HK1063726B - Pharmaceutical compositions enhancing the immunogenicity of poorly immunogenic antigens - Google Patents

Description

Pharmaceutical composition for enhancing immunogenicity of low-immunogenicity antigen

Technical Field

The present invention relates to human medicine, in particular to protective and/or therapeutic vaccines conferring protection against infectious and autoimmune diseases and cancer; the invention particularly provides vaccine compositions that induce or enhance an immune response to antigens of low immunogenicity.

Prior Art

The poor results achieved to date in the use of vaccines for the prevention and treatment of a group of infectious diseases, cancer and autoimmune diseases are due to a combination of factors. Wherein, mainly: low immunogenicity of the relevant antigens, no knowledge of how to manipulate immune system regulation, and escape strategies for pathogens and tumors, and host immunosuppression, among others.

In the prior art, low immunogenic antigens are known to be those peptides, polypeptides and proteins (or their corresponding DNA sequences) present in tumor and normal tissues or associated with pathogens, which produce chronic infections by evading the action of the immune system.

Among the low immunogenic antigens, growth factor receptors with kinase activity at tyrosine residues have been shown to be closely related to the development of tumors and tumor metastasis, and in some cases they have been shown to be of value as an indicator of poor prognosis of cancer, for example, the epidermal growth factor receptor (EGF-R), also known as HER-1; epidermal growth factor receptor 2 (HER-2); and platelet derived growth factor receptor (PDGF-R), as is the case.

Overexpression of these receptors in certain classes of tumors (mainly of epithelial origin) has been the target of interest in cancer immunotherapy, this being tumors of the breast, bladder, ovary, vulva, colon, lung, brain, prostate and head and neck. The presence of EGF-R has been shown to indicate a poor prognosis of Breast Cancer (Perez R et al, 1984, Breast Cancer and Treatment 4: 189-. Although the role of the EGF/EGF-R system in the regulation of tumor growth is still unknown, it has been suggested that EGF-R expression in tumor cells provides a mechanism for autocrine stimulation which leads to such cell proliferation disorders (Schlessinger J et al, (1983) Crit Rev Biochem14 (2): 93-111).

Due to its high expression in tumors, the epidermal growth factor receptor has become the target of interest for Passive Immunotherapy (PI) using monoclonal antibodies in their native form in combination with drugs, toxins and radioisotopes (Vollmar AM et al, (1987) J Cell Physiol 131: 418-425). Several Clinical studies on monoclonal antibodies (MAbs) are in progress, some of which have shown promising results, that is in Clinical studies on MAb C225 in breast, pancreatic and renal cells (stage II) and head and neck (stage III) (MendelsohnJ et al, (1999) American Society of Clinical Oncology Meeting). Another phase II clinical trial showing good results was performed in head and neck tumors against Mab IOR egf/r3 (Crombet et al, (2000) Cancer Biotherapy and radiopharmaceutical, manuscript accepted).

On the other hand, Specific Active Immunotherapy (SAI) using EGF-R as the target of interest has never been developed because of its low immunogenicity as a self-molecule and its widespread expression in tissues, which prevents immunologists from considering this option (Disis ML and CheeverMA, (1996) Current Opinion in Immunology 8: 637-642). SAI is preferred over PI because PI does not activate specific cellular effector pathways of the immune response and its effect depends on the half-life of the antibody used, and continuous reinfusion is often necessary to achieve the desired effect.

In order to develop an effective vaccine, carriers and adjuvants must act by appropriately modulating the immune system to overcome the low immunogenicity of the relevant antigens and to cooperate with evasive strategies against pathogens and tumors. For this reason, the search for new carriers and adjuvant systems currently constitutes an important field of research.

Over the past few years, new theories and emerging knowledge about immune system regulation have opened up new experimental areas to develop more effective carriers and adjuvants. Fearon et al (Science, Vol.272, pp50-53, 1996) have taught that protective immunity is the result of an interaction between two key systems: innate immunity, and acquired immunity. The cells responsible for adaptive immunity are unable to distinguish between structures that require an immune response and those that do not, and therefore they need to be directed by cells of the innate immune system. The necessary link between innate and adaptive immunity is provided by Antigen Presenting Cells (APCs), where Dendritic Cells (DCs) are the most potent inducers of immune responses, including primary and secondary DCs. In particular, DCs are critical because they are the only APC that can activate T lymphocytes of the female type.

Molecules associated with innate immunity have recently been identified and may be considered as a new generation of carriers and adjuvants because they mature DCs and mediate cross-presentation of antigens associated therewith.

There is a series of work in this respect in the prior art. Giroir, (Crit named, Vol.5, pp 780-789, 1993), Cella et al (Nature, Vol.388, pp782-787, 1997) and Hailman et al (J Exp Med, Vol.79, pp 269-277, 1994) teach that the interaction between Lipopolysaccharide (LPS) and the innate immune recognition system is the most potent of all and stimulates the production of cytokines and pro-inflammatory mediators in monocytes, macrophages and neutrophils, thereby additionally increasing the expression of adhesion molecules. These inflammatory cytokines are important in response to infection and tumor, but their excessive secretion leads to septic shock, which can be fatal to the patient, and prevents the use of LPS as a vaccine adjuvant. The response is mediated by the complex formed between LPS and a binding protein called Lipopolysaccharide Binding Protein (LBP), which in turn interacts with the CD14 molecule. This molecule facilitates the interaction of LPS with a signaling molecule called the Toll receptor (TLR). Much evidence points to Toll receptor 4(TLR4) as a Toll family of molecules involved in LPS signaling.

Ulrich et al (published in Vaccine Design: The subbunit and adjuvantpropaac p 495, MF Powel and MJ Newman, Plenum Press, New York, 1995), Tholen et al (Vaccine, Vol.16, p 708, 1998), De Becker et al (Int Immunol, Vol.12, pp 807-815, 2000) teach The presence of a non-toxic LPS derivative, i.e., monophosphoryl lipid A (MPLA), which has adjuvant activity in The cellular and humoral pathways of immune response and has been administered to humans in several clinical trials. Although it was indicated that MPLA retained the immunostimulatory properties of LPS, the above authors have demonstrated that MPLA induced DC migration and functional maturation in vivo, but at lower levels than observed for LPS.

Tamura et al (Science, Vol.278, pp 117-120, 1997) and Binder et al (Nature Immunol, Vol.1, pp 151-155, 2000) reported that Heat Shock Proteins (HSP) are potent vectors that stimulate cellular immunity through the phenomenon of cross-presentation of antigens to their chaperones. HSPs obtained from tumors show interesting antitumor effects in different models. The identification of CD91 as a receptor for HSP gp96 likely reflects the presence of specific capture pathways of HSPs by DCs, which have evolved to effectively recruit peptides associated with antigens, infectious agents, or damaged cells and present them to the major histocompatibility complex type I (MHC I). However, the use of HSPs as vaccine vectors has the inconvenience of being obtained from the original source, e.g. from a tumor. This makes the procedure complicated and expensive, and it is never known exactly what antigen produced the effect.

