WO2008107641A1 - Liposome preparation - Google Patents

Liposome preparation Download PDF

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Publication number
WO2008107641A1
WO2008107641A1 PCT/GB2008/000679 GB2008000679W WO2008107641A1 WO 2008107641 A1 WO2008107641 A1 WO 2008107641A1 GB 2008000679 W GB2008000679 W GB 2008000679W WO 2008107641 A1 WO2008107641 A1 WO 2008107641A1
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WIPO (PCT)
Prior art keywords
preparation according
preparation
polypeptide
liposome
disease
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PCT/GB2008/000679
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French (fr)
Inventor
Andrew William Heath
Peter Laing
Andrew Bacon
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Adjuvantix Limited
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Publication of WO2008107641A1 publication Critical patent/WO2008107641A1/en

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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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a liposome preparation comprising an adjuvant and antigen for use in vaccination and also a method for the formulation of the preparation.
  • Vaccines protect against a wide variety of infectious diseases. There are also vaccines in development for the treatment of various non-infectious diseases such as autoimmune and neurodegenerative diseases and various cancers. Many vaccines are produced by inactivated or attenuated pathogens which are injected into an individual. The immunised individual responds by producing both a humoral and cellular response. For example, some influenza vaccines are made by inactivating the virus by chemical treatment with formaldehyde, likewise the SaIk polio vaccine comprises whole virus inactivated with propionolactone For many pathogens chemical or heat inactivation while it may give rise to vaccine immunogens that confer protective immunity also gives rise to side effects such as fever and injection site reactions.
  • subunit vaccines have been the focus of considerable research in recent years. The emergence of new pathogens and the growth of antibiotic resistance have created a need to develop new vaccines and to identify further candidate molecules useful in the development of subunit vaccines. Likewise the discovery of novel vaccine antigens from genomic and proteomic studies is enabling the development of new subunit vaccine candidates, particularly against bacterial pathogens and cancers.
  • subunit vaccines tend to avoid the side effects of killed or attenuated pathogen vaccines, their 'pure' status means that subunit vaccines do not always have adequate immunogenicity. Many candidate subunit vaccines have failed in clinical trials in recent years that might otherwise have succeeded were a suitable adjuvant available to enhance the immune response to the purified antigen.
  • An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells.
  • the receptor CD40 plays an important co-stimulatory role in the activation of B-cells during the cognate interaction of antigen-specific T and B-cells that gives rise to an antibody response.
  • the CD40 signal is pivotal to the expression of T cell help and immunoglobulin class-switching in both humans and mice (9-12).
  • ligation of CD40 is also very important in activation of macrophages and of dendritic cells to express co-stimulatory antigens and thus in the generation of helper T cell priming by these antigen-presenting cells (13-15).
  • a stimulatory CD40- polypeptide for example an antibody with antigen (either covalently or non-covalently) we can achieve both of these aims together, using 500-fold less antibody, generating a surprisingly effective antigen-specific stimulus.
  • Vaccines increasingly are required to be 'multivalent' (e.g. containing antigens from several different strains of a pathogen as for influenza virus and the polysaccharide vaccines against S. pneumoniae), or containing multiple proteins from a single pathogen that are additive or synergistic in the protective immune response they generate (as is the case for a number of vaccines under development - e.g. for H. pylori, tuberculosis etc.).
  • a 'multivalent' e.g. containing antigens from several different strains of a pathogen as for influenza virus and the polysaccharide vaccines against S. pneumoniae
  • multiple proteins from a single pathogen that are additive or synergistic in the protective immune response they generate
  • There is also an economic and public health case for combining vaccines against different pathogens in a 'multivalent' format e.g.
  • DTP-Hib DTP-Hib in order to increase uptake by the population, and to minimise the increasing 'aluminium burden' associated with the introduction of new vaccines based on alum as adjuvant.
  • a reduction in the total number of injections required to induce protection against the widest possible range of infections also reduces the costs of vaccination programmes, and is more profitable for the manufacturers, and these are additional factors driving the move to multivalent vaccines.
  • Subunit vaccines based upon recombinant proteins or synthetic peptides have very much reduced immunogenicity compared with whole cell vaccines, and require more potent adjuvants than are currently available to enable them to work effectively. Pure polysaccharide antigens are ineffective in major target populations, and polysaccharide conjugate vaccines are expensive to produce, especially in a multivalent form which is often required.
  • conjugation of CD40 antibody with protein antigens is an effective means of enhancing immunogenicty, and we have also shown that high doses of CD40 antibody can enhance the response to T-independent, polysaccharide antigens.
  • CD40 mab to antigen is a relatively costly process and relatively toxic.
  • a liposome preparation comprising: i) a CD40 binding polypeptide; and ii) at least one antigenic molecule wherein said preparation is for use as a vaccine.
  • the CD40 binding polypeptide and antigenic molecule are encapsulated in a single liposome preparation.
  • Encapsulated is intended to indicate a physical association of the encapsulated substance with a liposome vesicle. This could be an association with the outer hydrophobic lipid moiety or more likely located in the internal hydrophilic core. The association may be via electrostatic interactions or covalent linkage.
  • Liposomes are lipid based vesicles which encapsulate a selected agent which is then introduced into a subject. The liposome is manufactured either from pure phospholipid or a mixture of phospholipid and phosphoglyceride. Typically, liposomes can be manufactured with diameters of less than 200nm. Furthermore the biochemical nature of liposomes confers permeability across blood vessel membranes to gain access to selected tissues. Liposomes typically contain an aqueous core which can contain a therapeutic agent or the like, e.g. antigen.
  • said liposome preparation comprises at least two antigenic molecules.
  • said preparation comprises first and second liposome populations wherein said first population comprises encapsulated CD40 binding protein and said second population comprises an encapsulated antigenic molecule.
  • said first liposome population consists of a CD40 binding polypeptide.
  • said second liposome population comprises of at least two antigenic molecules.
  • said CD40 binding polypeptide and said two antigenic molecules are encapsulated in a liposome.
  • At least one antigenic molecule is a polypeptide.
  • polypeptide is a polypeptide isolated from a virus.
  • said viral polypeptide is isolated from a viral pathogen selected from the group consisting of: Human Immunodeficiency Virus (HIV1 & 2); Human T Cell
  • Leukaemia Virus (HTLV 1 & 2); Ebola virus; human papilloma virus (e.g. HPV-2, HPV-5, HPV-8 HPV-16, HPV-18, HPV-31 , HPV-33, HPV-52, HPV-54 and HPV-56); papovavirus; rhinovirus; poliovirus; herpesvirus; adenovirus; Epstein Barr virus; influenza virus, hepatitis B and C viruses.
  • human papilloma virus e.g. HPV-2, HPV-5, HPV-8 HPV-16, HPV-18, HPV-31 , HPV-33, HPV-52, HPV-54 and HPV-56
  • papovavirus e.g. HPV-2, HPV-5, HPV-8 HPV-16, HPV-18, HPV-31 , HPV-33, HPV-52, HPV-54 and HPV-56
  • papovavirus e.g. HPV-2, HPV-5, HPV
  • polypeptide is a polypeptide isolated from a bacterial pathogen.
  • said polypeptide is isolated from a bacterial pathogen selected from the group consisting of: Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Mycobacterium tuberculosis; Streptococcus group B; Streptoccocus pneumoniae; Helicobacter pylori; Neisseria gonorrhea; Streptococcus group A; Borrelia burgdorferi; Coccidiodes immitis; Histoplasma sapsulatum; Neisseria meningitidis ; Shigella flexneri; Escherichia coli; Haemophilus influenzae; Clostridium tetani; Bordetella pertussis; Listeria monocytogenes; Corynebacterium diptheriae.
  • a bacterial pathogen selected from the group consisting of: Staphylococcus aureus; Staphylococcus epidermidis; Enterococc
  • polypeptide is tetanus toxoid or diptheria toxoid.
  • polypeptide is a polypeptide isolated from a parasitic pathogen.
  • polypeptide is isolated from a parasite selected from the group consisting of: Trypanosoma spp, Leishmania spp, Schistosoma spp or Plasmodium spp.
  • polypeptide is a polypeptide isolated from a fungal pathogen.
  • polypeptide is isolated from a yeast pathogen, for example of the genus Candida spp, preferably the species Candida albicans.
