WO2005102394A1 - Acide urique utilise comme adjuvant - Google Patents

Acide urique utilise comme adjuvant Download PDF

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Publication number
WO2005102394A1
WO2005102394A1 PCT/EP2005/003248 EP2005003248W WO2005102394A1 WO 2005102394 A1 WO2005102394 A1 WO 2005102394A1 EP 2005003248 W EP2005003248 W EP 2005003248W WO 2005102394 A1 WO2005102394 A1 WO 2005102394A1
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composition
uric acid
polynucleotide
antigen
final product
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PCT/EP2005/003248
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English (en)
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Kevin Michael Brindle
Alistair Mcfarlane Moore
Lindy Louise Thomsen
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Glaxo Group Limited
Cambridge University Technical Services Limited
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Publication of WO2005102394A1 publication Critical patent/WO2005102394A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/53—DNA (RNA) vaccination
    • 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

Definitions

  • the present invention provides a novel adjuvant for polynucleotide vaccines, and in particular the present invention provides polynucleotide vaccines that comprise, or are administered in association with, a composition that is a breakdown product of a purine, which in particular, may be uric acid.
  • the polynucleotide vaccines of the present invention are vaccines that encode an antigen against which it is desired to generate an immune response, and in particular the polynucleotide vaccine may be a DNA vaccine.
  • Also provided by the present invention is the use of uric acid in the manufacture of a polynucleotide vaccine composition for the purpose of enhancing the immune response against the specific antigen that is encoded by the polynucleotide vaccine.
  • Vaccine compositions, kits comprising separate polynucleotide composition and adjuvant compositions for separate administration, methods of manufacture of the vaccines and kits, and methods of treatment of individuals with the vaccine compositions of the present invention, are provided.
  • Uric acid or 2,6,8-trihydroxypurine, in its purified form exists as a microcrystalline powder, is colourless, odourless, tasteless, almost insoluble in water, and decomposes above 250 °C (Sharp, D.W.A. (ed.) (2003) The Penguin Dictionary of Chemistry, 3rd edition. Penguin Books Ltd, London).
  • Uric acid has the following molecular structure:
  • uric acid is formed as the end product of purine catabolism (Hitchings, G.H. (1978) Uric acid: Chemistry and synthesis. In: Uric Acid (Kelley,
  • Uric acid is the end product of the catabolism of adenosine and guanosine in humans.
  • Uric acid is produced from xanthine (by the enzyme xanthine oxidase), which is produced from either guanine (the first breakdown product of guanosine) or hypoxanthine (which is the breakdown product of inosine, the first breakdown product of adenosine (Mathews, C.K., van Holde, K.E., & Ahern, K.G. (2000). Biochemistry, 3rd edition. Benjamin/Cummings, San Francisco, CA. p. 803).
  • Uric acid has been shown to be a strong scavenger of carbon-centred and peroxyl radicals (Muraoka, S. & Miura, T. (2003) Pharmacology & Toxicology 93: 284- 9), as well as reactive nitrogen oxide species such as nitric oxide (NO) and peroxynitrite (ONOO " ) (Ghafourifar, P. et al. (1999) Journal of Biological Chemistry 274: 31185-8).
  • NO nitric oxide
  • ONOO " peroxynitrite
  • uric acid is also thought to prevent the formation of tolerance to nitroglycerin (glyceryl trinitrite), which is used in the therapy of cardiovascular diseases such as angina, congestive heart failure and hypertension. Tolerance is mediated by the oxidative actions of superoxide, peroxynitrite and protein kinase C; uric acid is postulated to protect against this (Abou-Mohamed, G. et al. (2004) Journal of Pharmacology and Experimental Therapeutics 308: 289-99).
  • nitroglycerin glyceryl trinitrite
  • Nitric oxide (NO) has been shown to modulate the production of uric acid in humans via its influence on xanthine oxidase activity, and a cyclic and repeating relationship between NO and uric acid levels has been suggested (Lee, Y. J. et al. (2003) Metabolism-Clinical and Experimental 52: 1448-53).
  • An elevated level of uric acid, or serum uric acid has been linked with several disease conditions and metabolic disorders, either pathogenically or as a prognostic indicator. Examples include hyperuricemia (an abnormally high accumulation of uric acid in the blood; Martin, E.A. (ed.) (1998) The Oxford Concise Colour Medical
  • lowered serum uric acid level can be pathogenic (and prognostic), as in multiple sclerosis (Hooper, D.C. et al. (1998) PNAS USA 95: 675-80) and the precursor condition optic neuritis (Spitsin, S. et al. (2001) Multiple Sclerosis 7: 313-9).
  • Therapeutic administration of uric acid in humans may boost anitoxidant capacity and aid in control of multiple sclerosis (Mousavizadeh, K. et al. (2003) Trends in Pharmacological Sciences 24: 563-4).
  • Gout is characterised by the accumulation of monosodium urate crystals in the peripheral joints, leading to neutrophil activation and subsequent inflammation of the joint.
  • Monosodium urate crystals are thought to activate neutrophils via a receptor- mediated process, involving in particular the Fc-receptor CD 16 and tyrosine kinase-dependent signal transduction (Desaulniers, P. et al. (2001) Journal of Leukocyte Biology 70: 659-68).
  • inosine can modulate immune reactions, whilst a number of authors claim that it is anti-inflammatory [for example Marton, A. et al.,(2001) Int. J. Mol. Med. 8:617-21); Liaudet, L. et al. (2002) Annals of Surgery 235:568-78)], one recent paper [Idzko, M et al.
  • Vaccines have for many years included substances that have a direct or indirect stimulatory effect on the immune system, termed "adjuvants", such that the magnitude or quality of the immune response is altered or augmented.
