WO2007041285A2

WO2007041285A2 - Complexes of inactivated pepsin fraction and heat shock protein

Info

Publication number: WO2007041285A2
Application number: PCT/US2006/038045
Authority: WO
Inventors: Harry H. Zhabilov
Original assignee: Viral Genetics, Inc.
Priority date: 2005-09-29
Filing date: 2006-09-29
Publication date: 2007-04-12
Also published as: WO2007041285A3

Abstract

The present invention relates to a method of modulating immune system activity comprising administering to a patient an effective amount of a complex of an inactivated pepsin fraction (IPF) component and a heat shock protein (HSP) peptide component.

Description

COMPLEXES OF INACTIVATED PEPSIN FRACTION AND HEAT SHOCK

PROTEIN CROSS-REFERENCE TO RELATED APPLICATION This application is related to U.S. Provisional Patent Application No. 60/644,054, filed

January 18, 2005, Zhabilov, entitled "Inactivated Pepsin Fraction, Pharmaceutical Compositions, and Methods for Detecting and Treating Diseases," and U.S. Patent Application No. 10/336,512, filed June 3, 2003, Zhabilov et al., entitled "Compositions and Methods for Detecting and Treating Acquired Immunodeficiency Syndrome," both of which are commonly assigned with the present invention and the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to compositions containing a complex of an inactivated pepsin fraction (IPF) component and a heat shock protein (HSP) peptide component (IPF-HSP complex) and methods for producing specific immunity to tumor peptides.

BACKGROUND OF THE INVENTION

There are a variety of technologies that modulate the immune system to treat various conditions. For example, commonly owned U.S. Provisional Application No. 60/644,054, Zhabilov, discloses methods for isolating and preparing an inactivated pepsin fraction (IPF) useful for detecting and treating infectious and autoimmune diseases and provides methods for early disease detection and treatment of viral infections. Furthermore, commonly owned U.S. Patent Application No. 10/336,512, Zhabilov, discloses the use of thymus cell preparations referred to as a Thymus Factor (TF) to detect HIV-I infection, and to reduce viral load.

Current data indicates that immune protection against cancer requires the generation of potent cellular immune responses against a unique tumor antigen expressed by malignant cells. As a consequence successful immune protection requires (1) a unique antigen in the tumor cells (tumor specific antigen) and (2) induction of potent T cell immune response targeted to the tumor antigen. However, tumor-associated antigens are often recognized by immune cells as a self-molecule and so the immune system is not activated. This presents at least two obstacles for targeting these tumor-associated molecules as the basis for a vaccine. The first obstacle is the unresponsiveness of the immune system to self-molecules, which restricts its ability to generate potent cellular immune responses. The second obstacle is targeting potent cellular immune response specifically to tumor cells and not to normal cells that express the target antigen.

To overcome these obstacles, heat shock proteins (HSPs) have been shown to elicit specific protective immunity against tumor cells. Specific immunogenicity of tumor-derived HSP preparation has been demonstrated with fibrosarcomas, lung carcinoma, prostate cancer, spinal cell carcinoma and melanoma in mice and rats of different haplotypes. These tumors include chemically induced, UV-induced and spontaneous tumors. Efficacy has been demonstrated in prophylactic and therapeutic models. The structural basis of this broad phenomenon appears to lie in HSP-peptide complexes. For example, HSP has been known to load peptides onto major Major Histocompatibility Complex proteins (MHC). At the cell surface, the loaded MHC is monitored by cells of the immune system that react to foreign peptides, e.g., fragments of viral proteins of altered constituents of a cancerous cell. HSP alone appear to be without variation, but when coupled with peptides, e.g., attached in HSP binding sites, conveys antigenic intelligence to the immune system. HSP-peptide complex preparations are highly immunogenic and isolated from given cells are associated with a range of peptides including self and antigenic peptides, generated within the given cells.

High doses of heat shock protein, such as gp69, have been shown to modulate immune response by suppressing tumor immunity, inhibiting onset of encephalomyelitis and inhibiting murine autoimmune diabetes, e.g., see Chandawarkar, et al., "Immune modulation with high- dose heat shock protein gp96: therapy of murine autoimmune diabetes and encephalomyelitis," International Immunology, Vol. 16, No. 4, pp.615-624 (2004).

The heat shock protein, gp96, is localized in the endoplasmic reticulum (ER) and has been shown to have a variety of roles in mammalian organisms. It has also been observed to be released into the extra-cellular space during necrotic cell death. Extra-cellular gp96 has been known to activate dendrites and macrophages by modulating inflammatory cytokines and inducing maturation of dendrites.

The ability of gp96 to transfer antigenic peptides/MHC, to initiate T-cell mediated anti- tumor responses and uptake and processing of tumor antigens by dendrite cells, makes it an ideal candidate for triggering an immune response in an organism in response to an infection. Often associated with the activation of an immune response, the receptor CD91 mediates uptake of gp96 by dendrites. CD91 is also known as the α2 macroglobulin (α2 M) receptor expressed on phagocytes. The presentation of gp96 associated peptide by antigen-presenting cells (APCs), e.g., dendrite cells, is induced by α2 macroglobulin.

