WO2012037551A2 - Agent biologique cellulaire principal et vaccin à long peptide synthétique wt1 - Google Patents

Agent biologique cellulaire principal et vaccin à long peptide synthétique wt1 Download PDF

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WO2012037551A2
WO2012037551A2 PCT/US2011/052139 US2011052139W WO2012037551A2 WO 2012037551 A2 WO2012037551 A2 WO 2012037551A2 US 2011052139 W US2011052139 W US 2011052139W WO 2012037551 A2 WO2012037551 A2 WO 2012037551A2
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Prior art keywords
cancer
antigen
cells
long peptide
chosen
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PCT/US2011/052139
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WO2012037551A3 (fr
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Neil Berinstein
Paul Naylor
James Egan
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Irx Therapeutics, Inc.
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Publication of WO2012037551A2 publication Critical patent/WO2012037551A2/fr
Publication of WO2012037551A3 publication Critical patent/WO2012037551A3/fr

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    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A61K39/001153—Wilms tumor 1 [WT1]
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Definitions

  • the present invention relates to vaccine immunotherapy. More specifically, the present invention relates to compositions and methods for treating cancer and other persistent disease with synthetic long peptide vaccines to tumor associated antigens such as WT1 in combination with a primary cell derived biologic.
  • Immune suppression or depletion involves a reduced capacity of the immune system to respond.
  • Such suppression can be drug-induced, i.e., by drug treatment, virus-induced, e.g., as in AIDS, or induced by a disease state such as cancer.
  • the immune system in this condition is effectively turned off.
  • a disease state such as cancer, the body is not able to protect itself against tumor antigens, thus allowing a tumor to grow and possibly metastasize.
  • a variety of tumor immunization strategies have been developed. All of these strategies are complex and deviate significantly from the conventional immunization strategies used for infectious diseases (see, e.g., Weber, 2000).
  • One such tumor immunization strategy involves THERATOPE® (Biomira), a sialyl Tn polysaccharide mucin antigen conjugated with keyhole limpet hemocyanin (KLH) and administered with DETOX® mycobacterium adjuvant and low dose cyclophosphamide (Maclean, 1996).
  • KLH keyhole limpet hemocyanin
  • KLH keyhole limpet hemocyanin
  • Use of this vaccine in patients with metastatic breast and ovarian cancer has yielded major clinical responses (i.e., greater than 50% tumor reduction) in only a low percentage of patients.
  • More recent therapeutic cancer vaccines that are currently being developed in the clinic are GlaxoSmithKlines
  • dendritic cell-mediated therapy e.g., wherein dendritic cells were pulsed with oligopeptide fragments of prostate-specific membrane antigens (PSMA).
  • PSMA prostate-specific membrane antigens
  • the dendritic cells (with or without the priming PSMA antigens) were then administered to patients with metastatic prostate cancer. Major clinical responses were obtained in only a low percentage of patients (Murphy, 1999; see also, Tjoa, 2000).
  • T cell mediated anti-cancer immune response primarily in the lymph nodes draining the site of the tumor or immunization, followed by T cell activation and migration to the peripheral sites.
  • the uptake of the antigen by tissue macrophages, neutrophils and/or dendritic cells and presentation of processed peptides in combination with MHC class I and class II antigens to the T cells in the lymph node are crucial to a complete immune response.
  • the kinetics of the immune response includes two phases. The first is the draining of the antigen and soluble proteins to the lymph nodes, where an initial immune activation occurs. Twenty four to forty eight hours later, antigen-presenting cells (APCs), most particularly dendritic cells, migrate from the site of immunization via the draining lymphatic ducts to the lymph node, where a second wave of presentation of antigen and activation occurs. More specifically, the APCs interact in the lymph node with precursor T helper cells via engagement of co-stimulatory receptors as well as T cell receptors to yield T helper 1 (Th1 ) cells and/or T helper 2 (Th2) cells.
  • APCs antigen-presenting cells
  • the ratio of these subsets controls subsequent development of either cell-mediated or humoral (antibody) immune responses (Th1 biasing towards DTH/cytotoxicity, whereas Th2 biases towards antibody production). Following the induction of these activated T cells, the immune response subsides, leaving predominately memory T cells, which are capable of responding upon re-exposure to antigen.
  • cytokines that bias the response in the direction of humoral or cellular immunity.
  • Locally produced cytokines such as IL-1 , IL-2, IFN- ⁇ , GM-CSF, IL-6, TNF-a, IL-I2 and IL-8, are associated with the recruitment of immune system cells, antigen uptake, dendritic cell maturation, dampening of T regulatory cell activity, T cell education and proliferation, and the development of Th1 cells (Naylor, 2003).
  • the interdependence of the response means that the activity of any given cytokine depends on the occurrence of precursor events such that the simultaneous presence of multiple cytokines can have different effects at both the injection site and the draining lymph nodes, depending on the kinetics of cell responses to different cytokines.
  • Adjuvants have thus been developed to accelerate, enhance, and prolong the immune response to vaccination and reduce the amount of antigen needed per dose. Adjuvants have been used for almost 100 years. Le Moignic and Pinoy first recognized that Salmonella typhimurium suspended in mineral oil potentiated immune responses in 1916. In 1926, Ramon demonstrated that an antitoxin response could be augmented by a large range of substances such as agar, tapioca, lethicin, starch oil, saponin, and breadcrumbs, and Attorney Docket No.: 3115-00170
  • Quil A was determined to have adjuvant properties in 1936 by Thibaud and Richou.
  • Quil A is a triterpenoid saponin extracted from the bark of the South American Molina soap tree Quillaja saponaria.
  • Freund also developed adjuvants from emulsions. Little progress has been made since then. As we begin to understand the immune system as described above, more progress is being made with adjuvants.
  • Several adjuvants have been developed for vaccines, including cancer vaccines, but still only alum has really had success worldwide.
  • Alum is a Th2 adjuvant; however, adjuvants providing a Th1 response are also desired.
  • Adjuvants are especially needed for population groups that do not sufficiently respond to conventional vaccines due to impaired immune response, such as elderly or immunosuppressed patients. Adjuvants have the potential to overcome immunotolerance; however, none have been very successful in doing so to date. Therefore, there remains a need for an effective adjuvant for various diseases including cancer.
  • WT1 Wilms tumor suppressor gene 1
  • WT1 is believed to have a role in various cancers, including leukemia.
  • WT1 is a 449 amino acid zinc finger transcription factor that is normally involved in cell growth and development.
  • WT1 has also been found to act as a tumor suppressor.
  • WT-1 is a potentially pivotal cancer antigen because it is expressed in low levels in adult tissues but at high levels in many types of cancer. More significantly, not only is it over-expressed in a majority of solid tumors but the over-expression appears to be required for leukemogenesis as well as the uncontrolled proliferation of many solid tumors.
  • Several vaccines have been developed to WT1 that have been made of synthetic long peptides corresponding to a length of amino acids in WT1 .
  • compositions comprise one or more of a WT1 polynucleotide, a WT1 polypeptide, an antigen-presenting cell presenting a WT1 polypeptide, an Attorney Docket No.: 3115-00170 antibody that specifically binds to a WT1 polypeptide; or a T cell that specifically reacts with a WT1 polypeptide.
  • Such compositions may be used, for example, for the prevention and treatment of metastatic diseases.
  • immune responses to WT1 mediate tumor protection and regression in various animal models.
  • the primary cell-derived biologic IRX-2 also previously known as a natural cytokine mixture (NCM), as disclosed in United States Patents 5,632,983 and 5,698,194, issued to Applicant, has been used to immunize patients and/or potentiate immunization, such as cancer patients or other patients with other antigen-producing lesions or disease states. More specifically, IRX-2 has been previously shown in United States Patent No. 5,698,194 to be effective in promoting T cell development and function in aged, immunosuppressed mice. IRX-2 was shown to decrease the proportion of immature T cells and increase the proportion of mature T cells in the thymus. The IRX-2 included IL-1 , IL-2, IL-6, IL-8, IFN- ⁇ , and TNF-a.