  • adjuvants substances that have a direct or indirect stimulatory effect on the immune system
  • General information about the use of adjuvants is provided in Powell, M.F. & Newman, M.J. (eds.) (1995) Vaccine Design - The Subunit and Adjuvant Approach. Plenum Press, New York and London. Shi et al.
  • uric acid can act as an adjuvant for a protein vaccine antigen.
  • Uric acid purified from the cytosol of ultraviolet light-damaged BALB/c 3T3 cells could boost cytotoxic T-lymphocyte (CTL) killing responses in splenocytes from mice primed with particulate HIV gpl20 antigen.
  • CTL cytotoxic T-lymphocyte
  • Commercially obtained pure uric acid had a similar effect, and was also able to boost killing in CTLs from mice primed against particulate ovalbumin.
  • Uric acid was shown by Shi et al.
  • dendritic cells including CD86 and CD80 (also known as B7.1 and B7.2, the ligands for essential CD28 and CTLA-4 receptor-mediated activatory co-stimulation of T-cells).
  • CD86 and CD80 also known as B7.1 and B7.2, the ligands for essential CD28 and CTLA-4 receptor-mediated activatory co-stimulation of T-cells.
  • concentrations at which dendritic cells were stimulated corresponded to the point at which uric acid would reach saturation, and crystallisation to form monosodium urate crystals would occur. Indeed, preformed monosodium urate crystals were shown to be highly stimulatory.
  • polynucleotide vaccines where the vaccine comprises a polynucleotide that encodes the antigen and facilitates antigen production in the host cells of the vaccinee, are themselves a relatively recent development. Necessarily therefore, less is known about polynucleotide vaccine adjuvants.
  • the adjuvant strategy for polynucleotide vaccines often involves the co-expression of immune modifiers, such as cytokines, together with the antigen.
  • polynucleotide vaccine adjuvants include small molecules such as tucerasol (WO 00/12121), imidazoquinoline amines (WO 02/24225, WO 03/077944) and inducible nitric oxide synthase (iNOS) inhibitors (WO 03/030935).
  • tucerasol WO 00/12121
  • imidazoquinoline amines WO 02/24225
  • WO 03/077944 inducible nitric oxide synthase
  • iNOS inducible nitric oxide synthase
  • the present invention provides novel immunogenic compositions or vaccines comprising (a) a polynucleotide component that encodes an antigen against which it is desired to generate an immune response, and (b) an adjuvant component comprising an immune stimulatory quantity of an intermediate or final product of purine catabolism, or a derivative thereof.
  • the polynucleotide component encoding the immunogenic compositions or vaccine antigen is any polynucleotide or vector that is capable of directing expression of the said antigen in the cells of the host vaccinee.
  • the vector may be a live or attenuated viral or bacterial vector which delivers the foreign sequence that encodes the vaccine antigen.
  • the immunogenic compositions or vaccines comprise a polynucleotide vector which is a DNA plasmid vector.
  • the plasmid vector may be delivered to the vaccinee in liquid form, or in the form of dense micro-beads suitable for ballistic delivery into the skin, or formulated on the surface of dense micro-beads suitable for ballistic delivery into the skin, or coated onto microneedles.
  • the intermediate or final product of purine catabolism that forms the adjuvant composition is uric acid.
  • the intermediate or final products of purine catabolism may be selected from inosine, hypoxanthine or xanthine, or salts, solvates or physiologically active derivatives thereof.
  • the adjuvant component comprises a combination of two or more intermediates or final products of purine catabolism.
  • the adjuvant compositions may comprise a combination of uric acid and inosine, uric acid and hypoxanthine, or uric acid and xanthine.
  • the adjuvant is a salt of uric acid, such as the monosodium salt.
  • the physical presentation of the uric acid, or other product of purine catabolism, in the polynucleotide vaccine of the present invention depends upon the form of the vaccine to be administered.
  • the uric acid, or other product of purine catabolism may be in solution or in crystalline form.
  • the uric acid may be in the form of a crystal or in the form of a crystal formed of one of its salts, for example the adjuvants of the present invention may be a crystal form of the monosodium salt of uric acid.
  • the immunogenic compositions or vaccines of the present invention may be in solid form, such that the polynucleotide may be in a "dry" form and co-formulated with the uric acid.
  • the polynucleotide antigen and the uric acid, or other product of purine catabolism may be in dry solid solution within a solid, or glassy, matrix.
  • the solid matrix may be a carbohydrate, or sugar, in solid form.
  • the polynucleotide and uric acid in its crystalline form, or crystals formed of uric acid salt, are provided on the surface of microbeads suitable for ballistic delivery into the epidermis
  • the solid immunogenic composition or vaccine formulation may comprise a protein antigen and uric acid.
  • the solid vaccine may comprise the antigen and uric acid, or salt thereof, in a solid matrix such as a sugar.
  • a lyophilised vaccine formulation for example, in a lyophilised vaccine formulation.
  • a method of stabilising a protein in its dry state such as in its lyophilised form, by co-formulating said protein with uric acid, and optionally further comprising a stabilising sugar.
  • This stabilised formulation and method has the additional advantage of enhancing the immune responses raised by the antigen.
  • an immunogenic composition or vaccine formulation comprising a polynucleotide which encodes an antigen, uric acid or salt thereof (or other breakdown product of a purine) in a dry form wherein the polynucleotide is stabilised.
  • this polynucleotide formulation may be lyophilised, optionally in the presence of a sugar.
  • the uric acid, or other breakdown product of purine is either in a crystalline form before administration to the patient, otherwise the crystals may be caused to form in the body of the vaccinee after administration of the vaccine.
  • the dose of the uric acid, or other product of purine catabolism, in the vaccines of the present invention is sufficient to enhance the immune response against the antigen, and in one embodiment is sufficiently high in concentration that crystallisation of uric acid occurs, to any appreciable extent, after administration.