  • said polypeptide is a polypeptide associated with a disease; preferably said disease associated polypeptide is a cancer specific polypeptide.
  • cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • cancer includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • sarcoma is art recognized and refers to malignant tumors of mesenchymal derivation.
  • tumour rejection precursors are known in the art and are predominantly expressed by cancer cells.
  • the tumour rejection antigens MAGE, BAGE, GAGE and DAGE families of tumour rejection antigens see Schulz et al Proc Natl Acad Sci USA, 1991, 88, pp991-993).
  • These precursor proteins are expressed by cancer cells and are typically processed to smaller peptides that act as tumour rejection antigens. These typically are nonapeptides but can vary in size from 8-30 amino acids.
  • said antigenic molecule is a peptide.
  • said peptide is 8-30 amino acids in length.
  • said peptide is 8-18 amino acids in length; preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acids in length.
  • said peptide comprises a tumour rejection antigen.
  • at least one antigenic molecule is a polysaccharide; preferably a capsular bacterial polysaccharide antigen.
  • the capsule is a protective structure that surrounds the surface of many bacteria.
  • the polysaccharides that form the capsule are variable in structure, for example Streptococcus pneumoniae have at least 80 capsular serotypes. These capsular polysaccharides are major antigenic determinants.
  • said capsular polysaccharide is S.pneumoniae PS3 and/or PS14 or a combination thereof.
  • said antigenic molecule is a lipopolysaccharide.
  • said preparation comprises two or more antigenic molecules isolated from a pathogen to provide a multivalent vaccine preparation; preferably said multivalent vaccine is a mixture of polypeptide and/or peptide and/or polysaccharide provided as an admixture.
  • said preparation comprises two or more disease specific antigenic molecules to provide a multivalent vaccine preparation; preferably said multivalent vaccine is a mixture of polypeptide and/or peptide and/or polysaccharide and/or glycosphingolipid (ganglioside) provided as an admixture.
  • said multivalent vaccine is a mixture of polypeptide and/or peptide and/or polysaccharide and/or glycosphingolipid (ganglioside) provided as an admixture.
  • said disease is cancer.
  • said multivalent vaccine comprises antigenic molecules isolated from different pathogens to provide a multivalent vaccine that is protective against two or more disease pathogens.
  • said CD40 binding polypeptide is an antibody; preferably a monoclonal antibody, or active binding part thereof.
  • said antibody is a humanised or chimeric antibody.
  • a chimeric antibody is produced by recombinant methods to contain the variable region of an antibody with an invariant or constant region of a human antibody.
  • a humanised antibody is produced by recombinant methods to combine the complementarity determining regions (CDRs) of an antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody.
  • CDRs complementarity determining regions
  • Chimeric antibodies are recombinant antibodies in which ail of the V-regions of a mouse or rat antibody are combined with human antibody C-regions.
  • Humanised antibodies are recombinant hybrid antibodies which fuse the complimentarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V- regions. The C-regions from the human antibody are also used.
  • the complimentarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V- region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.
  • a Fab fragment is a multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, covalently coupled together and capable of specifically binding to an antigen.
  • Fab fragments are generated via proteolytic cleavage (with, for example, papain) of an intact immunoglobulin molecule.
  • a Fab 2 fragment comprises two joined Fab fragments. When these two fragments are joined by the immunoglobulin hinge region, a F(ab') 2 fragment results.
  • An Fv fragment is multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically binding to an antigen.
  • a fragment could also be a single chain polypeptide containing only one light chain variable region, or a fragment thereof that contains the three CDRs of the light chain variable region, without an associated heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi specific antibodies formed from antibody fragments, this has for example been described in US patent No 6,248,516.
  • Fv fragments or single region (domain) fragments are typically generated by expression in host cell lines of the relevant identified regions.
  • immunoglobulin or antibody fragments are within the scope of the invention and are described in standard immunology textbooks such as Paul, Fundamental Immunology or Janeway et al. lmmunobiology (cited above). Molecular biology now allows direct synthesis (via expression in cells or chemically) of these fragments, as well as synthesis of combinations thereof. A fragment of an antibody or immunoglobulin can also have bispecific function as described above.
  • domain antibodies are the smallest binding part of an antibody (approximately 13kDa). Examples of this technology is disclosed in US6, 248, 516, US6, 291 , 158, US6.127, 197 and EP0368684 which are all incorporated by reference in their entirety.
  • said CD40 binding polypeptide is CD154, the natural ligand for CD40.
  • said CD40 binding polypeptide is mycobacterial heat shock protein 70, or a fragment thereof.
  • mycobacterial heat shock proteins available to the skilled man.
  • said CD 40 binding protein is a nematode polyladder protein or a fragment thereof.
  • said liposome preparation includes a carrier and optionally a further adjuvant.
  • said adjuvant is selected from the group consisting of: cytokines selected from the group consisting of GMCSF, interferon gamma, interferon alpha, interferon beta, interleukin 12, interleukin 23, interleukin 17, interleukin2,interleukin1 ,TGF, TNF ⁇ , TNF ⁇ .
  • said adjuvant is a CD28 binding polypeptide.
  • binding polypeptide is an antibody, preferably a monoclonal antibody or binding fragment thereof.
  • said polypeptide is the natural ligand of CD28, CD80/CD86, or modified variants thereof that bind CD28.
  • said adjuvant is a TLR agonist such as CpG oligonucleotides, flagellin, monophosphoryl lipid A, poly I:C and derivatives thereof.
  • said adjuvant is a bacterial cell wall derivative such as muramyl dipeptide (MDP) and/or trehelose dycorynemycolate (TDM)
  • MDP muramyl dipeptide
  • TDM trehelose dycorynemycolate
  • a carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter.
  • Some antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid.
  • Such antigens contain B-cell epitopes but no T cell epitopes.
  • the protein moiety of such a conjugate (the "carrier” protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B- cells to differentiate into plasma cells and produce antibody against the antigen.
  • Helper T-cells can also stimulate other immune cells such as cytotoxic T-cells, and a carrier can fulfil an analogous role in generating cell-mediated immunity as well as antibodies.
  • Certain antigens which lack T-cell epitopes such as polymers with a repeating B-cell epitope (e.g. bacterial polysaccharides), are intrinsically immunogenic to a limited extent. These are known as T-independent antigens. Such antigens benefit from association with a carrier such as tetanus toxoid, under which circumstance they elicit much stronger antibody responses.
  • Carrier conjugation of bacterial polysaccharides is used to produce a number of 'conjugate vaccines' against bacterial infections such as Haemophilus influenzae (Hib) and group-C meningococci.
  • a process for the formation of a liposome preparation comprising: i) forming a first liposome preparation comprising a CD40 binding polypeptide; ii) forming a second preparation comprising at least one antigenic molecule; iii) contacting first and second preparations under conditions that allow the fusion of said preparations to provide combined liposome preparation wherein said first and second preparations fuse to form a liposome preparation wherein the CD40 binding polypeptide and antigenic molecule are substantially encapsulated in said combined liposome preparation.
  • said antigenic molecule us selected from the group consisting of: a polypeptide, a peptide, polysaccharide or a lipopolysaccharide molecule.
  • a method to vaccinate a subject with respect to at least one disease or condition comprising administering an effective amount of a liposome preparation according to the invention to said subject.
  • said disease is caused by a viral pathogen.
  • said disease is caused by a bacterial pathogen.
  • said disease is caused by a parasitic pathogen.
  • said disease is caused by a fungal pathogen, for example Candida spp.
  • said disease is cancer
  • a method to reduce toxicity of a vaccine in a subject comprising: i) forming a first liposome preparation comprising a CD40 binding polypeptide; ii) forming a second preparation comprising at least one antigenic molecule; iii) contacting first and second preparations under conditions that allow the fusion of said preparations to provide combined liposome preparation wherein said first and second preparations fuse to form a liposome preparation wherein the CD40 binding polypeptide and antigenic molecule are substantially encapsulated in said combined liposome preparation; and iv) administering the fused preparation to a subject requiring immunisation against an infectious or non-infectious disease.
  • kits comprising: i) CD40 binding protein; ii) at least two antigenic molecules; and iii) liposomes; and optionally a list of instructions detailing the formation of liposome preparations according to the invention.
