/18 and E6/E7 antigens associated with cervical cancers, MUCl-KLH antigen associated with breast carcinoma (e.g., GenBank Accession No. J03651), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession No. X98311), gplOO (e.g., GenBank Accession No. S73003) or MARTl antigens associated with melanoma, and the PSA antigen associated with prostate cancer (e.g., GenBank Accession No. X14810). The p53 gene sequence is known (See e.g., Harris et al. (1986) MoI. Cell. Biol., 6:4650-4656) and is deposited with GenBank under Accession No. M 14694. Cancers include, but are not limited to, carcinomas, lymphomas, leukemias, and sarcomas.
Allergens
Allergens include, but are not limited to, pollen, a mold spore allergen, a plant allergen, a non-human animal allergen, a human allergen, an insect allergen, a bacterial allergen, a viral allergen, a food allergen, an industrial chemical allergen, an aeroallergen (e.g., airborne pollen, airborne fungal spores, and the like), and a drug allergen. In the context of this invention, the term "allergen" refers to an antigen that can trigger an allergic response which is mediated by IgE antibody. The method and compositions of this invention extend to a broad class of such allergens and fragments of allergens or haptens acting as allergens. These can include all the specific allergens that can cause an IgE-mediated response in allergic subjects. This invention is therefore useful for the treatment of allergic diseases in humans, other primates, and mammalian subjects, such as dogs, cats, and horses. Allergic diseases that are amenable to treatment using the compositions and methods of the instant invention include, but are not limited to, allergic diseases due to IgE; allergic rhinitis (hay fever); allergic asthma; atopic dermatitis; anaphylaxis; food allergy; drug allergy; urticaria (hives); angioedema; and allergic conjunctivitis.
Allergens include, but are not limited to, environmental aeroallergens; weed pollen allergens; grass pollen allergens; tree pollen allergens; house dust mite allergens; storage mite allergens; mold spore allergens; animal allergens (examples by species - cat, dog, guinea pig, hamster, gerbil, rat, mouse); animal allergens (examples by source - epithelial, salivary, urinary proteins); food allergens, including but not limited to the following common examples: crustaceans, nuts, such as peanuts, and citrus fruits; insect allergens (other than mites listed above); venoms, including, but not limited to, hymenoptera, yellow jacket, honey bee, wasp, hornet, and fire ant venoms; other environmental insect allergens from cockroaches, fleas, mosquitoes, etc.; bacteria such as streptococcal antigens; parasites such as ascaris antigen; viral antigens; drug allergens, such as antibiotics, e.g., penicillins and related compounds; other antibiotics; whole proteins such as hormones (e.g., insulin), enzymes (e.g., streptokinase); all drugs and their metabolites capable of acting as incomplete antigens or haptens; industrial chemicals and metabolites capable of acting as haptens and stimulating the immune system, including the following non-limiting examples: the acid anhydrides (such as trimellitic anhydride) and the isocyanates (such as toluene diisocyanate); occupational allergens such as flour in Baker's asthma, castor bean, coffee bean, and industrial chemicals described above.
Antigens can be synthesized chemically or enzymatically, can be produced recombinantly, can be isolated from a natural source, or a combination of the foregoing. Antigens may be isolated from natural sources using standard methods of protein purification known in the art, including, but not limited to, high performance liquid chromatography, exclusion chromatography (e.g., size exclusion chromatography), gel electrophoresis, affinity chromatography, or other purification technique. Where the antigen is a peptide, one may employ solid phase peptide synthesis techniques, where such techniques are known to those of skill in the art. See Jones, The Chemical Synthesis of Peptides (Clarendon Press, Oxford)(1994). For example, a peptide can be produced through the sequential additional of activated monomelic units to a solid phase bound growing peptide chain. Well-established recombinant DNA techniques can be employed for production of polypeptide antigens. In some embodiments, the antigen is pure, e.g., at least about S0%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%, or greater than 98%, pure.
Additional agents
In some embodiments, a subject immunogenic composition comprises, in addition to PIKA and an antigen, one or more additional agents, e.g., immunomodulatory agents, earners, and the like.
In an embodiment of particular interest, the present invention provides for an immunogenic composition and method of use, where the immunogenic composition comprises PKA, an antigen or vaccine together with another immunomodulating substance, including adjuvants, where suitable immunomodulating substances include, but are not limited to: an aluminum composition such as aluminum hydroxide; oil-in- water emulsions compositions or emulsions comprising an immunogenic substances, including Complete Freund's Adjuvant; an oil-in-water emulsion containing dried, heat- killed Mycobacterium tuberculosis organisms; Incomplete Freund's Adjuvant; emulsions including mycobacterial cell wall components; emulsions including squalene (MF-59); detoxified endotoxins, lipid A derivatives including monophosphoryl lipid A-microbial (MPL); haptens; nitrocellulose-absorbed protein; saponins including particulate immunomodulators isolated from the bark of Quillaja Saponoria for example QS21; endogenous human immunomodulators; bacterial derived adjuvants including unmethylated CpG dinucleotides; oligodeoxynucleotides (e.g., synthetic oligonucleotides) containing unmethylated CpG dinucleotides; liposomes (e.g., liposomes made of biodegradable materials such as phospholipids); biodegradable polymer microspheres (e.g., microspheres made from a variety of polymers such as polylactic-co- glycolic acid (PLGA), polyphosphazene and polyanhydrides); Interlukin-2; Bacillus Calmette Guerin; Granulocyte Monocyte-Colony Stimulating Factor; Montanide ISA-51; Keyhole limpet hemocyanin; DNA; proteins; encapsulated antigens; immune stimulating complexes (ISCOM's); cholera toxin, choleral toxin derivatives; zonula occludens toxin; escherichia coli heat-labile enterotoxin; labile toxin, labile toxin derivatives; pertussis toxin, pertussis toxin derivatives; muramyl dipeptide derivatives; seppic series of montanide adjuvants; poly-di(carboxylatophenoky)phosphazene and leishmania elongation factor.
When the subject immunogenic composition is administered in conjunction with an adjuvant, the subject composition of the invention can be administered before and/or after, and/or simultaneously with the adjuvant. For example the subject composition of the invention may be administered with the initial administration of the antigen, followed by a boost dose of vaccine comprising subject composition of the invention and/or the adjuvant. Alternatively the initial dose of vaccine administered may exclude the subject composition of the invention but an immunogenic substance comprising the subject composition of the invention is subsequently administered to the patient.