  • the vaccines of the present invention are particularly adapted, by the formulation with the adjuvants described herein, to the provision of highly potent immune responses, including cell mediated immune responses.
  • the immunogenic compositions or vaccines of the present invention are also highly stable compositions, in that the stability of the polynucleotides in the vaccine is enhanced by the presence of uric acid, or other product of purine catabolism.
  • An additional advantage of the present invention is the provision of a vaccine/adjuvant composition that does not have the toxicity issues associated with the persistence of potentially toxic adjuvants in the body of the vaccinee.
  • FIG. 1 Effect of vaccination with and without uric acid on mean tumour size.
  • FIG 2. Effect of various parameters on tumour regression after vaccination with the vaccines of the present invention.
  • FIG 4. Day 11 ELISPOT data.
  • FIG 5. Day 14 ELISPOT data.
  • the present invention provides novel polynucleotide immunogenic compositions or vaccines comprising (a) a polynucleotide component that encodes an antigen against which it is desired to generate an immune response, and (b) an adjuvant composition comprising an immune stimulatory quantity of an intermediate or final product of purine catabolism, or a derivative thereof.
  • the compositions of the present invention may be immunogenic compositions in that they are, after administration to a mammal, capable of generating an immune response, such as an antibody response or generation of T-cells that proliferate or secrete cytokines after stimulation with antigen, in that mammal which is specific for the antigen encoded by the polynucleotide component.
  • compositions of the present invention may be vaccine compositions, in that they are capable, after administration to a mammal, of generating an immime response in said mammal which is sufficient to afford a degree of protection against an infection or disease (prophylaxis), or ameliorate the symptoms of or eradicate an existing infection or disease.
  • Elements of the present text which refer to vaccines or immunogenic compositions may be interchanged accordingly.
  • the polynucleotide elements forming part of the vaccines of the present invention are vectors which, when administered to a vaccinee in an appropriate form, drive expression of an antigen in the cells of the vaccinee, thereby generating an immune response against the antigen.
  • the vectors or polynucleotide elements of the vaccines of the present invention which encode the antigen against which it is desired to generate an immune response, are operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • the term "operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.
  • the vectors may be, for example, plasmids, artificial chromosomes, live or attenuated bacterial, viral or phage vectors.
  • Promoters and other expression regulation signals that form part of the polynucleotide vectors may be selected to be compatible with the host cell for which expression is designed.
  • mammalian promoters include the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, and the ⁇ -actin promoter.
  • Viral promoters such as the SV40 large T antigen promoter, human cytomegalovirus (CMV) immediate early (IE) promoter, rous sarcoma virus LTR promoter, adenovirus promoter, or a HPN promoter, particularly the HPN upstream regulatory region (URR) may also be used. All these promoters are well described and readily available in the art.
  • suitable viral vectors include herpes simplex viral vectors, vaccinia or alpha- virus vectors and retroviruses, including lentiviruses, human and simian adenoviruses and adeno-associated viruses.
  • the polynucleotide is in the form of a D ⁇ A plasmid vector comprising covalently closed circular D ⁇ A, in a super-coiled or open circular form, comprising an expression cassette having a promoter region and a coding region.
  • the coding region encodes an antigen which, once expressed in the host cells of the vaccinee, generates an immune response.
  • the coding region, or an additional coding region may encode for an immunostimulatory cytokine such as IL-2, GM-CSF or lF ⁇ - ⁇ .
  • a vaccine composition comprising a compound of Formula (I):
  • physiologically functional derivative refers to any pharmaceutically acceptable derivative of an adjuvant of the present invention (formed, for example, by addition of alkyl, alkenyl, alkynyl, aryl or polysaccharide groups to oxidised nitrogen atoms of the purine skeleton of uric acid or intermediate of the purine catabolism pathway), which upon administration to a mammal is itself capable of enhancing the immune response against the antigen encoded by the polynucleotide, or is capable of indirectly doing so through the action of a breakdown product formed from the derivative in situ after administration to the body.
  • solvate refers to a complex of variable stoichiometry formed by a solute (in this invention, a compound of formula (I) or a salt or physiologically functional derivative thereof) and a solvent.
  • solvents for the purpose of the invention may not interfere with the biological activity of the solute.
  • suitable solvents include, but are not limited to, water, methanol, ethanol and acetic acid.
  • the solvate is boric acid.
  • the salts of the present invention are pharmaceutically acceptable salts.
  • Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the compounds of this invention.
  • Salts of the compounds of the present invention may comprise salts derived from a nitrogen on a substituent in the compound of formula (I).
  • Representative salts include the following salts: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, ⁇ -methylglucamine, ox
  • Suitable salts of uric acid, or derivatives thereof include most metals including sodium, potassium, lithium, calcium, magnesium, zinc. Ammonium salts are also known and a guanidinium salt. These salts may be solvated with water. Salts of acids with uric acid may be used if basic groups are attached to the uric acid molecule. Uric acid may be manufactured according to a method of preparation given in The Merck Index: H
  • the vaccination methods and compositions according to the present application be adapted for protection or treatment of mammals against a variety of disease states such as, for example, viral, bacterial or parasitic infections, cancer, allergies and autoimmune disorders.
  • Rl can be hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, aryl;
  • R2 can be hydrogen, alkyl, cycloalkyl, heteroalkyl;
  • R3 can be hydrogen, alkyl, heteroalkyl, cycloalkyl, aralkyl, sugars (ribose etc);
  • R4 can be hydrogen, alkyl, aryl, heteroalkyl, alkenyl.
  • the polynucleotide sequences referred to in this application, which are to be expressed within a mammalian system in order to induce an antigenic response may encode for an entire protein, or merely a shorter peptide sequence that is capable of initiating an antigenic response.