Hartmann et al (Proc Natl Acad Sci USA, Vol.96, pp 9305-9310, 1999), Hemmi et al (Nature, Vol.6813, pp 740-5, 2000), Sparwasser et al (Eur.J.Immunol, Vol.12, pp 3591-3597, 2000), Hochreiter et al (int. Arch.Allergy Immunol, Vol.124, 406-410, 2001) and Deng et al (Arthritis Res, Vol.3, pp 48-53, 2001) showed that in molecules associated with innate immunity, DC maturation inducers were identified, and CpG sequences of bacterial DNA were found. Cellular responses to CpG sequences have recently been demonstrated to be mediated by TLR9, suggesting that the receptor is able to distinguish bacterial DNA from its own. Induction of Cytotoxic T Lymphocytes (CTL) against different soluble antigens has been reproduced in mice genetically modified (negative for CD40, CD4 or MHC II markers). It follows that CpG-mediated CTL-activation occurs in the absence of help from CD4T cells, thereby conferring adjuvant properties to these classes of molecules. However, the ability of CpG sequences to shift the response pattern from Th2 to Th1 in vivo is entirely dependent on antigenic properties and immunological conditions, which is particularly effective when they are proteins. This would constitute an obstacle to the efficient use of CpG oligonucleotides as adjuvants, mainly in immunocompromised hosts. Bacterial CpG sequences have also been described as likely to induce arthritis.

Jeannin et al (Nature Immunol., Vol.6, pp 502-509.2000) and Miconnet et al (J.Immunol., Vol.166, pp 4612-4619.2001) found important immunostimulatory properties in particular in the OmpA protein of the gram-negative bacterium Klebsiella pneumoniae (Klebsiella pneumoniae). Experiments with this protein expressed by recombinant techniques showed that the protein binds and induces complete DC maturation using the TLR2 molecule as a signaling molecule. Another important property exhibited by the protein is its ability to direct antigen through the class I presentation pathway, provided that the antigen is hydrophobically or covalently coupled. In fact, this is a major limitation as a vaccine carrier, since covalent binding techniques have the inconvenience of chemically modifying the protein itself and the antigen, whereas hydrophobic binding can only be used for hydrophobic antigen subsets.

Lowell in U.S. patent 5,726,292 describes an immune enhancing system that increases the immunogenicity of peptides, polypeptides and proteins, which can be considered as the closest prototype to the present invention. In the above-mentioned patents, the composition is characterized in that the antigen is chemically modified by means of the addition of at least one cysteine residue and then bound to an aliphatic fatty acid or a hydrophobic peptide. The modified antigen is then complexed with the proteosome by dialysis or lyophilization processes. In particular, these compositions do not include glycosides.

Disclosure of Invention

The novelty of the present invention resides in providing a formulation that confers immunogenicity to peptides, polypeptides, proteins and their corresponding DNA sequences and target cells of vaccine interest without the need for structural alteration of the antigen by conjugation of the antigen to small proteoliposomes of neisseria meningitidis (VSSP) carrying innate immune potent ligands and gangliosides.

The invention shows that the immune enhancement vector is composed of very small proteoliposomes (VSSP) obtained by combining Outer Membrane Protein Complex (OMPC) of gram-negative bacterium Neisseria meningitidis with ganglioside.

The formulations to which the present invention relates are particularly effective in selecting low immunogenic antigens and administering them to immunocompromised hosts.

It is an object of the present invention to provide immunogenic compositions comprising peptides, polypeptides, proteins, their corresponding DNA sequences, target cells or their lysates as antigens, and very small proteoliposomes (VSSP) formed by hydrophobic linking of the Outer Membrane Protein Complex (OMPC) of neisseria meningitidis (gram-negative bacteria) with gangliosides. Furthermore, the present invention teaches that these compositions can be formulated alone or as an emulsion with Incomplete Freund's Adjuvant (IFA) and can also be lyophilized.

It is another object of the present invention to provide immunostimulatory compositions capable of generating an antigen-specific immune response, even in immunocompromised hosts, such as cancer or patients with chronic viral infections. For such patients, administration of the vaccine compositions described herein can reconstitute immune system function.

Furthermore, the vaccine compositions described herein constitute a solution to the problem of immunogenicity of growth factor receptors and their impact in tumor therapy, since said receptors exhibit tyrosine kinase activity, whereas gangliosides specifically bound thereto in membrane molecular clusters are simultaneously presented to the host immune system in the context of the red flag signal caused by VSSP and are required for efficient activation of Dendritic Cells (DC) and for generating cross-presentation. These vaccine compositions avoid the use of protein-coupled chemistry techniques that generate new pseudodominant immune epitopes, and in addition they present their components to the immune system, mimicking their naturally occurring molecular associations in tumor cells.

On the other hand, this solution allows the use of receptor global structures, thus solving the problem of immunodominant genetic constraints, as opposed to other solutions which use derivatized peptides and may present more constraints in this respect.

More specifically, the present invention provides vaccine compositions for the treatment of cancer. The vaccine compositions comprise as active ingredients one or more growth factor receptors, or an extracellular domain thereof, which may or may not comprise a transmembrane domain, and very small proteoliposomes (VSSPs) as vaccine carriers from the neisseria meningitidis outer membrane protein complex, and gangliosides specifically associated therewith, thereby forming membrane molecule clusters. The vaccine composition may further comprise a suitable adjuvant.

The vaccine compositions of the invention are useful in active immunotherapy, in particular in the treatment of tumors such as prostate, colon, lung, breast, ovarian, head and neck, vulva and bladder cancers, gliomas and in the treatment of non-infectious chronic diseases.

Disclosure of Invention

The present invention relates to pharmaceutical compositions capable of enhancing the immunogenicity of low immunogenic antigens comprising:

(A) one or more low immunogenic antigens;

(B) a vaccine carrier which is a proteoliposome derived from the outer membrane protein complex of a gram-negative bacterial strain and which carries gangliosides therein; and

(C) one or more adjuvants.

The compositions of the invention enhance the immunogenicity of low immunogenic antigens, which may be peptides, polypeptides, proteins and their corresponding nucleic acid sequences, as well as target cells of vaccine interest or lysates thereof and combinations thereof.

Among the low immunogenic antigens, a growth factor receptor or an extracellular domain thereof may be used. The extracellular domain of the growth factor receptor may or may not contain its transmembrane region.

Growth factor receptors that may be used to increase immunogenicity are HER-1, HER-2, PDGF-R or any variant thereof containing the extracellular domain, lacking or having a transmembrane region thereof.

Proteoliposomes useful as vaccine vectors in the present invention are obtained from the Outer Membrane Protein Complex (OMPC) of gram-negative bacterial strains, preferably neisseria meningitidis, which may be wild-type or genetically modified.

In the composition of the invention, the proteoliposomes carrying gangliosides therein are obtained by hydrophobic incorporation of said gangliosides in the Outer Membrane Protein Complex (OMPC) of neisseria meningitidis. GM1 and GM3 gangliosides or N-glycosylated (N-glycosylated) variants thereof may be used for this purpose.