  • NCM natural cytokine mixture
  • the present invention provides for a composition for treating cancer or other disease, including a primary cell-derived biologic having the cytokines IL-1 , IL-2, IL-6, IL-8, TNF-a, and IFN- ⁇ , and a synthetic long peptide of a tumor associated antigen such as WT1 .
  • the present invention provides for a composition for treating cancer or other disease, including a synthetic long peptide vaccine of a tumor associated antigen such as WT1 .
  • the present invention provides for a therapeutic vaccine to cancer or other disease, including synergistic amounts of a primary cell-derived biologic having the cytokines IL-1 , IL-2, IL-6, IL-8, TNF-a, and IFN- ⁇ , and a synthetic long peptide of a tumor associated antigen.
  • a primary cell-derived biologic having the cytokines IL-1 , IL-2, IL-6, IL-8, TNF-a, and IFN- ⁇
  • a synthetic long peptide of a tumor associated antigen
  • the present invention provides for a therapeutic vaccine to cancer or other disease, including synergistic amounts of a primary cell-derived biologic having the cytokines IL-1 , IL-2, IL-6, IL-8, TNF-cc, and IFN- ⁇ , and a synthetic long peptide including at least one Class I MHC binding epitope and at least one Class II MHC binding epitope of a tumor associated antigen.
  • the present invention provides for a method of treating cancer or other disease by activating Tc effector cells, activating Th effector cells, and promoting antigen presenting cells differentiation, maturation and antigen presentation.
  • the present invention provides for a method of reversing immune suppression and gaining immunity to cancer by administering synergistic amounts of a primary cell-derived biologic including the cytokines IL-1 , IL-2, IL-6, IL-8, TNF-cc, and IFN- ⁇ , and a synthetic long peptide vaccine to a tumor associated antigen, stimulating the production of na ' ive T cells, maturing immature dendritic cells, and allowing presentation by resulting mature dendritic cells of the exogenous antigens in the cancer vaccine to the na ' ive T cells, thereby reversing immune suppression and gaining immunity to the cancer.
  • a primary cell-derived biologic including the cytokines IL-1 , IL-2, IL-6, IL-8, TNF-cc, and IFN- ⁇
  • a synthetic long peptide vaccine to a tumor associated antigen
  • the present invention also provides for a method of treating cancer by administering an effective amount of a synthetic long peptide of a tumor associated antigen.
  • FIGURE 1 is a mapping of sequences for HLA on WT1 , regions where significant numbers of HLA haplotypes are present are indicated with a bar, note the hot spot around RMF (bold) and the lack of hot spot abound CMT (bold);
  • FIGURE 2 is a chart of the design of overlapping peptides to capture as many predictive epitopes as possible, the red numbers in the box indicate positive Attorney Docket No.: 3115-00170 epitopes for at lease 1 HLA, the specific numbers represent the amino acid position of the sequences (either 9 mer for HLA-A and HLA-B or 15-mer for HLA-DR) that should be included in the sequence of a long peptide in order to capture the most epitopes, thus, there area three possible overlapping sequences that would capture the most epitopes;
  • FIGURE 3 is a chart of the sequences for initial evaluation for T cell responses
  • FIGURE 4 is a chart of long peptides from WT-1 that are predicted to have overlapping HLA and HLA-DR;
  • FIGURE 5 shows the location of peptides in WT-1 sequence as defined in FIGURE 4, note the overlap regions in order to maximize the epitopes in the 30-33 aa sequences;
  • FIGURES 6A-6E show finer mapping of the HLA-A and HLA-B and HLA- DR regions of WT-1 , the location of the 1 st and last aa of each sequence is indicated on the figure, prediction is based on sequence indicated rather than top 8-10 epitopes in the whole protein, boxes represents regions with the best overlap;
  • FIGURE 7 is a graph comparing two short peptides, as immunogens, HPVshort being the dominant epitope from HPV E7 protein and RMFshort being the predicted a dominant epitope from WT-1 ;
  • FIGURE 8 is a graph comparing the primary and expansion ELISpot response of primary and expanded cell populations from mice immunized with the RMFshort peptide in Montanide (S+M) with or without IRX-2, as indicated in the x-axis;
  • FIGURE 9A is a graph of response to IRX-2 with mice immunized with short peptides
  • FIGURE 9B is a graph of response to IRX-2 with mice immunized with long peptides
  • FIGURE 10 is a set of four graphs showing the epitope specific ELISpot response of long peptide immunized mice, the specific epitope being indicated at the top of each graph;
  • FIGURE 1 1 is a graph of response to PMF peptide.
  • the present invention relates to compositions and methods of immunotherapy and treating cancer or other antigen-producing persistent lesions or disease states. More specifically, the invention relates to compositions and methods for eliciting an immune response to antigens associated with a cancer or other antigen- producing disease or lesion, wherein a primary cell-derived biologic comprising IL-1 , IL- 2, IL-6, IL-8, IFN- ⁇ , and TNF-a, as well as a synthetic long peptide vaccine to a tumor associated antigen such as WT1 is administered to a patient in an amount effective to stimulate an immune response to the antigen in the patient.
  • a primary cell-derived biologic comprising IL-1 , IL- 2, IL-6, IL-8, IFN- ⁇ , and TNF-a
  • a synthetic long peptide vaccine to a tumor associated antigen such as WT1
  • the antigen is preferably an exogenous antigen and the primary cell-derived biologic acts as an adjuvant with the antigen to stimulate an immune response to the antigen in a patient.
  • the primary cell-derived biologic can also be used in combination with other adjuvants as described herein.
  • the term "adjuvant” denotes a composition with the ability to enhance the immune response to a particular antigen. Such ability is manifested by a significant increase in immune-mediated protection. To be effective, an adjuvant must be delivered at or near the site of antigen. Enhancement of immunity is typically manifested by either a significant increase (usually greater than 10 fold) in the titer of antibody raised to the antigen and/or enhancement of cellular immunity, which can be measured by a positive skin test, cytotoxic T cell assay, ELISPOT assay for IFN-y or IL- 2, or T cell infiltration into the tumor.
  • the cytokine compositions of the present invention are particularly suited to enhance T cell-mediated immune responses.
  • the adjuvant effects of the cytokine compositions of the invention include the generation of na ' ive T cells, the promotion of dendritic cell differentiation and maturation, the stimulation of monocytes and macrophages, and in the case of cancer patients, increased lymphocyte infiltration into tumors, tumor fragmentation, tumor regression, and reduction of sinus histiocytosis in the lymph nodes.
  • adjuvant system denotes combining adjuvants to work together to obtain optimally effective and safe formulation in which each part of the vaccine, the antigen and adjuvant(s), works together to produce an immune response.
  • Examples of such adjuvant systems are AS02,3 and 4 formulated vaccines made by Glaxo Smith Kline (GSK).
  • Vaccine refers to a vaccine including at least one antigen, and preferably more than one antigen, that is administered to a patient to create an immune response and preferably T cell response to the antigen(s) delivered.
  • the vaccine can include various other components such as a carrier or other stimulatory molecules.
  • the vaccines can be DNA-based vaccines, peptide-based vaccines, or protein-based vaccines encoded in various viral/bacterial vectors or cells.
  • an effective amount refers to an amount of primary cell derived biologic that is needed to achieve the desired result of the present invention, namely, producing an immune response to an antigen by a synergistic manner.
  • One skilled in the art can determine the effective amount of the primary cell derived biologic that should be given to a particular patient.