In dendrites, CD91 receptor binds and internalizes gp96, which then induces the expression of co-stimulatory molecules, and causes the release of interleukin 12 (IL- 12) and tumor necrotic factor α (TNFα) by the APC. T-cells specific for foreign or altered antigens are likely to be activated. Heat shock proteins are known to bind peptide. Also, heat shock proteins purified from given cells have been shown to chaperone a large number of peptides derived from the cells from which they were isolated. This phenomenon is known as the "antigenic repertoire" of that cell. Early studies have shown that fractioned tumor cell lysates have the capacity to reduce tumor cell growth in mice. Immunization of mice with heat shock proteins, such as HSP70, HSP90 and gp96, isolated from murine tumor cells have been shown to induce anti-tumor immunity and tumor-specific cytolytic T-cells. The immunity has been shown to result from tumor-derived peptides associated with the heat shock protein rather than from the heat shock proteins themselves. Also, it has been reported that calreticulin, HSPl 10 and grpl70 can also be used in heat shock protein-based cancer immunotherapy. Preliminary clinical trials have demonstrated the induction of cancer-specific CD8+ T-cells responses in patients immunized with gp96-peptide prepared from the patients' own tumor.

The immunological effects of heat shock protein isolated and purified from tumor cells have been shown to have various effects. The induction of immunity to methylcholanthrane- induced fibrosarcoma by the administration of gp96 isolated from the tumor has been shown to display consistent dose restriction: two intradermal administrations of <lμg gp96 is ineffective; two doses of lμg induce immunity and provide optimal protection against tumor growth; and two doses of lOμg do not protect. The lack of protection at high doses of tumor-derived gp96 as an active ingredient were theorized to be due to an antigen specific down-regulation of turn or- specific immunity that can be adoptively transferred by CD4 T-cells purified from animal treated with high doses of tumor derived gp96.

DESCRIPTION OF THE INVENTION

Embodiments of the invention are discussed in detail below. While specific exemplary embodiments are discussed, it should be understood that this is done for illustration purposes only. A person skilled in the relevant art. will recognize that other components and configurations can be used without parting from the spirit and scope of the invention.

In an exemplary embodiment the present invention relates to a method of modulating immune system activity comprising administering to a patient an effective amount of a complex of an inactivated pepsin fraction (IPF) component and a heat shock protein (HSP) peptide component.

As used herein, the present invention may be directed to modulating immune system activity, which includes treating, decreasing, increasing, attenuating or modulating any conditions that benefit from an enhancement of immune system activity. Immune conditions can include immune diseases or disorders. Immune disorders may include Allergies, Auto- Immune, DiGeorge Syndrome, Familial Mediterranean Fever, Immune Deficiency, and Multiple Chemical Sensitivity.

Immune system disease or disorder may include at least one of Agammaglobulinemia, Anaphylaxis, Antiphospholipid Syndrome, Ataxia Telangiectasia, Autoimmune Diseases, Common Variable Immunodeficiency, DiGeorge Syndrome, Electrosensitivity, Familial Mediterranean Fever, Graft vs Host Disease, Granulomatous Disease, Chronic, HIV Infections, Hypersensitivity, Hypersensitivity, Immediate, IgA Deficiency, Immune Complex Diseases, Immune System Diseases, Immunologic Deficiency Syndromes, Lambert-Eaton Myasthenic Syndrome, Lambert-Eaton Myasthenic Syndrome, Latex Hypersensitivity, Lymphoproliferative Disorders, Multiple Chemical Sensitivity, Purpura, Schoenlein-Henoch, Samter's Syndrome, Severe Combined Immunodeficiency, Sick Building Syndrome, Sjogren's Syndrome, and Wiskott-Aldrich Syndrome.

In one aspect of the invention, auto-immune disorder may comprise Addison's, Ankylosing Spondylitis, Antiphospholipid Syndrome, Barth Syndrome, Graves' Disease, Hemolytic Anemia, IgA Nephropathy, Lupus Erythematosus, Systemic, Microscopic Polyangiitis, Multiple Sclerosis, Myasthenia Gravis, Myositis, Osteoporosis, Pemphigus, Psoriasis, Rheumatoid Arthritis, Sarcoidosis, Scleroderma and Sjogren's Syndrome. Examples of allergies may include Asthma, Food, Hay Fever - Rhinitis, Hives, Latex and Sinusitis. In yet another embodiment, the patient may have AIDS or AIDS Related Complex, multiple sclerosis, hepatitis, herpes, rheumatoid arthritis, autoimmune diabetes, encephalomyelitis or another autoimmune disease.

In another exemplary embodiment, the present invention may encompass a cancer preventive or therapeutic vaccine. The inactivated pepsin fraction (IPF) as used herein is described in commonly owned

U.S. Provisional Application N. 60/644,054, Zhabilov, which discloses methods for isolating, preparing and using the inactivated pepsin fraction (IPF) for detecting and treating infectious and autoimmune diseases.