  • Figure 1 Retention of CD40 binding by liposomally entrapped anti-CD40 antibody; ELISA plates were coated with anti-human IgG, blocked with 3% BSA, and then incubated with recombinant murine CD40-human IgGI (R&D systems). After washing, various liposomal preparations containing entrapped pneumococcal type 3 polsaccharide (PS) and/or CD40 mAb at varying concentrations (6 or 20 ⁇ g per ml) were added to the plate in two-fold dilutions. Liposomal binding to CD40-was detected using HRP labelled goat anti-rat IgG;
  • PS pneumococcal type 3 polsaccharide
  • FIG. 2 To assess liposomal binding to cell surface CD40, CD154 transfected (filled histograms) or CD40 transfected (open histograms) L929 cells were stained with liposomal preparations at a 1/10 dilution in FACS buffer. Binding was detected using FITC labelled anti-rat IgG.
  • Figure 3 Responses at day 30 to PS3 in mice immunised once with liposomes incorporating PS3, PS14 and CD40 (solid lines), or PS14, PS3 and control mAb (broken lines)
  • Figure 4 Recovery of type 3 pneumococcal colony forming units from lungs one day after challenge with 10 5 colony forming units of type 3 Streptococcus pneumoniae, and 5 months after immunisation. Each dot represents one mouse. Left, Control antibody liposomes, middle, CD40 antibody liposomes, right, PBS immunised.
  • Figure 5 Immune responses against Tetanus toxoid induced by TT/DT co-encapsulated in liposomes (3 ⁇ g DT, 1 ⁇ g TT) along with either 10 ⁇ g or 1mg of CD40mAb (solid lines) or isotype control mAb (dashed lines). On the right hand side the effect of 1 mg of free antibody mixed with 1 ⁇ g TT are shown. All results day 14 post-injection except for the bottom graph, which is day 28
  • Figure 6 Increase in spleen size following immunisation with liposomally entrapped or free CD40 antibodies.
  • the equivalents of 2, 10 and 50ug of antibody were injected intraperitoneally into mice.
  • the percentage increase in spleen weight (in comparison with controls) at day 5 post injection is shown.
  • Free antibody hatched bars; liposomally entrapped, stippled bars.
  • Figure 7 Responses at day 30 to PS3 in mice immunised once with a mixture of liposomes incorporating PS3 and liposomes incorporating CD40 (solid lines), or liposomes incorporating PS3 and liposomes incorporating control mAb (broken lines)
  • the lipidic materials Egg PC (99% purity, Avant Product Number: 840051) and DOPE (Avanti Product number: 850725) were obtained from Avanti ® Polar Lipids, whilst DOTAP was obtained from Merck Chemicals Ltd.
  • Pneumococcal polysaccharide type 3 powder PS, ATTC Number: 169-X
  • antibody materials anti CD40 antibody Barr TA and Heath AW (2001) Immunology 102, 39-43)
  • control isotype matched antibody (ref CRL-2382, ATCC) were obtained from Dr A Heath University of Sheffield.
  • CRM 197 and tetanus toxoid were provided by Dr Peter Laing.
  • liposomes incorporating CD40 antibody (population 1) and one or more antigens (population 2) can be prepared by methods described above, such as the dehydration-rehydration method; and if the liposomal preparations are produced from (preferably) phosphatidyl ethanolamine and palmitoylhomocysteine (at 8:2 molar ratio) along with 40% cholesterol, then the liposomal populations can be fused by reduction of the pH of the liposome suspension to 4.5 with vigorous mixing for two minutes, followed by neutralisation. pH reduction could be by a number of means, most simply by the addition of concentrated HCI with neutralisation by the addition of concentrated NaOH. Liposomes constructed from other materials will also fuse under acid conditions, but with efficiencies which vary depending on the lipid content. The presence of cholesterol helps to prevent leakage of the liposome contents.
  • the anti-mouse CD40 antibody 10C8 (rat IgGI) has been described previously (Barr and Heath).
  • the hybridoma producing the isotype control (20C2, anti-human IL12) was purchased from ATCC. Hybridomas were grown in bioreactors and antibody purified from the supernatant by protein G affinity by Sheffield Antibody Resource Centre. Immunisation
  • mice BALB/c female mice, aged 8-10 weeks (Harlan UK) were immunised sub-cutaneously with liposomal preparations as described for each experiment.
  • ELISA plates were coated overnight with goat anti-human IgG (BD Pharmingen) at 10 ⁇ g/ml in PBS. They were then blocked with 3% BSA, and incubated at room temperature with recombinant murine CD40-human IgGI (1 ⁇ g/mL, R&D systems) for 30 minutes, washed with PBS-Tween, and then incubated at RT with doubling dilutions of liposomal preparations in PBS. After 30 minutes, plares were washed again, and incubated with HRP labelled goat anti-rat IgG for 30 minutes. After further washes liposomal binding to CD40 was assessed following the addition of substrate. There was no binding of the anti-human coating reagent to either rat IgG, or anti-rat IgG (or vice- versa).
  • ELISA plates (Costar) were coated with polysaccharides or proteins at 10 ⁇ g/ml on carbonate buffer overnight at 4 C. Plates were blocked for one hour with 1% fish gelatin
  • Murine L929 fibroblasts stably transfected with either CD40 or CD154 (REF) were stained with liposomes as follows. Cells (10 6 per tube) were pelleted in FACS buffer
  • CD40 antibody conjugates are thought to bind directly to antigen specific B cells, and it was unknown whether simple liposomal formulation of antibody would allow it to bind CD40 from the liposomal surface
  • assessments were performed to assess the effectiveness of simple liposomal formulations, produced without the need for antibody-lipid covalent attachment, in binding to CD40.
  • Assessment of CD40 binding by the liposomes was performed by flow cytometric analysis on CD40 transfected or normal L929 cells, and by ELISA assay using plates coated with recombinant murine CD40-Fc, and with detection in both cases by anti-rat conjugates (the antibody is rat anti-mouse CD40).
  • the two assays were consistent in that the CD40mAb conjugated liposomes clearly were able to bind to both recombinant CD40-Fc ( Figure 1a), and to cell expressed CD40 (Fig 1b).
  • type 3 pneumococcus is one of the most virulent strains, and type 14 is one of the most prevalent.
  • Mice were immunised with liposomes containing 10 ⁇ g of antibody and of each polysaccharide, and then antibody responses against PS3 and PS14 assayed by ELISA.
  • mice immunised with PS3/PS14/CD40 or the equivalent control liposomes were challenged 5 months later by intratracheal instillation of (10 5 ) live type 3 pneumococci. Twenty-four hours later blood and lung samples were taken in order to assess levels of bacteraemia and bacterial lung counts. There was no bacteraemia in any of the animals, however two of the five CD40/PS3/PS14 animals were clearly protected against lung colonisation with bacterial numbers either undetectable, or extremely low (see figure below, showing bacterial numbers recovered from the lungs). None of the control animals were protected.
  • Tetanus toxoid (TT) and diptheria toxoid (DT), or the mutant form of diptheria toxin, CRM197 are currently administered as components of multivalent formulations, such as DTP (along with a pertussis component) or DTP-Hib (with pertussis and Haemophilus influenzae conjugate). These vaccines are administered many times in order to achieve and maintain sufficient immune responses (the DT components are given five times in the UK before age 18). Clearly improvement in the immunogenicity of the DT components, which could lead to a reduction in the number of doses required would be highly desirable.

Abstract

We describe a liposome preparation comprising an adjuvant and antigen for use in vaccination and also a method for the formulation of the preparation.

Description

Liposome Preparation
The invention relates to a liposome preparation comprising an adjuvant and antigen for use in vaccination and also a method for the formulation of the preparation.
Vaccines protect against a wide variety of infectious diseases. There are also vaccines in development for the treatment of various non-infectious diseases such as autoimmune and neurodegenerative diseases and various cancers. Many vaccines are produced by inactivated or attenuated pathogens which are injected into an individual. The immunised individual responds by producing both a humoral and cellular response. For example, some influenza vaccines are made by inactivating the virus by chemical treatment with formaldehyde, likewise the SaIk polio vaccine comprises whole virus inactivated with propionolactone For many pathogens chemical or heat inactivation while it may give rise to vaccine immunogens that confer protective immunity also gives rise to side effects such as fever and injection site reactions. In the case of bacteria, inactivated organisms tend to be so toxic that side effects have limited the application of such crude vaccine immunogens (e.g. the cellular pertussis vaccine). Many modern vaccines are therefore made from protective antigens of the pathogen, separated by purification or molecular cloning from the materials that give rise to side-effects. These latter vaccines are known as 'subunit vaccines'.