In certain embodiments the subject immunogenic composition may be administered with antibodies, cytokines, chemokines or other co- stimulatory molecules for example: an interleukin, e.g., IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-IO, IL- 12, IL- 15, and the like; a colony stimulating factor (e.g., Neupogen® (filgrastim; G-CSF); Neulasta (pegfilgrastkα); granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor); an interferon (e.g., IFN-γ, EFN-α, IFN-β, EFN-ω; IFN-τ); a chemokine (e.g., IP-IO; Mig; Groα/IL-8, RANTES; MIP-I α; MlP-lβ; MCP-I; PF-4; and the like); a tumor necrosis factor; a stem cell factor; a cytokine (e.g. TNF-alpha and the like) and the like. In some embodiments, the antibodies, cytokines, chemokines, etc. are produced in vitro using a subject method, as described in more detail below.
In a related embodiment the present invention provides for an immunogenic substance comprising a PIKA, an antigenic substance or substances, plus a suitable carrier. The caπier may be for example an oil-and-water emulsion, suspension, a lipid vehicle, aluminum salt, cochleates, ISCOMs, liposomes, live bacterial vectors, live viral vectors, microspheres, nucleic acid vaccines, polymers, polymer lings, sodium fluoride, transgenic plants, virosomes, virus like particles, and other delivery vehicles known in the art.
PIKA may be used in conjunction with a delivery complex. Where the delivery complex is a substance associated with a targeting means e.g. a molecule that results in higher affinity binding to target cell such as dendritic cell surfaces and/or increased cellular uptake by target cells. Examples of delivery complexes include but are not limited to; nucleic acid delivery acids associated with: a sterol (e.g. cholesterol), a lipid (e.g. cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by a target cell specific receptor). Preferred complexes may be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex may be cleavable under appropriate conditions within the cell.
In one embodiment of interest, the composition comprising PDCA does not include poly- L-lysine or a derivative thereof. Kits
In certain embodiments, the invention provides a kit comprising a subject immunogenic composition. In some embodiments, the invention provides a kit comprising a composition comprising PIKA; and a delivery system for in vivo delivery to an individual.
In certain embodiments, the invention provides a kit comprising a subject immunogenic composition, which includes an enriched immune cell population (e.g., isolated mature dendritic cells, which are optionally antigen-primed) and may further be provided in combination with PIKA (e.g., in the same or different compositions).
In certain embodiments, the invention provides a kit comprising a subject immunogenic composition, which includes an enriched immune cell population and/or one or more co- stimulatory molecules (e.g., a co-stimulatory molecule produced by a cell in the enriched immune cell population, e.g., a cytokine, a chemokine, an antibody, etc.) and may further be provided in combination with PIKA (e.g., in the same or different compositions), hi some embodiments, a subject kit comprises an enriched immune cell population, one or more factors (e.g., a cytokine, a chemokine, and antibody, etc.), where the one or more factors can be factors produced by the enriched immune cell population; PIKA; and an antigen. In some embodiments, the kit further provides a delivery system (e.g., one or more of a syringe; a needle; a system for intravenous infusion; etc.) for delivering the immunogenic composition to an individual.
In some embodiments, a subject kit comprises a mature dendritic cell, e.g., an enriched mature DC population. In some embodiments, a subject kit comprises a mature, antigen- primed dendritic cell, e.g., an enriched, mature, antigen-primed DC population. In some embodiments, a subject kit comprises a mature dendritic cell (which may be antigen primed); and PIKA. In some embodiments, a subject kit comprises a mature, antigen- primed dendritic cell. In some embodiments, a subject kit comprises a mature dendritic cell (which may be antigen primed); and an antigen. In some embodiments, a subject kit comprises a mature, antigen-primed dendritic cell. In some embodiments, a subject kit comprises a mature dendritic cell (which may be antigen primed); and one or more factors (e.g., one or more of a cytokine, a chemokine, an antibody, etc.). In some embodiments, a subject kit comprises a mature, antigen-primed dendritic cell. In some embodiments, a subject kit comprises a mature dendritic cell (which may be antigen primed); PIKA; and an antigen. In some embodiments, a subject kit comprises a mature, antigen-primed dendritic cell. In some embodiments, a subject kit comprises a mature dendritic cell (which may be antigen primed); PIKA; and one or more factors (e.g., one or more of a cytokine, a chemokine, an antibody, etc.). In some embodiments, a subject kit comprises a mature, antigen-primed dendritic cell. In some embodiments, a subject kit comprises a mature dendritic cell (which may be antigen primed); an antigen; and one or more factors (e.g., one or more of a cytokine, a chemokine, an antibody, etc.).
In certain embodiments, the invention provides a kit comprising PIKA and an antigen in separate foπnulations.
In a related embodiment, the invention provides for a kit comprising PIKA and an immunogenic compound where the immunogenic substance is an antigen.
In some embodiments, a subject kit comprises a subject immunogenic composition in a sterile liquid (e.g., aqueous) formulation, where the formulation is sterile, and is provided in a sterile container, a sterile vial, or a sterile syringe.
In some embodiments, a subject kit comprises a subject immunogenic composition formulated for injection. In some embodiments, a subject kit comprises a subject immunogenic composition in a sterile liquid formulation, contained within a sterile syringe; and a needle. In some embodiments, a subject kit comprises a subject immunogenic composition in a sterile liquid formulation in a unit dosage amount (e.g., a single dose), contained within a sterile syringe; and a needle.
In some embodiments, a subject kit comprises a subject immunogenic composition, lyophilized and in a sterile container; and a container comprising a sterile liquid for reconstitiition of the lyophilized composition. In some embodiments, the kit further comprises instructions for reconstitution of the lyophilized composition.
In some embodiments a subject kit comprises an immunogenic composition formulated for administration rectally, vaginally, nasally, orally (including inhalation), ophthalmically, topically, pulmonary, ocularly or transdermally and an appropriate delivery device for example, inhaler, suppository, applicator or the like.
In some embodiments a subject kit comprises an immunogenic composition formulated for administration in-vitro together with an applicator or the like.
A subject kit in some embodiments will further include instructions for use, including e.g., dosage amounts and dosage frequencies. Instructions are in some embodiments printed directly on the kit. In other embodiments, instructions are printed material provided as a package insert. Instructions can also be provided in other media, e.g., electronically in digital or analog form, e.g., on an audio cassette, an audio tape, a compact disc, a digital versatile disk, and the like.