As used herein, heat-shock proteins may be referred to as HSPs or stress proteins, and may be used as molecular chaperones for protein molecules. They can be cytoplasmic proteins and can perform functions in various intra-cellular .processes. The HSPs can also be referred to according to their molecular weights, for example Hsp70 and Hsp90, each of which define families of chaperones. Although some members of each family are listed here, it should be noted that some species may express additional chaperones, co-chaperones, and heat shock proteins not listed. Additionally, many of these proteins may have multiple splice variants (Hsp90α and Hsp90β, for instance) or conflicts of nomenclature (Hsp72 is sometimes called Hsp70). Examples include heat shock proteins having an approximate molecular weight (kDa) of 10 kDa, e.g., HsplO, 20-30 kDa, e.g., Hsp27, 40 kDa, e.g., Hsp40, 60 kDa, e.g., Hsp60, 70 kDa, e.g., Hsp70, Hsc70, Hsp72, Grp78 and BiP, 90 kDa, e.g., Hsp90 and Grp94, and 100 kDa, e.g., HsplO4, Hspl lO.

In an exemplary embodiment, the HSP may comprise a modified cellular shock protein purified from either cancer or normal cell tissue. The tissue used may be that of the patient. The HSP may be prepared in a variety of manners, e.g., as set forth in Chandawarkar, et al.,

"Immune modulation with high-dose heat shock protein gp96: therapy of murine autoimmune diabetes and encephalomyelitis," International Immunology, Vol. 16, No. 4, pp. 615-624 (2004).

The HSP-IPF complex may be prepared in a variety of manners. In an exemplary embodiment, the HSP-IPF complex is prepared by spontaneous binding, e.g., covalently bonded.

The present invention also relates to a method of preparing HSP-IPF complexes. The isolated IPF and the isolated HSP are diluted in a buffer solution and an adjuvant is added. In one aspect of the invention, the solution is maintained at a temperature of about +4 Celsius and incubated for a period of 12 hours. In one aspect, further chemicals, e.g., stabilizers, are added.

In an exemplary embodiment, the IPF is isolated from lyophilized pepsin and the HSP component used is gp96, readily lyophilized (P14625- human tumor rejection antigen-gp96). The IPF and gp96 are diluted in a buffer solution and after an incubation period of 12 hours aluminum phosphate is added as an adjuvant at a temperature of about +4 Celsius. In another aspect, further stabilizer may be added, e.g, sodium caprylate or sodium acetyltryptophanata.

In one aspect, the complex is prepared, by adding 12 milligrams (mg) of IPF (6 mg per ml) and 5.5 micrograms (μg) of gp96 (2.75 micrograms per ml) per 2 ml vial (or for a human dose, e.g., of about 100 micrograms per treatment, four times at weekly intervals. Adjuvant such as aluminum phosphate is added in an amount of about 0.004 M. In another aspect, about 0.004M Sodium Caprylate and/or 0.004 M Sodium Acetyltryptophanate is added to stabilize the solution.

In another exemplary embodiment, the IPF-HSP complex may be in a variety of forms, e.g., a pharmaceutical composition. In one aspect, the pharmaceutical composition may comprise the IPF-HSP complex and a pharmaceutically effective carrier, e.g., buffered saline, water, aluminum hydroxide, or another suitable adjuvant.

In one exemplary embodiment, the composition further comprises an antigen and an adjuvant, which potentiates the immune response to the antigen. The adjuvant may be an aluminum-containing compound, e.g., aluminum hydroxide (AH) or aluminum phosphate (AP) labeled with ²⁶Al. The AH adjuvant may be crystalline aluminum oxyhydroxide, AlOOH, and the AP adjuvant may be amorphous aluminum hydroxyphosphate. The adjuvant may be administered in an amount of no more than 0.85 mg aluminum per dose. In another exemplary embodiment, the adjuvants are substances that when mixed with antigens enhance the antibody response to the antigen itself. Example of adjuvants may include mineral oil which increase antigen persistent and recruit macrophages to the site of injection.

In yet another exemplary embodiment, the complex is administered with at least one other polynucleotide, like a molecular adjuvant, for cancer preventive or therapeutic vaccine. The cancer can be either primary or metastatic and may include renal cell carcinoma (kidney cancer), melanoma, pancreatic cancer, non-Hodgkin's lymphoma, lung carcinoma, prostate cancer, colon cancer, breast cancer, spinal cell carcinoma, soft tissue sarcoma or fibrosarcoma.

Molecular adjuvant may include various cytokines from interleukin type or adjuvant emulsion MF59 that are approved for clinical use. In an exemplary embodiment, the molecular adjuvant may be interleukin 2 (IL-2). The

IL-2 may be used to increase cellular immune response to activate normal human lymphocytes by directly promoting cellular functions selected from the group comprising of IL-2 stimulated T-cells, which exhibit enhanced cytotoxicity and produce lymphokins, e.g., INF-γ TNF-β and TGF-β; B-cells growth factors, e.g., IL-4 and IL-6 and GM-CSF. IL-2 may also induce lymphokine-activated killer (LAK) activity which is predominantly due to NK cells or increase production of T-cell clones.