  • IRX-2 also known as “citoplurikin” is a leukocyte-derived, natural primary cell-derived biologic produced by purified human white blood cells (mononuclear cells) stimulated by phytohemagglutinin (PHA) and ciprofloxacin (CIPRO).
  • PHA phytohemagglutinin
  • CIPRO ciprofloxacin
  • IL-1 interleukin 1 ⁇
  • IL-2 interleukin 2
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • TNF-a tumor necrosis factor a
  • IFN- ⁇ ⁇ -interferon
  • the IRX-2 used in the present invention includes these six critical cytokines.
  • IRX-2 has also previously been referred to as an "NCM", a natural cytokine mixture, defined and set forth in United States Patent Nos. 6,977,072 and 7,153,499.
  • NCM a natural cytokine mixture, defined and set forth in United States Patent Nos. 6,977,072 and 7,153,499.
  • the terms IRX-2, primary cell-derived biologic, and NCM are used interchangeably herein.
  • IRX-2 is prepared in the continuous presence of a 4- aminoquinolone antibiotic and with the continuous or pulsed presence of a mitogen, which in the preferred embodiment is PHA. Other mitogens, however, can also be used.
  • the IRX-2 produced for administration to patients contains a concentration of IL- 1 ⁇ that ranges from 60 - 6,000 pcg/mL, more preferably, from 150 - 1 ,800 pcg/mL; a concentration of IL-2 that ranges from 600-60,000 pcg/mL, more preferably, from 3,000- 12,000 pcg/mL, and concentrations of IFN- ⁇ and TNF-a that range from 200-20,000 pcg/mL, more preferably, from 1 ,000-4,000 pcg/mL.
  • IRX-2 can also contain a concentration of IL-6 that ranges from 60-6,000 pcg/mL, more preferably, from 300-2,000 pcg/mL; a concentration of IL-8 that ranges from 6000-600,000 pcg/mL, more preferably from 20,000-180,000 pcg/mL; a concentration of TNF-cc that ranges from 200-20,000 peg/ml, more preferably, from 1 ,000-4,000 pcg/mL.
  • Recombinant, natural or pegylated cytokines can be used, or IRX- 2 can include a mixture of recombinant, natural or pegylated cytokines.
  • IRX-2 can contain only the above cytokines; however, other cytokines can be included.
  • the IRX-2 of the present invention can further include other recombinant, natural or pegylated cytokines such as IL-7, IL-12, IL-15, GM-CSF (at a concentration that ranges from 100- 10,000 pcg/mL, more preferably from 500-2,000 pcg/mL), and G-CSF.
  • the method of making IRX-2 is disclosed in the above cited patents as well as in U.S. Provisional Patent Application No. 61 /044,674.
  • Also encompassed by the present invention are derivatives, fragments and peptides related to the cytokines disclosed herein, wherein such derivatives, fragments and peptides retain the biological activity of their respective cytokines.
  • compositions for promoting immune suppression such as chemical inhibitors; nonsteroidal anti-inflammatory drugs (NSAIDS); zinc; and combinations thereof.
  • the chemical inhibitor can be any chemotherapeutic agent that is not immunosuppressive (preferably used at low doses) and that has immunomodulatory effects so as to increase immunity and/or an immune response, e.g., by inhibiting immune suppression or suppressor mechanisms in the body.
  • the chemical inhibitor is an anti-neoplastic agent, including but not limited to alkylating agents, antimetabolites and antibiotics.
  • the chemical inhibitor can also be an immunomodulating agent such as thalidomide.
  • the chemical inhibitor can also be in a salt or other complex form.
  • the chemical inhibitor is the alkylating agent cyclophosphamide (CY).
  • the NSAID is preferably indomethacin (INDO), which is both a Coxl and Coxll inhibitor.
  • the NSAID can also be ibuprofen or Coxll inhibitors such as celecoxib and rofecoxib, or combinations thereof.
  • the four components used together i.e. chemical inhibitor, NSAID, primary cell derived biologic, and zinc
  • the chemical inhibitor inhibits T regulatory cells; the NSAID reverses local immune suppression by prostaglandins the primary cell derived biologic activates dendritic cells, stimulates T cells, and protects T cells from apoptosis; and zinc provides key nutrients for T cell function as shown in FIGURE 2. This combined action encourages immune response to both endogenous and exogenous antigens.
  • the term "endogenous antigen” denotes an antigen that is produced and situated in vivo, i.e., within an organism such as a patient, such that, after the administration of the cytokine composition of the invention in vivo, the cytokines act as an adjuvant with the antigen within the patient to stimulate an immune response to the antigen.
  • exogenous antigen denotes an antigen that is produced, i.e., isolated or generated, in vitro, i.e., outside of an organism to be treated, and is administered to the organism (i.e., a patient) in vivo, such that, after the administration of the cytokine composition of the invention in vivo, the cytokines act as an adjuvant with the antigen within the patient to stimulate an immune response to the antigen.
  • the exogenous antigen can be a chemically synthesized or genetically engineered compound or molecule or can be an endogenous antigen that has been extracted from its in vivo environment and isolated in vitro.
  • the extracted antigen can be processed or otherwise modified for re-introduction in vivo.
  • the exogenous antigen can be administered either in a separate pharmacological preparation from the cytokine composition of the invention or in the same preparation.
  • the IRX-2 composition of the invention is effective in combination with exogenous prostate-specific membrane antigens (PSMA) in promoting immune responses in both mice and humans.
  • PSMA prostate-specific membrane antigens
  • Other exogenous antigens can be combined with IRX-2 as further described below.
  • tumor associated antigen denotes a protein or peptide or other molecule capable of inducing an immune response to a tumor. This can include, but is not limited to, PSMA peptides, MAGE peptides (Sahin, 1997; Wang, 1999), Papilloma virus peptides (E6 and E7), MAGE fragments, NY ESO-1 or other similar antigens. Previously, these antigens were not considered to be effective in treating patients based either on their size, i.e., they were considered too small, or they Attorney Docket No.: 3115-00170 were previously thought to lack immunogenic properties (i.e., they were considered to be self antigens).
  • SLPs synthetic long peptides
  • An SLP is more specifically defined as containing two or more MHC I or MHC II epitopes or the combination of at least one or more MHC I and one or more MHC II epitopes.
  • the synthetic long peptides in the present invention are preferably to any portion of WT1 , or to any immunogenic portion of WT1 (epitopes), or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera and/or T-cell lines or clones is not substantially diminished.
  • Synthetic long peptides vaccines offer several advantages over minimal peptide sequence vaccines, which are explained below. One of the key factors in vaccine efficacy is the context in which peptides are presented to the immune system.
  • Synthetic long peptides are not able to bind directly to MHC class I but need to be taken up and processed by dendritic cells and then presented to cytotoxic T cells.
  • minimal cytotoxic T cells peptides (8-10 amino acids), are readily loaded exogenously in MHC class I molecules and can be presented by both professional APC (DC) and non-professional APC (T cells and B cells) in vivo.
  • DC professional APC
  • T cells and B cells non-professional APC
  • the presentation of cytotoxic T cells peptide epitopes on non-activated B cells induces a transient cytotoxic T cells response which is followed by a subsequent deletion of these CD8+ T-cells.
  • loading of minimal MHC class I binding peptides directly onto DC can convert a cytotoxic T cell tolerizing peptide into a peptide that triggers the expansion of a tumor-protective cytotoxic T cell response.
  • Synthetic long peptides are primarily processed and presented by professional APC and therefore Attorney Docket No.: 3115-00170 stimulate cytotoxic T cells predominantly within the strong stimulatory environment of the inflamed draining lymph.