Formulations
A subject immunogenic composition is provided in any of a variety of formulations. For example, a subject immunogenic composition may be prepared as an injectable, dry power, liquid solution, for example: aqueous or saline solution or as: a suspension, cream, emulsion, tablet, coated tablet, microcapsule, suppositoiy, drops, pill, granules, dragee, capsule, gel, syrup or slurry.
For example, for use in vitro, a subject immunogenic composition (e.g., a composition comprising PIKA) can be provided in any of a variety of formulations. For example, a subject immunogenic composition may be prepared as an injectable, dry power, liquid solution, for example: aqueous or saline solution or as: a suspension, cream, emulsion, tablet, coated tablet, microcapsule, suppository, drops, pill, granules, dragee, capsule, gel, syrup or slurry. A subject immunogenic composition may be microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized aerosols, pellets for implantation into the skin, or dried onto a sharp object (e.g., a needle) to be scratched into the skin.
A subject immunogenic composition which includes an enriched immune cell population (e.g., isolated mature dendritic cells, which are optionally antigen-primed) and may further be provided in combination with PIKA (e.g., in the same or different compositions), is provided in any of a variety of formulations. For example, a subject immunogenic composition may be prepared as an injectable, liquid solution, for example: aqueous or saline solution or as: a suspension, cream, emulsion, microcapsule, drops, gel, syrup or slurry.
A subject immunogenic composition may be microencapsulated, encochleated, contained in liposomes, for implantation into the skin.
In a further embodiment the subject immunogenic substance may be delivered alone or in conjunction with a dispersion system. In some embodiments the dispersion system is selected from the group consisting of for example: macromolecular complexes, nanocapsules, microspheres, beads and lipid based systems. Lipid based systems optionally include oil-in-water emulsions, micelles, mixed micelles or liposomes.
In certain embodiments a subject immunogenic composition comprising the PIKA is in the form of a pharmaceutically acceptable solution, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients. The composition may contain additives for example: disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers and the like. In certain embodiments a subject immunogenic composition comprising PIKA is administered in its neat form or in the form of a pharmaceutically acceptable salt.
The immunogenic composition of the present invention may be employed in such forms, both sterile and non-sterile, such as capsules, liquid solutions, liquid drops, emulsions, suspensions, elixirs, creams, suppositories, gels, soft capsules, sprays, inhalants, aerosols, powders, tablets, coated tablets, lozenges, microcapsules, suppositories, dragees, syrups, slurries, granules, enemas or pills. Any inert carrier can be used, such as saline, or phosphate buffered saline, stabilizers, propellants, encased in gelatin capsule or in a microcapsule or vector that aids administration or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties for use in the methods of the present invention.
In certain embodiments, the PIKA composition or an antigenic compound or an immunogenic composition comprising the PIKA and the antigenic compound is freeze- dried (lyophilized) for long term stability and storage in a solid form. The freeze-dried method is known to those skilled in the art.
In one aspect of particular interest, the invention provides for the PEKA composition or an immunogenic composition wherein the immunogenic composition, or the PIKA composition contained in the immunogenic composition, is in a solid or liquid form or in solution or in suspension or in emulsion.
A subject immunogenic composition may be administered to an individual by means of a pharmaceutical delivery system for the inhalation route (oral, intratracheal, intranasal). Thus, a subject immunogenic composition may be formulated in a form suitable for administration by inhalation. The pharmaceutical delivery system is one that is suitable for respiratory therapy by topical administration of a subject bacterial composition to mucosal linings of the bronchi. This invention can utilize a system that depends on the power of a compressed gas to expel the bacteria from a container. An aerosol or pressurized package can be employed for this purpose. As used herein, the term "aerosol" is used in its conventional sense as referring to very fine liquid or solid particles caπies by a propellant gas under pressure to a site of therapeutic application. When a pharmaceutical aerosol is employed in this invention, the aerosol contains the immunogenic composition, which can be dissolved, suspended, or emulsified in a mixture of a fluid earner and a propellant. The aerosol can be in the form of a solution, suspension, emulsion, powder, or semi-solid preparation. Aerosols employed in the present invention are intended for administration as fine, solid particles or as liquid mists via the respiratory tract of a subject. Various types of propellants known to one of skill in the art can be utilized. Examples of suitable propellants include, but are not limited to, hydrocarbons or other suitable gas. In the case of the pressurized aerosol, the dosage unit may be determined by providing a value to deliver a metered amount.
There are several different types of inhalation methodologies which can be employed in connection with the present invention. A subject immunogenic composition can be formulated in basically three different types of formulations for inhalation. First, a subject immunogenic composition can be formulated with low boiling point propellants. Such formulations are generally administered by conventional meter dose inhalers (MDI's). However, conventional MDI's can be modified so as to increase the ability to obtain repeatable dosing by utilizing technology which measures the inspiratory volume and flow rate of the subject as discussed within U.S. Patents 5,404,871 and 5,542,410.
Alternatively, a subject immunogenic composition can be formulated in aqueous or ethanolic solutions and delivered by conventional nebulizers. In some embodiments, such solution formulations are aerosolized using devices and systems such as disclosed within U.S. Patent 5,497,763; 5,544,646; 5,718,222; and 5,660,166.
Furthermore, a subject immunogenic composition can be formulated into dry powder formulations. Such formulations can be administered by simply inhaling the dry powder formulation after creating an aerosol mist of the powder. Technology for carrying such out is described within U.S. Patent 5,775,320 and U.S. Patent 5,740,794. Formulations suitable for intranasal administration include nasal sprays, nasal drops, aerosol formulations; and the like.
In some embodiments, a subject immunogenic composition is formulated as a sustained release (e.g. a controlled release formulation). For example, in some embodiments, a subject immunogenic composition is formulated into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections or as implants. Such implants will generally employ known inert materials such as biodegradable polymers. Injectable depot forms are made by forming microencapsule matrices of a subject immunogenic composition in biodegradable polymers such as polylactide-polyglycolide. Examples of other suitable biodegradable polymers include poly(ortho esters) and poly( anhydrides). Depot injectable formulations are also prepared by entrapping the composition in liposomes or microemulsions which are compatible with body tissue. Deliver}' release systems also include the following examples: polymer based systems, microcapsules, lipids, hydrogel release systems, sylastic systems, peptide systems, peptide based systems, wax coatings, compressed tablets, partially fused implants, Other forms of sustained release are known by those skilled in the art.