The complex may be administered in a variety of manners, e.g., orally, intradermally, intramuscularly, intravenously or by intranasal spray. In one embodiment, the complex is administered intramuscularly. Also, doses may be administered at least daily, weekly or monthly, for as long as treatment is required. In exemplary embodiments, the complex is administered intramuscularly once a week for four weeks, once a week for six weeks, three times a week for six weeks, or three times a week for three weeks.

Dosing duration may vary based on a variety of factors, e.g., if used as a therapeutic or preventive vaccine. For example, dosing may vary for a 2 ml vial of HSP-IPF complex composition containing 12 mg of IPF and 2.77μg of gp96. In an exemplary embodiment, the complex may be used as a therapeutic vaccine (TCV, VGV- TC) and a total of 18 vials are administered three vials a week for six weeks. After three months, a total of nine vials are administered once a week for nine weeks. In another exemplary embodiment, the complex may be used as preventive vaccine (PCV, VGV- PC) and a total of 10 vials are administered three vials a week for three weeks.

The HSP may be administered via the complex in a variety of doses, e.g., from about 1.0 to about 200 μg. As used herein low doses of HSP may be from about 1.0 to about 25 μg and

high doses of HSP may be from about 26 to 200 μg. In one aspect of the invention, a low dose

may be about 1.0, 10 or 25 μg, and a high dose may be about 26, 48, 50, 75, 100, 150 or 200 μg. The amount designation of low or high doses may vary based on the frequency of administration. For example, an amount of about 25 μg may be deemed high if administered more frequently or may be deemed low if administered less frequently.

The IPF may be administered via the complex in a variety of doses, e.g., from about 1 to about 25 mg of per 1 ml of the composition. In an exemplary embodiment, the IPF is administered in about 2 mg/ml, 4 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml or 12 mg/ml.

In another embodiment, the IPF-HSP complex is administered as pharmaceutical composition comprising about 10 μg/ml HSP, 6 mg/ml IPF, and optionally including 0.016 M Aluminum Phosphate (2.26 mg/ml AlPO₄) (or 0.5 mg/ml Al³⁺), 0.14 M NaCl, 0.004 M CH₃COONa, and/or 0.004 M KCl.

In yet another embodiment, the IPF-HSP complex is administered as pharmaceutical composition comprising about 100 μg/ml HSP, 4 mg/ml IPF, and optionally including 0.016 M Aluminum Phosphate (2.26 mg/ml AlPO₄) (or 0.5 mg/ml Al³⁺), 0.14 M NaCl, 0.004 M CH₃COONa, and/or 0.004 M KCl.

In an alternative aspect, the formulation may include about 10 μg/ml HSP, 4 mg/ml IPF,

2.26 mg/ml Aluminum Phosphate, 0.5 mg/ml Aluminum, 12.9 mg/ml sodium citrate, and 4.1 mg/ml sodium acetate. In one embodiment, the formulation may include about 100 μg/ml HSP, 4 mg/ml IPF, 2.26 mg/ml Aluminum Phosphate, 0.5 mg/ml Aluminum, 12.9 mg/ml sodium citrate, and 4.1 mg/ml sodium acetate.

In yet a further embodiment, the formulation may comprise per vial, about 10 μg HSP, 8 mg IPF, 4.52 mg Aluminum Phosphate, 1.0 mg Aluminum, 25.8 mg sodium citrate, and 8.2 mg sodium acetate. In another embodiment, the formulation may comprise per vial, about 100 μg HSP, 8 mg IPF, 4.52 mg Aluminum Phosphate, 1.0 mg Aluminum, 25.8 mg sodium citrate, and

8.2 mg sodium acetate.

In another exemplary embodiment, the HSP component may be gp96, e.g., Glycoprotein - 96 (MW 94 kDa) purified from mouse cells, in an amount of about lmg/ml and having 95% purity as determined by SDS - PAGE. In another aspect the IPF-gp96 complex is a liquid suspension containing gp96 purified native protein and IPF purified protein fragment.

These formulations may further include IL-2, in an amount from about 1000000 to 3000000 IU per vial.

In one aspect of the invention, the formulation may contain low dose concentrations, e.g., about 10 or 25 μg, of HSP effective to induce pro-inflammatory responses, e.g., recognition of non-self-heat shock proteins to induce inflammatory responses. This may be useful to treat cancer and infectious diseases. Examples of a HSP component that may be used at low dose concentrations include Hsp70, Hsp90, gp96, calreticulin, Hspl lO, grpl70, covalent Hsp-ahtigen complexes or non-self-Hsp60. In yet another aspect of the invention, the formulation may contain high dose concentrations, e.g., 100 μg, of HSP effective to induce regulatory immunity, e.g., recognition of conserved epitopes to induce regulatory responses. Examples of a HSP component that may be used at high dose concentrations include gp96, self-Hsp60 or self-HSp70.