  • Another advantage of synthetic long peptides vaccines over minimal cytotoxic T cells peptide vaccines is the increased duration of in vivo epitope presentation in the antigen draining lymph node, which is important for clonal expansion and for IFN- ⁇ production by effector T-cells.
  • the present invention is directed to a composition for treating cancer including a primary cell-derived biologic (IRX-2) having the cytokines IL- 1 , IL-2, IL-6, IL-8, TNF-cc, and IFN- ⁇ , and a synthetic long peptide of WT1 .
  • IRX-2 acts as an adjuvant alone or in combination with other adjuvants to the synthetic long peptide, i.e. it stimulates an immune response to the synthetic long peptide.
  • IRX-2 acts in a synergistic manner with the synthetic long peptide of WT1 to create a greater immune response than can be achieved by administering the synthetic long peptide alone.
  • IRX-2 provides advantages over previous adjuvants because of its affect of completely "turning on” the immune system of a patient whose immune system is in any way suppressed from its full function. Without reversing suppression of the immune system, antigen cannot be presented effectively to treat cancer and other diseases.
  • the mechanism by which IRX-2 "turns on” the immune system is further described below.
  • the composition can include the synthetic long peptide of WT1 without the IRX-2.
  • compositions of the present invention can be used to treat many different types of diseases such as cancers, such as, but not limited to, head and neck squamous cell carcinoma (H&NSCC), breast cancer, colorectal cancer, stomach cancer, pancreatic cancer, lung cancer, brain cancer, colon cancer, ovarian cancer, tongue cancer, pharynx cancer, prostate cancer, and melanoma.
  • diseases such as cancers, such as, but not limited to, head and neck squamous cell carcinoma (H&NSCC), breast cancer, colorectal cancer, stomach cancer, pancreatic cancer, lung cancer, brain cancer, colon cancer, ovarian cancer, tongue cancer, pharynx cancer, prostate cancer, and melanoma.
  • non-cancerous persistent lesions such as infectious lesions that produce an antigen in vivo, e.g., Attorney Docket No.: 3115-00170 cutaneous or systemic candidiasis, papilloma virus-associated venereal warts, or cervical dysplasia
  • the IRX-2 is essentially as described above in the definitions section and can optionally include additional cytokines other than the six critical cytokines of IL-1 , IL- 2, IL-6, IL-8, TNF-cc, and IFN- ⁇ , and the composition can optionally further include the various other compounds that are administered in the typical IRX-2 regimen, i.e., a chemical inhibitor, an NSAID, and zinc also as described above.
  • the present invention also provides for a therapeutic vaccine to cancer or other disease, including synergistic amounts of a primary cell-derived biologic (IRX-2) having the cytokines IL-1 , IL-2, IL-6, IL-8, TNF-cc, and IFN- ⁇ , and a synthetic long peptide of WT1 .
  • IRX-2 primary cell-derived biologic
  • the cancer treated by the vaccine is prostate or ovarian cancer, but other cancers can be treated as described above.
  • the IRX-2 and the synthetic long peptide of WT1 act in a synergistic manner to achieve therapeutic results not obtainable with either component alone.
  • the synthetic long peptide of WT1 can be used in a vaccine without the IRX-2.
  • the vaccine can also include other antigens or other combinations of vaccines.
  • Common antigens used in cancer vaccines include AFP, alpha-actinin-4, ARTC1 , BAGE, BCR-abl, B-RAF, CA 15-3, CA 19-9, CA-1 25, CASP-5, CASP-8, beta.- catenin, carcinoembryonic antigen, modified carcinoembryonic antigen, carcinoma- associated mutated mucins, cdc27, CDK4, CDKN2A, CEA, chromogranin A, COA-1 , cyclin dependent kinase-4, dek-can fusion protein, EFTUD, elongation factor 2, epidermal growth factor receptor EGFRvlll, Epstein Barr Virus EBNA gene product, ETA, ETV6-AML1 fusion protein, FLT3-ITD, FN1 , GAGE, gangliosides, GPNMB, gp75/TRP-1 , gp100, H1
  • SSX2 fusion protein TA 90, TAG, TGF- ⁇ anti-apoptotic factor, TGF- RII, thyroglobulin, TRAG-3, triosephosphate isomerase, TRP2, TRP2-INT2, tumor protein D52, tyrosinase, WT1 , fragments thereof, derivatives thereof, and combinations thereof. Any other suitable antigen can also be used.
  • the antigens administered can be the antigens themselves in the form of naturally occurring proteins, recombinant proteins, chemically synthesized proteins, synthetic long peptides or combinations thereof.
  • the antigens can be encoded in a viral or bacterial vector, i.e. a nucleic acid vector.
  • the antigen is encoded in nucleic acid material, such as DNA or RNA.
  • viral vectors used for encoding nucleic acid include, but are not limited to, retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, and poxvirus.
  • poxvirus include vaccinia, modified vaccinia Ankara (MA), NYVAC, avipox, TROVAX, fowlpox, and canarypox.
  • canarypox include ALVAC and ALVAC(2).
  • One skilled in the art can encode the antigens in the desired vector.
  • compositions can further include co-stimulatory molecules to stimulate T cells.
  • T cells require two different signals in order to become fully activated.
  • the first signal is antigen-specific and comes from the T cell receptor interacting with peptide-MHC molecules on the membrane of antigen presenting cells.
  • the second signal which is the co-stimulatory signal, is antigen non-specific and is achieved by the interaction between co-stimulatory molecules expressed on the membrane of antigen presenting cells and the T cell.
  • Co-stimulatory molecules can be found naturally in the body; however, in an immune suppressed patient, additional molecules can be administered.
  • co-stimulatory molecules examples include abatacept, belatacept, CD28-SuperMAB, B7/CD28 co-stimulatory molecules, TNF superfamily co-stimulatory molecules, SLAM family co-stimulatory molecules as well as others.
  • compositions can also include compounds for blocking suppressive or negative regulatory immune mechanisms, such as anti-CTLA-4, anti-PD-1 or anti-PDL- 1 .
  • CTLA-4 Cytotoxic T-Lymphocyte Antigen 4
  • the CTLA-4 family of receptors, when engaged, inhibits activation of T cells, promotes cell cycle arrest, and decreased cytokine production. This is an important function in a normally functioning immune system to Attorney Docket No.: 3115-00170 prevent excessive immune responses.
  • inhibiting the CTLA-4 receptor in an immune suppressed patient is desired in order to remove some of the suppression and stimulate the activation of T cells.
  • compositions can further include a Treg depleting molecule.
  • Tregs or regulatory T cells, distinguish between self-antigens and non-self antigens and actively suppress immune activation. Depletion of Tregs can reduce suppression of the immune system in an immune suppressed patient. This depletion can be accomplished by administering a Treg depleting molecule such as, but not limited to, denileukin difitox (ONTAK®, Eisai Inc.).
  • optimal cellular immune response requires the activation and recruitment of both Th and Tc effector cells.
  • optimal antitumor response requires the presentation of tumor antigen epitopes displayed on MHC II molecules of mature APC to naive Th cells, and the presentation of tumor antigen on MHC I molecules of mature APC to na ' ive Tc cells.
  • IRX-2 is an extremely effective promoter of the differentiation and maturation of dendritic cells and other APC, and of their antigen presentation capabilities.
  • the present invention also encompasses a therapeutic vaccine for cancer or other disease, including synergistic amounts of a primary cell- derived biologic having the cytokines IL-1 , IL-2, IL-6, IL-8, TNF- ⁇ , and IFN- ⁇ , and a synthetic long peptide including at least one Class I MHC binding epitope and at least one Class II MHC binding epitope of a tumor associated antigen such as WT1 .