Methods
In some embodiments, the present invention provides methods of inducing or enhancing an immune response. Both in vitro and in vivo methods of inducing or enhancing an immune response are provided. In some embodiments, a subject method comprises both in vitro and in vivo steps.
In vitro methods
In certain embodiments, a subject method involves use of PIKA (e.g., an immunogenic composition comprising PIKA) for in vitro stimulation. In vitro methods include, but are not limited to, increasing the number and/or activity of natural killer (NK) cells in vitro; increasing the number and/or activity of B cells in vitro; increasing the number and/or activity of dendritic cells in vitro; increasing the number and/or activity of antigen- primed antigen presenting cells (APCs) in vitro; increasing the number and/or activity of antigen-specific CDS+ T cells in vitro; and increasing the number and/or activity of antigen-specific CD4+ T cells in vitro. A subject in vitro method generally involves contacting a cell or a population of cells in vitro with a subject immunogenic composition (e.g., PIKA in the absence of antigen; PIKA plus antigen; etc.). hi some embodiments, a subject in vitro method involves contacting a cell or population of cells in vitro with an immunogenic composition comprising PIKA, where the immunogenic composition does not comprise an antigen, hi other embodiments, a subject in vitro method involves contacting a cell or population of cells in vitro with an immunogenic composition comprising PIKA and one or more antigens.
Suitable cells for use in a subject in vitro method include, but are not limited to, BMDCs, peripheral blood cells, dendritic cells, natural killer cells, CD4+ T lymphocytes, CD8 T lymphocytes, B cells, and the like. Suitable tissue sources for the cells include spleen, afferent lymph, bone marrow, and blood, including peripheral blood, fetal blood, and umbilical cord blood. The tissue source may be treated prior to culturing to enrich the proportion of a particular cell type relative to other cell types. The method of treatment may depend on the particular tissue source, and the type of cell to be enriched. For example, spleen or bone marrow could be treated so as to separate leukocytes from other cell types. Treatment of blood would involve cell separation techniques to separate leukocytes from other cells types including red blood cells (RBCs). Removal of RBCs may be accomplished by standard methods known to those skilled in the art. Those skilled in the art are familiar with methods of isolating DCs from tissues. See, e.g., U.S. Patent No. 6,194,204. Kits for isolating DCs are commercially available and can be used to obtain DCs. StemSep™ is an example of a DC isolation kit.
In some embodiments, the source of the cells is a mammal, e.g., a human, a non-human primate, a canine, a feline, an ungulate, etc. In other embodiments, the source of the cells is an avian animal, e.g., a chicken, a turkey, a goose, etc. In some embodiments, the source of the cells is an autologous source, e.g., the cells are obtained from an individual who is also a prospective recipient of cell. In other embodiments, the source of the cells is an allogeneic source, e.g, the cells are obtained from an individual of the same species, where the individual is not genetically identical to a prospective recipient. In other embodiments, the source of the cells is a xenogeneic source, e.g., the cells are obtained from an individual of a species that is different from the species of a prospective recipient.
In some embodiments, a subject in vitro method involves contacting, in vitro, a mixed cell population or an enriched cell population with an effective amount of a subject immunogenic composition (e.g., a composition comprising PDCA without antigen; a composition comprising PKA and an antigen; etc.). In some embodiments, the method further comprises collecting a desired cell population (e.g., an activated cell population; an antigen-specific cell population; a population of antigen-primed antigen presenting cells; etc.).
In some embodiments, an "effective amount" of a subject immunogenic composition is an amount that provides for an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or an at least about 1000-fold, or greater, increase in the number of NK cells, compared to the number of NK cells in the starting cell population.
In some embodiments, an "effective amount" of a subject immunogenic composition is an amount that provides for an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or an at least about 1000-fold, or greater, increase in the number of B cells, compared to the number of B cells in the starting cell population.
In some embodiments, an "effective amount" of a subject immunogenic composition is an amount that provides for an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or an at least about 1000-fold, or greater, increase in the number of antigen-primed APCs, compared to the number of antigen- primed APCs in the starting cell population.
In some embodiments, an "effective amount" of a subject immunogenic composition is an amount that provides for an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or an at least about 1000-fold, or greater, increase in the number of antigen-primed B cells, compared to the number of antigen- primed B cells in the starting cell population.
In some embodiments, an "effective amount" of a subject immunogenic composition is an amount that provides for an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50- fold, at least about 100-fold, at least about 500-fold, or an at least about 1000-fold, or greater, increase in the number of mature APCs, compared to the number of mature APCs in the starting cell population. In some embodiments, an "effective amount" of a subject immunogenic composition is an amount that provides for an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or an at least about 1000-fold, or greater, increase in the number of antigen-primed DCs, compared to the number of antigen- primed DCs in the starting cell population.
In some embodiments, an "effective amount" of a subject immunogenic composition is an amount that provides for an at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or two-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or an at least about 1000-fold, or greater, increase in the number of mature DCs, compared to the number of mature DCs in the starting cell population.
As noted above, in some embodiments, a subject in vitro method further comprises collecting a population of cells generated by the step of contacting a population of cells with a subject immunogenic composition, thereby generating an "enriched" cell population, or an "isolated" cell population. For example, an "enriched" or an "isolated" cell population comprises one or more desired cell types (e.g., activated cells; antigen- primed cells; etc.) in a proportion that is substantially higher than the proportion of the cell type(s) in the starting population (e.g., in the cell population before contacting with a subject immunogenic composition). For example, "isolated" antigen-primed DCs can be at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, pure, e.g., free of cells other than DCs, and/or at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about S0%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% free of non-antigen-primed DCs. As another example, "isolated" mature DCs can be at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, pure, e.g., free of cells other than mature DCs, and/or at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% free of immature DCs.
In some embodiments, a subject in vitro method further comprises collecting a particular desired cell type(s) and collecting one or more factors produced by the desired cell type(s). Factors of interest include, but are not limited to, cytokines, chemokines, antibodies, and the like.
In vivo methods
In one aspect of interest, the invention provides for a method for eliciting and/or enhancing immune responses to an immunogenic compound, comprising administering to a host a subject immunogenic composition. In some embodiments, the host is a human. In other embodiments, the host is a non-human animal, e.g., a non-human mammal, an avian species, etc.
In certain embodiments, the PIKA composition can be used in the context of an immune regulator. Optionally, the immune regulator composition may contain an adjuvant. Immune regulator classes included are anti-infectious diseases, anti cancer, anti-allergy and anti-autoimmune diseases.