Combining HSP and IPF in a complex contributes to a variety of synergistic effects. In one exemplary embodiment, the HSP may mediate one or more of the following effects: suppressing tumor immunity or eliciting protective immunity against tumor cells, chaperoning immune enhancing agents and peptides, activating dendrites and macrophages by modulating inflammatory cytokines and inducing maturation of dendrites, modulating release of IL- 12 and tumor necrosis factor α (TNF α), inducing anti-tumor activity and tumor-specific cytolytic T-cells or inducing cancer-specific CDS⁺ T-cell response. In another exemplary embodiment, the IPF component may mediate one or more of the following phenotypic effects: increasing the CD4 + CD45 RO + CD62 L population, increasing the CD4 + CD45 RA + CD62 L population, inducing a second CD4 + population having lower CD4 intensity but no increase in SSC, inducing a parallel increase in absolute CD4 cell counts, or increasing the CD8 + CCR5 + population.

In yet another aspect of the invention, the IPF component may mediate one or more of the following functional effects over time: increasing the IFN-γ containing CD3 + CD4 + cells post stimulation in vitro, decreasing the IL-4 containing CD3 +CD4 + cells post stimulation, or increasing the IFN-γ containing CD3 + CD8 + cells over time. EXAMPLES

EXAMPLE l

Preparing the IPF-HSP complex

The IPF can be extracted and purified according to commonly owned U.S. Provisional Application No. 60/644,054, filed January 18, 2005, Zhabilov, entitled "Inactivated Pepsin Fraction, Pharmaceutical Compositions, and Methods for Detecting and Treating Diseases." For the HSP component, gp69 can be extracted and purified from fibrosarcoma cells as set forth in Chandawarkar, et al., "Immune modulation with high-dose heat shock protein gp96: therapy of murine autoimmune diabetes and encephalomyelitis," International Immunology, Vol. 16, No. 4, pp. 615-624 (2004). After the two components are isolated, then the gp69 can be covalently bonded to the IPF as described below. EXAMPLE 2 Efficacy of immunizing/vaccinating Murine hosts against HT29 tumors

The IPF is isolated from lyophilized pepsin and gp96 is obtained, readily lyophilized (P14625- human tumor rejection antigen-gp96). 12 mg of IPF (6 mg per ml) and 96 μg of gp96

(48 micrograms per ml) per 2 ml vial are diluted in a buffer solution and after an incubation period of 12 hours, 0.004M Sodium Caprylate and 0.004 M Sodium Acetyltryptophanate are added. The solution is maintained at a temperature of about +4 Celsius.

Two basic groups consisting of ten athymic nude mice each are tested and analyzed for tumor growth and other physiological conditions.

For Group 1, ten mice are injected with 1 ml of IPF-gp96 complex at days 1, 3, 5 and 7. Ten days after last the injection, the mice are injected with HT29 (human colorectal adenocarcinoma) cells and tumor measurements are observed for days 17 to 35.

For Group II, ten mice are injected with buffer solution at days 1, 2, 5 and 7. Ten days after the last injection, the mice are injected with HT29 cells and tumor measurements are observed for days 17 to 35.

The mice in both groups are analyzed. No melanoma cells are detected in the first group but all the mice in the second group develop melanoma cells. EXAMPLE 3

Efficacy of therapeutically treating Murine hosts against HT29 tumors

The IPF is isolated from lyophilized pepsin and gp96 is obtained, readily lyophilized

(P14625- human tumor rejection antigen-gp96). 12 mg of IPF (6 mg per ml) and 5.5 μg of gp96

(2.75 micrograms per ml) per 2 ml vial are diluted in a buffer solution and after an incubation period of 12 hours, 0.004M Sodium Caprylate and 0.004 M Sodium Acetyltryptophanata are added. The solution is maintained at a temperature of about +4 Celsius.

For Group 1, at day 17 from the start of the experiment, ten mice are injected with HT29 for tumor development. After five days, the mice are injected with 1 ml IPF-gp69 solution on days 22, 24, 26 and 28 cells and tumor measurements are observed for days 17 to 35.

For Group II, at day 17 from the start of experiment, ten mice are injected with HT29 for tumor development. After five days, the mice are injected with buffer solution at days 22, 24, 26 and 28 and tumor measurements are observed for days 17 to 35. The mice in both groups are analyzed. No melanoma cells are detected in the first group but all the mice in the second group develop melanoma cells. EXAMPLE 4

For both Examples 2 and 3, the following protocol is used.

Culture of Human Cancer Cell Line: 1. Thaw out frozen (liquid nitrogen) aliquot of HT29 cells (from ATCC).

2. Disperse into 75 cm² flask containing McCoy's 5A media supplemented with 10% fetal bovine calf serum (FBS) and incubate at 37 Celsius in humidified atmosphere of 5% CO₂. 3. As cells become 90% confluent, expand cultures to 150 cm² flasks. a. Supplement or renew culture media as needed. b. Freeze down vials of cell line for future use (SOP #11,000).