The compositions of the invention additionally contain an adjuvant, which may be an oily adjuvant or a natural or recombinant polypeptide.

Preferred oily adjuvants are incomplete freund's adjuvant or Montanide ISA 51. Likewise, when a polypeptide adjuvant is used, it may be a cytokine, such as granulocyte-macrophage colony stimulating factor, or rennin.

The compositions of the invention are useful in the prevention and treatment of cancer, particularly prostate, colon, lung, breast, ovarian, head and neck, vulvar and bladder cancer and glioma, as well as non-infectious chronic diseases.

Likewise, they are useful in the prevention and treatment of viral and bacterial infectious diseases, in addition to the treatment of acquired immunodeficiency syndrome, and in the treatment of autoimmune diseases.

The present invention provides formulations that confer immunogenicity to low immunogenic peptides, recombinant or natural proteins, lysates, whole cells and nucleic acids. An immunostimulatory agent may be defined as an agent that stimulates both humoral and cellular responses to a particular antigen. In addition, these preparations have the characteristic of reconstituting immunity in immunocompromised individuals, such as individuals with cancer and chronic viral infections or certain types of autoimmune disease.

The invention describes an immunopotentiating vehicle consisting of very small proteoliposomes (VSSP) obtained by combining the Outer Membrane Protein Complex (OMPC) of the gram-negative bacterial strain neisseria meningitidis with gangliosides incorporated therein. The OMPC fraction is subjected to dialysis for 2 to 15 days, whereby glycosylated and/or acetylated gangliosides are incorporated. The result of incorporating gangliosides into the outer membrane complex is a non-vesicular preparation; the resulting non-vesicular formulations are very small in molecular size, invisible under electron microscopy, soluble and exhibit high floatability.

VSSPs of the invention exhibit surprising immunological properties, such as a surprising ability to cause dendritic cell maturation and immune reconstitution in immunosuppressed patients. VSSPs were obtained from Cuba patents 131/93 and 130/97, U.S. patents 5,788,985 and 6,149,921, and Estevez et al (Vaccine, Vol.18, pp 190-.

The antigenic peptides used in the present invention may be synthetic or extracted from several sources. The preferred size of the peptide may be between 7-25 amino acids, depending on the type of T cell to be stimulated. However, the length may vary between 3-50 amino acids. The peptide may be neutral or may be positively or negatively charged. The hydrophobicity of the peptides may also vary.

Also, the present invention discloses that the recombinant proteins used herein can be expressed in different expression systems, such as bacterial, yeast, plant and mammalian cells. A preferred embodiment of the invention claims the use of Neisseria meningitidis as an expression system, wherein the protein of interest is expressed on the outer membrane of the bacterium itself. This allows the protein of interest to be directly a component of the OMPC. In this case, expression of the entire protein is equally effective, or a portion of its polypeptide or peptide is inserted at one or more junctions of outer membrane proteins of N.meningitidis, such as TBP, Opa, Opc, and P1, P2, and P3 pore proteins.

Particular embodiments of the present invention show that the antigen from these vaccine compositions may be a growth factor receptor with tyrosine kinase activity, which is overexpressed in tumor tissue; or their extracellular domains, with or without a transmembrane region; and they have a specific relationship with gangliosides expressed in the tumor cell membrane. This is particularly the case for the HER-1, HER-2 and PDGF receptors.

The growth factor receptors to which the present invention relates are obtained by recombinant techniques and Polymerase Chain Reaction (PCR) in mammalian cell expression plasmids following typical procedures described in Molecular biology publications (Sambrook J, Fritsch E.F., Maniatis T, Molecular Cloning A Laboratory Manual, second edition, Cold spring harbor Laboratory Press, 1989). Plasmids containing these genes and encoding the receptor or variants thereof are stably transfected into mammalian cells, such as HEK 293(ATCC CRL1573), NIH-3T3(ATCC CRL 1658) and CHO. The receptor or variant thereof is expressed on the membrane of the transfected cell line or secreted into the supernatant, in any case.

These antigens are extracted from the cell membranes of mammals expressing them or from cell culture supernatants and purified by chromatography. Then, they were filtered under sterile conditions and lyophilized. They were stored at 4 ℃. The optimal amount of these antigens in the vaccine formulation is 1-1000. mu.g per dose.

For this particular type of antigen, VSSP used in the vaccine formulation comprises gangliosides selected from those that specifically bind to growth factor receptors to form membrane molecule clusters, such as GM3 and GM 1. The amount of VSSP in the vaccine composition is 1-1000 μ g based on the amount of gangliosides in each dose of vaccine.

Preferred vaccine compositions of the invention containing as antigen a growth factor receptor to be enhanced in immunogenicity may be prepared by different routes: (a) a given amount of VSSP solution was added to the lyophilized growth factor receptor or its extracellular domain (with or without transmembrane region) (1-100mg protein) to give a receptor/ganglioside mass ratio in the range of 0.1/1 to 1/1. The mixture was stirred at 4-20 ℃ for 5 minutes-24 hours. The formulation was stored at 4 ℃ until administration to the host.

Just prior to administration to a host, the above formulation was added to IFA at a volume/volume ratio of between 40/60 and 60/40 and stirred for 10-30 minutes at room temperature. This volume ratio covers a sufficient suitable range for the type of emulsion required according to the route of inoculation to be employed.

(b) Another equally convenient way of doing this is to store the lyophilized growth factor receptor or its extracellular domain (with or without a transmembrane region) and the VSSP solution in separate containers at 4 ℃. Adding a given amount of VSSP solution to the growth factor receptor immediately prior to administration to the host; the vaccine composition is then prepared in the same manner as described in a).

C) A third approach to manipulation is to combine more than one growth factor receptor or its extracellular domain (with or without a transmembrane region) with the corresponding VSSP solution in a vaccine composition. The amount of each antigen in the vaccine composition, and the ratio between them, will be in the range of 1-1000 μ g per dose of vaccine. Likewise, the amount of each ganglioside in VSSP in the vaccine composition will be in the range of 1-1000 μ g per dose of vaccine.

To prepare the combination vaccine, the growth factor receptor or its extracellular domain (with or without a transmembrane region) that is a component thereof is lyophilized in the amounts described by the above respective items. Subsequently, a given amount of VSSP solution was added so that the receptor/ganglioside mass ratio was in the range of 0.1/1 to 1/1. The mixture is then stirred at 4-20 ℃ for 5 minutes-24 hours. The formulation was stored at 4 ℃ until administration to the host.

Immediately prior to administration to a host, the above formulation was mixed with IFA by shaking at room temperature for 10-30 minutes at a volume/volume ratio of between 40/60 and 60/40. These volume ratios cover the appropriate range of emulsions of the type required depending on the route of inoculation to be employed.

D) Another way of preparing the combination vaccine described in c) is by the method described in b).

On the other hand, a multiple antigen system as cells taken from established tumor cell lines or directly from cancer patients may also be used in the formulation of the invention. Cell inactivation is accomplished by gamma irradiation therapy or treatment with mitomycin C. Another equally convenient alternative is to use a tumour cell lysate obtained by mechanical lysis or by viral infection of tumour cells.

