WO2010065157A2 - Inactivated pepsin fragments for modulating immune system activity against human malignant tumor cells - Google Patents

Description

INACTIVATED PEPSIN FRAGMENTS FOR MODULATING IMMUNE SYSTEM ACTIVITY AGAINST HUMAN MALIGNANT

TUMOR CELLS

FIELD OF THE INVENTION

[0001] The present invention relates to compositions and methods comprising an inactivated pepsin fragment ("IPF^") for modulating immune system activity. More specifically, embodiments of the present invention are directed to compositions and methods to elicit specific immunity to recognize peptides associated with tumors and malignancies. The present invention also relates to compositions and methods comprising IPF for treating infections such as infection by the human immunodeficiency virus.

BACKGROUND OF THE INVENTION

[0002| Pepsin is a proteolytic enzyme produced in the mucosal lining of the stomach and acts to degrades protein, together with chymotrypsin and trypsin. During digestion, these enzymes, each of which is effective in severing links between particular types of amino acids (e.g. phenylalanine, tryptophan and tyrosine), collaborate to break down dietary proteins to their components, i.e.. peptides and amino acids.

|00031 Current studies indicate that immune protection against cancer requires the generation of a potent cellular immune response against a unique tumor antigen expressed by a malignant cell. Thus, successful immune protection would first require identifying a unique antigen in the tumor cells (tumor specific antigen) and then inducing a potent T-eell response targeted to the tumor antigen. These tumor-associated antigens, however, would still be recognized by immune cells as 'self molecules, and so no true activation of the immune system would occur. Thus, two obstacles in targeting these tumor-associated molecules as a vaccine include the unresponsiveness of the immune system to "sell^" molecules, w hich restricts its ability to generate potent cellular immune responses, ami preventing the generated immune response from being directed to normal cells that express the target antigen.

|0004| Proteins that show promise in overcoming these problems include heat shock proteins (HSPs). HSPs include a collection of ubiquitously expressed cytoprotective proteins, which are expressed by cells under conditions of cell stress, such as increased temperature. \ iral infection and oxidativ e stress. Certain HSPs have been shown to have immunomodulatory effects, such as the induction of cytokines and the promotion of cell activation and maturation (see. I'ockley AG. Lancet 363 (9382) 469-476( 2003)).

|0005] For example. Zheng el al. (2001 ) report that cell surface targeting of HSP gp96 induces dendritic cell maturation and antitumor immunity as demonstrated by the expression of immune factors such as intcrleukins and certain cell surface antigens (e.g., CD40, CDSO and MHC class 11 antigens). It has been known for some time that heat shock proteins bind peptide and that heat shock proteins purified from cells chaperone a large number of peptides dei i\ cd from the cells from which they are isolated. This is the so-called "antigenic repertoire^" of that cell. Studies have demonstrated that immunizing mice with I ISP70. HSP90 and gρ% isolated from murine tumor cells induces anti-tumor immunity and tumor-specific cytolytic T-cells. These studies also show that the immunity results from tumor-derived peptides associated with the heat shock protein rather than from the heat shock proteins themselves. More recently, studies reported the use of calreticulin. HSP l I O and grp PO in heat shock protein-based cancer immunotherapy. Specific immunogenicity of tumor-derived heat shock protein preparations have been studied in relation to fibro sarcomas, lung carcinoma, prostate cancer, spinal cell carcinoma and melanomas in mice and rats of different haplotypes. These studies included chemically-induced tumors, UV-induccd tumors, and spontaneous tumors. Heat shock proteins show promise in that preparations isolated from a given cell may be associated with a range of peptides, including self and antigenic peptides and in that HSP-peptidc complexes are highly immunogenic. |0006| Certain heat shock proteins demonstrate "superantigen" activity. They are capable of activating large numbers of T-lymphocytes in a major histocompatibility complex- restricted manner. This polyclonal activation of certain T-cell subsets may be responsible for some of the immunomodulatory effects. These components have been reported to stimulate immune responses Io certain neoplasms and may be involved in the pathogenesis of certain autoimmune diseases.

|0007| Gp% is a HSP of particular interest. Gp96 is a 96 kDa glycoprotein localized to the endoplasmic reticulum, which can also be found at the cell surface. Gp96 is released into the extra cellular space during necrotic cell death and activates dendrite cells and macrophages by realizing inflammatory cytokines and inducing dendrites cells to mature. Gp96 has the ability to transfer antigenic peptides for their MHC-class I-restricted presentation and allows gp96 to function as an efficient messaging system alerting the immune system of an infection. This includes the receptor-mediated uptake of gp96 by dendrite cells. The receptor is CD91. which is known as the α2 macroglobulin (α2M) receptor expressed on phagocytes. The presentation of gp96-associated peptide by antigen-presenting cells ("APCs^" ) is induced by α2 macroglobulin. Gp96 is bound by CD91 on dendrite cells and internalized. Gp96 induces the expression of co-stimulatory molecules and the release of inlerleukin 12 (IL- 12) and tumor necrotic factor α ("TNFα") by the APC.

[0008] Certain infections, such as by the human immunodeficiency virus, have also presented challenges in targeting the disease-causing organism and neutralizing it. Typically, infection with the human immunodeficiency virus, HIV-I, eventually causes acquired immunodeficiency syndrome (AIDS) and an associated syndrome, AlDS-related complex (ARC). Neutralizing this virus has proved difficult, largely because its structure obstructs immune system access to viral epitopes and its genetic material is highly variable. Accordingly, researchers have been seeking prophylactic and therapeutic methods for preventing or controlling HIV which are not dependent upon antibody- mediated immunity. [0009] The I NY retrovirus replicates in certain immune system cells, specifically the CD4+ subset of T-lymphocytes (pre-Th cells arising in the thymus). In the usual course of a cell-medialetl immune response to an intracellular pathogen such as a virus, dendritic cells (antigen-presenting cells) carrying antigen fragments and secreted cytokines activate these CD4- T-cells. Activated cells, called T-hclper or Th cells, in turn secrete their own cytokines and stimulate macrophages. CD4+Th cells also propagate cellular immune response by binding chemotactic cytokines (chemokines, CCs) to their CC surface receptors. It is by this route lhat HIV-I infection of these cells is enabled because, in addition to binding chemokines. these CC receptors act together with the CD4+ surface glycoprotein as co-receptors for HIV-I and mediate entry of the virus into the CD4+Th cell. There, the virus usurps the native genetic material for viral replication while destroying cell functions essential for building immunity; the increasing destruction of these cells appears to be responsible for the eventual collapse of the cell-mediated immune system often seen in terminal AIDS patients.

[000101 It has been recognized that denying entry into CD4+ cells to the HIV-I virus could at least slow the progress of the infection and alleviate, if not cure, the disease and/or its symptoms. The complex mechanism by which the virus crosses the cell membrane has been widely investigated. Broadly, the entry of human immunodeficiency virus into, for example. CD4 - ThI cells (T-helpcr type 1 cells), is dependent upon a sequential interaction of the gpl 20 gρ41 subunits of the viral envelope glycoprotein gpl όO with the CD4+Thl cell surface glycoprotein and the cell surface receptor CCR5. On binding of up 120 with its cell surface binding sites, a conformational change in the latent gp41 subunit through an intermediate state to an active state is initiated, inducing fusion of the \ iral and cellular membranes and transport of the virus into the cell (Weissenhom et al., Nature, 3S^~:426-30 ( 1997)).