4. Continue to expand cμlture until sufficient cells are available for injection into mice (i.e. 2x10⁶ HT29 cells per mouse). B. Establishment of Tumors.

1. Receive (SOP 1910, SOP 1920) 40 male Nu/Nu mice and house using filter-topped cages supplied with autoclaved bedding.

2. Note: All mouse handling procedures should be carried out in laminar flow hood to prevent contamination of animals. 3. Implantation: a. Ear tag mice (SOP 810) for identification purposes b. Record initial weight. c. inject cancer cells subcutaneously (SOP #1610) in both flanks, 1x10⁶ cells/flank in 0.1 ml (DAY 17) 4. Staging: a. Record tumor measurements every Monday, Wednesday, and Friday for until tumor become 100 mm3. b. To reduce variability, the tumors will be measured by one technician. C. Treatment Regimen

1. Treatment: a. Record mouse weights b. Record tumor measurements c. Sort mice into 2 treatment groups (Group 1 Therapeutic and

Group 2 Vehicle) of 10 mice each based upon tumor size d. Start dosing regimen

2. Monitoring: a. Record mouse weights M, W, F. b. Record signs of distress daily c. Record tumor measurements M, W, F. d. Dose mice when required. EXAMPLE 5

From Examples 2 and 3, immune responses are measured. Methods of CD4 cell determination

All bloods samples are analyzed for their CD4 cell counts by flow cytometry. Triple labeling methods are utilized whereby CD45 monoclonal antibody is used to gate lymphocytes (based on their high expression of CD45 and low side scatter characteristics). Within this gate, the double positive CD3+ CD4+ cells are analyzed. This method of CD4 determination therefore excludes any cells (lymphocytes) which expresses only the CD4 marker. Observation of CD4 cell

After the last injection, all of the mice from Group I (Examples 2 and 3) show a new population of CD4 cells which seem to be excluded in the routine CD4 cell flow cytometric assays of Group II (Examples 2 and 3). This is due to the fact that (as explained above) the flow cytometric method employed would exclude these cells due to their non-expression of CD3 marker. These "new" cells are apparent on a dot plot of SSC versus CD4. An intermediate population of CD4 cells which are not monocytes, and a population of brightly fluorescent CD4+ population (true Theiper cells) are observed. Use of other monoclonal antibodies confirm these cells do not comply either to naϊve or memory status (by the expression of CD45RA or CD45RO expression, respectively) and they do not appear to express TCR of alpha or beta subtype.

It is proposed that these "new" cells are in fact dendritic cells which are CD4+ that are responsible for the immune regulatory activity of IPF. Such cells are known to secrete IL 12 which enhances CMI to infectious organisms and swing the regulatory arm of immunity to a beneficial THI phenotype. This would account (at least in part) for the other markers of immune reactivation induces by IPF, e.g., increased secretion of INF-γ by CD8 cells upon stimulation in vitro with HIV recombinant p24, improved functions of both CD4 and CD8 cells in response to a polyclonal activator and decreased in CD8+CD38+ cells (bad prognosis marker) over time. Overall a measured immune response may give evidence of two actions:

(1) Cytotoxic effect against tumor cells. Cytotoxic T lymphocytes (CTLs) are effectors CD8+ that can mediate the lyses of target cells bearing antigenic peptides associated with a MHC molecule. Other cytotoxic cells include gamma/delta chain and CD4+ NK 1.1+ cells.

(2) Increase antibody production. It could be assumed that the IPF is binding to gp96 identified as a major Epitope recognized by antibodies in the patient's body, as antigen presenting molecules, and this new super antigen stimulates immune response using a non- conventional antigen processing pathway.

From Examples 2 and 3, one or more of the following observations may be made:

• increase in the CD4 + CD45 RO + CD62 L population • increase in the CD4 + CD45 RA + CD62 L population

• appearance of a second CD4 + population having lower CD4 intensity but no increase in SSC. This implies a second CD4 cells population. Preliminary analysis of this population in isolation does not reveal these cells to be memory or naϊve cells. • there is a parallel increase in absolute CD4 cell counts when this phenomenon appears.

• increase in the CD8 + CCR5 + population also in parallel to the above mentioned parameters.

Since some functional assays are conducted it is interesting to note the following change in parallel to the phenotypic change above: • increase in the IFN-γ containing CD3 + CD4 + cells post stimulation in vitro

• decrease in the IL-4 containing CD3 +CD4 + cells post stimulation

• significant increase in the IFN-γ containing CD3 + CD8 + cells over time.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the above- described exemplary embodiments, but should instead be defined only in accordance with the following and their equivalents.

REFERENCES

The following are hereby incorporated by reference:

Chandawarkar, et al., "Immune modulation with high-dose heat shock protein gp96: therapy of murine autoimmune diabetes and encephalomyelitis," International Immunology, Vol. 16, No. 4, pp. 615-624 (2004).

Derky CS, "Task force on recurrent respiratory papillomatosis," Arch Otolaryngol Head Neck Surg 121: 1386-1391 (1995).

