WO2008151389A1 - Chemically modified macromolecules - Google Patents

Abstract

The present invention relates generally to chemically modified macromolecules for use in immunotherapy. In particular, the present invention relates to dendrimers having one or more adjuvant groups conjugated to the surface thereof. The invention further relates to combinations comprising a dendrimer having one or more adjuvant groups conjugated to the surface thereof, together with an antigen.

Description

CHEMICALLY MODIFIED MACROMOLECULES

FIELD OF THE INVENTION

The present invention generally relates to chemically modified macromolecules for use in immunogenic compositions and combinations. In particular, 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.

BACKGROUND OF THE INVENTION

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

There are currently significant unmet therapeutic needs in both the treatment and prevention of many infectious diseases, autoimmune diseases, allergies and cancer. Vaccines, both preventative and therapeutic, will likely play a significant role in addressing these unmet needs, although to date, lack of immunogenicity has been a recurrent problem with many potential vaccines.

The incidence of many types of tumors continues to rise, with the incidence of breast cancer in women currently 1 in 8 and other adenocarcinomas (lung, kidney, colon) also occurring at high frequencies. With the exception of a few treatable cancers, the introduction of new drugs and the conventional approaches such as surgery, cytotoxic therapy, radiotherapy or combination therapies have not greatly improved the prognosis. Immunotherapy may prove to be a desirable alternative.

Several tumor associated antigens, for example MUCl, and their 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). Importantly, although 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).

In many vaccines, 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. However, 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. The role of 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; Apostolopoulos et al., 2000; Lofthouse et al., 1997; Apostolopoulos et al., 1996a).

It has been demonstrated that oxidized mannan-MUCl fusion protein conjugates (MFP) produce strong cellular responses (CTL, IFNγ, IL- 12) in mice and are protected from a MUCl+ve tumor challenge (Apostolopoulos et al., 1996b). Studies into the mechanism of action indicated that the oxidized mannan conjugate is endocytosed by the mannose receptor and subsequently escapes from the endosome into the cytoplasm and this was primarily due to the aldehyde residues of the oxidized mannan. It has also been shown that the aldehydes in oxidized mannan are crucial for the MFP immunogenicity (Apostolopoulos et al., 2000b). The oxidized mannan conjugates stimulated lymphocytes to proliferate and induced TNF-α and IL- 12 from macrophages whereas reduced mannan (no aldehydes) did not induce these cytokines (Apostolopoulos et al., 2000a). However, although initial clinical trials using the i.m. injection of MFP, showed that some cancer patients made antigen specific IFNγ (intracellular staining), proliferative T cell responses and some had detectable cytotoxicity against MUCl targets, these responses were not intense enough to induce clinical responses.

Furthermore, there are several disadvantages associated with current protocols. First, for non- glycosylated antigens, incorporation of new glycosylation sites entails the expression of recombinant proteins in the yeast (Lam et al., 2005; Levitz and Specht, 2006; Zhong et al., 2005). This protocol requires complicated characterization and optimization procedures based on genetic engineering methodology. Secondly, direct attachment of multiple mannose residues to the antigen with the use of α-D-mannopyranosylphenyl isothiocyanate (Agnes et al., 1998) generates a potential risk of irreversibly modifying lysine residues present in immunodominant epitopes, which may subsequently compromise antigen immunogenicity.

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 -

groups. 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.

Given the therapeutic or prophylactic benefits which could be attained by an enhanced immune response to an antigen, there remains a need for new methods and agents for potentiating, enhancing or amplifying the immune response induced by an antigen in a subject. It has now been found that a dendrimer molecule which is modified by the conjugation of adjuvant groups, such as those containing a mannose or aldehyde functionality, at the exterior surface may enhance the immunogenic effect of a given antigen.

SUMMARY OF THE INVENTION

It is to be recognised throughout this specification , unless the context requires otherwise, the word "comprise", or variations thereof such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element or integer or a group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The present invention is predicated on the finding that a dendrimer which comprises one or more adjuvant groups conjugated to its exterior surface may advantageously enhance the immunogenicity of a co-administered antigen

Accordingly, in a first aspect, 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.

These 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.

In a second aspect the invention provides an immunogenic combination comprising: (i) an antigen, and

(ii) a dendrimer comprising one or more adjuvant groups conjugated to the surface of the dendrimer.

In a third aspect the invention provides an immunogenic composition comprising: (i) an antigen,

(ii) a dendrimer comprising one or more adjuvant groups conjugated to the surface of the dendrimer; and (iii) a pharmaceutically acceptable carrier.

In a fourth aspect, 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.

In a fifth aspect, the present invention provides a method of enhancing the cell mediated immunity of a subject, said method comprising:

(i) contacting ex vivo dendritic cells obtained from a subject with an immunogenic molecule, composition or combination according to the invention for a time and under conditions sufficient to mature said dendritic cells; and

(ii) introducing the activated dendritic cells autologously to the subject or syngeneically to another subject in order that T cell and/or B cell activation occurs.

Where more than one adjuvant group is present on the dendrimer, the adjuvant groups may the same or different. In particular embodiments of the invention, the adjuvant group is selected from a group which contains a mannose group or an aldehyde functional group. In further embodiments thereof, the adjuvant groups may be all mannose-containing groups, all aldehyde-containing groups or a combination of both.

In certain embodiments, the compositions or combinations according to the invention may further comprise one or more additional adjuvants which are not conjugated to the dendrimer.

In some embodiments, 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.

Thus, in a further embodiment, 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.

In some embodiments where the antigen is conjugated to or otherwise associated with the dendrimer, 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.

Where the antigen is delivered separate to the dendrimer, the antigen and dendrimer may be administered either as a single formulation or composition containing the separate components, or as separate formulations. BRIEF DESCRIPTION OF THE FIGURES

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 μg/ml], OVA_323-339 [10 μg/ml]), MDO (40 μg/ml) and OVA (40 μg/ml) were co-cultured with 2 x 10⁴ 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. (C) DCs (8.4 x 10⁵) were treated with control peptides (0.5 μg/ml SIINFEKL and 10 μg/ml OVA_323-339), titrated OVA (40 - 640 μg/ml) and 40 μg/ml MDO conjugates of various mannosylation levels (Ox - 3Ox) for 18 h. Cell survival was determined by typan blue staining. (D, E) The capacity of these DCs to stimulate OTI and OTII T cells was compared. Data shown are representative of at least two separate experiments. ** P < 0.01 (SIINFEKL-, OVA_323-339- or MDO- versus OVA- pulsed DCs).

Fig. 3 provides graphical representation demonstrating that MDO induces maturation of BMDCs. DCs stimulated with PBS, OVA (40 μg/ml), MDO (40 μg/ml) and LPS (1 μg/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 μg/ml) were analyzed by FACS after FITC labeling. The binding of MDO or OVA was determined by MFI values corresponding to specific concentrations. B. Histograms of FITC-labeled BMDCs, which were pre-incubated with 40 μg/ml of MDO and OVA, were shown. Data shown are representative of two different experiments.

