Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Utility Patent
1997-09-24
2001-01-02
Bansal, Geetha P. (Department: 1642)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C424S277100, C424S282100, C514S013800
Utility Patent
active
06168804
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to slow release formulations containing peptides for use as vaccines. More particularly this application relates to liposome and microsphere formulations containing short epitope-containing peptides for raising a specific Th1 cell-mediated immune response to provide protective immunity against a disease state associated with the antigen.
BACKGROUND OF THE INVENTION
Short synthetic peptides are poor immunogens in vivo. To enhance the immunogenicity of short peptides, they are usually conjugated to large carrier proteins and associated with immunological adjuvants. However, immune responses induced by such conjugates can be mainly directed against the epitopes in the carrier proteins, and may also induce epitopic suppression. Further, alum, the only immunoadjuvant approved for human immunization, is a poor inducer of cell-mediated immune responses, which are believed to mediate rejection of certain cancers, as described by R. Bomford, “The Comparative Selectivity of Adjuvants for Humoral and Cell-Mediated Immunity,”
Clin. Exp. Immunol.,
39:435-441, 1980.
The two types of T lymphocytes that can be distinguished based on function and surface markers are helper (Th) and cytotoxic (Tc) cells. Th cells express the CD4 surface protein and upon contact with foreign antigen, activate antibody-producing B cells and Tc. Tc express the CD8 protein, and are thought to control infection by elimination of cells expressing foreign antigens. Both Tc and Th lymphocytes recognize antigens as short peptides that are bound to cell surface class I and class II major histocompatibility complex (MHC) molecules, respectively.
The mechanism by which antigens are processed into peptides differs for class I and class II MHC presentation. Antigens taken into the cell via endocytosis are digested into peptides that then bind to class II, but not class I, MHC molecules. In contrast, antigens that are synthesized within the cytoplasm or endoplasmic reticulum selectively bind to class I MHC molecules. Thus, vaccines containing subunit antigens, which gain access to only the endocytic pathway of the cell, prime the MHC class II restricted Th, but not class I restricted Tc.
Microencapsulation of antigens into liposomes has been used as an approach to enhance the immunogenicity of proteins without the use of traditional adjuvants. Liposomes in the blood stream are generally taken up by the liver and spleen, and are easily phagocytosed by macrophages, one of the primary APCs. They serve as effective vehicles for antigen delivery to macrophages. Liposomes also allow co-entrapment of immunomodulatory molecules along with the antigens, so that such molecules may be delivered to the site of antigen encounter, allowing modulation of the immune system towards protective responses.
Mucins are complex (M
r
≧200 kD) glycoproteins produced by a variety of normal and malignant epithelial cells. They can be expressed as cell-surface and/or secreted molecules. Mucins comprise 50-90% (by weight) oligosaccharide structures that are linked to core peptides through O-glycosidic bonds. The core peptides of several human mucins have been characterized by cDNA cloning. Cancer-associated mucins tend to have fewer and shorter oligosaccharide structures in comparison to the normal mucins. As a result, certain core-peptide epitopes of the cancer-associated mucins can become exposed to the immune system, whereas such structures in normal mucins remain ‘cryptic’. Further, the loss of polarity of cancer cells and their increased expression of mucins can also facilitate exposure of the mucin epitopes to the immune system. Thus certain ‘cryptic self’ epitopes of cancer-associated mucins are believed to be immunogenic in humans and have been used for immunotherapy of carcinomas. See Longenecker et al., “Prospects for Mucin Epitopes in Cancer Vaccines,”
J. Tumor Marker Oncol.,
5:11-26, 1993.
A human mucin associated with breast, ovarian and pancreatic carcinomas, MUC1, mucin, is a known potential target for active specific immunotherapy. Peptide sequences in the variable number tandem repeat (VNTR) domain of this mucin have been shown to induce humoral and cell-mediated immune responses in cancer patients. Cytotoxic T cells have been generated from lymph nodes of pancreatic, breast and ovarian carcinoma patients and have demonstrated their specificity for peptide epitopes in VNTR region of the MUC1 mucin on cancer cells. Accordingly, peptide epitopes in the VNTR domain are suitable targets for directing immune responses against MUC1
+
human tumors. With this in view, rodent tumor models expressing human MUC1 have been developed for evaluation of the immune responses against MUC1 sequences and their effect on tumor growth. Immunization with a recombinant vaccinia virus expressing MUC1 induced protection against tumor challenge with MUC1
+
tumor cells in a rat model is described by Hareuveni et al., “Vaccination Against Tumor Cells Expressing Breast Cancer Epithelial Tumor Antigen,”
Proc. Natl. Acad. Sci. U.S.A.,
87:9498-9502, 1990. The immunogenicity of MUC1 synthetic peptides showed that formulations which induced strong cell mediated immune responses inhibited tumor growth in a MUC1-transfected mouse mammary carcinoma model. More recently, V. Aspostolopoulos et al., “Immunization with Cellular and Synthetic Antigens,”
Cancer Res.
54:5186-5193, 1994, have reported that cell-mediated immune responses of “Th1 type,” but not antibody responses, correlate with rejection of MUC1-transfected 3T3 cells in mice.
SUMMARY OF THE INVENTION
The present invention provides slow release antigen delivery systems that preferentially raise cell-mediated immune responses of the Th1 type against short T-cell epitope-containing peptides, for example those containing from about 11 to 34 amino acids, without the use of traditional carrier proteins and immunoadjuvants. Preferably, the peptide contains 12-25 amino acids. The antigen delivery system of this invention is a composition comprising a peptide contained within a liposome, or other slow-release delivery vehicle, that releases the peptide at a rate of less than 50 nanogram/ml/hour and preferably from about 10 to 2% of the encapsulated peptide is released over 24 hr period at 37° C.
The composition further contains a Th1 specific immunomodulatory agent associated with the slow-release delivery vehicle, preferably displayed on the surface of the delivery vehicle. The preferred Th1 specific cell mediating immunomodulator is monophosphoryl lipid A (MPLA). The compositions of this invention avoid the use of common immunomodulators completely, and exclusively use Th1 type specific immunomodulatory agents, so as to raise a specific Th1 cell mediated immune response and substantially avoid production of antibodies to the T cell antigen.
In the method of this invention, the rate of release of the antigenic peptide from the liposomal formulation into which it is encapsulated is controlled to a rate of a few nanograms per ml per hr, preferably about 10 to 2% release over a 24 hr period at 37° C. Therefore, in the practice of this invention, the liposome, or other slow release vehicle, used to encapsulate the epitope-containing peptide is selected to afford release rate characteristics that enable release of the peptide at such low rates. The very low release rate of the peptide from the liposomal formulation into the subject's body tissue and/or fluids aids in the avoidance of a humoral immune response to the antigenic peptide.
The slow-release compositions of this invention provide a vehicle effective in treatment of any disease state most effectively treated by a specific immune response of the cell-mediated Th1 type, wherein substantially no humoral immune response is raised by administration of the composition to the patient. Thus, by the practice of the methods of this invention, the problems associated with a humoral immune response in the treatment of certain disease states are substantially avoided. For instance the responses of the im
Kwon Glen
Samuel John
Bansal Geetha P.
Fish & Richardson P.C.
University of Alberta
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