The invention relates to an immunogenic complex comprising at a least one glycoside and at least one lipid, integrated into an iscom complex or matrix, and at least one antigen which antigen is integrated into the iscom complex or coupled on to or mixed with the iscom complex or iscom matrix complex, characterised in that it also comprises at least one enzyme. It also relates to such a complex further comprising at least one peptide which specifically binds to a receptor expressed on a cell capable of antigen presentation, which cell expresses MHC Class I or Class II and to compositions comprising the complexes.

Description of the Invention

SUMMARY OF THE INVENTION

It has now turned out that when combining iscoms and an enzyme, especially CTA1 and its derivatives, their adjuvant effects are enhanced, some of their limitations and disadvantages are overcome and that the over all effect unexpectedly may be synergistic. Surprisingly the enzymatic activity of CTA1 is kept intact in the complex. This novel formulation is non-toxic and is highly immuogenic by a variety of mucosal and systemic routes.

The main object of the invention is to provide an immunogenic complex comprising at least one glycoside, at least one lipid and at least one antigen which antigen is integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex, characterized in that it also comprises at least one enzyme.

Another object of the invention is to provide immunogenic iscom complexes, comprising at least one glycoside, at least one lipid and at least one antigen, into which an enzyme preferably A1 subunits of a bacterial enterotoxin have been integrated.

Another object is to provide immunogenic iscom complexes into which both enzymes and peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation, have been integrated.

Another object is to provide iscom complexes on to which antigens and enzymes and/or peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation, have been coupled.

Another object is to provide iscom complexes mixed with antigens and enzymes and/or peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation.

Another object of the invention is to provide immunogenic iscom matrix complexes, comprising at least one glycoside and at least one lipid on to which antigens, enzymes and/or peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation have been coupled.

Another object is to provide iscom matrix complexes mixed with antigens and enzymes and/or peptides or proteins, which specifically binds to a receptor expressed on a cell capable of antigen presentation.

Still another object is to provide a complex where the enzyme and a peptide or protein which specifically binds to a receptor is bound together into a fusion protein which is integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex.

Still another object is to provide a complex where the enzyme, the peptide or protein which specifically binds to a receptor and an antigen is bound together into a fusion protein, which is integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex.

Another object is to provide a composition comprising the new complexes according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a immunogenic complex comprising at least one glycoside, at least one lipid and at least one antigen which antigen is integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex, characterized in that it also comprises an enzyme.

The enzyme is preferably an enzyme that confers enzymatic ADP-ribosylating activity as it has turned out that such an enzyme has unexpected adjuvant activity in combination with iscom and iscom matrix complexes. If the enzyme is toxic it is preferred that the toxic put be deleted from the enzyme. Thus, the enzyme may be an enzyme with ADP-ribosylating activity from which a toxic part has been deleted. Especially the enzyme is a native or mutant bacterial toxin, preferably an enterotoxin and specifically a subunit of a toxin that confers enzymatic ADP-ribosylating activity. The enzyme may be selected from Cholera toxin (CT), E. Coli heat labile enterotoxin (LT), Pertussis, Clostridia, Shigella and Peudomonas toxins. Most preferably the enzyme is at least one A1 subunit of a bacterial enterotoxin wherein said enterotoxin is selected from the group consisting of cholera toxin (CT) and E. Coli heat labile enterotoxin (LT). Such enzymes and subunits and the production thereof are described in U.S. Pat. No. 5,917,026.

According to a preferred form of the invention, the immunogenic complex further comprises at least one peptide or protein, which specifically binds to a receptor expressed on a cell capable of antigen presentation. Preferably the cell expresses MHC Class I or Class II antigen. The antigen-presenting cell may be belong the group consisting of lymphocytes, macrophages, dendritic cells, Langerhans cells and epithelial cells.

Iscom contains at least one glycoside, at least one lipid and at least one type of antigenic substances, especially proteins and peptides and can be produced as described in EP 0 109 942 B1, EP 0 242 380 B1 and EP 0 180 564 B1.

Iscom matrices contain at least one glycoside and at least one lipids. Matrices have an is immunostimulating effect on administration together with antigenic substances, and can be produced as described in EP 0 436 620 B1.

The enzyme and/or the antigen and/or the peptide or protein which specifically binds to a receptor may be integrated into an iscom complex. It is also possible to couple one or mote of these substances an to an iscom complex already containing antigens or on to an iscom matrix complex.

Further is it possible to mix on or more of these substances with iscom or iscom matrices. In such a case the iscom complex may already contain one or more antigens and/or one or more enzymes and/or one or more peptides or proteins, which specifically bind to a receptor.