The immunopotentiating agent of the present invention can be advantageously used in DNA vaccines and RNA vaccines. The immunogenicity of retroviral and adenoviral vectors used as vaccine vectors is also increased when combined with the formulations of the invention. These vectors contain a gene encoding the subject antigenic protein. Typically, when a different antigen system is combined with the VSSP previously obtained, a different immunogenic formulation is obtained. Antigens introduced directly into the outer membrane of neisseria meningitidis by recombinant techniques and those incorporated into proteoliposomes during dialysis have been incorporated at the end of VSSP preparation. Nevertheless, the modified proteoliposomes can also be used for other unincorporated antigens. This can lead to multivalent vaccines.

The protein antigen preparation is obtained by: 10-1000. mu.g of the antigenic peptide or protein is mixed with a given amount of VSSP such that the total mass ratio of protein/ganglioside is in the range of 1-3. The formulation was stored at 4 ℃ until administration to the host. Another equally convenient route of operation is to store the antigen solution and VSSP solution separately at 4 ℃ and mix them immediately prior to administration.

The nucleic acid-containing preparation is obtained by: VSSP is mixed directly with a DNA or RNA solution. The mixing process was carried out at 4 ℃ using 2-100. mu.g of nucleic acid per 0.1mg of ganglioside in VSSP. This approach is feasible due to the lack of nucleases in VSSP formulations.

In a particularly advantageous method shown by the present invention, a live viral vector (vaccinia, avipox or other virus) IV (intravenous) comprising the DNA sequence of the protein of interest is administered to the host in an amount of from 10⁶-5×10⁷pfu. VSSP is administered by intramuscular, subcutaneous, intradermal, oral, or intranasal routes 12 hours before and 12 hours after administration of the viral vector.

The preparation containing the target cell of interest or a lysate thereof is obtained by: the respective cultures were first pelleted by centrifugation and the cell pellets were then resuspended in a given amount of VSSP to give 10 per 0.1mg of ganglioside³-5×10⁶And (4) cells. The amounts were mixed directly by shaking at 4-20 ℃ for 5-24 hours. The formulation was stored at 4 ℃ until administration to the host.

Another equally convenient way of handling is to store the cell suspension or its corresponding lysate and VSSP solution separately at 4 ℃ and mix them together immediately prior to administration.

The formulations described in the present invention, wherein the antigen is mixed with or incorporated into VSSP, may be administered alone or as an emulsion with Incomplete Freund's Adjuvant (IFA). The emulsion is prepared immediately prior to administration to the host. Each formulation was mixed with adjuvant at room temperature with shaking at a volume ratio of 40/60 to 60/40 for 10-30 minutes. The volume ratio range covers a sufficient suitable range for the type of emulsion required according to the route of inoculation to be employed.

In another preferred embodiment of the invention, the formulation in which the antigen is mixed with or incorporated into VSSP is lyophilized prior to administration either alone or as an emulsion with Incomplete Freund's Adjuvant (IFA).

The vaccine composition of the present invention may be administered to a patient by parenteral route (intramuscular, intradermal, subcutaneous) or by direct application to the mucosa.

Examples

Example 1. obtaining antigens for vaccine compositions: extracellular domain (ECD) of mouse EGF-R (ECD-mEGF-R)

The gene encoding ECD-mEGF-R was amplified from complementary dna (cdna) from mouse liver using PCR technology. PCR was performed as follows: mu.g of cDNA was mixed with 10pmole of each specific primer. Thereafter, 0.2mM of each dNTP and 1U of Taq polymerase were added. A total of 30 PCR cycles were performed: 9 ℃,1 minute (except for the first cycle, which lasts 3 minutes); 56 ℃ for 1 minute; 72 ℃,1 minute, and 30 seconds (which last 5 minutes except for the last cycle). The amplified gene was cloned into a mammalian cell expression vector pcDNA3 (Ampr, f' ori, ColE ori, CMV promoter, SV40 ori, SV40pa, neomycin, Invitrogen), after which the HEK-293 cell line was stably transfected with this plasmid. Transfection is carried out by conventional methods and cells are grown in selective media. The ECD-mEGF-R was obtained from the supernatant of a HEK-293/ECD-mEGF-R cell line stably expressing the ECD-mEGF-R.

The ECD-mEGF-R obtained in the culture supernatant was purified by Affinity Chromatography techniques by coupling the ligand to a matrix (Affinity Chromatography Principles and methods 3: 12, Pharmacia fine Chemicals); it is then filter sterilized and lyophilized.

Example 2a vaccine composition comprising ECD-mEGF-R, VSSP-GM3 and Incomplete Freund's Adjuvant (IFA) was obtained, all components being combined immediately prior to administration.

Proteoliposomes from neisseria meningitidis Outer Membrane Protein Complex (OMPC) containing incorporated GM3 ganglioside were obtained as described in us patent No 6,149,921.

The OMPC complex from neisseria meningitidis used for this purpose was supplied by the "carlos j. finlay" study (c. campa et al, EP 301992). 10mg of the OMPC complex was dispersed in 0.5% sodium deoxycholate and 0.1% sodium lauryl sulfate solution, which also contained 10mg of NGcGM3, by gentle mixing overnight at 4 ℃. The soluble OMPC-NGcGMP 3 was separated from the detergent by dialysis for 14 days using a 3.5kDa membrane.

The dialysate was centrifuged at 100,000g for 1 hour and the immunogenic complex present in the supernatant was filter sterilized.

The extent of ganglioside incorporation into proteins was determined using Bio-Rad reagents for proteins and resorcinol for sialic acid. By this method, 1mg of NGcGM3 was incorporated per mg of OMPC.

The amount of vaccine vector previously prepared was 120 μ g per dose of vaccine, based on the amount of ganglioside incorporated into the proteoliposomes.

To prepare the immunogenic material, 1mg of ECD-mEGF-R was lyophilized and stored at 4 ℃ until immunization. Just prior to administration to mice, 2.4mg of VSSP-GM3 (based on the amount of ganglioside) was added to the antigen to a volume of 1ml and the two components were mixed for 15 minutes at room temperature. Then, 1ml of IFA was added thereto and mixed by shaking at room temperature for 20 minutes.

Example 3 a vaccine composition comprising ECD-mEGF-R, VSSP-GM3 and IFA was obtained by combining part of the components and preserving the mixture until administration.

Proteoliposomes from neisseria meningitidis Outer Membrane Protein Complex (OMPC) containing incorporated GM3 ganglioside were obtained as described in us patent No 6,149,921. The amount of vaccine carrier used was 120 μ g per dose of vaccine, based on the amount of ganglioside incorporated.

To prepare the immunogenic material, 1mg of ECD-mEGF-R was lyophilized and then 2.4mg of VSSP-GM3 (based on the amount of ganglioside incorporated) was added in a volume of 1 ml. The two components were mixed at room temperature for 15 minutes and the mixture was stored at 4 ℃ until immunization. Immediately before administration to mice, 1ml of IFA was added and mixed by shaking at room temperature for 20 minutes.

