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WO2008151389A1 - Macromolécules modifiées chimiquement - Google Patents

Macromolécules modifiées chimiquement Download PDF

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Publication number
WO2008151389A1
WO2008151389A1 PCT/AU2008/000864 AU2008000864W WO2008151389A1 WO 2008151389 A1 WO2008151389 A1 WO 2008151389A1 AU 2008000864 W AU2008000864 W AU 2008000864W WO 2008151389 A1 WO2008151389 A1 WO 2008151389A1
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Prior art keywords
dendrimer
cells
mdo
ova
antigen
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PCT/AU2008/000864
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English (en)
Inventor
Geoffrey Allan Pietersz
Martha Kalkanidis
Kuo-Ching Sheng
Vasso Apostolopoulos
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Macfarlane Burnet Institute For Medical Research And Public Health Limited
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Priority claimed from AU2007903219A external-priority patent/AU2007903219A0/en
Application filed by Macfarlane Burnet Institute For Medical Research And Public Health Limited filed Critical Macfarlane Burnet Institute For Medical Research And Public Health Limited
Publication of WO2008151389A1 publication Critical patent/WO2008151389A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • 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
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention generally relates to chemically modified macromolecules for use in immunogenic compositions and combinations.
  • the invention relates to immunogenic compositions and combinations comprising an antigen and one or more synthetic dendrimers wherein the dendrimers incorporate or are conjugated to one or more adjuvant groups to enhance or potentiate the immunogenic effect of the antigen.
  • the invention further relates to the modified dendrimers, methods for preparing them and molecules, compositions and combinations comprising them, as well as their use in immunotherapy.
  • MUCl tumor associated antigens
  • cytotoxic T cell epitopes have been identified and used as targets for immunotherapy (Tan et al, 1997).
  • MUCl is overexpressed in adenocarcinomas, especially cancers of the breast and colon, and has thus been identified as a potential target for immunotherapy for antibodies, antibody-drug immunoconjugates and cytotoxic T cells (Karanikas et al, 1997; Pietersz et al, 1997; Sutton et al, 1994).
  • MUCl is a high molecular weight glycoprotein with multiple 20 amino acid repeats denoted 'variable number of tandem repeats' (VNTR).
  • MUCl is also present on non-cancerous cells, thus far the immunization experiments in MUCl transgenic mice, monkeys and humans have shown no evidence of autoimmune disease (Vaughan et al, 2000; Karanikas et al, 1997, 2001; Ko et al, 2003).
  • an adjuvant is necessary to enhance the ability of the antigen to induce an effective immune response.
  • Alum is an example of a common adjuvant and is used in the diphtheria and tetanus toxoid vaccines.
  • alum can in some cases combine with the antigen to form a toxic complex. Therefore, many vaccines, such as the influenza vaccine, are non-adjuvanted for this reason.
  • Antigen mannosylation has been recognised as a possible means to enhance antigen immunogenicity. Mannosylation results in enhanced antigen uptake by antigen presenting cells (APCs) via receptor-mediated endocytosis, and subsequent MHC class I and class II presentation to T cells.
  • APCs antigen presenting cells
  • mannose residues in antigen delivery has also been demonstrated by the mannose polymer, mannan, which enhanced antigen-specific ThI /cytotoxic T lymphocyte (CTL) and Th2/antibody responses in oxidized and reduced forms, respectively (Apostolopoulos et al, 1995; Davis et al., 2002; Lees et al., 2000;ENSopoulos et al., 2000; Lofthouse et al., 1997; protestopoulos et al., 1996a).
  • CTL cytotoxic T lymphocyte
  • oxidized mannan-MUCl fusion protein conjugates produce strong cellular responses (CTL, IFN ⁇ , IL- 12) in mice and are protected from a MUCl+ve tumor challenge (Apostolopoulos et al., 1996b).
  • CTL oxidized mannan-MUCl fusion protein conjugates
  • the oxidized mannan conjugates stimulated lymphocytes to proliferate and induced TNF- ⁇ and IL- 12 from macrophages whereas reduced mannan (no aldehydes) did not induce these cytokines (Apostolopoulos et al., 2000a).
  • reduced mannan no aldehydes
  • IFN ⁇ intracellular staining
  • proliferative T cell responses and some had detectable cytotoxicity against MUCl targets, these responses were not intense enough to induce clinical responses.
  • Dendrimers are well defined highly branched macromolecules characterized by a central core bearing one or multiple reactive sites, at which subsequent layers or 'generations' of monomers (or repeat units) are attached, and an 'exterior surface' of functional terminal - A -
  • Selective conditions of manufacture can control the number of generations of repeat units and allow for the formation of dendrimers of varying size and shape, including radially symmetrical as well as radially unsymmetrical "wedge"-shaped molecules with varying and controlled patterns of terminal groups.
  • the present invention provides a dendrimer for use in an immunogenic combination wherein the dendrimer comprises one or more adjuvant groups conjugated to the surface of the dendrimer.
  • adjuvant groups may be conjugated directly (for example by way of covalent, hydrogen or electrostatic bonding) to a terminal functional, or reactive, group of the outer layer or generation of monomers of repeating units of the dendrimer, or may be attached by a suitable chemical linker group.
  • the invention provides an immunogenic combination comprising: (i) an antigen, and
  • a dendrimer comprising one or more adjuvant groups conjugated to the surface of the dendrimer.
  • an immunogenic composition comprising: (i) an antigen,
  • a dendrimer comprising one or more adjuvant groups conjugated to the surface of the dendrimer; and (iii) a pharmaceutically acceptable carrier.
  • the present invention provides a method of inducing an immune response in a subject comprising administering to said subject an antigen together with a dendrimer which comprises one or more adjuvant groups conjugated to the exterior surface of the dendrimer.
  • the present invention provides a method of enhancing the cell mediated immunity of a subject, said method comprising:
  • the adjuvant groups may the same or different.
  • the adjuvant group is selected from a group which contains a mannose group or an aldehyde functional group.
  • the adjuvant groups may be all mannose-containing groups, all aldehyde-containing groups or a combination of both.
  • compositions or combinations according to the invention may further comprise one or more additional adjuvants which are not conjugated to the dendrimer.
  • the antigen is present as an entity discrete from the dendrimer, i.e., unconjugated. In other embodiments the antigen and dendrimer are combined in a single molecule.
  • the present invention provides an immunogenic molecule comprising an antigen conjugated to a dendrimer, wherein said dendrimer has one or more adjuvant groups conjugated to the surface thereof.
  • the antigen may be located at the surface of the dendrimer. In other embodiments the antigen is located at the core of the dendrimer, typically by covalent bonding. In further embodiments, the antigen is conjugated to the core of the dendrimer at an unreacted reactive site of the core, a protected reactive site that can be deprotected or a thiol group at the core arising from reduction of a disulfide linkage within the core.
  • the antigen and dendrimer may be administered either as a single formulation or composition containing the separate components, or as separate formulations.
  • Fig. 1 provides a gel photograph of the optimization of MDO conjugation with various amounts of MD.
  • OVA/SPDP was reacted with 0, 4, 8, 12 and 16 fold molar excess of MD.
  • These conjugates (before gel filtration chromatography) were analyzed by 15% SDS-PAGE using coomassie staining in wells 1 - 5 (OVA, MDO 4x, MDO 8x; MDO 12x and MDO 16x).
  • the molecular weights of MDO conjugates ranged from 64 - 98 kDa.
  • Unreacted MD appeared at 6 and 16 kDa.
  • OVA (43 kDa) was fully reacted during MDO conjugation, as no free OVA was detected.
  • Fig. 2 provides graphical representation demonstrating that MDO-pulsed BMDCs induce high levels of MHC-class I and II presentation CD4 + and CD8 + T cell proliferation.
  • A, B Titrated (500 - 4000) DCs pre-pulsed with control peptides (SIINFEKL [1 ⁇ g/ml], OVA 323-339 [10 ⁇ g/ml]), MDO (40 ⁇ g/ml) and OVA (40 ⁇ g/ml) were co-cultured with 2 x 10 4 OTI or OTII T cells in quadruplicates. T cell proliferation was monitored and peak values on day 2 (OTI) or day 3 (OTII) corresponding to each stimulant were compared.
  • Fig. 3 provides graphical representation demonstrating that MDO induces maturation of BMDCs.
  • DCs stimulated with PBS, OVA (40 ⁇ g/ml), MDO (40 ⁇ g/ml) and LPS (1 ⁇ g/ml) in duplicates for 18 h were analyzed for their CD40, CD80 and CD86 expression by MFI in the histogram.
  • A. MFI values of CD40, CD80 and CD86 were plotted against the corresponding stimulant.
  • B Histograms on CD40, CD80 and CD86 expression of a replicate from each stimulant condition are shown. The shaded area represents cells stained with the isotype control antibody. Data shown are representative of two different experiments. * P ⁇ 0.05, ** P ⁇ 0.01 (LPS or MDO versus PBS-pulsed DCs).
  • Fig. 4 provides graphical representation demonstrating that MDO binds BMDCs with a high avidity.
  • A. BMDCs which were incubated with titrated OVA and MDO (0.04 - 40 ⁇ g/ml) were analyzed by FACS after FITC labeling. The binding of MDO or OVA was determined by MFI values corresponding to specific concentrations.
  • Fig. 5 provides graphical representation demonstrating that MDO induces high levels of cellular and humoral immunity in immunized mice.
  • Mice (n 4) immunized with PBS, OVA (25 ⁇ g), MDO (12.5 or 25 ⁇ g) were sacrificed after 3 immunizations, and levels of A.
  • OVA 323 . 339 (CD4 epitope)-, SIINFEKL (CD8 epitope)- and OVA-specific T cell IFN ⁇ responses (evaluated by the ELISpot assay in triplicates) and B. the total IgG level to OVA (determined at 1 :400 serum dilution in the ELISA) were compared. Data shown are representative of two experiments. SFU: spot forming unit; * P ⁇ 0.05, ** P ⁇ 0.01.
  • Fig. 6 provides a graph demonstrating that regional LN cells isolated from mice injected with MDO, but not OVA, induce OTI T cell proliferation.
  • Popliteal LN cells isolated from groups of 4 mice injected separately with PBS, OVA (25 ⁇ g) and MDO (25 ⁇ g) into footpads were evaluated for their capacities in OTI T cell stimulation in vitro.
  • LN cells (2 x 10 4 ) isolated from injected mice were co-cultured with 5 x 10 4 OTI T cells in quadruplicates. T cell proliferation was monitored from days 1 - 4 and peak proliferation on day 2 was compared.
  • Fig. 8 provides graphical representation showing that MDO binds to DCs.
  • DCs cultured with GM-CSF/IL-4 (100 ng/ml) and Flt-3 ligand (300 ng/ml) were harvested at day 6.