[0001 1] Accordingly, numerous binding experiments have been conducted in an effort to find antiviral ligands thai will effectively compete with the HIV-I for CD4+ gp and/or CCR5 binding sites, or that will preferentially block gpl20 and/or gp41 binding domains. In one example, a reported structure (X-ray crystallography) comprising an HlV- I gp l 20 core complex ed w ith a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokinc-receptor binding, is said to reveal a ( D4-gp l 20 interface, a conserved binding site for the chcmokine receptor, evidence for a conformational change on CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for viral immune evasion, "which should guide efforts to intervene^" (Nature 393 (6686):632-l, 1998). Also, it has been shown that inhibition of the change in structure of gp41 from its intermediate to active state with peptides used as competitors for critical cell receptors may reduce viral load, and that w hile gpl20 masks epitopes on the gp41 subunit in its latent state, gp41 may be vulnerable to neutralizing antibodies in its transient or intermediate state (Molecular Membrane Bio/o&v 16:3-9. 1999).

BRIEF DKSCRIPTION OF THE DRAWINGS

[00012] Some aspects of the present invention are generally shown by way of reference to the accompanying drawings in which:

[00013] FlG. 1 illustrates the porcine pepsinogen sequence, and major and minor sequences of this pepsinogen.

|00014] FlG. 2 is a photograph of an electrophoresis gel showing an inactivated pepsinogen fragment ("IPF^") in the 45.0 kDa band. SDS-PAGE was used for determination of molecular w eight of the components of IPF. Column 1 included weight standards shown in the box to the left. The next two columns show #5, bovine albumin, at 1 :300 dilution and 1 :500 dilution. The remaining columns show samples 16-21 at various concentrations. Sample 16 was obtained from porcine pepsin Sigma p7000 1 : 10000 purified and methylated. Samples 17 and 21 were prepared as with Sample 16. Sample 18 w as porcine pepsin Sigma p70()0 1 : 10000, diluted in buffer at pH 3.2, so that pepsin is an activ e protease. Sample 19 was obtained from porcine pepsin Sigma p7000 1 : 10000 and purified, but unmethylated. Sample 20 was prepared as with Sample 19. The next columns, in order, show methylated IPF sample #16 at a 1:1 dilution, methylated IPF sample #17 at a 1 : 1 dilution, untreated pure (active) pepsin sample #18 at a 1 : 10 dilution, unmethylated IPF sample #19 at a 1:10 dilution, unmethylated IPF sample #20 at a 1 :10 dilution, and methylated IPF sample #21 at a 1 : 10 dilution. IPF samples # 16. 17. and 21 include as a major component a protein/peptide migrating with an apparent molecular weight of 45 kD. These samples show highly specific binding between IPF and gp4I , gpl20. and CD4 at different concentrations.

[00015] FlG. 3 is a Biacore graph showing a HPLC (High Performance Liquid

Chromatography) chromatogram of an isolated IPF in accordance with once aspect of the present invention.

(00016| Figures 4, 5. 6. and 7 illustrate exemplary binding of four samples of IPF with gp41. gρl2(). human CD4. and human serum at 3 different dilutions;

|00017| Figure 8 is a photograph of an electrophoresis agarose gel showing bound

IPF and gp4 l .

[00018) Figure 9 is a graph showing binding of an IPF with heat shock protein gp% at dilutions of IPF 1/2500. IPF 1 /500 and IPF 1/100, according to one embodiment of the invention.

[00019J Figure 10 shows amino acid normalization for the 45 kDa IPF fragment according to one embodiment of the invention.

[00020| Figure 1 1 shows amino acid sequencing data according to one embodiment of the invention.

SUMMARY OF THE DISCLOSURE

[000211 The present invention includes methods and compositions comprising modified cellular shock protein gp% which preferably includes an irreversible pepsin fraction IPF and more preferably includes an IPFgp96 complex. In another preferred embodiment, a complex of lPFμρ% may be combined with at least one other polynucleotide like a molecular adjuvant, such as IL-2, to increase cellular immune response.

|00022] Preliminary clinical trials have demonstrated the induction of cancer- specific CD8+ T-cells responses in 6 12 patients immunized with gp96-peptide prepared from their own tumor. The capacity of tumor-derived heat shock proteins to induce specific and protective immunity might have profound effects on the treatment and management of patients with malignant disease. For example, studies have shown that induction of immunity to methylcholanthrane-induced fibro sarcoma by the administration of gp96 isolated from the tumor displays consistent dose restriction: two intradermal administrations of - I μg gp96 was found to be ineffective; two doses of l μg was found to induce immunity and provide optimal protection against tumor growth; and two doses of 1 Oμg was found not to protect.

|00023] The lack of protection at high doses of tumor-derived gp96 is an active, antigen specific down-regulatkni of tumor-specific immunity that can be adoptively transferred by CD4 T-cells puri fied from animal treated with high doses of tumor derived gp96. These findings are exciting as they suggest that immunization with heat shock proteins that arc chaperoning clinically relevant peptides might be an effective strategy for down regulating several diseases including autoimmunity.

[00024] The addition of IL-2 to activate normal human lymphocytes directly promotes several other cellular functions as well as proliferation. IL-2 stimulated T-cells exhibit enhanced cytotoxicity and produce lymphokins such as INF-γ TNF-β and TGF-β; B-cells grow th factors such as IL-4 and IL-6 and GM-CSF. IL-2 also induces lymphoid nc-activated killer (LΛK) activity, which is predominantly due to NK cells.

[00025] The present invention also encompasses a cancer preventive or therapeutic vaccine comprising a IPFgp% complex, and more specifically complexes of IPF-I gp96; IPF-2gp96: IPF-3gp96, IPF-4gp%. IPF-5gp96, singly or in combinations thereof, which may be mixed with one or more polynucleotides encoding a molecular adjuvant. Any molecular adjuvant that increases cellular immune response may be used like cytokine IL-2. In the preferred embodiments, the compositions and methods of the present invention comprise binding betw een IPF fragments and receptors of gp96, such as for example. CD9 I . in vivo. Administration may be via an intramuscular injection. The cancer to be treated may be primary or metastatic and the patients to be treated may have multiple different types of cancer.

[00026] IPF has been shown to have an ability to modulate ThI immunity, cytokine secretion and γlFN. Placebo and controlled double blind assay using two groups of rats show immunological changes following active therapy, which were sustained over time. These changes include: I ) increase in the CD4+ CD45RO+ CD62 L population; 2) increase in the CD4+ CD45RA- CD62 L population; 3) appearance of a second CD4+ population having lower CD4 intensity but no increase in SSC, implying a second CD4 cell population. Preliminary studies of this population in isolation reveals that these cells are not memory or naive cells: 4) a parallel increase in absolute CD4 cell counts; 5) an increase in CDS+ CCR5+ population.

[00027| In addition, functional assays showed the following: 1) an increase in the

IFN-[y] containing CD3+ CD4 - cells post stimulation in vitro; 2) a decrease in the IL-4 containing CD3+ CD4+ cells post-stimulation; 3) a significant increase in the IFN-γ containing CD3- CD8+ cells o\ er time.

|00028] Also, numerous binding experiments have been conducted in an effort to find antiviral ligands that will effectively compete with the HIV-I for CD4+ gp and/or CCR5 binding sites, or that will preferentially block gpl20 and/or gp41 binding domains. In one example, a reported structure (X-ray crystallography) comprising a HIV-I gpl20 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding, is said to reveal a CD4-gpl20 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change on CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for viral immune evasion, "which should guide efforts to intervene" (λ'aiure 393 (6686):632-l , 1998). Also, it has been shown that inhibition of the change in structure of gp41 from its intermediate to active state with peptides used as competitors for critical cell receptors may reduce viral load, and that while gp I 20 masks epitopes on the gp4 l subunit in its latent state, gp41 may be vulnerable to neutralizing antibodies in its transient or intermediate state (Molecular Membrane Biology 16:3-9. IW⁾).

|00029j Other embodiments of the present invention are generally directed to providing pharmaceutical compositions comprising IPF (IPF-I, IPF-2, IPF-3, IPF-4, IPF- 5 and/or 1PF-6) and methods for preventing, treating, and diagnosing HIV-I infections and HIV- I related conditions such as AIDS (Acquired immune Deficiency Syndrome) and ARC (AIDS Related Complex) with these compositions.