Example 4 obtaining a combination vaccine comprising ECD-HER-1, ECD-HER-2, VSSP-GM3 and IFA

Proteoliposomes from neisseria meningitidis Outer Membrane Protein Complex (OMPC) containing incorporated GM3 ganglioside were obtained as described in us patent No 6,149,921. The amount of vaccine carrier used was 120 μ g per dose of vaccine, based on the amount of ganglioside incorporated into the proteoliposomes.

To prepare the immunogenic material, 1mg ECD-HER-1 and 1mg ECD-HER-2 were lyophilized together and stored at 4 ℃ until immunization. Just prior to administration to mice, 2.4mg VSSP-GM3 (based on the amount of gangliosides) was added in a 1ml volume. All components were mixed at room temperature for 15 minutes. Then, 1ml of IFA was added thereto and mixed by shaking at room temperature for 20 minutes.

Example 5 inducing a specific immune response against autologous EGF-R with the immunizing composition.

Mice of strain C57BL/6 were immunized with a vaccine composition comprising ECD-mEGF-R/VSSP-GM3 and IFA prepared as described in example 2. The dose of immunogenic agent is 50 μ g per mouse, based on the amount of antigen in the composition. The immunization scheme is as follows: every 15 days 3 doses were administered intramuscularly (i.m.) and blood was taken at 0, 21, 35 and 56 days after the primary immunization (group II). As a reference group, mice from the same strain were immunized with 50. mu.g of ECD-mEGF-R chemically bound to KLH, and Complete Freund's Adjuvant (CFA) and IFA, following the same immunization protocol (group I). The obtained sera were analyzed by ELISA technique to detect ECD-mEGF-R. The ELISA was performed as follows: after plating with 10. mu.g/ml ECD-mEGF-R and blocking the plate with PBS/5% calf serum, sera from control and immunized animals were incubated at different serial dilutions. Next, alkaline phosphatase-bound mouse anti-IgG antibody (specific for Fc) (Sigma) was added. All the above incubations were placed at 37 ℃ for 1 hour, and each of the steps was followed by 3 washes with PBS/0.05% Tween 20. The reaction was developed by adding 1mg/ml of substrate (p-nitrophenyl phosphate) in diethanolamine buffer (pH 9.8). After 30 minutes the absorbance at 405nm was measured in an ELISA plate reader.

100% of mice immunized with the vaccine composition of the invention induce an antibody response specific for ECD-mEGF-R; this response was stronger during the course of immunization and achieved antibody titers as high as 1/160000, while the pre-immune sera did not recognize ECD-mEGF-R. The isotype of the antibody response is essentially of the IgG type.

The subpopulation distribution of the induced antibody responses was determined by ELISA. 20.21% of the antibody was IgG2a, 36.03% was IgG1, and 38.93% was IgG2b, and the shift to the Th1 response pattern was estimated compared with the control group (FIG. 1).

Although this vaccine composition induced higher antibody titers for the formulation compared to the composition where ECD-mEGF-R was chemically bound to KLH and CFA was used as adjuvant, and the subpopulation trending to Th1 mode, was favorable for the vaccine efficacy.

Mice immunized with ECD-mEGF-R/VSSP-GM3/IFA showed no signs of clinical toxicity, and biochemical tests performed on sera taken from the animals showed no differences from non-immunized animals (Table 1).

TABLE 1

Group of	Reaction animal	IgG titre
										Day 21	1/100	1/500	1/1000	1/2500	1/5000	1/10000	1/20000
I	8/10	1	3	1		1	1	1
									II	10/10		1	1	2	5	1
	Day 35	1/100	1/1000	1/2500	1/5000	1/10000	1/20000	1/40000
									I	10/10		2		2	1		5
II	10/10		1			2	1	6
										Day 56	1/1000	1/5000	1/10000	1/20000	1/40000	1/80000	1/160000
I	10/10	1	1	1	2	1	4
									II	10/10		1		1	2	4	2

Group I animals immunized with ECD-mEGF-R/KLH/CFA and IFA

Group II animals immunized with ECD-mEGF-R/VSSP-GM3/Montanide-ISA 51

Example 6 recognition of human EGF-R expressing cells by sera from mice immunized with ECD-HER-1/VSSP-GM3/IFA

A431 cell line expressing the human epidermal growth factor receptor (10000 cells/well) was incubated for 30 minutes at room temperature with: c57BL/6 mouse preimmune serum (a) diluted to 1/5; the monoclonal antibody ior EGF-R3 directed against EGF-R at a concentration of 10. mu.g/ml as a positive control (B); and serum (C) from immunized C57BL/6 mice diluted to 1/5.

Excess antibody that did not bind to the receptor or bound to the receptor in a non-specific manner was removed by washing with phosphate buffer/0.5% calf serum solution. The immunodetection of the cells was carried out by: cells were incubated with anti-mouse secondary antibody diluted to 1/50 conjugated fluorescein isothiocyanate for 30 minutes at room temperature. Fluorescence intensity was measured in a Flow Cytometer (FC). Sera from mice immunized with the vaccine preparation recognized cells expressing EGF-R, at an intensity comparable to the experimental positive control, compared to preimmune sera from the same animals (FIG. 2).

Example 7 cytolytic Activity of sera from ECD-HER-1/VSSP-GM3/IFA immunized mice

A431 cell line (3X 10)⁶Cells) with radioactive sodium chromate⁵¹Cr was incubated for 1 hour and washed 3 times with medium to eliminate excess radioactive salts. To load a load⁵¹Cr cells were incubated with:

i)50 μ g/ml monoclonal antibody ior-t3 (MAb against CD3, as a negative control);

ii) 50. mu.g/ml monoclonal antibody ior EGF-R3 (MAb against EGF-R, as positive control);

iii) C57BL/6 mouse preimmune serum diluted to 1/20;

iv) sera from C57BL/6 mice immunized with ECD-HER-1/VSSP-GM3/IFA diluted to 1/20.

After 1 hour incubation at 37 ℃, 40 μ l rabbit complement was added and incubated at 37 ℃. Then, willThe tubes were centrifuged and 100. mu.l of the supernatant was measured in a gamma counter⁵¹Cr release, measured by antibody and complement mediated cell lysis. The total incorporation was measured by means of total dissolution with detergent.

Serum from mice immunized with the vaccine composition caused 80% lysis of A431 cells expressing EGF-R, on the other hand, pre-immune serum from the mice caused only 35% lysis (FIG. 3).

Example 8 neutralizing Capacity of sera from ECD-HER-1/VSSP-GM3/IFA immunized mice

Sera from mice immunized with the vaccine composition of the invention were assayed to determine their ability to inhibit EGF binding to its receptor on the a431 cell membrane. To achieve this effect, a431 cells were grown in culture plates to confluence. Once confluency was reached, a pool of immune sera at different dilutions (1/5, 1/10, 1/20, 1/40) was added followed by EGF-cells at 100000 cpm/well¹²⁵I. The volume of each well was filled to 500. mu.l with PBS/1% BSA. The plates were incubated at room temperature for 1 hour, after which the reaction was stopped by the addition of 2mL of cold PBS/1% BSA. The liquid in each well was then removed and the wells were gently washed with PBS/1% BSA; to each well was added 300. mu.l NaOH 2M. After 30 minutes at room temperature, 200. mu.l of each well was removed and the reading recorded in a gamma counter.