The development of subunit vaccines has been the focus of considerable research in recent years. The emergence of new pathogens and the growth of antibiotic resistance have created a need to develop new vaccines and to identify further candidate molecules useful in the development of subunit vaccines. Likewise the discovery of novel vaccine antigens from genomic and proteomic studies is enabling the development of new subunit vaccine candidates, particularly against bacterial pathogens and cancers. However, although subunit vaccines tend to avoid the side effects of killed or attenuated pathogen vaccines, their 'pure' status means that subunit vaccines do not always have adequate immunogenicity. Many candidate subunit vaccines have failed in clinical trials in recent years that might otherwise have succeeded were a suitable adjuvant available to enhance the immune response to the purified antigen. An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells. The receptor CD40 plays an important co-stimulatory role in the activation of B-cells during the cognate interaction of antigen-specific T and B-cells that gives rise to an antibody response. The CD40 signal is pivotal to the expression of T cell help and immunoglobulin class-switching in both humans and mice (9-12). In addition to its importance in T and B-cell interactions, ligation of CD40 is also very important in activation of macrophages and of dendritic cells to express co-stimulatory antigens and thus in the generation of helper T cell priming by these antigen-presenting cells (13-15). In recent studies we have shown that ligation of CD40 by antibodies can effectively replace the CD40 signals ordinarily made during intercellular interaction in the immune response (see WO03/063899; WO2004/052396 and WO2004/041866 the contents of which are incorporated by reference in their entirety). We have shown that large doses of anti-CD40 (500μg/mouse) are able to induce strong, class-switched antibody responses against T independent (Tl) antigens including pneumococcal polysaccharides (1,2) and to a lesser extent, T cell dependent antigens when injected mixed with antigen. We therefore sought a means: i) to reduce the dose of antibody required; and ii) to enhance the adjuvant effect. We found that by joining together a stimulatory CD40- polypeptide, for example an antibody with antigen (either covalently or non-covalently) we can achieve both of these aims together, using 500-fold less antibody, generating a surprisingly effective antigen-specific stimulus.
Unfortunately the production of antibody antigen conjugates is a process which may be difficult to control, and which has to be specially adapted for each antigen. We have therefore sought an alternative method which would allow for the close-association of CD40 antibody and antigen, without the need for physical conjugation.
Vaccines increasingly are required to be 'multivalent' (e.g. containing antigens from several different strains of a pathogen as for influenza virus and the polysaccharide vaccines against S. pneumoniae), or containing multiple proteins from a single pathogen that are additive or synergistic in the protective immune response they generate (as is the case for a number of vaccines under development - e.g. for H. pylori, tuberculosis etc.). There is also an economic and public health case for combining vaccines against different pathogens in a 'multivalent' format (e.g. DTP-Hib) in order to increase uptake by the population, and to minimise the increasing 'aluminium burden' associated with the introduction of new vaccines based on alum as adjuvant. A reduction in the total number of injections required to induce protection against the widest possible range of infections also reduces the costs of vaccination programmes, and is more profitable for the manufacturers, and these are additional factors driving the move to multivalent vaccines.
Subunit vaccines based upon recombinant proteins or synthetic peptides have very much reduced immunogenicity compared with whole cell vaccines, and require more potent adjuvants than are currently available to enable them to work effectively. Pure polysaccharide antigens are ineffective in major target populations, and polysaccharide conjugate vaccines are expensive to produce, especially in a multivalent form which is often required. We have shown that conjugation of CD40 antibody with protein antigens is an effective means of enhancing immunogenicty, and we have also shown that high doses of CD40 antibody can enhance the response to T-independent, polysaccharide antigens. Unfortunately the conjugation of CD40 mab to antigen is a relatively costly process and relatively toxic.
We disclose a method for producing "virtual conjugates" of CD40 binding proteins, for example a CD40 specific antibody, with antigen, which produce enhanced immunogenicity in the absence of physical conjugation, and which also allow for the production of multivalent vaccines. The method for producing these "virtual conjugates" was to entrap antibody and antigen together in liposomes. A further potential advantage of this approach is the additional adjuvant effect already present in the liposomal formulation of antigens.
According to an aspect of the invention there is provided a liposome preparation comprising: i) a CD40 binding polypeptide; and ii) at least one antigenic molecule wherein said preparation is for use as a vaccine.
Preferably the CD40 binding polypeptide and antigenic molecule are encapsulated in a single liposome preparation.
"Encapsulated" is intended to indicate a physical association of the encapsulated substance with a liposome vesicle. This could be an association with the outer hydrophobic lipid moiety or more likely located in the internal hydrophilic core. The association may be via electrostatic interactions or covalent linkage. Liposomes are lipid based vesicles which encapsulate a selected agent which is then introduced into a subject. The liposome is manufactured either from pure phospholipid or a mixture of phospholipid and phosphoglyceride. Typically, liposomes can be manufactured with diameters of less than 200nm. Furthermore the biochemical nature of liposomes confers permeability across blood vessel membranes to gain access to selected tissues. Liposomes typically contain an aqueous core which can contain a therapeutic agent or the like, e.g. antigen.
In a preferred embodiment of the invention said liposome preparation comprises at least two antigenic molecules.
In a preferred embodiment of the invention said preparation comprises first and second liposome populations wherein said first population comprises encapsulated CD40 binding protein and said second population comprises an encapsulated antigenic molecule.
In a preferred embodiment of the invention said first liposome population consists of a CD40 binding polypeptide.
In a further preferred embodiment of the invention said second liposome population comprises of at least two antigenic molecules.
In a preferred embodiment of the invention said CD40 binding polypeptide and said two antigenic molecules are encapsulated in a liposome.
In a preferred embodiment of the invention at least one antigenic molecule is a polypeptide.
In a preferred embodiment of the invention said polypeptide is a polypeptide isolated from a virus.
Preferably said viral polypeptide is isolated from a viral pathogen selected from the group consisting of: Human Immunodeficiency Virus (HIV1 & 2); Human T Cell
Leukaemia Virus (HTLV 1 & 2); Ebola virus; human papilloma virus (e.g. HPV-2, HPV-5, HPV-8 HPV-16, HPV-18, HPV-31 , HPV-33, HPV-52, HPV-54 and HPV-56); papovavirus; rhinovirus; poliovirus; herpesvirus; adenovirus; Epstein Barr virus; influenza virus, hepatitis B and C viruses.
In a further preferred embodiment of the invention said polypeptide is a polypeptide isolated from a bacterial pathogen.
Preferably said polypeptide is isolated from a bacterial pathogen selected from the group consisting of: Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Mycobacterium tuberculosis; Streptococcus group B; Streptoccocus pneumoniae; Helicobacter pylori; Neisseria gonorrhea; Streptococcus group A; Borrelia burgdorferi; Coccidiodes immitis; Histoplasma sapsulatum; Neisseria meningitidis ; Shigella flexneri; Escherichia coli; Haemophilus influenzae; Clostridium tetani; Bordetella pertussis; Listeria monocytogenes; Corynebacterium diptheriae.
In a preferred embodiment of the invention said polypeptide is tetanus toxoid or diptheria toxoid.
In a preferred embodiment of the invention said polypeptide is a polypeptide isolated from a parasitic pathogen.
Preferably said polypeptide is isolated from a parasite selected from the group consisting of: Trypanosoma spp, Leishmania spp, Schistosoma spp or Plasmodium spp.
In a further preferred embodiment of the invention said polypeptide is a polypeptide isolated from a fungal pathogen.
Preferably said polypeptide is isolated from a yeast pathogen, for example of the genus Candida spp, preferably the species Candida albicans.
In a yet further preferred embodiment of the invention said polypeptide is a polypeptide associated with a disease; preferably said disease associated polypeptide is a cancer specific polypeptide.
As used herein, the term "cancer" refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "cancer" includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "carcinoma" also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation.
Tumour rejection precursors are known in the art and are predominantly expressed by cancer cells. For example, the tumour rejection antigens MAGE, BAGE, GAGE and DAGE families of tumour rejection antigens, see Schulz et al Proc Natl Acad Sci USA, 1991, 88, pp991-993). These precursor proteins are expressed by cancer cells and are typically processed to smaller peptides that act as tumour rejection antigens. These typically are nonapeptides but can vary in size from 8-30 amino acids.