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 μg), MDO (12.5 or 25 μg) were sacrificed after 3 immunizations, and levels of A. OVA₃₂₃. ₃₃₉ (CD4 epitope)-, SIINFEKL (CD8 epitope)- and OVA-specific T cell IFNγ 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 μg) and MDO (25 μg) into footpads were evaluated for their capacities in OTI T cell stimulation in vitro. LN cells (2 x 10⁴) isolated from injected mice were co-cultured with 5 x 10⁴ OTI T cells in quadruplicates. T cell proliferation was monitored from days 1 - 4 and peak proliferation on day 2 was compared. While LN cells isolated from PBS immunized mice were used as the background control, a portion of these cells pulsed with SIINFEKL (1 μg/ml) were included as positive control (not shown). ** P < 0.01. Fi g. 7 provides graphical representation demonstrating that mice pre-immunized with MDO significantly delay or reject B16-0VA melanoma development. Mice (n = 5) pre-immunized with PBS, OVA and MDO of specified doses were inoculated subcutaneously with 10⁶ B 16- OVA tumor cells on abdomens. Tumor sizes were measured every 2 - 3 days within 14 days after tumor inoculation. Data shown are representative of two experiments.

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 10⁵) 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. A. In the homogeneous GM-CSF/IL-4 culture, CDl Ic⁺ DCs (> 85%) bound to MDO with a much higher avidity than OVA, which also showed a weak level of DC binding. B. 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.

At day 6, stimulants including PBS (negative control), MDO (40 μg/ml), LPS (1 μg/ml) and CpG1668 (10 μg/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. A. Myeloid DCs, including GM-CSF/IL-4 DCs, CD24^high and CDl lb^hlgh Flt-3 L DC subpopulations, are responsive to MDO and LPS (positive control), as well as CpG (not shown). B. CpG, but not MDO nor LPS, induced maturation of FU-3L cultured B220⁺ DCs. The shaded area represents unstained cells. Both MDO and LPS stimulate myeloid-like GM-CSF/IL-4 as well as CD24^high and CDl lb^high Flt-3L DCs; however, both stimulants fail to induce plasmacytoid-like Flt-3L B220⁺ DCs in comparison to CpG.

Fig. 10 provides graphical representation showing that MDO-induced DC maturation is dependent on TLR4. At day 6, stimulants including PBS (background control), MDO (40 μg/ml), LPS (1 μg/ml) and CpG1668 (10 μg/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 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 10³) BMDCs (A) and Flt3-L DCs (B) pulsed with MDO (40 μg/ml), OVA (40 μg/ml) or control peptides (SIINFEKL [1 μg/ml] and OVA_323-339 [10 μg/ml]) were seeded with 2 x 10⁴ OTI or OTII T cells. T cell proliferation was monitored for 5 days with ³H-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 NH₄Cl 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 10³) preloaded with 40 μg/ml MDO in the presence of titrated malondialdehyde (25 - 400 μg/ml), with 40 μg/ml OVA and with 40 μg/ml OVA plus 400 μg/ml malondialdehyde, were incubated with 2 x 10⁴ OTI T cells. T cell proliferation was monitored using the [³H]thymidine incorporation assay. Peak proliferation induced by DCs on day 2 from each pulsing condition was compared. Malondialdehyde used in 100 - 400 μg/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.

Titrated DCs (1 - 4 x 10³) preloaded with 20 μg/ml MDO or MDO-acetal (derived from homogeneous fraction 1 or heterogeneous fraction 2 during FPLC purification) were incubated with 2 x 10⁴ 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. Titrated DCs (1 - 4 x 10³) preloaded with 20 μg/ml MDO or MDO-acetal (fractions 1 and 2) were incubated with 2 x 10⁴ OTI T cells. T cell proliferation was monitored. Peak proliferation induced by DCs on day 2 from each pulsing condition was compared. 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). DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers but not the exclusion of any other integer or step or group of integers or steps.

The singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise.

Dendrimers

The term "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. 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 diaminoC₂-Ci₂alkanes such as ethylene diamine, 1 ,4-diaminobutane, 1,6-diaminohexane. Other suitable cores may be cleavable, for example a diaminoC_2-i₂alkane having a disulfide (S-S) linkage within the alkane chain, such as cystamine (₂HN-(CH₂)₂-S-S-(CH₂)₂-NH₂). 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. In further embodiments of such cores, 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. Whilst linear cores, such as those described above, are contemplated, it will be appreciated that 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).

In certain embodiments, the dendrimer or each independent "branch" extending from the core has at least 2 generations of monomer or repeat units (Generation 2). In further embodiments, 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. In further embodiments, 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. These functional group modifications can be readily carried out by methods known in the art. Dendrimers with surface amino groups can be readily functionalised to carboxylic acid groups by using acid anhydrides such as succinic or glutaric anhydride. Similarly, amino groups maybe functionalised to thiol groups by reaction with S- acetylmercaptosuccinic anhydride (SAMSA) or N-hydroxysuccinimidyl acetylthioacetate (SATA) followed by hydroxylamine. Alternatively, aminogroups can be modified with N- hydroxysuccinimidyl pridyldithiopropionate (SPDP) followed by reduction with dithiothreitol (DTT). 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.

In still further exemplary embodiments of the invention, the dendrimer is a Generation 3- Generation7 PAMAM dendrimer. Such 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. Exemplary, but non- limiting, 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). Alternatively, 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. In doing so, this leaves a reactive site at the core which may be utilised for conjugation of the antigen to the dendrimer. 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).

Methods for the preparation of 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.

The term "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, Apostolopoulos V, Pietersz GA, Receptor-mediated delivery of antigens to dendritic cells: anticancer applications, MoI Pharm. 2007, 4:58-72.; Pietersz GA, Pouniotis DS, Apostolopoulos V, Design of peptide-based vaccines for cancer, Curr Med Chem. 2006, 13:1591 -607).

Similarly, 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.

Many 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. Certain 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. 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. patent application Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1. CpG, poly-I:C, plasmid DNA, RNA, siRNA and antisense oligos may also provide suitable adjuvants as appropriate.

Advantageously, in some embodiments 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.

As used herein, the term "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.

As used herein, 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)-(CH₂)2-S-CH₂-CH(OCH₃)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.

The term "mannose" refers to α-D-mannopyranosyl, or its oxidized form. Mannose groups can be conjugated to dendrimers via reactive end groups on the exterior surface of the dendrimer. In certain embodiments, the end functional groups on the exterior surface of the dendrimer to which the adjuvant groups are attached are amino groups. A chemical group containing α-D-mannopyranosyl, or its oxidized form, may also advantageously comprise a "linker" moiety through which the α-D-mannopyranosyl ring (or oxidized form) is attached to the dendrimer surface, for example an isothiocyanate (S=C=N-R-, where R is a hydrocarbon group such as alkylene or phenylene) linker. In a typical example, the mannose-containing group is α-D-mannopyranosylphenyl iosothiocyanate. Mannose and other sugars can be added to dendrimers using a number of methodologies depending on the dendrimer functional groups. Using techniques in carbohydrate chemistry a variety of functional groups or linkers can be added to the anomeric carbon of the sugar. 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.

In some embodiments of the invention, 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).

Cleavage of a cystamine core, or use of a dendrimer having an unreacted reactive site on the core allows for the conjugation of the antigen to the dendrimer molecule. Such dendrimer- antigen conjugates (as well as conjugates where the antigen is conjugated to the dendrimer surface) are also referred to herein as "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).