Finn, O., "Cancer Vaccines: Between the Idea and the Reality," Nature Reviews: Immunology, 3: 630-641, Nature Publishing Group (2003), <http://www.nature.com/reviews/immunol>.

Goldman, B., "Heat Shock Proteins' Vaccine Potential: From Basic Science Breakthroughs to Feasible Personalized Medicine," Antigenics (2002), <http://www.antigenics.com/whitepapers/hsp_potential.html>.

Kovalchin, J.T., et al., "Determinants of Efficacy of Immunotherapy with Tumor- Derived Heat Shock Protein gp96, " Cancer Immunity, VoI 1 , p. 7- 16 (April 27, 2001 ).

Murray P and Young RA, "Stress and Immunological recognition in hostpathogen interaction," J Bacteriol 13: 114-119 (1992).

Panjwani, N. N., Popova, L, Febbraio, M and Srivastava, P. K., "CD91 is common receptor for heat shock proteins gp96, HSP 90, .HSP70 and calreticulin," Immunity 14:303 (2001).

Sigal LJ, Crotty S, Andino R and Rock KL, "Cytotoxic T-cell immunity to virus-infected non-haematopoietic cell requires presentation of exogenous antigen," Nature 398:77-80 (1999).

Srivastava, P., "Interaction of heat shock proteins with peptides and antigen presenting cells," Annu. Rev. Immunol, 20:395 (2002). Suto, R. and Srivastava, P.K, "A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides," Science 269; 1585 (1995).

Suzue K and Young RA., "Heat shock proteins as immunological carriers and vaccines. In: Stress-inducible Cellular Response," ed. U. Fiege, Birkhauser 451-465 (1996). Vabulas,R. M., Wagner, H. and Schild, H., "Heat shock proteins as a ligands of toll-like receptors," Curr. Topics Microbiol.Immunol. 270;169 (2002).

Zhabilov et al., "Compositions and Methods for Detecting and Treating Acquired Immunodeficiency Syndrome," U.S. Patent Application No. 10/336,512, filed June 3, 2003.

Zhabilov, "Inactivated Pepsin Fraction, Pharmaceutical Compositions, and Methods for Detecting and Treating Diseases," U.S. Provisional Patent Application N. 60/644,054, filed January 18, 2005.

Claims

OUTLINE OF THE INVENTION

1. A method of modulating immune system activity comprising administering to a patient an effective amount of a complex of an inactivated pepsin fraction (IPF) component and a heat shock protein (HSP) peptide component.

2. The method of claim 1, wherein the HSP comprises a modified cellular heat shock protein purified from either cancer or normal cell tissue.

3. The method of claim 1, wherein modulating immune system activity comprises treating, decreasing, increasing, attenuating or modulating a condition that benefits from an enhancement of immune system activity.

4. The method of claim 1, wherein the IPF-HSP complex is administered to treat an immune diseases or disorders.

5. The method of claim 4, wherein the immune disorder is selected from the group consisting of Allergies, Auto-Immune, DiGeorge Syndrome, Familial Mediterranean Fever, Immune Deficiency, and Multiple Chemical Sensitivity.

6. The method of claim 4, wherein the immune system disease or disorder is selected from the group consisting of Agammaglobulinemia, Anaphylaxis, Antiphospholipid Syndrome, Ataxia Telangiectasia, Autoimmune Diseases, Common Variable Immunodeficiency, DiGeorge Syndrome, Electrosensitivity, Familial Mediterranean Fever, Graft vs Host Disease, Granulomatous Disease, Chronic, HIV Infections, Hypersensitivity, Hypersensitivity, Immediate, IgA Deficiency, Immune Complex Diseases, Immune System Diseases, Immunologic Deficiency Syndromes, Lambert-Eaton Myasthenic Syndrome, Lambert-Eaton Myasthenic Syndrome, Latex Hypersensitivity, Lymphoproliferative Disorders, Multiple Chemical Sensitivity, Purpura, Schoenlein-Henoch, Samter's Syndrome, Severe Combined Immunodeficiency, Sick Building Syndrome, Sjogren's Syndrome, and Wiskott-Aldrich Syndrome.

7. The method of claim 4, wherein the disorder comprises auto-immune disorder selected from the group consisting of Addison's, Ankylosing Spondylitis, Antiphospholipid Syndrome, Barth Syndrome, Graves' Disease, Hemolytic Anemia, IgA Nephropathy, Lupus Erythematosus, Systemic, Microscopic Polyangiitis, Multiple Sclerosis, Myasthenia Gravis, Myositis, Osteoporosis, Pemphigus, Psoriasis, Rheumatoid Arthritis, Sarcoidosis, Scleroderma and Sjogren's Syndrome.

8. The method of claim 5, wherein the allergy is selected from the group consisting of Asthma, Food, Hay Fever - Rhinitis, Hives, Latex and Sinusitis.

9. The method of claim 4, wherein the disease is selected from the group consisting of AIDS or AIDS Related Complex, multiple sclerosis, hepatitis, herpes, rheumatoid arthritis, autoimmune diabetes, encephalomyelitis or another autoimmune disease.