Thus, the invention relates to a composition comprising iscoms wherein one or more antigens, one or more enzymes or one or more receptor binding peptides or proteins are integrated into, coupled on to or mixed with the iscom complex.

The invention also relates to a composition comprising matrix and one or more antigens, one or more enzymes and one or more receptor binding peptides or proteins coupled on to or mixed with the matrix complex.

The antigen-presenting cells having receptors to which the peptide can bind, are suitably cells capable of antigen presentation especially cells expressing MHC Class I and Class II and may be lymphocytes, such as B-lymphocytes, T-cells, monocytes, macrophages, dendritic cells, Langerhans cells, and epithelial and endothelial cells.

The peptide is a peptide that binds to receptors of the above cells, preferably to an Ig or Fc receptor expressed by said antigen-presenting cell and most preferably to receptors of B-lymphocytes.

Examples of specific targeting peptides are peptides capable of binding to receptors of:

(i) granulocyte-macrophage colony-stimulating factor (GM-CSF) capable of binding to the GM-CSF receptor .alpha./.beta.heterodimer present on monocytes, neutrophils, eosinophils, fibroblasts and endothelial cells, (ii) CD4 and CD8 expressed on T cells which together with the T cell receptor (TcR) act as co-receptors for MHC class II and MHC class I molecules, respectively. MHC class I are expressed on most nucleated cells, whereas MHC class II molecules are expressed on dendritic cells, B cells, monocytes, macrophages, myeloid and erythroid precursor cells and some epithelial cells, (iii) CD 28 and CTLA-4, two homodimeric proteins expressed mainly on T cells which bind to B7 expressed on B cells, (iiii) CD40 present mainly on the surface of mature B cells which interact with gp39 expressed on T cells, (iiiii) different isotypes of the Ig heavy chain constant regions which interact with a number of high or low affinity Fc receptors present on mast cells, basophils, eosinophils, platelets, dendritic cells, macrophages, NK cells and B cells.

According to a particularly preferred embodiment of the invention, said peptide is constituted by protein A or a fragment thereof in single or multiple copies, such as one or more D subunits thereof.

According to the invention, the enzyme and the peptide which specifically binds to a receptor may be bound together into a fusion protein, which may be integrated into an iscom complex or coupled on to or mixed with an iscom complex or iscom matrix complex.

The fusion proteins comprise a sub-unit of a native or mutant bacterial toxin that confers enzymatic ADP-ribosylating activity, and, lined thereto, a peptide. The peptide is preferably such that the resulting fusion protein is in possession of water solubility and capability of targeting the fusion protein to a specific cell receptor different from receptors binding to the native toxin; thereby mediating intracellular uptake of at least said subunit.

An antigen may also be incorporated in the fusion protein. Thus , the antigen, the receptor binding peptide or protein and the enzyme may be used as a single molecule or as different combinations in fusion proteins for integration into iscoms or coupling on to iscoms and/or matrices or mixing iscoms and/or matrices. One or more antigens, one or more receptor binding peptides or proteins and one or more enzymes may be used as single molecules or in the fusion protein.

The integration of the substances and the coupling thereof on to iscoms or iscom matrices maybe done as described in EP 0 109 942 B1, EP 0 242 380 B1 and 0 180 564 B1.

Although the invention is by no means limited hereto it will be exemplified in the following mainly with reference to the sub-unit A1 of cholera toxin or a mutant thereof.

Preferably the fusion protein comprises the A1 subunit of cholera toxic and is fused to one or more copies of protein A or a fragment thereof, such as the D region of said protein A.

One fusion protein denoted CTA1-DD consisting of CTA1 linked to DD, a dimer of the D-region of protein A, binds to soluble immunoglobulins as well as the Ig-receptor on B cells. The results demonstrate that is molecule lacks enterotoxic activity, but still effectively ADP-ribosylates target protein. When used as a parenteral adjuvant CTA1-DD enhances anti-KLH antibody responses and increases KLH T cell priming.

These results demonstrate the possibility to circumvent the toxic effects of CT simply by removing the CTB pentamer, thus excluding the potential interaction resulting in toxicity between the epithelial cell GM1-receptor and CT. The strategy of targeting of the immunomodulating activity of CTA1 to defined cell populations can be expanded to include essentially any given cell type, enabling specific modulation of cellular responses controlled by cAMP, provided that a suitable targeting molecule is available. CTA1 alone is highly insoluble in physiological aqueous solutions. Thus, the targeting molecule used as fusion partner in this invention also has the important function to enhance solubility of the CTA1 entity.