Typically, 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.

In a typical example the antigen is conveniently conjugated to the dendrimer with succinimidyl-3-2(-pyridyldithio)propionate (SPDP) or analogue, such as succinimydyl 6-(3-

[2-pyridyldithio]-propionamido hexanoate (LC-SPDP). 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).

Other suitable modifier or linker agents will vary with the nature of the reactive group at the core and the antigen and will be apparent within the art.

It will be recognised that an antigen may also be conjugated to the dendrimer at the surface of the dendrimer in accordance with the methodology described herein.

In some embodiments, immunogenic molecules contemplated herein, i.e., where the antigen is conjugated to the dendrimer, may be comprised of a single dendrimer molecule conjugated to a single antigen molecule. In other embodiments a dendrimer molecule may be conjugated to 1, 2, 3, 4 or more antigen molecules. In still other embodiments an antigen molecule may be conjugated to 1, 2, 3, 4 or more dendrimer molecules. Antigens

As used herein, 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. For example, such screens can be performed using methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, 1988. In one illustrative example, 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, I¹²⁵ labelled Protein A.

An "epitope" as used herein, 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. As used herein, 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. As used herein, 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. Generally 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.

As used herein, a "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. Typically 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.

As used herein, a "B cell epitope" is any epitope which is capable of eliciting the production of antibodies when administered to a subject. In certain embodiments, 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.

As used herein, the term "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. Reference hereinafter to a "protein" includes 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. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non- naturally occurring. A 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.

Advantageously, in some embodiments of the invention, 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. Alternatively, 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. For example, 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). Contact with a tumor polypeptide (100 ng/ml-100 μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days will typically result in at least a two fold increase in proliferation of the T cells. Contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN-γ.) is indicative of T cell activation (see Coligan et al. Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998)). 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. Within preferred embodiments, 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.

For therapeutic purposes, 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. For example, 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. Alternatively, 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.

Those skilled in the art are aware that optimum T cell activation requires cognate recognition of antigen/MHC by the T cell receptor (TcR), and a co-stimulation involving the ligation of a variety of cell surface molecules on the T cell with those on an antigen presenting cell (APC). The costimulatory interactions CD28/B7, CD40L/CD40 and OX40/OX40L are preferred, but not essential for T cell activation. Other costimulation pathways may operate.

For determining the activation of a CTL or precursor CTL or the level of epitope- specific activity, standard methods for assaying the number of CD8⁺ T cells in a specimen can be used. Examples of assay formats include a cytotoxicity assay, such as for example the standard chromium release assay, the assay for IFN-γ 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). To produce tetramers, the carboxyl terminus of an MHC molecule, such as, for example, the HLA A2 heavy chain, is associated with a specific peptide epitope or polyepitope, and treated so as to form a tetramer complex having bound thereto a suitable reporter molecule, preferably a fluorochrome such as, for example, fluoroscein isothiocyanate (FITC), 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. Following binding, and washing of the T cells to remove unbound or non-specifically bound Tetramer, 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. 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.

Because CD4⁺ T-helper cells function in cell mediated immunity (CMI) as producers of cytokines, such as, for example IL-2, to facilitate the expansion of CD8⁺ T cells or to interact with the APC thereby rendering it more competent to activate CD8⁺ T cells, 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.

Examples of 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, either solus or in a vaccine composition to elicit T cell and B cell activation, clonal expansion or CMI, 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. However, by way of example, the immunogenic molecules of the invention may be administered to a subject in an amount ranging from 1 to 10,000 μg/kg body weight, typically within the range of 10 to 1000 μg/kg, for example within the range of 10 to 100 μg/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. In general, 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. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. 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.

In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such 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. Thus, 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.

The formulation of such compositions is 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.

These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, buffers, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the 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. The phrase

"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. In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intradermally, subcutaneously, intravenously, intramuscularly, or even intraperitoneally. 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. In certain embodiments, 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). In all cases 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. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include 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. In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, 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). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, 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.

In certain embodiments, 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. Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al. J Controlled Release 52(1-

2):8\-7, 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are also well-known in the pharmaceutical arts. Likewise, illustrative transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045.

In another embodiment of the invention, the 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.

Without being bound by any theory or mode of action, 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.

In a further aspect, the present invention relates to chemically modified dendrimers for use in vaccines. As used herein, 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. In particular embodiments there is provided 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. Said method 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.

For such an ex vivo application, 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.

In a related embodiment, 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.

Accordingly, 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 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. Accordingly, 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., Crγptococcus spp., Curvularia spp., Epidermophyton spp., Exophiala jeanselmei, Exserohilum spp., Fonsecaea compacta, Fonsecaea pedrosoi, Fusarium oxysporum, Fusarium solani, Geotrichum candidum, Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Hortaea werneckii, 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 spp., Zygomcete fungi, Absidia corymbifera, Rhizomucor pusillus and Rhizopus arrhizus, Bacillus anthracis, Bordetella pertussis, Vibrio cholerae, Escherichia coli, Shigella dysenteriae, Clostridium perfringens, Clostridium botulinum, Clostridium tetani, Corynebacterium diphtheriae and Pseudomonas aeruginosa.

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.

In a related aspect, 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.

Accordingly, 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 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.

Accordingly, 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 myeloid leukaemia, colorectal cancers, cutaneous t-cell lymphoma, dermatofibrosarcoma-protuberans, desmoplastic-small-round-cell-tumor, ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconi anaemia, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers, germ cell tumors, gestational-trophoblastic-disease, glioma, gynaecological cancers, haematological malignancies, hairy cell leukaemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, Langerhan's-cell-histiocytosis, laryngeal cancer, leiomyosarcoma, leukaemia, li-fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer-(nsclc), ocular cancers, oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral-neuroectodermal-tumors, pituitary cancer, polycythemia vera, prostate cancer, rare-cancers-and-associated-disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous-cell- carcinoma-(skin), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom 's-macroglobulinemia or Wilms' tumor.

Within certain embodiments of the invention, the immunogenic combination of the invention induces an immune response predominantly of the ThI type. High levels of ThI -type cytokines (e.g., IFN -γ, TNFα., IL-2 and IL- 12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes ThI- and Th2-type responses. Within certain embodiments, 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.

In some embodiments of the invention, 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 kDa lipoprotein or Ag85, Neisseria meningitidis class I outerprotein, chlamydia trachomatis surface protein and Listeria surface protein.

Further examples of 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; Apostolopoulos, 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 antigens (eg MAGE series antigens).

According to another embodiment of this invention, 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. 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). In general, 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. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al. Nature Med 4:594-600, 1998).

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. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL- 13 and/or TNFα to cultures of monocytes harvested from peripheral blood. Alternatively, 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,

IL-3, TNF.ct, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells. 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 Fcγ. 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)..

In further aspects of the present invention, the immunogenic combinations described herein may be used for the treatment of cancer or a pathogenic infection. Within such methods, 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. Accordingly, 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.

Within certain embodiments, 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. For example, 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.

The combinations of the invention 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:

(a) 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; (b) parenteral administration, e.g. subcutaneous, intramuscular or intravenous injection as a sterile solution or suspension; (c) topical application e.g. creams, ointments, gels, lotions etc.

In further aspects, 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.