  • MHC I and II epitopes need not be limited to an SLP but can be any form of combination of MHC I and MHC II epitopes likely to be taken up and presented together by APC, such as mixtures of individual MHC I and II binding Attorney Docket No.: 3115-00170 epitopes and combinations of individual epitopes incorporated into liposomes or nanoparticles.
  • compositions can include angiogenesis-associated antigens in order to prevent or inhibit angiogenic processes occurring near or within tumors.
  • Angiogenesis-associated antigens can include, but are not limited to, VEGF, VEGF receptor, EGFR, bFGF, PDGF-B, PD-ECGF, TGFs including TGF-cc, endoglin, Id proteins, various proteases, nitric oxide synthase, aminopeptidase, thrombospondins, k- ras, Wnt, cyclin-dependent kinases, microtubules, heat shock proteins, heparin-binding factors, synthases, collagen receptors, integrins, and surface proteoglycan NG2.
  • compositions can further include potentiating agents CD80, ICAM-1 , and LFA-3 individually or in combination, collectively known as TRICOM, for potentiating the effect of the primary cell-derived biologic and cancer vaccine as well as the other components described above.
  • potentiating agents include IL-12 and GM-CSF; IL-12, GM-CSF, and TNF-cc; CD80 and IL-12; and CD86, GM-CSF, and IL-12.
  • the present invention also encompasses the use of both an endogenous antigen and an exogenous antigen, i.e., wherein the composition is administered to a patient having an endogenous antigen in vivo and wherein the cytokines act as an adjuvant with both the exogenous and endogenous antigens to stimulate immune responses in the patient.
  • the endogenous antigen can be present in regional lymph nodes or at the tumor site.
  • the present invention is also directed to a composition including the primary cell-derived biologic as described above, which itself acts as an adjuvant, and at least one other adjuvant in order to stimulate the immune system of a patient.
  • Exogenous antigen(s) such as in the cancer vaccines and the other components described above can also be included in the composition.
  • the composition is a multi-adjuvant composition that further enhances the effect of the antigens.
  • the primary cell-derived biologic not only potentiates the effect of the antigens as described above, but also the effect of the adjuvants, i.e. the primary cell-derived biologic acts synergistically with the adjuvants.
  • TLR-independent adjuvants act as delivery systems and help to concentrate antigens, target them to antigen-presenting cells, and colocalize antigens and immune potentiators.
  • TLR-dependent adjuvants directly stimulate the immune system through activation of TLRs.
  • the composition of the present invention can be used with a TLR-independent adjuvant, a TLR-dependent adjuvant, or combinations thereof. Using each kind of adjuvant can stimulate different parts of the immune system at once.
  • a TLR-independent adjuvant can traffic antigen and TLR-dependent adjuvants to APCs to stimulate antigen uptake and stability, while the TLR-dependent adjuvant directly enhances immunity through activation of TLR signaling and reduces potential toxicity of administering TLR-dependent adjuvants on their own. Also, using multiple TLR-dependent adjuvants can result in a synergistic effect of the adjuvants.
  • the adjuvants can be, but are not limited to, the following adjuvants.
  • TLR- independent adjuvants Alum (aluminum phosphate/hydroxide) is a mineral salt with various indications.
  • AS03 GSK; squalene (10.68 mg), DL-alpha-tocopherol (1 1 .86 mg), and polysorbate 80 (4.85 mg) is an oil in water emulsion that is used for pandemic influenza.
  • MF59 Novartis; 4-5% w/v squalene, 0.5% w/v Tween 80, 0.5% Span 85, optionally: varying amounts of muramul tripeptide phosphatidyl-ethanolamine (MTP- PE)
  • MTP- PE muramul tripeptide phosphatidyl-ethanolamine
  • Provax Biogen pie; squalene plus pluronic L121
  • Montanide Steppic SA; Bioven; Cancervax; mannide oleate and mineral oil
  • TiterMax (CytRx; squalene plus CRL-8941 ) is a water in oil emulsion.
  • Advax (Vaxine Pty; nanocrystalline particles of inulin) is a biopolymer that is used in vaccines against Hepatitis B (prophylactic and therapeutic), influenza, anthrax, shigella, Japanese encephalitis, rabies, bee venom, allergy, and cancer immunotherapy.
  • QS21 Antigenics; fraction of Quil A
  • Quil A (Statens Serum Institute; purified fraction of Quillaia saponaria) is a plant-derived composition used in various treatments.
  • ISCOM CSL; Isconova; saponin plus sterol plus, optionally, phospholipid
  • Liposomes Attorney Docket No.: 3115-00170
  • TLR-dependent adjuvants Ampligen (Hemispherx; synthetic specifically configured double-stranded RNA containing regularly occurring regions of mismatching) works by activation of TLR3 and is used as a vaccine against pandemic flu.
  • AS01 GSK; MPL, liposomes, and QS-21 ) works by M PL-activation of TLR4, liposomes provide enhanced antigen delivery to APCs, QS-21 provides enhancement of antigen presentation to APCs and induction of cytotoxic T cells, and this is used as a vaccine against malaria and tuberculosis.
  • AS02 (GSK; MPL, o/w emulsion, and QS-21 ) works by M PL-activation of TLR4, the o/w emulsion provides innate inflammatory responses, APC recruitment and activation, enhancement of antigen persistence at injection site, presentation to immune-competent cells, elicitation of different patterns of cytokines, and the QS-21 provides enhancement of antigen presentation to APCs and induction of cytotoxic T cells, and this is used as a vaccine against malaria, tuberculosis, HBV, and HIV.
  • AS04 (GSK; MPL, aluminum hydroxide/aluminum phosphate) works by MPL- activation of TLR4, alum provides a depot effect, local inflammation, and increase of antigen uptake by APCs, and this is used as a vaccine for HBV, HPV, HSV, RSV, and EBV.
  • MPL RC-529 (Dynavax; MPL) works by activation of TLR4 and is used as a vaccine against HBV.
  • E6020 (Eisa/Sanofi Pasteur; synthetic phospholipid dimer) works by activation of TLR4.
  • TLR-technology (Vaxinnate; antigen and flagellin) works by activation of TLR5 and is used in vaccines against influenza.
  • PF-3512676 (CpG 7909) (Coley/Pfizer/Novartis; immunomodulating synthetic oligonucleotide) works by activation of TLR9 and is used in vaccines against HBV, influenza, malaria, and anthrax.
  • ISS Denssionax; short DNA sequences
  • IC31 Intercell; peptide and oligonucleotide
  • TLR9 formation of an injection site depot, and enhancing of antigen uptake into APCs and is used as a vaccine against influenza, tuberculosis, malaria, meningitis, allergy, and cancer indications.
  • the primary cell-derived biologic and adjuvant combinations can be used for other indications than those currently recommended for the above adjuvants, and they especially can be used for cancer Attorney Docket No.: 3115-00170 indications as described herein.
  • the primary cell-derived biologic and adjuvant combination can be used to enhance the immune system and treat any of the diseases indicated specifically for the adjuvants or for any other disease described herein.
  • the present invention can include the primary cell-derived biologic and another adjuvant as described above that has a different mechanism of action than that of the primary cell-derived biologic.
  • the primary cell-derived biologic acts to effectively "turn on” the immune system by maturing immature dendritic cells, stimulate the production of na ' ive T cells, and effectively present antigen to the na ' ive T cells.
  • the mechanism of action is further described below.
  • the primary cell-derived biologic and adjuvant can produce a synergistic response in the immune system by stimulating multiple areas of the immune system at once.
  • the present invention provides for a method of treating cancer, by administering the composition described above including effective amounts of the primary cell-derived biologic including the cytokines IL-1 , IL-2, IL-6, IL-8, TNF-cc, and IFN- ⁇ , and a synthetic long peptide of a tumor associated antigen such as WT1 .