The immune serum pool showed inhibition of EGF-¹²⁵I binds to its receptor in the a431 cell membrane. The degree of inhibition was dependent on serum dilution (figure 4).

Example 9 longevity of ECD-HER-1/VSSP-GM3/IFA immunized mice

Mice of the C57/BL6 strain immunized with ECD-mEGF-R/VSSP-GM3/IFA (3 50 μ g dose every 15 days, i.m.) were i.m. inoculated with 100000 Lewis cells and the mice were observed to determine their lifespan. Lewis cells were derived from murine lung adenocarcinoma expressing EGF-R. The longevity of these mice was compared to groups immunized with ECD-mEGF-R/CFA (3 doses of 50. mu.g every 15 days, subcutaneously). Mice immunized with the ECD-mEGF-R/VSSP-GM3/IFA vaccine composition showed a significantly higher lifespan than the control (p < 0.05) (FIG. 5).

Example 10 obtaining a vaccine composition containing the P3 chimeric monoclonal antibody (P3cMAb), VSSP (GM3) and IFA

Proteoliposomes from neisseria meningitidis outer membrane protein complexes containing GM3[ VSSP (GM3) ] ganglioside were obtained as described in cuba patent 130/97 and us patent No.6,149,921. VSSP (GM3) was stored at a concentration of 4.8mg/ml in Tris/HCl solution pH 8.9 at 4 ℃ until use. To prepare the immunogenic material, a solution containing 2mg/ml of the chimeric monoclonal antibody P3 (P3cMAb) (U.S. Pat. No.5,817,513) was mixed with the prepared VSSP (GM3) in phosphate buffer at a ratio of 1/1 (v/v). The mixing process included magnetic stirring at room temperature for 15 minutes. Thereafter, IFA was added at a ratio of 1/1 (v/v). The mixture was shaken at room temperature for 15 minutes until an emulsion was obtained.

Another equally convenient procedure was to mix a solution containing 2mg/ml P3cMAb in phosphate buffer with the prepared VSSP (GM3) at a ratio of 1/1 (v/v). The mixing was done by magnetic stirring for 15 minutes at room temperature and the resulting solution was filter sterilized through 0.2 μm cellulose acetate membrane. After metering, the mixture is filled into a container and sealed, and the preparation is stored at 4 ℃ for 1 year. Just prior to administration to a host, the IFA formulation was added at a ratio of 1/1(v/v) and emulsified by shaking at room temperature for 15 minutes.

Example 11 obtaining a vaccine composition containing peptides from the P3cMAB heavy chain variable region (CDR3/VH-P3) and VSSP (GM3)

Proteoliposomes from neisseria meningitidis outer membrane protein complexes containing GM3[ VSSP (GM3) ] ganglioside were obtained as described in cuba patent 130/97 and us patent No.6,149,921. VSSP (GM3) was stored at a concentration of 4.8mg/ml in Tris/HCl solution pH 8.9 at 4 ℃ until use.

Immediately prior to administration to a host, an immunogenic material is prepared: the lyophilized CDR3/VH-P3 peptide was first dissolved in phosphate buffer to a concentration of 4 mg/ml. This solution was then mixed with VSSP (GM3) formulation at a ratio of 1/1 (v/v). The mixing was done by magnetic stirring at room temperature for 15 minutes.

Another equally convenient procedure is to first dissolve the lyophilized CDR3/VH-P3 peptide in phosphate buffer to a concentration of 4 mg/ml. This solution was then mixed with VSSP (GM3) formulation at a ratio of 1/1 (v/v). The mixing was done by magnetic stirring for 15 minutes at room temperature and the resulting solution was filter sterilized through 0.2 μm cellulose acetate membrane. After metering, the mixture is filled into a container and sealed, and the preparation is stored at 4 ℃ for 1 year.

Example 12 obtaining a vaccine composition containing B16 melanoma tumor cell lysate, VSSP (GM3) and IFA

Proteoliposomes from neisseria meningitidis outer membrane protein complexes containing GM3[ VSSP (GM3) ] ganglioside were obtained as described in cuba patent 130/97 and us patent No.6,149,921. VSSP (GM3) was stored at a concentration of 2.4mg/ml in Tris/HCl solution pH 8.9 at 4 ℃ until use.

To prepare the immunogenic material, a suspension (50X 10) of the murine melanoma B16 cell line was prepared⁶Cells/ml) were subjected to 5 freeze/thaw cycles, alternately incubated in a liquid nitrogen bath and a 37 ℃ distilled water bath. The resulting lysate was centrifuged at 500 Xg for 10 min. The resulting pellet was resuspended in VSSP (GM3) at a ratio corresponding to 10X 10⁶Cell pellet of cells/2.4 mg of GM3 in VSSP. The mixture was shaken at room temperature for 10 minutes. The formulation was then added to IFA at a ratio of 1/1 (v/v). The mixture was shaken at room temperature for about 15 minutes until an emulsion was obtained.

Example 13 obtaining a vaccine composition containing melanoma B16 cells, VSSP (GM3) and IFA

Proteoliposomes from neisseria meningitidis outer membrane protein complexes containing GM3[ VSSP (GM3) ] ganglioside were obtained as described in cuba patent 130/97 and us patent No.6,149,921. VSSP (GM3) was stored at a concentration of 2.4mg/ml in Tris/HCl solution pH 8.9 at 4 ℃ until use.

To prepare the immunogenic material, a suspension (50X 10) of the murine melanoma B16 cell line was prepared⁶Cells/ml) was centrifuged at 300 Xg for 10 min. The resulting pellet was resuspended in VSSP (GM3) to a concentration of 10X 10⁶Cells/2.4 mg GM3 in VSSP. The mixture was shaken at room temperature for 10 minutes. The formulation was then added to IFA at a ratio of 1/1 (v/v). The mixture was shaken at room temperature for about 15 minutes until an emulsion was obtained.

Example 14 obtaining a vaccine composition comprising a plasmid carrying a gene encoding the extracellular domain of the human EGF receptor (ECD-HER-1), VSSP (GM3) and IFA

Proteoliposomes from neisseria meningitidis outer membrane protein complexes containing GM3[ VSSP (GM3) ] ganglioside were obtained as described in cuba patent 130/97 and us patent No.6,149,921. VSSP (GM3) was stored at a concentration of 4.8mg/ml in Tris/HCl solution pH 8.9 at 4 ℃ until use.

The vector for insertion of the DNA of interest is the pcDNA3 mammalian expression plasmid, which contains the SV40 origin of replication, and the human Immediate Early Cytomegalovirus Promoter (IECP). A gene encoding the extracellular domain of human EGF receptor (ECD-HER-1) was inserted into the plasmid. The resulting plasmid (ECD-HER-1/pcDNA3) was used in an immunogenic formulation.