A subject immunogenic composition is in some embodiments delivered parenterally by injection, such as intramuscular, intraperitoneal, intravenous, subcutaneous or intradermal injection. In other embodiments the immunogenic composition is administered intradermally in ways other than by injection, for example, without breaching the epithelial barrier by mechanical means. In other embodiments, the immunogenic composition is delivered rectally, vaginally, nasally, orally (including inhalation), ophthalmically, ocularly, topically, pulmonary or transdermally. In certain embodiments, when the immunogenic composition comprising PIKA may be used for the treatment of cancer. In some embodiments, the immunogenic composition is delivered to enhance the biodistribution and hence enhance the therapeutic benefit.
In certain embodiments, when the mode of administration of the immunogenic composition comprising PIKA is for the treatment of cancer tumors, the delivery is by injection directly into the tumor, or adjacent to the tumor. In some embodiments, the immunogenic composition is delivered evenly over or throughout the tumor to enhance the biodistribution and hence enhance the therapeutic benefit.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Exemplary injection media which can be used in the present invention include a buffer with or without dispersing agents and/or preservatives, and edible oil, mineral oil, cod liver oil, squalene, mono-, di- or triglyceride, and a mixture thereof.
A subject immunogenic composition is administered in an "effective amount" that is, an amount of a subject immunogenic composition that is effective in a selected route of administration to elicit, induce, or enhance an immune response. In some embodiments, an immune response is elicited to antigens produced by a pathogenic microorganism. In some embodiments, the amount of a subject immunogenic composition is effective to limit an infection, and/or to eradicate an infection, and/or to reduce a symptom associated with infection, by a pathogenic organism.
For example, in some embodiments, administration of a subject immunogenic composition to an individual is effective to treat an infectious disease, where treating an infectious disease, encompasses one or more of reducing the number of pathogenic agents in the individual (e.g., reducing viral load, reducing bacterial load, reducing the number of protozoa, reducing the number of helminths) and/or reducing a parameter associated with the infectious disease, including, but not limited to, reduction of a level of a product produced by the infectious agent (e.g., a toxin, an antigen, and the like); and reducing an undesired physiological response to the infectious agent (e.g., fever, tissue edema, and the like).
The exact amount of such compositions required will vary from subject to subject, depending on the species, age, weight, and general conditions of the subject, the severity of the disease, infection, or condition that is being treated or prevented, the particular compound used, its mode administration, and the like. An appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. Following an initial administration, subjects may receive one or several booster immunizations adequately spaced.
In some embodiments, serial doses of a subject immunogenic composition are administered. The second dose of a subject immunogenic composition is administered to the individual after the individual has been immunologically primed by exposure to the first dose. The booster may be administered days, weeks or months after the initial immunization, depending upon the patient's response and condition. For example, the booster dose is administered from about 2 days to about 12 months after the initial dose, e.g., from about 2 days to about 7 days, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 8 weeks, from about 8 weeks to about 6 months, or from about 6 months to about 12 months after the initial dose. The present invention further contemplates the use of a third, fourth, fifth, sixth or subsequent booster immunization, using, e.g., a third, fourth, fifth, sixth, or subsequent dose.
In certain embodiments the means of administration may comprise a combination of alternative routes, for example: systemically administered dose (e.g. peritoneal, intramuscular, subcutaneous or intradermal administration) may be followed by mucosally delivered dose (e.g. intranasal, inhalation) or vice versa. In certain embodiments the composition of the administered immunogenic composition in-vitro may vary between the original administration and the boost and/or between booster doses. At least one of the doses administered as part of the overall protocol would comprise the PIKA.
In another aspect of interest, the invention provides for a method for eliciting and/or enhancing an immune response to an immunogenic compound in an individual (a "recipient" or a "recipient individual"), comprising administering to the individual an enriched cell population and/or factors produced by an enriched cell population, where the enriched cell population and/or factors are produced by a subject in vitro method. Thus, in some embodiments, the present invention provides a method of increasing an immune response to an antigen in an individual, the method comprising administering an immunogenic composition comprising an enriched immune cell population enriched in a selected immune cell type to the individual, where the enriched cell population is generated by a method comprising contacting a starting cell population obtained from a donor individual with PIKA composition. Administration of the enriched cell population increases the immune response to the antigen in the recipient individual. In some embodiments, the immunogenic composition comprising an enriched cell population further comprises PIKA. In some embodiments, the immunogenic composition comprising an enriched cell population further comprises one or more factors (e.g., one or more of a chemokine, a cytokine, an antibody, etc.), where the one or more factors are produced by the enriched cell population in vitro. In some embodiments, the immunogenic composition comprising an enriched cell population further comprises PDCA and one or more factors (e.g., one or more of a chemokine, a cytokine, an antibody, etc.), where the one or more factors are produced by the enriched cell population in vitro. In some embodiments, a subject method provides for preferential induction of a ThI immune response, e.g., a ThI immune response is induced preferentially over a Th2 immune response. In some embodiments, the source of the starting cells is an autologous source, e.g., the starting cell population is obtained from a donor individual who is the same as the recipient individual, where the recipient individual is the individual to whom the enriched cell population is administered. In other embodiments, the source of the starting cell population is an allogeneic source, e.g, the starting cell population is obtained from a donor individual of the same species as the recipient individual, where the donor individual is not genetically identical to the recipient individual. In other embodiments, the source of the starting cell population is a xenogeneic source, e.g., the starting cell population is obtained from a donor individual of a species that is different from the species of the recipient individual.
In some embodiments, multiple doses of an immunogenic composition comprising an enriched cell population are administered to a recipient individual. In some embodiments, none of the multiple doses comprises PIKA, e.g., each dose of an immunogenic composition comprises an enriched cell population, without PIKA added. In some embodiments, at least one of the multiple doses comprises PIKA. For example, in some embodiments, multiple doses of an immunogenic composition are administered, where at least one of the multiple doses comprises an enriched cell population and PIKA (administered in the same formulation or in different foπnulations). In some embodiments, at least one of the multiple doses comprises antigen. For example, in some embodiments, multiple doses of an immunogenic composition are administered, where at least one of the multiple doses comprises an enriched cell population and antigen (administered in the same formulation or in different formulations). In some embodiments, at least one of the multiple doses comprises PIKA and antigen. For example, in some embodiments, multiple doses of an immunogenic composition are administered, where at least one of the multiple doses comprises an enriched cell population, PIKA, and antigen (administered in the same formulation or in different formulations). In some embodiments, at least one of the multiple doses comprises a factor produced by the enriched cell population. For example, in some embodiments, multiple doses of an immunogenic composition are administered, where at least one of the multiple doses comprises an enriched cell population and a factor produced by the enriched cell population (administered in the same formulation or in different formulations).