The CTA1 moiety in CTA1-DD is targeted to B cells primarily, and away from the GM1-receptor on e.g. the gut epithelial cells. Furthermore, using this construct it has been demonstrated that (i) the enzymatic activity of CTA1 was retained in CTA1-fusion proteins provided that CTA1 was fused at its carboxy terminus; (ii) CTA1 in the fusion protein exerts its ADP-ribosyltransferase activity in target cells through a pathway for entry that is different from the surface ganglioside GM1-receptor; and that. (iii) CTA1-DD displays a strong immunopotentiating activity.

Similarly, CTA1 may be fused to other targeting molecules such as e.g. CD4 to access MHC II expressing cells or any other ligand that specifically can bind to a receptor present on the cell surface. Using this approach CTA1 will not interact with the GM1-receptor present on most mammalian cells including gut epithelial cells because the CTB portion is lacking in the construct. There fore, CTA1 is given a narrow spectrum of cellular interactions via specific binding to surface Ig or Fc-receptors thereby targeting CTA1 to primary B cells, and macrophages and other Fc-receptor carrying cells.

Fusion proteins may be produced by general biotechnological methods known in the art. Fusion protein CTA1DD may be produced as described in U.S. Pat. No. 5,917,026. Fusion proteins with CTA1DD any be produced using the vector described in FIG. 7 (see Original Patent) or as described in references 8, 29.

The pharmaceutical compositions may comprise one or more immunogenic complexes according to the invention, together with one or more excipients that are acceptable in pharmaceutical or veterinary products, whereby complexes and components to be mixed therewith may be placed in separate compartments.

The compositions according to the invention will in practice normally be administered orally but may be given topically, or by rectal administration or by injection.

For oral administration tablets and capsules may contain conventional excipients, such as binders, for example syrup, sorbitol, or polyvinyl pyrrolidone; fillers, for example lactose, microcrystalline cellulose, corn starch, calcium phosphate or sorbitol; lubricants, for example magnesium stearate, stearic acid, polyethylene glycol or silica; desintegrants, for example potato starch or sodium starch glycolate, or surfactants, such as sodium lauryl sulphate.

Oral liquid preparations can be in the form of for example water or oil suspensions, solutions, emulsions, syrups or elixirs, or can be supplied as a dry product for constitution with water or another suitable vehicle before use.

A composition according to the invention can be formulated for parenteral administration by injection or continuous infusion. Compositions for injection can be provided in unit dose form and can take a form such as suspension, solution or emulsion in oil or aqueous carriers and can contain formulating agents, such as suspending, stabilizing and/or disperging agents. Alternatively, the active constituent can be present in powder form for constitution with a suitable carrier, for example sterile pyrogen-free water, before use.

The compositions according to the invention can contain between 0.1 and 99% by weight of the active constituent, suitably from 30 to 95% for tablets and capsules and 3 to 50% for liquid preparations.

The experimental part shows that iscoms containing a fusion protein comprising CTA1-DD linked to the OVA 323-339 peptide epitope, used as a model antigen, were highly immunogenic when given by the subcutaneous, oral or nasal routes, inducing a wide range of systemic T cell dependent immune responses. No toxicity was observed by any route indicating that rationally designed combined vectors consisting of CTA1-DD and iscoms S may provide the basis of potent and safe mucosal vaccines.

Thus, iscoms containing OVA peptide fused to CTA1-DD were immunogenic when given by a variety of routes, including the oral, nasal and parenteral routes. The responses induced included DTH and serum IgG antibodies in vivo, antigen-specific T cell proliferation and .gamma.IFN production in vitro. Despite the fact that it was not possible to detect IL5 production when CTA1-DD-ISCOMS primed lymphocytes were restimulated with OVA in vitro, immunised mice were primed for the production of both IgG2a and IgG1 isotypes, indicating that Th1 and Th2 cells were primed in vivo.