  • Cells (5 x 10 5 ) were pelleted and incubated with MDO and OVA for 30 min. Cells were washed and labeled with anti-mouse CDl Ic and rabbit anti-OVA antibodies. After 20 min incubation, cells were treated with FITC-conjugated anti-rabbit antibody and analyzed by flow cytometry.
  • the Flt-3L culture yielded > 90% CDl Ic + population (not shown) that was divided into 3 heterogeneous subpopulations (CD24 high , CDl lb high and double-negative[CD220 + ]). They all bound to MDO, but not OVA.
  • the shaded area represents cells only treated with primary and secondary antibodies.
  • Fig. 9 provides graphical representation showing that MDO induces maturation of myeloid, but not plasmacytoid, DCs.
  • Fig. 10 provides graphical representation showing that MDO-induced DC maturation is dependent on TLR4.
  • stimulants including PBS (background control), MDO (40 ⁇ g/ml), LPS (1 ⁇ g/ml) and CpG1668 (10 ⁇ g/ml) were added into C3H/He and C3H/HeJ DC cultures.
  • DCs were harvested and analysed for CD40 and CD86 expression by flow cytometry. While the maturation effect of MDO and LPS in C3H/He DCs was greatly diminished in TLR4-defective C3H/HeJ DCs, the effect of CpG remained unchanged.
  • the shaded area represents cells stained with the isotype control antibody.
  • Fig. 11 provides graphical representation showing that MDO-pulsed BMDCs and FU3-L DCs induce OVA-specific CD4 + and CD8 + T cell proliferation.
  • Titrated (1 - 4 x 10 3 ) BMDCs (A) and Flt3-L DCs (B) pulsed with MDO (40 ⁇ g/ml), OVA (40 ⁇ g/ml) or control peptides (SIINFEKL [1 ⁇ g/ml] and OVA 323-339 [10 ⁇ g/ml]) were seeded with 2 x 10 4 OTI or OTII T cells.
  • T cell proliferation was monitored for 5 days with 3 H-thymidine incorporation. Peak proliferation on day 2 and day 3 for OTI and OTII T cells was compared. The data shown is representative of three experiments.
  • Fig. 12 provides graphical representation showing the effect of NH 4 Cl on OT-II Tcell proliferation and costimulatory molecule expression on DCs.
  • Fig. 13 provides graphical representation showing that CD24 hl DCs are the primary subset after 10 days of Flt-3L culture.
  • Fig. 14 provides graphical representation showing that GM-CSF/IL-4 DCs cross-present MDO to OTI T cells in the presence of malondialdehyde.
  • DCs (4 x 10 3 ) preloaded with 40 ⁇ g/ml MDO in the presence of titrated malondialdehyde (25 - 400 ⁇ g/ml), with 40 ⁇ g/ml OVA and with 40 ⁇ g/ml OVA plus 400 ⁇ g/ml malondialdehyde, were incubated with 2 x 10 4 OTI T cells. T cell proliferation was monitored using the [ 3 H]thymidine incorporation assay. Peak proliferation induced by DCs on day 2 from each pulsing condition was compared. Malondialdehyde used in 100 - 400 ⁇ g/ml resulted in cross- presentation of MDO by DCs.
  • GM-CSF/IL-4 DCs did not cross-present OVA to OTI T cells in the presence of malondialdehyde.
  • Fig. 15 provides graphical representation showing that GM-CSF/IL-4 DCs presents MDO- acetal to OTII T cells resulting in proliferation of the OTII T cells.
  • DCs (1 - 4 x 10 3 ) preloaded with 20 ⁇ g/ml MDO or MDO-acetal (derived from homogeneous fraction 1 or heterogeneous fraction 2 during FPLC purification) were incubated with 2 x 10 4 OTII T cells. T cell proliferation was monitored and peak proliferation induced by DCs on day 3 from each pulsing condition was compared. DCs preloaded with MDO and MDO-acetal (fraction 1) were highly stimulatory to OTII T cells.
  • Fig. 16 provides graphical representation showing that MDO-acetal promotes cross- presentation of OVA by GM-CSF/IL-4 DCs to OTI T cells.
  • DCs pre-loaded with MDO-acetal (fraction 1) induced a higher level of OTI T cell proliferation than those with MDO and MDO-acetal (fraction 2).
  • dendrimer as used herein is to be understood in its broadest sense and includes within its scope all forms and types of dendrimers known in the art.
  • Typical dendrimers contemplated herein have a core moiety with 1, 2, 3 or more reactive sites, at least one layer, typically 2, 3, 4, 5, 6, 7, 8, 9 or 10, of branched repeat units or monomers, and reactive terminal groups at the outer layer or surface suitable for conjugation to one or more adjuvant groups. It will be understood that the dendrimers contemplated herein are suitable for administration to a subject.
  • the "surface” of the dendrimer refers to the outermost layer or generation of repeat units of each branch of the dendrimer.
  • the dendrimers contemplated herein may be composed of a single monomer type or repeat unit or two or more monomer types or repeat units (see for example Per Antoni, et al).
  • Exemplary but non-limiting dendrimers contemplated herein include dendrimers based on polyamidoamine (PAMAM), poylysine, poly(etherhydroxylamine) (PEHAM) or polypropyleneimine (PPI) repeating units or monomers.
  • PAMAM polyamidoamine
  • PHAM poly(etherhydroxylamine)
  • PPI polypropyleneimine
  • One class of dendrimer specifically contemplated herein are PAMAM based dendrimers.
  • the core moieties may be any compound having at least one reactive site to which monomer or repeat units may be covalently attached.
  • Some exemplary suitable cores include those having 2, 3 or 4 reactive groups (selected from, for example, amino, carboxyl, thiol or hydroxy groups) to which the layers or generations of repeat units or monomers can be attached.
  • Typical examples include non-cleavable diaminoC 2 -Ci 2 alkanes such as ethylene diamine, 1 ,4-diaminobutane, 1,6-diaminohexane.
  • Suitable cores may be cleavable, for example a diaminoC 2- i 2 alkane having a disulfide (S-S) linkage within the alkane chain, such as cystamine ( 2 HN-(CH 2 ) 2 -S-S-(CH 2 ) 2 -NH 2 ).
  • Cleavable cores such as those having a disulfide bond, may advantageously provide a convenient point of attachment for the antigen by cleavage of the disulfide linkage to form a reactive thiol group.
  • Other cores contemplated herein contain only one reactive site from which the dendrimer is generated.
  • these may also advantageously contain a protected disulfide linkage, such as a pyridyldithio group, which may be cleaved to form a reactive thiol group to which an antigen may be conjugated.
  • a protected disulfide linkage such as a pyridyldithio group
  • the core is not necessarily a linear moiety with a single reactive group at each end.
  • Other "non-linear" core moieties are also contemplated, and include trihydroxypropylamine, or aromatic moieties such as 1,3,5-benzenetricarboxylic acid and benzhydrylamine (BHA).
  • the dendrimer or each independent "branch" extending from the core has at least 2 generations of monomer or repeat units (Generation 2).
  • the dendrimer is at least a Generation 3 dendrimer, i.e., has at least 3 layers or generations of monomer or repeat units attached to the central core.
  • the dendrimer is a Generation 3 to Generation 10 dendrimer, for example a Generation 4, 5 or 6 dendrimer.
  • Suitable reactive terminal groups on the dendrimer surface through which the adjuvant groups (and optionally an antigen) may be conjugated include amino-, hydroxy-, carboxy- and thio- groups.
  • the functional groups may be part of the monomer or repeat unit of which the outer layer or generation of the dendrimer is comprised, or may be introduced by further chemical modification of the outer monomer or repeat unit. Suitable methods for doing so are with the art of organic synthesis.
  • Some particular exemplary functional terminal groups are amidoethanol, amidoethylethanolamine, amino, carboxylate or succinamic acid, tris(hydroxymethyl)amidomethane, and 3-carbomethoxypyrrilidione groups.
  • the end group functions can be modified to other reactive functionalities to enable modification with various pendant groups.
  • Dendrimers with surface amino groups can be readily functionalised to carboxylic acid groups by using acid anhydrides such as succinic or glutaric anhydride.
  • amino groups maybe functionalised to thiol groups by reaction with S- acetylmercaptosuccinic anhydride (SAMSA) or N-hydroxysuccinimidyl acetylthioacetate (SATA) followed by hydroxylamine.
  • SAMSA S- acetylmercaptosuccinic anhydride
  • SATA N-hydroxysuccinimidyl acetylthioacetate
  • aminogroups can be modified with N- hydroxysuccinimidyl pridyldithiopropionate (SPDP) followed by reduction with dithiothreitol (DTT).
  • SPDP N- hydroxysuccinimidyl pridyldithiopropionate
  • Dendrimers with ester functionalities can be reacted with hydrazine hydrate to generate hydrazides.
  • Bromoacetyl groups can be introduced by reaction of aminodendrimers with N-hydroxysuccinimidyl bromoacetate.
  • Dendrimers may also be functionalised with alkyne groups such that other pendant groups such as sugars with azide groups maybe added using Click chemistry.
  • the dendrimer is a Generation 3- Generation7 PAMAM dendrimer.
  • a dendrimer typically has a diaminoalkane core or disulfide-containing diamino alkane core such as a cystamine core, which may be advantageously cleaved to provide a point of attachment for the antigen.
  • PAMAM dendrimers which may incorporate any of the functional terminal groups noted above include:
  • PAMAM dendrimer 1,12-diaminododecane (Generations 2, 3, 4, 5, 6 or7); PAMAM dendrimer, 1,4-diaminobutane core (Generations 2, 3, 4, 5, 6 or 7); PAMAM dendrimer, 1,6-diaminobutane core (Generations 2, 3, 4, 5, 6 or 7); PAMAM dendrimer, cystamine core (Generations 2, 3, 4, 5, 6 or 7); PAMAM dendrimer, ethylenediamine core (Generations 2, 3, 4, 5, 6 or 7).
  • Dendrimers contemplated for use herein may be formed by reaction at all of the reactive sites of a core moiety, for example by reaction at both amino groups of a diaminoalkane core (optionally with a disulfide linkage).
  • dendrimers may be formed by building successive generations or layers of monomer or repeat units at only one, or only some of the available number of reactive sites. This requires protection of the site(s) which remain unreacted, i.e. do not serve as a point of attachment for successive monomer or repeating units to form dendritic branches, with a suitable protecting group which may be subsequently removed once the dendrimer has been assembled.
  • Suitable protecting groups for reactive sites such as amino, thiol or hydroxy groups, are known within the art (see for example, Greene and Wuts, Protective Groups in Organic Synthesis, 1999 and Krapcho and Kuell, Synthetic Comntun., 20:2559, 1990).