All scientific citations, patents, patent applications and manufacturer's technical specifications referred to hereinafter are incorporated herein by reference in their entirety.

It is to be understood that unless otherwise indicated, the subject invention is not limited to specific formulation components, manufacturing methods, biological materials or reagents, dosage regimens and the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The invention will now be described with reference to the following Examples which are included for the purpose of illustrating certain embodiments of the invention and are not intended to limit the generality hereinbefore described.

EXAMPLES

The following abbreviations are used in the examples:

ABTS 2, 2-Azino-di-[3-ethylbenzthiozoline sulphonate]

ACK lysis buffer Buffered ammonium chloride lysis solution

BMDC Bone marrow derived dendritic cells

BSA Bovine Serum Albumin CHO aldehyde group

DCs Dendritic cells eGFP Enhanced green fluorescence protein

ELISA Enzyme-Linked Immunosorbent Assay

ELISPOT Enzyme linked immunospot FACS Fluorescence activated cell sorter

FITC Fluorescein isothiocyanate

GST Glutathione sepharose transferase kDa Kilodaltons

LPS Lipopolysaccharide mPBS Mouse phosphate buffered saline

MD Mannosylated dendrimer

MDO Mannosylated dendrimer OVA

MUCl MUCIN 1

NaCl Sodium chloride O/N overnight

OVA Ovalbumin

OxMan Oxidised Mannan

PBS Phosphate buffered saline PEI Polyethylenimine

PI Propidium iodide

SFB Succinimidyl 4-formyl benzoate]

SPDP Succinimidyl 3-(2-pyridyldithio)propionate

Example 1 - Preparation of OVA (O), Mannosylated Dendrimer (MD) and MDO

OVA PREPARATION

Commercially available OVA (Sigma, St. Louis, USA) contained a high level of endotoxin.

When 100 μg/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 4⁰C for 30 min, placed at 3O⁰C 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 -2O⁰C. 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.

MD PRODUCTION The procedures to produce MD and MDO are illustrated in Scheme 1 below. The dendrimer is treated with α-D-mannopyranosylphenyl isothiocyanate, which reacts with the surface amino groups of the dendrimer to introduce mannose residues onto the dendrimer giving the mannosylated dendrimer. 84 μl of 31.33 mg/ml of α-D-mannopyranosylphenyl isothiocyanate (Sigma) dissolved in DMSO was slowly added into a solution containing 44 μl of 10% (w/v in methanol) generation 4 PAMAM dendrimer (Dendritic Nanotechnologies, Mt. Pleasant, USA.) and 372 μl of 0.2 M sodium bicarbonate buffer (pH 9.0). The solution was mixed on the rotating wheel for 16 h at room temperature (RT) and then dialyzed overnight into PBS with 6000 - 8000 MWCO (molecular weight cut-off) tubing at 4⁰C. Through dialysis, unconjugated α-D-mannopyranosylphenyl isothiocyanate (M.W. 313.3) was removed. Mannose residues present in dendrimer were quantified by a colorimetric assay (Monsigny et al, 1988).

CONJUGATION OF OVA TO MD

To prepare MDO, 100 μl of 0.2 M tris(2-carboxyethyl)phosphine (TCEP) (Pierce, Gainesville, USA) in PBS was added to the MD solution and left for 30 min at RT to generate the reduced form of MD. The solution was then dialyzed overnight into PBS / 2 mM EDTA at 4⁰C with 6000 - 8000 MWCO tubing and TCEP was removed from the solution. The sulfhydryl groups were quantified as previously described (Ellman, 1959, Bulaj, 1998). OVA was modified by the addition of 18 μl of 20 mg/ml N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (Pierce) in DMSO to 500 μl 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 4⁰C 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. 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.

(NH₂)54 ^■*— *^■ (H₂N)₃₂ C C (NH₂)₃₂ Generation 4 Dendrimer α-D-Mannopyranosylphenyl

(A)

Mannosylated Dendrimer (MD)

OVA

Mannosylated Dendrimer OVA (MDO)

SCHEME 1 x, y and z represent the numbers of the unmannosylated amine group on the molecules

CHARACTERIZATION OF DENDRIMER CONJUGATES

As shown in Scheme. 1, generation 4 cystamine dendrimer was partially mannosylated by reacting with a 30 fold molar excess of α-D-mannopyranosylphenyl isothiocyanate. The resorcinol assay revealed the presence of 18 - 20 mannose residues per dendrimer molecule.

Reduction of the disulfide core of the mannosylated cystamine dendrimer by TCEP provided the thiol derivative. The sulfhydryl groups were quantitated and revealed that 90% of (mannosylated) dendrimer could be recovered. In addition, 2 - 4 activated disulfide groups were introduced on OVA (measured by the pyridine-2-thione assay) by reaction with a 10 fold molar excess of SPDP. Finally, the OVA-SPDP solution was reacted with reduced MD (based on the dendrimer molar concentration) to produce MDO. 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. To optimize MDO formation, 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.

Example 2 - Preparation of MUCl-GST Mannosylated Dendrimer (MD) with Aldehydes

Preparation of MD is as described above.

INCORPORATION OF MANNOSE AND ALDEHYDE GROUPS ONTO OVA (Using amino dendrimers)

METHOD 1 (Scheme 2):

CONJUGATION OF MD TO SFB

10 mM dithiothreitol (DTT) (final) in PBS was added to the MD solution (1.5 mg dendrimer) and left for 30 min at RT to generate the reduced form of MD. The solution was then loaded on a PD-IO column that was equilibrated with PBS, to remove excess DTT. The sulfhydryl groups were quantitated as previously described. The reduced MD was then treated with 46 μl of 10 mg/ml aldrithiol dissolved in DMSO, and mixed for 30 min at RT. The solution was loaded on a PD-IO column that was equilibrated with PBS, to remove excess aldrithiol. The solution was then treated with 78 μl 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).

CONJUGATION OF MD-SFB TO OVA

10 mM DTT (final) in PBS was added to the OVA-SPDP solution and left for 30 min at RT. The solution was then loaded on a PD-IO column that was equilibrated with PBS. The number of sulfhydryl groups present was quantitated as described previously. The reduced OVA- SPDP was subsequently added to the MD-SFB solution (OVA-SPDPrMD-SFB; 1:12) to form MDO-SFB. The sample was concentrated using a 30,000 MWCO Amicon Ultra Centrifugal filter device. Excess MD-SFB and OVA were removed by gel filtration using a Superdex-75 column (0.5 cm x 30 cm). In this study the concentration of MDO-SFB was calculated based on OVA.

-W -

Dendrimer— (NH₂)64 +

3Ox a-D-mannopyranosyl isothiocyanate M₂₀D

_Ml0d__s__s

lOx Aldrithiol

M₂₀D = M_1Od-S-S-(IM₁₀ OVA/SPDP + DTT →- OVA/SH

SCHEME V. METHOD 2 (Scheme 3):

CONJUGATION OF MD TO ACETAL

48 μl of 10 mg/ml SPDP was added to MD (2.2 mg dendrimer) and mixed O/N at RT. The solution was then loaded on a PD-IO column that was equilibrated with PBS, to remove excess SPDP. The solution was brought to pH 4 (to selectively reduce the disulfide groups of SPDP), and 10 mM dithiothreitol (final) in PBS was added to the MD-SPDP solution and left for 30 min at RT. The solution was then loaded on a PD-10 column that was equilibrated with PBS, to remove excess DTT. The number of sulfhydryl groups present was quantitated as described previously. The solution was brought to pH 8.0 and then it was treated with 73 μl 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.