The immune responses induced by iscoms containing the OVA peptide fused to the intact CTA1-DD construct were markedly superior to those found after immunisation with iscoms containing the CTA1-R72K-DD construct which contains a point mutation that abolishes the enzymatic activity of CTA1. This confirms our previous findings that ADP-ribosylating function is essential for the adjuvant property of the CTA1-DD Vector (8) and indicates that a significant proporon of the combined ISCOMS-CTA1DD structure also depends on targeting this activity to the immune system. Nevertheless, iscoms containing the enzymatically inert CTA1-R72K-DD molecule did retain some adjuvant activity when given by mucosal or parenteral routes. This may reflect the well-established adjuvant properties of the iscoms themselves, perhaps enhanced by the ability of the DD fragment to target them in vivo, presumably to B lymphocytes. Thus, in addition to being targeted to DC and/or macrophages like conventional iscoms (14-16, 17), the new, combined vector may have the additional ability to interact with B cells, creating a second potential source of APC for T cell priming. In addition to its potent APC targeting properties, the intact CTA1-DD-ISCOMS adjuvant has the great advantage of being able to activate these cells, creating a costimulatory microenvironment for efficient T cell priming. Iscoms induce DC and/or macrophages to produce proinflammatory cytokines such as IL1, IL6 and IL12 in vivo (15, 18-22), while CTA1-DD is a potent co-activator of B cells (8). For these reasons, at least three important features of CTA1-DD-ISCOMS were considered to contribute to their immunogenicity. First they can physically target antigen and adjuvant to distinct APC populations in vivo and via distinct mechanisms. In the case of iscoms, this probably involves phagocytic uptake by mononuclear cells, whereas CTA1-DD involves receptor-mediated binding and uptake by surface immunoglobulin (8). Secondly, the vector contains two active adjuvants, Quil A and the ADP-ribosylating enzyme CTA1, which can stimulate the relevant cells that have taken up the vector. Lastly, insertion of the antigenic construct into the rigid iscoms particle ensures that the antigen and the adjuvants ate delivered directly to the same APC, focussing their effects for optimal T cell priming.

Extremely low doses of OVA peptide were able to prime systemic immunity by both mucosal and parenteral routes using the CTA1-DD-ISCOMS vector, with as little as 150 ng or 750 ng peptide equivalent being effective by the subcutaneous and oral routes respectively. Secondly, although the antigenic epitope used was delivered as part of a large fusion protein inserted in an iscoms particle, it induced strong immune responses that could be recalled with intact OVA protein. This indicates that CTA1-DD fusion protein and the iscoms vector did not interfere with the antigen processing mechanisms, which normally generate this class II MHC-restricted epitope.

Taken together the results suggest that the combined vector gains access to physiologically relevant antigen processing pathways in an extremely efficient manner. Lastly, it is important to emphasise that no toxicity was observed in mice given the combined adjuvant vectors by any route. This contracts with the toxicity occasionally seen using vectors containing intact Quil A (12, 27, 28), but extends our previous findings that the Quadri A fraction of Quil A and the CTA1-DD fusion protein are themselves lacking significant toxicity, despite their potent adjuvant activities. Thus the combined vector should provide a safe means of inducing mucosal and systemic immunity.

One surprising finding from the study was at the free CTA1-DD fusion protein also had some adjuvant activity when given by the oral route (FIG. 3 (see Original Patent)). As confirmed here, previous studies had shown that this material was active by parenteral and subcutaneous routes (8), but it was considered it unlikely that it would be able to gain access to the B cells necessary for its adjuvant effects when given into the harsh environment of the intestine. However, it is now shown that oral immunization with CTA1-DD containing a defined peptide epitope induces a wide range of immune responses, which interestingly included marked levels of .gamma.IFN, despite other claims that CT based adjuvants stimulate predominantly Th2 dependent responses by this route. The responses induced by free CTA1-OVAp-DD were not as high as those, which occurred, when the fusion protein was inserted in iscoms, underling the added potency of the combined vector. However, the enzymatically inactive CTA-R72K-OVAp protein was unable to induce any response above that generated by peptide alone by the oral or parenteral routes, indicating that the adjuvant properties of the intact CTA1-DD material were dependent on its ADP ribosylating activity, even when give by the oral route.

Subcutaneous immunisation gives a synergistic between the CTA1-DD and iscom adjuvant effect in proliferation for CTA1-OVAp-DD-ISCOMS over the sum of the proliferation levels of CTA1-OVAp-DD and CTA1-OVAp-R72K-DD (FIG. 2A, see Original Patent). Similarly oral immunisation gives a synergistic effect in IgG2a induction for (FIG. 3D, see Original Patent) and in proliferation and .gamma.IFN induction for (FIGS. 4A and 4B, see Original Patent). Also, intranasal administration gives a synergistic effect in proliferation and .gamma.IFN induction as can be seen from FIG. 5 (see Original Patent).

Together, the results are encouraging evidence that by combining the distinctive adjuvant properties of iscoms and the novel, non-toxic CTA1-DD derivative, it may prove possible to construct effective, safe and stable subunit vaccines which are active by mucosal routes.
 

Claim 1 of 13 Claims

1. An immunogenic complex comprising: at least one glycoside and at least one lipid, integrated into an iscom complex or matrix; at least one antigen integrated into the iscom complex or coupled onto or mixed with the iscom complex or iscom matrix complex; and an adjuvant comprising (i) an isolated subunit of an A1 subunit of a bacterial enterotoxin selected from the group consisting of cholera toxin and E. coli heat labile enterotoxin, and (ii) a fragment of protein A comprising the D region of protein A, said fragment having the ability to bind to Ig or Fc receptors on an antigen-presenting cell.
 

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