  • dendrimers are well known in the art and are extensively described in the patent and scientific literature, for example, US Patent Nos 4,289,872, 4,376,861,4,410,688, 4,507,466, 4,515,920, 4,517,122, 4,558,120, 4,568,737, 4,587,329, 4,599,400, 4,600,535, WO/88/01178, WO/88011709 and WO/8801180 the contents of which are incorporated herein by reference. Suitable dendrimers are also commercially available from suppliers such as Sigma-Aldrich and Dendritic Nanotechnologies Inc.
  • adjuvant group refers to any chemical group, salt, complex or ion which accelerates, enhances, amplifies, potentiates or prolongs the immune response elicited or induced by an antigen. In some embodiments, this may allow for the administration of an amount of antigen which less than what is otherwise required to elicit the desired immune response. In other embodiments this may achieve a desired immune response which the antigen alone cannot effect.
  • Suitable adjuvant groups contemplated by the present invention include chemical groups containing a group selected from mannose and aldehyde functional groups. Other adjuvant groups contemplated include ligands that bind to C-type lectin receptors on antigen presenting cells. These can be simple sugars (e.g.
  • lactose or galactose or oligosaccharides, or peptide mimetics thereof, or peptide ligands that bind to these receptors.
  • Other adjuvant groups include toll-like receptor ligands, for example Pam3Cys, CpG and lipids (Eriksson EM, Jackson DC, Recent advances with TLR2-targeting lipopeptide-based vaccines. Curr Protein Pept Sci., 2007 8:412-7; Proudfoot O, strigopoulos V, Pietersz GA, Receptor-mediated delivery of antigens to dendritic cells: anticancer applications, MoI Pharm. 2007, 4:58-72.; Pietersz GA, Pouniotis DS, tendopoulos V, Design of peptide-based vaccines for cancer, Curr Med Chem. 2006, 13:1591 -607).
  • an "adjuvant" in the context of a discrete entity optionally administered in conjunction with the antigen and dendrimer combination of the invention refers to any organic or inorganic compound, molecule, complex or salt which accelerates, enhances amplifies, potentiates or prolongs the immune response elicited or induced by an antigen. This may allow for the administration of an amount of antigen less than what is otherwise required to elicit the desired immune response or achieve a desired response which the antigen alone cannot effect.
  • adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A.
  • Freund's Incomplete Adjuvant and Complete Adjuvant Difco Laboratories, Detroit, Mich.
  • Merck Adjuvant 65 Merck and Company, Inc., Rahway, N.J.
  • AS-2 SmithKline Beecham, Philadelphia, Pa.
  • aluminum salts such as aluminum hydroxide gel (alum) or aluminum
  • Cytokines such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
  • Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention may include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhanzyn.RTM.) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S.
  • AGPs aminoalkyl glucosaminide 4-phosphates
  • the adjuvant contains an aldehyde functional group. Suitable examples thereof include malondialdehyde or a protected form thereof, e.g., malondialdehyde dimethyl acetal, glycoaldehyde or glycoaldehyde dimer, tucaresol, aliphatic or aromatic mono- di- or polyaldehydes.
  • the adjuvant group or optional adjuvant may or may not elicit immunogenic activity in its own right.
  • conjugated includes any means of association between the adjuvant group and the dendrimer, and/or the antigen and the dendrimrer and includes covalent bonding as well as non-covalent bonding or forces such as hydrogen bonding, ionic bonding, Van der Waals forces and electrostatic interactions.
  • a group containing an aldehyde functional group is intended to include any chemical group which contains an aldehyde functional moiety (CHO).
  • a typical example of an aldehyde-containing group is succinimidyl 4-formylbenzoate (SFB).
  • the dendrimers can be functionalised to include aliphatic or aromatic aldehydes.
  • Amino groups can be modified with N-hydroxysuccinimde, isocyanates, isothiocyanates or suitably functionalised derivatives aliphatic or aromatic aldehydes. Alternatively, these maybe reacted with suitably functionalised acetals or dithianes that can be subsequently deprotected to the aldehyde.
  • Dendrimers may also be modified with glycolaldehyde to susequently rearrange to an aliphatic aldehyde via Armadori rearrangement. Furthermore, dendrimers with hydroxymethyl end groups may also be oxidised to aldehydes using Swern oxidation. Dendrimers synthesised or modified with vicinal diol or aminols (eg via acylation with serine) groups can be readily oxidised with periodate to introduce aldehydes. "Masked aldehydes" or “aldehyde precursor” groups include any chemical group which contains a moiety which may be converted to an aldehyde functional moiety, for example in vivo conversion to an aldehyde moiety.
  • Suitable masked aldehydes and aldehyde precurosor groups would be known to one skilled in the art and include acetals.
  • a typical example of an acetal group contemplated herein, as attached to the primary amime end group, is -C( O)-(CH 2 )2-S-CH 2 -CH(OCH 3 )2.
  • Aldehyde or aldehyde precursor/masked groups may additionally or alternatively be introduced by partial oxidation of some or all of the mannose residues with, for example, sodium periodate.
  • Mannose refers to ⁇ -D-mannopyranosyl, or its oxidized form.
  • Mannose groups can be conjugated to dendrimers via reactive end groups on the exterior surface of the dendrimer.
  • the end functional groups on the exterior surface of the dendrimer to which the adjuvant groups are attached are amino groups.
  • the mannose-containing group is ⁇ -D-mannopyranosylphenyl iosothiocyanate.
  • Mannose and other sugars can be added to dendrimers using a number of methodologies depending on the dendrimer functional groups.
  • a linker containing a variety of functional groups can be added via an ether linkage or thioether linkage at the anomeric carbon of sugar.
  • These functional groups may include and not limited to thiols, carboxylic acids, azides or isothiocyanates.
  • the linkers may be aliphatic or aromatic. Other methods for the conjugation of a mannose group onto the surface of the dendrimer would be known to those skilled in the art of organic synthesis.
  • the dendrimer has at least 10 % of the surface functional end groups conjugated to an adjuvant group. In further embodiments, the dendrimer has at least 20 or 25% of the surface functional groups conjugated to an adjuvant group. In still further embodiments, the dendrimer has at least 30, 40, 50, 60, 70, 75, 80, 90 or 95% of the surface functional groups conjugated to an adjuvant group. In yet other embodiments, each surface functional group is conjugated to an adjuvant group.
  • the adjuvant groups may be randomly distributed over the surface or alternatively may be introduced in a controlled or defined manner, for example in an alternating arrangement over the dendrimer surface through the selective use of protecting groups. Suitable methods therefore would be known to those skilled in the art (see for example US Patent No. 5,229,490).
  • dendrimer- antigen conjugates (as well as conjugates where the antigen is conjugated to the dendrimer surface) are also referred to herein as "immunogenic molecules".
  • immunogenic molecules Methods and reagents for the conjugation of macromolecules to antigens such as polypeptides are known to those skilled in the art (see for example Hermanson, G.T., Bioconjugate Techniques, Second Edition (Academic Press 2008) and the references cited therein).
  • the antigen and/or an unreacted reactive site of the core of the dendrimer is modified with a bifunctional agent to provide a linker between the dendrimer and antigen.
  • exemplary types of heterobifunctional crosslinker agents include groups selected from those reacting with a primary and/or secondary amine, e.g. N-hydroxysuccinimide (NHS); those reacting with a sulfhydryl group, e.g. haloacetamide (such as bromo or chloroacetamide), maleimide or a pyridylthio; and those reacting with a carboxyl group, e.g. hydrazide.
  • the antigen is conveniently conjugated to the dendrimer with succinimidyl-3-2(-pyridyldithio)propionate (SPDP) or analogue, such as succinimydyl 6-(3-
  • LC-SPDP [2-pyridyldithio]-propionamido hexanoate
  • This provides an amine-reactive N- hydroxysuccinimide (NHS) ester portion which may react with free amino groups on a dendrimer core or polypeptide and an exchangeable disulfide group for reaction with free sulfhydryls on a dendrimer core (e.g. reduced cystamine core) or polypeptide (e.g., cystine residue).
  • NHS N- hydroxysuccinimide
  • an antigen may also be conjugated to the dendrimer at the surface of the dendrimer in accordance with the methodology described herein.
  • immunogenic molecules contemplated herein may be comprised of a single dendrimer molecule conjugated to a single antigen molecule.
  • a dendrimer molecule may be conjugated to 1, 2, 3, 4 or more antigen molecules.
  • an antigen molecule may be conjugated to 1, 2, 3, 4 or more dendrimer molecules.
  • the term "antigen" includes any molecule that stimulates, a cytotoxic T cell response and/or a T helper response and/or B cell response (also referred to herein as an immune or immunogenic response), without the aid of an additional adjuvant, when administered to a subject.
  • Antigens specifically contemplated by the present invention may be a polypeptide, which term includes glycosylated polypeptides, T-cell epitopes, including T helper epitopes and CTL eptiopes, and B cell epitopes, carbohydrate, or other agent capable of eliciting a T cell and/or humoural immune response. Screening for immunogenic activity can be performed using techniques well known to the skilled artisan.
  • a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, I 125 labelled Protein A.
  • epitopes is a fragment of a polypeptide that itself is immunologically reactive (i.e., specifically binds) with the B-cells and/or T-cell surface antigen receptors that recognize the polypeptide.
  • Epitopes may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones.
  • antisera and antibodies are "antigen-specific” if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared using well- known techniques.
  • a "T helper epitope” can also be defined as a “Th epitope” or CD4 + T helper epitope” and includes any epitope capable of enhancing or stimulating a CD4 + T cell response when administered to a subject.
  • theT helper epitopes contemplated herein are at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.
  • CTL epitope can also be defined as a “cytotoxic T cell epitope” or “CD8 + CTL epitope” and includes any epitope which is capable of enhancing or stimulating a CD8 + T cell response when administered to a subject.
  • CTL epitopes contemplated herein are typically at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.
  • B cell epitope is any epitope which is capable of eliciting the production of antibodies when administered to a subject.
  • the B cell epitope is capable of eliciting neutralizing antibodies, and in a particular embodiment, high titer neutralizing antibodies.
  • B cell epitopes contemplated herein are typically at least about 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.
  • polypeptide is used in its conventional meaning, i.e., as a sequence of amino acids.
  • the naturally occurring or recombinant polypeptides of the present invention therefore, should be understood to also encompass peptides, oligopeptides and proteins.
  • the protein may be glycosylated (i.e. comprise a carbohydrate entity) or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • polypeptides include a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • Reference to a "carbohydrate entity” or a “glycosylated entity” includes a synthetically or naturally modified entity.
  • Exemplary polypeptides contain at least one CTL epitope and/or one T-helper epitope and/or one B cell epitope. As indicated above, the terms peptides, oligopeptides and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • polypeptide may be an entire protein, or a subsequence thereof.
  • Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising epitopes, i.e., antigenic determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response in an HLA-independent manner.
  • the present invention contemplates polypeptides comprising at least about 5, 10, 15, 20, 25, 50, 75, 90 or 100 contiguous amino acids, or more, including all intermediate lengths.
  • the peptide is a single epitope or multiple epitope peptide with a cysteine residue at the C or N terminal, either synthetically incorporated or natively present, to facilitate conguation to a free sulfhydryl group on the dendrimer.
  • recombinant proteins native or multiepitope
  • may be generated with 1 or more cysteine residues native or introduced by genetic engineering for conguation of the peptide to the dendrimer.
  • T cells are considered to be specific for an antigen contemplated by the present invention if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide.
  • T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al. Cancer Res 54: 1065- 1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques.
  • T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA).
  • a tumor polypeptide 100 ng/ml-100 ⁇ g/ml, preferably 200 ng/ml-25 ⁇ g/ml
  • 3-7 days will typically result in at least a two fold increase in proliferation of the T cells.
  • T cells that have been activated in response to a tumor polypeptide, polynucleotide or polypeptide-expressing APC may be CD4 + and/or CD8 + .
  • Tumor polypeptide-specific T cells may be expanded using standard techniques.
  • the T cells are derived from a patient, a related donor or an unrelated donor, and are administered to the patient following stimulation and expansion.
  • CD4 + or CD8 + T cells that proliferate in response to a specific polypeptide can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways.
  • the T cells can be re-exposed to the polypeptide, or a short peptide corresponding to an immunogenic portion of such a polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a tumor polypeptide.
  • T cell growth factors such as interleukin-2, and/or stimulator cells that synthesize a tumor polypeptide.
  • one or more T cells that proliferate in the presence of the tumor polypeptide can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.
  • T cell receptor T cell receptor
  • APC antigen presenting cell
  • assay formats include a cytotoxicity assay, such as for example the standard chromium release assay, the assay for IFN- ⁇ production, such as, for example, the ELISPOT assay.
  • MHC class 1 Tetramer assays can also be utilized, particularly for CTL epitope-specific quantitation of CD8 + T cells (Altman et al. Science 274:94-96, 1996; Ogg et al. Curr Opin Immunol 70:393-396, 1998).
  • the carboxyl terminus of an MHC molecule such as, for example, the HLA A2 heavy chain
  • a suitable reporter molecule preferably a fluorochrome such as, for example, fluoroscein isothiocyanate (FITC), phycoerythrin, phycocyanin or allophycocyanin.
  • FITC fluoroscein isothiocyanate
  • phycoerythrin phycocyanin or allophycocyanin.
  • Tetramer formation is achieved, for example, by producing the MHC-peptide fusion protein as a biotinylated molecule and then mixing the biotinylated MHC-peptide with deglycosylated avidin that has been labeled with a fluorophore, at a molar ratio of 4: 1.
  • the Tetramers produced bind to a distinct set of CD8 + T cell receptors (TcRs) on a subset of CD8 + T cells derived from the subject (eg in whole blood or a PBMC sample), to which the peptide is HLA restricted. There is no requirement for in vitro T cell activation or expansion.
  • the number of CD8 + cells binding specifically to the HLA-peptide Tetramer is readily quantified by standard flow cytometry methods, such as, for example, using a FACSCalibur Flow cytometer (Becton Dickinson).
  • the Tetramers can also be attached to paramagnetic particles or magnetic beads to facilitate removal of non-specifically bound reporter and cell sorting. Such particles are readily available from commercial sources (e.g. Beckman Coulter, Inc., San Diego, CA, USA) Tetramer staining does not kill the labeled cells ; therefore cell integrity is maintained for further analysis.
  • MHC Tetramers enable the accurate quantitative analyses of specific cellular immune responses, even for extremely rare events that occur at less than 1% of CD8 + T cells (Bodinier et al. Nature Med 6:707-710, 2000; Ogg et al. Curr Opin Immunol /0.393-396, 1998).
  • the total number of CD8 + cells in a sample can also be determined readily, such as, for example, by incubating the sample with a monoclonal antibody against CD8 conjugated to a different reporter molecule to that used for detecting the Tetramer.
  • a monoclonal antibody against CD8 conjugated to a different reporter molecule to that used for detecting the Tetramer.
  • Such antibodies are readily available (eg. Becton Dickinson).
  • the relative intensities of the signals from the two reporter molecules used allows quantification of both the total number of CD8 + cells and Tetramer- bound T cells and a determination of the proportion of total T cells bound to the Tetramer.
  • cytokine production is an indirect measure of T cell activation. Accordingly, cytokine assays can also be used to determine the activation of a CTL or precursor CTL or the level of cell mediated immunity in a human subject. In such assays, a cytokine such as, for example, IL-2, is detected or production of a cytokine is determined as an indicator of the level of epitope- specific reactive T cells.
  • cytokine assay formats used for determining the level of a cytokine or cytokine production are essentially as described by Petrovsky et al. J Immunol Methods 186: 37-46, 1995, which assay reference is incorporated herein.
  • the cytokine assay can be performed on whole blood or PBMC or buffy coat.
  • Subjects to be treated in accordance with the present invention include any subjects requiring an induced therapeutic or prophylactic immune response.
  • Subjects include mammalian subjects: humans, primates, livestock animals (including cows, horses, sheep, pigs and goats), companion animals (including dogs, cats, rabbits, guinea pigs), and captive wild animals. Human subjects are particularly contemplated. Laboratory animals such as rabbits, mice, rats, guinea pigs and hamsters are also contemplated as they may provide a convenient test system. Non-mammalian species such as birds, amphibians and fish may also be contemplated in certain embodiments of the invention.
  • the effective amount of immunogenic combination comprising the modified dendrimer and antigen to be administered will vary, depending upon the nature of the antigen, the nature of the dendrimer, the route of administration, the weight, age, sex, or general health of the subject immunized, and the nature of the immune response sought. All such variables are empirically determined by art-recognized means.
  • An effective amount of dendrimer/antigen combination as described herein is intended to include an amount which, when administered according to the desired dosing regimen, at least partially attains the desired therapeutic or prophylactic effect (immune response).
  • This may advantageously alleviate, eliminate or reduce the severity or frequency one or more symptoms, of, prevent or delay the onset of, inhibit the progression of, reduce the severity of or halt or reverse (partially or altogether) the onset or progression of a particular disease, disorder or condition against which an accelerated, prolonged, enhanced, induced or amplified immune response is desirable.
  • the amount of the dendrimer and antigen combination that may be administered either as separate entities or a single molecule will depend upon a number of factors including the immune status of the subject and the severity of any disease or condition being treated.
  • the immunogenic molecules of the invention may be administered to a subject in an amount ranging from 1 to 10,000 ⁇ g/kg body weight, typically within the range of 10 to 1000 ⁇ g/kg, for example within the range of 10 to 100 ⁇ g/kg body weight.
  • the dendrimers should be used at a concentration so as to avoid any toxic side effects.
  • the optimum dose to be administered and the preferred route for administration may be established using animal models, such as, for example, by injecting a mouse, rat, rabbit, guinea pig, dog, horse, cow, goat or pig, with an appropriate formulation comprising the antigen and the dendrimer and then monitoring the immune response using any conventional assay. Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques.
  • the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous) or intranasally (e.g., by aspiration). Preferably, between 1 and 10 doses may be administered over a 52 week period.
  • a suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor or anti-pathogen immune response, and is at least 10-50% above the basal (i.e., untreated) level.
  • Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro.
  • Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non-vaccinated patients.
  • an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non- treated patients.
  • Increases in pre-existing immune responses to a tumor protein generally correlate with an improved clinical outcome.
  • Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.
  • the combination of the dendrimer and antigen may be administered separately (simultaneously or sequentially), or together, either as a single formulation comprising the antigen and dendrimer as discrete entities, or as a single entity (immunogenic molecule), wherein the antigen is conjugated to the dendrimer.
  • the combination may be administered in a single dose or a series of doses.
  • the combination is advantageously presented as composition, preferably as a pharmaceutical composition, with one or more pharmaceutically acceptable carriers, diluents, adjuvants, excipients or additives.
  • the present invention also relates to the use of a an antigen, and a dendrimer comprising one or more adjuvant groups conjugated to the surface of the dendrimer in the manufacture of a medicament for inducing an immune response in a subject.
  • compositions are well known to those skilled in the art, see for example. Remington's Pharmaceutical Sciences, 18 th Edition, Mack Publishing, 1990.
  • the composition may contain any suitable carriers, diluents additive, adjuvants or excipients.
  • compositions of the invention may optionally also include other supplementary physiologically active agents as appropriate.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the immunogenic combination may be administered by any suitable mode including, for example, intramuscular injection, intravenous administration, nasal administration via an aerosol spray, intradermal, subcutaneous and oral administration.
  • suitable mode including, for example, intramuscular injection, intravenous administration, nasal administration via an aerosol spray, intradermal, subcutaneous and oral administration.
  • Such approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363.
  • solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms.
  • Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No. 5,466,468).
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing or consisting of, for example, water, saline, buffered saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • a solvent or dispersion medium containing or consisting of, for example, water, saline, buffered saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and
  • microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.
  • the delivery of drugs using intranasal microparticle resins Takenaga et al. J Controlled Release 52(1-
  • compositions disclosed herein may be formulated in a neutral or salt form.
  • Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the antigen and/or dendrimer) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the biological effects of the antigen and modified dendrimer are exerted through their ability to stimulate and mature dendritic cells. It is the dendritic cells which then activate CD4 + and CD8 + T cells in the draining lymph nodes.
  • a vaccine refers to a preparation, such as the combinations of the invention, to induce, stimulate or otherwise provide an immune response against a particular disease, parasite or condition.
  • a vaccine may be prophylactic to prevent or retard infection by the parasite or development of a disease and/or its symptoms, or may be therapeutic, so as to treat an existing infection or disease.
  • the invention also provides a method of inducing an immune response in a subject comprising administering to said subject an immunogenic combination according to the invention.
  • a method of enhancing the cell mediated immunity of a subject by administration of an immunogenic combination of the invention to a subject's own dendritic cells and reintroducing the cells to the subject comprises contacting the dendritic cells, obtained from a subject with and for a time and under conditions sufficient to mature said dendritic cells.
  • Said dendritic cells are then capable of conferring epitope specific activation of T cells and/or B cells.
  • the T cell may be a CTL or CTL precursor cell or a CD4 + T helper cell.
  • the subject from whom the dendritic cells are obtained may be the same subject or a different subject to the subject being treated.
  • the subject being treated can be any subject carrying a latent or active infection by a pathogen, such as, for example, a parasite, bacterium or virus or a subject who is otherwise in need of obtaining vaccination against such a pathogen or desirous of obtaining such vaccination.