(00030] In an exemplai > embodiment the present invention relates to a method of modulating immune system activ ity comprising administering to a patient an effective amount of a composition containing inactivated pepsin fragment (IPF).

[00031| The isolation, purification and characterization and a variety of other uses, e.g.. diagnosis and treatment of HIV infection and related diseases such as AIDS and ARC, of the inactivated pepsin fragment or fraction (IPF) as used herein is described in commonly owned U.S. Provisional Patent Application No. 60/644,054, filed January 18, 2005, Zhabikn . the contents of which arc incorporated herein by reference in their entirety. |00032| At least 8 isozymes of pepsinogen have been identified in gastric epithelial cells, and these have been categorized into two immunologically-separablc types (pepsins A and C). The mature, active enzymes arc roughly 325 amino acids with a mass of approximately 35 kDa. Pepsins are synthesized as inactive pre-proenzymes, consisting of a signal peptide, activation peptide and active enzyme. The signal peptide is cleaved as the protein is inserted into endoplasmic reticulum and the resulting proenzyme - pepsinogen - is transported io the Golgi and condensed into secretory granules. Pepsinogens are secreted in a form such that the activation peptide assumes a compact structure that occludes the active site. On exposure to an acidic (pH < 4) environment such as occurs in the lumen of the stomach, the activation peptide unfolds, allowing the active site to clip it off, yielding mature, catalytically active pepsin.

(00033] Structurally, the activ e site is located in a deep cleft within the molecule located betw een two homologous portions of the structure, the N-terminal lobe (residues 1-172) and the C-tenninal lobe (residues 173-327). Optimal activity of pepsins is at pH of 1.8 to 3.5. depending on the isoform. They are reversibly inactivated at about pH 5 and irreversibly inactivated at about pH 6 to 7 or 7 to 8. See, Yuji O. Kamatari, Christopher M. Dobson, and Takashi Konno, "Structural dissection of alkaline-denatured pepsin." Protein Science (2003 ). 12:717-724.

[00034| According to F.iure/ Protein, accession number NP_999038, a sequence of swine porcine pepsinogen is -

000 mkwllllslv vised vkvp Ivrkkslrqn likngklkdf lkthkhnpas kyfpeaaali 061 gdeplcnyld teyfgtigig tpaqdftvif dtgssnlwvp svycsslacs dhnqfnpdds 121 stfcatsqel sitygtgsmt gilgydtvqv ggisdtnqif glsetepgsf lyyapfdgil 181 glaypsisas gatpvfdnlw dqglvsqdlf svylssndds gsvvllggid ssyytgslnw 241 vpvsvegywq itldsitmdg etuiesggcq aivdtgtsll tgptsaiain iqsdigasen 301 sdgemviscs sidslpdivf tingx qypls psayilqddd sctsgfegmd vptssgelwi 361 lgdvfirqyy tvfdrannkv glapva 100035] According to Kamatari et al.. after the N-terminal 60 bases of pepsinogen are cleaved off to produce pepsin, the N-terminal lobe of pepsin protein includes residues 1-172. and the C-tcrminal lobe includes the remaining residues 173-326. IPF-6 according to the invention differs from pepsin. It includes a major component having an apparent MW of 45 kD when subjected to SDS-PAGE as shown in Figure 2. It is unclear whether the 45 kD IPF-6 peptide is actually larger than pepsin, e.g. due to dimerization or other bonding with itself or another peptide, or whether the 45 kD apparent molecular weight is an artifact due to chemical modification of the protein such as methylation. The term "45 kD ll'F-6" is used here to refer to the IPF-6 molecule obtained and assayed as described here, whether its actual molecular weight is 45 kD or another figure.

[00036| Surprisingly, preparations isolated from pepsin provide highly sensitive, specific, and therapeutic biological preparations. The starting material may be pure, active porcine pepsin A. An exemplary starting material is Sigma porcine pepsin P 7000, which has a concentration of 1 : 10000. Other starting material preparations are acceptable and may be effective according to the invention, if treated and isolated according to the invention. For example, porcine pepsin with other concentrations may be used e.g. 1 :60000. The starting material is preferably white to yellow, not brown, and may have a milk-like smell.

[00037] Other natural or recombinant sources of pepsin may be used, or other similar aspartic proteases, provided that the protease is inactive at alkaline, neutral, near- neutral, or mildly acidic pH. has a pi in those ranges, and has a sequence with substantial homology to the IPF 45kD fragment reported here. Substantial homology means at least about 50. 60. 70. 80. 90. or 95"« identity of amino acid residues in the relevant portions of the molecule, or structural homology. Pepsin shares structural homology with HIV and other aspartie proteases. Campos and Sancho, "The active site of pepsin is formed in the intermediate conformation dominant at mildly acidic pH," FEBS Letters Vol. 538:89- 95 (2003). [00038] The invention provides several uses for the peptides. Examples of possible uses inelude a diagnostic assay and a therapeutic agent. IPF causes a dramatic rise in cytokines (e.g. interlcukin 9 and 10) and antibodies to p24 antigen in HIV patients.

[00039| The properties of the peptides identified as part of the IPF herein include

MW. inactivity at neutral pH. us present in blood, and their partial sequence data. Also, the IPF fragments migrate as a single main peak in HPLC, such as shown in the chromatogram of Figure I for IPF-6. with a particular retention time. Because pepsin is inactivated during isolation of IPF. the preparation is stable and does not degrade significantly over time.

|00040] The inactivated pepsin fragments (IPF) of the invention may be referred to as irreversibly-inactivated. This is due to its treatment at neutral pH. As noted above, inactivation occurs above about pH 5. and becomes irreversible above about pH 6, and proteolytic activity is lost by such treatment. Maximal activity of pepsin as an enzyme is between 2 - 4 pH. The inventiv e method increases the pH of diluted pepsin to above 5, above about 6. and desirably in the range of pH 6.6 - 6.8 before precipitation and during use. Thus IPF formulation pepsin fragments are irreversibly inactivated.

[000411 The treatment, fragmentation, and isolation procedures inactivate pepsin and cut the pepsin chain into separate peptide fragments. IPF may be isolated from active pepsin. For example. Sigma porcine pepsin P7000 may be used. This is a pepsin A from porcine gastric mucosa, is a powder with 800-2,500 units/mg protein. The CAS number is 9001 -75-6. and the EC number is 3.4.23.1. According to Sigma, it preferentially cleaves C-terminal to Phe, Leu and GIu. It does not cleave at VaI, Ala or GIy. Optimum pH is 2-4. Stable at 6O⁰C. Pepsin is irreversibly inactived at pH >6 and has a mol wt 35 kDa. See. Harlow, E., and Lane. D.. Antibodies: A Laboratory Manual , Cold Spring Harbor, NV ( 1988), 626-628: and Merck 13, 7225. |00042] The IPF 45 kD peptide and IPF 15kD peptide have amino acid sequences homologous to pepsin, meaning that at least about 5, 10, 15, or 20 of the amino acid residues in the peptide are identical to those of pepsin.