Note: 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.

CONJUGATION OF MD-ACETAL TO OVA

10 mM DTT (final) in PBS was added to the MD-Acetal solution and left for 30 min at RT (to reduce the disulfide bond of the cystamine core of the dendrimer). The solution was then concentrated 3 times by sequential addition of PBS using a 5,000 MWCO Amicon Ultra Centrifugal Filter Device. The OVA-SPDP solution was subsequently added to the reduced MD-Acetal solution (OVA-SPDP:MD-Acetal; 1:12) to form MDO-Acetal. The sample was concentrated using a 30,000 MWCO Amicon Ultra Centrifugal filter device. Excess of the reduced MD-Acetal and OVA were removed by gel filtration using a Superdex-75 column (0.5 cm x 30 cm). In this study the concentration of MDO-Acetal was calculated based on OVA. Dendπmer — (NH₂)_M +

3Ox α-D-mannopyranosyl isothiocyanate M₂₀D

SCHEME 3

PREPARATION OF MDO USING THE CARBOXY DENDRIMER (cMDO) (SCHEME 4)

PREPARATION OF MANNOSE

12 μl Ethylenediamine was added to a solution of 5.6 mg of α-D-mannopyranosyl isothiocyanate dissolved in 100 μl of DMSO and 200 μl of 0.2 M sodium bicarbonate (pH 9.0), and the solution was mixed O/N at RT. The solution was then placed on the freeze-dryer O/N and the semi-solid was then dissolved in 150 μl dH₂O:DMSO (1:1). PREPARATION OF cMD

74 μl of 30 mg/ml N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) was added to a solution of 55 μl of 10% (w/v in water) generation 4 PAMAM succinamic acid dendrimer in 716.5 μl of 0.05 M MES buffer (pH 6.2). 154.5 μl of 37.33 mg/ml mannose (prepared above) was added to the solution and was mixed O/N at RT. 116 μl of 10 mg/ml glycine was added to the solution, and was mixed for 30 min at RT. The solution was then loaded on a PD-10 column that was equilibrated with PBS, to remove excess EDAC, mannose and glycine.

CONJUGATION OF cMD TO OVA

10 mM DTT (final) in PBS was added to the cMD solution and left for 30 min at RT. The solution was then loaded on a PD-10 column that was equilibrated with PBS, to remove excess DTT. The OVA-SPDP solution was subsequently added to the reduced cMD solution (OVA-SPDPXMD; 1:6) to form cMDO. The sample was concentrated using a 30,000 MWCO Amicon Ultra Centrifugal filter device. Excess of the reduced cMD and OVA were removed by gel filtration using a Sperdex-75 column (0.5 cm x 30 cm). In this study the concentration of cMDO was calculated based on OVA.

Dendrimer- (COOH)₆₄

cMDO

SCHEME 4

INCORPORATION OF MANNOSE AND ALDEHYDE GROUPS ONTO OVA (Using carboxy dendrimers)

PREPARATION OF cMD-diol (SCHEME 5)

74 μl of 30 mg/ml N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) was added to a solution of 55 μl of 10% (w/v in water) generation 4 PAMAM succinamic acid dendrimer in 770.2 μl of 0.05 M MES buffer (pH 6.2). 77.3 μl of 37.33 mg/ml mannose (prepared above) and 23.5 μl of 30 mg/ml 3-amino-l,2-propanediol were added to the solution and was mixed O/Ν at RT. 116 μl of 10 mg/ml glycine was added to the solution, and was mixed for 30 min at RT. The solution was then loaded on a PD-IO column that was equilibrated with PBS, to remove excess EDAC, mannose, 3-amino-l,2-propanediol and glycine. CONJUGATION OF cMD-diol TO OVA (SCHEME 5)

10 mM DTT (final) in PBS was added to the cMD-diol solution and left for 30 min at RT. The solution was then loaded on a PD-IO column that was equilibrated with PBS, to remove excess DTT. The OVA-SPDP solution was subsequently added to the reduced cMD-diol solution (OVA-SPDP:cMD-diol; 1:6) to form cMDO-diol. The sample was concentrated using a 30,000 MWCO Amicon Ultra Centrifugal filter device. Excess of the reduced cMD-diol and OVA were removed by gel filtration using a Superdex-75 column (0.5 cm x 30 cm). In this study the concentration of cMDO-diol was calculated based on OVA.

Dendrimer-(COOH)₆₄ + 32 x EDAC + 32 x (1) + 32 x H₂N_N

cMDO-diol

SCHEME 5

PREPARATION OF cMDO-CHO (SCHEME 6)

40 μl of 0.2 M sodium periodate was added to 400 μl of cMDO-diol, and the solution was left for 1 h at 4°C. The solution was then loaded on a PD-IO column that was equilibrated with PBS, to remove excess sodium periodate. 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). In this study the concentration of cMDO-CHO was calculated based on OVA.

cMDO-diol

NaIO₄

SCHEME 6

INCORPORATION OF MUCl ONTO MD (SCHEME 7)

PREPARATION OF MUCl-SPDP

9 μl of 10 mg/ml SPDP was added to 500 μl of 4.3 mg/ml MUCl and mixed for 2 h at RT. The solution was then loaded on a PD-IO column that was equilibrated with PBS, to remove excess SPDP. 10 mM dithiothreitol (final) in PBS was added to the MUCl-SPDP solution and left for 30 min at RT. The solution was then loaded on a PD-IO column that was equilibrated with PBS, to remove excess DTT. The number of sulfhydryl groups present was quantitated as described previously. PREPARATION OF MD-DTNB

10 mM dithiothreitol (DTT) (final) in PBS was added to the MD solution (5 mg dendrimer) and left for 30 min at RT to generate the reduced form of MD. The solution was then loaded on a PD-IO column that was equilibrated with PBS, to remove excess DTT. The reduced MD was then treated with 200 μl of 5 mg/ml 5,5'-dithiobis(2-nitrobenzoic acid) DTNB in phosphate buffer pH 7.5, and mixed for 15 min. The sulfhydryl groups were quantitated as previously described. The solution was then concentrated using a 5,000 MWCO Amicon Ultra

Centrifugal Filter Device. Then it was loaded on a PD-10 column that was equilibrated with PBS, to remove excess DTNB.

PREPARATION OF MD-MUCl

The reduced MUCl-SPDP was subsequently added to the MD-DTNB solution (MUCl- SPDP:MD-DTNB;1:12) to form MD-MUCl. 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.

Dendrimer— (NH₂J₆₄ +

3Ox a-D-mannopyranosyl isothiocyanate M₂₀D

DTT 30 min

MUC1/SH

M₂₀D = Mi_Od-S-S-dM,o

0 M 10*— S+ S- MUCl MUCl/SPDP + DTT-^ MUC1/SH

SCHEME 7

Example 3 - Preparation of Cells

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.