In preferred embodiment of the invention said antigenic molecule is a peptide.
In a preferred embodiment of the invention said peptide is 8-30 amino acids in length.
In a preferred embodiment of the invention said peptide is 8-18 amino acids in length; preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acids in length.
In a preferred embodiment of the invention said peptide comprises a tumour rejection antigen. In a further preferred embodiment of the invention at least one antigenic molecule is a polysaccharide; preferably a capsular bacterial polysaccharide antigen.
The capsule is a protective structure that surrounds the surface of many bacteria. The polysaccharides that form the capsule are variable in structure, for example Streptococcus pneumoniae have at least 80 capsular serotypes. These capsular polysaccharides are major antigenic determinants.
In a preferred embodiment of the invention said capsular polysaccharide is S.pneumoniae PS3 and/or PS14 or a combination thereof.
In a preferred embodiment of the invention said antigenic molecule is a lipopolysaccharide.
In a preferred embodiment of the invention said preparation comprises two or more antigenic molecules isolated from a pathogen to provide a multivalent vaccine preparation; preferably said multivalent vaccine is a mixture of polypeptide and/or peptide and/or polysaccharide provided as an admixture.
In a preferred embodiment of the invention said preparation comprises two or more disease specific antigenic molecules to provide a multivalent vaccine preparation; preferably said multivalent vaccine is a mixture of polypeptide and/or peptide and/or polysaccharide and/or glycosphingolipid (ganglioside) provided as an admixture.
Preferably said disease is cancer.
In a preferred embodiment of the invention said multivalent vaccine comprises antigenic molecules isolated from different pathogens to provide a multivalent vaccine that is protective against two or more disease pathogens.
In a preferred embodiment of the invention said CD40 binding polypeptide is an antibody; preferably a monoclonal antibody, or active binding part thereof.
Preferably said antibody is a humanised or chimeric antibody. A chimeric antibody is produced by recombinant methods to contain the variable region of an antibody with an invariant or constant region of a human antibody.
A humanised antibody is produced by recombinant methods to combine the complementarity determining regions (CDRs) of an antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody.
Chimeric antibodies are recombinant antibodies in which ail of the V-regions of a mouse or rat antibody are combined with human antibody C-regions. Humanised antibodies are recombinant hybrid antibodies which fuse the complimentarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V- regions. The C-regions from the human antibody are also used. The complimentarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V- region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.
Various fragments of immunoglobulins are known in the art, i.e., Fab, Fab2, F(ab')2l Fv, Fc, Fd, scFvs, etc. A Fab fragment is a multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, covalently coupled together and capable of specifically binding to an antigen. Fab fragments are generated via proteolytic cleavage (with, for example, papain) of an intact immunoglobulin molecule. A Fab2 fragment comprises two joined Fab fragments. When these two fragments are joined by the immunoglobulin hinge region, a F(ab')2 fragment results. An Fv fragment is multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically binding to an antigen. A fragment could also be a single chain polypeptide containing only one light chain variable region, or a fragment thereof that contains the three CDRs of the light chain variable region, without an associated heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi specific antibodies formed from antibody fragments, this has for example been described in US patent No 6,248,516. Fv fragments or single region (domain) fragments are typically generated by expression in host cell lines of the relevant identified regions. These and other immunoglobulin or antibody fragments are within the scope of the invention and are described in standard immunology textbooks such as Paul, Fundamental Immunology or Janeway et al. lmmunobiology (cited above). Molecular biology now allows direct synthesis (via expression in cells or chemically) of these fragments, as well as synthesis of combinations thereof. A fragment of an antibody or immunoglobulin can also have bispecific function as described above.
Alternatively said fragments are "domain antibody fragments". Domain antibodies are the smallest binding part of an antibody (approximately 13kDa). Examples of this technology is disclosed in US6, 248, 516, US6, 291 , 158, US6.127, 197 and EP0368684 which are all incorporated by reference in their entirety.
In an alternative preferred embodiment of the invention said CD40 binding polypeptide is CD154, the natural ligand for CD40.
In a further alternative preferred embodiment of the invention, said CD40 binding polypeptide is mycobacterial heat shock protein 70, or a fragment thereof.
There are many examples of mycobacterial heat shock proteins available to the skilled man. For example, Mycobacterium tuberculosis NCBl database entries P0A5B9 or CAA41306.
In yet another preferred embodiment of the invention said CD 40 binding protein.is a nematode polyladder protein or a fragment thereof.
In our co-pending application WO2004/064864 we disclose polypeptides with CD40 binding activity. We incorporate by reference specifically the nucleic acid and amino acid sequences of the polyladder proteins disclosed therein.
In a preferred embodiment of the invention said liposome preparation includes a carrier and optionally a further adjuvant.
In a preferred embodiment of the invention said adjuvant is selected from the group consisting of: cytokines selected from the group consisting of GMCSF, interferon gamma, interferon alpha, interferon beta, interleukin 12, interleukin 23, interleukin 17, interleukin2,interleukin1 ,TGF, TNF α, TNF β. In an alternative preferred embodiment of the invention said adjuvant is a CD28 binding polypeptide.
Preferably said binding polypeptide is an antibody, preferably a monoclonal antibody or binding fragment thereof.
In a preferred embodiment of the invention said polypeptide is the natural ligand of CD28, CD80/CD86, or modified variants thereof that bind CD28.
In a further alternative embodiment of the invention said adjuvant is a TLR agonist such as CpG oligonucleotides, flagellin, monophosphoryl lipid A, poly I:C and derivatives thereof.
In a preferred embodiment of the invention said adjuvant is a bacterial cell wall derivative such as muramyl dipeptide (MDP) and/or trehelose dycorynemycolate (TDM)
The term carrier is construed in the following manner. A carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter. Some antigens are not intrinsically immunogenic yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid. Such antigens contain B-cell epitopes but no T cell epitopes. The protein moiety of such a conjugate (the "carrier" protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B- cells to differentiate into plasma cells and produce antibody against the antigen. Helper T-cells can also stimulate other immune cells such as cytotoxic T-cells, and a carrier can fulfil an analogous role in generating cell-mediated immunity as well as antibodies. Certain antigens which lack T-cell epitopes, such as polymers with a repeating B-cell epitope (e.g. bacterial polysaccharides), are intrinsically immunogenic to a limited extent. These are known as T-independent antigens. Such antigens benefit from association with a carrier such as tetanus toxoid, under which circumstance they elicit much stronger antibody responses. Carrier conjugation of bacterial polysaccharides is used to produce a number of 'conjugate vaccines' against bacterial infections such as Haemophilus influenzae (Hib) and group-C meningococci.
According to a further aspect of the invention there is provided a process for the formation of a liposome preparation comprising: i) forming a first liposome preparation comprising a CD40 binding polypeptide; ii) forming a second preparation comprising at least one antigenic molecule; iii) contacting first and second preparations under conditions that allow the fusion of said preparations to provide combined liposome preparation wherein said first and second preparations fuse to form a liposome preparation wherein the CD40 binding polypeptide and antigenic molecule are substantially encapsulated in said combined liposome preparation.
Methods to fuse liposome preparations are known in the art; for example see MoI
Membr Biol 1999 16 279-296; Connor et at. Proc Nat Acad Sciences (USA) 1984 81 1715-1718 both of which are incorporated by reference in their entirety.
In a preferred method of the invention said antigenic molecule us selected from the group consisting of: a polypeptide, a peptide, polysaccharide or a lipopolysaccharide molecule.
According to an aspect of the invention there is provided a method to vaccinate a subject with respect to at least one disease or condition comprising administering an effective amount of a liposome preparation according to the invention to said subject.
In a preferred method of the invention said disease is caused by a viral pathogen.
In an alternative preferred method of the invention said disease is caused by a bacterial pathogen.
In a further preferred method of the invention said disease is caused by a parasitic pathogen.
In a yet further preferred method of the invention said disease is caused by a fungal pathogen, for example Candida spp.
In a further preferred method of the invention said disease is cancer.