[00043] As used herein, the present invention may be directed to modulating immune system activity, which includes treating, decreasing, increasing, attenuating or modulating any condition thai may 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.

|00044] Immune system disease or disorder may include at least one of

Agammaglobulinemia. Anaphy laxis. Antiphospholipid Syndrome, Ataxia Telangiectasia, Autoimmune Diseases, Common Variable Immunodeficiency, DiGeorge Syndrome, Electroscnsitiviiy. 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-Hcnoch, Samter's Syndrome, Severe Combined Immunodeficiency. Sick Building Syndrome, Sjogren's Syndrome, and Wiskott-Aldrich Syndrome.

(00045) 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.

|⁽⁾0046| In another exemplary embodiment, the present invention may encompass a cancer preventive or therapeutic vaccine.

[00047| In yet another exemplary embodiment, the IPF 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 (kidnc> cancer), melanoma, pancreatic cancer, non-Hodgkin"s lymphoma, lung carcinoma, prostate cancer, spinal cell carcinoma, soft tissue sarcoma or fibrosarcoma.

[00048| Compositions comprising the IPF fractions disclosed herein may also be used to treat hepatitis, multiple sclerosis, lupus, and herpes simplex. Anecdotal observations, backed up by blood work, indicate a reduction in severity of symptoms in patients with these diseases, ami predicts efficacy in other viral and autoimmune diseases.

[00049| In an exemplar) embodiment. IPF peptides, e.g. IPF1-6, show specific binding and indicate usefulness as a) a diagnostic and b) a therapeutic for HlV, and other diseases. The binding of this protein material with envelope proteins in several infectious diseases as well as direct binding to CD4 cells indicates that IPF can stimulate an immunological reaction, for example by promoting the formation of superantigens which increase production of specific antibodies.

[00050| Diagnostic methods using IPF may be performed by combining IPF with test and control sera and conducting 2-D electrophoresis in 1% agarose gels, following the techniques set forth in Zluibilov et al. (US 2004/0018639, filed 3 June 2003 by Zhabilov. Mai n P. et al. and incorporated herein by reference), with modifications apparent to a person of ordinary skill. Therapeutic methods using IPF disclosed herein arc performed by administering IPF pharmaceutical compositions to a subject having a disease susceptible to treatment with IPF. The formulations, dosages, dosing regimen, and routes of administering may be those described in Zhabilov et al. or other examples apparent to a person of ordinary skill.

[000511 In another exemplary embodiment, the IPF composition may be in a variety of forms, e.g., a pharmaceutical composition. In one aspect, the pharmaceutical composition may comprise the IPF disclosed herein and a pharmaceutically effective earner, e.g.. buffered saline, water, aluminum hydroxide, or another suitable adjuvant.

[00052] In yet another exemplary embodiment, the composition may contain preservativ es, vehicles, buffers, tonicity adjusters, chelating agents, antioxidants and or other material. Examples of preservatives include Phenylethyl alcohol USP, Sorbic Acid NF. Sodium Propionate. Sodium Bcnzoate NF, and Benzyl alcohol NF. Examples of Vehicles include Purified Water LSP. Hydroxy Ethyl Cellulose NF, Polyethylene Glycol NF. Povidone USP. Hydro\\ propyl M ethyl cellulose F4M USP, Dextran 70 USP, Poloxamer NF. Polyoxyl-40-Su arate USP and Aluminum Chloride. Examples of buffers include Sodium Phosphate (mono, di and tribasic), Sodium Carbonate, Sodium Biphosphate. Sodium Bicarbonate USP. Citric Acid Monohydrate USP,Acetic Acid, Sodium Citrate USP, Phosphoric Acid, Glacial Acetic Acid USP, Sodium Hydroxide NF, Sodium Acetate USP, Potassium Citrate USP, Hydrochloric Acid NF, and Potassium Phosphates: (mono, di and tribasic). Examples of tonicity adjusters include Sodium Chloride USP. Dextrose USP. Glycerin USP, Potassium Chloride USP and Mannitol USP. Examples of chelating agents include Edetate Disodium USP, Edetate Monosodium. F.detic Acid NF.

Edetate Trisodium. Examples of antioxidants include Sodium Metabisulfite NF. Sodium Bisulfite, Sodium Thiosulfate USP, and Acetylcysteine USP. Other material may include Polysorbates (20-85) NF, Pluronic Fl 68, Pluronic F 127. and Polyethylene Glycol 300. 400, 6000 NF. [00053] The IPF compositions disclosed herein may be administered in a variety of manners, e.g.. orally, by inhalation, intradermally, intramuscularly, subcutaneously or intravenously. It may be in the form of an injectable solution or formulation, tablet, liquid formulation, lyophilized or aerosolized receptors.

[00054] In one embodiment, the IPF compositions disclosed herein are administered intramuscularly. Also, doses may be administered at least daily, weekly or monthly, for as long as treatment is required. In exemplary embodiments, the IPF is administered intramuscularly once a week for six week, twice weekly for eight weeks, or as sixteen injections with two injections on consecutive days per week for eight weeks.

[000551 The IPF disclosed herein may be administered via the composition 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 IPI is administered in about 8 mg or about 4 mg per 2 ml of formulation. The IPF may be administered in an amount of 57 μg per 1 kg of body weight of the patient. In mice. e.g.. IPF can be administered from about 0.1 to about 0.5 mg'kg of body weight and in πibbils. about 1.24 mg/kg, twice weekly for eight weeks.

[00056] The IPF disclosed herein may have 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 o\^' I L- 12 and tumor necrosis factor α (TNFα), inducing anti-tumor activity and tumor-specific cytolytic T-cells and inducing cancer-specific CDS^' T-cell response.

|00057] The IPF components disclosed herein may have one or more of the following phcnotypic effects: increasing the CD4 + CD45 RO + CD62 L population, increasing in CD4 ÷ CD45 RΛ - 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 when this phenomenon appears, and increasing the CD8 ÷ CCR5 - population.

|00058] The IPF component may have one or more of the following functional effects over time: increasing tin.¹ IFN-γ containing CD3 + CD4 + cells post stimulation in vitro, decreasing the 1L-4 containing CD3 +CD4 + cells post stimulation, and increasing the IFN-γ containing CD3 + CDS * cells over time.

DETAILED DESCRIPTION OF THE INVENTION

[00059] Pepsins (of which there are several isozymes) are the principal proteases in gastric secretions of adult mammals. They belong to the family of aspartic proteases and arc synthesized and secreted by cells in the gastric mucosa as inactive enzyme- precursors consisting of a signal peptide, an activation peptide and an occluded active enzyme. En route to the lumen of the stomach for protein digestion, the signal peptide is cleaved to yield the inactive proenzyme pepsinogen, which, on exposure to a low gastric pH (<4), cleaves in turn to yield mature, catalytically active pepsin.

[00060] Porcine pepsin w as one of the first enzymes to be studied, and is perhaps the best-understood aspartic protease. It has 327 amino acid (aa) residues, and a molecular mass of 34kDa (PNAS (U.S.) 70:3437-39 1973). Proteolytic activity of pepsin is at its highest at a pH of about 1 .8 to 3.5; it is inactivated at a pH of about 5 and irreversibly inactivated (denatured) at a pH of about 6-7. Owing to their importance, amino acid residues associated with the substrate binding (active) site have been a research focal point. Howcu r. it is apparently still not clear how much functional activity, if any. is influenced b\ the remainder of the peptide.