Generation of bone-marrow derived DCs (BMDCs) - Bone marrow (BM) cells from femurs and tibias of C57BL/6 mice were collected and treated with ACK lysis buffer (0.15 MNH₄Cl,

1 mM KHCO₃ and 0.1 mM Na₂EDTA) to lyse erythrocytes. Cells were washed and cultured at

2 x 10⁶ cells/3 ml in 6 well plates with complete RPMI media (2% Hepes buffer, 0.1 mM 2- mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 10% fetal calf serum) supplemented with 10 ng/ml GM-CSF and IL-4 (Pharmingen, San Diego, USA). By this culture method, the purity of BMDCs was consistently > 80% CDl Ic⁺. T cell purification - Splenocytes from OTI or OTII mice were collected, washed and incubated in ACK lysis buffer at 37⁰C for 5 min to lyse erythrocytes. 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 4⁰C 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 4⁰C for 25 min.

Cells bound to the beads were removed by magnetic attraction. The purity of T cells was consistently > 94%.

Example 4 : Cellular Responses - Mannosylated Antisen Dendrimers

T CELL PROLIFERATION To test the ability of MDO and OVA to stimulate in vitro T cell responses, BMDCs were pulsed with (i) 10 μg/ml of the control peptide (H-2K^b-restricted SIINFEKL or I-A^b-restricted OVA_323-339 ISQAVHAAHAEINEAGR), (ii) 40 μg/ml of OVA and (iii) 40 μg/ml of MDO, respectively, for 18 h. DCs were irradiated, washed and seeded at specified cell numbers (0 - 4000) into the 96-well plates containing 2 x 10⁴ OTI or OTII T cells in triplicates with a final volume of 200 μl 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 ³H-thymidine (Amersham, Beaconsfield, UK) uptake. Briefly, cells in each well were pulsed with 1 μCi of ³H-thymidine for 16 h. These cells were then harvested and radioactivity measured by the Packard TopCount scintillation counter (PerkinElmer, Boston, USA) in counts per minute (CPM). For comparison, peak proliferation of OTI (on day 2) and OTII (on day 3) is shown. 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). The magnitude of 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.

BMDC MATURATION

Stimulants, including MDO (40 μg/ml), OVA (40 μg/ml), LPS (1 μg/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 10⁵ 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.

MDO moderately matures BMDCs - In comparison to PBS, 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). 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.

BINDING OF MDO AND OVA TO BMDCS

To test whether enhanced T cell stimulation was due to increased binding of MDO to DCs,

BMDCs were incubated with MDO and OVA at specified doses and detected with the anti- OVA antibody. To determine the binding avidities of MDO and OVA to DCs, 5 x 10⁵ cells were palleted and incubated with 100 μl of titrated MDO and OVA (0.04 - 40 μg/ml) at 4°C for 30 min. Cells were washed and incubated with 100 μl of the rabbit polyclonal anti-OVA antibody at 4⁰C 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.

Binding avidity of MDO to DCs - A high avidity of MDO in comparison to OVA was evidenced by the MFI in FACS analysis. At the lowest concentration (0.04 μg/ml), the enhanced binding of MDO was readily detectable in comparison to OVA (Fig. 4A). When the concentration increased 4-fold, the binding of MDO to DCs was further increased, with a much higher slope in comparison to OVA, which showed a much lower level of DC binding (Fig. 4A). At the highest concentration (40 μg/ml) equivalent to that used in the antigen- presentation assay, DCs labeled with MDO were far more fluorescent than those labeled with OVA (Figure 4B).

Example 5 : In vivo Responses - Mannosylated Antiεen Dendrimers

Materials and Methods Cross-presentation assay - Groups of 4 mice were separately injected with MDO (25 μg), OVA (25 μg) and an equivalent volume of PBS into hind footpads. Twenty hours post- injection, popliteal lymph node (LN) cells were isolated. A portion of LN cells derived from the PBS group was pulsed with SIINFEKL (1 μg/ml) for 1 h at 37⁰C as the positive control. Due to scarcity of APCs present in LNs, a high number (2 x 10⁴) of LN cells were seeded into 96-well round bottom plates, together with 5 x 10⁴ OTI T cells per well in quadruplicates. 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. Immunization of mice - To evaluate the immune responses in vivo, four groups of mice (n = 9) were injected intradermal^ at the base of a tail with OVA (25 μg), MDO (12.5 μg), MDO (25 μg) and the equivalent volume of PBS on days 0, 11 and 18. Mice (n = 4) were bled on day 28 for antibody detection and sacrificed next day for the ELISpot assay. The remaining mice (n = 5) in each group were given another boost injection on day 38 and challenged with B16-0VA tumor cells on day 45.

ELISA - Serial dilutions of mouse sera were performed in 1% (w/v) BSA in 50 μl PBS in 96- well ELISA plates (Corning, New York, USA). The plates were pre-coated with 50 μl of 10 μg/ml OVA in the coating buffer (0.05 M Na₂CO₃, pH 9.6) at 4⁰C overnight and blocked with 100 μl of 3% (w/v in PBS) bovine serum albumin (BSA) at 37⁰C for 1 h to prevent nonspecific binding. After incubation for 1 h at 37⁰C, the plates were washed 10 times with 0.05% (v/v in PBS) of Tween20 (Sigma). 50 μl of 0.1% (v/v in 1% BSA/PBS) horseradish peroxidase linked anti-mouse IgG (Amersham) was added into each well and incubated at 37⁰C for 1 h. The plates were again washed 10 times with 0.05% Tween20/PBS and 50 μl of the ABTS (2,2'-Azino-bis[3-ethylbenzthiazoline-6-sulfonic acid]) substrate solution (0.03% ABTS and 0.08 % H₂O₂ in ABTS buffer [0.1 M Na₂HPO₄ and 0.08 M citric acid, pH 4.5]) was added into each well. The reaction was developed for 30 min and read with 405 nm absorbance.

ELISpot assay - Splenocytes collected from each mouse were seeded at 5 x 10⁵ cells in the presence of 20 μg/ml SIINFEKL, OVA_323-33Q, OVA or 2 μg/ml of ConA as the positive control and media as the background control, in a total volume of 100 μl in triplicates, into 96- well MultiScreen filter plates (Millipore, Billerica, USA). These plates were pre-coated with 70 μl of 5 μg/ml anti-mouse IFNγ antibody (AN 18) (Mabtech, Stockholm, Sweden). After 18 h of culture, the plates were washed 6 times with PBS and 0.05% Tween20/PBS and submerged in ddH₂O for 2 min to lyse the cells. Seventy μl of 1 μg/ml biotinylated anti-mouse IFNγ 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 μl 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. Seventy μl of 0.1% (v/v in PBS) AP conjugate substrate (Bio-Rad Laboratories, Foster City, USA) prepared under manufacturer's instructions was added into each well and left to develop in dark for 30 min. The plates were then washed with tap water, dried overnight and read by the ELISpot counter. All spots produced in response to peptides or OVA were subtracted with ones produced when no peptide or OVA was present.

Tumor challenge - Pre-immunized mice were shaved on the abdominal area and inoculated subcutaneously with 1 x 10⁶ B 16-0 VA melanoma cells. Tumor growth was monitored every 2 - 3 days. The size of a tumor (mm²) was calculated by multiplication of two perpendicular diameters determined by callipers.