Preferably said subject is human. According to a further aspect of the invention there is provided a method to reduce toxicity of a vaccine in a subject comprising: i) forming a first liposome preparation comprising a CD40 binding polypeptide; ii) forming a second preparation comprising at least one antigenic molecule; iii) contacting first and second preparations under conditions that allow the fusion of said preparations to provide combined liposome preparation wherein said first and second preparations fuse to form a liposome preparation wherein the CD40 binding polypeptide and antigenic molecule are substantially encapsulated in said combined liposome preparation; and iv) administering the fused preparation to a subject requiring immunisation against an infectious or non-infectious disease.
According to a further aspect of the invention there is provided a kit comprising: i) CD40 binding protein; ii) at least two antigenic molecules; and iii) liposomes; and optionally a list of instructions detailing the formation of liposome preparations according to the invention.
Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. An embodiment of the invention will now be described by example only and with reference to the following figures:
Figure 1: Retention of CD40 binding by liposomally entrapped anti-CD40 antibody; ELISA plates were coated with anti-human IgG, blocked with 3% BSA, and then incubated with recombinant murine CD40-human IgGI (R&D systems). After washing, various liposomal preparations containing entrapped pneumococcal type 3 polsaccharide (PS) and/or CD40 mAb at varying concentrations (6 or 20 μg per ml) were added to the plate in two-fold dilutions. Liposomal binding to CD40-was detected using HRP labelled goat anti-rat IgG;
Figure 2: To assess liposomal binding to cell surface CD40, CD154 transfected (filled histograms) or CD40 transfected (open histograms) L929 cells were stained with liposomal preparations at a 1/10 dilution in FACS buffer. Binding was detected using FITC labelled anti-rat IgG.
Figure 3: Responses at day 30 to PS3 in mice immunised once with liposomes incorporating PS3, PS14 and CD40 (solid lines), or PS14, PS3 and control mAb (broken lines)
Figure 4: Recovery of type 3 pneumococcal colony forming units from lungs one day after challenge with 105 colony forming units of type 3 Streptococcus pneumoniae, and 5 months after immunisation. Each dot represents one mouse. Left, Control antibody liposomes, middle, CD40 antibody liposomes, right, PBS immunised.
Figure 5: Immune responses against Tetanus toxoid induced by TT/DT co-encapsulated in liposomes (3μg DT, 1μg TT) along with either 10μg or 1mg of CD40mAb (solid lines) or isotype control mAb (dashed lines). On the right hand side the effect of 1 mg of free antibody mixed with 1μg TT are shown. All results day 14 post-injection except for the bottom graph, which is day 28
Figure 6: Increase in spleen size following immunisation with liposomally entrapped or free CD40 antibodies. The equivalents of 2, 10 and 50ug of antibody were injected intraperitoneally into mice. The percentage increase in spleen weight (in comparison with controls) at day 5 post injection is shown. Free antibody, hatched bars; liposomally entrapped, stippled bars. Figure 7 Responses at day 30 to PS3 in mice immunised once with a mixture of liposomes incorporating PS3 and liposomes incorporating CD40 (solid lines), or liposomes incorporating PS3 and liposomes incorporating control mAb (broken lines)
Materials and Methods
Production of liposomes
The lipidic materials Egg PC (99% purity, Avant Product Number: 840051) and DOPE (Avanti Product number: 850725) were obtained from Avanti® Polar Lipids, whilst DOTAP was obtained from Merck Chemicals Ltd. Pneumococcal polysaccharide type 3 powder (PS, ATTC Number: 169-X) and antibody materials anti CD40 antibody (Barr TA and Heath AW (2001) Immunology 102, 39-43) and control isotype matched antibody (ref CRL-2382, ATCC) were obtained from Dr A Heath University of Sheffield. CRM 197 and tetanus toxoid were provided by Dr Peter Laing.
Fusion of pre-formed liposomes Separate populations of liposomes incorporating CD40 antibody (population 1) and one or more antigens (population 2) can be prepared by methods described above, such as the dehydration-rehydration method; and if the liposomal preparations are produced from (preferably) phosphatidyl ethanolamine and palmitoylhomocysteine (at 8:2 molar ratio) along with 40% cholesterol, then the liposomal populations can be fused by reduction of the pH of the liposome suspension to 4.5 with vigorous mixing for two minutes, followed by neutralisation. pH reduction could be by a number of means, most simply by the addition of concentrated HCI with neutralisation by the addition of concentrated NaOH. Liposomes constructed from other materials will also fuse under acid conditions, but with efficiencies which vary depending on the lipid content. The presence of cholesterol helps to prevent leakage of the liposome contents.
Antibody
The anti-mouse CD40 antibody 10C8 (rat IgGI) has been described previously (Barr and Heath). The hybridoma producing the isotype control (20C2, anti-human IL12) was purchased from ATCC. Hybridomas were grown in bioreactors and antibody purified from the supernatant by protein G affinity by Sheffield Antibody Resource Centre. Immunisation
BALB/c female mice, aged 8-10 weeks (Harlan UK) were immunised sub-cutaneously with liposomal preparations as described for each experiment.
ELISA assays
CD40 binding of liposomes
ELISA plates were coated overnight with goat anti-human IgG (BD Pharmingen) at 10μg/ml in PBS. They were then blocked with 3% BSA, and incubated at room temperature with recombinant murine CD40-human IgGI (1 μg/mL, R&D systems) for 30 minutes, washed with PBS-Tween, and then incubated at RT with doubling dilutions of liposomal preparations in PBS. After 30 minutes, plares were washed again, and incubated with HRP labelled goat anti-rat IgG for 30 minutes. After further washes liposomal binding to CD40 was assessed following the addition of substrate. There was no binding of the anti-human coating reagent to either rat IgG, or anti-rat IgG (or vice- versa).
Anti-polvsaccharide and DT/TT responses
ELISA plates (Costar) were coated with polysaccharides or proteins at 10μg/ml on carbonate buffer overnight at 4 C. Plates were blocked for one hour with 1% fish gelatin
(Sigma), and after washing with PBS-Tween, serial dilutions of immune sera were made. After a furtherih incubation at RT, and washing, HRP labelled goat anti-mouse immunoglobulin, or anti-mouse IgG were added and the plates incubated for a further hour prior to final wash and the addition of substrate. After 20 minutes incubation plates were read in an Anthos Labtec EIA plate reader.
Flow cytometry
Murine L929 fibroblasts stably transfected with either CD40 or CD154 (REF) were stained with liposomes as follows. Cells (106 per tube) were pelleted in FACS buffer
(PBS, 3% BSA) and were resuspended in liposomal preparations diluted 1/10 in FACS buffer. After 30 min incubation at 4C, the cells were washed twice with FACS buffer, and were then incubated with FITC labelled anti-rat IgG (BD Pharmingen) at 10μg/ml in
FACS buffer for 30 minutes at 4 C, washed twice in FACS buffer and then analysed on a FACScalibur™ analyser (Becton Dickinson) running Cellquest™ software . EXAMPLES
Liposomal entrapment of CD40mabs
Because CD40 antibody conjugates are thought to bind directly to antigen specific B cells, and it was unknown whether simple liposomal formulation of antibody would allow it to bind CD40 from the liposomal surface, initial experiments were performed to assess the effectiveness of simple liposomal formulations, produced without the need for antibody-lipid covalent attachment, in binding to CD40. Assessment of CD40 binding by the liposomes was performed by flow cytometric analysis on CD40 transfected or normal L929 cells, and by ELISA assay using plates coated with recombinant murine CD40-Fc, and with detection in both cases by anti-rat conjugates (the antibody is rat anti-mouse CD40). The two assays were consistent in that the CD40mAb conjugated liposomes clearly were able to bind to both recombinant CD40-Fc (Figure 1a), and to cell expressed CD40 (Fig 1b).