[000611 The family of aspartic proteases (aspartases) is characterized by aspartic acid residues at their active (catalytic) sites. Human pepsin, for example, has two active site aspartate residues (coded "D^" or "Asp^"). This family also includes the HIV protease (and its numerous variants), comprising two identical chains each having a single active- site aspartate residue. Essential for maturation of the newly synthesized virus, which is expressed as a polyprotein. this protease has become a popular target for researchers attempting to block HIV replication.

[00062| Certain embodiments of the present invention are generally directed to providing isolated peptides characterized by, respectively, the amino acid sequences TLTSGGGAIALPPSMAAPPLGPVAPLTGAIHAPTXG (SEQ ID: NO 1), a fragment of approximately 45 kDa: TLSTΛTGGAIPPVAAMPPGLVAPTHGPAIHP (SEQ ID: NO 2): NXVPVSYEGYXQITLDS1TX (SEQ ID: NO. 3), a fragment of approximately 13.5 kDa: MTLTTGSGAIAPAM I'PGLPPHTGAIHAPM (SEQ ID: NO. 4); and CCATSGPCGAVMILTPHL I A (SEQ. ID: NO. 5), and a significant in vitro binding affinity for gp%. The peptido. referred to herein as, respectively, IPF-I , IPF-2, IPF-3, IPF-4 and IPF-5 (Inactivated Pepsinogen Fragments- 1-5), were isolated from porcine pepsinogen, purified, and irreversibly inactivated for use in cancer therapeutic procedures.

[00063] The present in\ ention also encompasses a cancer preventive or therapeutic vaccine comprising IPFgp96. and more specifically IPF-I gp96; IPF-2gp96; IPF-3 gp96, IPF-4gp96 and lPF-5gp96 or combinations thereof, which may be mixed with one or more polynucleotides encoding a molecular adjuvant. Any molecular adjuvant that increases cellular immune response may be used like cytokine IL-2. Administration may be via an intramuscular injection. The cancer to be treated may be primary or metastatic and the patients to be treated may have multiple different types of cancer.

[00064| The heat shock protein, e.g.. gp96. may be prepared according to suitable methods known in the art, such as according to the methods set forth in Chandawarkar, et al. (hit 7 Immunology, Vol. 16. No. 4, 615-624 (2004)), incorporated in its entirety by this reference. Complexes comprising HSP and IPF may be prepared according to suitable methods know n in the art. such as disclosed in application No. PCT/US2006/038045, also incorporated in its entirety by this reference. [00065| Figure 9 is a graph showing binding of an IPF with heat shock protein gp% at dilutions of IPF 1/2500. IPF 1/500 and IPF 1/100, according to one embodiment of the invention. A number ol approaches may be used to detect complexing between IPF and gρ%. By way ol^' example, one such approach may be to obtain specific antibodies against IPF and gp% and to build a sandwich immunoassay by suitable methods know n in the art to detect the presence of these protein complexes. To detect binding of gp°6 to IPF, the antigen may be coated on a plate to capture proteins from the sample and then report the binding with a specific detection antibody. The secondary (or detection) antibody may be directly labeled with MSD SULFO-TAG NHS ester or a SULFO-TAG-labelcd anti-species, e.g.. anti-mouse if the detection antibody is raised in mouse and the detection antibody is raised in a different species than the capture or primary antibody . Antibodies specific to IPF-gp96 complexes may also be used.

|00066] The cancer pre\ entivc vaccine may comprise a clear liquid opalescent suspension of spontaneous precipitate IPF (e.g., IPF-I, IPF-2, IPF-3, IPF-4 and/or IPF-5) and gp96 molecules with IL2 n> adjuvant and may comprise complexes of IPF-I, IPF-2, IPF-3. IPF-4 and IPF-5 and gp⁽>6. Activity is preferably 1 :0.6 measured by the ability of IPF to bind with gp96 (e.g., three molecules of IPF bound two molecules of gp96). The cancer preventive vaccine may be injected intramuscularly one injection per week for six weeks. The immune response probably gives evidence of two actions: 1) cytotoxic effect against tumor cells (cytotoxic T lymphocytes (CTLs) are effectors of CD8+ that can mediate the lysis of target cells bearing antigenic peptides associated with a MHC molecule. Other cytotoxic cells include gamma/delta chain and CD4+ NK 1.1+ cells); and 2) increased antibody production.

|00067] Other embodiments of the present invention are generally directed to prov iding an isolated anth iral peptide characterized by the amino acid sequence GDEPLENYLDTEYF (SEQ ID:NO 6) (-Gly-Asp-Glu-Pro-Leu-Glu-Asn-Tyr-Leu-Asp- Thr-Glu-Tyr-Phe-) ("IPF-6^") and a significant in vitro binding affinity for HIV-I gp 120, gp 41 and human CD4 cells. The peptide has anti -retroviral activity in vivo, particularly anti-HlV- l

The peptide, referred to herein as IPF-6 (Inactivated Pepsinogen Fragment-6). was isolated from poreinc pepsinogen, purified, and irreversibly inactivated for use in HlY- I prophylactic, therapeutic and diagnostic procedures. IPF-6 is expected to have anti-retroviral activity //; vivo, particularly inhibition of HIV-I entry into human CD4÷ cells.

[ϋ0068| The exemplified peptide was obtained from porcine pepsinogen (Fig. 1) by isolation from a 45 kDa band of IPF preparation under gel electrophoresis (Fig.2). It may also be derived from pepsinogen from any other source containing this sequence, or from any other peptides or proteins containing this sequence whereby suitable source pepsinogens are readily av ailable commercially. Common laboratory methods and reagents for selectively cleav ing intact proteins and for isolating and sequencing the cleaved peptides, such as the Hrdman degradation process, may be used. The peptide may also be produced by peptide synthesis, using conventional methods. Moreover, genetically engineered constructs expressing the sequence of interest are generally preferred, although chemical sy ntheses may also be used.

(00069| The peptides in the IPF fractions may be isolated and concentrated by any one of sev eral techniques w ell-know n to those skilled in the art, such as ammonium sulfate precipitation. The produced peptide isolate may be purified by standard processes such as gel filtration and RP-I I PLC. and inactivated by exposure to a neutral-to-alkaline env ironment of about pH 6.5 or greater or as otherwise known in the art. The peptide may also be alkylated to increa.se immunogenicity if desired, for example, by the process described for methylation in I S. Patent Application Publication US 2004/0018639 Al . A HPLC ehromatogram of the purified, inactivated IPF-6 product in one embodiment of the invention is shown in Fig. 3.

(0007Oj Homologues or analogues of the sequence which conserve at least critical binding site amino acid structures and functions and also conserve any distal structural/functional residues essential for binding activity as described herein may be substituted for the IPF of SEQ ID: NOS. 1 -6 (IPF-l -IPF-6. Variants of the sequences, including chemically modified derivatives, having a high sequence similarity will be generally preferred, provided that binding activity is not significantly adversely affected. Residues superfluous to the disclosed function of the peptides of the invention may be deleted or added with the same proviso. Modified sequences may be evaluated for conserved binding activity b\ . lor example, following the binding assays described herein or in the literature. Numerous databanks are accessible for protein sequence analysis, such as those which combine sequence similarity with fold recognition to predict functional equivalents. Binding properties (affinity, specificity, etc.) may also be evaluated by the binding assa\ > described below or other conventional assays, as known in the art.

|00071) BIACORE assa>s were used for binding affinity. See Figure 6. This technology measures mass concentration of biomolccules close to a surface. The surface is made specific by attaching one of the interacting partners. Sample containing the other partner(s) flow s over the surface: when molecules from the sample bind to the interactant attached to the surface, the local concentration changes and an SPR (surface plasmon resonance) response is measured. The response is directly proportional to the mass of molecules that bind to the surface. The SPR response can be expressed by resonance units (RU). One RU represents change of 0.0001 in the angle of the intensity minimum which is equivalent to a change in concentration of 1 pg/ mm.