10. The method of claim 1, further comprising a cancer preventive or therapeutic vaccine.

11. The method of claim 1, wherein the complex is administered with at least one other polynucleotide for cancer preventive or therapeutic vaccine.

12. The method of claim 11, wherein polynucleotide comprises a molecular adjuvant.

13. The method of claim 12, wherein the molecular adjuvant comprises interleukin 2 (IL-2).

14. A pharmaceutical composition comprising a complex of an inactivated pepsin fraction (IPF) component and a heat shock protein (HSP) peptide component and a pharmaceutically effective carrier.

15. The composition of claim 14, wherein the pharmaceutically effective carrier is selected from the group consisting of buffered saline, water, aluminum hydroxide, or another suitable adjuvant.

16. The composition of claim 14, wherein the composition comprises from about 1.0 to about 200 μg of HSP per 1 ml of the composition.

17. The composition of claim 14, wherein the composition comprises from about 1 to about 25 mg of IPF per 1 ml of the composition.

18. The composition of claim 14, wherein the composition comprises about 10 μg/ml HSP, 4 mg/ml IPF, and optionally including 0.016 M Aluminum Phosphate (2.26 mg/ml AlPO₄) (or 0.5 mg/ml Al³⁺), 0.14 M NaCl, 0.004 M CH₃COONa, and/or 0.004 M KCl.

19. The composition of claim 14, wherein the composition comprises about 100 μg/ml HSP, 4 mg/ml IPF, and optionally including 0.016 M Aluminum Phosphate (2.26 mg/ml AlPO₄) (or 0.5 mg/ml Al³⁺), 0.14 MNaCl, 0.004 M CH₃COONa, and/or 0.004 M KCl.

20. The composition of claim 14, wherein the composition comprises about 10 μg/ml HSP, 4 mg/ml IPF, 2.26 mg/ml Aluminum Phosphate, 0.5 mg/ml Aluminum, 12.9 mg/ml sodium citrate and 4.1 mg/ml sodium acetate.

21. The composition of claim 14, wherein the composition comprises about 100 μg/ml HSP, 4 mg/ml IPF, 2.26 mg/ml Aluminum Phosphate, 0.5 mg/ml Aluminum, 12.9 mg/ml sodium citrate and 4.1 mg/ml sodium acetate.

22. The composition of claim 14, wherein the composition comprises per vial, about 10 μg HSP, 6 mg IPF, 4.52 mg Aluminum Phosphate, 1.0 mg Aluminum, 25.8 mg sodium citrate and 8.2 mg sodium acetate.

23. The composition of claim 14, wherein the composition comprises per vial, about 100 μg HSP, 8 mg IPF, 4.52 mg Aluminum Phosphate, 1.0 mg Aluminum, 25.8 mg sodium citrate and 8.2 mg sodium acetate.

24. The composition of any one of claims 14 to 23, wherein the composition further comprises IL-2 in an amount from about 1000000 to 3000000 IU per vial.

25. The composition of claim 14, wherein the HSP is in an amount of about 10 or 25

μg-

26. The composition of claim 25, wherein the HSP is selected from the group consisting of Hsp70, Hsp90, gp96, calreticulin, Hspl lO, grρl70, covalent Hsp-antigen complexes and non-self-Hsp60.

27. A method of using the composition of claim 26, wherein the composition induces pro-inflammatory responses.

28. The method of claim 27, wherein the pro-inflammatory response comprises recognizing of non-self-heat shock proteins leading to inflammatory responses.

29. A method of using the composition of claim 25, wherein the composition is used to treat cancer or infectious diseases.

30. The composition of claim 14, wherein the HSP is in an amount of from about 50 to 100 μg.

31. The composition of claim 30, wherein the HSP is selected from the group consisting of gp96, self-Hsp60 and self-HSp70.

32. A method of using the composition of claim 31, wherein the composition induces regulatory immunity comprising recognizing of conserved epitopes.

33. A method of using the composition of claim 32, wherein the composition is used to treat autoimmunity, vascular disease or transplant rejection.

34. A method of making a HSP-IPF complex comprising the steps of:

(a) combining an isolated IPF and an isolated HSP in a buffer solution, and

(b) adding an adjuvant.

35. The method of claim 34, wherein the solution of step (a) is incubated for a period of about 12 hours before adding the adjuvant from step (b).

36. The method of claim 34, wherein the temperature of the solution is maintained at about +4 Celsius.

37. The method of claim 34, further comprising adding a stabilizer after step (b).

38. The method of claim 34, wherein the HSP component comprises gp96.

39. The method of claim 34, wherein the adjuvant comprises aluminum phosphate.

40. The method of claim 39, wherein the adjuvant comprises sodium caprylate and/or sodium acetyltryptophanate.

41. The method of claim 34, wherein 12 milligrams (mg) of PF and 5.5 micrograms (μg) of gp96 are added per 2 ml vial.

42. The method of claim 41, wherein 0.004 M of adjuvant is added per 2 ml vial.