  • the antigens which may be used in the vaccines or immunogenic compositions may be surface exposed antigens derived from viral or bacterial pathogens.
  • antigenic peptide or “immunogen” is intended to encompass all peptide or protein sequences which are capable of inducing an immune response within the animal concerned.
  • the polynucleotide sequence will encode for a full protein that is associated with the disease state, as the expression of full proteins within the animal system is more likely to mimic natural antigen presentation, and thereby evoke a full immune response.
  • Antigens which are capable of eliciting an immune response against a human pathogen include those in which the antigen or antigenic composition is derived from any of a range of viral, bacterial, parasitic and yeast sources.
  • Viral antigen sources include: HIN-1 (such as tat, nef, gpl20 or gpl60, gp40, p24, gag, env, vif, vpr, vpu, rev); human herpes viruses (such as gH, gL gM gB gC gK gE or gD or derivatives thereof, or Immediate Early proteins such as ICP27 , ICP 47, IC P 4, ICP36 from HSN1 or HSN2); cytomegalo virus, especially human (such as gB or derivatives thereof); Epstein Barr virus (such as gp350 or derivatives thereof); Varicella Zoster Virus (such as gpl, II, III and IE63); hepatitis
  • Bacterial sources include: Neisseria spp. such as N. gonorrhea and N. meningitidis (e.g. transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesins); S. pyogenes (for example M proteins or fragments thereof, or C5A protease); S. agalactiae, S. mutans; H. ducreyi; Moraxella spp. such as M.
  • Neisseria spp. such as N. gonorrhea and N. meningitidis (e.g. transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesins); S. pyogenes (for example M proteins or fragments thereof, or C5A protease); S. agalactiae, S. mutans; H. ducreyi; Moraxella spp. such as M.
  • catarrhalis also known as Branhamella catarrhalis; antigens include high and low molecular weight adhesins and invasins
  • Bordetella spp. including B. pertussis (for example pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica;
  • Mycobacterium spp. including M. tuberculosis (for example ESAT6, Antigen 85A, 85B or 85C, MPT 44, MPT59, MPT45,
  • enterotoxic E. coli for example colonization factors, heat-labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof
  • enterohemorragic E. coli and enteropathogenic E. coli for example shiga
  • cholera for example cholera toxin or derivatives thereof
  • Shigella spp. including S. sonnei, S. dysenteriae and S.flexnerii
  • Yersinia spp. including Y. enterocolitica (for example a Yop protein), Y. pestis and Y. pseudotuberculosis
  • Campylobacter spp. including C. jejuni (for example toxins, adhesins and invasins) and C. coli
  • Salmonella spp. including S. typhi, S. paratyphi, S. choleraesuis and S. enteritidis
  • Listeria spp. including L.
  • H pylori for example urease, catalase, vacuolating toxin
  • Pseudomonas spp. including P. aeruginosa
  • Staphylococcus spp. including S. aureus and S. epidermidis
  • Enterococcus spp. including E. faecalis and E. faecium
  • Clostridium spp. including C. tetani (for example tetanus toxin and derivatives thereof), C. botulinum (for example botulinum toxin and derivatives thereof), and C.
  • Bacillus spp. including R. anthracis (for example botulinum toxin and derivatives thereof); Corynebacterium spp., including C. diphtheriae (for example diphtheria toxin and derivatives thereof); Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B.
  • B. burgdorferi for example OspA, OspC, DbpA, DbpB
  • B. garinii for example OspA, OspC, DbpA, DbpB
  • B. afzelii for example OspA, OspC, DbpA, DbpB
  • Ehrlichia spp. including E. equi and the agent of the Human Granulocytic Ehrlichiosis
  • Rickettsia spp. including R. rickettsii
  • Chlamydia spp. including C. trachomatis (for example MOMP, heparin-binding proteins), C. pneumoniae (for example MOMP, heparin-binding proteins), and C. psittaci
  • Leptospira spp. including L. interrogans
  • Treponema spp. including T.
  • Parasitic sources include: Plasmodium spp., including P. falciparum; Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34); Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; Trypanosoma spp., including T. cruzi; Giardia spp., including G. lamblia; Leshmania spp., including L. major; Pneumocystis spp., including P.
  • Trichomonas spp. including T. vaginalis
  • Schisostoma spp. including S. mansoni.Yeast sources include: Candida spp., including C. albicans; and Cryptococcus spp., including C. neoformans.
  • Proteins for M tuberculosis also include fusion proteins and variants thereof in which at least two, or at least three, polypeptides of M.
  • tuberculosis are fused into a larger protein.
  • Some specific fusions include Ral2-TbH9-Ra35, Erdl4-DPN-MTI, DPN-MTI-MSL, Erdl4-DPN-MTI-MSL- mTCC2, Erdl4-DPN-MTI-MSL, DPN-MTI-MSL-mTCC2, and TbH9-DPN-MTI (WO 99/51748).
  • Chlamydia include, for example, the High Molecular Weight Protein (HWMP) (WO 99/17741), ORF3 (EP 366 412), and putative membrane proteins (Pmps).
  • HWMP High Molecular Weight Protein
  • ORF3 ORF3
  • Pmps putative membrane proteins
  • Other Chlamydia antigens of the vaccine formulation can be selected from the group described in WO 99/28475.
  • bacterial antigens derived from Streptococcus spp. including S. pneumoniae (e.g. PsaA, PspA, streptolysin, choline-binding proteins) and the protein antigen Pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins, J.B. et al.
  • antigens derived from Haemophilus spp. include H. influenzae type B (for example PRP and conjugates thereof), non typeable H influenzae (for example OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin derived peptides (US 5,843,464) or multiple copy variants or fusion proteins thereof).
  • the antigens that may be used in the present invention may further comprise antigens derived from parasites that cause malaria.