|00072| The exact com ersion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the concentration change. A.ssays tracking the binding of IPF with CD4 cells, gp41, gpl 20, and human sera are very important to detect the formation of the super antigen responsible for the specific immune response. That is, the high response of IPF fragments, measured in Response Units, indicates a high utility as a specific binding agent for components of HIV. [00073| IPF-6 demonstrates binding in vitro with nonglycolysed fragment 579-

601 of the HIV- I envelope protein gp4l subυnit, with gpl20 HIV-I subunit, with human CD4+ cells and with human serum under gel electrophoresis (Biocor method) (see, Figs. 4-8). The assays for these bindings were conducted by The Institute for Molecular Medicine using standard protocols. The spontaneous binding of IPF-6 with the gp41 subunit is a particularly important biological property. Separately, under simple agarose electrophoresis. IPF-6 and gp-J l move in opposite directions. However, when they are mixed prior to electrophoresis. gp4 I changes direction and takes the direction of IPF-6. Quantitative analysis showed that lhe binding capacity ratio of IPF-6 to gp41 was 1 :0.66. That is. three molecules of IPF-6 bound two molecules of gp41 to form a complex which may function in vivo as, for example, a supcrantigen with significant anti-HlV-1 biological activity. Such antigen can be used as a bioassay reagent, in the production of mono- or polyclonal antibodies, in the manufacture of vaccines, and in other applications wherein antigens are conventionally employed.

[00074] While the mechanism of these binding events is not completely understood, it is contemplated that exposure of HIV-I to the IPF-6 of the present invention will effectively block gp41 and gpl20 domains essential for viral entry into CD4+ cells and inhibit viral infection, in vivo and in vitro. It is also contemplated that the IPF-6 of the present invention will effectively compete with HlV-I for its CD4+ cell surface binding sites and inhibit virus entry into these cells, in vivo and in vitro. Various in vitro protocols are know n in the art for predicting in vivo antiviral activity of compounds lor inhibiting replication of HlV, and these protocols may be used in connection with the practice o( the present invention. Exemplary art-recognized anti- HIV screening assays arc cited in U.S. patent 5,869,522, issued 9 February 1999 to Boyd ct ai, including those described in J. Virol. Methods, 33:87-100,1991; J.Natl. Cancer Inst., 81 :577-586. I 992: J.Med. Chan 35: 1978- 1986, 1992, and Boyd, M.R., in AIDS Etiology: Diagnosis. Treatment, and Pm anion, pp305-3 l 9 (Lippincott, 1988, DeVita, V.T., Jr., et a!., eds). 100075 j In accordance w ith one aspect of the present invention, IPF-6 is used to diagnose viral infection, particularly HIV-I infection. Bioassays suitable for this purpose are well-known and routine. Typical of these are assays based on competitive binding between, for example, a known amount of a viral protein and a biological sample to be tested for the same viral protein, using an excess of antiviral reagent capable of specifically binding with the known protein, such as an antibody. A mixture of these is incubated and the amount of bound complex calculated and compared to that in a control mixture lacking the sample. The presence, if any, and amount of the viral protein in the sample can then be determined. There arc numerous variations on this process, such as sandwich assays, assays with immobilized reagent, assays using labeled reagent (e.g., ELISA. RlA. FIA), and so on. Whatever the variation, whether for detecting or quantifying complex, or for additional reagents, or other modification, they all require a binding agent for the unknown sample. Any of these routine binding assays for quantifying or identifying an unknown sample may thus be used in the practice of the present invention by substituting IPF-6 as the antiviral binding agent for samples to be tested for HIY- ] gpl20, gp4 l . or infected CD4+T-cells.

|00076] In accordance with another aspect of the present invention, IPF-6 is used as a prophylactic or therapeutic to prevent or to treat HIV infections. (Herein the term "HIV infections^" refers to AIDS and ARC in addition to viral infection per se unless otherwise noted). For in Wvo use. the IPF compositions disclosed herein may be prepared for administration by mixing it at the desired degree of purity with a pharmaceutical Iy- acceptable carrier suitable for the route of administration, as well-known in the art. Although the IPF compositions disclosed herein are desirably administered with an adjuvant in sonic applications, in situations where a series of IPF doses are administered, boosters with the respective IPF may not require adjuvant. Intramuscular or subcutaneous injections are presently the contemplated route for both therapeutic and prophylactic administration of the IPF compositions disclosed herein. However, intravenous delivery, delivery via catheter or other surgical tubing, or other parenteral route may also be used. Alternative routes include oral routes for administering tablets, liquid formulations and the like, as well as inhalation routes. Liquid formulations reconstituted from powder formulations may be utilized. The IPF compositions disclosed herein may also be administered \ ia microspheres, liposomes, or other microparticulates, and via delivery systems or sustained release formulations dispersed in certain tissues including blood.

|00077| The dosage administered of the IPF compositions as disclosed herein will depend upon the properties of the formulation employed, e.g., its binding activity and in vivo plasma half-life, the concentration of IPF in the formulation, the administration route, the site and rate of dosage, the clinical tolerance of the patient involved, the patient^'s condition, and other considerations, as known in the art. Different dosages may be utilized during a scries of sequential treatments. The practitioner may administer an initial dose and then boost with relatively smaller doses of IPF. The dosages of the IPF compositions disclosed herein may be combined with other HIV antivirals such as AZT.

EXAMPLES

[00078] Isolation and Purification of Irreversibly-Inactive Pepsin Fraction

[00079| The following Examples are examples of methods for isolating, purifying, and characterizing the IPF compositions disclosed herein from active pig pepsinogen.

EXAMPLE I: Isolation and Innervation of Pepsinogen Fragment

[00080| All the buffers and solutions used in this section were sterilized by filtration. If needed, the buffer-. (0.2 N or 0.1 N HCl) were used to adjust the solutions. All the chemicals, including the distilled water, for the preparation of the buffers and solutions w ere USP Grade. The ratio of the pepsin to the buffers was 1 :4 (weight weight).

[000811 IPF was isolate! from active pepsin (Sigma 1 :10000) by ammonium sulfate precipitation with ccntπ (ligation at about 4⁰C. The lyophilized pepsin powder was dissolved in 0.14M sodium chloride (NaCl), 0.05M sodium acetate (CH₃COONa . 3H₂O), 0.05M sodium citrate (CJ IsO₇Na₂ . 2H₂O). and 0.20N HCl (pH 2.8-3.2) buffer. The pH of the active pepsin suspension w as then increased to 6.2-6.6 and the suspension was incubated for 30 minutes. The suspension was then precipitated with a saturated solution of (NH₄J₂SO₄. After degradation, the mixture was centrifugcd (8000 RPM at 4⁰C) for 60 minutes and the supernatant discarded. The pellet was dissolved in a minimum quantity of 0.14 M NaCl. and the resulting solution was dialyzed for 18 hr against dialysis buffer: 0.1 M NaCl. 0.1 M sodium acetate, and 0.02M thimerozal USP, pH 6.8.

EXAMPLE II: Purifictioii and Recovery of Irreversibly Inactivated Pepsinogen

Fragment

[00082| The purification of IPF included the following steps: dialysis, centrifugation. gel filtration, and reversed-phase HPLC.