  • the subject being treated may also be treated for a tumour that they are carrying or may be vaccinated against developing a tumour.
  • the dendritic cells are preferably contained in a biological sample obtained from a subject, such as, for example, blood, PBMC or a buffy coat fraction derived therefrom.
  • Another aspect of the invention provides a method of providing or enhancing immunity against a pathogen in an uninfected subject comprising administering to said subject an immunogenic combination of the invention for a time and under conditions sufficient to provide immunological memory against a future infection by the pathogen.
  • the invention provides a method of enhancing or conferring immunity against a pathogen in an uninfected subject comprising contacting ex vivo dendritic cells obtained from the subject with an immunogenic combination of the invention for a time and under conditions sufficient to confer epitope specific activity on T cells and/or B cells.
  • this aspect of the invention provides for the administration of a prophylactic or therapeutic vaccine to the subject, wherein the active agent of said vaccine (i.e. the antigen/dendrimer combination) induces immunological memory via memory T cells in an uninfected individual.
  • the active agent of said vaccine i.e. the antigen/dendrimer combination
  • the embodiments of vaccination protocols described herein for enhancing the cell mediated immunity of a subject apply mutatis mutandis to the induction of immunological memory against the pathogen in a subject.
  • the present invention contemplates inducing, providing or enhancing immunity against the following pathogens human: immunodeficiency virus (HIV), the human papilloma virus, Epstein-Barr virus, the polio virus, the rabies virus, the Ebola virus, the influenza virus, the encephalitis virus, smallpox virus, the rabies virus, the herpes viruses, the sendai virus, the respitory syncytial virus, the othromyxoviruses, the measles viruses, the vesicular stomatitis virus, visna virus and cytomegalovirus, Acremonium spp., Aspergillus spp., Basidiobolus spp., Bipolaris spp., Blastomyces dermatidis, Candida 5pp., Cladophialophora carrionii, Coccoidiodes immitis, Conidiobolus spp., Cr ⁇ ptococcus spp., Cur
  • capsulatum Histoplasma capsulatum var. duboisii, Hortaea wasneckii, Lacazia loboi, Lasiodiplodia theobromae, Leptosphaeria senegalensis, Madurella grisea, Madurella mycetomatis, Malassezia furfur, Microsporum spp., Neotestudina rosatii, Onychocola canadensis, Paracoccidioides brasiliensis, Phialophora verrucosa, Piedraia hortae, Piedra iahortae, Pityriasis versicolor, Pseudallesheria boydii, Pyrenochaeta romeroi, Rhizopus arrhizus, Scopulariopsis brevicaulis, Scytalidium dimidiatum, Sporothrix schenckii, Trichophyton spp., Trichosporon
  • Another aspect of the invention provides a method of providing or enhancing immunity against a cancer in a subject comprising administering to said subject an immunogenic combination of the invention for a time and under conditions sufficient to provide immunological memory against the cancer.
  • the invention provides a method of enhancing or conferring immunity against a cancer in a subject comprising contacting ex vivo dendritic cells obtained from said subject with an immunogenic combination of the invention for a time and under conditions sufficient to confer epitope specific activity on T cells.
  • this aspect of the invention provides for the administration of a prophylactic or therapeutic vaccine to the subject, wherein the active agent of said vaccine (i.e. the immunogenic combination of the invention) induces immunological memory via memory T cells in an individual.
  • the active agent of said vaccine i.e. the immunogenic combination of the invention
  • the embodiments of vaccination protocols described herein for enhancing the cell mediated immunity of a subject apply mutatis mutandis to the induction of immunological memory against the cancer in a subject.
  • the present invention contemplates inducing, providing or enhancing immunity against the following cancers ABLl protooncogene, AIDS related cancers, acoustic neuroma, acute lymphocytic leukaemia, acute myeloid leukaemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukaemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukaemia, chronic
  • the immunogenic combination of the invention induces an immune response predominantly of the ThI type.
  • High levels of ThI -type cytokines e.g., IFN - ⁇ , TNF ⁇ ., IL-2 and IL- 12
  • high levels of Th2-type cytokines e.g., IL-4, IL-5, IL-6 and IL-10
  • a patient will support an immune response that includes ThI- and Th2-type responses.
  • ThI -type cytokines in which a response is predominantly ThI -type, the level of ThI -type cytokines will increase to a greater extent than the level of Th2-type cytokines.
  • the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann et al. Ann Rev Immunol 7:145-173, 1989.
  • the antigen is associated with infectious diseases, for example viral antigens such as the hepatitis B virus (HBV) envelope Ag pre S2 protein, the hepatitis C virus (HCV) core antigen, HIV-gpl20/160 envelope glycoprotein, influenza nucleoprotein, rabies virus G protein, respiratory syncyticial virus (RSV) F and G proteins, Epstein Barr virus (EBV) gp340 and nucleoantigen 3A, Varicella zoster virus IE62 and gpl, Rubella virus capsid protein, human rhinovirus (HRV) capsid protein, papillomavirus peptides from oncogene E6 and E7, and antigens from various infectious microorganisms including the Plasmodium falciparum circumsporozoite protein, Leishmania major surface glycoprotein (gp63), Bordetella pertussis surface protein, Streptococcus M protein, Mycobacterium tuberculosis 38
  • infectious diseases for example viral
  • antigens contemplated herein include cancer-associated antigens such as any one of the human mucin antigens MUCl (VNTR as well as non-VNTR, MUC2, MUC3, MUC4, MUC5, MUC6, MUC7, MUC8, MUC9, MUClO, MUCH, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, and MUC19 (Pietersz et al., Vaccine, 2000, 18:2059-71; Marjolijn, JL et al, 1990; Crocker, G and Price, MR, 1987; tendopoulos, V et al, 1993; and Bobek, LA et al, 1993), carcinoembryonic antigen (CEA), survivin, Cripto-1, telomerase, claudin 7, Her2/Neu, Pim-1, p53, NM23, prostate specific antigen (PSA) and melanoma-specific antigen
  • an immunogenic combination described herein is delivered to a host via antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs.
  • APCs antigen presenting cells
  • Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti- tumor effects or anti-pathogen effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype).
  • APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
  • the present invention uses dendritic cells or progenitors thereof as antigen-presenting cells.
  • Dendritic cells are highly potent APCs (Banchereau et al. Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor or anti-pathogen immunity (see Timmerman et al. Ann Rev Med 50:501- 529, 1999).
  • dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate nave T cell responses.
  • Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention.
  • secreted vesicles antigen-loaded dendritic cells called exosomes
  • exosomes antigen-loaded dendritic cells
  • Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor- infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid.
  • dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL- 13 and/or TNF ⁇ to cultures of monocytes harvested from peripheral blood.
  • cytokines such as GM-CSF, IL-4, IL- 13 and/or TNF ⁇
  • CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF,
  • Dendritic cells are conveniently categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fc ⁇ . receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CDI l) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).
  • cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CDI l) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).
  • the immunogenic combinations described herein may be used for the treatment of cancer or a pathogenic infection.
  • the pharmaceutical compositions described herein are administered to a subject, typically a warmblooded animal, preferably a human.
  • a subject may or may not be afflicted with cancer or a pathogenic infection.
  • the above pharmaceutical combinations may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer or to prevent infection by a pathogen or to treat a pathogenic infection.
  • immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors or pathogens with the administration of immune response-modifying agents, such as the immunogenic combinations provided herein.
  • Immunogenic combinations of the invention are readily modified for diagnostic purposes.
  • modification may be by addition of a natural or synthetic hapten, an antibiotic, hormone, steroid, nucleoside, nucleotide, nucleic acid, an enzyme, enzyme substrate, an enzyme inhibitor, biotin, avidin, polyethylene glycol, a peptidic polypeptide moiety (e.g. tuftsin, polylysine), a fluorescence marker (e.g. FITC, RITC, dansyl, luminol or coumarin), a bioluminescence marker, a spin label, an alkaloid, biogenic amine, vitamin, toxin (e.g. digoxin, phalloidin, amanitin, tetrodotoxin), or a complex-forming agent.
  • a natural or synthetic hapten an antibiotic, hormone, steroid, nucleoside, nucleotide, nucleic acid, an enzyme, enzyme substrate, an enzyme inhibitor, biotin, avidin, polyethylene glyco
  • compositions may also be presented for use in veterinary compositions. These may be prepared by any suitable means known in the art. Examples of such compositions include those adapted for:
  • oral administration external application (e.g. drenches including aqueous and nonaqueous solutions or suspensions), tablets, boluses, powders, granules, pellets for admixture with feedstuffs, pastes for application to the tongue;
  • parenteral administration e.g. subcutaneous, intramuscular or intravenous injection as a sterile solution or suspension;
  • topical application e.g. creams, ointments, gels, lotions etc.
  • the invention provides a process for preparing an immunogenic molecule of the invention which comprises the steps of preparing a modified dendrimer comprising one or more adjuvant groups, typically mannose and/or aldehyde groups, conjugated to the surface of the dendrimer and conjugating an antigen to said dendrimer.
  • the antigen may be conjugated either to a surface terminal group or the core of the dendrimer.
  • Further aspects provide a process for preparing an immunogenic composition comprising the step of combining a modified dendrimer with an antigen wherein the antigen is optionally conjugated to the dendrimer, together with a pharmaceutically acceptable carrier.
  • BMDC Bone marrow derived dendritic cells
  • OVA Commercially available OVA (Sigma, St. Louis, USA) contained a high level of endotoxin.
  • OVA When 100 ⁇ g/ml of OVA was tested, it contained > 6 E.U./ml of endotoxin, equivalent to > 1 ng/ml of LPS (EML, Melbourne, Australia). Prior to experimental use, endotoxin was removed. Briefly, 1% (v/v in the OVA solution) of Triton X-114 (BDH, Kilsyth, Australia) was added. The solution was gently mixed on a rotating wheel at 4 0 C for 30 min, placed at 3O 0 C for 10 min and centrifuged at 2500 rpm for 10 min. The upper layer of the OVA solution was collected and the above procedure was repeated three times. The OVA solution was dialyzed overnight in PBS, filtered and kept frozen at -2O 0 C. Following this protocol, the final OVA product was certified by EML and contained an extremely low level of lipopolysaccharide (LPS) ( ⁇ 0.06 E.U./ml) (equivalent to ⁇ 0.01 ng/ml).
  • DENDRIMER The generation 4 PAMAM dendrimer with 64 amino-groups, having a cystamine core, is commercially available (Dendritic Nanotechnologies,inc, Mt. Pleasant, USA or Sigma Aldrich) or alternately can be synthesized via a multistep process wherein cystamine is reacted with methyl acrylate and the resulting Micheal addition adduct is further reacted with ethylenediamine to form a Generation 0 adduct with 4 amidoamine groups. Repeating the steps builds successive generations or layers until the desired number is attained. The dendrimer can then be fully characterized by NMR and mass spectrometry.