  • antigens from Plasmodium falciparum include RTS,S and TRAP.
  • RTS is a hybrid protein comprising substantially all the C-terminal portion of the circumsporozoite (CS) protein of P. falciparum linked via four amino acids of the preS2 portion of hepatitis B surface antigen to the surface (S) antigen of hepatitis B virus. Its full structure is disclosed in the International Patent Application No. PCT/EP92/02591, published under Number WO 93/10152 claiming priority from UK patent application No. 9124390.7.
  • Other plasmodia antigens that are likely candidates to be components of a multistage malaria vaccine are P.
  • An embodiment of the present invention is a malaria vaccine wherein the antigenic preparation comprises a combination of the RTS,S and MSP-1 antigens.
  • the invention contemplates the use of an anti-tumour antigen and may be useful for the immunotherapeutic treatment of cancers.
  • tumour rejection antigens such as those for prostrate, breast, colorectal, lung, pancreatic, renal or melanoma cancers.
  • exemplary antigens include MAGE 1, MAGE 3 and MAGE 4, or other MAGE antigens such as disclosed in WO99/40188, PRAME, BAGE, Lü (also known as NY Eos 1) SAGE and HAGE (WO 99/53061) or GAGE (Robbins, P.F. & Kawakami, Y. (1996) Current Opinion in Immunology 8: 628-36; Nan den Eynde, BJ.& Boon, T. (1997) International Journal of ' Clinical and Laboratory Research 27: 81-6. Coneale, P. et al.
  • MAGE antigens for use in the present invention may be expressed as a fusion protein with an expression enhancer or an immunological fusion partner.
  • the MAGE protein may be fused to Protein D from Haemophilus influenzae B.
  • the fusion partner may comprise the first one third of Protein D.
  • fusion proteins that may contain cancer specific epitopes include bcr / abl fusion proteins.
  • prostate antigens are utilised, such as Prostate Specific Antigen (PSA), PAP, PSCA (Reiter, R.E. et al. (1998) PNAS USA 95: 1735 -40), PSMA or the antigen known as Prostase.
  • PSA Prostate Specific Antigen
  • PAP PAP
  • PSCA Reiter, R.E. et al. (1998) PNAS USA 95: 1735 -40
  • PSMA Prostase
  • Prostase is a prostate-specific serine protease (trypsin-like), and has been described by Nelson, P.S. et al. (1999; PNAS USA 96: 3114- 9).
  • the nucleotide sequence and deduced polypeptide sequence of the mature protein, and homologues are disclosed in (PNAS USA (1999) 96: 3114-9) and in International Patent Applications WO 98/12302 (and also the corresponding granted patent US 5,955,306), WO 98/20117 (and also the corresponding granted patents US 5,840,871 and US 5,786,148) (prostate-specific kallikrein) and WO 00/04149 (P703P).
  • the present invention provides antigens comprising prostase protein fusions based on prostase protein and fragments and homologues thereof ("derivatives"). Such derivatives are suitable for use in therapeutic vaccine formulations that are suitable for the treatment of prostate tumours.
  • the fragment will contain at least 20, or at least 50, or at least 100, contiguous amino acids as disclosed in the above referenced patent and patent applications.
  • a further prostate antigen for use in the present invention is known as P501S, sequence ID No. 113 of WO98/37814.
  • -mmunogenic fragments and portions encoded by the gene thereof comprising at least 20, at least 50, or in another embodiment at least 100, contiguous amino acids as disclosed in the above referenced patent application, are contemplated.
  • a particular fragment is PS 108 (WO 98/50567).
  • Other prostate specific antigens are known from WO98/37418, and WO/004149. Another is STEAP (Hubert, R.S. et al. (1999) PNAS USA 96: 14523-8).
  • tumoxir associated antigens useful in the context of the present invention include: Plu-1 (Lu, P.J. et al. (1999) Journal of Biological Chemistry 274: 15633-45), HASH -1, HasH-2, Cripto (Salomon, D.S. et al. (1999) Bioessays 21: 61 -70; US patent 5654140), and Criptin (US patent 5 981 215). Additionally, antigens particularly relevant for vaccines in the therapy of cancer also comprise tyrosinase and survivin.
  • the present invention is also useful in combination with breast cancer antigens such as Muc-1, Muc-2, EpCAM, HER 2 / Neu, mammaglobin (US patent 5668267) or those disclosed in WO 00/52165, WO99/33869, WO99/19479, WO 98/45328.
  • HER / 2 neu antigens are disclosed, ter alia, in US patent 5,801,005.
  • the HER / 2 neu comprises the entire extracellular domain (comprising approximately amino acids 1-645), or fragments thereof, and at least an immunogenic portion of or the entire intracellular domain (approximately the 580 C-terminal amino acids), hi particular, the intracellular portion should comprise the phosphorylation domain or fragments thereof.
  • ECD PD ECD ⁇ PD
  • HER / 2 neu as used herein can be derived from rat, mouse or human.
  • the antigens may also be associated with tumour-support mechanisms (e.g. angiogenesis, tumour invasion), for example tie 2.
  • Vaccines of the present invention may also be used for the prophylaxis or therapy of chronic disorders in addition to allergy, cancer or infectious diseases. Such chronic disorders are diseases such as asthma, atherosclerosis, and Alzheimer's and other auto-immune disorders. Vaccines for use as a contraceptive may also be considered.
  • Antigens relevant for the prophylaxis and the therapy of patients susceptible to or suffering from Alzheimer's neurodegenerative disease are, in particular, the N- terminal 39 -43 amino acid fragment of the ⁇ -amyloid precursor protein and smaller fragments). This antigen is disclosed in the International Patent Application No. WO 99/27944 (Athena Neurosciences).
  • Potential self-antigens that could be included as vaccines for auto-immxme disorders or as a contraceptive vaccine include: cytokines, hormones, growth factors or extracellular proteins, such as a 4-helical cytokine, like IL13.
