Ganglioside immunostimulating complexes and uses thereof

Details Ganglioside immunostimulating complexes and uses thereof Ganglioside immunostimulating complexes and uses thereof

2000-02-10

2004-11-09

Eyler, Yvonne (Department: 1642)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Particulate form

C424S210100, C424S193100, C424S277100, C424S489000, C424S484000

Reexamination Certificate

active

06814981

ABSTRACT:

The present invention relates generally to an immunostimulating complex comprising one or more gangliosides and more particularly to an immunostimulating complex comprising at least one of the gangliosides GM2, GD2, GD3 or GT3. The present invention is useful, inter alia, as a prophylactic and/or therapeutic agent in the treatment of tumours, and more particularly, melanomas.
The bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Throughout the specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of element or integer.
The incidence of malignant melanoma has been increasing rapidly over the last ten years with the survival rate of patients diagnosed with melanoma decreasing with increasing primary tumour thickness (Batch et al., 1982). Patients with regional lymphnode metastasis generally have a five year survival rate of less than 35% following dissection (Coit et al., 1991).
The identification of tumour associated antigens has provided further scope for tumour related therapies. Tumour antigens, both those which are unique to tumour cells and those which are shared with normal cells, have emerged as potential targets for active immunisation. One example of a class of tumour antigens are the gangliosides. Gangliosides are sialated glycosphingolipids consisting of hydrophilic (sugar) and hydrophobic (ceramide) portion. They are prevalent in brain and other neural crest derived cells and are also found on the tumours originating from these cells—astrocytomas, neuroblastomas and melanomas (Lloyd, 1993). Their pattern of cell surface expression is altered during the process of the malignant transformation (Halcomori, 1985). More than 70 gangliosides have been identified although not all of these are found on tumours.
In terms of developing a vaccine, the induction of an effective immune response to carbohydrate antigens requires the co-presentation of carbohydrate epitopes (B epitopes) and T epitopes derived from protein to antigen presenting cells. This has traditionally been achieved by chemical conjugation of carbohydrate to carrier protein, the best known example being the
haemophilus influenzae
type b vaccines where a carbohydrate antigen is conjugated to either diphtheria toxoid, tetanus toxoid or
Neisseria meningitidis
major outer membrane protein. However, the use of chemical conjugation in the preparation of such vaccines presents inherent problems in that the structure of conformational epitopes may be altered thereby reducing or eliminating their antigenicity. In addition, chemical conjugation procedures are expensive, time consuming, very difficult to perform under uniform conditions and generally involve substantial losses of material. Further, in stimulating the immune response to such an antigen, adjuvants have commonly been used to increase immunogenicity of the antigen. One of the difficulties associated with the use of adjuvants stems from the unwanted reactivity which they may also induce.
Dose-site reactivities are a major concern for both veterinary and human use of adjuvant in vaccine preparation. One way of avoiding toxicity is via the use of immunostimulating complexes. Immunostimulating complexes are typically small, cage like structures 30 to 40 nanometers in diameter which retain their structure on freeze drying. The size can vary, however, depending on mode of preparation, composition and method used for measurement. The final formulation of a typical immunostimulating complex with an optimal amount of immunogenic protein or polypeptide is a weight ratio of saponin, cholesterol, phospholipids and protein or polypeptide (5:1:1:1). Such a typical immunostimulating complex is estimated to contain around 60% by weight saponin, around 10% each for cholesterol and phospholipids and the remainder protein or polypeptides. Proteins or polypeptides can be incorporated into the immunostimulating complex matrix either directly or by chemical coupling to a carrier protein (eg. influenza envelope protein) after incorporation of the carrier protein into the immunostimulating complex.
The saponin adjuvants, a group of surface active glycosides of plant origin, are highly effective adjuvants. However, although effective they nevertheless exhibit toxicity. Such toxicity is reducable by incorporation of saponin into an immunostimulating complex. This is due to the association of the saponin adjuvant with cholesterol in the complex thereby reducing its ability to bind to cholesterol in cell membranes. Further, a lesser amount of saponin is required to generate a similar level of adjuvant effect.
In work leading up to the present invention, the inventors have found that the phospholipid component of an immunostimulating complex can be replaced with ganglioside molecules since gangliosides are structurally related to phospholipids. Both molecules have two acyl chains joined through glycerol to a head group. Although not intending to limit the operations of the invention to any one mode of action, it is believed that because the nature of a ganglioside molecule is a fatty acid chain with a polar head group, the fatty acid chain immerses in the immunostimulating complex leaving the polar head group exposed, said head group thereby functioning as an exposed B cell epitope. Such a formulation can present the antigenic portion of the ganglioside at the surface of the immiunostimulating complex structure.
Accordingly, one aspect of the present invention relates to an immunogenic immunostimulating complex comprising a saponin preparation, a sterol, a protein epitope together with a phospholipid and at least one ganglioside or at least one ganglioside alone.
Saponin preparations, sterols and phospholipids suitable for use in the present invention are described in detail in PCT/AU95/00670, the disclosures of which are incorporated herein by reference.
An immunogenic immunostimulating complex in accordance with the present invention may be prepared by techniques which are well known to persons skilled in the art, and which are described in detail in the publications of Cox and Coulter, 1992 and Morein et al., Australian Patent Specifications No. 558258, 589915, 590904 and 632067 the disclosures of which are incorporated by reference herein. Since the preparation of the immunogenic immunostimulating complexes of the present invention does not involve chemical conjugation, the risk of altering the conformation of the ganglioside or protein epitope is minimized.
Multiple proteins and gangliosides can be incorporated into a single immunostimulating complex. This permits the preparation of immunostimulating complexes wherein the ratio of protein components to gangliosides varies between individual immunostimulating complexes.
Reference hereinafter to “gangliosides” should be read as including reference to all forms of gangliosides and derivatives thereof. Derivatives include fragments, parts, portions, chemical equivalents, mutants, homologs and analogs. Chemical equivalents of a ganglioside can act as a functional analogue of the ganglioside. Chemical equivalents may not necessarily be derived from a ganglioside but may share certain conformational similarities. Alternatively, chemical equivalents may be specifically designed to mimic certain physiochemical properties of the ganglioside. Chemical equivalents may be chemically synthesised or may be detected following, for example, natural product screenings.
Homologs of gangliosides contemplated herein include, but are not limited to, gangliosides derived from different species.
Preferably, said ganglioside is selected from GM2, GD2, GD3 and/or GT3 (Lloyd, 1993) or derivatives thereof.
Accordingly, in this preferred embodiment the present invention relates to an immunogenic immunostimulating complex comprising a saponi

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