Subjects suitable for treatment
Subjects suitable for treatment with a subject method of inducing an immune response to a microbial pathogen, and methods of treating or preventing an infection with a microbial pathogen, include individuals who have been infected with a pathogenic microorganism; individuals who are susceptible to infection by a pathogenic microorganism, but who have not yet been infected; and individuals who are at risk of becoming infected with a pathogenic microorganism, but who have not yet been infected. Suitable subjects include infants, children, adolescents, and adults.
Subjects suitable for treatment with a subject method of inducing an immune response to a microbial pathogen, and methods of treating or limiting an infection with a microbial pathogen, include pediatric target population, e.g., individuals between about 1 year of age and about 17 years of age, including infants (e.g., from about 1 month old to about 1 year old); children (e.g., from about 1 year old to about 12 years old); and adolescents (e.g., from about 13 years old to about 17 years old).
Subjects suitable for treatment with a subject method of inducing an immune response to a microbial pathogen, and methods of treating or limiting an infection with a microbial pathogen, include neonates, e.g., an individual (e.g., a human neonate) from one day to about 14 days old, e.g., from about 1 day to about 2 days old, from about two days to about 10 days old, or from about 10 days to about 14 days old.
In a particular embodiment, the subject is a human child about ten years or younger, e.g., about five years old or younger, and the immunogenic compositions are administered at any one or more of the following times: two weeks, one month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, S months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, or 21 months after birth, or at 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years of age. In some embodiments, a subject immunogenic composition is administered to an individual in the age range of from about 6 months to about 6 years, where the individual receives a first dose at about 6 months of age, and subsequent booster doses, e.g., 2-3 subsequent booster doses, at, e.g., 2 years of age, 4 years of age, and 6 years of age.
In a particular embodiment, the subject is a human adult from about 17 years old to 49 years old. In some embodiments, the subject is an elderly human adult from 50 to 65 years old, 65 to 75 years old, 75 to S5 years old or over 85 years old.
In some embodiments, a subject immunogenic composition is administered to an individual shortly after contact (e.g., shortly after confirmed or suspected contact) with an actual or potential source of the microbial pathogen, for example, an individual who is known to have or suspected to have an infection with a microbial pathogen. For example, in some embodiments, a subject immunogenic composition is administered to an individual within about 1 hour, within about 2 hours, within about 5 hours, within about 8 hours, within about 12 hours, within about 18 hours, within about 24 hours, within about 2 days, within about 4 days, within about 7 days, within about 2 weeks, or within about one month after contact with an individual who is known to have or suspected to have an infection with a microbial pathogen.
In some embodiments, a subject immunogenic composition is administered to an individual that is known or may be suspected of being a carrier or a microbial pathogen whether or not they are showing symptoms of the infection.
Subjects suitable for treatment with a subject method of inducing an immune response to a microbial pathogen, and methods of treating or limiting an infection with a microbial pathogen, include CD4+ T cell-deficient individuals ("CD4+-deficient" individuals), e.g., individuals who have lower than normal numbers of functional CD4+ T lymphocytes. As used herein, the term "normal individual" refers to an individual having CD4+ T lymphocyte levels and function(s) within the normal range in the population, for humans, typically 600 to 1500 CD4+ T lymphocytes per mm3 blood. CD4+-deficient individuals include individuals who have an acquired immunodeficiency, or a primary immunodeficiency. An acquired immunodeficiency may be a temporary CD4+ deficiency, such as one caused by radiation therapy, or chemotherapy.
Also suitable for treatment with the methods of the invention are individuals with healthy, intact immune systems, but who are at risk for becoming CD4+ deficient ("at- risk" individuals). At-risk individuals include, but are not limited to, individuals who have a greater likelihood than the general population of becoming CD4+ deficient. Individuals at risk for becoming CD4+ deficient include, but are not limited to, individuals at risk for HIV infection due to sexual activity with HIV-infected individuals; intravenous drug users; individuals who may have been exposed to HIV-infected blood, blood products, or other HlV-contaminated body fluids; a baby who has passed through the birth canal of an HIV-infected individual; babies who are being nursed by HIV- infected mothers; and the like.
Subjects suitable for treatment with a subject method for treating cancer include individuals who have been infected with a carcinogenic substance, individuals who are susceptible to cancer but who have not yet been diagnosed with cancer; and individuals who are at risk of contracting cancer, but who have not yet been diagnosed with cancer. Suitable subjects include infants, children, adolescents, and adults.
Subjects suitable for treatment with a subject method for treating cancer include individuals who have been diagnosed with cancer; individuals who were previously treated for cancer, e.g., by chemotherapy or radiotherapy, and who are being monitored for recurrence of the cancer for which they were previously treated; and individuals who have undergone bone marrow transplantation or any other organ transplantation.
Subjects suitable for treatment with the formulations and methods of the instant invention for treating allergy include any individual who has been diagnosed as having an allergy. Subjects amenable to treatment using the methods and agents described herein include individuals who are known to have allergic hypersensitivity to one or more allergens. Subjects amenable to treatment include those who have any of the above-mentioned allergic disorders. Also amenable to treatment are subjects that are at risk of having an allergic reaction to one or more allergens. Also suitable are individuals who failed treatment with one or more standard therapies for treating an allergic disorder.
Subjects suitable for treatment include individuals living in industrialized nations; individuals living developing countries; individuals living in rural areas; individuals living in relatively isolated areas; and the like.
The target population for a subject immunogenic composition will vary, depending on the microbial pathogen
The above disclosure generally describes the present invention. The following examples will be of assistance to the understanding of the present invention. These examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
EXAMPLES
The following examples are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like. Example 1 - PIKA Induces an Innate Immune Response In-vivo
Balb/c mice were injected intra-peritoneal with PIKA of molecular size 66kDa to 660 kDa (500 μg/mouse), and sera were collected prior to injection (hour 0) as well as 1, 2 and 5 hours following the administration of PIKA. The levels of cytokines in the sera were determined by a standard ELISA test known to those skilled in the art. As shown in Figures 1, 2, 3 and 4 the injection of PIKA resulted in the production of TNF-α, IL-6, IFN-γ and IL-12ρ40.