  • TCEP tris(2-carboxyethyl)phosphine
  • OVA was modified by the addition of 18 ⁇ l of 20 mg/ml N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (Pierce) in DMSO to 500 ⁇ l of 10 mg/ml OVA in PBS, and mixed for 16 h at RT to generate OVA-SPDP. The solution was then dialyzed overnight at 4 0 C to remove unconjugated SPDP (M.W. 312.37). The number of activated disulfide groups present in OVA-SPDP was quantified as described previously (Carlsson et al., 1978). OVA-SPDP was subsequently added to the reduced MD solution (OVA-SPDP:MD; 1:12) to form MDO.
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • the increase in OD at 343 nm was a measure of the number of MD residues incorporated on OVA.
  • the sample was concentrated using a centrifugal filtration device (Amicon, 30,000 MWCO). Excess MD and OVA was removed by gel filtration chromatography using a Superdex-75 column (0.5 cm x 30 cm) (Pharmacia Biotech, Piscataway, USA). Following this preparation, MDO was tested for the endotoxin level and contained ⁇ 0.06 E.U./ml (equivalent to ⁇ 0.01 ng/ml) of LPS (EML). In this study, the concentration of MDO was calculated based on OVA.
  • MD Mannosylated Dendrimer
  • MDO Mannosylated Dendrimer OVA
  • SCHEME 1 x, y and z represent the numbers of the unmannosylated amine group on the molecules
  • Each MDO molecule therefore contained 2 - 4 MD molecules (i.e., 18 - 40 mannose residues) as determined by measuring the release of pyridine-2-thione at absorbance of 343 nm.
  • OVA- SPDP was reacted with various amounts of MD (4, 8, 12 and 16-fold molar excess) and analyzed by gel electrophoresis (Fig. 1.)
  • the reaction with a 12-fold excess of MD gave conjugates with molecular weight ranging from ⁇ 64 - 98 kDa, indicating that each OVA was linked to 2 - 4 MD molecules.
  • MDO conjugates were purified by gel filtration to remove unreacted OVA and MD.
  • the solution was then treated with 78 ⁇ l of 10 mg/ml succinimidyl 4-formylbenzoate (SFB) dissolved in DMSO.
  • the solution was adjusted to pH 5.0 and the solution was mixed O/N at RT.
  • the solution was then concentrated 3 times by sequential addition of PBS using a 5,000 MWCO Amicon Ultra Centrifugal Filter Device.
  • the number of aldehyde groups was measured by spectrophotometry by measuring the release of pyridine-2-thione when treated with 3-(2-pyridyldithio)propionyl hydrazide (PDPH).
  • PDPH 3-(2-pyridyldithio)propionyl hydrazide
  • M 20 D M 1O d-S-S-(IM 10 OVA/SPDP + DTT ⁇ - OVA/SH
  • the solution was brought to pH 8.0 and then it was treated with 73 ⁇ l of 14.3 mg/ml 1,1- dimethoxy-2-bromoethane and mixed for 8 days at RT.
  • the solution was then concentrated 3 times by sequential addition of PBS using a 5,000 MWCO Amicon Ultra Centrifugal Filter Device.
  • MD is reacted with 1,1- dimethoxy-2-bromoethane to introduce masked aldehyde groups. These groups can be converted to aldehyde in mild acidic conditions.
  • the reduced MUCl-SPDP was subsequently added to the MD-DTNB solution (MUCl- SPDP:MD-DTNB;1:12) to form MD-MUCl.
  • MD-DTNB solution MUCl- SPDP:MD-DTNB;1:12
  • the sample was concentrated using a 30,000 MWCO Amicon Ultra Centrifugal filter device.
  • Excess MD-DTNB and reduced MUCl-SPDP were removed by gel filtration using a Superdex-75 column (0.5 cm x 30 cm). In this study the concentration of MD-MUCl was calculated based on MUCl.
  • mice Animals - C57BL/6, OTI and OTII mice, aged 6-10 weeks, used throughout this study, were purchased from the animal facilities of the Walter and Eliza Hall Institute. C57BL/6 mice were used as wild type mice. OTI and OTII mice were donors of peptide-specific T cells.
  • BMDCs bone-marrow derived DCs
  • BM Bone marrow
  • Splenocytes were washed, counted and incubated with antibody cocktail containing in-house-produced rat anti-mouse Gr-I (RB6- 8C5), anti-B220 (RA3-6B2), anti-CDl lb (Ml/70.15), anti-erythrocyte (TER-119) and anti-
  • MHC-class II M5/114 at 4 0 C for 30 min. Cells were washed and unwanted cells were depleted with 2 rounds of bead separation. In each round, cells were incubated with BioMag goat anti-rat magnetic beads (Qiagen, Hilden, Germany) (8 beads per cell) at 4 0 C for 25 min.
  • T cells bound to the beads were removed by magnetic attraction. The purity of T cells was consistently > 94%.
  • BMDCs were pulsed with (i) 10 ⁇ g/ml of the control peptide (H-2K b -restricted SIINFEKL or I-A b -restricted OVA 323-339 ISQAVHAAHAEINEAGR), (ii) 40 ⁇ g/ml of OVA and (iii) 40 ⁇ g/ml of MDO, respectively, for 18 h.
  • control peptide H-2K b -restricted SIINFEKL or I-A b -restricted OVA 323-339 ISQAVHAAHAEINEAGR
  • DCs were irradiated, washed and seeded at specified cell numbers (0 - 4000) into the 96-well plates containing 2 x 10 4 OTI or OTII T cells in triplicates with a final volume of 200 ⁇ l per well.
  • Control wells which contained T cells alone or T cells with non- pulsed DCs were invariably included in all experiments performed. In these wells, T cells did not proliferate. Proliferation of T cells was monitored between days 1 and 5 by 3 H-thymidine (Amersham, Beaconsfield, UK) uptake. Briefly, cells in each well were pulsed with 1 ⁇ Ci of 3 H-thymidine for 16 h.
  • MDO-pulsed BMDCs stimulate a high level of OTII, but not OTI, T cell proliferation in vitro - BMDCs that were pulsed with MDO or with OVA, did not stimulate OTI T cell proliferation (Fig. 2A). However, MDO-pulsed DCs were much more efficient in stimulating OTII T cell proliferation compared to OVA-pulsed DCs (Fig. 2B).
  • OTII T cell proliferation correlated with the numbers of seeded DCs, although it appeared DCs pulsed with MDO reached optimal T cell stimulation when used at 2000 cells per well (Fig. 2B). In this condition, MDO-pulsed DCs were also more stimulatory to OTII T cells than surface- loaded DCs.
  • Stimulants including MDO (40 ⁇ g/ml), OVA (40 ⁇ g/ml), LPS (1 ⁇ g/ml) as the positive control and PBS as the background buffer control, were separately added into the BMDC culture at day 6. After 18 h incubation, 5 x 10 5 cells derived from each stimulant condition were pelleted and stained individually with standardized in-house anti-CD40, anti-CD80 and anti-CD86 that had been conjugated to FITC, together with anti-CD l ie- APC (Pharmingen). The live BMDC population was gated, based on the PI (negative) and CDl Ic (positive) staining in the dot plot. The mean fluorescence intensity (MFI) of FITC-labelled cells was determined in the histogram for DC maturation states.
  • MFI mean fluorescence intensity
  • MDO moderately matures BMDCs -
  • MDO stimulated moderate upregulation of CD40, CD80 and CD86, while LPS greatly upregulated expression of these markers (Fig. 3).
  • the maturation effect of MDO on DCs was not due to LPS contamination, since MDO was certified containing less than 0.06 E.U./ml (equal to ⁇ 0.01 ng/ml) endotoxin (EML, Melbourne, Australia).
  • EML E.U./ml
  • EML Europayl
  • Melbourne, Australia EML, Melbourne, Australia
  • This dosage of LPS was at two orders of magnitude below the stimulatory dose (1 ng/ml) (25).
  • the LPS present in MDO was non-stimulatory to DCs.
  • BMDCs were incubated with MDO and OVA at specified doses and detected with the anti- OVA antibody.
  • 5 x 10 5 cells were palleted and incubated with 100 ⁇ l of titrated MDO and OVA (0.04 - 40 ⁇ g/ml) at 4°C for 30 min. Cells were washed and incubated with 100 ⁇ l of the rabbit polyclonal anti-OVA antibody at 4 0 C for 30 min. Cells were washed and stained with the FITC-conjugated anti- rabbit Ig antibody (Silenus, Melbourne, Australia) and anti-CD 1 Ic-APC. The MFI of FITC- labelled CDl Ic + cells was determined in the histogram.
  • mice T cell proliferation was monitored for 4 days and peak proliferation of T cells induced by LN cells derived from PBS, MDO and OVA injected mice was compared.
  • ELISA - Serial dilutions of mouse sera were performed in 1% (w/v) BSA in 50 ⁇ l PBS in 96- well ELISA plates (Corning, New York, USA).
  • the plates were pre-coated with 50 ⁇ l of 10 ⁇ g/ml OVA in the coating buffer (0.05 M Na 2 CO 3 , pH 9.6) at 4 0 C overnight and blocked with 100 ⁇ l of 3% (w/v in PBS) bovine serum albumin (BSA) at 37 0 C for 1 h to prevent nonspecific binding. After incubation for 1 h at 37 0 C, the plates were washed 10 times with 0.05% (v/v in PBS) of Tween20 (Sigma).
  • ELISpot assay - Splenocytes collected from each mouse were seeded at 5 x 10 5 cells in the presence of 20 ⁇ g/ml SIINFEKL, OVA 323-33 Q, OVA or 2 ⁇ g/ml of ConA as the positive control and media as the background control, in a total volume of 100 ⁇ l in triplicates, into 96- well MultiScreen filter plates (Millipore, Billerica, USA). These plates were pre-coated with 70 ⁇ l of 5 ⁇ g/ml anti-mouse IFN ⁇ antibody (AN 18) (Mabtech, Sweden).
  • the plates were washed 6 times with PBS and 0.05% Tween20/PBS and submerged in ddH 2 O for 2 min to lyse the cells. Seventy ⁇ l of 1 ⁇ g/ml biotinylated anti-mouse IFN ⁇ antibody (R4-6A2) (Mabtech) was added into each well and left at RT for 2 h. The plates were washed 6 times with PBS and 0.05% Tween20/PBS. Seventy ⁇ l of 0.1% (v/v in PBS) Streptavidin-ALP (Mabtech) was added into each well and left at RT for 2 h. The plates were again washed as mentioned above.