  • Cytokines include, for example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL20, IL21, TNF, TGF, GM-CSF, MCSF and OSM.
  • 4-helical cytokines include IL2, IL3, IL4, IL5, IL13, GM-CSF and MCSF.
  • Hormones include, for example, luteinising hormone (LH), follicle stimulating hormone (FSH), chorionic gonadotropin (CG), VGF, GHrelin, agouti, agouti related protein and neuropeptide Y.
  • the vaccines of the present invention are particularly suited for the immunotherapeutic treatment of diseases, such as chronic conditions and cancers, but also for the therapy of persistent infections. Accordingly the vaccines of the present invention are particularly suitable for the immunotherapy of infectious diseases, such as tuberculosis (TB), HIV infections such as AIDS, and hepatitis B (HepB) virus infections.
  • infectious diseases such as tuberculosis (TB), HIV infections such as AIDS, and hepatitis B (HepB) virus infections.
  • the immunogenic compositions or vaccines are administered to a vaccinee.
  • the vaccinee is a mammal, and in one embodiment a human, to whom the vaccines or immunogenic compositions have been administered.
  • the polynucleotide of the vaccines and the adjuvants of the present invention may be administered simultaneously or separately.
  • the polynucleotide and the adjuvant may be co-formulated in a single composition, or alternatively may be separately formulated in distinct compositions.
  • the at least two compositions are administered in functional cooperation, and may be administered at substantially the same time, or alternatively be administered at different time points separated by, in different embodiments, within 30 minutes to 1 hour apart, or within 1 and 2 hours apart, or within 12-36 hours apart, such as 24 hours apart; or the two compositions may, substantially, be administered the next following day.
  • the polynucleotide may be administered before the adjuvant.
  • the vaccine compositions may further be provided in a single composition, comprising both the polynucleotide and the adjuvant, wherein the adjuvant composition is in a delayed release formulation which allows the release of the adjuvant composition at the site of injection within 30 minutes to 1 hour after administration, or within 1 and 2 hours after admimstration, or within 12-36 hours after administration, such as 24 hours after administration.
  • a kit comprising two compositions, the polynucleotide containing composition and the adjuvant containing composition, for separate administration.
  • the separate admimstration may be separated by administration site or time, or both.
  • a method of raising an immune response in an individual against an antigen comprising administering to that individual a polynucleotide composition encoding the antigen, followed by administering to said individual an adjuvant composition comprising a product of purine catabolism, such as uric acid, or salt thereof.
  • the adjuvant composition is administered within 12 to 36 hours after the administration of the polynucleotide.
  • the polynucleotide is a DNA plasmid vector, or alternatively in the form of a viral vector, such as a simian adenovirus vector.
  • Also provided by the present invention is a method of therapeutically treating a patient having a tumour, comprising administering to that patient a polynucleotide composition encoding a tumour associated antigen, followed by administering to said patient an adjuvant composition comprising a product of purine catabolism, such as uric acid, or salt thereof.
  • an adjuvant composition comprising a product of purine catabolism, such as uric acid, or salt thereof.
  • the adjuvant composition is administered within 12 to 36 hours after the administration of the polynucleotide.
  • the polynucleotide is a DNA plasmid vector, or alternatively in the form of a viral vector, such as a simian adenovirus vector.
  • plasmids of the vaccines are prevented from replicating within the mammalian vaccinee and integrating within the chromosomal DNA of the host, as such the plasmid may be produced without an origin of replication that is functional in eukaryotic cells.
  • the immunogen component comprising a vector which comprises the nucleotide sequence encoding an antigenic peptide can be administered in a variety of manners.
  • the vector can be administered in a naked form (that is, as a naked nucleotide sequence not in association with liposomal formulations, with viral vectors or transfection facilitating proteins) suspended in an appropriate medium, for example a buffered saline solution such as PBS, and then injected intramuscularly, subcutaneously, intraperitonally or intravenously (Brohm, et al. (1998) Vaccine 16 ⁇ 949-54, the disclosure of which is included herein in its entirety by way of reference). It is additionally possible for the vectors to be encapsulated by, for example, liposomes or within polylactide co-glycolide (PLG) particles for administration via the oral, nasal or pulmonary routes in addition to the routes detailed above.
  • PEG polylactide co-glycolide
  • intradermal administration of the immunogen component for example via use of gene-gun (particularly particle bombardment) administration techniques.
  • Such techniques may involve coating of the immunogen component on to dense micro-beads, such as gold beads, which are then administered under high pressure into the epidermis, such as, for example, as described in Haynes, J.R. et al. (1996; Joxraial of Biotechnology 44: 37-42).
  • the adjuvant component may be co-formulated on the dense microbeads, or on separate populations of microbeads, or alternatively the polynucleotide vaccine may be administered ballistically on microbeads and the adjuvant administered separately via systemic or local delivery, possibly at the site of polynucleotide delivery by intradermal or subcutaneous injection.
  • a patch comprising a plurality of needles, being in the range of 30-1000 micrometers in length, the external surface of which is coated with a solid reservoir medixim.
  • the solid reservoir medium in this context would comprise the vaccines of the present invention in solid form. Microneedles of this form are described in WO 02/07813 and WO
  • the adjuvants and vaccines of the present invention may be administered via a variety of different administration routes, such as intramuscular, subcutaneous, intraperitoneal, intradermal, or topical routes.
  • the adjuvant or polynucleotide components may be administered via the subcutaneous, intradermal or topical routes. In one embodiment, the administration of both components, the polynucleotide and adjuvant, is by the same route.
  • the polynucleotide is administered by ballistic delivery (gene gun) into the epidermis or dermis, and the adjuvant composition is delivered in the vicinity of the polynucleotide either topically or by intradermal or subcutaneous injection.