Statistical analysis - All data are shown as the mean ± standard error of the mean (SEM). The data generated in this study were analyzed by student's t test. Significance of difference was determined by the P value (* P < 0.05, ** P < 0.01).

Results

MD as an OVA delivery vehicle enhances in vivo OVA-speciβc immune responses - Mice immunized with MDO and OVA demonstrated various levels of cellular and antibody responses analyzed by the ELISpot assay and ELISA, respectively. As shown in 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 μg of MDO produced a significant level of IFNγ. Mice immunized with OVA and MDO generated different levels of SIINFEKL-specifϊc CD8⁺ T cell response, in comparison to the non-responding PBS group. Only 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 IFNγ and such a response to MDO appeared to be dose-dependent. It was noted only mice immunized with 25 μg of MDO developed competent T cell responses (CD4, CD8 and OVA-specific IFNγ responses). While a decrease of the immunization dosage to 12.5 μg 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). The ConA response was invariably included as an internal positive control in all ELISpot assays performed (not shown). In evaluation of the antibody response, mice immunized with MDO (25 μg) 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. To test this hypothesis, popliteal lymph node cells isolated after 20 h footpad injection with MDO and OVA were used to stimulate in vitro 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

MD enhanced cross-presentation of the OVA peptide SIINFEKL in the lymph milieu.

MDO immunization delays or prevents growth of B16-OVA melanoma - To evaluate the therapeutic potential of MDO, 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). On day 14, the average tumor sizes in mice immunized with 12.5 and 25 μg/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 μg) offered better tumor protection than that with a lower dose (12.5 μg). Mice were sacrificed before the tumor reached 225 mm². 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.

Example 6 : Analysis of Results - Mannosylated Antiεen Dendrimers

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).

It has been previously shown that mannosylated antigens are internalized and processed following the endocytic pathway, leading to enhanced MHC-class II presentation of antigenic peptides. It has now been shown that MD enhances the delivery of OVA into both MHC-class

I and II presentation pathways in BMDCs, which are dependent on acidic lysomal processing.

The increased mannosylation on dendrimer OVA enhances MHC-class II, but not MHC-class

I, presentation, suggesting the presence of divergent routing mechanisms. While mannose binding receptors such as DC-SIGN and MR mediate MDO delivery and processing in acidic lysosomes, resulting in MHC-class II presentation, there is an unknown routing mechanism targeting MHC-class I loading compartments, leading to cross presentation. The use of MD as an OVA delivery vehicle reduces the threshold of OVA concentration required for effective cross presentation by BMDCs.

Although DCs pulsed with OVA are also stimulatory to OTII T cells, the level of proliferation induced is much lower than DCs loaded with MDO. In disagreement with our findings, Burgdorf et al., recently demonstrated the capability of OVA to induce cross-presentation in BMDCs, leading to OTI T cell proliferation (Burgdorf et al, 2006). Curiously, those BMDCs were pre-matured with LPS for 24 h and then pulsed with the OVA antigen. Also, the number of BMDCs seeded in the proliferation assay was high (4 x 10⁵ DCs versus 2 x 10⁵ T cells) without appropriate titration. In our experience, either when OVA was used without Triton- Xl 14 pretreatment for LPS depletion or when BMDCs carrying a very small amount of peptide were seeded in high numbers, OTI T cells would proliferate extensively.

In evaluating the binding avidity of MDO to DCs, BMDCs were incubated with MDO and OVA and labeled with anti-OVA and the following FITC. Both MDO and OVA bound to the DC surface when determined by flow cytometry analysis. The observation of weak OVA binding to DCs is in agreement with previous studies. It is worth noting that, OVA is often glycosylated and contains 3 - 7 branched mannose residues (Mao et al., 2003). Although the OVA (A2512-5G, Sigma, USA) used in this study is purified with a reduced content of mannose, it is demonstrated that such OVA also binds to DC surfaces. In comparison to OVA, 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).

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. 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.

To explain the in vivo MDO-induced CD8⁺ T cell responses, it was demonstrated that MDO induced in vivo cross-presentation that cannot be accomplished by in vitro BMDCs. LN cells derived from mice injected with MDO were able to stimulate in vitro OTI T cell proliferation, suggesting that the SIINFEKL peptide was indeed presented in the lymph milieu by APCs after immunization (Fig. 7). Without limiting by theory, it is suggested that 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).

The therapeutic efficacy of MDO was explored in the highly aggressive B 16-0 VA melanoma disease model. Mice pre-immunized with OVA or control PBS, grew B 16-0 VA tumors in a much faster rate than those pre-immunized with MDO. After 3 months of challenge, one and two mice in groups pre-immunized with 12.5 and 25 μg of MDO respectively did not grow any tumors and appeared healthy with no indication of metastasis. Since induction of a tumor antigen-specific CD8⁺ T cell response is crucial for tumor protection in mice (Ikeda et al, 2004; Pietersz et al, 1998), the anti-tumor effect of MDO correlates with the ELISpot results. 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. In this study, for the first time, 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.

Example 7 : Mannosylated Antigen Dendrimers with Aldehydes

MDO binds to DCs (refer to Fig. 8) :

DCs cultured with GM-CSF/IL-4 (100 ng/ml) and Flt-3 ligand (300 ng/ml) were harvested at day 6. Cells (5 * 10⁵) 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. A. In the homogeneous GM-CSF/IL-4 culture, CDl Ic⁺ DCs (> 85%) bound to MDO with a much higher avidity than OVA, which also showed a weak level of DC binding. B. 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):

At day 6, stimulants including PBS (negative control), MDO (40 μg/ml), LPS (1 μg/ml) and CpG1668 (10 μg/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.

Both MDO and LPS stimulate myeloid-like GM-CSF/IL-4 as well as CD24^high and CDl lb^high Flt-3L DCs; however, both stimulants fail to induce plasmacytoid-like Flt-3L B220⁺ DCs in comparison to CpG. This is most likely due to the lack of TLR4 expression on plasmacytoid DCs (Binding of C-type lectins does not induce maturation.)

MDO-induced DC maturation is dependent on TLR4 (refer to Fig. 10): At day 6, stimulants including PBS (background control), MDO (40 μg/ml), LPS (1 μg/ml) and CpG 1668 (10 μg/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).. Titrated (1 - 4 x 10³) BMDCs (A) and FU3-L DCs (B) pulsed with MDO (40 μg/ml), OVA (40 μg/ml) or control peptides (SIINFEKL [1 μg/ml] and

OVA_323-339 [10 μg/ml]) were seeded with 2 x 10⁴ OTI or OTII T cells. T cell proliferation was monitored for 5 days with ³H-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.

The effect of NH₄Cl on:

(A) OVA_323-339 and MDO-mediated OT-I T cell proliferation, and

(B) CD40, CD80 and CD86 expression, (refer to Fig. 12).

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).

DCs (4 x 10³) preloaded with 40 μg/ml MDO in the presence of titrated malondialdehyde (25 - 400 μg/ml), with 40 μg/ml OVA and with 40 μg/ml OVA plus 400 μg/ml malondialdehyde, were incubated with 2 x 10⁴ OTI T cells. T cell proliferation was monitored using the [³H]thymidine incorporation assay. Peak proliferation induced by DCs on day 2 from each pulsing condition was compared. Malondialdehyde used in 100 - 400 μg/ml resulted in proliferation of OTI T cells ie cross-presentation of MDO by DCs.