  • R4-6A2 biotinylated anti-mouse IFN ⁇ antibody
  • Tumor challenge Pre-immunized mice were shaved on the abdominal area and inoculated subcutaneously with 1 x 10 6 B 16-0 VA melanoma cells. Tumor growth was monitored every 2 - 3 days. The size of a tumor (mm 2 ) was calculated by multiplication of two perpendicular diameters determined by callipers.
  • mice immunized with MDO and OVA demonstrated various levels of cellular and antibody responses analyzed by the ELISpot assay and ELISA, respectively.
  • Fig. 5A in testing the CD4 + helper T cell response to the MHC-class II restricted OVA 323-339 peptide, only mice immunized with 25 ⁇ g of MDO produced a significant level of IFN ⁇ .
  • Mice immunized with OVA and MDO generated different levels of SIINFEKL-specif ⁇ c CD8 + T cell response, in comparison to the non-responding PBS group.
  • mice immunized with MDO induced an enhanced CD8 + T cell response compared to OVA. Moreover, when the OVA-specific response was evaluated, mice immunized with MDO, but not OVA, generated significant levels of IFN ⁇ and such a response to MDO appeared to be dose-dependent. It was noted only mice immunized with 25 ⁇ g of MDO developed competent T cell responses (CD4, CD8 and OVA-specific IFN ⁇ responses). While a decrease of the immunization dosage to 12.5 ⁇ g reduced the specific CD4 + T cell response, the CD8 + T cell response was maintained and the OVA-specific T cell response remained at an intermediate level (Fig. 5A).
  • mice immunized with MDO 25 ⁇ g generated a higher level of OVA-specific IgG than that of the OVA immunized mice (Fig. 5B).
  • MDO induces cross-presentation in vivo -
  • the ability of MDO to stimulate a strong SIINFEKL-specific CD8 + response in vivo that was absent in vitro led us to investigate its capability in inducing cross-presentation of SIINFEKL in the lymph environment after immunization.
  • popliteal lymph node cells isolated after 20 h footpad injection with MDO and OVA were used to stimulate in vitro OTI T cells.
  • OTI T cells As shown in Fig. 6, while LN cells derived from mice injected with MDO induced significant SIINFEKL-specific OTI T cell proliferation in contrast to those derived from mice injected with OVA, indicating
  • pre-immunized mice were challenged subcutaneously with B 16-0 VA melanoma cells.
  • B16-0VA melanoma is a well-established tumor model which expresses chicken OVA as the surrogate tumor antigen. Mice pre-immunized with OVA did not prevent B 16-0 VA tumor growth (Fig. 7). In contrast, mice immunized with MDO exhibited much delayed or no tumor growth within 14 days post-challenge (Fig. 8).
  • mice immunized with 12.5 and 25 ⁇ g/ml of MDO were 29% and 13% of the average tumor size in mice immunized with OVA, respectively. It was noted that immunization with a higher dose (25 ⁇ g) offered better tumor protection than that with a lower dose (12.5 ⁇ g). Mice were sacrificed before the tumor reached 225 mm 2 . All mice in which B 16-0 VA tumors did not grow continued to survive 3 months after challenge, with no sign of tumor growth.
  • a mannosylated dendrimer In contrast to previous methods of antigen mannosylation, a mannosylated dendrimer has been used to incorporate multiple mannose residues ( ⁇ 40) with minimal modification of native lysine residues of the targeted antigens. The extent of modification with mannose is carefully monitored with the use of quantitative biochemical assays. Moreover, the presence of a significant level of endotoxin in commercial OVA is noted. After endotoxin removal, both OVA and MDO contain a low level of LPS, which is not sufficient to stimulate DCs (Sheng et al, 2006).
  • MDO-treated DCs exhibit a much higher level of binding at all doses tested, suggesting that the presence of endogenous mannose residues on OVA is not sufficient for effective DC binding, and the addition of MD to OVA greatly promotes its binding to DCs.
  • the high avidity of MDO to DCs is most likely due to enhanced recognition of carbohydrate recognition domains (CRDs) on mannose-binding receptors such as C-type lectins, which are abundantly present on DC surfaces (Ng et al., 1998).
  • MDO In addition to enhanced recognition and presentation of the OVA antigen, MDO also stimulates maturation of BMDCs, which is evidenced by elevated expression of costimulatory molecules including CD40, CD80 and CD86.
  • costimulatory molecules including CD40, CD80 and CD86.
  • the enhanced costimulation provided by DCs may in part explain the robust OTII T cell proliferation.
  • Such an associated adjuvanticity of MDO is clearly desirable for boosting immune responses.
  • CD8 + DCs may play a significant role.
  • CD8 + DCs, but not other DC subpopulations, in lymphoid organs are primary APCs capable of cross-presenting soluble or cell-associated antigens, due to the presence of specialized antigen processing machinery.
  • Peripheral myeloid DCs can acquire the CD8 + phenotype upon migration to the LN after taking up foreign antigens (Merad et al, 2000). Footpad injection with polymannose (mannan) has been shown to primarily induce CD8 + DC maturation in the LN (Sheng et al, 2006). Taken together, it is possible that MDO, on one hand, is internalized by the APCs at the injection site, transported by myeloid DCs into the LN and finally cross-presented by CD8 + DCs. On the other hand, MDO may also cause maturation of CD8 + DCs, facilitating ThI cell priming (den Haan et al, 2004).
  • MDO stimulates a high level of CD8 + T cell response which may offer tumor protection in mice, while low levels of the CD8 + T cell and antibody responses induced by OVA do not provide any tumor protection. Immunization with a higher dose of MDO stimulates both CD4 and CD8 responses, resulting in distinctive protection from rapid development of B 16-0 VA melanoma. These results perhaps indicate the cooperative helper CD4 response is required for competent anti-tumor immunity in addition to the CD8 response (Knutson et al, 2005). In addition, the capacity of MDO to stimulate a distinctive level of the humoral response proposes the potential role of MD conjugated antigens in targeting infectious diseases and cancer, which can impact on the vaccine design.
  • a dendrimer is used to provide a link between an antigen and multiple mannoses.
  • This alternative approach to antigen mannosylation promotes antigenicity through (i) increased binding and recognition of antigens by DCs, (ii) enlarged quantity of antigens processed through the endocytic pathway leading to MHC-class II presentation, (iii) enhanced T cell costimulation due to induction of DC maturation, and (iv) induction of antigen cross-presentation in vivo.
  • DCs cultured with GM-CSF/IL-4 (100 ng/ml) and Flt-3 ligand (300 ng/ml) were harvested at day 6.
  • Cells (5 * 10 5 ) were pelleted and incubated with MDO and OVA for 30 min. Cells were washed and labeled with anti -mouse CDl Ic and rabbit anti-OVA antibodies. After 20 min incubation, cells were treated with FITC-conjugated anti-rabbit antibody and analyzed by flow cytometry.
  • the Flt-3L culture yielded > 90% CDl Ic + population (not shown) that was divided into 3 heterogeneous subpopulations (CD24 high , CDl lb high and double-negative[CD220 + ]). They all bound to MDO, but not OVA.
  • the shaded area represents cells only treated with primary and secondary antibodies.
  • MDO induces maturation of myeloid DCs, but not plasmacytoid DCs (refer to Fig. 9):
  • stimulants including PBS (negative control), MDO (40 ⁇ g/ml), LPS (1 ⁇ g/ml) and CpG1668 (10 ⁇ g/ml) were added into GM-CSF/IL-4 and FU-3L cultures. After 18 h incubation, DCs were harvested and analysed for CD40, CD80, CD86 and MHC-class II expression by flow cytometry.
  • MDO-induced DC maturation is dependent on TLR4 (refer to Fig. 10): At day 6, stimulants including PBS (background control), MDO (40 ⁇ g/ml), LPS (1 ⁇ g/ml) and CpG 1668 (10 ⁇ g/ml) were added into C3H/He and C3H/HeJ DC cultures. After 18 h incubation, DCs were harvested and analysed for CD40 and CD86 expression by flow cytometry. While the maturation effect of MDO and LPS in C3H/He DCs was greatly diminished in C3H/HeJ DCs, the effect of CpG remained unchanged. The maturation effect of MDO and LPS observed in wild type C3H/He DCs is greatly diminished in TLR4-defective C3H/HeJ DCs. Thus, it is likely that MDO-induced DC maturation is largely dependent on TLR4.
  • MDO-pulsed BMDCs and FU3-L DCs induce OVA-specific CD4 + and CD8 + T cell proliferation (refer to Fig 11).
  • OVA 323-339 [10 ⁇ g/ml] were seeded with 2 x 10 4 OTI or OTII T cells. T cell proliferation was monitored for 5 days with 3 H-thymidine incorporation. Peak proliferation on day 2 and day 3 for OTI and OTII T cells was compared. The data shown is representative of three experiments.
  • CD24 hi DCs are the primary subset after 10 days of Flt-3L culture(refer to Fig. 13).
  • MDO but not OVA, stimulates cross-presentation in GM-CSF/IL-4 cultured DCs in the presence of malondialdehyde (refer to Fig 14).
  • MDO-acetal is processed by GM-CSF/IL-4 DCs, leading to OTII T cell proliferation. This suggests that, similar to MDO, MDO-acetal can be processed and delivered into the MHC- class II pathway.
  • DCs (1 - 4 x 10 3 ) preloaded with 20 ⁇ g/ml MDO or MDO-acetal (derived from homogeneous fraction 1 or heterogeneous fraction 2 during FPLC purification) were incubated with 2 x 10 4 OTII T cells. T cell proliferation was monitored and peak proliferation induced by DCs on day 3 from each pulsing condition was compared. DCs preloaded with MDO and MDO-acetal (fraction 1) were highly stimulatory to OTII T cells.
  • MDO-acetal promotes aldehydes promotes cross presentation of OVA by GM-CSF/IL- 4 DCs, leading to a higher level of OTI T cell proliferation (refer to Fig 16).
  • MUCl proteins/peptides - Mannosylated MUCl dendrimers are prepared according to the methods described herein. Additional MUCl proteins and peptides include protein fragments, peptides and fusion proteins.
  • An example of a MUCl fusion protein is a GST-MUCl fusion protein (MUClFP) comprising glutathione S transferase (GST) and 5 VNTR (variable number of tandem repeats) repeats, i.e. Glutathione-S-transferase- (PDTRPAPGSTAPPAHGVTSA) 5 .
  • GST-MUCl fusion protein MUClFP
  • GST glutathione S transferase
  • VNTR variable number of tandem repeats
  • PDTRPAPGSTAPPAHGVTSA Glutathione-S-transferase-
  • Another example is a fusion protein comprising GST and an N-terminal extracellular region of human MUCl (amino acids 33-103), i.e
  • NFP SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAP ATEPASGSAATW (designated NFP).