  • the composition is effective at treating cancer because the primary cell-derived biologic and the antigens interact in a synergistic manner to present antigens to T cells and activate the immune system.
  • the primary cell-derived biologic acts to stimulate the production of na ' ive T cells, mature immature dendritic cells, and allow presentation by resulting mature dendritic cells of the exogenous antigens in the cancer vaccine to the na ' ive T cells.
  • the mechanism of action is further described below.
  • the cancer vaccine would not be as effective in generating immunity to the cancer in the patient because the immune system would remain suppressed.
  • the administration step can be achieved as further described below, and preferably by perilymphatic injection once daily.
  • T cells in the patient can also be co-stimulated by administering co- stimulatory molecules as described above to generate antigen non-specific signals to T cells.
  • Inhibition of T cells can be prohibited by administering anti-CTLA-4 as described above in order to further stimulate the T cells as described above.
  • Tregs can be depleted by administering Tregs depleting molecules in order to further reduce Attorney Docket No.: 3115-00170 suppression of the immune system as described above.
  • Induction and continued development of blood vessels in tumors can be prevented by administering angiogenesis-associated antigens as described above.
  • Potentiating agents can be administered that potentiate the effect of the components of the composition as described above.
  • the method of treating cancer can further include performing a therapy such as chemotherapy, radiation, anti-angiogenic therapy, and combinations thereof.
  • a therapy such as chemotherapy, radiation, anti-angiogenic therapy, and combinations thereof.
  • Each of these therapies is more effective when performed in combination with the composition of the present invention than when performed alone.
  • the present invention also provides for a method of reversing immune suppression and gaining immunity to cancer by administering the composition described above including synergistic amounts of the primary cell-derived biologic including the cytokines IL-1 , IL-2, IL-6, IL-8, TNF-cc, and IFN- ⁇ , and a synthetic long peptide vaccine to a tumor associated antigen such as WT1 , stimulating the production of na ' ive T cells, maturing immature dendritic cells, and allowing presentation by resulting mature dendritic cells of the exogenous antigens in the cancer vaccine to the na ' ive T cells, thereby reversing immune suppression and gaining immunity to the cancer.
  • the primary cell-derived biologic and the cancer vaccine work together in order to effectively turn on the immune system in the immune suppressed patient and stimulate immunity to the cancer based on the exogenous antigens.
  • T cells in the patient can also be co-stimulated by administering co-stimulatory molecules as described above to generate antigen nonspecific signals to T cells.
  • Inhibition of T cells can be prohibited by administering anti- CTLA-4 as described above in order to further stimulate the T cells as described above.
  • Tregs can be depleted by administering Tregs depleting molecules in order to further reduce suppression of the immune system as described above.
  • Induction and continued development of blood vessels in tumors can be prevented by administering angiogenesis-associated antigens as described above.
  • Potentiating agents can be administered that potentiate the effect of the components of the composition as described above.
  • the method of reversing immune suppression and gaining immunity to cancer can further include performing a therapy such as chemotherapy, radiation, anti- angiogenic therapy, and combinations thereof.
  • a therapy such as chemotherapy, radiation, anti- angiogenic therapy, and combinations thereof.
  • Each of these therapies is more effective when performed in combination with the composition of the present invention than when performed alone.
  • the primary cell-derived biologic of the invention acts as an adjuvant, i.e., stimulates or enhances the immune response of a patient to a particular antigen.
  • the IRX-2 compositions and methods of the invention are particularly suited to stimulate T cell-mediated immune responses.
  • Immune responses promoted by the compositions and methods of the invention include the induction or generation of na ' ive T cells, the differentiation and maturation of dendritic cells, allowing for proper presentation of antigen to T cells (e.g., in the lymph nodes), and the activation of monocytes and macrophages.
  • immune responses promoted by the compositions and methods of the invention include tumor infiltration by lymphocytes, tumor fragmentation and regression as well as a reduction in sinus histiocytosis (when present).
  • the primary cell-derived biologic induces immune production and blocks immune destruction.
  • the mechanism of action of the primary cell-derived biologic is further described in U.S. Patent Application No. 12/323,595 to Applicants.
  • compositions and methods of the present invention aid in overcoming immune depression/suppression in patients by inducing the production of na ' ive T cells.
  • na ' ive T cells denotes newly produced T cells, which T cells have not yet been exposed to antigen. Such T cells are non-specific, yet are capable of becoming specific upon presentation of antigen by a mature dendritic cell having antigen, such as tumor peptides, exposed thereon.
  • the compositions and methods of the invention replenish or generate new T cells.
  • the present compositions and methods allow for lymphocyte infiltration into the tumors with significant tumor fragmentation and regression.
  • Such infiltration is important in order to Attorney Docket No.: 3115-00170 maximize clinical response and for the greatest increase in survival rate.
  • lymphocyte:granulocyte or macrophage infiltration of a 90:10 ratio is optimal and T and/or B cell infiltration is preferably diffuse and intense and not peripheral.
  • Tumor reduction and fragmentation in histological samples reflects a good immune response and is indicative of an adjuvant effect by the compositions of the invention.
  • lymph node changes also indicate an effective immune response, such as lymph node enlargement, i.e., not just reversal of tumor-induced reduction of size but overall increase in size compared to the normal node size, as well as increased T and B cell areas.
  • lymph nodes of cancer patients often contain an intrasinusoidal accumulation of large histiocytes, also termed sinus histiocytosis (SH).
  • SH is believed to be the accumulation of immature dendritic cells, which have ingested and processed tumor antigens but are unable to mature and present these tumor peptides to naive T cells.
  • cytokine compositions and methods of this invention reduced SH in the lymph nodes of cancer patients and produced the various lymph changes described above, again indicating an adjuvant effect by the compositions of the invention.
  • cytokine compositions of the present invention promote dendritic cell maturation. Furthermore, the cytokine compositions of the invention also unblock the dendritic cell defect that leads to SH, i.e., by promoting DC maturation, and thus specifically, in cancer patients, the compositions of the invention provide multiple adjuvant effects, i.e., in unblocking DCs in SH in the lymph nodes and in promoting DC maturation generally.
  • the cytokine compositions of the invention also provide a further adjuvant effect by acting as potent activators of monocytes/macrophages.
  • Monocytes are Attorney Docket No.: 3115-00170 precursors to both DCs and macrophages in the body and thus an agent that promotes monocyte/macrophage activation has an adjuvant effect on immune responses in vivo.
  • the primary cell-derived biologic also blocks immune destruction by protecting the activated T cells from apoptosis.
  • One of the mechanisms of tumor escape involves targeted elimination of CD8+ effector T cells through apoptosis mediated by tumor-derived microvesicles (MV).
  • Immunosuppressive MV have been found in neoplastic lesions, sera, ascites and pleural effusions obtained from cancer patients and have been linked to apoptosis and TCR alterations in effector T cells in these patients.
  • MV-driven elimination of effector T cells which are necessary for anti-tumor host defense, contributes to tumor escape and cancer progression. Therefore, protection of anti-tumor effector cells from functional impairments and death is a major objective of immune therapy.
  • Clinical and experimental data show that certain cytokines, especially survival cytokines using the common receptor ⁇ chain, are able to protect activated T cells from tumor-induced death and enhance their anti-tumor activity.
  • the primary cell-derived biologic protects T cells from apoptosis.
  • the expression of anti-apoptotic signaling molecules i.e. JAK-3 and phosphor-Akt
  • the expression of pro- apoptotic molecules i.e. SOCS-2
  • Activation of caspases in CD8+ and CD4+ T lymphocytes is decreased and cFLIP expression is increased.
  • Inhibition of the PI3K/Akt survival pathway is counteracted by IRX-2.
  • the T cells are protected from both extrinsic apoptosis (MV-induced and FasL-induced apoptosis) and intrinsic mitochondrial apoptosis.