  • the dose of admimstration of the adjuvant will also vary, but may, for example, range in a liquid form of the vaccine between about 5 ⁇ g per ml to about 5 mg per ml, and may be between 25 ⁇ g per ml to about 1 mg per ml, and may be between 50 to 500 ⁇ g per ml. In a liquid form between 0.5 and 1 ml of the vaccine may be administered to a human vaccinee.
  • a total mass of the adjuvant may also be in the range of 5 ⁇ g to about 5 mg per dose, and may be between 25 ⁇ g to about 1 mg per dose, and may be between 50 to 500 ⁇ g per dose.
  • a dosing schedule may be one where sufficient uric acid, or other product of purine catabolism, is administered to a vaccination site which results in high enough localised concentration such that crystallisation occurs and crystals of uric acid, or a salt thereof, can then mediate the adjuvant effect.
  • the adjuvant component is uric acid such a concentration is likely to be greater than or equal to 70 micrograms of uric acid per ml of solvent in a localised concentration at the site of administration.
  • Administration of the adjuvant may be repeated with each subsequent or booster administration of the nucleotide sequence.
  • the dose of the polynucleotide encoding the antigen will depend on the route of administration and will be readily determined by the man skilled in the art. Conventionally speaking for gene gun applications the dose will be between 0.5 and lOO ⁇ g per administration, and for intramuscular administration of "naked" DNA between 10 and 2000 ⁇ g per administration.
  • This experiment investigated the effect of uric acid admimstration on solid E.G7-OVA tumour growth in vivo following DNA vaccination by gene gxxn performed according to two different immunisation schedules.
  • mice Male C57B1 6 mice (Charles River Ltd., Thanet, UK) were used throughout all experiments. All animals were shaved on the lower back immediately prior to tumour cell implantation, and on the abdomen immediately prior to initial gene gun application.
  • the ovalbumin-transfected murine lymphoma line E.G7-OVA (Moore, M.W. et al. (1988) Cell 54: 777-85) was used in this experiment. 5 x 10 6 live E.G7-OVA cells at nominal passage number 23 were injected subcutaneously into shaved skin of the lower back in 0.1 ml phosphate buffered saline. Date of implantation was designated day 0 ("dO"). Palpable tumours formed at the implant site were measured using callipers, and tumour size in mm 2 (representing the longest measured diameter multiplied by the diameter perpendicular to it) was recorded every two days up to the tumour end point for each animal. Tumour end points were defined as the points at which tumours either exceeded 250 mm 2 or became ulcerated (all such animals were euthanased for ethical reasons), or tumoxxrs regressed completely.
  • Cartridges were cut from dried tubing. Samples from each batch of prepared cartridges were tested to ensure a DNA loading of 0.5 ⁇ g DNA per cartridge. Briefly, cartridge DNA was eluted by immersion of cartridges in DNAse/RNAse-free water and incubation at 37 °C for 30 min, followed by momentary centrifugation at 14,000 rpm. A supernatant sample (70 ⁇ l) was then transfened into a quartz capillary (Pharmacia Biotech), and the DNA concentration determined using a GeneQuant II DNA/RNA calculator (path length 10 mm, blanked against purified water; Pharmacia Biotech).
  • Txxmour size was recorded for each animal every two days up to tumour end points. Plotted growth curves showing mean tumour size over time for each group are presented in Figure 1. The control groups ("pVacl d2, d4 + borate d4" and "pVacl d7 + borate d8";
  • Fig 1 show typical uninliibited txxmour growth. Immunisation with pVacl.OVA(cyt) via the d2, d4 schedule delays tumour growth, and immxxnisation at d7 only has a similar effect. Admimstration of uric acid at d4 following immunisation at d2 and d4 appears to alter the kinetics of tumour growth. The administration of uric acid at d8 following a single immunisation at d7
  • mice (group 6; "pVacl.OVA(cyt) d7 + UA d8") has the greatest effect on tumour growth, with three of the six mice having tumours that regressed completely by day 31 post- implantation (no tumour re-growth was subsequently observed). Of the remaining mice in group 6, two had tumours which exceeded 250 mm 2 (and were therefore euthanased for ethical reasons), and one had a tumour which appeared to have anested growth but did not regress (this animal was euthanased on day 31 due to txxmour ulceration).
  • Example 2 Variation of adjuvantsolution preparation and administration protocol, and its effect on solid E.G7-OVA tumour growth in vivo following DNA vaccination and adjuvant administration
  • aspects of the adjuvant solution preparation and administration protocols were varied, and the effect on solid E.G7-OVA tumour growth in vivo observed following DNA vaccination by gene gun and adjuvant administration.
  • a precipitation time of at least 24 hours was required to illicit an adjuvant effect, and this was improved after 48 hours. There was no further improvement after 72 hours compared to 48 hours.
  • the degree of crystallisation allowed to occur in the adjuvant solution seems, therefore, to influence the adjuvanticity of the solution independently of the prepared uric acid concentration.
  • experiment C the time of adjuvant administration relative to DNA vaccination was varied. An adjuvant effect was observed when the uric acid solution was administered 24 hours post- vaccination.
  • experiment D the site of adjuvant injection was varied, and compared to injections of control borate buffer prepared as described in section 2.1.3 but without addition of uric acid.
  • Adjuvant effect was observed when the adjuvant solution was administered at the site of DNA vaccination, but not when it was injected at a remote site (the flank). There was some indication of adjuvant effect when the solution was injected at the tumour growth site, but may not be significant and has not been observed in a repeat experiment.
  • Example 3 Measurement of cytokine production following immunisation and administration of uric acid adjuvant solution against a growing tumour.
  • This experiment investigated levels of cytokine production following DNA vaccination by gene gun and administration of uric acid adjuvant solution against a growing solid E.G7-OVA tumour.