For the preparation of the immunogenic material, the ECD-HER-1/pcDNA3 plasmid solution was adjusted to a concentration of 2mg/ml in phosphate buffer. Then, it was mixed with VSSP (GM3) preparation at a ratio of 1/1 (v/v). Mix for 5 minutes at room temperature with shaking. Subsequently, the formulation was added to IFA at a ratio of 1/1 (v/v). The mixture was shaken at room temperature for about 15 minutes until an emulsion was obtained.

Example 15 Induction of dendritic cell maturation in vitro by VSSP (GM3) formulations

Human dendritic cells are obtained from monocytes isolated from peripheral blood and cultured in recombinant human GM-CSF (hr) (5)0ng/ml) and hr-IL4(1000U/ml) for 7 days. On day 7, the obtained dendritic cells were allowed to react with or withoutWas contacted with VSSP (GM3) (1. mu.g/ml) for 18 hours. As a control, dendritic cells were incubated with 0.1. mu.g/ml LPS purified from Neisseria meningitidis strain 44/76 or with MPLA (Sigma). The phenotype of each preparation was determined by flow cytometry.

As shown in table 2, treatment with VSSP (GM3) resulted in increased CD11c expression, as well as appreciable changes in the DC CD83 maturation marker. An increase in the level of expression of HLA-DR molecules is observed. VSSP (GM3) induces an increase in the number of cells expressing CD86 molecules. VSSP (GM3) and LPS showed the same ability to induce maturation of treated DCs. On the other hand, the detoxified variant of LPS, MPLA, was ranked lower.

TABLE 2 different formulations induce dendritic cell maturation in vitro

	CD11c	CD86	CD83	HLA-DR	CD40
						Culture medium	37,5	12,5	5,5	401,5	9
LPS	57,1	35,4	14,6	672,7	15,8
						VSSP	60	29,6	13,3	656,4	14,1
MPLA	40,1	15,4	5,6	415,5	9,7

Mean fluorescence intensity values, measured by FACS

Example 16 Induction of specific humoral immune response to P3 chimeric MAb (P3cMAb) in connection with administration of vaccine compositions

Mice of strain C57BL/6 were immunized with the vaccine composition described in example 10. 50 μ g of the chimeric monoclonal antibody was inoculated by i.m. injection, and 2 doses (one dose every 14 days) were administered. Serum samples were taken 21 days after the first immunization. As a control group, mice of the same strain were similarly immunized with P3cMab adjuvanted with IFA or alum. The obtained sera were analyzed by ELISA technique to determine the presence of anti-P3 cMAb antibodies.

100% of the mice immunized with the vaccine composition of the invention produced higher titers of specific IgG antibodies against P3cMAb compared to the control group (Table 3).

TABLE 3 antibody response induced against P3C MAb in C57BL/6 mice immunized with different formulations

Preparation	Reaction animal	Specific IgG titre (mean)
			P3c MAb/VSSP/IFA	5/5	8000
P3c MAb/IFA	4/5	4000
			P3c MAb/Alum	2/5	1000

Example 17 Induction of a proliferative cellular response specific for the CDR3/VH-P3 peptide, in connection with administration of a vaccine composition

Mice of strain C57BL/6 were immunized with the vaccine composition described in example 11. 100 μ g of peptide was inoculated by i.m. injection and 4 doses (one dose every 14 days) were administered. As a control group, mice of the same strain were similarly immunized with CDR3/VH-P3 peptide adjuvanted with IFA or alum. Inguinal lymph nodes were removed from the animals 7 days after the last dose administration and lymphocytes were isolated by means of organ perfusion. Lymphocytes were grown with CDR3/VH-P3 peptide (50. mu.g/ml) for 96 hours. During the last 18 hours of culture, cells were exposed to 1 μ Ci of tritiated thymidine (Amersham, United Kingdom), then harvested and tested for beta-emission (cpm) in a scintillation counter (LKB Wallac, Finland). The level of cell proliferation was determined as the Stimulation Index (SI). The results of this measurement are shown in table 4.

TABLE 4 Induction of a proliferative cellular response specific for the CDR3/VH-P3 peptide in C57BL/6 mice immunized with different formulations

	CDR3/VH-P3/VSSP(GM3)	CDR3/VH-P3/IFA	CDR 3/VH-P3/alum
				50μg/ml	4±0,2	1,8±0,1	1,6±0,2

A stimulation index value. It can be seen that only the peptides contained in the formulation with VSSP (GM3) induced specific antigen proliferation.

Example 18 Induction of a cytotoxic cellular response specific for ECD-mEGF-R in association with administration of a vaccine composition comprising recombinant APV, ECD-mEGF-R/APV, VSSP (GM3) and IFA

Proteoliposomes from neisseria meningitidis outer membrane protein complexes containing GM3[ VSSP (GM3) ] ganglioside were obtained as described in cuba patent 130/97 and us patent No.6,149,921. VSSP (GM3) was stored at a concentration of 2.4mg/ml in Tris/HCl solution pH 8.9 at 4 ℃ until use.

The viral vector for insertion of the DNA of interest is fowlpox virus (APV). The gene encoding the extracellular domain of the murine EGF receptor (ECD-mEGF-R) was inserted into APV by means of homologous recombination. Adjusting the solution of the ECD-mEGF-R/APV recombinant vector to 10⁸pfu/ml concentration.

VSSP (GM3) emulsion was prepared simultaneously and the carrier solution was added to IFA at a ratio of 1/1 (v/v). The mixture was shaken at room temperature for about 15 minutes. Subsequently, Balb/c mice were IP immunized with 200. mu.l of ECD-mEGF-R/APV solution and then i.m. inoculated with 100. mu.l of VSSP (GM 3)/IFA. Control mice received ECD-mEGF-R/APV and phosphate buffered saline (STFS). Mice were sacrificed at 21 days after the start of the experiment to obtain the corresponding splenocytes, administered 2 doses each every 2 weeks. CD8T cells were isolated from splenocytes using magnetic bead technology. Bone marrow-derived dendritic cells (bmDCs) previously pulsed with ECD-mEGF-R immunodominant peptide ` NYGTNRTGL ` were stimulated at a ratio of 10: 1 (T: bmDCs) in the presence of IL-2(50u/ml) for 5 days. At the end of the stimulation, a cytotoxicity test was performed in which Cr was assessed when different amounts of the cells were exposed to a P815 cell line pulsed with the 'NYGTNRTGL' peptide⁵¹Released (table 5).

TABLE 5 cytotoxic cellular response specific for ECD-mEGF-R

	Immunization with ECD-mEGFR/APV + VSSP (GM3)		Immunization with ECD-mEGFR/APV + STFS
						P815+ peptides	P815	P815+ peptides	P815
Cr51Release (%)
					100∶1	78	8	43	9
50∶1	51	6	25	7
					25∶1	29	5	14	5

VSSP (GM3) administered to animals enhanced the ability to induce cytotoxic T cells 2-fold compared to APV.