The amount and timing of the production of each cytokine varied. Following the administration of PQCA, TNF-α reached the peak level after 1 hour, IL-6, IL-12p40 and IFN-γ after 2 hours.
Example 2 - PIKA Induces Proliferation and Activation of Natural Killer Cells
Splenocytes from Balb/c mice were incubated in the presence or absence of PIKA of molecular size 66kDa to 660 kDa (250 μg/ml) for 3 days. Cells were harvested, washed and stained with surface antibodies. Lymphocytes were first gated, and the percentages of NK cells were assayed as well as the expression of CD25 on NK cells were determined by a flow cytometry test known to those skilled in the art.
The data are depicted in Figure 5. PIKA markedly enhanced the proliferation of NK cells (68.94%) compared with un-stimulated splenocytes (42.01%). Furthermore, PIKA significantly induced the expression of CD25 on NK cells (Ly49+) (37.40%) compared with un-stimulated splenocytes (5.40%). These results demonstrate PIKA's role in the proliferation and activation of NK cells.
Example 3 - PIKA Induces Proliferation and Activation of B Cells
Splenocytes from Balb/c mice were incubated in the presence or absence of PIKA of molecular size 66kDa to 660 kDa (250 μg/ml) for 3 days. Cells were harvested, washed and stained with surface antibodies. Lymphocytes were first gated, and the percentages of B cells were assayed as well as the expression of CD25 on B cells were determined by a flow cytometry test known to those skilled in the art.
The data are depicted in Figure 6. PIKA increased the percentage of CDl 9+ B cells (18.03%) compared with un-stimulated splenocytes (14.0%), furthermore, PIKA markedly induced the expression of CD25 on CD19+ B cells (25.46%) compared with unstimulated cells (3.25%). These results demonstrate PIKA's role in the proliferation and activation of B cells.
Example 4 - PIKA Induces Maturation of Bone Marrow Derived Dendritic Cells
This example demonstrates that PQCA of molecular size 66kDa to 660 kDa promotes the maturation of bone marrow-derived dendritic cells (BMDCs) including up-regulation of the co-stimulatory molecules CD80, CD86, and CD40 (Figures 7, 8, and 9, respectively), and the induction of cytokines such as IL-12p70, IL-12ρ40 and IL-6 (Figures 10, 11, and 12, respectively).
BMDCs from C57BL/6 mice were prepared by removing all muscle tissue from the femora of C57BL/6 mice, the bones λvere then placed in a 60-mm dish with 75% alcohol for 1 minute, washed twice with Hanks' solution, and transferred into a fresh dish. Both ends of the bones were cut with scissors, and the marrow was flushed out using a 2 ml syringe and a 25 gauge needle until the bones became white. The bone marrow cell suspensions were centrifuged (700 g*8 min at 40C), and red cells were lysed with ammonium chloride. The cells were washed twice with Hanks' solution, and cell concentration was adjusted to 2^106 cells/ml and cultured with complete RPMI 1640 medium in the presence of rmGM-CSF (20 ng/ml) and rmIL-4 (5 ng/ml) in a 25 cm" culture flask. After 72 hours, the flask was gently shaken and all of the supernatants were replaced with a complete RPMI 1640 medium containing rmGM-CSF (20 ng/ml) and rmIL-4 (5 ng/ml), and cultured for another 72 hours.
At the end of the culturing, the cells were harvested and washed twice with PBS, and the Fc receptors were blocked with anti-CD 16/CD32 monoclonal antibodies at 40C for 20 minutes and stained with fluorescence labeled antibodies. All incubations were conducted on ice and washed twice with cold staining buffer after each step (PBS containing 0.1% BSA and 0.05% NaN3). The cells were collected on a FACSCalibur (BD Bioscience, Mountain View, CA, USA), and the data was analyzed using CellQuest software (BD Bioscience, Mountain View, CA, USA).
For the detection of cytokines in the sera or culture supernatants, IL-6 OptEIA ELISA (detection range, 31.1-2000 pg/ml), IL-12P40 OptEIA ELISA (detection range, 15.6-1000 ρg/ml), IL-12P70 OptEIA ELISA (detection range, 62.5-4000 pg/ml), IFN-γ OptEIA ELISA (detection range, 3.1-200 pg/ml) and TNF-α OptEIA ELISA (detection range, 15.6-1000 pg/ml) (all from BD PharMingen) were used according to the manufacture's protocols .
To determine whether PIKA could induce the maturation of DCs, immature BMDCs were prepared were harvested at 6 days. After washing, immature BMDCs were cultured with PIKA for 18 hours. The cells were collected, washed, and incubated with fluorescence labeled anti-CDSO, anti-CD86 and anti-CD40 antibodies. The expression of the molecules on BMDCs was determined by FACS. PIKA markedly increased the percentages of CDSO+ and CD40+ cells and the MFI of CD80, CD86 and CD40 on BMDCs (Figures 7, 8 and 9), indicating that PIKA promoted the maturation of BMDCs.
The cytokines produced by DC are likely to influence the differentiation of ThI and Th2 cells. To determine whether PIKA could activate DCs to produce cytokines BMDCs were stimulated with or without PIKA at different concentrations (50 μg/ml and 250 μg/ml, respectively) for 18 hours. PIKA significantly induced the production of IL-12p70 (Figure 10), IL-12ρ40 (Figure 11) and IL-6 (Figure 12) by BMDCs in a dose-dependent manner. There was an undetectable amount of IL-12p40 and IL-12p70 production in unstimulated BMDCs. Similar results were observed in IL-6 production by BMDCs. The conclusion of this study is that PIKA facilitates the activation and maturation of dendritic cells. Example 5 - PIKA Induces the Maturation of Dendritic Cells and Natural Killer Cells Derived from Human Blood Samples
This example demonstrates that PIKA either of of molecular size 66kDa to 660 kDa or 66kDa to 1,200 kDa facilitates the activation and maturation of human dendritic cells and human natural killer cells in vitro.
Immature dendritic cells derived from human peripheral blood mononuclear cells (PBMCs) are isolated, washed and incubated either with PIKA or in the absence of PIKA. Analysis of the molecules expressed by the cultured dendritic cells is used to determine the presence of mature dendritic cells. Comparing results from immature dendritic cells cultured in the presence of PIKA with those cultured in the absence of PDCA provides an indication of the ability of PIKA to facilitate the maturing of immature dendritic cells.