Co-entrapment of polysaccharide antigens with CD40 mAbs
There are over 90 different pneumoccocal capsular polysaccharide types. We incorporated type 3 and type 14 (PS3 and PS14) into multilamellar DRV liposomes. These polysaccharides were chosen as type 3 pneumococcus is one of the most virulent strains, and type 14 is one of the most prevalent. Mice were immunised with liposomes containing 10μg of antibody and of each polysaccharide, and then antibody responses against PS3 and PS14 assayed by ELISA. Antibody (IgG) responses against PS3 30 days after a single immunisation with CD40/PS3/PS14 liposomes, were very strongly enhanced in comparison with responses generated by control antibody/PS3/PS14 liposomes. There was no significant difference in IgM responses against PS3, but in vaccination against T independent antigens a strong IgG response is highly desirable. In addition, obtaining strong responses after a single immunisation is a major aim of all vaccination programmes, albeit a usually unattainable aim. There was little detectable response against the PS14 polysaccharide until after a booster dose was given, when it two of the five mice given CD40/PS3/PS14 responded more strongly to PS14 than the controls. Pneumococcal Challenge Studies
Mice immunised with PS3/PS14/CD40 or the equivalent control liposomes were challenged 5 months later by intratracheal instillation of (105) live type 3 pneumococci. Twenty-four hours later blood and lung samples were taken in order to assess levels of bacteraemia and bacterial lung counts. There was no bacteraemia in any of the animals, however two of the five CD40/PS3/PS14 animals were clearly protected against lung colonisation with bacterial numbers either undetectable, or extremely low (see figure below, showing bacterial numbers recovered from the lungs). None of the control animals were protected. Taking a reduction of >2 log™ from the average lung count in control animals as indicative of protection, there was a significant difference in the level of susceptibility of the CD40/PS3/PS14 immunised mice versus isotype control/PS3/PS14 immunised mice (p<0.05, Yate's corrected Chi2)
TT and DT co-formulation
Tetanus toxoid (TT) and diptheria toxoid (DT), or the mutant form of diptheria toxin, CRM197, are currently administered as components of multivalent formulations, such as DTP (along with a pertussis component) or DTP-Hib (with pertussis and Haemophilus influenzae conjugate). These vaccines are administered many times in order to achieve and maintain sufficient immune responses (the DT components are given five times in the UK before age 18). Clearly improvement in the immunogenicity of the DT components, which could lead to a reduction in the number of doses required would be highly desirable. Furthermore, while we had shown (above) that responses against TI-2 antigens could be enhanced by liposomal CD40 antibody, protein antigens such as TT and DT are T dependent antigens, and thus are inherently capable of generating T cell help through CD154-CD40 binding. We envisaged, therefore, that proof of an adjuvant effect with these antigens would be much harder to obtain. Liposomes were formulated containing 3μg of CRM197 (DT) and 1μg of TT, along with 10μg CD40mAb, At day 14, following a single immunisation, antibody responses against TT were very strongly enhanced in the CD40 group as compared with the isotype control liposome group. This significant enhancement in responses to TT remained at days 28 and 45 post- immunisation. Assessment of the toxicity of CD40 liposome preparations
We have shown previously that chemical conjugation of CD40mAbs to antigen enhances the adjuvant effect of the antibody, allowing closes of antibody used to be reduced to sub-toxic levels (higher doses induce polyclonal antibody increases and increases in spleen size.) As we have shown (above) that the adjuvant effect of CD40 antibody, at the low dose, can be enhanced by liposomal entrapment as well as by conjugation, we were interested in assessing the effects of entrapment on toxicity. It was theoretically possible that entrapment might increase the toxic effects of the antibody by enhancing receptor cross-linking. In fact, entrapment within liposomes served to decrease the toxicity of the CD40 antibody. Thus, at a 50μg dose of free CD40mAb there is a significant increase in spleen weight at day 5, this effect is much reduced by the administration of the same amount of CD40mAb encapsulated in liposomes.

Claims

Claims 1. A liposome preparation comprising: i) a CD40 binding polypeptide; and ii) at least one antigenic molecule wherein said preparation is for use as a vaccine.
2. A preparation according to claim 1 wherein said preparation comprises at least two antigenic molecules.
3. A preparation according to claim 1 or 2 wherein said preparation comprises first and second liposome populations wherein said first population comprises an encapsulated CD40 binding protein and said second population comprises an encapsulated antigenic molecule.
4. A preparation according to any of claims 1-3 wherein said first liposome population consists of a CD40 binding polypeptide.
5. A preparation according to claim 3 or 4 wherein said second liposome population comprises two antigenic molecules.
6. A preparation according to any of claims 2-5 wherein said CD40 binding polypeptide and said at least two antigenic molecules are encapsulated in a liposome.
7. A preparation according to any of claims 1-6 wherein at least one antigenic molecule is a polypeptide.
8. A preparation according to claim 7 wherein said polypeptide is a polypeptide isolated from a viral pathogen.
9. A preparation according to claim 8 wherein said polypeptide is isolated from a viral pathogen selected from the group consisting of: Human Immunodeficiency Virus 1 & 2; Human T Cell Leukaemia Virus 1 & 2; Ebola virus; human papilloma virus HPV-2, HPV-5, HPV-8 HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54 and HPV-56; papovavirus; rhinovirus; poliovirus; herpesvirus; adenovirus; Epstein Barr virus; influenza virus, hepatitis B and C viruses.
10. A preparation according to claim 7 wherein said polypeptide is a polypeptide isolated from a bacterial pathogen.
11. A preparation according to claim 10 wherein said polypeptide is isolated from a bacterial pathogen selected from the group consisting of: Staphylococcus aureus; Staphylococcus epidermidis; Enterococcus faecalis; Mycobacterium tuberculosis; Streptococcus group B; Streptoccocus pneumoniae; Helicobacter pylori; Neisseria gonorrhea; Streptococcus group A; Borrelia burgdorferi; Coccidiodes immitis; Histoplasma sapsulatum; Neisseria meningitidis ; Shigella flexneri; Escherichia coli; Haemophilus influenzae; Clostridium tetani; Corynebacterium diphtheriae; Bordetella pertussis; Listeria monocytogenes.
12. A preparation according to claim 11 wherein said polypeptide is tetanus toxoid or diptheria toxoid.
13. A preparation according to claim 7 wherein said polypeptide is a polypeptide isolated from a parasitic pathogen.
14. A preparation according to claim 13 wherein said polypeptide is isolated from a parasite selected from the group consisting of: Trypanosoma spp, Leishmania spp, Schistosoma spp or Plasmodium spp.
15. A preparation according to claim 7 wherein said polypeptide is a polypeptide isolated from a fungal pathogen.
16. A preparation according to claim 15 wherein said polypeptide is isolated from a yeast pathogen.
17. A preparation according to claim 16 wherein said yeast pathogen is Candida albicans.
18. A preparation according to claim 7 wherein said polypeptide is a polypeptide associated with a disease.
19. A preparation according to claim 18 wherein said disease is cancer or Alzheimer's disease.
20. A preparation according to any of claims 1-6 wherein said antigenic molecule is a peptide.
21. A preparation according to claim 19 wherein said peptide is 8-30 amino acids in length.
22. A preparation according to claim 21 wherein said peptide is 8-18 amino acids in length.
23. A preparation according to claim 22 wherein said peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acids in length.
24. A preparation according to any of claims 20-23 wherein said peptide comprises a tumour rejection antigen.
25. A preparation according to any of claims 1-6 wherein at least one antigenic molecule is a polysaccharide.
26. A preparation according to claim 25 wherein said polysaccharide is a bacterial capsular polysaccharide antigen.
27. A preparation according to claim 26 wherein said capsular polysaccharide is PS3 or PS 14 or a combination thereof.
28. A preparation according to claim 25 wherein said antigenic molecule is a lipopolysaccharide.
29. A preparation according to any of claims 1-17 wherein said preparation comprises two or more antigenic molecules isolated from a pathogen to provide a multivalent vaccine preparation.
30. A preparation according to claim 29 wherein said multivalent vaccine is a mixture of polypeptide and/or peptide and/or polysaccharide provided as an admixture.
31. A preparation according to any of claims 18-25 wherein said preparation comprises two or more disease specific antigenic molecules to provide a multivalent vaccine preparation.
32. A preparation according to claim 31 wherein said multivalent vaccine is a mixture of polypeptide and/or peptide and/or polysaccharide and/or lipopolysaccharide and/or a ganglioside provided as an admixture.
33. A preparation according to claim 31 or 32 wherein said disease is cancer.
34. A preparation according to claim 31 or 32 wherein said disease is Alzheimer's disease.
35. A preparation according to claim 29 or 30 wherein said multivalent vaccine comprises antigenic molecules isolated from different pathogens to provide a multivalent vaccine that is protective against two or more disease pathogens.