[00083| After dialysis, the low molecular weight dialysate was centrifuged at

15.000 rpm at 4 C for 60 minutes (Bcckman rotor) with precipitation of the residual ammonium sulfate. The product was purified by gel filtration to recover purified IPF from the crude mixture, and thai purified by filtration on Bio-gel P lO or Scphadex G-75 gels (from Pharmacia Uppsala. Sw eden), or 0.2μ SFCA membrane (Nalgene Labware, Rochester. NY.). Further purification was achieved by reversed phase high-performance liquid chromatography in an RP-HPLC system GOLD (Beckman) on C- 18 columns (RP Ultrasphere 10mm Spherical XOA Preparative 21.2x150mm) using gradient 30% acetonitrile diluted in sterile w ater, HPLC-gradc at 15% methanol HPLC-grade mobile phase. Detection 254 nm; flow rate 0.850 ml/min., solvent at pH 6.8. The final purification step included sterile filtration with Nalgen filters 0.45μ. The HPLC elution profile of the product showed one isolated peak, IPF(see Fig. 3).

EXAMPLE I I I: Determination of Molecular Weight [00084] Molecular weight was determined by silver stained 13% non-reducing

SDS-PAGE using the Laemmli method (Nature 227-680, 1970). The molecular weight standard demonstrated one peptide with a molecular weight of 45.000 KD (Fig. 2). This band was isolated, and HPLC chromatogram (Fig. 3) confirmed a single peptide in the band.

EXAMPLE IV: Assessment of Binding Activity

[00085] Samples of IPF (*≠ I 8. 19. 20, and 21 ) were used to detect binding with gpl 20. gp41. CD4+ cells, and scrum from a healthy patient. New chips were coated with these proteins and Biacore assays for binding activity were performed. These samples were diluted to 1 :2000. 1 :500 and 1 : 100. The results are shown in Figs. 4, 5, 6, and 7. Sample #21 bound to all target proteins better than the other samples. The assay used a Biacore (Biacore AB. L^'ppsala. Sweden) system based on sensor chips which provide surface conditions for attaching molecules of interest, a microfluidic flow system for delivering samples to the surface, and a surface plasma response (SPR) which detects mass concentration at the surface. SPR-based biosensors monitor interactions by measuring the mass of molecules bound to the surface. This response is expressed by resonance units (RU), \vhcrcb\ a change in concentration of 1 pg/mm is equivalent to a change of 0.0001 in the angle of intensity minimum, which equals one RU. The exact conversion factor between RL^' depends upon the properties of the sensor surface and the nature of the molecule responsible for the change in concentration. The assays demonstrate the formation of superantigen for provoking immune response.

EXAMPLE V: UV Absorption

|00086| Circular dichroism (CD) provides information about the secondary structure of optically active materials. The far-UV or amide region (170-250nm) is dominated by contributions of the peptide bonds, whereas CD bands in the near-UV region (25()-3O()nm) originate horn the aromatic amino acids. The UV region of IPF was in the ranue of 252-260nm. EXAM PLE VI: Methylation of IPF

[00087] The purified IPF w as methylated to form a methylated IPF preparation as follows. IPF w as mixed with CH2BrCOOH in sufficient quantity to produce a final solution of 0.2 M CH2BrCOOl 1. For example, 2.78g CH2BrCOOH was dissolved in 10 ml distilled w ater and added to 100 ml IPF from the HPLC purification step, containing 70()mg IPF (7nig ml). The pH of the mixture was adjusted to and maintained at 7.2 with 0.1 M NaOH. The mixture was allowed to incubate from 6 to 8 hours. The IPF protein in the resulting aqueous fragment was concentrated by ammonium sulfate precipitation, using techniques known in the art. To 10ml of the methylated IPF was added an equal volume of saturated ammonium sulfate. The mixture was refrigerated at 4 C for 12 hours and then centiϊfuged at 15000 rpm for 60 minutes. The pellet was removed and dissolved in final buffer containing 0. I M NaCl, 0.1 M sodium citrate, and 0.02M thiodiglycol. The mixture was then dialyzed against the same buffer, to remove ammonium sulfate, for 24 hours.

|00088] Methylated IPF is lyophilized to form a slightly yellow powder. It is stable at room temperature bui is preferably stored under refrigeration. The molecular weight of the IPF fragment was determined by silver stained 13% non-reducing SDS- PAGE (sodium dodecyl suli^'ak-polyacrylamide gel electrophoresis) using the Laemmli method (Laemmli. U.K., Nature. 227:680 (1970)). Standards were: bovine serum albumin (66.000 molecular weight. MW): porcine heart fumarose (48,500 MW); bovine erythrocytes carbonic anhydrase (29.000 MW); bovine milk beta-lactoglobulin (18,400 MW); and bovine milk α-lactalbumin ( 14,200 MW). The isoelectric point of the IPF composition is about 6.2 as determined by isoelectric focusing. Pepsin, in contrast, has a low pi.

KXAMPLE VII: Adjuvant The IPF may be form u ta ted in an aluminum hydroxide adjuvant. 1 ml of an IPF formulation eυnlains: 4 mg IPI-^". 0.016 M AlPO.sub.4 (or 0.5 mg Al.sup.3+), 0.14 M NaCI, 0.004 M CH.sub.3COO\a. 0.004 M KCl. pH 6.2. ALUM-aluminum hydroxide; Al (OH) 3. Aluminum hydroxide is a widely used adjuvant, especially in commercial products such as vaccines. It is very well suited for strong antigens. Many sources of aluminum hydroxides arc available, e.g. Alhydrogel, Accurate Chemical & Scientific Co.. Westburv. New York.

EXAMPLE VII: Preparation of IPF Injection for Treating HIV

[00089| The inactivated pepsin fragment suspension may be prepared for injecting a preparation of highly purified inactivated pepsin fragment, such as the IPF-6 fragment with a molecular weight of 45 KDa.

[00090| For example, the formulation may comprise (w/v) 0.4% inactivated pepsin fragment. 0.23¹O aluminum phosphate U.S.P.. 1.29% sodium citrate U.S.P., 0.41% sodium acetate I \S. P. and water for injection to 100%. For a 1000 ml batch: place 900 ml of U. S. P. sterile water into container, preferably glass. Add 12.9g sodium citrate and mix until dissolved. Add 4.1 sodium acetate and mix until dissolved. Add 4g inactivated pepsin fragment, mix until a IK -HiOgCnOUS clear solution is obtained. Filter the resulting solution through a sterile 02.μιu filter into a sterile depyrogenated 2 liter container with a sterile magnetic stirrer. Sterile filter 55 ml of 0.016 M trisodium phosphate solution into the above 2 liter sterile container. Sterile filter 50 ml of 0.016 M aluminum chloride solution into the 2 liter container, with the aluminum chloride being dispensed at a steady, drop by drop rate. Stir the resulting inactivated pepsin fragment suspension for 30 minutes at room temperature. Continue stirring for another 6 hours at 4⁰C. The sterile inactivated pepsin fragment suspension is ready to be filled into sterile 3 ml borosilicate vials.

|000911 The final I ml of the final IPF formulation may contain: 4 mg IPF (purity preferably ^■ 96% ±0.290): 2.26 mg 0.016M AlPO₄ (or 0.5 mg Af ⁵); 4.1 mg 0.004M CH₃COONa (sodium acetate): and 12.9 mg C₆H₅O₇ (sodium citrate); pH 6.2. In yet a further embodiment, the formulation may comprise per vial, about 8 mg IPF, 4.52 mg aluminum phosphate. 1.0 mg aluminum. 25.8 mg sodium citrate and 8.2 mg sodium acetate. In one regimen. 2 ml of this formulation makes up one vial with the dosage per patient per da> being 16 vials. During the regimen, the patient should be monitored to assess the effectiveness of the regimen. CD+4 cell counts are useful and common methodology for evaluating H IV infection, as are assays for antibody or T-cell titers.