  • the protection from extrinsic MV-induced apoptosis is further accomplished by preventing down-regulation of JAK3, ⁇ 3- ⁇ , and STAT5; inhibiting dephosphorylation of Akt-1 /2; and maintaining balanced ratios of Bax/Bcl-2, Bax-Bcl-xL, and Bim/Mcl-1 .
  • the protection from MV-induced apoptosis is also accomplished by preventing induction of the activity of caspase-3 and caspase-7. More specifically, the induction of the active cleaved form of caspase-3 is blocked, as is the loss of mitochondrial membrane potential. Nuclear DNA fragmentation is inhibited. Protection from intrinsic apoptosis by the primary cell-derived biologic is shown by its protection of activated T cells from staurosporine-induced apoptosis. Attorney Docket No.: 3115-00170
  • the cytokines of the primary cell-derived biologic protect the activated T cells from apoptosis in a synergistic manner.
  • the combination of the cytokines in the primary cell-derived biologic produces a greater affect than is seen by administering individual cytokines alone.
  • compositions and methods of the present invention stimulate the immune system via multiple effects, including the in vivo maturation of dendritic cells resulting in effective peptide antigen presentation as well as activation of monocytes and macrophages and the production of na ' ive uncommitted T cells.
  • the proper presentation of antigen leads to T and B cell clonal expansion, creating immunity in the patient.
  • the effects noted above result in the infiltration, e.g., of lymphocytes, into tumors (e.g., via hematogenous spread) and tumor reduction and/or destruction.
  • the result is increased survival due to immunologic memory.
  • the cytokine composition of the present invention is injected around lymphatics that drain into lymph nodes regional to a lesion, such as a tumor or other persistent lesions being treated. More specifically, local perilymphatic injections or other injections that are known to those of skill in the art are administered to provide sufficient localization of the immunotherapy preparation.
  • a ten (10) day injection scheme for administration of the compositions of the invention is preferred, but a twenty (20) day injection protocol can be used. Bilateral injections are effective. Where radical neck dissection has occurred, contralateral injection is effective.
  • exogenously provided synthetic or extracted antigens such as tumor antigen and peptides (see Bellone, 1998) can be administered into the pre-primed or co-primed regional or distal lymph node, either in a separate preparation or as part of the cytokine composition of the invention.
  • Endogenous suppression of T cells which can be caused by, e.g., cancer or other immunosuppressive diseases, can be blocked by the co-administration of low Attorney Docket No.: 3115-00170 dose cyclophosphamide (CY) and a non-steroidal anti-inflammatory drug (NSAID) (i.e., in combination with the cytokine compositions of the invention).
  • NSAID non-steroidal anti-inflammatory drug
  • the NSAID is preferably indomethacin (INDO) but ibuprofen or Coxll inhibitors such as celecoxib (CELEBREX®) or rofecoxib (VIOXX®) or combinations thereof can also be used.
  • Zinc and multi-vitamins can also be added as agents to help restore T cell immunity.
  • the dose of zinc is 15 to 75 mg.
  • a standard multivitamin can be administered.
  • the zinc can be an available gluconate.
  • the cytokine compositions of the invention can be administered prior to or after surgery, radiotherapy, chemotherapy, or combinations thereof.
  • the compositions of the invention can be administered during the recurrence of tumors, i.e., during a period where tumor growth is occurring again after a period where tumors were thought to have disappeared or were in remission.
  • the cytokine compositions of the present invention are administered and dosed to promote optimal immunization either to exogenous or endogenous antigen, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, and body weight.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to promote immunization, leading to, e.g., tumor reduction, tumor fragmentation and leukocyte infiltration, delayed recurrence or improved survival rate, or improvement or elimination of symptoms, including increased T cell counts.
  • compositions of the present invention can be administered in various ways.
  • the cytokines or exogenous antigens used in the compositions of the invention can be administered in their standard forms or as pharmaceutically acceptable derivatives and can be administered alone or as active ingredients in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles.
  • the compositions of the invention can be administered intra- or subcutaneously, or peri- or intralymphatically, intranodally or intrasplenically or intramuscularly, intraperitoneal ⁇ , and intrathorasically.
  • the patient being treated is a warm-blooded animal and, in Attorney Docket No.: 3115-00170 particular, mammals including man.
  • the pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.
  • the doses can be single doses or multiple doses over a period of several days.
  • they are generally formulated in a unit dosage injectable form (e.g., solution, suspension, or emulsion).
  • a unit dosage injectable form e.g., solution, suspension, or emulsion.
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, liposomes, polymers, polymeric microspheres, polysialic acid, poly lactic glycolic acid microspheres, poly amino acid nanogel systems, protein grafted co-polymers, hydroxyethyl starch or vegetable oils.
  • polyol for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like
  • suitable mixtures thereof liposomes, polymers, polymeric microspheres, polysialic acid, poly lactic glycolic acid microspheres, poly amino acid nanogel systems, protein grafted co-polymers, hydroxyethyl starch or vegetable oils.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as solvent systems for the compositions of the invention.
  • various additives which enhance the stability, sterility, and isotonicity of the compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • Sterile injectable solutions can be prepared by incorporating the cytokines or exogenous antigens utilized in practicing the present invention in the required amount of the appropriate solvent with several of the other ingredients, as desired.
  • a pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicles, additives, and diluents; or the cytokines and/or exogenous antigens utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres. Examples of delivery systems useful in the present invention include those disclosed in U.S. Pat. Nos.
  • compositions and methods of the invention are useful for the treatment of antigen-producing diseases such as cancer, infectious diseases or persistent lesions, as discussed above.
  • the compositions and methods promote immunization against the antigens produced by these diseases by stimulating immune responses in patients in vivo, which immune responses help to alleviate or eliminate the symptoms and effects of the disease in the patient.
  • a murine model is used for proof of principle studies comparing long and short peptides.
  • the model is attractive because there is a high degree of homology between murine and human protein (-95%) (Buckler 1991 ) which means that generating an immune response will require breaking tolerance in mice as it will in humans.
  • These studies will utilize two peptides that have been identified as CTL epitopes in both mouse and human (Oka 2000, 2008). The epitopes are:
  • RFM aa 126-134 (RMFPNAPYL) for C57BI/6 and HLA-A * 0201 (most common sequence used for mouse studies)
  • CMT (aa 235-243 (CMTWNQMNL) for C57BI/6 and HLA-A2402 & HLA- A * 0201 (Lower affinity for C57/BI6 than RFM)
  • CYT aa 235-243 modified
  • CYTWNQMNL mouse epitope binding not yet experimentally assessed
  • XXXXNXXXL contains the anchor motifs for H-2D b so this should be a mouse epitope
  • Analyze sequence and HLA mapping The first step will involve analyzing the WT1 amino acid sequence to identify clusters of HLA class I and II binding sites. Good fit, high affinity binding sites will be mapped and the ten SLPs (30 amino acids or greater) with greatest collection of such sites will be defined. The ends of these sites will be selected based upon immunologic and biochemical considerations.
  • the ultimate objective will be to evaluate these WT1 SLPs and immunoadjuvant/ immuomodulator combinations in a phase l/l I clinical trial.
  • the design of the clinical design will be based on the results of the preclinical experiments and formulation studies being undertaken. Subsequent clinical development will be dependant on the safety and immunogenicity seen in this trial.
  • FIGURE 2 presents the same information in a format that allows the design of long peptides that encompass overlapping HLA's. Based on this initial analysis, 10 sequences have been identified for further evaluation (FIGURES 4 and 5). A finer mapping of the regions is found in FIGURES 6A-6E.
  • the RMF region contains HLA-A, HLA-B and HLA-DR and as such is a "hot spot".