Example 19 immune reconstitution Properties of VSSP vaccine vectors

In a phase I clinical trial, VSSP vaccine vectors described herein were administered to patients with metastatic melanoma by i.m. injection. The patient received 9 doses (200 μ g of NGcGM3 in VSSP) within 6 months. The first 5 doses were administered within the first 2 months and the remaining 4 doses were administered monthly.

Patients were bled on day 0 (before dose 1 administration) and bled again on day 56 (dose 5). Blood was also drawn from 8 healthy volunteers. Corresponding Peripheral Mononuclear Cells (PMC) were obtained from the samples by Ficoll gradient method and the% of CD3+, CD4+ and CD8+ cells was determined by flow cytometry. As shown in table 6, the relative expression of CD3, CD4, and CD8T cell markers in the PMC of 3 patients was lower than the mean expression of healthy donors on day 0.

It can be seen that at day 56, the relative expression levels of CD3, CD4, and CD8T cell markers in the PMC of the same patient were restored to conventional levels, i.e., after receiving 4 VSSP (nggm 3) injections.

TABLE 6 relative expression of T cell markers in PMCs of melanoma patients and healthy controls. Effect of VSSP (NGcGM3) on marker normalization.

	% of total PMC day 0			56 th day accounting for% of total PMC
								CD3+	CD4+	CD8+	CD3+	CD4+	CD8+
Healthy controls	70	45	25	-	-	-
							EM	30	25	5	70	48	22
SJ	26	40	4	70	40	18
							VD	15	28	4	70	56	20

Brief description of the drawings

FIG. 1 shows the subpopulation distribution of antibodies induced by immunization with ECD-mEGF-R/VSSP-GM 3/IFA.

Sera from C57BL/6 mice immunized with ECD-mEGF-R/KLH/CFA (I) or ECD-mEGF-R/VSSP-GM3/IFA (II) were assayed by ELISA to determine the subpopulation distribution of IgG produced by immunization.

FIG. 2 identification of EGF-R expressing cells by sera from mice immunized with ECD-HER-1/VSSP-GM 3/IFA.

A431 line cells were incubated with: mouse C57BL/6 preimmune serum (A); monoclonal antibody ior egf-r3 as positive control (B); and immunized mouse C57BL/6 serum. For immunodetection, an anti-mouse secondary antibody conjugated to a fluorophore was used. Fluorescence intensity was measured in a flow cytometer.

FIG. 3 cytolytic activity of sera from mice immunized with ECD-HER-1/VSSP-GM 3/IFA.

To load a load⁵¹Cr a431 cells were incubated with complement and: I) monoclonal antibody ior-t3 (against CD3 as negative control); II) monoclonal antibody iorEGF-R-R3 (for EGF-R, as positive control); III) preimmune sera from C57BL/6 mice; IV) sera from C57BL/6 mice immunized with ECD-HER-1/VSSP-GM 3/IFA; v) equal number of identical cells were lysed with detergent to determine total⁵¹The amount of Cr is added. Results are expressed as% specific lysis.

FIG. 4 neutralizing capacity of sera obtained from mice immunized with ECD-HER-1/VSSP-GM 3/IFA.

A431 cells were incubated with 1/5, 1/10, 1/20 and 1/40 diluted serum pools from mice immunized with ECD-HER-1/VSSP-GM3/IFA or with the same dilution of preimmune serum pools. Adding EGF to each well¹²⁵I (100000com) by contacting the cell with EGF¹²⁵I incubated together to determine total binding. CPM was measured in a gamma counter.

FIG. 5 lifespan of Lewis tumor implanted mice immunized with ECD-mEGF-R/VSSP-GM 3/IFA.

100000 Lewis tumor cells were implanted into C57BL/6 mice immunized as described in example 9 and observed to determine their lifespan. Mice of the same strain of the control group were immunized with ECD-mEGF-R/CFA and implanted with tumors in the same manner.

Claims (21)

1. A pharmaceutical composition for enhancing the immunogenicity of a low immunogenic antigen comprising:

(A) one or more low immunogenic antigens;

(B) a vaccine carrier is a proteoliposome derived from the outer membrane protein complex of a gram-negative bacterial strain Neisseria meningitidis, into which gangliosides have been incorporated; and

(C) optionally, one or more adjuvants.

2. The composition of claim 1, wherein the low immunogenic antigen is selected from the group consisting of peptides, polypeptides, proteins and their corresponding nucleic acid sequences, and target cells of vaccine interest or lysates thereof and combinations thereof.

3. The composition of claim 2, wherein the low immunogenic antigen is a growth factor receptor or an extracellular domain thereof.

4. The composition of claim 3, wherein the extracellular domain of a growth factor receptor comprises or does not comprise a transmembrane region.

5. The composition of claim 3 or 4, wherein the growth factor receptor is HER-1 or HER-2.

6. The composition of claim 1, wherein the vaccine carrier protein liposomes are obtained from outer membrane protein complexes of wild-type or genetically modified neisseria meningitidis strains.

7. The composition of claim 1, wherein said vaccine carrier protein liposome having incorporated therein a ganglioside is obtained by hydrophobically incorporating said ganglioside into the outer membrane protein complex of neisseria meningitidis.

8. The composition of claim 7, wherein the ganglioside that is hydrophobically incorporated into the outer membrane protein complex of Neisseria meningitidis is GM1, GM3, or an N-glycosylated variant thereof.

9. The composition of claim 1, wherein the adjuvant is an oily adjuvant, or a native or recombinant polypeptide.

10. The composition of claim 9, wherein said oily adjuvant is incomplete freund's adjuvant.

11. The composition of claim 10, wherein said incomplete freund's adjuvant is Montanide ISA 51.

12. The composition of claim 9, wherein the polypeptide adjuvant is a cytokine or rennet.

13. The composition of claim 12, wherein the cytokine is granulocyte-macrophage colony stimulating factor.

14. Use of a composition according to any one of claims 1 to 13 for the preparation of a medicament for the prevention and treatment of cancer as well as non-infectious chronic diseases.

15. The use of claim 14, wherein the cancer is selected from the group consisting of prostate, colon, lung, breast, ovary, head and neck, vulva, bladder and brain cancers and gliomas.

16. Use of a composition according to any one of claims 1 to 13 for the preparation of a medicament for the prevention and treatment of viral and bacterial infectious diseases.

17. Use of a composition according to any one of claims 1 to 13 for the preparation of a medicament for the treatment of acquired immunodeficiency syndrome.

18. Use of a composition according to any one of claims 1 to 13 for the manufacture of a medicament for the treatment of an autoimmune disease.

19. Use of very small proteoliposomes VSSP obtained by combining the outer membrane protein complex OMPC of the gram-negative bacterial strain neisseria meningitidis with gangliosides incorporated therein for the preparation of a medicament for therapy to reconstitute immunity in immunosuppressed patients.

20. The use of claim 19, wherein said miniproteoliposomes into which gangliosides are incorporated are obtained by hydrophobically incorporating said gangliosides into the outer membrane protein complex of neisseria meningitidis.

21. The use of claim 20, wherein the ganglioside that hydrophobically incorporates into the outer membrane protein complex of neisseria meningitidis is GM1, GM3, or an N-glycosylated variant thereof.