  • mice Female C57B1/6 mice (Charles River Ltd., Thanet, UK) were shaved on the flank immediately prior to tumour cell implantation, and on the abdomen immediately prior to initial gene gun application.
  • Tumour cells 1 x 10 6 live E.G7-OVA cells were injected subcutaneously into shaved skin of the flank in 0.1 ml phosphate buffered saline.
  • Intracellular cytokine staining 10 ⁇ l of anti-mouse CD28 antibody / anti-mouse CD49d antibody solution was added to 4 x 10 splenocytes and the mixture incubated for 10 minutes at room temperature. 1 ml of SIINFEKL peptide (300nM) was added followed by incubation at 37° C for 1 hour in a humidified atmosphere with 5% CO 2 . Brefeldin A (20 ⁇ l) was added and the tubes placed in a waterbath for 6 hours at 37° C, before storage overnight at 4° C.
  • SIINFEKL peptide 300nM
  • Brefeldin A 20 ⁇ l
  • Splenocytes were resuspended in 250 ⁇ l of BD cellfix reagent (Becton Dickinson) then analysed on a FACSCalibur flow cytomoter. Blood samples were resuspended in 250 ⁇ l of lysis buffer (Beckman Coulter) diluted 1 part in 25 parts PBS, left at room temperature for 2 minutes, and then 250 ⁇ l of fixative (Beckman Coulter) added. Blood cells were washed and resuspended in flow cytometry buffer then analysed on a FACSCalibur flow cytometer.
  • ELISPOT data from splenocytes collected at days 11 and 14 post tumour-implantation are shown in Fig. 4 and 5 respectively.
  • the results of intracellular cytokine staining from splenocytes collected at day 14 are illustrated in Fig. 6.
  • Tetramer staining from splenocyte and blood samples taken at day 14 is shown in Fig. 7.
  • Splenocytes stimulated ex vivo with the CD8-recognised peptide SIINFEKL showed IFN-gamma and IL2 production at both days 11 (Fig. 4 a, b) and 14 (Fig. 5 a, b).
  • Cytokine production in empty vector-immunised groups may be attributable to CD8 + cells activated by tumour-derived antigen or the inflammatory effects of uric acid admimstration.
  • IFN-gamma and IL2 production was increased in groups that received the uric acid adjuvant compared with those that received only borate buffer.
  • Splenocytes stimulated with the CD4-recognised peptide TEWT (Fig. 4 c, d and
  • Fig. 5 c, d showed little production of either cytokine, and there were only marginal increases in groups immunised with ova-encoding DNA compared with empty vector controls.
  • Intracellular cytokine production at day 14 in CD8 + spleen-derived T-cells detected by ICS (Fig. 6) reflected the pattern that was detected by the ELISPOT assay.
  • Groups immunised with ova-encoding DNA produced higher levels of intracellular cytokines (particularly IFN-gamma) than empty vector controls, and production appeared highest in the group that received the uric acid adjuvant.
  • Tetramer staining of spleen and blood derived cells taken at day 14 (Fig.
  • cytokine production by immune cells is quantified ex vivo in tumour-bearing mice that have been immxxnised by DNA vaccination against a tumoxxr associated antigen with or without subsequent administration of the uric acid adjuvant.
  • the SIINFEKL peptide an ovalbumin-derived CD8-stimulatory sequence, was used to quantify ex vivo CD8 + cell populations for cytokine capture (ELISPOT) assays, while the ovalbumin-derived CD4-stimulatory peptide TEWT was used to quantify CD4 + cells.

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Abstract

La présente invention concerne un adjuvant pour vaccins polynucléotidiques qui comprennent, ou sont administrés conjointement avec, une composition qui est un produit de dégradation d'une purine pouvant être en particulier de l'acide urique. Les vaccins polynucléotidiques de l'invention sont des vaccins codant pour un antigène contre lequel il est souhaitable de déclencher une réponse immunitaire, le vaccin polynucléotidique pouvant être en particulier un vaccin à ADN. Cette invention concerne également l'utilisation d'acide urique pour la fabrication d'une composition de vaccin polynucléotidique devant accentuer la réponse immunitaire contre l'antigène spécifique codé par ledit vaccin. Sont décrits des compositions de vaccin, des kits renfermant une compositions polynucléotidique séparée et des compositions adjuvantes pour administration distincte, des procédés de fabrication des vaccins ainsi que des kits, et des méthodes de traitement au moyen des compositions de vaccin de la présente invention.
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Publication number Priority date Publication date Assignee Title
WO2012112691A1 (fr) * 2011-02-15 2012-08-23 Immune Design Corp. Procédés d'amélioration des réponses immunitaires spécifiques d'un immunogène avec des vaccins vectorisés
WO2014064229A1 (fr) * 2012-10-25 2014-05-01 Novartis Ag Nicotinamide en tant qu'adjuvant

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WO2004100984A1 (fr) * 2003-05-13 2004-11-25 The University Of Massachusetts Molecules endogenes d'adjuvant et utilisations de celles-ci

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WO2004100984A1 (fr) * 2003-05-13 2004-11-25 The University Of Massachusetts Molecules endogenes d'adjuvant et utilisations de celles-ci
US20050025790A1 (en) * 2003-05-13 2005-02-03 The University Of Massachusetts An Agency Of The Commonwealth Of Massachusetts Endogenous adjuvant molecules and uses thereof

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012112691A1 (fr) * 2011-02-15 2012-08-23 Immune Design Corp. Procédés d'amélioration des réponses immunitaires spécifiques d'un immunogène avec des vaccins vectorisés
WO2014064229A1 (fr) * 2012-10-25 2014-05-01 Novartis Ag Nicotinamide en tant qu'adjuvant

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