MDO modified with aldehyde (refer to Fig 15)

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.

Titrated DCs (1 - 4 x 10³) preloaded with 20 μg/ml MDO or MDO-acetal (derived from homogeneous fraction 1 or heterogeneous fraction 2 during FPLC purification) were incubated with 2 x 10⁴ 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 (produces 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).

Titrated DCs (1 - 4 x 10³) preloaded with 20 μg/ml MDO or MDO-acetal (fractions 1 and 2) were incubated with 2 x 10⁴ OTI T cells. T cell proliferation was monitored. Peak proliferation induced by DCs on day 2 from each pulsing condition was compared. 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). This indicates the influence of aldehyde on the adjuvanticity of the MDO conjugate. Example 8 - Cellular responses - Mannosylated MUCl Dendrimers (MD-MUCl)

MUCl proteins/peptides - Mannosylated MUCl dendrimers (MD-MUCl) 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. Another example is a fusion protein comprising GST and an N-terminal extracellular region of human MUCl (amino acids 33-103), i.e. Glutathione-S¹- transferase-

SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAP ATEPASGSAATW (designated NFP). An alternative version of the MUClFP antigen (p VNTR), not containing GST, is useful for measuring VNTR-specific immune responses in ELISpot and ELISA assays. In addition 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^band 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.

Mice - C57BL/6, HLA-A*0201/K^b transgenic mice (HLA-A2/K^b), human MUCl transgenic mice and MUCl -specific TCR transgenic mice are useful for the experiments described herein. HLA-A2/K^b transgenic 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).

Generation of bone-marrow derived DCs (BMDCs) - Bone marrow (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 MNH₄Cl, 1 mM KHCO₃ and 0.1 mM Na₂EDTA) to lyse erythrocytes. Cells are washed and cultured at 2x10⁶ cells/3 ml in 6 well plates with complete RPMI media (2%

Hepes buffer, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/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 37⁰C 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 4⁰C 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.

T cell proliferation assay

To test the ability of MD-MUCl and MUClFP to stimulate T cell responses, 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 μl per well. Control wells contain T cells alone or T cells with non-pulsed DCs. Proliferation of T cells is monitored between days 1 and 5 by ³H-thymidine (Amersham, Beaconsfield, UK) uptake. Briefly, cells in each well are pulsed with 1 μCi of ³H-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 37⁰C 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.

Immunization of 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 μl 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 Na₂CO₃, pH 9.6) at 4⁰C overnight and blocked with 100 μl of 3% (w/v in PBS) bovine serum albumin (BSA) at 37⁰C for 1 h to prevent non-specific binding. After incubation for 1 h at 37⁰C, the plates are washed 10 times with 0.05% (v/v in PBS) of Tween20 (Sigma). 50 μl of 0.1% (v/v in 1% BSA/PBS) horseradish peroxidase linked anti-mouse IgG (Amersham) is added into each well and incubated at 37⁰C for 1 h. The plates are again washed 10 times with 0.05% Tween20/PBS and 50 μl of the ABTS (2,2'-Azino-bis[3-ethylbenzthiazoline-6-sulfonic acid]) substrate solution (0.03% ABTS and 0.08 % H₂O₂ in ABTS buffer [0.1 M Na₂HPO₄ 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 10⁵ cells in the presence of a MUCl protein fragment, peptide or fusion protein (e.g. 20 μg/ml MUClFP or or MUClK- or NFP or pVNTR or MUC1A2 12-20 or 950-958) or 1 μg/ml of ConA as the positive control and media as the background control, in a total volume of 100 μl in triplicates, into 96-well MultiScreen filter plates (Millipore, Billerica, USA). These plates are pre-coated with 70 μl of 5 μg/ml anti-mouse IFNγ antibody (AN 18) (Mabtech, Stockholm, Sweden). After 18 h of culture, the plates are washed 6 times with PBS and 0.05% Tween20/PBS and submerged in ddH₂O for 2 min to lyse the cells. Seventy μl of 1 μg/ml biotinylated anti-mouse IFNγ antibody (R4-6A2) (Mabtech) is added into each well and left at RT for 2 h. The plates are washed 6 times with PBS and 0.05% Tween20/PBS. Seventy μl of 0.1% (v/v in PBS) Streptavidin-ALP (Mabtech) is added into each well and left at RT for 2 h. The plates are again washed. Seventy μl of 0.1% (v/v in PBS) AP conjugate substrate (Bio-Rad Laboratories, Foster City, USA) prepared under manufacturer's instructions is added into each well and left to develop in dark for 30 min. The plates are then washed with tap water, dried overnight and read by the ELISpot counter. All spots produced in response to peptides or MUClFP are subtracted with ones produced when no peptide or MUCl FP is present.

Prophylactic and therapeutic tumor studies

In prophylactic tumor protection studies, groups of mice are immunized at least once (e.g. twice at a 2- week interval) before being challenged with 1x10⁶ 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). For therapeutic studies, mice are injected subcutaneously with IxIO⁵ 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.

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.

Detection of MUCl expression on MUCl -expressing tumor cells

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 37⁰C 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).

The ability of mice to mount an anti-MUCl response in vivo can be measured using ELISA or ELISpot assays as described above. Furthermore, 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 Apostolopoulos et al., 2006). REFERENCES

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Claims

CLAIMS:

1. A dendrimer for use in an immunogenic combination wherein the dendrimer comprises one or more adjuvant groups conjugated to the surface of the dendrimer.

2. The dendrimer according to claim 1 wherein the dendrimer is selected from the group consisting of PAMAM, polylysine, PEHAM or polypropyleneimine based dendrimers.

3. The dendrimer according to claim 1 wherein the dendrimer is a PAMAM dendrimer.

4. The dendrimer according to claim 1 wherein the adjuvant groups are selected from mannose and aldehyde groups.

5. The dendrimer according to claim 1 wherein the dendrimer's core moiety contains a cleavable disulfide bond.

6. The dendrimer according to claim 5 wherein the disulfide bond is cleaved to form a sulfhydryl group.

7. An immunogenic combination comprising: (i) an antigen; and (ii) a dendrimer according to any one of claims 1 to 6.

8. The immunogenic combination according to claim 1 wherein the antigen is conjugated to the dendrimer.

9. The combination according to claim 8 wherein the antigen is conjugated to the core of the dendrimer.

10. The combination according to claim 8 or 9 wherein the antigen is conjugated to the dendrimer via a disulfide bond.

11. The combination according to any one of claims 7 to 10 wherein the antigen is a polypeptide.

12. The combination according to any one of claims 7 to 11 further comprising a pharmaceutically acceptable carrier.

13. A method of inducing an immune response in a subject comprising administering to said subject a combination according to any one of claims 7 to 12.

14. A method of enhancing the cell mediated immunity of a subject, said method comprising: (ii) contacting ex vivo dendritic cells obtained from a subject with a combination according to any one of claims 7 to 12 for a time and under conditions sufficient to mature said dendritic cells; and (ii) introducing the activated dendritic cells to the subject or to another subject in order that T cell and/or B cell activation occurs.