  • the RMF region will be used for pre-clinical evaluation with IRX2.
  • Multiple long peptides from WT1 with multiple embedded class I and or II epitopes can be identified. Ends of the peptides can be modified to optimize their biochemical features. Lowering the predictive scores for mapping can result in additional epitopes, however, the value may be suspect. Not all predicted peptides may pass the immunogenicity validation studies. Screening in vitro for processing is critical for confirming optimal peptide sequences.
  • the C57B/6 model is a relevant model for responses to tolerogenic self tumor antigen.
  • FIGURE 8 The results for the expansion assay are presented in FIGURE 8 as ELISpots using the spleen cells incubated with the RMFshort peptide pulsed EL-4 cells. It can be seen that effector cells generated by culture with peptide pulsed EL-4 irradiated cells showed far greater activity than those seen in the primary Attorney Docket No.: 3115-00170 immunized population. Thus, when the expansion protocol was used, it was possible to demonstrate that mice immunized with the short peptide with IRX-2 had a stronger T cell response than mice receiving only the short peptide in Montanide. Spleen cells incubated with irradiated EL-4 cells that were not pulsed with peptide had equal or fewer spots, consistent with peptide specific expansion in the presence of IL-2 (data not shown).
  • an effective therapeutic vaccine against cancer can be formulated by combining the primary cell derived biologic IRX-2 with a synthetic long peptide including at least one Class I MHC binding epitope and at least one Class II MHC binding epitope of the tumor associated antigen WT1 .
  • mice immunized with the long peptide recognized multiple epitopes
  • the 4 best scoring CTL/CD8 binding epitopes in the long peptide were used as target epitopes in the ELISpot assay.
  • Mice immunized with the long peptide and spleen cells 9-16 days after a booster immunization were evaluated for peptide specific ELISpot response.
  • the peptides evaluated in the ELISpot included the long peptide and 4 of the peptides representing epitopes predicted to be positive in the C57BI/6 mice (see TABLE 2). The results are presented for mice from 3 separate experiments based on the presence of a strong ELISpot response to the long peptide in order to evaluate the contribution of the individual epitopes to the response.
  • mice Na ' i ' ve mice from the three experiments were included as negative controls.
  • the mice are numbered sequentially and ranked based on the magnitude of the increase over the media control for the long peptide. The results shown are for the epitope specific increase. Values greater than 50 spots (2 x average of na ' ive mice) are defined as positive. See TABLE 2 for comparison of response and prediction scores.
  • mice that were positive for the long peptide recognized multiple epitopes whether defined by the number of mice with an ELISpot response greater than media (ie % positive mice) or ranked by magnitude of the response (ie greater than 2x the na ' ive mouse controls; > 50 ELISpots).
  • peptide-based vaccines include safety, specificity and ease of cGMP production.
  • limitations with small peptide vaccines including MHC restriction, lack of helper activity and weak presentation of antigen by APC due to exchange into the MHC as compared to cross presentation processing.
  • only limited clinical anti-tumor activity has been documented to date with these types of peptide vaccines.
  • the limitations can be somewhat overcome by using multiple peptide vaccines which include both class I and class II epitopes, the processing issue can only be overcome by larger constructs such as recombinant protein or longer peptides with overlapping MHC I and II epitopes.
  • IRX-2 is a multi- targeted primary cell-derived biologic that shapes the adaptive immune response toward effective T cell activation.
  • the synergistic activity of the cytokines in IRX-2 is directed towards both the antigen presenting cells and the activated T cells resulting in a novel T cell immune modulator.
  • Preclinical studies have demonstrated IRX-2 antitumor activity in tumor models especially in conjunction with cyclophosphamide and chemo-radiation. Enhancement of the response of mice to several vaccine constructs has also been demonstrated.
  • IRX-2 With respect to mechanism of action of IRX-2, among the many immune activities demonstrated for IRX-2 in vitro are activation of antigen Attorney Docket No.: 3115-00170 presenting cells (dendritic, monocytes/macrophages and Langerhans cells) and T cells. While the T cell activity of cytokines such as IL-1 , IL-2 and IFN- ⁇ are well defined, only recently has the activity on APCs been appreciated. In addition to increasing the activation of DCs as measured by surface expression of many activation markers, IRX-2 reversed the depressed antigen processing activity of dendritic cells from cancer patients (Schilling et al., 2010).
  • IRX-2 has also been shown to reverse tumor induced death of T cells thus providing an additional mechanism of action for IRX-2 in a tumor vaccine model (Czystowska et al., 201 1 ).

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Abstract

Cette invention concerne une composition utilisée pour traiter le cancer ou d'autres maladies et constituée d'un agent biologique cellulaire principal, notamment les cytokines IL-1, IL-2, IL-6, IL-8, TNF-α et IFN‑γ, et le long peptide synthétique d'un antigène associé aux tumeurs. Cette invention concerne une composition contenant un vaccin à long peptide synthétique dirigé contre un antigène spécifique de tumeurs. Cette invention concerne aussi des vaccins thérapeutiques utilisés dans le traitement du cancer ou d'autres maladies. Cette invention concerne également une méthode de traitement du cancer ou d'autres maladies consistant à activer les cellules effectrices Tc, à activer les cellules effectrices Th et à promouvoir la différenciation des cellules présentatrices de l'antigène, leur maturation et la présentation de l'antigène. Cette invention concerne par ailleurs une méthode d'inversion de la suppression immunitaire et d'amplification de l'immunité vis-à-vis du cancer. Cette invention concerne en outre une méthode de traitement du cancer consistant à administrer une quantité efficace d'un long peptide synthétique d'un antigène associé aux tumeurs.
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US9492517B2 (en) 2000-10-27 2016-11-15 Irx Therapeutics, Inc. Vaccine immunotherapy
US8784796B2 (en) 2000-10-27 2014-07-22 Irx Therapeutics, Inc. Vaccine immunotherapy for treating hepatocellular cancer in immune suppressed patients
US9789173B2 (en) 2000-10-27 2017-10-17 Irx Therapeutics, Inc. Vaccine immunotherapy for treating cervical cancer in immune suppressed patients
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US9539320B2 (en) 2009-05-15 2017-01-10 Irx Therapeutics, Inc. Vaccine immunotherapy
US9566331B2 (en) 2009-05-15 2017-02-14 Irx Therapeutics, Inc. Vaccine immunotherapy
US9333238B2 (en) 2009-12-08 2016-05-10 Irx Therapeutics, Inc. Method of immunotherapy for treament of human papillomavirus infection
US9931378B2 (en) 2009-12-08 2018-04-03 Irx Therapeutics, Inc. Method of immunotherapy for treatment of human papillomavirus infection
US9987357B2 (en) 2013-02-04 2018-06-05 Oncomed Pharmaceuticals, Inc. Methods and monitoring of treatment with a WNT pathway inhibitor
WO2017040666A3 (fr) * 2015-08-31 2017-05-11 Oncomed Pharmaceuticals, Inc. Polythérapie pour le traitement d'une maladie
US11419927B2 (en) 2016-06-02 2022-08-23 Ultimovacs As Vaccine in combination with an immune checkpoint inhibitor for use in treating cancer
US12006354B2 (en) 2017-05-24 2024-06-11 Novartis Ag Antibody-IL2 engrafted proteins and methods of use in the treatment of cancer
US12006348B2 (en) 2017-09-07 2024-06-11 Cue Biopharma, Inc. T-cell modulatory multimeric polypeptide with conjugation sites and methods of use thereof
CN113395976A (zh) * 2018-10-05 2021-09-14 株式会社癌免疫研究所 良性肿瘤的预防或治疗药
CN114908051A (zh) * 2022-05-12 2022-08-16 洪纪宪 一种逆转生物年龄制剂及其制备方法

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