  • MUClFP antigen p VNTR
  • p VNTR MUClFP antigen
  • a recombinant protein or fusion protein without the VNTR can also be used.
  • Peptides of the invention are derived from both the VNTR region and the non-VNTR region of MUCl.
  • An example of a H2- K b -restricted MUCl epitope is SAPDTRPAP (MUC lK b )
  • An example of an HLA-A2-restricted MUCl epitope is STAPPAHGV (MUC1A2).
  • MUClK b and MUC 1A2 are epitopes derived from the VNTR region.
  • Other MUCl peptides, 12-20 (LLLLTVLTV) and 950-958 (STAPPVHNV) are HLA-A2 CTL epitopes from the non- VNTR region.
  • Peptides 31-55 (TGSGHASSTPGGEKETSATQRSSVP) and 51-70 (RSSVPRSSVPSSTEKNAVSMTSSVL) correspond to the extracellular N-terminal region of MUCl.
  • HLA-A2/K b mice express both HLA-A2 and H2-K b .
  • An example of a MUCl -specific TCR transgenic mouse has been described by Beatty et al. (2008) The FASEB Journal 22:lb450. Human MUCl transgenic mice have been described previously and are H2 d (Acres et al. (2000) Cancer Immunol Immunother 48(10):588-594).
  • BMDCs bone-marrow derived DCs
  • BM cells are collected from femurs and tibias of C57BL/6 and HLA-A2/K b transgenic mice and treated with ACK lysis buffer (0.15 MNH 4 Cl, 1 mM KHCO 3 and 0.1 mM Na 2 EDTA) to lyse erythrocytes. Cells are washed and cultured at 2x10 6 cells/3 ml in 6 well plates with complete RPMI media (2%
  • Hepes buffer 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 mM glutamine and 10% fetal calf serum) supplemented with 10 ng/ml GM-CSF and IL-4 (Pharmingen, San Diego, USA).
  • T cell purification - Splenocytes from MUCl -specific TCR transgenic mice or mice immunised with MUCl are collected, washed and incubated in ACK lysis buffer at 37 0 C for 5 min to lyse erythrocytes.
  • Splenocytes are washed, counted and incubated with antibody cocktail containing in-house-produced rat anti-mouse Gr-I (RB6-8C5), anti-B220 (RA3-6B2), anti-CDl lb (Ml/70.15), anti-erythrocyte (TER-119) and anti-MHC-class II (M5/114) at 4 0 C for 30 min.
  • Cells are washed and unwanted cells depleted with 2 rounds of bead separation. In each round, cells are incubated with BioMag goat anti-rat magnetic beads (Qiagen, Hilden, Germany) and cells bound to the beads are removed by magnetic attraction.
  • BMDCs are pulsed with (i) control peptide (e.g. MUC lK b or MUC 1A2); (ii) MUClFP or pVNTR; and (iii) MD-MUCl, respectively, for 18 h.
  • DCs are then irradiated, washed and seeded at specified cell numbers (0 - 4000) into 96-well plates containing MUCl -specific TCR transgenic T cells or in a total volume of 200 ⁇ l per well.
  • Control wells contain T cells alone or T cells with non-pulsed DCs.
  • T cells Proliferation of T cells is monitored between days 1 and 5 by 3 H-thymidine (Amersham, Beaconsfield, UK) uptake. Briefly, cells in each well are pulsed with 1 ⁇ Ci of 3 H-thymidine for 16 h. These cells are then harvested and radioactivity measured by the Packard TopCount scintillation counter (PerkinElmer, Boston, USA) in counts per minute (CPM).
  • Cross-presentation assay - Groups of C57BL/6 and HLA-A2/K b transgenic mice are separately injected with MD-MUCl, MUClFP or an equivalent volume of PBS into hind footpads. Twenty hours post-injection, popliteal lymph node (LN) cells are isolated. A portion of LN cells derived from the PBS group is pulsed with a Kb-restricted epitope or HLA-A2- restricted epitope for 1 h at 37 0 C as the positive control. LN cells, comprising APCs, are seeded into 96-well round bottom plates, together with MUCl -specific TCR transgenic T cells. T cell proliferation is monitored for up to 5 days and peak proliferation of T cells induced by LN cells derived from PBS-, MD-MUCl- and MUClFP- injected mice is compared.
  • LN popliteal lymph node
  • mice - C57BL/6 and HLA-A2/K b transgenic mice are injected intradermally at least once at the base of a tail with MUClFP, MD-MUCl or the equivalent volume of PBS. Some mice are bled for antibody detection and culled for the ELISpot assay. Other mice are optionally given a boost injection prior to challenge with B16 tumor cells transfected with MUCl (e.g. B16-MUC1).
  • ELISA - Serial dilutions of mouse sera are performed in 1% (w/v) BSA in 50 ⁇ l PBS in 96- well ELISA plates (Corning, New York, USA).
  • the plates are pre-coated with, for example, MUClFP, pVNTR, NFP or peptides 31-55/51-70, in coating buffer (0.05 M Na 2 CO 3 , pH 9.6) at 4 0 C overnight and blocked with 100 ⁇ l of 3% (w/v in PBS) bovine serum albumin (BSA) at 37 0 C for 1 h to prevent non-specific binding.
  • coating buffer 0.05 M Na 2 CO 3 , pH 9.6
  • BSA bovine serum albumin
  • the plates are again washed 10 times with 0.05% Tween20/PBS and 50 ⁇ l of the ABTS (2,2'-Azino-bis[3-ethylbenzthiazoline-6-sulfonic acid]) substrate solution (0.03% ABTS and 0.08 % H 2 O 2 in ABTS buffer [0.1 M Na 2 HPO 4 and 0.08 M citric acid, pH 4.5]) is added into each well.
  • the reaction is developed for 30 min and read with 405 nm absorbance.
  • ELISpot assay - Splenocytes collected from each mouse are seeded at 5 x 10 5 cells in the presence of a MUCl protein fragment, peptide or fusion protein (e.g. 20 ⁇ g/ml MUClFP or or MUClK- or NFP or pVNTR or MUC1A2 12-20 or 950-958) or 1 ⁇ g/ml of ConA as the positive control and media as the background control, in a total volume of 100 ⁇ l in triplicates, into 96-well MultiScreen filter plates (Millipore, Billerica, USA).
  • MUCl protein fragment, peptide or fusion protein e.g. 20 ⁇ g/ml MUClFP or or MUClK- or NFP or pVNTR or MUC1A2 12-20 or 950-958
  • 1 ⁇ g/ml of ConA as the positive control and media as the background control
  • mice are immunized at least once (e.g. twice at a 2- week interval) before being challenged with 1x10 6 B16-MUC1 cells (from Dr. Jianlin Gong, University of Boston, USA) by subcutaneous injection into the abdomen. High levels of MUCl expression are maintained by culturing B16-MUC1 cells in 1.2 mg/ml G418/gentamycin (Invitrogen, California, USA).
  • mice are injected subcutaneously with IxIO 5 B16-MUC1 tumor cells and subsequently immunized at least once intradermally at the base of the tail with MD-MUCl or various other versions of MUCl (including MUCl DNA).
  • An example of such a protocol comprises immunization on days 3 and 7 after inoculation of B 16-MUCl cells. Tumor growth is subsequently monitored by measuring the two perpendicular diameters using calipers and the results expressed as the product of the two perpendicular diameters.
  • RMAMUCl Another example of a tumor cell line that expresses MUCl is RMAMUCl .
  • the RMA tumor cell line is C57BL/6-derived (H-2 b ).
  • RMAMUCl cells are RMA cells transfected with MUCl cDNA.
  • Tumors are removed from mice at the end of tumor challenge experiments. Single suspensions of tumor cells are prepared by teasing and flushing the tumor mass with RPMI media followed by treatment with 0.73% NH4C1 for 10 min at 37 0 C to lyse red blood cells. Cells are washed, resuspended in RPMI media and cultured for 7 days. MUCl expression on adherent cells is determined by staining with anti-MUCl monoclonal antibody (BC2) for 45min at 4°C followed by the addition of anti-mouse F-(ab )2-FITC antibody (Chemicon, Melbourne, Australia) for a further incubation of 45 min at 4°C. MUCl expression is detected by flow cytometry (FACS Canto, NJ, USA).
  • mice to mount an anti-MUCl response in vivo can be measured using ELISA or ELISpot assays as described above.
  • the in vivo anti-tumour efficacy can be measured using human MUCl transfected cell lines in a number of types of mice (C57BL/6, BALB/c, DBA/2, HLA-A2/Kb and MUCl transgenic mice (Apostolopoulos et al., 1996b; and may havepoulos et al., 2006).
  • Aldehyde-mannan antigen complexes target the MHC class I antigen-presentation pathway. Eur J Immunol 2000b. 30: 1714-1723.

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Abstract

La présente invention porte d'une manière générale sur des macromolécules modifiées chimiquement pour une utilisation en immunothérapie. En particulier, la présente invention porte sur des dendrimères ayant un ou plusieurs groupes adjuvants conjugués à la surface de ceux-ci. L'invention porte en outre sur des combinaisons comprenant un dendrimère ayant un ou plusieurs groupes adjuvants conjugués à la surface de celui-ci, conjointement avec un antigène.
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US9877984B2 (en) 2013-12-23 2018-01-30 Massachusetts Institute Of Technology Controllably degradable compositions and methods

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WO1997028809A1 (fr) * 1996-02-07 1997-08-14 Novavax, Inc. Proprietes adjuvantes de dendrimeres de polyamidoamines
WO1998013378A1 (fr) * 1996-09-26 1998-04-02 Rijksuniversiteit Te Leiden Peptides mannosyles
AU2002214938B2 (en) * 2000-11-03 2006-04-13 Centro De Ingenieria Genetica Y Biotecnologia Method for obtaining antigenic structures enhancing specific cross reactivity
WO2004041310A1 (fr) * 2002-11-08 2004-05-21 Danmarks Fødevareforskning Preparation de conjugues de dendrimere et de glucide chimiquement bien definis
WO2004053072A2 (fr) * 2002-12-06 2004-06-24 Northwest Biotherapeutics, Inc. Administration de cellules dendritiques partiellement matures in vitro pour le traitement de tumeurs

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120093761A1 (en) * 2009-04-01 2012-04-19 University Of Miami Vaccine compositions and methods of use thereof
US9764012B2 (en) * 2009-04-01 2017-09-19 University Of Miami Vaccine compositions and methods of use thereof
US20180099032A1 (en) * 2009-04-01 2018-04-12 University Of Miami Vaccine compositions and methods of use thereof
US9877984B2 (en) 2013-12-23 2018-01-30 Massachusetts Institute Of Technology Controllably degradable compositions and methods
US10736914B2 (en) 2013-12-23 2020-08-11 Massachusetts Institute Of Technology Controllably degradable compositions and methods

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