Natural killer cells obtained from human PBMCs are washed and incubated either with PIKA or in the absence of PIKA. Analysis of the number of natural killer cells and the proportion of natural killer cells expressing selected surface molecules is a means to determine the proliferation and activation of natural killer cells. Comparing results from natural killer cells cultured in the presence of PIKA with those cultured in the absence of PIKA provides an indication of the ability of PIKA to facilitate the proliferation and activation of natural killer cells.

Claims

1. A method of inducing activation of a dendritic cell (DC) in an individual, the method comprising: administering to the individual an effective amount of an immunogenic composition, wherein the immunogenic composition comprises a population of polyriboinosinic-polyribocytidylic acid (PIC) molecules, at least one antibiotic, and at least one positive ion.
2. The method of claim 1, wherein the immunogenic composition further comprises at least one antigen, and wherein the DC is primed with the antigen.
3. The method of claim 2, wherein the antigen is an autoantigen, a tumor-associated antigen, an allergen, and/or an antigen associate with a microbial pathogen.
4. A method of increasing an immune response to an antigen in an individual, the method comprising: administering an immunogenic composition comprising an immune cell population enriched in a selected immune cell type to the individual, wherein the enriched immune cell population is generated by a method comprising contacting a starting cell population with an immunogenic composition in vitro, wherein the immunogenic composition comprises a population of polyriboinosinic- polyribocytidylic acid (PIC) molecules, at least one antibiotic, and at least one positive ion, wherein said administering of the enriched immune cell population increases the immune response to the antigen.
5. The method of claim 4, wherein the starting cell population comprises immature dendritic cells, and wherein the selected cell type is a mature, activated dendritic cell (DC).
6. The method of claim 5, wherein the immunogenic composition further comprises at least one antigen, and wherein the DC is primed with the antigen.
7. The method of claim 6, wherein the antigen is an autoantigen, a tumor-associated antigen, an allergen, and/or an antigen associate with a microbial pathogen.
8. The method of claim 4, wherein the starting cell population comprises a B cell, wherein the selected cell type is a B cell, and wherein said contacting increases the proportion of activated B cells in the enriched cell population.
9. The method of claim 4, wherein the starting cell population comprises a B cell, wherein the selected cell type is a natural killer (NK) cell, and wherein said contacting increases the proportion of NK cells in the enriched cell population.
10. The method of claim 4, wherein the composition comprising the enriched cell population further comprises at least one co-stimulatory molecule.
11. The method of claim 10, wherein the co-stimulatory molecule is a cytokine.
12. The method of claim 10, wherein the co-stimulatory molecule is a chemokine.
13. The method of claim 4, wherein said antigen is a tumor-associated antigen, and wherein said increasing reduces the number of cancer cells in the individual.
14. The method of claim 4, wherein said antigen is an allergen, and wherein said increasing reduces at least one parameter associated with an allergic response to the allergen.
15. The method of claim 4, wherein said antigen is associated with a pathogen, and wherein said increasing reduces the number of pathogens in the individual.
16. The method of claim 4, wherein the individual is a mammal.
17. The method of claim 16, wherein said mammal is a human.
18. The method according to any one of claims 1 to 17, wherein the PIC molecules have a molecular weight in a range of from about 66,000 to about 1,200,000 Dalton.
19. An immunogenic composition comprising: a) a population of polyriboinosinic-polyiibocytidylic acid (PIC) molecules, at least one antibiotic, and at least positive ion; and b) an enriched immune cell population.
20. The immunogenic composition according to claim 19, wherein the enriched immune cell population comprises mature dendritic cells.
21. The immunogenic composition according to claim 19, further comprising at least one antigen.
22. The immunogenic composition according to any one of claims 19 to 21 , for use in medicine.
23. The immunogenic composition according to any one of claims 19 to 22, for use in inducing activation of a dendritic cell (DC) in an individual.
24. The immunogenic composition according to any one of claims 19 to 22, for use in increasing an immune response to an antigen in an individual.
25. The immunogenic composition according to any one of claims 19 to 24, wherein the PIC molecules have a molecular weight in a range of from about 66,000 to about 1,200,000 Dalton.
26. Use of a population of polyriboinosinic-polyribocytidylic acid (PIC) molecules, at least one antibiotic, and at least one positive ion, in the preparation of an immunogenic composition for inducing activation of a dendritic cell (DC) in an individual.
27. Use of an immune cell population enriched in a selected immune cell type, for increasing an immune response to at least one antigen in an individual wherein the enriched immune cell population is prepared by contacting a starting cell population with a population of polyriboinosinic-polyribocytidylic acid (PIC) molecules, at least one antibiotic and at least one positive ion,
28. The use according to claim 27, wherein the starting cell population comprises immature dendritic cells, and wherein the selected cell type is a mature, activated dendritic cell (DC).
29. The use according to any one of claims 26 to 28, wherein the immunogenic composition further comprises at least one antigen, and wherein the DC is primed with the antigen.
30. The use according to claim 29, wherein the antigen is an autoantigen, a tumor- associated antigen, an allergen, and/or an antigen associate with a microbial pathogen.
31. The use according to any one of claims 27 to 30, wherein the starting cell population comprises a B cell, wherein the selected cell type is a B cell, and wherein said contacting increases the proportion of activated B cells in the enriched cell population.
32. The use according to any one of claims 27 to 30, wherein the starting cell population comprises a B cell, wherein the selected cell type is a natural killer (NK) cell, and wherein said contacting increases the proportion of NK cells in the enriched cell population.
33. The use according to any one of claims 27 to 32, wherein the composition comprising the enriched cell population further comprises at least one co- stimulatory molecule.
34. The use according to claim 33, wherein the co-stimulatory molecule is a cytokine.
35. The use according to claim 33, wherein the co-stimulatory molecule is a chemokine.
36. The use according to any one of claims 27 to 35, wherein said antigen is a tumor- associated antigen, and wherein said increasing reduces the number of cancer cells in the individual.
37. The use according to any one of claims 27 to 35, wherein said antigen is an allergen, and wherein said increasing reduces at least one parameter associated with an allergic response to the allergen.
38. The use according to any one of claims 27 to 35, wherein said antigen is associated with a pathogen, and wherein said increasing reduces the number of pathogens in the individual.
39. The use according to any one of claims 26 to 38, wherein the individual is a mammal.
40. The use according to any one of claims 26 to 37, wherein the individual is a human.
41. The use according to any one of claims 26 to 40, wherein the PIC molecules have a molecular weight in a range of from about 66,000 to about 1,200,000 Dalton.
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