EXAMPLE \ I I: IPF Formulation and Adminstration

[00092| The following jv an example of a contemplated IPF formulation, dosage and administration schedule, w hich may be used with the IPF compositions disclosed herein:

|00093] The patient is administered an intramuscular injection containing 8 mg of the IPF composition (preferably 2 ml of a formulation containing 6 mg/ml of IPF in a pharmaceutically acceptable solution) or 57 μg of IPF protein per kg body weight of the patient. Each treatment course consists of 16 injections, with two injections on consecutive days per week for 8 weeks. Three months after the last injection, if the patient^'s condition warrants, the treatment regimen is repeated. The treatment regimen may be repeated until satisfactory results arc obtained, e.g., a halt or delay in the progress of the infection or disease, an alleviation of the infection or disease, or a cure is obtained. Preferably, in this application. IPF will be formulated with an aluminum hydroxide adjuvant.

EXAMPLE VIV

|00094| Table I includes data from a preliminary study of a 54 year-old male patient with 4" stage pancreatic carcinoma treated with two cycles of treatment with IPF, which were administered to tin- patient in September, 2008. Unenhanced images of the liver were obtained at 5mm inter\ al and thickness. Following bolus of IV infusion of 125 ml of nonionic contrast (Isox ur 370) parameters used were: abdomen and pelvis 5 mm thick helical scans: axial reconstructions were obtained at 2.5 mm slice thickness and 2 mm slice intcn al; patient received oral contast. Prior examination was on April 18, 2008. In this study, the full treatment comprises three cycles of 16 vials of IPF each cycle.

[00095| In the second examination, tumor measurement was obtained with a CT scan clone in November 200S. ( T scan of the abdomen with limited imaging through the lower lungs showed that the patient had two very small focal densities in the right middle lobe likle atelectasis. No pleural effusion were seen. In the liver is significant for multiple hypodesnse lesions consistent with metastases. Many of these appear to decrease slightly in size when compared to the prior examination. The lesion in the posterior right lobe currently measures 35 x 22 mm. Previously it measured 43 x 33 mm. A lesion in the anterior left lobe currently measures 18 x 17 mm. previously it measured 26 x 25. The other lesions all appeared to decrease in size. There was a hypodense lesion seen in the spleen slightly decreased consistent with a metastatic lesion. The pancreatic mass was not well demonstrated due to adjacent stomach. It measured approximately 40 x 48 mm slightly decreased in size. The patient has had a prior cholecystectomy. Left adrenal masses were demonstrated suggesting metastases not significantly changed. The kidneys showed normal excretion of contrast bilaterally. A large amount of ascites is present new compared w ith prior examination.

|00096] No definite retroperitoneal lymphadenopathy or pelvic lympadenopathy was demonstrated. Two small focal right middle lobe lung densities were likely atelectasis. Multiple hypodense lesions consistent with metastases all were slightly decreased when compared with the prior examination. Splenic lesion had decreased. Left adrenal masses not significantly changed. Large amount of ascites present new. Pancreatic mass poorly demonstrated bu likely slightly decreased. The finding on the CT scan show ed that most of the lesion in the liver, pancreas and spleen had decreased in size. The patient^'s CA 19-9 at 19. returned to normal. [00097| The IPF composition used in this study comprised IPF-IL2 adjuvant, specifically the study was done using SEQ ID: NOS. 1 and 2. The Roche Modular El 70 CA 19-9 electiOchcmiluminescvnt immunoassay was used.

|00098] T VBLE 1

[00099| A person skillet! in the art would appreciate that exemplary embodiments described hereinabove arc merely illustrative of the general principles of the present invention. Other modifications or variations may be employed that are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations may be utilized

accordance with the teachings herein. Accordingly, the drawings and description are illustrative and not meant to be a limitation thereof. -

[0001001 Moreover, all tei ins should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Thus, it is intended that the invention cover all embodiments and variations thereof as long as such embodiments and variations come within the scope of the appended claims and their equivalents.

Claims

CLAIMSWhat is claimed:

1. An isolated irreversibly inactivated anti-cancer pepsin characterized by the amino acid sequence of SEQ ID: NO. 1

2. A composition comprising the isolated irreversibly inactivated anti-cancer pepsin of claim I . further comprising a carrier.

3. A composition for inferring immunity against malignant human cells, said composition comprising the composition of claim 2 combined with IL2

4. A therapeutic composition comprising a complex of the composition of claim 1 bound to human gp96.

5. A therapeutic composition comprising a complex of the composition of claim 1 bound to a receptor disposed on human gp96.

6. The composition of claim 5. where in the receptor is CD91.

7. A 45kD pepsinogen fragment consisting of the isolated irreversibly inactivated anti-cancer pepsin of claim I , wherein said pepsinogen fragment has a binding affinity for human gp96.

8. A composition comprising the therapeutic composition of claim 4, said composition further comprising IL2.

9. An isolated irreversibly inactivated anti-cancer pepsin characterized by the amino acid sequence of SEQ ID: NO. 2.

10. A composition comprising the isolated irreversibly inactivated anti-cancer pepsin of claim 9. further comprising a carrier.

1 1. A therapeutic composition comprising a complex of the composition of claim 9 bound to human gp96.

12. A composition comprising the therapeutic composition of claim 1 1, said composition further comprising IL2.

13. A thirty one amino acid pepsinogen fragment having a binding affinity for human gp96.

14. An isolated irreversibly inactivated anti-cancer pepsin characterized by the amino acid sequence of SEQ ID: NO. 3.

15. A composition comprising the isolated irreversibly inactivated anti-cancer pepsin of claim 14. lurthcr comprising a carrier.

16. A therapeutic composition comprising a complex of the composition of claim 14 bound to human μp96.

17. The therapeutic composition of claim 16. wherein the irreversibly inactivated anti-cancer pepsin ol claim has a molecular weight of approximatley 13.5kD.

18. The composition of claim 16 further comprising IL2.

19. A composition comprising an IPF gp96 complex, wherein said composition demonstrates supcraiitigcn activ ity.

20. A complex of gp96 and IPF. wherein the complex activates major histocompatibility eomρlcx(HMC)- restricted manner.

21. An isolated irreversibly inactivated anti-cancer pepsin characterized by the amino acid sequence of SEQ ID: NO. 4.

22. A composition comprising the isolated irreversibly inactivated anti-cancer pepsin of claim 21 . Uirther comprising a carrier.

23. A therapeutic composition comprising a complex of the composition of claim 21 bound to human gp%.

24. An isolated irreversibly inactivated anti-cancer pepsin characterized by the amino acid sequence of SEQ ID: NO. 5.

25. A composition comprising the isolated irreversibly inactivated anti-cancer pepsin of claim 24. lurthcr comprising a carrier.

26. A therapeutic compt sition comprising a complex of the composition of claim 24 bound to human ;.;p96.

27. The composition ol^'daim 26. further comprising IL2.

28. A method of treating cancer, said method comprising the step of administering to a patient the therapeutic composition of claim 4.

29. A method of treating cancer, said method comprising the step of administering to a patient the therapeutic composition of claim 1 1.

30. A method of treating cancer, said method comprising the step of administering to ii patient the therapeutic composition of claim 16.

31. A method of treat ing cancer, said method comprising the step of administering to a patient the therapeutic composition of claim 23.

32. A method of treating cancer, said method comprising the step of administering to a patient the therapeutic composition of claim 26.