36. A preparation according to any of claims 1-35 wherein said CD40 binding polypeptide is an antibody.
37. A preparation according to claim 36 wherein said antibody is a monoclonal antibody, or active binding part thereof.
38. A preparation according to claim 37 wherein said monoclonal antibody is a humanised or chimeric antibody.
39. A preparation according to claim 37 or 38 wherein said active binding parts of are selected from the group consisting of: Fab, Fab2, F(ab')2, Fv, Fc, Fd, scFvs.
40. A preparation according to any of claims 1-35 wherein said CD40 binding polypeptide is CD154 or fragment thereof
41. A preparation according to any of claims 1-35 wherein said CD40 binding polypeptide is mycobacterial HSP70 or a fragment thereof
42. A preparation according to any of claims 1-35 wherein said CD40 binding polypeptide is a nematode polyladder protein or fragment thereof
43. A preparation according to any of claims 1-35 wherein said preparation includes a carrier.
44. A preparation according to any of claims 1-43 wherein said preparation includes a further adjuvant.
45. A preparation according to claim 44 wherein said adjuvant is a cytokine selected from the group consisting of: GMCSF1 interferon gamma, interferon alpha, interferon beta, interleukin 12, interleukin 23, interleukin 17, interleukin2, interleukini , TGF1TNFa; TNFβ.
46. A preparation according to claim 44 wherein said adjuvant is a CD28 binding polypeptide.
47. A preparation according to claim 46 wherein said binding polypeptide is an antibody, preferably a monoclonal antibody or binding fragment thereof.
48. A preparation according to claim 46 wherein said polypeptide is the natural ligand of CD28, CD80/CD86, or modified variants thereof that bind CD28.
49. A preparation according to claim 44 wherein said adjuvant is TLR agonists such as CpG oligonucleotides, flagellin, monophosphoryl lipid A, poly I:C and derivatives thereof.
50. A preparation according to claim 44 wherein said adjuvant is a bacterial cell wall derivative such as muramyl dipeptide (MDP) and/or trehelose dycorynemycolate (TDM)
51. A process for the formation of a liposome preparation comprising: i) forming a first liposome preparation comprising a CD40 binding polypeptide; ii) forming a second preparation comprising at least one antigenic molecule; iii) contacting first and second preparations under conditions that allow the fusion of said preparations to provide combined liposome preparation wherein said first and second preparations fuse to form a liposome preparation wherein the CD40 binding polypeptide and antigenic molecule are substantially encapsulated in said combined liposome preparation.
52. A process according to claim 51 wherein said antigenic molecule is selected from the group consisting of: a polypeptide, a peptide or a polysaccharide molecule.
53. A method to vaccinate a subject with respect to at least one disease or condition comprising administering an effective amount of a liposome preparation according to any of claims 1-50 to said subject.
54. A method according to claim 53 wherein said disease is caused by a viral pathogen.
55. A method according to claim 53 wherein said disease is caused by a bacterial pathogen.
56. A method according to claim 53 wherein said disease is caused by a parasitic pathogen.
57. A method according to claim 53 wherein said disease is caused by a fungal pathogen.
58. A method according to claim 53 wherein said disease is cancer.
59. A method according to any of claims 53-58 wherein said subject is human.
60. A method to reduce toxicity of a vaccine in a subject comprising: i) forming a first liposome preparation comprising a CD40 binding polypeptide; H) forming a second preparation comprising at least one antigenic molecule; iii) contacting first and second preparations under conditions that allow the fusion of said preparations to provide combined liposome preparation wherein said first and second preparations fuse to form a liposome preparation wherein the CD40 binding polypeptide and antigenic molecule are substantially encapsulated in said combined liposome preparation; and iv) administering the fused preparation to a subject requiring immunisation against an infectious or non-infectious disease.
61. A kit comprising: i) CD40 binding protein; ii) at least two antigenic molecules; iii) liposomes; and optionally a list of instructions detailing the formation of liposome preparations according to any of claims 1-50.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010034974A2 (en) * 2008-09-24 2010-04-01 Adjuvantix Limited Tb vaccine
US20110033494A1 (en) * 2008-04-01 2011-02-10 Innate Therapeutics Limited Anti-Infective Agents and Uses Thereof
CN112920267A (en) * 2021-03-09 2021-06-08 北京康乐卫士生物技术股份有限公司 Monoclonal neutralizing antibody against human papillomavirus 31 and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001015728A1 (en) * 1999-08-27 2001-03-08 University Health Network Method for activating cytotoxic t-lymphocytes (ctls) in vivo : composition comprising antibody anti cd40 (or cd40l or cd40 binding protein) and an antigen
US20030170236A1 (en) * 1999-05-17 2003-09-11 Polly Matzinger Identification and use of high efficacy vaccine antigens which modulate antigen presenting cells
WO2003074712A1 (en) * 2002-03-02 2003-09-12 The Scripps Research Institute Dna vaccines encoding cea and a cd40 ligand and methods of use thereof
WO2005013896A2 (en) * 2003-07-09 2005-02-17 Vaxdesign Corporation Programmed immune responses using a vaccination node
EP1616579A1 (en) * 1998-05-23 2006-01-18 Leiden University Medical Center CD40 binding molecules and CTL peptides for treating tumors

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1616579A1 (en) * 1998-05-23 2006-01-18 Leiden University Medical Center CD40 binding molecules and CTL peptides for treating tumors
US20030170236A1 (en) * 1999-05-17 2003-09-11 Polly Matzinger Identification and use of high efficacy vaccine antigens which modulate antigen presenting cells
WO2001015728A1 (en) * 1999-08-27 2001-03-08 University Health Network Method for activating cytotoxic t-lymphocytes (ctls) in vivo : composition comprising antibody anti cd40 (or cd40l or cd40 binding protein) and an antigen
WO2003074712A1 (en) * 2002-03-02 2003-09-12 The Scripps Research Institute Dna vaccines encoding cea and a cd40 ligand and methods of use thereof
WO2005013896A2 (en) * 2003-07-09 2005-02-17 Vaxdesign Corporation Programmed immune responses using a vaccination node

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
GURUNATHAN S ET AL: "CD40 ligand/trimer DNA enhances both humoral and cellular immune responses and induces protective immunity to infectious and tumor challenge", JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, vol. 161, no. 9, 1 November 1998 (1998-11-01), pages 4563 - 4571, XP002345704, ISSN: 0022-1767 *
ITO D ET AL: "Induction of CTL responses by simultaneous administration of liposomal peptide vaccine with anti-CD40 and anti-CTLA-4 mAb.", JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 1 FEB 2000, vol. 164, no. 3, 1 February 2000 (2000-02-01), pages 1230 - 1235, XP002489906, ISSN: 0022-1767 *
JEURISSEN AXEL ET AL: "Essential role for CD40 ligand interactions in T lymphocyte-mediated modulation of the murine immune response to pneumococcal capsular polysaccharides.", JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 MAR 2002, vol. 168, no. 6, 15 March 2002 (2002-03-15), pages 2773 - 2781, XP002489907, ISSN: 0022-1767 *
NINOMIYA A ET AL: "Intranasal administration of a synthetic peptide vaccine encapsulated in liposome together with an anti-CD40 antibody induces protective immunity against influenza A virus in mice", VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 20, no. 25-26, 19 August 2002 (2002-08-19), pages 3123 - 3129, XP004374551, ISSN: 0264-410X *
WU Z Q ET AL: "In vivo polysaccharide-specific IgG isotype responses to intact Streptococcus pneumoniae are T cell dependent and require CD40- and B7-ligand interactions.", JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 JUL 1999, vol. 163, no. 2, 15 July 1999 (1999-07-15), pages 659 - 667, XP002489908, ISSN: 0022-1767 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110033494A1 (en) * 2008-04-01 2011-02-10 Innate Therapeutics Limited Anti-Infective Agents and Uses Thereof
US8709448B2 (en) * 2008-04-01 2014-04-29 Innate Immunotherapeutics Ltd. Anti-infective agents and uses thereof
WO2010034974A2 (en) * 2008-09-24 2010-04-01 Adjuvantix Limited Tb vaccine
WO2010034974A3 (en) * 2008-09-24 2010-08-12 Adjuvantix Limited Tuberculosis vaccines targeted to cd40
CN112920267A (en) * 2021-03-09 2021-06-08 北京康乐卫士生物技术股份有限公司 Monoclonal neutralizing antibody against human papillomavirus 31 and application thereof

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